Rsubread/CHANGELOG0000644000175200017520000007624614141550405014573 0ustar00biocbuildbiocbuild6 Nov 2021 - 2.8.1 (1) Fixed a bug that caused error when featureCounts runs in Windows on BAM files larger than 4GB. 12 Oct 2021 - 2.7.5 (1) Fixed a bug that caused error when the programs are compiled in a 32-bit Windows platform. 10 Oct 2021 - 2.7.4 (1) Polished for the next release. 6 Jul 2021 - 2.7.3 (1) Updated the user manual to include the "--countReadPairs" option in the example. 12 Jun 2021 - 2.7.2 (1) Fixed a bug in featureCounts that cannot process multiple BAM files when "_R_CHECK_LENGTH_1_CONDITION_" is set to "verbose". 24 May 2021 - 2.7.1 (1) Fixed a bug in cellCounts reporting wrong "assigned cell number" in the summarisation data-frame, and fixed a bug in cellCounts that generated no results when the sample names are numbers. 8 May 2021 - 2.5.9 (1) Updated NEWS and cellCounts Rd files. (2) Updated users guide. 29 Mar 2021 - 2.5.8 (1) Fix a few bugs and match the Subread-2.0.2 release. 23 Mar 2021 - 2.5.7 (1) Make the results always consistent no matter if the aligner was run or not, in cellCounts. 5 Mar 2021 - 2.5.6 (1) Raise the limit to the maximum length of a line in the annotation file because some new annotation files from NCBI have very long lines. (2) Improve cellCounts to use less memory in the counting step and run faster. 3 Feb 2021 - 2.5.5 (1) Increase chunk size and modify indel-merging in cellCounts. 11 Jan 2021 - 2.5.4 (1) Fix a bug in cellCounts that reads the sample sheet with multiple FASTQ files in a unpredictable order. 4 Jan 2021 - 2.5.3 (1) Allow the user to specify the UMI-number cutoff to cellCounts. (2) Update the document of cellCounts, describing the new format of the output object. 16 Dec 2020 - 2.5.2 (1) Use the two-step UMI merging algorithm in cellCounts. 14 Dec 2020 - 2.5.1 (1) Support FASTQ input in cellCounts. 25 Oct 2020 - 2.3.19 (1) Updated cellCounts.Rd and users guide. 22 Oct 2020 - 2.3.17 (1) Fixed the error caused by the new time-getting function on Windows. 21 Oct 2020 - 2.3.16 (1) Updated users guide and NEWS file. 20 Oct 2020 - 2.3.15 (1) Remove warnings from a deprecated function and string bondary checks. 16 Oct 2020 - 2.3.14 (1) Remove cellCounts-related parameters from align(), subjunc() and featureCounts(). 14 Oct 2020 - 2.3.13 (1) Largely change cellCounts() to remove bugs and improve the cell-rescuing algorithm. 25 Aug 2020 - 2.3.12 (1) Change the structure of the returned object from cellCounts(). 19 Aug 2020 - 2.3.11 (1) Keep results from cellCounts() consistent when run in multiple threads. 8 Aug 2020 - 2.3.10 (1) Add "Assigned.Reads" output in cellCounts(). 24 July 2020 - 2.3.9 (1) Fix the error when compiled by gcc-10. 21 July 2020 - 2.3.8 (1) Largely change the isPairedEnd option in featureCounts() and added countReadPairs option. 18 July 2020 - 2.3.7 (1) Report mapped and all reads in cellCounts per sample. 11 July 2020 - 2.3.6 (1) Improve cellCounts to be more conservative in mapping reads. 7 July 2020 - 2.3.5 (1) Improve the screen outputs and removed redundant error messages. 2 July 2020 - 2.3.4 (1) Update the Email addresses of two authors. 24 June 2020 - 2.3.3 (1) Update GPL license to GPL (>=3) instead of GPL-3 only. 17 May 2020 - 2.3.2 (1) Check the ambient-RNA profile in cellCounts and do not do cell-barcode rescue if no ambient-RNA profile can be built. 2 May 2020 - 2.3.1 (1) Fix the '\r\n' end-of-line issue in the chr alias file. (2) Support multiple feature types in GTF annotations. 17 April 2020 - 2.1.5 (1) Update users guide and NEWS. 17 Apr 2020 - 2.1.4 (1) Fix warnings in Windows Mingw32. 15 Apr 2020 - 2.1.3 (1) Add the "aligner" option for cellCounts. 18 Feb 2020 - 2.1.2 (1) Use raw file names in column names in featureCounts() and other functions. 9 Dec 2019 - 2.1.1 (1) Update the ambient-RNA cell-barcode detection algorithm in cellCounts(). 25 Oct 2019 - 1.99.8 and 1.99.9 (1) Update manual pages using the standard format. 21 Oct 2019 - 1.99.7 (1) Update users guide. 16 Oct 2019 - 1.99.6 (1) Updated NEWS file. 11 Oct 2019 - 1.99.5 (1) Fixed bug in long read assignment when the input format is BAM. 8 Oct 2019 - 1.99.4 (1) Disabled the error message in featureCounts about the long read-record lengths but automatically switch into the "safe mode". 16 Sep 2019 - 1.99.3 (1) Fixed a bug of showing misleading chromosome names that are unique in the index or unique in the annotation file in subjunc() and align() when an alias file is provided. 3 Sep 2019 - 1.99.2 (1) Fixed a bug about sorting the BAM or SAM input files. 2 Sep 2019 - 1.99.1 (1) Updated users guide. (2) Added SingleCell to bioc views. 29 Aug 2019 - 1.99.0 (1) Delete .BBSoption file for that it causes a NOTE. (2) Increase version number to 1.99.0 for the next major release. 29 Aug 2019 - 1.35.25 (1) Remove warning messages when building on 32-bit Windows. (2) Issue warning messages in some functions if the system is 32-bit. 28 Aug 2019 - 1.35.24 (1) Re-submit for the latest changes 26 Aug 2019 - 1.35.23 (1) Remove "UnsupportedPlatforms" to make the package available for Windows users. 26 Aug 2019 - 1.35.22 (1) Add support to the Windows platform (Rstudio + Rtools) 21 Aug 2019 - 1.35.21 (1) Fix a bug in featureCounts() that was introduced in the 1.35.20 version 21 Aug 2019 - 1.35.20 (1) Add "cellCounts" function for scRNA read mapping and counting. 19 Jul 2019 - 1.35.19 (1) Update users guide and help pages for flattenGTF and featureCounts. 19 Jul 2019 - 1.35.18 (1) Synchronize with the 1.34.5 version in the release branch. 11 Jul 2019 - 1.35.17 (1) Add "Read_Type" and "Singleton" categories into the statistics from the featureCounts() function. 8 Jul 2019 - 1.35.15 (1) Use match.arg() to test the arguments to the method parameter in flattenGTF and the to the readShiftType parameter in featureCounts. 3 Jul 2019 - 1.35.14 (1) Add the method option to the flattenGTF() function. 26 Jun 2019 - 1.35.12 (1) Update help page for align and subjunc. 25 Jun 2019 - 1.35.11 (1) Update documentation regarding memory use and running time. 24 Jun 2019 - 1.35.10 (1) Use proper way to calculate the free memory size on Mac OS. 19 Jun 2019 - 1.35.9 (1) Disable memory checks on Mac OS. 14 Jun 2019 - 1.35.8 (1) Improve buildindex() to largely reduce peak memory use. 7 Jun 2019 - 1.35.7 (1) Add nreads=-1 to qualityScores() function for using all the reads in the input file. 6 Jun 2019 - 1.35.6 (1) Fix a bug in the qualityScores() function that returned fewer columns than the length of the longest read that is sampled. 6 Jun 2019 - 1.35.5 (1) Fix a typo in scanFasta(): the "Occurance" column in the returned object has been changed to "Occurrence". 6 Jun 2019 - 1.35.4 (1) Fix a bug in scanFasta(): the old parameter spliter, comma, was used in a part of the function. 19 May 2019 - 1.35.3 (1) Reduce the lengths of column names in the returned objects from featureCounts(), align(), subjunc() and propmapped(). 13 May 2019 - 1.35.2 (1) Add the check on the normalised file name lengths. (2) Terminate featureCounts() if both single- and paired-end reads are found in the same input file. (3) Add a warning message for specifying isPairedEnd=TRUE on single-end input files. 7 May 2019 - 1.35.1 (1) Change the splitor to '\027' for passing arguments to the C functions. (2) Change the maximum length of file paths to 1000 bytes. 12 Apr 2019 - 1.33.41 (1) Update users guide. 11 Apr 2019 - 1.33.40 (1) Update NEWS.Rd. 11 Apr 2019 - 1.33.39 (1) Fix a bug in the new voting algorithm that do not work with long indel detection. 11 Apr 2019 - 1.33.38 (1) Fix a bug in the new voting algorithm that can lead to infinit loop. 11 Apr 2019 - 1.33.37 (1) Change the total length of parameters for many functions from 1000 bytes to 10240 bytes. 10 Apr 2019 - 1.33.36 (1) Improve the speed of voting by removing the backward scanning and minor changes. 9 Apr 2019 - 1.33.35 (1) Change the shifting-indel read voting algorithm to only disable indel detection at locations where the shifting-indel events are found. 9 Apr 2019 - 1.33.34 (1) Improve mapping algorithm in align() and subjunc() by treating junctions and indels in different ways, and detect shifting-indel reads then disallowing indels in them. (2) Fix a bug in align() on DNA-seq mode. 8 Apr 2019 - 1.33.33 (1) Improve mapping algorithm in align() and subjunc() by preferring no-indel alignments, learning mean TLEN from the first 1000 read-pairs and removing symmetric indels in the voting table. 5 Apr 2019 - 1.33.32 (1) Amend documents for simReads() and scanFasta() to reflect the latest changes. 29 Mar 2019 - 1.33.31 (1) align() and subjunc() now write the summary mapping statistics for each sample to a file (as well as returning the same statistics, combined over samples in a data.frame). 28 Mar 2019 - 1.33.30 (1) Enable single-end read simulation in simReads(). 25 Mar 2019 - 1.33.29 (1) Amend the document of simReads() and scanFasta() to remove a warning message. 21 Mar 2019 - 1.33.28 (1) Change the type of "expression.levels" in simReads() from data.frame to vector. 20 Mar 2019 - 1.33.27 (1) Fix simReads() to correctly treat the coordinates as "1-based" coordinates. 20 Mar 2019 - 1.33.26 (1) Change simReads() to draw the numbers of read-pairs generated from transcripts from a multinomial distribution. 15 Mar 2019 - 1.33.25 (1) Add hidden file .Rbuildignore in package folder to tell R not to include the .BBSoptions file in package builds. 14 Mar 2019 - 1.33.24 (1) Shorten package Description slightly. (2) Add 5 BiocViews suggested by BiocCheck. (3) Use "fragment" instead of "insertion" in simReads arguments, code and documentation. 13 Mar 2019 - 1.33.23 (1) Some code polishing for simReads. (2) Simplify error messages from simReads. (3) Add comments about read order to Details section of align.Rd 12 Mar 2019 - 1.33.22 (1) Edit Value and Details sections of align.Rd. (2) Update references in buildindex.Rd and add a 'see also' to align.Rd. (3) Add packages mentioned in NAMESPACE to Import field of DESCRIPTION. 11 Mar 2019 - 1.33.21 (1) Add comments to Details section of propmapped.Rd to alert users that align and subjunc return the same statistics. 11 Mar 2019 - 1.33.20 (1) The summary data.frames returned by align and subjunc now have the field names as row.names instead of a first column. The column names are now the basenames of the bam files instead of full paths. 10 Mar 2019 - 1.33.19 (1) propmapped now returns file names as row.names instead of as column. (2) Remove unnecessary as.character() in default arguments for align() and subjunc(). (3) Use doi link as page numbers and Epub date as volume in CITATION 9 Mar 2019 - 1.33.18 (1) Add a strategy in simReads() to iteratively find the best value of N based on the wanted sample size. 8 Mar 2019 - 1.33.17 (1) Change featureCounts() to allow RNAME = "*" for reads reported as mapped and having non-empty CIGAR strings in the SAM or BAM input. (2) Check the integrity of the index in align() and subjunc(). 28 Feb 2019 - 1.33.16 (1) Edits to package citation file. 27 Feb 2019 - 1.33.15 (1) Add BAM format support in removeDupReads(). 25 Feb 2019 - 1.33.14 (1) Update citation info. 25 Feb 2019 - 1.33.13 (1) Update users guide. 5 Feb 2019 - 1.33.12 (1) Update processExons.Rd file. 3 Feb 2019 - 1.33.11 (1) Remove irregular calls of rand(), srand() and removed the modification of the R stack limit. (2) Register the ".C" functions when the Rsubread package is loaded. 2 Jan 2019 - 1.33.8 (1) Remove .tex file from vignettes directory. (2) Update the vignette. 15 Dec 2018 - 1.33.7 (1) Add "low.transcripts" option to simReads() to avoid generating reads from transcripts that have less than one expected reads. 14 Dec 2018 - 1.33.6 (1) Update the document for simReads() and scanFasta(): "contig" is changed to "transcript". (2) Change the simReads() function to generate quality strings of "H"s for non-sequencing error reads. 5 Dec 2018 - 1.33.5 (1) Add biocViews tag 'ImmunoOncology' to the biocViews. 5 Dec 2018 - 1.33.4 (1) Indicate the uniqueness of sequences in the returned data frame from scanFasta() (2) Minor changes on the screen outputs from scanFasta() and genReads() 1 Dec 2018 - 1.33.3 (1) Change functions generateSimulatedReads() and summarizeContigs() to simReads() and scanFasta(). (2) Add warning and error messages to simReads() and scanFasta() for duplicate contig sequences and duplicate contig names. (3) Support gzipped FASTA format input in align(), subjunc() and buildindex(). 26 Nov 2018 - 1.33.2 (1) Add generateSimulatedReads() and summarizeContigs() functions. 8 Nov 2018 - 1.33.1 (1) Improve help page for align and subjunc. 25 Oct 2018 - 1.31.23 (1) Fix a bug in findCommonVariants() running in Mac OS X. 23 Oct 2018 - 1.31.22 (1) Update users guide. 19 Oct 2018 - 1.31.21 (1) Update help page for featureCounts. (2) Update users guide. 18 Oct 2018 - 1.31.20 (1) Fix the format of NEWS file. 15 Oct 2018 - 1.31.19 (1) Add the read shifting operation into featureCounts(). 12 Oct 2018 - 1.31.18 (1) Update NEWS. 29 Sept 2018 - 1.31.17 (1) Further change the order of locations in the index. (2) Remove two screen output messages from featureCounts(). 28 Sept 2018 - 1.31.16 (1) Change the order of locations of the same subread in the index, making the mapping results consistant no matter how many buckets are in the index. 26 Sept 2018 - 1.31.15 (1) Update align.Rd and buildindex.Rd. 26 Sept 2018 - 1.31.14 (1) Update users guide. 20 Sep 2018 - 1.31.13 (1) Changed the return format of align() and subjunc() to include more information (2) Fixed the issue of featureCounts(), align() and subjunc() not quiting when the annotation format is wrong. 18 Sep 2018 - 1.31.12 (1) align() and subjunc() return the mapping summary data frame. The summary includes total reads, mapped reads, indels, etc. 11 Sep 2018 - 1.31.11 (1) Fixed a big that featureCounts didn't allow gzipped annotation. 10 Sep 2018 - 1.31.10 (1) Fixed all bugs found in pre-release tests. 8 Sep 2018 - 1.31.9 (1) Fixed some bugs found in pre-release tests. 4 Sep 2018 - 1.31.8 (1) Largely improved the speed of align and subjunc (2) Minor changes on the screen outputs of align, subjunc and featureCounts (3) Improve documentation for 'read2pos' option in featureCounts. (4) Bug fixes 17 Aug 2018 - 1.31.7 (1) Default value of 'countMultiMappingReads' parameter in featureCounts is changed to TRUE. 11 July 2018 - 1.31.6 (1) Fix a typo in atgcContent.Rd file. 22 June 2018 - 1.31.5 (1) Change default setting of buildindex() to enable one-block full index is generated by default. 15 May 2018 - 1.31.4 (1) Provide download link for human reference genome in builindex() help page and users guide. 11 May 2018 - 1.31.3 (1) Update users guide. (2) Update help page for align, subjunc, featureCounts and flattenGTF. 10 May 2018 - 1.31.2 (1) Update users guide. (2) Place users guide under 'doc' directory under 'inst'. (3) Update help page for buildindex() function. 9 May 2018 - 1.31.1 (1) Added the 'sortReadsByCoordinates' option to align() and subjunc(). (2) Added the flattenGTF() function. (3) Allowed featureCounts() to apply different strandness checks on different input files. (4) Added the 'additionalAttributes' option to featureCounts(). 27 April 2018 - 1.29.6 (1) Removed warning messages from the installation of the package. 23 April 2018 - 1.29.5 (1) Update users guide. 19 April 2018 - 1.29.4 (1) Update NEWS file. 10 Apr 2018 - 1.29.3 (1) Largedly improved the speed of featureCounts on the BAM files that have reads overhanging two gzipped blocks. This includes BAM files from STAR and Picard. 28 Mar 2018 - 1.29.2 (1) Added 'reportReadsPath' option to featureCounts(). (2) Allowed tilde (~) in input or output file names for all the functions. (3) Enabled multi-threaded BAM compression in align() and subjunc(). (4) Added 'keepReadOrder' option to align() and subjunc() for multi-threaded BAM compression. (5) Changed screen output of some functions by removing the full paths from the file names shown on screen. (6) Fixed a bug in featureCounts() on long-read mode. 21 Mar 2018 - 1.29.1 (1) Fixed two bugs in featureCounts related to the BAM files generated by using STAR or Picard packages. (2) Added sublong() function for long read mapping. (3) Added ‘nonOverlap’ and ‘nonOverlapFeature’ options to featureCounts(). (4) Added gzipped GTF, SAF and VCF file support to featureCounts(), align(), subjunc() and exactSNP() functions. (5) Default value of maxReads argument in exactSNP() is changed to 1000000. (6) Update users guide. 25 Oct 2017 - 1.27.9 (1) Update help page for featureCounts. 20 Oct 2017 - 1.27.8 (1) updated users guide. 19 Oct 2017 - 1.27.7 (1) C code update from Yang. 18 Oct 2017 - 1.27.6 (1) Add ‘verbose’ option to propmapped(). (2) Add ‘fracOverlapFeature’ to featureCounts(). (3) Updated NEWS file. 14 July 2017 - 1.27.4 (1) C code from SF Subread 1.5.3. 11 July 2017 - 1.27.3 (1) Count long reads in featureCounts. (2) Add ‘verbose’ option to featureCounts. (3) Detailed assignment results can be saved to text file or SAM/BAM file by featureCounts. (4) Filters included in featureCounts counting summary are ordered by their order being applied in counting. (5) Update users guide. 1 May 2017 - 1.27.2 (1) Added txUnique() function. (2) C code in align() and subjunc() has -u option removed. (3) New option in featureCounts to allow counting of reads in each read group individually. (4) Align() and subjunc() report percentage of uniquely mapped reads, multi-mapping reads and unmapped reads. 27 April 2017 - 1.27.1 (1) Added promoterRegions() function. (2) Modified getInBuiltAnnotation() function. 24 April 2017 - 1.25.3 (1) Updated NEWS file. 15 March 2017 - 1.25.2 (1) Incorporate SF Subread 1.5.2. (2) Update users guide. 9 March 2017 (1) Align and subjunc function can take user provided annotation for exon-exon junction detection. (2) Dynamic programming parameters added to subjunc. (3) Updated users guide. 12 Dec 2016 - 1.25.1 (1) Update help page for buildindex function. 14 Oct 2016 - 1.23.8 (1) Updated users guide. 14 Oct 2016 - 1.23.7 (1) Fixed some warnings from C code. 12 Oct 2016 - 1.23.6 (1) Updated NEWS file. 11 Oct 2016 - 1.23.5 (1) Fix annotation issue for detectionCall function. (2) Improved featureCounts help page. 24 Aug 2016 - 1.23.4 (1) Incorporate changes from SF Subread 1.5.1. (2) New parameters in featureCounts: fracOverlap, tmpDir (3) Fractional counts can be generated for multi-overlapping reads. 28 July 2016 - 1.23.3 (1) Coerce file name parameters in align and subjunc to be of character type. 9 June 2016 - 1.23.2 (1) Incoporate further changes from Subread 1.5.0-p3. 27 May 2016 - 1.23.1 (1) Incoporate changes from Subread 1.5.0-p3. 15 Apr 2016 - 1.21.12 (1) Add hg38 annotation. 11 Apr 2016 - 1.21.11 (1) Incorporate more C-code update from Subread 1.5.0-p2. 6 Apr 2016 - 1.21.10 (1) Incorporate C-code update from Subread 1.5.0-p2. 30 Mar 2016 - 1.21.9 (1) processExons() and detectionCallAnnotation() functions return NULL object. (2) Update documentation for processExons() and detectionCallAnnotation() functions. (3) Update DESCRIPTION for the package. 24 Mar 2016 - 1.21.8 (1) Removed 'PE_orientation' parameter from featureCounts. 1 Feb 2016 - 1.21.7 (1) 'ran' and 'dan' were added as possible values for type parameter in align(). 29 Jan 2016 - 1.21.6 (1) add parameter '--nonSplitOnly' to featureCounts. 18 Dec 2015 - 1.21.5 (1) Incorporate changes made in SourceForge Subread 1.5.0-p1 release. 1 Dec 2015 (1) Change order of parameters of featureCounts function. 16 Nov 2015 - 1.21.4 (1) Fix usage section in Rd file for repair function. 12 Nov 2015 (1) Add repair function (2) New parameters for featureCounts -- juncCounts, genome and maxMOp. (3) Removed 'DNAseq' option from subjunc() function. (4) Parameter 'reportFusions' is changed to 'detectSV' in align() function. (5) Update users guide. 20 Oct 2015 (1) Default value of TH_subread in buildindex() was changed to 100. Devel version changed to 1.21.0 after Bioc 3.2 release. 12 Oct 2015 - 1.19.8 (1) updated users guide. 9 Oct 2015 - 1.19.7 (1) Added parameters "type" and "complexIndels" to align. (2) Added parameter "complexIndels" to subjunc. 8 Oct 2015 - 1.19.6 (1) C code update. 14 Sept 2015 - 1.19.5 (1) Check if input and output file names in align() and subjunc() are valid. 30 July 2015 - 1.19.3 (1) Added 'largestOverlap' option to featureCounts. 15 July 2015 (1) Changed 'minReadOverlap' to 'minOverlap' in featureCounts. 23 June 2015 - 1.19.2 (1) Changed parameter 'sortByName' to 'autosort' in featureCounts. 19 June 2015 - 1.19.1 (1) Added 'sortByName' and "fraction" parameters for featureCounts. (2) Improved the reporting of meta-features that have excessive number of features in featureCounts output. 27 March 2005 (1) Improved documentation for propmapped function. 24 March 2015 - 1.17.2 (1) Changes to Makevars files. 31 Oct 2014 - 1.17.1 (1) Updated citation info. (2) Updated users guide. 10 Oct 2014 - 1.15.12 (1) updated users guide. 9 Oct 2014 - 1.15.11 (1) Fixed a bug in featureCounts for read extension. (1) When full index is built, subreads can be extracted from every position of the read and only one subread set is required for mapping. (2) By default, the maximum allowed mismatches in the mapped reads are 3. (3) The soft-clipping bug in subread/subjunc is fixed. (6) Bug fix for Subindel in generating CIGAR for assembled contigs. (7) Subjunc requires the number of consensus subreads be at least 30% of the extracted subreads when reporting a hit for any exonic read. 7 June 2014 - 1.15.9 (1) Fixed a bug for reporting unmapped reads in SAM output. 18 June 2014 - 1.15.8 (1) Bug fix to scientifc notation issue in featureCounts. 12 June 2014 - 1.15.7 (1) updated C code. 29 May 2014 - 1.15.6 (1) Fixed the codoc mismatch bug for subjunc. (2) Update users guide. 27 May 2014 - 1.15.5 (1) Add maxMismatches parameter to align and subjunc functions. 12 May 2014 -- 1.15.4 (1) Fixed a bug in featureCounts when dealing with a very big annotation file. 9 May 2014 -- 1.15.3 (1) Added 'ignoreDup' option to featureCounts. 2 May 2014 -- 1.15.2 (1) Added read2pos option to featureCounts. 1 May 2014 -- 1.15.1 (1) Added following parameters to featureCounts: readExtension5,readExtension3,minReadOverlap and countSplitAlignmentsOnly. (2) Changed the order of parameters used by featureCounts. 8 April 2014 - 1.13.25 (1) Fixed formatting issue for buildindex function in the vignette. (2) Added release news. 7 April 2014 - 1.13.24 (1) Added return value in sam2bed.c (2) Improved documentation for featureCounts. 1 April, 2014 - 1.13.23 (1) Updated citations in the vignette 31 March, 2014 - 1.13.22 (1) Updated citation for featureCounts. 28 Mar, 2014 - 1.13.21 (1) Improved vignette and users guide. 27 March, 2014 - 1.13.20 (1) Fixed a bug in featureCounts (2) Fixed a bug in propmapped 25 March, 2014 - 1.13.19 (1) Improved output info of featureCounts. 19 March, 2014 - 1.13.18 (1) Updated users manual. 13 March, 2014 - 1.13.17 (1) Subjunc can now output multiple best mapping locations for a read. (2) Default memory size was changed to 8000MB in buildindex function. (3) buildindex function can now build a full index with no gaps. (4) buildindex now supports the generation of a single-segment index. (5) Default input format and output format were changed to 'gzFASTQ' and 'BAM', respectively, for align and subjunc functions. (6) A new argument 'countPrimaryAlignmentsonly' was added into featureCounts. 21 Feb, 2014, 1.13.14 (1) Re-wrote the propmapped function. 21 Jan, 2014 (1) Improved documentation for the Phred offset parameter. 20 Dec, 2013 - 1.13.13 (1) Documentation improvement. 18 Dec, 2013 - 1.13.12 (1) Fixed a bug in featureCounts for processing long header lines in SAM/BAM files. 9 Dec, 2013 - 1.13.11 (1) Added recommended alignment settign in users guide. (2) Updated biocviews terms. 7 Dec, 2013 - 1.13.10 (1) Removed 'doc' directory under Rsubread/inst/ 5 Dec, 2013 - 1.13.9 (1) Fixed a file input issue for exactSNP. 1 Dec, 2013 - 1.13.8 (1) Re-wrote the qualityScores function. (2) Align and subjunc functions allow input of multiple library files at the same time. (3) Align and subjunc functions support gzipped FASTQ input. (4) Align and subjunc output uniquely mapped reads only by default (tieBreakHamming option is turned on by default as well). (5) exactSNP can use annotated SNPs to improve SNP-calling performance. 16 Nov, 2013 - 1.13.7 (1) Fixed a bug in featureCounts in dealing with unpaired reads in SAM/BAM files. 15 Nov, 2013 -1.13.6 (1) featureCounts outputs summary info after read assignments (giving reasons for those unassigned reads). (2) featureCounts automatically re-orders paired-end reads if reads from the same pair are not adjacent to each other ('PEReadsReordering' option is not needed any more). It can also deal with those read pairs that have only one end included in SAM/BAM files. (3) Subjunc has an improved performance in mapping the exon-spanning reads in which junction locations are very close to (1-2bp away from) the ends of reads. (4) Fixed a bug related to FreeBSD installation. (5) Fixed a bug for running on 32-bit machines for a few functions. 11 Nov, 2013 - 1.13.5 (1) Added an example for mapping paired-end reads in the vignette (2) Added an example for featureCounts in the vignette 7 November, 2013 - 1.13.4 (1) featureCounts automatically detects the type of input read files ('file.type' argument removed) (2) subjunc function supports the alignment of genomic DNA-seq reads ('DNAseq' option). It detects junctions and fusions (eg chimera) and report them in the mapping results (optional fields in the SAM/BAM file are used to store the mapping results of second alignments). (3) subjunc function can report non-canonical exon-exon junctions and fusions (eg. chimera) for RNA-seq read data('reportAllJunctions' option), in addition to the reporting of canonical exon-exon junctions. (4) align function reports fusions when mapping gDNA-seq data ('reportFusions' option). 25 Oct, 2013 - 1.13.3 (1) Changed inst/doc to vignettes/ (2) Created 'usersguide' directory under 'inst' to store users guide. 25 Oct, 2013 - 1.13.1 (1) Fixed a bug for featureCounts 14 Oct, 2013 - 1.11.18 (1) Updated users guide. 11 Oct, 2013 - 1.11.17 (1) Updated NEWS, vignette and users guide. 6 Oct, 2013 - 1.11.16 (1) Updated C code with Subread v1.4.0 (2) Modified arguments for exactSNPs, featureCounts and Subjunc. 28 Aug, 2013 - 1.11.15 (1) Fixed a bug for processing long head lines in index building. (2) Fixed a bug for reporting strands for junctions discovered from long reads. 17 Aug, 2013 - 1.11.14 (1) Added support for SunOS. 12 Aug, 2013 - 1.11.13 (1) Added support for freeBSD. 8 Aug, 2013 - 1.11.12 (1) Remove -T parameter from removeDupReads function (2) Fixes a bug in featureCounts. 3 Aug, 2013 - 1.11.11 (1) The maximum allowed length of chromosome names and gene identifiers was increased to 100 for featureCounts function. (2) Documentation improvement for featureCounts. 1 Aug, 2013 - 1.11.10 (1) Added getInBuiltAnnotation function. (2) Improved featureCounts interface. 31 July, 2013 (1) Minor edits on the documentation. 30 July, 2013 - 1.11.9 (1) Add an option to align to support the conversion of color-space bases to base-space bases. (2) Documentation improvement. 3 July, 2013 - 1.11.8 (1) Update createAnnotationFile() function. 25 June, 2013 - 1.11.7 (1) Fixed the RGui output problem. 18 June, 2013 - 1.11.6 (1) Incorporated the changes made in Subread 1.3.5 patches 1-4. 6 June, 2013 - 1.11.5 (1) Incorporated the changes made in Subread 1.3.5 4 June, 2013 - 1.11.4 (1) Modified featureCounts to let it exclude multi-mapping reads from read summarization in its default setting. (2) Added 'countMultiMappingReads' option to allow multi-mapping reads to be counted if required. 2 Jun, 2013 (1) Made changes to the isStrandSpecific parameter of featureCounts. 30 May, 2013 - 1.11.3 (1) Added a users guide. 9 May, 2013 (1) Add more options for featureCounts. April 28, 2013 - 1.11.2 (1) featureCounts now supports multi-threading. April 22, 2013 (1) sam2bed functions now output strandness information. April 17, 2013 - 1.11.1 (1) Allowed multiple equally-best mapping locations to be reported for reads March 31, 2013 - 1.9.8 (1) Document improvement March 29, 2013 - 1.9.7 (1) Changes to the configure file and Makevars.Linux (2) Changes to sorted-hashtable.c March 28, 2013 - 1.9.6 (1) Fix the multithreading issue (2) Documentation improvements March 27, 2013 - 1.9.5 (1) Updated CITATION file. (2) Updated documentation. (3) Modified the arguments of featureCounts() function (4) featureCounts() function now accepts BAM files as input. March 14, 2013 - 1.9.3 (1) Mac support was added. (2) Subread version 1.3.1 was used. (3) Fix warnings related to C functions. August 24, 2012 - 1.7.7 (1) Align and subjunc functions are now updated using the latest Subread package (1.3.0 on sourceforge.net). (2) Major updates to SNP calling function and the function for removing duplicated reads. August 23, 2012 (1) Temporary files generated by functions will be temporarily stored in the current working directory. June 30, 2012 (1) Add mm10 annotation file to featureCounts function. June 26, 2012 - 1.7.6 (1) Add an option to featureCounts to count fragments for paired-end sequencing data. (2) Disable the running of example included in createAnnotationFile.Rd. June 14, 2012 - 1.7.5 (1) featureCounts function now accepts a data frame as the annotation input. June 8, 2012 - 1.7.4 (1) Change static memory allocation to dynamic memory requests for a number of functions to reduce memory use when the package is loaded. (2) The buildindex function no longer requies an allocation of 1GB continous memory block. May 31, 2012 - 1.7.3 (1) Tim Triche added createAnnotationFile function to convert annotations to the format which can be used by featureCounts. April 27, 2012 - 1.7.2 (1) Bug fix to incorrect CIGAR strings. April 3, 2012 - 1.7.1 (1) Bug fix to parameter passing from R to C for align and subjunc (2) Fix memory leakage for align and subjun. March 26, 2012 - 1.5.8 (1) Bug fixes to align and callSNPs function. March 22, 2012 - 1.5.7 (1) Major updates to align and subjunc functions. March 21, 2012 - 1.5.6 (1) Add snpCalls function. (2) Add removeDupReads function. Feb 15, 2012 - 1.5.5 (1) Bug fix to subjunc parameters passed to the underlying C program. February 14, 2012 - 1.5.4 (1) Fixed the bug of mapping reads out of chromosome boundary. Junuary 17, 2012 - 1.5.3 (1) Align function can now report uniquely mapped reads only. (2) Buildindex function now includes the reference sequences in the built index by default. (3) Subjunc now supports up to 16bp INDELs. (4) Subjunc supports extra splicing donor/receptor sites (GC-AG and GT-AT), in addition to the canonical one(GT-AG). November 7, 2011 - 1.5.2 (1) Bug fix to chr3 length issue. November 2, 2011 - 1.5.1 (1) Bug fix to the flag field in SAM output September 28, 2011 - 1.2.1 (1) propmapped function now returns a data frame. (2) remove file name restraint in detectionCall function. (3) output of featureCounts function was changed to a list. (4) fixed the incorrect use of ppois function in detectionCall function August 30, 2011 - 1.2.0 (1) Add subjunc function which can be used to find exon junctions. (2) Add functions for determining detection p values for each gene in RNA-seq data. July 26, 2011 - 1.1.1 featureCounts function now supports human data and allows users to provide their annotation data. July 22, 2011 - 1.1.0 Add featureCounts function, which counts number of mapped reads for each feature. 1.0.0 Package release. Rsubread/CHANGE_IN_RELEASED_VERSION0000644000175200017520000000033614136047615017263 0ustar00biocbuildbiocbuild18 Nov 2020 - 2.4.2 (1) Amended the author information in the document of the simReads() function. 11 Nov 2020 - 2.4.1 (1) Fixed the bug in the align() and subjunc() in generating coordinate-sorted BAM files in Windows. Rsubread/DESCRIPTION0000644000175200017520000000270214142061021015041 0ustar00biocbuildbiocbuildPackage: Rsubread Version: 2.8.1 Date: 2021-11-06 Title: Mapping, quantification and variant analysis of sequencing data Author: Wei Shi, Yang Liao and Gordon K Smyth with contributions from Jenny Dai Maintainer: Wei Shi , Yang Liao and Gordon K Smyth Description: Alignment, quantification and analysis of RNA sequencing data (including both bulk RNA-seq and scRNA-seq) and DNA sequenicng data (including ATAC-seq, ChIP-seq, WGS, WES etc). Includes functionality for read mapping, read counting, SNP calling, structural variant detection and gene fusion discovery. Can be applied to all major sequencing techologies and to both short and long sequence reads. URL: http://bioconductor.org/packages/Rsubread License: GPL (>=3) Imports: grDevices, stats, utils, Matrix biocViews: Sequencing, Alignment, SequenceMatching, RNASeq, ChIPSeq, SingleCell, GeneExpression, GeneRegulation, Genetics, ImmunoOncology, SNP, GeneticVariability, Preprocessing, QualityControl, GenomeAnnotation, GeneFusionDetection, IndelDetection, VariantAnnotation, VariantDetection, MultipleSequenceAlignment git_url: https://git.bioconductor.org/packages/Rsubread git_branch: RELEASE_3_14 git_last_commit: 4465874 git_last_commit_date: 2021-11-05 Date/Publication: 2021-11-07 NeedsCompilation: yes Packaged: 2021-11-07 23:32:33 UTC; biocbuild Rsubread/NAMESPACE0000755000175200017520000000056314136047615014600 0ustar00biocbuildbiocbuilduseDynLib(Rsubread) exportPattern("^[^\\.]") importFrom("grDevices", "dev.off", "pdf") importFrom("stats", "approxfun", "density", "dgamma", "dpois", "lowess", "ppois", "runif", "rmultinom", "dmultinom", "p.adjust") importFrom("utils", "download.file", "read.csv", "read.delim", "write.table", "head", "read.table") importFrom("Matrix", "readMM") Rsubread/R/0000755000175200017520000000000014136047615013553 5ustar00biocbuildbiocbuildRsubread/R/RsubreadUsersGuide.R0000644000175200017520000000025214136047615017444 0ustar00biocbuildbiocbuildRsubreadUsersGuide <- function() { f <- system.file("doc","SubreadUsersGuide.pdf",package="Rsubread") system(paste(Sys.getenv("R_PDFVIEWER"),f,"&")) return(f) } Rsubread/R/align.R0000755000175200017520000002120214136047615014770 0ustar00biocbuildbiocbuild.stop_quietly <- function() { opt <- options(show.error.messages = FALSE) on.exit(options(opt)) stop() } .load.delete.summary <- function(bam.name){ sumfile <- paste0(bam.name,".summary") if(!file.exists(sumfile)){ # stop("Summary file ",sumfile," was not generated! The program terminated wrongly!") .stop_quietly() } tmp.frame <- read.delim(sumfile, header=FALSE, row.names=1, stringsAsFactors=FALSE) # file.remove(sumfile) return(tmp.frame) } align <- function(index,readfile1,readfile2=NULL,type="rna",input_format="gzFASTQ",output_format="BAM",output_file=paste(readfile1,"subread",output_format,sep="."),phredOffset=33,nsubreads=10,TH1=3,TH2=1,maxMismatches=3,unique=FALSE,nBestLocations=1,indels=5,complexIndels=FALSE,nTrim5=0,nTrim3=0,minFragLength=50,maxFragLength=600,PE_orientation="fr",nthreads=1,readGroupID=NULL,readGroup=NULL,keepReadOrder=FALSE,sortReadsByCoordinates=FALSE,color2base=FALSE,DP_GapOpenPenalty=-1,DP_GapExtPenalty=0,DP_MismatchPenalty=0,DP_MatchScore=2,detectSV=FALSE,useAnnotation=FALSE,annot.inbuilt="mm10",annot.ext=NULL,isGTF=FALSE,GTF.featureType="exon",GTF.attrType="gene_id",chrAliases=NULL) { if(!.is.64bit.system()) cat("WARNING: your system seems to be 32-bit. Rsubread supports 32-bit systems to a very limited level.\nIt is highly recommended to run Rsubread on a 64-bit system to avoid errors.\n\n") extra.params <- .retrieve.tmp.parameters() isBCLinput <- FALSE isScRNAFastqinput <- FALSE isScRNABAMinput <- FALSE if("isBCLinput" %in% names(extra.params))isBCLinput <- extra.params[["isBCLinput"]] if("isScRNAFastqinput" %in% names(extra.params))isScRNAFastqinput <- extra.params[["isScRNAFastqinput"]] if("isScRNABAMinput" %in% names(extra.params)) isScRNABAMinput <- extra.params[["isScRNABAMinput"]] .check_string_param(input_format,"input_format") .check_string_param(output_format,"output_format") .check_string_param(PE_orientation,"PE_orientation") .check_string_param(annot.inbuilt,"annot.inbuilt") .check_string_param(GTF.featureType,"GTF.featureType") .check_string_param(GTF.attrType,"GTF.attrType") .check_string_param(readGroupID,"readGroupID") .check_string_param(readGroup,"readGroup") out.base.names <- basename(output_file) if(any(duplicated(out.base.names))){ out.table.cols <- output_file }else{ out.table.cols <- out.base.names } #out.table.cols <-gsub("[[:punct:]]+", ".", out.table.cols) #out.table.cols <-gsub(" ", ".", out.table.cols) readfile1 <- as.character(readfile1) if(!isScRNAFastqinput)readfile1 <- .check_and_NormPath(readfile1, mustWork=TRUE, opt="readfile1") output_file <- .check_and_NormPath(output_file, mustWork=FALSE, opt="output_file") index <- .check_and_NormPath(index, mustWork=FALSE, opt="index") if((!is.null(annot.ext)) && is.character(annot.ext)) annot.ext = .check_and_NormPath(annot.ext, mustWork=TRUE, opt="annot.ext") if(!is.null(chrAliases)) chrAliases = .check_and_NormPath(chrAliases, mustWork=TRUE, opt="chrAliases") if(length(readfile1) != length(output_file)) stop("The number of input file names is different from the number of output file names.") if(!is.null(readfile2)){ readfile2 <- as.character(readfile2) readfile2 <- .check_and_NormPath(readfile2, mustWork=TRUE, "readfile2") if(length(readfile1) != length(readfile2)) stop("The number of file names for the first reads is different from the number of file names for the second reads.") } if(!all(file.exists(dirname(output_file)))) stop("Invalid path was found in output file name(s).") opt <- paste("-i",index,sep=.R_param_splitor) type_C <- -1 if(is.character(type)){ if(tolower(type) == "rna") type_C <- 0 if(tolower(type) == "dna") type_C <- 1 } if(is.numeric(type)){ if (type == 0 || type == 1) type_C <- type } if(type_C == -1) stop("Invalid value was provided for data type.") opt <- paste(opt,"--type",type_C,sep=.R_param_splitor) if(tolower(input_format) == "sam") opt <- paste(opt,"--SAMinput",sep=.R_param_splitor) if(tolower(input_format) == "bam") opt <- paste(opt,"--BAMinput",sep=.R_param_splitor) if(tolower(output_format) == "sam") opt <- paste(opt,"--SAMoutput",sep=.R_param_splitor) opt <- paste(opt,"-n",nsubreads,"-m",TH1,"-p",TH2,"-M",maxMismatches,"-T",nthreads,"-I",indels,sep=.R_param_splitor) if(complexIndels) opt <- paste(opt,"--complexIndels",sep=.R_param_splitor) if(isScRNAFastqinput) opt <- paste(opt,"--scRNA_FQinput",sep=.R_param_splitor) if(isScRNABAMinput) opt <- paste(opt,"--scRNA_BAMinput",sep=.R_param_splitor) if(!unique) opt <- paste(opt,"--multiMapping",sep=.R_param_splitor) if(isBCLinput) opt <- paste(opt,"--BCLinput",sep=.R_param_splitor) opt <- paste(opt,"-B",nBestLocations,"-d",minFragLength,"-D",maxFragLength,"-S",PE_orientation,"--trim5",nTrim5,"--trim3",nTrim3,sep=.R_param_splitor) if(!is.null(readGroupID)) opt <- paste(opt,"--rg-id",readGroupID,sep=.R_param_splitor) if(!is.null(readGroup)) opt <- paste(opt,"--rg",readGroup,sep=.R_param_splitor) if(color2base) opt <- paste(opt,"-b",sep=.R_param_splitor) if(keepReadOrder) opt <- paste(opt,"--keepReadOrder",sep=.R_param_splitor) if(sortReadsByCoordinates) opt <- paste(opt,"--sortReadsByCoordinates",sep=.R_param_splitor) opt <- paste(opt,"-G",DP_GapOpenPenalty,"-E",DP_GapExtPenalty,"-X",DP_MismatchPenalty,"-Y",DP_MatchScore,sep=.R_param_splitor) if(detectSV) opt <- paste(opt,"--sv",sep=.R_param_splitor) flag <- FALSE if(useAnnotation){ annot.display <- "R data.frame" if(is.null(annot.ext)){ switch(tolower(as.character(annot.inbuilt)), mm9={ ann <- system.file("annot","mm9_RefSeq_exon.txt",package="Rsubread") annot.display <- "inbuilt (mm9)" }, mm10={ ann <- system.file("annot","mm10_RefSeq_exon.txt",package="Rsubread") annot.display <- "inbuilt (mm10)" }, hg19={ ann <- system.file("annot","hg19_RefSeq_exon.txt",package="Rsubread") annot.display <- "inbuilt (hg19)" }, hg38={ ann <- system.file("annot","hg38_RefSeq_exon.txt",package="Rsubread") annot.display <- "inbuilt (hg38)" }, { stop("In-built annotation for ", annot.inbuilt, " is not available.\n") } ) # end switch } else{ if(is.character(annot.ext)){ ann <- annot.ext annot.display <- paste0(basename(ann), " (", ifelse(isGTF,"GTF","SAF"),")") } else{ annot_df <- as.data.frame(annot.ext,stringsAsFactors=FALSE) if(sum(c("geneid","chr","start","end", "strand") %in% tolower(colnames(annot_df))) != 5) stop("One or more required columns are missing in the provided annotation data. Please refer to help page for annotation format.\n") colnames(annot_df) <- tolower(colnames(annot_df)) annot_df <- data.frame(geneid=annot_df$geneid,chr=annot_df$chr,start=annot_df$start,end=annot_df$end,strand=annot_df$strand,stringsAsFactors=FALSE) annot_df$chr <- as.character(annot_df$chr) fout_annot <- file.path(".",paste(".Rsubread_align_UserProvidedAnnotation_pid",Sys.getpid(),sep="")) oldScipen <- options(scipen=999) write.table(x=annot_df,file=fout_annot,sep="\t",row.names=FALSE,quote=FALSE) options(oldScipen) ann <- fout_annot flag <- TRUE } } opt <- paste(opt,"-a",ann,sep=.R_param_splitor) if(isGTF) opt <- paste(opt,"-F","GTF","--gtfFeature",GTF.featureType,"--gtfAttr",GTF.attrType,sep=.R_param_splitor) else opt <- paste(opt,"-F","SAF",sep=.R_param_splitor) if(!is.null(chrAliases)) opt <- paste(opt,"-A",chrAliases,sep=.R_param_splitor) opt <- paste(opt, "--exonAnnotationScreenOut", annot.display, sep=.R_param_splitor) } if(phredOffset == 33) opt <- paste(opt,"-P",3,sep=.R_param_splitor) else opt <- paste(opt,"-P",6,sep=.R_param_splitor) for(i in 1:length(readfile1)){ opt_files <- paste("-r",readfile1[i],sep=.R_param_splitor) if(!is.null(readfile2)) opt_files <- paste(opt_files,"-R",readfile2[i],sep=.R_param_splitor) opt_files <- paste(opt_files,"-o",output_file[i],sep=.R_param_splitor) cmd <- paste("subread-align",opt_files,opt,sep=.R_param_splitor) n <- length(unlist(strsplit(cmd,.R_param_splitor))) C_args <- .C("R_align_wrapper",as.integer(n),as.character(cmd),PACKAGE="Rsubread") summary.data <- .load.delete.summary(output_file[i]) if(i == 1){ return.summary <- summary.data }else{ return.summary <- data.frame(return.summary, summary.data) } } if(flag) file.remove(fout_annot) names(return.summary) <- out.table.cols # basename(output_file) return.summary } Rsubread/R/atgcContent.R0000755000175200017520000000152514136047615016155 0ustar00biocbuildbiocbuildatgcContent <- function(filename, basewise=FALSE) { filename <- .check_and_NormPath(filename, mustWork=T, opt="filename") sequence_file <- paste("./.Rsubread_atgcContent_sequence_pid",Sys.getpid(),sep="") perc_file <- paste("./.Rsubread_atgcContent_percentage_pid",Sys.getpid(),sep="") .C("retrieve_sequence", as.character(filename), as.character(sequence_file), PACKAGE="Rsubread") .C("atgcContent", as.character(sequence_file), as.character(perc_file), as.integer(basewise)) data <- as.matrix(read.csv(perc_file, header=T)) file.remove(sequence_file) file.remove(perc_file) if (basewise){ percentage <- data percentage <- as.matrix(percentage[-1,]) percentage <- as.matrix(t(percentage)) colnames(percentage) <- 1:ncol(percentage) return(percentage) } else { return(data) } } Rsubread/R/buildindex.R0000755000175200017520000000176214136047615016036 0ustar00biocbuildbiocbuildbuildindex <- function(basename,reference,gappedIndex=FALSE,indexSplit=FALSE,memory=8000,TH_subread=100,colorspace=FALSE) { if(!.is.64bit.system()) cat("WARNING: your system seems to be 32-bit. Rsubread supports 32-bit systems to a very limited level.\nIt is highly recommended to run Rsubread on a 64-bit system to avoid errors.\n\n") basename <- .check_and_NormPath(basename, mustWork=F, opt="basename") reference <- .check_and_NormPath(reference, mustWork=T, opt="reference") opt <- paste("-o",basename,"-f",TH_subread,"-M",memory,reference,sep=.R_param_splitor) if(gappedIndex == FALSE) opt <- paste("-F",opt,sep=.R_param_splitor) if(indexSplit == FALSE) opt <- paste("-B",opt,sep=.R_param_splitor) if(colorspace) opt <- paste("-c",opt,sep=.R_param_splitor) cmd <- paste("subread-buildindex",opt,sep=.R_param_splitor) n <- length(unlist(strsplit(cmd,.R_param_splitor))) C_args <- .C("R_buildindex_wrapper",argc=as.integer(n),argv=as.character(cmd),PACKAGE="Rsubread") } Rsubread/R/cellCounts.R0000644000175200017520000031443614136047615016024 0ustar00biocbuildbiocbuild.write.tmp.parameters <- function(param){ temp.file.prefix <- file.path(".",paste(".Rsubread_PARAM_cellCounts_",Sys.getpid(),sep="")) param$PMR_CREATE_TIME <- Sys.time() saveRDS(param, temp.file.prefix) } .retrieve.tmp.parameters <- function(){ temp.file.prefix <- file.path(".",paste(".Rsubread_PARAM_cellCounts_",Sys.getpid(),sep="")) if(!file.exists(temp.file.prefix)) return (NA) rv <- readRDS(temp.file.prefix) file.remove(temp.file.prefix) if( Sys.time() - rv$PMR_CREATE_TIME > 5) return (NA) # the file should be created no longer than 5 seconds ago rv } .index.names.to.sheet.BAM.mode<-function(fqtab, fname){ lines <- paste(fqtab$BAMFile, fqtab$SampleName , sep=",") fileConn<-file(fname) writeLines(c("EMFileVersion,4","[data]","BAMFile,Sample_Name",lines), fileConn) close(fileConn) } .index.names.to.sheet.FASTQ.mode<-function(fqtab, fname){ lines <- paste(fqtab$BarcodeUMIFile, fqtab$ReadFile, fqtab$SampleName , sep=",") fileConn<-file(fname) writeLines(c("EMFileVersion,4","[data]","BarcodeUMIFile,ReadFile,Sample_Name",lines), fileConn) close(fileConn) } .index.names.to.sheet.raw.dir.mode<-function(dirname, nametab, fname, sample.name=NA){ lanes <- c() index.names <- c() sample.names <- c() index.seq <- c() is_dual_index <- c() for(cli in 1:nrow(nametab)){ if(nametab$InputDirectory[cli]!=dirname)next if((!is.na(sample.name)) && as.character(nametab$SampleName[cli])!=sample.name)next seqs <- .convert.sample_index.id.to.seq(nametab$IndexSetName[cli]) for(seq in seqs){ is_dual_index <- c(is_dual_index , nchar(seq)>12) lanes <-c(lanes, nametab$Lane[cli]) index.names <-c(index.names, nametab$IndexSetName[cli]) sample.names <- c(sample.names, as.character(nametab$SampleName[cli])) index.seq <- c(index.seq, seq) } } sdf<-data.frame(Lane=lanes, Sample_ID=index.names, Sample_Name=sample.names, index=index.seq, Sample_Project=rep("cellCounts", length(lanes)), stringsAsFactors=F) fileConn<-file(fname) lines <- paste( sdf$Lane, sdf$Sample_ID, sdf$Sample_Name, sdf$index, sdf$Sample_Project ,sep=",") writeLines(c("EMFileVersion,4","[data]","Lane,Sample_ID,Sample_Name,index,Sample_Project",lines), fileConn) close(fileConn) if(any( is_dual_index != is_dual_index [1] )) stop("Dual-index and single-index samples cannot be mixed in the sample data-frame.") return(is_dual_index[1]) } .convert.sample_index.id.to.seq <- function(spid){ seqs <- .one.convert.sample_index.id.to.seq(spid) if(any(is.na(seqs))){ spid <- gsub("-","_", spid) seqs <- .one.convert.sample_index.id.to.seq(spid) } if(any(is.na(seqs)))stop(paste("Unable to find sample index for", spid)) seqs } .one.convert.sample_index.id.to.seq <- function(spid){ spseqs <- NA if(spid == "SI_001") spseqs <- c('TCGCCATA', 'GTATACAC', 'AATGGTGG', 'CGCATGCT') if(spid == "SI_002") spseqs <- c('TATCCTCG', 'GCGAGGTC', 'CGCTTCAA', 'ATAGAAGT') if(spid == "SI_003") spseqs <- c('TGACGTCG', 'CTTGTGTA', 'ACGACCGT', 'GACTAAAC') if(spid == "SI_004") spseqs <- c('ATCTAGCT', 'GAGCGTAC', 'TCAGCCTG', 'CGTATAGA') if(spid == "SI_005") spseqs <- c('CCGTTCCC', 'ATACAGTT', 'TGTAGTAA', 'GACGCAGG') if(spid == "SI_006") spseqs <- c('TCAATTGG', 'AGTTAGAA', 'GAGCGCTT', 'CTCGCACC') if(spid == "SI_007") spseqs <- c('CTGCCTTG', 'ACTAGCCC', 'GGCTAGAT', 'TAAGTAGA') if(spid == "SI_008") spseqs <- c('GGCAGAAA', 'ACGGTTCT', 'CATTCGTC', 'TTACACGG') if(spid == "SI_P01_A1") spseqs <- c('TTGTAAGA', 'GGCGTTTC', 'CCTACCAT', 'AAACGGCG') if(spid == "SI_P01_B1") spseqs <- c('CTAGCTGT', 'GCCAACAA', 'AGGCTACC', 'TATTGGTG') if(spid == "SI_P01_C1") spseqs <- c('GATGCAGT', 'AGACTTTC', 'TTCTACAG', 'CCGAGGCA') if(spid == "SI_P01_D1") spseqs <- c('AGCTGCGT', 'GTGGAGCA', 'TCTATTAG', 'CAACCATC') if(spid == "SI_P01_E1") spseqs <- c('CGCCCGTA', 'GTTTGCCT', 'TAAGTTAG', 'ACGAAAGC') if(spid == "SI_P01_F1") spseqs <- c('TGACTAGT', 'GATAGGTA', 'CCGTACAG', 'ATCGCTCC') if(spid == "SI_P01_G1") spseqs <- c('CATATGCG', 'ATGCGATT', 'TCCGCTAC', 'GGATACGA') if(spid == "SI_P01_H1") spseqs <- c('TCTTGTCC', 'CGGGAGTA', 'GTCACAGG', 'AAACTCAT') if(spid == "SI_P01_A2") spseqs <- c('AGCCCTTT', 'TCTTAGGC', 'GTGAGAAG', 'CAAGTCCA') if(spid == "SI_P01_B2") spseqs <- c('GGTCGAGC', 'TTAGATTG', 'CCCACCCA', 'AAGTTGAT') if(spid == "SI_P01_C2") spseqs <- c('CCGAGAAC', 'TGCTCTGT', 'GTAGTGCG', 'AATCACTA') if(spid == "SI_P01_D2") spseqs <- c('ACATTCCG', 'GACACAAT', 'CTGCGGTA', 'TGTGATGC') if(spid == "SI_P01_E2") spseqs <- c('TCATCAAG', 'GTTGGTCC', 'AGGCTGGT', 'CACAACTA') if(spid == "SI_P01_F2") spseqs <- c('TGCCCGCT', 'GCAAACGC', 'CATTGATA', 'ATGGTTAG') if(spid == "SI_P01_G2") spseqs <- c('CGGTGAGC', 'ATAACCTA', 'TCCGAGCG', 'GATCTTAT') if(spid == "SI_P01_H2") spseqs <- c('CCGAACTC', 'AACGGTCA', 'TTATTGGT', 'GGTCCAAG') if(spid == "SI_P01_A3") spseqs <- c('AAAGCATA', 'GCCTTTAT', 'CTGCAGCC', 'TGTAGCGG') if(spid == "SI_P01_B3") spseqs <- c('TCATCCTT', 'ATTGGACG', 'CAGCTTAC', 'GGCAAGGA') if(spid == "SI_P01_C3") spseqs <- c('ACGTTACA', 'TTACCTAC', 'GACGACGG', 'CGTAGGTT') if(spid == "SI_P01_D3") spseqs <- c('GAGCACGC', 'CGAAGTTG', 'TTCGTGAA', 'ACTTCACT') if(spid == "SI_P01_E3") spseqs <- c('TCTGCAGG', 'CGGCTCCA', 'AACAAGTC', 'GTATGTAT') if(spid == "SI_P01_F3") spseqs <- c('TTAGGACC', 'AGTCTGTA', 'GCCTCCGT', 'CAGAATAG') if(spid == "SI_P01_G3") spseqs <- c('TACGAGTT', 'ATGTCCAG', 'GCTATAGC', 'CGACGTCA') if(spid == "SI_P01_H3") spseqs <- c('TTGGGCTT', 'GACAAACC', 'ACACCTAA', 'CGTTTGGG') if(spid == "SI_P01_A4") spseqs <- c('CATGGCAG', 'AGAACGCC', 'GTCTTTGA', 'TCGCAATT') if(spid == "SI_P01_B4") spseqs <- c('GACAGGCT', 'CCTCTAAC', 'AGGGACTG', 'TTATCTGA') if(spid == "SI_P01_C4") spseqs <- c('ACATTGGC', 'GAGCCCAT', 'CTTAGTCA', 'TGCGAATG') if(spid == "SI_P01_D4") spseqs <- c('AAATCGTC', 'GCGATCGG', 'CTTCGAAT', 'TGCGATCA') if(spid == "SI_P01_E4") spseqs <- c('GTAAACAT', 'TATCCTGA', 'AGCTGACG', 'CCGGTGTC') if(spid == "SI_P01_F4") spseqs <- c('GCATGATA', 'CGTCCTCT', 'AACGACAC', 'TTGATGGG') if(spid == "SI_P01_G4") spseqs <- c('CCTGCGGT', 'GTACAACG', 'AGCTTCTC', 'TAGAGTAA') if(spid == "SI_P01_H4") spseqs <- c('TTCCATCT', 'ACTGGAGC', 'CGGTCGTG', 'GAAATCAA') if(spid == "SI_P01_A5") spseqs <- c('CAGTCTGG', 'TCACACTC', 'ATTGGGAA', 'GGCATACT') if(spid == "SI_P01_B5") spseqs <- c('TCATGCGA', 'ATCGTACT', 'CATCAGTG', 'GGGACTAC') if(spid == "SI_P01_C5") spseqs <- c('TCAGTCAA', 'CACTGACT', 'ATGCATTC', 'GGTACGGG') if(spid == "SI_P01_D5") spseqs <- c('GTCGACTC', 'AGACGGAT', 'CCTTTAGA', 'TAGACTCG') if(spid == "SI_P01_E5") spseqs <- c('CCGTTGAA', 'TATGCTCT', 'ATCCAAGG', 'GGAAGCTC') if(spid == "SI_P01_F5") spseqs <- c('TCTGACTA', 'GTACGGCT', 'CGGTTTAG', 'AACACAGC') if(spid == "SI_P01_G5") spseqs <- c('ATGAAGTA', 'GAAGCTCG', 'TCTTTCGT', 'CGCCGAAC') if(spid == "SI_P01_H5") spseqs <- c('ATAGTATG', 'TATAAGGA', 'GGCTCCAC', 'CCGCGTCT') if(spid == "SI_P01_A6") spseqs <- c('CTTTCGAC', 'ACGGGACT', 'TGCATCTG', 'GAACATGA') if(spid == "SI_P01_B6") spseqs <- c('GCGCACCT', 'AACGCGAA', 'CTATTTGG', 'TGTAGATC') if(spid == "SI_P01_C6") spseqs <- c('CGCTCAGG', 'GAGGTTTA', 'ACTCAGAC', 'TTAAGCCT') if(spid == "SI_P01_D6") spseqs <- c('GAAGTCTT', 'TGCAGGGC', 'ATGCCAAA', 'CCTTATCG') if(spid == "SI_P01_E6") spseqs <- c('TGATGGCT', 'GCCATTTG', 'ATTGAAAC', 'CAGCCCGA') if(spid == "SI_P01_F6") spseqs <- c('GACTTCCT', 'TGAGGAAG', 'ATGCCGGC', 'CCTAATTA') if(spid == "SI_P01_G6") spseqs <- c('GTGCGACA', 'TCAGTGTT', 'AGCACTGG', 'CATTACAC') if(spid == "SI_P01_H6") spseqs <- c('AAGCATAA', 'CCCATCGC', 'TTAGCGCT', 'GGTTGATG') if(spid == "SI_P01_A7") spseqs <- c('CTCATCAT', 'TAACGTCC', 'AGGTCATA', 'GCTGAGGG') if(spid == "SI_P01_B7") spseqs <- c('TCCACACG', 'CTTCTGTT', 'GAATGCAC', 'AGGGATGA') if(spid == "SI_P01_C7") spseqs <- c('GGCGGAAT', 'ACACCGGG', 'CATAATCC', 'TTGTTCTA') if(spid == "SI_P01_D7") spseqs <- c('CCGGATCC', 'GGTCGCAT', 'TTAACGTG', 'AACTTAGA') if(spid == "SI_P01_E7") spseqs <- c('AAGACGTG', 'CCATGTGT', 'GTTCACAA', 'TGCGTACC') if(spid == "SI_P01_F7") spseqs <- c('GGTTAGAC', 'CAAACTTT', 'ACCCGAGA', 'TTGGTCCG') if(spid == "SI_P01_G7") spseqs <- c('GCCGGTAA', 'TGACTGCC', 'ATTACCGG', 'CAGTAATT') if(spid == "SI_P01_H7") spseqs <- c('TGGCACGA', 'AACGGGTG', 'CTAATTCT', 'GCTTCAAC') if(spid == "SI_P01_A8") spseqs <- c('GACTGTTC', 'ATGATACG', 'CCACAGAA', 'TGTGCCGT') if(spid == "SI_P01_B8") spseqs <- c('ACGTTCAC', 'TGCCCAGA', 'CAAGGTCT', 'GTTAAGTG') if(spid == "SI_P01_C8") spseqs <- c('TTTATCCC', 'GCACGTTT', 'CAGGAAGA', 'AGCTCGAG') if(spid == "SI_P01_D8") spseqs <- c('AATCTTTG', 'GGATGAGT', 'CTCAAGAC', 'TCGGCCCA') if(spid == "SI_P01_E8") spseqs <- c('GCTTACAT', 'TAGGGTGC', 'AGCCTATG', 'CTAACGCA') if(spid == "SI_P01_F8") spseqs <- c('AGTTGGGA', 'TACATTCT', 'CCAGAAAG', 'GTGCCCTC') if(spid == "SI_P01_G8") spseqs <- c('AAGTACTC', 'GGAACTCT', 'TCCCTGAG', 'CTTGGAGA') if(spid == "SI_P01_H8") spseqs <- c('AAGAGCGG', 'TCATAGCA', 'GGCCCATC', 'CTTGTTAT') if(spid == "SI_P01_A9") spseqs <- c('GAGTGCGT', 'CTCCAACA', 'ACAACTTG', 'TGTGTGAC') if(spid == "SI_P01_B9") spseqs <- c('AAGCGTGT', 'CTTGACCG', 'TGATTAAC', 'GCCACGTA') if(spid == "SI_P01_C9") spseqs <- c('AGATCGGT', 'CATCGTCG', 'GTCATATA', 'TCGGACAC') if(spid == "SI_P01_D9") spseqs <- c('CAAGGGAC', 'ACCTACTG', 'GGGACACA', 'TTTCTTGT') if(spid == "SI_P01_E9") spseqs <- c('AGTAAGCA', 'TACCGCGG', 'CCGGTAAT', 'GTATCTTC') if(spid == "SI_P01_F9") spseqs <- c('AGTTAGTT', 'GTACTTAA', 'CACGCACG', 'TCGAGCGC') if(spid == "SI_P01_G9") spseqs <- c('TTGACTTC', 'GCCGAAGT', 'CAATGGCA', 'AGTCTCAG') if(spid == "SI_P01_H9") spseqs <- c('GGAATATG', 'ACCTGCCA', 'CTTCATAC', 'TAGGCGGT') if(spid == "SI_P01_A10") spseqs <- c('ACAGCAAC', 'TTTCGCGA', 'CGCAATTT', 'GAGTTGCG') if(spid == "SI_P01_B10") spseqs <- c('ACCATTAA', 'CTGGACGT', 'GAACGGTC', 'TGTTCACG') if(spid == "SI_P01_C10") spseqs <- c('CGTGCTAA', 'TCACTCCT', 'ATCTGATC', 'GAGAAGGG') if(spid == "SI_P01_D10") spseqs <- c('CTTATTTG', 'GCGGGCAT', 'AGATAACA', 'TACCCGGC') if(spid == "SI_P01_E10") spseqs <- c('GCACCAGT', 'CGCAGGAG', 'TAGTACCA', 'ATTGTTTC') if(spid == "SI_P01_F10") spseqs <- c('TCGACAAT', 'GAATACTG', 'ATTCGTGC', 'CGCGTGCA') if(spid == "SI_P01_G10") spseqs <- c('CGGAGACT', 'TCCTATGA', 'ATACTGAG', 'GATGCCTC') if(spid == "SI_P01_H10") spseqs <- c('GACCGCCA', 'TCGAATTG', 'ATTTCAGC', 'CGAGTGAT') if(spid == "SI_P01_A11") spseqs <- c('CTTTCCTT', 'TAGGTAAA', 'ACCAGTCC', 'GGACAGGG') if(spid == "SI_P01_B11") spseqs <- c('TCCAGATA', 'GATTCGCT', 'CGACATAG', 'ATGGTCGC') if(spid == "SI_P01_C11") spseqs <- c('GTTTGTGG', 'ACCGAACA', 'TAGACGAC', 'CGACTCTT') if(spid == "SI_P01_D11") spseqs <- c('GCTACTTC', 'CACCTCAG', 'ATATGAGA', 'TGGGAGCT') if(spid == "SI_P01_E11") spseqs <- c('ATCGCCAT', 'TCACGGTA', 'GGGTTTCC', 'CATAAAGG') if(spid == "SI_P01_F11") spseqs <- c('GAACCCGG', 'AGCAGTTA', 'TCGTAGAT', 'CTTGTACC') if(spid == "SI_P01_G11") spseqs <- c('AGGGCGTT', 'CTATACGC', 'TACATAAG', 'GCTCGTCA') if(spid == "SI_P01_H11") spseqs <- c('TCTCGACT', 'AGGATCGA', 'CACGATTC', 'GTATCGAG') if(spid == "SI_P01_A12") spseqs <- c('TTATGGAA', 'ACTACTGT', 'CGGGAACG', 'GACCTCTC') if(spid == "SI_P01_B12") spseqs <- c('GAAAGACA', 'CGCTACAT', 'ACGCTTGG', 'TTTGCGTC') if(spid == "SI_P01_C12") spseqs <- c('TAAGCCAC', 'CCGTTATG', 'GGTAATGT', 'ATCCGGCA') if(spid == "SI_P01_D12") spseqs <- c('GCTGTGTA', 'AGAAACGT', 'CACTCAAC', 'TTGCGTCG') if(spid == "SI_P01_E12") spseqs <- c('CGCTATCC', 'ACGCGGAA', 'TAAATCGT', 'GTTGCATG') if(spid == "SI_P01_F12") spseqs <- c('AATTGAAC', 'TGGACCCT', 'CCAGTGGA', 'GTCCATTG') if(spid == "SI_P01_G12") spseqs <- c('CATGCGTA', 'ACCCGCAC', 'TGATATCG', 'GTGATAGT') if(spid == "SI_P01_H12") spseqs <- c('TGTGTATA', 'GTCCAGGC', 'CAATCCCT', 'ACGAGTAG') if(spid == "SI_3A_A1" || spid == "SI_P03_A1") spseqs <- c('AAACGGCG', 'CCTACCAT', 'GGCGTTTC', 'TTGTAAGA') if(spid == "SI_3A_B1" || spid == "SI_P03_B1") spseqs <- c('AGGCTACC', 'CTAGCTGT', 'GCCAACAA', 'TATTGGTG') if(spid == "SI_3A_C1" || spid == "SI_P03_C1") spseqs <- c('AGACTTTC', 'CCGAGGCA', 'GATGCAGT', 'TTCTACAG') if(spid == "SI_3A_D1" || spid == "SI_P03_D1") spseqs <- c('AGCTGCGT', 'CAACCATC', 'GTGGAGCA', 'TCTATTAG') if(spid == "SI_3A_E1" || spid == "SI_P03_E1") spseqs <- c('ACGAAAGC', 'CGCCCGTA', 'GTTTGCCT', 'TAAGTTAG') if(spid == "SI_3A_F1" || spid == "SI_P03_F1") spseqs <- c('ATCGCTCC', 'CCGTACAG', 'GATAGGTA', 'TGACTAGT') if(spid == "SI_3A_G1" || spid == "SI_P03_G1") spseqs <- c('ATGCGATT', 'CATATGCG', 'GGATACGA', 'TCCGCTAC') if(spid == "SI_3A_H1" || spid == "SI_P03_H1") spseqs <- c('AAACTCAT', 'CGGGAGTA', 'GTCACAGG', 'TCTTGTCC') if(spid == "SI_3A_A2" || spid == "SI_P03_A2") spseqs <- c('AGCCCTTT', 'CAAGTCCA', 'GTGAGAAG', 'TCTTAGGC') if(spid == "SI_3A_B2" || spid == "SI_P03_B2") spseqs <- c('AAGTTGAT', 'CCCACCCA', 'GGTCGAGC', 'TTAGATTG') if(spid == "SI_3A_C2" || spid == "SI_P03_C2") spseqs <- c('AATCACTA', 'CCGAGAAC', 'GTAGTGCG', 'TGCTCTGT') if(spid == "SI_3A_D2" || spid == "SI_P03_D2") spseqs <- c('ACATTCCG', 'CTGCGGTA', 'GACACAAT', 'TGTGATGC') if(spid == "SI_3A_E2" || spid == "SI_P03_E2") spseqs <- c('AGGCTGGT', 'CACAACTA', 'GTTGGTCC', 'TCATCAAG') if(spid == "SI_3A_F2" || spid == "SI_P03_F2") spseqs <- c('ATGGTTAG', 'CATTGATA', 'GCAAACGC', 'TGCCCGCT') if(spid == "SI_3A_G2" || spid == "SI_P03_G2") spseqs <- c('ATAACCTA', 'CGGTGAGC', 'GATCTTAT', 'TCCGAGCG') if(spid == "SI_3A_H2" || spid == "SI_P03_H2") spseqs <- c('AACGGTCA', 'CCGAACTC', 'GGTCCAAG', 'TTATTGGT') if(spid == "SI_3A_A3" || spid == "SI_P03_A3") spseqs <- c('AAAGCATA', 'CTGCAGCC', 'GCCTTTAT', 'TGTAGCGG') if(spid == "SI_3A_B3" || spid == "SI_P03_B3") spseqs <- c('ATTGGACG', 'CAGCTTAC', 'GGCAAGGA', 'TCATCCTT') if(spid == "SI_3A_C3" || spid == "SI_P03_C3") spseqs <- c('ACGTTACA', 'CGTAGGTT', 'GACGACGG', 'TTACCTAC') if(spid == "SI_3A_D3" || spid == "SI_P03_D3") spseqs <- c('ACTTCACT', 'CGAAGTTG', 'GAGCACGC', 'TTCGTGAA') if(spid == "SI_3A_E3" || spid == "SI_P03_E3") spseqs <- c('AACAAGTC', 'CGGCTCCA', 'GTATGTAT', 'TCTGCAGG') if(spid == "SI_3A_F3" || spid == "SI_P03_F3") spseqs <- c('AGTCTGTA', 'CAGAATAG', 'GCCTCCGT', 'TTAGGACC') if(spid == "SI_3A_G3" || spid == "SI_P03_G3") spseqs <- c('ATGTCCAG', 'CGACGTCA', 'GCTATAGC', 'TACGAGTT') if(spid == "SI_3A_H3" || spid == "SI_P03_H3") spseqs <- c('ACACCTAA', 'CGTTTGGG', 'GACAAACC', 'TTGGGCTT') if(spid == "SI_3A_A4" || spid == "SI_P03_A4") spseqs <- c('AGAACGCC', 'CATGGCAG', 'GTCTTTGA', 'TCGCAATT') if(spid == "SI_3A_B4" || spid == "SI_P03_B4") spseqs <- c('AGGGACTG', 'CCTCTAAC', 'GACAGGCT', 'TTATCTGA') if(spid == "SI_3A_C4" || spid == "SI_P03_C4") spseqs <- c('ACATTGGC', 'CTTAGTCA', 'GAGCCCAT', 'TGCGAATG') if(spid == "SI_3A_D4" || spid == "SI_P03_D4") spseqs <- c('AAATCGTC', 'CTTCGAAT', 'GCGATCGG', 'TGCGATCA') if(spid == "SI_3A_E4" || spid == "SI_P03_E4") spseqs <- c('AGCTGACG', 'CCGGTGTC', 'GTAAACAT', 'TATCCTGA') if(spid == "SI_3A_F4" || spid == "SI_P03_F4") spseqs <- c('AACGACAC', 'CGTCCTCT', 'GCATGATA', 'TTGATGGG') if(spid == "SI_3A_G4" || spid == "SI_P03_G4") spseqs <- c('AGCTTCTC', 'CCTGCGGT', 'GTACAACG', 'TAGAGTAA') if(spid == "SI_3A_H4" || spid == "SI_P03_H4") spseqs <- c('ACTGGAGC', 'CGGTCGTG', 'GAAATCAA', 'TTCCATCT') if(spid == "SI_3A_A5" || spid == "SI_P03_A5") spseqs <- c('ATTGGGAA', 'CAGTCTGG', 'GGCATACT', 'TCACACTC') if(spid == "SI_3A_B5" || spid == "SI_P03_B5") spseqs <- c('ATCGTACT', 'CATCAGTG', 'GGGACTAC', 'TCATGCGA') if(spid == "SI_3A_C5" || spid == "SI_P03_C5") spseqs <- c('ATGCATTC', 'CACTGACT', 'GGTACGGG', 'TCAGTCAA') if(spid == "SI_3A_D5" || spid == "SI_P03_D5") spseqs <- c('AGACGGAT', 'CCTTTAGA', 'GTCGACTC', 'TAGACTCG') if(spid == "SI_3A_E5" || spid == "SI_P03_E5") spseqs <- c('ATCCAAGG', 'CCGTTGAA', 'GGAAGCTC', 'TATGCTCT') if(spid == "SI_3A_F5" || spid == "SI_P03_F5") spseqs <- c('AACACAGC', 'CGGTTTAG', 'GTACGGCT', 'TCTGACTA') if(spid == "SI_3A_G5" || spid == "SI_P03_G5") spseqs <- c('ATGAAGTA', 'CGCCGAAC', 'GAAGCTCG', 'TCTTTCGT') if(spid == "SI_3A_H5" || spid == "SI_P03_H5") spseqs <- c('ATAGTATG', 'CCGCGTCT', 'GGCTCCAC', 'TATAAGGA') if(spid == "SI_3A_A6" || spid == "SI_P03_A6") spseqs <- c('ACGGGACT', 'CTTTCGAC', 'GAACATGA', 'TGCATCTG') if(spid == "SI_3A_B6" || spid == "SI_P03_B6") spseqs <- c('AACGCGAA', 'CTATTTGG', 'GCGCACCT', 'TGTAGATC') if(spid == "SI_3A_C6" || spid == "SI_P03_C6") spseqs <- c('ACTCAGAC', 'CGCTCAGG', 'GAGGTTTA', 'TTAAGCCT') if(spid == "SI_3A_D6" || spid == "SI_P03_D6") spseqs <- c('ATGCCAAA', 'CCTTATCG', 'GAAGTCTT', 'TGCAGGGC') if(spid == "SI_3A_E6" || spid == "SI_P03_E6") spseqs <- c('ATTGAAAC', 'CAGCCCGA', 'GCCATTTG', 'TGATGGCT') if(spid == "SI_3A_F6" || spid == "SI_P03_F6") spseqs <- c('ATGCCGGC', 'CCTAATTA', 'GACTTCCT', 'TGAGGAAG') if(spid == "SI_3A_G6" || spid == "SI_P03_G6") spseqs <- c('AGCACTGG', 'CATTACAC', 'GTGCGACA', 'TCAGTGTT') if(spid == "SI_3A_H6" || spid == "SI_P03_H6") spseqs <- c('AAGCATAA', 'CCCATCGC', 'GGTTGATG', 'TTAGCGCT') if(spid == "SI_3A_A7" || spid == "SI_P03_A7") spseqs <- c('AGGTCATA', 'CTCATCAT', 'GCTGAGGG', 'TAACGTCC') if(spid == "SI_3A_B7" || spid == "SI_P03_B7") spseqs <- c('AGGGATGA', 'CTTCTGTT', 'GAATGCAC', 'TCCACACG') if(spid == "SI_3A_C7" || spid == "SI_P03_C7") spseqs <- c('ACACCGGG', 'CATAATCC', 'GGCGGAAT', 'TTGTTCTA') if(spid == "SI_3A_D7" || spid == "SI_P03_D7") spseqs <- c('AACTTAGA', 'CCGGATCC', 'GGTCGCAT', 'TTAACGTG') if(spid == "SI_3A_E7" || spid == "SI_P03_E7") spseqs <- c('AAGACGTG', 'CCATGTGT', 'GTTCACAA', 'TGCGTACC') if(spid == "SI_3A_F7" || spid == "SI_P03_F7") spseqs <- c('ACCCGAGA', 'CAAACTTT', 'GGTTAGAC', 'TTGGTCCG') if(spid == "SI_3A_G7" || spid == "SI_P03_G7") spseqs <- c('ATTACCGG', 'CAGTAATT', 'GCCGGTAA', 'TGACTGCC') if(spid == "SI_3A_H7" || spid == "SI_P03_H7") spseqs <- c('AACGGGTG', 'CTAATTCT', 'GCTTCAAC', 'TGGCACGA') if(spid == "SI_3A_A8" || spid == "SI_P03_A8") spseqs <- c('ATGATACG', 'CCACAGAA', 'GACTGTTC', 'TGTGCCGT') if(spid == "SI_3A_B8" || spid == "SI_P03_B8") spseqs <- c('ACGTTCAC', 'CAAGGTCT', 'GTTAAGTG', 'TGCCCAGA') if(spid == "SI_3A_C8" || spid == "SI_P03_C8") spseqs <- c('AGCTCGAG', 'CAGGAAGA', 'GCACGTTT', 'TTTATCCC') if(spid == "SI_3A_D8" || spid == "SI_P03_D8") spseqs <- c('AATCTTTG', 'CTCAAGAC', 'GGATGAGT', 'TCGGCCCA') if(spid == "SI_3A_E8" || spid == "SI_P03_E8") spseqs <- c('AGCCTATG', 'CTAACGCA', 'GCTTACAT', 'TAGGGTGC') if(spid == "SI_3A_F8" || spid == "SI_P03_F8") spseqs <- c('AGTTGGGA', 'CCAGAAAG', 'GTGCCCTC', 'TACATTCT') if(spid == "SI_3A_G8" || spid == "SI_P03_G8") spseqs <- c('AAGTACTC', 'CTTGGAGA', 'GGAACTCT', 'TCCCTGAG') if(spid == "SI_3A_H8" || spid == "SI_P03_H8") spseqs <- c('AAGAGCGG', 'CTTGTTAT', 'GGCCCATC', 'TCATAGCA') if(spid == "SI_3A_A9" || spid == "SI_P03_A9") spseqs <- c('ACAACTTG', 'CTCCAACA', 'GAGTGCGT', 'TGTGTGAC') if(spid == "SI_3A_B9" || spid == "SI_P03_B9") spseqs <- c('AAGCGTGT', 'CTTGACCG', 'GCCACGTA', 'TGATTAAC') if(spid == "SI_3A_C9" || spid == "SI_P03_C9") spseqs <- c('AGATCGGT', 'CATCGTCG', 'GTCATATA', 'TCGGACAC') if(spid == "SI_3A_D9" || spid == "SI_P03_D9") spseqs <- c('ACCTACTG', 'CAAGGGAC', 'GGGACACA', 'TTTCTTGT') if(spid == "SI_3A_E9" || spid == "SI_P03_E9") spseqs <- c('AGTAAGCA', 'CCGGTAAT', 'GTATCTTC', 'TACCGCGG') if(spid == "SI_3A_F9" || spid == "SI_P03_F9") spseqs <- c('AGTTAGTT', 'CACGCACG', 'GTACTTAA', 'TCGAGCGC') if(spid == "SI_3A_G9" || spid == "SI_P03_G9") spseqs <- c('AGTCTCAG', 'CAATGGCA', 'GCCGAAGT', 'TTGACTTC') if(spid == "SI_3A_H9" || spid == "SI_P03_H9") spseqs <- c('ACCTGCCA', 'CTTCATAC', 'GGAATATG', 'TAGGCGGT') if(spid == "SI_3A_A10" || spid == "SI_P03_A10") spseqs <- c('ACAGCAAC', 'CGCAATTT', 'GAGTTGCG', 'TTTCGCGA') if(spid == "SI_3A_B10" || spid == "SI_P03_B10") spseqs <- c('ACCATTAA', 'CTGGACGT', 'GAACGGTC', 'TGTTCACG') if(spid == "SI_3A_C10" || spid == "SI_P03_C10") spseqs <- c('ATCTGATC', 'CGTGCTAA', 'GAGAAGGG', 'TCACTCCT') if(spid == "SI_3A_D10" || spid == "SI_P03_D10") spseqs <- c('AGATAACA', 'CTTATTTG', 'GCGGGCAT', 'TACCCGGC') if(spid == "SI_3A_E10" || spid == "SI_P03_E10") spseqs <- c('ATTGTTTC', 'CGCAGGAG', 'GCACCAGT', 'TAGTACCA') if(spid == "SI_3A_F10" || spid == "SI_P03_F10") spseqs <- c('ATTCGTGC', 'CGCGTGCA', 'GAATACTG', 'TCGACAAT') if(spid == "SI_3A_G10" || spid == "SI_P03_G10") spseqs <- c('ATACTGAG', 'CGGAGACT', 'GATGCCTC', 'TCCTATGA') if(spid == "SI_3A_H10" || spid == "SI_P03_H10") spseqs <- c('ATTTCAGC', 'CGAGTGAT', 'GACCGCCA', 'TCGAATTG') if(spid == "SI_3A_A11" || spid == "SI_P03_A11") spseqs <- c('ACCAGTCC', 'CTTTCCTT', 'GGACAGGG', 'TAGGTAAA') if(spid == "SI_3A_B11" || spid == "SI_P03_B11") spseqs <- c('ATGGTCGC', 'CGACATAG', 'GATTCGCT', 'TCCAGATA') if(spid == "SI_3A_C11" || spid == "SI_P03_C11") spseqs <- c('ACCGAACA', 'CGACTCTT', 'GTTTGTGG', 'TAGACGAC') if(spid == "SI_3A_D11" || spid == "SI_P03_D11") spseqs <- c('ATATGAGA', 'CACCTCAG', 'GCTACTTC', 'TGGGAGCT') if(spid == "SI_3A_E11" || spid == "SI_P03_E11") spseqs <- c('ATCGCCAT', 'CATAAAGG', 'GGGTTTCC', 'TCACGGTA') if(spid == "SI_3A_F11" || spid == "SI_P03_F11") spseqs <- c('AGCAGTTA', 'CTTGTACC', 'GAACCCGG', 'TCGTAGAT') if(spid == "SI_3A_G11" || spid == "SI_P03_G11") spseqs <- c('AGGGCGTT', 'CTATACGC', 'GCTCGTCA', 'TACATAAG') if(spid == "SI_3A_H11" || spid == "SI_P03_H11") spseqs <- c('AGGATCGA', 'CACGATTC', 'GTATCGAG', 'TCTCGACT') if(spid == "SI_3A_A12" || spid == "SI_P03_A12") spseqs <- c('ACTACTGT', 'CGGGAACG', 'GACCTCTC', 'TTATGGAA') if(spid == "SI_3A_B12" || spid == "SI_P03_B12") spseqs <- c('ACGCTTGG', 'CGCTACAT', 'GAAAGACA', 'TTTGCGTC') if(spid == "SI_3A_C12" || spid == "SI_P03_C12") spseqs <- c('ATCCGGCA', 'CCGTTATG', 'GGTAATGT', 'TAAGCCAC') if(spid == "SI_3A_D12" || spid == "SI_P03_D12") spseqs <- c('AGAAACGT', 'CACTCAAC', 'GCTGTGTA', 'TTGCGTCG') if(spid == "SI_3A_E12" || spid == "SI_P03_E12") spseqs <- c('ACGCGGAA', 'CGCTATCC', 'GTTGCATG', 'TAAATCGT') if(spid == "SI_3A_F12" || spid == "SI_P03_F12") spseqs <- c('AATTGAAC', 'CCAGTGGA', 'GTCCATTG', 'TGGACCCT') if(spid == "SI_3A_G12" || spid == "SI_P03_G12") spseqs <- c('ACCCGCAC', 'CATGCGTA', 'GTGATAGT', 'TGATATCG') if(spid == "SI_3A_H12" || spid == "SI_P03_H12") spseqs <- c('ACGAGTAG', 'CAATCCCT', 'GTCCAGGC', 'TGTGTATA') if(spid == "SI_T2_1") spseqs <- c('GGGTGATC', 'TTACCGAT', 'AATGACGA', 'CCCATTCG') if(spid == "SI_T2_2") spseqs <- c('GGGTCGAA', 'ATCCGCCC', 'TCTATAGT', 'CAAGATTG') if(spid == "SI_T2_3") spseqs <- c('GCTGATAT', 'TGCCGAGC', 'AAATTGCG', 'CTGACCTA') if(spid == "SI_T2_4") spseqs <- c('ACTTCTGA', 'TTCATCTT', 'CGACGACG', 'GAGGAGAC') if(spid == "SI_T2_5") spseqs <- c('GAATACAA', 'AGCATACC', 'TCGGGTTT', 'CTTCCGGG') if(spid == "SI_T2_6") spseqs <- c('TATTGAGA', 'GTAGTCAG', 'CGCCATTC', 'ACGACGCT') if(spid == "SI_T2_7") spseqs <- c('AAATCTGT', 'GTCCAACC', 'TCTGGCTG', 'CGGATGAA') if(spid == "SI_T2_8") spseqs <- c('CCTTGAAC', 'GAAATCGG', 'TGGCCTCT', 'ATCGAGTA') if(spid == "SI_GA_A1" || spid == "SI_P2_A1") spseqs <- c('GGTTTACT', 'CTAAACGG', 'TCGGCGTC', 'AACCGTAA') if(spid == "SI_GA_A2" || spid == "SI_P2_A2") spseqs <- c('TTTCATGA', 'ACGTCCCT', 'CGCATGTG', 'GAAGGAAC') if(spid == "SI_GA_A3" || spid == "SI_P2_A3") spseqs <- c('CAGTACTG', 'AGTAGTCT', 'GCAGTAGA', 'TTCCCGAC') if(spid == "SI_GA_A4" || spid == "SI_P2_A4") spseqs <- c('TATGATTC', 'CCCACAGT', 'ATGCTGAA', 'GGATGCCG') if(spid == "SI_GA_A5" || spid == "SI_P2_A5") spseqs <- c('CTAGGTGA', 'TCGTTCAG', 'AGCCAATT', 'GATACGCC') if(spid == "SI_GA_A6" || spid == "SI_P2_A6") spseqs <- c('CGCTATGT', 'GCTGTCCA', 'TTGAGATC', 'AAACCGAG') if(spid == "SI_GA_A7" || spid == "SI_P2_A7") spseqs <- c('ACAGAGGT', 'TATAGTTG', 'CGGTCCCA', 'GTCCTAAC') if(spid == "SI_GA_A8" || spid == "SI_P2_A8") spseqs <- c('GCATCTCC', 'TGTAAGGT', 'CTGCGATG', 'AACGTCAA') if(spid == "SI_GA_A9" || spid == "SI_P2_A9") spseqs <- c('TCTTAAAG', 'CGAGGCTC', 'GTCCTTCT', 'AAGACGGA') if(spid == "SI_GA_A10" || spid == "SI_P2_A10") spseqs <- c('GAAACCCT', 'TTTCTGTC', 'CCGTGTGA', 'AGCGAAAG') if(spid == "SI_GA_A11" || spid == "SI_P2_A11") spseqs <- c('GTCCGGTC', 'AAGATCAT', 'CCTGAAGG', 'TGATCTCA') if(spid == "SI_GA_A12" || spid == "SI_P2_A12") spseqs <- c('AGTGGAAC', 'GTCTCCTT', 'TCACATCA', 'CAGATGGG') if(spid == "SI_GA_B1" || spid == "SI_P2_B1") spseqs <- c('GTAATCTT', 'TCCGGAAG', 'AGTTCGGC', 'CAGCATCA') if(spid == "SI_GA_B2" || spid == "SI_P2_B2") spseqs <- c('TACTCTTC', 'CCTGTGCG', 'GGACACGT', 'ATGAGAAA') if(spid == "SI_GA_B3" || spid == "SI_P2_B3") spseqs <- c('GTGTATTA', 'TGTGCGGG', 'ACCATAAC', 'CAACGCCT') if(spid == "SI_GA_B4" || spid == "SI_P2_B4") spseqs <- c('ACTTCATA', 'GAGATGAC', 'TGCCGTGG', 'CTAGACCT') if(spid == "SI_GA_B5" || spid == "SI_P2_B5") spseqs <- c('AATAATGG', 'CCAGGGCA', 'TGCCTCAT', 'GTGTCATC') if(spid == "SI_GA_B6" || spid == "SI_P2_B6") spseqs <- c('CGTTAATC', 'GCCACGCT', 'TTACTCAG', 'AAGGGTGA') if(spid == "SI_GA_B7" || spid == "SI_P2_B7") spseqs <- c('AAACCTCA', 'GCCTTGGT', 'CTGGACTC', 'TGTAGAAG') if(spid == "SI_GA_B8" || spid == "SI_P2_B8") spseqs <- c('AAAGTGCT', 'GCTACCTG', 'TGCTGTAA', 'CTGCAAGC') if(spid == "SI_GA_B9" || spid == "SI_P2_B9") spseqs <- c('CTGTAACT', 'TCTAGCGA', 'AGAGTGTG', 'GACCCTAC') if(spid == "SI_GA_B10" || spid == "SI_P2_B10") spseqs <- c('ACCGTATG', 'GATTAGAT', 'CTGACTGA', 'TGACGCCC') if(spid == "SI_GA_B11" || spid == "SI_P2_B11") spseqs <- c('GTTCCTCA', 'AGGTACGC', 'TAAGTATG', 'CCCAGGAT') if(spid == "SI_GA_B12" || spid == "SI_P2_B12") spseqs <- c('TACCACCA', 'CTAAGTTT', 'GGGTCAAG', 'ACTGTGGC') if(spid == "SI_GA_C1" || spid == "SI_P2_C1") spseqs <- c('CCACTTAT', 'AACTGGCG', 'TTGGCATA', 'GGTAACGC') if(spid == "SI_GA_C2" || spid == "SI_P2_C2") spseqs <- c('CCTAGACC', 'ATCTCTGT', 'TAGCTCTA', 'GGAGAGAG') if(spid == "SI_GA_C3" || spid == "SI_P2_C3") spseqs <- c('TCAGCCGT', 'CAGAGGCC', 'GGTCAATA', 'ATCTTTAG') if(spid == "SI_GA_C4" || spid == "SI_P2_C4") spseqs <- c('ACAATTCA', 'TGCGCAGC', 'CATCACTT', 'GTGTGGAG') if(spid == "SI_GA_C5" || spid == "SI_P2_C5") spseqs <- c('CGACTTGA', 'TACAGACT', 'ATTGCGTG', 'GCGTACAC') if(spid == "SI_GA_C6" || spid == "SI_P2_C6") spseqs <- c('ATTACTTC', 'TGCGAACT', 'GCATTCGG', 'CAGCGGAA') if(spid == "SI_GA_C7" || spid == "SI_P2_C7") spseqs <- c('GTCTCTCG', 'AATCTCTC', 'CGGAGGGA', 'TCAGAAAT') if(spid == "SI_GA_C8" || spid == "SI_P2_C8") spseqs <- c('GTTGAGAA', 'AGATCTGG', 'TCGATACT', 'CACCGCTC') if(spid == "SI_GA_C9" || spid == "SI_P2_C9") spseqs <- c('GCGCAGAA', 'ATCTTACC', 'TATGGTGT', 'CGAACCTG') if(spid == "SI_GA_C10" || spid == "SI_P2_C10") spseqs <- c('TCTCAGTG', 'GAGACTAT', 'CGCTTAGC', 'ATAGGCCA') if(spid == "SI_GA_C11" || spid == "SI_P2_C11") spseqs <- c('GAGGATCT', 'AGACCATA', 'TCCTGCGC', 'CTTATGAG') if(spid == "SI_GA_C12" || spid == "SI_P2_C12") spseqs <- c('TCTCGTTT', 'GGCTAGCG', 'ATGACCGC', 'CAAGTAAA') if(spid == "SI_GA_D1" || spid == "SI_P2_D1") spseqs <- c('CACTCGGA', 'GCTGAATT', 'TGAAGTAC', 'ATGCTCCG') if(spid == "SI_GA_D2" || spid == "SI_P2_D2") spseqs <- c('TAACAAGG', 'GGTTCCTC', 'ATCATGCA', 'CCGGGTAT') if(spid == "SI_GA_D3" || spid == "SI_P2_D3") spseqs <- c('ACATTACT', 'TTTGGGTA', 'CAGCCCAC', 'GGCAATGG') if(spid == "SI_GA_D4" || spid == "SI_P2_D4") spseqs <- c('CCCTAACA', 'ATTCCGAT', 'TGGATTGC', 'GAAGGCTG') if(spid == "SI_GA_D5" || spid == "SI_P2_D5") spseqs <- c('CTCGTCAC', 'GATCAGCA', 'ACAACAGG', 'TGGTGTTT') if(spid == "SI_GA_D6" || spid == "SI_P2_D6") spseqs <- c('CATGCGAT', 'TGATATTC', 'GTGATCGA', 'ACCCGACG') if(spid == "SI_GA_D7" || spid == "SI_P2_D7") spseqs <- c('ATTTGCTA', 'TAGACACC', 'CCACAGGG', 'GGCGTTAT') if(spid == "SI_GA_D8" || spid == "SI_P2_D8") spseqs <- c('GCAACAAA', 'TAGTTGTC', 'CGCCATCG', 'ATTGGCGT') if(spid == "SI_GA_D9" || spid == "SI_P2_D9") spseqs <- c('AGGAGATG', 'GATGTGGT', 'CTACATCC', 'TCCTCCAA') if(spid == "SI_GA_D10" || spid == "SI_P2_D10") spseqs <- c('CAATACCC', 'TGTCTATG', 'ACCACGAA', 'GTGGGTGT') if(spid == "SI_GA_D11" || spid == "SI_P2_D11") spseqs <- c('CTTTGCGG', 'TGCACAAA', 'AAGCAGTC', 'GCAGTTCT') if(spid == "SI_GA_D12" || spid == "SI_P2_D12") spseqs <- c('GCACAATG', 'CTTGGTAC', 'TGCACCGT', 'AAGTTGCA') if(spid == "SI_GA_E1" || spid == "SI_P2_E1") spseqs <- c('TGGTAAAC', 'GAAAGGGT', 'ACTGCTCG', 'CTCCTCTA') if(spid == "SI_GA_E2" || spid == "SI_P2_E2") spseqs <- c('GTGGTACC', 'TACTATAG', 'ACAAGGTA', 'CGTCCCGT') if(spid == "SI_GA_E3" || spid == "SI_P2_E3") spseqs <- c('AGGTATTG', 'CTCCTAGT', 'TCAAGGCC', 'GATGCCAA') if(spid == "SI_GA_E4" || spid == "SI_P2_E4") spseqs <- c('TTCGCCCT', 'GGATGGGC', 'AATCAATG', 'CCGATTAA') if(spid == "SI_GA_E5" || spid == "SI_P2_E5") spseqs <- c('CATTAGCG', 'TTCGCTGA', 'ACAAGAAT', 'GGGCTCTC') if(spid == "SI_GA_E6" || spid == "SI_P2_E6") spseqs <- c('CTGCGGCT', 'GACTCAAA', 'AGAAACTC', 'TCTGTTGG') if(spid == "SI_GA_E7" || spid == "SI_P2_E7") spseqs <- c('CACGCCTT', 'GTATATAG', 'TCTCGGGC', 'AGGATACA') if(spid == "SI_GA_E8" || spid == "SI_P2_E8") spseqs <- c('ATAGTTAC', 'TGCTGAGT', 'CCTACGTA', 'GAGCACCG') if(spid == "SI_GA_E9" || spid == "SI_P2_E9") spseqs <- c('TTGTTTCC', 'GGAGGAGG', 'CCTAACAA', 'AACCCGTT') if(spid == "SI_GA_E10" || spid == "SI_P2_E10") spseqs <- c('AAATGTGC', 'GGGCAAAT', 'TCTATCCG', 'CTCGCGTA') if(spid == "SI_GA_E11" || spid == "SI_P2_E11") spseqs <- c('AAGCGCTG', 'CGTTTGAT', 'GTAGCACA', 'TCCAATGC') if(spid == "SI_GA_E12" || spid == "SI_P2_E12") spseqs <- c('ACCGGCTC', 'GAGTTAGT', 'CGTCCTAG', 'TTAAAGCA') if(spid == "SI_GA_F1" || spid == "SI_P2_F1") spseqs <- c('GTTGCAGC', 'TGGAATTA', 'CAATGGAG', 'ACCCTCCT') if(spid == "SI_GA_F2" || spid == "SI_P2_F2") spseqs <- c('TTTACATG', 'CGCGATAC', 'ACGCGGGT', 'GAATTCCA') if(spid == "SI_GA_F3" || spid == "SI_P2_F3") spseqs <- c('TTCAGGTG', 'ACGGACAT', 'GATCTTGA', 'CGATCACC') if(spid == "SI_GA_F4" || spid == "SI_P2_F4") spseqs <- c('CCCAATAG', 'GTGTCGCT', 'AGAGTCGC', 'TATCGATA') if(spid == "SI_GA_F5" || spid == "SI_P2_F5") spseqs <- c('GACTACGT', 'CTAGCGAG', 'TCTATATC', 'AGGCGTCA') if(spid == "SI_GA_F6" || spid == "SI_P2_F6") spseqs <- c('CGGAGCAC', 'GACCTATT', 'ACTTAGGA', 'TTAGCTCG') if(spid == "SI_GA_F7" || spid == "SI_P2_F7") spseqs <- c('CGTGCAGA', 'AACAAGAT', 'TCGCTTCG', 'GTATGCTC') if(spid == "SI_GA_F8" || spid == "SI_P2_F8") spseqs <- c('CATGAACA', 'TCACTCGC', 'AGCTGGAT', 'GTGACTTG') if(spid == "SI_GA_F9" || spid == "SI_P2_F9") spseqs <- c('CAAGCTCC', 'GTTCACTG', 'TCGTGAAA', 'AGCATGGT') if(spid == "SI_GA_F10" || spid == "SI_P2_F10") spseqs <- c('GCTTGGCT', 'AAACAAAC', 'CGGGCTTA', 'TTCATCGG') if(spid == "SI_GA_F11" || spid == "SI_P2_F11") spseqs <- c('GCGAGAGT', 'TACGTTCA', 'AGTCCCAC', 'CTATAGTG') if(spid == "SI_GA_F12" || spid == "SI_P2_F12") spseqs <- c('TGATGCAT', 'GCTACTGA', 'CACCTGCC', 'ATGGAATG') if(spid == "SI_GA_G1" || spid == "SI_P2_G1") spseqs <- c('ATGAATCT', 'GATCTCAG', 'CCAGGAGC', 'TGCTCGTA') if(spid == "SI_GA_G2" || spid == "SI_P2_G2") spseqs <- c('TGATTCTA', 'ACTAGGAG', 'CAGCCACT', 'GTCGATGC') if(spid == "SI_GA_G3" || spid == "SI_P2_G3") spseqs <- c('CCTCATTC', 'AGCATCCG', 'GTGGCAAT', 'TAATGGGA') if(spid == "SI_GA_G4" || spid == "SI_P2_G4") spseqs <- c('GCGATGTG', 'AGATACAA', 'TTTCCACT', 'CACGGTGC') if(spid == "SI_GA_G5" || spid == "SI_P2_G5") spseqs <- c('GAGCAAGA', 'TCTGTGAT', 'CGCAGTTC', 'ATATCCCG') if(spid == "SI_GA_G6" || spid == "SI_P2_G6") spseqs <- c('CTGACGCG', 'GGTCGTAC', 'TCCTTCTT', 'AAAGAAGA') if(spid == "SI_GA_G7" || spid == "SI_P2_G7") spseqs <- c('GGTATGCA', 'CTCGAAAT', 'ACACCTTC', 'TAGTGCGG') if(spid == "SI_GA_G8" || spid == "SI_P2_G8") spseqs <- c('TATGAGCT', 'CCGATAGC', 'ATACCCAA', 'GGCTGTTG') if(spid == "SI_GA_G9" || spid == "SI_P2_G9") spseqs <- c('TAGGACGT', 'ATCCCACA', 'GGAATGTC', 'CCTTGTAG') if(spid == "SI_GA_G10" || spid == "SI_P2_G10") spseqs <- c('TCGCCAGC', 'AATGTTAG', 'CGATAGCT', 'GTCAGCTA') if(spid == "SI_GA_G11" || spid == "SI_P2_G11") spseqs <- c('TTATCGTT', 'AGCAGAGC', 'CATCTCCA', 'GCGGATAG') if(spid == "SI_GA_G12" || spid == "SI_P2_G12") spseqs <- c('ATTCTAAG', 'CCCGATTA', 'TGGAGGCT', 'GAATCCGC') if(spid == "SI_GA_H1" || spid == "SI_P2_H1") spseqs <- c('GTATGTCA', 'TGTCAGAC', 'CACGTCGG', 'ACGACATT') if(spid == "SI_GA_H2" || spid == "SI_P2_H2") spseqs <- c('TAATGACC', 'ATGCCTTA', 'GCCGAGAT', 'CGTATCGG') if(spid == "SI_GA_H3" || spid == "SI_P2_H3") spseqs <- c('CCAAGATG', 'AGGCCCGA', 'TACGTGAC', 'GTTTATCT') if(spid == "SI_GA_H4" || spid == "SI_P2_H4") spseqs <- c('GCCATTCC', 'CAAGAATT', 'TTGCCGGA', 'AGTTGCAG') if(spid == "SI_GA_H5" || spid == "SI_P2_H5") spseqs <- c('CCACTACA', 'GATTCTGG', 'TGCGGCTT', 'ATGAAGAC') if(spid == "SI_GA_H6" || spid == "SI_P2_H6") spseqs <- c('TAGGATAA', 'CCTTTGTC', 'GTACGCGG', 'AGCACACT') if(spid == "SI_GA_H7" || spid == "SI_P2_H7") spseqs <- c('AGCTATCA', 'CATATAAC', 'TCAGGGTG', 'GTGCCCGT') if(spid == "SI_GA_H8" || spid == "SI_P2_H8") spseqs <- c('TTGTTGAT', 'GCTCAACC', 'CAAAGTGG', 'AGCGCCTA') if(spid == "SI_GA_H9" || spid == "SI_P2_H9") spseqs <- c('ACACTGTT', 'CAGGATGG', 'GGCTGAAC', 'TTTACCCA') if(spid == "SI_GA_H10" || spid == "SI_P2_H10") spseqs <- c('GTAATTGC', 'AGTCGCTT', 'CACGAGAA', 'TCGTCACG') if(spid == "SI_GA_H11" || spid == "SI_P2_H11") spseqs <- c('GGCGAGTA', 'ACTTCTAT', 'CAAATACG', 'TTGCGCGC') if(spid == "SI_GA_H12" || spid == "SI_P2_H12") spseqs <- c('GACAGCAT', 'TTTGTACA', 'AGGCCGTG', 'CCATATGC') dualseqs <- NA if(spid == "SI-TT-A1" || spid == "SI_TT_A1") dualseqs <- c('GTAACATGCG','AGTGTTACCT','AGGTAACACT') if(spid == "SI-TT-A2" || spid == "SI_TT_A2") dualseqs <- c('GTGGATCAAA','GCCAACCCTG','CAGGGTTGGC') if(spid == "SI-TT-A3" || spid == "SI_TT_A3") dualseqs <- c('CACTACGAAA','TTAGACTGAT','ATCAGTCTAA') if(spid == "SI-TT-A4" || spid == "SI_TT_A4") dualseqs <- c('CTCTAGCGAG','TATCTTCATC','GATGAAGATA') if(spid == "SI-TT-A5" || spid == "SI_TT_A5") dualseqs <- c('GTAGCCCTGT','GAGCATCTAT','ATAGATGCTC') if(spid == "SI-TT-A6" || spid == "SI_TT_A6") dualseqs <- c('TAACGCGTGA','CCCTAACTTC','GAAGTTAGGG') if(spid == "SI-TT-A7" || spid == "SI_TT_A7") dualseqs <- c('TCCCAAGGGT','TACTACCTTT','AAAGGTAGTA') if(spid == "SI-TT-A8" || spid == "SI_TT_A8") dualseqs <- c('CGAAGTATAC','GAACTTGGAG','CTCCAAGTTC') if(spid == "SI-TT-A9" || spid == "SI_TT_A9") dualseqs <- c('AAGTGGAGAG','TTCCTGTTAC','GTAACAGGAA') if(spid == "SI-TT-A10" || spid == "SI_TT_A10") dualseqs <- c('CGTGACATGC','ATGGTCTAAA','TTTAGACCAT') if(spid == "SI-TT-A11" || spid == "SI_TT_A11") dualseqs <- c('CGGAACCCAA','GATTCGAGGA','TCCTCGAATC') if(spid == "SI-TT-A12" || spid == "SI_TT_A12") dualseqs <- c('CACCGCACCA','GACTGTCAAT','ATTGACAGTC') if(spid == "SI-TT-B1" || spid == "SI_TT_B1") dualseqs <- c('ACAGTAACTA','ACAGTTCGTT','AACGAACTGT') if(spid == "SI-TT-B2" || spid == "SI_TT_B2") dualseqs <- c('TCTACCATTT','CGGGAGAGTC','GACTCTCCCG') if(spid == "SI-TT-B3" || spid == "SI_TT_B3") dualseqs <- c('CACGGTGAAT','GTTCGTCACA','TGTGACGAAC') if(spid == "SI-TT-B4" || spid == "SI_TT_B4") dualseqs <- c('GTAGACGAAA','CTAGTGTGGT','ACCACACTAG') if(spid == "SI-TT-B5" || spid == "SI_TT_B5") dualseqs <- c('TCGGCTCTAC','CCGATGGTCT','AGACCATCGG') if(spid == "SI-TT-B6" || spid == "SI_TT_B6") dualseqs <- c('AATGCCATGA','TACGTAATGC','GCATTACGTA') if(spid == "SI-TT-B7" || spid == "SI_TT_B7") dualseqs <- c('GCCTTCGGTA','CCAACGATTT','AAATCGTTGG') if(spid == "SI-TT-B8" || spid == "SI_TT_B8") dualseqs <- c('GCACTGAGAA','TATGCGTGAA','TTCACGCATA') if(spid == "SI-TT-B9" || spid == "SI_TT_B9") dualseqs <- c('TATTGAGGCA','CAGGTAAGTG','CACTTACCTG') if(spid == "SI-TT-B10" || spid == "SI_TT_B10") dualseqs <- c('GCCCGATGGA','AATCGTCTAG','CTAGACGATT') if(spid == "SI-TT-B11" || spid == "SI_TT_B11") dualseqs <- c('TCTTACTTGC','TGACCTCTAG','CTAGAGGTCA') if(spid == "SI-TT-B12" || spid == "SI_TT_B12") dualseqs <- c('CGTCAAGGGC','TAGGTCACTC','GAGTGACCTA') if(spid == "SI-TT-C1" || spid == "SI_TT_C1") dualseqs <- c('TGCGCGGTTT','CAAGGATAAA','TTTATCCTTG') if(spid == "SI-TT-C2" || spid == "SI_TT_C2") dualseqs <- c('CAATCCCGAC','CCGAGTAGTA','TACTACTCGG') if(spid == "SI-TT-C3" || spid == "SI_TT_C3") dualseqs <- c('ATGGCTTGTG','GAATGTTGTG','CACAACATTC') if(spid == "SI-TT-C4" || spid == "SI_TT_C4") dualseqs <- c('TTCTCGATGA','TGTCGGGCAC','GTGCCCGACA') if(spid == "SI-TT-C5" || spid == "SI_TT_C5") dualseqs <- c('TCCGTTGGAT','ACGTTCTCGC','GCGAGAACGT') if(spid == "SI-TT-C6" || spid == "SI_TT_C6") dualseqs <- c('ACGACTACCA','ACGACCCTAA','TTAGGGTCGT') if(spid == "SI-TT-C7" || spid == "SI_TT_C7") dualseqs <- c('CGCGCACTTA','CCTGTATTCT','AGAATACAGG') if(spid == "SI-TT-C8" || spid == "SI_TT_C8") dualseqs <- c('GCTACAAAGC','CACGTGCCCT','AGGGCACGTG') if(spid == "SI-TT-C9" || spid == "SI_TT_C9") dualseqs <- c('TATCAGCCTA','GTTTCGTCCT','AGGACGAAAC') if(spid == "SI-TT-C10" || spid == "SI_TT_C10") dualseqs <- c('AGAATGGTTT','GAGGGTGGGA','TCCCACCCTC') if(spid == "SI-TT-C11" || spid == "SI_TT_C11") dualseqs <- c('ATGGGTGAAA','CTTGGGAATT','AATTCCCAAG') if(spid == "SI-TT-C12" || spid == "SI_TT_C12") dualseqs <- c('TCGTCAAGAT','GCAACTCAGG','CCTGAGTTGC') if(spid == "SI-TT-D1" || spid == "SI_TT_D1") dualseqs <- c('TGCAATGTTC','GCTTGTCGAA','TTCGACAAGC') if(spid == "SI-TT-D2" || spid == "SI_TT_D2") dualseqs <- c('TTAATACGCG','CACCTCGGGT','ACCCGAGGTG') if(spid == "SI-TT-D3" || spid == "SI_TT_D3") dualseqs <- c('CCTTCTAGAG','AATACAACGA','TCGTTGTATT') if(spid == "SI-TT-D4" || spid == "SI_TT_D4") dualseqs <- c('GCAGTATAGG','TTCCGTGCAC','GTGCACGGAA') if(spid == "SI-TT-D5" || spid == "SI_TT_D5") dualseqs <- c('TGGTTCGGGT','GTGGCAGGAG','CTCCTGCCAC') if(spid == "SI-TT-D6" || spid == "SI_TT_D6") dualseqs <- c('CCCAGCTTCT','GACACCAAAC','GTTTGGTGTC') if(spid == "SI-TT-D7" || spid == "SI_TT_D7") dualseqs <- c('CCTGTCAGGG','AGCCCGTAAC','GTTACGGGCT') if(spid == "SI-TT-D8" || spid == "SI_TT_D8") dualseqs <- c('CGCTGAAATC','AGGTGTCTGC','GCAGACACCT') if(spid == "SI-TT-D9" || spid == "SI_TT_D9") dualseqs <- c('TGGTCCCAAG','CCTCTGGCGT','ACGCCAGAGG') if(spid == "SI-TT-D10" || spid == "SI_TT_D10") dualseqs <- c('ATGCGAATGG','ACAAGTGTCG','CGACACTTGT') if(spid == "SI-TT-D11" || spid == "SI_TT_D11") dualseqs <- c('CGAATATTCG','CTGGAAGCAA','TTGCTTCCAG') if(spid == "SI-TT-D12" || spid == "SI_TT_D12") dualseqs <- c('GAATTGGTTA','ACTCTAGTAG','CTACTAGAGT') if(spid == "SI-TT-E1" || spid == "SI_TT_E1") dualseqs <- c('TTATTCGAGG','CTGTCCTGCT','AGCAGGACAG') if(spid == "SI-TT-E2" || spid == "SI_TT_E2") dualseqs <- c('ATGGAGGGAG','ATAACCCATT','AATGGGTTAT') if(spid == "SI-TT-E3" || spid == "SI_TT_E3") dualseqs <- c('ACCAGACAAC','AGGAACTAGG','CCTAGTTCCT') if(spid == "SI-TT-E4" || spid == "SI_TT_E4") dualseqs <- c('AACCACGCAT','ATTCAGGTTA','TAACCTGAAT') if(spid == "SI-TT-E5" || spid == "SI_TT_E5") dualseqs <- c('CGCGGTAGGT','CAGGATGTTG','CAACATCCTG') if(spid == "SI-TT-E6" || spid == "SI_TT_E6") dualseqs <- c('TTGAGAGTCA','AACCTGGTAG','CTACCAGGTT') if(spid == "SI-TT-E7" || spid == "SI_TT_E7") dualseqs <- c('GTCCTTCGGC','TCATGCACAG','CTGTGCATGA') if(spid == "SI-TT-E8" || spid == "SI_TT_E8") dualseqs <- c('GAGCAAGGGC','ATTGACTTGG','CCAAGTCAAT') if(spid == "SI-TT-E9" || spid == "SI_TT_E9") dualseqs <- c('TGTCCCAACG','TCGATGTCCA','TGGACATCGA') if(spid == "SI-TT-E10" || spid == "SI_TT_E10") dualseqs <- c('CACAATCCCA','ATATCCACAA','TTGTGGATAT') if(spid == "SI-TT-E11" || spid == "SI_TT_E11") dualseqs <- c('TCCGGGACAA','GTGAATGCCA','TGGCATTCAC') if(spid == "SI-TT-E12" || spid == "SI_TT_E12") dualseqs <- c('CGTCCACCTG','CATTCATGAC','GTCATGAATG') if(spid == "SI-TT-F1" || spid == "SI_TT_F1") dualseqs <- c('AAGATTGGAT','AGCGGGATTT','AAATCCCGCT') if(spid == "SI-TT-F2" || spid == "SI_TT_F2") dualseqs <- c('AAGGGCCGCA','CTGATTCCTC','GAGGAATCAG') if(spid == "SI-TT-F3" || spid == "SI_TT_F3") dualseqs <- c('GAGAGGATAT','TTGAAATGGG','CCCATTTCAA') if(spid == "SI-TT-F4" || spid == "SI_TT_F4") dualseqs <- c('CCCACCACAA','ACCTCCGCTT','AAGCGGAGGT') if(spid == "SI-TT-F5" || spid == "SI_TT_F5") dualseqs <- c('CGGCTGGATG','TGATAAGCAC','GTGCTTATCA') if(spid == "SI-TT-F6" || spid == "SI_TT_F6") dualseqs <- c('TTGCCCGTGC','GCGTGAGATT','AATCTCACGC') if(spid == "SI-TT-F7" || spid == "SI_TT_F7") dualseqs <- c('AATGTATCCA','AATGAGCTTA','TAAGCTCATT') if(spid == "SI-TT-F8" || spid == "SI_TT_F8") dualseqs <- c('CTCCTTTAGA','GACATAGCTC','GAGCTATGTC') if(spid == "SI-TT-F9" || spid == "SI_TT_F9") dualseqs <- c('GTCCCATCAA','CGAACGTGAC','GTCACGTTCG') if(spid == "SI-TT-F10" || spid == "SI_TT_F10") dualseqs <- c('CCGGCAACTG','CGGTTTAACA','TGTTAAACCG') if(spid == "SI-TT-F11" || spid == "SI_TT_F11") dualseqs <- c('TTCACACCTT','TAGTGTACAC','GTGTACACTA') if(spid == "SI-TT-F12" || spid == "SI_TT_F12") dualseqs <- c('GAGACGCACG','CTATGAACAT','ATGTTCATAG') if(spid == "SI-TT-G1" || spid == "SI_TT_G1") dualseqs <- c('TGTAGTCATT','CTTGATCGTA','TACGATCAAG') if(spid == "SI-TT-G2" || spid == "SI_TT_G2") dualseqs <- c('CATGTGGGTT','GATTCCTTTA','TAAAGGAATC') if(spid == "SI-TT-G3" || spid == "SI_TT_G3") dualseqs <- c('ATGACGTCGC','AGGTCAGGAT','ATCCTGACCT') if(spid == "SI-TT-G4" || spid == "SI_TT_G4") dualseqs <- c('GCGCTTATGG','GCCTGGCTAG','CTAGCCAGGC') if(spid == "SI-TT-G5" || spid == "SI_TT_G5") dualseqs <- c('ATAGGGCGAG','TGCATCGAGT','ACTCGATGCA') if(spid == "SI-TT-G6" || spid == "SI_TT_G6") dualseqs <- c('GCGGGTAAGT','TAGCACTAAG','CTTAGTGCTA') if(spid == "SI-TT-G7" || spid == "SI_TT_G7") dualseqs <- c('GTTTCACGAT','TTCGGCCAAA','TTTGGCCGAA') if(spid == "SI-TT-G8" || spid == "SI_TT_G8") dualseqs <- c('TAAGCAACTG','CTATACTCAA','TTGAGTATAG') if(spid == "SI-TT-G9" || spid == "SI_TT_G9") dualseqs <- c('CCGGAGGAAG','TGCGGATGTT','AACATCCGCA') if(spid == "SI-TT-G10" || spid == "SI_TT_G10") dualseqs <- c('ACTTTACGTG','TGAACGCCCT','AGGGCGTTCA') if(spid == "SI-TT-G11" || spid == "SI_TT_G11") dualseqs <- c('GATAACCTGC','CATTAGAAAC','GTTTCTAATG') if(spid == "SI-TT-G12" || spid == "SI_TT_G12") dualseqs <- c('CTTGCATAAA','ATCAGGGCTT','AAGCCCTGAT') if(spid == "SI-TT-H1" || spid == "SI_TT_H1") dualseqs <- c('ACAATGTGAA','CGTACCGTTA','TAACGGTACG') if(spid == "SI-TT-H2" || spid == "SI_TT_H2") dualseqs <- c('TAGCATAGTG','CGGCTCTGTC','GACAGAGCCG') if(spid == "SI-TT-H3" || spid == "SI_TT_H3") dualseqs <- c('CCCGTTCTCG','GACGGATTGG','CCAATCCGTC') if(spid == "SI-TT-H4" || spid == "SI_TT_H4") dualseqs <- c('AGTTTCCTGG','TGCCACACAG','CTGTGTGGCA') if(spid == "SI-TT-H5" || spid == "SI_TT_H5") dualseqs <- c('AGCAAGAAGC','TTGTGTTTCT','AGAAACACAA') if(spid == "SI-TT-H6" || spid == "SI_TT_H6") dualseqs <- c('CCTATCCTCG','GAATACTAAC','GTTAGTATTC') if(spid == "SI-TT-H7" || spid == "SI_TT_H7") dualseqs <- c('ACCTCGAGCT','TGTGTTCGAT','ATCGAACACA') if(spid == "SI-TT-H8" || spid == "SI_TT_H8") dualseqs <- c('ATAAGGATAC','ATAGATAGGG','CCCTATCTAT') if(spid == "SI-TT-H9" || spid == "SI_TT_H9") dualseqs <- c('AGAACTTAGA','CGAGTCCTTT','AAAGGACTCG') if(spid == "SI-TT-H10" || spid == "SI_TT_H10") dualseqs <- c('TTATCTAGGG','AAAGGCTCTA','TAGAGCCTTT') if(spid == "SI-TT-H11" || spid == "SI_TT_H11") dualseqs <- c('ACAATCGATC','TGACGGAATG','CATTCCGTCA') if(spid == "SI-TT-H12" || spid == "SI_TT_H12") dualseqs <- c('TGATGATTCA','GTAGGAGTCG','CGACTCCTAC') if(spid == "SI-NN-A1" || spid == "SI_NN_A1") dualseqs <- c('GCCTTGTCAA','GATCCAATCA','TGATTGGATC') if(spid == "SI-NN-A2" || spid == "SI_NN_A2") dualseqs <- c('AACTTCTTTG','ATAAGCGGTA','TACCGCTTAT') if(spid == "SI-NN-A3" || spid == "SI_NN_A3") dualseqs <- c('TGGTTATAGA','GAGCTCATAA','TTATGAGCTC') if(spid == "SI-NN-A4" || spid == "SI_NN_A4") dualseqs <- c('GTAGTGCATC','ATTTGACGGT','ACCGTCAAAT') if(spid == "SI-NN-A5" || spid == "SI_NN_A5") dualseqs <- c('GCTTGATTTA','CGGGTGTGAC','GTCACACCCG') if(spid == "SI-NN-A6" || spid == "SI_NN_A6") dualseqs <- c('GAAGTTTCGC','TTTCAAAGCA','TGCTTTGAAA') if(spid == "SI-NN-A7" || spid == "SI_NN_A7") dualseqs <- c('GCAGTTGTTT','ACACTACTTT','AAAGTAGTGT') if(spid == "SI-NN-A8" || spid == "SI_NN_A8") dualseqs <- c('GCCGCAAGTA','AAAGGTCCGC','GCGGACCTTT') if(spid == "SI-NN-A9" || spid == "SI_NN_A9") dualseqs <- c('AAGTTGTATG','TGTACAATGC','GCATTGTACA') if(spid == "SI-NN-A10" || spid == "SI_NN_A10") dualseqs <- c('AGCAACCTTT','TTCAGCGCTA','TAGCGCTGAA') if(spid == "SI-NN-A11" || spid == "SI_NN_A11") dualseqs <- c('GACGAGGGTT','TTACTTTGAC','GTCAAAGTAA') if(spid == "SI-NN-A12" || spid == "SI_NN_A12") dualseqs <- c('TGAACAGTCG','ATTTCTCAGA','TCTGAGAAAT') if(spid == "SI-NN-B1" || spid == "SI_NN_B1") dualseqs <- c('CGAAAGGAGT','ACACCAAACA','TGTTTGGTGT') if(spid == "SI-NN-B2" || spid == "SI_NN_B2") dualseqs <- c('GAATGACTTG','TGAGCTCGCT','AGCGAGCTCA') if(spid == "SI-NN-B3" || spid == "SI_NN_B3") dualseqs <- c('GCCAGGGATT','CAAGTTTAGG','CCTAAACTTG') if(spid == "SI-NN-B4" || spid == "SI_NN_B4") dualseqs <- c('CTTTGTCCGC','GCTGTCCCGT','ACGGGACAGC') if(spid == "SI-NN-B5" || spid == "SI_NN_B5") dualseqs <- c('AGCTTGGGTG','AGCGTATAGT','ACTATACGCT') if(spid == "SI-NN-B6" || spid == "SI_NN_B6") dualseqs <- c('TCGCACCGGT','TAGCCTCAGG','CCTGAGGCTA') if(spid == "SI-NN-B7" || spid == "SI_NN_B7") dualseqs <- c('TTTAGGTAGG','ACTGAGGGAC','GTCCCTCAGT') if(spid == "SI-NN-B8" || spid == "SI_NN_B8") dualseqs <- c('GTTCGAGCGG','CTGTGGGTTT','AAACCCACAG') if(spid == "SI-NN-B9" || spid == "SI_NN_B9") dualseqs <- c('AGGGTATGGC','GAGTCCGACT','AGTCGGACTC') if(spid == "SI-NN-B10" || spid == "SI_NN_B10") dualseqs <- c('TACCCACTAT','TGGAACATAT','ATATGTTCCA') if(spid == "SI-NN-B11" || spid == "SI_NN_B11") dualseqs <- c('TTCAGGCTAC','CTGGGTATTG','CAATACCCAG') if(spid == "SI-NN-B12" || spid == "SI_NN_B12") dualseqs <- c('AGGAATAGAT','TGCGGTCACG','CGTGACCGCA') if(spid == "SI-NN-C1" || spid == "SI_NN_C1") dualseqs <- c('ACAGCGCTGT','GAGGGTCATC','GATGACCCTC') if(spid == "SI-NN-C2" || spid == "SI_NN_C2") dualseqs <- c('GACGTACGGA','ACCTCGCTTT','AAAGCGAGGT') if(spid == "SI-NN-C3" || spid == "SI_NN_C3") dualseqs <- c('CTTAGATGCA','CTATTCGACT','AGTCGAATAG') if(spid == "SI-NN-C4" || spid == "SI_NN_C4") dualseqs <- c('ACTCCTCAAC','ATTCACGGGA','TCCCGTGAAT') if(spid == "SI-NN-C5" || spid == "SI_NN_C5") dualseqs <- c('TCCATACTGT','GAGCGACTTC','GAAGTCGCTC') if(spid == "SI-NN-C6" || spid == "SI_NN_C6") dualseqs <- c('GCCAGAATGG','GAATTGTATC','GATACAATTC') if(spid == "SI-NN-C7" || spid == "SI_NN_C7") dualseqs <- c('ATTGCAAGAC','CGGTGACCAT','ATGGTCACCG') if(spid == "SI-NN-C8" || spid == "SI_NN_C8") dualseqs <- c('CAACATATCG','AACTAAAGGC','GCCTTTAGTT') if(spid == "SI-NN-C9" || spid == "SI_NN_C9") dualseqs <- c('CAGGGACCTC','AATAGCAGCA','TGCTGCTATT') if(spid == "SI-NN-C10" || spid == "SI_NN_C10") dualseqs <- c('ATTTGGGAAT','TCAGCCGAGT','ACTCGGCTGA') if(spid == "SI-NN-C11" || spid == "SI_NN_C11") dualseqs <- c('TGGATAATTG','TTTGTGATCA','TGATCACAAA') if(spid == "SI-NN-C12" || spid == "SI_NN_C12") dualseqs <- c('CAGATTATGA','GTGCCTGTAC','GTACAGGCAC') if(spid == "SI-NN-D1" || spid == "SI_NN_D1") dualseqs <- c('AATGGTAAGC','CTGTGAAGTT','AACTTCACAG') if(spid == "SI-NN-D2" || spid == "SI_NN_D2") dualseqs <- c('CATCCCTCGG','AGGGTAGGCG','CGCCTACCCT') if(spid == "SI-NN-D3" || spid == "SI_NN_D3") dualseqs <- c('CTGGTCCGTC','ACGAATAAGG','CCTTATTCGT') if(spid == "SI-NN-D4" || spid == "SI_NN_D4") dualseqs <- c('GTCGTAAATT','GTAGTAGGCT','AGCCTACTAC') if(spid == "SI-NN-D5" || spid == "SI_NN_D5") dualseqs <- c('CAACACTGAT','CAAACCAGGG','CCCTGGTTTG') if(spid == "SI-NN-D6" || spid == "SI_NN_D6") dualseqs <- c('CAGACTGAAT','TGCCTAACCG','CGGTTAGGCA') if(spid == "SI-NN-D7" || spid == "SI_NN_D7") dualseqs <- c('CGGACACGCT','TCACGATATA','TATATCGTGA') if(spid == "SI-NN-D8" || spid == "SI_NN_D8") dualseqs <- c('CTCGGCTCTC','TATACTTCCA','TGGAAGTATA') if(spid == "SI-NN-D9" || spid == "SI_NN_D9") dualseqs <- c('AAGCCTATCA','GTAGTGAACA','TGTTCACTAC') if(spid == "SI-NN-D10" || spid == "SI_NN_D10") dualseqs <- c('ACTGTGCCAA','TAACCGTTAT','ATAACGGTTA') if(spid == "SI-NN-D11" || spid == "SI_NN_D11") dualseqs <- c('CTACTCCCAT','TACGTTGCGG','CCGCAACGTA') if(spid == "SI-NN-D12" || spid == "SI_NN_D12") dualseqs <- c('AGACTCTAAC','GTGTCGATGT','ACATCGACAC') if(spid == "SI-NN-E1" || spid == "SI_NN_E1") dualseqs <- c('TGAGCCGGCA','TGCTGCATGT','ACATGCAGCA') if(spid == "SI-NN-E2" || spid == "SI_NN_E2") dualseqs <- c('TAGCTTGCGT','GCTGCTAAAG','CTTTAGCAGC') if(spid == "SI-NN-E3" || spid == "SI_NN_E3") dualseqs <- c('GTACACCGGG','AACAGTCGCG','CGCGACTGTT') if(spid == "SI-NN-E4" || spid == "SI_NN_E4") dualseqs <- c('CGCGTAGACG','AAAGTAGCTG','CAGCTACTTT') if(spid == "SI-NN-E5" || spid == "SI_NN_E5") dualseqs <- c('CCGATATATT','AACTATCCGA','TCGGATAGTT') if(spid == "SI-NN-E6" || spid == "SI_NN_E6") dualseqs <- c('TTGGGCGGGA','ATGTATCTGT','ACAGATACAT') if(spid == "SI-NN-E7" || spid == "SI_NN_E7") dualseqs <- c('GTTTATGAGG','GCCAAACAGC','GCTGTTTGGC') if(spid == "SI-NN-E8" || spid == "SI_NN_E8") dualseqs <- c('TTGGATACTC','CGTACTGAAG','CTTCAGTACG') if(spid == "SI-NN-E9" || spid == "SI_NN_E9") dualseqs <- c('GCTATCTATC','AAAGATGGAT','ATCCATCTTT') if(spid == "SI-NN-E10" || spid == "SI_NN_E10") dualseqs <- c('ATGTGATCGC','TGACCTACCT','AGGTAGGTCA') if(spid == "SI-NN-E11" || spid == "SI_NN_E11") dualseqs <- c('TCCTCACATG','AACAGATTCA','TGAATCTGTT') if(spid == "SI-NN-E12" || spid == "SI_NN_E12") dualseqs <- c('GCCTCCTAAT','CTCCTCCTGT','ACAGGAGGAG') if(spid == "SI-NN-F1" || spid == "SI_NN_F1") dualseqs <- c('CGAGGCTGAT','TCGACTTTCT','AGAAAGTCGA') if(spid == "SI-NN-F2" || spid == "SI_NN_F2") dualseqs <- c('CTCATGACAT','TGTTCCGCAC','GTGCGGAACA') if(spid == "SI-NN-F3" || spid == "SI_NN_F3") dualseqs <- c('AACGCGGCAA','GCCCGTTTAC','GTAAACGGGC') if(spid == "SI-NN-F4" || spid == "SI_NN_F4") dualseqs <- c('AACACCTCTC','GATTGTATGA','TCATACAATC') if(spid == "SI-NN-F5" || spid == "SI_NN_F5") dualseqs <- c('AAATAACGCG','ATAGAGGAGC','GCTCCTCTAT') if(spid == "SI-NN-F6" || spid == "SI_NN_F6") dualseqs <- c('GAAATCTTGT','TAGACGCCAC','GTGGCGTCTA') if(spid == "SI-NN-F7" || spid == "SI_NN_F7") dualseqs <- c('TAGATGGACT','CCAGGGATAT','ATATCCCTGG') if(spid == "SI-NN-F8" || spid == "SI_NN_F8") dualseqs <- c('CAATCTGTAT','TCCCATCGTT','AACGATGGGA') if(spid == "SI-NN-F9" || spid == "SI_NN_F9") dualseqs <- c('GCGAAAGTAT','GTGTCACAGA','TCTGTGACAC') if(spid == "SI-NN-F10" || spid == "SI_NN_F10") dualseqs <- c('CCCAGAGCTT','ATGCTGTAAT','ATTACAGCAT') if(spid == "SI-NN-F11" || spid == "SI_NN_F11") dualseqs <- c('GACTACAGGA','AGTGACAAAG','CTTTGTCACT') if(spid == "SI-NN-F12" || spid == "SI_NN_F12") dualseqs <- c('CAATTCATGG','ACGTAGTTCG','CGAACTACGT') if(spid == "SI-NN-G1" || spid == "SI_NN_G1") dualseqs <- c('TGGGAAGGGC','ACTAACGATG','CATCGTTAGT') if(spid == "SI-NN-G2" || spid == "SI_NN_G2") dualseqs <- c('TACTTCGCAT','AACGGTGTCG','CGACACCGTT') if(spid == "SI-NN-G3" || spid == "SI_NN_G3") dualseqs <- c('TAAACTGGCA','CCGACCTCTT','AAGAGGTCGG') if(spid == "SI-NN-G4" || spid == "SI_NN_G4") dualseqs <- c('TTGAAGAACT','CAACACCAGG','CCTGGTGTTG') if(spid == "SI-NN-G5" || spid == "SI_NN_G5") dualseqs <- c('TCCCTCGTCA','ATCAGGTGTG','CACACCTGAT') if(spid == "SI-NN-G6" || spid == "SI_NN_G6") dualseqs <- c('ACTGGCCTAA','CGCTCCCTTG','CAAGGGAGCG') if(spid == "SI-NN-G7" || spid == "SI_NN_G7") dualseqs <- c('TCATAATGCA','GATGAGGCGA','TCGCCTCATC') if(spid == "SI-NN-G8" || spid == "SI_NN_G8") dualseqs <- c('CGCCACGTTA','CTTTCATAGG','CCTATGAAAG') if(spid == "SI-NN-G9" || spid == "SI_NN_G9") dualseqs <- c('TGGACTAAGC','CTTAACATCG','CGATGTTAAG') if(spid == "SI-NN-G10" || spid == "SI_NN_G10") dualseqs <- c('AAGCCCGGCA','GAGTTTCCGT','ACGGAAACTC') if(spid == "SI-NN-G11" || spid == "SI_NN_G11") dualseqs <- c('ATAGTGGCGG','TACTGTTGTT','AACAACAGTA') if(spid == "SI-NN-G12" || spid == "SI_NN_G12") dualseqs <- c('CCAACCGGAC','GTTCTATCTA','TAGATAGAAC') if(spid == "SI-NN-H1" || spid == "SI_NN_H1") dualseqs <- c('TTACAGAGGG','GTTTATGGCA','TGCCATAAAC') if(spid == "SI-NN-H2" || spid == "SI_NN_H2") dualseqs <- c('TCCAACAACG','GTCCGGGTTT','AAACCCGGAC') if(spid == "SI-NN-H3" || spid == "SI_NN_H3") dualseqs <- c('AAAGCATGTC','AGATACTACT','AGTAGTATCT') if(spid == "SI-NN-H4" || spid == "SI_NN_H4") dualseqs <- c('AGAAGTCGTT','AGGACATTTA','TAAATGTCCT') if(spid == "SI-NN-H5" || spid == "SI_NN_H5") dualseqs <- c('GCTAGCGTTC','TCGCGTGGTG','CACCACGCGA') if(spid == "SI-NN-H6" || spid == "SI_NN_H6") dualseqs <- c('CAACTAGCTT','TCAACTAATC','GATTAGTTGA') if(spid == "SI-NN-H7" || spid == "SI_NN_H7") dualseqs <- c('AGGGCCAGTT','TAGGGCAAAT','ATTTGCCCTA') if(spid == "SI-NN-H8" || spid == "SI_NN_H8") dualseqs <- c('CTCTATGCGT','CCTCTGATGC','GCATCAGAGG') if(spid == "SI-NN-H9" || spid == "SI_NN_H9") dualseqs <- c('GTCCATACAA','AGCCACTCAC','GTGAGTGGCT') if(spid == "SI-NN-H10" || spid == "SI_NN_H10") dualseqs <- c('AGGGATGCAA','CTCATATCCT','AGGATATGAG') if(spid == "SI-NN-H11" || spid == "SI_NN_H11") dualseqs <- c('TTCAAGTCCT','CAGCAGCTAA','TTAGCTGCTG') if(spid == "SI-NN-H12" || spid == "SI_NN_H12") dualseqs <- c('CTCCAACCTA','GCACCGCATC','GATGCGGTGC') if(spid == "SI-NT-A1" || spid == "SI_NT_A1") dualseqs <- c('ATTTACCGCA','GACAATAAAG','CTTTATTGTC') if(spid == "SI-NT-A2" || spid == "SI_NT_A2") dualseqs <- c('TTGTCGTAGA','CAATGTAGCA','TGCTACATTG') if(spid == "SI-NT-A3" || spid == "SI_NT_A3") dualseqs <- c('AGTCCTGCGG','TGACACAAGT','ACTTGTGTCA') if(spid == "SI-NT-A4" || spid == "SI_NT_A4") dualseqs <- c('TTGTTCATGT','TGACAGCTGA','TCAGCTGTCA') if(spid == "SI-NT-A5" || spid == "SI_NT_A5") dualseqs <- c('TCAGGAAGGA','GAACGTGCTT','AAGCACGTTC') if(spid == "SI-NT-A6" || spid == "SI_NT_A6") dualseqs <- c('CTGTTAGAGG','CCACGCTTCG','CGAAGCGTGG') if(spid == "SI-NT-A7" || spid == "SI_NT_A7") dualseqs <- c('AGATGAGAAT','GTCGACGGGT','ACCCGTCGAC') if(spid == "SI-NT-A8" || spid == "SI_NT_A8") dualseqs <- c('CCAAAGCCGG','ACCGTGCACA','TGTGCACGGT') if(spid == "SI-NT-A9" || spid == "SI_NT_A9") dualseqs <- c('AGTCATAATG','AGGCTTGAAA','TTTCAAGCCT') if(spid == "SI-NT-A10" || spid == "SI_NT_A10") dualseqs <- c('TTCATCAGAG','TTGTCGTCTC','GAGACGACAA') if(spid == "SI-NT-A11" || spid == "SI_NT_A11") dualseqs <- c('GAAGCGCGAA','CAGCGAAATT','AATTTCGCTG') if(spid == "SI-NT-A12" || spid == "SI_NT_A12") dualseqs <- c('ATCCGCCGAA','GCTACAGAAT','ATTCTGTAGC') if(spid == "SI-NT-B1" || spid == "SI_NT_B1") dualseqs <- c('CTTATTGTGG','AGCTGTGGGT','ACCCACAGCT') if(spid == "SI-NT-B2" || spid == "SI_NT_B2") dualseqs <- c('ATTCGTTGGG','CATCAGGAGC','GCTCCTGATG') if(spid == "SI-NT-B3" || spid == "SI_NT_B3") dualseqs <- c('GTGGCCTCAT','TCGAAAGTGA','TCACTTTCGA') if(spid == "SI-NT-B4" || spid == "SI_NT_B4") dualseqs <- c('CTATGGCATC','CTCTGAGCGC','GCGCTCAGAG') if(spid == "SI-NT-B5" || spid == "SI_NT_B5") dualseqs <- c('AAACCACAGT','CCGCAAATGG','CCATTTGCGG') if(spid == "SI-NT-B6" || spid == "SI_NT_B6") dualseqs <- c('ATGTATCCAC','CAGGCTGAGG','CCTCAGCCTG') if(spid == "SI-NT-B7" || spid == "SI_NT_B7") dualseqs <- c('GATGAGTCTG','TATGTAACCG','CGGTTACATA') if(spid == "SI-NT-B8" || spid == "SI_NT_B8") dualseqs <- c('CTCACAATAA','CCGTGTTTAA','TTAAACACGG') if(spid == "SI-NT-B9" || spid == "SI_NT_B9") dualseqs <- c('TCTGTCGCAA','ATCCGAACTG','CAGTTCGGAT') if(spid == "SI-NT-B10" || spid == "SI_NT_B10") dualseqs <- c('CATCGAGAAG','GTGCATCCGC','GCGGATGCAC') if(spid == "SI-NT-B11" || spid == "SI_NT_B11") dualseqs <- c('ACGTATTGGG','CAATCGCAAA','TTTGCGATTG') if(spid == "SI-NT-B12" || spid == "SI_NT_B12") dualseqs <- c('CTTACGCGAC','CCTGTCGGAT','ATCCGACAGG') if(spid == "SI-NT-C1" || spid == "SI_NT_C1") dualseqs <- c('GATAGTGAAG','CGTTCGCCAG','CTGGCGAACG') if(spid == "SI-NT-C2" || spid == "SI_NT_C2") dualseqs <- c('CTGTGCAATG','ATGGTGCTTT','AAAGCACCAT') if(spid == "SI-NT-C3" || spid == "SI_NT_C3") dualseqs <- c('AAAGCTGAGC','CAGGAACGAG','CTCGTTCCTG') if(spid == "SI-NT-C4" || spid == "SI_NT_C4") dualseqs <- c('GTCCAAGTCG','GTCATGGCAC','GTGCCATGAC') if(spid == "SI-NT-C5" || spid == "SI_NT_C5") dualseqs <- c('GATTTCCATC','CTTATGGTCA','TGACCATAAG') if(spid == "SI-NT-C6" || spid == "SI_NT_C6") dualseqs <- c('CTAAATGGAT','AATCACATAC','GTATGTGATT') if(spid == "SI-NT-C7" || spid == "SI_NT_C7") dualseqs <- c('ATTTCAGTTC','GCAGCATTAA','TTAATGCTGC') if(spid == "SI-NT-C8" || spid == "SI_NT_C8") dualseqs <- c('TTGCGACGTC','TTAATCCACA','TGTGGATTAA') if(spid == "SI-NT-C9" || spid == "SI_NT_C9") dualseqs <- c('GTACTGTCCA','TTTGTTGGAA','TTCCAACAAA') if(spid == "SI-NT-C10" || spid == "SI_NT_C10") dualseqs <- c('AAACGGTTTA','TCTTCGTTAC','GTAACGAAGA') if(spid == "SI-NT-C11" || spid == "SI_NT_C11") dualseqs <- c('CGATAGTTCT','GCGATTTCCT','AGGAAATCGC') if(spid == "SI-NT-C12" || spid == "SI_NT_C12") dualseqs <- c('TACTTTAGCT','GAGTTATTTG','CAAATAACTC') if(spid == "SI-NT-D1" || spid == "SI_NT_D1") dualseqs <- c('CTGGGATTAA','AGTAAAGTTC','GAACTTTACT') if(spid == "SI-NT-D2" || spid == "SI_NT_D2") dualseqs <- c('TACACTATTC','ATTGCAACGT','ACGTTGCAAT') if(spid == "SI-NT-D3" || spid == "SI_NT_D3") dualseqs <- c('AGAGCTATTT','CTGGCACCCA','TGGGTGCCAG') if(spid == "SI-NT-D4" || spid == "SI_NT_D4") dualseqs <- c('AGGTCGTTAT','AGGTTATCCA','TGGATAACCT') if(spid == "SI-NT-D5" || spid == "SI_NT_D5") dualseqs <- c('GCTGGACAGG','TAATCTCTGT','ACAGAGATTA') if(spid == "SI-NT-D6" || spid == "SI_NT_D6") dualseqs <- c('ACTTAGATGG','AATGTTGAGT','ACTCAACATT') if(spid == "SI-NT-D7" || spid == "SI_NT_D7") dualseqs <- c('AGGCGCGAGT','TGTGTCTATA','TATAGACACA') if(spid == "SI-NT-D8" || spid == "SI_NT_D8") dualseqs <- c('GAGGTCGTAC','CAACTGCGGG','CCCGCAGTTG') if(spid == "SI-NT-D9" || spid == "SI_NT_D9") dualseqs <- c('CCCAATGAGC','AGTTTCTGAT','ATCAGAAACT') if(spid == "SI-NT-D10" || spid == "SI_NT_D10") dualseqs <- c('ACATTCAGGG','GTCCTGAGAG','CTCTCAGGAC') if(spid == "SI-NT-D11" || spid == "SI_NT_D11") dualseqs <- c('AGAAACGGTG','TCACCCAGCA','TGCTGGGTGA') if(spid == "SI-NT-D12" || spid == "SI_NT_D12") dualseqs <- c('AGTTGCAAGT','ATACTTCATG','CATGAAGTAT') if(spid == "SI-NT-E1" || spid == "SI_NT_E1") dualseqs <- c('GCCTAGAAAT','ACTACTCCGG','CCGGAGTAGT') if(spid == "SI-NT-E2" || spid == "SI_NT_E2") dualseqs <- c('TACAAAGATG','CCGATGACGT','ACGTCATCGG') if(spid == "SI-NT-E3" || spid == "SI_NT_E3") dualseqs <- c('GCAGGCTTAG','CGTCTATTGC','GCAATAGACG') if(spid == "SI-NT-E4" || spid == "SI_NT_E4") dualseqs <- c('CAGCTCCAAT','TAGGAGGGTG','CACCCTCCTA') if(spid == "SI-NT-E5" || spid == "SI_NT_E5") dualseqs <- c('TTTGGAGAAA','CAGCCCTTGA','TCAAGGGCTG') if(spid == "SI-NT-E6" || spid == "SI_NT_E6") dualseqs <- c('TGTCGCTTTC','CCTAAAGGGT','ACCCTTTAGG') if(spid == "SI-NT-E7" || spid == "SI_NT_E7") dualseqs <- c('GTGTTGCGAG','CACTTTGAGC','GCTCAAAGTG') if(spid == "SI-NT-E8" || spid == "SI_NT_E8") dualseqs <- c('GTCTGTTCTG','TCTGATGCTT','AAGCATCAGA') if(spid == "SI-NT-E9" || spid == "SI_NT_E9") dualseqs <- c('TCGCTCAATC','GCTTACGTGA','TCACGTAAGC') if(spid == "SI-NT-E10" || spid == "SI_NT_E10") dualseqs <- c('CGTTACTAGC','AATGGCCATT','AATGGCCATT') if(spid == "SI-NT-E11" || spid == "SI_NT_E11") dualseqs <- c('GTGCAGATTT','TAAAGGCGAT','ATCGCCTTTA') if(spid == "SI-NT-E12" || spid == "SI_NT_E12") dualseqs <- c('GTTACCGAAA','CGTTTGAATC','GATTCAAACG') if(spid == "SI-NT-F1" || spid == "SI_NT_F1") dualseqs <- c('CGGTATGGAA','TGCAACTGGT','ACCAGTTGCA') if(spid == "SI-NT-F2" || spid == "SI_NT_F2") dualseqs <- c('TCGGTTTAGT','TAAAGTGTTC','GAACACTTTA') if(spid == "SI-NT-F3" || spid == "SI_NT_F3") dualseqs <- c('CTTTGCTTTG','ACATTCATTC','GAATGAATGT') if(spid == "SI-NT-F4" || spid == "SI_NT_F4") dualseqs <- c('ATGATCGCAC','GCGGTATGTA','TACATACCGC') if(spid == "SI-NT-F5" || spid == "SI_NT_F5") dualseqs <- c('CGGCGTGTTA','TGAGACGCGG','CCGCGTCTCA') if(spid == "SI-NT-F6" || spid == "SI_NT_F6") dualseqs <- c('TCATTTCCGC','ACAGCTGTTG','CAACAGCTGT') if(spid == "SI-NT-F7" || spid == "SI_NT_F7") dualseqs <- c('TTGTGCCCAG','TTGCGAGTTG','CAACTCGCAA') if(spid == "SI-NT-F8" || spid == "SI_NT_F8") dualseqs <- c('AGCCAACGTG','TCAAAGGAAC','GTTCCTTTGA') if(spid == "SI-NT-F9" || spid == "SI_NT_F9") dualseqs <- c('GATCCATACT','ATCGAGAGAA','TTCTCTCGAT') if(spid == "SI-NT-F10" || spid == "SI_NT_F10") dualseqs <- c('TTTCCATGAA','CGGTTCTAAC','GTTAGAACCG') if(spid == "SI-NT-F11" || spid == "SI_NT_F11") dualseqs <- c('TCTTTATCGG','CCCAATAGCG','CGCTATTGGG') if(spid == "SI-NT-F12" || spid == "SI_NT_F12") dualseqs <- c('GAACCCGATG','TCTATCCGGG','CCCGGATAGA') if(spid == "SI-NT-G1" || spid == "SI_NT_G1") dualseqs <- c('GATGTCTGTG','CGTGCTACCG','CGGTAGCACG') if(spid == "SI-NT-G2" || spid == "SI_NT_G2") dualseqs <- c('TGTTAGACTA','TCATGACGAC','GTCGTCATGA') if(spid == "SI-NT-G3" || spid == "SI_NT_G3") dualseqs <- c('TAGAACGCTT','GTCGCAAACG','CGTTTGCGAC') if(spid == "SI-NT-G4" || spid == "SI_NT_G4") dualseqs <- c('AATTTGACGT','AGAAGTGAGG','CCTCACTTCT') if(spid == "SI-NT-G5" || spid == "SI_NT_G5") dualseqs <- c('GAGTGGCCAC','GATGCACAGT','ACTGTGCATC') if(spid == "SI-NT-G6" || spid == "SI_NT_G6") dualseqs <- c('ATCAGTTACG','TACAATCTCT','AGAGATTGTA') if(spid == "SI-NT-G7" || spid == "SI_NT_G7") dualseqs <- c('TGTAAGCTTT','CGGACTAGTC','GACTAGTCCG') if(spid == "SI-NT-G8" || spid == "SI_NT_G8") dualseqs <- c('GCTTCGGTCT','CGCGGGTTAG','CTAACCCGCG') if(spid == "SI-NT-G9" || spid == "SI_NT_G9") dualseqs <- c('AGTCTAAGAC','CTGAAGTGAA','TTCACTTCAG') if(spid == "SI-NT-G10" || spid == "SI_NT_G10") dualseqs <- c('TGAAACATTC','CCTCCTGCCT','AGGCAGGAGG') if(spid == "SI-NT-G11" || spid == "SI_NT_G11") dualseqs <- c('CCTCACGAAA','TGCCCTGTGC','GCACAGGGCA') if(spid == "SI-NT-G12" || spid == "SI_NT_G12") dualseqs <- c('CTACGGGCTT','AGGCGTCCCT','AGGGACGCCT') if(spid == "SI-NT-H1" || spid == "SI_NT_H1") dualseqs <- c('GCGGCGTAAA','CACGCGATCA','TGATCGCGTG') if(spid == "SI-NT-H2" || spid == "SI_NT_H2") dualseqs <- c('TCTCCTTCGG','TGTCCGTCGC','GCGACGGACA') if(spid == "SI-NT-H3" || spid == "SI_NT_H3") dualseqs <- c('CCTTACCCAT','TCCCGGCAAC','GTTGCCGGGA') if(spid == "SI-NT-H4" || spid == "SI_NT_H4") dualseqs <- c('AAGGAACATC','TATCTGTGGG','CCCACAGATA') if(spid == "SI-NT-H5" || spid == "SI_NT_H5") dualseqs <- c('GTGGGAACTT','GCACTTACAG','CTGTAAGTGC') if(spid == "SI-NT-H6" || spid == "SI_NT_H6") dualseqs <- c('ACCGCACCAC','TCGGATTATA','TATAATCCGA') if(spid == "SI-NT-H7" || spid == "SI_NT_H7") dualseqs <- c('ACCTTTATCT','CGAGTGCCAA','TTGGCACTCG') if(spid == "SI-NT-H8" || spid == "SI_NT_H8") dualseqs <- c('AGTAGCCCGT','TTCGGGACCT','AGGTCCCGAA') if(spid == "SI-NT-H9" || spid == "SI_NT_H9") dualseqs <- c('AGATAGCATA','CATTTCCGGA','TCCGGAAATG') if(spid == "SI-NT-H10" || spid == "SI_NT_H10") dualseqs <- c('TGGCGTTAAA','TGTTAAGATG','CATCTTAACA') if(spid == "SI-NT-H11" || spid == "SI_NT_H11") dualseqs <- c('TCAGGCGAAA','TCTCTGGAAG','CTTCCAGAGA') if(spid == "SI-NT-H12" || spid == "SI_NT_H12") dualseqs <- c('AAGACATAGC','ATGTGGACGT','ACGTCCACAT') if(is.na(spseqs ) && !is.na(dualseqs )){ return(c(paste0(dualseqs[1], dualseqs[2]),paste0(dualseqs[1], dualseqs[3])) ) }else return(spseqs) } .rstest <- function(r, coef) r * (1 + 1/r)^(1 + coef) .averaging_transform <- function(r, nr){ d <- c(1, r[ 2:length(r) ] - r[1:(length(r)-1)]) dr <- c( 0.5 * (d[2:length(d)] + d[1:(length(d)-1)]),d[ length(d) ]) nr/dr } .do.EXACT.DEBUG <- FALSE #.do.EXACT.DEBUG <- TRUE .simple.Good.Turing.Freq<-function( raw.freq ){ n_zero <- sum(raw.freq == 0) times <- table(raw.freq[raw.freq>0]) obs <- sort(as.numeric(names(times))) times <- times[as.character(obs)] nobs <- length(obs) if(nobs < .min_mySGT_input_size){ cat(sprintf("Warning: not enough element in the observation array : %d < %d.\nThe `Rescued' element in the returned object will be set to NA.\n", nobs, .min_mySGT_input_size)) return(NA) }else{ if(nobs != length(times)) stop("The oservation array doesn't match the frequency array.") total_observed <- sum(obs * times) tval <- matrix(0, ncol=2, nrow=nobs) ks <- c(obs[2:nobs], 2*obs[nobs] - obs[nobs-1]) tval[,1] <- log(obs) tval[,2] <- log( .averaging_transform(obs,times)) meanx <- mean(tval[,1]) meany <- mean(tval[,2]) slope <- sum( (tval[,1]-meanx) * (tval[,2]-meany) ) / sum((tval[,1]-meanx) ^2) intercept <- meany - slope * meanx obs.in.next <- (obs+1) %in% obs SD <- ( 1+(1:nobs) ) / times *(c(times[2:nobs], 0)* (1+c(times[2:nobs],0) /times))^.5 SD [!obs.in.next] <- 1 GT <- (obs+1)/obs * c(times[2:nobs],0)/times GT [!obs.in.next] <- 0 y <- .rstest(obs,slope) LGT <- y / obs xy.sim <- ( abs(GT-LGT) * (1:nobs) / SD ) > 1.65 min.no.turing <- min(which(!xy.sim)) r <- GT if(any(!xy.sim))r[min.no.turing:nobs] <- LGT[min.no.turing:nobs] p_zero <- times[1] / total_observed rs <- (1-p_zero) * obs * r / sum(r*times*obs/total_observed) total_rs <- sum(rs * times) puniq <- (1-p_zero) * rs / total_rs pres <- rep(p_zero / n_zero, length(raw.freq)) pres [raw.freq>0] <- puniq[match( raw.freq[raw.freq>0], obs )] ret <- list(p0=p_zero, p=pres) if(.do.EXACT.DEBUG){ print("DEBUG_EXACT") skf <- "/tmp/del4-YangLiao-GoodTuring-Pr-vals.txt" if(file.exists(skf))file.remove(skf) sink(skf) for(rr in pres){ cat(sprintf("%.20g\n",rr)) } sink() } ret } } .min_mySGT_input_size <- 5 .smoothed <- function(i,intercept,slope) 2.718281828^(intercept + slope * log(i)) # Simple Good Turing .mySGT <- function(obs.per.spe){ obs.tab <- table(obs.per.spe[obs.per.spe>0]) obs <- sort( as.numeric(names(obs.tab))) obs.tab <- obs.tab[ as.character(obs) ] sgtr <- .mySGTsorted(as.numeric(names(obs.tab)), obs.tab) res <- obs.per.spe res[obs.per.spe!=0] <- sgtr$p[match(as.character(obs.per.spe[obs.per.spe!=0]), names(obs.tab))] n_zero <- sum(obs.per.spe==0) res[obs.per.spe==0] <- sgtr$p0/n_zero res } .mySGTsorted<-function( obs, times ){ if(any(obs != sort(obs)))stop("Observations have to be sorted.") nobs <- length(obs) if(nobs < .min_mySGT_input_size) stop("Not enough element in the observation array.") if(nobs != length(times)) stop("The oservation array doesn't match the frequency array.") total_observed <- sum(obs * times) p_zero <- 0 if(1 %in% obs) p_zero <- times[which(1==obs)[1]] / total_observed tval <- matrix(0, ncol=2, nrow=nobs) ks <- c(obs[2:nobs], 2*obs[nobs] - obs[nobs-1]) tval[,1] <- log(obs) tval[,2] <- log(2*times / (ks - c(0, obs[1:(nobs-1)]))) meanX <- mean(tval[,1]) meanY <- mean(tval[,2]) slope <- sum( (tval[,1]-meanX) * (tval[,2]-meanY) ) / sum((tval[,1]-meanX) ^2) intercept <- meanY - slope * meanX y <- (1+obs) * .smoothed(1+obs, intercept, slope ) / .smoothed(obs, intercept, slope) x <- (1+obs) * c((times[2:nobs] / times[1:(nobs-1)]), y[nobs]) xy.sim <- abs(x-y) <= 1.96*( (obs+1)^2 * c(times[2:nobs], 0)/ times^2 * (1+ c(times[2:nobs], 0) / times ))^0.5 obs.in.next <- (obs+1) %in% obs min.inv <- min(which( xy.sim | !obs.in.next )) r<-x if(min.inv>0)r[min.inv:nobs] <- y[min.inv:nobs] list(p0=p_zero, p=(1-p_zero) * r / sum(r*times)) } .cellCounts_try_cellbarcode <- function( input.directory, sample.sheet, cell.barcode.list, nreads.testing, input.mode ){ # the three parameters can only be one string, not strings! if(is.null(sample.sheet)) sample.sheet<-"." cmd <- paste0(c(input.directory, sample.sheet, cell.barcode.list, as.character(nreads.testing), input.mode), collapse=.R_param_splitor) rvs <- as.integer(rep(0,5)) C_args <- .C("R_try_cell_barcode_wrapper",nargs=as.integer(5),argv=as.character(cmd),retv=rvs ,PACKAGE="Rsubread") return_val <- ifelse(C_args$retv[1]==0,"FINISHED","ERROR") tested_reads <- C_args$retv[2] good_sample <- C_args$retv[3] good_cell <- C_args$retv[4] if(return_val=="ERROR"){ return(NA) } return(c(tested_reads, good_sample, good_cell)) } .dataURL <- function(uri){ sprintf("http://shilab-bioinformatics.github.io/cellCounts/Barcodes/%s",uri) } .find_best_cellbarcode <- function( input.directory, sample.sheet=NULL, input.mode="bcl"){ barcode.database.file <- path.expand("~/.Rsubread/cellCounts/known_barcode_sets.txt") if(!file.exists(barcode.database.file)){ dir.create("~/.Rsubread/cellCounts", recursive=TRUE) rr <- download.file(.dataURL("known_barcode_sets.txt"), barcode.database.file) if(rr!=0)stop("ERROR: the barcode database cannot be retrieved from the Internet. You may still run cellCounts by specifying a local barcode list file to the `cell.barcode.list` option.") } bcb.sets <- read.delim(barcode.database.file, stringsAsFactors=F, header=T) cat(sprintf("Found %d known cell barcode sets.\n", nrow(bcb.sets))) max.cell.good <- -1 for(libf in bcb.sets$File){ listfile <- path.expand(paste0("~/.Rsubread/cellCounts/",libf)) if(!file.exists(listfile)){ rr <- download.file(.dataURL(libf), listfile) if(rr!=0)stop("ERROR: the barcode list cannot be retrieved from the Internet. You may still run cellCounts by specifying a local barcode list file to the `cell.barcode.list` option.") } cat(sprintf("Testing the cell barcodes in %s.\n", libf)) barcode_res <- .cellCounts_try_cellbarcode(input.directory[1], sample.sheet[1], listfile, 30000, input.mode) if(length(barcode_res)<3)stop("ERROR: the input sample cannot be processed.") sample.good.rate <- barcode_res[2]/barcode_res[1] cell.good.rate <- barcode_res[3]/barcode_res[1] max.cell.good <- max(max.cell.good, cell.good.rate) cat(sprintf("Cell barcode supporting rate : %.1f%%.\n", cell.good.rate*100.)) # sample index isn't tested anymore # if(input.mode=="bcl" && sample.good.rate < 0.5)cat(sprintf("WARNING: there are only %.1f%% reads having known sample indices. Please check if the sample sheet is correct.\n", sample.good.rate*100.)) if(cell.good.rate > 0.6){ cat(sprintf("Found cell-barcode list '%s' for the input data: supported by %.1f%% reads.\n", libf, cell.good.rate*100.)) return(listfile) } } stop(sprintf("ERROR: no known cell barcode set was found for the data set. The highest percentage of cell-barcode matched reads is %.1f%%\n", max.cell.good*100.)) } library(Matrix) .read.sparse.mat <- function (fn){ cat("Loading matrix from",fn,"\n") mtx <- readMM(paste0(fn, ".spmtx")) coln <- read.delim(paste0(fn, ".BCtab"), stringsAsFactors=F, header=F)$V1 rown <- read.delim(paste0(fn, ".GENEtab"), stringsAsFactors=F, header=F)$V1 colnames(mtx) <- coln rownames(mtx) <- rown mtx } .use.DEBUG.data <- NA # .use.DEBUG.i <- 1 .get.multi.nomial <- function(n, size, prob){ if(.do.EXACT.DEBUG){ .use.DEBUG.i <- 1 ret <- read.table(paste0("/tmp/del4-YangLiao-multi-nomial-SIZE",size,"-",.use.DEBUG.i,".txt"), header=F) ret <- t(ret) #.use.DEBUG.i <<- .use.DEBUG.i+1 #print(ret[1:5,1:5]) ret }else{ rmultinom(n, size, prob ) } } .simu.multinomial <- function(candi.mat, gene.profile.freq , times=10000){ bcsizes <- sort(unique( colSums(candi.mat) )) ret.nUMI.LLH.tab <- list() N10000.LLH <- NA Old_bs_one <- NA log_E_GTE <- log(gene.profile.freq) if(.do.EXACT.DEBUG){ tff<-"/tmp/del4-YangLiao-for-multi-nomial-steps.txt" if(file.exists(tff))file.remove(tff) sink(tff) for(bcsize_one in bcsizes){ UMI_step_diff <- NA if(!any(is.na(Old_bs_one))) UMI_step_diff <- bcsize_one - Old_bs_one st1 <- NA if(is.na(Old_bs_one)) { st1 <- bcsize_one }else if(UMI_step_diff >= 1000){ st1 <- UMI_step_diff } if(!is.na(st1))cat(st1,"\n") Old_bs_one <- bcsize_one } sink() system("python /usr/local/work/liao/subread/scripts/Cellranger-replicate/CrepPY-multi-nomial.py") } Old_bs_one <- NA N1000.FstLLH <- c() if(.do.EXACT.DEBUG) M50.gene.ids <- read.table("/tmp/del4-YangLiao-50M-gene-nos.txt", header=F)$V1 all.steps.len <- 0 for(bcsize_one in bcsizes){ UMI_step_diff <- NA if(!is.na(Old_bs_one)) UMI_step_diff <- bcsize_one - Old_bs_one if(is.na(Old_bs_one)){ }else if(UMI_step_diff >= 1000){ }else{ step_len <- bcsize_one - Old_bs_one all.steps.len <- step_len + all.steps.len } Old_bs_one <- bcsize_one } if(5000 <= all.steps.len)print("WARNING: SMALL STEPS >= 5000!") Old_bs_one <- NA M50.in.loop.K <- 0 for(bcsize_one in bcsizes){ UMI_step_diff <- NA if(!any(is.na(Old_bs_one))) UMI_step_diff <- bcsize_one - Old_bs_one if(any(is.na(N10000.LLH))){ N10000 <- .get.multi.nomial(n=times, size=bcsize_one, prob=gene.profile.freq ) print("======== N10000 and PROF DIM =======") print(dim(N10000)) print(length(gene.profile.freq)) N10000.LLH <- apply(N10000, 2, function(x) dmultinom(x, prob=gene.profile.freq, log =T )) }else if(UMI_step_diff >= 1000){ UMI_step_diff_N10000 <- .get.multi.nomial(n=times, size=UMI_step_diff, prob=gene.profile.freq ) N10000 <- N10000 + UMI_step_diff_N10000 N10000.LLH <- apply(N10000, 2, function(x) dmultinom(x, prob=gene.profile.freq, log =T )) }else{ step_len <- bcsize_one - Old_bs_one for(curi in (Old_bs_one+1):bcsize_one){ if(.do.EXACT.DEBUG){ UMI_step_indices_N10000 <- M50.gene.ids[1+M50.in.loop.K + (0:9999) * all.steps.len ] if(curi == 588){ tff<-"/tmp/del4-YangLiao-DEBUG-588-10KJ.txt" if(file.exists(tff))file.remove(tff) sink(tff) for(pi in 1:10000){ cat(pi," ", N10000[UMI_step_indices_N10000[pi],pi],"\n") } sink() } M50.in.loop.K <- 1+M50.in.loop.K }else{ UMI_step_indices_N10000 <- sample(1:nrow(candi.mat), size=times, prob=gene.profile.freq, replace=T) } for(idi in 1:times){ N10000[ UMI_step_indices_N10000[idi] ,idi ] <- N10000[ UMI_step_indices_N10000[idi] ,idi ]+1 } UMI_step_values_N10000 <- rep(0, times) for(idi in 1:times){ UMI_step_values_N10000[idi] <- N10000[UMI_step_indices_N10000[idi] ,idi ] } #print(UMI_step_values_N10000) N10000.LLH <- N10000.LLH + log_E_GTE[ UMI_step_indices_N10000 ] + log((curi) / UMI_step_values_N10000) if(.do.EXACT.DEBUG && curi == 588)for(uii in 1:30){ tff<-"/tmp/del4-YangLiao-DEBUG-1-30-E_GTE.txt" if(file.exists(tff))file.remove(tff) sink(tff) for(i in 1:30){ cat(sprintf("%.5g %.5g %.5g\n", N10000.LLH[i], log_E_GTE[ UMI_step_indices_N10000 [i]], log((curi) / UMI_step_values_N10000[i]))) } sink() } } } ret.nUMI.LLH.tab[[ bcsize_one ]] <- N10000.LLH N1000.FstLLH <- c( N1000.FstLLH, N10000.LLH[1] ) Old_bs_one <- bcsize_one } if(.do.EXACT.DEBUG){ tff<-"/tmp/del4-YangLiao-10K-LLH-from-R.txt" if(file.exists(tff))file.remove(tff) sink(tff) for(flh in N1000.FstLLH){ cat(sprintf("%.6g\n", flh)) } sink() } ret.nUMI.LLH.tab } .is.no.candBC <- function(fn){ info <- file.info(fn) return(info$size < 2) } .cellCounts.rescue <- function( BAM.name, FC.gene.ids, sample.no ){ fname <- sprintf("%s.scRNA.%03d", BAM.name, sample.no) nozero.anywhere.genes <- read.delim(paste0(fname,".no0Genes"), stringsAsFactors=F, header=F)$V1 ambient.accumulate <- read.delim(paste0(fname,".AmbSum"), stringsAsFactors=F) ambient.accumulate <- ambient.accumulate[ match(FC.gene.ids , ambient.accumulate$GeneID), ] ambient.accumulate$UMIs[is.na(ambient.accumulate$UMIs)] <- 0 ambient.accumulate <- ambient.accumulate$UMIs names(ambient.accumulate) <- FC.gene.ids ambient.accumulate <- ambient.accumulate[ names(ambient.accumulate) %in% nozero.anywhere.genes] resc.bc.fn <- paste0(fname,".RescCand") #print(summary(ambient.accumulate)) rstfq <- .simple.Good.Turing.Freq(ambient.accumulate) if(any(is.na(rstfq)) || .is.no.candBC(paste0(resc.bc.fn,".BCtab"))){ return(NA) }else{ gte <- rstfq$p # This function returns "times" log-likelihoods. rescue.candidates <- as.matrix(.read.sparse.mat(resc.bc.fn)) rescue.candidates <- rescue.candidates[ match(names(ambient.accumulate), rownames(rescue.candidates)), ] rownames(rescue.candidates) <- names(ambient.accumulate) rescue.candidates [is.na(rescue.candidates )] <- 0 # Compute observed log-likelihood of barcodes being generated from ambient RNA # Compute the multinomial log PMF for many barcodes -- log-likelihoods # Only use the "non-zero anywhere" genes. log.like.cands <- apply(rescue.candidates, 2, function(x) dmultinom(x, prob=gte, log =T )) # Simulate log likelihoods # This step is to build like 10000 sets of N_GENES vectors from the multinomial distribution of "gte" # See what are their log-likelihoods against "gte" -- most should be very small. simu.pvalues <- .simu.multinomial( rescue.candidates, gte ) # Compute p-values : the p-value is the chance of a barcode is actually from ambient RNA (ie smaller the p-value, more likely this barcode is for a real cell) actual.pvalues <- rep(0, ncol(rescue.candidates) ) names(actual.pvalues) <- colnames(rescue.candidates) for(candi in names(log.like.cands)){ cand_umis <- sum(rescue.candidates[,candi]) cand.simu.pvs <- simu.pvalues[[ cand_umis ]] cand.actual.pv <- log.like.cands[candi] actual.pvalues[candi] <- (1+sum(cand.simu.pvs < cand.actual.pv))/(length(cand.simu.pvs)+1) } # p-value => FDR actual.FDR <- p.adjust(actual.pvalues, method='BH') if(.do.EXACT.DEBUG){ tff<-"/tmp/del4-YangLiao-final-FDR.txt" if(file.exists(tff))file.remove(tff) sink(tff) for(candi in names(log.like.cands)) cat(sprintf("%s %.10g %.10g\n", candi, actual.pvalues[candi], actual.FDR[candi])) sink() } # select cells that has FDR < cutoff FDR.Cutoff <- 0.01 head(actual.FDR) Rescured.Barcodes <- names(actual.FDR)[actual.FDR <= FDR.Cutoff] head(Rescured.Barcodes) rescue.candidates[,Rescured.Barcodes, drop=FALSE] } } .match.exons <- function(annot.tab, counts){ annot.str <- paste(annot.tab$GeneID, annot.tab$Chr, annot.tab$Start, annot.tab$End, ifelse(annot.tab$Strand == "+", "P","N"), sep=":fc@R@Spl:") ret <- matrix(0, ncol=ncol(counts), nrow=nrow(annot.tab)) colnames(ret) <- colnames(counts) rownames(ret) <- annot.str ret[ rownames(counts),] <- counts ret } .load.one.scSample <- function( BAM.name, FC.gene.ids, sample.no, use.meta.features, annot.tab, umi.cutoff){ set.seed(0) fname <- sprintf("%s.scRNA.%03d", BAM.name, sample.no) cat("Loading high-conf matrix from '",fname,"'\n") highconf <- as.matrix(.read.sparse.mat(paste0(fname,".HighConf"))) rescued <- NA if(is.null(umi.cutoff)) rescued <- .cellCounts.rescue(BAM.name, FC.gene.ids, sample.no) if(use.meta.features){ if(!any(is.na(rescued))) rescued <- rescued[rowSums(rescued)>0,] ncolRescued <- 0 if(any(is.na(rescued))){ ret <- matrix(0,ncol=ncol(highconf), nrow=length(FC.gene.ids)) colnames(ret) <- colnames(highconf) }else{ ret <- matrix(0,ncol=ncol(highconf)+ncol(rescued), nrow=length(FC.gene.ids)) colnames(ret) <- c( colnames(highconf), colnames(rescued) ) } rownames(ret) <- FC.gene.ids ret[rownames(highconf), colnames(highconf) ] <- highconf if(!any(is.na(rescued)))ret[rownames(rescued), colnames(rescued) ] <- rescued list(Counts=ret, HighConfidneceCell=colnames(ret) %in% colnames(highconf)) }else{ fcmat <- .match.exons(annot.tab,highconf) rownames(fcmat)<-NULL list(Counts=fcmat, HighConfidneceCell=rep(T, ncol(highconf))) } } .load.all.scSamples <- function( BAM.name, FC.gene.ids, use.meta.features, annot.tab, umi.cutoff){ sum.tab <- read.delim(paste0(BAM.name,".scRNA.SampleTable"), stringsAsFactors=F) ret <- list() for(roiw in 1:nrow(sum.tab)){ sname <- as.character(sum.tab$SampleName[roiw]) sid <- sum.tab$Index[roiw] count.tab <- .load.one.scSample(BAM.name, FC.gene.ids, sid, use.meta.features, annot.tab, umi.cutoff) ret[[sprintf("Sample.%d",sid)]] <- count.tab } ret[["Sample.Table"]] <- sum.tab ret } .del.temp.files <- function(pfx){ if(nchar(pfx)<16) stop("Prefix should be longer.") flist <- dir(".", "^[.]Rsubread", all.files=TRUE) to.delete <- flist[grepl(pfx, flist)] cat(paste(flist,";"),"Delete temporary files :", paste(to.delete, collapse=","),"\nthat have a prefix of",pfx,"\n") for(df in to.delete){ if(grepl(pfx, df)){ file.remove(df) #cat ("Goinng to remove",df,"\n") }else{ stop("Wrong selection of files to delete") } } } .extract.sample.table.cols <- function(rdir, smr, input.mode="bcl", umi.cutoff=NULL){ total.cells <- c() hiconf.cells <- c() res.cells <- c() umis <- c() umi.statistics <- data.frame(MinUMI=NULL, MedianUMI=NULL, MaxUMI=NULL, MeanUMI=NULL,stringsAsFactors=F)[,0] for(spi in 1:nrow(smr[["Sample.Table"]])){ samplename <- as.character(smr[["Sample.Table"]]$SampleName[spi]) sampleno <- sprintf("Sample.%d", spi) total.cells <- c(total.cells,length(smr[[sampleno]][["HighConfidneceCell"]])) hiconf.cells <- c(hiconf.cells,sum(smr[[sampleno]][["HighConfidneceCell"]])) res.cells <- c(res.cells, sum(!(smr[[sampleno]][["HighConfidneceCell"]]))) cell.umis <- colSums(smr[[sampleno]][["Counts"]]) umis <- c(umis,sum(cell.umis)) umi.statistics <- rbind(umi.statistics, list(MinUMI=min(cell.umis), MedianUMI=median(cell.umis), MaxUMI=max(cell.umis), MeanUMI=mean(cell.umis))) } ret <- NULL if(is.null(umi.cutoff)){ if(input.mode=="fastq" || input.mode=="bam") ret <- cbind( SampleName=as.character(smr[["Sample.Table"]]$SampleName), TotalCells=total.cells, HighConfidenceCells=hiconf.cells, RescuedCells=res.cells, TotalUMI=umis, umi.statistics, smr[["Sample.Table"]][,c("TotalReads","MappedReads","AssignedReads")],stringsAsFactors=F ) else ret <- cbind( SampleName=as.character(smr[["Sample.Table"]]$SampleName), InputDirectory=rep(rdir, nrow(smr[["Sample.Table"]])), TotalCells=total.cells, HighConfidenceCells=hiconf.cells, RescuedCells=res.cells, TotalUMI=umis, umi.statistics, smr[["Sample.Table"]][,c("TotalReads","MappedReads","AssignedReads")],stringsAsFactors=F ) }else{ if(input.mode=="fastq" || input.mode=="bam") ret <- cbind( SampleName=as.character(smr[["Sample.Table"]]$SampleName), TotalCells=total.cells, TotalUMI=umis, umi.statistics, smr[["Sample.Table"]][,c("TotalReads","MappedReads","AssignedReads")], stringsAsFactors=F) else ret <- cbind( SampleName=as.character(smr[["Sample.Table"]]$SampleName), InputDirectory=rep(rdir, nrow(smr[["Sample.Table"]])), TotalCells=total.cells, TotalUMI=umis, umi.statistics, smr[["Sample.Table"]][,c("TotalReads","MappedReads","AssignedReads")], stringsAsFactors=F) } } .scan.fastq.dir <- function(dirname){ ddf <- list.files(dirname , recursive=TRUE) ddf <- ddf[grepl("L[0-9][0-9][1-9]_R1_[0-9][0-9][1-9].fastq.gz$",ddf) &(!grepl("Undetermined_S",ddf)) &!grepl("/fork0/", ddf)] sample.sheet <- data.frame(stringsAsFactors=F) for(df1 in sort(ddf)){ df1 <- file.path(dirname,df1) r1_part <- substr(df1, nchar(df1)-12, 9999) r1_pref <- substr(df1, 1, nchar(df1)-15) df2 <- paste0(r1_pref, "R2", r1_part) sample.name <- unlist(strsplit(df2, "\\/")) sample.name0<- sample.name sample.name <- sample.name[length(sample.name)] if(substr(sample.name, nchar(sample.name)-22, nchar(sample.name)-22)=='S') sample.name <- substr(sample.name, 1, nchar(sample.name)-24) else if(substr(sample.name, nchar(sample.name)-23, nchar(sample.name)-23)=='S') sample.name <- substr(sample.name, 1, nchar(sample.name)-25) else stop(paste("ERROR: Unable to parse the file name,",sample.name)) if(substr(sample.name,1,1)=="_") sample.name <- substr(sample.name,2,999) if(substr(sample.name,1,1)=="_") sample.name <- substr(sample.name,2,999) #if(substr(sample.name,1,1)!="S")stop(sprintf("Unable to parse the FASTQ file name : '%s'", sample.name0)) sample.sheet <- rbind(sample.sheet, data.frame( SampleName=as.character(sample.name), BarcodeUMIFile=df1, ReadFile=df2, stringsAsFactors=F)) } sample.sheet } .SCRNA_FASTA_SPLIT1 <- "|Rsd:cCounts:mFQs|" .SCRNA_FASTA_SPLIT2 <- "|Rsd:cCounts:1mFQ|" .new_cellCounts <- function(index, sample,input.mode="BCL", cell.barcode=NULL, aligner="align", annot.inbuilt="mm10",annot.ext=NULL,isGTFAnnotationFile=FALSE,GTF.featureType="exon",GTF.attrType="gene_id",useMetaFeatures=TRUE, umi.cutoff=NULL, nthreads=10, reportedAlignmentsPerRead =1, unique.mapping=FALSE, maxMismatchBases=3, minVotesPerRead=3, subreadsPerRead=10, maxDiffToTopVotes=2, minMappedLength=40, onlyDetectBarcode=FALSE, ...){ .remove.temp.files <- T index <- .check_and_NormPath(index, mustWork=F, opt="index name") if(!is.null(aligner)) aligner <- match.arg(aligner,c("subjunc","align")) if(!is.null(umi.cutoff)){ umi.cutoff <- as.numeric(umi.cutoff) if(umi.cutoff < 0.0) stop("UMI cutoff must be a positive number.") } BAM_is_Rerun_Persample <- is.null(aligner) sample.info.idx <- sample fc <- list() temp.file.prefix <- file.path(".",paste(".Rsubread_cCounts_Tmp_for_Pid_",Sys.getpid(),"_Rproc",sep="")) raw.fc.annot <- NA df.sample.info <- data.frame(stringsAsFactors=F) sample.1 <- paste0(temp.file.prefix,".samplesheet") annlist <- .get.annotation.file(annot.inbuilt, annot.ext, isGTFAnnotationFile) ann <- annlist$ann annot.screen.output <- annlist$screen delete.annot.file <- annlist$delete if(input.mode=="FASTQ" || input.mode == "FASTQ-dir"){ if(input.mode == "FASTQ-dir") sample.info.idx <- .scan.fastq.dir(sample) print(sample.info.idx) if(!("BarcodeUMIFile" %in% colnames(sample.info.idx) && "ReadFile" %in% colnames(sample.info.idx) )) stop("You need to provide BC+UMI and Genomic sequence files") combined.fastq.names <- "" for(rowi in 1:nrow(sample.info.idx)){ df1 <- as.character(sample.info.idx[rowi,"BarcodeUMIFile"]) df2 <- as.character(sample.info.idx[rowi,"ReadFile"]) R1.file.name <- .check_and_NormPath(df1, mustWork=TRUE, "The barcode FASTQ file in sample.info.idx") R2.file.name <- .check_and_NormPath(df2, mustWork=TRUE, "The genomic read FASTQ file in sample.info.idx") combined.fastq.names <- paste0( combined.fastq.names,.SCRNA_FASTA_SPLIT1, R1.file.name, .SCRNA_FASTA_SPLIT2,".",.SCRNA_FASTA_SPLIT2, R2.file.name) } combined.fastq.names <- substr(combined.fastq.names, nchar(.SCRNA_FASTA_SPLIT1)+1, 9999999) if(is.null(cell.barcode)){ cell.barcode <- .find_best_cellbarcode(combined.fastq.names, "N/A", input.mode="fastq") }else{ cell.barcode <- .check_and_NormPath(cell.barcode, mustWork=T, opt="cell.barcode") } if(onlyDetectBarcode){ cat("Barcode is",cell.barcode,"\n") return(NA) } .index.names.to.sheet.FASTQ.mode(sample.info.idx, sample.1) opt <- c("--inputMode","FASTQ","--cellBarcodeFile", cell.barcode,"--dataset", combined.fastq.names, "--sampleSheetFile", sample.1, "--index", index, "--annotation", ann, "--geneIdColumn", GTF.attrType, "--annotationType", GTF.featureType, "--threads", nthreads, "--output", temp.file.prefix, "--maxMismatch", maxMismatchBases, "--minVotesPerRead", minVotesPerRead, "--subreadsPerRead", subreadsPerRead, "--reportedAlignmentsPerRead", reportedAlignmentsPerRead, "--maxDiffToTopVotes", maxDiffToTopVotes, "--minMappedLength", minMappedLength) if(isGTFAnnotationFile)opt <- c(opt, "--isGTFannotation") if(!unique.mapping)opt <- c(opt, "--reportMultiMappingReads") cmd <- paste(opt,collapse=.R_param_splitor) n <- length(unlist(strsplit(cmd,.R_param_splitor))) C_args <- .C("R_cellCounts",as.integer(n),as.character(cmd),PACKAGE="Rsubread") bam.for.FC <- c() raw.fc.annot<-read.delim(paste0(temp.file.prefix,".Annot"), header=T, stringsAsFactors=F) some.results <- .load.all.scSamples(temp.file.prefix, as.character(raw.fc.annot$GeneID), useMetaFeatures, raw.fc.annot, umi.cutoff) for(spi in 1:nrow(some.results[["Sample.Table"]])){ samplename <- as.character(some.results[["Sample.Table"]][["SampleName"]][spi]) fc[["counts"]][[samplename]] <- some.results[[sprintf("Sample.%d", spi)]][["Counts"]] # only one sample. if(is.null(umi.cutoff))fc[["cell.confidence"]][[samplename]] <- some.results[[sprintf("Sample.%d", spi)]][["HighConfidneceCell"]] } df.sample.info <- .extract.sample.table.cols(NA,some.results,input.mode="fastq", umi.cutoff=umi.cutoff) }else if(input.mode=="BCL"){ sample.info.idx$SampleName <- as.character(sample.info.idx$SampleName) if('IndexSetName' %in% colnames(sample.info.idx))sample.info.idx$IndexSetName <- as.character(sample.info.idx$IndexSetName) dirs <- unique( sample.info.idx$InputDirectory ) fc[["counts"]] <- list() for(dirname in dirs) .check_and_NormPath(dirname, mustWork=TRUE, "InputDirectory in sample.info.idx") # check files before the slow mapping/counting step. dirno <- 1 for(dirname in dirs){ unique.samples <- unique( as.character(sample.info.idx$SampleName[ sample.info.idx$InputDirectory == dirname ] )) if(is.null(cell.barcode)){ .index.names.to.sheet.raw.dir.mode(dirname, sample.info.idx, sample.1) cell.barcode <- .find_best_cellbarcode(dirname, sample.1) }else{ cell.barcode <- .check_and_NormPath(cell.barcode, mustWork=T, opt="cell.barcode") } if(onlyDetectBarcode){ cat("Barcode is",cell.barcode,"\n") return(NA) } full_dirname <- .check_and_NormPath(dirname, mustWork=TRUE, "InputDirectory in sample.info.idx") is_dual_index <- .index.names.to.sheet.raw.dir.mode(dirname, sample.info.idx, sample.1) generate.scRNA.BAM <- TRUE opt <- c("--cellBarcodeFile", cell.barcode,"--dataset", dirname, "--sampleSheetFile", sample.1, "--index", index, "--annotation", ann, "--geneIdColumn", GTF.attrType, "--annotationType", GTF.featureType, "--threads", nthreads, "--output", temp.file.prefix, "--maxMismatch", maxMismatchBases, "--minVotesPerRead", minVotesPerRead, "--subreadsPerRead", subreadsPerRead, "--maxDiffToTopVotes",maxDiffToTopVotes, "--minMappedLength", minMappedLength) if(isGTFAnnotationFile)opt <- c(opt, "--isGTFannotation") if(!unique.mapping)opt <- c(opt, "--reportMultiMappingReads") cmd <- paste(opt,collapse=.R_param_splitor) n <- length(unlist(strsplit(cmd,.R_param_splitor))) C_args <- .C("R_cellCounts",as.integer(n),as.character(cmd),PACKAGE="Rsubread") dirno <- dirno +1 if(any(is.na(raw.fc.annot))) raw.fc.annot<-read.delim(paste0(temp.file.prefix,".Annot"), header=T, stringsAsFactors=F) some.results <- .load.all.scSamples(temp.file.prefix, as.character(raw.fc.annot$GeneID), useMetaFeatures, raw.fc.annot, umi.cutoff) for(spi in 1:nrow(some.results[["Sample.Table"]])){ samplename <- as.character(some.results[["Sample.Table"]][["SampleName"]][spi]) fc[["counts"]][[samplename]] <- some.results[[sprintf("Sample.%d", spi)]][["Counts"]] # only one sample. if(is.null(umi.cutoff))fc[["cell.confidence"]][[samplename]] <- some.results[[sprintf("Sample.%d", spi)]][["HighConfidneceCell"]] } stt <- .extract.sample.table.cols(full_dirname,some.results, umi.cutoff=umi.cutoff) df.sample.info <- rbind(df.sample.info, stt) } } .del.temp.files(substr(temp.file.prefix,4,99)) fc } cellCounts <- function(index, sample,input.mode="BCL", cell.barcode=NULL, aligner="align", annot.inbuilt="mm10",annot.ext=NULL,isGTFAnnotationFile=FALSE,GTF.featureType="exon",GTF.attrType="gene_id",useMetaFeatures=TRUE, umi.cutoff=NULL, nthreads=10, nBestLocations =1, unique.mapping=FALSE, ...){ .remove.temp.files <- T if(!is.null(aligner)) aligner <- match.arg(aligner,c("subjunc","align")) if(!is.null(umi.cutoff)){ umi.cutoff <- as.numeric(umi.cutoff) if(umi.cutoff < 0.0) stop("UMI cutoff must be a positive number.") } BAM_is_Rerun_Persample <- is.null(aligner) sample.info.idx <- sample fc <- list() temp.file.prefix <- file.path(".",paste(".Rsubread_cCounts_Tmp_for_Pid_",Sys.getpid(),"_Rproc",sep="")) raw.fc.annot <- NA df.sample.info <- data.frame(stringsAsFactors=F) sample.1 <- paste0(temp.file.prefix,".samplesheet") if(input.mode=="BCL"){ sample.info.idx$SampleName <- as.character(sample.info.idx$SampleName) if('IndexSetName' %in% colnames(sample.info.idx))sample.info.idx$IndexSetName <- as.character(sample.info.idx$IndexSetName) dirs <- unique( sample.info.idx$InputDirectory ) fc[["counts"]] <- list() # if(is.null(umi.cutoff))fc[["cell.confidence"]] <- list() for(dirname in dirs) .check_and_NormPath(dirname, mustWork=TRUE, "InputDirectory in sample.info.idx") # check files before the slow mapping/counting step. dirno <- 1 for(dirname in dirs){ unique.samples <- unique( as.character(sample.info.idx$SampleName[ sample.info.idx$InputDirectory == dirname ] )) if(is.null(cell.barcode)){ .index.names.to.sheet.raw.dir.mode(dirname, sample.info.idx, sample.1) cell.barcode <- .find_best_cellbarcode(dirname, sample.1) }else{ cell.barcode <- .check_and_NormPath(cell.barcode, mustWork=T, opt="cell.barcode") } full_dirname <- .check_and_NormPath(dirname, mustWork=TRUE, "InputDirectory in sample.info.idx") if(is.null(aligner)){ generate.scRNA.BAM <- FALSE if(!all(file.exists(paste0( unique.samples,".bam" )))) stop("No aligner is specified but the BAM file does not exist. Please specify 'align' or 'subjunc' as the aligner.") for(samplename in unique.samples){ is_dual_index <- .index.names.to.sheet.raw.dir.mode(dirname, sample.info.idx, sample.1, samplename) one.bam.name <- paste0(samplename, ".bam") .write.tmp.parameters(list(sampleSheet=sample.1, umi.cutoff=umi.cutoff, cellBarcodeList=cell.barcode, generate.scRNA.BAM=generate.scRNA.BAM, BAM_is_Rerun_Persample=BAM_is_Rerun_Persample,Is_Dual_Index=is_dual_index, BAM_file_no = dirno)) one.raw.fc <- featureCounts(one.bam.name, annot.inbuilt=annot.inbuilt, annot.ext=annot.ext, isGTFAnnotationFile=isGTFAnnotationFile, GTF.featureType=GTF.featureType, GTF.attrType=GTF.attrType, useMetaFeatures=useMetaFeatures, nthreads=nthreads, strandSpecific=1, ...) dirno <- dirno +1 if(any(is.na(raw.fc.annot))) raw.fc.annot<- one.raw.fc$annotation cat("Processing sample '",samplename,"'\n") one.result <- .load.all.scSamples(paste0(samplename,".bam"), as.character(raw.fc.annot$GeneID), useMetaFeatures, raw.fc.annot, umi.cutoff) fc[["counts"]][[samplename]] <- one.result[["Sample.1"]][["Counts"]] # only one sample. if(is.null(umi.cutoff))fc[["cell.confidence"]][[samplename]] <- one.result[["Sample.1"]][["HighConfidneceCell"]] stt <- .extract.sample.table.cols(full_dirname,one.result, umi.cutoff=umi.cutoff) df.sample.info <- rbind(df.sample.info, stt) } }else{ is_dual_index <- .index.names.to.sheet.raw.dir.mode(dirname, sample.info.idx, sample.1) generate.scRNA.BAM <- TRUE .write.tmp.parameters(list(isBCLinput=TRUE)) if(aligner=="align"){ drna.type <- "rna" align(index, full_dirname, output_file=temp.file.prefix, nthreads=nthreads, type=drna.type, useAnnotation=TRUE, annot.inbuilt=annot.inbuilt, annot.ext=annot.ext, isGTF=isGTFAnnotationFile, GTF.featureType=GTF.featureType, GTF.attrType=GTF.attrType, ...) }else if(aligner=="subjunc"){ subjunc(index, full_dirname, output_file=temp.file.prefix, nthreads=nthreads, ...) } .write.tmp.parameters(list(sampleSheet=sample.1, umi.cutoff=umi.cutoff, cellBarcodeList=cell.barcode, generate.scRNA.BAM=generate.scRNA.BAM,BAM_is_Rerun_Persample=BAM_is_Rerun_Persample,Is_Dual_Index=is_dual_index, BAM_file_no = dirno)) dirno <- dirno +1 raw.fc<-featureCounts(temp.file.prefix, annot.inbuilt=annot.inbuilt, annot.ext=annot.ext, isGTFAnnotationFile=isGTFAnnotationFile, GTF.featureType=GTF.featureType, GTF.attrType=GTF.attrType, useMetaFeatures=useMetaFeatures,nthreads=nthreads,strandSpecific=1, ...) if(any(is.na(raw.fc.annot))) raw.fc.annot<-raw.fc$annotation some.results <- .load.all.scSamples(temp.file.prefix, as.character(raw.fc.annot$GeneID), useMetaFeatures, raw.fc.annot, umi.cutoff) for(spi in 1:nrow(some.results[["Sample.Table"]])){ samplename <- as.character(some.results[["Sample.Table"]][["SampleName"]][spi]) fc[["counts"]][[samplename]] <- some.results[[sprintf("Sample.%d", spi)]][["Counts"]] # only one sample. if(is.null(umi.cutoff))fc[["cell.confidence"]][[samplename]] <- some.results[[sprintf("Sample.%d", spi)]][["HighConfidneceCell"]] } stt <- .extract.sample.table.cols(full_dirname,some.results, umi.cutoff=umi.cutoff) df.sample.info <- rbind(df.sample.info, stt) } } } else if(input.mode == "BAM"){ if(is.null(cell.barcode)){ cell.barcode <- .find_best_cellbarcode(sample$BAMFile, "N/A", input.mode="bam") }else{ cell.barcode <- .check_and_NormPath(cell.barcode, mustWork=T, opt="cell.barcode") } unique.samples <- unique(as.character(sample.info.idx$SampleName)) for(this.sample in unique.samples){ .index.names.to.sheet.BAM.mode(data.frame(BAMFile="COMBINED.INPUT",SampleName=as.character(this.sample),stringsAsFactors=F), sample.1) BAM.names <- paste(sample.info.idx$BAMFile[ as.character(sample.info.idx$SampleName) == this.sample ], collapse=.SCRNA_FASTA_SPLIT1) .write.tmp.parameters(list(isScRNABAMinput=TRUE)) align(index, BAM.names, output_file=temp.file.prefix, nthreads=nthreads, useAnnotation =TRUE,annot.inbuilt=annot.inbuilt, annot.ext=annot.ext, isGTF=isGTFAnnotationFile, GTF.featureType=GTF.featureType, GTF.attrType=GTF.attrType,...) generate.scRNA.BAM <- TRUE .write.tmp.parameters(list(BAM_is_ScRNA_BAM=TRUE, sampleSheet=sample.1, umi.cutoff=umi.cutoff, cellBarcodeList=cell.barcode, generate.scRNA.BAM=generate.scRNA.BAM,BAM_is_Rerun_Persample=BAM_is_Rerun_Persample)) raw.fc<-featureCounts(temp.file.prefix, annot.inbuilt=annot.inbuilt, annot.ext=annot.ext, isGTFAnnotationFile=isGTFAnnotationFile, GTF.featureType=GTF.featureType, GTF.attrType=GTF.attrType, useMetaFeatures=useMetaFeatures,nthreads=nthreads,strandSpecific=1, ...) if(any(is.na(raw.fc.annot))) raw.fc.annot<-raw.fc$annotation some.results <- .load.all.scSamples(temp.file.prefix, as.character(raw.fc.annot$GeneID), useMetaFeatures, raw.fc.annot, umi.cutoff) for(spi in 1:nrow(some.results[["Sample.Table"]])){ samplename <- as.character(some.results[["Sample.Table"]][["SampleName"]][spi]) fc[["counts"]][[samplename]] <- some.results[[sprintf("Sample.%d", spi)]][["Counts"]] # only one sample. if(is.null(umi.cutoff))fc[["cell.confidence"]][[samplename]] <- some.results[[sprintf("Sample.%d", spi)]][["HighConfidneceCell"]] } } df.sample.info <- .extract.sample.table.cols(NA,some.results, input.mode="bam", umi.cutoff=umi.cutoff) } else if(input.mode == "FASTQ" || input.mode == "FASTQ-dir"){ if(input.mode == "FASTQ-dir") sample.info.idx <- .scan.fastq.dir(sample) print(sample.info.idx) if(!("BarcodeUMIFile" %in% colnames(sample.info.idx) && "ReadFile" %in% colnames(sample.info.idx) )) stop("You need to provide BC+UMI and Genomic sequence files") combined.fastq.names <- "" for(rowi in 1:nrow(sample.info.idx)){ df1 <- as.character(sample.info.idx[rowi,"BarcodeUMIFile"]) df2 <- as.character(sample.info.idx[rowi,"ReadFile"]) R1.file.name <- .check_and_NormPath(df1, mustWork=TRUE, "The barcode FASTQ file in sample.info.idx") R2.file.name <- .check_and_NormPath(df2, mustWork=TRUE, "The genomic read FASTQ file in sample.info.idx") combined.fastq.names <- paste0( combined.fastq.names,.SCRNA_FASTA_SPLIT1, R1.file.name, .SCRNA_FASTA_SPLIT2,".",.SCRNA_FASTA_SPLIT2, R2.file.name) } combined.fastq.names <- substr(combined.fastq.names, nchar(.SCRNA_FASTA_SPLIT1)+1, 9999999) if(is.null(cell.barcode)){ cell.barcode <- .find_best_cellbarcode(combined.fastq.names, "N/A", input.mode="fastq") }else{ cell.barcode <- .check_and_NormPath(cell.barcode, mustWork=T, opt="cell.barcode") } bam.for.FC <- c() if(is.null(aligner)){ unique.samples <- unique(as.character(sample.info.idx$SampleName)) bam.for.FC <- paste0(sample.info.idx$SampleName,".bam") generate.scRNA.BAM <- FALSE for(samplename in unique.samples){ leftover.bam <- paste0(samplename, ".bam") .index.names.to.sheet.FASTQ.mode(sample.info.idx[ as.character(sample.info.idx$SampleName) == samplename, ][1,], sample.1) .write.tmp.parameters(list(BAM_is_ScRNA_Fastq=TRUE, sampleSheet=sample.1, umi.cutoff=umi.cutoff, cellBarcodeList=cell.barcode, generate.scRNA.BAM=generate.scRNA.BAM,BAM_is_Rerun_Persample=BAM_is_Rerun_Persample)) raw.fc<-featureCounts(leftover.bam, annot.inbuilt=annot.inbuilt, annot.ext=annot.ext, isGTFAnnotationFile=isGTFAnnotationFile, GTF.featureType=GTF.featureType, GTF.attrType=GTF.attrType, useMetaFeatures=useMetaFeatures,nthreads=nthreads,strandSpecific=1, ...) if(any(is.na(raw.fc.annot))) raw.fc.annot<-raw.fc$annotation some.results <- .load.all.scSamples(leftover.bam, as.character(raw.fc.annot$GeneID), useMetaFeatures, raw.fc.annot, umi.cutoff) fc[["counts"]][[samplename]] <- some.results[[sprintf("Sample.%d", 1)]][["Counts"]] # featureCounts treats each left-over BAM file as a single run and has only one sample if(is.null(umi.cutoff))fc[["cell.confidence"]][[samplename]] <- some.results[[sprintf("Sample.%d", 1)]][["HighConfidneceCell"]] newrow<- .extract.sample.table.cols(NA,some.results,input.mode="fastq", umi.cutoff=umi.cutoff) df.sample.info <- rbind(df.sample.info,newrow) } }else{ .write.tmp.parameters(list(isScRNAFastqinput=TRUE)) drna.type <- "rna" max.MM <- 3 if(F)for(iii in 1:17){ print("WARNING: USING DNA MODE align") drna.type <- "dna" } if(F)for(iii in 1:17){ print("WARNING: USING Max.MM = 10 align") max.MM <- 10 } if(aligner == "align"){ align(index, combined.fastq.names, maxMismatches=max.MM, type=drna.type, output_file=temp.file.prefix, nthreads=nthreads, useAnnotation =TRUE, annot.inbuilt=annot.inbuilt, annot.ext=annot.ext, isGTF=isGTFAnnotationFile, GTF.featureType=GTF.featureType, GTF.attrType=GTF.attrType, nBestLocations = nBestLocations, unique=unique.mapping) }else if(aligner=="subjunc"){ subjunc(index, combined.fastq.names, maxMismatches=max.MM, output_file=temp.file.prefix, nthreads=nthreads, useAnnotation =TRUE, annot.inbuilt=annot.inbuilt, annot.ext=annot.ext, isGTF=isGTFAnnotationFile, GTF.featureType=GTF.featureType, GTF.attrType=GTF.attrType, nBestLocations = nBestLocations, unique=unique.mapping) } bam.for.FC <- temp.file.prefix generate.scRNA.BAM <- TRUE .index.names.to.sheet.FASTQ.mode(sample.info.idx, sample.1) .write.tmp.parameters(list(BAM_is_ScRNA_Fastq=TRUE, sampleSheet=sample.1, umi.cutoff=umi.cutoff, cellBarcodeList=cell.barcode, generate.scRNA.BAM=generate.scRNA.BAM,BAM_is_Rerun_Persample=BAM_is_Rerun_Persample)) raw.fc<-featureCounts(bam.for.FC, annot.inbuilt=annot.inbuilt, annot.ext=annot.ext, isGTFAnnotationFile=isGTFAnnotationFile, GTF.featureType=GTF.featureType, GTF.attrType=GTF.attrType, useMetaFeatures=useMetaFeatures,nthreads=nthreads,strandSpecific=1, ...) if(any(is.na(raw.fc.annot))) raw.fc.annot<-raw.fc$annotation some.results <- .load.all.scSamples(temp.file.prefix, as.character(raw.fc.annot$GeneID), useMetaFeatures, raw.fc.annot, umi.cutoff) for(spi in 1:nrow(some.results[["Sample.Table"]])){ samplename <- as.character(some.results[["Sample.Table"]][["SampleName"]][spi]) fc[["counts"]][[samplename]] <- some.results[[sprintf("Sample.%d", spi)]][["Counts"]] # only one sample. if(is.null(umi.cutoff))fc[["cell.confidence"]][[samplename]] <- some.results[[sprintf("Sample.%d", spi)]][["HighConfidneceCell"]] } df.sample.info <- .extract.sample.table.cols(NA,some.results,input.mode="fastq", umi.cutoff=umi.cutoff) } } fc[["annotation"]] <- raw.fc.annot fc[["sample.info"]] <- df.sample.info if(.remove.temp.files).del.temp.files(substr(temp.file.prefix,4,99)) fc } Rsubread/R/detectionCall.R0000644000175200017520000001447114136047615016457 0ustar00biocbuildbiocbuilddetectionCall <- function(dataset, species="hg", plot=FALSE) { dataset <- .check_and_NormPath(dataset, mustWork=T, opt="dataset") if (file.exists(dataset) == FALSE) { stop("File for the given dataset doesn't exist!\n") } if (species == "mm"){ exon_file <- system.file("annot","mm9_NCBI_exon_GC.txt",package="Rsubread") if(exon_file == ""){ ret_df <- download.file("http://bioinf.wehi.edu.au/software/Rsubread/mm9_NCBI_exon_GC.txt",file.path(system.file("annot",package="Rsubread"),"mm9_NCBI_exon_GC.txt")) if(ret_df != 0) stop("Failed to download file http://bioinf.wehi.edu.au/software/Rsubread/mm9_NCBI_exon_GC.txt") exon_file <- system.file("annot","mm9_NCBI_exon_GC.txt",package="Rsubread") } ir_file <- system.file("annot","mm9_NCBI_binned_integenic_region_GC.txt",package="Rsubread") if(ir_file == ""){ ret_df <- download.file("http://bioinf.wehi.edu.au/software/Rsubread/mm9_NCBI_binned_integenic_region_GC.txt",file.path(system.file("annot",package="Rsubread"),"mm9_NCBI_binned_integenic_region_GC.txt")) if(ret_df != 0) stop("Failed to download file http://bioinf.wehi.edu.au/software/Rsubread/mm9_NCBI_binned_integenic_region_GC.txt") ir_file <- system.file("annot","mm9_NCBI_binned_integenic_region_GC.txt",package="Rsubread") } } if(species == "hg"){ exon_file <- system.file("annot","hg19_NCBI_exon_GC.txt",package="Rsubread") if(exon_file == ""){ ret_df <- download.file("http://bioinf.wehi.edu.au/software/Rsubread/hg19_NCBI_exon_GC.txt",file.path(system.file("annot",package="Rsubread"),"hg19_NCBI_exon_GC.txt")) if(ret_df != 0) stop("Failed to download file http://bioinf.wehi.edu.au/software/Rsubread/hg19_NCBI_exon_GC.txt") exon_file <- system.file("annot","hg19_NCBI_exon_GC.txt",package="Rsubread") } ir_file <- system.file("annot","hg19_NCBI_binned_integenic_region_GC.txt",package="Rsubread") if(ir_file == ""){ ret_df <- download.file("http://bioinf.wehi.edu.au/software/Rsubread/hg19_NCBI_binned_integenic_region_GC.txt",file.path(system.file("annot",package="Rsubread"),"hg19_NCBI_binned_integenic_region_GC.txt")) if(ret_df != 0) stop("Failed to download file http://bioinf.wehi.edu.au/software/Rsubread/hg19_NCBI_binned_integenic_region_GC.txt") ir_file <- system.file("annot","hg19_NCBI_binned_integenic_region_GC.txt",package="Rsubread") } } temp_header <- "." temp_header <- paste(temp_header,paste("/.Rsubread_pid",Sys.getpid(),sep=""), sep="") temp_header <- paste(temp_header, "_", sep="") temp_header <- paste(temp_header, dataset,sep="") ##temp_header <- dataset .C("detectionCall", as.character(dataset), as.character(exon_file), as.character(ir_file), as.character(temp_header), PACKAGE="Rsubread") ########################################################## ### After processing SAM file and Annotation file in C#### ### The result will be processed here for statistics #### ########################################################## ### ALL CHROMOSOMES chrs <- c("chr1", "chr10", "chr11","chr12", "chr13","chr14","chr15","chr16", "chr17","chr18","chr19", "chr2","chr20","chr21","chr22","chr3","chr4", "chr5","chr6", "chr7", "chr8","chr9", "chrX","chrY") bg_file <- paste(temp_header, "_mapping_binned_integenic_region_GC.txt", sep="") signal_file <- paste(temp_header, "_mapping_exon_GC.txt", sep="") if ((file.exists(bg_file) == FALSE) || (file.exists(signal_file) == FALSE)) { print("Mapping results for this dataset are not available.\n") } else { ###################################################### ########## INTEGENIC REGION EXPRESSION DATA ########## ###################################################### bg <- read.delim(bg_file, header=TRUE) bg$percent <- bg$gcnum / (bg$chr_stop - bg$chr_start + 1 - bg$nnum) bg$sqrtnreads <- sqrt(bg$nreads) lowessfit <- lowess(x=bg$percent, y=bg$sqrtnreads) lowessfun <- approxfun(x=lowessfit$x, y=lowessfit$y) ########################################## ########## GENE EXPRESSION DATA ########## ########################################## exon <- read.delim(signal_file, header=TRUE) exon <- exon[exon$chr %in% chrs, ] exon$length <- exon$chr_stop - exon$chr_start + 1 # filter out exons which are covered by other exons exon <- exon[exon$nnum + exon$gcnum + exon$atnum >= 0.9*exon$length, ] # group data into gene level gene_id <- sort(unique(exon$entrezid)) gene_nreads <- tapply(exon$nreads, factor(exon$entrezid), sum) gene_length <- tapply(exon$length, factor(exon$entrezid), sum) gene_gcnum <- tapply(exon$gcnum, factor(exon$entrezid), sum) gene_nnum <- tapply(exon$nnum, factor(exon$entrezid), sum) gene_percent <- gene_gcnum / (gene_length - gene_nnum) gene <- cbind(matrix(gene_id), matrix(gene_length),matrix(gene_percent), matrix(gene_nreads)) gene <- as.data.frame(gene) colnames(gene) <- c("entrezid", "length","GC","nreads") ### For each gene, get the read intensity of same GC content, then calculate its p_values # predict the background intensity of a gene using previously fitted lowess function # and translate it back from sqrt scale to normal scale gene$background <- (lowessfun(gene$GC))^2 gene$background[is.na(gene$background)] <- 0 # calculate the expected nreads under null hypothesis for each gene gene$expected_nreads <- gene$background*gene$length / 2000 gene$p_value <- ppois(gene$nreads, gene$expected_nreads, lower.tail=FALSE) + runif(nrow(gene))*dpois(gene$nreads, gene$expected_nreads) ########################################################### ########## DENSITY PLOT OF P_VALUE FOR EACH GENE ########## ########################################################### if (plot){ plot_output_file <- paste(dataset, "_p_value_density_plot.pdf", sep="") pdf(plot_output_file) plot(density(gene$p_value), xlab = "p_value", ylab = "density", main="Density Plot of P_values for Each Gene(Binsize=2000)", col=1) dev.off() } ############################################## ########## CLEAN UP AND DATA EXPORT ########## ############################################## unlink(bg_file) unlink(signal_file) return(gene) } } Rsubread/R/detectionCallAnnotation.R0000644000175200017520000000105614136047615020505 0ustar00biocbuildbiocbuilddetectionCallAnnotation <- function(species="hg", binsize=2000) { if (species=="hg"){ exonfile <- "hg19_exon.txt" irfile <- "hg19_integenic_region.txt" } if (species == "mm"){ exonfile <- "mm9_exon.txt" irfile <- "mm9_integenic_region.txt" } if ((file.exists(exonfile) == FALSE) || (file.exists(irfile) == FALSE)){ print("Source files specified doesn't exist!") } else { .C("detectionCallAnnotation", as.character(exonfile), as.character(irfile), as.character(species), as.integer(binsize), PACKAGE="Rsubread") } NULL } Rsubread/R/exactSNP.R0000755000175200017520000000235014136047615015366 0ustar00biocbuildbiocbuildexactSNP <- function(readFile,isBAM=FALSE,refGenomeFile,SNPAnnotationFile=NULL,outputFile=paste0(readFile,".exactSNP.VCF"),qvalueCutoff=12,minAllelicFraction=0,minAllelicBases=1,minReads=1,maxReads=1000000, minBaseQuality=13,nTrimmedBases=3,nthreads=1) { if(length(readFile) > 1L) stop("only one input file is allowed.") readFile <- normalizePath(readFile, mustWork=T) refGenomeFile <- normalizePath(refGenomeFile, mustWork=T); outputFile <- normalizePath(outputFile, mustWork=F) if(!is.null(SNPAnnotationFile))SNPAnnotationFile <- normalizePath(SNPAnnotationFile, mustWork=T) opt <- paste("-i",readFile,sep=.R_param_splitor) if(isBAM) opt <- paste(opt,"-b",sep=.R_param_splitor) if(!is.null(SNPAnnotationFile)) opt <- paste(opt,"-a",SNPAnnotationFile,sep=.R_param_splitor) opt <- paste(opt,"-g",refGenomeFile,"-o",outputFile,"-Q",qvalueCutoff,"-f",minAllelicFraction,"-n",minAllelicBases,"-r",minReads,"-x",maxReads,"-s",minBaseQuality,"-t",nTrimmedBases,"-T",nthreads,sep=.R_param_splitor) cmd <- paste("SNPcalling",opt,sep=.R_param_splitor) n <- length(unlist(strsplit(cmd,.R_param_splitor))) C_args <- .C("R_SNPcalling_wrapper",as.integer(n),as.character(cmd),PACKAGE="Rsubread") } Rsubread/R/featureCounts.R0000755000175200017520000003551414136047615016540 0ustar00biocbuildbiocbuild.R_param_splitor <- "\027" .R_flist_splitor <- "\026" .filepath_maximum_len <- 990L .stop_quietly <- function() { opt <- options(show.error.messages = FALSE) on.exit(options(opt)) stop() } .is.64bit.system <- function(){ if(grepl("[-_]64",Sys.info()[["machine"]]))return(TRUE) if(grepl("aarch64",Sys.info()[["machine"]]))return(TRUE) return(FALSE) } .check_string_param <- function(argu, opt=NULL){ if(!is.null(argu)){ if(any(grepl(.R_param_splitor, argu))){ stop(paste0("Error: the argument for ",opt," contains the internal splitor of featureCounts (\\027). The \\027 character is unallowd in this parameter.")) } } } .check_and_NormPath<- function(files, mustWork=F, opt=NULL){ if(any(is.na(files)) || any(is.null(files)) || any(class(files) != "character")){ if( is.null(opt) ){ if(any(is.na(files)) || any(is.null(files))){ stop("Error: the file name is NA or NULL.") }else{ stop(paste0("Error: the file name must be a character vector. The current input is ",class(files))) } }else{ if(any(is.na(files)) || any(is.null(files))){ stop(paste0("Error: the argument to '",opt,"' is NA or NULL.")) }else{ stop(paste0("Error: the argument to '",opt,"' must be a character vector. The current input is ",class(files))) } } } rv <- normalizePath(files, mustWork = mustWork) if(any(grepl(.R_param_splitor, rv))){ stop(paste0("Error: the file path to '",opt,"' contains the internal splitor of featureCounts (\\027). The \\027 character is unallowd in the file names or in the paths.")) } if(any(grepl("\t", rv))){ stop(paste0("Error: the file path to '",opt,"' contains character(s). The TAB character is unallowd in the file names or in the paths.")) } if(any(grepl(.R_flist_splitor, rv))){ stop(paste0("Error: the file path to '",opt,"' contains the internal splitor of featureCounts (\\026). The \\026 character is unallowd in the file names or in the paths.")) } if(any(nchar(rv)>=.filepath_maximum_len)){ errv <- rv[ nchar(rv)>=.filepath_maximum_len ] stop(paste0("Error: the file name and path of ",opt," is longer than ",.filepath_maximum_len," bytes. Beaware that all the file names are normalized to include the full path to the original file. The value is '", errv[1],"'")) } return(rv) } .flatten.and.numeric <- function(logicarr){ paste0(as.numeric(logicarr),collapse="") } .get.annotation.file <- function(annot.inbuilt, annot.ext, isGTFAnnotationFile){ delete.annot.file <- FALSE annot.screen.output <- 'R data.frame' if(is.null(annot.ext)){ switch(tolower(as.character(annot.inbuilt)), mm9={ ann <- system.file("annot","mm9_RefSeq_exon.txt",package="Rsubread") annot.screen.output <- 'inbuilt (mm9)' cat("NCBI RefSeq annotation for mm9 (build 37.2) is used.\n") }, mm10={ ann <- system.file("annot","mm10_RefSeq_exon.txt",package="Rsubread") annot.screen.output <- 'inbuilt (mm10)' cat("NCBI RefSeq annotation for mm10 (build 38.1) is used.\n") }, hg19={ ann <- system.file("annot","hg19_RefSeq_exon.txt",package="Rsubread") annot.screen.output <- 'inbuilt (hg19)' cat("NCBI RefSeq annotation for hg19 (build 37.2) is used.\n") }, hg38={ ann <- system.file("annot","hg38_RefSeq_exon.txt",package="Rsubread") annot.screen.output <- 'inbuilt (hg38)' cat("NCBI RefSeq annotation for hg38 (build 38.2) is used.\n") }, { stop("In-built annotation for ", annot.inbuilt, " is not available.\n") } ) # end switch } else{ if(is.character(annot.ext)){ annot.ext <- .check_and_NormPath(annot.ext, mustWork=T, opt="external annotation") ann <- annot.ext annot.screen.output <- paste0(basename(ann), " (", ifelse(isGTFAnnotationFile, "GTF", "SAF"), ")"); } else{ annot_df <- as.data.frame(annot.ext,stringsAsFactors=FALSE) if(sum(c("geneid","chr","start","end", "strand") %in% tolower(colnames(annot_df))) != 5) stop("One or more required columns are missing in the provided annotation data. Please refer to help page for annotation format.\n") colnames(annot_df) <- tolower(colnames(annot_df)) annot_df <- data.frame(geneid=annot_df$geneid,chr=annot_df$chr,start=annot_df$start,end=annot_df$end,strand=annot_df$strand,stringsAsFactors=FALSE) annot_df$chr <- as.character(annot_df$chr) fout_annot <- file.path(".",paste(".Rsubread_UserProvidedAnnotation_pid",Sys.getpid(),sep="")) oldScipen <- options(scipen=999) write.table(x=annot_df,file=fout_annot,sep="\t",row.names=FALSE,quote=FALSE) options(oldScipen) ann <- fout_annot delete.annot.file <- TRUE } } return(list(ann=ann, screen=annot.screen.output, delete=delete.annot.file)) } featureCounts <- function(files,annot.inbuilt="mm10",annot.ext=NULL,isGTFAnnotationFile=FALSE,GTF.featureType="exon",GTF.attrType="gene_id",GTF.attrType.extra=NULL,chrAliases=NULL,useMetaFeatures=TRUE,allowMultiOverlap=FALSE,minOverlap=1,fracOverlap=0,fracOverlapFeature=0,largestOverlap=FALSE,nonOverlap=NULL,nonOverlapFeature=NULL,readShiftType="upstream",readShiftSize=0,readExtension5=0,readExtension3=0,read2pos=NULL,countMultiMappingReads=TRUE,fraction=FALSE,isLongRead=FALSE,minMQS=0,splitOnly=FALSE,nonSplitOnly=FALSE,primaryOnly=FALSE,ignoreDup=FALSE,strandSpecific=0,juncCounts=FALSE,genome=NULL,isPairedEnd=FALSE,countReadPairs=TRUE,requireBothEndsMapped=FALSE,checkFragLength=FALSE,minFragLength=50,maxFragLength=600,countChimericFragments=TRUE,autosort=TRUE,nthreads=1,byReadGroup=FALSE,reportReads=NULL,reportReadsPath=NULL,maxMOp=10,tmpDir=".",verbose=FALSE) { delete.annot.file <- FALSE if(!.is.64bit.system()) warning("your system seems to be 32-bit. Rsubread supports 32-bit systems to a limited level only.\nWe recommend that Rsubread be run on 64-bit systems to avoid any possible problems.\n\n",call.=FALSE) extra.params <- .retrieve.tmp.parameters() sampleSheet <- NULL generate.scRNA.BAM <- TRUE cellBarcodeList <- NULL umi.cutoff <- NULL BAM_is_ScRNA_Fastq <- FALSE BAM_is_ScRNA_BAM <- FALSE BAM_is_Rerun_Persample <- FALSE Is_Dual_Index <- FALSE BAM_file_no <- 0 if("sampleSheet" %in% names(extra.params)) sampleSheet <- extra.params[["sampleSheet"]] if("generate.scRNA.BAM" %in% names(extra.params)) generate.scRNA.BAM <- extra.params[["generate.scRNA.BAM"]] if("cellBarcodeList" %in% names(extra.params)) cellBarcodeList <- extra.params[["cellBarcodeList"]] if("BAM_is_ScRNA_Fastq" %in% names(extra.params)) BAM_is_ScRNA_Fastq <- extra.params[["BAM_is_ScRNA_Fastq"]] if("BAM_is_ScRNA_BAM" %in% names(extra.params)) BAM_is_ScRNA_BAM <- extra.params[["BAM_is_ScRNA_BAM"]] if("BAM_is_Rerun_Persample" %in% names(extra.params)) BAM_is_Rerun_Persample <- extra.params[["BAM_is_Rerun_Persample"]] if("umi.cutoff" %in% names(extra.params)) umi.cutoff <- extra.params[["umi.cutoff"]] if("Is_Dual_Index" %in% names(extra.params)) Is_Dual_Index <- extra.params[["Is_Dual_Index"]] if("BAM_file_no" %in% names(extra.params)) BAM_file_no <- extra.params[["BAM_file_no"]] .check_string_param(annot.inbuilt, "annot.inbuilt") .check_string_param(GTF.featureType, "GTF.featureType") .check_string_param(GTF.attrType, "GTF.attrType") .check_string_param(GTF.attrType.extra, "GTF.attrType.extra") .check_string_param(readShiftType, "readShiftType") .check_string_param(sampleSheet, "sampleSheet") .check_string_param(cellBarcodeList, "cellBarcodeList") if(length(countReadPairs)>1){ if(length(countReadPairs) != length(files))stop("The argumet for countReadPairs is a vector, but it has a different length to the files vector.") if(length(isPairedEnd)>1 && length(countReadPairs)!=length(isPairedEnd))stop("The argumets for countReadPairs and isPairedEnd are both vectors, but they have different lengths.") } if(length(isPairedEnd)>1){ if(length(isPairedEnd) != length(files))stop("The argumet for isPairedEnd is a vector, but it has a different length to the files vector.") countReadPairs <- ifelse( isPairedEnd, countReadPairs, F ) }else{ if(!isPairedEnd) countReadPairs <- F } if(length(GTF.featureType)>1) GTF.featureType<-paste(GTF.featureType, collapse=",") out.base.names <- basename(files) if(any(duplicated(out.base.names))){ out.col.names <- files }else{ out.col.names <- out.base.names } #out.col.names <-gsub("[[:punct:]]+", ".", out.col.names) #out.col.names <-gsub(" ", ".", out.col.names) if(!is.character(files)) stop("files must be a character vector of file paths") files <- .check_and_NormPath(files, mustWork=T, opt="files") if(!is.null(annot.ext) && is.character(annot.ext)) annot.ext <- .check_and_NormPath(annot.ext, mustWork=T, opt="annot.ext") if(!is.null(chrAliases))chrAliases <- .check_and_NormPath(chrAliases, mustWork=T, opt="chrAliases") if(!is.null(genome)) genome <- .check_and_NormPath(genome, mustWork=T, opt="genome") if(!is.null(cellBarcodeList)) cellBarcodeList <- .check_and_NormPath(cellBarcodeList, mustWork=T, opt="cellBarcodeList") if(!is.null(sampleSheet)) sampleSheet <- .check_and_NormPath(sampleSheet, mustWork=T, opt="sampleSheet") if(!is.null(reportReadsPath)){ reportReadsPath <- .check_and_NormPath(reportReadsPath, mustWork=T, opt="reportReadsPath") }else reportReadsPath <- ' ' strandSpecific<-as.character(strandSpecific) strandSpecific<-paste(strandSpecific, collapse=".") strandSpecific<-gsub(",", ".", strandSpecific) annlist <- .get.annotation.file(annot.inbuilt, annot.ext, isGTFAnnotationFile) ann <- annlist$ann annot.screen.output <- annlist$screen delete.annot.file <- annlist$delete if(readShiftSize < 0){ stop("The value of the readShiftSize parameter should not be negative.") } readShiftType <- match.arg(readShiftType, c("upstream","downstream","left","right")) if(readExtension5 < 0){ stop("The value of the readExtension5 parameter should not be negative.") } if(readExtension3 < 0){ stop("The value of the readExtension3 parameter should not be negative.") } fout <- file.path(".",paste(".Rsubread_featureCounts_pid",Sys.getpid(),sep="")) files_C <- paste(files,collapse=.R_flist_splitor) if(nchar(files_C) == 0) stop("No read files provided!") genome_C <- genome if(is.null(genome)) genome_C <- " " chrAliases_C <- chrAliases if(is.null(chrAliases)) chrAliases_C <- " " read2pos_C <- read2pos if(is.null(read2pos)) read2pos_C <- 0 split_C <- 0 if(splitOnly) split_C <- 1 if(nonSplitOnly) split_C <- 2 PE_orientation <- "fr" if(is.null(reportReads)) { reportReads_C <- 0 } else if(reportReads == "CORE") { reportReads_C <- 10 } else if(reportReads == "SAM") { reportReads_C <- 50 } else if(reportReads == "BAM") { reportReads_C <- 500 } else stop("Invalid value was provided for reportReads parameter.") do_detection_calls <- FALSE max_missing_bases_in_read <- -1 max_missing_bases_in_feature <- -1 GTF.attrType.extra_str <- " " if(!is.null(nonOverlap)) max_missing_bases_in_read <- nonOverlap if(!is.null(nonOverlapFeature)) max_missing_bases_in_feature <- nonOverlapFeature if(!is.null(GTF.attrType.extra))GTF.attrType.extra_str <- paste(GTF.attrType.extra, collapse="\t") if(is.null(sampleSheet)) sampleSheet<-' ' if(is.null(cellBarcodeList)) cellBarcodeList<-' ' #print(.flatten.and.numeric(countReadPairs)) #print(.flatten.and.numeric(isPairedEnd)) cmd <- paste("readSummary",ann,files_C,fout,.flatten.and.numeric(countReadPairs),minFragLength,maxFragLength,0,as.numeric(allowMultiOverlap),as.numeric(useMetaFeatures),nthreads,as.numeric(isGTFAnnotationFile),strandSpecific,reportReads_C,as.numeric(requireBothEndsMapped),as.numeric(!countChimericFragments),as.numeric(checkFragLength),GTF.featureType,GTF.attrType,minMQS,as.numeric(countMultiMappingReads),chrAliases_C," ",as.numeric(FALSE),14,readExtension5,readExtension3,minOverlap,split_C,read2pos_C," ",as.numeric(ignoreDup),as.numeric(!autosort),as.numeric(fraction),as.numeric(largestOverlap),PE_orientation,as.numeric(juncCounts),genome_C,maxMOp,0,as.numeric(fracOverlap),as.character(tmpDir),"0",as.numeric(byReadGroup),as.numeric(isLongRead),as.numeric(verbose),as.numeric(fracOverlapFeature), as.numeric(do_detection_calls), as.numeric(max_missing_bases_in_read), as.numeric(max_missing_bases_in_feature), as.numeric(primaryOnly), reportReadsPath, GTF.attrType.extra_str, annot.screen.output, readShiftType,readShiftSize, sampleSheet, cellBarcodeList ,.flatten.and.numeric(isPairedEnd), as.numeric(generate.scRNA.BAM), ifelse(BAM_is_ScRNA_Fastq, 4, ifelse(BAM_is_ScRNA_BAM, 5,3)), as.numeric(BAM_is_Rerun_Persample), ifelse(is.null(umi.cutoff), -1, umi.cutoff),ifelse(Is_Dual_Index,"Dual-Index","Single-Index"),as.numeric(BAM_file_no),sep=.R_param_splitor) #print(cmd) n <- length(unlist(strsplit(cmd, .R_param_splitor ))) C_args <- .C("R_readSummary_wrapper",as.integer(n),as.character(cmd),PACKAGE="Rsubread") if(file.exists(fout)){ add_attr_numb <- 0 if(!is.null(GTF.attrType.extra)) add_attr_numb <- length(GTF.attrType.extra) x <- read.delim(fout,stringsAsFactors=FALSE, sep="\t") colnames(x)[1:6] <- c("GeneID","Chr","Start","End","Strand","Length") colnames(x)[(7 + add_attr_numb ): (add_attr_numb + 6+length(out.col.names)) ] <- out.col.names x_summary <- read.delim(paste(fout,".summary",sep=""), stringsAsFactors=FALSE, sep="\t") colnames(x_summary)[2:ncol(x_summary)] <- out.col.names if(juncCounts){ x_jcounts <- read.delim(paste(fout,".jcounts",sep=""), stringsAsFactors=FALSE, sep="\t") colnames(x_jcounts)[-(1:8)] <- out.col.names } file.remove(fout) file.remove(paste(fout,".summary",sep="")) if(juncCounts) file.remove(paste(fout,".jcounts",sep="")) if(delete.annot.file) file.remove(ann) if(ncol(x) <= (6 + add_attr_numb)){ cat("No count data were generated.\n") .stop_quietly() } y <- as.matrix(x[,-c(1:(6 + add_attr_numb))]) colnames(y) <- colnames(x)[-c(1:(6 + add_attr_numb))] rownames(y) <- x$GeneID if(juncCounts) z <- list(counts=y,counts_junction=x_jcounts,annotation=x[,1:(6 + add_attr_numb)],targets=out.col.names,stat=x_summary) else z <- list(counts=y,annotation=x[,1:(6 + add_attr_numb)],targets=out.col.names,stat=x_summary) z }else{ cat("No counts were generated.\n") .stop_quietly() } } Rsubread/R/findCommonVariants.R0000644000175200017520000000332414136047615017501 0ustar00biocbuildbiocbuildfindCommonVariants <- function(VCF_files) { VCF_files <- .check_and_NormPath(VCF_files, mustWork=T, opt="VCF_files") fout <- file.path(".",paste(".Rsubread_featureCounts_pid",Sys.getpid(),sep="")) files_C <- paste(VCF_files,collapse=.R_param_splitor) cmd <- paste('-o', fout, files_C, sep=.R_param_splitor) n <- 2 + length(VCF_files) C_args <- .C("R_mergeVCF",as.integer(n),as.character(cmd),PACKAGE="Rsubread") x <- try(read.delim(fout,stringsAsFactors=FALSE, comment.char="#"),silent=TRUE) file.remove(fout) if(class(x)!='try-error') { colnames(x)[1:8] <- c("Chr","Pos","Reference","Identity","Alternative","Quality", "Filter","Info") x } else{ NA } } .findCommonVariants_inner <- function(VCF_files) { stop("Unused function, to be removed.") for(vcf_file in VCF_files) { print(paste("Doing", vcf_file)) vcf_body <- read.delim(vcf_file, comment.char='#', header=FALSE) nky <- 0 for(row_no in 1:nrow(vcf_body)) { alts <- strsplit(as.vector(vcf_body$V5[row_no]), ',') alts <- alts[[1]] nky <- nky + length(alts) } print(paste("NROW =", nky)) data_mat <- matrix(ncol = 3, nrow = nky) n2 <- 1 for(row_no in 1:nrow(vcf_body)) { vcf_row <- vcf_body[row_no,] key_values <- strsplit(as.vector(vcf_row$V8), ';') key_values <- key_values[[1]] key_type <- ifelse('INDEL' %in% key_values, 'INDEL','SNP') alts <- strsplit(as.vector(vcf_row$V5), ',') alts <- alts[[1]] chro <- vcf_row$V1 pos <- vcf_row$V2 qual <- vcf_row$V6 for(alt in alts) { ky = paste(key_type, chro, pos, alt) data_mat[n2,1] <- ky data_mat[n2,2] <- qual data_mat[n2,3] <- as.vector(vcf_row$V8) n2<-n2+1 } print(paste("CUR_ROW" , n2)) n2<-n2+1 } } } Rsubread/R/flattenGTF.R0000644000175200017520000000207714136047615015702 0ustar00biocbuildbiocbuildflattenGTF <- function(GTFfile,GTF.featureType="exon",GTF.attrType="gene_id",method="merge"){ .check_string_param(GTF.featureType,"GTF.featureType") .check_string_param(GTF.attrType,"GTF.attrType") .check_string_param(method,"method") method <- match.arg(method,c("merge","chop")) GTFfile <- .check_and_NormPath(GTFfile, mustWork=TRUE, opt="GTFfile") fout <- file.path(".",paste0(".Rsubread_flattenGTF_pid",Sys.getpid())) cmd <- paste("RflattenGTF","-a",GTFfile,sep=.R_param_splitor) cmd <- paste(cmd,"-g",GTF.attrType,sep=.R_param_splitor) cmd <- paste(cmd,"-t",GTF.featureType,sep=.R_param_splitor) cmd <- paste(cmd,"-o",fout,sep=.R_param_splitor) if(method == 'chop') cmd <- paste(cmd,"-C",sep=.R_param_splitor) n <- length(unlist(strsplit(cmd,.R_param_splitor))) C_args <- .C("R_flattenGTF_wrapper",n,cmd,PACKAGE="Rsubread") if(file.exists(fout)){ z <- read.delim(fout,stringsAsFactors=FALSE,colClasses=c("character","character","integer","integer","character")) file.remove(fout) z }else{ warning("No output was generated.") data.frame() } } Rsubread/R/getInBuiltAnnotation.R0000755000175200017520000000202414136047615020000 0ustar00biocbuildbiocbuildgetInBuiltAnnotation <- function(annotation="mm10") { .check_string_param(annotation,"annotation") switch(tolower(as.character(annotation)), mm9={ ann <- system.file("annot","mm9_RefSeq_exon.txt",package="Rsubread") message("NCBI RefSeq annotation for mm9 (build 37.2).") }, mm10={ ann <- system.file("annot","mm10_RefSeq_exon.txt",package="Rsubread") message("NCBI RefSeq annotation for mm10 (build 38.1).") }, hg19={ ann <- system.file("annot","hg19_RefSeq_exon.txt",package="Rsubread") message("NCBI RefSeq annotation for hg19 (build 37.2).") }, hg38={ ann <- system.file("annot","hg38_RefSeq_exon.txt",package="Rsubread") message("NCBI RefSeq annotation for hg38 (build 38.2).") }, { stop("In-built annotation for ", annotation, " is not available.\n") } ) # end switch x <- read.delim(ann, sep="\t", quote="", colClasses=c("character","character","integer","integer","character"), comment.char="") x } Rsubread/R/processExons.R0000644000175200017520000000564614136047615016404 0ustar00biocbuildbiocbuildprocessExons <- function(filename = "human_seq_gene.md", species="hg") { .check_string_param(species,'species') if (species == "mm"){ filename <- "mouse_seq_gene.md" } if (file.exists(filename) == FALSE){ print("Souce file specified doesn't exist!") } else { x <- read.delim(filename, stringsAsFactors=FALSE) if (species == "hg"){ y <- x[x$group_label=="GRCh37.p2-Primary Assembly" | x$group_label=="GRCh37.p2-non-nuclear", ] } else { if (species == "mm"){ y <- x[x$group_label=="MGSCv37-C57BL/6J",] } } y <- y[y$feature_type=="CDS" | y$feature_type=="UTR",] y <- y[,c("chromosome","chr_start","chr_stop","chr_orient","feature_id")] if (species == "hg"){ chr_max <- 22 } else { chr_max <- 19 } for(i in 1:chr_max){ index <- y$chromosome %in% i y$chromosome[index] <- rep(paste("chr",i,sep=""),sum(index)) } index <- y$chromosome %in% "X" y$chromosome[index] <- rep("chrX",sum(index)) index <- y$chromosome %in% "Y" y$chromosome[index] <- rep("chrY",sum(index)) ###NT <- unique(y$chromosome[grep("NT_",y$chromosome)]) ###for(i in NT){ ### index <- y$chromosome %in% i ### i1 <- unlist(strsplit(i,"|",fixed=TRUE)) ### i1 <- i1[2] ### y$chromosome[index] <- rep(i1,sum(index)) ###} z <- y[substr(y$chromosome,1,3) == "chr",] z$feature_id <- substring(z$feature_id, 8, 1000000L) ### sort utr and cds according to their chromosome and chr_start sort.z <- z[order(z$chromosome, z$chr_start),] ### specify file header according to species if (species == "mm"){ header <- "mm9_" } else { header <- "hg19" } utr_cds_file <- paste(header, "utr_cds.txt", sep="") exon_file <- paste(header,"exon.txt",sep="") gene_file <- paste(header,"gene.txt",sep="") ir_file <- paste(header,"integenic_region.txt",sep="") if (length(unique(z$feature_id)) > 1000000){ print("The input file contains too many genes.\n") } else { write.table(sort.z,file=utr_cds_file, quote=FALSE,sep="\t",row.names=FALSE, col.names=FALSE) .C("processExons", as.character(utr_cds_file), as.character(exon_file), as.character(gene_file), PACKAGE="Rsubread") gene <- read.delim(gene_file, stringsAsFactors=FALSE) colnames(gene) <- c("entrezid","chromosome", "chr_start","chr_stop") sort.gene <- gene[order(gene$chromosome, gene$chr_start),] file.remove(gene_file) write.table(sort.gene,file=gene_file, quote=FALSE,sep="\t",row.names=FALSE, col.names=FALSE) exon <- read.delim(exon_file, stringsAsFactors=FALSE) colnames(exon) <- c("entrezid","chromosome", "chr_start","chr_stop") sort.exon <- exon[order(exon$chromosome, exon$chr_start),] file.remove(exon_file) write.table(sort.exon,file=exon_file, quote=FALSE,sep="\t",row.names=FALSE, col.names=FALSE) .C("generate_ir_file", as.character(gene_file), as.character(ir_file), PACKAGE="Rsubread") file.remove(gene_file) file.remove(utr_cds_file) } } NULL } Rsubread/R/promoterRegions.R0000644000175200017520000000416514136047615017102 0ustar00biocbuildbiocbuildpromoterRegions <- function(annotation="mm10", upstream=3000L, downstream=2000L) # Create a SAF data.frame of genewise promoter regions # Gordon Smyth # Created 24 April 2017. Last modified 22 Oct 2020. { # annotation can be a SAF format data.frame or can be the name of a genome with built-in annotation if(is.character(annotation)) { .check_string_param(annotation,'annotation') annotation <- getInBuiltAnnotation(annotation) } else { if(!is.data.frame(annotation)) stop("annotation should be character string or data.frame") if(!all(c("GeneID", "Chr", "Start", "End", "Strand") %in% names(annotation))) stop("annotation data.frame is not in SAF format") } # Remove unassembled contigs N <- grep("^N",annotation$Chr) annotation <- annotation[-N,] # Check upstream and downstream limits upstream <- max(upstream[1],0L) upstream <- as.integer(min(.Machine$integer.max %/% 2L,upstream)) downstream <- max(downstream[1],0L) downstream <- as.integer(pmin(.Machine$integer.max %/% 2L,downstream)) # Combine Chr and GeneID annotation$ChrGeneID <- paste(annotation$Chr,annotation$GeneID,sep=".") # Get start of each gene o <- order(annotation$ChrGeneID,annotation$Start) anno.start <- annotation[o,] isdup <- duplicated(anno.start$ChrGeneID) exon.first <- which(!isdup) anno.start <- anno.start[exon.first,] # Get end of each gene o <- order(annotation$ChrGeneID,annotation$End) anno.end <- annotation[o,] exon.last <- c(exon.first[-1]-1L,nrow(annotation)) anno.end <- anno.end[exon.last,] # Assemble start and end into one data.frame ann.gene <- anno.start ann.gene$End <- anno.end$End # Compute promoter region for + strand prom.start <- pmax(ann.gene$Start-upstream,1L) prom.end <- pmin(ann.gene$Start+downstream,ann.gene$End) # Compute promoter region for - strand neg <- ann.gene$Strand=="-" prom.start[neg] <- pmax(ann.gene$End[neg]-downstream,ann.gene$Start[neg]) prom.end[neg] <- ann.gene$End[neg]+upstream # Output ann.gene$Start <- prom.start ann.gene$End <- prom.end ann.gene$ChrGeneID <- NULL o <- order(ann.gene$Chr,ann.gene$Start) ann.gene[o,] } Rsubread/R/propmapped.R0000755000175200017520000000255014136047615016052 0ustar00biocbuildbiocbuildpropmapped <- function(files,countFragments=TRUE,properlyPaired=FALSE,verbose=FALSE) { out.base.names <- basename(files) if(any(duplicated(out.base.names))){ out.table.rows <- files }else{ out.table.rows <- out.base.names } #out.table.rows <-gsub("[[:punct:]]+", ".", out.table.rows) #out.table.rows <-gsub(" ", ".", out.table.rows) files <- .check_and_NormPath(files, mustWork=T, opt="files") if(any(duplicated(files)))stop("Error: duplicated input files are provided. No result is generated.") fout <- file.path(".",paste(".Rsubread_propmapped_pid",Sys.getpid(),sep="")) for(i in 1:length(files)){ opt <- paste("-i",files[i],sep=.R_param_splitor) if(countFragments) opt <- paste(opt,"-f",sep=.R_param_splitor) if(properlyPaired) opt <- paste(opt,"-p",sep=.R_param_splitor) if(verbose) opt <- paste(opt,"-V",sep=.R_param_splitor) opt <- paste(opt,"-o",fout,sep=.R_param_splitor) cmd <- paste("propmapped",opt,sep=.R_param_splitor) n <- length(unlist(strsplit(cmd,.R_param_splitor))) C_args <- .C("R_propmapped_wrapper",as.integer(n),as.character(cmd),PACKAGE="Rsubread") } x1 <- read.csv(fout,header=FALSE,row.names=1,stringsAsFactors=FALSE) rownames(x1) <- out.table.rows file.remove(fout) colnames(x1) <- c("NumTotal","NumMapped","PropMapped") x1 } Rsubread/R/qualityScores.R0000755000175200017520000000305614136047615016554 0ustar00biocbuildbiocbuildqualityScores <- function(filename,input_format="gzFASTQ",offset=33,nreads=10000) { .check_string_param(input_format,"input_format") if(length(filename)>1) stop("The qualityScores function only allows one input file.") filename <- .check_and_NormPath(filename, mustWork=T, opt="filename") if (file.exists(filename) == FALSE) stop("Can not find the input file. Pleaes check whether the file name is correct.") score_file = paste(".Rsubread_qualityScores_score_pid",Sys.getpid(),sep="") opt <- paste("-i",filename,"-o",score_file,sep=.R_param_splitor) if(tolower(input_format) == "gzfastq") opt <- paste(opt,"--gzFASTQinput",sep=.R_param_splitor) if(tolower(input_format) == "sam") opt <- paste(opt,"--SAMinput",sep=.R_param_splitor) if(tolower(input_format) == "bam") opt <- paste(opt,"--BAMinput",sep=.R_param_splitor) if(nreads<=0){ if(nreads==-1) nreads <- "100000000000" else stop("ERROR: nreads cannot be zero or negative.") } opt <- paste(opt,"--phred-offset",offset,"--counted-reads",nreads,sep=.R_param_splitor) cmd <- paste("qualityScores",opt,sep=.R_param_splitor) n <- length(unlist(strsplit(cmd,.R_param_splitor))) C_args <- .C("R_qualityScores_wrapper",as.integer(n),as.character(cmd),PACKAGE="Rsubread") scores <- read.csv(score_file,header=FALSE,stringsAsFactors=FALSE) scores <- scores[, colSums( is.na(scores) ) < nrow(scores) ] # remove all-NA columns scores <- as.matrix(scores) colnames(scores) <- 1:ncol(scores) file.remove(score_file) scores } Rsubread/R/removeDupReads.R0000755000175200017520000000124014136047615016623 0ustar00biocbuildbiocbuildremoveDupReads <- function(inputFile,threshold=50,outputFile,outputFormat="BAM") { SAMfile <- .check_and_NormPath(inputFile, mustWork=T, opt="SAMfile") outputFile <- .check_and_NormPath(outputFile, mustWork=F, opt="outputFile") cmd <- paste("removeDupReads","-i",SAMfile,"-r",threshold,"-o",outputFile,sep=.R_param_splitor) if(outputFormat=="SAM") cmd <- paste(cmd, "-S", sep=.R_param_splitor) else if(outputFormat!="BAM") stop("ERROR: Unknown output format! Only 'BAM' or 'SAM' are accepted!") n <- length(unlist(strsplit(cmd, .R_param_splitor))) C_args <- .C("R_removeDupReads_wrapper",as.integer(n),as.character(cmd),PACKAGE="Rsubread") } Rsubread/R/repair.R0000755000175200017520000000236014136047615015164 0ustar00biocbuildbiocbuildrepair <- function(inFiles,inFormat="BAM",outFiles=paste(inFiles,"repair",sep="."),addDummy=TRUE,fullData=TRUE,compress=FALSE,nthreads=8) { .check_string_param(inFormat,"inFormat") inFiles <- .check_and_NormPath(inFiles, mustWork=T, opt="inFiles") outFiles <- .check_and_NormPath(outFiles, mustWork=F, opt="outFiles") if(length(inFiles) != length(outFiles)) stop("Number of input files is different from number of output files.") if(!all(file.exists(inFiles))) stop("One or more input files cannot be found.") if(!all(file.exists(dirname(outFiles)))) stop("Invalid path was found in output file names.") for(i in 1:length(inFiles)){ opt <- paste("-i",inFiles[i],sep=.R_param_splitor) if(tolower(inFormat) == "sam") opt <- paste(opt,"-S",sep=.R_param_splitor) if(!addDummy) opt <- paste(opt,"-d",sep=.R_param_splitor) if(!fullData) opt <- paste(opt,"-t",sep=.R_param_splitor) if(compress) opt <- paste(opt,"-c",sep=.R_param_splitor) opt <- paste(opt,"-T",nthreads,"-o",outFiles[i],sep=.R_param_splitor) cmd <- paste("repair",opt,sep=.R_param_splitor) n <- length(unlist(strsplit(cmd,.R_param_splitor))) C_args <- .C("R_repair_wrapper",as.integer(n),as.character(cmd),PACKAGE="Rsubread") } invisible() } Rsubread/R/sam2bed.R0000755000175200017520000000075414136047615015224 0ustar00biocbuildbiocbuildsam2bed <- function(samfile,bedfile,readlen) { .check_string_param(readlen,"readlen") samfile <- .check_and_NormPath(samfile, mustWork=T, opt="samfile") bedfile <- .check_and_NormPath(bedfile, mustWork=F, opt="bedfile") opt <- paste("-n",readlen,samfile,bedfile,sep=.R_param_splitor) cmd <- paste("sam2bed",opt,sep=.R_param_splitor) n <- length(unlist(strsplit(cmd,.R_param_splitor))) C_args <- .C("R_sam2bed_wrapper",as.integer(n),as.character(cmd),PACKAGE="Rsubread") } Rsubread/R/simReads.R0000644000175200017520000001444214136047615015452 0ustar00biocbuildbiocbuild.simFragments <- function(transcript.lengths, transcript.expressions=NULL, library.size=1e6, fragment.length.min=100L, fragment.length.max=500L, fragment.length.mean=180, fragment.length.sd=40) # Randomly generate fragment lengths and starting positions for RNA-seq simulation. # Assume gamma distribution for fragment lengths. # Gordon Smyth # Created 13 March 2019. Last modified 15 March 2019. { # Default to equal expression levels if(is.null(transcript.expressions)) transcript.expressions <- rep_len(1,length(transcript.lengths)) # To conserve memory, use integers transcript.lengths <- as.integer(transcript.lengths) fragment.length.min <- as.integer(fragment.length.min) fragment.length.max <- as.integer(fragment.length.max) # First generate number of fragments from each transcript # We will assume that any transcript with length >= fragment.length.min has a chance of being sequenced effective.transcript.lengths <- pmax(transcript.lengths - fragment.length.min + 1, 0) prob <- effective.transcript.lengths * transcript.expressions prob <- prob / sum(prob) # Randomly allocate total number of fragments (library size) to transcripts n.fragments <- drop(rmultinom(1L, size=library.size, prob=prob)) # Parameter values for gamma distribution alpha <- ( fragment.length.mean / fragment.length.sd )^2 beta <- fragment.length.mean / alpha # Probability distribution of fragment lengths frag.len <- seq.int(fragment.length.min,fragment.length.max) frag.len.p <- dgamma(frag.len,shape=alpha,scale=beta,log=TRUE) frag.len.p <- exp(frag.len.p - mean(frag.len.p)) frag.len.n <- length(frag.len) # Expand out transcript.lengths to library.size out <- matrix(0L,library.size,3) colnames(out) <- c("Transcript","FragmentLength","StartPosition") ntranscripts <- length(transcript.lengths) out[,"Transcript"] <- rep.int(seq_len(ntranscripts), n.fragments) TraLen <- rep.int(transcript.lengths, n.fragments) # Randomly assign fragment lengths out[,"FragmentLength"] <- sample.int(frag.len.n, size=library.size, replace=TRUE, prob=frag.len.p) out[,"FragmentLength"] <- frag.len[out[,"FragmentLength"]] # Rerun for short transcripts i <- which(out[,"FragmentLength"] > TraLen) if(length(i)) { FragLenShort <- TraLenShort <- TraLen[i] WhichFragMax <- max(TraLenShort) - fragment.length.min + 1L for (j in seq_len(WhichFragMax)) { k <- which(TraLenShort == frag.len[j]) n <- length(k) if(n) FragLenShort[k] <- sample.int(j, size=n, replace=TRUE, prob=frag.len.p[1:j]) } out[i,"FragmentLength"] <- frag.len[FragLenShort] } # Generate start position out[,"StartPosition"] <- 1L + as.integer( runif(library.size) * (TraLen - out[,"FragmentLength"]) + 0.5 ) list(n.fragments=n.fragments, read.positions=out) } simReads <- function(transcript.file, expression.levels, output.prefix, library.size=1000000, read.length=75, truth.in.read.names=FALSE, simulate.sequencing.error=TRUE, quality.reference=NULL, paired.end=FALSE, fragment.length.min=100L, fragment.length.max=500L, fragment.length.mean=180, fragment.length.sd=40, simplify.transcript.names=FALSE){ expression.levels[ is.na(expression.levels) ]<-0 if(paired.end && fragment.length.min < read.length)stop("The minimum fragment length cannot be lower than the output read length.") if(read.length<1 || read.length>250)stop("The output read length must be between 1 and 250.") if(library.size<1) stop("At least one read should be generated.") if(any(expression.levels<0))stop("No negative expression values are allowed.") if(sum(expression.levels)==0)stop("The wanted expression levels are all zero.") if(library.size>100*1000*1000) stop("The current version cannot generate more than 100 million reads/fragments in a single run") transcript.file <- .check_and_NormPath(transcript.file, mustWork=TRUE, opt="transcript.file") output.prefix <- .check_and_NormPath(output.prefix, mustWork=FALSE, opt="output.prefix") fasta.meta <- scanFasta(transcript.file, simplify.transcript.names, quiet=T) if(length(expression.levels)!=nrow(fasta.meta))stop("The wanted expression levels do not match the transcripts in the input fasta file.") if(simulate.sequencing.error){ if(is.null(quality.reference)){ if(read.length==75) quality.reference <- system.file("qualf","ref-quality-strings-20k-75bp-ERR1_59-SRR3649332.txt",package="Rsubread") if(read.length==100) quality.reference <- system.file("qualf","ref-quality-strings-20k-100bp-ERR2_70-SRR3045231.txt",package="Rsubread") if(is.null(quality.reference)) stop("To simulate sequencing errors in reads that are neither 100-bp nor 75-bp long, you need to provide a file containing reference quality strings of the same length as the output reads.") }else{ quality.reference <- .check_and_NormPath(quality.reference, mustWork=TRUE, opt="quality.reference") } }else{ quality.reference <- NULL } sf <- .simFragments(fasta.meta$Length, expression.levels, library.size, fragment.length.min, fragment.length.max, fragment.length.mean, fragment.length.sd ) C_args <- .C("R_genSimReads_at_poses", transcript.file, output.prefix, as.character(quality.reference), fasta.meta$TranscriptID, sf$read.positions[,'Transcript'], sf$read.positions[,'StartPosition'], sf$read.positions[,'FragmentLength'], as.integer(read.length), as.integer(library.size), nrow(fasta.meta), as.integer(simplify.transcript.names), as.integer(truth.in.read.names), as.integer(paired.end), PACKAGE="Rsubread") data.frame(fasta.meta[,1:2], NReads=sf$n.fragments) } scanFasta <- function(transcript.file, simplify.transcript.names=FALSE, quiet=FALSE){ fout_sum <- file.path(".",paste(".Rsubread_sumfile_pid",Sys.getpid(),sep="")) transcript.file <- .check_and_NormPath(transcript.file, mustWork=T, opt="transcript.file") cmd <- paste("RscanFasta","--summarizeFasta","--transcriptFasta",transcript.file,"--outputPrefix", fout_sum, sep=.R_param_splitor) if(quiet) cmd<-paste(cmd, "--quiet", sep=.R_param_splitor) if(simplify.transcript.names) cmd<-paste(cmd, "--simpleTranscriptId", sep=.R_param_splitor) n <- length(unlist(strsplit(cmd,.R_param_splitor))) C_args <- .C("R_generate_random_RNAseq_reads",as.integer(n), as.character(cmd),PACKAGE="Rsubread") fout_sum <-paste0(fout_sum,".faSummary") summ <- read.delim(fout_sum, stringsAsFactors=FALSE, header=T) file.remove(fout_sum) summ } Rsubread/R/subjunc.R0000755000175200017520000001762714136047615015367 0ustar00biocbuildbiocbuildsubjunc <- function(index,readfile1,readfile2=NULL,input_format="gzFASTQ",output_format="BAM",output_file=paste(readfile1,"subjunc",output_format,sep="."),phredOffset=33,nsubreads=14,TH1=1,TH2=1,maxMismatches=3,unique=FALSE,nBestLocations=1,indels=5,complexIndels=FALSE,nTrim5=0,nTrim3=0,minFragLength=50,maxFragLength=600,PE_orientation="fr",nthreads=1,readGroupID=NULL,readGroup=NULL,keepReadOrder=FALSE,sortReadsByCoordinates=FALSE,color2base=FALSE,DP_GapOpenPenalty=-1,DP_GapExtPenalty=0,DP_MismatchPenalty=0,DP_MatchScore=2,reportAllJunctions=FALSE,useAnnotation=FALSE,annot.inbuilt="mm10",annot.ext=NULL,isGTF=FALSE,GTF.featureType="exon",GTF.attrType="gene_id",chrAliases=NULL) { if(!.is.64bit.system()) cat("WARNING: your system seems to be 32-bit. Rsubread supports 32-bit systems to a very limited level.\nIt is highly recommended to run Rsubread on a 64-bit system to avoid errors.\n\n") extra.params <- .retrieve.tmp.parameters() isBCLinput <- FALSE isScRNAFastqinput <- FALSE if("isBCLinput" %in% names(extra.params))isBCLinput <- extra.params[["isBCLinput"]] if("isScRNAFastqinput" %in% names(extra.params))isScRNAFastqinput <- extra.params[["isScRNAFastqinput"]] .check_string_param(input_format,"input_format") .check_string_param(output_format,"output_format") .check_string_param(PE_orientation,"PE_orientation") .check_string_param(annot.inbuilt,"annot.inbuilt") .check_string_param(GTF.featureType,"GTF.featureType") .check_string_param(GTF.attrType,"GTF.attrType") .check_string_param(readGroupID,"readGroupID") .check_string_param(readGroup,"readGroup") out.base.names <- basename(output_file) if(any(duplicated(out.base.names))){ out.table.cols <- output_file }else{ out.table.cols <- out.base.names } #out.table.cols <-gsub("[[:punct:]]+", ".", out.table.cols) #out.table.cols <-gsub(" ", ".", out.table.cols) readfile1 <- as.character(readfile1) if(!isScRNAFastqinput)readfile1 <- .check_and_NormPath(readfile1, mustWork=TRUE, opt="readfile1") output_file <- .check_and_NormPath(output_file, mustWork=FALSE, opt="output_file") index <- .check_and_NormPath(index, mustWork=FALSE, opt="index") if((!is.null(annot.ext)) && is.character(annot.ext)) annot.ext = .check_and_NormPath(annot.ext, mustWork=TRUE, opt="annot.ext") if(!is.null(chrAliases)) chrAliases = .check_and_NormPath(chrAliases, mustWork=TRUE, opt="chrAliases") if(length(readfile1) != length(output_file)) stop("The number of input file names is different from the number of output file names.") if(!is.null(readfile2)){ readfile2 <- as.character(readfile2) readfile2 <- .check_and_NormPath(readfile2, mustWork=TRUE, "readfile2") if(length(readfile1) != length(readfile2)) stop("The number of file names for the first reads is different from the number of file names for the second reads.") } if((!isScRNAFastqinput) && !all(file.exists(readfile1))) stop("One or more input files cannot be found (readfile1).") if(!is.null(readfile2)){ if(!all(file.exists(readfile2))) stop("One or more input files cannot be found (readfile2).") } if(!all(file.exists(dirname(output_file)))) stop("Invalid path was found in output file name(s).") opt <- paste("-i",index,sep=.R_param_splitor) if(tolower(input_format) == "sam") opt <- paste(opt,"--SAMinput",sep=.R_param_splitor) if(tolower(input_format) == "bam") opt <- paste(opt,"--BAMinput",sep=.R_param_splitor) if(tolower(output_format) == "sam") opt <- paste(opt,"--SAMoutput",sep=.R_param_splitor) if(isBCLinput) opt <- paste(opt,"--BCLinput",sep=.R_param_splitor) if(isScRNAFastqinput) opt <- paste(opt,"--scRNA_FQinput",sep=.R_param_splitor) opt <- paste(opt,"-n",nsubreads,"-m",TH1,"-p",TH2,"-M",maxMismatches,"-T",nthreads,"-I",indels,sep=.R_param_splitor) if(complexIndels) opt <- paste(opt,"--complexIndels",sep=.R_param_splitor) if(!unique) opt <- paste(opt,"--multiMapping",sep=.R_param_splitor) opt <- paste(opt,"-B",nBestLocations,"-d",minFragLength,"-D",maxFragLength,"-S",PE_orientation,"--trim5",nTrim5,"--trim3",nTrim3,sep=.R_param_splitor) if(!is.null(readGroupID)) opt <- paste(opt,"--rg-id",readGroupID,sep=.R_param_splitor) if(!is.null(readGroup)) opt <- paste(opt,"--rg",readGroup,sep=.R_param_splitor) if(color2base) opt <- paste(opt,"-b",sep=.R_param_splitor) if(keepReadOrder) opt <- paste(opt,"--keepReadOrder",sep=.R_param_splitor) if(sortReadsByCoordinates) opt <- paste(opt,"--sortReadsByCoordinates",sep=.R_param_splitor) opt <- paste(opt,"-G",DP_GapOpenPenalty,"-E",DP_GapExtPenalty,"-X",DP_MismatchPenalty,"-Y",DP_MatchScore,sep=.R_param_splitor) if(reportAllJunctions) opt <- paste(opt,"--allJunctions",sep=.R_param_splitor) flag <- FALSE if(useAnnotation){ annot.display <- "R data.frame" if(is.null(annot.ext)){ switch(tolower(as.character(annot.inbuilt)), mm9={ ann <- system.file("annot","mm9_RefSeq_exon.txt",package="Rsubread") annot.display <- "inbuilt (mm9)" }, mm10={ ann <- system.file("annot","mm10_RefSeq_exon.txt",package="Rsubread") annot.display <- "inbuilt (mm10)" }, hg19={ ann <- system.file("annot","hg19_RefSeq_exon.txt",package="Rsubread") annot.display <- "inbuilt (hg19)" }, hg38={ ann <- system.file("annot","hg38_RefSeq_exon.txt",package="Rsubread") annot.display <- "inbuilt (hg38)" }, { stop("In-built annotation for ", annot.inbuilt, " is not available.\n") } ) # end switch } else{ if(is.character(annot.ext)){ ann <- annot.ext annot.display <- paste0(basename(ann), " (", ifelse(isGTF,"GTF","SAF"),")") } else{ annot_df <- as.data.frame(annot.ext,stringsAsFactors=FALSE) if(sum(c("geneid","chr","start","end", "strand") %in% tolower(colnames(annot_df))) != 5) stop("One or more required columns are missing in the provided annotation data. Please refer to help page for annotation format.\n") colnames(annot_df) <- tolower(colnames(annot_df)) annot_df <- data.frame(geneid=annot_df$geneid,chr=annot_df$chr,start=annot_df$start,end=annot_df$end,strand=annot_df$strand,stringsAsFactors=FALSE) annot_df$chr <- as.character(annot_df$chr) fout_annot <- file.path(".",paste(".Rsubread_subjunc_UserProvidedAnnotation_pid",Sys.getpid(),sep="")) oldScipen <- options(scipen=999) write.table(x=annot_df,file=fout_annot,sep="\t",row.names=FALSE,quote=FALSE) options(oldScipen) ann <- fout_annot flag <- TRUE } } opt <- paste(opt,"-a",ann,sep=.R_param_splitor) if(isGTF) opt <- paste(opt,"-F","GTF","--gtfFeature",GTF.featureType,"--gtfAttr",GTF.attrType,sep=.R_param_splitor) else opt <- paste(opt,"-F","SAF",sep=.R_param_splitor) if(!is.null(chrAliases)) opt <- paste(opt,"-A",chrAliases,sep=.R_param_splitor) opt <- paste(opt, "--exonAnnotationScreenOut", annot.display, sep=.R_param_splitor) } if(phredOffset == 33) opt <- paste(opt,"-P",3,sep=.R_param_splitor) else opt <- paste(opt,"-P",6,sep=.R_param_splitor) for(i in 1:length(readfile1)){ opt_files <- paste("-r",readfile1[i],sep=.R_param_splitor) if(!is.null(readfile2)) opt_files <- paste(opt_files,"-R",readfile2[i],sep=.R_param_splitor) opt_files <- paste(opt_files,"-o",output_file[i],sep=.R_param_splitor) cmd <- paste("subjunc",opt_files,opt,sep=.R_param_splitor) n <- length(unlist(strsplit(cmd,.R_param_splitor))) C_args <- .C("R_junction_wrapper",as.integer(n),as.character(cmd),PACKAGE="Rsubread") summary.data <- .load.delete.summary(output_file[i]) if(i ==1){ return.summary <- summary.data }else{ return.summary <- data.frame(return.summary, summary.data) } } if(flag) file.remove(fout_annot) names(return.summary) <- out.table.cols # basename(output_file) return.summary } Rsubread/R/sublong.R0000755000175200017520000000213514136047615015353 0ustar00biocbuildbiocbuildsublong <- function(index, readFiles, outputFiles, outputFormat="BAM" , nthreads=1) { .check_string_param(outputFormat,"outputFormat") readFiles <- .check_and_NormPath(as.character(readFiles), mustWork=T, opt="readFiles") outputFiles <- .check_and_NormPath(as.character(outputFiles), mustWork=F , opt="outputFiles") if(length(readFiles) != length(outputFiles)) stop("The number of input file names is different from the number of output file names.") if(!all(file.exists(readFiles))) stop("One or more input files cannot be found.") if(!all(file.exists(dirname(outputFiles)))) stop("Invalid path was found in output file name(s).") for(i in 1:length(readFiles)){ opt <- paste("-i",index,sep=.R_param_splitor) if(tolower(outputFormat) == "sam") opt <- paste(opt,"--SAMoutput",sep=.R_param_splitor) opt <- paste(opt,"-r",readFiles[i],"-T",nthreads,"-o",outputFiles[i],sep=.R_param_splitor) cmd <- paste("Rsublong",opt,sep=.R_param_splitor) n <- length(unlist(strsplit(cmd,.R_param_splitor))) C_args <- .C("R_sublong_wrapper",as.integer(n),as.character(cmd),PACKAGE="Rsubread") } } Rsubread/R/txUnique.R0000644000175200017520000000225114136047615015520 0ustar00biocbuildbiocbuildtxUnique <- function(GTF_Annotation_File, Feature_Type = "exon", Gene_ID_Attribute = "gene_id", Transcript_ID_Attribute = "transcript_id"){ .check_string_param(Feature_Type, "Feature_Type") .check_string_param(Gene_ID_Attribute,"Gene_ID_Attribute") .check_string_param(Transcript_ID_Attribute,"Transcript_ID_Attribute") GTF_Annotation_File <- .check_and_NormPath(GTF_Annotation_File, mustWork=T, opt="GTF_Annotation_File") fout <- file.path(".",paste(".Rsubread_txUnique_pid",Sys.getpid(),sep="")) opt <- paste("-a",GTF_Annotation_File,sep=.R_param_splitor) opt <- paste(opt,"-o",fout,sep=.R_param_splitor) opt <- paste(opt,"-g",Gene_ID_Attribute,sep=.R_param_splitor) opt <- paste(opt,"-t",Transcript_ID_Attribute,sep=.R_param_splitor) cmd <- paste(opt,"-f",Feature_Type,sep=.R_param_splitor) n <- length(unlist(strsplit(cmd,.R_param_splitor))) C_args <- .C("R_txUnique_wrapper",as.integer(n),as.character(cmd),PACKAGE="Rsubread") if(file.exists(fout)){ z <- read.delim(fout) z <- z[ order(z[,'Gene_ID'], z[,'Transcript_ID']) ,] file.remove(fout) z }else{ print("No output was generated.") NA } } Rsubread/build/0000755000175200017520000000000014142061021014431 5ustar00biocbuildbiocbuildRsubread/build/vignette.rds0000644000175200017520000000032714142061021016772 0ustar00biocbuildbiocbuilduQK0m?M4\p ƍ!Ƹ(M!<8*RcNޛIBAqtCHs Fa_"Q[ lw*rΔbłfEFz,b8Xc1S}ZE(xĝYٽ3%3^C %Nm_?'3n#3ZRZ>D(׾_[1{Rsubread/configure0000755000175200017520000000133714142061021015245 0ustar00biocbuildbiocbuild#!/bin/sh #cp src/Makevars.in src/Makevars UNAME=`uname` IS_CYG=`uname |grep -i cygwin |wc -l` echo @@@@@ The operating system is $UNAME. version_no=`cat DESCRIPTION |grep Version|cut -f2 -d' '` echo SUBREAD_VER = \"\\\"Rsubread $version_no\\\"\" > src/Makevars if test $IS_CYG = 1 then cat src/Makevars.Linux >> src/Makevars elif test $UNAME = "Linux" then # This is a Linux machine! cat src/Makevars.Linux >> src/Makevars elif test $UNAME = "FreeBSD" then cat src/Makevars.FreeBSD >> src/Makevars elif test 1 = ` echo $UNAME |grep "MSYS_NT" |wc -l ` then cat src/Makevars.Windows >> src/Makevars elif test $UNAME = "SunOS" then cat src/Makevars.SunOS >> src/Makevars else cat src/Makevars.MacOS >> src/Makevars fi Rsubread/configure.win0000644000175200017520000000032114136047615016046 0ustar00biocbuildbiocbuildecho @@@@@ The operating system is Windows. version_no=`cat DESCRIPTION |grep Version|cut -f2 -d' '` echo SUBREAD_VER = \"\\\"Rsubread $version_no\\\"\" > src/Makevars cat src/Makevars.Windows >> src/Makevars Rsubread/inst/0000755000175200017520000000000014136047615014327 5ustar00biocbuildbiocbuildRsubread/inst/CITATION0000644000175200017520000000106214136047615015463 0ustar00biocbuildbiocbuildcitEntry( entry = "Article", title = "The {R} package {Rsubread} is easier, faster, cheaper and better for alignment and quantification of {RNA} sequencing reads", author = "Yang Liao and Gordon K. Smyth and Wei Shi", journal = "Nucleic Acids Research", year = 2019, volume = 47, issue = 8, pages = "e47", doi = "10.1093/nar/gkz114", textVersion = "Liao Y, Smyth GK and Shi W (2019). The R package Rsubread is easier, faster, cheaper and better for alignment and quantification of RNA sequencing reads. Nucleic Acids Research 47(8), e47." ) Rsubread/inst/NEWS.Rd0000644000175200017520000000715414136047615015401 0ustar00biocbuildbiocbuild\name{Rsubreadnews} \title{Rsubread News} \encoding{UTF-8} \section{Version 2.6.0}{\itemize{ \item Improved the speed of cellCounts and also reduced its memory use. \item Added a parameter 'umi.cutoff' to cellCounts to call all the cells that had a total UMI count greater than the specified threshold. \item Added support for FASTQ-format read input in CellCounts. }} \section{Version 2.4.0}{\itemize{ \item The 'isPairedEnd' parameter in featureCounts() is now used to check if the type of input reads (paired-end or single-end) is correctly specified. \item A new parameter 'countReadPairs' was added to featureCounts to specify if read pairs should be counted for paired-end read data. \item Changes to the input parameters and output of cellCounts() function. CellCounts will generate a Sample Sheet for samples included in the scRNA-seq data, based on the sample index set name provided by the user. Structure of the List object returned by cellCounts() is also simplified. cellCounts() now also outputs a BAM file and a gzipped FASTQ file including raw reads for each sample. }} \section{Version 2.2.0}{\itemize{ \item Improve cellCounts() on the identification of cell barcodes arising from ambient RNAs. }} \section{Version 2.0.0}{\itemize{ \item Rsubread package is ported to Windows OS. \item New function cellCounts(): generate UMI counts for Chromium 10X single-cell RNA-seq data. \item flattenGTF() function can merge or chop overlap features. \item Check and display the amount of memory available on the computer before starting read mapping. \item Optimize the data structure used in buildindex() function to reduce its memory use. \item qualityScores() function can optionally retrieve quality scores from all the reads. \item File paths included in column names of objects returned by featureCounts(), align(), subjunc() and propmapped() functions are removed or shortened where appropriate. \item featureCounts() will be terminated if both single-end and paired-end reads are found in the same input file. \item Limit on the length of input file names is increased to 1000 bytes for all functions. }} \section{Version 1.34.0}{\itemize{ \item New functions: simReads() and scanFasta(). simReads() generates simulation RNA-seq reads for transcripts. \item align() and subjunc() estimate fragment length from mapped read pairs and use the estimated length to assist reporting the best alignment. \item align() and subjunc() prefer alignments with no indels included over indel-containing alignments when same number of matched bases are found. \item align() and subjunc() check if index files were successfully loaded before starting read mapping. \item align() and subjunc() detect indels arising from incorrect shifting of seed sequence when being mapped to low-complexity region and exclude such indels from read re-alignment and indel reporting. \item buildindex(), align() and subjunc() support gzipped FASTA format. \item featureCounts() allows mapped reads to have ‘*’ as their chromosome name. \item removeDupReads() supports BAM-format input and output. }} \section{Version 1.32.0}{\itemize{ \item New function flattenGTF() that merges overlapping features into a single interval. \item New parameter for align() and subjunc(): sortReadsByCoordinates. \item New parameters for featureCounts(): readShiftType, readShiftSize and additionalAttributes. \item Specify strand protocol for each library individually in featureCounts(). \item Much improved speed of align() and subjunc(). \item align() and subjunc() return mapping statistics. \item Default setting of buildindex() is changed to building a one-block full index. }} Rsubread/inst/annot/0000755000175200017520000000000014136047615015446 5ustar00biocbuildbiocbuildRsubread/inst/annot/hg19_RefSeq_exon.txt0000644000175200017520003326615014136047615021275 0ustar00biocbuildbiocbuildGeneID Chr Start End Strand 653635 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100040400 NT_166424.1 301131 301814 + Rsubread/inst/doc/0000755000175200017520000000000014142061021015054 5ustar00biocbuildbiocbuildRsubread/inst/doc/Rsubread.R0000644000175200017520000000410414142061021016745 0ustar00biocbuildbiocbuild### R code from vignette source 'Rsubread.Rnw' ################################################### ### code chunk number 1: Rsubread.Rnw:70-73 ################################################### library(Rsubread) ref <- system.file("extdata","reference.fa",package="Rsubread") buildindex(basename="reference_index",reference=ref) ################################################### ### code chunk number 2: Rsubread.Rnw:90-92 ################################################### reads <- system.file("extdata","reads.txt.gz",package="Rsubread") align.stat <- align(index="reference_index",readfile1=reads,output_file="alignResults.BAM",phredOffset=64) ################################################### ### code chunk number 3: Rsubread.Rnw:99-103 ################################################### reads1 <- system.file("extdata","reads1.txt.gz",package="Rsubread") reads2 <- system.file("extdata","reads2.txt.gz",package="Rsubread") align.stat2 <- align(index="reference_index",readfile1=reads1,readfile2=reads2, output_file="alignResultsPE.BAM",phredOffset=64) ################################################### ### code chunk number 4: Rsubread.Rnw:125-135 ################################################### ann <- data.frame( GeneID=c("gene1","gene1","gene2","gene2"), Chr="chr_dummy", Start=c(100,1000,3000,5000), End=c(500,1800,4000,5500), Strand=c("+","+","-","-"), stringsAsFactors=FALSE) ann fc_SE <- featureCounts("alignResults.BAM",annot.ext=ann) fc_SE ################################################### ### code chunk number 5: Rsubread.Rnw:142-144 ################################################### fc_PE <- featureCounts("alignResultsPE.BAM",annot.ext=ann,isPairedEnd=TRUE) fc_PE ################################################### ### code chunk number 6: Rsubread.Rnw:164-166 ################################################### x <- qualityScores(filename=reads,offset=64,nreads=1000) x[1:10,1:10] ################################################### ### code chunk number 7: Rsubread.Rnw:191-192 ################################################### propmapped("alignResults.BAM") Rsubread/inst/doc/Rsubread.Rnw0000644000175200017520000002277714136047615017352 0ustar00biocbuildbiocbuild%\VignetteIndexEntry{Rsubread Vignette} %\VignetteDepends{} %\VignetteKeywords{Read mapping} %\VignettePackage{Rsubread} \documentclass[12pt]{article} \usepackage{hyperref} \newcommand{\R}[1]{{\texttt{#1}}} \newcommand{\C}[1]{{\texttt{#1}}} \textwidth=6.2in \textheight=8.5in \oddsidemargin=0.2in \evensidemargin=0.2in \headheight=0in \headsep=0in \begin{document} \title{Rsubread package: high-performance read alignment, quantification and mutation discovery} \author{Wei Shi} \date{2 January 2019} \maketitle \section{Introduction} This vignette provides a brief description to the Rsubread package. For more details, please refer to the Users Guide which can brought up in your R session via the following commands: \\ \noindent\R{> library(Rsubread)}\\ \R{> RsubreadUsersGuide()}\\ The Rsubread package provides facilities for processing short and long reads generated from the sequencing technologies. These facilities include quality assessment, read alignment, read summarization, exon-exon junction detection, absolute expression calling and SNP discovery. The Subread aligner (\R{align} function) is a highly efficient and accurate aligner for mapping genomic DNA and RNA sequencing reads. It adopts a novel mapping paradigm called ``seed-and-vote". Under this paradigm, a number of 16mers (called seeds or subreads) are extracted from each read and they were mapped to the reference genome to vote for the mapping location of the read. Read mapping performed under this paradigm has been found to be more efficient and accurate than that carried out under the conventional ``seed-and-extend" paradigm (Liao et al. 2013). Another aligner included in Rsubread is the Subjunc aligner (\R{subjunc} function). It was also developed under the ``seed-and-vote" paradigm, but different from the Subread aligner it performs full alignment for exon spanning reads and reports exon-exon junctions in addition to the read mapping results. The Subread aligner is recommended for gene-level expression analysis. For other types of RNA-seq analyses such as alternative splicing analysis, the Subjunc aligner should be used. An important step in processing next-gen sequencing data is to assign mapped reads to genomic features such as genes, exons and genomic windows. This package includes a general-purpose read summarization function \R{featureCounts} that takes mapped reads as input and assigns them to genomic features. In-built annotations are provided for users convenience. Different from microarray technologies, the next-gen sequencing technologies do not provide Present/Absent calls for genomic features such as genes. We have developed an algorithm to use the background noise measured from the RNA-seq data to call absolutely expressed genes. The function \R{detectionCall} returns a detection p value for each gene from the read mapping results. We have also developed a new SNP calling algorithm which is being implemented in function \R{exactSNPs}. Our results showed that it compared favorably to competing methods, but was an order of magnitude faster. This package also includes some other useful functions such as quality assessment (\R{qualityScores}, \R{atgcContent}), duplicate read removal (\R{removeDupReads}) and mapping percentage calculation (\R{propmapped}). \section{Read alignment} An index needs to be built first and then alignments can be carried out. Building the index is an one-off operation. The generated index can be re-used in subsequent read alignments.\\ {\noindent\bf Step 1: Index building \\} The Rsubread package includes a dummy reference sequence that was generated by concatenating 900 100bp reads that were taken from a pilot dataset generated from the SEquencing Quality Control (SEQC) project. We further extracted 100 reads from the same dataset and combine them with the 900 reads to make a read dataset for mapping. Below is the command for building an index for the reference sequence: \begin{scriptsize} <<>>= library(Rsubread) ref <- system.file("extdata","reference.fa",package="Rsubread") buildindex(basename="reference_index",reference=ref) @ \end{scriptsize} The generated index files were saved to the current working directory. Rsubread creates a hash table for indexing the reference genome. Keys in the hash table are the 16bp sequences and hash values are their corresponding chromosomal locations. Color space index can be built by setting the \R{colorsapce} argument to \R{TRUE}.\\ A unique feature of Rsubread is that it allows users to control the computer memory usage in read mapping process. Users can do this by tuning the amount of memory (in MB) to be used in read mapping.\\ {\noindent\bf Step 2: read mapping\\} After the index was successfully built, we map the read dataset (including 1,000 reads) to the reference sequence: \begin{scriptsize} <<>>= reads <- system.file("extdata","reads.txt.gz",package="Rsubread") align.stat <- align(index="reference_index",readfile1=reads,output_file="alignResults.BAM",phredOffset=64) @ \end{scriptsize} Map paired-end reads: \begin{scriptsize} <<>>= reads1 <- system.file("extdata","reads1.txt.gz",package="Rsubread") reads2 <- system.file("extdata","reads2.txt.gz",package="Rsubread") align.stat2 <- align(index="reference_index",readfile1=reads1,readfile2=reads2, output_file="alignResultsPE.BAM",phredOffset=64) @ \end{scriptsize} \section{Counting mapped reads for genomic features} The \R{featureCounts} function is a general-purpose read summarization function that assigns mapped reads (RNA-seq or gDNA-seq reads) to genomic features such as genes, exons, promoters, gene bodies and genomic windows. This function takes as input a set of files that contain read mapping results and an annotation file that includes genomic features. It automatically detects the format of input read files (supported formats include SAM and BAM). Input reads can be name-sorted or location-sorted. Users do not need to resort the reads before feeding them to \R{featureCounts}. In-built NCBI RefSeq gene annotations for genomes mm9, mm10, hg19 and hg38 are provided for convenience. These annotations include chromosomal coordinates of exons of each gene. When these annotations are used for summarization, only reads overlapping with exons will be counted by \R{featureCounts}. Users can use {\texttt{getInBuiltAnnotation}} function to retrieve these annotations. Below gives the example code of assigning reads and fragments, generated in the last section, to two artificial genes. Assign single end reads to genes: \begin{scriptsize} <<>>= ann <- data.frame( GeneID=c("gene1","gene1","gene2","gene2"), Chr="chr_dummy", Start=c(100,1000,3000,5000), End=c(500,1800,4000,5500), Strand=c("+","+","-","-"), stringsAsFactors=FALSE) ann fc_SE <- featureCounts("alignResults.BAM",annot.ext=ann) fc_SE @ \end{scriptsize} Assign fragments (read pairs) to the two genes: \begin{scriptsize} <<>>= fc_PE <- featureCounts("alignResultsPE.BAM",annot.ext=ann,isPairedEnd=TRUE) fc_PE @ \end{scriptsize} \section{Finding exon junctions} The RNA-seq technology provides a unique opportunity to identify the alternative splicing events that occur during the gene transcription process. The \R{subjunc} function can be used to detect exon-exon junctions. It first extracts a number of subreads (16mers) from each read, maps them to the reference genome and identifies the two best mapping locations for each read (representing potential locations of exons spanned by the read). Then, it builds a junction table including all putative junctions. Finally, it carries out a verification step to remove false positives in junction detection by realigning all the reads. The donor (`GT') and receptor sites(`AG'), are required to be present when calling exon-exon junctions. Output of this function includes the discovered exon-exon junctions and also read mapping results. \section{Base quality scores} Quality scores give the probabilities of read bases being incorrectly called, which is useful for examining the quality of sequencing data. The \R{qualityScores} function can be used to quickly retrieve and display the quality score data extracted from a read file. \begin{scriptsize} <<>>= x <- qualityScores(filename=reads,offset=64,nreads=1000) x[1:10,1:10] @ \end{scriptsize} \section{GC content} The \R{atgcContent} function returns fractions of A, T, G and C bases at each base location of reads or in the entire dataset. %\begin{scriptsize} %<<>>= %## Fraction of A,T,G and C bases in all the reads %x <- atgcContent(filename=reads) %x %## Fraction of A,T,G and C bases at each read location %xb <- atgcContent(filename=reads,basewise=TRUE) %xb[,1:5] %@ %\end{scriptsize} \section{Mapping percentage} Function \R{propmapped} returns the proportion of mapped reads included in a SAM/BAM file. For paired end reads, it can return the proportion of mapped fragments (ie. read pairs). \begin{scriptsize} <<>>= propmapped("alignResults.BAM") @ \end{scriptsize} \section{Citation} Yang Liao, Gordon K Smyth and Wei Shi (2013). The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Research, 41(10):e108.\\ \noindent{Yang Liao, Gordon K Smyth and Wei Shi (2014). featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics, 30(7):923-30} \section{Authors} Wei Shi and Yang Liao \\ Bioinformatics Division \\ The Walter and Eliza Hall Institute of Medical Research \\ 1G Royal Parade, Parkville, Victoria 3052 \\ Australia \\ \section{Contact} Please post to the Bioconductor Support site (\url{https://support.bioconductor.org/}) if you have any questions or suggestions. \end{document} Rsubread/inst/doc/Rsubread.pdf0000644000175200017520000032712214142061021017325 0ustar00biocbuildbiocbuild%PDF-1.5 % 45 0 obj << /Length 2231 /Filter /FlateDecode >> stream xڍYKPDVC]v\l`zEiw'>@Ɂh4u ̫ʖis[׋yaLMr *rkקN>Mp4+Eʇ O?0 nI;E@r"o5 1!.mi?xoOmE 7=4MpG)IW\"wEezƁj2/%m$[[%%2e -E ˄? 3d iYX:,PU\J7(25ifΏ܎>^+"r[>q4-$Ӏ]rnR3y:z"IPPY}݄PFC7qYet/۝cEP9.XEQ^d"{'VᄣZH~G?\b/> B1@7̳街'oN O^Ȫe"WP{`qGl>f5XE@Mo%,㒽pň~͢]r, l[UȁKTKݯ1"|4.RO Xv_( 9m6 jîu2tL41q4 .u] )ZT&p#U?jjUE՝q{Xse꼮ꩉ/D~E5&#1qMp bʱρJlKmʨiaK;2/ZZlnoT\aY<9@"XLQ/%`/QSMEL.R^@JZ}ͯ^ܪ|Gi DV_9RNm~UN\kkBE~Tla}C !K` eQث {TE⢵N7\3 +$/3|kLIV» "Ln8tWm;R׷Caխ|g-ʲEDx}P.(;CJ|Xe9.q-WHy^({ծBF]kEUN3{V/J=bS;8yiZZy8ХWSAƣ5% /( bd!+;9xHG"o$hw  hpgm*]EHɍpH{gV$@G.G1H|X]TNn?L-t$!rG> jg|藝CY]TN"PfXK~w@ar䶺Ⰼ/bd.m.pT{eiiTr9~V/d|P IkSv_Q[/1v9÷z!r}JKge>yԢ' L̮&NpKWR_|%u2:ƺ2(wp2> stream xڝn=_a gנ)g7CCSDj,K+\Ct}43~ͫ^yn/nUdEmV7Eͥ8Kh G.gQk+#t'ç1 <ܴ41>xvgߜg]re,;c#-b 4[Yj3O2_2"GV/_.ҋdI(k<DER":fS&y^ (RgKN9#Ya4&$ՑpC% v((kkuuCopXR bP)'μH&~!j3&Pefh<C/GlTguۈ><)&4MiEeFzNd>g6r'a2+% JQ))[=AEW;K̼ӝ4kSFmY,q!S?*À!q5yO>*"pS8."L`i* <~GfȝS7%,CY^Qdp09A{+e4\/^|͟A@͵*Ε{VDÑHBs!#γ?vOxc-*!Bk(nPȜ#V $?Đ0ջS9ԏKrA뚾7ciA%wqTmU]!meϜI]fМ# f/ɔ\_ruct ("f _l~^%Fw3LK~p-E%t)mh H22 `Ӱ̨V$+^//Ұ2hBнڞdO*-=aI0Tx2[_*aB-@-/9*RSOCg{! oGp*,\sIC{g7`#98w)[c5홴~&x/{ ʲBO<3g̛J˞Ce!Nޖab"g }gؙ*W5T ch|U- Xe! fl42#XEv+V75<,ϋHܫRZ=9ؐgic13C_A'=aıjTb@AV{ 1%ف)jdb0G}O UR["/pS3L8RqOc:Fg6siEVc]{q9ܹ_@6Zv76|QCkΘ˺F$[xCI^ qao'+ #|_ 2 |g_"nWxkĖ%0&en% _t꿲 endstream endobj 61 0 obj << /Length 1533 /Filter /FlateDecode >> stream xYK6W@4U`/ -CꞒ-b[[K̐n,]z, 8Go&b=JXp1.F6fz nG|)}eH)x*R0v~ތEoWFhu5~?T9R,Fo/R9F,|}0Bk5/ocO=c3uPEu͉A~r=tUF_yb/]{޿ė{޻h~K(uNJs8vxk:8gO$;-XWTxP NTGM͡-z9};y2MyyRy'Sk/ؾd`ۇ\C`2%6'[M!lݶG[,˦M}Y@t_U $m}uc uW-k[[ԙϱS/a?z_mVɶ;H#_/fC&L 92}!h`#XN|5 ȟoeV#X.AvmG?Us ʏ| ,il*ì9ɦŞV84kdfZDŽx/VgX$Qc"yG RL/pF;O}Iv_]p1Rg,CvhXE< ߎ2o>[!Zq5AS@ E!Va.?_ۚBɚS3$Ԍѻ?N}s(4~Sev- ^_Tû endstream endobj 65 0 obj << /Length 1972 /Filter /FlateDecode >> stream xڭYKo8W{HIIm.vi${j CX[[v#9M<(Q+Mr$rș77WKPf=+Dx{WguߕS_ߡQ,Ku?1Y(Zo4E 92fS2Ժ#~(5!{y:[z |>lO׺؋I\3uW<U['쯉u'3lgơP Ch G_ϗЖN 0NAL<Ӝ1w bN4)V.&qj[S Oo"B˼ZK ׿c 7:3p,3)`R}guNX d S" 軡nTK%.]hðhLk~Oi~o+@+k 5ws`y W㎬LTr&^-xL*Ι{ڔj-vԂŌߦ]UF{xXKZH]X l޻{cJ+~+˼_yǤsK7#˝܌H˄0j{;3O|ACg+ꄮ_R&i ؔ/7]cu5p0v,f<@#_ lZChB=Q5#QX84:;XݚKgE%;JnIuwVq݆!kҰNq{ƍ-CgYf潡=N(ا˭Ua9@eF$S%޵=־Ru](f?CP1S߹2M87apH+|"&쯫̔va(~;P%s5{#V[1e6| <䰪rf)y8up2ϸf7EϜpA- yk cdQ ӡ&N3z"Jq8ӻN8J]1 @A|։s] [_*7Ea# V $}h|?>U:Uܡ ) h*#Kڵ=+р̷^o30t"B(d7%@ċctmջW`l endstream endobj 69 0 obj << /Length 1767 /Filter /FlateDecode >> stream xYYoF~ϯRАڃH H%>% KF2@?;Ԓ Z\{Ρg7߉$b3u;a=pl]7a"t˲˝;k-tDžce]Wp3g>O?:&V9c jjN3,` |>Z@v*L-IU;jLk#Qx%t32sUUF@2<,%tJ<$juMf,RIBP!rŝz *NyQ۩e's~ l Hk 3//,)F>rã}wx~WO=F^3 Pz u])Lg p q9-3)ou/4PX[1^ZR@zzi`ڞzf׀yNdJLJ.&_Z4<41|֯hD=i5i\[#0L_)4&34Q ]J(ϩUɌ~wN MSNqwJϡ!Hc~,iGJE>jac`iCR%kRϠa=w {?#)B v҈` G-7Xa8#tZt#8#]1-~ 3?8m(V׵fWl5-QK>[w>D:gQ-6ǁ NAdE6zDUJKMG+=GҷR}6͇9j =4{v}hq M}_#Jo@cj5|ei@6u,1`'nfhNj{5Ӛ f0oDB (GF=- )j_muc2]^uKP᱗D/jv^l~ VW,C ˜NׄS!MҼ$sS;?A/&} bp+J zyjM}P F7EaRbUYP`6w8G z 3C.XC~}j9G?zu`ktS!cS醡O0tXׅH{ΰZ* O0C 16IPQ:cr8WlAMY#!N~ҫ3,}Y'bsn/0I\Dt:%@qEPBlO3TOz8ӹ-lYmгu!x$D yX#`;"+;K w5;Jx—w5K 1PFpG\}`,oXzB㇆јSjqe"[ x</"έ+0 h 1/a8|P釄6|#䣩906&)os_;zp95џ`0 dE4$ݓN?9{VHHx|/ W¤,0]W.?NNh-Bw[ åc1:r]qiZwWwk*LCBH,sc@?a 'GoG4++uԵ Ҷ , ށ_ߐRħn4_ ##Xx^XwLl .FĘ(M"F:4B0OD&mqL_0?A > stream xڵYYoF~ϯ Q.ES v"E>4EASDȏ\K.*NԞs7Cb$l|9I?$UNɟއg8 _Ly (YXMy!\oKo|xTGY ſ?O4#c3_l ^oaRoxoXb/ Cf: YgAW/ wHp8CVV"*P#*Vp1 ?2tWvy(0.Q6Yyg>#o?G0̢' )zvgG%2?hT/H!}ɫe\I"c:7'r2qJxYQzV*AmPT.4'c6{, \:O{>¦qfyU.%P /)zOGOIJR[=uED|[^P:8BB#zaEcc#og e %S (lq':8Ok->"4\/vpݦ=:> Y_p0N+nRI9˯im̑1\EƲݐ #pl$78N(܉8JrZo'M计.WZa@YW VN!1 U+Yq˙=at@`$@z."oERD(A,Kᴳkr,~ Ɉ!$ =xi2UY9HZ[i) Y͜5&NY?l{jo Z&рsO>x:D endstream endobj 77 0 obj << /Length 1587 /Filter /FlateDecode >> stream xXYo6~ϯ0P+)dbXd][-'l9$객M]p曃3C]N׾, ̖gď<'며*u%*L]su}W.Nw=挌y1N߳'|bG+5k1ፀ{cިE @?=?|Diă`pN/ 5ި %UUḰDEkFb`^@![ gutj;Ga{qUT]@.=plboXB:P;S9.xUEy@zʘCU3` $#Ns 3.qwL.7#=o#x׻K"ùH)[CRdU&e+ "%-]K.(o Y-l&]z8,9`Y}ǩ-}ϳb0M\I-k7s葘hɏ4$  КbNOjpܝ)? 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f~^RuS7lV~?{!y$.哵`)Im6@I}$X2{[qsxo..@< e Y k6j IWALRE1a݀4Dlj24PO/=ɷIH2*)˂yqS=G(X 1/|_] 4BU{gGֽi3'%8`,jpRs)uml}7z|wXiHU$ii۔4wQVWv !MԤ),IA>nQ!_A}sv2a+G>ljj~X L11zṆdѸB=:|s7@]J ӷ3(GzU۷OMe02,k/RmW/M4Bð k$>VLf vQG6xʹmn ˲0@ZGW;Y]u3=KIEBv?D5S$-{~,&פ ,n 5>LQ2֌@[-0%ut 3*VijV2k2j hܒpo.N+8Vc e0o5 vu+8(v#aLBoux-ڃG R'g-A6d?Sisci-ms$- P7-DAd *PwrRj"8F]8X+{E&P+3Q E\ɮJY=N%Di44}v= oKbbQZ@fhtYId /@cG; +T]vGbW @yipwQeb|Bh:uY!.\sl`'2ϊ=yҗ4 Q&Mwo7wiwZWlw9Ochr_dummy GGGACGGGGGGGGGGGGGGGGGGGGGCCCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGAGGGGGGGGGGGGGGGGGGGCGCGGGGGGGGCGGT GGTTGAGAACCCTTCCTGTATGTTTTGAATGTGAGGTACTGGGGAAAAAGGGGGGGGTGGGGGGGGGGGGTGTTTTTTGGGTGTGGGGGCGGGTGTGTGT CTTCCTTCGTGTGCTTGAAAATTGGAGCCCGGCCCCCCGGGCCCACAAGACCCCTGTGGGGGGTGGGGGGGGGGGTGGGGGGGGGGCGCAGGGTGTGCTG TTTTTACGTATGCATATATATGATTATTCTTTTGGTAATTCGGGTTTTTTTTAAAAATATAAAAAAAAAAAAAAATAGAGAAATATATTTGTAAAAAATA AAAAATTGCCATTGGATTACACTAATGTGGTCATCTGTTTTTTTTAAAGTTTGGTATTAGGGGGGGGGGGGGGGGGGTGGCGGGGTGTGGTGTTAGCTGT GAACCATGTGCTAATTTTTCACAATTATTGACCTGTATTGATTGCCACTGTAGTTTGGTATTTTCCTTTACTTTTGTGGGCTGGTTTCCTGCTTCCCTGG CGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGCGGGGGCGGGCGGGCGCGGGTGGGCTGTGCGGCCGGCAGGGCCTCTGTG GGCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGTGGGGCGGAGGGGGGTGTGTGCGTGTGCTGGCTGGGGGTG TTTGCACGGGCTGTTCCTGTTTTCGGGGGGATTTTTTTTTTTCTTCTCCCCCCCCCCCCTGTATACACATATCTTTCTTTCTCTTCTCTCTCTTCTCACG TTGGACTTTCTTTTCGCCGTGGCTGCGGGACCCCCTTGGGTTTTTTTTGGTACTGCCTTGGGGGGGGCACACGTGGTGGGCGCGGGGGGGGGTGGCGGTG CAGCAAACCCTCGCCGAAAGGAGGGACTCACGGAGGGGGGGCGGCGGTCGTTGGGGGGGGGGGGGCGTCTCGGGCGGCGCGTGTGTGAGCACTGACTCCA TGTCACTTGTGCTTTGCTCTTGTCCCACGGGTCTTTCACTTTTGATATGAGCCGGGGTGGACGTGGGTTGGGTGGGGGGGGGTGGGGGGTGTGGGGGGGG AGCATCTGAATACAGTTTTCAGGACCTTCCAACGAGGATTCCTGGGGAGTTTTTTTTTGGGGGGTGTTTTTGTCTTTCTTTCTCCCCTTGTGGTGGTGTG CGGCGGGGGGGGGGGGGGGGGTTTGTGGGGAAAAAGTGGGGGGGGGTCTTGTTGGGGGCGGGGGGGGGAGTAGGACGCCTTTTTAAAAAGGCAGAGGTTC CATGCACTGGGTTTTTAAACTGAAATACAAAGAAAGCCATTTTGAAATTGGGTTTAGGGAATTTTTTCAGATCGGGGGGGGGGGGGTGGTGCGTGTGGGT GATTTTGCGTAGCTGGGTTTGGTTTAATCCACCTCAAATTGCTGGTTTGTTTGGGAAGGATTTGGGGGGGGGGGGGGAGGGGGGTGGTTTGTTGTGGGGG GTTGTGGTGTGCTGGAAAAGTCCCATAGCCTTGGCCACTGGATCGGGGGGGAGGGTGGGCGGGGGGGGGGGGGGTGGGGGGGGGGGTGGGGGGGGGGGGG GAGACAGTTATCACATCTGTGGGAGACGAAGAAGGGGGAGGGGGGGACGAGTGGGTGATCGTCGGGGGGGGTGTGGGGGGGGGCTGGTGGTGGTCGGGGT CTTTTGGGTGCAGTGTGGGCTGGCCGCACGCCCCCCCCTCAACCCGGGGGGGGGGGGGCGCGCCGGGGGGGGGGGGTGCGGTGGGGGGGGTGGGGTGGTG CTTATCAAATTTATTATTCCAATGGCACTAGGTAAAGGTGGGGGGGGGGTGGGGGGGAACACAGGGGGGGGAAGGGGGGTGTGGGGGTGTGGGGGAGGTG TTGAACTGCTCCTGTTTTTGAGCTGCGATGCGCTTCTGGTCCCTCTTCCCGCCGGCGGGGGGGGGGGGGGGGGGGGGTGTGGTGGGTGTGGTCGTGTGGT CAACATTTTAGCAGTTCTTAAGCATCTAGCTACATCTGGTATGTTGGGGGGTGGTTTTTAAGATTGGCTAGGGGGACACGGGTGTGTTTTTGTTGGTGGG CTTCACTTGGCTTATTCCCTGAGCCTGTAGGTCTCAATTAAAAAACAAAACAAAACAAAAATCCACTTTTTCTGCAAGGGCTTTCCTAACTAATACAATA CCCAAAGCGTGGTATTTTGCCACCACATCAGGACAAGCCTGTCCTTTTGCACGCTCCCCGGGGGGTGTTGGGTGCGCGCGTTCTCACTCCGCTTCTCTTT CGCCTGCACCGCAGCTCCTGGCGCCGGGGTCGCCTCTTACTGGGGGGGGGGGGGTGGAGAGGGCGGGGGGGGGGGTGGGGGGGGGTGTGGCAGTGAAGCG GAAAGAAGGAAAAAAATGCGGGGGGGGGGGAGGGGAAGAAGGGGGGGGGGGCGCGGGCTGTCTCAGGACGCTGGTGGGGGGGGGGAGGGGCAGAGGCGCG CTGGTACTGGTTCTTTGCTGCCCTGTAATCAATATGGCATCTCACCCCCGACAAACCAACCAGCGCCAGAGGGGAGGGGGGGTGTCCGGGGGAGGGGCGT CTTTGTCTTGTGGTTTTGGTACTACCTTTGGCCACGCGGGGAAAAGGAGGGGGGGGGGGGGTGTGTGGTTTTTTGTTGCATACACGGGGTGCGTCTCGTG GGAACACTTGCCTTTGAGCGGGTCTTCAATGGCAAACAAAAAAAGGGTGGGGTGGGGCCGCGACTCTCTCTCGTCGTTGTGTTGTGGGTGGGGGGGTGTT GGGAAATTCTGCAGGCCTTGTAGTTTTTTTTAAGCCCCCCTCCGCAACCGGGGGGATGGGGTTGTTTTTTTTGTGTGGGGCTTGTGTGGGGGTGTGGGTG CGCGCCTGTAATCCCAGTTACTTAGGAGGGGGGGGGGGGGGGGGAGAACAAAAAACATCGGGGGGGGGGGGGGGGGGGGGGGGGTGGTTGTCTGTCAAGG TGAGCTGCAGGGCTTCTGAGTCAGACTGGCTGGGGGGGGGTCCTCAGCGGGCCCCCGTGGCGGCGCGTCTTTTTGTGTGTTGTGGTTATATTCACAAAAG CCACAGCCAAGGGAACGGGCTTGGCGGGATCAGCGGGGGGGGGAGAGACTGTGTGGGGTGGCTCTTGTTGGTGGGGGGGGGGGGGTGAGGTGGGGGGGTG ATCAAATGTGAGGGACAGGACGGGGCGGGGCGGGGGGGGGGGACCCCCGGGGGAGGGGGGGGAAAGGGGGGAAAGGGGTGGCCGTCGCGCTGTGTGGGGG CAGCGTTTGGTTTAATTGGAGGGACTTTATCTTCCGGGCGGCCGCGGTTCTTCTTTTCTCTCTCGGGGGTGCCAGCGCCCAACAGAAAGCGCACCACAGC GGGGGAAGGTGAGGGAGAGCTTCTGTACAAGGTCATCTTTCGTCTGGGGGGGGGGCGGCCGGCCGCGCCACGCACGAGCGGGGCGGGTGCGGGGGGGGGG TGGGGGTTGGGTGGAGAAGGAAACCTACTACTGACCTCAGCCCCACCACATCGCCCATAGCTTGCCCCTCTCCGCCGCACCTGGACCGGCACCACAGGGA TGTCACCTCTCCTCTCATCTGGGGTCAGGCAGGGGTTGGGGGGGGGGGGAGGGCAGAGGGGGGGGGGGGGGTGTCTGTGGGTGTTGTGTGGCGCCAGTAG GCCGCCTCTTGGGAGAAAAAGTTCGCGCTCTTTACAAACCCACTCGTCAGGGGGGTGTCCCTCCCCGCCCCCCCCTTTGTATGAACAATTTTTTTTGTCT CAGCTATCTAATGCAGGATTCCATCAAAAAAGTCATCAGGGGGAAATCCCCGTTTTTCCACTAGCCCCCGCCTTCATCTGCGCTTGCCCCAGTGTGGTCC CGGGGGTGAGGAAGGGGAGGGCCAGGGGCGCCCCGGGGAAAAAAAAAAAAAGTTCCTTTTTTTTGGGTGGGGTGTGGGGTGGGGGGGGGGGGGGGGGGTG CTTGGGAGCATTAAAACCCAGACACCAAGACACACCCAAAGAAGACCATCCCACTCTGTGGTGTGTTGTTCTGCACATCATTATTTTCTTGTTAATGTGT GCCCCACCTACCTCCTCATTGGCACCCAAGCATGAAAAGGGAAAGGTGGGGGGTTCCACCCCACCACCCGCGTGGTGGGGGGGGGGGGGTGGCGGGGGCG GCTTCCTGGCCGGGGAAACAAGACGGGCCCCGCGGGGGGGGGGGCTGCACTCGCTTCGGCGTCGCGTAGGGTGTGAGCCCACGACCCACATGCGGAGTGT TGTCCCTGTTGTAAGGAAGCCAGGTCTTCTCTCTTCATTCCTCTTTGGAGAGGGGCAAACTCAGGGGAGGGGAGGGGGGGGGTAGCGGTGTGGGGGGGGA CGGAAGAGCCTTGGGAGCACGGGGGGGGGGGGGGGGGGAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGAGAGGGGGGGAGGGTGGGGACAGAGAGAG AACAAACCCGTTTTATGATTTACTCATTATCGCCTTTTTTGAGGGTTGTGGTTATCTAACAGAAGAGGTGTACATTTGTTTATTCTATACTCTAATAATA CTGGTCTGCACAGGTCTCCATGTTGCTCTTTTTCACTGGGATAGACCTCCCTGCACCAAGCATGGTATGTAAATGAGGAGAGCGTGTGTTTTTTTTTGAG GTAGAGGCTTACTAGAAGTGTGAAAACGGAGGGTTGGGTTTAGGCGGCAGGGGGTTTTTTGGGGGTGCTGTGGAGATTGAATGTGGTGAGGAGGTGGTGC TGTAGAGATGGGGTTTCACCATGTTGCCCAGGTTGGTTTCAAACTCCTGGGTTCAAACGATCTGCCCACCTCATGTTCCCAAAGTGCTGGGGTCACAGGC GGCATTTTTAATCTTAGAGCGAAAGCCTATAATCACTGCGCCCGCTTCTTAAGGGGGGGGGGGGGGGTGGGGGTTTGTTTGGAGGTGGGGGGGGGGTGTG GTTTTTGTGCAGCTGACTTTTCTGACCCCTGTTTACGTTGTTTTCATCTCATCTTCGTGAAAACAAAACAACAGAGACGAGCGGGGGGGGGCAGGTGGGA GCGAAGACCATGACCACATCCCACCCGGAGCAGGGGATCGAGGTGGGGGGGGAGGTTTGGTTTTTTGAGGAGCTGGGTGGGGCTGTGTTCGCGTAGGACG CAAGTTTGTCCTCAATAGTGTCCTCCATGATGTCCTTGATAATTGGGGGCCACCGCGGGGGGGGGGGTGGGTGTGTGCGGGCGTGGGTCTTCTTGTAGTG CTCAAACTGGAGGGTAACCCTGTCACCTGTGTCCGGGGCGGTGGGGGTGCAGGGGCTGAGAGTGGGGGCGGGGGTCGCGGGGAGGGACGCGGCTGCAGTC GTCCCAGGTATGCTTCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTCCTTTTGTTCAAACACACCCCCACAATCAGAGAGGGGGGGGAGACGGTGGCGGA GGTGCTTCTGGTGCGGGCTCAGGAGGAAGTGAGGATGCAGGTGTTGGAAGCTGGAGGGAAGGCTGTCCTTGTGTTGTTGGTGGCAGAGACTCTACTGGGA GGCTGGAGTGCAGTGACCACGAACTCGGCTCACTCGAAACCCCTCTCCTCCCCATCTTACAGAATTTCTTCCCCCCCCCTCTCCTGTCGCGGGGGTGTGG GCCAATTACTACCCCTTCCTTGTAAAATGAAGTAACCAAGCCCCAGGGGGGGGGGGGGGGCCGCCCGCACCGCGCGGCGCGTCGTGTGGGGGGGGGGTGG GGGATGTATGTCCCCCCCGGGGGGGGGGGGAAGAAGGGAAGGGGGGGGGGGAAAGAACATGGGCGGGGGGGGGGGGGGGGGGGGGGGGGAGCGGGGGGGA GTTGGCACAGGGCCTGTCCTGTGTTTTCACACAGGCCTGTGGCCCCTCGTGGGTAAAGGGGGGGGGGGGGGGGTTGGGGGGGGGGGGGAGTGGGCGCGGG GGACTCCTACCCTTCTGCTCTGAGTGCGCAAGTCTTCCTGGACTCTTCCGGGTGGGGGGTGTGGTTTTGGTAAGGTGTGTGTGTTATGGCGTGGTGCTGG TGTCTGTCCACCTCATCCCTCCGTGTATCTGAGGGGGTAAAGGTGAGGTCTTTATTGTTTCACTGCCTAATTTTCTCACACACATTCGCTGTAGCGATGG CTTGGCTGCCTGGATTTCCATAGGTAGAATAACTTTGCTGCTTTCCCCCCCCCACAGACCGCAGGACTTCACAACATTCAGTGTCAACACACAGAGACGG GTTGGAGTGAATGTGCCCATAACCCTGTGCTCCCATAAGTCTGTGTGCCCGTGGTCTTTTGTCTTTTGGGGGGGGTTTTTGTGGTGTGGTGTTTTGTCAT CACACTTCCTTAAAACCCAAATCAGCAAGGAATGAACAATTTTGTTTGGCATGGGTGAAATTAGCACTGTGTGGCTCGGGTGGGGTGGCCGCGGGGAGAG GGCCGGGCCACCCCTCCCACGGCGCGACCGCAGGTGCGGAGGGGGGGGGGGGGTGGTGAGGGGCGGGGAGAGCTGTATCGTGGCATGTCTTCTCGACGCG ACTGGATGAGTTCCTGTTCAGTGACCTGCAAGTACTGGAGGGGGTGCTGCTCTACAATAACCACATCATGGCGGGGGTGGGGGGGGTGGTGTGGTGGGCG TCCACCGCGTCCTCCTCCCCGCCCACCTCTGCTCATGCCTCCAAGATCAAATCCCCCCCTTTCCCCGCCCCGGGTTTTCGGGCGACTCATGCTCCGGGTT CTGAGCAACACCACAGCCATTGCTGAGGCCGGGGGTCGCATGGGGCACAAGGTTTGACTTGTGGTTGGCTAGCGGTGCGTGTGTTCACTGTAGTTGGTGG CTAAAATTGATGTCTTTTGGTTTATGAAATCAGTAATTTTTGATATTTCCCCAGTTCTTTTTAATGGGGTCAATAATGGGCATTTGTGTTTTTGTGTGGG CGCAGCTGCAGGATGCCATGTAAAGGTGTCAGGTGGAGCTCACCTTGCCTGTAGGGGGCAGCGGGCTAACGGGGTGTGTTTATGGGTGTGTGGGGGGGAG GAAGGAGCAGCTGATGCCCAGAGCGGGGGCAGGGGGGGGGGGGGGGGTGTTTTGTCGTCCCCGCCCGCGGGGGGGGGCGCCTCCACACGCGAGGCGATCG GGGGTCCCGGGGGGGGGCAGGGGGGGAGGGCGCGGGGCCGGGGGGGGGGGGGCGGGGGGGGGGGGGGGGCGGGGGGGGGGGGCGGGGTGTGGCGGGGGTC GTTCAAGTCAATTGATCTACCCAACAGGTGGGCGGGTCTCTGGTTTAGCAGTGTTTCATAGCGGTTGGGGGGGGGGGTACGGGAGGGGAGGGGGCGACAT AGGGTCTTCACGAAGATCTGCATCCCACCTCTGAGACGGGGCAACCGGGGGGGGGGGGGATGTTTTTTTGTGTTTTGTGTGAGGTCGGGGGCGCGCGCGC GAAAAAGTAGGAGTTGAAGCCAAACAGCCAAATTCTGCCATTTGGGAGGGGGGAAGGGGGCAGCTGGTCAAGAATGGGGGGGAAAAAACAGCAGTGTTTG CAGGTGTCTCCTCTATCTTAGGGGCCATCAATTTCTTTACAACAATTATCAATATAAAAAACCCCCGCCAAAACCCAACTCCAAAAACCACCACCTCCTT GGGTTATTTAGCTGTAATCGCAGTGTGCTACACATTTCTCTCCAAAGGGGGTTCTTCTCATCCGTTTTAAACGCCAATATGAGAGGGGGCGGGGGGTGTG TGCAGATGGAGGTCCTCATGAATTAAGTGCCATGCTTTGTGGGAGGGTGGGGGGGGGGCAACTGTGGAGATCATCGGGTAGGGGGTGGCGCGGGGTGGGG TCACAACTCACTCCCTGACTCAACCAGAGGGGGGCCCAGGGAGATCACGAGGGTGGGGGGGTGGGGGGCGCGCGGCGCCGAGAGGGGTGATATCGTGTTA TGTGCCTCAAATTGGTCTCTGCAGGCCTTCGGTTGTACTTGGAAAATTCAAATAACACCCGCGTGTTTGGGGGCTTTCGGGGGGGGGGGGGCGGCACAAC CAGGAGGGGGATTCAGCAGCCGCCTTATCACTTTACTGCAAGGGTCTGGTCTTTTTTGTAGCTGCCCTGGCTTTTGGAGGGGGTGGTCTAGGGGAGGGGG GAAACCCATTGCTGTTAAACTGGTGAAGATAAAACAAGAAATCCTCCCTGAAGAAAGAATGAAATGGCAAAGAAGGGTTTTTTTGTGATTTACACCCCTG CCCACTTATTCTACACCTCTCATGTCTCTTCACCGTGGCAGACTAGGGGGAAGAACAACACAGAGTGTTTTTCTTGTCTCCTCGTTTGACGCCTCCGCCC GGGGAGGGCGATGCCAGCGTGGGACTCTTTGGAAGGCCTATCAATCACAGGTGCGCTAAAATTAAAAGGGGGGGTGGGTGGGTTGTGGGGGAGGGTGGTG CTGCCCAACACCTCACCTACCCGTTTCCTGCTGCACAAAGTCTCCTTCACTCTCATTTTTTTGTTTCATATGTTGTAGTGGGGGGGGGGGGGGGCGCGGA GTGGAGACTAAGCCCTGTCGAGACACTTGCCTTCTTTACCCAGCTAATCTGTAGGGGTGGGCGCATGTGTCGCTGAGTGACTAAACGAACGATTAATAAT CTTCCAATTCAATTCGTTGCCTTTTGAGGCCACTGGGGGCCATGAAACGCCCTGGCATCTCTTGTGGTGGGGGGGGGTGGGGGGGCGTGTCTTGTTTTCT CAACACTGACTGCGGGGCATGGCCCCCCCCCTCCCCTTTGGCGGCGGGGGGGCGGGGGCAGGTCGGCGCCCCCGCCGGCGCGGGGGGGCGTGCGCCCTCT TGTTCCCCATGGTTGTGGACAGGGGGGGGGGGGCCCCGGGGGGGGGGGGCCCCCGCATGGGGGGGGTGGGGAGGGGGGGGGGGGGGAGCGGAGAGGGTGC GTCCAACTTCTCCCGGAGCAGCCGGGGGGGAGGGGGCGGGGGGCGGGGACAAGAGGGGAGGGGGACCATGGCGGGGGGGGGGGGGGGGGTGAGCGCGATC GGGGATGTTAAGAAGCAGGGCGTAGACCTTTCTTTAACAGGTAGGAGAGGACAGGGTTTGGGGGGTAATGGGTTTGTTAATACCACACAAACTGCTCACC AAAAGACCAGGTCTACAGTGATAGAGCAAAGCATCAAAGAATCTTTAAGGGAGGGTTAAAAAAAAAAAAAAAAAAAAGGTTGGTTGCCTCTGCTTTTTTG CAGCTTCAGGCCTTCTGGAAGCATGGAGTGCAGGTGTGGGCTCTAGGCTCGGGTGAGGTGGTGTAGGGGGGTGCACGGGGGGAGGGGGGCGACGGGAGGG AGCTCTCAAAGAAGCCTCATCATCTTTAGCCTGTATTGTTAACAACTGCCTGGTTTATGCTACATGTGGGCCGGGGTTGTTTGTTTTTTTTTTTGTGTAA CAAACCCCAGTGTGCCCTGCCCCTGCTGTTTTGTTTCTTGTCTTTGTGTTCTGTGGTACTTGCTGAGAGAAAAGAAAAGTGAGCGAAGCAGACGGAGGTG CAGGGAGTGAAGGAGGATAATTGGGGAAAACATCCAAAGAGGGGGGGAAAGGGGTGGGTTTTGTGTGTTGGCCCCCCCAGAGCTAGGGGGGGTTTTCTGT CTTACGTTTAGTGAGGGAGAGATTTGGTATATGATTGAGATGGGGGCTAGTTTTTGTCATGTGAGAAGAAGCAGGCCGGGTGGCTGAGGGGGGGCGGTGG AGCAGTTCTACCGTACAACCCTAACATAACCATTCTTAATTTAACTATTTATATTATACTAACTACTACCGCATTCCTTCTACTCAACTTAAACTCCAGC CCCCAGTTTACCGTGGTGAAATTGAACTTACTCTGAAACAGATGAAAAGGGACATGGAAAATAGGAGGAAGGGCGGGGGGGGGGGTGGGGGGCGCTCGCT TACTTCTCCTATCTCTCCCAGTCCTAGCTGCTGGCATTACTATACTACTAACAGACCGCAACCTCCACAACACCCTCTTCGACCCCCCCGGGGGAGGGGG CGCCTCCTGAGTCTGCTCAGCCTGGCACAGGCCCCCGGAAAGGGACCGGGGGGGGGGGGGGGTTGTGTCGCTTGTCTTCTCTCCGCGGGTGTGGGGGGGG CCTTGGGATCTCCAGCGATGTAATCGAAAAGGGCCGCCCCATCTCAAAGGTCCCGGGGGGGGGGGGGGGGGGGGGGGGGGGGCTGTTCTTCTCGGCGCGC GGGAGGGCCTACGGATGGGTCCGTAGAGGGCCCCCCCCCCTCCTTTCTCCCTGGGCCCCCTCCCCCTCTTTTCCTTTTTTCTCCTTTTGCTCCTCACACT AGCAGGGCGAGGCGCAGAGGGCGTTGCGGACTCATGCCCCCGGCGGGCGGGGGGGGGGCCAAGGCCTTCTGGTGTAACGGTTTGTGGATCACCAATTTTG GAGAAAGTCAAGCAAAGCCAAGGAGAAGAAGCAGAAGCGGGTGGGGGGGGGGGGAGGGGAGGGTGGCGGGTGTGGTGTGGGTGTGCGGGCGCGGAGAGCG CAAAGATTTCTGTCCTTGGGGCCAGAAGGTCCTAGGTTCAAGCTCTTACTCCATCAGCTTTAGCTGCGCCACCTTGGGGAGGGCTCTTAACCACTCTGGG CTGGGCTTGTCCATGCGGTGACCCTCCTTCAGCAGCTTGAAAAGTTCCTCCACAGGCACACCGGGGGATGGGGGGCCGCTCACAGGGAAGGACTCTCACA CGCGATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGGTTTCAGGGACAAAACCACCCCCCCCGCGGGGTTTTTTTTTTTGTTTTTTTTTGATTGAGGGTG TCTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGGTGTCTGTGCGCGCGTGTGTGGGGTGTGGGGGGGGCGTGTGTTTGGTGGGTGGTTTTAGGGTGGGG GTTCAGGAAAGAAAGCTGGAATAATACTACGTAACTTTGTCCAGGTTACTTTGACTGTAACACGTTTTTGGTGGATTTCTTTTCCTCAAAGAACTCTCTA TTCTGCCTAGTTTTTATGTGAAAATATTCCGGTTTCTCTTAAAACCCCCGCTAGGGGGAGCGGGGGGGGGGGGGGGGTGGGGCGGCGCGAGATGACACTA GCATAGTTTGCCTGGAGAATCCACTCACGGTCATAAAGAGGAAGTTGGTGGTGGGGGGGGGGGGGAGGGGTGGGGGGGAGGGCGGGGGGCGCGGCGCGGG GCTGCCCTCACCCTCTCTGAACTGACCATTGTCCTGCTGTCTCAGAGGGCTGCGCTTCCCCACCTCCAGATAAAGCCAGTATTGATAATGGCCCCGGGGG CTGCCCGCTGCACTTTGGCCTCATCACTCTCCCCCCCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGTGCGGGGGTGGC GCCATGCACTACTCACCAGACGCCTCAACCGCCTTTTTCTCAATCGCCCCCCCCCCACCCGTGGGGGATATTTATTGGGGTGTTATTGCCGCCTCTCACA GAAGAATGTGGCAAAGCCTTTAATGTATTCTCAACCCTTACTAAACATAAGAGAATTTCTAATGGAGGGAAACCCTACAAAAGGTTTGTATGTGGGGTGG CCAGATTCTGACCATATTGGCATACTCATCTTGGACTTCCAGCTTCCAGAACTATGAGAAAATAAATGTCTGTTGTTCAAGACTGGGAGAGGGGGTCGGT CAAATCTCCAGGAAGACCCCGTTTCCTATGTGAGGCCATTGCCCAGCCCACGTTTGCCTCCTTTTCCTCCCTCTGCCACACGCGAATTACAGATTGGACG GTTTTAGCATTGGAGTAGGTTTAGGTTATGTACGTAGTCTAGGCCATATGTGTTGGAGATTGAGACTAGTAGGGCTAGGGCCACCGCTGCTTCGCAGGGG GCCCATAGAGGTCCACAAGCGCATGGGAGGGGGGGGGGACAACTTGGACAGCTACCCCCTCGGCCACCCCCTTAAAATGAGATTGGGAAGGGGGGTCACG CAAGCTTCGCCTCCCGGGTTCACACCATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGGCTATAGGCACCCCGCCCCCCGCCCCGGGCTATTTTTTTTTT CTTGACTTCTGCAGTATTTTAATGTCCTTCCTCCCCCCCCCCCGCGTGGGGGGGGCCAGGGGGGGTGGGCTCCGCCGTGCCCCTCCGCCGCTCCGCCGGT GTTGGTTTATTGGCCTACAGAGTTGGACACACACACAAATGCGGGGGTAAATATTTGTCAGGTTTTCTAAATATGGGTGCTCACTCTGTATAGAGCTAGT CGATCATATCCTCTGTCATAGTAATCCCGGCGGGGGGGGGGGGCCCAGGGGTGGGGTCCCCCGTTAATATCCTGTGGTTGCGTGGTGTGGGGTTTTTTTT CGCAGGTCTGCAAGGCCCTCACTCCGGCCATACACCTCCCCACAGGGAATCTCGGGCCGTCAACGGGGCCCCCCCCCCCCCCCCCCCCCCCCGGCGCGCT GGAATGTACCAGAAGCAGACATTTGTGTGCTGAGGGTTCCCCTCAAACACCCTCTCATATACTTTTTGGGGTTTTTTTGTGTTGTTTTTATAATTTGGCT CTTAGGGCTAGGATGATGATTAATAAGAGGGATGACATAACTATTAGTGGCAGGGTGGTTGTGGGGGGGGGGGGGGGTGGTGGGGCGGTGGGAGAGCGGG CCTTAATAAGCCTGGTGCGCCCCGTCTCAGATTGGAAGAGGGGGTCAGTAGGAATGGCGGGACGGAGATAGTCTGTCGGTGCTCGTGTTGATATAAAACT AGGAACTCCGGGAAGTCAATGGTCCCGGTTCCCTCTTTCTTCACCCCCTTTTTTTTTATTTTTTGCGCTGTCTTTGTTGGGGGGGTGTGCGCGGGGGTGT TTACAAGGAAATCTGTGTCAAAACAGATAACAGTGCATAAATCAATCCAAATTTAGACCCTGGCAACCAGTTCCCCATTGCTCATCTCAGGGAGTCTGTC GAAGAAGATGATGTGTCAGAGGGCTCTGAAGTGCCCGAGAGTGACCGTCCTGCAGGGGCCCAGCACCCCCAGGTTTACGGGGCGGGGGGTGGGTCAGTGG GCCGAGAGCAAGCGAGGGCTCTGAGGAGTGCCAGCACTCTGTCACCTCTCACCAGCGCTTTTGGGGGCCGGGGGAGGGGGGGTGATTGGTGCGGGGTGGT CAGACCGAGAGCAGCCTGCCGGGGCGGAGGAAGGAGCGCCCCCCCTCCCTGAACGGGGTCCCCCTCGCTGGGGGCGCGGGGCGGGGGGTGAGAGGGGGGG GGAGTGCAGTGGTGTGATCTTGGCTCCGGTGGAACCCCCCCCCCCCGGGGTTTAGTGGTTTCTCTCCCCCTTCGTCCCACCGCCTTCTGTGGGGTGGGCT CCATTATCGAAGAATTCACAAAAAACAATAGCCTCATCATCCCCCCCCACACAAAGGAAGGGGGGGGGGGGGTGTGGGAGGGGGGGGGGCGCGAGCTCGC CACTTTGGGTGGCCAAGGCGGGCAGATCACTTGAGGTCAGGAGGTCGAGACCAGCCTGGCGAACATGGGGGAAAACCGCCTCTATTAAAAATACAACAAA GGGATGTTAGCACCAGGTATTTACAGAACAGCCCGGGGGGGGGGCGGGGAGGGGGGGGGGGGGCGAATATTGGGGGGGGGGACGAGAGGCGTGCGACACA CGAAACCTTGCAATGCCATGAACTGGTCCATGGTTATCATCAAAAGTTCCATGCTAAGGGGGGTGGGGGGAGGGGGGGCGGGGGGCAGAGGAGGAGCGCG TGGAAGAGGAGTTGCAAGTTTAAATTTGTAACCTGACTCTGGGTTCTGTTCTAGGAATAGTGCATGTTTTAGAGGCTTTGCCAGTTGGGATACATTGTTG CGAAGCTGTCCTCTTTCTCTGAGTCGCTTTTGGGTGGGGGGACAGAAGCCGGTTGGGGGGGGCGGTGGGGGCGGCGGTGGCGCTGCATGCGCGCACACGC GTTAAAGGAGCCACTTATTAGTAATGTTGATAGTAGAATGATGGCTAGGGGGGGGATTAAAATCAAAATACGCCCTGGTTCGGGGCGGTCGGGGCGGTGG GGAGGCCAAGCGGGCCAAGGAGGAGGGCAAGAAGAAGAAGGAGGAAGAGAAGGAGCGTAAGGAAAAGGAGAGGCGGGGGGGGGGGGGGGGGGGGGGGGAG GTCAACCTCGCTTCCCCACCCTTACTAACATTAACGAAAATAAACCCACCCCACCAAAACCCACAACCGGGGGGGTGTGGTGCAGGGGATGGCGGGGACG GACAGTTCGACCTCTTACTCTCTCTTGTGGGTTTGAGGGAACCACCAACATGGGAAGAGGGGGGGGGGGGAGGGAGGGGGAGGAGAGGATGTCACGTCTG CAGCACTGCCGCGGGACCCTCTGCCGCCGCCGAACCGTGTGTAGTGCCGCTGTGTGGTGGGGGTGTGCTCGCGTCACTATGTGTGCTGTGTGTACGTGCG CGCCATGCTGCCCTATGACCAGTACCTGCACCGCTTTGCTGCGTACTTCCAGCAGGGGGGCATGGGGGTCACAGGGGGAAACATCACCACAACTCGGACG CTGGGATCTTGTACTGGCGTGGATTCTGCATAATGGTGATCACACGGTTCACCCCCGCCCCTGCGGGGGTTTCCCCCCCCTCTTGGTGGGGGCTGTGTGT GTGAAACCCCATCTCAGAAGGTACGGAAATTGGCCAGGTGTGGGGGTGGGGGGGGGGGGGTGTCCAGCGCACCTGGGTGTGGCGGTGGGGGGGGGGGGGG GCCTTGGATAAGGGCTGCTCCCAATTCCGCCTGGGCGGCCTGGGGGGGGACAAGCGGGGCGTGGACTTGTTACAAGCGCTATGGGGGCGCGAGTGCGTGG CACAGCCCTATACTCCCTCTACATATTTACCACAACACAATGGGGCTCACTCACCCACCACATTAACCACATAAAACACTCATTCACACCAGAACACAAC CCAGGATTGTGGATGATCTGAAAGATGAAGCCGAACAGTACAGAAAAATGGTGATGGAGACAATTGAGAAAATTATGGGCAATTTTGGGGCAGCAGATAT GTGAGAACAGCAGCCCCACGGCCCCCCTGGGGGGGGGGGGGGTGTCCCCCGGCGTGGGGGGGGTGGGGAGGGGGGGGGGGGGAGTGGAGGTGGGCGGTAG CAGGACGAGGCATACATGGATGTGTGGAAGCTTTGGGGACGGGAGGGGGGGGTGTGGGGTGGGGGGTGGTCTCGCCGGAGGTTGTTGTACTCGTGTTGTG CACACACACACATCACGGGGGGGGGGGGGGGGCGCGGGGGGGGGGGGGGGGGGGGGGGGGTGGGGTGTGTTATACGGGGGGGGGAGGGTGGTGGTGGTGG CAGCCTGTTGAGGTGGACGCCACAGAAAAGGGGGGGGAGAGGGGGGGGGGGTGGGGTGCGGTGGGGGGGGTGGGGGGGGGGGGGGGGGGGGGGGGGGGCG GCCAGTATTACTTCAAACCCATGATGAAGGAGTTCCGGGTTGAGCCATCCTCACAGATGATCGAGGGGGGGGGAGGGGCGGGAGACAGGAGAAACAAACA CTTGTTTCGGACTATAGAGTGCTGATGGCAGGGGTTGGGGGGGGGTTGGGTAAATATGTTGTGGTCTCCTTAATTTTTCTCATTATGGGTTACATAGGTG GGAGCTGTGTGTTTGGTGGGCAGACTTGTCCTGGGCTGTGGGTGGGGGGGGGGTTGGGGGGGGTGCTCGGGGGGGGGGGGGTCGTGTAGTGGGGGTGCGT CTTGGGGGATCAGGAGCGAAGTTGGGCGGGACTTCAGGGATCCGCCCCCCTCGCCCTTTCCCCCCCCCCCGCCCGGGCCCGCAACCCACACACGCGCGCT GCAGTTCTAAGTATAACTATGAGAGACCTGAGACATAGTCTAAAGCTGTTTGAGGCTGGCAGTTGAGTGTTGTGAGCGGGATGGGGGGGTTGGGGGGTGG TTCCAAACGTGTTTACAGGTTCTCTTAAGCAATGTTGTATTTGCAGGCTTTTCTGAATACCAAATCTGCTTTTTGTAAAGCGTAAAAACATCACAAAGGA GTTGGGGCCTAAGGTGATAGGGTGGACATGCCTGGAGGGGCCTGGCCAGCTTCTTACCACTACTTGTGTATTCTTGGTCCCGGGGTGATGGGGGCGGGGG GCTTCCTGTTCAAAACCCCGTAGATGCCACCGGACACCCGGTCTGGGCACTGGGGTTCCTCAACGGGGAGGGGGGGCCCGGGGCGGGGGGGGGGTGGGGG CAGCCCCCTGCCCCATCTTCTGGCACAGGCCATCCCGACTGCATGTCCCTGTTTGCCACCGCTCTGATCATGAAGCCACCCGCAGCGTCGACGCAGCGTT AGCTGCCATCAAGTATTTCCTCACGCAAGCAACCGCATCCATAATCCTTCTAATAGCTATCCTCTTTCACAATTTACTCTCTCTGACATTGACTCTAACA ATGAGCCTGTCCTGCTGGAGGAACCAGTAGTCCTGGCATTGGCTGAAAAGTATGGCCGATCTCCAGCTCAGATCTTTCTCGTGGGGGGGGGGCGGCGGGA GAGATTACCCAAGGCACCCCTCTGACATCCGGGCTGCTTTTTTTCTCATGACAAAAAACAGCCCCCCTCTTCCTCAAATAATCAACGATGTAGAGAGGGG CTTCACAGAAAAACATGTCCCAGACACGCAGCACCGACGCCCAGGGGAGGGGGCGGGGGGAGATGAGCATGAAACACTACGTCCTGTAGAGGAGCAGGGT TTTCTCTTAATTTTTCCATTCCCAGGCCGGTTAGCTCCCCCCAAAAAAAGGGGTTTTGGGGTTCCCACACCCCCCCCCCTCCACACCCCTTCCTCCCTCC GGGTAGGAGGCTTCTTTCTACCCTACAGAAAGACTCACACTCCCCATTTACCATGAACAACGGATGTCGAGGCACTTTTGTGGACACAACACTATGCATG GGCTGTGATACCTCGGCCCGGTTTTTACCGATGTCCTCCTCCCGCAGGTAGAACTGGGGGGAAAGATCAGCCCACTGGGCAGTGCCCTGGGCATGTAGGG GGACAGCACGAGATTTTACCATGCTCAGAAAAGGGCACACTTAAAAACTATGAAGTGTTTATCTCTGGAATTTTCCATGTAATATTTTTCGACCATGGCT CTGAGAATAGGGGAAATCAGTGAATGAAGCCTCCTATGATGGCAAATACAGCTCCTTTTTGTTGGGGAATTGGGGGGGGGGGGGTTCACAGTGGAGGGTG GGGGCTTGACACCCTTAGTTTAATGCCTTCAACACAAGGACAGGAGAGGGAAAAAAACACTAGACACCAGCAGGGGGAGCGAGGGGGGACAGGGGCGTCC CAGGCTGGAGTGTAATGGCGTGATCTCAGCTCGGTACAACATCTGCTTCCTGGGGTGAGGGGATTTTCTCTCCTCTGGCTGTCACACAGACGGGGTGACA CCCAACCGAAATTTTTAATGCAGGTTTGGTAGTTTAGGACCTTTTGGGGTTTTTGGGGTTTGTTTTTTTTATAAAATTAAAATCGCATGGGTGGTTTTTG GTCTTCAGCTCACCAGGCCGGAGCCAGGGGAAGAGACCAAGGGAGGAGGCTCCAAGAAACCCTGGGGGGGGGGGGGAAGCGCACAAAGACACGGACGGGC CAAGTTCTGGCATTTCACATCGGACTGTACCATTGTCCTTCTCAATCTCATCTAGATCCAGCTGGGATTCTCGGGGGGCGATCTGTGGTGGTCGTGGTGG GGGGGAGCGTAACTTCTCACTCCTTCAGTGTGGGCTGCACAAGTGACTTCCTTCCCTATGGGATCGTTATAACAAAAGACTGTAACAAGGGCTATGGGAG GAGGAGCCCTCCCTGCCCCACCGTCCTCCAGAGAATGCGCCCCTCCTGGCCCTCCCTGCCCCACCGTCCTCCCGAGAATGCGCGATCGGGAGAGGGGGTC GCCTGACTGGCATTGTATTAGCAAACTCATCACTAGACATCGTACTACACGCCACCGTCCCCGTGTTTGGGCCTCTTTCTCACTTCTCTTTTCTATAGCG CTCACACTCATTCTCAACCCCCTGACAAAACACATAGCCTACCCCTTCCTTGTACTATCCCTTTTGGGGATAAATATTATAAGTCTCCATCTGCCGGTCG GAAGCCTGCAGCCATGCGACCCCCCCCCCCCCCCCGCGCCCCCCCCCCCCCCCCGCCGCGCCGCGGGGGGGCGTCCGCCGCCCCCCGCGCCCCCCCCGGG CGTTTACCCGCGCTTCATTGAATTTCTTCACTTTGACATTCAGAGGACTGGGGGGGGAAAACAACACGGCTCCACACCCCCCCGCGGCGTGTAGGCGGTC GCGTTGTGCAGCGGGTAAGCGCACGGCCGAGGGGGGATGGGGGGGGGCGGGGGGGGGGGGGGGGGGGTGGGGGGGTGGGTTACGACGGTGATTGCGCGCG GAGGGCTGGGATATTCCTCATGGTGGCAGCGGCCATAGCGAAAGCCTCCTGTAATATGCACCCATCTCCTTCCCGTGTGTAAGAGAACTTAAAAAATTAA CCTCGAGCCGCTTTAACTGGGCCTCCTGGACAGCACTCAGCTTCTCTGCCTCATTCAGTCTCTTCCTTGCAAAACCCGAGGAGGGTCTGTTGCTGGCTGC CCTGTTTGCACCTGCTGAAGCCCCAAGATGCTTTTCCACTATTATGTAGAAGTGGAAAGGAACAGCACCAGGAAACTGCCACCAATTACACATGATGAGG AAGCTTTTCATACACACTATGGAGAGCCCACGCCCCACATAACCCTTTAGGAAACAGTTACATGGCTTGGCTATACAGGGGCGCTGCCTCTGTCTGCCCA GCCTGCCCTTGGCCGTGTCATCCAGCCACGTGTCCACCGGTTCGCGAGGTGGACGCGCGGCACAGTCTCCACAAACTTATATCTTTTTTCTCACCTCTGT GATGCCTGAGAGGTGCAGCCTGCCGATGCCGGCCCGGGGGGAGACACGGGTGTTGCCGTGTGTCGTTTTTTTTTTGGGGGGGGTGTGTGGGCTGGGGGTG GGGAGAGCAATGTGTCAGGCCGGGAGCTCCTCTCAGCTCCTTCCCGGCAGGCGCCCCCGTCCTCTGGGGGGGCGGACGCGCGCGTGTGCGTCGTCGCGGG CGGGGCCGCATGTCCATGAAGGAGGTGGACGAGCAGATGGTGAGCGGGGAGAGGAAGGACAGAAGCTCCCTCGTGTGGGGGGGCGCTCCCCACACCAGAG TGGACTTTCTGGACTACAAGTCCAACTTTGAGCCCTTCTTCATGATGATCGCCCCGAGTCGGGGAGGGGGGTGTGGTGGGAGAGGGGGGCACGACGACGG CCCAGGAGTTCACTGACTCAGAGATCCTCCCGAAGGTTTTTCCCCTTTGGCCCGCGATCTCTTCCCCACAATTTTTTTTTCGCAGAGCCACACCACACAC GGCATGGGAACAGGAGCAGGCGCCTACATCCTAACTCGATTTGCTCTAAACAACCCTGAGATGGTGGAGGGCCTTGTCCTTATCAAAGTGAAACCTTGTG CCCATTCCAGGGCGCCCTGCCCTTCACAAAGAAAAGAGAACTCTCCCCGGGGGTCCCCGCCGGCTTTTCCGGGGTGGGTGGCGTGCCTGCACTGGGTGCG CGAACTCAAGCAATCCTCCCAGCTCAGCCTCCCAAAGTGCTAGCATTACATGTGTGAGCCATCATGCCTGGTCTCTGTCTGTGTCATTCTCCCTGTCTCT TGTCTGTCACCTCCTCTGTCGGAGGCATGTCTGTCATGGGAGGGGGTTTCCCCCCCCTGGGCGCCTCCTTTTCCCTCTTCTCTTTTTTTTCTCGCGCTGG GGCCGCCCTCTCGGAGGTGGAACCGCCGTGCATCCCGTACCTGGGGCTGATCCTGCAGGACCTGACCTTCGTTCACATGGGTCATCCAGAGTATATCGCT CTGACTTCTTCTGCTTAACGAGGAAAGGGGAGGGCACAGGGCAGGGGGGCCCCCGGGGGGGGGGGGGGCAACGAGAGCGGCAGACGCGGCGCGGGCCACG ACGCAGCCATCCATCTTCTACAGCACGCACACCCCACTCTCTCCCCCCGTCAATATGTCTCTCTCCGCTGGGGGAGTTCATAAATTTTGTGCTTGATTAA CCCACATTCCAGATGATAATGGCATTATCACAGCCTGCACTAAGAAGCACATTGCGGGCCGTTGGGTGGCCAGGCACGAGGGCGGCTCTCTCTGTGTGGT CGCGAATTGAGCTAATTGGGGTGTGTGGGCAGCCACGGCCTGGGGGGGGGGGCCCCCCTCTGTGCTGTCCCCCGGTGGGCTCTTACACGTGTCCAGGCCG CACGCCTGGCTAATTTTTGTTATTTTTAGTAGAGACAGCATTTTTCCCTCTTTGTCCGGCCTGGTTTTGGGTCCTTGTCTCTCGCGCCACCCCCCCTCGG TGAGAATAGGGGAAATCAGTGAATGAAGCCTCCTATGATGGCAAATACAGCTNNTATTGATAGGACTGAGTGGTAGGNGGGGGCAACGTGGACGGGGGTG GCCCCTCTTCGTCTGATCCGTCCTAATCACAGCAGTCCTACTTCTCCTATCTCTGCCAGTCCTAGCTGCTGGCATCACTATAATACTAAAAGAACGCAAC CCGCCTGCCGCCCCCTGTCCAGGGTCCCTCCCCGCCCCAGGGGAATAAAGAAGAATGGGGGGGGGGGGGTGGCGGGGGAGGGCTGGGCAGGCAGACGTCG GTGCTGGCAAGGGATTGGGTTTGTGTGGGTGTCTCTAGCCTGCAGAGTGCAGTGAGTGAGAGTCCTTGGGGGCGCGGCGGGGCCTGTGGGTGCGCGTGGT CCAGCCTCCGTACAACGAGTCCTTCCCGGGTCCAGACCCCCCCGTGGGGCCCGGGCTGGGGGGGGCGACTGTCTCAGTGCGGGAGAGCTGGTGATGTGGC CAGCCGGTCCCTCCGTTTGGGGTCCCTGACTTCCCGCAACAGAAGATTATATGGTTTGGATCTGTGTCCCCACCCAAATCTCATGGTCAGTTGTAATCTG CAAGCTTGAAGTCCTCACCGGAGTCATTGTAGACAATGGGCTCAAAGACGTTGCCCTGGGGGGGGGGTTTTTCGTGGCGGGGCGGTTGTGCGCGCTCTGT TGCTTCTTTCAAAATTGAATAAAAGGATGAAAATCCACGCCCTCGCCCGAGGGGCACCCCCAATAGGGCCGTGTGGGGGACACACACCCCGCGGTGCGCG GGCAAATCTTACCCCGCCTGTTTACAAAAAACATCACCTCTAGCATCACCAGTATTAGAGGCACCGCCTGCCCAGTGAGACACGTTTAACGGCCGCGGGA GCTGCTGGTTGGTGCTGGGTTCCATGCGCTCCCACCTCCTGCACTGTCCTGCGGTCCTACGAGGCTTTTGTGACAGAGGCGTCCCTCCACACCTCAATCT CACGCTTTCCGCACTGAGCTGCAGCTGAATGTGCTGGTCGCTCCTGTCCCTTGTCCCATCCACTGTGCCATCCGGGAGGGTCCTCAGGGAGTGTGCGCCG GCAAGATAGGTGGGAGGACCAACAGATGGACATGGGGGTAAGGGAGGCTTGGAGAGGGATTGCACTGGCCCTGGGGGGATGTGTTGGACAGACGTGCGAA GCTGGGGCATCTGCAGAGGGCTCATTTAAGATGACATCATCTTTGATGTACTTTATTCCACAGTAGTACACGTCATATGGTTGAAGTGTAAAATATTTGT GAACCTGCGAAAGGGAGGCATCCATCCTGAGCTATGATGGTAGCATGTACATGAAGATCATCATGCCCATGGTCATGCATACTGAGGCAGAGGATGTGTG GAACGCCTGAACGCAGGCACATACTTCCTATTCTACACCCTAGTAGGCTCCCTTCCCCTACTCATCGCACTGATTTATACTCACACCACCCCAGGCTCAC GCAAAAGTTGGAAAGCTTAGATCTGTCACCTTAGAAGCGTCTCAGATCGGGAGAGCGGGGGGGGGGGGAGGAGGTGGGAGAGACAGGAACTCGTGGTGTG CGGGGATTTTAGGTGGTGGTGGTTCTGCTGACCGGGGGATTTAGAGAAAGGTAATGGAGTGGTTGAATCAAAAGGTGGGGGGTGGTTCTCTGCTGTGAGT GGTTTATGTGAGTTCTGGAGACCTACTGTAAATTCTGCACTGGTCTTCTCTGCGTTGCCTGGTGAATGGGGGCTGCTGTACTTACTGTGTTCGCCGGCAG GGACGATGGGCATGAAACTGTGGTTTGCTCCACAGATTTCAGAGCATTGACCGTAGTATACCCCCGGTCGGGGTGGGGGGGTGAGAGAGTTGGTTTTGTG GTCTGGAGCTGGCTGGCCGGGACCTGACTGACTACCTTATGAAGAATCTTCCCGAGCGGGGGTGCAGCTTTCCCCCCACAGGCGGGGGGGGGAGCGGGTG GAAAGCCCAGGCACTTCGGGACAACTCCACCATGGGCTATATGATGGCCAAAAAGCACCTGGAGATCAACCCTGACCACCCCATTGTGGAGACGCTGCGG GCTTGCCAATCCGCTGTCAGCAACTTGTTTTTTGTGGTGGGGGGGGGGGGTGGGGGGGGGGGGGGGGGGGGGGGTGGGGGTGTTTTTCGCGCGGGGGACG CGGGGTCGGAGGAAAAGGTTGGGGAACAGCTAAATAGGTTGTTGTTGATTTGGTTAAAAAATAGTAGAGGGATGATGCTAATAATTAGGCTGTGGGGGGG TGTGCACATACGTGAGTGTGGGGGTGTGTAGCGTGCTGATCTGTGAGTTCCAGGGTTCACCACCTTCCTGAAGATCACGCGCCCTCTCCGTGCGCCCTCT TTCCTTTGCAGCTTCAAATCCTTCAGTGATTATTCTAGGATGAAGGCCTTCAGAAATGTAGAGATCCGCCTGGTTTAGCAGCTTTCTCATGAGGACGGGG TCCACACTGGAGCTTCCTGTGGGTTGTCTTGTTCAATTTCCATCCTGGAAAGGGACTTTCCCTTTTCTTATGAGTTCAGTGATTGGAAGGGCGGGTGGTG CAGCATGGGGGTAGCTGCCCCCATGCTTCAATTACCTCCCACCAGGTCCCTCCCATGACACGCGGGGGGCGGGGGTGCGCGACTTTCATATTGGTGTTGT CGTCAAATGTACCCTAGAGCTGACGGTTTTTTTTTTAGGGGTACAGCTACATCATAGGTAGTGATGTTGATGTGTCTTCACCACGTGTCTTGTGTGCGAG GGTTCTTGTTGGGCTTTTAGTGCTACTGGTGCTCTTGAAGGACAGATGTTCCGGGAAACTGGGAGGGTTATTTCACTGAGTGAGCAGAATCTGGTGGGAT GCTGGAAAGAGTTTGCTCCAGGAAGGCAGCGTTCTGGCTGACAGCATCCACTAGCAGGGTGGAGTCCATATGCACAGACTAGGCGTGCTTCAGGAGAGCT CGCAGTGAAGCCCCGTCTCTATTAAAAATGCAAAAAAATTGGCCGGGCGTGGTGGCGGGCACCTGTGGTCCCAGCTATTCGGGAGGGTGTGGCGGGGGTG GAAGAACCCTGTCACCGCTGTCCCCGTTGTCCTGAAAAGACTGAAGGCCAAGGAAGAGGAGTGGCGGGGGGGGGGGGCGGGGGGCGGTCAGACGAGGTGG CAAAAAGGGAGACCAAGGAAGTGCAGACCAATGAGCTGAAAGCAGAGAAGGCATCTCAAAAAGTCACCAAGAAGACCAAAACTGTCCAGTGTAAAGCGCA TGCCGATGTACTTGGACTGCAGGTGCTCCAGGGGGGTTTTGGGGGGGGTGGGGGGGGGGGGGGGGGGGGAGTGTGGTGTGGAGGGAGAGGTGCGCTGGCG ATGCCATGCCTGACTCGCCCACCCCAATGCACCTGAGTGGGGTAGAAGCTCATGACGTGCAGCTCGCGACACTCGTCTGAGAGGTGTCGCGCCCACGTCG CTGACATAGGGGTAAGGTATGGGAGAGGTAGGTGGATCCAGGGGAAAGGAGGGGGGGGGGGGGGGGTGGGGGGGGGGGGGGGGTGTGGGGGGCGCGAGCG GTTGGAGTCCCTGAGCGAGGGCTCCCCAGATGTGGTGACATGACACTCACCGCTCTGTGCGTGCAGCGCAGGGGCTGTGGGACAGTGTAGCAACACAGCG CATCATTGTCCCCTCCATCCTGCAGTCCCAGGACAGCCTGCACACCGGCGGGCACCCCGGACCGTGTTCCCGGACCCCGACTCCTTCCTGGCGTCGCCAC GCAGAACTACGTGATACCATCAATACTTCTTGTGATATTGAGCTCCTGGCAGCTTGCAGAGAAGAATTTCATAGGAGACTAAAAGTGTATCATGCTTGGA GCCGCCGCGGCTTTTTGCCCGCCGCGGCAGATCGGGAGAGGGGGTGTGTAGGGGGGAGGGGGGAGCGGTCGTTTTGTGTGGTGTTTGTTGTGTGCACTAA GGGACGTGTTGGTGCTACAGCAAGGAAATAGGGGAGTGTGGAGGGGGTGTGGGACATCGTAAGTGGGGAGGGGTGTTGGGCTGGGGGAGGTGGGGAGCGG CTCGTGTTCGCCTCTGTCTACATCTACAGATACTTCTTCCTTGCGCAGCTGGCCCGAGATAACTTCTTCAGATCGGATGAGAGGGGGTGGAGGACAGTGG CACCCACAACGAAACCATGGCAAGCGGTGGATGCAGGTACGGCCCGCTGTGGTTGTATTCCGTGAGGGGTGGGGAGAGGGGTGATGGGGGGGGGCGCGGG CAGTGAGGTAGAAGACCATGATGGAGAAGGTGATGTGGCTGGAGATGATGATGATGACGATGATGATTCACCTGATCCTGAAAGGCCAGATGATTCTGAA CCGGCTGAAGGACACACAGGACCCGGGGCCCGGGGGGGGGGGGGGGGGGGGTGTGCGATATGGGGGGGGGGTGGTCGCGGCGGGGGGAGGGTGGGTGGGG GAAGGCCTTAGAGGCAACTCCAGGTCAAAGGTGGGGAACCAAGGACTAAGATCCTGCTCAAAGGCCCAGAGAGACCGGGACCAGTGTCAGAGCCTGGAGT GGAAGTCCTCTAAGCCGTACAAAGCCATCATTGGCCGTGTGAGGGCTCTTTTGACCAGTATGGTTCTACACCCACCACATTTTAAAGTGGTGTGTTTTCA GTGGAATTCTGGGTGTTAGGCCCGCCATTCGCTTGACTCACGCCTTCGCCGGAGGATCTTTTGCAGGGGACCGAGGGGGAGAAAAGTGGGGCAGAGCGGG GCACTTACTGGGAATTCCTCGTTCATGGGGGATAATTGCAAACCCCCCTCCCCCCCACGCAAGGGGGGTGTACGGGTGCACTCCCCCTGCGCGGGCGGGT AGACGCTGCTCCGCGGGGGATGGAGAAGCCCCGCCCCTCCCCAACGCCCTTAATCTTGATGGAATTAGAGGAAAACAGCCGAAAAACCCGCGGGCGCTGG TGCGCAGTGAGTAGAGCTTGAAGGCGCAGCCCGGCGCGGGGGGCGAGCGCGTGGCGCACACACCGGCGGGCCTAGAGGTGTCTCTGGCGATGGCGTGCGG CGCAGATGCTGCATCTCTGTGGTTTAGATCTAACTTAACCCTTTATGAATGGTTGAAGCTGCTCAACTCAAAAAGTAGAATTTTTCGTCATTCTGTTTTA GCGAGGGCTGGCGGGGCGCGGGGAGAAGCCAGATCGGAAGAGCGGTTCAGCAGGAATGACGAGACCGATCTCGTATGCCGTCGTCTGTTTGTAAATGACA AGCAGTTAAGGCAGGAGTGTGATGGATGAGAAAACCTTTGAGAAGCAGGACATGGAAGACATGGGAGGAAGCAAATCATAATAATGCCTTTGAGTGTGAG GGGCTGAGGAGGGGGGTCGGAGGCACTTGGTTTGGACCCGAACGGGCGGGGGGGAGATCGCGGGCGGGGGGCGTCGGACAGGCAGGAGTCGAGGGTCGTC TGAGGAGGCTGTGCTGTGTGCCAATGTTTCGTTTGCCTCAGACAGGTATCTCTTCGTTATCAGAAGAGTTGCTTCATTTCTTCTGGGGGCGGAAAAAAGA CCGCGGCTGCGCCACTCCAAATCCATCGACGGGGGGGGGGTTTTCCCCGCGCGCCCACTCCGCCACGGGCGTGCGGGGGGTGGGGGGGGTGTGCGGCGGT CGGCCCCTGCCGCCCCGACCCTTCTCCCACCGCCGCCGCCCCCACGGGGCGGGTTGGGAGAGGGGTTCAGCAGGAATGCTGAGGACGATGTCTGATGTCT GGGGGCTTGGGGTAGAGAGGACAGGTTTTGGGCCCAAGGGGGGTTTGGGGGGGGGGGGTGGGGTTGGGGGGTGGGGGGGGGGGGGGGGGGTGTTGTGGGT GTCGGATCTTTGGCTGATGGGAAAGCGAAGAAGAGAAGAGGGATGTGTTCCGCATACTCTTCACTGGGCATGGTGACGTAGCGCTCCATCGTACATAGAT GAGCCAACCGCGCCGTCTGTCCCTGGCAAGCCAGCGGGGGGGGGTTTGGGGGGGGGGGGGGGGGGGGGGTGGGGGGGGGGGGGGGGGGGCGCAGAGATCA GTCAGGTGGCCTCCTCCCCCTCGACCCCCTACCGCACCTTGGGTACCAAGCCTTTGCCAAAAAAAGAAAAATAATGAAAGCGAAAGGAGATTTTTAAACG CCACCAGAGACTGCAACCATCCACAGTCACTGTCCCTCTGCACTGAGAACCAGCTCCAGAGCAGATGGCAGCATGGGCATCCTGGTCTGGGGGGGGGCTG GAATTATTATTCAAAATTTTAGCACAAGATCCATTCTCACTGTTACCAACGTGACACAGGAGCACTTCGGCAATTATACTTGTGTGGCTGCCAACAAGCT TCTAACCTTCCTGACAACTTGAAGAAGCTTCCGGAGCTTGGCCATGACCAAGCCCGGCCGGCAGGGTAATGCCTCGGGCGGTCTGGTATCACCCAGTACG CTGATCTGCACTAATATGGCCCCGCCTGTGGCCCGGCCCGTGGAAGGCCCGCATTTTGAACAGGGTTTTGTTTTTTGCAAATCTTCTACCCCTGCCCCAC TGGCGAATCTGGAGCTCCCGGTGCAAAAGTCACAGAGGAAGAGAGGTTGTAACCAGGAAGTACGTAGGTGATGGGGAGAAACTGTGAACGGAGTAGCAAA CACCTACACCCCTTATCCCAATACTAGTTATTATCGAAACCCTCAGCCTACTCATTTAACCAATAACACTTGGCGTACGCCTCAACGGTAACCATACTGG TGACCATCCATGGGGACCTGTACTATGAGGGGAAGGAGTTCGAGACACGACTGGAGGGGGAGGAGACACGGGGTATGTGTGGTTGGGCACGGGGTTCTCT CGCATGAAACAGTCCGGGGGGGGGGGGGGGGAGGTTCGGGGGGCAGGAGCGCGGGGGTGGGGGAGGGGGGTGTGGGGAGGAGGGGGGCGAGGGGCAGCGG CGCCGGTGATCCGATGCAGGGGGTTAAAAAGAAAGCGAGAGTTCAGGGGCTCTGGCGATAAAGGTGGCCGGGGGCCGGGGGGGGGGGGGGGGGGGGCGGG GAACAATGTAGGTGGTGTGCGGAAGGTGGAAACTTGATTTGTTGTGACTTTTGCCATAATGCTTTCTGCAAGAAATGCATTCTACGCAACCTTGGTCGAA GCAGACCTCCTCATTCTAACCTGAATCGGAGGACAACCAGTAAGCTACCCTTTTTCCATCATTTGTGAAGTAGCATACGTACTATATTTCACAACAAATC TTTATATCTTCTTCGAAACCACACTTATCCCCACCTTGGCTATCATCACCCGATGAGGGAACCCGCCCGAACGCCCGTAACGCGGGCCACACCTTCTGTT CTTGTAGATGATCACAAGATATTTTTTTGTGATGCACTCTCAAAAGACTCTGAAAGACTAGGGGTATTTTTTTTTTGTTTGTTTTGTTTTGTTTTTACTT GCGGGGTGTTGCCGGGGACTCTGACCCCATCAATCAAGGTCTCCTCTTCCAGCAGGCGAAAGGTGCCCCATGCTGGTGGGTACAGCCTCAGCGAGATCGG GCCCCTGGCCCCTTGCTGTTCCCAAGTGCACGATGCTGCTGTGACTGAGGAGTGGATGATGCTCGTGTGGGCAGATAGTGTGTGGGGTGTGTGCGGAGTG CCTGTTTAGTCTGCAGCTTCTGTCGAATCTTCTGCATCTGCAGATCAGAGTTGGGAGAGCGGGGGGGGGGGGGGGGTGGGGGGGAGGGGGGCGGCGAGCG GAGCAGTGGGGGGAAATGGAAGAACAGGGACTGGCCAAGGGGAAATGTCTGGGTTGGGTTGTTTGGGGGTTTGCTTTTTTTAAATGTGTGTCCTCATGTA GCTGTGCTGACCTCCTGAGCGGCAGACACCTTCCAGGAGCCCTGAGGGTGGCAGGAGCTAACCCCAACCAGCAGGCAACTAACACGGAATTGGCAGATCG GGGCTCAGCCCTGCCTGGTTCCTGCAGCACGTCATCGGCCGGGGCCGGGAGAGGGCACGCAGGGCCGTCTTTCTTGTCTCTTAGTGGGGTTCTGGGGGGT GCGAAACTCTCCAGCTTTACATATGTGAGGTAGTGCTTCTCCCGGGTTTAATGTGGAAGTTGTTCCATTTTTATCCAAAAGTTGATTATCTGTGCAAAAC GAAAGAAGGAAAACATGACGTCTCCCGCCAAATTCAAAAAGGATAAGGAGATCATCGCAGAGGAAGGTACTCAGGGGAGAAAGAACGGTGCGCTCACCTC CGGGCTTGGAGTCGGCTGGGGGGGGGGCCACCCTCTCCCCCCCCCGGCGCTCCTTTGTGGGGGCTCTGTTGTTTCCTGTGGGCGGGGCGGACAGCGCGGG CGCGCCGCCACAATGGTGCGCATGAATGTCCTGGCAGATGCTCTCAAGAGTATCAACAATGCCGAAAAGAGAGGCAAACGCACGGTGCTTATTAGGCCGT TTTGCCTATGGGCAGACCGGGGCTGGGAAATCCTATACCATGATGGGGCGACAGGGGCGCGGGGAGGGGGGGGGGGGGGGCGCGGGGGGAGCGGAGACGG CCAGAAATTATGTTGAATTCCAAGGGCTACACCAAGTCCATTGATATTTGGTCTGTAGGCTGCATTCTGGCAGAAATGCTTTCTTACAGGGCCATCTTTC GTTTGGTTTAATCCACCTCAACTGCCTGCTATGATGGATAAGATTGAGAGAGTGAGGAGAAGGCTTACGTTTAGTGAGGGAGAGATTCGGTGTATGTTTG ATTTAATCCTTCCAACAAGCCTTTAGGCAAGGGGGGGGGTTTTGTTTGTGGCCGTTTTCAGATGAGGGTATTATGTGGAGCGGAGGAGGGGCGCGGTGTG CTACACAGCTGGAAGTTTAGGTTGCAGGTTATTAAAAAGATCGGGGGAGGGGGGGAGGGGGAGAGGGGGGGGGAGGAGAGGGCGTGTCTTGTCGTTTTTG CCAGGTCAAAAATGCGAATCTTGGCATCAGGGACACCTCGGCAGAAGCGAGACTTTGGGTACGGCTTGTTCTTACAATAACGGTGACAAAGGGGGGGGGG CAGCCTCCCAAGTAGCTGAGGCTACAGACGGGCAGGACCATGCCCCACTAATTTTTTTTTTTTTTTTAGAAACAGAGTCTCACTTTTTTGCCCCCGCTGG GGAGCAAGTTTCATCAGCCCTTGGGGAAAAAAGGGCCCCCTCGGGGGACCCCTTTACTCGGTGTAAACGGGACTCTTTGGGGGGGGGGTGTGGGGGTGGG TTAGAAATGGCAAATATTGATGATTGTGTGCTATTGATTGGTGCAGGATACTTGGTATACGAGTAAATACTTGAGACTCGTGTCACTTGTTAAATTTTCT GGCTGGAGAGCCAGCCCTGGGAGTTCCTGGTCGGCTCCGACGCCCTCTGCTGCAACTTGGCTCGTGCGCTACTTTCAGACACTGTCTCGCACACTCACTC AGAATGTGGACAGCTACACCGAGGCGGGGGAGGGAAAAGAATATCTCCTCCCGCCGGGGCAGAGGGGGGGCCCCCAACCGGCTGGGCGCAGAGAGGAACT CTGGGCTCTTCCCTGTTCACTCGCCGTTACTGAGGGGATCCTGGTTTGTTTCTTTTTTTCCGCTGTCTAATATGCTTAAATTCAGCAGATCGGGAGAGCG CAAGGCACCCCTCTGACATCCGGCCTGCTTTTTCTCACATGACAAAAAATAGCCCCCATTTCCATCATATAACAAATCTCTTCCTCACTTAAAGCAAGAG GGGGCGGCAGAGGCAACTTCAGTGTCCCTGTTAGAGCAACACGGGTCCCTCCGTGGGGGGCTGGGTGCGGCCCCCTGTCGTGTATTTCCTCCCTAGGGAG GGAGAACGCGAGGGGAGTGAGCATTACAGAGTCTTTAGGGGGGCAGGTAGATTTGGGTTTAGGCGCCGCCCTGCCCCTTTTTTTTCTGTGTGGGTGGACG CTTCTACAGGGACCGTCACAGTGATGGAATCAGAGTCAAAGAGCTTCACGACCACCTCCGTGACACCGGGAGGGACGTGCGGGGGGGAGGGGTGGGGGTG GGAGGAGGCTGCTCCTCCCGTGGTCCTGATCTTTAGATCGGGAGAGGGGGTCAGCAGGGGTGGCGAGAAGGCGGCTGGTTGTTGTGGGTCTCTTTTGGTG GGGAGAGGGAGCAGGTGCAGCTGGACCAGGAGACAGGCTTCTCCATCACTGAACTGCAGAGGGAGCAGGGGCAGCTGGGGCAGGGGGAGTGAGTCTCATG CTGGGACCAGGTGATCTCGCCTTGGTGTTTAGGGAATGCTCATCCCTCCAACCTTGGCTTAGGATGATGCGTGGGGGAATAAAGTGTTCCTCTCATCAGG CGCCGCCGCACAGGGCCCGTGGCCCTGGGGGCCCGGGTGGGGCTGGGCAGCAGGGCGGCGGCCGGGGTTGGCACGAAGGAGTGCGTGCCCCCCGCGCGAG CGGGATCACCTGGCATGTCATCGCCCATCCTACCCAGCACAGATCGGAAGAGCGGGTCAGCAGGAATGCTGAGACGGATCTCGCATGCTGTGTTCTGGTT CCTGTGGCTTCCAAGTTCAGTCCTGGAGCCCCAGGTGGATCTGGGTCACAACCAAATCAAAAATTGGGGCACCCCGAAGCTCTTTCTGCTGGCACAGGCT GGCATTTCCTGCTGAACGTTTCCCCGAAGAGTCAGCCCCCGCCTTCTTGGTCTCTCCCTCTCTCTTTGTGTGACTGGGGTGGGAGGACGGGGGGGGGGGG GTCAAATATTCCCAAAATGAAGGAAGCAGGTTCTGACAGGTCACAGATACTACAGCAGGTAATAGCTGCACCCGGGGGGGGGGGGGGGTGTTGACTGGGC CCTCTCTCTGGACCAGCTTCTTGTAGACAGTGTCTGGGAAGGACCTCAGGGGACAGAACTTGCCGGTGCCCTCGGCGCACATGAAGACGCGGTTCTCATA CACTGGCCCTGCCTTGAGAAGAGACACAGGTCTCCCGTCCCTGCACCAGCTGAGAGAGACTTGCCACAAAGCACAAGGGTGGCAGAGAGTTATGTATGAA GGGTAATTGAGGAGTATGCTAAGATTTTGCGTAGCTGGGTTTGGTTTAATCCACCTCAACTGCCAGATCGGAAGAGCGGGGGAGCAGGGGTGCGGAGAGG CCTGTATGGCAAGAGGGGCAAGTACCAGGAGGCTGAGGCATTGTGGAAGCGGGCGCTGAGAGCGGAGGAGGGGGTCATGAGGAGGGGCGAGAGCGAAGTC GCCAGGTTTTGGTTGGTGCAAACATTCGGCCTCCCCGACACATGTTTCTCAAAGCACCCTCGCCAGAGAGGGGAGTGCCCCCACCTCGCAGTCTGGGTGT GCCAGAGGCCGCGCCGCGGGAGGCGGCACAGGGCCCGGGGCGTGGGGCGCAGCGGTGGGGGGGCGAGGGCGCTCAGGTGGGTGGGGCGGGTGTGCAGGGA ATCCCCACTATGCACATTTTATTTCTCCAACATACTCGGGTTCTAACCTCGCATCACACACCGCCCCATCCCCCCTACTCCGCCTACTCTCTGTCGCTGA CTGGCAAACCTCACGCAAGGCGAAGACCAGTACTATCTGCGGGGCACCACCGTGGGGTGTCACCCCTCTCTCTTCGACGGTGCTCTCCTCGTGTCTTGTG CGGAGCTTCCCGAACCTCTTCAGCCGCCCGGGGGCGCTCCCCCGGGCCGCGCGGGGGGGGGGGGGTAGCGGCGGCGGGGGTGCCGCGCTCGCGCCCGCGC CGCCTCCTCAGATTCATTGAACTAGGTCTGTCCCAATGTATGGGATGGCGGATAGTAAGTTTGTAATTACTGTGGCCCCTCAGAATGATATTTTGCCTCA CAAATTTTCAGGGTCCTACCTGTCACTCCCGGGTTCTTAGGTGGAACCTCCGAAGGTGTGGGCTCCGAGCGTGGTTAGTAAACTTTTTTAAAAGAGGGGG GTTCAGGGGAGAGTGCGTCATATGTTGTTCCTAGGGAGATTGTAGGGGGGAGGGGATGGGGGGAGAGGGTGTGGCGGGGGGGCGAGACGCGACACGCGCG CCCTGCTGGCCGGGAGCCCCTCCCCGGGGAGTTCTTGGTGAAGTCCTTCCCGGGCCTCCTTGTGTTTTTGCCTCATTCCTATTGTCACACAGTTCACGAG GGTGCCCAGTGCTCCGCTCAAGGGAGGCTGGATCTGGGGCCCACCTACAGAGAGGGCTCCGGGGGGGGGCGGGGGGCGTGTGTGGTTTGGGCGGCACGCG GGGGGATAAAGGGCTATTAATCCGTTTCTCAGGAACACGAGGCAGGGGAGAGGTTAAGGGAACCAGCCCTCTCTCGGTGCGGTTTTGAGTGTGTGTGGGG GCGCCCCTCCTGGTTCCCTGTCCCTCCCCTGCGCTCCTGCGAGACCAGATATAAAACTAGCTGCCAAACCAGCCTGTGGCGAGGGCACTGTCTATCTCCG CTAACTGGGCCCCCACTCCAGGTCCAGAGGGGCTGTGCTCACTGCACAGAAATCAATCACCCTCCGCTCCCCCGCCCTGTAGCGGTGTGTGTAGGGGGGC GTCACCAAGACCCTACTTCTAACCTCCCTGTTCTTATGAATTCGAACAGCATACCCCCGATTCCGCTACGACCAACTCATACACCTCCTATGAAAAAAAT CAGCAACTTCCTGGGGGCCATGTGGCTGATTTCCATCACCTTCCGCTCCCGTGGCTTCCGCGGACCTGGGCCCCCCCACCTCCTCGCGCGGAGGGGTGGG GAGCCCTGGGCCCTACTGACCATGGCGCAGGACAGCTGTCCTTCAGAAAGCACACGGTCCATCCCCGTGGGGCGCGTTCTCTCGCCGAAGTCCGCATGCC GTGAATCTCAAGATACTAATCCTCACATCATTTGCCTTCTCATATTTCCCTCGTCTTGACCATCTGGTGCCTGTTCTGCAGTTTAACATTCTGCCGCAAT TACAGTTCTATGGGTTTTGATGAGCACAGTAATTTTTTCAGCACCAACGGCAGGTTCTCCAAAAGGGAGGGGGGGGGGTGGGGGGGGGGGGGAGAGAGAG GAAAAATGCATTTCAGAGCCAAAGATGGTGATGATGGGGGGGAGGGAGGTGGTGGGGAAAAGAGAGGGGGGAGGGGGGGGGGGGGGGGGGGAGGGTGTGC TACTTGGACCGCATGCCTTTTTATAGCCAAATTTCTGTGTATTTCGGAAATGGGTTTTGCGTTAATGGATATTTTTGTTATAACTAGACTTCTCAGATTA GGTAGACAGTGCCTTTCTTGGTCCCTTTGAAGGCTTCTGGCAGGTTGTTCATGTCATTGAATGGGAGTTCTACGTGTTCATAGGGCGTTAGGATGCGTGT GGAAGGAGAAGGCACAGGACCGGGTGGAAGGGGGGCGGGGGGGGGGGCGCGCGGGGGGCCGCGCGCCACGCGGGCGGGGGGGGGGGGGCGCGCGGGGTGG TCCGAATCAGTGGTGGGAACGACAAACAAGGGTTCCCCCCGTAGCAGGGGGGGTTGAGCCCAGGGCGCGGGCGCGCGCGTCTGGGGGAGGGGGGGGACGT GTCCACCCCCCTGTGTCCCCCCCCCCCTCTCCCCGCCCGCCCCCCCACCACACCCCACTGCGCCACGCGCGCACACACCGTCTCGCGCACTCTCGTCATC CACCCGTCTATGTAGCAAAATAGTGGGAAGATTTATAGGTAGAGGCGACCAACCCTACCGCGCTGGGGTGTGGGTGTGGTTCTGGTATGATGATTTGGTT CCCAAGCCACCAGCAGCTGCTTCCAAAATCCCTATGCTATTACAGTGGGAATTACATCATTTAAAAAGCCTGATTATTCCAGGCTTCTAATCTTTCATAT CACTGCAGCAGATCATTTCATATTGCTTCCGTGGAGTGTGGCGAGTCAGCTAAATACTTTGACGCCGGTGGGGGTAGGGGTGGTTATGGGTGCGGGGGTG GGGGGCGTGTGGAGAGCCCCGGGGGGGGCTGCCCCGGGCCCCCGGGTTTGGAAAACTGAGAAGAGGGCAGGTAGGGAGGGGGGGCGGGGGGCGGGGCGCC CCCTGGGGCCCGGGATGGAGTGGCACCAACAGGGAAGGGGGGGGGGCCCAGGGGATGATGCGCGGCCCACTCCACCCGCTGTGCTCAAACTTCGGGCGGA CACGAAGGGGAGGGCCCGGTTCTTCTTTGGGCCCCGCGGGTGGTTGGTCCGGCGGCGGGGGGTCACGGCCCCCTTCTTTGAGCGTCCTGCGGGGTCTTCT GGAAGGTCAAGTTGTTGAGACAACTACTTTATTCTTGGGATGACTGTGGAGGTGGGGGGGGTGAGGCTTGTTTGCCAGATTTTCGTTAGTGGTTCACTAG ACACGCTGCAGGTGCACCAGGAGCTGCGAGAGGAGCTGGGCCAGGGGAGGAGACCGGGGGGGGGCGGCGGTGGGGGGCCGGGGGGGGGGGGGGGGGGGTG CGGGAATGAAGAGGCGTAGCCATGTTCACACTCTCGCTCCCGGCGCGCCACAAAAAACGGCGGCGCCCCAAGAAGTGGGCGGGTGGTGTCACTTTCCCCC CTGGGAGGTAGCAGCCCCTCTCTGGAGGGCACCCACCCATTCCCCATGCCCCCGCCCTCCTGGGAGGGCAGTGCCGTGAGATCGGGAGAGAGGGTCGTCA GAGACCACTTTGCCACCATCCGAACCGCCTCCCAGATCGGGAGGGGGGGGTAGTAGGGGAGTGTGGGAAGGAGCGGTTGGTGGCGCTCGTGTTTTGTTGT CCTCACTTGACCTTGATTACGTAAACAGTTTGACATTGTCTCTGTGCCCCCCAGCGGGGGCTCCTCACGTCAACAGCCGCGCGCGCGCGTGCCGAGTGTG GCAGGTAGAGGCTTACTAGAAGTGTGAAAACGTAGGCTTGGATTAAGGCGACAGGGGTGAGACGGGGGGAGGGGGTGTCGGGCGGGGGGGGAGAGGGATG ACACGAGCATATTTCACCTCCGCTACCATAATCATCGCTATCCCCACCGGCGGCAAAGTATTTAGCTGACTCGCCACACTCCACGGGGGGAATATGAAAT CACAAGAACACCTCTGATTACTCCTGCCATCATGACCCTTGGCCATAATATGATTTATCTCCACAGATCGGGAGAGGGGTTCATCAGGAGGGATGAGACG CTCAAACTTTGTCTACATAAAGCATATCATAAAAAAAACCCCTATCCATATCTAGTTATTTTTCATACTCCATACTGGCATTCTCCTCTTTTCTACTATT CGCAGCACCTACAACTACAGCCTCTCCATCACCGTCAAGAAGACATCTAGTCAGCTTGTCACTATGCATGACCTGAAGCAGGGCGCGAGTGCGGAAGGGG GCGGAGTTGGCGGAGCTGCGGTGGCCCCGGGCAGCCCAGTCCGAGTTGTTGGGCACCTTCACGGTGATGGCGCGCTCTGTGGCCAGCCGCTCGATGGGGG CGCGCGCGTCCCCCGAAGAGGGGGACGGCGGAGCGAGCGCACGGGGGCGGGGGGGGGGGCGGGTACCCCCCCCACCCGTGTTGTACCACTGCGCACGGTG CGGCAGAGCTGTAGGGGGGGAGGAGGAGGAGGAAGAGGAGGAGAAGGTAGCTACAGCAAGCTGGGTAGCAGGCAGATCCAAAGGATATCATGAAGTTTCT GCGGAGACCAAGATCAAACGGCCACTGGGTGTGAAGCTCAGATCTCTCACGCCCTCTCTGTGGCGGGGAAGAGTCATAAACAGGGGGCCCAGTGTCGCCG GTTCCCCTTTCAAATCCCAGTGCCGCTTTGTTTTCTTTCCAGATAGGAAGAGGGGTGCAGCAGGGAGTGCGAGGCAGAGCTGTGTGTTCGGCGTGTGTTT TCCTACTGTAAATATATGGTGTGCTCGTGTGTCTACGTCTATTCCTACTGTAAATATATGGTGTGCTCACACGATAAAACCTCGGAAGCCAATTGAGTTC ATTTTGTTTATTTTTTTAGAGACAGGGTCTCACTATGTTGGCCCGGGTGGGGCTCCAATCCTGGGGTTAAGGGGGCCTCCCCGAGTGGGAGAGGGGGTCA TCCGGGTCTTTGAAGGCAACGTCTTCTTTATCTGTTGCGATGACATCTTTCAGGAGGGGAAGGGCACAGTCTTGCAGATCGGAAGAGAGGTAGAGAAGGA TGCTGTTCCACACCCTGTAGTTGTGAGTATTTGTCCATGCTGTGACAAAAGTTGAGGTCGGGAGAGCGGGGGATGTGGGAGGGCGCGAGAGGCGCGCCGT GGTTCTATTCTCTATTTCTACTGTGTGTCGTTAAACAAACGGCGGGGAGCAGAGGGGCTGTTTTGGGGGGGTGGCTGTGTAACCTGTGTGTGTGTGTTGT AGCGCATGGGGGAAGGCGCTGTCCTGTTGGAACTGTGCAGCGATTCTGAGATCGGGAGAGGGGTGCTGTAGGGGTGGGTGGGGCGAGTATGCGTGTGACG CAAAAATGGTACACTGCAATTCTAAACATGTCCAAAGCCTACTTGGAGAGTGAGAATGTACTGGAACCTTCACCAGCCAACATATTGCAGGATAACTTCC GGGTAGGTCTGGTAGCCCATGAGCACTCGGTCCACGGCCCGGGCGCGGGGGGCCGCGGTCCGCCGGGGGCTGCGCCTCTTCTGGGGGTGTTTCTGTATAA CGCGCAGACTTCAGGCACCCAAGAGATCGGGAGAGGGGGTCAGCAGGGGGGGCGCGGGCGCAGCCGTGTGTCGTGTGTGTGGTGTCTGTCAACACTCTCA CCGGACCCAAGATGAAAACCCCGTAGTCCACTTCTTCAAGAACATTGTGACGCCTCGCACACCACCCCCGGCGCAGGGAAAGGGGGGAGGGCGGGCCCTG TTGGCATAGGTTTGCTTAGGTAGCCGGCTTCTAGAATGTAGATCCGTGGGGGGGGGAGGTTGGACTGGGCCCCACGCTGGGTCTCCTCACACATCAGGAT GTGCTTTCTCGTGTTACATCGCGCCATCATTGGTATATGGTTAGTGTGTTGGTTAGTAGGCGTAGTATGAGGAGCGTTATGGAGAGTAAGTGTGAGCACA GGGCATCTCAGAAGAAGCACGTGAACCCGGTGCAGGCCCTCAGAGAGTTCAAAGCTATGGACAGCATAGAAGTCTGCCCAGGCCGGTCGACTGTGTGTGT GCACGTTGTAGAGCAGGGAGAGAGACTGATTCATGCGCTCATAAATCACACGGGGGTGTGTCATAAACTGGGAACGGGTCTGGGGGGCTTTCTTGGGGTG CTGGGGCCTCGGTCTGTGGCTTAGAACTCGGTGTCCACCACGCGGGAAGCTGGGCTGCCTGAGTCAGGGCTCGACGGAGAGAGAGAGGTGGCCTACCTCT CCGGTGCAAAGCACAGTGCCTGACGTGGAGCAGACACTTGACAAAGACACAGTGAGTGGTCGGCTGGGGGAGGGGGAGGGGGGGAGTGGGGCGGCGCCCC GGGGTTGCAGACTTTCACCCACAGAATATGGTCCAGGAGCCAGTCGATGGGGTTGTTCCGGGAGAACATGAGAATGAAATCTACAATCATGCTCAGGACC TGGAACTGCTGGCACCGCTACTTTGTCGGGAGTGTTTTGGGCCAAAACCCCCCCTCAACATGCTCATTTTTTAACAGCCACGCATGCTCTCACTGTTGTG CGCTTCCCCCTCCTCGGCCACCTTCCCGGCGGAAGCAGCGAGGAGGGAGCAGATCGGAAGAGCGGTTCAGCAGGAATGCTGAGAGCGAGCGCGTATGCAG CCTGGCTTAGCTGTTATCATCTTCCCCAAACTGAAGGGCCCACCGTCCTCGCGCTCCAGCGGCGGGGGCGCTGGGGGGGGTGCGTGTGCGGTGCGAGGGG AGGTGATGAGGCTGACGGGGGGCGGGGGCCAACACACGGTGGTGGGCTTTGGTGAGGTGGGACCAAAGCGGCGGGGGGGGGAGAGGGGCGGTGAGGTGCA CCCTGGGCCACAGCAGAAATAATGTCTCACTAATTTTTGCATTACTAGCACTTAGCACAGATCGGAAGAGCGGTTCAGCAGGAATGACGAGAAGGGTGTC CGCGGGTCTGTCTCTTGCTTCAACAGTGTTTGGACGGAACAGATCCGGGGGGGTCCTTTCTCTCCCCCCGCCCCCCGCCTCTTCTTACTCTCGTCTCTCC AGACCAAGCATCCGGGCCTGACCAAGGACACAGACGGCGGGGGTTTGTACACGGGGGGCCCCCCCCCCCCGGGGCCGCCCCTCCCGTAGCGGGTTGGTCA ATGGAAACCAGGTCTGATTACTGTCTTCCAGTAGCTGATTACAACTACAACCCCCCCCCCCAGGGGGGGGGGGGGGGAGAGGGGGGGGGGGGGGGGGTGG GGTTGTCCTCCGACTCAGGTTAGAATGAGGAGGTCTGCGGCTAGGAGTCAATAAAGTGATTGGCTTAGTGGGCGAAATATTTTGCTTTGTTGTTTGGGTA GTGAGCTGAAGCCACACATCAATGGGCCCTTCACCCCTGACCTGGCTCACCCTGTGGGAGAAGTGGGAGCGGGGGGCGGGGGGGGAGGAGGGCGGCTGGG CTTGGTGATGGTGATCTCCTTCTCTTTTGACGTGATGACCTCGATGGCCGCCATGAAAAGGGCGGGGGGGGGGGGGAGACCCCGAAAACATAGTGGCGCT GCTTCCTGTACAGCACGGCGCTCCCCAACCACGCCACCCGAGGGGTCAAACAGGGAGGACCTTTCCTCCCCCCTTCCCCCCGCGGACACGGCCCGGGTCG ATACACATGCCTCCATGCATATATGTGTGTGATAATACACATGCATTTATGTGCGTGTGTGTATACACATGCATATATTTACTAATCCTATCTGCCACAA CGTTCACGCACAGCTGCAGGGCCTGGCTCTCCTCGTAGGGCATGGGCCGGGCTTCAGGCATACGGATGGGAAGGGGGAGGGGGCGAGCCCACCGCGCGCG CTCCTCCATCATGTCTGGAGTTACGCTGTTCTTTATGTATTGAGAGAACTGTTTCTTGTAAGCATCTTCATCTTCTTCCATTAAGAAGCGCATGTAATCT CTGCAGTCTTCATCTAGCCACACATCAAAAGACATTTGTTCTATCAATGTTTTGAGATCGGAAGAGCGGGTGAGCAGGAAGGACGAGACAGATCTCGTCT TTGGGATGATTCAAGCCCATTACACTTACCATTCTATTCTCATAAAAGAGCGCACAACCTTGCATGCACGGTTCACAGTTGGGTTCTGGCTCCTGTGGGA GTCCATTTTGTGAAGCCCTGTTGTGCCACAGAGTGTAGCCAGGGCCCCCTGCAGCTAGTCCTAGTGAACCTCAACCCCGGGGCCTGCATGGGGCAGGCCC CGCGGGCCCAGTCCAGCTTCTCGGCCACAGTGCCTCCGTTCACCCGGATCTCCATCAGGTGGGGCTTCTTTTGGCGGCGAGGCAGGAGGCGCATATGGGA CCTTCACAACTTTCTTCAGGGAAGTCTTCATATCATCCTTGTTGGAAACCTGCTCAATCTCATCTGGAGGGAAAATCTTCTTCATGCTACCCCGAGTCAC CGCGCGCCTGTTGTCTTAGCTACTCGGGGGGGTGGGGCAGGAGGGGCGGTTTTAAAAAGGGGGGGGGGGGGGGAGGGGGGGGGGTGTGCAGCCCCCGCGC GCAGCACCGTGTTGATGTGAGCAGTGTGCGGGAACGGGTGGGCCGCGTGGGGCAAGGCAGTGCCGGGCTCTTGGGGGGGGGGCTGAGCGTGCAGGATGTG CTGGCACCCAGCCTCTGAGGGAAGGAGGGATGGGGCATAGTGGGAGACCCAGCCAAGAGCTGAGGGGAAGGGCAGAGAGCGGGAGAGCGGGTCATCAGGG CGGGCCCTTCCTCTTCCAGGGGGTGGGAGGGTGGAGGTGGGGCTAGGACCAACCCTCAAGAAAGAAAAGAGGTTAAGGGGGGGGGTTTGCTGAATGTCTA TATGCATTTCAAAGCCCCCAATATTTCTATGCCTGATGTTGATCTAAATCTCAAAGGACCCAAAATCAAGGGGGATGTGGATGTGTCTGTGCCTGAGGTA CTGCAGTGTCACCTGACTGTACTACCGCAGAGCCAACTTTACATCTTGGCACGTTTTTCGCTACGGGGGGGGGGGGGGGCAGCGGTGAGGGGAGAGTGAT GGAACACAGCCACACCCGTGATGATGTGGGTAAAAAGTTCCATTTTTGCCGCAATGGGGGGGGAGGGAGAGAGGGGGCGCAGGGGGGAGTGCAGAGCGCG GGAACATTCAGGTATATAAATAAGGTAAATGCTTTCCAACGTACAGTATATCATGTTCTTACATTGCGTGGAAAAAGAACCTACATGTATTGTTCAACAT GCCAAGAATGTTTTCATTAATCAAGAACGAAAGTCGGGGAAAGGGGGAGGGGGTGTGGGGGGGCGGGGGGGGAGAGGAGAGCTTGTGTGTGCTCTTCTTG CTCTTCCAGCCCCAGCACGTCCACGAAGCGCCCCCGGGCGGCGGCCCCCCCCCCGGGCAGGCCCTTTTCTCGCATATTTGGGGGGTGTTTCCCTCGCTTT GCACGGTGAAGAGACATGAGAGGGGTAGAATAAGCGGGGGGGGTCGGGGGAGGGGGTGTGGAGGGGGGGGGGGGAGGGCGGGAGATGTGTGGCGCTTGTT GGCCTCCGTTGCCCTCGGCCGATCGAAAGGGGGTCGGGGTTAGATCCCCGGATCTGGGGTGGGGGGGGGGGGGTGGGGGTGGGGGGGGGGTGCGGGAGGG GGTTCACAAGCCATGGTCGGGGTCCGGGGGGGACAGAAATGGATTTTCTAGATCGGAAGAGCGGTGCAGCGGGGGTGTGGAGAGCGGTCTCGTACGCGGG CTTTAGTGTTGTGTATGGTTATCATTTGTTTTGAGGTTAGTTTGGTTAGTCATTGTTGGGTGGTGATTAGTCGGGTGTTGGTGGGATATTTGGGGGGGGG GCAACACAGGGGCCTGGCAGCTGGCTCCGAGGGCAACACAGGGGCCTGGCCTCTCGAGGGCTGGTGATTGAGGGGCGCGGGCGGGCGGGTGGGGAGAGCG GCCGGACTCACTTCTTACACTCCGGTGGGGGGGGGGGTGGTTTGGGGCGCATGGCCTCCATTTCCAGTTAGCTTAAAACATAGGTCACGCTGGATTTCTG CTGGGCAGCACCAGTGGTGAGGAAAGTGACTTGGAGAGGGAGGTAAGTGACAGCGAGGCAGGGGGGGGCGCCCAGGGGTCGGGAGAGCGGGTCGCGAGGA TTCTGACCCCTGGAGGAGCCACTGTGGAAGCAGAGCAATCGCCATGGAGTTTGTGATGAAGCAGGCTCTAGGAGGGGCCACCAAGGACATGGGGAAGATG CAGGCCCTGGCCGAGGGCCGCGAGCTGCGGCAGCGCGTGGTGGAACTTGAGACGCAAGATCGGAAGAGCGGTTCAGAGGGAAAGACGAGACCGAGCTCGT CTGCAAGTTGAATGTCTTGCTGAATGTGTCTAGGGGTTCATCTTCAGTAATCGACATTCCACTAGTGCCATAGTTAACTTCATGACATGTAGACATTACA CAACTTGGTCACAGGCAGACAGGTTGGCCACCACCGTGGGGATGACATCAGCGGGGGCGGCGACGGGGGCGGGGGGGGGGGGGGTGCCGCCACTCCACAT CCACAGTCCATCTCTGACCTCCTTATATAGTAAATCACATAATGTCCACACAACCACACACAAACACACTTGCACAGGCACACGCCAACAACTAAGTAAC GGCAGGTCCGCAGGTGCCCGGGTGCCAGGGTGGATTCGCACTTGCTCCCACTGGAAGTCATCGTACTGGGCCTGGCTGGTCTCGCAGGGCACTGCTGTGT CGAAACCAGACGAGCTACCTAAGAACAGCTAAAAGAGCACACCCGTCTATGTAGCAAAATAGTGGGGAGATTTTTAGGGAGAGGGGAGAAAACACACGCG GTCCTGGAAGACATCTTTACTTTATGGAGACAGGTGGAAACCAAAGTTCGAGCTAAAATCCGTAAGATGAAGGTGACAACAACAGTCACCCGGCATGACA CCCATGCCCTGCTCCGCTTCGCATGCCACCATCAGCCTGGGGGATGTGCCTGGTCATGGCAGCACCCATTCAAACATAAAGGCTTCTCCTTTCTTGCCTC CAGCTGAGCTCTTCAGAAATGCGTCACATTCAGCGTTCCCTTCCTTCGCTTCCTCCACTACCTATGACATAGCGGGGGGAAGAGAGACACACGAGAGAGG GGAAAATTCTGTGGGATTAATTATTGCCATTCTCATCCCAGTGGCAGTGGGTGTGTGTTTTGGGGGGGGGGGAAGGGTCGTTTTTGTTTTGGAAAGAGAG CGGAGACAGGCCGCCGCCGCCGCTGTCGCTGTCGGAGCTGGGCGCGCTGGGCAGGGACAGGCCGCCGGGCGGGTGCAAGTGGCGGGCGTGGGTGTTGCAG GAAAATTCCATTTTAAAAACGTATTCACTTTACTGATTACTGTGCAATTTGAATTAAGTCACGATTCTTTAGTAAATGGAGGTGAGAATCTCAGATTCAA CTGGGTCTGGGCTCCATCCTGCGCCCAAAGACAGATCGGGAGAGGGGGTCAGGAGGGAGGGCGCGACAGAGCTCGCGTGTGTGTGTTTTTTGTTTATATA GTTTGAGCATCTGCCCAGTTCCTAGCAATGGTGGTCACTCGGATGCGTCTCTGCCCACTTGAATGCTGATACTGAGTCACAAAATGGATTGTACCACGCC CACAAATGTCTTCTGGTCTAGTTCTGTAAGGCAGATCGGAAGAGCGGTTCAGCAGGGAGGCCGAGAGCGATGTCGTATGGTGCTTTTTTCTTTGTAGACA GCAAAAGAAGGAAGATTGGGAGCGCATGGTTTAGGGTGGGGTGGAGTGGAAGGGCAAATGGGGACCATACTAGACTGCAGAACTAACTAACAGGGCACGG GCCCCTCCTGCCTGCTGAAGTCACTGAGACCCTGCCACACCTCTCCTCGCCACTGTCACCACTCAGGGCACCACTGTACAGATCGGAAGAGCGGGTGTGC GTCATCTCGTGGCTGGACGCCAACACCTTGGGCGGGAAGGGCGGGTTTGGGAACAGATCGGGAGGGGGGGGTGGCAGGGGAGAGGGGAGAGAGAGTCTCG CTTGCCCCTTCTTTGATAAGGAAACAGTAACGACAGGAATTCCAGTTTTACTGGGAAACAGCCAACCCCATGCTCAAAGCCTACCCTCTGCCCGTCTTGT AATACAGATCATTCATAGAAAGAAGGAAGAGAAGGGGGACCCTAATATAGGGCTTAGTTTTTTAAGATGAAGTCTTGCTCTGTCACCTAGGCTGGAGTAC CGTGGGCATATTTCCGAGCAGTGGCTCCCTTTGGGGGGGGGAGGGGGGGGTCCCTTTTCTCTTGGGTGTGGTTTTTTATATGGGGTGCGTTCTGCACACT CTGGGACTCTGGGTCCCCTACTGCAGACACTTTCCTGTGAGCCAGAAGTGTATAAAGGGCTGGTGGGTGACCATCCTTTGGGGAAGGTCATAGGGGGGAA GCGAAGGGGCCTGCGGCGTATTCGATGTTGAAGCCTGAGACTAGTTCGGACTCCCCTTCGGCAAGGTCGAAGGGGGTTCGGTTGGTCTCTGCTAGTGTGG CCGGCACACCCCCATCTGGCACTTCACCACATCCTGGACCTCTCGGCGCCGGGAGCAGTGCACAGCAGTGATGACAAAGACCTTGGTGGGGTTGCAGACA GCACGTGGAGATAGTGCCAGGTGGCTCAGTGCCACCTATGCCTGTGGTGAAGTGTGGCTGGTGTCCCCTTCCCCTGCTGACCTTGGTGGAATGACAATAA GCGGGCTTGATGTGGGGAGGGGTGTTTAAGGGGTTGGCTAGGGTATAATTGTCTGGGTCGCCTAGGAGGTCTGGTGAGAATAGTGTTAAGCTCGGAAGAG CAGATCCCAAACTTGACTGTTGTGTCATCCAGGCAGACAGTCTGTGTGAGGAAGGCCGCTCCAATTGTGCTCTCCTGGTACTCGTGAAACTGTCCCTTGA TCAGAATTTCCAGAGAGCTCTTCCTGGCTGAAAAGATGTCCAAGGATCATCTCCCGGGGGGGGGGGGTGGGGGGTGTTTGTGTGTGGGGTGTGTGGGGGG TTGCATTCATAGGAGCCCAGGTGGTTCTTCATGAAGTACGGGTCCTTGTTGATGTCGATGGTGTCCAGGGCCCGCTGCCGGAGGGGGGCGCTCGCGGTGG GCGGGATGGCGGGGGCACTTCTGGGTCCCAGGGTGCACGGGATCCGGAGATCGGGAGGGGGGGGTAGCAGGAATGCTGAGAACGAAGTGGTCTGTAGTCT GGCAACTCTGGTGTTACAGTTGTGTATCTTGGATCCAACCTGGGGATGAAATCCTGAAAGGGTTTAGATGCACCAGTACCAAAAACATCAAGGACTTTTC AGCGGCTGCACCATCGGGATGTCCTGATACAACACTTGGGGGGGGAGGTAGGGGGAGGGGGGGGGGGGGGGGGGGGGGGGCGGGTCGGGGTCGGCGGTGT GTAGTGGTGAGGGTGTTTATTATAATAATGTTTGTGTATTCGGCTATGAAGAATAGGGCGAAGGGGCCTGCGGGGTATTTGATGTTGGAGCCTGAGACTA CTTTAACACAAAGATTATGTTTTTGTTCAGCAGATGGGGAGGGGGGGGGTGGGGGAGGGGGGGAGAGCGGGCGGGTGTGTCTGGGTTGCTCTGTGTGTGC GTCACAGTTAGGACGGGAGATGGTGAGATCGCAGATGGGAGATCGGAAGAGGGGGTCAGCAGGAATGCCGAGAGAGATCTCGTGTGTCTGTGTATTCTGT GCTACTTTAGCAAGATGTGGCCTTTCACAAAAGAGGTAAGAGTGACCAAATAGAATTTAGATCGGAAGAGCGGTTCAGCAGTAGTGCCGAGACCGATGTG GTTTTATGGGGAATGATGAGGGCTGGTCAGTTCTCCTCATGACAAAAGTCAAACCGACTTCCCTGTGTTGCGTGTGAAGCTTGTTAGTGGACAGAGAGAA TAGGCAGTGCCAGCTATGGCCCCCCAGCAGACAACAAGGCACAGTGGGGCTTGGGGACTGGGGGGGGCCAGCCCTGTCCGCCTGTCCTTGACATCAGTGG CTCGGGAAGGGTTGGTGTTCATCCGCTTGCGGGGGGAAGCCAGGTACTTCAACTTGTTTCTGTTGAAATTGCCAGAAATGTTGATGGCTTTACAGCGTAC CAGAAAAATGGGTGAAGGATAAACCGTTGAGACGTGGCCCCACTGGTAGGATGGTCCTCTTGTACTTCGTGTGCTCCGACCCCTGGGGACGATGACACAC GCTTGTTCCTTCTCCACTGGGACAGCTAGGGAGGCGGGGGGGGGTTTCAGCAGGATGGGAGAGGGGGGGTGCGGGGGTGATGCAGGGGAGGTGGCACGCT CAGCATCACTGCGTCCATCAGCTTGAGGACCTCAGCGCCCGTCTTGAACTGGTAGCTTTGGTCCCGGCTGGTGAGTAGGGAAATGTGTTGGTGCACATGG CTCCATCTCTACCAGCGTCCCCTCCCGTCTGCACGGGGCAGTCCTCCTGGGCTTGACCTCTCTGTGTCCACAGCTGTGCGCGAGGCAGCCTCACTCTCTC GGGCGCTGTTCCCTTGACACTTCGTTCAGCGTCTTCAAGTACATGGCTCTGTACAGCCTGAACCAGTTCATCTCCGTCCTGATACTCTACACTCTCCACA TGAATTTGGGGTAAGAGGAAGTGAGATCTCCGCTTGCAGGTCAGCCCCTGCCTTGCAGGGCGGGCTGGCTTGACTCAGGCCCTGTGAGATAGAGGGCCCA CTGTCCCCAGGGCTGGGGTTCCTCCACCACCGTTCCAGTGCCTCCTGCCACATCCTCCCGGCAATCAGTCTGTCCATCTTGCCTGTTGTTTATTGTCTTC CGGGGCCACAGCTAACTCCTCCCCCATTTCCACCCTGGAAGGGCCGCCCCGCAGGGAGACTGTTCTGTAGCCCCGGTCCAGGCCACTCCCCCCACCACAC CTGCCTCCTCGGAAGCCTCCTCGCCCTCCAAAGCCTCCCCGGCCTCTGCCACCAAATCCTCCTCGGCCTCCTCTACCACTACCTCGTCCTCCAAAGCCGC GCCCCCTAACTCAGCCTCTGCTTCAGCTCGGTTCCCATTTCCTGCCTCTACCCCCCAACTCCTTATAAAGAGCCCCGTGAGCTAAGACTAAGGAGAGGAT CGATTTCATAGACCCAGTTCACACAGCGTCCTCTGGGATTCACAAGCCTGAACCTGCAATGACAGAGGAAGGTTTACCCGCAAATGGGGTTGGGAAAGAT CTGGTGTGCATCACAAAAGAGTAGCCGATCCCCCCCGGGGGCCCCCCCCCGGGGGGGGGGGGGGGGGGGGTGGGGGTGGGGTGGGGGGGCGACTACTCCT ACGCAATGGTCCCTCGCATGCAAGAAGTCAAAGAGCTCCTCCGTGCAATCCTCTTCTGTATGTGATCGAGAGGATACACGCTCATCACAAGACTTTTGCC TCTTCATGTTCTTGTTTAGGTGATACTATTTTCTGAGGTGGTCGGAAGAAAATGTACTCGGGGTAGCTGCTCCAGATCTTGTCGTTGCGGGGCTGGTGTG CGCCGCTTAGTGGTATGTGCTTTCACGAGCGTCGTAAGGTCACAGCTGTACACCTTTTTGACATGCTTCAAGTCTGGCTTACAGTCCTTTGGGACCATCT GTGCCGTTGACGTGGACAGCGCCCCTCCCTTAAGATGCCCCCTTGCCGTTGCCATGAGCCGCTGTGACTCACGCGTGCACTGGGGCTTGCTTGGTGCTCT GGATGATGAGGCTATTGTTTTTTGTGAATTCTTCGATAATGGCCCATTTGGGGCAAAAGACGGTTGGCGGGGGGGGGGGTCCCTGGGGGGGGGGGGGTCG TCTGCAAATCACACCCTCCCCCCACGAGTTCCTCCTTTGAGGCCAGCAGCACCCGAGGGAGGGCAGGGGCTGCACAGAGACCAGAGACAGGAAAACCACA GATCTGTCCTGGGCGGGGACCAGGAGGGAGCGGGGTCGGGATCACCTGCCAGTGTGCAGACTCTGGGACTGCGTGCTGTCTGCGGACCATATGTGTAGGG GGCCGGGGGGCGGAGAGCGGACGCAAATACTCACGGCGGAGCGGTCCGAACCCCGGAGGGCGGCGGGAGGGGGGGGGCGCCGGCCGCGCCGCCACCCGCC GGCCAGCCCCCCAACTATGAGATGCTCGAAGAGGGGGACGGGGGGGGTGGGGGGGGGGGGGGGCCCACACCGCTCTCCCCCTCGCCGCCCGCGGTGTGAC TTACAAGTAGAAAGACCATGCTTGAGGACGACAGAAGTTCACTAAGGATGCACGATTGCTCTGAGAAGTTTTTAAGCTTCGTGTTCGCGCTGCTCCGCGT CCCACTTCGGGCCGAAAGCACGGTACAGAGGGTGGGGGGGGGGGGTGTTGGGGTGCCCCCCCCCCCCCCCCCGCCGTGCGGTTTCACGGGTGGTGGTGTG GTGCATCTGAAGTATGTACAATAAAACACCAACACAGAGATCGGGAGAGGGGGGGTGCCGGAGAGGGGGGGCGGGGGGTCTTCGTCGCGTCTCTCTGTGT GGCTGAGGCAGGAGAATGGCTTGAGGCCAGGAGGTGGGGGGTGGAGGGGGGCGGGGGTGGCCCCACGCACTCCAGGCCGGGGGGTCGGGGGGGGGGGGGG GTGGGGTCCGCGCAGTCCGCCCGGGGGGTTAAAACCCGCGGGGGGGCCGGGCGCGTGCGGGGGCGCGGGGGGGGGTTCCCGTCCCTCCTCGCACCCCACG CCGGCCACATGATCCTGTCCTACATGGAGGACCACCTGAAGAAAAAGAACAGGGGGGGGGAGGGGGGGGGAGCGCTGTGGGCCCACCAGGCGGGGCGCCA GGAGTCTGTCTTCCACCTGCTGGTGCCCAGTGTCATCCTGGTCTTGCTGGCCGTGGGGGGGCCCTTTTTTTCTCGGGTGGGGGGGGGGGGGGCGCCATGG CGCTCAGGGTGCGTGCCCTTCGGGTTAGCAAGACAGTCTTGTCGCTGGGATGGAGAATGACGGGCACACCCCGGTCCACCCACACATCTGTGTGGAGTGC CTGGGAGGGTCGAGCCGTCACCTTGCTGGGGGTTTTAGATCGGGAGAGCGGGTCAGCAGGAGTGCTGAGAAGGAGCTCGTATGTCGCCTTCTGTTTGTGT GCTAGTGACTAGTTTCTTTGCTTTTTGTGTTGTTCCGTATGCTAACATGTGCATGGGAAAAATTTAGAATAGCCAGGGGGAGTGGGGAGGGGGGTGGCAG CAAGACAGGCCTTCCCCAACCACAGAGCCCCCTGCGCCACCCAGCCCCGCGCTCTCACCCGTGCCCAAGAGCCCGTCTGTGGACAGTGACAACGTGCGAG CGTGAGCACTGCTCACTTACAGTTTCCCGGGGCTGAGTCCAGGGCCAAAGCTGGGCCCCTGGGAGGCAGGATAGCCCACCACGCAGGGCCTGCTGGGGCG TCCCTCTCTCCTACTCCTGCTCGCATCTGCTATAGTGGAGGCCGGGGCGGGGAGAGGGTGGACAGTTTACCCTTCCTTTGCAGGGAACTAATTCCACCCT CGACGGCCTCCTCTCTGCCCCGGGAGGTGGTGCCCTGGGGGGAGGGGCCGGGGCCCGCGAACGGGAGAGCGGGGGGGCGGGAGGGACGGTGCAGGTGTAG GCTGGCCCTGCGGCCCCGCCCCATAGGGGCAGCTCCTGGAAGACAAAATTCAGCATGATGGAAGACTGCTCCCTGTGAGGTCTGAGATGCGTATTGGCCG CTGAGTACCACACACTTGGCCCTCTAGCATGGGGAGGGCCTGTCTGGTGCACCTGGGGGGAAGGCTGGTTTTCTGGTGCAGAACCAGAGATCGGGAGACG CAGATAGTCTGAGAAAAAGTGTCGCCTTATTAAAGGACCGGGGGGGAGGGGCGACAAATTTTTCTGACTGGTGTATGTGGTATTGTGTTGTTGATAGGTG AAGAAAACAGGTCCCCCCCGGGGGGCTGCTGCCTAAGGCACCCCCCTCAGGACGGTGGTGGCGTGTCACCCCCGGTGGGGCGGCCCTCGTGTGGGGCGTG CTGAGAACCAAAATGAACGAAAATCTGTTCGCTTCATTCATTGCCCCCACAATCCTAGGCCTACCCGCCGCAGTACTGATCATTCTATTTCCCCCTCTAT CGTCCGAGCTGCTGGTGCGGGGCAGCCCTGGCCCTAGGCTTCCTAACCCTGGAAGCGAGAGGATGAGAGGAGGCCGGCGAGAGCGGGAGAGCGGTGCTGA GGCCAGCAGCAGGCCAGTACAATATGCTGCAGCATAATTTGTCAGGCCAACCTTCACACCATATTTTGGCAGTTCGTGTGCATCCGCTGCGCCGCCTATT GGGACCCTCACCGTAGCCCTCCCTGCAGGCATCAAGCAGATCCTGCCACACCAGATGACCACGGGAGGGGCCGGGGCGGGGGGGGGGCGGGGGTGGCGCT CTCCCCTAAGCCTAGAATGACTCCGTACACGGCTCCCCTCTCTGCACCCATGGGAATTAATCCCACCGTGCCCCGGGCCTCCGCTCTGGTACAGACAGGG GTCGCATTCCAGATCATCCACTGGGAAAAGCATCACTTCTTACACGTGGGTTTTAGAGATCGGGAGAGGGGGTCTGCAGGAAGGGCGAGGACCGCGCTCG AAAGTGACATGTGCAAGAGTCTTGATAATCCCCCAAACCTGTATCTTGGAAGATCGGGAGAGGGGGTCAGCAGGGGGGAGGAGAGCGAGGTCTGCTATAG CCCACCTCGGCCTCCCAAAATGCTGGGATTACAGGGGTGAGATCGGAAGAGCGGGTCAGCAGGAGTGCCGAGAGCGCTCTCGTATGCTGTCGTCTTTTTT GGCCAGGCGGGCGGGCGTGGGACAGACCTCGGCATCCCCAGCCTCCGGGCCCTCGCCCTGGCCTGGCTCCCCCTCACCATCCGACTCCTCCAGCAGCCCC CGACTACGGCGGACTAATCTTCAACTCCTACATACTTCCCCCATTATTCCTAGAACCAGGCGACCTGCGACTCCTTGACGTTGACAATCGAGTAGTACTC GCGCAATGAAGGTGAAGGCCGGCGCGCTCGCCGGCCGAGGTGGGGTCCCGAGGCCTCTCCCGTCCGCCGCGGGGGCGCCCCCCGGCCGCGTCGCCCGCCG GTCGCAGTCTCCAGTGATGAGCATCCCCCGTTGGGGGCTTCGGGGGGTCTTCTCTGCGGCTAATGCAGTTCTGGAGATACACACAGACTAGGCGTGGTCT CGAGTAGCTGGGATCACAGGCATGTGCCACCACACTCAGCTAATTTTTTATTTTTAGTGCAGACGGTGTTTCACCATGTTGGCCAGGCTGCTGTCAAACT GAGCGCTTGCTACAGAGTGTGAAAAACTCGATGCTCCAGATGGAGATGGGTGAGGGGGCCGCGAGATCGGGAGAGGGGGGCTGCAGGGGTGCCGCGAGCG CATACACACACACACACACGCACACACACGCACACATGTTGCCGCTCCTGTCAATTTATGTACAAAACAGGGGGAGGAGGAAGGGGGGGGGCTGGGGGGT TCTAGACAGACCTCTGTTGAGCAGTCAGAGTCTTCCCTGACTCCACAATGCCCCTTTTCTTTGGTGGCCCGTATGGCCACTGGGCCCCCCCCGTGTCCGC CCTAGCAAGAGCAGAAGTTTCAAGTAGCTCCGAGTCAGAGGCGCGGGTGCCTCAGCGAGGAGAGGGGTGGTGTGGGGGAGCGTTTTCCTGTACTGAGCCG AAACAACTGGTTCTTGCCAGCCTCTAGAGAAATCCCCGAACACTCAGCCCTGACACGCTGATACACTCCCACCACTCGAGGGTGGCGCGCGCGCGCTGTG CGAGTACGACGTGGCCTACGGCGAGAGGCACCCTGTTCGTGAGGATCGGGGGAGGGGGGGCGACGGGGGGGGGAGGGGGGCGCAGGACGTGCTGGGGTGT CAACGTAGTACGTGTCGTGTAGTACGATGTCTAGTGATGAGTTTGCTAATACAATGCCAGTCAGGCCACCTACGGTGAAAAGAAAGATGAATCCTAGGGC TGGGATTCGTGGGACGTGGTTTGCTCTTCTATCACCTGCTTCACAATTTCTGCCAGTCTTAGTCGATCTGTTTTATTTCTCAATCCACGAGCATTCTCGC CCAATACCATCATCAACCTCATAATCTGAGATGTCACTATCACTGATTCGCTGAGATCTTTGAGATCGGGAGAGCGGGTGAGAGGGAATGCGGAGAGCGA CCGAGCTCAAGCAATCCTCCTGCCTCGGCCTCCCCAAGTGCTGAGAGCGGGAGAGCGGGTCTGCGGGGAGGTGGGGGCAGGGCGGGGTCGTGCGTCTGTG GGTGTACTTGCTTCCTCGTTGTAAAGTCCGTTTCTTTCTGTAGGTCACAGTCAAAACACGGAGAGCCATGGCTACAACTTGAGCAAGTACACCACGCGGT GAGAGATTGGGCAGGGGGGGGGGGGGGGGGCGGGCCCGGGGGGGGGGGGGGGGGGGCGCGCGGGGGGGGGTCGGGTGTGTGGGTGGGGGGGGGTGGTGCG CCCTGGCGGAGCGCTGAGAAGACGGTCGAAATTTTTTATTTTGAGGGAGGAAAAAACGGAGGGTGGGGGAGGGGGTGGTGGGGGAGAGTGGGGGTGAAGA GCAGGGACAGCCGCCGTGCGCATGCGTCACGGGCCCGCGCCCCGCCGCCCCCTTCCCCTCGCCTAGCGCGCGGCGGAACGCTGGGGACGCGACGTGAGCG TGGCAGTTAACTGCTTTTCTCTTTCAAACTCCTTCAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACAGATCTCGTATGCTGTCTTTTGCTTGTGAT GTCCAGTAGCTGCCTCGCTGCTCGAAGGCTCCGGTCTTTTTCGTCGCGGACCCGGCGCCAGCCCAGGGAGGGGGGGGGGGCGGAGAGCGCGGAACCCACC CCAACCCCCAGTCAGTGTTTGAGCCTCCTCGTTCCCCTCACGCACCCGCTCACGCACCCTCGGTGAGTCCTTGGTGATGATTTTGAGATCGGAAGAGCTG GTGAGAGTGAGTAGTAGAATGTTTAGTGAGCCTAGGGTGTTGTGAGTGTAAATTAGTGCGATGAGTAGGGGAAGGGAGCCGTGTTGCCATTATTAACTCT CCTGCTTCTCCGTGGCCCCCAGCTCCTCTCCGGCCAGGCCCCCAGCAGCCCATGAAACAGAAAGCAAATTTCACCAGAGACACACATGGAAGCCCTACGT GAAGAAATGCATTCACACAGTCGCAGAGATGCAGGCCTTGCCAGTGGTGTGCCGGGCGCGGGTCCTGTCTGGCGGCGGCGTGTCGTGTCTCGGGGTTGAA CAGCTGTCTCTTACTTTTAACCAGTGAAATTGACCTGCCCGTGAAGAGGCGGGAGATCGGAGGAGCGGGTCAGCAGGGATGCCGAGGCGGCTCTCGTATG GCCTTCACCTCTTGGACCTTTCCTGGTTTGGGCTCCTGAGGTCGGGAGAGAGGGGCTGTCGGGGGGGGGGGAACGGGACTGGCTGTTTGTGGCGTTCTTC GCTTGACATCTCCATCACCAAATCTCCAGGAAGACCCTGTTTCCTATGTGAGGCCATTGCCCAGCCCACGTTTGCCTCCTTTTCCTCTCTCTGCCACACG AGAATAGCCTTTGGAAGGAACTCCTTTGTGGCTGCGTGGTGCCTGGAGTGGGGGGGCAAGAGCGGGAGAGAGGAGACAAGTGAGAAGTTACAGATTTTAG GTTAATGACATTCAATTCTAAATGATACATTGGAATTGTGCCTTTTCTACCACACTGGATTAAATAAACTTGTCAAAATATGGTTTTGTCATTTTCTGAG GTCTGTAGAGTTCCGTGACTTCCTGAAGATAGCCCTGGATAAGAACCCAGAAACCCGACCCAGTGCCGCGCAGCTGCTGGGGCATCCCTTTGTCCGCAGC GAGCACCCCCTGCCATCAGGGGGGTTGAAATCCCTCTCCCCCAGGAGCCCTGCTGCTTTGCTTGGTGGTAGGGCAGGAGAGCAAACAAACAGTCATGGTC ATTTGAGGTGCTTGAATGTTCTTGGGATGAGCTTTGGAACAAAGTCCAGAGATCGGAAGAGCGGGTGAGCAGGGAGGGCGAGCGAGGACATGTGTTTGCG CAAGGTGAACAGACTCTGGCATGTTGGTACAATGTAGGTAAGGGAAGTAGGTAGGCCCGCGTCGGTACGTCGGGGCAAGGGTTGGGTGTGTCGGCAGCGT TTCAGGTGGCCCTTGGGCACATTGACAGAGTTGCCCTTCCCTTCTTTGCAACACCAGGCTTCCTATAGCACCCGGTTGCATGCTTTGCAGATAGGTGGCA AGCCAATGTGCCGGAAGGTTTGCTGGCCACTGTCACGGTCTGTCTGACAGATCGGGAGAGAGGGGGTGCAGGAGGGCGGGGGAGGACGCTCTCTCGTGTG CTTCTATAGCTGTCTCGGTCGATGCCCCACAATTTTACATTTGTCTTTGCTTTGACAGTGGCTGTGCTCGGTGTGTCATATATAAAAGCAAGTTCTCTCG GGCTGCTGCGCTTCGCGAGGTCTTGCTCCCTTGGGACCTGGTCTCCCATCTGACCCTCCAGGCCTTAGCTTGCCTCACATGTCAGGGCAGTGATCCACGA CGGGGGAAGGTTAATGGTTGATATTGCTAGGGTGGCGCTTCCAATTAGGTGCATGAGTAGGTGGCCTGCAGTAATGTTAGCGGTTAGACGTACGCCCAGG GTTGGTTCCACAAGTTAAGGCACTTCCGGCTGCTTTGGTGGCAGCGTGGTTCCTCCTCTCCTTTTTTAAGGCATGTGTCCTCTCAGAGTAGTCAAGCTTT GCCATCTCGATTCCCGACTCTCTAGGGAGGCTGCCTTTGGGAATTGCCAGGTCACGGGGGCATGTGAAATTAGCAGAGGGTCTGGAGGACCTGCAGAGAG GGGGAGGTTAGTTGTGGCAATAAAAATGATTAAGGATACTAGTATAAGAGATCAGGTTCGTCCTTTTGTGTTGTGTGTGGTTATCATTTGTTTTTGGGGT CGTGGTGCCGCGCGCTGTCGTGGCCGCCGGACTTGTACTTGTACTTGTTGAGCTCCGCCGTGATGCGCTTGTTTTGCTCTTCCAGGTCGGGCACGCTCCT GGAGAGCACGCCATGAAGGCCTCGGGCACGCTACGAGAGTACAAGGGAGTGGGGGGCTGCCTGCCCACCCCCCAATGCCACACGACGCGCCTCTACCGCC GACTGTGAGTGCGTTCGTAGTTTGAGTTTGCTAGGCAGAATAGTAATGAGGATGTAAGTCCGTGGGCGATTATGAGAATGACTGCGCCGGTGAAGCGTCA CTTGACCTCTCGGTGCCTGCGACGGGCTGACTGCTGGGAATGAAGCAGGATCAATGATGGTATAAACATCAAGGGTAAGAAGCGGAGTGTAGGCAGATCT GAATCACCAGGGCAACGCTTTCTTTTCAGTGCCAGTGTGACTATGGTGGCCCGTGCACCTCCTAGGCCGTGCACGCAGGGAGGTCGCCACGCAGACACAG GGAAGTGCTTGGTGAGGGCTTCCAGCAGCTCCTGCTTCTTCAGCCCACTCTTCAGCCCGTAAGCCCGGGCGGCCTTCTTTAGCATGGGGCCAGTGGAATT CGGGCATGTGAGAGGGGTGCGTCCCCAGGTCCCCCAGAGGGCCCTGTCGCCGAGGACCTTTCTGAAGGAAGCAGAAGACGACATTTCCTCTACTTCACAC GGGTTTTCATCTTGGGTCCGGCCGTGTGGCTTCTGGGGCAGGGGGGCGGAGTGGGCAGTTCTTGCCGGGTGGTGTGGGGCCTCGCCAGAGCTCCGCTTTG CGCATCCTCCGCATCCACATCCGCATCGTCGAGATCGGGAGGGAGGGTGTGTAGGAGTGGCGAGAGCGAGCGTAGTGTTGCGTGGTTTGTGTGTATAAAA CTTTTTTGCCCTTTTTGCCCTTTTTATCACCTTGTTCTGAAACAGCTGCAGGCTCCGTCTTATCACGTCCAAACCCATTTCTCTGTGTGGGGTGGGCGGA CTCGAACTCGCCCTCCTCCTCGGCTGTGGCATCCTGGTACTGCTGGTACTCGGACACCAGGTCATTCATGTTGCTCTCGGCCTCGGTGAACTCCATCTCG GGTGCCTGCAATCGTTCCATCGACCACTTTTTAATGATGGAGGAGACTTGGGAGATCGGAAGAGCGGTTCAGCAGGAATGAGGAGACAGATCTAGTACGC GTCTGATCCGTCCTAATCACAGCAGTCCTACTTCTCCTATCTCTCCCAGTCCTAGCTGCTGGCATCACTATACTACTAACAGACCGCAACCTCAACACCA CGAAGGCAAAGCCCTTGGGGAACTGCTCCTCGGGCGGGGGATAATGGCAGAAAACCAGACCGCGCTCGCGATCGGGAGAGCGGGGGCGCAGCAGTGCGTG GAACTTCTGGATGTTTTCTTGGGCCTGGAGACTGATGGTGAAGAGCTAGCTGGGGCGATCGGTGGCGGGAACCCTGGAGGGGCTCTCCGTGAGGTTCGTA CGCACATACTCTGCTGCTAGGAATGTAATTGGTACAATGACTCTAGAAACCTCTTTGGCACTATCTACCAAAAGTGAGTATGTGCGAGATCGGAAGAGCG GAAAAGCTGTGGTGAGAGCAACATCACTCAAAGCCTGAGATCGGGAGAGCGGGTCAGCAGGGAGGGGGAGGACGGAGACGGCGTGCGTGTGTCTCTTGTT TTTGAAGTATACTTGAGGAGGGTGACGGGCGGTGTGTACGCGCTTCAGGGGCCTGTTCAACTAAGCACTCTACTCTCAGTTTACTGCTAAATCCACCTTC GGTAGGGCCAGCAGGGCAATCCTGAAGAATGACGATGCTCTGCTGCCGCCATTTTGACCCGGTTGGGAAGAGGGTTTTTTGGAGGAGGGGGGAGTTTTTG TTCCTGTTCATGCTTGGGCGGGAGTCTGGCCCAGCCTGGGCCAGAGCGCTCACCCATCACCACCCTTAAATAACACCATCTTAGCTCCGACAATAAAATT GCCGGGTACCTGGACTCAGCGGTAGCAACCTCGCCCCTTGCAACCAAGGCAGACTGAGCGCCAGAGAGGGCGTTTCCAACTCACAAATGCAGGCTCAACA CCGCCTTCCGCGCCGCCTCCTTCTCCGCCTTGGCCGACGGCTTCCCGGGGGTTCGGTGGCCGCCGCCGCCGCCCCAGCCTGGGTGGGCCCCCTGCGCTTC GCATGGGATGGCAATGTCCACATAGCGCAGAGGAGAATCTGTGTTACCCAGCGCAATGGTAGGGAGGTTAACATAAGATGCCTCCGCGAGGGGTTGTGGG CTGCGTTGGTGGGGTGGCCTTGGAGCCCTGGTCTGTGCCTGCCAGCCGCTAGGAGGGGGGCGCCTGGTACACTTTGGTGGTGTGGTCCGCAGCGGCACAG CTGTGCCCTGCGGCTGCTTGCAGCCACGGAACAGCTCACCGCGCTGCTGGGCCTGAGGCCCCACCCCGGCATGCGGCTGGCTGGGAGACACGAGGTCCGG CCGAGGAGGTTAGTTTGATTAGTCATTGTTGGGGGGGGATTAGTCGAGATGGGGGGGGGGGGGGTGCAGGAGGGGCGAGGCGGAGCTCGCTGTGCGCGCG GTTGACTTGTTGGGTGCAGCTGCAGCCCAGGAGCAGGTGCGGTGGGAGTCTCGAGGCGGGATAGCTGGAAGATGGCTCAGGAGAGGGGAGCTGGGTGGAT ATTAGAACTATACAGGAGAGACAGAAAGCTTTGATTGCTAAGATCGGAGGAGGGGGGTTGCAGGGAGGGGGAGGGTGAGAGGTCGAGCCGGCAGGTGTGT CGAGCCTACAGATGATAGGATGTTTCATGTGGTGTATGCATCGGGGTAGTCCGAGTAACGTCGGGGCATTCCGGATAGGCCGCGAAAGTGTTGTGGGAGG GGCGTCCTCATACCAGGATAAATTAAGGGTCATTTGCTGCAAGGGAAACCTCGTGAAGGAGTCCGGCACTGAACACCAACGGGGCCCGTCCACCTCCCGC CCCCAAGGCCAAGACATGCACATTCATTCTCCGTGGCGGCGCCGAGCAGTTTATGGAGGAGACAGAGCGGTCTCTGCAACATGTCATCATGTGCGCGATG TACAGACTCCATGGAGATGTCAGGGGGGCCATCTTTTAGGGCCCCGCAGGAGGAGGGGATGTGCCAACAGAATGCCCCGGGTGGCGGGGGTCTTCCCACA CAAGTTTGCATTAGAAAGTGAAGAAGTGGGGGCAAGCCCAGTGTCATGGCTTGACACTGGAAGCGCGTGGGAGGTGGCCAGGGAGGGGTGTGGGGGGGGA GGAGCTTGAGGCTGTGAGACATGCCAGCTCCCTCTGACATCCCCCTGTCTTTACCAGCCCTGACCATGTACTCGATCAGGGTGGGGAATTAGACCTGGAT CTTGTGGTGCTGCGTGGTCGAGGTGGGGCCCGGGATGCTCGATCTGCAGGCCCTTTACCAGGGGTGGGTGAGCGGTTTCCCCGAAGATCCAGACGGCGTG CTTTAACGAGAGGGTGCCTGCTTCGTGCTATAAAGCCAAAAGATCGGAAGAGCGGGTCAGCAGGAAGGCCGAGAACGCGCATGTATGCTGTGTGTGTGTT GGCCAGCAGCAGCTACAACATGGTCATCCGGGGGGGCAAATAGACCAACCGCCTGCAGGAGGCGCTGTAAATCTTCAAGAGTGTCTGGGACACACGATGG CTTTTTGGTCTCCACAAACACTAAGGTCAGTGAATCCTTGCCTGGTGGATTTATGAGGGCAAGCTGAAATGACAGTTTGTCTGTGTGTTCAACCCCTGTG CGAGAAGGAGTGTATGAGTGTGAGTGTGTGTGCATGGATGTCGGGGCACTTGGCGTCTGACNTCTTCCCGACTCAAGTGTGTAAGCACCTCACACATTNC CACACGATGGCATTCTCGCTGGCCTTGTACTTGGCCTTCCCCTTCATGAGATCGGGAGAGCGGGTGCGGAGCGAGGGCGAGACGGCACGCGTCGCTCGTG TGCTAATCTTGTCCCTAAGTAAGTTTCTTCCTGCTCCTTTTGTATCTTCCTTTCTTGTCTTAGATCGGAAGAGCGGGTCTGCAGGAGTGCGGAGAGAGAT GTCTATATCAACCAACACCTTTTCTGGGGTCTGATGAGCGTCGGCATCGGGCGCCTTTACCCGGGGGTCGGGTCTTCCCGCGGCGCGAGTGTGGCTTATC CTCCTATAGCCACAATGTGGGCATTCATTAGCTATGCCAATCAGTTGCTCAGACTCGGGCTTTGAACCGGGGCTAACATTACAAAGAAGTGGAAGGCATG GCCTCGGTCAGTATTCTTCATTGTTGGTTCAGGATGCCATTATTCAGGCCCAGGTGTGCTATGCGTCCTGCAGAATCCGGCTAGAATATGTGAAGGAGCT TGCTTGGGCTGGGGGGGGACTCTGTCCGGAAGTCCTCATTGATGTAGGTGAAGCTGGTGACAGGGACAGCCTTGCTAGGGGGCACAGCTGTGCACAGCGG GGCAGAATTTGCCACAGGAAACGACAGAGATTGGGAGGAGGGGGGGGCAGGGAGGGGGGGAAGAAGTGCTTTTTTTTGGGGTGTTTTCTCTGTATCTAAA GTGAGGACCCAGGACCCCTTCCTGCCATACCCACCAAGGTCCTGCCCACAGGGCCAGGCTGCCTCTTGCGCCACTACTCGAGACCCTGCCCAGAGGGGCG CTTCATCCCACCAGTCACACGGCAGATGGTTTCTTTGCCCGAAAGATCAGTGACATGGACAAAAGTGTCATTGAAGGCTGCACGAGCGGGAGAGTGGTGC CCGGGTTTTAGTATTTAACCAAGAGCCTTTTAAATATTGAAAACCCATAGTTCAGAAAATGTTAGTATTGCTGCCCTTCTTCACATAAATTTTTTTTTAA AACGCACTCACAGTCGCATCATAATCCTCTCTCAAGGACTTCAAACTCTACTCCCACTAATAGCTTTTTGATGACTTCTAGCAAGCCTCGCTAACCTCGC GCCTTCTGATCCATCATGGGGATGTTGGAGTGCACTGGCAGGGTGAGGTACTTGCTCTCGTGCATGCCCCGGGAGATGGGAAGAGCGGGTTGGCAGGAAT GCCAGGGTCGCAGCTGAACTCCACAGTGGTACCCACAGGGGAGGTGGGGGGGGTGCTGCTGGGGTCGGGAGAGTGGGGGAGACGGAGGGAGAGACGGTCC CAGACGCAGTACGTGCTGGATGTGCAGTACTCCCCCACGGCCCGGATTCATGCCTCCCACGCTGTGGTGAGGGAGGGAGACACGCTGGTGCTGACGTGTG CAGGACACGGATGGGACTGGGCATACAGACGAATGGGGCTGACTCATCTGGCTTCTTCCTGAGCAGGGGATTTGATGGTTGTTTCTGCAGAGGTGGCACG CTTATTAAGACGTGCATAGTAGGGATACCATTTTTGGAGTCCAAAAAGAGCTCGGTGAGTAGAGGAAGCTGTGATTTGCTGGTTTGATATACTTCCACCT TGGAGGTGCTGGTAGCTGAGGGGACGGGCGAGTGAGAGGGGAGGGAGGGAAGTCCTGGGAGGATCCTGAGCTGCTGTTGCAGTCTAAGCCTCTAATCAGT GCAGGTGCCTGCTACCACGCCCGGCTAATTTGTTTTGTATTTTTAGTCGAGACGGGGGTTCACCATGTTGGCCAGGATGGGCTTGATTTCTTGACCTCGT CAGGGGTCTCCTCACAGAGCTCGGCTCCCAGGGGTGCCTCCTCCCCCGGCAGGCGCTCCTGTTTCGGGCACATTTTCTCTTTTCGCCCCCGCGTCCGCCT GTTGACATAGGCTTCCTGATCTAGCCAATTCTATTGCCAAAGCTCCCCAGTGAATGGCAGCTTTAATCACGGCAGGTAGGGAGGAGAGGGTATTCGGTAG GGCAAGTCCAATCTGCAGAGTGGCCAAATGAGGAGTCCCCCCCCCGCCAAACCTCAGAAAACCCATACGATGGTGTTAGAAAAGTAGCCCGCGGGGGTGG GGAATACTGCTACAACCCCAGCCACAACCCCGAGGAGCAGCTCTCAGGTCGGGAGAGGGGGTCTGCAGGGACGTGGAGAGCGCGACACAGATCGGGCACT TCGAGCTCCAGGGGACTGGAGATGGGGGCGTGGGGGCCGCCGCCCGCGGCGGACTCGGCGCTCCCCGTGGGACCGGGGCGCCACCACGCCCCTCGTCGCG CGGATCTCTTCGTCTTTGCAGCGTAGCCCGAGTCGGGCACCGCGGGGGGGGCGCCGCCGCCCCCGCGGGCGGCCGCCAGGCACGTGGGGCGCCGCCTCTT TTTTAAGATGCTTTGGAGCAGACAGCTTTTGCCGTTCTCCCTCCCTTTTCAAGACTTTGTTGGGGGGGCTATGGCAGACAGTGCGGGAGAGTGGCGGGGG CTGAACTGCCGCCTGGTCTGCAGCCTCTGGCGGGACCTCATCGACCTCGTGACCCTCTGGTAACGCAAGAGCCTGCGAGAGGGCGTGCGCGCGTAGTAGT CCACAAGAACCCAGCAGACAATCACATCTTCTGTTAAGAGGATGGGGGAGGGGGCGTGGAGGGCGGTCGAGAGAGACGTGCGCTGTTGTCGCGTGTGTTC ACCATGGTCCAATCAACCCTTGCCCTTTTGAACACATGAAGCTGGAGATGTTTGTCAGATGTCACAGGGGCAGTTAGAACCTCGAGTTGGACAAGATCGG CTCACCTGGACCACTGTATCCTGCCACTGTCCTTCCTGGTCTCGCACTGCCACTGCATGGCCTCCTGTCACTGTGAATCGTGGCCCAGTCTCAGTTTGTA CTCCCCGCACACGGACTGTGTGGATGCGGCGGGGGGCCCATTTTGTGGTTGGGGGGTGGGGGGCCCAACCACTCTTTCCGCTCTTTTGTGTGGGGGTTTT GGCGGATCCAGGGGTGGTGGATGGCGATGTCCATGGTGGCTAGGGGGGTGGGGGAGAGCGGGAGAGCGCGTCAGCAGGCAGGAGGAGACACAATCCTGTG ATGGGCTCAAGGTGGACAAAGCTCTTGCTTGGGGAGAACACAAGATAAGCAGTGTGATGTGCCTCTTCGTGTTCTTCTGACACCCACTGGTAGACGTAGA AGAATGTACTCCAGGATGGCCTCACGCTCATACAGGTAGCCATCTGGGGTGACAACAGGATCGCGGGAAGGCTGGAGGGGGAGACAACACAGGTCGAGGG AAGGATGCGTGCTCTCTGCTCTCCCTCCTCTAAGACGCAAGGCAGCCCCCCGAGGAAGGGGGCTCATGTTCCCCAGAGAGGCTGGCTTCCAGGATGCTAC GGCAGCAGAAGCTAAAGCAGGCCAAACTGGTGGAGCAGTACAGAGAACAGAGCTGGATGACTATGGCCAATTTGGAGAAAGAGCTCCAGGAGATGGAGGC GTCTGAATCTGCCAACGTGGTGTTGGAGGCTCCACCTTGAAAAGGGCCACAGTCAGGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGAACGATATCG GCTGACTGACCGCACAAGACATACCTAATAGCCAGTTCCTGGGAGCAATGCAACATCCTTGAGGCATTGCTGGATAAACAACTGCTAGTTCCTGCCTGAG CCTGGATGTCTGCCGTCCTGGGTTTGAAAGAACGCCCTTTCGTTGATCGGGGGAGGGGGGCGGCGGGGGGGCGGCGAAGGAGCTCTGAGTGTGTGTGTTT CGCCAACATTCTGTTTCCAGCATCAGGCAGTTGATGCCATTCTTCAGATCGGGAGAGCGGTTCAGCAGGAAGGCGGAGAGGGATATCGTATGTGCACTAC CAGTGAGGCTGAAAGGGGGGGCTATGGAAGAGCGGTAGGGAGTCCACGGAGAAGATGCAGTGAATGCTTGCATGCATTCACACGAGATCGGAAGAGCGGT CTTCTTCCTCCTTGGTGGCTTCTTCCTTGGCCGCCTCCTCCTTCGCCTCTTCCTGCCTCCTCCCGGCTTCCGCCGCCGCCACTCCAGAGATCGGAAGAGC GATGAGCTGCAGCTGGCTCATGGCCTTCTTAGAGCAGAGAGAGGAGTATGTCATTTTACTAAGTTCCTAAACAAACATTTATGCAGGCAACACTCCTTGC CCCGTGTACCCTGTGACAGTGACTGTCCCCCCCTCCTGTGTTAGTGGTGCCCTTACTGCCGTCGCTCTCCCCCTCGCGTGTGGGGTGTGTTAGTGCAGGG GGAATGAGCACACATATGATGAGAGCAGGGTTGGCAACTGTGGGAAGAATCCATGTGTATGCGTGTGCATTTTCTACCCGTTGCTTTATGATCGGAAGAG CCTGAACCAAACAAACCTGTTGGTTGGGAGACTGCCCAGACATGATTGATGACGGGGTCCCGCCGGCTTGCCCCCTCCCGCTCACACCGCTAACGCGGCG ATTTTTAACGTGTAGGTTGAGAACAAGCCTAAAGGATTCCCACAGCTGGGAGCCAGCAAGAGAGCTTGGAGTCGCCTCTCTAGACCAGAGCTAGCGCGAC CTTCTCCTCCATGTCTAGGCTCTGTTCAGCAGGCCGCGTGTCCTGCTGGGATGGATGCTTCTCATTGGAGTTGGCCTCTGGGCGGCCGGGGCTGGCAGGG TTGGTTTCCGCGGGAACACAGAGGACAGGGGGGGGGGGGTCTGGGGTGGGGTCCCACTCTCGGTATGTCCTTCAACCTCTCACTGTTCCCACGGGGCGCA CGGGGAAGTCCTTCAGATATAGGTGAGGCTGGGCTCACCCAGATCGGAAGAGCGGTTCAGCAGGAGTGCCGAGAGCGATCTCGTATGCCGTCTTCTGCTT CCCTTCTCAACCTTCTTATCTAAGATTTCTTTCATGAGCTTGCAGAGGTTCTCAAACTTTGACTTGCTCTCTTTCATCTTCTTCTTCTCCTCCTCATCCT CACGGAAGCAGTAGGCATCCCAGCGTTCTGAGAGGTTCTTGCGGGCACCCCCGGGGGCTATGTCCCCCCGCCCCATGCTCCACAGCGCTCACGGGGACAA CCAAGTCACACCCTGGAGGGGAGCTGGGCGTGCTCATCCTCCCCTCCCTCCCAGGGGGGCTTCTGAAGACCCTCTGGACAGGCCCCCCAACCCTCACTTG TAGAGACCTTCCCGCAGTGACACCCCGGGGGGGGGGTCGCAGGAGGCCGGGAGGAGGGGGAAGAAGTAGGCCTCACACACCTGCCTAGGTTGGACGGTGT GTTCTTGCCGTGCTTTGTCAGGATCGGCCCCTGCTCGGCGGGGGGGTCTGAGGGAAAGGCCGCGCTAGGGGTGGGGACAGCTCGTATCTCTCGCGGAGGG CTTGTTGTTGCATGGGATGGCAATGTCCACATAGCGCAGAGGAGAATCTGTGTTACACAGCGCAATGGTAGGTAGGTTAACATAAGATGCCTCTGTGTGG ATCTACGTGCTCATCGTGGAGGTGCCAGGGGTCACGCTCTTTGACAAAGCCGACCCGCTGGTGACCGCCCACGCCCTCTACTTTTTTTTAACCGGGGACA CGGAGAGACACCTCATAGGTTTTTACGTGCCGCCCCTGAGGGGCCTGGTAGACAAAGCAAATAGTAGGTGCTTGGGAACAATATAGGAACTTGACATCAA CGGGGTAGTCCGAGTAACGTGGGGGAATTCCGGATAGGACGCGCAAGTGTTGTGGGAAGAAAGTTAGATTTAGGGCGTTGAATATGATAGTGAGATGGAG GGCTTAAGCATGTGGACCCCTTCGGTGTGTCTGGCCTTCCTCCATCGTCCTGCCCTTTGGCCTTTTGGTTTGAAGCCACAGGTGGGGCTTTTGGCCTTTG CTCCTCTCCTCCTCCTCGCGCCCGTCTCCGCAGCTCCTGCGTCCTCCCCGGGTGCGGGCTCACACCACACTCCCCATGCCCGGCGCCCGCAAGCGGTGAG CACAAACCAGGTAATGCCAGTTTGCCAGCAGCTATTTGCCTATAGAGATGAGTCTGTCCTGGTCATAACTGTGTGTTCACGGGGTCCAGGCTTTTGGGGG GGTGGCCCCAGCTCCCCCAGCCTCTGCAGCGCCAGCGTCTCTGTCTTCTCCCCAGGAGTGCTGGACCTGAGGGACATGGTAGGCTGTGACTTCATCCCGG GTCCGAACTAGTCTCAGGCTTCAACATCGAATACGCCGCAGGCCCCTTCGCCCTATTCTTCATAGCCGAATACACAAACATTATTATAATAAACACCCTC GCGATGAGGACTAGGATGATGCAACCGGGGGATCGGGAGAGGGGGTCAGCAGGAATGTCGAGGCGGAACTAGTAGTGTGTGTTTTTGTTTTGAAAAAAAC GAGCGATGCTGGGTCAGGAGAAGATCCTCACCGCCGACGGGCCGTGGGGTTCCCCCCCCTCGCCCTCCCACCGGGACACACTCGTCACGTCTCCGGCACA AAATGTATTTATTTATTGGTATGTGCAATGACAAACTTGGTATTTTCCCATGTTGACATTATGTATGTTGTAGAATTTAGTGTTTGTCTAAGTACACACA CCGGCGCAAGGCCTTCTTGCACTGGTACACGGGCGAGGGCATGGACGAGATGGAGTTCACCGAGGCCGAGAGCAACATGAATGACCTGGTATCTGAGTAC TGGCGCTGTTGGCCGAGGCGGAGCGCAAAGTGAAGAACTCGCAGTCCTTCTTCTCTGGCCTCTTTGGAGGCTCATCCAAAATAGAGGAAGCATGCGAAAT CGATCACCTGCACTGCTGCCCCCAAGACACTGTGTGTGACCTGATCCAGAGTAAGTGCCTCTCCCAGGGGGAAGCTACCTCGGACCCCCCCACTACACTG GGGGGGTGATGTGTGGACAGGGAGCTCAGGGGCTTCAGGGCGGGAGGGGGAGGCAGGGTGGCATTCATGGAGAAGGGTGAGGGCCCAGCGAGGTGCGGAG GGGTGGCTTCCTGGCAGAGCTCTGCAAAGCTGGAGGAGGCTTCATTATTACTTCCAACGAGGGTTCCTCTTCACTCCCTCCTTCTCTCTCCCAATTCCAC GTTTAAATTGCACCCAAGTCTACATGATTCAAAAATAATTTTCTTGTGCCATGGCTAATATTAAATACCATGTTTTTATTTTTACTCAAATCTTTGTGTC TTGAGAAGCTGGGTAACAAGTACTGAGAGAACAAAAGCTTGTGGGTCAGCGGGCCCCACAAAGATGTGAGTGCAGACAGGGTCGGCGCTGGGGGAGACAG CGTTAATCGCTTTGTCAAAGTCTTCATGTTTTTTGATCAGAGCCTCTACGCTGTCCAGTGAGTCTCCTTTGTCTTCGGTATTCAAGCAGGCCTCCCGGGC CGGCGCTGTAGCAGGGATCCTCCCGCACTATCACGTGCGTCACGCCAGGGCCTGCGTAGGGCGGTCCGTAGTAGCTGCGGAGATACGTGGTCTCGCGTGC GGATCCCAGGATTCAGTATTCCTGGCCCAGAGGGCCCTTGCTGGCTACTGGGGGTTAGTTTGCAGTCCTGTGTGCTTCTCTCTCTTATGAGTGTGTCCCT GTGGGATTTGGAGGCAGACAAGCCTGAGTCCAGTTCCCCGTTTAGCACTCATTAGCTGTGTGACTCTGGGGGAGTCCCTTAATCCTACACAGCGCGCGCT CTGCGACTTGGGGCAGGGGGTCCTAGGGGTGGTCTGGCCAGGAGCCGCGGGGGCGGAGTGCGCGGTGCCCAGGAGCACAGCTGCGCTCTCAGGGACGACA CACGAAGGGTAGCAAGTCACTCTTGTGGGTCCAAATGTTTTTTTGGGCCCGGGGGGGGGGACACGAAAACGGGGCCCCCTCTTTTTCCCCTCCTTTCTAT CAAGTTCCAGCCTCTGACCCTGGAGCAGCACAAGGGCTTCATGATGGAGCCCGATTGGCCCCTGTACCCGCCGGCGGCCCACGCCTGTGCGGCGCGCCTA CTTTTGTTAACCTTGGCTATGACTGGCCAGATTCTCCTTTTCGTAAACAACATACAATTGATCTGTTCGTTTTTTTTATGCTTGGAATCATTAGGATCAA CTTAATCCCATCCACCCTCCTCTCCCTAGGAGGCCTGGCCCCGCTAGATCGGGAGAGCGGGTTAGCAGGGGGGGCGAGACGGACGATGGTGTCGGAGTGT CACGGGGTCTGGGATCTCCACCGTCTTCTGCTCCTCAGTCAGGCCGGCAGATCGGAAGAGCGGGTCTGCAGGGATGACGAGAGCGACATCTGCTGCAGTC GCAGTACCTATGGGAGAAGGAGGGCAGTGTGCCACCCCTGAAGATGACCCCGGAGAGGGCCCTGGTCTTTCCTTTCCTCCACCAGCGCGACAGTGGCACC CTTTTGTGAAGTTCGCCGATGCCCACCGCAGCATCGAGAAGTTCGGCATTCGGCTTCTGAAAACCATCAAGCCGATGCTGACGGATATGCACACGTACTT CGGGAAAAGCACTTCCAGCAGCAGGAGCAAGAGCGGCAAGAGCGCAGAAAGCGTCTGGAGGAGATCATGAAGAGGACTCGGAAGTCAGACGTTTCTGAAA CTCATACTAGGCCTACTAACCAACACACTAACCATATACCAATGATGGCGCGATGTAACACGAGAAAGCACATACCAAGGCCACCACACACCACCTGTCC GGGACCGTGCCAAGCGGAGGGTCAGGCGGGGCTGAACCTGGGCGAGGCGCGGAGAGGCGGGGGGGGAGAGGTTTGGGTGGGGCTGCGGGGGGGGCGCACA CGTGCTCGGGGAGTCCTCAGTGGCAGTCCCCAGTGGGGGGGAGGGGGTGCCCCGCCTGGTGGCCGGGCTGGGGGAAATACCTTTTTTTTGTCGTCGGTGG CCTCCTCGTCGTCTCCAGCCCCAGCCCCACCCTGGCCCTTCTTGGCATTCTTCCTCTTCACGCGGCCCGGGCGGCCACCCCCGTAGGGAGAGCGCAGAGA GGAGGAATCCTTCCCACAGCCAGTAGATGATTACTTCGTGGAGCCTCCGCAGGCTGAAGAGGAAGAGGAAACGGTCTCACATCCAAGCTGCCCTACACTT CGCGTGTGGGGTCCAGCAGCACGGTGGAGAGCGGCACACGGCTGAGCTTGAAGCTGTTGCGGTTACGACAGGTGTGGGTGCGCCGGGGGGGGATGGGGTG GCAGATTTGTGGTGCGTTCTGAGCCGTCTGGCCTGCGCCAAGATGCTTCAAAGTATTATTAAAAACATATGGATCCCTATGAAGCCCTACGACACCAAAG CCACGCCAACGCCTCCATGTCCCACACCATCCTCCTGAAGGCAGCCGAACTTGGGATGGTGTACGACTATTACACATACATATGTATTAATCTGGAGTTT CACCTACTCCATGCTGGGCCACACCACCAAAAGGTCCTGGGAGGCCGAGCGAGTCATGGAGTGGCTCAAGACCCCGGAGGGGGGCGGCATGTTCCTGGGT GGAGGCTGGGGTGTCTGCACGAAGTCAGGCCAGCGTCCTCCTCCACAGCCTGTCACTGCCCCCTCCCCAGATCGGAAGAGCGGTTCAGAAGGAATGCCGA CACGAGTCATGTACAGGGTTGAAAGAGAAATAAGGCAAGATCGGGGGAGGGGGGCAGAGGGGGGGAGGCGAGCGGGCGCGCGCTGAGGGGGTGTTGTTGT CTCGATGGTGTCACTGGGCTCAACCTCGAGGGTGATGGTCTTACCAGTCAGAGTCTTCACGAAGATCTGCAGATCGGGAGAGAGGTTCAGCAGGAACGCT CGAGAACTCGTGTCACCTTTCCTGACGCCTGGCAGGGGTATCGCGACTCCATCCTCCTGCCCAGCATCCTGATCAATTAGTGCCAGGTTGCCAAACTCTG GGCTGAGACACCGGCTGGCAGGGCTGGAGAAAGTGGCCCACCAGCTGCAGGCACTACGGCGGGGGGGGGGGAACGGCGGGGGGGGGGTGGGACCCGGGTG AAAATTCTGTGTTCTGATTTTTGTCTCTCCCAAACACAAACAGTCCCCCCCTCTCTTCTTTTCTCCTCCTTCTTCTTCTTCTTCTTTCTCTCTTTCTTCC CGGCACAGGAACAACACGCATGGTGTTGGTGAAGCCGGAGCCAGGGCGCCATCCGGTCAGCACAATGACCACATCTCCCTTCTTGAAGAAGCCTCTGGCA GCCATGAGCAGGCCCATCATACATTTCGGCAGTGGCTATGAGGGCCGGTATTTTTGTGGAAAACTGGACCGGTTCCCCAACCCAGCTGATTACTGATTGG GGGATATGAAGCACCGCCAGGTCCTTTGAGATCGGGAGAGCGGGGCAGTAGGGAGGGCGAGGGCGCGCGCGCATGGCGTCGCTTTGTTTTTGTAATAAGA TGGACTTCCTGGTGGAGAAGGACCAGGAGTACTGCGTGTGTGAAATGCCTTGCAACCTGACCCGCTATGGGATAGAGCTGGACAGGGCCTGGATCTCGTG TGTGCCTGCAGCTCCTTCCTCAACGGCTGGCTCATGGGTCTGCCCTGGCTGGGGGTTCTTGGGCGGGCGGGACCGAGGAAGGGGGGCAGTGAGCGGCGGG GCGCTAGATCGGTATTTTCCCCTACCACTTTAAGATTACGCTTCAGGCCAGTGTTAGGCTTTCTGGTGGGGTGGGAGGCAACAGGCGCGCTCCTCGTGCG TCAGTGCTGACTGCCCGCAAACTATGACTCAACTGCTTTTCCCTCAGCTTTTCCATCAGCTGTCTCACCTCCTCCCCACTCCTTTTCATATTCTCCTCTC CTGGTGATGACATAGAGAACCTTTAGATTGCATTAATGTTCAACATTTCCCACCCCCTCTCTCTGTCCCCGCCGATCGGAAGAGCGGGTCAGCAGGCATG GCCGGTTGGGGCCGGCTGTGATTGTTATCTTGGTGCTGCAGAGGACAGCAGAAGAGGAGATTGGGTCAGAAACATGCCCTGCCGCCACAGATCACAGTGC CACTGCTGCGTCAACCCTGTCCTCTACAGCTTCATCAATCGCAACTACAGGTACGAGCTGATGAAGGCCTTCATCTTCAAGTACTCGGGCAAAAACGATC GCTGCTGCCCCTGCCTGCTGTGAACACCTGCCCAGCCCTTCCTTGTCTGCTGAGGTGCTCAGACCAGAGTGCCATTAAATACCAACTGATGTCACGGAGC CAGGCAGCTAGGTGATGGCAAGAGATGTTCACTTTTGGATATTTTCCCGTTTGTGGGGTTTGTCCACACGCTGGGAGGGGCCCTCCCCGGGGAAAGACTC CACCCTTCTGCCCAATGATTGTGCGATGAAATCTTTGCTCAATGATTAGATCCTTGGTACGCTCATTTTTCATGCGAGATGCATGTGCCCGCCGCTCTCG GCAGGCTTCCTCCAGGCGGAGGTCCTGGGGAAGTGGGGGGGCCAGGGCAGCTGCCGCCTCCCCCACTATGTAGCATCTGATTCGTCATCTCTCATGATGG CCCAGATCCTGAGACCCTGGCTGAGAACTTGGCCCAGCCTGCTGCTTAAAGGCACCATGGGGACCTGGGTTGCCCTCAGACCCAAGCCATTGTTAGCAGC CGAGGATGAACCTGAGCAGGAAGGTGGAGGTGTCCATGATGATGTTGCTGGAGAGAGTGAAGGTCTCCGCGGTGATGAGGAGACGCACTGGGAGGTAGAG CCCCAACCCGCTTTGGGGGAGCTTAGCCCCCTGCAGATCGGAAGAGCGGTTCAGCAGGAATGACGAGACCGCTGTCGTTTGTCGGCGTCTGTCTGTAAAC GGACACCACAAATCGGTCCACCTCAGCCTGGGGGGGCAGCCAGCCAAGCTCCCGTGTTGTGGGTGTGACAGCCACCCCTCCGACGTGCGGGGGGGCGTCG CCTCACTTCCTTTGAGCCTGAGGCACTTGGTAACCTCGTGGAGGGGATGGATTTCCATAAGTTTTACTTTGAGAATCGTGAGTGGGTTCGTGCTGCTGAT AGTGAACAGTGAACCTGGCTTTATCATGGCCTTCCACTAGGTCACTGGCACAGATCGGAAGAGCGGGTCGGCAGGAGTGCTGAGACGGATCGTGCTGGTG CGGCGAAGCCCGCAGCCCCTATCCGCTGCACCTGCCAGATCGGGAGAGCGGTTCAGCAGGAAGGCCGAGACAGAGCTCGGCTTCTGGCTTTTGTTTGTGT CACGGAGCCACCCCCACGATGGGAGAGGGATGCGCGCAGGCCCACAGAACCCTCAGGACTCCCGTGGCCCGAGCACACACAGCGGGCCTGTGGGGGGAGC CCGCCTTTCAGCCTCCCGAGCAGCTGGGACCGCAGGCGCCCGCACCACGCCCAGCTAATTTTTTTGCATTTTGTTCAGTAGAGACAGGGTTTCACCGTGT CACAAGCGCATGTTTTTTCTTTTTCTTTTATTTTTTTCTTTTGAGACAGAGTCTCGCTCTGTCGCCACGGGCGGAGGGGGGTGGGGGGGCGTGTTCTGTG TCTACACCCTAGTAGGCTCCCTTCCCCTACTCATCGCACTAATTTACACTCACAACACCCTAGGCTCACTAAACATTCTACTACTCACTCTCACTGCGCA GGAGGCAGTGCCCAACATCCCCCTGGTGCCCGATGAGGAGCTGGACGCTTTGAAGATCAAGATCTCCCAGATCAAGAGTGACATCCAGAGAGAGAAGAGG CCTCTGTGCCATCCTTCTCGATGAGCGCCTGCACAAAGGTCAGCGACGGCTCTAGGGCATGGGTGTCCCGGGGGCAGGTGTCCAGACTGCGGGCGCCCAG CGGGAACTCAGGCTGACCCGACACCTAACTCCCGGCGAGTGGGACCAGCAGGAGCCTGGAAGAGCGCGCGCACCGAGGCGGAAGTTGGGCACCGGGGGGC GTTTGAAGTCCTTGAGAGAGGATTATGATGCGACTGTGAGTGCGTTCGTAGTTTGAGTTTGCTAGGCAGAATAGTAATGAGGATGTAAGCGCGTGGGCGA GGCCCAGATGGCATCATAGGCCAGCGGTGCCTCCTGGAAGCCTCCTGTCTCCTCAGGGTGTCTTTTCAGTCGCTTGGTTAGTTTCTCCACAAATTCCTGG GGGAGCAGCTGGGTCACCTGAGAGTCGGGCATCCAGAGTTCGAGCTGACGAGGTGCGACTGCAGTGCCAGAGTGGCAGGGGGGTTTTTTGTGGGTGTGTC GCCAAAGTGCAGCGGGCAGCAGCCGGGGTTGGGGGCAGCTTGCGGGCCCAGGTGGAGCGATTGCGGGTGGAGCTGCAGCGGGAGCGGCGGCGGGGGGAGG GCTGGACACACTTGCAAACTCTTGCGCTGTAATGTCAGTCTGGACTCTGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACGGATGTAGTATGTGGC TGCTGTGACTGCAGGGCATATTTGTCACATTCTTCTCTGCTTATTTTGTGTTTTACAGCAAGATTCTCTGCAGTCATTGCCATGGGGAGCTGGACATGCT CTGAATTCGCGTGTGGCAGAGGGAGGAAAAGGAGGCAAACGCGGGCTGGGCAGCTGTTAGAGTCCAACGTGGAGATCGGAAGAGCGGGTCAGCAGGCATG TGTGTCTCTGGGTTCACAATTTGAAATTGGCCATGGATGCTCTGGCCTGAAGCCCCAGTTTTGGGGGTGGATGTAGGTGGCTCTTGGTCCTAGGGTCACG GGCAAAGCCTTCAGCCAGAAGTCCAAACTGGTGTCGCACCGGCGCATCCGCACGGGCGGGCGGGCCTGCGCCTGTCCCGGTCGAGTGGTGTAGCGGTCTA GTTGACTTTGCCAGTCAGCTCCTACAGTGATCAGTTGGCCACCTCTAGATCTGTGTTTGCCGGTCTAGACGTTTTCGGGGGCCTTCCTTGTATGGCGTGG GTGGGCGTGGGGTGAGGGGCCCACTGAGGGCGTGACTGGCGGCGGGGCCTGGGAGGCAGGGCTGGCGAGCATGGACAGGACGCAGTGTGTGTCTGCCGGT CTGCTGCCTACCCCCGTGCGCCTCGGCGCCCCCTCGCAGCCGCCCGCGCCGCCGCCGCTGGACAACGCGAGGCACGGGGGGCGGTAGGTCGGTAGAGTGT GAAATGGAAATGTAGCAGAGTTTCAAAACTAGAAATGTACTAGAGATGGAAGCTGTATTCTTTACTAAAGGCATCAGCATTACTGTTAACTTCCAGGCTT CATATACCCTTTGAACTAACTATTCCACTTCTATGTAAGCACAATTATATGCCAGTAACACTCATTTTAACGCTACTCTAGTGGGGAAAAATTGAGATCG CAGGCATGTGCCACTGTGCTTGGCTAATTTTTTTATTTTTTGTAGGAACAGGGCCTCATTATGTTGTCCAGGCTGGTCTTGAACTCTTGACTTCATCACA GTCTGAAGCCAATGCTGTCTGGTTGCGCCATTTTTGTGCTTTTATGTTTAATTTTATGAGGGCCACGGGTCTGTGTTCGACTCAGCCTCAGGGACGACTC GCCATGCCTGCCCTCCTGGTACTGTTGTATTCCCCTAGGCCCCCTCACATGTGTATTCAGCAGCTCTATGGCGTTGGCTCTGAGCGCGGTGGGTCAGGGG AAAATGGCGAAACCCCGTCTCTACCAAAAATACAAAAATTAGCCGGGCCCGGTGGCGGGCGCCTGAGATCGGAAGAGCGGGTCAGCAGGAGTGCCGAGAC CCAAAACTTAGCCAGGCATGGTGGTGCGTGCATGTAGTCCCAGCCACTCAGGAGGCTGAAAGATCGGAAGAGCGGTTCAGCAGGAGTGACGAGACCGACG GAGATTCACTACATTCCTTCTGTGCTGATCGGCGGGCACGGACGGGGGGGGCGGCGCGCCGCCCGCGTGGGGGGGGGCCACACCAGGGGTGACCTGTGCG GGAAGGAGAACGAGATGGACTGGGACGACTCTGCCCTGACCATCACCGTCAACCCCATGGAGACCTATGAGGACCAGCACAGCAGTGAGGAGGAGGAGGA CTTGAAGCCTGCCTCATTTCCTCCTCTCCTGCCTGTGAAAGCAGAGAGAGCTCTTCTTTGGGAGAGCTGCGGCATCACATCGCTTGGGCCCCTTACCCAT CTGCCTTCTTATCAGCTTTAGGCAAGTTCTTTGCTTGACGTTCTGCCTCTTCCAGATCGGAAGAGCGGTTCAGCGGGAGTGCCGAGACGGATCTCGTGTG GTCGGAGCCGCCCGCGCTGCCCCCCAAGCCTCTGGCAGCGCGATCCAGCCACCTGGAGAATGGGGCCCGGAGGAGTGACCCCGGCCCGCGGCCCCGCGCG AAGTAGGAGAGTGATATTTGATCAGGAGAACGTGGTTACTAGCACAGAGAGTTCTCCCAGTAGGTTAATAGTGGGGGGTAAGGCGAGGTTAGCGAGGCTT TCTCGCTGCTCTGACCTTAGGTAGAGTCCTCCACAGAAGCATCAAAGTGGACTGGCACATATGGGCTCCCTTCACCGGCCACCATTGTGGTGCTCTTCCT GTTCGAGACCAGCCTGGGCAATATGGCGGAACCCTCATCTCTACTAAACAGAGGTTGGCCGGGTATGCCTGTAATCCCACCTACTCAGGAGGTGGAGTGT CGATGTAAAACAGCTGGGAGCAGTGGGAGCTGTGGCAAACTAAAGTGCCCAAGCCGCATTTAAAAAGGGCATCTGTTCAGCTCCATCTGCTTGCTGCGGT CTCCCGCTGCTACTGCCGCGGCGGCGGGAACCTCTCCAAGGCTCCGGGCGGGCCCTTTTGTTTTTTTTCTCTGGTTGGCGCAGGGCGTTCTGTGGGGGCC TATTCTCCCTCTACATATTTACCACAACACAATGGGGCTCACTCACCCACCACATTAACAACATAAAACCCTCATTCACACGAGAAAACACCCTCATGTT GCCTCGCACACCCCCTGCGCGAGGCAGCTCCTCAATGGATACAAACAGCGAGCGTCTCAATGGATACATTCTCAGGGCCAGCCAATGAGCGTGCTGCGGA CTCGGGGCCACGCGCGCGTCCCCCGAAGAGGGGGACGGCGGAGCGAGCGCACGGGGGCGGCGGCGACGGTGGCTATAGATCGGGAGAGGGGTGCAGCAGG CTCTGCCCACCTCTCTCATGCTGAATCACATCCATGGTGAGCATTTTGATGGACAAAGTGGCACATCCAGTGGAGCGGTGGTAGATCCTGATAGACATGG CTTGCTCTGTGCAGTGGCATGATCTCCGCTCATTTCAGCCTTCACCACCCGGGCTCAAGGGATTCTCCCGCCTCTGTCTCCTGAGTAGCTGGGACTACAG GGAAAAGCACTTCTCCTCACCTTCTCCATGGTGACACTTGGCTTCTATGATGTTCTACAGATCGGAAGAGCGGTTCAGCAGGAATGCTGAGACCGATCTC CTCCAGTGAGGTCTTCTCAACCCCTCCCATGCTCACGGATCGCGGGCATTTCGTTACCATGCAGGCCCCATTGAGCAGAGCTGAGCTGGAGCACGAGAGC CCGAGGTCAAGGTTTAAAAGATCCCCTAGAAGATCACCTTGAGAGGGGATAACCTGAGGCTGTTCCAGGTTCGTTGCAGTGGTAGTGGCAACAGGGCTGT GGGTGATCAGCAATGGCATCTCTTTGGAGCTAACACCCCCTGCAGGGGAGAGGGGTCTGTGATCGCTTAAGCATTAATTGTGCTCTTTCTACCACCTTGA CCATGAACATGCATCTGTGTGCAGGGAGGTCAGAGGAGCCCAGAGATGCGAGATGGCGTCACTGAAGGGAGGTGAGGCTGGAGAGGAAGGGGAGGTGTGG GGGATCCCTAATTTGAAGACTTTGACACAGAAGATCGGGGGAGGGGGTGTGCGGGGGGGGCGCGGGGGGCCGCGTCTTCTGGTGGTGTGTTTCTTGTGTG GGAGCCCACAGCGTCTCTGTGACTTGGCAGGACCCAGCTCCACTGAATCAGAGTCCAGAAAAAGATCAATTTCAAAAAGAAAGTCTCATCTGGATCTCCT GCAAGTTCAGGTTTTCATGGTGGTCATTTCGTACATATGAGAGGGGTGCCTTTTCGTGCAACTGAAAATGACATTGCTAATTAGATCGGAGGAGCGGGTC TTTGTCACCAAGACCATACTTCTAACCTCCCTGTTCTTATGAATTCGAACAGCATACCCCCGATTCCGCTACGACCAACTCATACACCTCCTATGAAAAA GGTTCTAGGAATAATGGGGGAAGTATGTAGGAGTTGAAGATTAGTCCGCCGTAGTCGGGGTATTCGTTGGGTCTCTGCCATTGGGGGTCACTTGATTTTT GCCTTGTCAGCAGACTCTCCGCCCATCATGCTGCCACCGCCCCTCCCCCCACTGCTGCCCAAAGAAGAGGGGGGGGGGGGCGGGGGGGGGGGGGGGGGAT GCCGCCTTCCGGAGGAGGACGGTGGGGCGCGAGGGAAGCCATCGCGGACTAAATAAGGGAGGCTGCAGCGGCTGCGCGTGCGCCCAGAGGCGCAGCGGGA GGGTAGAGTACCTGTACTTTTGGCACCATTGAAACCAGCTCTGGCAGATCGGGAGAGCGGGGGAGCAGGCGTGGCGAGAGAGCAGTAGGATGTCGTGGTG GTTCCGCTGCCCTGAGGCACTCTTCCAGCCTTCCTTCCTGGGCATGGAGTCCTGAGATCGGAAGAGCGGTTCAGCAGGTATGCGGAGAGCGATCTCGTAT TTGCACTCCAGCCTGGGCGACAAGAGCGAAACTGCATCTCAAAAACAAAGATGCCTAGCTTTCACCTTGTTGCTGTTTTTCTAAATTTTTGAATAATAAA GAGAATATTCCCATGCCATCACCATTTATGATGGAGTAGGGCATGTTTGCAGTTTTGCGAGAGAGAGGGGGAGGGGGGGGGCGGGTGTGAGGAGGTGGGG CGCTTCTGCTCCTCGCTCTCCCGGGTCCTCAGGTCCTCCACCAGCTTGTCCTCCACGATGCTGGCGCCGCGATCGAAGAAGCCCTCCACCATCTTGAAGA CTAGGATGAGGATGGATAGTAATAGGGCAAGGACGCCTCCTAGCTTGTTAGGGACGGATCGGAGAATTGTGTAGGCGAATAGGAAATATCATTTGGAGAT GGCAGACCTGCATGCCGTCCCAAGGGGCATGTACGATGGAGATCGGGAGAGCGGGTCAGCAGGAAGGCCGAGACAGACCTCGCATGTAGGGTTTTTGTGT CAATGGCGTGATCTCGGTTCACTGCAACTTCTGCCTCCTGGACTCAAGTGACTCTCTTGCCTCAGCCTCCTGAGTAGCTGTGACTACAGGCACGTGCCGC TGTTCATGCTGATTGCAGTGATGATGCACGTCCTCCTGGGCCAGTCCACGGGGTGCGCCTGTACACACTGGGCGTGTGCGGATACATGGGCATGAATGCA CTGTACCGCACCAGCTGGGAGGACATCCCGTGGCAGGGGGAGAGGATCGCCCGGGGGCCCGCGAGGGAGGGGGGGTGGGGAGGGGTGGCGTGAGCGGGCT CACAGGGCTGGCCTCCCCTAGAGGGTGGGCCTCAACCACCTGCTCCCGCCACGCACCGGTTAGTGAGACAGGGCTGCCACGGCAACCGCCACGCCCCCCT TCAGGATCTCGAACCTGCGGAAGGAGGACCAGTCTGTGGACTTCTGCCACGTCCAGCTGGACATACAGATCAGGGAGGCTGGCGTGGCAGGTCACTCAGG AGAAGGTGGCCTCCAGAATCGCCGAGGGCTTCAGCGACACTGCGCTCATCATGGTAGACAACACCAAGTTTACGATGGGCTGTGTAGCGCGTACGATATA CATATTCATCGGCGTAAATCTAACTTTCTTCCCACAATACTTTCTTGGGCCCAGTGGTCAGGGCGGAGGGGCGGGAGGGGGAGGGACAGACGCGTGACTG GCAGGCTGCGACGTAAATGATAGCTGTGACATCGTTAAAGCACTGGATCCATTTTCTCCTCTCATCCCTCTGGCCACCAACATCAAACATGTGGAAGTTT TTTGGTCAGCAAGGAGTACCAGGCAGTGTATGGACTCAGGGCAGGCCTGCAAACAAGTGCACTGTGGAGATCGGGAGAGCGGGTCAGCAGGAGTGACGAG CCAGCCTATCCATGCTGTATATGCTACCTGCCCTTTAGTCATCAACATCTGCTCAACATTGTCGTGGCTCAATGGTCTGTGATCACCTCAAGACGATGAG GACAGGGCCCAAATAACTCCCCCAGGTCCTGCTTTCCATTCTCTTGTGCATGGACCTAGAAGTGGAGCTGCTGGGTCCTATGGTTGCTCTGTGCTTCATT TAGCCATCACCAGCACAACAAACGGGGCAGGGCTTTCCATGGTGGGGCTGGTCAGAAGGGAATCTCTGATAAGAGGCCCACAGGCAGGGAGAGCGAAGTA CTGTTCTTGCCCCCCGCGTCCCCTCTGCTGTGCCTAAACACCTCTCAGGAACACTTGGCAGCACGCCATTGTCAGTTCCAGGTCCACTCCTCCCCTGCGT CACCGGTGACTGGTGCGGCACCTAGCATGTTTGACAGGCGGGGGGTGCGAGGGACGAGATCGGGAGAGGGGGTGAGCAGGAGTGAGGAGAGCGAGCTCGT CGTTGAACAAACGAACCTTTAATAGCGGCTGCACCATTGGGATGTCCTGATCCAACATCGAGGTCGTAAACCCTATTGTTGTTATGGACTCTAGAATAGG CTTTGCAGAAGTCATATGAAAGGGAGGTGTTCTCCAAGGGTCAGGCGGTGGGGAGGGGGCGGAAGATTTTTTCGCCTTCTTTTTTCTCGCGGTGTCTCTA CGAAGGATCAAAGATGTTCATGAAAACACCACCATCTCCCAAGGAAGTGAATCAGCCCCTGGCCCCAGTACACCCCATTAAATCAAAGGAGCAGGATTTG GCACTTCAGCCTGGGTGACAGTGCGAGACTCCATCTCGAAATAATAATAATAATAATAGAGACGGGGGCTTGCTGTGTTTCTCAGGTTGGTCTCGACCTC CAGATGTCTTGTCTTTTCCATTGGTTGCCCCAGCACCTTGTCGAGATCGGAAGAGAGGGTCAGCAGGAATGCGGAGACCGATCGTGTATGGTGTGTGTTG CGGTGGTGGCCGTAGCGGTTCCTCCTGGCCCTGTTAATGTCGGGGCCCGGCCGGGGGGGGAGGGGGCGCTAGCACACGCGCTTGCTGCGGTCGGTGCGTG GGTTTCTCCAGTCACAAACTGTTTTGAGCAGAGGCACAGCATCCATTGACCAATCATAACTGAGGGTATCTGGACATAGCATCAGCCAGAGGTGCTTGGT CTGGTCTCGAGCTCCTGACCTCAGGTGATCCGCCTGCTTTGGCCTCCCAGAGTGCTGGGATTACAGGTGTGGGCCACCGTGCCTGGCCTAACAATGAGAT CTGGCCTGCTGCCTCTTCCTCTCCGTGCTGGTGATGCTGTGGCTGAGCTGCTCCACCCTGGTGAACGCGCCTGGCCAGCACCCTAAGTTCACGCATCTGA GCGAGTTCCAAGAGCCAGGCAGGTGGTGATGACTGCAGGCAGACCTTCAGGAATGGCTGCTACCGCCAGGGCCACTGCCATTTTAAAGGAGTAAATAGCA GAGACCAGATGGGTTGCCCCAGGATCCGGCTGCCAGCCCTGAGGCCAAGCACGGCTGGAGACCCACGACCTGGGCTGCCGTTGCCCTGAGCTGCTGCGTC CCTCTGTGAAGGCCCCATCTAGCGCCTTGATGGTCTTGACAAGATTAGCTGTGCTGTGTGCCACCTCCTTGTGTGAGTGTACAAACTGGCGCTTGGGATC GAAGATGCCTAATGTACCCAATACACAACCAGCAATAATGAAACCAACAGAGGAACATCCAGCTTATACACAGATTGCAAAGGAACATGCATTGGCCCAA CGGCCCAGCTCCACGTCCAAGAAGTAGTTCACAGATCGGGAGAGCGGTTCAGCAGGAATGCGGAGACGGATCTCGTATGCTGTGTTCTGTTTGTATAAAC CTGGCATCAGGCTGGGCTCTCTCAGAAGCCCCCAGAACAAAGGACAGAGAATTTAAGCACAGAAATCCCAGACCTGTTGTTCTCTCCATCCAGCGTGACC CAACGTTGGCAATGTGCTCCCTGTGGGCACCATGCCTGAGAGATCGGAAGAGCGGTTCAGCAGGAAGGCCGAGACAGATCTCGTATGCGGTCTTCTGCTT AGAGGAGGGCATCTTCTTGCGACAGCACTGTCTGGGTTAAGTGCCCAGTGAGGGCATGGTGTGGGGAGCTGGCCTCAGAAGATCGGAGGAGCGGTTCTGC CTCGCTGCAGAGCCCGCACCACGGCCACCGCCTCCTCCCCGCTCTCTGGGCAGTGCTCTCGCACCCAGGCTTGAAGCTCCTCGGGCAGGATGGTGAGGGA TGAACAGCGATAGTATTATTCCTTCTAGGCATAGTAGGGAGGATATGAGGTGTGAGCGATATACTAGTATTCCTAGAAGTGAGATGGTAAATGCTAGTAT CTAAGACCCCCGAAACCAGACGAGCTACCTAAGAACAGCTAAAAGAGCACACCCGTCTATGTAGCAAAATAGTGGGAAGATTTATAGGTAGAGGCGACAA CCTCCATGTATGGCCTCTGCCTGCTGTCTCACATGTCCCTTCTGGTGGTCACTTGGGCTCTAGGAGTATACGTCACCTCAGACCATTTGGGCGAAATACT GTCAAATGTATGGAAACATTTTGTGCCCTACTCTCCATACCCCGTGTACTCAAATTCTCTACTGTATGAATTATGCTTTAAGTAGAATTCAGTGCCAAGG CTGTAGCATCACTACGTAGAGTCACATCGGCATTATGAATCTTGTATTCAGCCAGAATGCTCTTCACAGTTTCAGCATCAAGCTCACTCTGGGGGCAAGT CATGAGTAAAGTGAAGCTTCTGTCTTTGTTACTGTACCAGGTGACAACAGCTGAGTGTCCCTCCCTGGGCAGGCACTATTGGCGCTTTGTGATCGGCAGA CCCGTCTAAACTGGACATCCCTCTTGCCTGCATCCTTCACAAAGAACTACAAGCCTGTTGTACAGACCACAGGCAATGCCAGGATTGTCCAGGAGCTTCT CCGCAGGTGGTCCTCCTCGTTGACCCACACCAGAGATCGGAAGAGCGGTTCAGCAGGAAGGCCGAGACCGATCTCGTATGTGGTCTTCTTCTTGATAAAT TCGCCATCCACCACGGGCCCAAAGGCGATGTGGGAGCGGGCAGGCTGCACGTCCTGGTGCACCCGCTCCCGTGAGGGCTTCCGGCGCGGAGACTCTACAG CGTCGCTGTCCTGTGCAAACCAGAAGCAAAGCTCCATCTGTGAAAGACATTCTTTCCTGGAGGAGGGGTTTCATGAATCTCTCTCATTTCTACACAACTC CCGAGACCCAGACGAGGAGTGAGGAATGAGAGAGACCAAAGTTCCTGCCTCCGCTGGGCTCACATTCAGATGTGAGCTATACAGTGTGGTGGCCACTAGC GCAGGAGAGAGACCCAGGAAGCCCGGCAGAGCCCTGAGCCCCACTGCAATTCCTCCTAGGGCTGCACAATCATGTGGCCTTAGGGCCAACTGTGCAAGAT GCATTTTCAAAAGTCACATCATACAGTCTTAATTCCCCTTTATGATACGCATATCCATTTTTCAACCATCGGATTGTAGGGATGGGCTTTCCTGTGGCAA AAAAAATATCAGCCTGTCTCCTAGGTCAGTCCCCTCCTGGACCCACCCGCTCCCCTTTTTTCTTAGCCTGCAGTGCCATAGGAGTAATCATGGGGGGAAC CCGAGGCCTCCTCCTAGCCGCCGGGGCCTCCAGCCCTTCACGGTTCGGCCACTCTCCACGTCCACAAGGACCCTCATGCTCTTCACTGTCCGGCCCATAC CAGGAATGCCCCTTACTCAGTAGCAGGCGGACACGGCCACGGGGGAACACACCCCCCTTCTTGGGGGGAACTTGTTTTTGGTTGTCCCCACTCAGTATGC GGTGACAAGTGCTATGAAGACAACCAAACTTAGAGTGATCAAATAGAGGAACTATGGGAACAGCACCGCGGTTTCAAGGGGAGCACCCCTGAGGCGAGAT GAGTCCCTTGTCATCCCCTCCACCAGGTCCGAGGGGAAGCAGGCTGGCCGACGGGGCAGGCGCACATACCTGAAGGAGCCGCAGGCAGCCCGGCCACAGA CTGGACCTCTCCACGACACCGTCCTCGCTGGGGGGGAAGGGGCAGGCAGATCGGAAGAGCGGGTCAGCAGGAGTGCTGAGAGCGATCACGTCTGCCGTCT CTTCCTGCTATGTTCGATCAGATATAGAAGAGGAACAACTAGCATATGGTTTCACAGAACTCCCTGATGCGCCATATGAATCTGATTCTTGAGAACGTCT GTTTCGCTGGATAGTAGGTAGGGACAGTGGGAATCTCGTTCATCCATTCATGCGCGTCACTAATTAGATGACGAGGCATTTGGCTACCTTAAGAGAGTCA GAAAGTCCTCCAGGAGAGGATGGGTTGGGGTGCTTGTAAGTGGGCTTCTGTAGAGATTTGGCATGGAGATTTGCCATGAAGAGTGGGTGGTTATATTTGA CGCTGAGCTTGGTGACGCTGCTCATGGAGCCCCGGCGCGAGTGGGTGGGCGCCTTCTCCTTTCGCTGCACCCCAGCCCCGTCTTTGTGCCGCTGAGTACA TGCACCTGTAATCCCAGCTACTCGGGAGACTGAGGCAGGAGAATTACTTGAACCCAGGAGGTGGAGGTTGCAGTGAGCCGAGATCGTGAGATCGGAAGAG CCCATCTGTAAAATTTGTATAACGATAACCTACCTCGCGTATCTACAGTGCCTAGCATAGTGCCCAGATCGGAAGAGCGGGTCAGCAGGAAGGCCGAGAC TGAAGGCATCTCTCATCCTCGTCTTGAAACCCTGAATCTCAAAGGGAACAGCATCCACATGGTGACAGGTCTGGACGCCGAGAAGAGATCGGAAGAGCGG GGCAGCTACAGTGGGGAGCTCCAGCTGAGCGTGGATTGTGCCAACAAGACCGAGAGTGACCTCAGCATCGAGGTCGAGTTCGAGATAGGGAGAGAGGTTC CTTCCAGATGCAACTCAGCCCTTTTTGTTTTTTTTCTATTTAACTGCAAAAGACAGCAGACAAATCAGCCCTCTTTCAGACACTCATTTCAGCTTCTCTG GCCGCCTCGGCCGCCGGGAGCCCCGTGGAGCCCCCGCCGCCGCGCCGCCCCGTGCACCGGACGATGAGGGCACGTGGCGCGGGGCGAGGACTGTGCGAGA ATCCCGAGGCCTCTCCAGTCCGCCGAGGGCGCACCACCGGCCCGTCTCGCCCGCCGCGCCGGGGAGGTGGAGCACGAGCGCGCGTGTGAGGACTCGCACG GCCGGGGGGGCCGGGACGGAGCGGTCGCCGGCCCCCACCGGAGAGACGGGGGGAGAGTGCGTGGACACGCGGGCGGGGGGGGGTGGGGGCGCGGGGTGGG GCCGCCGCCATCAAGAGGATCGTGGCAGATCGGGAGAGCGGGTCAGCAGGAAGGGCGAGACCGATCTTGTATGGCGGCGTCTGTGTGTGAGAAAAGAAAG CAAAAACATCACCTCTAGCATCACCAGTATTAGAGGCACCGCCTGCCCAGTGACACAAGATCGGGAGAGGGGTTCAGCAGGGCTGCGGAGACGGAGCGTG GAAAGGCTGTGCCTGAGGATGGACAATGTCCCCAGTAGCGCCTTCGGGGGGGGGGAGTTGGGGGGAGTGGTGGGGGGGCGTGTATTGGGCGGGTGCGCTC GTTCCGGAATGGTGCCAGGGGCAGAGGGGGCAATGCCGGGGCCCAGGTCGGCACTGTACATGCGGTCGTTGGCAATGCCGGGCGGGTCGGGCAGGTAGGA GTCAAAGCAGTGGCTGAAATGCAGTTTGGGGAACTCCTTGCTGCTGTGAGGAAGGCCCAGGCCAAGGTGATGCTCTTGTGAGAGGAGAAGGAGCACGCTG GGAAGATGTAGGTGGCCAGGGCCCCCATGGAGTAGAGGAAGGCAAACACGGCCACGGGGGCGAAGAATTTGGCGGAGCGGGGGAGCGGGGGAGCAGGGAT GGGGTGGTAAGGAGCTCAAGGGCACGCACTGGGAATGGCCTCACAAGGCAGCCTTGAGACGCACAGGGCTACAACTGACCACCCTCCACCACAGGCATGT GCAAAGTCGAAAGAGAAGACAGAGGGGGCCTGGGGTTGGGGCGGCCCACTGCAGGCAGCATCCCCACAGTGGGGCCCCTGGGTCCGGCCTGGACACCTCT CGACTGGAAAGGAATGACTCCCGGGGGGGGAGGGCTCCCGGCCTTTGTCCAGCTGAGGCCCAGCCGCGGGGGGGTGTCCACATATTCACCAGCGCCTTGG CGCCTGTGTTGGGTTGACAGTGAGGGTAATAATGACTTGTTGGGATCGGGAGAGCGGGTCTGCAGGAGGGCCGAGGGCGCCATAGCTGTCGGCGCTCTTC TATGGCTGAACCTGCGCAGAGTAAGATAATCAGGCACAAGATTCGACCTCACATTACTACTTTGCGCAAATACACATACGGGAAGCTGATACTGGCGTGG CATCACTGTCCTCTTGATCCTTCAATCCCAGCTCTTGGCTAGAACTCTTGCCCTGTTCTGCTGACTGTTCATGGCTTTCCTCTTTTGACTTTAGTACTGA TTGTGAAACCCTTTTCTAGGGCATGAGCCCTCATATTCTTATTGAAAATATCACTCCCAGTGAAATAGAAAACACCACAGTAATACTGATCTTTGGGTAT GTTGGAAGTAAGCAGCCTTGCCCAAACATCCAGTGCAGTGGCCTCCAGAGATCGGAAGAGCGGTTCAGCAGGAGTGCCGAGACGGATCTCGTATGCGGTC GTTGCCATGCCTGCTCTCGCCCCACCAGAAGTTGTCATGGACCCAGCTTTGGCAGCACAGTATGAGCACGACTTAGAGGTTGCTCAGACAACTGCTCAGA GTGACCTTGTGGTGGGAGAGCAGAGGTGGACGCGGCCGAGAGCCCTACAGAGAAGCTGGCTGGTAGGACCCGCAGGGACGAGCTGACCAGGCTTGTGGTC AGTGAAGCGCGTTCATGTTGGTCCAGCGGCTGCGCAGCGTCACATCTGCGCCCAGCGCCAGCAGCTGCTGCGAGAGGCGCACGGGTGCCGCGGGGTCCCC GCTCTGTTGCCCAGGCTGGAGTGCAGTGGCGCGATCTTGGCTCACCGCAACCTCCGCCTCCAGGGTACGGGCTATTCTCCCACCTCGGCCTCCCAAGTAG CCTGTCCCAACACCATCTGACAAGACTCCACGGGACTCACCAACCTCCAACACACCGAGCTCCTCCTCTGTGATACCCTGCCGCCCACAGTGCTTCTGCT CCTCTGGGCTCAGTCTTTTGAGGTAAGTGGAATTAGAGGGCCTTGCTTCTCCTCTTTCCATTAGTCTTGCTACACCCCGTTTCCAGTTGCGGTGCATCTA GTTGTGCAGAGCAGTTAATGCTGGGACATCTGCTGGTGCATTTAAAAAATGATTGCCATTTTGAAGAGATCGGAAGAGCGGTTCAGCAGGAGTGCGGAGA CAAACTCAATTCAGGAGGCCTTCCCTCCCCACCACCATCAACCTCTTCAAAACCTACTCCCTCCCTCTAAGAGATCGGAAGAGCGGTTCAGCAGGAGTGC CGCAGGGCTCTACCGGCAATAGCCGAGGCATCAACTCTTTCACTGCGGGCTCCGCTGGGACCCACGAGATCGGAAGAGCGTTCAGCAGGAATGCTGAGAG AACAAACACTAGGAGGTGTTTTGGCAATTTAAACATATTCCAGGTTAAGCAGCATCAGAATGGACACCAGGGTAGACCATAACCTCAGAAAGATCGGAAG GCGGGATTGATGTGCAACAAGTGTCTTTGGTTATAAATTATGATCTACCTACCAATCGTGAAAACTATATTCACAGAATTGGCAGAGGGGGTCGATTTGG GGTGATAAAGACAAACTCGCACCCCCACTGGAGGGTTCAGGAGGAAGAAGGAATCACAAGATGGAGAGAAGCGTGCGTGTGTGTGGCAGATCGGAAGAGC TGAACCTACGAGTACACCGACTACGGCGGGCTAATTTTCACATCATACATACTTCCACCCTTATTCCTCTAACCAGGCGGCCTGTGTGTCGTCTACGTTG GACGGAGTCTTGCTCTGTCACCCAGGCTGGAGTGCAGTGGCACAATCACAGCTCACTGCAGCCTCAACCTCCCTGTAGCTCACTGTAGCCTTGATCCTCC CTGAACTTGAGTCCCCAGATTGTCAAACTACTGGTCAGCTATTGAGAATTTTAGAACTCAGGTCTTTGATTTGAAGTAGGGAACATAGTGGCGCACACAG AAAGATCTTTATGGCCCTGACAACCACCTCGTTGAGTGGCATCGGGCACCCACGACCCCGGGGAGCGGGAGAGAGGGTGTGCAGGAGTACAGAGGCGACA ATTATGTGGACCACTGCAAAGAACAGAACGTGCCCGTGCCCAAGGAGCCCATCATCTTCAGCAAGTTTGCCAGCTCCATCGTGGGGCCCTATGATGAGGT CGGGAGTCATGCCACGTGAGGCTGGTGCCAGGTGTCCGAGGCAAGTGGCAGGGGATGCTCTCAGCTGACACCGTGTGCTCCAGAGAGCGGGAGAGCGGTG TACGGGGGAAGGCGCTTTGTGAAGTAGGCCTTATTTCTCTTGTCCTTTCGTACAGGGAGGAATTTGAAGTAGATAGAAACCGACCTGGATTACTCCGGTC GGAGGAGTCAGTCCTGCCCAAGACCCCTCGGGCCTCTGCAGCACCCCCATGGGGGGGCCCCTCTATGTGGGGGGATGTTTTGTCCCGCACGCGAGTGGGG CTTTGACCGCGATGATGTGGCTTTGAAGAACTTTGCCAAATACTTTCTTCACCAATCTCATGAGGAGAGGGAACATGCTGAGAAACTGATGACGCTGCAG GTTCATGCCTGTAATCCCAGCACTTTGGGAAGGTGAGGCAGGGGGGTCATCTGTGGGCAGGAGTTCAAGACCAGAGATCGGAAGAGGGGTGCAGCAGGAG GTGGCACCCATGCTCCTCACTGCGACATCACATCCTCCCCAGCCCCGATGGCCCAGCTCCCAGGCACACTGGCCTCAGGGAACCCAGGATTGGCCAGGAA TGCCAGGTTGTTCTCGGCTTCCAGCCTCAGGTTGGTTTCATCCTGGAGCTTCTGCCTCACAGTGGCCAGGTCCTGTGCCAGATTGTCCCTCTCACACTCC TCTGGACAATACTTTGATTTTGTAGGAGTGGAGGAGGGCTCTTGGGAGGGGGGGGGGGGAAAAACCCCCGGGGTGTTTCTGTTTGGTGTGGGGGGTTTCG GCGCAGGTGTTTGCGGAAGTTTCTCTGGATTACAGACGCGGAATCATTCTCCCGTTTCCGGCGCTTCCTCTCCGCGTAGCCGCTGAACACAGAGCTGGCT GGGACAGCTACCGGCGCATCCTTATGGGCAGCACGCTGAGGAAACGCAAGATGTACGAGGAGTTCCGCAGCAAGGTCTCCATCCGAGAGTGCCTGGAGAA TGCTGACTGCACCTCCCATCAAAAGGGACTCCCTGGGGCAGAGTGGGCCGGCCCCCTACCCCCGAGGGAGGCAACCTCCTGGGCGGGGGCAGACCTTGGG GCGCGCGCAGGTCGCGCAGCTCGCGCTGGAAGAGCTCGCCGACGCGCGACGGCTCAGCGCGGCGCTGTCGCAGCGCGGCGCGCTCGGCGTGCCACGCGCG GAGGGGTGTCTAGGGGGACTGGCAGAGTGTGAGAGGGACCGTGAGGGGGCTCTTGATGGGAGTGGAAGAAGTGCTGAGGGTCTGAGAGGGGAGATGCATG CAGCTAAGCCCCATCTGCCCCGAGGAGCGGCTCTTTGGGGAGGAACCCCAGTCCAACCCCCCGCACTGCTTTAGAGCTACGGCAAATATGCTCCACGCGC TGAGGTTTTGGTGGCCATTCTGGATCAACCAAGAGCTCCTGCGCCAGATACATGTACTTTGCCTTTGGTGTCTTCTTTCTACAGCCCAGCCCCCCGATCG CTATAATCCCAGCTACACAGGAGGCTGAGGTGGGTAGACTGCTTGAGCCCAGGAGGTCAAGGGGAGAGAGCCGGGGAGGGGGGCCGCAGGGAGGAGCGCG GGGAAAATGCCGGAAGACTCCAGGGTGGGAGGAAGCGCGTCCCAAGGCCCAGGATCTGCGAGGCGACTTGGGGAAAACGCAGGCAGGACCTGCTGAGGCT CACTAGCAGAGACCAACCGAACCCCCTTCGACCTTGCCGAAGGGGAGTCCGAACTAGTCCCAGGCTTCAACATCGAGATCGGAAGGCGGTTCAGCAGGAG CCAGAAGCCATGCCATAGAGGCTGCTCTGAGGCATGTTTTGGGAGCCAGGGAACTGAGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATGTC AACTGGTGGGCAAGAACTCTCGAACTATAGTCTCTATCAGCCGAGGGCACTCCAGGACCCCTGTGGGGGCAGGGCCTACCCCTAGTCTATACAAAGTACC GCGGTATCCAGGCGGCTCGGGCCTGCTTTGAACACTCTAATTTTTTCAAAGTATACGCTTCGGGCCCCGCGGGACACTCAGCTAAGAGCATCGAGGGGGC GCCAACACAGCAGCCATTCAAGCAATCCTATACAACCGTATCGGCGATGTTGGTGTGCTTCTTCCCGTTGTCTGGTTTATCTCTCCCCCCGCGCGCGTGG AGGAACTGAGGCGCATGCAGGAGATGCTGCAGAGGATGAAGCAGCAGATGCAGGACCAGTGACGCTCGCCGCGGAGACAACGTCCGGGTCCGGGGTGCCC CACTGTGGGTGGAGGCAGGGAGGGGCAGCTGGGCCATGCTTTTAGCTTCCTTAGGCTGGGAGGGATGATGAACCCTCATCTTTTACCTCCTTCTCCCATG CGCTCCACATTTGGCTGATTATTTCAACCGAAACTGTTATCAGGATGATATTAACAGGTCAAATTTGTTGGGGCATAAAGGTGAAGTGGCAGAAGAATTT CAAAAACATGTCCTTCTCCGTTTAACACTGCACAGCCCGTGCCACCGCATGCAGCCTGTCGCACCAGATCGGAAGAGCGGTTCAGCAGGAGTGCCGAGAT TGTCTGTCCACTTGGTGGCTGGTGGGTGGGGCAGGAGATCGGGAGAGCGGTTGAGCAGGAATGCCGAGATCGATCTTGTAGTGCGTCTTCTGTTTGTATA CGTTGGTGGAGTACCAGGAGCTGACGGGCGCTCGCATCCAGATCTCCCAGAAGGGCGAGTTCCTGCCAGGCACGCGGCACAGGCGGGGTCGCCATCCCGG TGAAGATACTCCCTACAACTACATGGGTACCCCAGAGACTATGACGAATCACTGTACAGCATCCAAGGGCATCAACAGAGAATCTTTATCGTCGGCGCCG CTTCCATGACGGGCCCCTTTCTCAGGAGGTCAAACACCAAATAGAGGTTGTCCTCAGCTGGGTCATCCAGGACCTCGATCAGTTCGCCAGATCGGGAGAG CGAGTCCTGGGGCTTCATCACATCGGGCAGCTCTGGCGGGCTGGCAAACGGGCCGATGCACCGGGCGTCAACGGCGTCATCTGCGCGGAAGGGCAGTGTC GGAAAAGGGCATACAGGACTAGGAAGCAGATAAGGAAAATGATTATGAGGGCGTGATCATGAAAGGTGATAAGCTCTTCTATGATAGGGGAAGTAGCGTC GTCAATTTCGTGCCAGCCACCGCGGTCACACGATTAACCCAAGTCAATAGAAGCCGGCGTAAAGAGTGTTTAGATCGGAAGAGTGGTTCAGCAGGCATGC TGGCTACCACACCAGCCAGCAGCTCCATTGACAAGGCCGTGCAGCTGTTCCTGTGTGTCCTGCTTCTTTTGGTGGGGACCAGCCTGTGGCGGGAGGAGGC GGACAACCCTACTGCACAGCCAATCGTGCAGCTCCTTGGTGAGATGCAGGACCACCAGGAGCACCCAGGCTTGGGCAGCAACTGCTGTGTGTCATGCGTT TCTCGGCCTGTTTCCGTAGCCTCATGAGCTGTTTCTTCTTCCGGTAGTGGATCTTGGCTTTCTCTTTCCTCTTCTCCTCCAGGGGGGCTGGTACTGCCTG Rsubread/inst/qualf/0000755000175200017520000000000014136047615015437 5ustar00biocbuildbiocbuildRsubread/inst/qualf/ref-quality-strings-20k-100bp-ERR2_70-SRR3045231.txt0000644000175200017520000755124014136047615025605 0ustar00biocbuildbiocbuildCCCFFFFFHHHHHJJJJJIJJJJJJGIHIIJJJJJJJIJIIIJJJJJJIJJIJGHJJJJJJIJJHFHHHFFFEEFEEEEEEEEFEDDDDDADDEEDCDEE CCCFFDFFHHHHHJJIJJJJJIIGHEEHIIIJIIJJJBEGIHGJDIHECCDDDDCAACDAADDDDDDB@D>@>DB@>B?@@CCDDB849@DBCDDCCCCA CCCFFFFFHHHHHJJJJJJJJJJJJJJJGIIJJJJJJIJJJJJJJJJJJJHJJJIJJIJIJJJJIJJJIBHEF@CFF@CADE@CDECCCDCCCCCDCCC@ @CCFFFFDHHHHHJJJJIJJJJIJJIIGIFEGG>BGGG/=>BBBD?BDCD>ADDD@CCA@CCB??BCCC>&5>BBB9@99@D5<>(+:(:>:@@C9>B9> CCCFFFFFHHHHFIGDHJJJICGFHIJJJJJIJJJJEHIFHGJJCHIIHJJGHHFFDDDDDDDD5>?CDDD@DD::CDDCDDDD>CACDDCDDDDDDDD> CCCFFFFFHHHHHJIJIIIHJIJIJJIIGIIIIIFHG?GHFHGHIJJJJJGHHGIJJEHIJHDDD;@;AB9:>ACD:A:ACA>C>>BDDDDCC@###### @@CFFFFFHHHHHJIIJIJJJIJJIJIIGIIIJDIGBHIJJJHIHHGCHEFBHEHFBDFEDECECCCDCDCDDDDABDACDDDCCE:ACBD@BDDDACDC CC@FFFFFHHHHHJJJGIGIIIJFHHIJIIEFHCFFHIIJJIIJIGIJIJFGHIJIJIIJJJJJIJJJH?HHHED?EDEEEEEDDDDDDDDDDDDDDDED ?@@DFFDFHHGHFGIGGIGJFIIIIICGGIIJJJHIIGIIJJIIJJGIHIIJJIJJJJJJGIIHHHGHFDFDEFDDDDDBBDD@CDD8ABDDDEDBDDDA CCCFFFFDHHHHFIIJGFGIIJJJJIIFHIIIGIGGEHIJJJHDHHHGFEFFBDDBDDDDD?BBDCDDBDDDD>C>BDDCDCDDDDDBDDDDDBDDDDDD C@CFFFFFHHHGHJJJJJJJJJJJJJJJJJJJJJJJJGJIJFHHJJFHGIIIHHFFFFFFEEEEEEBDDDBDDDDACDDEEDDDDDDDDACDDDCDCD@C CCCFFFFFHHHHHIJJIIJJJIIJJJJIJJIGIGGIIGIIJJHGHIIIJJHIJIJJJJJJJHIJJJJJJJIHHFFFFFEEEEEEDDDDDCDDEDDDDDDC +:1A=BDEACFFFHGDEBF?AFF+:2A;;A@CC?@??BB9BB3@@)-5>34?@C 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@?@DDDDDF?FFHIGG@CC@DGA############################################################################# B@;DBDDDAHHHABEEGGG<:CCFDHGB@466A(;(55;(;;>CA958(5(;:(3>(3:338A<8739@@>>448@B@59<340-<&&3+:>4@A BBCFFFFFCFHFHGIJJJJJJJJGDEGGHGHIFEGHGIJJJIJJJJIIGJJJJJJHHIIJHHHGHFFFFFEEEEEEDDDDDDDDDDDDDDDDDDDD>CCC B@@FFFDDFFH4?FCGGEGHEHJJIGJJGBGEGGG?AFGEHIGEGGI?E??ED>C;@ACCC@BBDAC>4+>@DD9?CDDDBDD)98@CC CCCFDFFFHHHHGJJJEIJIIJJIJIEIHJJIGEGIGGHIJJJIJGJJHH?EHFFFFDDDDDDDC?BDDDDDDDCA?BBCCDDDDDDDDCDDDDDCCDDD CCCFFFFFHHHHHIJIJGIJJJIJIJGGGIIGIHIEDECBDCEEEEEEDDCDDDDDDBBD;CDDDC?AC8;9:CCCDDDCDCBB>BDBCD<HHIE?FDGFA9?CGHDCGH;8B?DGI@@BFG@FCCC@;@C=);4.;,;;;;(;6;;?@BB;@<@B?B?CC@CC@ CCCFFFFFHHHHHJJJJJJIIJJIJJJIJJJJJJHIJJJJJBGHGIIIJJHIJJJJJJJJJJJJHHFDDDDDDDDEDDDDDEEDDEDDDDDDDDDDDDED CCCDFFFFHHHFHFGEHIJJJEIJJIJJJJJIJI@HIJIICIIJJIIIIGFGIGIJIGGIJHHHFBBBBDDBDBBCCDDDBB;5@CD>>;>@CD@DEED @@@DDFFFHFHHHJIIIJIG@HGJIJGIJIIGIDDHH?DGIIFHGEDGHGEFE@BEHEE3?DD@CDEDDBDB::>>CCCDDDCCCCDCBDDD@BBDDCCC ?@?DDFDFHFFFHIGGEGEHHFGGI@EGBHIIJFI@7DCGHIGGG3@GGGEGH?AEHFBBBD?CC>:@C@CD4:?@@CCDCCDDC BC@DDEFFHGHHHJJJJJJJJIJJ;FHGIJJJ0DGIIJHIJGIIGIJJIEIJIIH@EHEFFDD>CEDDCDDDCDCACDCCCC=BD>@BBDD@<@BDDDDD @@@DDFADFHFFDEGGGIJACCFHIJJGEHFHHIIGG@ACCA?BBBDDCDBDD==CHGBEDEEEABDDCDDDDDCA==A>A>ADA>(85)&)0<@@CCC??######### CCCFBDFFHHHHHJJJJJJJJJIJJIJJJJJJJHIJJFHIIJJIIGJJJJHIIJJJJJIJJJGIJJJJJJJHHHHHFFFFFEDDBDDDDDDDD>>5:@CCDCAC3>93@>38@780950055>>::A#################### CCCFFFFFHHHHHIJJJIJJJJJIJIJJJJJJJJJJJJHIJJJJGIJJJIJJIIJJFJHF>A@DDDDDDDDDDCCCDEEDEDDDDDDCDDDDDDDDDDDD CCCFFFFFHHHHHJJJJJJJJIJJJJJJJJJJJJIIJJJJIIIIJJJJIIGIIHJJJJJJJJIJJJIJJJJIJHHEFFEEEEEEDDDDDDDDDDDDDDCD CCCFFFFFHHHHHJJJJJJJJJJJJJJJJJJJJJIIJIIECGEGGHGHFHEHGFFFFCCDDDDDDEEDDDDDDBDB?ABBCDDDB?BDDEDDDDDDDCCC @?@DDBDDDDDCDEGB:CF@AFHBCHE@HEGIGG9D?FB9@?FCGHDGHE8@HBB287A3+<7=DHEE)=?A;C@@DB6?C@BCD>;5;A@C>CDDD05CDDDAB?ACCDDECBDB@C@ C@@FFDFFHHHHHHIJJIJJIIJGJJJJIJJIJJJJJIJIJJIJJJIJJJGIJJJJJJJJJJJIJHGICHHHFBD@DFEEFEEEEEDDDCCDDDDDDDDD 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CCCFFFFFHHHHHJIJJJJJJJJJIJJJJIIIHJGIJJEHIJGIIIIJIHJEIJIHFF@=BC?BDBBDBDDDDBD CCCFFFFFHHFHHJJJHHJJJJJJJJJJJJJJJJJJJJJJHGIJJJIHIJJJJJJJJJJJIJIJJIJJJJJJHHH CCCFFFFEHHGHHIJJJJJJJJJJJJJJJJJJIJIJJJJJIJIGIIJJJJJJIIJJHIJJGIHGHGHHHAHHFFF CCCFFFFFHHHHGJFGIJJIJJJJJJJJJHIJJJJJJJJJFIJJIJFHHJJIJJJJJIIJJIGGJJJJIIGEFFH CCCFFFFFHHHHHJJIJJIIJJIJJJJJJJGIIJJJJJIJJIJJJJJJGHFFDDDDDDDDBDDDDDDDDDDDDDD B@@FFFFFHHHHHJJJJJJIJIJJJIJJJJIJJIJJJJJJJIJJGIJ=@DDHIJGHHEEHEFFFFFDACCDDDDD Rsubread/man/0000755000175200017520000000000014136047615014125 5ustar00biocbuildbiocbuildRsubread/man/RsubreadUsersGuide.Rd0000644000175200017520000000144414136047615020166 0ustar00biocbuildbiocbuild\name{RsubreadUsersGuide} \alias{RsubreadUsersGuide} \title{View Rsubread Users Guide} \description{Users Guide for Rsubread and Subread} \usage{ RsubreadUsersGuide() } %\arguments{} \value{Character string giving the file location.} \details{ The Subread/Rsubread Users Guide provides detailed description to the functions and programs included in the Subread and Rsubread software packages. It also includes case studies for analyzing next-gen sequencing data. The Subread package is written in C and it can be downloaded from \url{http://subread.sourceforge.net}. The Rsubread package provides R wrappers functions for many of the programs included in Subread package. } \seealso{ \code{\link{vignette}} } \author{Wei Shi} %\examples{} \keyword{documentation} Rsubread/man/align.Rd0000755000175200017520000004256514136047615015525 0ustar00biocbuildbiocbuild\name{align} \alias{align} \alias{subjunc} \title{Align Sequence Reads to a Reference Genome via Seed-and-Vote} \description{The \code{align} function can align both DNA and RNA sequencing reads. \code{Subjunc} is an RNA-seq aligner and it reports full alignment of each read (\code{align} reports partial alignment for exon spanning reads).} \usage{ align( # index for reference sequences index, # input reads and output readfile1, readfile2 = NULL, type = "rna", input_format = "gzFASTQ", output_format = "BAM", output_file = paste(readfile1,"subread",output_format,sep="."), # offset value added to Phred quality scores of read bases phredOffset = 33, # thresholds for mapping nsubreads = 10, TH1 = 3, TH2 = 1, maxMismatches = 3, # unique mapping and multi-mapping unique = FALSE, nBestLocations = 1, # indel detection indels = 5, complexIndels = FALSE, # read trimming nTrim5 = 0, nTrim3 = 0, # distance and orientation of paired end reads minFragLength = 50, maxFragLength = 600, PE_orientation = "fr", # number of CPU threads nthreads = 1, # read group readGroupID = NULL, readGroup = NULL, # read order keepReadOrder = FALSE, sortReadsByCoordinates = FALSE, # color space reads color2base = FALSE, # dynamic programming DP_GapOpenPenalty = -1, DP_GapExtPenalty = 0, DP_MismatchPenalty = 0, DP_MatchScore = 2, # detect structural variants detectSV = FALSE, # gene annotation useAnnotation = FALSE, annot.inbuilt = "mm10", annot.ext = NULL, isGTF = FALSE, GTF.featureType = "exon", GTF.attrType = "gene_id", chrAliases = NULL) subjunc( # index for reference sequences index, # input reads and output readfile1, readfile2 = NULL, input_format = "gzFASTQ", output_format = "BAM", output_file = paste(readfile1,"subjunc",output_format,sep = "."), # offset value added to Phred quality scores of read bases phredOffset = 33, # thresholds for mapping nsubreads = 14, TH1 = 1, TH2 = 1, maxMismatches = 3, # unique mapping and multi-mapping unique = FALSE, nBestLocations = 1, # indel detection indels = 5, complexIndels = FALSE, # read trimming nTrim5 = 0, nTrim3 = 0, # distance and orientation of paired end reads minFragLength = 50, maxFragLength = 600, PE_orientation = "fr", # number of CPU threads nthreads = 1, # read group readGroupID = NULL, readGroup = NULL, # read order keepReadOrder = FALSE, sortReadsByCoordinates = FALSE, # color space reads color2base = FALSE, # dynamic programming DP_GapOpenPenalty = -1, DP_GapExtPenalty = 0, DP_MismatchPenalty = 0, DP_MatchScore = 2, # detect all junctions including gene fusions reportAllJunctions = FALSE, # gene annotation useAnnotation = FALSE, annot.inbuilt = "mm10", annot.ext = NULL, isGTF = FALSE, GTF.featureType = "exon", GTF.attrType = "gene_id", chrAliases = NULL) } \arguments{ \item{index}{ character string giving the basename of index file. Index files should be located in the current directory.} \item{readfile1}{ a character vector including names of files that include sequence reads to be aligned. For paired-end reads, this gives the list of files including first reads in each library. File format is FASTQ/FASTA by default. See \code{input_format} option for more supported formats.} \item{readfile2}{ a character vector giving names of files that include second reads in paired-end read data. Files included in \code{readfile2} should be in the same order as their mate files included in \code{readfile1} .\code{NULL} by default.} \item{type}{ a character string or an integer giving the type of sequencing data. Possible values include \code{rna} (or \code{0}; RNA-seq data) and \code{dna} (or \code{1}; genomic DNA-seq data such as WGS, WES, ChIP-seq data etc.). Character strings are case insensitive.} \item{input_format}{ character string specifying format of read input files. \code{gzFASTQ} by default (this also includes FASTQ, FASTA, gzipped FASTQ and gzipped FASTA formats). Other supported formats include \code{SAM} and \code{BAM}. Character values are case insensitive.} \item{output_format}{ character string specifying format of output file. \code{BAM} by default. Acceptable formats include \code{SAM} and \code{BAM}.} \item{output_file}{ a character vector specifying names of output files. By default, names of output files are set as the file names provided in \code{readfile1} added with an suffix string.} \item{phredOffset}{ numeric value added to base-calling Phred scores to make quality scores (represented as ASCII letters). Possible values include \code{33} and \code{64}. By default, \code{33} is used.} \item{nsubreads}{ numeric value giving the number of subreads extracted from each read.} \item{TH1}{ numeric value giving the consensus threshold for reporting a hit. This is the threshold for the first reads if paired-end read data are provided.} \item{TH2}{ numeric value giving the consensus threhold for the second reads in paired-end data.} \item{maxMismatches}{ numeric value giving the maximum number of mis-matched bases allowed in the alignment. \code{3} by default. Mis-matches found in soft-clipped bases are not counted.} \item{unique}{ logical indicating if only uniquely mapped reads should be reported. A uniquely mapped read has one single mapping location that has less mis-matched bases than any other candidate locations. By default, multi-mapping reads will be reported in addition to uniquely mapped reads. Number of alignments reported for each multi-mapping read is determined by the \code{nBestLocations} parameter.} \item{nBestLocations}{ numeric value specifying the maximal number of equally-best mapping locations that will be reported for a multi-mapping read. 1 by default. The allowed value is between 1 to 16 (inclusive). In the mapping output, `NH' tag is used to indicate how many alignments are reported for the read and `HI' tag is used for numbering the alignments reported for the same read. This argument is only applicable when \code{unique} option is set to \code{FALSE}.} \item{indels}{ numeric value giving the maximum number of insertions/deletions allowed during the mapping. \code{5} by default.} \item{complexIndels}{ logical indicating if complex indels will be detected. If \code{TRUE}, the program will try to detect multiple short indels that occurs concurrently in a small genomic region (indels could be as close as 1bp apart).} \item{nTrim5}{ numeric value giving the number of bases trimmed off from 5' end of each read. \code{0} by default.} \item{nTrim3}{ numeric value giving the number of bases trimmed off from 3' end of each read. \code{0} by default.} \item{minFragLength}{ numeric value giving the minimum fragment length. 50 by default.} \item{maxFragLength}{ numeric value giving the maximum fragment length. 600 by default.} \item{PE_orientation}{ character string giving the orientation of the two reads from the same pair. It has three possible values including \code{fr}, \code{ff} and \code{rf}. Letter \code{f} denotes the forward strand and letter \code{r} the reverse strand. \code{fr} by default (ie. the first read in the pair is on the forward strand and the second read on the reverse strand).} \item{nthreads}{ numeric value giving the number of threads used for mapping. \code{1} by default.} \item{readGroupID}{ a character string giving the read group ID. The specified string is added to the read group header field and also be added to each read in the mapping output. \code{NULL} by default.} \item{readGroup}{ a character string in the format of \code{tag:value}. This string will be added to the read group (RG) header in the mapping output. \code{NULL} by default.} \item{keepReadOrder}{ logical indicating if the order of reads in the BAM output is kept the same as that in the input file. (Reads from the same pair are always placed next to each other regardless of this option).} \item{sortReadsByCoordinates}{ logical indicating if reads will be sorted by their mapping locations in the mapping output. This option is applicable for BAM output only. A BAI index file will also be generated for each BAM file so the generated BAM files can be directly loaded into a genome browser such as IGB or IGV. \code{FALSE} by default.} \item{color2base}{ logical. If \code{TRUE}, color-space read bases will be converted to base-space bases in the mapping output. Note that the mapping itself will still be performed at color-space. \code{FALSE} by default.} \item{DP_GapOpenPenalty}{ a numeric value giving the penalty for opening a gap when using the Smith-Waterman dynamic programming algorithm to detect insertions and deletions. The Smith-Waterman algorithm is only applied for those reads which are found to contain insertions or deletions. \code{-1} by default.} \item{DP_GapExtPenalty}{ a numeric value giving the penalty for extending the gap, used by the Smith-Waterman algorithm. \code{0} by default.} \item{DP_MismatchPenalty}{ a numeric value giving the penalty for mismatches, used by the Smith-Waterman algorithm. \code{0} by default.} \item{DP_MatchScore}{ a numeric value giving the score for matches used by the Smith-Waterman algorithm. \code{2} by default.} \item{detectSV}{ logical indicating if structural variants (SVs) will be detected during read mapping. See below for more details.} \item{reportAllJunctions}{ logical indicating if all discovered junctions will be reported. This includes exon-exon junctions and also gene fusions. Presence of donor/receptor sites is not required when \code{reportAllJunctions} is \code{TRUE}. This option should only be used for RNA-seq data.} \item{useAnnotation}{ logical indicating if gene annotation information will be used in the mapping. \code{FALSE} by default.} \item{annot.inbuilt}{ a character string specifying an in-built annotation used for read summarization. It has four possible values including \code{"mm10"}, \code{"mm9"}, \code{"hg38"} and \code{"hg19"}, corresponding to the NCBI RefSeq annotations for genomes `mm10', `mm9', `hg38' and `hg19', respectively. \code{"mm10"} by default. See \code{\link{featureCounts}} function for more details on the in-built annotations.} \item{annot.ext}{ A character string giving name of a user-provided annotation file or a data frame including user-provided annotation data. If the annotation is in GTF format, it can only be provided as a file. If it is in SAF format, it can be provided as a file or a data frame. The provided annotation file can be a uncompressed or gzip compressed file. If an annotation is provided via \code{annot.ext}, the \code{annot.inbuilt} parameter will be ignored. See \code{\link{featureCounts}} function for more details about this parameter.} \item{isGTF}{ logical indicating if the annotation provided via the \code{annot.ext} argument is in GTF format or not. \code{FALSE} by default. This option is only applicable when \code{annot.ext} is not \code{NULL}.} \item{GTF.featureType}{ a character string denoting the type of features that will be extracted from a GTF annotation. \code{"exon"} by default. This argument is only applicable when \code{isGTF} is \code{TRUE}.} \item{GTF.attrType}{ a character string denoting the type of attributes in a GTF annotation that will be used to group features. \code{"gene_id"} by default. The grouped features are called meta-features. For instance, a feature can be an exon and several exons can be grouped into a gene (meta-feature). This argument is only applicable when \code{isGTF} is \code{TRUE}.} \item{chrAliases}{ a character string providing the name of a comma-delimited text file that includes aliases of chromosome names. This file should contain two columns. First column contains names of chromosomes included in the SAF or GTF annotation and second column contains corresponding names of chromosomes in the reference genome. No column headers should be provided. Also note that chromosome names are case sensitive. This file can be used to match chromosome names between the annotation and the reference genome.} } \details{ \code{align} and \code{subjunc} are R versions of programs in the Subread suite of programs (\url{http://subread.sourceforge.net}) that implement fast, accurate sequence read alignment based on the "seed-and-vote" mapping paradigm (Liao et al. 2013; Liao et al. 2019). \code{align} is the R version of the command-line program \code{subread-align} and \code{subjunc} is the R version of the command-line program \code{subjunc}. \code{align} is a general-purpose aligner that can be used to align both genomic DNA-seq reads and RNA-seq reads. \code{subjunc} is designed for mapping RNA-seq reads only. The main difference between \code{subjunc} and \code{align} for RNA-seq alignment is that \code{subjunc} reports discovered exon-exon junctions and it also performs full alignments for every read including exon-spanning reads. \code{align} instead reports the largest mappable regions for each read and soft-clips the remainder of the read. \code{subjunc} requires the presence of donor and receptor sites when calling exon-exon junctions. It can detect up to four junction locations in a junction read. \code{align} and \code{subjunc} can be run with either a full index or a gapped index (see \code{\link{buildindex}} for more details). Maximum mapping speed is achieved when the full index is used. With a full index built for human/mouse genome, \code{align} maps ~14 million reads per minute with 10 threads. \code{subjunc} is slightly slower than \code{align}. Both \code{align} and \code{subjunc} require 17.8GB of memory for mapping. With a gapped index built for human/mouse genome, \code{align} maps ~9 million reads per minute with 10 threads. \code{align} requires 8.2GB of memory for mapping and \code{subjunc} requires 8.8GB of memory. The \code{detectSV} argument of \code{align} can be used to detect structural variants (SVs) in genomic DNA sequencing data. The \code{reportAllJunctions} argument of \code{subjunc} function can be used for the detection of SVs in RNA-seq data, as well as the detection of non-canonical junctions that do not have the canonical donor/receptor sites. For each library, breakpoints detected from SV events are saved to a file with suffix name `.breakpoints.txt', which includes chromosomal coordinates of SV breakpoints and numbers of supporting reads. The BAM/SAM output includes extra fields to describe the complete alignments of breakpoint-containing reads. For a breakpoint-containing read, mapping of its major sequence segment is described in the main fields of BAM/SAM output whereas mapping of its minor sequence segment, which does not map along with the major segment due to the presence of a breakpoint, is described in the extra fields including 'CC'(Chr), 'CP'(Position),'CG'(CIGAR) and 'CT'(strand). Note that each breakpoint-containing read occupies only one row in the BAM/SAM output. } \value{ For each library, mapping results including both aligned and unaligned reads are saved to a file. Indels discovered during mapping are saved to a VCF format file (\code{.indel.vcf}). \code{subjunc} also outputs a BED format file that contains list of discovered exon-exon junctions (\code{.junction.bed}). If \code{detectSV} or \code{reportAllJunctions} options are set to \code{TRUE}, structural variants discovered during read mapping will be reported and saved to a file (\code{.breakpoints.txt}), which includes chromosomal coordinates of breakpoints and number of supporting reads for each breakpoint. Other than outputting data to files, \code{align} and \code{subjunc} also return a \code{data.frame} that includes mapping statistics for each library, such as total number of reads, number of mapped reads, number of uniquely mapped reads, number of indels and number of junctions (\code{subjunc} only). } \references{ Yang Liao, Gordon K Smyth and Wei Shi (2019). The R package Rsubread is easier, faster, cheaper and better for alignment and quantification of RNA sequencing reads. \emph{Nucleic Acids Research}, 47(8):e47. \url{http://www.ncbi.nlm.nih.gov/pubmed/30783653} Yang Liao, Gordon K Smyth and Wei Shi (2013). The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. \emph{Nucleic Acids Research}, 41(10):e108. \url{http://www.ncbi.nlm.nih.gov/pubmed/23558742} } \author{Wei Shi and Yang Liao} %\note{} \seealso{ \code{\link{buildindex}} } \examples{ # Build an index for the artificial sequence included in file 'reference.fa'. ref <- system.file("extdata","reference.fa",package="Rsubread") buildindex(basename="./reference_index",reference=ref) # align a sample read dataset ('reads.txt') to the sample reference reads <- system.file("extdata","reads.txt.gz",package="Rsubread") align.stat <- align(index = "./reference_index", readfile1 = reads, output_file = "./Rsubread_alignment.BAM", phredOffset = 64) align.stat } % Add one or more standard keywords, see file 'KEYWORDS' in the % R documentation directory. %\keyword{} Rsubread/man/atgcContent.Rd0000755000175200017520000000254114136047615016672 0ustar00biocbuildbiocbuild\name{atgcContent} \alias{atgcContent} \title{Calculate Percentages of Nucleotides in Reads} \description{Calculate percentages of nucleotides A, T, G and C in the input FASTQ or FASTQ file. } \usage{ atgcContent( filename, basewise = FALSE) } \arguments{ \item{filename}{ a character string giving the name of input FASTQ/FASTA file} \item{basewise}{ logical specifying how the percentages are calculated. If \code{TRUE}, nucleotide percentages will be calculated for each base position in the read across all the reads. By default, percentages are calculated for the entire dataset.} } \details{ Sequencing reads could contain letter "N" besides "A", "T", "G" and "C". Percentage of "N" in the read dataset is calcuated as well. The \code{basewise} calculation is useful for examining the GC bias towards the base position in the read. By default, the percentages of nucleotides in the entire dataset will be reported. } \value{ A named vector containing percentages for each nucleotide type if \code{basewise} is \code{FALSE}. Otherwise, a data matrix containing nucleotide percentages for each base position of the reads. } %\references{} \author{Zhiyin Dai and Wei Shi} %\note{} %\seealso{} %\examples{} % Add one or more standard keywords, see file 'KEYWORDS' in the % R documentation directory. %\keyword{} %\keyword{}% __ONLY ONE__ keyword per line Rsubread/man/buildindex.Rd0000755000175200017520000001214214136047615016546 0ustar00biocbuildbiocbuild\name{buildindex} \alias{buildindex} \title{Build Index for a Reference Genome} \description{An index needs to be built before read mapping can be performed. This function creates a hash table index for the reference genome, which can then be used by Subread and Subjunc aligners for read alignment.} \usage{ buildindex( # basic input/output options basename, reference, # options for the details of the index gappedIndex = FALSE, indexSplit = FALSE, memory = 8000, TH_subread = 100, colorspace = FALSE) } \arguments{ \item{basename}{ a character string giving the basename of created index files.} \item{reference}{ a character string giving the name of a FASTA or gzipped FASTA file containing the sequences of all chromosomes and contigs.} \item{gappedIndex}{ logical indicating whether a gapped index or a full index should be built. A gapped index contains 16mers (subreads) that are extracted every three bases from a reference genome, whereas a full index contains subreads extracted from every chromosomal location of a genome. The index provides a hash table mapping the sequences of all subreads to their corresponding chromosomal locations. Default value is \code{FALSE} (i.e., a full index is built). } \item{indexSplit}{ logical indicating whether the index can be split into multiple blocks. The block size is determined by the value of \code{memory}. \code{FALSE} by default (ie. a single-block index is generated). } \item{memory}{ a numeric value specifying the amount of memory (in megabytes) used for storing the index during read mapping. 8000 MB by default. Note that this option is ignored when \code{indexSplit} is \code{FALSE}. } \item{TH_subread}{ a numeric value specifying the threshold for removing highly repetitive subreads (16bp mers). 100 by default. Subreads will be excluded from the index if they occur more than threshold number of times in the genome.} \item{colorspace}{ logical specifying the mode of the index. If \code{TRUE}, a color space index will be built. Otherwise, a base space index will be built.} } \details{ This function generates a hash-table index for a reference genome, in which keys are subreads (16mers) and values are their chromosomal locations in the reference genome. The built index can then be used by Subread (\code{\link{align}}) and \code{\link{subjunc}} aligners to map reads (Liao et al. 2019; Liao et al. 2013). Index building is an one-off operation. Highly repetitive subreads (or uninformative subreads) are excluded from the hash table so as to reduce mapping ambiguity. \code{TH_subread} specifies the maximal number of times a subread is allowed to occur in the reference genome to be included in hash table. Maximum mapping speed can be achieved by building a full index for the reference genome. By default \code{buildindex} builds a full index. Building a gapped index will significantly reduce the memory use, at a modest cost to read mapping time. It is recommended to use a gapped index on a personal computer due to the limited amount of computer memory available. Memory use can be further reduced by splitting an index to multiple blocks. The amount of memory to be used in read mapping is determined at the index building stage. To build a full index for human/mouse genome, \code{buildindex} function requires 15GB memory. When using a full index to map reads to human/mouse genome, \code{\link{align}} and \code{\link{subjunc}} requires 17.8GB memory. To build a gapped index for human/mouse genome, \code{buildindex} function only requires 5.7GB memory. When using a gapped index to map reads to human/mouse genome, \code{\link{align}} requires 8.2GB memory and \code{\link{subjunc}} requires 8.8GB memory. Sequences of reference genomes can be downloaded from public databases. For instance, primary assembly of human genome GRCh38/hg38 or mouse genome GRCm38/mm10 can be downloaded from the GENCODE database via the following links: \url{ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_28/GRCh38.primary_assembly.genome.fa.gz} \url{ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M18/GRCm38.primary_assembly.genome.fa.gz} } \value{ No value is produced but index files are written to the current working directory. } \references{ Yang Liao, Gordon K Smyth and Wei Shi (2019). The R package Rsubread is easier, faster, cheaper and better for alignment and quantification of RNA sequencing reads. \emph{Nucleic Acids Research}, 47(8):e47. \url{http://www.ncbi.nlm.nih.gov/pubmed/30783653} Yang Liao, Gordon K Smyth and Wei Shi (2013). The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. \emph{Nucleic Acids Research}, 41(10):e108. \url{http://www.ncbi.nlm.nih.gov/pubmed/23558742} } \author{Wei Shi and Yang Liao} %\note{} \seealso{\code{\link{align}}} \examples{ # Build an index for the artifical sequence included in file 'reference.fa' ref <- system.file("extdata","reference.fa",package="Rsubread") buildindex(basename="./reference_index",reference=ref) } % Add one or more standard keywords, see file 'KEYWORDS' in the % R documentation directory. %\keyword{} %\keyword{}% __ONLY ONE__ keyword per line Rsubread/man/cellCounts.Rd0000644000175200017520000002156514136047615016540 0ustar00biocbuildbiocbuild\name{cellCounts} \alias{cellCounts} \title{Map and quantify single cell RNA-seq data generated by 10X Genomics} \description{Process raw 10X scRNA-seq data and generate UMI counts for each gene in each cell.} \usage{ cellCounts( # input data index, sample, input.mode = "BCL", cell.barcode = NULL, # specify the aligner used for read mapping aligner = "align", # parameters used by featureCounts for assigning and counting UMIs annot.inbuilt = "mm10", annot.ext = NULL, isGTFAnnotationFile = FALSE, GTF.featureType = "exon", GTF.attrType = "gene_id", useMetaFeatures = TRUE, # user provided UMI cutoff for cell calling umi.cutoff = NULL, # number of threads nthreads = 10, # dealing with multi-mapping reads in the alignment step nBestLocations = 1, unique.mapping = FALSE, # other parameters passed to align, subjunc and featureCounts functions ...) } \arguments{ \item{index}{ A character string providing the base name of index files created for a reference genome by the \code{\link{buildindex}} function.} \item{sample}{ A data frame or a character string providing sample-related information, including location of the data, sample names and index set names. See the Details section below for more details.} \item{input.mode}{ A character string specifying the input mode. The supported input modes include \code{BCL}, \code{FASTQ} and \code{FASTQ-dir}. \code{BCL} is the BCL format of raw reads generated by the sequencers such as Illumina sequencers. \code{FASTQ} is the FASTQ format of sequencing reads. \code{FASTQ-dir} is a directory where FASTQ-format reads are saved. \code{FASTQ-dir} is useful for providing \code{cellCounts} the FASTQ data generated by \code{bcl2fastq} program or \code{bamtofastq} program (developed by 10X). \code{BCL} by default.} \item{cell.barcode}{ A character string giving the name of a text file (can be gzipped) that contains the set of cell barcodes used in sample preparation. If \code{NULL}, a cell barcode set will be determined for the input data by \code{cellCounts} based on the matching of cell barcodes sequences of the first 100,000 reads in the data with the three cell barcode sets used by 10X Genomics. \code{NULL} by default.} \item{aligner}{ Specify the name of the aligner used for read mapping. Currently only the \code{\link{align}} function (the Subread aligner) in this package is supported. \code{\link{align}} by default.} \item{annot.inbuilt}{ Specify an inbuilt annotation for UMI counting. See \code{\link{featureCounts}} for more details. \code{mm10} by default.} \item{annot.ext}{ Specify an external annotation for UMI counting. See \code{\link{featureCounts}} for more details. \code{NULL} by default.} \item{isGTFAnnotationFile}{ See \code{\link{featureCounts}} for more details. \code{FALSE} by default.} \item{GTF.featureType}{ See \code{\link{featureCounts}} for more details. \code{exon} by default.} \item{GTF.attrType}{ See \code{\link{featureCounts}} for more details. \code{gene_id} by default.} \item{useMetaFeatures}{ Specify if UMI counting should be carried out at the meta-feature level (eg. gene level). See \code{\link{featureCounts}} for more details. \code{TRUE} by default.} \item{umi.cutoff}{ Specify a UMI count cutoff for cell calling. All the cells with a total UMI count greater than this cutoff will be called. If \code{NULL}, a bootstrapping procedure will be performed to determine this cutoff. \code{NULL} by default. } \item{nthreads}{ A numeric value giving the number of threads used for read mapping and counting. \code{10} by default.} \item{nBestLocations}{A numeric value giving the maximum number of reported alignments for each multi-mapping read. \code{1} by default.} \item{unique.mapping}{A logical value specifying if the multi-mapping reads should not be reported as mapped (i.e. reporting uniquely mapped reads only). \code{FALSE} by default.} \item{...}{other parameters passed to \code{\link{align}} and \code{\link{featureCounts}} functions.} } \details{ This function takes as input scRNA-seq reads generated by the 10X platform, maps them to the reference genome and then produces UMI (Unique Molecular Identifier) counts for each gene in each cell. The \code{\link{align}} read mapping function and the \code{\link{featureCounts}} quantification function, both included in this package, are utilised by this function. Sample demultiplexing, cell barcode demultiplexing and read deduplication are carried out before generating the UMI counts. \code{cellCounts} can process multiple datasets at the same time. The sample information should be provided to \code{cellCounts} via the \code{sample} parameter. If the input format is \code{BCL} (ie. \code{input.mode="BCL"}), the provided sample information should include the location where the read data are stored, flowcell lanes used for sequencing, sample names and names of index sets used for indexing samples. These information should be saved to a \code{data.frame} object and then provided to the \code{sample} parameter. Below shows an example of this data frame: \preformatted{ InputDirectory Lane SampleName IndexSetName /path/to/dataset1 1 Sample1 SI-GA-E1 /path/to/dataset1 1 Sample2 SI-GA-E2 /path/to/dataset1 2 Sample1 SI-GA-E1 /path/to/dataset1 2 Sample2 SI-GA-E2 /path/to/dataset2 1 Sample3 SI-GA-E3 /path/to/dataset2 1 Sample4 SI-GA-E4 /path/to/dataset2 2 Sample3 SI-GA-E3 /path/to/dataset2 2 Sample4 SI-GA-E4 ... } It is compulsory to have the four column headers shown in the example above when generating this data frame for a 10X dataset. If more than one datasets are provided for analysis, the \code{InputDirectory} column should include more than one distinct directory. Note that this data frame is different from the Sample Sheet generated by the Illumina sequencer. The \code{cellCounts} function uses the index set names included in this data frame to generate an Illumina Sample Sheet and then uses it to demultiplex all the samples. If the input format is \code{FASTQ}, a \code{data.frame} object containing the following three columns, \code{BarcodeUMIFile}, \code{ReadFile} and \code{SampleName}, should be provided to the \code{sample} parameter. Each row in the data frame represents a sample. The \code{ReadFile} column includes names of FASTQ files that contain read data for the samples. Each FASTQ file corresponds to a sample. The read data included in these FASTQ files only contain genomic sequences of the reads. The cell barcode and UMI sequences of these reads can be found in the corresponding FASTQ files included in the \code{BarcodeUMIFile} column. Finally, if the input format is \code{FASTQ-dir}, a character string, which includes the path to the directory where the FASTQ-format read data are stored, should be provided to the \code{sample} parameter. The data in this directory are expected to be generated by the \code{bcl2fastq} program or the \code{bamtofastq} program (a program developed by 10X). } \value{ The \code{cellCounts} function returns a \code{List} object to R. It also outputs three gzipped FASTQ files and one BAM file for each sample. The three gzipped FASTQ files include cell barcode and UMI sequences (R1), sample index sequences (I1) and the actual genomic sequences of the reads (R2), respectively. The BAM file includes location-sorted read mapping results. The returned \code{List} object contains the following components: \item{counts}{a \code{List} object including UMI counts for each sample. Each component in this object is a matrix that contains UMI counts for a sample. Rows in the matrix are genes and columns are cells.} \item{annotation}{a \code{data.frame} object containing a gene annotation. This is the annotation that was used for the assignment of UMIs to genes during quantification. Rows in the annotation are genes. Columns of the annotation include \code{GeneID}, \code{Chr}, \code{Start}, \code{End} and \code{Length}.} \item{sample.info}{a \code{data.frame} object containing sample information and quantification statistics. It includes the following columns: \code{SampleName}, \code{InputDirectory} (if the input format is \code{BCL}), \code{TotalCells}, \code{HighConfidenceCells} (if \code{umi.cutoff} is \code{NULL}), \code{RescuedCells} (if \code{umi.cutoff} is \code{NULL}), \code{TotalUMI}, \code{MinUMI}, \code{MedianUMI}, \code{MaxUMI}, \code{MeanUMI}, \code{TotalReads}, \code{MappedReads} and \code{AssignedReads}. Each row in the data frame is a sample.} \item{cell.confidence}{a \code{List} object indicating if a cell is a high-confidence cell or a rescued cell (low confidence). Each component in the object is a logical vector indicating which cells in a sample are high-confidence cells. \code{cell.confidence} is included in the output only if \code{umi.cutoff} is \code{NULL}.} } \author{Yang Liao and Wei Shi} \seealso{ \code{\link{buildindex}}, \code{\link{align}}, \code{\link{featureCounts}} } %\examples{} Rsubread/man/detectionCall.Rd0000755000175200017520000000167114136047615017176 0ustar00biocbuildbiocbuild\name{detectionCall} \alias{detectionCall} \title{Determine Detection P Values for Each Gene in an RNA-seq Dataset} \description{Use GC content adjusted background read counts to determine the detection p values for each gene} \usage{ detectionCall( dataset, species = "hg", plot = FALSE) } \arguments{ \item{dataset}{ a character string giving the filename of a SAM format file, which is the output of read alignment.} \item{species}{ a character string specifing the species. Options are \code{hg} and \code{mm}.} \item{plot}{ logical indicating whether a density plot of detection p values will be generated.} } \value{A data frame which includes detection p values and annotation information for each gene.} %\references{} \author{Zhiyin Dai and Wei Shi} %\note{} %\seealso{} % Add one or more standard keywords, see file 'KEYWORDS' in the % R documentation directory. %\keyword{} %\keyword{}% __ONLY ONE__ keyword per line Rsubread/man/detectionCallAnnotation.Rd0000755000175200017520000000256414136047615021233 0ustar00biocbuildbiocbuild\name{detectionCallAnnotation} \alias{detectionCallAnnotation} \title{Generate Annotation Data Used for Calculating Detection P Values} \description{This is for internal use only. This function generates the annotation files for calculating p values in the \code{detectionCall} function.} \usage{ detectionCallAnnotation( species = "hg", binsize = 2000) } \arguments{ \item{species}{ a character string specifying the species to analyase} \item{binsize}{ an integer value specifying the bin-size of integenic region} } \details{ This is an internal function and should not be called by users directly. It takes as input the annotation files produced by \code{\link{processExons}} function, calculates GC percentages for each exon of genes and also for intergenic regions and add GC info into the annotations. The new annotation data are then saved to files which can be used by \code{\link{detectionCall}} function for calling absolutely expressed genes. } \value{ Two annotation files, which contain GC content for exons of genes and for intergenic regions respectively, are written to the current working directory. This function returns a \code{NULL} object. } %\references{} \author{Zhiyin Dai and Wei Shi} %\note{} %\seealso{} % Add one or more standard keywords, see file 'KEYWORDS' in the % R documentation directory. %\keyword{} %\keyword{}% __ONLY ONE__ keyword per line Rsubread/man/exactSNP.Rd0000755000175200017520000001071414136047615016107 0ustar00biocbuildbiocbuild\name{exactSNP} \alias{exactSNP} \title{Accurately and Efficiently call SNPs} \description{Measure background noises and perform Fisher's Exact tests to detect SNPs.} \usage{ exactSNP( # basic input/output options readFile, isBAM = FALSE, refGenomeFile, SNPAnnotationFile = NULL, outputFile = paste0(readFile, ".exactSNP.VCF"), # fine tuning parameters qvalueCutoff = 12, minAllelicFraction = 0, minAllelicBases = 1, minReads = 1, maxReads = 1000000, minBaseQuality = 13, nTrimmedBases = 3, nthreads = 1) } \arguments{ \item{readFile}{ a character string giving the name of a file including read mapping results. This function takes as input a SAM file by default. If a BAM file is provided, the \code{isBAM} argument should be set to \code{TRUE}. } \item{isBAM}{ logical indicating if the file provided via \code{readFile} is a BAM file. \code{FALSE} by default.} \item{refGenomeFile}{ a character string giving the name of a file that includes reference sequences (FASTA format).} \item{SNPAnnotationFile}{ a character string giving the name of a VCF-format file that includes annotated SNPs (the file can be uncompressed or gzip compressed). Such annotation can be downloaded from public databases such as dbSNP. Incorporating known SNPs into SNP calling has been found to be helpful. However note that the annotated SNPs may or may not be called for the sample being analyzed.} \item{outputFile}{ a character string giving the name of the output file to be generated by this function. The output file includes all the reported SNPs (in VCF format). It includes discovered indels as well.} \item{qvalueCutoff}{ a numeric value giving the q-value cutoff for SNP calling at sequencing depth of 50X. \code{12} by default. The q-value is calcuated as -log10(p), where p is the p-value yielded from the Fisher's Exact test. Note that this function automatically adjusts the q-value cutoff for each chromosomal location according to its sequencing depth, based on this cutoff.} \item{minAllelicFraction}{ a numeric value giving the minimum fraction of allelic bases out of all read bases included at a chromosomal location required for SNP calling. Its value must be within \code{0} and \code{1}. \code{0} by default.} \item{minAllelicBases}{ an integer giving the minimum number of allelic (mis-matched) bases a SNP must have at a chromosomal location. \code{1} by default.} \item{minReads}{ an integer giving the minimum number of mapped reads a SNP-containing location must have (ie. the minimum coverage). \code{1} by default.} \item{maxReads}{ an integer specifying the maximum depth a SNP-containing location is allowed to have. \code{1000000} by default. Any location having number of mapped reads higher than this threshold will not be considered for SNP calling. This option is useful for removing PCR artefacts.} \item{minBaseQuality}{ a numeric value giving the minimum base quality score (Phred score) read bases should satisfy before being used for SNP calling. \code{13} by default(corresponding to base calling p value of 0.05). Read bases with quality scores less than 13 will be excluded from analysis.} \item{nTrimmedBases}{ a numeric value giving the number of bases trimmed off from each end of the read. \code{3} by default.} \item{nthreads}{ a numeric value giving the number of threads/CPUs used. \code{1} by default.} } \details{ This function takes as input a SAM/BAM format file, measures local background noise for each chromosomal location and then performs Fisher's exact tests to find statistically significant SNPs . This function implements a novel algorithm for discovering SNPs. This algorithm is comparable with or better than existing SNP callers, but it is fast more efficient. It can be used to call SNPs for individual samples (ie. no control samples are required). Detail of the algorithm is described in a manuscript which is currently under preparation. } \value{ No value is produced but but a VCF format file is written to the current working directory. This file contains detailed information for discovered SNPs including chromosomal locations, reference bases, alternative bases, read coverages, allele frequencies and p values. } %\references{ %} \author{Yang Liao and Wei Shi} %\note{} %\seealso{} %\examples{} % Add one or more standard keywords, see file 'KEYWORDS' in the % R documentation directory. %\keyword{} %\keyword{}% __ONLY ONE__ keyword per line Rsubread/man/featureCounts.Rd0000755000175200017520000006657214136047615017266 0ustar00biocbuildbiocbuild\name{featureCounts} \alias{featureCounts} \title{Count Reads by Genomic Features} \description{Assign mapped sequencing reads to specified genomic features.} \usage{ featureCounts(files, # annotation annot.inbuilt = "mm10", annot.ext = NULL, isGTFAnnotationFile = FALSE, GTF.featureType = "exon", GTF.attrType = "gene_id", GTF.attrType.extra = NULL, chrAliases = NULL, # level of summarization useMetaFeatures = TRUE, # overlap between reads and features allowMultiOverlap = FALSE, minOverlap = 1, fracOverlap = 0, fracOverlapFeature = 0, largestOverlap = FALSE, nonOverlap = NULL, nonOverlapFeature = NULL, # Read shift, extension and reduction readShiftType = "upstream", readShiftSize = 0, readExtension5 = 0, readExtension3 = 0, read2pos = NULL, # multi-mapping reads countMultiMappingReads = TRUE, # fractional counting fraction = FALSE, # long reads isLongRead = FALSE, # read filtering minMQS = 0, splitOnly = FALSE, nonSplitOnly = FALSE, primaryOnly = FALSE, ignoreDup = FALSE, # strandness strandSpecific = 0, # exon-exon junctions juncCounts = FALSE, genome = NULL, # parameters specific to paired end reads isPairedEnd = FALSE, countReadPairs = TRUE, requireBothEndsMapped = FALSE, checkFragLength = FALSE, minFragLength = 50, maxFragLength = 600, countChimericFragments = TRUE, autosort = TRUE, # number of CPU threads nthreads = 1, # read group byReadGroup = FALSE, # report assignment result for each read reportReads = NULL, reportReadsPath = NULL, # miscellaneous maxMOp = 10, tmpDir = ".", verbose = FALSE) } \arguments{ \item{files}{ a character vector giving names of input files containing read mapping results. The files can be in either SAM format or BAM format. The file format is automatically detected by the function.} \item{annot.inbuilt}{ a character string specifying an in-built annotation used for read summarization. It has four possible values including \code{"mm10"}, \code{"mm9"}, \code{"hg38"} and \code{"hg19"}, corresponding to the NCBI RefSeq annotations for genomes `mm10', `mm9', `hg38' and `hg19', respectively. \code{"mm10"} by default. The in-built annotation has a SAF format (see below).} \item{annot.ext}{ A character string giving name of a user-provided annotation file or a data frame including user-provided annotation data. If the annotation is in GTF format, it can only be provided as a file. If it is in SAF format, it can be provided as a file or a data frame. See below for more details about SAF format annotation. If an annotation file is provided, it can be uncompressed or gzip compressed. Note that \code{annot.ext} will override \code{annot.inbuilt} if both provided.} \item{isGTFAnnotationFile}{ logical indicating whether the annotation provided via the \code{annot.ext} argument is in GTF format or not. \code{FALSE} by default. This option is only applicable when \code{annot.ext} is not \code{NULL}.} \item{GTF.featureType}{ a character string or a vector of character strings giving the feature type or types used to select rows in the GTF annotation which will be used for read summarization. \code{"exon"} by default. This argument is only applicable when \code{isGTFAnnotationFile} is \code{TRUE}. Feature types can be found in the third column of a GTF annotation.} \item{GTF.attrType}{ a character string giving the attribute type in the GTF annotation which will be used to group features (eg. exons) into meta-features (eg. genes). \code{"gene_id"} by default. This argument is only applicable when \code{isGTFAnnotationFile} is \code{TRUE}. Attributes can be found in the ninth column of a GTF annotation.} \item{GTF.attrType.extra}{ a character vector specifying extra GTF attribute types that will also be included in the counting output. These attribute types will not be used to group features. \code{NULL} by default.} \item{chrAliases}{ a character string giving the name of a chromosome name alias file. This should be a two-column comma-delimited text file. Chromosome name aliases included in this file are used to match chr names in annotation with those in the reads. First column in the file should include chr names in the annotation and second column should include chr names in the reads. Chr names are case sensitive. No column header should be included in the file.} \item{useMetaFeatures}{ logical indicating whether the read summarization should be performed at the feature level (eg. exons) or meta-feature level (eg. genes). If \code{TRUE}, features in the annotation (each row is a feature) will be grouped into meta-features, using their values in the \code{GeneID} column in the SAF-format annotation file or using the \code{GTF.attrType} attribute in the GTF-format annotation file, and then reads will be assiged to the meta-features instead of the features. See below for more details.} \item{allowMultiOverlap}{ logical indicating if a read is allowed to be assigned to more than one feature (or meta-feature) if it is found to overlap with more than one feature (or meta-feature). \code{FALSE} by default.} \item{minOverlap}{ \code{integer} giving the minimum number of overlapped bases required for assigning a read to a feature (or a meta-feature). For assignment of read pairs (fragments), number of overlapping bases from each read in the same pair will be summed. If a negative value is provided, then a gap of up to specified size will be allowed between read and the feature that the read is assigned to. \code{1} by default.} \item{fracOverlap}{ \code{numeric} giving minimum fraction of overlapping bases in a read that is required for read assignment. Value should be within range [0,1]. \code{0} by default. Number of overlapping bases is counted from both reads if paired end. Both this option and \code{minOverlap} option need to be satisfied before a read can be assigned.} \item{fracOverlapFeature}{ \code{numeric} giving minimum fraction of bases included in a feature that is required for overlapping with a read or a read pair. Value should be within range [0,1]. \code{0} by default.} \item{largestOverlap}{ If \code{TRUE}, a read (or read pair) will be assigned to the feature (or meta-feature) that has the largest number of overlapping bases, if the read (or read pair) overlaps with multiple features (or meta-features).} \item{nonOverlap}{ \code{integer} giving the maximum number of bases in a read (or a read pair) that are allowed not to overlap the assigned feature. \code{NULL} by default (ie. no limit is set).} \item{nonOverlapFeature}{ \code{integer} giving the maximum number of non-overlapping bases allowed in a feature during read assignment. \code{NULL} by default (ie. no limit is set).} \item{readShiftType}{ a character string indicating how reads should be shifted. It has four possible values including \code{upstream}, \code{downstream}, \code{left} and \code{right}. Read shifting is performed before read extension (\code{readExtension5} and \code{readExtension3}) and reduction (\code{read2pos}).} \item{readShiftSize}{ \code{integer} specifying the number of bases the reads will be shifted by. \code{0} by default. Negative value is not allowed.} \item{readExtension5}{ \code{integer} giving the number of bases extended upstream from 5' end of each read. \code{0} by default. Read extension is performed after read shifting but before read reduction. Negative value is not allowed.} \item{readExtension3}{ \code{integer} giving the number of bases extended downstream from 3' end of each read. \code{0} by default. Negative value is not allowed.} \item{read2pos}{ Specifying whether each read should be reduced to its 5' most base or 3' most base. It has three possible values: \code{NULL}, \code{5} (denoting 5' most base) and \code{3} (denoting 3' most base). Default value is \code{NULL}, ie. no read reduction will be performed. If a read is reduced to a single base, only that base will be considered for the read assignment. Read reduction is performed after read shifting and extension.} \item{countMultiMappingReads}{ logical indicating if multi-mapping reads/fragments should be counted, \code{TRUE} by default. `NH' tag is used to located multi-mapping reads in the input BAM/SAM files.} \item{fraction}{ logical indicating if fractional counts are produced for multi-mapping reads and/or multi-overlapping reads. \code{FALSE} by default. See below for more details.} \item{isLongRead}{ logical indicating if input data contain long reads. This option should be set to \code{TRUE} if counting Nanopore or PacBio long reads.} \item{minMQS}{ \code{integer} giving the minimum mapping quality score a read must satisfy in order to be counted. For paired-end reads, at least one end should satisfy this criteria. \code{0} by default.} \item{splitOnly}{ logical indicating whether only split alignments (their CIGAR strings contain letter 'N') should be included for summarization. \code{FALSE} by default. Example split alignments are exon-spanning reads from RNA-seq data. \code{useMetaFeatures} should be set to \code{FALSE} and \code{allowMultiOverlap} should be set to \code{TRUE}, if the purpose of summarization is to assign exon-spanning reads to all their overlapping exons.} \item{nonSplitOnly}{ logical indicating whether only non-split alignments (their CIGAR strings do not contain letter 'N') should be included for summarization. \code{FALSE} by default.} \item{primaryOnly}{ logical indicating if only primary alignments should be counted. Primary and secondary alignments are identified using bit 0x100 in the Flag field of SAM/BAM files. If \code{TRUE}, all primary alignments in a dataset will be counted no matter they are from multi-mapping reads or not (ie. \code{countMultiMappingReads} is ignored).} \item{ignoreDup}{ logical indicating whether reads marked as duplicates should be ignored. \code{FALSE} by default. Read duplicates are identified using bit Ox400 in the FLAG field in SAM/BAM files. The whole fragment (read pair) will be ignored if paired end.} \item{strandSpecific}{ an integer vector indicating if strand-specific read counting should be performed. Length of the vector should be either \code{1} (meaning that the value is applied to all input files), or equal to the total number of input files provided. Each vector element should have one of the following three values: \code{0} (unstranded), \code{1} (stranded) and \code{2} (reversely stranded). Default value of this parameter is \code{0} (ie. unstranded read counting is performed for all input files).} \item{juncCounts}{ logical indicating if number of reads supporting each exon-exon junction will be reported. Junctions are identified from those exon-spanning reads in input data. \code{FALSE} by default.} \item{genome}{ a character string giving the name of a FASTA-format file that includes the reference sequences used in read mapping that produced the provided SAM/BAM files. \code{NULL} by default. This argument should only be used when \code{juncCounts} is \code{TRUE}. Note that providing reference sequences is optional when \code{juncCounts} is set to \code{TRUE}.} \item{isPairedEnd}{ A logical scalar or a logical vector, indicating whether libraries contain paired-end reads or not. \code{FALSE} by default. For any library that contains paired-end reads, the 'countReadPairs' parameter controls if read pairs or reads should be counted.} \item{countReadPairs}{ A logical scalar or a logical vector, indicating if read pairs, instead of reads, should be counted for paired-end read data. \code{TRUE} by default. This parameter is only applicable for paired-end data. For single-end data, it is ignored.} \item{requireBothEndsMapped}{ logical indicating if both ends from the same fragment are required to be successfully aligned before the fragment can be assigned to a feature or meta-feature. This parameter is only appliable when \code{isPairedEnd} is \code{TRUE}.} \item{checkFragLength}{ logical indicating if the two ends from the same fragment are required to satisify the fragment length criteria before the fragment can be assigned to a feature or meta-feature. This parameter is only appliable when \code{isPairedEnd} is \code{TRUE}. The fragment length criteria are specified via \code{minFragLength} and \code{maxFragLength}.} \item{minFragLength}{ \code{integer} giving the minimum fragment length for paired-end reads. \code{50} by default.} \item{maxFragLength}{ \code{integer} giving the maximum fragment length for paired-end reads. \code{600} by default. \code{minFragLength} and \code{maxFragLength} are only applicable when \code{isPairedEnd} is \code{TRUE}. Note that when a fragment spans two or more exons, the observed fragment length might be much bigger than the nominal fragment length.} \item{countChimericFragments}{ logical indicating whether a chimeric fragment, which has its two reads mapped to different chromosomes, should be counted or not. \code{TRUE} by default.} \item{autosort}{ logical specifying if the automatic read sorting is enabled. This option is only applicable for paired-end reads. If \code{TRUE}, reads will be automatically sorted by their names if reads from the same pair are found not to be located next to each other in the input. No read sorting will be performed if there are no such reads found.} \item{nthreads}{ \code{integer} giving the number of threads used for running this function. \code{1} by default.} \item{byReadGroup}{ logical indicating if read counting will be performed for each individual read group. \code{FALSE} by default.} \item{reportReads}{ output detailed read assignment results for each read (or fragment if paired end). The detailed assignment results can be saved in three different formats including \code{CORE}, \code{SAM} and \code{BAM} (note that these values are case sensitive). Default value of this option is \code{NULL}, indicating not to report assignment results for reads. \code{CORE} format represents a tab-delimited text file that contains four columns including read name, status (assigned or the reason if not assigned), number of targets and target list. A target is a feature or a meta-feature. Items in the target lists is separated by comma. If a read is not assigned, its number of targets will be set as -1. For each input file, a text file is generated and its name is the input file name added with `.featureCounts'. When \code{SAM} or \code{BAM} format is specified, the detailed assignment results will be saved to SAM and BAM format files. Names of generated files are the input file names added with `.featureCounts.sam' or `.featureCounts.bam'. Three tags are used to describe read assignment results: XS, XN and XT. Tag XS gives the assignment status. Tag XN gives number of targets. Tag XT gives comma separated target list.} \item{reportReadsPath}{ a character string specifying the directory where files including detailed assignment results are saved. If \code{NULL}, the results will be saved to the current working directory.} \item{maxMOp}{ \code{integer} giving the maximum number of `M' operations (matches or mis-matches) allowed in a CIGAR string. \code{10} by default. Both `X' and `=' operations are treated as `M' and adjacent `M' operations are merged in the CIGAR string.} \item{tmpDir}{ a character string specifying the directory under which intermediate files are saved (later removed). By default, current working directory is used.} \item{verbose}{ logical indicating if verbose information for debugging will be generated. This may include information such as unmatched chromosomes/contigs between reads and annotation.} } \details{ \code{featureCounts} is a general-purpose read summarization function that can assign mapped reads from genomic DNA and RNA sequencing to genomic features or meta-features. The function takes as input a set of SAM or BAM files containing read mapping results. The files might be generated by \code{\link{align}} or \code{\link{subjunc}} or any suitable aligner. \code{featureCounts} accepts two annotation formats to specify the genomic features: SAF (Simplified Annotation Format) or GTF/GFF. The GTF/GFF format is specified at \url{https://genome.ucsc.edu/FAQ/FAQformat.html}. SAF is a Simplified Annotation Format with five columns. It can be either a data.frame or a tab-delimited text file in the following style: \preformatted{ GeneID Chr Start End Strand 497097 chr1 3204563 3207049 - 497097 chr1 3411783 3411982 - 497097 chr1 3660633 3661579 - 100503874 chr1 3637390 3640590 - 100503874 chr1 3648928 3648985 - 100038431 chr1 3670236 3671869 - ... } SAF annotation includes the following five required columns: \code{GeneID}, \code{Chr}, \code{Start}, \code{End} and \code{Strand}. The \code{GeneID} column includes identifiers of features. These identifiers can be integer or character string. The \code{Chr} column includes chromosome names of features and these names should match the chromosome names includes in the provided SAM/BAM files. The \code{Start} and \code{End} columns include start and end coordinates of features, respectively. Both start and end coordinates are inclusive. The \code{Strand} column indicates the strand of features (\code{"+"} or \code{"-"}). Column names in a SAF annotation should be the same as those shown in the above example (case insensitive). Columns can be in any order. Extra columns are allowed to be added into the annotation. In-built annotations, which were generated based on NCBI RefSeq gene annotations, are provided to faciliate convenient read summarization. We provide in-built annotations for the following genomes: \code{"hg38"}, \code{"hg19"}, \code{"mm10"} and \code{"mm9"}. The content of in-built annotations can be accessed via the \code{\link{getInBuiltAnnotation}} function. These annotations have a SAF format. The in-built annotations are a modified version of NCBI RefSeq gene annotations. We downloaded the RefSeq gene annotations from NCBI ftp server (eg. RefSeq annotation for \code{mm10} was downloaded from \url{ftp://ftp.ncbi.nlm.nih.gov/genomes/M_musculus/ARCHIVE/BUILD.38.1/mapview/seq_gene.md.gz}). We then used these annotations to create our in-built annotations. For each gene, we used its CDS (coding DNA sequence) and UTR (untranslated) regions provided in the original annotation to construct a list of exons, by merging and concatenating all CDSs and UTRs belonging to the same gene. Exons within each gene include all chromosomal bases included in the original CDS and UTR regions, but they include each base only once (they might be included multiple times in the original CDSs and UTRs). Also, exons within the same gene do not overlap with each other. Users may provide an external annotation for read summarization via the \code{annot.ext} argument. If the external annotation is in SAF format, it can be provdied as either a \code{data.frame} or a tab-delimited text file with proper column names included. If it is in GTF/GFF format, it should be provided as a file only (and \code{isGTFAnnotationFile} should be set to \code{TRUE}). \code{featureCounts} function uses the \code{GTF.attrType} attribute in a GTF/GFF annotation to group features to form meta-features when performing read summarization at meta-feature level. The argument \code{useMetaFeatures} specifies whether read summarization should be performed at feature level or at meta-feature level. A feature represents a continuous genomic region and a meta-feature is a group of features. For instance, an exon is a feature and a gene comprising one or more exons is a meta-feature. To assign reads to meta-features, \code{featureCounts} firstly groups into meta-features the features that have the same gene identifiers. \code{featureCounts} looks for gene identifiers in \code{GeneID} column of a SAF annotation or by using \code{GTF.attrType} attribute in a GTF/GFF annotation. Then for each read \code{featureCounts} searches for meta-features that have at least one feature that overlaps with the read. A read might be found to overlap with more than one feature within the same meta-feature (eg. an exon-spanning read overlaps with more than one exon from the same gene), however this read will still be counted only once for the meta-feature. RNA-seq reads are often summarized to meta-features to produce read counts for genes. Further downstream analysis can then be carried out to discover differentially expressed genes. Feature-level summarization of RNA-seq data often yields exon-level read counts, which is useful for investigating alternative splicing of genes. \code{featureCounts} provides multiple options to count multi-mapping reads (reads mapping to more than one location in the reference genome). Users can choose to ignore such reads by setting \code{countMultiMappingReads} to \code{FALSE}, or fully count every alignment reported for a multi-mapping read by setting \code{countMultiMappingReads} to \code{TRUE} (each alignment carries \code{1} count), or count each alignment fractionally by setting both \code{countMultiMappingReads} and \code{fraction} to \code{TRUE} (each alignment carries \code{1/x} count where \code{x} is the total number of alignments reported for the read). \code{featureCounts} also provides multiple options to count multi-overlapping reads (reads overlapping with more than one meta-feature when conducting meta-feature-level summarization or overlapping with more than one feature when conducting feature-level summarization). Users can choose to ignore such reads by setting \code{allowMultiOverlap} to \code{FALSE}, or fully count them for each overlapping meta-feature/feature by setting \code{allowMultiOverlap} to \code{TRUE} (each overlapping meta-feature/feature receives a count of 1 from a read), or assign a fractional count to each overlapping meta-feature/feature by setting both \code{allowMultiOverlap} and \code{fraction} to \code{TRUE} (each overlapping meta-feature/feature receives a count of \code{1/y} from a read where \code{y} is the total number of meta-features/features overlapping with the read). If a read is both multi-mapping and multi-overlapping, then each overlapping meta-feature/feature will receive a fractional count of \code{1/(x*y)} if \code{countMultiMappingReads}, \code{allowMultiOverlap} and \code{fraction} are all set to \code{TRUE}. When \code{isPairedEnd} is \code{TRUE}, fragments (pairs of reads) instead of reads will be counted. \code{featureCounts} function checks if reads from the same pair are adjacent to each other (this could happen when reads were for example sorted by their mapping locations), and it automatically reorders those reads that belong to the same pair but are not adjacent to each other in the input read file. } \value{ A list with the following components: \item{counts}{ a data matrix containing read counts for each feature or meta-feature for each library.} \item{counts_junction (optional)}{ a data frame including primary and secondary genes overlapping an exon junction, position information for the left splice site (`Site1') and the right splice site (`Site2') of a junction (including chromosome name, coordinate and strand) and number of supporting reads for each junction in each library. Both primary and secondary genes overlap at least one of the two splice sites of a junction. Secondary genes do not overlap more splice sites than the primary gene. When the primary and secondary genes overlap same number of splice sites, the gene with the smallest leftmost base position is selected as the primary gene. For each junction, no more than one primary gene is reported but there might be more than one secondary genes reported. If a junction does not overlap any genes, it has a missing value in the fields of primary gene and secondary gene. Note that this data frame is only generated when \code{juncCounts} is \code{TRUE}.} \item{annotation}{ a data frame with six columns including \code{GeneID}, \code{Chr}, \code{Start}, \code{End} and \code{Length}. When read summarization was performed at feature level, each row in the data frame is a feature and columns in the data frame give the annotation information for the features. When read summarization was performed at meta-feature level, each row in the data frame is a meta-feature and columns in the data frame give the annotation information for the features included in each meta feature except the \code{Length} column. For each meta-feature, the \code{Length} column gives the total length of genomic regions covered by features included in that meta-feature. Note that this length will be less than the sum of lengths of features included in the meta-feature when there are features overlapping with each other. Also note the \code{GeneID} column gives Entrez gene identifiers when the in-built annotations are used.} \item{targets}{ a character vector giving sample information.} \item{stat}{ a data frame giving number of successfully assigned reads in each library and also number of unassigned reads falling in each filtering category (unmapped, read type, singleton, mapping quality, chimera, fragment length, duplicate, multi-mapping, secondary alignment, split alignment, no features, overlapping length and assignment ambiguity). See Users Guide for more details.} } \references{ Yang Liao, Gordon K Smyth and Wei Shi (2019). The R package Rsubread is easier, faster, cheaper and better for alignment and quantification of RNA sequencing reads. \emph{Nucleic Acids Research}, 47(8):e47. \url{http://www.ncbi.nlm.nih.gov/pubmed/30783653} Yang Liao, Gordon K Smyth and Wei Shi (2014). featureCounts: an efficient general-purpose program for assigning sequence reads to genomic features. \emph{Bioinformatics}, 30(7):923-30. \url{http://www.ncbi.nlm.nih.gov/pubmed/24227677} } \author{Wei Shi and Yang Liao} %\note{} \seealso{ \code{\link{getInBuiltAnnotation}}} \examples{\dontrun{ # Summarize SAM format single-end reads using built-in RefSeq annotation for mouse genome mm10: featureCounts(files="mapping_results_SE.sam",annot.inbuilt="mm10") # Summarize single-end reads using a user-provided GTF annotation file: featureCounts(files="mapping_results_SE.sam",annot.ext="annotation.gtf", isGTFAnnotationFile=TRUE,GTF.featureType="exon",GTF.attrType="gene_id") # Summarize single-end reads using 5 threads: featureCounts(files="mapping_results_SE.sam",nthreads=5) # Summarize BAM format single-end read data: featureCounts(files="mapping_results_SE.bam") # Perform strand-specific read counting (strandSpecific=2 if reversely stranded): featureCounts(files="mapping_results_SE.bam",strandSpecific=1) # Summarize paired-end reads and counting fragments (instead of reads): featureCounts(files="mapping_results_PE.bam",isPairedEnd=TRUE) # Count fragments satisfying the fragment length criteria, eg. [50bp, 600bp]: featureCounts(files="mapping_results_PE.bam",isPairedEnd=TRUE, checkFragLength=TRUE,minFragLength=50,maxFragLength=600) # Count fragments that have both ends successfully aligned without checking the fragment length: featureCounts(files="mapping_results_PE.bam",isPairedEnd=TRUE,requireBothEndsMapped=TRUE) # Exclude chimeric fragments from fragment counting: featureCounts(files="mapping_results_PE.bam",isPairedEnd=TRUE,countChimericFragments=FALSE) }} % Add one or more standard keywords, see file 'KEYWORDS' in the % R documentation directory. %\keyword{} %\keyword{}% __ONLY ONE__ keyword per line Rsubread/man/findCommonVariants.Rd0000755000175200017520000000502414136047615020221 0ustar00biocbuildbiocbuild\name{findCommonVariants} \alias{findCommonVariants} \title{Finding the Common Variants Among All Input VCF Files} \description{ The common variants (inc. SNPs and indels) among all the input files are found. A data frame containing these common variants is returned. The data frame has a similar format as VCF files. } \usage{ findCommonVariants(VCF_files) } \arguments{ \item{VCF_files}{ a character vector giving the names of VCF format files.} } \details{ This function loads all variants (SNPs and indels) from the input VCF files, and find the common variants that are reported in all the VCF files. If a variant record in a input VCF file has multiple alternative sequences (split by `,'), each alternative sequence is treated as a single variant. Two variants in two VCF files are the same only if their genomic locations, their reference sequences, their alternative sequences and their variant types are identical. This function currently does not support other types of variants other than SNPs and indels. There are eight columns in the returned data frame: chromosome name, position, identity, reference sequence, alternative sequence, quality, filter and extra information. The input may have more columns; these columns are not included in the data frame. If the identity, the quality, the filter and the extra information for the same variant are different among the input VCF files, those information associated with the lowest quality value of this variant among the VCF files is reported in the resulted data frame. For example, if an SNP on chrX:12345 (A=>G) is reported in all the three input VCF files, and the quality scores in the three VCF files are 100, 10, 50 respectively, the identity, the quality, the filter and the extra information in the second VCF file are reported in the resulted data frame for this SNP. } \value{ A data frame containing the common variants among all the input VCF files is returned. The first eight columns are: chromosome name, position, identity, reference sequence, alternative sequence, quality, filter and extra information. If there are not any common variants, this function returns an NA value. } %\references{ %} \author{Yang Liao and Wei Shi} %\note{} %\seealso{} \examples{\dontrun{ # finding the common variants between to input VCF files: a.vcf and b.vcf library(Rsubread) findCommonVariants(c('a.vcf','b.vcf')) }} % Add one or more standard keywords, see file 'KEYWORDS' in the % R documentation directory. %\keyword{} %\keyword{}% __ONLY ONE__ keyword per line Rsubread/man/flattenGTF.Rd0000644000175200017520000000514414136047615016416 0ustar00biocbuildbiocbuild\name{flattenGTF} \alias{flattenGTF} \title{Flatten Features in GTF or GFF Annotation Files} \description{Convert a GTF/GFF annotation to SAF format and flatten overlapping features.} \usage{ flattenGTF( # basic input/output options GTFfile, GTF.featureType = "exon", GTF.attrType = "gene_id", # the option specifying the merging algorithm method = "merge") } \arguments{ \item{GTFfile}{ a character string giving the name of a GTF file as input.} \item{GTF.featureType}{ a character string giving the feature type used to select rows in a GTF annotation. \code{"exon"} by default. Feature types can be found in the third column of a GTF annotation.} \item{GTF.attrType}{ a character string giving the attribute type in a GTF annotation which will be used to group features. \code{"gene_id"} by default. Attributes can be found in the ninth column of a GTF annotation.} \item{method}{ a character string specifying the method for how to flatten the GTF file. The method can be either \code{"merge"} or \code{"chop"}. \code{"merge"} by default. See the \code{details} section for more information.} } \details{ This function locates features in a GTF annotation via \code{GTF.featureType} and then groups them into meta-features via \code{GTF.attrType}. When \code{method="merge"}, the overlapping features found in a meta-feature will be merged to form a single continuous feature encompassing all the overlapping features. If \code{method="chop"}, overlapping features will be chopped into multiple non-overlapping bins. Here is an example to illustrate the differences between the two methods. Say there are three exons belonging to the same gene and the coordinates of these exons are [100, 200], [150, 500] and [250, 400]. When running on \code{"merge"} mode, a single exon will be returned for this gene and the coordinates of this exon are [100, 500], representing the union of the three original exons. When running on \code{"chop"} mode, five non-overlapping bins will be returned, including [100, 149], [150, 200], [201, 249], [250, 400] and [401, 500]. Intervals of these bins are determined by start and end coordinates of the three original exons (100, 150, 200, 250, 400 and 500). Output of this function is a SAF format annotation which can be fed to \code{\link{featureCounts}} function for read counting. Description to SAF format annotation can also be found in \code{\link{featureCounts}}. } \value{ A \code{data.frame} including a SAF format annotation in which features included in each meta-feature are all distinct. } \author{Yang Liao and Wei Shi} \seealso{\code{\link{featureCounts}}} %\examples{} Rsubread/man/getInBuiltAnnotation.Rd0000755000175200017520000000247614136047615020531 0ustar00biocbuildbiocbuild\name{getInBuiltAnnotation} \alias{getInBuiltAnnotation} \title{Retrieve In-Built Annotations} \description{Retrieve an in-built annotation and save it to a data frame.} \usage{ getInBuiltAnnotation(annotation = "mm10") } \arguments{ \item{annotation}{ a character string specifying the in-built annotation to be retrieved. It has four possible values including \code{mm10}, \code{mm9}, \code{hg38} and \code{hg19}, corresponding to the NCBI RefSeq annotations for genomes `mm10', `mm9', `hg38' and `hg19', respectively. \code{mm10} by default.} } \details{ The \code{\link{featureCounts}} read summarization function provides in-built annotations for conveniently summarizing reads to genes or exons, and this function allows users to have access to those in-built annotations. For more information about these annotations, please refer to the help page for \code{\link{featureCounts}} function. } \value{ A data frame with five columns including \code{GeneID}, \code{Chr}, \code{Start}, \code{End} and \code{Strand}. } %\references{ %} \author{Wei Shi} %\note{} \seealso{ \code{\link{featureCounts}}} \examples{ x <- getInBuiltAnnotation("hg38") x[1:5,] } % Add one or more standard keywords, see file 'KEYWORDS' in the % R documentation directory. %\keyword{} %\keyword{}% __ONLY ONE__ keyword per line Rsubread/man/processExons.Rd0000755000175200017520000000344114136047615017114 0ustar00biocbuildbiocbuild\name{processExons} \alias{processExons} \title{Obtain Chromosomal Coordiates of Each Exon Using NCBI Annotation} \description{This is for internal use. It converts the NCBI exon annotations into data files for use in the \code{detectionCall} function.} \usage{ processExons( filename = "human_seq_gene.md", species = "hg") } \arguments{ \item{filename}{ a character string giving the name of input .md file (NCBI annotation file)} \item{species}{ a character string specifying the species} } \details{ This is an internal function and should not be called by users directly. It processes annotation of genes in human genome GRCh37/hg19 or mouse genome GRCm37/mm9. The annotation is available for download from the following links (these annotations include chromosomal coordinates of UTR and CDS regions of genes): \url{ftp://ftp.ncbi.nlm.nih.gov/genomes/H_sapiens/ARCHIVE/BUILD.37.2/mapview/seq_gene.md.gz} \url{ftp://ftp.ncbi.nlm.nih.gov/genomes/M_musculus/ARCHIVE/BUILD.37.2/mapview/seq_gene.md.gz} This function finds the chromosomal coordinates of intergenic regions (regions between neighbouring genes) and then outputs them to a file. It also outputs to a file chromosomal coordinates of exons of genes by concatenating UTRs with CDSs and merging overlapping CDSs within each gene. The generated annotation files will then be used by \code{\link{detectionCallAnnotation}} function to produce annotation data required by \code{\link{detectionCall}} function. } \value{ Two annotation files are written to the current working directory. This function returns a \code{NULL} object. } %\references{} \author{Zhiyin Dai and Wei Shi} %\note{} %\seealso{} % Add one or more standard keywords, see file 'KEYWORDS' in the % R documentation directory. %\keyword{} %\keyword{}% __ONLY ONE__ keyword per line Rsubread/man/promoterRegions.Rd0000755000175200017520000000414014136047615017614 0ustar00biocbuildbiocbuild\name{promoterRegions} \alias{promoterRegions} \title{Generate Annotation for Promoter Regions of Genes} \description{Create a SAF data-frame of genewise promoter regions.} \usage{ promoterRegions( annotation = "mm10", upstream = 3000L, downstream = 2000L) } \arguments{ \item{annotation}{ a data.frame containing gene annotation in SAF format or a character string giving the name of a genome with built-in annotation. If using built-in annotation, the character string should be one of the following: \code{mm10}, \code{mm9}, \code{hg38} or \code{hg19} corresponding to the NCBI RefSeq annotations for the genomes `mm10', `mm9', `hg38' and `hg19', respectively.} \item{upstream}{ an integer giving the number of upstream bases that will be inclued in the promoter region generated for each gene. These bases are taken immediately upstream (5' end) from transcriptional start site of each gene.} \item{downstream}{ an integer giving the number of downstream bases that will be inclued in the promoter region generated for each gene. These bases are taken immediately downstream (3' end) from transcriptional start site of each gene.} } \details{ This function takes as input a SAF format gene annotation and produces a SAF format data.frame containing the chromosomal coordinates of the specified promoter region for each gene. See \code{\link{featureCounts}} for definition of the SAF format. Regardless of the \code{upstream} or \code{downstream} values, the downstream end of the region never extends past the end of the gene and the upstream end never extends outside the relevant chromosome. Setting \code{downstream} to an infinite or large value will cause the body of each gene to be included. } \value{ A SAF format \code{data.frame} with columns \code{GeneID}, \code{Chr}, \code{Start}, \code{End} and \code{Strand}. } %\references{ %} \author{Gordon K Smyth} %\note{} \seealso{ \code{\link{featureCounts}}, \code{\link{getInBuiltAnnotation}}} \examples{ # To get whole gene bodies for the mouse genome: x <- promoterRegions("mm10", upstream = 0, downstream = Inf) head(x) } Rsubread/man/propmapped.Rd0000755000175200017520000000533414136047615016573 0ustar00biocbuildbiocbuild\name{propmapped} \alias{propmapped} \title{Calculate the Proportion of Mapped Reads or Fragments in SAM/BAM Files} \description{Number of mapped reads/fragments will be counted and fraction of such reads/fragments will be calculated.} \usage{ propmapped( files, countFragments = TRUE, properlyPaired = FALSE, verbose = FALSE) } \arguments{ \item{files}{ a character vector giving the names of SAM/BAM format files. Format of input files is automatically determined by the function.} \item{countFragments}{ logical indicating whether reads or fragments (read pairs) should be counted. If \code{TRUE}, fragments will be counted when paired-end read data are provided. This function automatically detects if the data are single end or paired end. For single end data, each read is treated as a fragment and therefore the value of this parameter should be set to \code{TRUE}.} \item{properlyPaired}{ logical indicating if only properly paired reads will be counted. This is only applicable for paired end data. \code{FALSE} by default.} \item{verbose}{ logical indicating if verbose information should be displayed.} } \details{ This function uses the \code{FLAG} field in the SAM/BAM to look for mapped reads and count them. Reads/fragments, which have more than one reported location, will be reported only once. When counting single end reads, counting reads has the same meaning as counting fragments (the results are identical). Note that \code{align} and \code{subjunc} return the same mapping statistics as \code{propmapped} as a by-product of the alignment process, so saving the output from \code{align} or \code{subjunc} will be faster and more informative than running \code{propmapped} separately. } \value{ A data frame containing the total number of reads, number of mapped reads and proportion of mapped reads for each library. } %\references{ %} \author{Wei Shi and Yang Liao} %\note{} %\seealso{} \examples{ # build an index using the sample reference sequence provided in the package # and save it to the current directory ref <- system.file("extdata","reference.fa",package="Rsubread") buildindex(basename="./reference_index",reference=ref) # align the sample read data provided in this package to the sample reference # and save the mapping results to the current directory reads <- system.file("extdata","reads.txt.gz",package="Rsubread") stat <- align(index="./reference_index",readfile1=reads,output_file="./Rsubread_alignment.BAM") stat # get the percentage of successfully mapped reads propmapped("./Rsubread_alignment.BAM") } % Add one or more standard keywords, see file 'KEYWORDS' in the % R documentation directory. %\keyword{} %\keyword{}% __ONLY ONE__ keyword per line Rsubread/man/qualityScores.Rd0000755000175200017520000000372314136047615017273 0ustar00biocbuildbiocbuild\name{qualityScores} \alias{qualityScores} \title{Extract Quality Score Data in a Sequencing Read Dataset} \description{Extract quality strings and convert them to Phred scores for generating boxplots. } \usage{ qualityScores( # basic input/output options filename, input_format = "gzFASTQ", offset = 33, nreads = 10000) } \arguments{ \item{filename}{ a character string giving the name of an input file containing sequence reads.} \item{input_format}{ a character string specifying format of the input file. \code{gzFASTQ} (gzipped FASTQ) by default. Acceptable formats include \code{gzFASTQ}, \code{FASTQ}, \code{SAM} and \code{BAM}. Character string is case insensitive.} \item{offset}{ a numeric value giving the offset added to the base-calling Phred scores. Possible values include \code{33} and \code{64}. By default, \code{33} is used.} \item{nreads}{ a numeric value giving the number of reads from which quality scores are extracted. \code{10000} by default. A value of \code{-1} indicates that quality scores will be extracted from all the reads.} } \details{ Quality scores of read bases are represented by ASCII characters in next-gen sequencing data. This function extracts the quality characters from each base in each read and then converts them to Phred scores using the provided offset value (\code{offset}). If the total number of reads in a dataset is \code{n}, then every \code{n}/\code{nreads} read is extracted from the input data. } \value{ A data matrix containing Phred scores for read bases. Rows in the matrix are reads and columns are base positions in each read. } %\references{} \author{Wei Shi, Yang Liao and Zhiyin Dai} %\note{} %\seealso{} \examples{ reads <- system.file("extdata","reads.txt.gz",package="Rsubread") x <- qualityScores(filename=reads,offset=64,nreads=1000) x[1:10,1:10] } % Add one or more standard keywords, see file 'KEYWORDS' in the % R documentation directory. %\keyword{} %\keyword{}% __ONLY ONE__ keyword per line Rsubread/man/removeDupReads.Rd0000755000175200017520000000235114136047615017345 0ustar00biocbuildbiocbuild\name{removeDupReads} \alias{removeDupReads} \title{Remove Sequencing Reads Mapped to Identical Locations} \description{Remove reads which are mapped to identical locations, using mapping location of the first base of each read.} \usage{ removeDupReads( # basic input/output options inputFile, threshold = 50, outputFile, outputFormat = "BAM") } \arguments{ \item{inputFile}{ a character string giving the name of a SAM or BAM format input file.} \item{threshold}{ a numeric value giving the threshold for removing duplicated reads, 50 by default. Reads will be removed if they are found to be duplicated equal to or more than \code{threshold} times.} \item{outputFile}{ a character string giving the base name of output files.} \item{outputFormat}{ a character string giving the format of the output file. \code{BAM} and \code{SAM} are accepted. \code{BAM} by default.} } \details{ This function uses the mapping location of first base of each read to find duplicated reads. Reads are removed if they are duplicated more than \code{threshold} number of times. } \value{ This function generates a BAM or SAM file including the remaining reads after duplicate removal. } \author{Yang Liao and Wei Shi} Rsubread/man/repair.Rd0000755000175200017520000000442214136047615015703 0ustar00biocbuildbiocbuild\name{repair} \alias{repair} \title{Re-Order Paired-End Reads to Place Reads} \description{Fast re-ordering of paired-end reads using read names and mapping locations.} \usage{ repair( inFiles, inFormat = "BAM", outFiles = paste(inFiles,"repair",sep="."), addDummy = TRUE, fullData = TRUE, compress = FALSE, nthreads = 8) } \arguments{ \item{inFiles}{ a character vector giving names of input files. These files are typically location-sorted BAM files.} \item{inFormat}{ a character string specifying format of input files. Supported formats include \code{BAM} and \code{SAM}.} \item{outFiles}{ a character string giving names of output files. Re-ordered reads are saved to BAM-format files.} \item{addDummy}{ logical indicating if a dummy read will be added to each singleton read which has a missing pair in the input. \code{TRUE} by default.} \item{fullData}{ logical indicating if sequences and base-calling quality scores of reads will be included in the output. \code{TRUE} by default.} \item{compress}{ logical indicating if compression should be turned on when generating BAM output. \code{FALSE} by default.} \item{nthreads}{ a numeric value giving number of CPU threads. 8 by default.} } \details{ This function takes as input paired-end BAM or SAM files, re-orders reads to make reads from the same pair be adjacent to each other and outputs the re-ordered reads into new BAM files. The function makes use of both read names and mapping information of reads (eg. mapping coordinates) to identify reads belonging to the same pair. This makes sure that all paired-end reads are correctly re-ordered, especially those multi-mapping read pairs that include more than one reported alignment in the input. The BAM files produced by this function are compatible with \code{\link{featureCounts}}, meaning that no further re-ordering needs to be performed by \code{\link{featureCounts}}. } \value{ No value is produced but BAM files with re-ordered reads are written to the current working directory. } %\references{} \author{Wei Shi and Yang Liao} %\note{} %\seealso{} %\examples{} % Add one or more standard keywords, see file 'KEYWORDS' in the % R documentation directory. %\keyword{} %\keyword{}% __ONLY ONE__ keyword per line Rsubread/man/sam2bed.Rd0000755000175200017520000000247314136047615015742 0ustar00biocbuildbiocbuild\name{sam2bed} \alias{sam2bed} \title{Convert a SAM Format File to a BED File} \description{This function performs SAM to BED conversion. Each read in the SAM input is converted into an interval in the BED file.} \usage{ sam2bed( samfile, bedfile, readlen) } \arguments{ \item{samfile}{ a character string giving the name of input file. Input format should be in SAM format.} \item{bedfile}{ a character string giving the name of output file. Output file is in BED format.} \item{readlen}{ a numeric value giving the length of reads included in the input file.} } \details{ This function converts a SAM format file to a BED format file, which can then be displayed in a genome browser like UCSC genome browser, IGB, IGV. This function does not find the read length from the SAM file, but requires the user to specify the read length. } \value{ No value is produced but a BED format file is written to the current working directory. This file contains six columns including chromosomal name, start position, end position, name(`.'), mapping quality score and strandness. } %\references{} \author{Wei Shi} %\note{} %\seealso{} %\examples{} % Add one or more standard keywords, see file 'KEYWORDS' in the % R documentation directory. %\keyword{} %\keyword{}% __ONLY ONE__ keyword per line Rsubread/man/simReads.Rd0000644000175200017520000001740114136047615016166 0ustar00biocbuildbiocbuild\name{simReads} \alias{simReads} \alias{scanFasta} \title{Generate Simulated Reads from a Set of Transcripts} \description{The \code{simReads} function generates simulated reads from a set of transcripts. Each transcript has a predefined abundance in the output.} \usage{ simReads( # the transcript database and the wanted abundances transcript.file, expression.levels, # the name of the output output.prefix, # options on the output library.size = 1000000, read.length = 75, truth.in.read.names = FALSE, # simulating sequencing errors simulate.sequencing.error = TRUE, quality.reference = NULL, # parameters for generating paired-end reads. paired.end = FALSE, fragment.length.min = 100, fragment.length.max = 500, fragment.length.mean = 180, fragment.length.sd = 40, # manipulating transcript names simplify.transcript.names = FALSE) scanFasta( # the file containing the transcript database transcript.file, # manipulating transcript names simplify.transcript.names = FALSE, # miscellaneous options quiet = FALSE) } \arguments{ \item{transcript.file}{ a character string giving the name of a file that contains the transcript names and sequences. The format can be FASTA or gzipped-FASTA. No duplicate sequence name is allowed in the file, and duplicate sequences will trigger warning messages.} \item{expression.levels}{ a numeric vector giving the relative expression levels of the transcripts, in the order of transcripts in \code{transcript.file}. The sum of the values must be positive, and no negative value is allowed.} \item{output.prefix}{ a character string giving the basename of all the output files.} \item{library.size}{ a numeric value giving the number of reads or read-pairs to be generated. One million by default.} \item{read.length}{a anumeric value giving the length of each read in the output. Maximum length is 250bp. Transcripts that are shorter than the read length will not be used for generating simulated reads. 75 by default.} \item{truth.in.read.names}{ logical indicating if the true mapping location of reads or read-pairs should be encoded into the read names. \code{FALSE} by default.} \item{simulate.sequencing.error}{ logical indicating if sequencing errors should be simulated in the output reads. If \code{TRUE}, the \code{quality.reference} parameter must be specified unless the output read length is 100-bp or 75-bp. If the output read length is 100-bp or 75-bp, the \code{quality.reference} parameter can be optionally omitted, and the function will use its inbuilt quality strings.} \item{quality.reference}{ a character string giving the name of a file that contains one or multiple sequencing quality strings in the Phred+33 format. The sequencing quality strings must have the same length as \code{read.length}.} \item{paired.end}{ logical indicating if paired-end reads should be generated. \code{FALSE} by default.} \item{fragment.length.min}{ a numeric value giving the minimum fragment length. The minimum fragment length must be equal to or greater than the output read length. 100 by default.} \item{fragment.length.max}{ a numeric value giving the maximum fragment length. 500 by default.} \item{fragment.length.mean}{ a numeric value giving the mean of fragment lengths. 180 by default.} \item{fragment.length.sd}{ a numeric value giving the standard deviation of fragment lengths. The fragment lengths are drawn for a truncated gamma distribution that is defined by \code{fragment.length.min}, \code{fragment.length.max}, \code{fragment.length.mean} and \code{fragment.length.sd}. 40 by default.} \item{simplify.transcript.names}{ logical indicating if transcript names should be simplified. If \code{TRUE}, the transcript names are truncated to the first \code{|} or space. \code{FALSE} by default.} \item{quiet}{ logical indicating if the warning message for repeated sequences should be suppressed in the \code{scanFasta} function. \code{FALSE} by default.} } \details{ \code{simReads} generates simulated reads from a set of transcript sequences at specified abundances. The input includes a transcript file in FASTA or gzipped-FASTA format, and a numeric vector that describes the wanted abundance for each transcript. The output of this function is one or two gzipped FASTQ files that contain the simulated reads or read-pairs. To have reads generated from it, a transcript must have a length equal to or greater than the output read length, and also equal to or greater than the minimum fragment length in case of paired-end reads. When generating paired-end reads, the fragment lengths are drawn from a truncated normal distribution with the mean and standard deviation specified in \code{fragment.length.mean} and \code{fragment.length.sd}; the minimum and maximum fragment lengths are specified in \code{fragment.length.min} and \code{fragment.length.max}. Substitution sequencing errors can be simulated in the reads by emulating the sequencing quality of a real High-Throughput Sequencing sample. When \code{simulate.sequencing.error = TRUE} and a set of Phred+33 encoded quality strings are provided to \code{simReads}, it randomly chooses a quality string for each output read, and substitutes the read bases with random base values at the probabilities described in the quality string. This function has inbuilt quality strings for generating 100-bp and 75-bp long reads, hence the \code{quality.reference} can be optionally omitted when \code{read.length} is 100 or 75. The \code{scanFasta} function checks and processes the FASTA or gzipped-FASTA file. It scans through the file that defines the transcript sequences and returns a data.frame of transcript names and sequence lengths. It additionally checks the transcript sequences for uniqueness. } \value{ \code{simReads} writes a FASTQ file, or a pair of FASTQ files if \code{paired.end}, containing the simulated reads. It also returns a \code{data.frame} with three columns: \tabular{lll}{ \code{TranscriptID} \tab character \tab transcript name\cr \code{Length} \tab integer \tab length of transcript sequence\cr \code{Count} \tab integer \tab simulated read count } \code{scanFasta} returns a \code{data.frame} with six columns: \tabular{lll}{ \code{TranscriptID} \tab character \tab transcript name\cr \code{Length} \tab integer \tab length of transcript sequence\cr \code{MD5} \tab character \tab MD5 digest of the transcript sequence\cr \code{Unique} \tab logical \tab is this transcript's sequence unique in the FASTA file?\cr \code{Occurrence} \tab integer \tab number of times this transcript's sequence was observed\cr \code{Duplicate} \tab logical \tab this transcript's sequence is a duplicate of a previous sequence. } Note that selecting transcripts with \code{Duplicate == FALSE} will ensure unique sequences, i.e., any sequence that was observed multiple times in the FASTQ file will be only included only once in the selection. } %\references{ %Yang Liao, Gordon K Smyth and Wei Shi. The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. %\emph{Nucleic Acids Research}, 41(10):e108, 2013. %} \author{Yang Liao, Gordon K Smyth and Wei Shi} \examples{ \dontrun{ # Scan through the fasta file to get transcript names and lengths transcripts <- scanFasta("GENCODE-Human-transcripts.fa.gz") nsequences <- nrow(transcripts) - sum(transcripts$Duplicate) # Assign a random TPM value to each non-duplicated transcript sequence TPMs <- rep(0, nrow(transcripts)) TPMs[!transcripts$Duplicate] <- rexp(nsequences) # Generate actual reads. # The output read file is my-simulated-sample_R1.fastq.gz # The true read counts are returned. true.counts <- simReads("GENCODE-Human-transcripts.fa.gz", TPMs, "my-simulated-sample") print(true.counts[1:10,]) } } Rsubread/man/sublong.Rd0000644000175200017520000000456314136047615016075 0ustar00biocbuildbiocbuild\name{sublong} \alias{sublong} \title{Align Long Sequence Reads to a Reference Genome via Seed-and-Vote} \description{This function aligns DNA-seq reads, generated by long-read sequencing technologies such as Nanopore and PacBio sequencers, to a reference genome.} \usage{ sublong( # basic input/output options index, readFiles, outputFiles, outputFormat = "BAM", nthreads = 1) } \arguments{ \item{index}{ a character vector giving the basename of index files. Index files should be located in the current directory. The provided index should be a full index and also it should have only one block. See \code{buildindex} for index building options.} \item{readFiles}{ a character vector giving the names of input files that contain long sequence reads. FASTQ and gzipped FASTQ formats are both accepted.} \item{outputFiles}{ a character vector specifying the names of output files that contain read mapping results.} \item{outputFormat}{ a character string specifying the format of output files. \code{BAM} by default. Acceptable formats include \code{SAM} and \code{BAM}. } \item{nthreads}{ an integer giving the number of threads used for mapping. \code{1} by default. Note that when more than one thread is used, the order of reads might be changed in the output.} } \details{ \code{sublong} is designed for the mapping of long reads. It performs full alignment of reads by performing seed-and-vote mapping followed by a bounded dynamic programming procedure. \code{sublong} is able to map reads as long as millions of bases. \code{sublong} is extremely fast. It takes less than 10 minutes to complete the mapping of more than 100,000 long reads generated from Nanopore MinION ultra-long sequencing protocol. The number of CIGAR operations (eg. insertion and deletion) reported for a long read may exceed the limit on the total number of operations allowed in a CIGAR string (up to 65,535 operations in a CIGAR string in BAM output and up to 99,900 operations in a CIGAR string in SAM output). If this limited is exceeded, the read will be soft clipped. } \author{Yang Liao and Wei Shi} \examples{ ref <- system.file("extdata","reference.fa",package="Rsubread") buildindex(basename="./full_index",reference=ref,gappedIndex=FALSE, indexSplit=FALSE) reads <- system.file("extdata","longreads.txt.gz",package="Rsubread") sublong("./full_index",reads,"./Long_alignment.BAM",nthreads=4) } Rsubread/man/txUnique.Rd0000644000175200017520000000362114136047615016240 0ustar00biocbuildbiocbuild\name{txUnique} \alias{txUnique} \title{Count Number of Bases Unique to Each Transcript} \description{For each transcript, number of unique bases and total number of bases will be reported. } \usage{ txUnique( # basic input/output options GTF_Annotation_File, Feature_Type = "exon", Gene_ID_Attribute = "gene_id", Transcript_ID_Attribute = "transcript_id") } \arguments{ \item{GTF_Annotation_File}{ a character string giving the name of a GTF file containing the transcript-level annotations.} \item{Feature_Type}{ a character string specifying the type of annotations. Only the annotations of the given type will be loaded from the GTF file. \code{exon} by default. The third column in the GTF file contains the type of the annotation in each line} \item{Gene_ID_Attribute}{ a character string specifying the attribute name in the 9-th column in the GTF file for the gene identifier. \code{gene_id} by default.} \item{Transcript_ID_Attribute}{ a character string specifying the attribute name in the 9-th column in the GTF file for the transcript identifier. \code{transcript_id} by default.} } \details{ This function compares the transcripts belonging to the same gene and then counts the number of bases unique to each transcript. It also reports the total number of bases for each transcript. When a transcript is found to contain overlapping exons, the overlapping exons will be merged and each overlapping base will be counted only once. } \value{ A data matrix in which each row represents a transcript. The matrix includes four columns: gene identifier, transcript identifier, number of bases unique to each transcript and total number of bases each transcript has. } %\references{} \author{Yang Liao and Wei Shi} %\note{} %\seealso{} %\examples{ %} % Add one or more standard keywords, see file 'KEYWORDS' in the % R documentation directory. %\keyword{} %\keyword{}% __ONLY ONE__ keyword per line Rsubread/src/0000755000175200017520000000000014142061021014121 5ustar00biocbuildbiocbuildRsubread/src/HelperFunctions.c0000644000175200017520000022772014136047615017427 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #include #include #include #include #include #include #include #ifdef MACOS #include #include #include #include #include #include #include #include #include #include #include #include #else #ifndef __MINGW32__ #include #include #include #include #endif #include #endif #include "subread.h" #include "input-files.h" #include "seek-zlib.h" #include "gene-algorithms.h" #include "HelperFunctions.h" void sorting_LLU_array_exchange(void * varr, int i, int j){ srUInt_64 *arr = varr; srUInt_64 t = arr[i]; arr[i]=arr[j]; arr[j]=t; } int sorting_LLU_array_compare(void * varr, int i, int j){ srUInt_64 *arr = varr; if(arr[i]>arr[j])return 1; if(arr[i]= 9999) { ret *=1024; break; } } } if(!rret) break; } fclose(mfp); return ret; } char * get_short_fname(char * lname){ char * ret = lname; int x1; for(x1 = strlen(lname)-2; x1>=0; x1--){ if(lname [x1] == '/'||lname [x1] == '\\'){ ret = lname + x1 + 1; break; } } return ret; } // This assumes the first part of Cigar has differet strandness to the main part of the cigar. // Pos is the LAST WANTED BASE location before the first strand jump (split by 'b' or 'n'). // The first base in the read actually has a larger coordinate than Pos. // new_cigar has to be at least 100 bytes. unsigned int reverse_cigar(unsigned int pos, char * cigar, char * new_cigar) { int cigar_cursor = 0; new_cigar[0]=0; unsigned int tmpi=0; int last_piece_end = 0; int last_sec_start = 0; unsigned int chro_pos = pos, this_section_start = pos, ret = pos; int is_positive_dir = 0; int read_cursor = 0; int section_no = 0; for(cigar_cursor = 0 ; ; cigar_cursor++) { if( cigar [cigar_cursor] == 'n' || cigar [cigar_cursor] == 'b' || cigar [cigar_cursor] == 0) { int xk1, jmlen=0, nclen=strlen(new_cigar); char jump_mode [13]; if(cigar [cigar_cursor] !=0) { sprintf(jump_mode, "%u%c", tmpi, cigar [cigar_cursor] == 'b'?'n':'b'); jmlen = strlen(jump_mode); } for(xk1=nclen-1;xk1>=0; xk1--) new_cigar[ xk1 + last_piece_end + jmlen - last_sec_start ] = new_cigar[ xk1 ]; new_cigar [nclen + jmlen + last_piece_end - last_sec_start ] = 0; memcpy(new_cigar , jump_mode, jmlen); memcpy(new_cigar + jmlen , cigar + last_sec_start, last_piece_end - last_sec_start); last_sec_start = cigar_cursor+1; if(is_positive_dir && cigar [cigar_cursor] !=0) { if(cigar [cigar_cursor] == 'b') chro_pos -= tmpi - read_cursor - 1; else chro_pos += tmpi - read_cursor - 1; } if((!is_positive_dir) && cigar [cigar_cursor] !=0) { if(cigar [cigar_cursor] == 'b') chro_pos = this_section_start - tmpi - read_cursor - 1; else chro_pos = this_section_start + tmpi - read_cursor - 1; } this_section_start = chro_pos; if(section_no == 0) ret = chro_pos; is_positive_dir = ! is_positive_dir; section_no++; tmpi=0; } else if(isalpha(cigar [cigar_cursor])) { if(cigar [cigar_cursor]=='M' || cigar [cigar_cursor] == 'S') read_cursor += tmpi; tmpi=0; last_piece_end = cigar_cursor+1; } else tmpi = tmpi*10 + (cigar [cigar_cursor] - '0'); if(cigar [cigar_cursor] == 0)break; } SUBREADprintf("REV CIGAR: %s => %s\n", cigar, new_cigar); return ret; } unsigned int find_left_end_cigar(unsigned int right_pos, char * cigar){ int delta_from_right = 0; int cigar_cursor = 0; unsigned int tmpi = 0; while(1){ int nch = cigar[cigar_cursor++]; if(nch == 0) break; if(isdigit(nch)){ tmpi = tmpi * 10 + nch - '0'; }else{ if(nch == 'M'||nch == 'D' || nch == 'N'){ delta_from_right +=tmpi; } tmpi = 0; } } return right_pos - delta_from_right; } char contig_fasta_int2base(int v){ if(v == 1) return 'A'; if(v == 2) return 'T'; if(v == 3) return 'G'; if(v == 4) return 'C'; return 'N'; } int contig_fasta_base2int(char base){ base = tolower(base); if((base) == 'a'){ return 1;} else if((base) == 't' || (base) == 'u'){ return 2;} else if((base) == 'g'){ return 3;} else if((base) == 'c'){ return 4;} else return 15 ; } int get_contig_fasta(fasta_contigs_t * tab, char * chro, unsigned int pos, int len, char * out_bases){ unsigned int this_size = HashTableGet( tab -> size_table, chro ) - NULL; if(this_size > 0){ if(this_size >= len && pos <= this_size - len){ char * bin_block = HashTableGet(tab -> contig_table, chro ); unsigned int bin_byte = pos / 2; int bin_bit = 4*(pos % 2), x1; for(x1 = 0 ;x1 < len; x1++) { int bin_int = (bin_block[bin_byte] >> bin_bit) & 0xf; if(bin_bit == 4) bin_byte++; bin_bit = (bin_bit == 4)?0:4; out_bases[x1] = contig_fasta_int2base(bin_int); } return 0; } } return 1; } void destroy_contig_fasta(fasta_contigs_t * tab){ HashTableDestroy( tab -> size_table ); HashTableDestroy( tab -> contig_table ); } int read_contig_fasta(fasta_contigs_t * tab, char * fname){ autozip_fp fp; int rv = autozip_open(fname, &fp); if(rv>=0){ tab -> contig_table = HashTableCreate(3943); tab -> size_table = HashTableCreate(3943); HashTableSetDeallocationFunctions(tab -> contig_table, free, free); HashTableSetDeallocationFunctions(tab -> size_table, NULL, NULL); HashTableSetKeyComparisonFunction(tab -> contig_table, fc_strcmp_chro); HashTableSetKeyComparisonFunction(tab -> size_table, fc_strcmp_chro); HashTableSetHashFunction(tab -> contig_table, fc_chro_hash); HashTableSetHashFunction(tab -> size_table, fc_chro_hash); char chro_name[MAX_CHROMOSOME_NAME_LEN]; unsigned int inner_cursor = 0, current_bin_space = 0; int status = 0; char * bin_block = NULL; chro_name[0]=0; while(1){ int nch = autozip_getch(&fp); if(nch<0)break; if(status == 0){ assert(nch == '>'); status = 1; }else if(status == 1){ if(inner_cursor == 0){ bin_block = calloc(sizeof(char),10000); current_bin_space = 10000; } if(nch == '|' || nch == ' ') status = 2; else if(nch == '\n'){ status = 3; inner_cursor = 0; }else{ chro_name[inner_cursor++] = nch; chro_name[inner_cursor] = 0; } }else if(status == 2){ if(nch == '\n'){ status = 3; inner_cursor = 0; } }else if(status == 3){ if(nch == '>' || nch <= 0){ char * mem_chro = malloc(strlen(chro_name)+1); strcpy(mem_chro, chro_name); HashTablePut(tab -> size_table , mem_chro, NULL + inner_cursor); HashTablePut(tab -> contig_table , mem_chro, bin_block); // SUBREADprintf("Read '%s' : %u bases\n", chro_name, inner_cursor); inner_cursor = 0; status = 1; if(nch <= 0) break; }else if(nch != '\n'){ int bin_bytes = inner_cursor / 2; int bin_bits = 4*(inner_cursor % 2); int base_int = contig_fasta_base2int(nch); if(bin_bytes >= current_bin_space){ unsigned int new_bin_space = current_bin_space / 4 * 5; if(current_bin_space > 0xffff0000 /5 * 4){ assert(0); return 1; } bin_block = realloc(bin_block, new_bin_space); memset(bin_block + current_bin_space, 0, new_bin_space - current_bin_space); current_bin_space = new_bin_space; } bin_block[bin_bytes] |= (base_int << bin_bits); inner_cursor++; } } } autozip_close(&fp); return 0; } return 1; } int RSubread_parse_CIGAR_Extra_string(int FLAG, char * MainChro, unsigned int MainPos, const char * CIGAR_Str, const char * Extra_Tags, int max_M, char ** Chros, unsigned int * Staring_Chro_Points, unsigned short * Section_Start_Read_Pos, unsigned short * Section_Length, int * is_junction_read){ int ret = RSubread_parse_CIGAR_string(MainChro, MainPos, CIGAR_Str, max_M, Chros, Staring_Chro_Points, Section_Start_Read_Pos, Section_Length, is_junction_read); char read_main_strand = (((FLAG & 0x40)==0x40) == ((FLAG & 0x10) == 0x10 ))?'-':'+'; int tag_cursor=0; //SUBREADprintf("EXTRA=%s\n", Extra_Tags); int status = PARSE_STATUS_TAGNAME; char tag_name[2], typechar=0; int tag_inner_cursor=0; char current_fusion_char[MAX_CHROMOSOME_NAME_LEN]; unsigned int current_fusion_pos = 0; char current_fusion_strand = 0; char current_fusion_cigar[max_M * 15]; current_fusion_cigar [0] =0; current_fusion_char [0]=0; while(1){ int nch = Extra_Tags[tag_cursor]; if(status == PARSE_STATUS_TAGNAME){ tag_name[tag_inner_cursor++] = nch; if(tag_inner_cursor == 2){ status = PARSE_STATUS_TAGTYPE; tag_cursor += 1; assert(Extra_Tags[tag_cursor] == ':'); } }else if(status == PARSE_STATUS_TAGTYPE){ typechar = nch; tag_cursor +=1; assert(Extra_Tags[tag_cursor] == ':'); tag_inner_cursor = 0; status = PARSE_STATUS_TAGVALUE; }else if(status == PARSE_STATUS_TAGVALUE){ if(nch == '\t' || nch == 0 || nch == '\n'){ if(current_fusion_cigar[0] && current_fusion_char[0] && current_fusion_pos && current_fusion_strand){ //SUBREADprintf("ENTER CALC:%s\n", current_fusion_char ); unsigned int left_pos = current_fusion_pos; if(current_fusion_strand!=read_main_strand) left_pos = find_left_end_cigar(current_fusion_pos, current_fusion_cigar); ret += RSubread_parse_CIGAR_string(current_fusion_char, left_pos, current_fusion_cigar, max_M - ret, Chros + ret, Staring_Chro_Points+ ret, Section_Start_Read_Pos+ ret, Section_Length + ret, is_junction_read); current_fusion_pos = 0; current_fusion_strand = 0; current_fusion_cigar [0] =0; current_fusion_char [0]=0; //SUBREADprintf("EXIT CALC:%s\n", current_fusion_char ); } tag_inner_cursor = 0; status = PARSE_STATUS_TAGNAME; }else{ if(tag_name[0]=='C' && tag_name[1]=='C' && typechar == 'Z'){ current_fusion_char[tag_inner_cursor++]=nch; current_fusion_char[tag_inner_cursor]=0; }else if(tag_name[0]=='C' && tag_name[1]=='G' && typechar == 'Z'){ current_fusion_cigar[tag_inner_cursor++]=nch; current_fusion_cigar[tag_inner_cursor]=0; }else if(tag_name[0]=='C' && tag_name[1]=='P' && typechar == 'i'){ current_fusion_pos = current_fusion_pos * 10 + (nch - '0'); }else if(tag_name[0]=='C' && tag_name[1]=='T' && typechar == 'Z'){ //SUBREADprintf("pos=%d %c -> %c\n", tag_cursor, current_fusion_strand, nch); current_fusion_strand = nch; //SUBREADprintf("spo=%d %c -> %c\n", tag_cursor, current_fusion_strand, nch); } } } if(nch == 0 || nch == '\n'){ assert(status == PARSE_STATUS_TAGNAME); break; } tag_cursor++; //SUBREADprintf("CUR=%d [%s], c=%d\n", tag_cursor, Extra_Tags, Extra_Tags[tag_cursor]); } return ret; } int RSubread_parse_CIGAR_string(char * chro , unsigned int first_pos, const char * CIGAR_Str, int max_M, char ** Section_Chromosomes, unsigned int * Section_Start_Chro_Pos,unsigned short * Section_Start_Read_Pos, unsigned short * Section_Chro_Length, int * is_junction_read) { unsigned int tmp_int=0; int cigar_cursor=0; unsigned short current_section_chro_len=0, current_section_start_read_pos = 0, read_cursor = 0; unsigned int chromosome_cursor=first_pos; int ret=0, is_first_S = 1; for(cigar_cursor=0; ; cigar_cursor++) { char ch = CIGAR_Str[cigar_cursor]; if(ch >='0' && ch <= '9') { tmp_int=tmp_int*10+(ch - '0'); } else { if(ch == 'S'){ if(is_first_S) current_section_start_read_pos = tmp_int; read_cursor += tmp_int; } else if(ch == 'M' || ch == 'X' || ch == '=') { read_cursor += tmp_int; current_section_chro_len += tmp_int; chromosome_cursor += tmp_int; } else if(ch == 'N' || ch == 'D' || ch=='I' || ch == 0) { if('N' == ch)(*is_junction_read)=1; if(ret < max_M) { if(current_section_chro_len>0) { Section_Chromosomes[ret] = chro; Section_Start_Chro_Pos[ret] = chromosome_cursor - current_section_chro_len; Section_Start_Read_Pos[ret] = current_section_start_read_pos; Section_Chro_Length[ret] = current_section_chro_len; ret ++; } }else assert(0); current_section_chro_len = 0; if(ch == 'I') read_cursor += tmp_int; else if(ch == 'N' || ch == 'D') chromosome_cursor += tmp_int; current_section_start_read_pos = read_cursor; if(ch == 0) break; } //printf("C=%c, TV=%d, CC=%d, RC=%d\n", ch, tmp_int, chromosome_cursor, current_section_chro_len); tmp_int = 0; is_first_S = 0; } if(cigar_cursor>100) return -1; } return ret; } void display_sections(char * CIGAR_Str) { //int is_junc=0; int Section_Start_Chro_Pos[FC_CIGAR_PARSER_ITEMS]; unsigned short Section_Start_Read_Pos[FC_CIGAR_PARSER_ITEMS]; unsigned short Section_Chro_Length[FC_CIGAR_PARSER_ITEMS]; int retv = 0;//RSubread_parse_CIGAR_string(CIGAR_Str, Section_Start_Chro_Pos, Section_Start_Read_Pos, Section_Chro_Length, &is_junc); int x1; SUBREADprintf("Cigar=%s ; Sections=%d\n", CIGAR_Str, retv); for(x1=0; x1190) return -1; char nch = Extra_Col[col_cursor]; if(nch == '\n' || nch == '\r') nch = 0; if(state == GECV_STATE_BEFORE) { if(GTF_extra_column_istoken_chr(nch)) { name_buffer[0] = nch; name_cursor = 1; state = GECV_STATE_NAME; } else if(nch != ' ' && nch != 0) { state = GECV_STATE_ERROR; } } else if(state == GECV_STATE_NAME) { if(nch == ' ' || nch == '=') { state = GECV_STATE_GAP; name_buffer[name_cursor] = 0; is_correct_name = (strcmp(name_buffer , Target_Name) == 0); //printf("CORR=%d : '%s'\n", is_correct_name, name_buffer); } else if(nch == '"') { name_buffer[name_cursor] = 0; is_correct_name = (strcmp(name_buffer , Target_Name) == 0); state = GECV_STATE_QVALUE; if(is_correct_name) value_cursor = 0; } else if(GTF_extra_column_istoken_chr(nch)) name_buffer[name_cursor++] = nch; else { state = GECV_STATE_ERROR; //printf("ERR2 : '%c'\n", nch); } } else if(state == GECV_STATE_GAP) { if(nch == '"') { state = GECV_STATE_QVALUE; if(is_correct_name) value_cursor = 0; } else if(nch != '=' && isgraph(nch)) { state = GECV_STATE_VALUE; if(is_correct_name) { Target_Value[0]=nch; value_cursor = 1; } } else if(nch != ' ' && nch != '=') state = GECV_STATE_ERROR; } else if(state == GECV_STATE_VALUE) { if(nch == ';' || nch == 0) { state = GECV_STATE_BEFORE; if(is_correct_name) { Target_Value[value_cursor] = 0; } is_correct_name = 0; } else{ if(value_cursor < TargVal_Size-1 && is_correct_name) Target_Value[value_cursor++] = nch; } } else if(state == GECV_STATE_QVALUE) { if(nch == '"') { state = GECV_STATE_QV_END; if(is_correct_name) Target_Value[value_cursor] = 0; is_correct_name = 0; } else { if(value_cursor < TargVal_Size-1 && is_correct_name) { if(nch !=' ' || value_cursor>0) Target_Value[value_cursor++] = nch; } } } else if(state == GECV_STATE_QV_END) { if(nch == ';' || nch == 0) state = GECV_STATE_BEFORE; else if(nch != ' ') state = GECV_STATE_ERROR; } if (GECV_STATE_ERROR == state){ Target_Value[0]=0; return -1; } if (nch == 0) { if(state == GECV_STATE_BEFORE && value_cursor>0) { int x1; for(x1 = value_cursor-1; x1>=0; x1--) { if(Target_Value[x1] == ' '){ value_cursor --; Target_Value[x1]=0; } else break; } if(value_cursor>0) return value_cursor; } Target_Value[0]=0; return -1; } col_cursor++; } } void hpl_test2_func() { char * extra_column = " gene_id \"PC4-013 \"; 013=ABCD ; PC4 = CCXX "; char * col_name = "gene_id"; char col_val[100]; int col_len = GTF_extra_column_value(extra_column, col_name, col_val, 100); SUBREADprintf("LEN=%d; KEY='%s'; VAL=\"%s\"\n", col_len, col_name, col_val); col_name = "013"; col_len = GTF_extra_column_value(extra_column, col_name, col_val, 100); SUBREADprintf("LEN=%d; KEY='%s'; VAL=\"%s\"\n", col_len, col_name, col_val); col_name = "PC4"; col_len = GTF_extra_column_value(extra_column, col_name, col_val, 100); SUBREADprintf("LEN=%d; KEY='%s'; VAL=\"%s\"\n", col_len, col_name, col_val); col_name = "XXX"; col_len = GTF_extra_column_value(extra_column, col_name, col_val, 100); SUBREADprintf("LEN=%d; KEY='%s'; VAL=\"%s\"\n", col_len, col_name, col_val); extra_column = "gene_id = \"PC4-013 ;=\" ;013 = AXXD ; PC4=x"; col_name = "013"; col_len = GTF_extra_column_value(extra_column, col_name, col_val, 100); SUBREADprintf("LEN=%d; KEY='%s'; VAL=\"%s\"\n", col_len, col_name, col_val); col_name = "gene_id"; col_len = GTF_extra_column_value(extra_column, col_name, col_val, 100); SUBREADprintf("LEN=%d; KEY='%s'; VAL=\"%s\"\n", col_len, col_name, col_val); col_name = "PC4"; col_len = GTF_extra_column_value(extra_column, col_name, col_val, 100); SUBREADprintf("LEN=%d; KEY='%s'; VAL=\"%s\"\n", col_len, col_name, col_val); extra_column = " gene_id\" PC4-013 ;= \"; XXX='123' ;013 :ABCD ; PC4 = CCXX= ;"; col_name = "013"; col_len = GTF_extra_column_value(extra_column, col_name, col_val, 100); SUBREADprintf("LEN=%d; KEY='%s'; VAL=\"%s\"\n", col_len, col_name, col_val); col_name = "XXX"; col_len = GTF_extra_column_value(extra_column, col_name, col_val, 100); SUBREADprintf("LEN=%d; KEY='%s'; VAL=\"%s\"\n", col_len, col_name, col_val); col_name = "PC4"; col_len = GTF_extra_column_value(extra_column, col_name, col_val, 100); SUBREADprintf("LEN=%d; KEY='%s'; VAL=\"%s\"\n", col_len, col_name, col_val); col_name = "gene_id"; col_len = GTF_extra_column_value(extra_column, col_name, col_val, 100); SUBREADprintf("LEN=%d; KEY='%s'; VAL=\"%s\"\n", col_len, col_name, col_val); extra_column = "gene_id \"653635\"; transcript_id \"TR:653635\";"; col_name = "gene_id"; col_len = GTF_extra_column_value(extra_column, col_name, col_val, 100); SUBREADprintf("LEN=%d; KEY='%s'; VAL=\"%s\"\n", col_len, col_name, col_val); } void hpl_test1_func() { display_sections(""); display_sections("*"); display_sections("5S10M2D10M800N12M3I12M450N12M12D99M6S"); display_sections("110M2I10M800N32M3I12M6S"); display_sections("200S110M2I10M800N32M3I12M200N40M"); display_sections("3M1663N61M1045N36M3D20M66N10M2D10M77N3M1663N61M1045N36M3D20M66N103M1663N61M1045N36M3D20M66N9M"); } void test_bam_reader(){ SamBam_FILE * bamfp = SamBam_fopen("/usr/local/work/liao/arena/Rsubread_Paper_OCT2016/RNAseq-SimHG38/Runs-100/STAR/STAR-Simulation-15M-DXC.bamAligned.out.bam", SAMBAM_FILE_BAM); assert(bamfp); while(1){ char linebuf[2000]; char * ret = SamBam_fgets(bamfp, linebuf, 1999, 1); if(!ret) break; SUBREADprintf("%s", linebuf); // linebuf has "\n" in the end. } SamBam_fclose(bamfp); } #ifdef HELPER_TEST_SAMBAM_READER main() { //hpl_test1_func(); test_bam_reader(); // hpl_test1_func(); } #endif char *str_replace(char *orig, char *rep, char *with) { char *result; // the return string char *ins; // the next insert point char *tmp; // varies int len_rep; // length of rep int len_with; // length of with int len_front; // distance between rep and end of last rep int count; // number of replacements if (!orig) return NULL; if (!rep) rep = ""; len_rep = strlen(rep); if (!with) with = ""; len_with = strlen(with); ins = orig; for (count = 0; NULL != (tmp = strstr(ins, rep)); ++count) { ins = tmp + len_rep; } tmp = result = malloc(strlen(orig) + (len_with - len_rep) * count + 1); if (!result) return NULL; while (count--) { ins = strstr(orig, rep); len_front = ins - orig; tmp = strncpy(tmp, orig, len_front) + len_front; tmp = strcpy(tmp, with) + len_with; orig += len_front + len_rep; // move to next "end of rep" } strcpy(tmp, orig); return result; } // rule: the string is ABC123XXXXXX... // This is the priroity: // First, compare the letters part. // Second, compare the pure numeric part. // Third, compare the remainder. int strcmp_number(char * s1, char * s2) { int x1 = 0; int ret = 0; while(1) { char nch1 = s1[x1]; char nch2 = s2[x1]; if((!nch1) || !nch2){return nch2?1:(nch1?(-1):0);} if(isdigit(nch1) && isdigit(nch2))break; ret = nch1 - nch2; if(ret) return ret; x1++; } int v1 = 0, v2 = 0; while(1) { char nch1 = s1[x1]; char nch2 = s2[x1]; if((!nch1) || !nch2){ if(nch1 || nch2) return nch2?(-1):1; break; } int is_chr1_digit = isdigit(nch1); int is_chr2_digit = isdigit(nch2); if(is_chr1_digit || is_chr2_digit) { if(is_chr1_digit && is_chr2_digit) { v1 = v1*10+(nch1-'0'); v2 = v2*10+(nch2-'0'); } else { ret = nch1 - nch2; return ret; } } else break; x1++; } if(v1==v2) return strcmp(s1+x1, s2+x1); else { ret = v1 - v2; return ret; } } int mac_str(char * str_buff) { #if defined(FREEBSD) || defined(__MINGW32__) return 1; #else #ifdef MACOS int mib[6], x1, ret = 1; size_t len; char *buf; unsigned char *ptr; struct if_msghdr *ifm; struct sockaddr_dl *sdl; for(x1 = 0 ; x1 < 40; x1++) { mib[0] = CTL_NET; mib[1] = AF_ROUTE; mib[2] = 0; mib[3] = AF_LINK; mib[4] = NET_RT_IFLIST; mib[5] = x1; if (sysctl(mib, 6, NULL, &len, NULL, 0) >=0) { buf = malloc(len); if (sysctl(mib, 6, buf, &len, NULL, 0) >=0) { ifm = (struct if_msghdr *)buf; sdl = (struct sockaddr_dl *)(ifm + 1); ptr = (unsigned char *)LLADDR(sdl); if(sdl -> sdl_nlen < 1) continue; char * ifname = malloc(sdl -> sdl_nlen + 1); memcpy(ifname, sdl -> sdl_data, sdl -> sdl_nlen); ifname[sdl -> sdl_nlen] = 0; if(ifname[0]!='e'){ free(ifname); continue; } free(ifname); sprintf(str_buff,"%02X%02X%02X%02X%02X%02X", *ptr, *(ptr+1), *(ptr+2), *(ptr+3), *(ptr+4), *(ptr+5)); ret = 0; break; } free(buf); } } return ret; #else #if defined(IFHWADDRLEN) struct ifreq ifr; struct ifconf ifc; char buf[1024]; int success = 0; int sock = socket(AF_INET, SOCK_DGRAM, IPPROTO_IP); if (sock == -1) { /* handle error*/ }; ifc.ifc_len = sizeof(buf); ifc.ifc_buf = buf; if (ioctl(sock, SIOCGIFCONF, &ifc) == -1) { /* handle error */ } struct ifreq* it = ifc.ifc_req; const struct ifreq* const end = it + (ifc.ifc_len / sizeof(struct ifreq)); for (; it != end; ++it) { strcpy(ifr.ifr_name, it->ifr_name); if (ioctl(sock, SIOCGIFFLAGS, &ifr) == 0) { if (! (ifr.ifr_flags & IFF_LOOPBACK)) { // don't count loopback if (ioctl(sock, SIOCGIFHWADDR, &ifr) == 0) { success = 1; break; } } } } close(sock); unsigned char mac_address[6]; if (success){ memcpy(mac_address, ifr.ifr_hwaddr.sa_data, 6); int x1; for(x1 = 0; x1 < 6; x1++){ sprintf(str_buff+2*x1, "%02X",mac_address[x1]); } return 0; } #endif return 1; #endif #endif } int rand_str(char * str_buff){ int ret = 1; FILE * fp = fopen("/dev/urandom","r"); if(fp){ int x1; for(x1=0; x1<6; x1++){ sprintf(str_buff + 2*x1 , "%02X", fgetc(fp)); } fclose(fp); ret = 0; } return ret; } int mathrand_str(char * str_buff){ myrand_srand((int)(miltime()*100)); int x1; for(x1 = 0; x1 < 6; x1++){ sprintf(str_buff+2*x1, "%02X", myrand_rand() & 0xff ); } return 0; } int mac_or_rand_str(char * str_buff){ return mac_str(str_buff) && rand_str(str_buff) && mathrand_str(str_buff); } #define PI_LONG 3.1415926535897932384626434L long double fast_fractorial(unsigned int x, long double * buff, int buflen){ if(x<2) return 0; if(buff != NULL && x < buflen && buff[x]!=0){ return buff[x]; } long double ret; if(x<50){ int x1; ret =0.L; for(x1 = 2 ; x1 <= x; x1++) ret += logl((long double)(x1)); }else{ ret = logl(x)*1.0L*x - 1.0L*x + 0.5L * logl(2.0L*PI_LONG* x) + 1.L/(12.L*x) - 1.L/(360.L* x*x*x) + 1.L/(1260.L* x*x*x*x*x) - 1.L/(1680.L*x*x*x*x*x*x*x); } if(buff != NULL && x < buflen) buff[x]=ret; return ret; } #define BUFF_4D 36 long double fast_freq( unsigned int tab[2][2] , long double * buff, int buflen){ int x0 = -1; if(buff && buflen > BUFF_4D * BUFF_4D * BUFF_4D * BUFF_4D && tab[0][0] < BUFF_4D && tab[0][1] < BUFF_4D && tab[1][0] < BUFF_4D && tab[1][1] < BUFF_4D ){ x0 = buflen + tab[0][0]* BUFF_4D * BUFF_4D * BUFF_4D + tab[0][1] * BUFF_4D * BUFF_4D + tab[1][0] * BUFF_4D + tab[1][1]; if(buff[x0]!=0) return buff[x0]; } long double ret = fast_fractorial(tab[0][0]+tab[0][1],buff,buflen)+fast_fractorial(tab[1][0]+tab[1][1],buff,buflen) + fast_fractorial(tab[0][0]+tab[1][0],buff,buflen)+fast_fractorial(tab[0][1]+tab[1][1],buff,buflen) - fast_fractorial(tab[0][0],buff,buflen) - fast_fractorial(tab[0][1],buff,buflen) - fast_fractorial(tab[1][0],buff,buflen) - fast_fractorial(tab[1][1],buff,buflen) - fast_fractorial(tab[0][0] + tab[0][1] + tab[1][0] + tab[1][1],buff,buflen); if(x0>=0) buff[x0] = ret; return ret; } double fast_fisher_test_one_side(unsigned int a, unsigned int b, unsigned int c, unsigned int d, long double * buff, int buflen){ unsigned int tab[2][2]; long double P0, P_delta, ret; tab[0][0]=a; tab[0][1]=b; tab[1][0]=c; tab[1][1]=d; int x_a, y_a, x_min=-1, y_min=-1; unsigned int min_a = 0xffffffff; for(x_a = 0; x_a < 2; x_a++) for(y_a = 0; y_a < 2; y_a++){ if(tab[x_a][y_a]0){ unsigned int Qa = min_a; unsigned int Qb = tab[x_min][!y_min]; unsigned int Qc = tab[!x_min][y_min]; unsigned int Qd = tab[!x_min][!y_min]; for(; ; ){ min_a --; Qb++;Qc++; P_delta -= logl(Qb*Qc); P_delta += logl(Qa*Qd); Qa--;Qd--; ret += expl(P_delta); // printf("%LG %LG %LG\n", ret, 1 - (ret - P0), expl(P_delta)); if(min_a < 1) break; } } double ret1 = ret, ret2 = 1 - (ret - P0); if(min(ret1, ret2) < 0){ return 0.0; } return min(ret1, ret2) ; } int load_features_annotation(char * file_name, int file_type, char * gene_id_column, char * transcript_id_column, char * used_feature_type, void * context, int do_add_feature(char * gene_name, char * transcript_name, char * chro_name, unsigned int start, unsigned int end, int is_negative_strand, void * context) ){ char * file_line = malloc(MAX_LINE_LENGTH+1); int lineno = 0, is_GFF_txid_warned = 0, is_GFF_geneid_warned = 0, loaded_features = 0; autozip_fp afp; int aret = autozip_open(file_name, &afp); if(aret < 0){ SUBREADprintf("Error: unable to open the annotation file : %s\n", file_name); return -1; } while(1){ int is_tx_id_found = 0, is_gene_id_found = 0, is_negative_strand = -1; char * token_temp = NULL, * feature_name, *transcript_id = NULL, * chro_name = NULL; char feature_name_tmp[FEATURE_NAME_LENGTH], txid_tmp[FEATURE_NAME_LENGTH]; feature_name = feature_name_tmp; unsigned int start = 0, end = 0; aret = autozip_gets(&afp, file_line, MAX_LINE_LENGTH); if(aret < 1) break; lineno++; if(is_comment_line(file_line, file_type, lineno-1))continue; if(file_type == FILE_TYPE_RSUBREAD) { feature_name = strtok_r(file_line,"\t",&token_temp); int feature_name_len = strlen(feature_name); if(feature_name_len > FEATURE_NAME_LENGTH) feature_name[FEATURE_NAME_LENGTH -1 ] = 0; chro_name = strtok_r(NULL,"\t", &token_temp); int chro_name_len = strlen(chro_name); if(chro_name_len > MAX_CHROMOSOME_NAME_LEN) chro_name[MAX_CHROMOSOME_NAME_LEN -1 ] = 0; char * start_ptr = strtok_r(NULL,"\t", &token_temp); char * end_ptr = strtok_r(NULL,"\t", &token_temp); if(start_ptr == NULL || end_ptr == NULL){ SUBREADprintf("\nWarning: the format on the %d-th line is wrong.\n", lineno); } long long int tv1 = atoll(start_ptr); long long int tv2 = atoll(end_ptr); if( isdigit(start_ptr[0]) && isdigit(end_ptr[0]) ){ if(strlen(start_ptr) > 10 || strlen(end_ptr) > 10 || tv1 > 0x7fffffff || tv2> 0x7fffffff){ SUBREADprintf("\nError: Line %d contains a coordinate greater than 2^31.\n", lineno); return -2; } }else{ SUBREADprintf("\nError: Line %d contains a format error. The expected annotation format is SAF.\n", lineno); return -2; } start = atoi(start_ptr);// start end = atoi(end_ptr);//end char * strand_str = strtok_r(NULL,"\t", &token_temp); if(strand_str == NULL) is_negative_strand = 0; else is_negative_strand = ('-' ==strand_str[0]); is_gene_id_found = 1; } else if(file_type == FILE_TYPE_GTF) { chro_name = strtok_r(file_line,"\t",&token_temp); strtok_r(NULL,"\t", &token_temp);// source char * feature_type = strtok_r(NULL,"\t", &token_temp);// feature_type if(strcmp(feature_type, used_feature_type)==0){ char * start_ptr = strtok_r(NULL,"\t", &token_temp); char * end_ptr = strtok_r(NULL,"\t", &token_temp); if(start_ptr == NULL || end_ptr == NULL){ SUBREADprintf("\nWarning: the format on the %d-th line is wrong.\n", lineno); } long long int tv1 = atoll(start_ptr); long long int tv2 = atoll(end_ptr); if( isdigit(start_ptr[0]) && isdigit(end_ptr[0]) ){ if(strlen(start_ptr) > 10 || strlen(end_ptr) > 10 || tv1 > 0x7fffffff || tv2> 0x7fffffff){ SUBREADprintf("\nError: Line %d contains a coordinate greater than 2^31.\n", lineno); return -2; } }else{ SUBREADprintf("\nError: Line %d contains a format error. The expected annotation format is GTF/GFF.\n", lineno); return -2; } start = atoi(start_ptr);// start end = atoi(end_ptr);//end if(start < 1 || end<1 || start > 0x7fffffff || end > 0x7fffffff || start > end) SUBREADprintf("\nWarning: the feature on the %d-th line has zero coordinate or zero lengths\n\n", lineno); strtok_r(NULL,"\t", &token_temp);// score is_negative_strand = ('-' == (strtok_r(NULL,"\t", &token_temp)[0]));//strand strtok_r(NULL,"\t",&token_temp); // "frame" char * extra_attrs = strtok_r(NULL,"\t",&token_temp); // name_1 "val1"; name_2 "val2"; ... if(extra_attrs && (strlen(extra_attrs)>2)){ int attr_val_len = GTF_extra_column_value(extra_attrs , gene_id_column , feature_name_tmp, FEATURE_NAME_LENGTH); if(attr_val_len>0) is_gene_id_found=1; if(transcript_id_column){ transcript_id = txid_tmp; attr_val_len = GTF_extra_column_value(extra_attrs , transcript_id_column , txid_tmp, FEATURE_NAME_LENGTH); if(attr_val_len>0) is_tx_id_found=1; else transcript_id = NULL; } } if(!is_gene_id_found){ if(!is_GFF_geneid_warned){ int ext_att_len = strlen(extra_attrs); if(extra_attrs[ext_att_len-1] == '\n') extra_attrs[ext_att_len-1] =0; SUBREADprintf("\nERROR: failed to find the gene identifier attribute in the 9th column of the provided GTF file.\nThe specified gene identifier attribute is '%s'.\nAn example of attributes included in your GTF annotation is '%s'.\n\n", gene_id_column, extra_attrs); } is_GFF_geneid_warned++; } if(transcript_id_column && !is_tx_id_found){ if(!is_GFF_txid_warned){ int ext_att_len = strlen(extra_attrs); if(extra_attrs[ext_att_len-1] == '\n') extra_attrs[ext_att_len-1] =0; SUBREADprintf("\nERROR: failed to find the transcript identifier attribute in the 9th column of the provided GTF file.\nThe specified transcript identifier attribute is '%s'.\nAn example of attributes included in your GTF annotation is '%s'.\n\n", transcript_id_column, extra_attrs); } is_GFF_txid_warned++; } } } if(is_gene_id_found){ do_add_feature(feature_name, transcript_id, chro_name, start, end, is_negative_strand, context); loaded_features++; } } autozip_close(&afp); free(file_line); if(is_GFF_txid_warned || is_GFF_geneid_warned)return -2; if(loaded_features<1){ SUBREADprintf("\nERROR: No feature was loaded from the annotation file. Please check if the annotation format was correctly specified, and also if the feature type was correctly specified if the annotation is in the GTF format.\n\n"); return -2; } return loaded_features; } HashTable * load_alias_table(char * fname) { FILE * fp = f_subr_open(fname, "r"); if(!fp) { print_in_box(80,0,0,"WARNING unable to open alias file '%s'", fname); return NULL; } char * fl = malloc(2000); HashTable * ret = HashTableCreate(1013); HashTableSetDeallocationFunctions(ret, free, free); HashTableSetKeyComparisonFunction(ret, fc_strcmp_chro); HashTableSetHashFunction(ret, fc_chro_hash); while (1) { char *ret_fl = fgets(fl, 1999, fp); if(!ret_fl) break; if(fl[0]=='#') continue; char * sam_chr = NULL; char * anno_chr = strtok_r(fl, ",", &sam_chr); if((!sam_chr)||(!anno_chr)) continue; sam_chr[strlen(sam_chr)-1]=0; if(sam_chr[strlen(sam_chr)-1]=='\r') sam_chr[strlen(sam_chr)-1]=0; char * anno_chr_buf = malloc(strlen(anno_chr)+1); strcpy(anno_chr_buf, anno_chr); char * sam_chr_buf = malloc(strlen(sam_chr)+1); strcpy(sam_chr_buf, sam_chr); // SUBREADprintf("ALIAS_LOAD : '%s' => '%s'\n", sam_chr_buf, anno_chr_buf); HashTablePut(ret, sam_chr_buf, anno_chr_buf); } fclose(fp); free(fl); return ret; } int rebuild_command_line(char ** lineptr, int argc, char ** argv){ int linecap = 1000,c; *lineptr = malloc(linecap); **lineptr = 0; for(c = 0; c linecap) { linecap += cline+500; *lineptr = realloc(*lineptr, linecap); } sprintf((*lineptr) + strlen(*lineptr), "\"%s\" ", argv[c]); } return strlen(*lineptr); } // The following code was downloaded from // https://openwall.info/wiki/people/solar/software/public-domain-source-code/md5 // on the 23rd of Nov, 2018. I (Yang LIAO) made minor changes to the code. // // It says "Public Domain" in the licence but the original author should be acknwoledged (only for expressing my gratitude): // Alexander Peslyak, better known as Solar Designer /* Any 32-bit or wider unsigned integer data type will do */ /* * The basic HelpFuncMD5 functions. * * F and G are optimized compared to their RFC 1321 definitions for * architectures that lack an AND-NOT instruction, just like in Colin Plumb's * implementation. */ #define F(x, y, z) ((z) ^ ((x) & ((y) ^ (z)))) #define G(x, y, z) ((y) ^ ((z) & ((x) ^ (y)))) #define H(x, y, z) (((x) ^ (y)) ^ (z)) #define H2(x, y, z) ((x) ^ ((y) ^ (z))) #define I(x, y, z) ((y) ^ ((x) | ~(z))) /* * The HelpFuncMD5 transformation for all four rounds. */ #define STEP(f, a, b, c, d, x, t, s) \ (a) += f((b), (c), (d)) + (x) + (t); \ (a) = (((a) << (s)) | (((a) & 0xffffffff) >> (32 - (s)))); \ (a) += (b); /* * SET reads 4 input bytes in little-endian byte order and stores them in a * properly aligned word in host byte order. * * The check for little-endian architectures that tolerate unaligned memory * accesses is just an optimization. Nothing will break if it fails to detect * a suitable architecture. * * Unfortunately, this optimization may be a C strict aliasing rules violation * if the caller's data buffer has effective type that cannot be aliased by * HelpFuncMD5_u32plus. In practice, this problem may occur if these HelpFuncMD5 routines are * inlined into a calling function, or with future and dangerously advanced * link-time optimizations. For the time being, keeping these HelpFuncMD5 routines in * their own translation unit avoids the problem. */ #define SET(n) (*(HelpFuncMD5_u32plus *)&ptr[(n) * 4]) #define GET(n) SET(n) /* * This processes one or more 64-byte data blocks, but does NOT update the bit * counters. There are no alignment requirements. */ static const void *body(HelpFuncMD5_CTX *ctx, const void *data, unsigned long size) { const unsigned char *ptr; HelpFuncMD5_u32plus a, b, c, d; HelpFuncMD5_u32plus saved_a, saved_b, saved_c, saved_d; ptr = (const unsigned char *)data; a = ctx->a; b = ctx->b; c = ctx->c; d = ctx->d; do { saved_a = a; saved_b = b; saved_c = c; saved_d = d; /* Round 1 */ STEP(F, a, b, c, d, SET(0), 0xd76aa478, 7) STEP(F, d, a, b, c, SET(1), 0xe8c7b756, 12) STEP(F, c, d, a, b, SET(2), 0x242070db, 17) STEP(F, b, c, d, a, SET(3), 0xc1bdceee, 22) STEP(F, a, b, c, d, SET(4), 0xf57c0faf, 7) STEP(F, d, a, b, c, SET(5), 0x4787c62a, 12) STEP(F, c, d, a, b, SET(6), 0xa8304613, 17) STEP(F, b, c, d, a, SET(7), 0xfd469501, 22) STEP(F, a, b, c, d, SET(8), 0x698098d8, 7) STEP(F, d, a, b, c, SET(9), 0x8b44f7af, 12) STEP(F, c, d, a, b, SET(10), 0xffff5bb1, 17) STEP(F, b, c, d, a, SET(11), 0x895cd7be, 22) STEP(F, a, b, c, d, SET(12), 0x6b901122, 7) STEP(F, d, a, b, c, SET(13), 0xfd987193, 12) STEP(F, c, d, a, b, SET(14), 0xa679438e, 17) STEP(F, b, c, d, a, SET(15), 0x49b40821, 22) /* Round 2 */ STEP(G, a, b, c, d, GET(1), 0xf61e2562, 5) STEP(G, d, a, b, c, GET(6), 0xc040b340, 9) STEP(G, c, d, a, b, GET(11), 0x265e5a51, 14) STEP(G, b, c, d, a, GET(0), 0xe9b6c7aa, 20) STEP(G, a, b, c, d, GET(5), 0xd62f105d, 5) STEP(G, d, a, b, c, GET(10), 0x02441453, 9) STEP(G, c, d, a, b, GET(15), 0xd8a1e681, 14) STEP(G, b, c, d, a, GET(4), 0xe7d3fbc8, 20) STEP(G, a, b, c, d, GET(9), 0x21e1cde6, 5) STEP(G, d, a, b, c, GET(14), 0xc33707d6, 9) STEP(G, c, d, a, b, GET(3), 0xf4d50d87, 14) STEP(G, b, c, d, a, GET(8), 0x455a14ed, 20) STEP(G, a, b, c, d, GET(13), 0xa9e3e905, 5) STEP(G, d, a, b, c, GET(2), 0xfcefa3f8, 9) STEP(G, c, d, a, b, GET(7), 0x676f02d9, 14) STEP(G, b, c, d, a, GET(12), 0x8d2a4c8a, 20) /* Round 3 */ STEP(H, a, b, c, d, GET(5), 0xfffa3942, 4) STEP(H2, d, a, b, c, GET(8), 0x8771f681, 11) STEP(H, c, d, a, b, GET(11), 0x6d9d6122, 16) STEP(H2, b, c, d, a, GET(14), 0xfde5380c, 23) STEP(H, a, b, c, d, GET(1), 0xa4beea44, 4) STEP(H2, d, a, b, c, GET(4), 0x4bdecfa9, 11) STEP(H, c, d, a, b, GET(7), 0xf6bb4b60, 16) STEP(H2, b, c, d, a, GET(10), 0xbebfbc70, 23) STEP(H, a, b, c, d, GET(13), 0x289b7ec6, 4) STEP(H2, d, a, b, c, GET(0), 0xeaa127fa, 11) STEP(H, c, d, a, b, GET(3), 0xd4ef3085, 16) STEP(H2, b, c, d, a, GET(6), 0x04881d05, 23) STEP(H, a, b, c, d, GET(9), 0xd9d4d039, 4) STEP(H2, d, a, b, c, GET(12), 0xe6db99e5, 11) STEP(H, c, d, a, b, GET(15), 0x1fa27cf8, 16) STEP(H2, b, c, d, a, GET(2), 0xc4ac5665, 23) /* Round 4 */ STEP(I, a, b, c, d, GET(0), 0xf4292244, 6) STEP(I, d, a, b, c, GET(7), 0x432aff97, 10) STEP(I, c, d, a, b, GET(14), 0xab9423a7, 15) STEP(I, b, c, d, a, GET(5), 0xfc93a039, 21) STEP(I, a, b, c, d, GET(12), 0x655b59c3, 6) STEP(I, d, a, b, c, GET(3), 0x8f0ccc92, 10) STEP(I, c, d, a, b, GET(10), 0xffeff47d, 15) STEP(I, b, c, d, a, GET(1), 0x85845dd1, 21) STEP(I, a, b, c, d, GET(8), 0x6fa87e4f, 6) STEP(I, d, a, b, c, GET(15), 0xfe2ce6e0, 10) STEP(I, c, d, a, b, GET(6), 0xa3014314, 15) STEP(I, b, c, d, a, GET(13), 0x4e0811a1, 21) STEP(I, a, b, c, d, GET(4), 0xf7537e82, 6) STEP(I, d, a, b, c, GET(11), 0xbd3af235, 10) STEP(I, c, d, a, b, GET(2), 0x2ad7d2bb, 15) STEP(I, b, c, d, a, GET(9), 0xeb86d391, 21) a += saved_a; b += saved_b; c += saved_c; d += saved_d; ptr += 64; } while (size -= 64); ctx->a = a; ctx->b = b; ctx->c = c; ctx->d = d; return ptr; } void HelpFuncMD5_Init(HelpFuncMD5_CTX *ctx) { ctx->a = 0x67452301; ctx->b = 0xefcdab89; ctx->c = 0x98badcfe; ctx->d = 0x10325476; ctx->lo = 0; ctx->hi = 0; } void HelpFuncMD5_Update(HelpFuncMD5_CTX *ctx, const void *data, unsigned long size) { HelpFuncMD5_u32plus saved_lo; unsigned long used, available; saved_lo = ctx->lo; if ((ctx->lo = (saved_lo + size) & 0x1fffffff) < saved_lo) ctx->hi++; ctx->hi += size >> 29; used = saved_lo & 0x3f; if (used) { available = 64 - used; if (size < available) { memcpy(&ctx->buffer[used], data, size); return; } memcpy(&ctx->buffer[used], data, available); data = (const unsigned char *)data + available; size -= available; body(ctx, ctx->buffer, 64); } if (size >= 64) { data = body(ctx, data, size & ~(unsigned long)0x3f); size &= 0x3f; } memcpy(ctx->buffer, data, size); } #define OUT(dst, src) \ (dst)[0] = (unsigned char)(src); \ (dst)[1] = (unsigned char)((src) >> 8); \ (dst)[2] = (unsigned char)((src) >> 16); \ (dst)[3] = (unsigned char)((src) >> 24); void HelpFuncMD5_Final(unsigned char *result, HelpFuncMD5_CTX *ctx) { unsigned long used, available; used = ctx->lo & 0x3f; ctx->buffer[used++] = 0x80; available = 64 - used; if (available < 8) { memset(&ctx->buffer[used], 0, available); body(ctx, ctx->buffer, 64); used = 0; available = 64; } memset(&ctx->buffer[used], 0, available - 8); ctx->lo <<= 3; OUT(&ctx->buffer[56], ctx->lo) OUT(&ctx->buffer[60], ctx->hi) body(ctx, ctx->buffer, 64); OUT(&result[0], ctx->a) OUT(&result[4], ctx->b) OUT(&result[8], ctx->c) OUT(&result[12], ctx->d) memset(ctx, 0, sizeof(*ctx)); } void Helper_md5sum(char * plain_txt, int plain_len, unsigned char * bin_md5_buff){ HelpFuncMD5_CTX ctx; HelpFuncMD5_Init(&ctx); HelpFuncMD5_Update(&ctx, plain_txt, plain_len); HelpFuncMD5_Final(bin_md5_buff, &ctx); } unsigned long long plain_txt_to_long_rand(char * plain_txt, int plain_len){ unsigned char md5v[16]; Helper_md5sum(plain_txt, plain_len, md5v); unsigned long long ret = 0; memcpy(&ret, md5v, sizeof(ret)); return ret; } void md5txt(char *s){ unsigned char md5v[16]; unsigned long long randv; Helper_md5sum(s, strlen(s), md5v); int x; randv = plain_txt_to_long_rand(s, strlen(s)); for(x=0;x<16;x++){ SUBREADprintf("%02X", md5v[x]); } #ifdef __MINGW32__ SUBREADprintf("\t'%s'\t%016I64X\t%I64u\t%.9f\n", s, randv, randv, randv*1./0xffffffffffffffffllu); #else SUBREADprintf("\t'%s'\t%016llX\t%llu\t%.9f\n", s, randv, randv, randv*1./0xffffffffffffffffllu); #endif } //#define TESTHelpermain main int TESTHelpermain(){ int xx; for(xx=0; xx<100; xx++){ char xt[10]; sprintf(xt, "%08d", xx); md5txt(xt); } return 0; } // The following code was retrieved from // https://github.com/google/omaha/blob/master/third_party/lzma/files/C/Helper_Sha256.c // Thank the authors -- Igor Pavlov and Wei Dai. /* Crypto/Helper_Sha256.c -- SHA-256 Hash 2010-06-11 : Igor Pavlov : Public domain This code is based on public domain code from Wei Dai's Crypto++ library. */ #define SHA256_DIGEST_SIZE 32 typedef struct { unsigned int state[8]; unsigned long long count; unsigned char buffer[64]; } CHelper_Sha256; /* define it for speed optimization */ /* #define _SHA256_UNROLL */ /* #define _SHA256_UNROLL2 */ void Helper_Sha256_Init(CHelper_Sha256 *p) { p->state[0] = 0x6a09e667; p->state[1] = 0xbb67ae85; p->state[2] = 0x3c6ef372; p->state[3] = 0xa54ff53a; p->state[4] = 0x510e527f; p->state[5] = 0x9b05688c; p->state[6] = 0x1f83d9ab; p->state[7] = 0x5be0cd19; p->count = 0; } #define rotlFixed(x, n) (((x) << (n)) | ((x) >> (32 - (n)))) #define rotrFixed(x, n) (((x) >> (n)) | ((x) << (32 - (n)))) #define S0(x) (rotrFixed(x, 2) ^ rotrFixed(x,13) ^ rotrFixed(x, 22)) #define S1(x) (rotrFixed(x, 6) ^ rotrFixed(x,11) ^ rotrFixed(x, 25)) #define s0(x) (rotrFixed(x, 7) ^ rotrFixed(x,18) ^ (x >> 3)) #define s1(x) (rotrFixed(x,17) ^ rotrFixed(x,19) ^ (x >> 10)) #define blk0(i) (W[i] = data[i]) #define blk2(i) (W[i&15] += s1(W[(i-2)&15]) + W[(i-7)&15] + s0(W[(i-15)&15])) #define Ch(x,y,z) (z^(x&(y^z))) #define Maj(x,y,z) ((x&y)|(z&(x|y))) #define a(i) T[(0-(i))&7] #define b(i) T[(1-(i))&7] #define c(i) T[(2-(i))&7] #define d(i) T[(3-(i))&7] #define e(i) T[(4-(i))&7] #define f(i) T[(5-(i))&7] #define g(i) T[(6-(i))&7] #define h(i) T[(7-(i))&7] #ifdef _SHA256_UNROLL2 #define R(a,b,c,d,e,f,g,h, i) h += S1(e) + Ch(e,f,g) + K[i+j] + (j?blk2(i):blk0(i));\ d += h; h += S0(a) + Maj(a, b, c) #define RX_8(i) \ R(a,b,c,d,e,f,g,h, i); \ R(h,a,b,c,d,e,f,g, i+1); \ R(g,h,a,b,c,d,e,f, i+2); \ R(f,g,h,a,b,c,d,e, i+3); \ R(e,f,g,h,a,b,c,d, i+4); \ R(d,e,f,g,h,a,b,c, i+5); \ R(c,d,e,f,g,h,a,b, i+6); \ R(b,c,d,e,f,g,h,a, i+7) #else #define R(i) h(i) += S1(e(i)) + Ch(e(i),f(i),g(i)) + K[i+j] + (j?blk2(i):blk0(i));\ d(i) += h(i); h(i) += S0(a(i)) + Maj(a(i), b(i), c(i)) #ifdef _SHA256_UNROLL #define RX_8(i) R(i+0); R(i+1); R(i+2); R(i+3); R(i+4); R(i+5); R(i+6); R(i+7); #endif #endif static const unsigned int K[64] = { 0x428a2f98, 0x71374491, 0xb5c0fbcf, 0xe9b5dba5, 0x3956c25b, 0x59f111f1, 0x923f82a4, 0xab1c5ed5, 0xd807aa98, 0x12835b01, 0x243185be, 0x550c7dc3, 0x72be5d74, 0x80deb1fe, 0x9bdc06a7, 0xc19bf174, 0xe49b69c1, 0xefbe4786, 0x0fc19dc6, 0x240ca1cc, 0x2de92c6f, 0x4a7484aa, 0x5cb0a9dc, 0x76f988da, 0x983e5152, 0xa831c66d, 0xb00327c8, 0xbf597fc7, 0xc6e00bf3, 0xd5a79147, 0x06ca6351, 0x14292967, 0x27b70a85, 0x2e1b2138, 0x4d2c6dfc, 0x53380d13, 0x650a7354, 0x766a0abb, 0x81c2c92e, 0x92722c85, 0xa2bfe8a1, 0xa81a664b, 0xc24b8b70, 0xc76c51a3, 0xd192e819, 0xd6990624, 0xf40e3585, 0x106aa070, 0x19a4c116, 0x1e376c08, 0x2748774c, 0x34b0bcb5, 0x391c0cb3, 0x4ed8aa4a, 0x5b9cca4f, 0x682e6ff3, 0x748f82ee, 0x78a5636f, 0x84c87814, 0x8cc70208, 0x90befffa, 0xa4506ceb, 0xbef9a3f7, 0xc67178f2 }; static void Helper_Sha256_Transform(unsigned int *state, const unsigned int *data) { unsigned int W[16]; unsigned j; #ifdef _SHA256_UNROLL2 unsigned int a,b,c,d,e,f,g,h; a = state[0]; b = state[1]; c = state[2]; d = state[3]; e = state[4]; f = state[5]; g = state[6]; h = state[7]; #else unsigned int T[8]; for (j = 0; j < 8; j++) T[j] = state[j]; #endif for (j = 0; j < 64; j += 16) { #if defined(_SHA256_UNROLL) || defined(_SHA256_UNROLL2) RX_8(0); RX_8(8); #else unsigned i; for (i = 0; i < 16; i++) { R(i); } #endif } #ifdef _SHA256_UNROLL2 state[0] += a; state[1] += b; state[2] += c; state[3] += d; state[4] += e; state[5] += f; state[6] += g; state[7] += h; #else for (j = 0; j < 8; j++) state[j] += T[j]; #endif /* Wipe variables */ /* memset(W, 0, sizeof(W)); */ /* memset(T, 0, sizeof(T)); */ } #undef S0 #undef S1 #undef s0 #undef s1 static void Helper_Sha256_WritecharBlock(CHelper_Sha256 *p) { unsigned int data32[16]; unsigned i; for (i = 0; i < 16; i++) data32[i] = ((unsigned int)(p->buffer[i * 4 ]) << 24) + ((unsigned int)(p->buffer[i * 4 + 1]) << 16) + ((unsigned int)(p->buffer[i * 4 + 2]) << 8) + ((unsigned int)(p->buffer[i * 4 + 3])); Helper_Sha256_Transform(p->state, data32); } void Helper_Sha256_Update(CHelper_Sha256 *p, const char *data, size_t size) { unsigned int curBufferPos = (unsigned int)p->count & 0x3F; while (size > 0) { p->buffer[curBufferPos++] = (char)*data; p->count++; data++; size--; if (curBufferPos == 64) { curBufferPos = 0; Helper_Sha256_WritecharBlock(p); } } } void Helper_Sha256_Final( unsigned char *digest , CHelper_Sha256 *p) { unsigned long long lenInBits = (p->count << 3); unsigned int curBufferPos = (unsigned int)p->count & 0x3F; unsigned i; p->buffer[curBufferPos++] = 0x80; while (curBufferPos != (64 - 8)) { curBufferPos &= 0x3F; if (curBufferPos == 0) Helper_Sha256_WritecharBlock(p); p->buffer[curBufferPos++] = 0; } for (i = 0; i < 8; i++) { p->buffer[curBufferPos++] = (char)(lenInBits >> 56); lenInBits <<= 8; } Helper_Sha256_WritecharBlock(p); for (i = 0; i < 8; i++) { *digest++ = (unsigned char)(p->state[i] >> 24); *digest++ = (unsigned char)(p->state[i] >> 16); *digest++ = (unsigned char)(p->state[i] >> 8); *digest++ = (unsigned char)(p->state[i]); } } void Helper_sha256sum(char * plain_txt, int plain_len, unsigned char * bin_md5_buff){ CHelper_Sha256 ctx; Helper_Sha256_Init(&ctx); Helper_Sha256_Update(&ctx, plain_txt, plain_len); Helper_Sha256_Final(bin_md5_buff, &ctx); } void sha256txt(char *s){ unsigned char sha256v[32]; Helper_sha256sum(s, strlen(s), sha256v); int x; for(x=0;x<32;x++){ SUBREADprintf("%02X", sha256v[x]); } SUBREADprintf("\t'%s'\n", s); } //#define TEST256Helpermain main int TEST256Helpermain(){ int xx; for(xx=0; xx<100; xx++){ char xt[10]; sprintf(xt, "%08d", xx); sha256txt(xt); } return 0; } // The code of Helper_erfinv() was retreived from // https://github.com/antelopeusersgroup/antelope_contrib/blob/master/lib/location/libgenloc/erfinv.c // I (Yang LIAO) adopted and modified it because it is under the New BSD license. // // Copyright (c) 2014 Indiana University // All rights reserved. // // Written by Prof. Gary L. Pavlis, Dept. of Geol. Sci., // Indiana University, Bloomington, IN // // This software is licensed under the New BSD license: // // Redistribution and use in source and binary forms, // with or without modification, are permitted provided // that the following conditions are met: // // Redistributions of source code must retain the above // copyright notice, this list of conditions and the // following disclaimer. // // Redistributions in binary form must reproduce the // above copyright notice, this list of conditions and // the following disclaimer in the documentation and/or // other materials provided with the distribution. // // Neither the name of Indiana University nor // the names of its contributors may be used to endorse // or promote products derived from this software without // specific prior written permission. // // THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND // CONTRIBUTORS "AS IS" AND ANY EXPRESS OR IMPLIED // WARRANTIES, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED // WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A // PARTICULAR PURPOSE ARE DISCLAIMED. IN NO EVENT SHALL // THE COPYRIGHT OWNER OR CONTRIBUTORS BE LIABLE FOR ANY // DIRECT, INDIRECT, INCIDENTAL, SPECIAL, EXEMPLARY, OR // CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT LIMITED TO, // PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF // USE, DATA, OR PROFITS; OR BUSINESS INTERRUPTION) // HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY, WHETHER // IN CONTRACT, STRICT LIABILITY, OR TORT (INCLUDING // NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE // USE OF THIS SOFTWARE, EVEN IF ADVISED OF THE // POSSIBILITY OF SUCH DAMAGE. #include #include #define MAXDOUBLE DBL_MAX #define CENTRAL_RANGE 0.7 double Helper_erfinv( double y) { double x=0,z,num,dem; /*working variables */ /* coefficients in rational expansion */ double a[4]={ 0.886226899, -1.645349621, 0.914624893, -0.140543331}; double b[4]={-2.118377725, 1.442710462, -0.329097515, 0.012229801}; double c[4]={-1.970840454, -1.624906493, 3.429567803, 1.641345311}; double d[2]={ 3.543889200, 1.637067800}; if(fabs(y) > 1.0) return (atof("NaN")); /* This needs IEEE constant*/ if(fabs(y) == 1.0) return((copysign(1.0,y))*MAXDOUBLE); if( fabs(y) <= CENTRAL_RANGE ) { z = y*y; num = (((a[3]*z + a[2])*z + a[1])*z + a[0]); dem = ((((b[3]*z + b[2])*z + b[1])*z +b[0])*z + 1.0); x = y*num/dem; } else if( (fabs(y) > CENTRAL_RANGE) && (fabs(y) < 1.0) ) { z = sqrt(-log((1.0-fabs(y))/2.0)); num = ((c[3]*z + c[2])*z + c[1])*z + c[0]; dem = (d[1]*z + d[0])*z + 1.0; x = (copysign(1.0,y))*num/dem; } /* Two steps of Newton-Raphson correction */ x = x - (erf(x) - y)/( (2.0/sqrt(M_PI))*exp(-x*x)); x = x - (erf(x) - y)/( (2.0/sqrt(M_PI))*exp(-x*x)); return(x); } double inverse_sample_normal(double p){ return Helper_erfinv(2*p-1)*1.4142135623730950488; } //#define TestNormalMain main void TestNormalMain(){ int i; for(i=0; i<=10; i++){ double ii = i*1./10; double p_0 = inverse_sample_normal(ii); SUBREADprintf("p of %.1f = %.40f\n\n" , ii, p_0); } } #ifndef MAKE_STANDALONE message_queue_t mt_message_queue; int safeRprintf(char * fmt,...){ va_list args; va_start(args , fmt); FILE * ofp = fopen("/tmp/del4-Rlog.txt", "a"); //fprintf(ofp, "NEW LOG : %p \"%s\"\n", fmt, fmt); fprintf(ofp, fmt, args); fclose(ofp); va_end(args); return 0; } #endif void msgqu_destroy(){ #ifndef MAKE_STANDALONE ArrayListDestroy(mt_message_queue.message_queue); subread_destroy_lock(&mt_message_queue.queue_lock); #endif } void msgqu_notifyFinish(){ #ifndef MAKE_STANDALONE subread_lock_occupy(&mt_message_queue.queue_lock); mt_message_queue.is_finished = 1; subread_lock_release(&mt_message_queue.queue_lock); #endif } void msgqu_init(int thread_mode){ #ifndef MAKE_STANDALONE mt_message_queue.is_thread_mode = thread_mode; if(!thread_mode) return; mt_message_queue.is_finished = 0; mt_message_queue.message_queue = ArrayListCreate(100); ArrayListSetDeallocationFunction(mt_message_queue.message_queue,free); subread_init_lock(&mt_message_queue.queue_lock); // subread_init_lock(&mt_message_queue.queue_notifier); #endif } void msgqu_main_loop(){ #ifndef MAKE_STANDALONE while(1){ subread_lock_occupy(&mt_message_queue.queue_lock); while(0< mt_message_queue.message_queue->numOfElements){ char * amsg = ArrayListShift(mt_message_queue.message_queue); Rprintf("%s", amsg); free(amsg); } if(mt_message_queue.is_finished) break; subread_lock_release(&mt_message_queue.queue_lock); usleep(40000); } #endif } #define MSGQU_LINE_SIZE 3000 void msgqu_printf(const char * fmt, ...){ va_list args; va_start(args , fmt); char * obuf = malloc(MSGQU_LINE_SIZE+1); vsnprintf( obuf, MSGQU_LINE_SIZE, fmt, args ); va_end(args); obuf[MSGQU_LINE_SIZE]=0; #ifdef MAKE_STANDALONE fputs(obuf, stderr); free(obuf); #else if(!mt_message_queue.is_thread_mode){ Rprintf("%s", obuf); free(obuf); return; } subread_lock_occupy(&mt_message_queue.queue_lock); ArrayListPush(mt_message_queue.message_queue, obuf); subread_lock_release(&mt_message_queue.queue_lock); // it will be freed in the Main thread after printing. #endif } #ifdef MAKE_TEST_MYRAND_TEST int main(){ myrand_srand(time(NULL)); int x; for(x = 0; x<100000; x++){ int mv = myrand_rand(); SUBREADprintf("%d\n", mv); } return 0; } #endif // retrieved from https://github.com/kokke/tiny-TNbignum-c/ // The original code was in public domain /* Functions for shifting number in-place. */ static void _lshift_one_bit(struct bn* a); static void _rshift_one_bit(struct bn* a); static void _lshift_word(struct bn* a, int nwords); static void _rshift_word(struct bn* a, int nwords); /* Public / Exported functions. */ void TNbignum_init(struct bn* n) { require(n, "n is null"); int i; for (i = 0; i < BN_ARRAY_SIZE; ++i) { n->array[i] = 0; } } void TNbignum_from_int(struct bn* n, DTYPE_TMP i) { require(n, "n is null"); TNbignum_init(n); /* Endianness issue if machine is not little-endian? */ #ifdef WORD_SIZE #if (WORD_SIZE == 1) n->array[0] = (i & 0x000000ff); n->array[1] = (i & 0x0000ff00) >> 8; n->array[2] = (i & 0x00ff0000) >> 16; n->array[3] = (i & 0xff000000) >> 24; #elif (WORD_SIZE == 2) n->array[0] = (i & 0x0000ffff); n->array[1] = (i & 0xffff0000) >> 16; #elif (WORD_SIZE == 4) n->array[0] = i; DTYPE_TMP num_32 = 32; DTYPE_TMP tmp = i >> num_32; /* bit-shift with U64 operands to force 64-bit results */ n->array[1] = tmp; #endif #endif } int TNbignum_to_int(struct bn* n) { require(n, "n is null"); int ret = 0; /* Endianness issue if machine is not little-endian? */ #if (WORD_SIZE == 1) ret += n->array[0]; ret += n->array[1] << 8; ret += n->array[2] << 16; ret += n->array[3] << 24; #elif (WORD_SIZE == 2) ret += n->array[0]; ret += n->array[1] << 16; #elif (WORD_SIZE == 4) ret += n->array[0]; #endif return ret; } void TNbignum_from_string(struct bn* n, char* str, int nbytes) { require(n, "n is null"); require(str, "str is null"); require(nbytes > 0, "nbytes must be positive"); require((nbytes & 1) == 0, "string format must be in hex -> equal number of bytes"); TNbignum_init(n); DTYPE tmp; /* DTYPE is defined in bn.h - uint{8,16,32,64}_t */ int i = nbytes - (2 * WORD_SIZE); /* index into string */ int j = 0; /* index into array */ /* reading last hex-byte "MSB" from string first -> big endian */ /* MSB ~= most significant byte / block ? :) */ while (i >= 0) { tmp = 0; sscanf(&str[i], SSCANF_FORMAT_STR, &tmp); //printf("SCAN_IN %d : v=%u\n", i, tmp); n->array[j] = tmp; i -= (2 * WORD_SIZE); /* step WORD_SIZE hex-byte(s) back in the string. */ j += 1; /* step one element forward in the array. */ } } void TNbignum_to_string(struct bn* n, char* str, int nbytes) { require(n, "n is null"); require(str, "str is null"); require(nbytes > 0, "nbytes must be positive"); require((nbytes & 1) == 0, "string format must be in hex -> equal number of bytes"); int j = BN_ARRAY_SIZE - 1; /* index into array - reading "MSB" first -> big-endian */ int i = 0; /* index into string representation. */ /* reading last array-element "MSB" first -> big endian */ while ((j >= 0) && (nbytes > (i + 1))) { sprintf(&str[i], SPRINTF_FORMAT_STR, n->array[j]); //printf("WRITE:%d %s\n" , i, str+i); i += (2 * WORD_SIZE); /* step WORD_SIZE hex-byte(s) forward in the string. */ j -= 1; /* step one element back in the array. */ } /* Count leading zeros: */ j = 0; while (str[j] == '0') { j += 1; } /* Move string j places ahead, effectively skipping leading zeros */ for (i = 0; i < (nbytes - j); ++i) { str[i] = str[i + j]; } /* Zero-terminate string */ str[i] = 0; } void TNbignum_dec(struct bn* n) { require(n, "n is null"); DTYPE tmp; /* copy of n */ DTYPE res; int i; for (i = 0; i < BN_ARRAY_SIZE; ++i) { tmp = n->array[i]; res = tmp - 1; n->array[i] = res; if (!(res > tmp)) { break; } } } void TNbignum_inc(struct bn* n) { require(n, "n is null"); DTYPE res; DTYPE_TMP tmp; /* copy of n */ int i; for (i = 0; i < BN_ARRAY_SIZE; ++i) { tmp = n->array[i]; res = tmp + 1; n->array[i] = res; if (res > tmp) { break; } } } void TNbignum_add(struct bn* a, struct bn* b, struct bn* c) { require(a, "a is null"); require(b, "b is null"); require(c, "c is null"); DTYPE_TMP tmp; int carry = 0; int i; for (i = 0; i < BN_ARRAY_SIZE; ++i) { tmp = (DTYPE_TMP)a->array[i] + b->array[i] + carry; carry = (tmp > MAX_VAL); c->array[i] = (tmp & MAX_VAL); } } void TNbignum_sub(struct bn* a, struct bn* b, struct bn* c) { require(a, "a is null"); require(b, "b is null"); require(c, "c is null"); DTYPE_TMP res; DTYPE_TMP tmp1; DTYPE_TMP tmp2; int borrow = 0; int i; for (i = 0; i < BN_ARRAY_SIZE; ++i) { tmp1 = (DTYPE_TMP)a->array[i] + (MAX_VAL + 1); /* + number_base */ tmp2 = (DTYPE_TMP)b->array[i] + borrow;; res = (tmp1 - tmp2); c->array[i] = (DTYPE)(res & MAX_VAL); /* "modulo number_base" == "% (number_base - 1)" if number_base is 2^N */ borrow = (res <= MAX_VAL); } } void TNbignum_mul(struct bn* a, struct bn* b, struct bn* c) { require(a, "a is null"); require(b, "b is null"); require(c, "c is null"); struct bn row; struct bn tmp; int i, j; TNbignum_init(c); for (i = 0; i < BN_ARRAY_SIZE; ++i) { TNbignum_init(&row); for (j = 0; j < BN_ARRAY_SIZE; ++j) { if (i + j < BN_ARRAY_SIZE) { TNbignum_init(&tmp); DTYPE_TMP intermediate = ((DTYPE_TMP)a->array[i] * (DTYPE_TMP)b->array[j]); TNbignum_from_int(&tmp, intermediate); _lshift_word(&tmp, i + j); TNbignum_add(&tmp, &row, &row); } } TNbignum_add(c, &row, c); } } void TNbignum_div(struct bn* a, struct bn* b, struct bn* c) { require(a, "a is null"); require(b, "b is null"); require(c, "c is null"); struct bn current; struct bn denom; struct bn tmp; TNbignum_from_int(¤t, 1); // int current = 1; TNbignum_assign(&denom, b); // denom = b TNbignum_assign(&tmp, a); // tmp = a const DTYPE_TMP half_max = 1 + (DTYPE_TMP)(MAX_VAL / 2); int overflow = 0; while (TNbignum_cmp(&denom, a) != LARGER) // while (denom <= a) { { if (denom.array[BN_ARRAY_SIZE - 1] >= half_max) { overflow = 1; break; } _lshift_one_bit(¤t); // current <<= 1; _lshift_one_bit(&denom); // denom <<= 1; } if (!overflow) { _rshift_one_bit(&denom); // denom >>= 1; _rshift_one_bit(¤t); // current >>= 1; } TNbignum_init(c); // int answer = 0; while (!TNbignum_is_zero(¤t)) // while (current != 0) { if (TNbignum_cmp(&tmp, &denom) != SMALLER) // if (dividend >= denom) { TNbignum_sub(&tmp, &denom, &tmp); // dividend -= denom; TNbignum_or(c, ¤t, c); // answer |= current; } _rshift_one_bit(¤t); // current >>= 1; _rshift_one_bit(&denom); // denom >>= 1; } // return answer; } void TNbignum_lshift(struct bn* a, struct bn* b, int nbits) { require(a, "a is null"); require(b, "b is null"); require(nbits >= 0, "no negative shifts"); TNbignum_assign(b, a); /* Handle shift in multiples of word-size */ const int nbits_pr_word = (WORD_SIZE * 8); int nwords = nbits / nbits_pr_word; if (nwords != 0) { _lshift_word(b, nwords); nbits -= (nwords * nbits_pr_word); } if (nbits != 0) { int i; for (i = (BN_ARRAY_SIZE - 1); i > 0; --i) { b->array[i] = (b->array[i] << nbits) | (b->array[i - 1] >> ((8 * WORD_SIZE) - nbits)); } b->array[i] <<= nbits; } } void TNbignum_rshift(struct bn* a, struct bn* b, int nbits) { require(a, "a is null"); require(b, "b is null"); require(nbits >= 0, "no negative shifts"); TNbignum_assign(b, a); /* Handle shift in multiples of word-size */ const int nbits_pr_word = (WORD_SIZE * 8); int nwords = nbits / nbits_pr_word; if (nwords != 0) { _rshift_word(b, nwords); nbits -= (nwords * nbits_pr_word); } if (nbits != 0) { int i; for (i = 0; i < (BN_ARRAY_SIZE - 1); ++i) { b->array[i] = (b->array[i] >> nbits) | (b->array[i + 1] << ((8 * WORD_SIZE) - nbits)); } b->array[i] >>= nbits; } } void TNbignum_mod(struct bn* a, struct bn* b, struct bn* c) { /* * Take divmod and throw away div part * */ require(a, "a is null"); require(b, "b is null"); require(c, "c is null"); struct bn tmp; TNbignum_divmod(a,b,&tmp,c); } void TNbignum_divmod(struct bn* a, struct bn* b, struct bn* c, struct bn* d) { /* * Puts a%b in d * and a/b in c * * mod(a,b) = a - ((a / b) * b) * * example: * mod(8, 3) = 8 - ((8 / 3) * 3) = 2 * */ require(a, "a is null"); require(b, "b is null"); require(c, "c is null"); struct bn tmp; /* c = (a / b) */ TNbignum_div(a, b, c); /* tmp = (c * b) */ TNbignum_mul(c, b, &tmp); /* c = a - tmp */ TNbignum_sub(a, &tmp, d); } void TNbignum_and(struct bn* a, struct bn* b, struct bn* c) { require(a, "a is null"); require(b, "b is null"); require(c, "c is null"); int i; for (i = 0; i < BN_ARRAY_SIZE; ++i) { c->array[i] = (a->array[i] & b->array[i]); } } void TNbignum_or(struct bn* a, struct bn* b, struct bn* c) { require(a, "a is null"); require(b, "b is null"); require(c, "c is null"); int i; for (i = 0; i < BN_ARRAY_SIZE; ++i) { c->array[i] = (a->array[i] | b->array[i]); } } void TNbignum_xor(struct bn* a, struct bn* b, struct bn* c) { require(a, "a is null"); require(b, "b is null"); require(c, "c is null"); int i; for (i = 0; i < BN_ARRAY_SIZE; ++i) { c->array[i] = (a->array[i] ^ b->array[i]); } } int TNbignum_cmp(struct bn* a, struct bn* b) { require(a, "a is null"); require(b, "b is null"); int i = BN_ARRAY_SIZE; do { i -= 1; /* Decrement first, to start with last array element */ if (a->array[i] > b->array[i]) { return LARGER; } else if (a->array[i] < b->array[i]) { return SMALLER; } } while (i != 0); return EQUAL; } int TNbignum_is_zero(struct bn* n) { require(n, "n is null"); int i; for (i = 0; i < BN_ARRAY_SIZE; ++i) { if (n->array[i]) { return 0; } } return 1; } void TNbignum_pow(struct bn* a, struct bn* b, struct bn* c) { require(a, "a is null"); require(b, "b is null"); require(c, "c is null"); struct bn tmp; TNbignum_init(c); if (TNbignum_cmp(b, c) == EQUAL) { /* Return 1 when exponent is 0 -- n^0 = 1 */ TNbignum_inc(c); } else { /* Copy a -> tmp */ TNbignum_assign(&tmp, a); TNbignum_dec(b); /* Begin summing products: */ while (!TNbignum_is_zero(b)) { /* c = tmp * tmp */ TNbignum_mul(&tmp, a, c); /* Decrement b by one */ TNbignum_dec(b); TNbignum_assign(&tmp, c); } /* c = tmp */ TNbignum_assign(c, &tmp); } } void TNbignum_isqrt(struct bn *a, struct bn* b) { require(a, "a is null"); require(b, "b is null"); struct bn low, high, mid, tmp; TNbignum_init(&low); TNbignum_assign(&high, a); TNbignum_rshift(&high, &mid, 1); TNbignum_inc(&mid); while (TNbignum_cmp(&high, &low) > 0) { TNbignum_mul(&mid, &mid, &tmp); if (TNbignum_cmp(&tmp, a) > 0) { TNbignum_assign(&high, &mid); TNbignum_dec(&high); } else { TNbignum_assign(&low, &mid); } TNbignum_sub(&high,&low,&mid); _rshift_one_bit(&mid); TNbignum_add(&low,&mid,&mid); TNbignum_inc(&mid); } TNbignum_assign(b,&low); } void TNbignum_assign(struct bn* dst, struct bn* src) { require(dst, "dst is null"); require(src, "src is null"); int i; for (i = 0; i < BN_ARRAY_SIZE; ++i) { dst->array[i] = src->array[i]; } } /* Private / Static functions. */ static void _rshift_word(struct bn* a, int nwords) { /* Naive method: */ require(a, "a is null"); require(nwords >= 0, "no negative shifts"); int i; if (nwords >= BN_ARRAY_SIZE) { for (i = 0; i < BN_ARRAY_SIZE; ++i) { a->array[i] = 0; } return; } for (i = 0; i < BN_ARRAY_SIZE - nwords; ++i) { a->array[i] = a->array[i + nwords]; } for (; i < BN_ARRAY_SIZE; ++i) { a->array[i] = 0; } } static void _lshift_word(struct bn* a, int nwords) { require(a, "a is null"); require(nwords >= 0, "no negative shifts"); int i; /* Shift whole words */ for (i = (BN_ARRAY_SIZE - 1); i >= nwords; --i) { a->array[i] = a->array[i - nwords]; } /* Zero pad shifted words. */ for (; i >= 0; --i) { a->array[i] = 0; } } static void _lshift_one_bit(struct bn* a) { require(a, "a is null"); int i; for (i = (BN_ARRAY_SIZE - 1); i > 0; --i) { a->array[i] = (a->array[i] << 1) | (a->array[i - 1] >> ((8 * WORD_SIZE) - 1)); } a->array[0] <<= 1; } static void _rshift_one_bit(struct bn* a) { require(a, "a is null"); int i; for (i = 0; i < (BN_ARRAY_SIZE - 1); ++i) { a->array[i] = (a->array[i] >> 1) | (a->array[i + 1] << ((8 * WORD_SIZE) - 1)); } a->array[BN_ARRAY_SIZE - 1] >>= 1; } /* void TNbignum_pow_then_mod( struct bn * m, int e, struct bn * modulus, struct bn * res ){ int xx; struct bn res, remove_times, remove_value, tmpv; TNbignum_from_string(&res, "01", 2); for(xx = 0; xx < 31; xx++){ TNbignum_mul( m, m, tmpv ); TNbignum_div( tmpv, modulus, remove_times); TNbignum_mul( remove_times, modulus, remove_value); TNbignum_sub( tmpv , remove_value, m); e = e >> 1; } } */ #ifdef HELPER_TEST_BIGNUM void main(){ char restxt[1030]; struct bn n,e,res; int exponent = 35; restxt[0]=0; char * modulus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bignum_from_string(&n, modulus,strlen(modulus) ); TNbignum_pow_then_mod(&m, exponent, &n, &res); // (m ^ e) mod N. E doesn't need to be a BIG NUMBER. TNbignum_to_string(&res, restxt, 1030); printf("RES=%s\n", restxt); } #endif #ifdef HELPER_TEST_CRC32 void main(){ long c32=0; c32 = crc32(c32, "Hello.",6); printf("CRC = %08lX\n", c32); c32=0; c32 = crc32(c32, "Hel",3); c32 = crc32(c32, "lo",2); c32 = crc32(c32, ".",1); c32 = crc32(c32, "",0); printf("CRC = %08lX\n", c32); } #endif #ifdef __MINGW32__ #include #endif int get_free_total_mem(size_t * total, size_t * free_mem){ #ifdef FREEBSD return -1; #endif #ifdef __MINGW32__ MEMORYSTATUSEX statex; statex.dwLength = sizeof (statex); GlobalMemoryStatusEx (&statex); (*total) = statex.ullTotalPhys; (*free_mem) = statex.ullAvailPhys; return 0; #else #ifdef MACOS mach_msg_type_number_t count = HOST_VM_INFO_COUNT; vm_statistics_data_t vmstat; int page_size = getpagesize(); if(KERN_SUCCESS != host_statistics(mach_host_self(), HOST_VM_INFO, (host_info_t)&vmstat, &count)) return -1; //printf("PSIZE=%d\nACT=%u; INACT=%u; FREE=%u\n", page_size, vmstat.active_count, vmstat.inactive_count, vmstat.free_count); size_t btlen = sizeof(*total); if(sysctl( (int[]) { CTL_HW, HW_MEMSIZE }, 2, total, &btlen, NULL, 0)) return -1; *free_mem = (vmstat.free_count + vmstat.inactive_count) * 1llu * page_size; return 0; #else struct sysinfo sinf; sysinfo(&sinf); size_t cached_mem = get_sys_mem_info("Cached:"); if(cached_mem<0)cached_mem=0; *free_mem = cached_mem + sinf.bufferram+sinf.freeram; *total = sinf.totalram; return 0; #endif #endif } void worker_master_mutex_init(worker_master_mutex_t * wmt, int all_workers){ memset(wmt,0,sizeof(worker_master_mutex_t)); wmt -> conds_worker_wait = malloc(sizeof(pthread_cond_t) * all_workers); wmt -> mutexs_worker_wait = malloc(sizeof(pthread_mutex_t) * all_workers); wmt -> mutex_with_master = calloc(sizeof(int), all_workers); wmt -> worker_is_working = calloc(sizeof(int), all_workers); wmt -> workers = all_workers; int x1; for(x1=0;x1 conds_worker_wait [x1], NULL); pthread_mutex_init(&wmt -> mutexs_worker_wait [x1], NULL); } } void worker_thread_start(worker_master_mutex_t * wmt, int worker_id){ pthread_mutex_lock(&wmt->mutexs_worker_wait[worker_id]); wmt -> mutex_with_master[worker_id] = 0; } void worker_master_mutex_destroy(worker_master_mutex_t * wmt){ int x1; for(x1=0;x1 workers;x1++){ pthread_mutex_destroy(&wmt -> mutexs_worker_wait [x1]); pthread_cond_destroy(&wmt -> conds_worker_wait [x1]); } free( wmt -> worker_is_working); free( wmt -> mutex_with_master); free( wmt -> conds_worker_wait); free( wmt -> mutexs_worker_wait); } int worker_wait_for_job(worker_master_mutex_t * wmt, int worker_id){ pthread_mutex_trylock(wmt->mutexs_worker_wait + worker_id); wmt->worker_is_working[worker_id] = 0; while(1){ pthread_cond_wait(&wmt->conds_worker_wait[worker_id], &wmt->mutexs_worker_wait[worker_id]); if(wmt -> all_terminate) pthread_mutex_unlock(&wmt->mutexs_worker_wait[worker_id]); if(wmt -> mutex_with_master[worker_id] == 0)break; // otherwise, it is a spurious wakeup } return wmt -> all_terminate; } void master_wait_for_job_done(worker_master_mutex_t * wmt, int worker_id){ if(!wmt -> mutex_with_master[worker_id] ){ while(1){ pthread_mutex_lock(&wmt->mutexs_worker_wait[worker_id]); if(!wmt->worker_is_working[worker_id])break; // In a rare situation, the worker hasn't been scheduled after the last notification. // Namely, the master will notify twice the worker when the worker thread hasn't started at all. The second notification will cause no condition signaled. // This test is to prevent such a situation. pthread_mutex_unlock(&wmt->mutexs_worker_wait[worker_id]); usleep(50); } if(wmt->worker_is_working[worker_id]) SUBREADprintf("ERROR 3: HOW CAN I HAVE THIS LOCK : %d?? TH_%d\n", wmt->worker_is_working[worker_id], worker_id); } wmt -> mutex_with_master[worker_id] = 1; } // master collects results between master_wait_for_job_done and master_notify_worker void master_notify_worker(worker_master_mutex_t * wmt, int worker_id){ if(!wmt -> mutex_with_master[worker_id])SUBREADprintf("ERROR 2: HOW CAN I NOT HAVE THE LOCK : %d ; TERM=%d\n", worker_id, wmt -> all_terminate); wmt -> worker_is_working[worker_id] = 1; wmt -> mutex_with_master[worker_id] = 0; pthread_cond_signal(&wmt->conds_worker_wait[worker_id]); pthread_mutex_unlock(&wmt->mutexs_worker_wait[worker_id]); } // this function must be called when the master thread has all the worker locks in control. // ie all the workers should be in the "wait_for_job" status. void terminate_workers(worker_master_mutex_t * wmt){ int x1; wmt -> all_terminate = 1; for(x1=0;x1 workers;x1++) master_notify_worker(wmt,x1); } void *windows_memmem(const void *haystack_start, size_t haystack_len, const void *needle_start, size_t needle_len) { const unsigned char *haystack = (const unsigned char *) haystack_start; const unsigned char *needle = (const unsigned char *) needle_start; const unsigned char *h = NULL; const unsigned char *n = NULL; size_t x = needle_len; /* The first occurrence of the empty string is deemed to occur at * the beginning of the string. */ if (needle_len == 0) return (void *) haystack_start; /* Sanity check, otherwise the loop might search through the whole * memory. */ if (haystack_len < needle_len) return NULL; for (; *haystack && haystack_len--; haystack++) { x = needle_len; n = needle; h = haystack; if (haystack_len < needle_len) break; if ((*haystack != *needle) || ( *haystack + needle_len != *needle + needle_len)) continue; for (; x ; h++ , n++) { x--; if (*h != *n) break; if (x == 0) return (void *)haystack; } } return NULL; } Rsubread/src/HelperFunctions.h0000644000175200017520000002643214136047615017431 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #ifndef __HELPER_FUNCTIONS_H_ #define __HELPER_FUNCTIONS_H_ #include #include "subread.h" #include "hashtable.h" #define PARSE_STATUS_TAGNAME 1 #define PARSE_STATUS_TAGTYPE 2 #define PARSE_STATUS_TAGVALUE 3 typedef struct{ int workers; int * mutex_with_master; pthread_cond_t *conds_worker_wait; pthread_mutex_t *mutexs_worker_wait; int all_terminate; int * worker_is_working; } worker_master_mutex_t; /**** How to run master and worker * Master: prepare_next_job -> wait_for_last_job_done -> collect_results -> notify_worker_do_next -> goto 0 * Worker: wait_for_next_job -> do_next_job -> goto 0 * The result is collected from worker while it is "wait_for_next_job": it releases the lock when "cond_wait" is called. * Master can prepare the next job while the worker is running on the last job, hence parallel. */ void worker_master_mutex_init(worker_master_mutex_t * wmt, int all_workers); void worker_thread_start(worker_master_mutex_t * wmt, int worker_id); void worker_master_mutex_destroy(worker_master_mutex_t * wmt); // 0 : a job is allocated; 1 : worker should terminate int worker_wait_for_job(worker_master_mutex_t * wmt, int worker_id); void master_wait_for_job_done(worker_master_mutex_t * wmt, int worker_id); // master collects results between master_wait_for_job_done and master_notify_worker void master_notify_worker(worker_master_mutex_t * wmt, int worker_id); // this function must be called when the master thread has all the worker locks in control. // ie all the workers should be in the "wait_for_job" status. void terminate_workers(worker_master_mutex_t * wmt); typedef struct{ HashTable * contig_table; HashTable * size_table; } fasta_contigs_t; #ifndef MAKE_STANDALONE typedef struct{ ArrayList * message_queue; int is_thread_mode; subread_lock_t queue_lock; subread_lock_t queue_notifier; int is_finished; } message_queue_t; extern message_queue_t mt_message_queue; #endif void msgqu_init(); void msgqu_destroy(); void msgqu_main_loop(); void msgqu_notifyFinish(); void msgqu_printf(const char * fmt, ...); int read_contig_fasta(fasta_contigs_t * tab, char * fname); int get_contig_fasta(fasta_contigs_t * tab, char * chro, unsigned int pos, int len, char * out_bases); void destroy_contig_fasta(fasta_contigs_t * tab); // This function parses CIGAR_Str and extract the relative starting points and lengths of all sections (i.e., the sections of read that are separated by 'N'). // CIGAR_Str is a CIGAR string containing 'S', 'M', 'I', 'D' and 'N' operations. Other operations are all ignored. The length of CIGAR_Str should be always less than 100 bytes or "-1" is returned. // Staring_Points and Section_Length are empty arrays to write the sections. The minimum length of each array is 6 items. // The length of a section is its length on the chromosome, namely 'I' is ignored but 'D' is added into the length. // This function ignores all sections from the 7-th. // This function returns the number of sections found in the CIGAR string. It returns -1 if the CIGAR string cannot be parsed. int RSubread_parse_CIGAR_string(char * chro , unsigned int first_pos, const char * CIGAR_Str, int max_M, char ** Section_Chromosomes, unsigned int * Section_Start_Chro_Pos,unsigned short * Section_Start_Read_Pos, unsigned short * Section_Chro_Length, int * is_junction_read); int RSubread_parse_CIGAR_Extra_string(int FLAG, char * MainChro, unsigned int MainPos, const char * CIGAR_Str, const char * Extra_Tags, int max_M, char ** Chros, unsigned int * Staring_Chro_Points, unsigned short * Section_Start_Read_Pos, unsigned short * Section_Length, int * is_junction_read); // This function try to find the attribute value of a given attribute name from the extra column string in GTF/GFF. // If the value is found, it returns the length of the value (must be > 0 by definition), or -1 if no attribute is found or the format is wrong. int GTF_extra_column_value(const char * Extra_Col, const char * Target_Name, char * Target_Value, int TargVal_Size); // Replacing `rep' with `with' in `orig'. // Rhe return value must be freed if it is not NULL. char *str_replace(char *orig, char *rep, char *with) ; // rule: the string is ABC123XXXXXX... // // This is the priroity: // // First, compare the letters part. // // Second, compare the pure numeric part. // // Third, compare the remainder. int strcmp_number(char * s1, char * s2); unsigned int reverse_cigar(unsigned int pos, char * cigar, char * new_cigar); unsigned int find_left_end_cigar(unsigned int right_pos, char * cigar); int mac_or_rand_str(char * char_14); double fast_fisher_test_one_side(unsigned int a, unsigned int b, unsigned int c, unsigned int d, long double * frac_buffer, int buffer_size); int load_features_annotation(char * file_name, int file_type, char * gene_id_column, char * transcript_id_column, char * used_feature_type, void * context, int do_add_feature(char * gene_name, char * transcript_id, char * chrome_name, unsigned int start, unsigned int end, int is_negative_strand, void * context) ); HashTable * load_alias_table(char * fname) ; char * get_short_fname(char * lname); // Rebuild a string containing the command line. // Return the string length (without the terminating \0) // You need to free(*lineptr) after all. int rebuild_command_line(char ** lineptr, int argc, char ** argv); // Calculate a full round of MD5 or SHA256. void Helper_md5sum(char * plain_txt, int plain_len, unsigned char * bin_md5_buff); typedef unsigned int HelpFuncMD5_u32plus; typedef struct { HelpFuncMD5_u32plus lo, hi; HelpFuncMD5_u32plus a, b, c, d; unsigned char buffer[64]; HelpFuncMD5_u32plus block[16]; } HelpFuncMD5_CTX; void HelpFuncMD5_Init(HelpFuncMD5_CTX *ctx); void HelpFuncMD5_Update(HelpFuncMD5_CTX *ctx, const void *data, unsigned long size); void HelpFuncMD5_Final(unsigned char *result, HelpFuncMD5_CTX *ctx); void Helper_sha256sum(char * plain_txt, int plain_len, unsigned char * bin_md5_buff); unsigned long long plain_txt_to_long_rand(char * plain_txt, int plain_len); // give me a p, I give you the value such that Pr( x < value ) == p in a 0/1 normal distribution. double inverse_sample_normal(double p); // big number functions // retrived from https://github.com/kokke/tiny-TNbignum-c // "This is free and unencumbered software released into the public domain." #include #include /* This macro defines the word size in bytes of the array that constitues the big-number data structure. */ #ifndef WORD_SIZE #define WORD_SIZE 4 #endif /* Size of big-numbers in bytes */ #define BN_MAXIMUM_BITS 4096 #define BN_ARRAY_SIZE ( BN_MAXIMUM_BITS / 8 / WORD_SIZE ) /* Here comes the compile-time specialization for how large the underlying array size should be. */ /* The choices are 1, 2 and 4 bytes in size with uint32, uint64 for WORD_SIZE==4, as temporary. */ #ifndef WORD_SIZE #error Must define WORD_SIZE to be 1, 2, 4 #elif (WORD_SIZE == 1) /* Data type of array in structure */ #define DTYPE uint8_t /* bitmask for getting MSB */ #define DTYPE_MSB ((DTYPE_TMP)(0x80)) /* Data-type larger than DTYPE, for holding intermediate results of calculations */ #define DTYPE_TMP uint32_t /* sprintf format string */ #define SPRINTF_FORMAT_STR "%.02x" #define SSCANF_FORMAT_STR "%2hhx" /* Max value of integer type */ #define MAX_VAL ((DTYPE_TMP)0xFF) #elif (WORD_SIZE == 2) #define DTYPE uint16_t #define DTYPE_TMP uint32_t #define DTYPE_MSB ((DTYPE_TMP)(0x8000)) #define SPRINTF_FORMAT_STR "%.04x" #define SSCANF_FORMAT_STR "%4hx" #define MAX_VAL ((DTYPE_TMP)0xFFFF) #elif (WORD_SIZE == 4) #define DTYPE uint32_t #define DTYPE_TMP uint64_t #define DTYPE_MSB ((DTYPE_TMP)(0x80000000)) #define SPRINTF_FORMAT_STR "%.08x" #define SSCANF_FORMAT_STR "%8x" #define MAX_VAL ((DTYPE_TMP)0xFFFFFFFF) #endif #ifndef DTYPE #error DTYPE must be defined to uint8_t, uint16_t uint32_t or whatever #endif /* Custom assert macro - easy to disable */ #define require(p, msg) assert(p && #msg) /* Data-holding structure: array of DTYPEs */ struct bn { DTYPE array[BN_ARRAY_SIZE]; }; /* Tokens returned by TNbignum_cmp() for value comparison */ enum { SMALLER = -1, EQUAL = 0, LARGER = 1 }; /* Initialization functions: */ void TNbignum_init(struct bn* n); void TNbignum_from_int(struct bn* n, DTYPE_TMP i); int TNbignum_to_int(struct bn* n); void TNbignum_from_string(struct bn* n, char* str, int nbytes); // warning: maxsize MUST >= 1026 void TNbignum_to_string(struct bn* n, char* str, int maxsize); /* Basic arithmetic operations: */ void TNbignum_add(struct bn* a, struct bn* b, struct bn* c); /* c = a + b */ void TNbignum_sub(struct bn* a, struct bn* b, struct bn* c); /* c = a - b */ void TNbignum_mul(struct bn* a, struct bn* b, struct bn* c); /* c = a * b */ void TNbignum_div(struct bn* a, struct bn* b, struct bn* c); /* c = a / b */ void TNbignum_mod(struct bn* a, struct bn* b, struct bn* c); /* c = a % b */ void TNbignum_divmod(struct bn* a, struct bn* b, struct bn* c, struct bn* d); /* c = a/b, d = a%b */ /* Bitwise operations: */ void TNbignum_and(struct bn* a, struct bn* b, struct bn* c); /* c = a & b */ void TNbignum_or(struct bn* a, struct bn* b, struct bn* c); /* c = a | b */ void TNbignum_xor(struct bn* a, struct bn* b, struct bn* c); /* c = a ^ b */ void TNbignum_lshift(struct bn* a, struct bn* b, int nbits); /* b = a << nbits */ void TNbignum_rshift(struct bn* a, struct bn* b, int nbits); /* b = a >> nbits */ /* Special operators and comparison */ int TNbignum_cmp(struct bn* a, struct bn* b); /* Compare: returns LARGER, EQUAL or SMALLER */ int TNbignum_is_zero(struct bn* n); /* For comparison with zero */ void TNbignum_inc(struct bn* n); /* Increment: add one to n */ void TNbignum_dec(struct bn* n); /* Decrement: subtract one from n */ void TNbignum_pow(struct bn* a, struct bn* b, struct bn* c); /* Calculate a^b -- e.g. 2^10 => 1024 */ void TNbignum_isqrt(struct bn* a, struct bn* b); /* Integer square root -- e.g. isqrt(5) => 2*/ void TNbignum_assign(struct bn* dst, struct bn* src); /* Copy src into dst -- dst := src */ size_t get_sys_mem_info(char * keyword); int get_free_total_mem(size_t * total, size_t * free_mem); void *windows_memmem(const void *haystack_start, size_t haystack_len, const void *needle_start, size_t needle_len); void sorting_LLU_array_exchange(void * arr, int i, int j); int sorting_LLU_array_compare(void * arr, int i, int j); #endif Rsubread/src/LRMbase-index.c0000644000175200017520000001705714136047615016711 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #include #include #include #include #include #include "LRMconfig.h" #include "LRMbase-index.h" #include "LRMfile-io.h" #define LRMgvindex_baseno2offset_m(base_number, index, offset_byte, offset_bit) {offset_byte = (base_number - index -> start_base_offset) >>2; offset_bit = base_number % 4 * 2;} void LRMgvindex_baseno2offset(unsigned int base_number, LRMgene_value_index_t * index, unsigned int * offset_byte, unsigned int * offset_bit) { // the base number corrsponding to the 0-th bit in the whole value array; unsigned int offset = (base_number - index -> start_base_offset); * offset_byte = offset >>2 ; * offset_bit = base_number % 4 * 2; } // return 'A', 'G', 'T' and 'C' int LRMgvindex_get(LRMgene_value_index_t * index, LRMgehash_data_t offset) { unsigned int offset_byte, offset_bit; LRMgvindex_baseno2offset_m(offset, index , offset_byte, offset_bit); if(offset_byte >= index-> values_bytes -1)return 'N'; unsigned int one_base_value = (index->values [offset_byte]) >> (offset_bit); //LRMprintf("RECV_BASE=%d (%d - %d)\n",one_base_value & 3, offset_byte , offset_bit); return LRMint2base(one_base_value & 3); } int LRMgvindex_match(LRMgene_value_index_t * index, LRMgehash_data_t offset, LRMgehash_key_t base_values) { unsigned int offset_byte, offset_bit; LRMgvindex_baseno2offset_m(offset, index , offset_byte, offset_bit); int i, ret = 0; for (i=0; i<16; i++) { unsigned char mask = 0x3 << (offset_bit); unsigned char one_base_value = (index->values [offset_byte] & mask) >> (8-offset_bit); if ( ((base_values >> (30 - i*2)) & 0x3) == one_base_value) ret |= 1 << i; offset_bit +=2; if(offset_bit >=8) { offset_bit = 0; offset_byte ++; } } return ret; } int LRMgvindex_load(LRMgene_value_index_t * index, const char filename []) { FILE * fp = fopen(filename, "rb"); int read_length; read_length = fread(&index->start_point,4,1, fp); if(read_length<1){ LRMprintf("ERROR: the array index is incomplete : %d", read_length ); return 1; } read_length = fread(&index->length,4,1, fp); if(read_length<1){ LRMprintf("Bad index\n"); return 1; } //LRMprintf ("\nBINDEX %s : %u ~ +%u\n",filename, index->start_point, index->length ); unsigned int useful_bytes, useful_bits; index -> start_base_offset = index -> start_point - index -> start_point%4; LRMgvindex_baseno2offset (index -> length+ index -> start_point, index ,&useful_bytes,&useful_bits); index -> values = malloc(useful_bytes+1); index -> values_bytes = useful_bytes+1; if(!index->values) { LRMprintf("Out of memory\n"); return 1; } read_length =fread(index->values, 1, useful_bytes+1, fp); if(read_length < useful_bytes){ LRMprintf("ERROR: the array index is incomplete : %d < %d.", read_length, useful_bytes+1 ); return 1; } fclose(fp); return 0; } void LRMgvindex_get_string(char *buf, LRMgene_value_index_t * index, unsigned int pos, int len, int is_negative_strand){ int i; if (is_negative_strand) for (i=len-1;i>=0;i--) { buf[i] = LRMgvindex_get (index, pos + len - 1 - i); switch(buf[i]) { case 'A': buf[i] = 'T'; break; case 'G': buf[i] = 'C'; break; case 'C': buf[i] = 'G'; break; default: buf[i] = 'A'; } } else for (i=0;i='0' && nch <='9'){ tmpi = tmpi * 10 + (nch - '0'); }else{ tmpi *= tmpi_sign; if(nch == 'M'){ int this_matched = LRMmatch_chro(read + read_chrsor, index, chro_cursor, tmpi, 0); if(show_txt){ unsigned txt_chro_cursor = chro_cursor; int txt_read_chrsor = read_chrsor; for(x1 = 0; x1 < tmpi ; x1++){ int knownval = LRMgvindex_get(index, txt_chro_cursor); int readval = read[txt_read_chrsor]; txt_chro_cursor ++; txt_read_chrsor ++; LRMprintf("%c[3%dm%c", 27, knownval == readval ? 7:1, readval); } } if(0 && abs(tmpi) > 22 && this_matched < tmpi * 0.6){ LRMprintf("Too many mismatched (%d%c) : %d / %d : read + %d\n", tmpi, nch, this_matched, tmpi, read_chrsor); } all_matched += this_matched; all_mismatched += ( tmpi - this_matched ); (*maplen) += tmpi; } if(nch == 'M' || nch == 'I' || nch == 'S'){ if(nch == 'I' && show_txt) for(x1 = 0; x1 < tmpi ; x1++) LRMprintf("%c[32m%c", 27, read[ read_chrsor + x1 ]); if(nch == 'S' && show_txt) for(x1 = 0; x1 < tmpi ; x1++) LRMprintf("%c[4m%c%c[0m", 27, read[ read_chrsor + x1 ], 27); read_chrsor += tmpi; } if(nch == 'M' || nch == 'D' || nch == 'N' || nch == 'S'){ if(( nch == 'N' || nch == 'D' ) && show_txt) LRMprintf("%c[36m//%c[37m", 27, 27); if(nch != 'S')chro_cursor += tmpi; } tmpi = 0; tmpi_sign = 1; } } if(show_txt){ char postxt[100]; LRMpos2txt(context, chro_cursor, postxt); LRMprintf("%c[37m\n", 27); LRMprintf("Ending Pos : Read + %d ( %s )\n", read_chrsor, postxt); } if(neg) LRMreverse_read(read, strlen(read)); return all_matched; } int LRMmatch_chro(char * read, LRMgene_value_index_t * index, unsigned int pos, int test_len, int is_negative_strand){ int ret = 0; int i; if ((unsigned int)(pos + test_len) >= index -> length + index -> start_point) return 0; if (pos > 0xffff0000) return 0; if (is_negative_strand) { for (i=test_len -1;i>=0;i--) { char tt = LRMgvindex_get (index, pos+test_len-1-i); switch(tt) { case 'A': ret += read[i] == 'T'; break; case 'T': ret += read[i] == 'A'; break; case 'G': ret += read[i] == 'C'; break; case 'C': ret += read[i] == 'G'; break; } } }else{ unsigned int offset_byte, offset_bit; LRMgvindex_baseno2offset_m(pos, index , offset_byte, offset_bit); if(offset_byte >= index-> values_bytes)return 0; char int_value = index->values [offset_byte]; for (i=0;i> offset_bit) & 3; char tv = read[i]; switch(tv){ case 'A': ret += tt==0; break; case 'G': ret += tt==1; break; case 'C': ret += tt==2; break; case 0: break; default: ret += tt==3; } offset_bit+=2; if(offset_bit==8) { offset_byte++; if(offset_byte == index-> values_bytes)return 0; int_value = index->values [offset_byte]; offset_bit = 0; } } } return ret; } void LRMgvindex_destory(LRMgene_value_index_t * index) { free(index -> values); } Rsubread/src/LRMbase-index.h0000644000175200017520000000302514136047615016704 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #ifndef __LRMBASE_INDEX_H_ #define __LRMBASE_INDEX_H_ #include "LRMconfig.h" int LRMgvindex_load(LRMgene_value_index_t * index, const char filename []); void LRMgvindex_destory(LRMgene_value_index_t * index); void LRMgvindex_baseno2offset(unsigned int base_number, LRMgene_value_index_t * index, unsigned int * offset_byte, unsigned int * offset_bit); int LRMgvindex_get(LRMgene_value_index_t * index, LRMgehash_data_t offset); int LRMmatch_chro(char * read, LRMgene_value_index_t * index, unsigned int pos, int test_len, int is_negative_strand); void LRMgvindex_get_string(char *buf, LRMgene_value_index_t * index, unsigned int pos, int len, int is_negative_strand); int LRMvalidate_mapping(LRMcontext_t * context, char * read, char * cigar, LRMgene_value_index_t * index, unsigned int pos, int rev, int * mapped_length, int show_txt); #endif Rsubread/src/LRMchro-event.c0000644000175200017520000010660514136047615016742 0ustar00biocbuildbiocbuild#include #include #include #include "LRMconfig.h" #include "LRMchro-event.h" #include "LRMfile-io.h" #include "LRMbase-index.h" #include "LRMhelper.h" #define JUNCTION_CONFIRM_WINDOW 14 #define ceq(c,t) ((c)[0]==(t)[0] && (c)[1]==(t)[1]) #define c2eq(ch1, ch2, tg1, tg2) ((ceq(ch1, tg1) && ceq(ch2, tg2)) || (ceq(ch1, tg2) && ceq(ch2, tg1)) ) #define LRMis_donor_chars(cc) (((cc)[0]=='G' && (cc)[1]=='T') || \ ((cc)[0]=='A' && (cc)[1]=='G') || \ ((cc)[0]=='A' && (cc)[1]=='C') || \ ((cc)[0]=='C' && (cc)[1]=='T') ||\ ((cc)[0]=='G' && (cc)[1]=='C')) int LRMpaired_chars(char * ch1, char * ch2){ if (c2eq(ch1, ch2, "GC", "AG") || c2eq(ch1, ch2, "GT", "AG") || c2eq(ch1, ch2, "CT", "AC")) { if ( ceq(ch1, "GC") || ceq(ch1, "CT") || ceq(ch1, "GT")) return 1; } return 0; } int LRMscanning_events_compare(void * arr, int l, int r){ void ** arrr = (void **) arr; LRMcontext_t * context = arrr[0]; int * event_ids = arrr[1]; LRMevent_t * body_l = context -> event_space+event_ids[l]; LRMevent_t * body_r = context -> event_space+event_ids[r]; if(body_l->small_side > body_r->small_side)return 1; if(body_l->small_side < body_r->small_side)return -1; if(body_l->large_side > body_r->large_side)return 1; if(body_l->large_side < body_r->large_side)return -1; if(body_l->event_type > body_r->event_type) return 1; if(body_l->event_type < body_r->event_type) return -1; if(body_l -> indel_length > body_r -> indel_length) return -1; // same length, but L is del and R is ins -- prefer del than ins if(body_l -> indel_length < body_r -> indel_length) return 1; return -1; } void LRMscanning_events_merge(void * arr, int start, int items, int items2){ void ** arrr = (void **) arr; int * records = arrr[1]; int read_1_ptr = start, read_2_ptr = start+items, write_ptr; int * merged_records = malloc(sizeof(int) * (items+items2)); for(write_ptr=0; write_ptr= start+items)||(read_2_ptr < start+items+items2 && LRMscanning_events_compare(arr, read_1_ptr, read_2_ptr) > 0)) merged_records[write_ptr] = records[read_2_ptr++]; else merged_records[write_ptr] = records[read_1_ptr++]; } memcpy(records + start, merged_records, sizeof(int) * (items+items2)); free(merged_records); } void LRMscanning_events_exchange(void * arr, int l, int r){ void ** arrr = (void **) arr; int * records = arrr[1]; int tmpi; tmpi = records[l]; records[l] = records[r]; records[r] = tmpi; } int LRMevents_build_entries(LRMcontext_t * context){ int x1,side_i; for(x1=0; x1 < context->event_number; x1++){ LRMevent_t * te = context->event_space+ x1; for(side_i = 0; side_i <2; side_i++){ unsigned int sidepos = side_i?te->large_side:te->small_side; int * entries_list = LRMHashTableGet(context -> events_realignment, NULL+sidepos); //LRMprintf("INSERT ENTRY : %u -> %p ; SRC: %u ~ %u\n", sidepos, entries_list, te->small_side, te->large_side); if(NULL == entries_list){ entries_list = malloc(sizeof(int) * 3); if(!entries_list){ LRMprintf("ERROR: NO MEMORY CAN BE ALLOCATED.\n"); assert(0); } entries_list[0]=2; entries_list[1]=0; LRMHashTablePut(context -> events_realignment , NULL+sidepos, entries_list); } int x2 = 0, inserted = 0; for(x2=1; x2< 1+ min( LRMMAX_EVENTS_PER_SITE , entries_list[0] ); x2++){ //#warning ">>>>>>>>> COMMENT NEXT LINE <<<<<<<<" //if( x1 + 1 == entries_list[x2] )LRMprintf("REPEATED ENTRY: %d\n"); if(0 == entries_list[x2]){ entries_list[x2] = x1+1; if( x2 < entries_list[0] )entries_list[x2+1]=0; inserted = 1; break; } } if((!inserted) && entries_list[0] < LRMMAX_EVENTS_PER_SITE){ int last_x1 = entries_list[0]; entries_list[0] = LRMMAX_EVENTS_PER_SITE; entries_list = realloc(entries_list, sizeof(int)*(1+LRMMAX_EVENTS_PER_SITE)); entries_list[last_x1] = x1+1; entries_list[last_x1+1] = 0; if(te -> small_side == 457511654 ) LRMprintf("INSERT_NEW EVENT : %d AT %u\n",x1, sidepos ); LRMHashTablePut(context -> events_realignment, NULL+sidepos, entries_list); } } } return 0; } void LRMevents_reorder_merge_next(LRMcontext_t * context, int *order_index){ LRMevent_t *prev_event = NULL, * new_space = malloc(sizeof(LRMevent_t) * 10000); int x1, new_space_size = 10000, new_space_used = 0; for(x1=0; x1 <=context->event_number; x1++){ LRMevent_t *this_event = NULL; if(x1 < context->event_number) this_event = context->event_space+order_index[x1]; if( x1 < context->event_number && prev_event!=NULL && prev_event->large_side == this_event->large_side && prev_event->small_side == this_event->small_side && prev_event->event_type == this_event->event_type && prev_event->indel_length == this_event->indel_length){ prev_event -> supporting_reads ++; }else{ if(new_space_size -1 < new_space_used){ new_space_size*=1.7; new_space = realloc(new_space, sizeof(LRMevent_t)*new_space_size); } if(prev_event) memcpy(new_space+(new_space_used++), prev_event, sizeof(LRMevent_t)); if(this_event){ prev_event = this_event; prev_event -> supporting_reads = 1; } } } free(context -> event_space); context -> event_space = new_space; context -> event_space_size = new_space_size; context -> event_number = new_space_used; } int LRMevents_reorder(LRMcontext_t * context){ int * order_index = malloc(context -> event_number*sizeof(int)); int x1=0; while(x1 event_number){ order_index[x1]=x1; x1++; } void * sort_arr[2]; sort_arr [0] = context; sort_arr [1] = order_index; LRMmerge_sort(sort_arr, context -> event_number, LRMscanning_events_compare, LRMscanning_events_exchange, LRMscanning_events_merge); //basic_sort(sort_arr, context -> event_number, LRMscanning_events_compare, LRMscanning_events_exchange); LRMevents_reorder_merge_next(context, order_index); if(0){ LRMprintf("Total events : %d\n", context -> event_number); for(x1=0; x1 event_number; x1++){ LRMevent_t * te = context->event_space+ x1; if(1 || te -> small_side == 457511654){ char pos1txt[100], pos2txt[100]; LRMpos2txt(context, te->small_side, pos1txt); LRMpos2txt(context, te->large_side, pos2txt); LRMprintf("SORTED EVENT: TYPE: %d - INS %d %s ~ %s, nsup=%d\n", te -> event_type, te -> indel_length, pos1txt, pos2txt, te->supporting_reads); } } } free(order_index); return 0; } int LRMchro_event_new(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context, LRMevent_t * new_event){ //#warning " ================ NO INDEL EVENTS ================" if(new_event -> event_type == LRMEVENT_TYPE_INDEL) return 0; LRMthread_lock(&context -> event_space_lock); if(context -> event_space_size < context -> event_number + 1 ){ context -> event_space_size *= 1.7; context -> event_space =realloc(context -> event_space, sizeof(LRMevent_t) * context -> event_space_size); if(!context -> event_space )return 1; } memcpy(context -> event_space+context -> event_number, new_event, sizeof(LRMevent_t)); context -> event_number++; //LRMprintf("Total events after adding : %d\n", context -> event_number); LRMthread_lockrelease(&context -> event_space_lock); return 0; } int LRMevents_search(LRMcontext_t * context, unsigned int testing_pos, int search_for_smaller_coordinate, int * event_ids_buff){ int * event_here = LRMHashTableGet(context -> events_realignment, NULL + testing_pos); if(NULL == event_here)return 0; int x1, ret = 0; for(x1 = 1; x1< 1+ min(LRMMAX_EVENTS_PER_SITE, event_here[0]); x1++){ int event_i = event_here[x1] - 1; if(0 > event_i)break; //LRMprintf("TESTING EVENT AT %u [%d] : %d \n", testing_pos, x1 , event_i); LRMevent_t * thise = context -> event_space + event_i; if((thise -> large_side == testing_pos && !search_for_smaller_coordinate)|| (thise -> small_side == testing_pos && search_for_smaller_coordinate) ) event_ids_buff[ret++]=event_i; } return ret; } #define LRMDP_score(x,y) dynamic_score_buffer[ (x)*dynamic_row_width + (y) ] #define LRMDP_move(x,y) dynamic_movement_BEFORE_buffer[ (x)*dynamic_row_width + (y) ] #define LRMSOFTCLIPPING_WINDOW 30 #define LRMSOFTCLIPPING_MATCHED 25 int LRMsoftclipping_moves(LRMcontext_t* context, LRMthread_context_t* thread_context, LRMread_iteration_context_t * iteration_context, char * move_buff, int moves, int bases_in_read, int to_large){ int ii; int included_read_length = 0; int last_M = 0x7fffffff; int window_end = moves - 1; int window_start = 0; int window_MX = 0, window_M=0; //LRMprintf("MOVES for %s =%s\n", iteration_context -> read_name, move_buff); for(ii = moves -1; ii>= 0; ii--){ if( move_buff[ii] == 'M' || move_buff[ii] == 'X' ){ window_MX++; if(move_buff[ii] == 'M') window_M++; } if(window_MX == LRMSOFTCLIPPING_WINDOW)break; } window_start = ii; if(window_MX ==LRMSOFTCLIPPING_WINDOW){ for(; window_start >=0 ; window_start--){ if(move_buff[window_start]=='M' || move_buff[window_start] =='X'){ window_MX ++; if(move_buff[window_start]=='M') window_M++; } if(window_MX > LRMSOFTCLIPPING_WINDOW){ while(1){ char nch = move_buff[window_end--]; if(nch == 'M' || nch == 'X'){ window_MX --; if(nch == 'M') window_M--; break; } } } //LRMprintf("M=%d, W = %d - %d, windows_MX=%d in %d\n", window_M, window_start, window_end, window_MX, bases_in_read); if(window_M < LRMSOFTCLIPPING_MATCHED) break; } } int smallwindow_Xs = 0; last_M = window_end; for(ii = window_end; ii>=0 && ii >= window_start; ii--){ if(move_buff[ii] == 'M') last_M = ii; if(move_buff[ii] == 'X' && window_M < LRMSOFTCLIPPING_MATCHED){ smallwindow_Xs++; if(smallwindow_Xs > 1) break; } } if(last_M > 0){ //LRMprintf("M=%d, last 'M' at %d, windows_MX=%d in %d\n", window_M, last_M, window_MX, bases_in_read); for(ii = moves -1; ii>= last_M; ii--){ if(move_buff[ii] == 'M' || move_buff[ii] == 'X' || move_buff[ii] == 'I') included_read_length++; } //assert( (last_M - 1) >=(bases_in_read - included_read_length)); //LRMprintf("last_M=%d, included_read_length=%d in %d\n", last_M, included_read_length, bases_in_read ); int Ss = (bases_in_read - included_read_length); if(Ss < 2 || last_M < 11){ for(ii = last_M - 1; ii > (last_M - 1) - Ss ; ii--){ if(ii<0){ LRMprintf("MINUS_MOVE : %s , last_M = %d, Ss = %d\n", iteration_context -> read_name, last_M, Ss); return -1; } move_buff[ii]='S'; } for(; ii >= 0; ii--) move_buff[ii]='.'; }else{ for(ii = last_M - 1; ii >= 0; ii--) move_buff[ii]='.'; int splen = sprintf(move_buff + last_M - 10, "%dS", Ss); if(to_large){ for(ii = 0; ii < splen/2; ii++){ int tv; tv = move_buff[ last_M - 10 + ii]; move_buff[ last_M - 10 + ii] = move_buff[ last_M - 10 + splen -1 - ii ]; move_buff[ last_M - 10 + splen -1 - ii ] = tv; } } move_buff[ last_M - 10+splen ] = '.'; } } return 0; } void LRMindel_dynamic_search_debug(LRMcontext_t* context, int * dynamic_score_buffer, char * dynamic_movement_BEFORE_buffer, int dynamic_row_width, int dynamic_rows, int *best_offset_history){ int rr, ii; LRMprintf(" "); for(ii=0;ii max_dynamic_indel_length); LRMprintf("\n"); for(rr=0; rr read_name, moves, rebuild_len, rlen, movtxt); } } int my_debug = 0; int LRMdynamic_in_middle(LRMcontext_t* context, LRMthread_context_t* thread_context, LRMread_iteration_context_t * iteration_context, int last_correct_base, int first_correct_base , unsigned int last_correct_base_on_chro, int expected_indels){ int moves, xx1; char * corrected_read = iteration_context -> read_text;//+ last_correct_base; int high_penalty_create_gap = 0; if(0) { char postxt[100]; LRMpos2txt(context, last_correct_base_on_chro, postxt); LRMprintf("Dynamic: at %s : %d - %d ; expected = %d\n", postxt, last_correct_base, first_correct_base, expected_indels); for(xx1 = 0 ; xx1 < first_correct_base - last_correct_base ; xx1++ ){ LRMprintf("%c", corrected_read[ xx1 + last_correct_base ]); } LRMprintf("\n"); for(xx1 = 0 ; xx1 < first_correct_base - last_correct_base - expected_indels; xx1++){ LRMprintf("%c", LRMgvindex_get(& context -> current_base_index, last_correct_base_on_chro + xx1)); } LRMprintf("\n"); } int dynamic_rows = first_correct_base - last_correct_base; int trying_indel_length ; if(abs(expected_indels) < 10) trying_indel_length = abs(expected_indels) * 2; else trying_indel_length = (abs(expected_indels) -10) * 3/2 + 20; trying_indel_length = max(trying_indel_length, 10); trying_indel_length = min(trying_indel_length, LRMINDEL_DYNAMIC_CHANNEL_TOLERANCE); int best_offset_history [dynamic_rows]; int score_match = context -> dynamic_programming_score_match; int score_mismatch = context -> dynamic_programming_score_mismatch; int score_create_gap = context -> dynamic_programming_score_create_gap * (1+high_penalty_create_gap); int score_extend_gap = context -> dynamic_programming_score_extend_gap; int dynamic_row_width = 2* trying_indel_length + 1; char postxt[100]; LRMpos2txt(context, last_correct_base_on_chro, postxt); //LRMprintf("DDDDD1 SIZE=%d ; last_correct = %d; first_correct = %d ; delta=%d ; pos=%u (%s)\n", dynamic_row_width * dynamic_rows, last_correct_base, first_correct_base, expected_indels, last_correct_base_on_chro, postxt); int * dynamic_score_buffer = (int *)thread_context -> dynamic_programming_score_buffer; char * dynamic_movement_BEFORE_buffer = thread_context -> dynamic_programming_movement_buffer; memset(dynamic_score_buffer, 0, sizeof(int) * dynamic_row_width * dynamic_rows); memset(dynamic_movement_BEFORE_buffer, 0, sizeof(char) * dynamic_row_width * dynamic_rows); char * indel_movement_buff = (char *) thread_context -> dynamic_programming_indel_movement_buf; int this_movement_start = thread_context -> dynamic_programming_indel_movement_start; LRMDP_score(0, trying_indel_length )=0; int read_cursor = last_correct_base, row_i, indel_i; unsigned int chro_cursor = last_correct_base_on_chro; int last_slope_offset = 0; if(1){ float slope = expected_indels *1. / dynamic_rows; for(xx1 = 0; xx1 < dynamic_rows; xx1++) best_offset_history[xx1] = 1* (int)(xx1 * slope); } for(; read_cursor < first_correct_base; read_cursor++){ row_i = read_cursor - last_correct_base; int slope_offset = row_i>0?best_offset_history[row_i-1]:0; int last_slope_delta = slope_offset - last_slope_offset; for(indel_i = dynamic_row_width-1 ; indel_i >=0; indel_i --){ // negative: deletion; positive: insertion int testing_indel = indel_i - trying_indel_length; if(1){ int score_from_del = -0x7fffffff, score_from_ins = -0x7fffffff, score_from_match = -0x7fffffff; int is_matched_base = toupper(corrected_read[read_cursor]) == toupper(LRMgvindex_get(& context -> current_base_index, chro_cursor - slope_offset - testing_indel)); if(row_i>0 && (indel_i-1+ last_slope_delta)>=0 && (indel_i-1+ last_slope_delta)< dynamic_row_width && LRMDP_score(row_i-1, indel_i-1 + last_slope_delta) > -0x7ffffff0) score_from_ins = LRMDP_score(row_i-1, indel_i-1 + last_slope_delta) + ( (LRMDP_move(row_i-1, indel_i-1+ last_slope_delta) == 'M' || LRMDP_move(row_i-1, indel_i-1 +last_slope_delta) == 'X')?score_create_gap:score_extend_gap); if(testing_indel < 0 || testing_indel < row_i)if(indel_i < dynamic_row_width-1 && LRMDP_score(row_i, indel_i+1) > -0x7ffffff0) score_from_del = LRMDP_score(row_i, indel_i+1) + ((LRMDP_move(row_i, indel_i+1) == 'M' || LRMDP_move(row_i, indel_i+1) == 'X')?score_create_gap:score_extend_gap); if((indel_i+ last_slope_delta)>=0 && (indel_i+ last_slope_delta)< dynamic_row_width && (row_i ==0 || LRMDP_score(row_i-1, indel_i + last_slope_delta) > -0x7ffffff0)){ score_from_match =(row_i > 0 ?LRMDP_score(row_i-1, indel_i + last_slope_delta): 0)+ (is_matched_base?score_match:score_mismatch); if(row_i == 0 && testing_indel > 0) score_from_match += score_create_gap + (testing_indel-1) * score_extend_gap; } int final_score = max(score_from_del, max(score_from_ins, score_from_match)); if(testing_indel + slope_offset > 0 && row_i < slope_offset + testing_indel){ LRMDP_score(row_i, indel_i) = score_create_gap + (score_extend_gap-1) * (testing_indel+slope_offset) ; LRMDP_move(row_i, indel_i) = 'I'; }else{ LRMDP_score(row_i, indel_i) = final_score; if(final_score < -0x7ffffff0) LRMDP_move(row_i, indel_i) = '?'; else LRMDP_move(row_i, indel_i) = score_from_del == final_score?'D':((score_from_ins == final_score)?'I': ( is_matched_base ?'M':'X')); } } } last_slope_offset = slope_offset; chro_cursor ++; } if(0) LRMindel_dynamic_search_debug(context, dynamic_score_buffer, dynamic_movement_BEFORE_buffer, dynamic_row_width, dynamic_rows, best_offset_history); row_i = first_correct_base - last_correct_base - 1; indel_i = trying_indel_length + expected_indels -(row_i >0?best_offset_history[row_i - 1]:0); moves = 0; while(row_i >= 0 && indel_i >=0 && indel_i < dynamic_row_width){ int slope_offset = best_offset_history[row_i-1]; int next_slope_offset = row_i > 1?best_offset_history[row_i-2]:0; int last_slope_delta = slope_offset - next_slope_offset; //#warning "========= DO NOT ASSERT =============" indel_movement_buff[ this_movement_start + moves] = LRMDP_move(row_i, indel_i); if(indel_movement_buff[ this_movement_start + moves]=='?')LRMprintf("Assertion_Error:%s\n", iteration_context -> read_name); assert(indel_movement_buff[ this_movement_start + moves]!='?'); if(indel_movement_buff[ this_movement_start + moves] == 'M' || indel_movement_buff[ this_movement_start + moves] == 'X'){ row_i--; indel_i += last_slope_delta; } else if(indel_movement_buff[ this_movement_start + moves] == 'D')indel_i++; else { indel_i --; indel_i += last_slope_delta; row_i--; } moves ++; if(row_i < 0 && indel_i < trying_indel_length) for(; indel_i < trying_indel_length; indel_i++) indel_movement_buff[ this_movement_start + ( moves++ ) ] ='D'; if(moves > max( LRMDYNAMIC_MAXIMUM_GAP_LENGTH * 15, 300 ) + context -> max_dynamic_indel_length ){ LRMprintf("ERROR: Dynamic programming moves more than %d\n", max( (int)(LRMDYNAMIC_MAXIMUM_GAP_LENGTH * 15), 300 ) + context -> max_dynamic_indel_length); return -1; } } indel_movement_buff[ this_movement_start + moves]=0; for(row_i = 0; row_i < moves/2; row_i++){ char tmp = indel_movement_buff[ this_movement_start + row_i]; indel_movement_buff[ this_movement_start + row_i] = indel_movement_buff[ this_movement_start + moves - row_i - 1]; indel_movement_buff[ this_movement_start + moves - row_i - 1] = tmp; } indel_movement_buff[ this_movement_start + (moves++)]='/'; indel_movement_buff[ this_movement_start + moves] = 0; LRMtest_move_buff( context, thread_context, iteration_context , indel_movement_buff + this_movement_start, moves , dynamic_rows); if(0)LRMprintf("MOVES = %s\n", indel_movement_buff + this_movement_start); return moves; } int LRMdynamic_to_ends(LRMcontext_t* context, LRMthread_context_t* thread_context, LRMread_iteration_context_t * iteration_context, int last_mapped_in_read, unsigned int last_correct_base_on_chro, int search_to_3end){ int moves = 0; int high_penalty_create_gap = 0; int last_correct_base = search_to_3end? last_mapped_in_read : 0; int first_correct_base = search_to_3end? iteration_context -> read_length : last_mapped_in_read; int head_SS = 0, tail_SS = 0; int this_movement_start = thread_context -> dynamic_programming_indel_movement_start; //LRMprintf("SEARCH_END_01 %s : start=%d, moves=%d\n", iteration_context -> read_name , this_movement_start, moves); char * indel_movement_buff = (char *) thread_context -> dynamic_programming_indel_movement_buf; int bases_in_read_no_SS = first_correct_base - last_correct_base; //if(bases_in_read_no_SS<3){ // return sprintf(indel_movement_buff + this_movement_start,"%dS/",bases_in_read_no_SS); //} if((!search_to_3end) && first_correct_base >= LRMDYNAMIC_MAXIMUM_GAP_LENGTH){ last_correct_base = first_correct_base - LRMDYNAMIC_MAXIMUM_GAP_LENGTH; head_SS = last_correct_base; //LRMprintf("HEAD_SKIP = %d\n", head_SS); } if(search_to_3end && iteration_context -> read_length - last_correct_base >= LRMDYNAMIC_MAXIMUM_GAP_LENGTH){ first_correct_base = last_correct_base + LRMDYNAMIC_MAXIMUM_GAP_LENGTH; tail_SS = iteration_context -> read_length - first_correct_base; } char * corrected_read = iteration_context -> read_text;// + first_correct_base; int bases_in_read = first_correct_base - last_correct_base; int trying_indel_length = LRMINDEL_DYNAMIC_CHANNEL_TOLERANCE/3; //if( bases_in_read > 1000 ) trying_indel_length = LRMINDEL_DYNAMIC_CHANNEL_TOLERANCE/2; //if( bases_in_read > 10000 ) trying_indel_length = LRMINDEL_DYNAMIC_CHANNEL_TOLERANCE; if(bases_in_read_no_SS<2) trying_indel_length = 1; //int best_offset_history [bases_in_read]; int best_offset_history[bases_in_read]; int score_match = context -> dynamic_programming_score_match; int score_mismatch = context -> dynamic_programming_score_mismatch; int score_create_gap = context -> dynamic_programming_score_create_gap * (1+high_penalty_create_gap); int score_extend_gap = context -> dynamic_programming_score_extend_gap; int dynamic_row_width = 2* trying_indel_length + 1; int * dynamic_score_buffer = (int *)thread_context -> dynamic_programming_score_buffer; char * dynamic_movement_BEFORE_buffer = thread_context -> dynamic_programming_movement_buffer; //LRMprintf("EEEEE1 %s: Memsize=4 * %d = %d x %d\n", iteration_context -> read_name, dynamic_row_width * bases_in_read , dynamic_row_width, bases_in_read); memset(dynamic_score_buffer, 0, sizeof(int) * dynamic_row_width * bases_in_read); memset(dynamic_movement_BEFORE_buffer, 0, sizeof(char) * dynamic_row_width * bases_in_read); LRMDP_score(0, trying_indel_length )=0; unsigned int chro_cursor ; int last_slope_offset = 0, read_i, indel_i, previous_base_in_read = 0; if(0){ char postxt[100]; LRMpos2txt(context, last_correct_base_on_chro, postxt); LRMprintf("EXTEND_UNKNOWN: %s\n", postxt); if(!search_to_3end){ int bb; bb = corrected_read[first_correct_base]; corrected_read[first_correct_base] = 0; LRMprintf("READ: %s\n", corrected_read); corrected_read[first_correct_base] = bb; LRMprintf("CHRO: "); for(chro_cursor = last_correct_base_on_chro - bases_in_read - 10; chro_cursor < last_correct_base_on_chro; chro_cursor ++){ bb = LRMgvindex_get(& context -> current_base_index, chro_cursor); LRMprintf("%c", bb); } LRMprintf("\n\n"); }else{ int bb; bb = corrected_read[first_correct_base]; corrected_read[first_correct_base] = 0; LRMprintf("READ: %s\n", corrected_read + last_correct_base); corrected_read[first_correct_base] = bb; LRMprintf("CHRO: "); for(chro_cursor = last_correct_base_on_chro; chro_cursor < last_correct_base_on_chro + 10 + bases_in_read; chro_cursor ++){ bb = LRMgvindex_get(& context -> current_base_index, chro_cursor); LRMprintf("%c", bb); } LRMprintf("\n\n"); } } for(read_i = 0; read_i < bases_in_read; read_i ++){ int this_base_in_read = search_to_3end? read_i :(bases_in_read - read_i -1); int this_base_value_in_read = corrected_read [ last_correct_base + this_base_in_read ]; int slope_offset = read_i>0?best_offset_history[previous_base_in_read]:0; int last_slope_delta = slope_offset - last_slope_offset; if(0 && ! search_to_3end)LRMprintf("GET READ_BASE='%c' LAST_OFF=%d SLP_OFF=%d LAST_DELTA=%d\n", this_base_value_in_read, last_slope_offset, slope_offset, last_slope_delta); int thisrow_max_score = -0x7fffffff, thisrow_max_indel_from_start = -0x7fffffff; for(indel_i = search_to_3end?dynamic_row_width-1 : 0; indel_i !=(search_to_3end?-1:dynamic_row_width); indel_i+=(search_to_3end?-1:1)){ int indel_from_start = slope_offset + ( indel_i - trying_indel_length); // if to_3end: +:ins, -:del; if !to_3end: +:del, -:ins unsigned int chro_location_after_indel = last_correct_base_on_chro + ( search_to_3end ?( read_i):(- read_i -1)) - indel_from_start; // if !to_3end: "DEL" from right is "INS" from left int this_base_value_in_chro = LRMgvindex_get(& context -> current_base_index, chro_location_after_indel); int score_from_del = -0x7fffffff, score_from_ins = -0x7fffffff, score_from_match = -0x7fffffff; int is_matched_base = toupper(this_base_value_in_read)== toupper(this_base_value_in_chro); if(search_to_3end){ if(read_i > 0 && indel_i-1 + last_slope_delta >=0 && indel_i-1 + last_slope_delta < dynamic_row_width && read_i > 0 && LRMDP_score(read_i-1, indel_i-1 + last_slope_delta) > -0x7ffffff0) score_from_ins = LRMDP_score(read_i - 1, indel_i-1 + last_slope_delta) + ( (LRMDP_move(read_i - 1, indel_i-1+ last_slope_delta) == 'I')?score_extend_gap:score_create_gap); if( indel_from_start < 0 || indel_from_start < read_i ) if(indel_i < dynamic_row_width-1 && read_i > 0 && LRMDP_score(read_i, indel_i+1) > -0x7ffffff0) score_from_del = LRMDP_score(read_i, indel_i+1) + ((LRMDP_move(read_i, indel_i+1) == 'D')?score_extend_gap:score_create_gap); if((indel_i+ last_slope_delta)>=0 && (indel_i+ last_slope_delta)< dynamic_row_width && (read_i ==0 || LRMDP_score(read_i-1, indel_i + last_slope_delta) > -0x7ffffff0)) score_from_match =(read_i > 0 ?LRMDP_score(read_i-1, indel_i + last_slope_delta): 0)+ (is_matched_base?score_match:score_mismatch); //if(read_i == 2) LRMprintf("INDEL_i=%d, F_INS=%d, F_DEL=%d, F_MAT=%d\n", indel_i, score_from_ins, score_from_del, score_from_match); }else{ if( indel_from_start > 0 || ( -indel_from_start < read_i ) ) if(indel_i>0 && LRMDP_score(read_i, indel_i-1) > -0x7ffffff0) score_from_ins = LRMDP_score(read_i, indel_i-1) + ( (LRMDP_move(read_i, indel_i-1) == 'I')?score_extend_gap:score_create_gap); if(indel_i < dynamic_row_width-1 && indel_i+1 + last_slope_delta >= 0 && indel_i+1 + last_slope_delta < dynamic_row_width && read_i > 0 && LRMDP_score(read_i - 1, indel_i+1+last_slope_delta) > -0x7ffffff0) score_from_del = LRMDP_score(read_i - 1, indel_i+1 + last_slope_delta) + ((LRMDP_move(read_i - 1, indel_i+1+last_slope_delta) == 'D')?score_extend_gap:score_create_gap); if((indel_i+ last_slope_delta)>=0 && (indel_i+ last_slope_delta)< dynamic_row_width && (read_i ==0 || LRMDP_score(read_i-1, indel_i + last_slope_delta) > -0x7ffffff0)) score_from_match =(read_i > 0 ?LRMDP_score(read_i-1, indel_i + last_slope_delta): 0)+ (is_matched_base?score_match:score_mismatch); //if(read_i == 4 && !search_to_3end) // LRMprintf("INDEL_i=%d, F_INS=%d, F_DEL=%d, F_MAT=%d\n", indel_i, score_from_ins, score_from_del, score_from_match); // LRMprintf("SCORE AT readi=%d, indel_i=%d = %d ;; MOVE = '%c'\n", read_i, indel_i-1+ last_slope_delta, LRMDP_score(read_i, indel_i-1+ last_slope_delta), LRMDP_move(read_i, indel_i-1+ last_slope_delta)); } if(read_i == 0 && indel_from_start > 0 && !search_to_3end) score_from_match += score_create_gap + (indel_from_start-1) * score_extend_gap; if(read_i == 0 && indel_from_start > 0 && search_to_3end) score_from_match += score_create_gap + (indel_from_start-1) * score_extend_gap; int final_score = max(score_from_del, max(score_from_ins, score_from_match)); //if(read_i == 4 && !search_to_3end)LRMprintf(" == READ_I %d; INDEL_I %d; INDEL_START %d; FINAL %d\n", read_i, indel_i, indel_from_start, final_score); if(indel_from_start < 0 && read_i < - indel_from_start && !search_to_3end){ LRMDP_score(read_i, indel_i) = score_create_gap - score_extend_gap * indel_from_start ; LRMDP_move(read_i, indel_i) = 'D'; } else if(indel_from_start > 0 && read_i < indel_from_start && search_to_3end) { LRMDP_score(read_i, indel_i) = score_create_gap + score_extend_gap * (indel_from_start -1) ; LRMDP_move(read_i, indel_i) = 'I'; } else if(final_score < -0x7ffffff0){ LRMDP_move( read_i, indel_i) = '?'; }else{ if(0 && ! search_to_3end)LRMprintf("#%d %c%c%c %c %d |", indel_i, indel_from_start?' ':'>', this_base_value_in_chro, indel_from_start?' ':'<', score_from_del == final_score?'D':((score_from_ins == final_score)?'I': ( is_matched_base ?'M':'X')), final_score); LRMDP_score( read_i, indel_i) = final_score; //LRMprintf(" !! READ_I %d; INDEL_I %d; INDEL_START %d; FINAL %d\n", read_i, indel_i, indel_from_start, final_score); if(final_score > thisrow_max_score && abs( indel_i - dynamic_row_width/2 ) < dynamic_row_width/3){ thisrow_max_score = final_score; thisrow_max_indel_from_start = indel_from_start; } LRMDP_move(read_i, indel_i) =(score_from_del == final_score?'D':((score_from_ins == final_score)?'I': ( is_matched_base ?'M':'X'))); } } //if(thisrow_max_indel_from_start < -0x7ffffff0) LRMprintf("ERROR READ\n"); //else LRMprintf(" READ\n"); assert(thisrow_max_indel_from_start > -0x7ffffff0); best_offset_history[this_base_in_read] = thisrow_max_indel_from_start; if(0 && ! search_to_3end)LRMprintf("\nSET %d-th BEST_FROM_START=%d ; SLOPE_OFFSET=%d ; MAX_SCORE=%d\n\n", this_base_in_read, thisrow_max_indel_from_start, slope_offset, thisrow_max_score); last_slope_offset = slope_offset; previous_base_in_read = this_base_in_read; } read_i = bases_in_read - 1; //LRMprintf("RESTART SEARCH: best_offset_history[read_i]=%d, best_offset_history[read_i-1]=%d\n", best_offset_history[ search_to_3end? bases_in_read - 1 : 0 ] - best_offset_history[ search_to_3end? bases_in_read - 2 : 1 ] ); if(bases_in_read_no_SS > 2) indel_i = best_offset_history[ search_to_3end? bases_in_read - 1 : 0 ] - best_offset_history[ search_to_3end? bases_in_read - 2 : 1 ] + trying_indel_length; else indel_i = 1; int error_in = 0; while(read_i >= 0 && indel_i >=0 && indel_i < dynamic_row_width){ int real_base_in_read = search_to_3end? read_i:(bases_in_read - (read_i+1)); int slope_offset; int next_slope_offset; if(search_to_3end){ slope_offset = (real_base_in_read>0)?best_offset_history[real_base_in_read - 1]:0; next_slope_offset = (real_base_in_read>1)?best_offset_history[real_base_in_read - 2]:0; }else{ slope_offset = (real_base_in_read read_name, read_i, LRMDP_score(read_i, indel_i), indel_i, LRMDP_move(read_i, indel_i), last_slope_delta); //LRMprintf("SEARCH_END_01 %s : start=%d, moves=%d\n", iteration_context -> read_name , this_movement_start, moves); if(indel_movement_buff[ this_movement_start + moves]=='?'){ error_in = 1; //LRMprintf("WRONG_READ_ERROR_MOVE : %s\n", iteration_context -> read_name); break; } if(indel_movement_buff[ this_movement_start + moves] == 'M' || indel_movement_buff[ this_movement_start + moves] == 'X'){ read_i--; if(read_i>=0)indel_i -= last_slope_delta; }else { if(search_to_3end){ if(indel_movement_buff[ this_movement_start + moves] == 'D')indel_i++; else { indel_i --; if(read_i>=0)indel_i -= last_slope_delta; read_i--; } }else{ if(indel_movement_buff[ this_movement_start + moves] == 'I')indel_i--; else { indel_i ++; if(read_i>=0)indel_i -= last_slope_delta; read_i--; } } } moves ++; //if(read_i<0) LRMprintf("END_UP: indel_i = %d > %d\n", indel_i, trying_indel_length); if(search_to_3end){ if(read_i < 0 && indel_i < trying_indel_length) for(; indel_i trying_indel_length) for(; indel_i > trying_indel_length; indel_i--) indel_movement_buff[ this_movement_start + (moves++)] = 'I'; if(moves > max( LRMDYNAMIC_MAXIMUM_GAP_LENGTH * 15, 300 ) + context -> max_dynamic_indel_length ){ LRMprintf("ERROR: Dynamic programming moves more than %d\n", max( (int)(LRMDYNAMIC_MAXIMUM_GAP_LENGTH * 15), 300 ) + context -> max_dynamic_indel_length); return -1; } } //LRMprintf("SEARCH_END_02 %s : start=%d, moves=%d\n", iteration_context -> read_name , this_movement_start, moves); if(0){ indel_movement_buff[this_movement_start + moves] = 0; LRMprintf("MOVE_ENDS [ %s E=%d] = %s\n",iteration_context -> read_name , error_in, indel_movement_buff); } if(error_in){ moves = sprintf(indel_movement_buff + this_movement_start, "%dS", bases_in_read); return moves; } indel_movement_buff[ this_movement_start + moves]=0; if(!search_to_3end){ for(read_i = 0; read_i < moves; read_i++){ char tmp = indel_movement_buff[ this_movement_start + read_i]; if(tmp == 'I') indel_movement_buff[ this_movement_start + read_i]='D'; else if(tmp == 'D') indel_movement_buff[ this_movement_start + read_i]='I'; } } error_in = LRMsoftclipping_moves(context,thread_context, iteration_context ,indel_movement_buff + this_movement_start, moves, bases_in_read, search_to_3end); if(error_in){ LRMprintf("SOFT CLIPPING ERROR : %d of %s\n", error_in, iteration_context -> read_name); return -1; } if(search_to_3end){ for(read_i = 0; read_i < moves/2; read_i++){ char tmp = indel_movement_buff[this_movement_start + read_i]; indel_movement_buff[ this_movement_start + read_i] = indel_movement_buff[this_movement_start + moves - read_i - 1]; indel_movement_buff[ this_movement_start + moves - read_i - 1] = tmp; } } if(head_SS>0){ int iii; for(iii = moves-1; iii >=0; iii--){ indel_movement_buff[ this_movement_start + iii + 10] = indel_movement_buff[ this_movement_start + iii]; } for(iii = 0; iii < 10; iii++) indel_movement_buff[ this_movement_start + iii] = '.'; moves += 10; iii = sprintf(indel_movement_buff + this_movement_start, "%dS",head_SS); indel_movement_buff[this_movement_start + iii] = '.'; } if(tail_SS>0)moves += sprintf(indel_movement_buff + this_movement_start + moves, "%dS",tail_SS); indel_movement_buff[ this_movement_start + (moves++)]='/'; indel_movement_buff[this_movement_start + moves] = 0; LRMtest_move_buff( context, thread_context, iteration_context , indel_movement_buff + this_movement_start, moves , bases_in_read_no_SS ); if(0)LRMprintf("MOVE_ENDS [ %s E=%d] = %s; rlen = %d\n",iteration_context -> read_name , error_in, indel_movement_buff + this_movement_start, bases_in_read); return moves; } Rsubread/src/LRMchro-event.h0000644000175200017520000000301314136047615016734 0ustar00biocbuildbiocbuild#ifndef __LRMCHRO_EVENT_H_ #define __LRMCHRO_EVENT_H_ #include "LRMconfig.h" int LRMevents_load_annot(LRMcontext_t * context); int LRMevents_filtering(LRMcontext_t * context); int LRMevents_reorder(LRMcontext_t * context); int LRMevents_build_entries(LRMcontext_t * context); int LRMevents_lookup(LRMcontext_t * context, unsigned int abs_pos, int event_type_masks, int search_large_side, LRMevent_t ** res); int LRMchro_event_new(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context, LRMevent_t * new_event); // high_penalty_for_creating_gap is for creating less CIGAR options (<65535) int LRMindel_dynamic_search(LRMcontext_t* context, LRMthread_context_t* thread_context, int expected_indels, unsigned int last_correct_base_on_chro, char * corrected_read, int last_correct_base, int first_correct_base, unsigned int * total_mismatched, int high_penalty_for_creating_gap); void LRMreverse_read_and_qual(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context); int LRMdynamic_to_ends(LRMcontext_t* context, LRMthread_context_t* thread_context, LRMread_iteration_context_t * iteration_context, int last_mapped_in_read, unsigned int last_correct_base_on_chro, int search_to_3end); int LRMdynamic_in_middle(LRMcontext_t* context, LRMthread_context_t* thread_context, LRMread_iteration_context_t * iteration_context, int last_correct_base, int first_correct_base , unsigned int last_correct_base_on_chro, int expected_indels); #endif Rsubread/src/LRMconfig.h0000644000175200017520000002315014136047615016133 0ustar00biocbuildbiocbuild#ifndef __LRMconfig_h_ #define __LRMconfig_h_ #ifndef SRINT_64_DEFINED #define SRINT_64_DEFINED typedef long long srInt_64; typedef unsigned long long srUInt_64; #endif #include #include #include "LRMseek-zlib.h" #include "LRMhashtable.h" #ifndef MAKE_STANDALONE #ifndef RUNNING_ENV #include #endif #endif #define LRMMAX_FILENAME_LENGTH 500 #define LRMMAX_READ_NAME_LEN 256 #define LRMMAX_CHROMOSOME_NAME_LEN 256 #define LRMMAX_EVENTS_PER_SITE 3 #define LRMMAX_READ_LENGTH 1200000 #define LRMMAX_THREADS 64 #define LRMMAX_SUBREAD_PER_READ_HARDLIMIT 1200000 #define LRMMAX_INDEL_TOLERANCE 16 #define LRMMAX_INDEL_SECTIONS 7 #define LRMMAX_CIGAR_OPTS_IN_READ 65500 #define LRMSEGMENT_CIGAR_SIZE 1000 #define LRMSOFTCLIPPING_MAX_MISMATCH 2 #define LRMJUMP_MISMATCH_TOLERANCE 4 #define LRMDYNAMIC_MAXIMUM_GAP_LENGTH (15000) // " * 250" is for RNA-seq mapping -- a much larger dynamic programming space. #define LRMINDEL_DYNAMIC_CHANNEL_TOLERANCE (150 * 250 / 250 ) #define LRMSUBREAD_INDEX_OPTION_INDEX_GAP 0x0101 #define LRMSUBREAD_INDEX_OPTION_INDEX_PADDING 0x0102 #define LRMMULTI_THREAD_OUTPUT_ITEMS (4096 * 3/5 *3) #define LRMIS_REVERSED_HALVES 1 #define LRMIS_PAIRED_MATCH 2 #define LRMIS_NEGATIVE_STRAND 4 #define LRMIS_BREAKEVEN_READ 8 #define LRMIS_IN_KNOWN_EXONS 16 #define LRM_EVENT_IS_GT_AT_DONOR 1 #define LRM_EVENT_IS_ANNOTATED 2 #define LRMEVENT_TYPE_JUNCTION 30 #define LRMEVENT_TYPE_INDEL 20 #define LRMBAM_COMPRESS_LEVEL Z_BEST_SPEED #define LRMGZIP_WINDOW_BITS -15 #define LRMSUBINDEX_VER2 201 #define LRMRUNNING_STEP_VOTING 10 #define LRMthread_lock_t pthread_mutex_t #define LRMthread_lock pthread_mutex_lock #define LRMthread_lockrelease pthread_mutex_unlock #define LRMthread_lockinit(a) pthread_mutex_init(a, NULL) #define LRMGENE_VOTE_SPACE (131 - 80 ) // 131 gave the bast recall & accuracy & speed #define LRMGENE_VOTE_TABLE_SIZE 64973 #define LRMMAX_LOCATIONS_PER_READ_HARDLIMIT (LRMGENE_VOTE_SPACE * LRMGENE_VOTE_TABLE_SIZE) typedef char LRMgene_quality_score_t; typedef unsigned int LRMgehash_key_t; typedef unsigned int LRMgehash_data_t; typedef struct { unsigned short items[LRMGENE_VOTE_TABLE_SIZE]; unsigned int pos [LRMGENE_VOTE_TABLE_SIZE][LRMGENE_VOTE_SPACE]; unsigned short votes [LRMGENE_VOTE_TABLE_SIZE][LRMGENE_VOTE_SPACE]; short masks [LRMGENE_VOTE_TABLE_SIZE][LRMGENE_VOTE_SPACE]; short indel_recorder [LRMGENE_VOTE_TABLE_SIZE][LRMGENE_VOTE_SPACE][LRMMAX_INDEL_SECTIONS*3]; char current_indel_cursor[LRMGENE_VOTE_TABLE_SIZE][LRMGENE_VOTE_SPACE]; unsigned short toli[LRMGENE_VOTE_TABLE_SIZE][LRMGENE_VOTE_SPACE]; unsigned int coverage_start [LRMGENE_VOTE_TABLE_SIZE][LRMGENE_VOTE_SPACE]; unsigned int coverage_end [LRMGENE_VOTE_TABLE_SIZE][LRMGENE_VOTE_SPACE]; } LRMgene_vote_t ; #define LRMMAX_SORTED_WINDOW_NO_HARD_LIMIT 3 typedef struct { unsigned int read_no_in_chunk; unsigned int read_length; char read_name[LRMMAX_READ_NAME_LEN]; char read_text[LRMMAX_READ_LENGTH]; char qual_text[LRMMAX_READ_LENGTH]; int is_reversed; unsigned int extracted_subreads; LRMgene_vote_t vote_table; unsigned int sorting_total_votes; unsigned int sorting_vote_locations[LRMMAX_LOCATIONS_PER_READ_HARDLIMIT]; unsigned int sorting_subread_nos[LRMMAX_LOCATIONS_PER_READ_HARDLIMIT]; unsigned short sorting_subread_votes[LRMMAX_LOCATIONS_PER_READ_HARDLIMIT]; unsigned int sorting_is_negative_strand[LRMMAX_LOCATIONS_PER_READ_HARDLIMIT]; unsigned int sorted_window_vote_start[LRMMAX_SORTED_WINDOW_NO_HARD_LIMIT]; unsigned int sorted_window_vote_stop[LRMMAX_SORTED_WINDOW_NO_HARD_LIMIT]; unsigned int sorted_window_total_votes[LRMMAX_SORTED_WINDOW_NO_HARD_LIMIT]; unsigned int sorted_window_is_negative_strand[LRMMAX_SORTED_WINDOW_NO_HARD_LIMIT]; unsigned int chain_tosmall_items; unsigned int chain_tolarge_items; unsigned int chain_total_items; unsigned int chain_cov_start[LRMMAX_SUBREAD_PER_READ_HARDLIMIT]; unsigned int chain_cov_end[LRMMAX_SUBREAD_PER_READ_HARDLIMIT]; unsigned int chain_chro_at_cov_start[LRMMAX_SUBREAD_PER_READ_HARDLIMIT]; ArrayList * chain_used_gaps; double subread_extract_gap; int subread_total; int total_matched_bases ; int current_alignment_no; unsigned int final_mapping_location; } LRMread_iteration_context_t; typedef struct { char filename [LRMMAX_FILENAME_LENGTH]; int file_type ; void * input_fp; // can be system (FILE * sam or fastq or fasta), (seekable_zfile_t *) unsigned long long read_chunk_start; } LRMgene_input_t; typedef struct{ unsigned int memory_block_size; unsigned int start_base_offset; unsigned int start_point; unsigned int length; unsigned char * values; unsigned int values_bytes; } LRMgene_value_index_t; struct LRMgehash_bucket { int current_items; int space_size; short * new_item_keys; LRMgehash_data_t * item_values; }; typedef struct { int version_number; unsigned long long int current_items; int buckets_number; char is_small_table; struct LRMgehash_bucket * buckets; int index_gap; int padding; } LRMgehash_t; typedef struct{ int thread_id; // SAM BAM output int output_buffer_item, output_buffer_pointer; LRMthread_lock_t output_lock; char * out_SAMBAM_buffer; int out_buff_used; int out_buff_capacity; z_stream bam_file_output_stream; // Chro events HashTable * events_fresh; // only used in voting step ; event_name "I:chrX:99881122:-5" -> supporting_reads unsigned int * events_realignment_counts; // event_ids -> n_supp unsigned int * dynamic_programming_score_buffer; char * dynamic_programming_movement_buffer; char * dynamic_programming_indel_movement_buf; char * final_cigar_string; unsigned int dynamic_programming_indel_movement_start; unsigned int mapped_reads; } LRMthread_context_t; typedef struct{ unsigned int small_side, large_side; unsigned int supporting_reads; short masks; char event_type; char indel_length; // >0: del; <0:ins } LRMevent_t; typedef struct { char user_command_line[10000]; char input_file_name [LRMMAX_FILENAME_LENGTH]; char output_file_name [LRMMAX_FILENAME_LENGTH]; char index_prefix [LRMMAX_FILENAME_LENGTH]; int threads; int is_SAM_output; int max_dynamic_indel_length; int max_read_indel_length; int multi_best_read_alignments; int is_Phred_64; int max_junction_distance; int max_mismatched_bases_in_subread; int min_voting_number; int min_matched_bases_in_alignment; int result_merge_tolerance; int show_read_validation; int max_cigars_in_read; int is_RNAseq_mode; // RUNNING STATUS double start_running_time; pthread_t running_threads[LRMMAX_THREADS]; LRMthread_context_t thread_contexts[LRMMAX_THREADS]; LRMthread_lock_t input_lock; LRMgene_input_t input_file; LRMgehash_t current_index; int current_index_padding; LRMgene_value_index_t current_base_index; seekable_position_t last_saved_zlib_pos; unsigned long long int last_saved_raw_pos; int input_exhausted; int processed_reads_in_chunk; int all_processed_reads; // Mapping results unsigned int mapped_reads; // Output char bam_file_tail_binary[200]; int bam_file_tail_length; int sam_bam_file_header_written; FILE * sam_bam_file; ArrayList * chromosome_size_list; HashTable * chromosome_size_table; LRMthread_lock_t sam_bam_file_lock; HashTable * sam_bam_chromosome_table; ArrayList * sam_bam_chromosome_list; // Chro Events LRMthread_lock_t event_space_lock; HashTable * events_realignment; // only used in realignment step ; entry_position -> [max_N_size, N, event_id1, event_id2, ..., event_idN] LRMevent_t * event_space; unsigned int event_space_size; unsigned int event_number; // Dynamic Programming int dynamic_programming_score_match, dynamic_programming_score_mismatch, dynamic_programming_score_create_gap, dynamic_programming_score_extend_gap; } LRMcontext_t; #ifdef MAKE_STANDALONE #define LRMprintf printf #define LRMputs puts #else #define LRMprintf Rprintf #define LRMputs(x) Rprintf("%s\n",(x)) #endif #define SEEKZLIBprintf LRMprintf #define abs(a) ((a)>=0?(a):-(a)) #define max(a,b) ((a)<(b)?(b):(a)) #define min(a,b) ((a)>(b)?(b):(a)) #define LRMint2base(c) (1413695297 >> (8*(c))&0xff) #define LRMbase2int(c) ((c)<'G'?((c)=='A'?0:2):((c)=='G'?1:3)) #define LRMpthread_create pthread_create #define LRMpthread_join pthread_join int LRMshow_conf(LRMcontext_t* context); int LRMrun_task(LRMcontext_t* context); int LRMfinalise(LRMcontext_t* context); int LRMdestroy_context(LRMcontext_t* context); int LRMprint_mapping_summary(LRMcontext_t* context); int LRMstart_thread(LRMcontext_t * context, int task ); int LRMwait_threads( LRMcontext_t * context ); int LRMmerge_threads( LRMcontext_t * context , int step); void LRMset_default_values_context(LRMcontext_t * context); int LRMinput_has_finished( LRMcontext_t * context ); int LRMload_index(LRMcontext_t* context); int LRMsave_input_pos(LRMcontext_t* context); int LRMrewind_input_pos(LRMcontext_t* context); int LRMmap_chunk_reads(LRMcontext_t* context); int LRMrealign_write_chunk_reads(LRMcontext_t* context); int LRMfinalise_chunk_reads(LRMcontext_t* context); int LRMiterate_reads( LRMcontext_t * context, int task ); double LRMmiltime(); void * LRMmap_chunk_reads_run(void * thread_args); void * LRMrealign_write_chunk_reads_run(void * thread_args); int LRMchunk_read_iteration(LRMcontext_t * context, int thread_id, int task); int LRMwrite_chunk_add_buffered_output(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context, int flags, char * chro_name, unsigned int chro_pos, int map_quality, char * cigar, int mis_matched); //void LRMwrite_chunk_destroy_write_buffer(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iter_context, LRMwritting_buffer_t * buf); int LRMFIXLENstrcmp(char * fixed_len, char * rname); #endif Rsubread/src/LRMfile-io.c0000644000175200017520000005101514136047615016206 0ustar00biocbuildbiocbuild#include #include #include #include #include #include #include #ifndef __MINGW32__ #include #endif #include #include #include #include #include #include "LRMfile-io.h" #include "LRMsorted-hashtable.h" int LRMgenekey2int(char key []){ int i; int ret; ret = 0; for (i=0; i<16; i++) ret |= (LRMbase2int(key[i]))<<(2*(15-i)); return ret; } int LRMgeinput_open(const char * filename, LRMgene_input_t * input){ int ret = 0; if(strlen(filename)>LRMMAX_FILENAME_LENGTH-2) return 1; strcpy(input->filename, filename); FILE * TMP_FP = fopen(filename, "rb"); if(TMP_FP == NULL) return 1; int id1, id2; id1 = fgetc(TMP_FP); id2 = fgetc(TMP_FP); if(id1 == 31 && id2 == 139) { fclose(TMP_FP); input->input_fp = malloc(sizeof(seekable_zfile_t)); input->file_type = LRMGENE_INPUT_GZIP_FASTQ; ret = LRMseekgz_open(filename, input->input_fp); }else{ input->file_type = LRMGENE_INPUT_FASTQ; input->input_fp = TMP_FP; fseek(input->input_fp, 0, SEEK_SET); } return ret; } void LRMgeinput_close(LRMgene_input_t * input){ if(input -> file_type == LRMGENE_INPUT_GZIP_FASTQ) LRMseekgz_close((seekable_zfile_t * ) input->input_fp); else fclose((FILE*)input->input_fp); } char * LRM__converting_char_table = "NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTNGNNNCNNNNNNNNNNNNAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTNGNNNCNNNNNNNNNNNNAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN "; void LRMreverse_read(char * InBuff, int read_len){ int i; for (i=0; i file_type == LRMGENE_INPUT_GZIP_FASTQ){ return LRMseekgz_next_char((seekable_zfile_t*)input -> input_fp); }else{ return fgetc((FILE*)input -> input_fp); } } int LRMgeinput_readline(LRMgene_input_t * input, int buf_len, char * linebuffer){ int ret =0; while(1){ char ch = LRMgeinput_getc(input); if (ch == '\n' || ch == EOF) break; if(ret < buf_len-1) linebuffer[ret++] = ch; } linebuffer[ret]=0; return ret; } #define SKIP_LINE { nch=' '; while(nch != EOF && nch != '\n') nch = LRMgeinput_getc(input); } int LRMgeinput_next_read(LRMgene_input_t * input, char * read_name, char * read_string, char * quality_string){ char nch; int ret; //READ NAME if (read_name == NULL){ SKIP_LINE; if(nch == EOF) return -1; } else { int ATnch = LRMgeinput_getc(input); assert(ATnch == '@' || ATnch <0); if(ATnch < 0) return -1; int retv = LRMgeinput_readline(input, LRMMAX_READ_NAME_LEN, read_name); if(retv<=0) return -1; int cursor = 1; while(read_name[cursor]) { if(read_name[cursor] == ' ' || read_name[cursor] == '\t') { read_name [cursor] = 0; break; } cursor++; } } // READ LINE ret = LRMgeinput_readline(input, LRMMAX_READ_LENGTH, read_string); // SKIP "+" do{ nch = LRMgeinput_getc(input); } while( nch == '\n' ); SKIP_LINE; // QUAL LINE if (quality_string) LRMgeinput_readline(input, LRMMAX_READ_LENGTH, quality_string); else SKIP_LINE; return ret; } int LRMfetch_next_read(LRMcontext_t * context, LRMthread_context_t * thread_context, unsigned int *read_len, char * read_name, char * read_text, char * qual_text, unsigned int * read_no_in_chunk){ int this_number = -1; int this_rlen = 0; LRMthread_lock(&context -> input_lock); this_rlen = LRMgeinput_next_read(&context -> input_file, read_name, read_text, qual_text); if(this_rlen > 0 ){ this_number = context -> processed_reads_in_chunk; context -> processed_reads_in_chunk ++; }else context -> input_exhausted = 1; LRMthread_lockrelease(&context -> input_lock); if(this_rlen && this_number>=0) { *read_no_in_chunk = this_number; *read_len = this_rlen; return 0; }else{ *read_no_in_chunk = -1; return 1; } } void LRMreverse_quality(char * InBuff, int read_len){ int i; if(!InBuff) return; if(!InBuff[0]) return; for (i=0; i= context -> max_cigars_in_read){ int_opt = 4; //'S' tmp_int = read_text_len - reported_read_len; cigar_int[num_opt ++] = (tmp_int << 4) | int_opt; LRMprintf("CIGAR_TRIMMED : %d bases\n", tmp_int); break; } } } return num_opt; } int LRMreg2bin(int beg, int end){ --end; if (beg>>14 == end>>14) return ((1<<15)-1)/7 + (beg>>14); if (beg>>17 == end>>17) return ((1<<12)-1)/7 + (beg>>17); if (beg>>20 == end>>20) return ((1<<9)-1)/7 + (beg>>20); if (beg>>23 == end>>23) return ((1<<6)-1)/7 + (beg>>23); if (beg>>26 == end>>26) return ((1<<3)-1)/7 + (beg>>26); return 0; } int LRMgenerate_bam_record_encode_read_qual(char * bin, char * read, char * qual, int rlen){ int w_ptr = 0, xk1; for(xk1 = 0; xk1 < rlen; xk1++){ int fourbit; for(fourbit=0;fourbit<15;fourbit++) if("=ACMGRSVTWYHKDBN"[fourbit] == read[xk1])break; if( xk1 % 2 == 0){ fourbit = fourbit << 4; bin[w_ptr]=0; } bin[w_ptr] |= fourbit; if(xk1 % 2 == 1)w_ptr++; } if(rlen %2) w_ptr++; for(xk1=0; xk1sam_bam_chromosome_table, chro_name) - NULL; chro_number -=1; memcpy(target_ptr+4, &chro_number, 4); memcpy(target_ptr+8, &chro_pos, 4); int name_len = strlen(iteration_context -> read_name)+1; memcpy(target_ptr+20, &iteration_context->read_length, 4); int next_non = -1; memcpy(target_ptr+24, &next_non, 4); memcpy(target_ptr+28, &next_non, 4); memset(target_ptr+32, 0, 4); //TLEN memcpy(target_ptr+36, iteration_context->read_name, name_len); int bin_ptr = 36+name_len; int cigar_opts = LRMgenerate_bam_record_encode_cigar(context, (int *)(target_ptr + bin_ptr), cigar, &mapped_length, iteration_context->read_length); int flag_nc = flags << 16 | cigar_opts; memcpy(target_ptr+16, &flag_nc, 4); bin_ptr += cigar_opts*4; int bin_mq_nl = (LRMreg2bin(chro_pos, chro_pos+mapped_length)<<16) | map_quality << 8 | name_len; memcpy(target_ptr+12, &bin_mq_nl, 4); //LRMprintf("READ TEXT %s : len=%d \n", iteration_context->read_name, iteration_context->read_length); bin_ptr += LRMgenerate_bam_record_encode_read_qual(target_ptr + bin_ptr, iteration_context->read_text, iteration_context -> qual_text, iteration_context->read_length); memcpy(target_ptr+bin_ptr, "NM",2); target_ptr[bin_ptr+2]='C'; target_ptr[bin_ptr+3]=mis_matched; memcpy(target_ptr, &bin_ptr, 4); // block len not include itself. bin_ptr += 4; return bin_ptr; } unsigned int LRM_CRC32(char * dat, int len){ unsigned int crc0 = crc32(0, NULL, 0); unsigned int ret = crc32(crc0, (unsigned char *)dat, len); return ret; } int LRMwrite_chunk_compress_bam_block(LRMcontext_t * context, LRMthread_context_t * thread_context, char * bin_buf, char * bam_buf, int bin_len){ char * compressed_buff = bam_buf + 18; int compressed_size ; unsigned int CRC32; thread_context -> bam_file_output_stream.avail_out = 66600; thread_context -> bam_file_output_stream.avail_in = bin_len; CRC32 = LRM_CRC32(bin_buf , bin_len); int Z_DEFAULT_MEM_LEVEL = 8; thread_context -> bam_file_output_stream.zalloc = Z_NULL; thread_context -> bam_file_output_stream.zfree = Z_NULL; thread_context -> bam_file_output_stream.opaque = Z_NULL; deflateInit2(&thread_context -> bam_file_output_stream, LRMBAM_COMPRESS_LEVEL, Z_DEFLATED, LRMGZIP_WINDOW_BITS, Z_DEFAULT_MEM_LEVEL, Z_DEFAULT_STRATEGY); thread_context -> bam_file_output_stream.next_in = (unsigned char*) bin_buf; thread_context -> bam_file_output_stream.next_out = (unsigned char*) compressed_buff; deflate(&thread_context -> bam_file_output_stream, Z_FINISH); deflateEnd(&thread_context -> bam_file_output_stream); compressed_size = 66600 -thread_context -> bam_file_output_stream.avail_out; //LRMprintf("COMPRESS: %d -> %d\n", bin_len, compressed_size); bam_buf[0]=31; bam_buf[1]=(char)139; bam_buf[2]=8; bam_buf[3]=4; memset(bam_buf+4, 0, 5); bam_buf[9] = 0xff; // OS int tmpi = 6; memcpy(bam_buf+10, &tmpi, 2); //XLSN bam_buf[12]=66; // SI1 bam_buf[13]=67; // SI2 tmpi = 2; memcpy(bam_buf+14, &tmpi, 2); //BSIZE tmpi = compressed_size + 19 + 6; memcpy(bam_buf+16, &tmpi, 2); //BSIZE memcpy(bam_buf+18+compressed_size, &CRC32, 4); memcpy(bam_buf+18+compressed_size+4, &bin_len, 4); return compressed_size + 26; } int LRMhash_strcmp(const void * s1, const void * s2){ return strcmp(s1, s2); } srUInt_64 LRMhash_strhash(const void * sv){ unsigned char *s = (unsigned char *)sv; unsigned long ret = 0; while(*s){ ret ^= (ret << 6); ret ^= *s; s++; } return ret; } int LRMreadline(FILE * fp, char * buf, int buflen){ int cr = 0; while(!feof(fp)){ char c = fgetc(fp); if(c == '\r'){ continue; }else if(c == '\n'){ break; }else if(cr < buflen-1) buf[cr++]=c; } buf[cr]=0; return cr; } int LRMload_offsets(LRMcontext_t * context){ char fn[LRMMAX_FILENAME_LENGTH+20]; FILE * fp; int padding = 0; unsigned int last_end = 0; LRMgehash_load_option(context -> index_prefix, LRMSUBREAD_INDEX_OPTION_INDEX_PADDING , &padding); assert(padding>16); sprintf(fn, "%s.reads", context->index_prefix); fp = fopen(fn, "r"); if(!fp){ LRMprintf("file not found :%s\n", fn); return 1; } context -> current_index_padding = padding; int chromosome_no = 0; while (!feof(fp)) { int i=0, step = 0, j=0; unsigned int this_offset =0; LRMreadline(fp, fn, LRMMAX_FILENAME_LENGTH-1); if (strlen(fn)<2)continue; char * chro_name = malloc(LRMMAX_CHROMOSOME_NAME_LEN); while (fn[i]) { if (fn[i] == '\t') { fn[i]=0; this_offset = (unsigned int)atoll(fn); step = 1; }else if (step) { if(jsam_bam_chromosome_table, chro_name, NULL + chromosome_no + 1); LRMArrayListPush(context->sam_bam_chromosome_list, chro_name); LRMHashTablePut(context->chromosome_size_table, chro_name, NULL + (this_offset - last_end + 16 - context->current_index_padding * 2)); LRMArrayListPush(context->chromosome_size_list, NULL + this_offset); chromosome_no++; last_end = this_offset; } fclose(fp); return 0; } #define LRMcheck_resize_buff if(new_need >= thread_context -> out_buff_capacity){\ thread_context -> out_buff_capacity = max(thread_context -> out_buff_capacity*2, new_need );\ thread_context -> out_SAMBAM_buffer=realloc(thread_context -> out_SAMBAM_buffer, thread_context -> out_buff_capacity);\ }\ #define LRMBAM_COMPRESS_BLOCK 63000 #define LRMBAM_COMPRESS_TRIGGER 53000 void LRMsambam_write_header(LRMcontext_t * context, LRMthread_context_t * thread_context){ if(context -> sam_bam_file_header_written) return; thread_context -> out_SAMBAM_buffer = malloc(3000000); thread_context -> out_buff_capacity = 3000000; thread_context -> out_buff_used = 0; int chro_no; if(!context -> is_SAM_output){ memcpy(thread_context -> out_SAMBAM_buffer,"BAM\1",4); thread_context -> out_buff_used = 8; } for(chro_no = -1; chro_no < context -> sam_bam_chromosome_list->numOfElements + 2; chro_no++){ char * header_line = malloc(10100); int wrlen = 0; if(chro_no >=0 && chro_no < context -> sam_bam_chromosome_list->numOfElements){ char * chro_name = LRMArrayListGet(context -> sam_bam_chromosome_list, chro_no); int chro_length = LRMHashTableGet(context -> chromosome_size_table, chro_name) - NULL; wrlen = sprintf(header_line, "@SQ\tSN:%s\tLN:%d\n",chro_name,chro_length); }else if(chro_no == -1){ wrlen = sprintf(header_line, "@HD\tVN:1.0\tSO:unsorted\n"); }else if(chro_no == context -> sam_bam_chromosome_list->numOfElements ){ wrlen = sprintf(header_line, "@PG\tID:subread-long-read-mapping\tPN:subread-long-read-mapping\tCL:%s\n", context -> user_command_line); } if(context -> is_SAM_output){ fwrite( header_line, 1, wrlen, context -> sam_bam_file); }else{ int new_need = thread_context -> out_buff_used + wrlen +1; LRMcheck_resize_buff; memcpy(thread_context -> out_SAMBAM_buffer + thread_context -> out_buff_used,header_line,wrlen); thread_context -> out_buff_used += wrlen; } free(header_line); } int new_need = thread_context -> out_buff_used + 10; LRMcheck_resize_buff; int BAM_text_len = thread_context -> out_buff_used-8; memcpy(thread_context -> out_SAMBAM_buffer+4, &BAM_text_len, 4); memcpy(thread_context -> out_SAMBAM_buffer + thread_context -> out_buff_used, &context ->sam_bam_chromosome_list->numOfElements , 4); thread_context -> out_buff_used +=4; for(chro_no = 0; chro_no < context -> sam_bam_chromosome_list->numOfElements ; chro_no++){ char * chro_name = LRMArrayListGet(context -> sam_bam_chromosome_list, chro_no); int chro_namelen = strlen(chro_name)+1; int new_need = thread_context -> out_buff_used + chro_namelen + 9; LRMcheck_resize_buff; memcpy(thread_context -> out_SAMBAM_buffer+thread_context -> out_buff_used, &chro_namelen, 4); thread_context -> out_buff_used +=4; memcpy(thread_context -> out_SAMBAM_buffer+thread_context -> out_buff_used, chro_name, chro_namelen); thread_context -> out_buff_used += chro_namelen; int chro_length = LRMHashTableGet(context -> chromosome_size_table, chro_name) - NULL; memcpy(thread_context -> out_SAMBAM_buffer+thread_context -> out_buff_used, &chro_length, 4); thread_context -> out_buff_used +=4; } if(!context -> is_SAM_output){ LRMwrite_chunk_check_buffer_write(context, thread_context, 1); } context -> sam_bam_file_header_written = 1; free(thread_context -> out_SAMBAM_buffer); } void LRMbam_generate_tail_binary(LRMcontext_t * context, LRMthread_context_t * thread_context){ if(context -> bam_file_tail_length>0)return; context -> bam_file_tail_length = LRMwrite_chunk_compress_bam_block(context, thread_context, "", context -> bam_file_tail_binary , 0); } int LRMwrite_chunk_add_buffered_output(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context, int flags, char * chro_name, unsigned int chro_pos, int map_quality, char * cigar, int mis_matched){ char * target_ptr; int cigar_len = strlen(cigar); int rname_len = strlen(iteration_context->read_name); int read_binlen, actural_target_len; read_binlen = cigar_len+rname_len+2.5*iteration_context->read_length+500; if(read_binlen + thread_context->out_buff_used >= thread_context -> out_buff_capacity){ thread_context -> out_buff_capacity = max( thread_context -> out_buff_capacity*1.3, read_binlen); thread_context -> out_SAMBAM_buffer=realloc(thread_context -> out_SAMBAM_buffer, thread_context -> out_buff_capacity); } target_ptr = thread_context -> out_SAMBAM_buffer + thread_context->out_buff_used; if(context -> is_Phred_64) LRMquality_64_to_33(iteration_context->qual_text) ; if(context->is_SAM_output){ actural_target_len = sprintf(target_ptr,"%s\t%d\t%s\t%u\t%d\t%s\t*\t0\t0\t%s\t%s\tNM:%d\n", iteration_context -> read_name, flags, chro_name, chro_pos + 1, map_quality, cigar, iteration_context->read_text, iteration_context->qual_text, mis_matched); }else{ actural_target_len = LRMgenerate_bam_record(context, thread_context, iteration_context, target_ptr, flags, chro_pos, chro_name, map_quality, cigar, mis_matched); } thread_context -> out_buff_used+=actural_target_len; LRMwrite_chunk_check_buffer_write(context, thread_context, 0); return 0; } int LRMwrite_chunk_check_buffer_write(LRMcontext_t * context, LRMthread_context_t * thread_context, int force_write){ if(force_write || thread_context -> out_buff_used > LRMBAM_COMPRESS_TRIGGER){ if(!context->is_SAM_output){ int write_cursor = 0; int compressed_cursor = 0; for(write_cursor = 0; write_cursor < thread_context -> out_buff_used; write_cursor += LRMBAM_COMPRESS_BLOCK){ char compressed_data [66666]; int bin_len = min(LRMBAM_COMPRESS_BLOCK,thread_context -> out_buff_used - write_cursor); int compressed_len = LRMwrite_chunk_compress_bam_block(context, thread_context, thread_context -> out_SAMBAM_buffer + write_cursor, compressed_data, bin_len); if(compressed_len > bin_len){ // very unlikely int new_needed_size = thread_context -> out_buff_used + (compressed_len - bin_len); if(new_needed_size > thread_context -> out_buff_capacity){ thread_context -> out_buff_capacity = new_needed_size; thread_context -> out_SAMBAM_buffer=realloc(thread_context -> out_SAMBAM_buffer, thread_context -> out_buff_capacity); } int x1; for(x1 = thread_context -> out_buff_used - 1; x1 >= write_cursor + bin_len; x1 --) thread_context -> out_SAMBAM_buffer[ x1 + (compressed_len - bin_len) ] = thread_context -> out_SAMBAM_buffer[ x1 ]; } memcpy(thread_context -> out_SAMBAM_buffer + compressed_cursor, compressed_data, compressed_len); compressed_cursor += compressed_len; } thread_context -> out_buff_used = compressed_cursor; } LRMthread_lock(&context->sam_bam_file_lock); fwrite(thread_context -> out_SAMBAM_buffer,1, thread_context -> out_buff_used, context -> sam_bam_file); LRMthread_lockrelease(&context->sam_bam_file_lock); thread_context -> out_buff_used = 0; } return 0; } void LRMpos2txt(LRMcontext_t * context, unsigned int linear, char * txt){ char * chro_name = NULL; int pos; int retv = LRMlocate_gene_position(context, linear, &chro_name, &pos); if(chro_name && !retv) sprintf(txt, "%s:%d", chro_name, pos+1); else strcpy(txt, "*"); } int LRMlocate_gene_position(LRMcontext_t * context, unsigned int linear, char ** chro_name, int * pos) { int n = 0; int total_offsets = context -> chromosome_size_list -> numOfElements; int jump_ns = total_offsets/4; //LRMprintf("LINEAR = %u\n", linear); (*chro_name)=NULL; (*pos) = -1; while (jump_ns > 5) { while(n+jump_ns < total_offsets && LRMArrayListGet(context->chromosome_size_list, n + jump_ns) - NULL <= linear) n+=jump_ns; jump_ns /=4; } for (; n < total_offsets; n++) { //LRMprintf("TESTING %u\n",(LRMArrayListGet(context->chromosome_size_list, n) - NULL) ); if ( (LRMArrayListGet(context->chromosome_size_list, n) - NULL) > linear) { *pos = linear; if(n>0)(*pos) -= (LRMArrayListGet(context->chromosome_size_list, n-1)-NULL); if( (*pos) < context -> current_index_padding ) return 1; else (*pos) -= context -> current_index_padding; (*chro_name) = LRMArrayListGet(context -> sam_bam_chromosome_list, n); return 0; } } return -1; } int LRMlocate_chro_length(LRMcontext_t * context, unsigned int linear, char ** chro_name, long long * end_pos){ int n = 0; int total_offsets = context -> chromosome_size_list -> numOfElements; int jump_ns = total_offsets/4; while (jump_ns > 5) { while(n+jump_ns < total_offsets && LRMArrayListGet(context->chromosome_size_list, n + jump_ns) - NULL <= linear) n+=jump_ns; jump_ns /=4; } for (; n < total_offsets; n++) { //LRMprintf("TESTING %u\n",(LRMArrayListGet(context->chromosome_size_list, n) - NULL) ); if ( (LRMArrayListGet(context->chromosome_size_list, n) - NULL) > linear) { (* chro_name) = LRMArrayListGet(context -> sam_bam_chromosome_list, n); (* end_pos) = LRMArrayListGet(context->chromosome_size_list, n)-NULL; if((* end_pos) > context -> current_index_padding) (* end_pos) -= context -> current_index_padding ; return 0; } } return -1; } Rsubread/src/LRMfile-io.h0000644000175200017520000000323514136047615016214 0ustar00biocbuildbiocbuild#ifndef __LRMFILEIO_H_ #define __LRMFILEIO_H_ #define LRMGENE_INPUT_FASTQ 1 #define LRMGENE_INPUT_GZIP_FASTQ 51 #define LRMMAX_LINE_LENGTH (LRMMAX_READ_LENGTH + 10) #include "LRMconfig.h" int LRMhash_strcmp(const void * s1, const void * s2); srUInt_64 LRMhash_strhash(const void * sv); int LRMgenekey2int(char key []); int LRMgeinput_open(const char * filename, LRMgene_input_t * input); void LRMsambam_write_header(LRMcontext_t * context, LRMthread_context_t * thread_context); void LRMbam_generate_tail_binary(LRMcontext_t * context, LRMthread_context_t * thread_context); int LRMwrite_chunk_check_buffer_write(LRMcontext_t * context, LRMthread_context_t * thread_context, int force_write); // return 0 if successful int LRMfetch_next_read(LRMcontext_t * context, LRMthread_context_t * thread_context, unsigned int *read_len, char * read_name, char * read_text, char * qual_text, unsigned int * read_no_in_chunk); // Return the length of this read or -1 if EOF. int LRMgeinput_next_read(LRMgene_input_t * input, char * read_name, char * read_string, char * quality_string); void LRMgeinput_close(LRMgene_input_t * input); // returns read length int LRMgeinput_readline(LRMgene_input_t * input, int buf_len, char * linebuffer) ; void LRMreverse_read(char * ReadString, int Length); void LRMreverse_quality(char * QualtyString, int Length); int LRMload_offsets(LRMcontext_t * context); int LRMlocate_gene_position(LRMcontext_t * context, unsigned int linear, char ** chro_name, int * pos); void LRMpos2txt(LRMcontext_t * context, unsigned int linear, char * txt); int LRMlocate_chro_length(LRMcontext_t * context, unsigned int linear, char ** chro_name, long long * chro_len); #endif Rsubread/src/LRMhashtable.c0000644000175200017520000007253414136047615016626 0ustar00biocbuildbiocbuild/*--------------------------------------------------------------------------*\ * -----===== HashTable =====----- * * Author: Keith Pomakis (pomakis@pobox.com) * Date: August, 1998 * Released to the public domain. * *-------------------------------------------------------------------------- * $Id: LRMhashtable.c,v 1.5 2019/08/28 22:31:15 cvs Exp $ \*--------------------------------------------------------------------------*/ #include #include #include #include #include #include "LRMconfig.h" #include "LRMhashtable.h" static int LRMpointercmp(const void *pointer1, const void *pointer2); static srUInt_64 LRMpointerHashFunction(const void *pointer); static int LRMisProbablePrime(srInt_64 number); static srInt_64 LRMcalculateIdealNumOfBuckets(HashTable *hashTable); ArrayList * LRMArrayListCreate(int init_capacity){ ArrayList * ret = malloc(sizeof(ArrayList)); memset(ret,0,sizeof(ArrayList)); ret -> capacityOfElements = init_capacity; ret -> elementList = malloc(sizeof(void *)*init_capacity); return ret; } void LRMArrayListDestroy(ArrayList * list){ srInt_64 x1; if(list -> elemDeallocator) for(x1 = 0;x1 < list->numOfElements; x1++) list -> elemDeallocator(list -> elementList[x1]); free(list -> elementList); free(list); } void * LRMArrayListGet(ArrayList * list, srInt_64 n){ if(n<0 || n >= list->numOfElements)return NULL; return list -> elementList[n]; } int LRMArrayListPush(ArrayList * list, void * new_elem){ if(list -> capacityOfElements <= list->numOfElements){ list -> capacityOfElements *=1.3; list -> elementList=realloc(list -> elementList, sizeof(void *)*list -> capacityOfElements); } list->elementList[list->numOfElements++] = new_elem; return list->numOfElements; } void LRMArrayListSetDeallocationFunction(ArrayList * list, void (*elem_deallocator)(void *elem)){ list -> elemDeallocator = elem_deallocator; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableCreate() - creates a new HashTable * DESCRIPTION: * Creates a new HashTable. When finished with this HashTable, it * should be explicitly destroyed by calling the HashTableDestroy() * function. * EFFICIENCY: * O(1) * ARGUMENTS: * numOfBuckets - the number of buckets to start the HashTable out with. * Must be greater than zero, and should be prime. * Ideally, the number of buckets should between 1/5 * and 1 times the expected number of elements in the * HashTable. Values much more or less than this will * result in wasted memory or decreased performance * respectively. The number of buckets in a HashTable * can be re-calculated to an appropriate number by * calling the HashTableRehash() function once the * HashTable has been populated. The number of buckets * in a HashTable may also be re-calculated * automatically if the ratio of elements to buckets * passes the thresholds set by HashTableSetIdealRatio(). * RETURNS: * HashTable - a new Hashtable, or NULL on error \*--------------------------------------------------------------------------*/ HashTable *LRMHashTableCreate(srInt_64 numOfBuckets) { HashTable *hashTable; int i; assert(numOfBuckets > 0); hashTable = (HashTable *) malloc(sizeof(HashTable)); if (hashTable == NULL) return NULL; hashTable->appendix1=NULL; hashTable->appendix2=NULL; hashTable->appendix3=NULL; hashTable->counter1=0; hashTable->counter2=0; hashTable->counter3=0; hashTable->bucketArray = (KeyValuePair **) malloc(numOfBuckets * sizeof(KeyValuePair *)); if (hashTable->bucketArray == NULL) { free(hashTable); return NULL; } hashTable->numOfBuckets = numOfBuckets; hashTable->numOfElements = 0; for (i=0; ibucketArray[i] = NULL; hashTable->idealRatio = 3.0; hashTable->lowerRehashThreshold = 0.0; hashTable->upperRehashThreshold = 15.0; hashTable->keycmp = LRMpointercmp; hashTable->valuecmp = LRMpointercmp; hashTable->hashFunction = LRMpointerHashFunction; hashTable->keyDeallocator = NULL; hashTable->valueDeallocator = NULL; return hashTable; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableDestroy() - destroys an existing HashTable * DESCRIPTION: * Destroys an existing HashTable. * EFFICIENCY: * O(n) * ARGUMENTS: * hashTable - the HashTable to destroy * RETURNS: * \*--------------------------------------------------------------------------*/ void LRMHashTableDestroy(HashTable *hashTable) { int i; for (i=0; inumOfBuckets; i++) { KeyValuePair *pair = hashTable->bucketArray[i]; while (pair != NULL) { KeyValuePair *nextPair = pair->next; if (hashTable->keyDeallocator != NULL) hashTable->keyDeallocator((void *) pair->key); if (hashTable->valueDeallocator != NULL){ // fprintf(stderr,"FREE %p\n", pair->value); hashTable->valueDeallocator(pair->value); } free(pair); pair = nextPair; } } free(hashTable->bucketArray); free(hashTable); } /*--------------------------------------------------------------------------*\ * NAME: * HashTableContainsKey() - checks the existence of a key in a HashTable * DESCRIPTION: * Determines whether or not the specified HashTable contains the * specified key. Uses the comparison function specified by * HashTableSetKeyComparisonFunction(). * EFFICIENCY: * O(1), assuming a good hash function and element-to-bucket ratio * ARGUMENTS: * hashTable - the HashTable to search * key - the key to search for * RETURNS: * bool - whether or not the specified HashTable contains the * specified key. \*--------------------------------------------------------------------------*/ int LRMHashTableContainsKey(const HashTable *hashTable, const void *key) { return (LRMHashTableGet(hashTable, key) != NULL); } /*--------------------------------------------------------------------------*\ * NAME: * HashTableContainsValue() * - checks the existence of a value in a HashTable * DESCRIPTION: * Determines whether or not the specified HashTable contains the * specified value. Unlike HashTableContainsKey(), this function is * not very efficient, since it has to scan linearly looking for a * match. Uses the comparison function specified by * HashTableSetValueComparisonFunction(). * EFFICIENCY: * O(n) * ARGUMENTS: * hashTable - the HashTable to search * value - the value to search for * RETURNS: * bool - whether or not the specified HashTable contains the * specified value. \*--------------------------------------------------------------------------*/ int LRMHashTableContainsValue(const HashTable *hashTable, const void *value) { int i; for (i=0; inumOfBuckets; i++) { KeyValuePair *pair = hashTable->bucketArray[i]; while (pair != NULL) { if (hashTable->valuecmp(value, pair->value) == 0) return 1; pair = pair->next; } } return 0; } /*--------------------------------------------------------------------------*\ * NAME: * HashTablePut() - adds a key/value pair to a HashTable * DESCRIPTION: * Adds the specified key/value pair to the specified HashTable. If * the key already exists in the HashTable (determined by the comparison * function specified by HashTableSetKeyComparisonFunction()), its value * is replaced by the new value. May trigger an auto-rehash (see * HashTableSetIdealRatio()). It is illegal to specify NULL as the * key or value. * EFFICIENCY: * O(1), assuming a good hash function and element-to-bucket ratio * ARGUMENTS: * hashTable - the HashTable to add to * key - the key to add or whose value to replace * value - the value associated with the key * RETURNS: * err - 0 if successful, -1 if an error was encountered \*--------------------------------------------------------------------------*/ int LRMHashTablePut(HashTable *hashTable, const void *key, void *value) { return LRMHashTablePutReplace(hashTable, key, value, 1); } int LRMHashTablePutReplace(HashTable *hashTable, const void *key, void *value, int replace_key) { srInt_64 hashValue; KeyValuePair *pair; assert(key != NULL); assert(value != NULL); hashValue = hashTable->hashFunction(key) % hashTable->numOfBuckets; pair = hashTable->bucketArray[hashValue]; while (pair != NULL && hashTable->keycmp(key, pair->key) != 0) pair = pair->next; if (pair) { if (pair->key != key) { if(replace_key) { if(hashTable->keyDeallocator) hashTable->keyDeallocator((void *) pair->key); pair->key = key; } } if (pair->value != value) { if (hashTable->valueDeallocator != NULL) hashTable->valueDeallocator(pair->value); pair->value = value; } } else { KeyValuePair *newPair = (KeyValuePair *) malloc(sizeof(KeyValuePair)); if (newPair == NULL) { return -1; } else { newPair->key = key; newPair->value = value; newPair->next = hashTable->bucketArray[hashValue]; hashTable->bucketArray[hashValue] = newPair; hashTable->numOfElements++; if (hashTable->upperRehashThreshold > hashTable->idealRatio) { float elementToBucketRatio = (float) hashTable->numOfElements / (float) hashTable->numOfBuckets; if (elementToBucketRatio > hashTable->upperRehashThreshold) LRMHashTableRehash(hashTable, 0); } } } return 0; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableGet() - retrieves the value of a key in a HashTable * DESCRIPTION: * Retrieves the value of the specified key in the specified HashTable. * Uses the comparison function specified by * HashTableSetKeyComparisonFunction(). * EFFICIENCY: * O(1), assuming a good hash function and element-to-bucket ratio * ARGUMENTS: * hashTable - the HashTable to search * key - the key whose value is desired * RETURNS: * void * - the value of the specified key, or NULL if the key * doesn't exist in the HashTable \*--------------------------------------------------------------------------*/ void *LRMHashTableGet(const HashTable *hashTable, const void *key) { srInt_64 hashValue = hashTable->hashFunction(key) % hashTable->numOfBuckets; KeyValuePair *pair = hashTable->bucketArray[hashValue]; while (pair != NULL && hashTable->keycmp(key, pair->key) != 0) pair = pair->next; return (pair == NULL)? NULL : pair->value; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableRemove() - removes a key/value pair from a HashTable * DESCRIPTION: * Removes the key/value pair identified by the specified key from the * specified HashTable if the key exists in the HashTable. May trigger * an auto-rehash (see HashTableSetIdealRatio()). * EFFICIENCY: * O(1), assuming a good hash function and element-to-bucket ratio * ARGUMENTS: * hashTable - the HashTable to remove the key/value pair from * key - the key specifying the key/value pair to be removed * RETURNS: * \*--------------------------------------------------------------------------*/ void LRMHashTableRemove(HashTable *hashTable, const void *key) { srInt_64 hashValue = hashTable->hashFunction(key) % hashTable->numOfBuckets; KeyValuePair *pair = hashTable->bucketArray[hashValue]; KeyValuePair *previousPair = NULL; while (pair != NULL && hashTable->keycmp(key, pair->key) != 0) { previousPair = pair; pair = pair->next; } if (pair != NULL) { if (hashTable->keyDeallocator != NULL) hashTable->keyDeallocator((void *) pair->key); if (hashTable->valueDeallocator != NULL) hashTable->valueDeallocator(pair->value); if (previousPair != NULL) previousPair->next = pair->next; else hashTable->bucketArray[hashValue] = pair->next; free(pair); hashTable->numOfElements--; if (hashTable->lowerRehashThreshold > 0.0) { float elementToBucketRatio = (float) hashTable->numOfElements / (float) hashTable->numOfBuckets; if (elementToBucketRatio < hashTable->lowerRehashThreshold) LRMHashTableRehash(hashTable, 0); } } } /*--------------------------------------------------------------------------*\ * NAME: * HashTableRemoveAll() - removes all key/value pairs from a HashTable * DESCRIPTION: * Removes all key/value pairs from the specified HashTable. May trigger * an auto-rehash (see HashTableSetIdealRatio()). * EFFICIENCY: * O(n) * ARGUMENTS: * hashTable - the HashTable to remove all key/value pairs from * RETURNS: * \*--------------------------------------------------------------------------*/ void LRMHashTableRemoveAll(HashTable *hashTable) { int i; for (i=0; inumOfBuckets; i++) { KeyValuePair *pair = hashTable->bucketArray[i]; while (pair != NULL) { KeyValuePair *nextPair = pair->next; if (hashTable->keyDeallocator != NULL) hashTable->keyDeallocator((void *) pair->key); if (hashTable->valueDeallocator != NULL) hashTable->valueDeallocator(pair->value); free(pair); pair = nextPair; } hashTable->bucketArray[i] = NULL; } hashTable->numOfElements = 0; LRMHashTableRehash(hashTable, 5); } /*--------------------------------------------------------------------------*\ * NAME: * HashTableIsEmpty() - determines if a HashTable is empty * DESCRIPTION: * Determines whether or not the specified HashTable contains any * key/value pairs. * EFFICIENCY: * O(1) * ARGUMENTS: * hashTable - the HashTable to check * RETURNS: * bool - whether or not the specified HashTable contains any * key/value pairs \*--------------------------------------------------------------------------*/ int LRMHashTableIsEmpty(const HashTable *hashTable) { return (hashTable->numOfElements == 0); } /*--------------------------------------------------------------------------*\ * NAME: * HashTableSize() - returns the number of elements in a HashTable * DESCRIPTION: * Returns the number of key/value pairs that are present in the * specified HashTable. * EFFICIENCY: * O(1) * ARGUMENTS: * hashTable - the HashTable whose size is requested * RETURNS: * long - the number of key/value pairs that are present in * the specified HashTable \*--------------------------------------------------------------------------*/ srInt_64 LRMHashTableSize(const HashTable *hashTable) { return hashTable->numOfElements; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableGetNumBuckets() - returns the number of buckets in a HashTable * DESCRIPTION: * Returns the number of buckets that are in the specified HashTable. * This may change dynamically throughout the life of a HashTable if * automatic or manual rehashing is performed. * EFFICIENCY: * O(1) * ARGUMENTS: * hashTable - the HashTable whose number of buckets is requested * RETURNS: * long - the number of buckets that are in the specified * HashTable \*--------------------------------------------------------------------------*/ srInt_64 LRMHashTableGetNumBuckets(const HashTable *hashTable) { return hashTable->numOfBuckets; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableSetKeyComparisonFunction() * - specifies the function used to compare keys in a HashTable * DESCRIPTION: * Specifies the function used to compare keys in the specified * HashTable. The specified function should return zero if the two * keys are considered equal, and non-zero otherwise. The default * function is one that simply compares pointers. * ARGUMENTS: * hashTable - the HashTable whose key comparison function is being * specified * keycmp - a function which returns zero if the two arguments * passed to it are considered "equal" keys and non-zero * otherwise * RETURNS: * \*--------------------------------------------------------------------------*/ void LRMHashTableSetKeyComparisonFunction(HashTable *hashTable, int (*keycmp)(const void *key1, const void *key2)) { assert(keycmp != NULL); hashTable->keycmp = keycmp; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableSetValueComparisonFunction() * - specifies the function used to compare values in a HashTable * DESCRIPTION: * Specifies the function used to compare values in the specified * HashTable. The specified function should return zero if the two * values are considered equal, and non-zero otherwise. The default * function is one that simply compares pointers. * ARGUMENTS: * hashTable - the HashTable whose value comparison function is being * specified * valuecmp - a function which returns zero if the two arguments * passed to it are considered "equal" values and non-zero * otherwise * RETURNS: * \*--------------------------------------------------------------------------*/ void LRMHashTableSetValueComparisonFunction(HashTable *hashTable, int (*valuecmp)(const void *value1, const void *value2)) { assert(valuecmp != NULL); hashTable->valuecmp = valuecmp; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableSetHashFunction() * - specifies the hash function used by a HashTable * DESCRIPTION: * Specifies the function used to determine the hash value for a key * in the specified HashTable (before modulation). An ideal hash * function is one which is easy to compute and approximates a * "random" function. The default function is one that works * relatively well for pointers. If the HashTable keys are to be * strings (which is probably the case), then this default function * will not suffice, in which case consider using the provided * HashTableStringHashFunction() function. * ARGUMENTS: * hashTable - the HashTable whose hash function is being specified * hashFunction - a function which returns an appropriate hash code * for a given key * RETURNS: * \*--------------------------------------------------------------------------*/ void LRMHashTableSetHashFunction(HashTable *hashTable, srUInt_64 (*hashFunction)(const void *key)) { assert(hashFunction != NULL); hashTable->hashFunction = hashFunction; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableRehash() - reorganizes a HashTable to be more efficient * DESCRIPTION: * Reorganizes a HashTable to be more efficient. If a number of * buckets is specified, the HashTable is rehashed to that number of * buckets. If 0 is specified, the HashTable is rehashed to a number * of buckets which is automatically calculated to be a prime number * that achieves (as closely as possible) the ideal element-to-bucket * ratio specified by the HashTableSetIdealRatio() function. * EFFICIENCY: * O(n) * ARGUMENTS: * hashTable - the HashTable to be reorganized * numOfBuckets - the number of buckets to rehash the HashTable to. * Should be prime. Ideally, the number of buckets * should be between 1/5 and 1 times the expected * number of elements in the HashTable. Values much * more or less than this will result in wasted memory * or decreased performance respectively. If 0 is * specified, an appropriate number of buckets is * automatically calculated. * RETURNS: * \*--------------------------------------------------------------------------*/ void LRMHashTableRehash(HashTable *hashTable, srInt_64 numOfBuckets) { KeyValuePair **newBucketArray; int i; assert(numOfBuckets >= 0); if (numOfBuckets == 0) numOfBuckets = LRMcalculateIdealNumOfBuckets(hashTable); if (numOfBuckets == hashTable->numOfBuckets) return; /* already the right size! */ newBucketArray = (KeyValuePair **) malloc(numOfBuckets * sizeof(KeyValuePair *)); if (newBucketArray == NULL) { /* Couldn't allocate memory for the new array. This isn't a fatal * error; we just can't perform the rehash. */ return; } for (i=0; inumOfBuckets; i++) { KeyValuePair *pair = hashTable->bucketArray[i]; while (pair != NULL) { KeyValuePair *nextPair = pair->next; srInt_64 hashValue = hashTable->hashFunction(pair->key) % numOfBuckets; pair->next = newBucketArray[hashValue]; newBucketArray[hashValue] = pair; pair = nextPair; } } free(hashTable->bucketArray); hashTable->bucketArray = newBucketArray; hashTable->numOfBuckets = numOfBuckets; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableSetIdealRatio() * - sets the ideal element-to-bucket ratio of a HashTable * DESCRIPTION: * Sets the ideal element-to-bucket ratio, as well as the lower and * upper auto-rehash thresholds, of the specified HashTable. Note * that this function doesn't actually perform a rehash. * * The default values for these properties are 3.0, 0.0 and 15.0 * respectively. This is likely fine for most situations, so there * is probably no need to call this function. * ARGUMENTS: * hashTable - a HashTable * idealRatio - the ideal element-to-bucket ratio. When a rehash * occurs (either manually via a call to the * HashTableRehash() function or automatically due the * the triggering of one of the thresholds below), the * number of buckets in the HashTable will be * recalculated to be a prime number that achieves (as * closely as possible) this ideal ratio. Must be a * positive number. * lowerRehashThreshold * - the element-to-bucket ratio that is considered * unacceptably low (i.e., too few elements per bucket). * If the actual ratio falls below this number, a * rehash will automatically be performed. Must be * lower than the value of idealRatio. If no ratio * is considered unacceptably low, a value of 0.0 can * be specified. * upperRehashThreshold * - the element-to-bucket ratio that is considered * unacceptably high (i.e., too many elements per bucket). * If the actual ratio rises above this number, a * rehash will automatically be performed. Must be * higher than idealRatio. However, if no ratio * is considered unacceptably high, a value of 0.0 can * be specified. * RETURNS: * \*--------------------------------------------------------------------------*/ void LRMHashTableSetIdealRatio(HashTable *hashTable, float idealRatio, float lowerRehashThreshold, float upperRehashThreshold) { assert(idealRatio > 0.0); assert(lowerRehashThreshold < idealRatio); assert(upperRehashThreshold == 0.0 || upperRehashThreshold > idealRatio); hashTable->idealRatio = idealRatio; hashTable->lowerRehashThreshold = lowerRehashThreshold; hashTable->upperRehashThreshold = upperRehashThreshold; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableSetDeallocationFunctions() * - sets the key and value deallocation functions of a HashTable * DESCRIPTION: * Sets the key and value deallocation functions of the specified * HashTable. This determines what happens to a key or a value when it * is removed from the HashTable. If the deallocation function is NULL * (the default if this function is never called), its reference is * simply dropped and it is up to the calling program to perform the * proper memory management. If the deallocation function is non-NULL, * it is called to free the memory used by the object. E.g., for simple * objects, an appropriate deallocation function may be free(). * * This affects the behaviour of the HashTableDestroy(), HashTablePut(), * HashTableRemove() and HashTableRemoveAll() functions. * ARGUMENTS: * hashTable - a HashTable * keyDeallocator * - if non-NULL, the function to be called when a key is * removed from the HashTable. * valueDeallocator * - if non-NULL, the function to be called when a value is * removed from the HashTable. * RETURNS: * \*--------------------------------------------------------------------------*/ void LRMHashTableSetDeallocationFunctions(HashTable *hashTable, void (*keyDeallocator)(void *key), void (*valueDeallocator)(void *value)) { hashTable->keyDeallocator = keyDeallocator; hashTable->valueDeallocator = valueDeallocator; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableStringHashFunction() - a good hash function for strings * DESCRIPTION: * A hash function that is appropriate for hashing strings. Note that * this is not the default hash function. To make it the default hash * function, call HashTableSetHashFunction(hashTable, * HashTableStringHashFunction). * ARGUMENTS: * key - the key to be hashed * RETURNS: * unsigned long - the unmodulated hash value of the key \*--------------------------------------------------------------------------*/ srUInt_64 LRMHashTableStringHashFunction(const void *key) { const unsigned char *str = (const unsigned char *) key; srUInt_64 hashValue = 0; int i; for (i=0; str[i] != '\0'; i++) hashValue = hashValue * 37 + str[i]; return hashValue; } static int LRMpointercmp(const void *pointer1, const void *pointer2) { return (pointer1 != pointer2); } static srUInt_64 LRMpointerHashFunction(const void *pointer) { return ((srUInt_64)( pointer - NULL )) ; } static int LRMisProbablePrime(srInt_64 oddNumber) { srInt_64 i; for (i=3; i<51; i+=2) if (oddNumber == i) return 1; else if (oddNumber%i == 0) return 0; return 1; /* maybe */ } static srInt_64 LRMcalculateIdealNumOfBuckets(HashTable *hashTable) { srInt_64 idealNumOfBuckets = hashTable->numOfElements / hashTable->idealRatio; if (idealNumOfBuckets < 5) idealNumOfBuckets = 5; else idealNumOfBuckets |= 0x01; /* make it an odd number */ while (!LRMisProbablePrime(idealNumOfBuckets)) idealNumOfBuckets += 2; return idealNumOfBuckets; } void LRMfree_values_destroy(HashTable * tab) { KeyValuePair * cursor; int bucket; for(bucket=0; bucket< tab -> numOfBuckets; bucket++) { cursor = tab -> bucketArray[bucket]; while (1) { if(!cursor) break; char * read_txt = (char *) cursor ->value; free(read_txt); cursor = cursor->next; } } LRMHashTableDestroy(tab); } void LRMHashTableIteration(HashTable * tab, void process_item(void * key, void * hashed_obj, HashTable * tab) ) { int i; for (i=0; i< tab ->numOfBuckets; i++) { KeyValuePair *pair = tab ->bucketArray[i]; while (pair != NULL) { process_item(( void * )pair -> key, pair -> value, tab); KeyValuePair *nextPair = pair->next; pair = nextPair; } } } Rsubread/src/LRMhashtable.h0000644000175200017520000005124314136047615016625 0ustar00biocbuildbiocbuild/*--------------------------------------------------------------------------*\ * -----===== HashTable =====----- * * Author: Keith Pomakis (pomakis@pobox.com) * Date: August, 1998 * Released to the public domain. * *-------------------------------------------------------------------------- * $Id: LRMhashtable.h,v 1.4 2019/08/28 22:31:15 cvs Exp $ \*--------------------------------------------------------------------------*/ #ifndef _HASHTABLE_H_LRM #define _HASHTABLE_H_LRM /* These structs should not be accessed directly from user code. * All access should be via the public functions declared below. */ typedef struct KeyValuePair_struct { const void *key; void *value; struct KeyValuePair_struct *next; } KeyValuePair; typedef struct { srInt_64 numOfBuckets; srInt_64 numOfElements; KeyValuePair **bucketArray; float idealRatio, lowerRehashThreshold, upperRehashThreshold; int (*keycmp)(const void *key1, const void *key2); int (*valuecmp)(const void *value1, const void *value2); srUInt_64 (*hashFunction)(const void *key); void (*keyDeallocator)(void *key); void (*valueDeallocator)(void *value); void * appendix1; void * appendix2; void * appendix3; srInt_64 counter1; srInt_64 counter2; srInt_64 counter3; } HashTable; typedef struct { void ** elementList; srInt_64 numOfElements; srInt_64 capacityOfElements; void (*elemDeallocator)(void *elem); } ArrayList; ArrayList * LRMArrayListCreate(int init_capacity); void LRMArrayListDestroy(ArrayList * list); void * LRMArrayListGet(ArrayList * list, srInt_64 n); int LRMArrayListPush(ArrayList * list, void * new_elem); void LRMArrayListSetDeallocationFunction(ArrayList * list, void (*elem_deallocator)(void *elem)); void LRMHashTableIteration(HashTable * tab, void process_item(void * key, void * hashed_obj, HashTable * tab) ); /*--------------------------------------------------------------------------*\ * NAME: * LRMHashTableCreate() - creates a new LRMHashTable * DESCRIPTION: * Creates a new LRMHashTable. When finished with this LRMHashTable, it * should be explicitly destroyed by calling the LRMHashTableDestroy() * function. * EFFICIENCY: * O(1) * ARGUMENTS: * numOfBuckets - the number of buckets to start the LRMHashTable out with. * Must be greater than zero, and should be prime. * Ideally, the number of buckets should between 1/5 * and 1 times the expected number of elements in the * LRMHashTable. Values much more or less than this will * result in wasted memory or decreased performance * respectively. The number of buckets in a LRMHashTable * can be re-calculated to an appropriate number by * calling the LRMHashTableRehash() function once the * LRMHashTable has been populated. The number of buckets * in a LRMHashTable may also be re-calculated * automatically if the ratio of elements to buckets * passes the thresholds set by LRMHashTableSetIdealRatio(). * RETURNS: * LRMHashTable - a new LRMHashtable, or NULL on error \*--------------------------------------------------------------------------*/ HashTable *LRMHashTableCreate(srInt_64 numOfBuckets); /*--------------------------------------------------------------------------*\ * NAME: * LRMHashTableDestroy() - destroys an existing LRMHashTable * DESCRIPTION: * Destroys an existing LRMHashTable. * EFFICIENCY: * O(n) * ARGUMENTS: * hashTable - the LRMHashTable to destroy * RETURNS: * \*--------------------------------------------------------------------------*/ void LRMHashTableDestroy(HashTable *hashTable); /*--------------------------------------------------------------------------*\ * NAME: * LRMHashTableContainsKey() - checks the existence of a key in a LRMHashTable * DESCRIPTION: * Determines whether or not the specified LRMHashTable contains the * specified key. Uses the comparison function specified by * LRMHashTableSetKeyComparisonFunction(). * EFFICIENCY: * O(1), assuming a good hash function and element-to-bucket ratio * ARGUMENTS: * hashTable - the LRMHashTable to search * key - the key to search for * RETURNS: * bool - whether or not the specified LRMHashTable contains the * specified key. \*--------------------------------------------------------------------------*/ int LRMHashTableContainsKey(const HashTable *hashTable, const void *key); /*--------------------------------------------------------------------------*\ * NAME: * LRMHashTableContainsValue() * - checks the existence of a value in a LRMHashTable * DESCRIPTION: * Determines whether or not the specified LRMHashTable contains the * specified value. Unlike LRMHashTableContainsKey(), this function is * not very efficient, since it has to scan linearly looking for a * match. Uses the comparison function specified by * LRMHashTableSetValueComparisonFunction(). * EFFICIENCY: * O(n) * ARGUMENTS: * hashTable - the LRMHashTable to search * value - the value to search for * RETURNS: * bool - whether or not the specified LRMHashTable contains the * specified value. \*--------------------------------------------------------------------------*/ int LRMHashTableContainsValue(const HashTable *hashTable, const void *value); /*--------------------------------------------------------------------------*\ * NAME: * LRMHashTablePut() - adds a key/value pair to a LRMHashTable * DESCRIPTION: * Adds the specified key/value pair to the specified LRMHashTable. If * the key already exists in the LRMHashTable (determined by the comparison * function specified by LRMHashTableSetKeyComparisonFunction()), its value * is replaced by the new value. May trigger an auto-rehash (see * LRMHashTableSetIdealRatio()). It is illegal to specify NULL as the * key or value. * EFFICIENCY: * O(1), assuming a good hash function and element-to-bucket ratio * ARGUMENTS: * hashTable - the LRMHashTable to add to * key - the key to add or whose value to replace * value - the value associated with the key * RETURNS: * err - 0 if successful, -1 if an error was encountered \*--------------------------------------------------------------------------*/ int LRMHashTablePut(HashTable *hashTable, const void *key, void *value); int LRMHashTablePutReplace(HashTable *hashTable, const void *key, void *value, int replace_key); /*--------------------------------------------------------------------------*\ * NAME: * LRMHashTableGet() - retrieves the value of a key in a LRMHashTable * DESCRIPTION: * Retrieves the value of the specified key in the specified LRMHashTable. * Uses the comparison function specified by * LRMHashTableSetKeyComparisonFunction(). * EFFICIENCY: * O(1), assuming a good hash function and element-to-bucket ratio * ARGUMENTS: * hashTable - the LRMHashTable to search * key - the key whose value is desired * RETURNS: * void * - the value of the specified key, or NULL if the key * doesn't exist in the LRMHashTable \*--------------------------------------------------------------------------*/ void * LRMHashTableGet(const HashTable *hashTable, const void *key); /*--------------------------------------------------------------------------*\ * NAME: * LRMHashTableRemove() - removes a key/value pair from a LRMHashTable * DESCRIPTION: * Removes the key/value pair identified by the specified key from the * specified LRMHashTable if the key exists in the LRMHashTable. May trigger * an auto-rehash (see LRMHashTableSetIdealRatio()). * EFFICIENCY: * O(1), assuming a good hash function and element-to-bucket ratio * ARGUMENTS: * hashTable - the LRMHashTable to remove the key/value pair from * key - the key specifying the key/value pair to be removed * RETURNS: * \*--------------------------------------------------------------------------*/ void LRMHashTableRemove(HashTable *hashTable, const void *key); /*--------------------------------------------------------------------------*\ * NAME: * LRMHashTableRemoveAll() - removes all key/value pairs from a LRMHashTable * DESCRIPTION: * Removes all key/value pairs from the specified LRMHashTable. May trigger * an auto-rehash (see LRMHashTableSetIdealRatio()). * EFFICIENCY: * O(n) * ARGUMENTS: * hashTable - the LRMHashTable to remove all key/value pairs from * RETURNS: * \*--------------------------------------------------------------------------*/ void LRMHashTableRemoveAll(HashTable *hashTable); /*--------------------------------------------------------------------------*\ * NAME: * LRMHashTableIsEmpty() - determines if a LRMHashTable is empty * DESCRIPTION: * Determines whether or not the specified LRMHashTable contains any * key/value pairs. * EFFICIENCY: * O(1) * ARGUMENTS: * hashTable - the LRMHashTable to check * RETURNS: * bool - whether or not the specified LRMHashTable contains any * key/value pairs \*--------------------------------------------------------------------------*/ int LRMHashTableIsEmpty(const HashTable *hashTable); /*--------------------------------------------------------------------------*\ * NAME: * LRMHashTableSize() - returns the number of elements in a LRMHashTable * DESCRIPTION: * Returns the number of key/value pairs that are present in the * specified LRMHashTable. * EFFICIENCY: * O(1) * ARGUMENTS: * hashTable - the LRMHashTable whose size is requested * RETURNS: * long - the number of key/value pairs that are present in * the specified LRMHashTable \*--------------------------------------------------------------------------*/ srInt_64 LRMHashTableSize(const HashTable *hashTable); /*--------------------------------------------------------------------------*\ * NAME: * LRMHashTableGetNumBuckets() - returns the number of buckets in a LRMHashTable * DESCRIPTION: * Returns the number of buckets that are in the specified LRMHashTable. * This may change dynamically throughout the life of a LRMHashTable if * automatic or manual rehashing is performed. * EFFICIENCY: * O(1) * ARGUMENTS: * hashTable - the LRMHashTable whose number of buckets is requested * RETURNS: * long - the number of buckets that are in the specified * LRMHashTable \*--------------------------------------------------------------------------*/ srInt_64 LRMHashTableGetNumBuckets(const HashTable *hashTable); /*--------------------------------------------------------------------------*\ * NAME: * LRMHashTableSetKeyComparisonFunction() * - specifies the function used to compare keys in a LRMHashTable * DESCRIPTION: * Specifies the function used to compare keys in the specified * LRMHashTable. The specified function should return zero if the two * keys are considered equal, and non-zero otherwise. The default * function is one that simply compares pointers. * ARGUMENTS: * hashTable - the LRMHashTable whose key comparison function is being * specified * keycmp - a function which returns zero if the two arguments * passed to it are considered "equal" keys and non-zero * otherwise * RETURNS: * \*--------------------------------------------------------------------------*/ void LRMHashTableSetKeyComparisonFunction(HashTable *hashTable, int (*keycmp)(const void *key1, const void *key2)); /*--------------------------------------------------------------------------*\ * NAME: * LRMHashTableSetValueComparisonFunction() * - specifies the function used to compare values in a LRMHashTable * DESCRIPTION: * Specifies the function used to compare values in the specified * LRMHashTable. The specified function should return zero if the two * values are considered equal, and non-zero otherwise. The default * function is one that simply compares pointers. * ARGUMENTS: * hashTable - the LRMHashTable whose value comparison function is being * specified * valuecmp - a function which returns zero if the two arguments * passed to it are considered "equal" values and non-zero * otherwise * RETURNS: * \*--------------------------------------------------------------------------*/ void LRMHashTableSetValueComparisonFunction(HashTable *hashTable, int (*valuecmp)(const void *value1, const void *value2)); /*--------------------------------------------------------------------------*\ * NAME: * LRMHashTableSetHashFunction() * - specifies the hash function used by a LRMHashTable * DESCRIPTION: * Specifies the function used to determine the hash value for a key * in the specified LRMHashTable (before modulation). An ideal hash * function is one which is easy to compute and approximates a * "random" function. The default function is one that works * relatively well for pointers. If the LRMHashTable keys are to be * strings (which is probably the case), then this default function * will not suffice, in which case consider using the provided * LRMHashTableStringHashFunction() function. * ARGUMENTS: * hashTable - the LRMHashTable whose hash function is being specified * hashFunction - a function which returns an appropriate hash code * for a given key * RETURNS: * \*--------------------------------------------------------------------------*/ void LRMHashTableSetHashFunction(HashTable *hashTable, srUInt_64 (*hashFunction)(const void *key)); /*--------------------------------------------------------------------------*\ * NAME: * LRMHashTableRehash() - reorganizes a LRMHashTable to be more efficient * DESCRIPTION: * Reorganizes a LRMHashTable to be more efficient. If a number of * buckets is specified, the LRMHashTable is rehashed to that number of * buckets. If 0 is specified, the LRMHashTable is rehashed to a number * of buckets which is automatically calculated to be a prime number * that achieves (as closely as possible) the ideal element-to-bucket * ratio specified by the LRMHashTableSetIdealRatio() function. * EFFICIENCY: * O(n) * ARGUMENTS: * hashTable - the LRMHashTable to be reorganized * numOfBuckets - the number of buckets to rehash the LRMHashTable to. * Should be prime. Ideally, the number of buckets * should be between 1/5 and 1 times the expected * number of elements in the LRMHashTable. Values much * more or less than this will result in wasted memory * or decreased performance respectively. If 0 is * specified, an appropriate number of buckets is * automatically calculated. * RETURNS: * \*--------------------------------------------------------------------------*/ void LRMHashTableRehash(HashTable *hashTable, srInt_64 numOfBuckets); /*--------------------------------------------------------------------------*\ * NAME: * LRMHashTableSetIdealRatio() * - sets the ideal element-to-bucket ratio of a LRMHashTable * DESCRIPTION: * Sets the ideal element-to-bucket ratio, as well as the lower and * upper auto-rehash thresholds, of the specified LRMHashTable. Note * that this function doesn't actually perform a rehash. * * The default values for these properties are 3.0, 0.0 and 15.0 * respectively. This is likely fine for most situations, so there * is probably no need to call this function. * ARGUMENTS: * hashTable - a LRMHashTable * idealRatio - the ideal element-to-bucket ratio. When a rehash * occurs (either manually via a call to the * LRMHashTableRehash() function or automatically due the * the triggering of one of the thresholds below), the * number of buckets in the LRMHashTable will be * recalculated to be a prime number that achieves (as * closely as possible) this ideal ratio. Must be a * positive number. * lowerRehashThreshold * - the element-to-bucket ratio that is considered * unacceptably low (i.e., too few elements per bucket). * If the actual ratio falls below this number, a * rehash will automatically be performed. Must be * lower than the value of idealRatio. If no ratio * is considered unacceptably low, a value of 0.0 can * be specified. * upperRehashThreshold * - the element-to-bucket ratio that is considered * unacceptably high (i.e., too many elements per bucket). * If the actual ratio rises above this number, a * rehash will automatically be performed. Must be * higher than idealRatio. However, if no ratio * is considered unacceptably high, a value of 0.0 can * be specified. * RETURNS: * \*--------------------------------------------------------------------------*/ void LRMHashTableSetIdealRatio(HashTable *hashTable, float idealRatio, float lowerRehashThreshold, float upperRehashThreshold); /*--------------------------------------------------------------------------*\ * NAME: * LRMHashTableSetDeallocationFunctions() * - sets the key and value deallocation functions of a LRMHashTable * DESCRIPTION: * Sets the key and value deallocation functions of the specified * LRMHashTable. This determines what happens to a key or a value when it * is removed from the LRMHashTable. If the deallocation function is NULL * (the default if this function is never called), its reference is * simply dropped and it is up to the calling program to perform the * proper memory management. If the deallocation function is non-NULL, * it is called to free the memory used by the object. E.g., for simple * objects, an appropriate deallocation function may be free(). * * This affects the behaviour of the LRMHashTableDestroy(), LRMHashTablePut(), * LRMHashTableRemove() and LRMHashTableRemoveAll() functions. * ARGUMENTS: * hashTable - a LRMHashTable * keyDeallocator * - if non-NULL, the function to be called when a key is * removed from the LRMHashTable. * valueDeallocator * - if non-NULL, the function to be called when a value is * removed from the LRMHashTable. * RETURNS: * \*--------------------------------------------------------------------------*/ void LRMHashTableSetDeallocationFunctions(HashTable *hashTable, void (*keyDeallocator)(void *key), void (*valueDeallocator)(void *value)); /*--------------------------------------------------------------------------*\ * NAME: * LRMHashTableStringHashFunction() - a good hash function for strings * DESCRIPTION: * A hash function that is appropriate for hashing strings. Note that * this is not the default hash function. To make it the default hash * function, call LRMHashTableSetHashFunction(HashTableStringHashFunction). * ARGUMENTS: * key - the key to be hashed * RETURNS: * long - the unmodulated hash value of the key \*--------------------------------------------------------------------------*/ srUInt_64 LRMHashTableStringHashFunction(const void *key); void LRMfree_values_destroy(HashTable * tab); #endif /* _HASHTABLE_H */ Rsubread/src/LRMhelper.c0000644000175200017520000000257714136047615016152 0ustar00biocbuildbiocbuild#include #include #include"LRMconfig.h" #include"LRMhelper.h" void LRMbasic_sort_run(void * arr, int start, int items, int compare (void * arr, int l, int r), void exchange(void * arr, int l, int r)){ int i, j; for(i=start; i< start + items - 1; i++) { int min_j = i; for(j=i + 1; j< start + items; j++) { if(compare(arr, min_j, j) > 0) min_j = j; } if(i!=min_j) exchange(arr, i, min_j); } } void LRMbasic_sort(void * arr, int items, int compare (void * arr, int l, int r), void exchange(void * arr, int l, int r)){ LRMbasic_sort_run(arr, 0, items, compare, exchange); } void LRMmerge_sort_run(void * arr, int start, int items, int compare (void * arr, int l, int r), void exchange(void * arr, int l, int r), void merge(void * arr, int start, int items, int items2)) { if(items > 6) { int half_point = items/2; LRMmerge_sort_run(arr, start, half_point, compare, exchange, merge); LRMmerge_sort_run(arr, start + half_point, items - half_point, compare, exchange, merge); merge(arr, start, half_point, items - half_point); } else { LRMbasic_sort_run(arr, start, items, compare, exchange); } } void LRMmerge_sort(void * arr, int arr_size, int compare (void * arr, int l, int r), void exchange(void * arr, int l, int r), void merge(void * arr, int start, int items, int items2)) { LRMmerge_sort_run(arr, 0, arr_size, compare, exchange, merge); } Rsubread/src/LRMhelper.h0000644000175200017520000000055714136047615016153 0ustar00biocbuildbiocbuild#ifndef __LRM_HELPER_H_ #define __LRM_HELPER_H_ void LRMbasic_sort(void * arr, int items, int compare (void * arr, int l, int r), void exchange(void * arr, int l, int r)); void LRMmerge_sort(void * arr, int arr_size, int compare (void * arr, int l, int r), void exchange(void * arr, int l, int r), void merge(void * arr, int start, int items, int items2)); #endif Rsubread/src/LRMseek-zlib.c0000644000175200017520000002762414136047615016560 0ustar00biocbuildbiocbuild#include #include "LRMconfig.h" #include "LRMseek-zlib.h" #define SEEKGZ_INIT_TEXT_SIZE (1024*1024) #define SEEKGZ_BINBUFF_SIZE (1*1024*1024) unsigned long long LRMseekgz_ftello(seekable_zfile_t * fp){ unsigned long long ret = ftello(fp -> gz_fp); ret -= fp -> stem.avail_in; return ret; } unsigned int LRMcrc_pos(char * bin, int len){ unsigned int crc0 = crc32(0, NULL, 0); unsigned int CRC32 = crc32(crc0, (unsigned char *) bin, len); return CRC32; } void LRMseekgz_binreadmore(seekable_zfile_t * fp){ if(feof(fp->gz_fp))return; assert(fp -> stem.avail_in >= 0); if(fp -> stem.avail_in < SEEKGZ_BINBUFF_SIZE / 2 ) { if(fp -> in_pointer > 0 && fp -> stem.avail_in > 0){ int i; for(i = 0 ; i < fp -> stem.avail_in ; i ++){ fp -> current_chunk_bin[i] = fp -> current_chunk_bin[i + fp -> in_pointer]; } } fp -> in_pointer = 0; int readlen = fread(fp -> current_chunk_bin + fp -> stem.avail_in, 1 , SEEKGZ_BINBUFF_SIZE - fp -> stem.avail_in , fp -> gz_fp); if(readlen>0) fp -> stem.avail_in += readlen; fp -> stem.next_in = (unsigned char *)fp -> current_chunk_bin; //SEEKZLIBprintf("READIN: %d, POS: %llu, CRC:%u\n", fp -> stem.avail_in , ftello(fp -> gz_fp) , crc_pos(fp -> current_chunk_bin , fp -> stem.avail_in)); } } int LRMseekgz_bingetc(seekable_zfile_t * fp){ LRMseekgz_binreadmore(fp); int ret = -1; if(fp -> stem.avail_in > 0) { ret = fp -> current_chunk_bin [ fp -> in_pointer ++]; fp -> stem.next_in = (unsigned char *)(fp -> current_chunk_bin + fp -> in_pointer); fp -> stem.avail_in --; if(ret<0) ret=256+ret; } return ret; } int LRMseekgz_skip_header(seekable_zfile_t * fp, int tail_size){ int id1, id2; if(tail_size){ for(id1=0; id10; XLEN--) LRMseekgz_bingetc(fp); } for(id1 = 3; id1 <=4; id1++){ if(FLG & (1< next_block_file_offset = LRMseekgz_ftello(fp); if(fp -> block_start_in_file_offset<1) fp -> block_start_in_file_offset = fp -> next_block_file_offset; fp -> next_block_file_bits = 0; fp -> dict_window_used = 0; fp -> dict_window_pointer = 0; fp -> is_the_last_chunk = 2; return 0; } int LRMseekgz_decompress_next_chunk(seekable_zfile_t * fp); int LRMseekgz_open(const char * fname, seekable_zfile_t * fp){ memset(fp, 0, sizeof(seekable_zfile_t)); fp -> gz_fp = fopen(fname, "rb"); if(NULL==fp -> gz_fp)return -1; fp -> current_chunk_bin = malloc(SEEKGZ_BINBUFF_SIZE); fp -> current_chunk_txt = malloc(SEEKGZ_INIT_TEXT_SIZE); fp -> current_chunk_txt_size = SEEKGZ_INIT_TEXT_SIZE; //fp -> txt_buffer_size = SEEKGZ_INIT_TEXT_SIZE; fp -> stem.zalloc = Z_NULL; fp -> stem.zfree = Z_NULL; fp -> stem.opaque = Z_NULL; fp -> stem.avail_in = 0; fp -> stem.next_in = Z_NULL; int ret = LRMseekgz_skip_header(fp,0); if(ret) return 1; ret = inflateInit2(&(fp -> stem), -15); if(ret) return 1; return 0; } void LRMseekgz_tell(seekable_zfile_t * fp, seekable_position_t * pos){ pos -> block_gzfile_offset = fp -> block_start_in_file_offset; pos -> block_gzfile_bits = fp -> block_start_in_file_bits; memcpy(pos -> dict_window, fp -> block_dict_window, fp -> block_dict_window_size); pos -> block_dict_window_size = fp -> block_dict_window_size; pos -> in_block_text_offset = fp -> in_block_offset; } void LRMseekgz_seek(seekable_zfile_t * fp, seekable_position_t * pos){ //#warning "COMMENT THIS LINE !!!!." //fprintf(stderr, "SEEK => %llu[%d] + %u ; WIN=%d CRC=%u\n", pos -> block_gzfile_offset, pos -> block_gzfile_bits, pos -> in_block_text_offset, pos -> block_dict_window_size, crc_pos( pos -> dict_window, pos -> block_dict_window_size)); fseeko(fp->gz_fp, pos -> block_gzfile_offset - (pos -> block_gzfile_bits?1:0), SEEK_SET); if(Z_OK!=inflateReset(&fp->stem)) SEEKZLIBprintf("FATAL: UNABLE TO INIT STREAM.\n\n\n"); if(pos -> block_dict_window_size>0){ if(pos -> block_gzfile_bits){ char nch = fgetc(fp->gz_fp); //fprintf(stderr, "SEEK 2 FPPOS:%llu, NCH=%d\n", ftello(fp->gz_fp) , nch); inflatePrime(&fp->stem, pos -> block_gzfile_bits, nch>>(8-pos -> block_gzfile_bits)); } if(Z_OK != inflateSetDictionary(&fp->stem, (unsigned char *)pos -> dict_window, pos -> block_dict_window_size)) SEEKZLIBprintf("FATAL: UNABLE TO RESET STREAM.\n\n\n"); } fp -> stem.avail_in = 0; fp -> in_pointer = 0; fp -> txt_buffer_used = 0; fp -> in_chunk_offset = 0; memcpy(fp -> block_dict_window, pos -> dict_window, pos -> block_dict_window_size); memcpy(fp -> dict_window, pos -> dict_window, pos -> block_dict_window_size); fp -> block_dict_window_size = fp -> dict_window_used = pos -> block_dict_window_size; fp -> dict_window_pointer = (pos -> block_dict_window_size block_dict_window_size:0; fp -> in_block_offset = 0; fp -> block_start_in_file_offset = pos -> block_gzfile_offset; fp -> block_start_in_file_bits = pos -> block_gzfile_bits; unsigned int chunk_end_block_offset=0; while(1){ LRMseekgz_decompress_next_chunk(fp); if(fp -> internal_error) break; chunk_end_block_offset += fp -> txt_buffer_used; if(chunk_end_block_offset >= pos -> in_block_text_offset){ fp -> in_chunk_offset = fp -> txt_buffer_used - (chunk_end_block_offset - pos -> in_block_text_offset); fp -> in_block_offset = pos -> in_block_text_offset; break; } assert(chunk_end_block_offset < fp -> current_chunk_txt_size && !feof(fp->gz_fp)); fp -> txt_buffer_used=0; } } int LRMseekgz_decompress_next_chunk(seekable_zfile_t * fp){ unsigned int this_chunk_size = 0; int loaded_blocks = 0; while(1){ LRMseekgz_binreadmore(fp); if(loaded_blocks > 0) //SEEKZLIBprintf("LOADED BLOCKS=%d\n", loaded_blocks); if(fp -> txt_buffer_used >= fp -> current_chunk_txt_size * 7 / 8){ //SEEKZLIBprintf("TRE ALLOC CHUNK_TXT: %d -> %d\n", fp -> current_chunk_txt_size, (int)(fp -> current_chunk_txt_size*1.5)); fp -> current_chunk_txt_size *= 1.5; assert(fp -> current_chunk_txt_size < 3*512*1024*1024); fp -> current_chunk_txt = realloc(fp -> current_chunk_txt, fp -> current_chunk_txt_size ); } fp -> stem.avail_out = fp -> current_chunk_txt_size - fp -> txt_buffer_used; int out_start = fp -> txt_buffer_used; fp -> stem.next_out = (unsigned char *)(fp -> current_chunk_txt + out_start); int inlen = fp -> stem.avail_in ; //fprintf(stderr,"INFLATING_0 : LEN=%u, CRC=%u\n", fp -> stem.avail_in , crc_pos( fp -> stem.next_in , fp -> stem.avail_in )); int ret = inflate(&(fp -> stem), Z_BLOCK); int have = ( fp -> current_chunk_txt_size - fp -> txt_buffer_used) - fp -> stem.avail_out; int is_chunk_end = 0; //#warning "COMMENT NEXT LINE!!!!!." //fprintf(stderr,"INFLATING: INLEN=%d , OLEN=%d, POS=%lld, RET=%d, TOOL=%s\n", inlen , have, LRMseekgz_ftello(fp), ret, zlibVersion()); if(ret != Z_OK && ret != Z_STREAM_END){ //any error #ifdef __MINGW32__ SEEKZLIBprintf("FATAL: INFLATE-ERROR=%d POS=%I64d\n", ret, LRMseekgz_ftello(fp)); #else SEEKZLIBprintf("FATAL: INFLATE-ERROR=%d POS=%lld\n", ret, LRMseekgz_ftello(fp)); #endif fp -> internal_error = 1; return -1; } fp -> in_pointer += inlen - fp -> stem.avail_in ; if(have > 0){ fp -> txt_buffer_used += have; int one_length = 0, one_src_start = 0, one_dst_start = 0; int two_length = 0, two_src_start = 0, two_dst_start = 0; int new_pntr = 0; if(have <= SEEKGZ_ZLIB_WINDOW_SIZE - fp -> dict_window_pointer){ one_length = 0; two_src_start = out_start; two_dst_start = fp -> dict_window_pointer; two_length = have; new_pntr = two_dst_start + two_length; }else if(have > SEEKGZ_ZLIB_WINDOW_SIZE - fp -> dict_window_pointer && have <= SEEKGZ_ZLIB_WINDOW_SIZE){ one_src_start = out_start + SEEKGZ_ZLIB_WINDOW_SIZE - fp -> dict_window_pointer; one_dst_start = 0; one_length = have - SEEKGZ_ZLIB_WINDOW_SIZE + fp -> dict_window_pointer; two_src_start = out_start; two_dst_start = fp -> dict_window_pointer; two_length = SEEKGZ_ZLIB_WINDOW_SIZE - fp -> dict_window_pointer; new_pntr = one_dst_start + one_length; }else{ one_src_start = out_start + have - fp -> dict_window_pointer; one_dst_start = 0; one_length = fp -> dict_window_pointer; two_src_start = out_start + have - SEEKGZ_ZLIB_WINDOW_SIZE; two_dst_start = fp -> dict_window_pointer; two_length = SEEKGZ_ZLIB_WINDOW_SIZE - fp -> dict_window_pointer; new_pntr = fp -> dict_window_pointer; } if(one_length > 0)memcpy(fp -> dict_window + one_dst_start, fp -> current_chunk_txt + one_src_start, one_length); //fprintf(stderr,"CPY: %d -> %d [%d] ; PNTR=%d, NEWPNTR=%d, have=%d\n", two_src_start, two_dst_start, two_length, fp -> dict_window_pointer, new_pntr, have); memcpy(fp -> dict_window + two_dst_start, fp -> current_chunk_txt + two_src_start, two_length); fp -> dict_window_pointer = new_pntr; fp -> dict_window_used = SEEKZLIBmin(fp -> dict_window_used + have, SEEKGZ_ZLIB_WINDOW_SIZE); is_chunk_end = (fp -> stem.data_type & 128) && !(fp -> stem.data_type & 64); if(is_chunk_end){ fp -> is_the_last_chunk = 1; unsigned long long file_pos_after_avail = LRMseekgz_ftello(fp); fp -> next_block_file_offset = file_pos_after_avail; fp -> next_block_file_bits = fp->stem.data_type & 7; } this_chunk_size += have; } loaded_blocks ++; if( 0 == fp -> stem.avail_in ) this_chunk_size = 0; if(Z_STREAM_END == ret || ((is_chunk_end || 0 == fp -> stem.avail_in) && fp -> txt_buffer_used >=10)){ if(Z_STREAM_END == ret){ LRMseekgz_skip_header(fp, 8); inflateReset(&fp->stem); } break; } } return 0; } int LRMseekgz_next_char(seekable_zfile_t * fp){ if(fp -> internal_error) return -1; while(fp -> in_chunk_offset >= fp -> txt_buffer_used){ if(feof(fp -> gz_fp) && fp -> stem.avail_in < 10 ) return EOF; else { fp -> txt_buffer_used = 0; fp -> in_chunk_offset = 0; int decompress_ret = LRMseekgz_decompress_next_chunk(fp); if(decompress_ret) return -1; } } fp -> in_block_offset ++; char retc = fp -> current_chunk_txt[fp -> in_chunk_offset++]; if(fp -> is_the_last_chunk && fp -> in_chunk_offset == fp -> txt_buffer_used){ fp -> in_block_offset = 0; fp -> block_start_in_file_offset = fp -> next_block_file_offset; fp -> block_start_in_file_bits = fp -> next_block_file_bits; if(1 == fp -> is_the_last_chunk){ fp -> block_dict_window_size = fp -> dict_window_used; if(fp -> dict_window_used < SEEKGZ_ZLIB_WINDOW_SIZE) memcpy(fp -> block_dict_window , fp -> dict_window, fp -> dict_window_used); else{ memcpy(fp -> block_dict_window , fp -> dict_window + fp -> dict_window_pointer, SEEKGZ_ZLIB_WINDOW_SIZE - fp -> dict_window_pointer); memcpy(fp -> block_dict_window + SEEKGZ_ZLIB_WINDOW_SIZE - fp -> dict_window_pointer, fp -> dict_window, fp -> dict_window_pointer); } }else fp -> block_dict_window_size = 0; fp -> is_the_last_chunk = 0; } return retc; } int LRMseekgz_gets(seekable_zfile_t * fp, char * buf, int buf_size){ int i=0; buf[0]=0; while(1){ if(i >= buf_size - 1){ buf[i]=0; return i; } int nch = LRMseekgz_next_char(fp); if(nch<0 || nch == '\n'){ if(i<1 && nch <0) return 0; buf[i] = '\n'; buf[i+1]=0; return i+1; }else buf[i++]=nch; } } void LRMseekgz_close(seekable_zfile_t * fp){ fclose(fp -> gz_fp); free(fp -> current_chunk_txt); free(fp -> current_chunk_bin); } Rsubread/src/LRMseek-zlib.h0000644000175200017520000000331714136047615016556 0ustar00biocbuildbiocbuild#ifndef __SEEK_ZLIB_H_LRM_ #define __SEEK_ZLIB_H_LRM_ #include #include #include #include #define SEEKGZ_ZLIB_WINDOW_SIZE (32*1024) typedef struct { FILE * gz_fp; char * current_chunk_txt; char * current_chunk_bin; z_stream stem; int current_chunk_txt_size; unsigned int in_pointer; unsigned int in_chunk_offset; unsigned int in_block_offset; //unsigned int txt_buffer_size; unsigned int txt_buffer_used; unsigned long long block_start_in_file_offset; unsigned int block_start_in_file_bits; unsigned long long next_block_file_offset; unsigned int next_block_file_bits; int is_the_last_chunk; int internal_error; unsigned int dict_window_pointer; unsigned int dict_window_used; char dict_window[SEEKGZ_ZLIB_WINDOW_SIZE]; unsigned int block_dict_window_size; char block_dict_window[SEEKGZ_ZLIB_WINDOW_SIZE]; } seekable_zfile_t; typedef struct{ char dict_window[SEEKGZ_ZLIB_WINDOW_SIZE]; unsigned long long block_gzfile_offset; unsigned int block_gzfile_bits; unsigned int block_dict_window_size; unsigned int in_block_text_offset; } seekable_position_t; #define SEEKZLIBmin(a,b) ( (a)<(b)?(a):(b) ) // returns 0 if OK; returns 1 if the file is not indexable; returns -1 if file doesn't exist. int LRMseekgz_open(const char * fname, seekable_zfile_t * fp); // returns length in bytes if OK (length includes the line break at the end); returns 0 if EOF int LRMseekgz_gets(seekable_zfile_t * fp, char * buf, int buf_size); void LRMseekgz_tell(seekable_zfile_t * fp, seekable_position_t * pos); void LRMseekgz_seek(seekable_zfile_t * fp, seekable_position_t * pos); int LRMseekgz_next_char(seekable_zfile_t * fp); void LRMseekgz_close(seekable_zfile_t * fp); #endif Rsubread/src/LRMsorted-hashtable.c0000644000175200017520000004304114136047615020113 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #include #include #include #include "LRMsorted-hashtable.h" #ifndef MACOS #ifndef FREEBSD #include #endif #endif #include #include "LRMfile-io.h" #define _gehash_hash(k) ((unsigned int)(k)) #define WITHOUT_CLUSTER_ORDERING 0 struct LRMgehash_bucket * LRM_gehash_get_bucket(LRMgehash_t * the_table, LRMgehash_key_t key) { int bucket_number; bucket_number = _gehash_hash(key) % the_table -> buckets_number; return the_table -> buckets +bucket_number; } #define INDEL_SEGMENT_SIZE 5 #define _index_vote(key) (((unsigned int)(key))%LRMGENE_VOTE_TABLE_SIZE) #define _index_vote_tol(key) (((unsigned int)(key)/INDEL_SEGMENT_SIZE)%LRMGENE_VOTE_TABLE_SIZE) #define is_quality_subread(scr) ((scr)>15?1:0) size_t LRMgehash_go_q(LRMgehash_t * the_table, LRMgehash_key_t raw_key, int offset, int read_len, int is_reversed, LRMgene_vote_t * vote, int indel_tolerance, int subread_number){ //LRMprintf("Q=%u, OFFSET=%d, B=%u ~ %u\n", raw_key, offset, low_border, high_border); // VER_1 // VER_2 struct LRMgehash_bucket * current_bucket; int i = 0, items; short *current_keys;//, *endp12; short key = raw_key / the_table->buckets_number; current_bucket = LRM_gehash_get_bucket (the_table, raw_key); items = current_bucket -> current_items; current_keys = current_bucket -> new_item_keys; if(!items) return 0; int imin=0, imax=items; int last_accepted_index = 0; while( imin < items ) { last_accepted_index=(imin+imax)/2; short current_key = current_keys[last_accepted_index]; if(current_key>key) { imax = last_accepted_index - 1; } else if(current_key5) ii_end=(indel_tolerance % INDEL_SEGMENT_SIZE)?(indel_tolerance - indel_tolerance%INDEL_SEGMENT_SIZE+INDEL_SEGMENT_SIZE):indel_tolerance; for (; last_accepted_indexitem_values[last_accepted_index] - offset; int iix, offsetX2, offsetX, datalen, datalen2; offsetX2 = offsetX = _index_vote_tol(kv); datalen = datalen2 = vote -> items[offsetX2]; unsigned int * dat2, *dat; dat = dat2 = vote -> pos[offsetX2]; //LRMprintf("You can find KV at %u\n", kv); for(iix = 0; iix<=ii_end; iix = iix>0?-iix:(-iix+INDEL_SEGMENT_SIZE)) { if(iix) { offsetX = _index_vote_tol(kv+iix); datalen = vote -> items[offsetX]; dat = vote -> pos[offsetX]; } if(!datalen)continue; for (i=0;i= -indel_tolerance && dist0 <= indel_tolerance ) { if(is_reversed == (0!=(vote -> masks[offsetX][i]&LRMIS_NEGATIVE_STRAND))) { if(offset < vote->coverage_end [offsetX][i] + 10){ unsigned char test_max = (vote->votes[offsetX][i]); test_max += 1; vote -> votes[offsetX][i] = test_max; if (offset +16 > vote->coverage_end [offsetX][i]) vote->coverage_end [offsetX][i] = offset+16; /* int toli = vote -> toli[offsetX][i]; if (dist0 != vote->current_indel_cursor[offsetX][i]) { toli +=3; if (toli < LRMMAX_INDEL_SECTIONS*3) { vote -> toli[offsetX][i] = toli; vote -> indel_recorder[offsetX][i][toli] = subread_number+1; vote -> indel_recorder[offsetX][i][toli+1] = subread_number+1; vote -> indel_recorder[offsetX][i][toli+2] = dist0; //LRMprintf("subread=#%d ,TOLI=%d, DIST0=%d, POS=%u \n", subread_number, toli, dist0, kv); if(toli < LRMMAX_INDEL_SECTIONS*3-3) vote -> indel_recorder[offsetX][i][toli+3]=0; } vote->current_indel_cursor [offsetX][i] = (char)dist0; } else vote -> indel_recorder[offsetX][i][toli+1] = subread_number+1; */ i = 9999999; } } } } if (i>=9999999){ break; } } if (i < 9999999) { if (datalen2 items[offsetX2] ++; dat2[datalen2] = kv; vote -> masks[offsetX2][datalen2]=(is_reversed?LRMIS_NEGATIVE_STRAND:0); vote -> votes[offsetX2][datalen2]=1; vote -> toli[offsetX2][datalen2]=0; // data structure of recorder: // {unsigned char subread_start; unsigned char subread_end, char indel_offset_from_start} // All subread numbers are added with 1 for not being 0. vote -> indel_recorder[offsetX2][datalen2][0] = vote -> indel_recorder[offsetX2][datalen2][1] = subread_number+1; vote -> indel_recorder[offsetX2][datalen2][2] = 0; vote -> indel_recorder[offsetX2][datalen2][3] = 0; vote -> current_indel_cursor [offsetX2][datalen2] = 0; vote -> coverage_start [offsetX2][datalen2] = offset; vote -> coverage_end [offsetX2][datalen2] = offset+16; //LRMprintf("subread=#%d ,NEW RECORD =%u\n", subread_number, kv); } } else i=0; } return 1; } short LRMindel_recorder_copy(unsigned short *dst, unsigned short * src) { short all_indels = 0; // memcpy(dst, src, 3*MAX_INDEL_TOLERANCE); return; int i=0; while(src[i] && (i<3*LRMMAX_INDEL_TOLERANCE-2)) { dst[i] = src[i]; i++; dst[i] = src[i]; i++; dst[i] = src[i]; all_indels = dst[i]; i++; } dst[i] = 0; return all_indels; } // Data Struct of dumpping: // { // size_t current_items; // size_t buckets_number; // struct // { // size_t current_items; // size_t space_size; // gehash_key_t item_keys [current_items]; // gehash_data_t item_values [current_items] // } [buckets_number]; // } // unsigned int LRMload_int32(FILE * fp) { int ret; int read_length; read_length = fread(&ret, sizeof(int), 1, fp); if(read_length<=0)assert(0); return ret; } long long int LRMload_int64(FILE * fp) { long long int ret; int read_length; read_length = fread(&ret, sizeof(long long int), 1, fp); if(read_length<=0)assert(0); return ret; } int LRMgehash_load_option(const char fname [], int option_no, int * result){ char tabname[LRMMAX_FILENAME_LENGTH]; char magic_chars[8]; int found = 0; sprintf(tabname, "%s.00.b.tab", fname); FILE * fp = fopen(tabname, "rb"); if(fp == NULL){ sprintf(tabname, "%s.00.c.tab", fname); fp = fopen(tabname, "rb"); } if(fp){ int rlen = fread(magic_chars,1,8,fp); if(rlen < 8){ LRMprintf("ERROR: Unable to read-in the index.\n"); return -1; } if(memcmp(magic_chars, "2subindx",7)==0) { while(1) { short option_key, option_length; rlen = fread(&option_key, 2, 1, fp); if(rlen<1){ LRMprintf("ERROR: Unable to read-in the index.\n"); return -1; } if(!option_key) break; rlen = fread(&option_length, 2, 1, fp); if(rlen<1){ LRMprintf("ERROR: Unable to read-in the index.\n"); return -1; } if(option_key == option_no){ *result = 0; rlen = fread(result ,option_length,1,fp); if(rlen<1){ LRMprintf("ERROR: Unable to read-in the index.\n"); return -1; } found = 1; } else fseek(fp, option_length, SEEK_CUR); } } fclose(fp); } return found; } int LRMgehash_load(LRMgehash_t * the_table, const char fname []) { int i, read_length; char magic_chars[8]; magic_chars[7]=0; the_table -> index_gap = 0; FILE * fp = fopen(fname, "rb"); if (!fp) { LRMprintf ("Table file '%s' is not found.\n", fname); return 1; } int rlen = fread(magic_chars,1,8,fp); if(rlen<8){ LRMprintf("ERROR: Unable to read-in the index.\n"); return -1; } if(memcmp(magic_chars+1, "subindx",7)==0) { if('2'==magic_chars[0]) the_table -> version_number = LRMSUBINDEX_VER2; else assert(0); while(1) { short option_key, option_length; rlen = fread(&option_key, 2, 1, fp); if(rlen<1){ LRMprintf("ERROR: Unable to read-in the index.\n"); return -1; } if(!option_key) break; rlen = fread(&option_length, 2, 1, fp); if(rlen<1){ LRMprintf("ERROR: Unable to read-in the index.\n"); return -1; } rlen = 999; if(option_key == LRMSUBREAD_INDEX_OPTION_INDEX_GAP) rlen = fread(&(the_table -> index_gap),2,1,fp); else if (option_key == LRMSUBREAD_INDEX_OPTION_INDEX_PADDING) rlen = fread(&(the_table -> padding),2,1,fp); else fseek(fp, option_length, SEEK_CUR); if(rlen<1){ LRMprintf("ERROR: Unable to read-in the index.\n"); return -1; } } assert(the_table -> index_gap); the_table -> current_items = LRMload_int64(fp); if(the_table -> current_items < 1 || the_table -> current_items > 0xffffffffllu){ LRMputs("ERROR: the index format is unrecognizable."); return 1; } the_table -> buckets_number = LRMload_int32(fp); the_table -> buckets = (struct LRMgehash_bucket * )malloc(sizeof(struct LRMgehash_bucket) * the_table -> buckets_number); if(!the_table -> buckets) { LRMputs("Error: out of memory"); return 1; } for (i=0; i buckets_number; i++) { struct LRMgehash_bucket * current_bucket = &(the_table -> buckets[i]); current_bucket -> current_items = LRMload_int32(fp); current_bucket -> space_size = LRMload_int32(fp); current_bucket -> space_size = current_bucket -> current_items; current_bucket -> new_item_keys = (short *) malloc ( sizeof(short) * current_bucket -> space_size); current_bucket -> item_values = (LRMgehash_data_t *) malloc ( sizeof(LRMgehash_data_t) * current_bucket -> space_size); if(!(current_bucket -> new_item_keys&¤t_bucket -> item_values)) { LRMputs("Error: out of memory"); return 1; } if(current_bucket -> current_items > 0) { read_length = fread(current_bucket -> new_item_keys, sizeof(short), current_bucket -> current_items, fp); if(read_length < current_bucket -> current_items){ LRMprintf("ERROR: the index is incomplete : %d < %u.\n",read_length, current_bucket -> current_items); return 1; } read_length = fread(current_bucket -> item_values, sizeof(LRMgehash_data_t), current_bucket -> current_items, fp); if(read_length < current_bucket -> current_items){ LRMprintf("ERROR: the index value is incomplete : %d < %u.\n",read_length, current_bucket -> current_items); return 1; } } } read_length = fread(&(the_table -> is_small_table), sizeof(char), 1, fp); assert(read_length>0); fclose(fp); return 0; } else assert(0); return 0; } void LRMtest2key(unsigned int kk, char * obuf){ int xx,oo=0; for(xx=0; xx<32;xx++){ obuf[oo++] = (kk & (1<> (xx*2)) & 3; int b2 = (k2 >> (xx*2)) & 3; if(b1!=b2) ret++; } return ret; } size_t LRMgehash_go_QQ(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context, LRMgehash_t * the_table, LRMgehash_key_t raw_key, int offset, int read_len, int is_reversed, LRMgene_vote_t * vote, int indel_tolerance, int subread_number){ struct LRMgehash_bucket * current_bucket; int i = 0, items; short *current_keys;//, *endp12; short key = raw_key / the_table->buckets_number; current_bucket = LRM_gehash_get_bucket (the_table, raw_key); items = current_bucket -> current_items; current_keys = current_bucket -> new_item_keys; if(!items) return 0; int imin=0, imax=items; int last_accepted_index = 0; while( imin < items ) { last_accepted_index=(imin+imax)/2; short current_key = current_keys[last_accepted_index]; if(current_key>key) imax = last_accepted_index - 1; else if(current_keyitem_values[last_accepted_index] - offset; int offsetX, datalen; offsetX = _index_vote(kv); datalen = vote -> items[offsetX]; unsigned int *dat; dat = vote -> pos[offsetX]; for (i=0;i masks[offsetX][i]&LRMIS_NEGATIVE_STRAND)) && offset < vote->coverage_end [offsetX][i] + 14){ vote -> votes[offsetX][i]++; if (offset +16 > vote->coverage_end [offsetX][i]) vote->coverage_end [offsetX][i] = offset+16; i = 9999999; } } if (i < 9999999) { if (datalen items[offsetX] ++; dat[datalen]=kv; vote -> masks[offsetX][datalen]=(is_reversed?LRMIS_NEGATIVE_STRAND:0); vote -> votes[offsetX][datalen]=1; vote -> coverage_start [offsetX][datalen] = offset; vote -> coverage_end [offsetX][datalen] = offset+16; } } else i=0; } return 1; } size_t LRMgehash_go_tolerance(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context, LRMgehash_t * the_table, LRMgehash_key_t key, int offset, int read_len, int is_reversed, LRMgene_vote_t * vote, int indel_tolerance, int subread_number, int max_MM){ if(max_MM >= 10) return 0; int ret = 0; ret+=LRMgehash_go_q(the_table, key, offset, read_len, is_reversed, vote, indel_tolerance, subread_number); int error_bases ; for (error_bases=1; error_bases <= max_MM; error_bases++) { assert(error_bases<5); int i, j; char error_pos_stack[10]; // max error bases = 10; LRMgehash_key_t mutation_key; for(i=0; i> 2*base_to_change) & 3; int new_index = mutation_stack[j]; int new_value; if(old_value <= new_index) new_value = 1+ new_index; else new_value = new_index; mutation_key = mutation_key & ~(0x3 << (2*base_to_change)); mutation_key = mutation_key | (new_value << (2*base_to_change)); } if(key != mutation_key ){ int dret=LRMgehash_go_q(the_table, mutation_key, offset, read_len, is_reversed, vote, indel_tolerance, subread_number); ret += dret; if(0 && LRMFIXLENstrcmp("@39076b1f-df29-4487-be51-4c30bf6c1cc4_Basecall_Alignment_template", iteration_context->read_name)==0){ char bin_mutation_key[53], bin_key[53]; LRMtest2key(mutation_key, bin_mutation_key); LRMtest2key(key, bin_key); LRMprintf("GO_TOLE_TEST: %s + %d DIST = %d HITS = %d :\nNEWKY: %s\nORGKY: %s\n", iteration_context->read_name , offset, LRMtest2key_dist(mutation_key,key), dret, bin_mutation_key, bin_key); } } // increase one in the mutation_stack mutation_stack[error_bases-1]++; for(i = error_bases-1; i>=0; i--){ if( mutation_stack[i]>2 ) { if(i == 0)break; else{ mutation_stack[i] = 0; mutation_stack[i-1]++; } } } if(mutation_stack[0]>2)break; } int is_end = 1; for (i = error_bases-1; i>=0; i--) if (error_pos_stack[i] < 16 - (error_bases - i)) { error_pos_stack[i] ++; for (j = i+1; j buckets_number; i++) { struct LRMgehash_bucket * current_bucket = &(the_table -> buckets[i]); if (current_bucket -> space_size > 0) { free (current_bucket -> new_item_keys); free (current_bucket -> item_values); } } free (the_table -> buckets); the_table -> current_items = 0; the_table -> buckets_number = 0; } void LRMprint_v(LRMcontext_t * context, LRMread_iteration_context_t * iteration_context, int min_votes){ LRMgene_vote_t * v = &iteration_context-> vote_table; LRMprintf(" ==== VOTING TABLE ========================= \n"); int iii, jjj; for(iii=0; iii < LRMGENE_VOTE_TABLE_SIZE; iii++){ for(jjj = 0; jjj < v -> items[iii]; jjj++){ unsigned int this_pos = v -> pos[iii][jjj]; int votes = v -> votes[iii][jjj]; if(votes >= min_votes){ char postxt[100]; int conf_start = v -> coverage_start[iii][jjj] , conf_end = v -> coverage_end [iii][jjj]; LRMpos2txt(context, this_pos, postxt); LRMprintf(" %d (%s) %3d - %3d : %16s ", votes,(v -> masks[iii][jjj]&LRMIS_NEGATIVE_STRAND)?"NEG":"POS", conf_start, conf_end, postxt); int ix; for(ix=0; v->indel_recorder[iii][jjj][ix]; ix+=3){ LRMprintf(" %d ~ %d : %d ", v->indel_recorder[iii][jjj][ix] , v->indel_recorder[iii][jjj][ix+1], v->indel_recorder[iii][jjj][ix+2]); } LRMprintf("\n"); } } } LRMprintf(" =========================================== \n\n"); } Rsubread/src/LRMsorted-hashtable.h0000644000175200017520000000436514136047615020126 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #ifndef _SORTED_HASHTABLE_H_ #define _SORTED_HASHTABLE_H_ #include #include #include "LRMconfig.h" #define LRMGEHASH_DEFAULT_SIZE 2000000000 #define LRMGEHASH_BUCKET_LENGTH 2291 #define LRMinit_gene_vote(a) {memset((a)->items, 0, LRMGENE_VOTE_TABLE_SIZE*sizeof( *((a)->items))); } size_t LRMgehash_go_q(LRMgehash_t * the_table, LRMgehash_key_t key, int offset, int read_len, int is_reversed, LRMgene_vote_t * vote, int indel_tolerance, int subread_number); size_t LRMgehash_go_tolerance(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context, LRMgehash_t * the_table, LRMgehash_key_t key, int offset, int read_len, int is_reversed, LRMgene_vote_t * vote, int indel_tolerance, int subread_number, int max_MM); void LRMgehash_destory(LRMgehash_t * the_table); void LRMfinalise_vote(LRMgene_vote_t * vote); int LRMgehash_load(LRMgehash_t * the_table, const char fname []); void LRMassign_best_vote(LRMgene_vote_t * vote, int i, int j); void LRMselect_best_vote(LRMgene_vote_t * vote); void LRMgehash_sort(LRMgehash_t * the_table); int LRMgehash_load_option(const char fname [], int option_no, int * result); void LRMprint_v(LRMcontext_t * context, LRMread_iteration_context_t * iteration_context, int min_votes); size_t LRMgehash_go_QQ(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context, LRMgehash_t * the_table, LRMgehash_key_t key, int offset, int read_len, int is_reversed, LRMgene_vote_t * vote, int indel_tolerance, int subread_number); #endif Rsubread/src/Makevars0000644000175200017520000000024414142060654015630 0ustar00biocbuildbiocbuildSUBREAD_VER = "\"Rsubread 2.8.1\"" PKG_CFLAGS=-DMAKE_FOR_EXON -D SUBREAD_VERSION=${SUBREAD_VER} # -Wno-unused-result # -w -g PKG_LIBS=-lz -lpthread -DMAKE_FOR_EXON Rsubread/src/Makevars.FreeBSD0000644000175200017520000000021614136047615017045 0ustar00biocbuildbiocbuildPKG_CFLAGS=-DMAKE_FOR_EXON -D MACOS -D FREEBSD -D SUBREAD_VERSION=${SUBREAD_VER} # -w PKG_LIBS=-pthread -DMAKE_FOR_EXON -D MACOS -D FREEBSD Rsubread/src/Makevars.Linux0000644000175200017520000000020114136047615016724 0ustar00biocbuildbiocbuildPKG_CFLAGS=-DMAKE_FOR_EXON -D SUBREAD_VERSION=${SUBREAD_VER} # -Wno-unused-result # -w -g PKG_LIBS=-lz -lpthread -DMAKE_FOR_EXON Rsubread/src/Makevars.MacOS0000644000175200017520000000017114136047615016575 0ustar00biocbuildbiocbuildPKG_CFLAGS=-DMAKE_FOR_EXON -D MACOS -D SUBREAD_VERSION=${SUBREAD_VER} # -w PKG_LIBS=-lpthread -DMAKE_FOR_EXON -D MACOS Rsubread/src/Makevars.SunOS0000644000175200017520000000017114136047615016642 0ustar00biocbuildbiocbuildPKG_CFLAGS=-DMAKE_FOR_EXON -D MACOS -D SUBREAD_VERSION=${SUBREAD_VER} # -w PKG_LIBS=-lpthread -DMAKE_FOR_EXON -D MACOS Rsubread/src/Makevars.Windows0000644000175200017520000000020014136047615017256 0ustar00biocbuildbiocbuildPKG_CFLAGS=-DMAKE_FOR_EXON -D SUBREAD_VERSION=${SUBREAD_VER} -D_FILE_OFFSET_BITS=64 # -w PKG_LIBS=-lz -lpthread -DMAKE_FOR_EXON Rsubread/src/R_wrapper.c0000644000175200017520000004015614136047615016254 0ustar00biocbuildbiocbuild/*************************************************************** The Subread and Rsubread software packages are free software packages: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #include #include #include #include #include #if (R_VERSION >= R_Version(2,3,0)) #define R_INTERFACE_PTRS 1 #define CSTACK_DEFNS 1 #ifndef __MINGW32__ #include #endif #include #endif #include "HelperFunctions.h" #define PARAM_SPLITTOR "\027" // ========== This part of code is to run everything in child threads; the main thread is only for screen output ============ struct R_child_thread_run_opt{ int (*func)(int , char * []); char ** args; int n; }; void * R_child_thread_child(void * aa){ struct R_child_thread_run_opt *opts = aa; opts->func(opts->n, opts->args); free(opts); msgqu_notifyFinish(); return NULL; } void R_child_thread_run(int (*func)(int , char *[]), int n, char **args, int is_thread_mode){ msgqu_init(is_thread_mode); if(is_thread_mode){ struct R_child_thread_run_opt *opts = malloc(sizeof(struct R_child_thread_run_opt)); opts -> func = func; opts -> n = n; opts -> args = args; pthread_attr_t thread_attr; pthread_attr_init(&thread_attr); pthread_attr_setstacksize(&thread_attr,32*1024*1024); pthread_t thread; pthread_create(&thread, &thread_attr, R_child_thread_child , opts); msgqu_main_loop(); pthread_join(thread, NULL); pthread_attr_destroy(&thread_attr); msgqu_destroy(); }else{ //fprintf(stderr,"RUN_WITH_THR N=%d, ARGS=%p\n", n, args); func(n, args); } } // ========== END: main thread screen output ============ extern void retrieve_sequence(char ** input, char ** output_seq); extern void atgcContent(char ** input, char ** output, int *basewise); extern int cellCounts_main(int argc, char ** argv); extern int detectionCall(int argc, char ** argv); extern int main_junction(int argc,char ** argv); extern int main_align(int argc,char ** argv); extern int main_buildindex(int argc,char ** argv); extern int sam2bed(int argc,char *argv[]); extern int propmapped(int argc,char *argv[]); extern int readSummary(int argc,char *argv[]); extern int main_snp_calling_test(int argc,char *argv[]); extern int main_repeated_test(int argc, char *argv[]); extern int main_read_repair(int argc,char *argv[]); extern int main_qualityScores(int argc, char *argv[]); extern int findCommonVariants(int argc, char *argv[]); extern int longread_mapping_R(int argc, char *argv[]); extern int TxUniqueMain(int argc, char *argv[]); extern int R_flattenAnnotations(int argc, char *argv[]); extern int gen_rnaseq_reads_main(int argc, char *argv[]); extern int do_R_try_cell_barcode_files(int argc, char ** argv); extern int simRead_at_main(char *fasta_name, char *output_name, char *qualstr_name, int all_transcripts, char ** trans_names_unique, int *trans_ids, int *start_poses, int *fra_lens, int read_length, int total_reads, int simplify_names, int truth_in_rnames,int do_paired_reads ); void R_try_cell_barcode_wrapper(int * nargs, char ** argv, int * retv){ int i,n = *nargs; if(n!=5) { SUBREADprintf("ERROR: must be 5 arguments, not %d.\n" ,n); retv[0]=-1; return; } char * r_argv = strdup(*argv); char ** c_argv = (char **) calloc(n+1+4,sizeof(char *)); // 4 for return variables. for(i=0;i<1+n;i++) c_argv[i] = (char *)calloc(MAX_FILE_NAME_LENGTH*MAX_SCRNA_FASTQ_FILES,sizeof(char)); strcpy(c_argv[0],"R_cell_barcode"); strcpy(c_argv[1],strtok(r_argv,PARAM_SPLITTOR)); for(i=2;i #include #include #include #include #include #include #include #include #ifndef __MINGW32__ #include #endif #include #include #include #include #include #include #include #include "subread.h" #include "HelperFunctions.h" #include "seek-zlib.h" #include "input-files.h" #define TRANSCRIPT_FASTA_LINE_WIDTH 1000 #define MAX_SIMULATION_READ_LEN 250 #define TRANSCRIPT_FASTA_LINE_INIT 3000 #define warn_if_untrue(exp) if(!(exp)){\ Rprintf("ERROR: unsatisfied assertion in %s at %d\n",__FILE__, __LINE__);\ } typedef struct { int read_length; ArrayList * quality_strings; ArrayList * transcript_names; HashTable * transcript_lengths; HashTable * transcript_sequences; HashTable * expression_levels; char fake_quality_string[MAX_SIMULATION_READ_LEN+3]; int is_paired_end; int simple_transcript_names; int truth_in_read_names; gzFile out_fps[2]; } RsimReads_context_t; void Rgrc_sequencing_error_read(char * seq, int qlen, char * qua){ int b; for(b=0; b0); warn_if_untrue(seq_no<=grc -> transcript_names -> numOfElements); char * seq, *seq_name = ArrayListGet(grc -> transcript_names, seq_no-1); char read_seq [grc -> read_length+1]; int trans_len = HashTableGet(grc->transcript_lengths, seq_name)-NULL-1; warn_if_untrue(trans_len>0); warn_if_untrue(seq_start_pos + grc -> read_length <= trans_len); if(seq_start_pos + grc -> read_length>trans_len){ Rprintf("ERROR: seq %s has %d bases; wanted %d\n", seq_name, trans_len, seq_start_pos + grc -> read_length ); } seq = HashTableGet( grc -> transcript_sequences, seq_name); warn_if_untrue(seq); // Rprintf("Extract: chro=%s; pos=%d; seq=%.8s; rseq=%.8s\n" , seq_name , seq_start_pos , seq, seq+seq_start_pos); memcpy(read_seq, seq + seq_start_pos, grc -> read_length); read_seq[grc -> read_length]=0; if(trans_negative) reverse_read(read_seq , grc -> read_length , GENE_SPACE_BASE); char * qual_str = NULL; if(grc->quality_strings->numOfElements>0){ qual_str = ArrayListRandom(grc->quality_strings); //SUBREADprintf("TESTQUAL\t%p\n", qual_str); Rgrc_sequencing_error_read(read_seq, grc -> read_length, qual_str); }else{ if(!grc->fake_quality_string[0]){ int xx; for(xx = 0; xx < grc -> read_length; xx++) grc->fake_quality_string[xx]='H'; grc->fake_quality_string[xx]=0; } qual_str = grc->fake_quality_string; } gzFile thisfp = (is_PE_second==1) ? grc -> out_fps[1] : grc -> out_fps[0]; if(grc->truth_in_read_names){ int R1_pos = is_PE_second?mate_pos:seq_start_pos; int R2_pos = is_PE_second?seq_start_pos:mate_pos; if(is_PE_second<0) gzprintf(thisfp, "@R%09llu:%s:%d\n%s\n+\n%s\n", rno, seq_name, 1+seq_start_pos, read_seq, qual_str); else gzprintf(thisfp, "@R%09llu:%s:%d:%d\n%s\n+\n%s\n", rno, seq_name, 1+R1_pos, 1+R2_pos, read_seq, qual_str); } else gzprintf(thisfp, "@R%09llu\n%s\n+\n%s\n", rno, read_seq, qual_str); // Rprintf("Extract - 2: chro=%s; pos=%d; rseq=%.8s; qual=%.8s\n" , seq_name , seq_start_pos , read_seq, qual_str); } void Rgrc_put_new_trans(RsimReads_context_t *grc, char * seq_name, char * lbuf, int lbuf_len){ // Rprintf("Putting: chro=%s ; seq=%.8s\n", seq_name, lbuf); warn_if_untrue(lbuf_len>0); HashTablePut( grc->transcript_sequences , seq_name, lbuf ); HashTablePut( grc->transcript_lengths, strdup(seq_name) , NULL+lbuf_len +1); } int init_grc_by_file(RsimReads_context_t *grc, char *fasta_name, char *output_name, char *qualstr_name, char ** input_order_transcript_names, int * input_order_transcript_per_read, int input_transcripts, int read_length, int all_reads, int simplify_names, int truth_in_rnames, int do_PE_reads){ memset(grc, 0, sizeof(RsimReads_context_t)); grc->transcript_sequences = StringTableCreate(100000); HashTableSetDeallocationFunctions(grc->transcript_sequences, free, free); grc -> expression_levels = StringTableCreate(100000); HashTableSetDeallocationFunctions(grc->expression_levels, free, NULL); grc -> transcript_lengths = StringTableCreate(100000); HashTableSetDeallocationFunctions(grc -> transcript_lengths ,free, NULL); grc -> transcript_names = ArrayListCreate(100000); ArrayListSetDeallocationFunction(grc -> transcript_names,free); grc -> quality_strings = ArrayListCreate(50000); ArrayListSetDeallocationFunction(grc -> quality_strings,free); grc -> simple_transcript_names = simplify_names; grc -> read_length = read_length; grc -> truth_in_read_names = truth_in_rnames; grc -> is_paired_end = do_PE_reads; autozip_fp auto_FP; int xk1, lbuf_cap=0, lbuf_used=0, this_seq_len=-1, total_dup=0; char * lbuf=NULL, * seq_name = NULL; for(xk1 = 0; xk1 < input_transcripts ; xk1++){ // Rprintf("INSERT : %d = %s\n", xk1, input_order_transcript_names[xk1]); ArrayListPush(grc -> transcript_names , strdup(input_order_transcript_names[xk1])); HashTablePut(grc -> expression_levels, strdup(input_order_transcript_names[xk1]), NULL +1); //if(xk1<100) Rprintf("TNAME [%d] = %s\n", xk1, input_order_transcript_names[xk1]); } for(xk1 = 0; xk1 < all_reads; xk1++){ warn_if_untrue(input_order_transcript_per_read[xk1]>0); warn_if_untrue(input_order_transcript_per_read[xk1] <= input_transcripts); int to_display = 0; //if(input_order_transcript_per_read[xk1] > 206650) to_display = 1; if(to_display) Rprintf("Inp %d . Req %d . StrP %p =%s ; xk1=%d/%d\n", input_transcripts , input_order_transcript_per_read[xk1], input_order_transcript_names[input_order_transcript_per_read[xk1]-1], input_order_transcript_names[input_order_transcript_per_read[xk1]-1], xk1, all_reads); int ov = HashTableGet( grc -> expression_levels, input_order_transcript_names[input_order_transcript_per_read[xk1]-1] ) -NULL; warn_if_untrue(ov>0); ov++; HashTablePutReplace(grc -> expression_levels, input_order_transcript_names[input_order_transcript_per_read[xk1]-1], NULL+ov, 0); } HelpFuncMD5_CTX md5ctx; HelpFuncMD5_Init(&md5ctx); HashTable * seq_duplicate_tab = StringTableCreate(100000); HashTableSetDeallocationFunctions(seq_duplicate_tab, free, NULL); int ret = autozip_open(fasta_name, &auto_FP); while(1){ char clinebuf[TRANSCRIPT_FASTA_LINE_WIDTH]; int rlength = autozip_gets(&auto_FP, clinebuf, TRANSCRIPT_FASTA_LINE_WIDTH -1); if(rlength < 1)break; if(rlength >= TRANSCRIPT_FASTA_LINE_WIDTH -1 || clinebuf[rlength]!='\0' || clinebuf[rlength-1]!='\n'){ SUBREADprintf("Error: The line width of the fasta file excessed %d bytes.\n", TRANSCRIPT_FASTA_LINE_WIDTH); ret = 1; break; } if(clinebuf[0]=='>'){ if(NULL != seq_name){ char * md5mem = malloc(33); unsigned char md5res[16]; HelpFuncMD5_Final(md5res, &md5ctx); int md5i;for(md5i=0;md5i<16;md5i++)sprintf(md5mem+2*md5i, "%02X", 0xff&(int)md5res[md5i]); char * had_tab = HashTableGet(seq_duplicate_tab, md5mem); long seq_exp = HashTableGet(grc->expression_levels, seq_name)-NULL; if(had_tab && seq_exp>1) total_dup++;//SUBREADprintf("Warning: duplicate sequence was found in '%s' and '%s'.\n", seq_name, had_tab); if(seq_exp>1)HashTablePut(seq_duplicate_tab, md5mem, 1+NULL); else free(md5mem); had_tab = HashTableGet(grc-> transcript_sequences, seq_name); if(had_tab){ SUBREADprintf("Error: duplicate sequence names were found in the input: '%s'.n", seq_name); return -1; } Rgrc_put_new_trans(grc, seq_name, lbuf, this_seq_len); } clinebuf[rlength-1]=0; if(grc->simple_transcript_names) for(xk1=1; xk1 0 lbuf_used += rlength -1; this_seq_len = lbuf_used; } } if(lbuf_used<1){ SUBREADprintf("Error: The fasta file did not end correctly! \n"); ret = 1; } if(NULL != seq_name && lbuf_used >0){ char * md5mem = malloc(33); unsigned char md5res[16]; HelpFuncMD5_Final(md5res, &md5ctx); int md5i;for(md5i=0;md5i<16;md5i++)sprintf(md5mem+2*md5i, "%02X", 0xff&(int)md5res[md5i]); long seq_exp = HashTableGet(grc->expression_levels, seq_name)-NULL; char * had_tab = HashTableGet(seq_duplicate_tab, md5mem); if(had_tab && seq_exp>1)total_dup++;// SUBREADprintf("Warning: duplicate sequence was found in '%s' and '%s'.\n", seq_name, had_tab); free(md5mem); had_tab = HashTableGet(grc-> transcript_sequences, seq_name); if(had_tab){ SUBREADprintf("Error: duplicate sequence names were found in the input: '%s'.\n", seq_name); return -1; } Rgrc_put_new_trans(grc, seq_name, lbuf, this_seq_len); } if(total_dup)SUBREADprintf("Warning: there are %d transcripts that have replicate sequences and the wanted expression levels are non-zero. You may use scanFasta() to find their names.\n", total_dup); autozip_close(&auto_FP); HashTableDestroy(seq_duplicate_tab); if(qualstr_name && qualstr_name[0]){ ret = autozip_open(qualstr_name, &auto_FP); if(ret){ SUBREADprintf("Error: cannot open reference quality file '%s'\n", qualstr_name); return -1; } while(1){ char linebuf[MAX_SIMULATION_READ_LEN+3]; int rline = autozip_gets(&auto_FP, linebuf, MAX_SIMULATION_READ_LEN); if(rline<1) break; if(rline==grc->read_length +1 || ( rline==grc->read_length && linebuf[rline-1]!='\n' ) ) { }else{ SUBREADprintf("Error: all your quality strings must be %d-byte long.\n",read_length); return -1; } char * qstr = malloc(rline); memcpy(qstr, linebuf, rline); if(qstr[rline-1]=='\n') qstr[rline-1]=0; ArrayListPush(grc -> quality_strings, qstr); } autozip_close(&auto_FP); } char outname[MAX_FILE_NAME_LENGTH+30]; sprintf(outname,"%s_R1.fastq.gz", output_name); grc->out_fps[0] = gzopen(outname, "wb"); if(grc->is_paired_end){ sprintf(outname,"%s_R2.fastq.gz", output_name); grc->out_fps[1] = gzopen(outname, "wb"); }else grc->out_fps[1]=NULL; if(grc->out_fps[0] == NULL || ( grc->out_fps[1]== NULL && grc->is_paired_end )){ SUBREADprintf("Error: cannot create the output file.\n"); return -1; } return 0; } int destroy_Rsim_context(RsimReads_context_t *grc){ gzclose(grc->out_fps[0]); if(grc->out_fps[1]) gzclose(grc->out_fps[1]); HashTableDestroy(grc->transcript_sequences); HashTableDestroy(grc->expression_levels); HashTableDestroy(grc->transcript_lengths); ArrayListDestroy(grc->transcript_names); ArrayListDestroy(grc->quality_strings); return 0; } #define A_LARGE_PRIME_FOR_MOD 24537224085139llu int simRead_at_main(char *fasta_name, char *output_name, char *qualstr_name, int all_transcripts, char ** trans_names_unique, int *trans_ids, int *start_poses, int *fra_lens, int read_length, int total_reads, int simplify_names, int truth_in_rnames,int do_paired_reads ){ warn_if_untrue(read_length<=MAX_SIMULATION_READ_LEN); warn_if_untrue(total_reads>0); warn_if_untrue(all_transcripts>0); RsimReads_context_t grc; myrand_srand(0); // in R, the seed doesn't matter. unsigned long long read_pick_i = myrand_rand()&0xffff; read_pick_i = (0x10000llu * read_pick_i) +(myrand_rand()&0xffff); read_pick_i = (0x10000llu * read_pick_i) +(myrand_rand()&0xffff); read_pick_i = (0x10000llu * read_pick_i) +(myrand_rand()&0xffff); int retv = init_grc_by_file(&grc, fasta_name, output_name, qualstr_name, trans_names_unique, trans_ids, all_transcripts, read_length, total_reads, simplify_names, truth_in_rnames, do_paired_reads); if(retv==0){ long long int read_i; for(read_i = 0 ; read_i < total_reads; read_i ++){ read_pick_i %= 1llu * total_reads; warn_if_untrue(start_poses[read_pick_i] >0); int start_offset = start_poses[read_pick_i] -1; // it is 1-based from R! int end_offset = start_offset + fra_lens[read_pick_i]; int is_R1_at_3End = myrand_rand() % 2; int pos_small = start_offset; int pos_large = end_offset - read_length; Rgen_one_read_here(&grc, trans_ids[read_pick_i], is_R1_at_3End?pos_large:pos_small, grc.is_paired_end?0:-1, is_R1_at_3End, read_i, is_R1_at_3End?pos_small:pos_large); if(grc.is_paired_end)Rgen_one_read_here(&grc, trans_ids[read_pick_i], is_R1_at_3End?pos_small:pos_large, 1,!is_R1_at_3End, read_i, is_R1_at_3End?pos_large:pos_small); read_pick_i += A_LARGE_PRIME_FOR_MOD; } } retv = retv || destroy_Rsim_context(&grc); return 0; } Rsubread/src/SNPCalling.c0000644000175200017520000016600514136047615016247 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #include #include #include #include #include #include #include #include #include #include #include #include #include "subread.h" #include "hashtable.h" #include "HelperFunctions.h" #include "gene-algorithms.h" #include "SNPCalling.h" #include "input-files.h" #define HUGE_PILE_HEIGHT 300 #define SNP_INTERVAL_SUP 0 extern unsigned int BASE_BLOCK_LENGTH; float LOG_10_2 ; int (*snp_progress_report_callback) (int,int,int); struct SNP_Calling_Parameters{ float supporting_read_rate; int empty_blocks; int max_supporting_read_number; int min_supporting_read_number; int min_alternative_read_number; int neighbour_filter_testlen; float neighbour_filter_rate; int bases_ignored_head_tail; int all_blocks; int is_phred_64; int is_BAM_file_input; int is_paired_end_data; int is_coverage_calculation; int use_soft_clipping_bases; int fisher_exact_testlen; int min_phred_score; int excellent_phred_score; int final_phred_score; int test_two_strands; int neighbour_abundance_test; int snp_interval; float cutoff_multiplex ; float cutoff_upper_bound ; pthread_spinlock_t * output_fp_lock; char pile_file_name[MAX_FILE_NAME_LENGTH]; int delete_piles; int disk_is_full; char background_input_file[MAX_FILE_NAME_LENGTH]; char subread_index[MAX_FILE_NAME_LENGTH]; char known_SNP_vcf[MAX_FILE_NAME_LENGTH]; unsigned int known_SNPs_number; gene_offset_t * subread_index_offsets; gene_value_index_t * subread_index_array; HashTable * cigar_event_table; char * rebuilt_command_line; srInt_64 all_mapped_bases; unsigned int fisher_normalisation_target; double start_time; unsigned int real_read_count; unsigned int reported_SNPs; unsigned int reported_indels; int supporting_read_excessed_reported; }; #define PRECALCULATE_FACTORIAL 2000000 long double * precalculated_factorial;// [PRECALCULATE_FACTORIAL]; double factorial_float_real(int a) { double ret = 0; while(a) ret += log(a--); return ret; } double factorial_float(int a) { if(a * The code used to calculate a Fisher p-value comes originally from a * JavaScript program * by T. Kadosawa (kadosawa@niaes.affrc.go.jp). * Retrieved from http://www.users.zetnet.co.uk/hopwood/tools/StatTests.java on 3/Jul/2012 * * @author David Hopwood * @date 2000/04/23 */ double fisher_exact_test(int a, int b, int c, int d) { if(a*1./c < b*1./d) return 1.1; // the abnormal level at this base should be at least as the noise level. //printf("FET: %d %d %d %d\n", a, b, c, d); if(1){ double ret = fast_fisher_test_one_side(a,b,c,d, precalculated_factorial, PRECALCULATE_FACTORIAL); //#warning ">>>>>> FOR ACCURACY MEASUREMENT ONLY <<<<<<<<" //SUBREADprintf("FISHER_RES %d %d %d %d %.19G %.19G\n", a,b,c,d, ret, log(ret)); return ret; }else{ int AA=a, BB=b, CC=c, DD=d; if (a * d > b * c) { a = a + b; b = a - b; a = a - b; c = c + d; d = c - d; c = c - d; } if (a > d) { a = a + d; d = a - d; a = a - d; } if (b > c) { b = b + c; c = b - c; b = b - c; } double p_sum = 0.0; double p = fisherSub(a, b, c, d); while (a >= 0) { p_sum += p; if (a == 0) break; --a; ++b; ++c; --d; p = fisherSub(a, b, c, d); } if(1){ // DON'T PUT IT HERE! double r1 = fast_fisher_test_one_side(AA,BB,CC,DD, NULL, PRECALCULATE_FACTORIAL); if(abs(r1-p_sum) / max(r1,p_sum) >= 0.01){ printf("BADR: FAST = %.7f != JAVA %.7f, %d %d %d %d\n", log(r1), log(p_sum),AA,BB,CC,DD); }else{ printf("GOODR: FAST = %.7f ~= JAVA %.7f, %d %d %d %d\n", log(r1), log(p_sum),AA,BB,CC,DD); } } return p_sum; } } unsigned int fisher_test_size; void mask_snp_bitmap(char * bm, unsigned int pos) { int bytes = (pos >> 3); int bits = pos & 0x7; bm [bytes] |= (1 << bits); } int is_snp_bitmap(char * bm, unsigned int pos) { int bytes = (pos >> 3); int bits = pos & 0x7; return bm [bytes] & (1 << bits); } void put_hash_to_pile(HashTable * merge_table, unsigned int* snp_voting_piles, struct SNP_Calling_Parameters * parameters, char * chro, int block_start ) { int bucket; KeyValuePair * cursor; for(bucket=0; bucket< merge_table -> numOfBuckets; bucket++) { cursor = merge_table -> bucketArray[bucket]; while (1) { if (!cursor) break; int base_N_qual = cursor->value - NULL; int POI_pos = cursor->key - NULL - 100; int base1 = (base_N_qual>>8) & 0xff; int qual1 = (base_N_qual&0xff)-1; int j; unsigned int supporting_bases = 0; for(j=0; j<4; j++) { supporting_bases += snp_voting_piles[POI_pos*4+j]; } if(supporting_bases < parameters->max_supporting_read_number) { if(qual1 < (parameters -> is_phred_64?'@':'!')+parameters->min_phred_score){ // low quality bases // the low-seq-quality bases are not inclued in voting. } else snp_voting_piles[POI_pos*4+base1]+=1; }else{ if(parameters -> supporting_read_excessed_reported < 100){ parameters -> supporting_read_excessed_reported++; SUBREADprintf("Warning: the depth exceeded the limit of %d at %s : %d\n", parameters->max_supporting_read_number, chro , block_start + POI_pos); if(parameters -> supporting_read_excessed_reported == 100) SUBREADprintf("Too many warnings.\nNo more warning messages are reported.\n"); } } cursor = cursor->next; } } } int read_tmp_block(struct SNP_Calling_Parameters * parameters, FILE * tmp_fp, char ** SNP_bitmap_recorder, unsigned int * snp_voting_piles, int block_no, unsigned int reference_len, char * referenced_genome, char * chro_name, unsigned int offset) { int last_read_id=-1,i; HashTable * merge_table = HashTableCreate(1000); unsigned long long int OVERLAPPED_BASES=0; unsigned long long int OVER_MISMA_BASES=0; unsigned long long int ALL_BASES=0; while(!feof(tmp_fp)) { int type_char = fgetc(tmp_fp), rlen=-1; if(type_char == EOF ) break; fseek(tmp_fp, -1 , SEEK_CUR); if(type_char == 200) // SNP { VCF_temp_read_t SNP_rec; rlen = fread(&SNP_rec, sizeof(SNP_rec),1 , tmp_fp); if(rlen < 1){ SUBREADputs("ERROR: the temporary file is broken."); return -1; } if(!(*SNP_bitmap_recorder)) { (*SNP_bitmap_recorder)=malloc((reference_len/8)+200); memset((*SNP_bitmap_recorder), 0 , (reference_len/8)+200); } if(SNP_rec.pos > block_no * BASE_BLOCK_LENGTH && SNP_rec.pos <= block_no * BASE_BLOCK_LENGTH + reference_len ) mask_snp_bitmap((*SNP_bitmap_recorder), SNP_rec.pos - block_no * BASE_BLOCK_LENGTH - 1); parameters -> known_SNPs_number ++; //printf("SNPat: %u\n", SNP_rec.pos); } else if(type_char == 100) // read { base_block_temp_read_t read_rec; unsigned short read_len; unsigned int first_base_pos; char read[MAX_READ_LENGTH]; char qual[MAX_READ_LENGTH]; int rlen = fread(&read_rec, sizeof(read_rec), 1, tmp_fp); if(rlen < 1){ SUBREADputs("ERROR: the temporary file is broken."); return -1; } rlen = fread(&read_len, sizeof(short), 1, tmp_fp); if(rlen < 1){ SUBREADputs("ERROR: the temporary file is broken."); return -1; } rlen = fread(read, sizeof(char), read_len, tmp_fp); if(rlen < read_len){ SUBREADputs("ERROR: the temporary file is broken."); return -1; } rlen = fread(qual, sizeof(char), read_len, tmp_fp); if(rlen < read_len){ SUBREADputs("ERROR: the temporary file is broken."); return -1; } first_base_pos = read_rec.pos - block_no * BASE_BLOCK_LENGTH; parameters->is_paired_end_data = read_rec.flags & 1; //SUBREADprintf("Loading bases at %u ; len=%d\n", first_base_pos, read_len); if(first_base_pos + read_len -1> reference_len || first_base_pos<1) { SUBREADprintf("WARNING: read length %u+%d out of boundary: %u at the %d-th block.\n", first_base_pos, read_len, reference_len, block_no); continue; } if(parameters->is_paired_end_data){ if( (last_read_id >>1) != (read_rec.read_number>>1) && last_read_id>=0 && merge_table -> numOfElements > 0) { put_hash_to_pile(merge_table, snp_voting_piles, parameters,chro_name, offset); HashTableDestroy(merge_table); merge_table = HashTableCreate(1000); } } last_read_id = read_rec.read_number; for(i=0;imin_phred_score)continue; if(true_value=='N' || true_value=='.') continue; if(i+first_base_pos > reference_len || i+first_base_pos<1) { SUBREADprintf("Warning: read out of boundary: %u >= %u\n", i+first_base_pos, reference_len); break; } if(parameters->is_paired_end_data){ // to remove overlapped bases between the two ends of the reads. unsigned int POI_pos = 100 + first_base_pos+i-1; unsigned int original_value = HashTableGet(merge_table,NULL+POI_pos)-NULL; if(0 == original_value)// no value has been put to this location { int base_N_qual = base_int; base_N_qual = base_N_qual << 8 | (1+qual[i]); HashTablePut(merge_table, NULL+POI_pos, NULL+base_N_qual); ALL_BASES++; } else { // the value has been in the merge_table. ALL_BASES++; OVERLAPPED_BASES++; int old_qual = (original_value & 0xff) - 1; int old_base = (original_value >> 8) & 0xff; if(old_base != base_int)OVER_MISMA_BASES++; if(old_qual < qual[i]) { int base_N_qual = base_int; base_N_qual = base_N_qual << 8 | (1+qual[i]); HashTablePut(merge_table, NULL+POI_pos, NULL+base_N_qual); } } } else { // if it is single-end, just put the read into the piles. int j; unsigned int supporting_bases = 0; for(j=0; j<4; j++) { supporting_bases += snp_voting_piles[(first_base_pos+i-1)*4+j]; } if(supporting_bases < parameters->max_supporting_read_number) { if(qual[i] < (parameters -> is_phred_64?'@':'!')+parameters->min_phred_score) // low quality bases { // the low-seq-quality bases are not inclued in voting. } else { snp_voting_piles[(first_base_pos+i-1)*4+base_int]+=1; } }else{ if(parameters -> supporting_read_excessed_reported < 100){ parameters -> supporting_read_excessed_reported++; SUBREADprintf("Warning: the depth exceeded the limit of %d at %s : %d\n", parameters->max_supporting_read_number, chro_name , offset + first_base_pos+i); if(parameters -> supporting_read_excessed_reported == 100) SUBREADprintf("Too many warnings.\nNo more warning messages are reported.\n"); } } } } } else assert(0); } if(parameters->is_paired_end_data && merge_table -> numOfElements > 0) put_hash_to_pile(merge_table, snp_voting_piles, parameters, chro_name, offset); HashTableDestroy(merge_table); return 0; } void set_sample_MM(unsigned int * tumor_M, unsigned int *tumor_MM , unsigned int *snp_voting_piles, int i, int true_i){ int j; for(j=0;j<4;j++) { if(j==true_i)(*tumor_M)+=snp_voting_piles[4*i + j]; else (*tumor_MM)+=snp_voting_piles[4*i + j]; } } void fishers_test_on_POI(struct SNP_Calling_Parameters * parameters, float * snp_fisher_VS, unsigned int * snp_voting_piles,unsigned int * snp_BGC_piles, char * referenced_genome, unsigned int reference_len, float * tumor_P) { int i; for(i=0; i fisher_exact_testlen;i=0) { char true_value = referenced_genome[i]; int true_value_int = (true_value=='A'?0:(true_value=='C'?1:(true_value=='G'?2:3))); for(j=0;j<4;j++) { if(j == true_value_int) POIbase_MAT = snp_voting_piles[ i * 4 + j ] ; else POIbase_MM += snp_voting_piles[ i * 4 + j ] ; } } if(i+parameters -> fisher_exact_testlen < reference_len_long) { int mm=0, pm=0; char true_value = referenced_genome[i + parameters -> fisher_exact_testlen ]; int true_value_int = (true_value=='A'?0:(true_value=='C'?1:(true_value=='G'?2:3))); for(j=0;j<4;j++) { if(j == true_value_int) pm = snp_voting_piles[ (i+parameters -> fisher_exact_testlen ) *4 + j ] ; else mm += snp_voting_piles[ (i+parameters -> fisher_exact_testlen ) *4 + j ] ; } unsigned int chro_pos = block_start + i + parameters -> fisher_exact_testlen; if(0 && chro_pos < 16084550+12 && chro_pos > 16084550-12){ SUBREADprintf("ADD-BASE-AT-RIGHT : %s : %u : KNOWN-SNP : %d a b c d : %d %d %d %d A 4 > C : %d\n" , chro_name, chro_pos, is_snp_bitmap(SNP_bitmap_recorder,i + parameters -> fisher_exact_testlen), POIbase_MM , ALLbase_MM, POIbase_MAT, ALLbase_MAT, (POIbase_MM *4 >= POIbase_MAT)); } if((!SNP_bitmap_recorder)||(!is_snp_bitmap(SNP_bitmap_recorder, i + parameters -> fisher_exact_testlen))) { ALLbase_MAT += pm; ALLbase_MM += mm; } } // test the middle base // a : mismatched at POI ; c : matched at POI if(i>=0 && POIbase_MM > 0){ int Known_SNP_here = (SNP_bitmap_recorder!=NULL) && is_snp_bitmap(SNP_bitmap_recorder,i); //if(Known_SNP_here) // SUBREADprintf("SNP known at %s:%d\n" , chro_name, block_start + i); //all_SNPs_at_pois += (Known_SNP_here)?1:0; float observed_coverage = (ALLbase_MM + ALLbase_MAT) *1./(1. + 2 * parameters -> fisher_exact_testlen) ; double p_cutoff = pow(10, -(observed_coverage/multiplex_base)); p_cutoff = min(parameters -> cutoff_upper_bound, p_cutoff); p_cutoff = max(1E-323, p_cutoff); int flanking_unmatched = ALLbase_MM; int flanking_matched = ALLbase_MAT; if(SNP_bitmap_recorder == NULL || ! Known_SNP_here){ flanking_unmatched -= POIbase_MM; flanking_matched -= POIbase_MAT; } unsigned int chro_pos = block_start + i; if(0 && strcmp("8", chro_name)==0 && chro_pos < 144762490 + 12 && chro_pos > 144762490-12){ SUBREADprintf("KNOWN-SNP-AT-PoI at %s : %u : KNOWN-SNP : %d a b c d : %d %d %d %d A 4 > C : %d\n" , chro_name, chro_pos, is_snp_bitmap(SNP_bitmap_recorder,i), POIbase_MM, ALLbase_MM , POIbase_MAT, ALLbase_MAT, (POIbase_MM *4 >= POIbase_MAT)); } float p_middle = fisher_exact_test(POIbase_MM, flanking_unmatched, POIbase_MAT, flanking_matched); if(0 && chro_pos < 16084550+12 && chro_pos > 16084550-12){ SUBREADprintf("TEST: %s : %u a,b,c,d=%d %d %d %d; FU=%d FM=%d; Goahead=%d; Tailleft=%d; p=%G; p-cut=%G\n KNOWN-SNP : %d A 4 > C : %d\n", chro_name, chro_pos ,POIbase_MM, ALLbase_MM,POIbase_MAT, ALLbase_MAT , flanking_unmatched, flanking_matched, 0, 0, p_middle, p_cutoff, is_snp_bitmap(SNP_bitmap_recorder,i), (POIbase_MM *4 >= POIbase_MAT)); } //#warning " ===================== If a known SNP is at POI, the Fisher's p-value is only compared with the 'upper bound' of the p-value. =====================" //#warning " ===================== '1 &&' is the switch ========================" if(all_result_needed || ( 0 && Known_SNP_here && p_middle < parameters -> cutoff_upper_bound ) || ( p_middle < p_cutoff && flanking_matched*20>(flanking_matched+ flanking_unmatched )*16)) snp_fisher_raw [i] = p_middle; else snp_fisher_raw [i] = -999; if(flanking_unmatched <0) SUBREADprintf("ERROR_AB: A=%d, B=%d, C=%d, D=%d, flanking_unmatched=%d\n", POIbase_MM, ALLbase_MM , POIbase_MAT, ALLbase_MAT,flanking_unmatched); assert(flanking_unmatched>=0); assert(flanking_matched>=0); //if(strcmp(chro_name, "chr20")==0 && block_no == 3 && i == 57566366-1-3*15000000) if(snp_filter_background_unmatched){ snp_filter_background_unmatched[i] = flanking_unmatched; snp_filter_background_matched[i] = flanking_matched; } fisher_test_size ++; }else if(i>=0 && all_result_needed) snp_fisher_raw[i]=1.1; if(i >= parameters -> fisher_exact_testlen) { int mm=0, pm=0; char true_value = referenced_genome[i - parameters -> fisher_exact_testlen]; int true_value_int = (true_value=='A'?0:(true_value=='C'?1:(true_value=='G'?2:3))); for(j=0;j<4;j++) { if(j == true_value_int) pm = snp_voting_piles[ (i-parameters -> fisher_exact_testlen) *4 + j ] ; else mm += snp_voting_piles[ (i-parameters -> fisher_exact_testlen) *4 + j ] ; } if((!SNP_bitmap_recorder)||!is_snp_bitmap(SNP_bitmap_recorder, i - parameters -> fisher_exact_testlen)) { ALLbase_MAT-=pm; ALLbase_MM-=mm; } } } // SUBREADprintf("BLOCK_FINISH; SNP found = %d\n", all_SNPs_at_pois); } int process_snp_votes(FILE *out_fp, unsigned int offset , unsigned int reference_len, char * referenced_genome, char * chro_name , char * temp_prefix, struct SNP_Calling_Parameters * parameters) { int block_no = (offset -1) / BASE_BLOCK_LENGTH, i, disk_is_full = 0; char temp_file_name[MAX_FILE_NAME_LENGTH]; FILE *tmp_fp; unsigned int * snp_voting_piles, *snp_BGC_piles = NULL; // offset * 4 + "A/C/G/T"[0,1,2,3] char * SNP_bitmap_recorder = NULL; double multiplex_base = parameters -> fisher_normalisation_target / parameters -> cutoff_multiplex; float * snp_fisher_raw, *pcutoff_list, *snp_fisher_BGC=NULL, *snp_fisher_VS=NULL; unsigned short * snp_filter_background_unmatched; unsigned short * snp_filter_background_matched; long long int reference_len_long = reference_len; char * sprint_line; sprintf(temp_file_name , "%s%s-%04u.bin", temp_prefix, chro_name, block_no); tmp_fp = f_subr_open(temp_file_name, "rb"); // if no temp file is here, do nothing if(!tmp_fp){ parameters->empty_blocks ++; return 0; } sprint_line = malloc(1000); sprint_line[0]=0; LOG_10_2 = log(2) / log(10); //printf("I %s %d\n", chro_name, offset); snp_voting_piles = (unsigned int *)SUBREAD_malloc(sizeof(unsigned int) * reference_len*4); snp_filter_background_matched = (unsigned short *)SUBREAD_malloc(sizeof(unsigned short) * reference_len*4); snp_filter_background_unmatched = (unsigned short *)SUBREAD_malloc(sizeof(unsigned short) * reference_len*4); pcutoff_list = (float *)SUBREAD_malloc(sizeof(float) * reference_len); snp_fisher_raw = (float *)SUBREAD_malloc(sizeof(float) * reference_len); if((!snp_fisher_raw)) { fatal_memory_size(); return -1; } memset(snp_voting_piles,0 ,sizeof(unsigned int) * reference_len*4); for(i=0; i delete_piles) unlink(temp_file_name); if(parameters -> background_input_file[0]) { snp_BGC_piles = (unsigned int *)SUBREAD_malloc(sizeof(unsigned int) * reference_len*4); snp_fisher_BGC = (float *)SUBREAD_malloc(sizeof(float) * reference_len); snp_fisher_VS = (float *)SUBREAD_malloc(sizeof(float) * reference_len); memset(snp_BGC_piles, 0, sizeof(int) *4 * reference_len); memset(snp_fisher_BGC, 0, sizeof(float) * reference_len); memset(snp_fisher_VS, 0, sizeof(float) * reference_len); sprintf(temp_file_name , "%sBGC-%s-%04u.bin", temp_prefix, chro_name, block_no); tmp_fp = f_subr_open(temp_file_name, "rb"); if(tmp_fp) { int min_phred_score_raw = parameters -> min_phred_score; parameters -> min_phred_score -= 3; read_tmp_block(parameters, tmp_fp,NULL , snp_BGC_piles,block_no, reference_len, referenced_genome, chro_name, offset); parameters -> min_phred_score = min_phred_score_raw; } fclose(tmp_fp); unlink(temp_file_name); } // Finding SNPs from the finished voting table char * base_is_reliable = NULL; if(parameters -> fisher_exact_testlen || parameters -> test_two_strands || parameters->neighbour_abundance_test || 1) { base_is_reliable = (char *)malloc(sizeof(char) * reference_len); memset(base_is_reliable,1 , sizeof(char) * reference_len); } if(parameters->neighbour_abundance_test) { int window_size = 7; int window_no = 0; for(i= -window_size;i=window_size) for(j=0;j<4;j++) { window_no-=snp_voting_piles[(i-window_size)*4+j]; } } } if(parameters -> test_two_strands) { for(i= 0;i fisher_exact_testlen]; int true_value_int = (true_value=='A'?0:(true_value=='C'?1:(true_value=='G'?2:3))); for(j=0;j<4;j++) { if(j==true_value_int) { pos_match += snp_voting_piles[i*4+j]; } else { pos_unmatch += snp_voting_piles[i*4+j]; } } float p_diff = fisher_exact_test(pos_match, neg_match, pos_unmatch, neg_unmatch); if(p_diff<0.15) base_is_reliable[i]=0; } } if(parameters -> neighbour_filter_testlen ) { int j; int a=0, b=0, c=0, d=0; long long int reference_len_long = reference_len; /** | This | All_Window * ----+------+------- * #mm | a | b * #pm | c | d **/ for(i= - parameters -> neighbour_filter_testlen;i neighbour_filter_testlen < reference_len_long) { char true_value = referenced_genome[i + parameters -> neighbour_filter_testlen]; int true_value_int = (true_value=='A'?0:(true_value=='C'?1:(true_value=='G'?2:3))); if(j == true_value_int) d += snp_voting_piles[ (i+parameters -> neighbour_filter_testlen) *4 + j ] ; else b += snp_voting_piles[ (i+parameters -> neighbour_filter_testlen) *4 + j ] ; } if(i>=0) { char true_value = referenced_genome[i]; int true_value_int = (true_value=='A'?0:(true_value=='C'?1:(true_value=='G'?2:3))); if(j == true_value_int) c = snp_voting_piles[ i * 4 + j ] ; else a += snp_voting_piles[ i * 4 + j ] ; } } // test the middle base if(i>=0 && a > 0){ float window_correct_rate = (d-c)*1./(d+b-a-c); if(window_correct_rate < parameters->neighbour_filter_rate) { base_is_reliable[i] = 0; } } for(j=0;j<4;j++) if(i >= parameters -> neighbour_filter_testlen) { char true_value = referenced_genome[i - parameters -> neighbour_filter_testlen]; int true_value_int = (true_value=='A'?0:(true_value=='C'?1:(true_value=='G'?2:3))); if(j == true_value_int) d -= snp_voting_piles[ (i-parameters -> neighbour_filter_testlen) *4 + j ] ; else b -= snp_voting_piles[ (i-parameters -> neighbour_filter_testlen) *4 + j ]; } } } if(parameters -> fisher_exact_testlen) { fishers_test_on_block(parameters, snp_fisher_raw, snp_voting_piles, referenced_genome, reference_len, multiplex_base, SNP_bitmap_recorder, snp_filter_background_matched, snp_filter_background_unmatched, 0, chro_name, BASE_BLOCK_LENGTH*block_no +1); if(parameters->background_input_file[0]) { fishers_test_on_block(parameters, snp_fisher_BGC, snp_BGC_piles, referenced_genome, reference_len, multiplex_base, SNP_bitmap_recorder, NULL, NULL, 1, chro_name, BASE_BLOCK_LENGTH*block_no +1); fishers_test_on_POI(parameters, snp_fisher_VS, snp_voting_piles, snp_BGC_piles, referenced_genome, reference_len, snp_fisher_raw); } } for(i=0;imin_supporting_read_number || !all_reads) continue; //if(base_is_reliable && !(base_is_reliable[i])) continue; if(1) { unsigned int POI_MM = 0; base_list[0]=0; supporting_list[0]=0; for(tested_int=0; tested_int <4; tested_int ++) { if(tested_int != true_value_int) { int midNexcellent_sup = snp_voting_piles[i*4+tested_int]; POI_MM += midNexcellent_sup; if(midNexcellent_sup >= parameters->min_alternative_read_number) { char int_buf[12]; sprintf(int_buf, "%u", midNexcellent_sup); if(snps>0) { base_list[snps*2-1] = ','; base_list[snps*2] = tested_int==0?'A':(tested_int == 1?'C':(tested_int == 2?'G':'T')); base_list[snps*2+1] = 0; strcat(supporting_list,","); strcat(supporting_list,int_buf); } else { base_list[0] = tested_int==0?'A':(tested_int == 1?'C':(tested_int == 2?'G':'T')); base_list[1] = 0; strcpy(supporting_list, int_buf); } snps++; } } } // SUBREADprintf("OUTTEXT: i=%d, all_reads=%d, Fisher-p=%f, SNPs=%d, POI-MM=%d\n", i, all_reads, snp_fisher_raw[i], snps, POI_MM); if(snps && POI_MM *1. / all_reads >= parameters->supporting_read_rate ) { if(snp_fisher_raw[i] >= 0. || parameters -> fisher_exact_testlen < 1) { float Qvalue = -1.0*log(max(1E-40,snp_fisher_raw[i]))/log(10); char BGC_Qvalue_str [120]; BGC_Qvalue_str[0]=0; if(snp_fisher_BGC) { float BGC_Qvalue = -1.0*log(max(1E-40,snp_fisher_BGC[i]))/log(10); float VS_Qvalue = -1.0*log(max(1E-40,snp_fisher_VS[i]))/log(10); int BGC_all_reads = 0; int BGC_alt_reads = 0; for(tested_int=0; tested_int <4; tested_int ++) { BGC_all_reads += snp_BGC_piles[i*4+tested_int] ; if(tested_int != true_value_int) BGC_alt_reads += snp_BGC_piles[i*4+tested_int] ; } sprintf( BGC_Qvalue_str, ";CTRL_DP=%d;CTRL_MM=%d;CTRL_QV=%.4f;VS_QV=%.4f",BGC_all_reads, BGC_alt_reads, BGC_Qvalue,max(0,VS_Qvalue)); } snprintf(sprint_line,999, "%s\t%u\t.\t%c\t%s\t%.4f\t.\tDP=%d;MMsum=%d;MM=%s;BGTOTAL=%d;BGMM=%d%s\n", chro_name, BASE_BLOCK_LENGTH*block_no +1 + i, true_value,base_list, Qvalue, all_reads, POI_MM, supporting_list , snp_filter_background_matched[i]+snp_filter_background_unmatched[i], snp_filter_background_unmatched[i], BGC_Qvalue_str); if(parameters->output_fp_lock) subread_lock_occupy(parameters->output_fp_lock); int sprint_line_len = strlen(sprint_line); int wlen = fwrite(sprint_line, 1, sprint_line_len,out_fp); if(wlen < sprint_line_len){ disk_is_full=1; break; } parameters->reported_SNPs++; if(parameters->output_fp_lock) subread_lock_release(parameters->output_fp_lock); } } // check the indels right after the POI location char event_token[100]; char event_token2[100]; snprintf(event_token, 99, "%s\t%u", chro_name, BASE_BLOCK_LENGTH*block_no + 1 + i); long long int indel_no = HashTableGet(parameters -> cigar_event_table, event_token) - NULL; for(tested_int = 0; tested_int < indel_no; tested_int ++) { snprintf(event_token2, 99, "%s\t%u\t%d", chro_name, BASE_BLOCK_LENGTH*block_no + 1 + i, tested_int); long long int tmpv = HashTableGet(parameters -> cigar_event_table, event_token2) - NULL; long long int event_id = 0xffffff&(tmpv >> 8); int indels = (tmpv & 0xff) - 0x80; if(parameters->output_fp_lock) subread_lock_occupy(parameters->output_fp_lock); fprintf(out_fp, "%s\t.\t", event_token); fwrite(referenced_genome + i, max(0, indels) + 2, 1, out_fp); fwrite("\t", 1, 1, out_fp); if(indels<0) { char ** seq_tab = (char **)parameters -> cigar_event_table-> appendix1; char * ins_seq = seq_tab[event_id]; fwrite(referenced_genome + i, 1, 1, out_fp); fwrite(ins_seq, 1, -indels, out_fp); } else fwrite(referenced_genome + i, 1, 1, out_fp); fwrite(referenced_genome + 1 + i + max(0,indels), 1, 1, out_fp); unsigned short * indel_sups = parameters -> cigar_event_table-> appendix2; int wlen = fprintf(out_fp, "\t1.0\t.\tINDEL;DP=%d;SR=%d\n",all_reads,indel_sups[event_id]); if(wlen < 10){ disk_is_full=1; break; } parameters->reported_indels++; if(parameters->output_fp_lock) subread_lock_release(parameters->output_fp_lock); } } } if(base_is_reliable) free(base_is_reliable); if(SNP_bitmap_recorder) free(SNP_bitmap_recorder); free(snp_filter_background_matched); free(snp_filter_background_unmatched); free(snp_voting_piles); if(snp_BGC_piles){ free(snp_BGC_piles); free(snp_fisher_BGC); free(snp_fisher_VS); } free(snp_fisher_raw); free(pcutoff_list); free(sprint_line); //SUBREADprintf("OVERLAPPED=%llu; MISMA=%llu; ALL_BASES=%llu\n",OVERLAPPED_BASES, OVER_MISMA_BASES, ALL_BASES); return read_is_error || disk_is_full; } int run_chromosome_search(FILE *in_fp, FILE * out_fp, char * chro_name , char * temp_prefix, chromosome_t * chromosomes , struct SNP_Calling_Parameters* parameters, int * task_no, int thread_no, int all_threads) { int chro_no; unsigned int offset=0, all_offset = 0; unsigned int chro_len=0; char * referenced_base; referenced_base = (char *) SUBREAD_malloc(sizeof(char)* BASE_BLOCK_LENGTH + 10000); if(!referenced_base) { fatal_memory_size(); return -1; } for(chro_no=0;chro_no < XOFFSET_TABLE_SIZE; chro_no++) { if(!chromosomes[chro_no].chromosome_name[0]) break; if(strcmp(chro_name , chromosomes[chro_no].chromosome_name)==0) { chro_len = chromosomes[chro_no].known_length; break; } } //if (all_threads>1) // SUBREADprintf("Thread #%d is processing chromosome %s in FASTA file; expected length is %u.\n", thread_no, chro_name, chro_len); if(chro_len) { //SUBREADprintf("Processing chromosome %s in FASTA file; expected length is %u.\n", chro_name, chro_len); while( 1 || all_offset <= chro_len - 1) { char nc = fgetc(in_fp); if(nc == ' ' || nc == '\r' || nc == '\n') continue; if(nc == '>') fseek(in_fp, -1, SEEK_CUR); if(nc != EOF && nc != '>') { nc = toupper(nc); referenced_base[offset++] = (nc=='A' || nc == 'G' || nc == 'C')?nc:'T'; all_offset ++; } if((nc == '>'||nc == EOF) && all_offset < chro_len) SUBREADprintf("WARNING: Chromosome is shorter than expected: %s\n", chro_name); if(offset == BASE_BLOCK_LENGTH || nc == EOF || nc == '>') { if(nc!=EOF && nc!='>') { long long int back_seek = ftello(in_fp); int xk2; for(xk2=0; xk2<10000; xk2++) { char ncx = fgetc(in_fp); if(ncx == EOF || ncx == '>') break; if(ncx == '\r' || ncx=='\n') continue; referenced_base[offset+xk2] = (ncx=='A' || ncx == 'G' || ncx == 'C')?ncx:'T'; } fseeko(in_fp, back_seek, SEEK_SET); } if( (*task_no) % all_threads == thread_no && all_offset <= chro_len) { //#warning "=== ONLY TEST ONE BLOCK , USE 'if(1)' IN RELEASE ===" //if(strcmp(chro_name,"chr7")==0 && all_offset == 60000000){ if(1){ parameters -> disk_is_full |= process_snp_votes(out_fp, all_offset, offset, referenced_base, chro_name , temp_prefix, parameters); print_in_box(89,0,0,"processed block %c[36m%s@%d%c[0m by thread %d/%d [block number=%d/%d]", CHAR_ESC, chro_name, all_offset, CHAR_ESC , thread_no+1, all_threads, 1+(*task_no)-parameters->empty_blocks, parameters->all_blocks); } if(parameters -> disk_is_full)break; } else if((*task_no) % all_threads == thread_no) { print_in_box(80,0,0,"Ignored in: %s@%d by thr %d/%d [tid=%d]\n", chro_name, all_offset, thread_no, all_threads, *task_no); // SUBREADprintf("LEN %u > %u\n", all_offset, chro_len); } offset = 0; (*task_no)++; } if(nc == EOF || nc == '>'){ if(nc == '>') fseek(in_fp, -1, SEEK_CUR); break; } } //printf("Current indels=%ld\n", parameters->cigar_event_table->numOfElements); } else { if(thread_no==0) print_in_box(89,0,0,"WARNING chromosome %c[36m%s%c[0m is not in the SAM header.", CHAR_ESC, chro_name, CHAR_ESC); while(!feof(in_fp)) { long long int fp0 = ftello(in_fp); char * ret = fgets(referenced_base, 2000, in_fp); if(!ret) break; if(referenced_base[0]=='>') { fseeko(in_fp, fp0, SEEK_SET); break; } } } free(referenced_base); return 0; } // This scan is driven by reading the FASTA file. // While reading the FASTA file, if you see a new sequence, then find the base blocks for that sequence. int parse_read_lists(char * in_FASTA_file, FILE * out_fp, char * temp_prefix, chromosome_t * chromosomes, struct SNP_Calling_Parameters * parameters, int all_threads, int thread_no) { char line_buffer [3000]; int task_no = 0; FILE *fp = f_subr_open(in_FASTA_file,"r"); long long int FASTA_size = ftello(fp); if(!fp) { SUBREADprintf("Referenced Genome not found or inaccessible: '%s'.\n", in_FASTA_file); return -1; } while(!feof(fp)) { int linelen = read_line(2999, fp, line_buffer, 0); if(line_buffer[0] == '>') { char chro_name [MAX_CHROMOSOME_NAME_LEN]; int i; for(i=0; i< linelen -1; i++) { if(line_buffer[i+1] == ' ' || line_buffer[i+1] == '|' || line_buffer[i+1] == '\t') break; else chro_name[i] = line_buffer[min(MAX_CHROMOSOME_NAME_LEN-1,i+1)]; } chro_name[min(MAX_CHROMOSOME_NAME_LEN-1,i)]=0; // if(strcmp(chro_name,"chr20")==0) if(run_chromosome_search(fp, out_fp, chro_name , temp_prefix, chromosomes, parameters, &task_no, thread_no, all_threads)) return -1; float finished_rate = ftello(fp)*1./FASTA_size; if(snp_progress_report_callback) snp_progress_report_callback(40,10,3000+finished_rate*7000); } } fclose(fp); return 0; } struct parse_read_thread_context { int thread_id; int all_threads; struct SNP_Calling_Parameters* parameters; char * temp_prefix; char * in_FASTA_file; chromosome_t * chromosomes; pthread_spinlock_t init_lock; FILE * out_fp; }; void *parse_read_lists_wrapper(void * vparam) { struct parse_read_thread_context *param = (struct parse_read_thread_context *)vparam; int my_thread_id = param -> thread_id; pthread_spin_unlock(¶m -> init_lock); parse_read_lists(param -> in_FASTA_file, param -> out_fp, param -> temp_prefix, param -> chromosomes, param -> parameters , param -> all_threads, my_thread_id); return NULL; } int parse_read_lists_maybe_threads(char * in_FASTA_file, char * out_BED_file, char * temp_prefix, chromosome_t * chromosomes, struct SNP_Calling_Parameters* parameters, int all_threads) { FILE * out_fp = f_subr_open(out_BED_file,"w"); int ret=0; if(!out_fp){ SUBREADprintf("Cannot open the output file: '%s'\n", out_BED_file); } fputs("##fileformat=VCFv4.0\n",out_fp); fprintf(out_fp, "##exactSNP_Commandline=%s\n", parameters -> rebuilt_command_line); fputs("##comment=The QUAL values for the SNPs in this VCF file are calculated as min(40, - log_10 (p_value)), where p_value is from the Fisher's Exact Test. The QUAL values for the Indels in this VCF file are always 1.0.\n", out_fp); fputs("##INFO=\n", out_fp); fputs("##INFO=\n", out_fp); fputs("##INFO=\n", out_fp); fputs("##INFO=\n", out_fp); fputs("##INFO=\n", out_fp); fputs("##INFO=\n", out_fp); fputs("#CHROM\tPOS\tID\tREF\tALT\tQUAL\tFILTER\tINFO\n", out_fp); if(all_threads < 2) { ret = parse_read_lists(in_FASTA_file, out_fp, temp_prefix, chromosomes, parameters , all_threads, 0); } else { pthread_t runners [200]; struct parse_read_thread_context param; param.parameters = parameters; pthread_spin_init(¶m.init_lock, PTHREAD_PROCESS_PRIVATE); pthread_spin_lock(¶m.init_lock); param.out_fp = out_fp; param.in_FASTA_file = in_FASTA_file; param.chromosomes = chromosomes; param.all_threads = all_threads; param.temp_prefix = temp_prefix; parameters -> output_fp_lock = malloc(sizeof(*parameters -> output_fp_lock)); pthread_spin_init(parameters -> output_fp_lock , PTHREAD_PROCESS_PRIVATE); int i; for(i=0; i output_fp_lock); } //fprintf(out_fp, "## Fisher_Test_Size=%u\n",fisher_test_size); fclose(out_fp); if(parameters -> disk_is_full){ unlink(out_BED_file); SUBREADputs("ERROR: cannot write into the output VCF file. Please check the disk space in the output directory."); ret = 1; } return ret; } int guess_quality_threshold(char * qual_file, float percentage) { char buff[100]; unsigned long long readns [100], all_readn = 0; FILE * qfp = f_subr_open(qual_file, "r"); int lineno = 0; if(!qfp) { SUBREADprintf("Unable to open file: %s\n", qual_file); return 0; } memset(readns, 0, 100*sizeof(long long)); while(!feof(qfp)) { int pos=0; unsigned long long readn = 0; read_line(100, qfp, buff, 0); while(buff[pos]!='\t')pos++; pos++; for(;buff[pos] && buff[pos]!='\n'; pos++) { readn *=10; readn += buff[pos]-'0'; } readns[lineno]=readn; all_readn += readn; lineno ++; if(lineno>64)break; } fclose(qfp); long long read_threshold = all_readn * percentage; for(lineno=99; lineno>1; lineno--) { read_threshold -= readns[lineno]; if(read_threshold<=0){ SUBREADprintf("Phred score threshold has been shifted to %d\n", lineno); return lineno; } } return 0; } char * _EXSNP_SNP_delete_temp_prefix = NULL; void EXSNP_SIGINT_hook(int param) { #ifndef MAKE_STANDALONE if(param==0) { #endif int xk1, last_slash = -1; if(_EXSNP_SNP_delete_temp_prefix != NULL) { char del2[MAX_FILE_NAME_LENGTH], del_suffix[MAX_FILE_NAME_LENGTH], del_name[MAX_FILE_NAME_LENGTH]; #ifdef MAKE_STANDALONE if(param) SUBREADprintf("\n\nReceived a terminal signal. The temporary files were removed.\n"); #endif for(xk1=0; _EXSNP_SNP_delete_temp_prefix[xk1]; xk1++) { if(_EXSNP_SNP_delete_temp_prefix[xk1]=='/') last_slash = xk1; else if(_EXSNP_SNP_delete_temp_prefix[xk1]=='\\') { SUBREADprintf("The file name is unknown.\n"); return; } } if(last_slash>=0) { memcpy(del2, _EXSNP_SNP_delete_temp_prefix, last_slash); del2[last_slash] = 0; strcpy(del_suffix , _EXSNP_SNP_delete_temp_prefix + last_slash + 1); } else { strcpy(del2,"."); strcpy(del_suffix , _EXSNP_SNP_delete_temp_prefix); } if(strlen(del_suffix)>8) { DIR *d; struct dirent *dir; d = opendir(del2); if (d) { while ((dir = readdir(d)) != NULL) { if(strstr(dir->d_name, del_suffix)) { //printf("%s\n", dir->d_name); strcpy(del_name, del2); strcat(del_name, "/"); strcat(del_name, dir->d_name); unlink(del_name); } } closedir(d); } } } #ifdef MAKE_STANDALONE if(param) exit(param); #else } #endif } int SNP_calling(char * in_SAM_file, char * out_BED_file, char * in_FASTA_file, char * temp_location, unsigned int known_read_count, int threads, struct SNP_Calling_Parameters* parameters) { char temp_file_prefix[MAX_FILE_NAME_LENGTH]; chromosome_t * known_chromosomes; unsigned int real_read_count=0; int i, fpos; signal (SIGTERM, EXSNP_SIGINT_hook); signal (SIGINT, EXSNP_SIGINT_hook); parameters -> output_fp_lock = NULL; fisher_test_size = 0; precalculated_factorial = (long double*)malloc(sizeof(long double)*PRECALCULATE_FACTORIAL * 2); for(i=0; isubread_index[0]) { char table_fn[MAX_FILE_NAME_LENGTH+80]; parameters -> subread_index_offsets = (gene_offset_t*)malloc(sizeof(gene_offset_t)); load_offsets (parameters -> subread_index_offsets, parameters->subread_index); sprintf(table_fn,"%s.00.b.array", parameters->subread_index); parameters -> subread_index_array = (gene_value_index_t*) malloc(sizeof(gene_value_index_t)); if(gvindex_load(parameters -> subread_index_array,table_fn)) return -1; } //Step 1:The SAM file is scanned to create a number of temp files "temp-snps-chrX-21-00000000-XXXXXX" and the related read positions/sequences are written into them (in 2-bit base coding). A read can contribute to two blocks if it crosses the border of the blocks. If there are indels in a read, each continuously mapped section is individually written into the temporary file. The quality scores are written companying the bases. The hierarchy of the data is: block -> read -> sections {bases, phred scores, CIGAR string, reported mapping quality} if(parameters->pile_file_name[0]) { fpos=0; while(1) { int fpos0 = fpos; char one_fn [MAX_FILE_NAME_LENGTH]; while(in_SAM_file[fpos]!=',' && in_SAM_file[fpos]!=0) fpos++; strncpy(one_fn, in_SAM_file+fpos0, fpos-fpos0); one_fn[fpos-fpos0]=0; if(get_known_chromosomes(one_fn, known_chromosomes)) return -1; if(!in_SAM_file[fpos]) break; fpos++; } strcpy(temp_file_prefix, parameters->pile_file_name); } else { parameters-> cigar_event_table = HashTableCreate(11011); HashTableSetHashFunction(parameters-> cigar_event_table ,HashTableStringHashFunction); HashTableSetDeallocationFunctions(parameters-> cigar_event_table, NULL, NULL); HashTableSetKeyComparisonFunction(parameters-> cigar_event_table, my_strcmp); char mac_rand[13]; mac_or_rand_str(mac_rand); sprintf(temp_file_prefix, "%s/temp-snps-%06u-%s-", temp_location, getpid(), mac_rand); _EXSNP_SNP_delete_temp_prefix = temp_file_prefix; print_in_box(89,0,0,"Split %s file into %c[36m%s*%c[0m ..." , parameters -> is_BAM_file_input?"BAM":"SAM" , CHAR_ESC, temp_file_prefix, CHAR_ESC); fpos=0; while(1) { int fpos0 = fpos; char one_fn [MAX_FILE_NAME_LENGTH]; while(in_SAM_file[fpos]!=',' && in_SAM_file[fpos]!=0) fpos++; strncpy(one_fn, in_SAM_file+fpos0, fpos-fpos0); one_fn[fpos-fpos0]=0; if(break_SAM_file(one_fn, parameters -> is_BAM_file_input, temp_file_prefix, &real_read_count, ¶meters->all_blocks, known_chromosomes, 1, parameters -> bases_ignored_head_tail, parameters->subread_index_array, parameters->subread_index_offsets, ¶meters -> all_mapped_bases, parameters-> cigar_event_table, parameters->known_SNP_vcf, NULL, 0,0, parameters -> use_soft_clipping_bases)) return -1; if(!in_SAM_file[fpos]) break; fpos++; } if(parameters -> background_input_file[0]) { char temp_file_prefix2[MAX_FILE_NAME_LENGTH+80]; sprintf(temp_file_prefix2, "%sBGC-", temp_file_prefix); if(break_SAM_file(parameters -> background_input_file, parameters -> is_BAM_file_input, temp_file_prefix2, NULL, NULL, known_chromosomes, 1, parameters -> bases_ignored_head_tail, parameters->subread_index_array, parameters->subread_index_offsets, NULL, NULL, NULL, NULL, 0,0, parameters -> use_soft_clipping_bases)) return -1; } } parameters -> real_read_count = real_read_count; char qfname[MAX_FILE_NAME_LENGTH+40]; sprintf(qfname, "%s.qStatic", temp_file_prefix); parameters -> final_phred_score = 0; if (parameters -> delete_piles) unlink(qfname); //Step 2:Each base blocks are load from the FASTA file into memory, then each temp file is scanned to create the SNP voting table. The sections in the temp files are scanned to create the SNP voting table. The voting table is compared with the sequences in the FASTA file to determine if each base is a SNP. The result is written into the bed file immediatly. if(parse_read_lists_maybe_threads(in_FASTA_file, out_BED_file, temp_file_prefix, known_chromosomes, parameters, threads)) return -1; free(known_chromosomes); free(precalculated_factorial); if(parameters->subread_index_array) { gvindex_destory(parameters->subread_index_array); free(parameters->subread_index_array); free(parameters->subread_index_offsets); } destroy_cigar_event_table(parameters-> cigar_event_table); return 0; } void print_usage_snp(char * myname) { SUBREADprintf("\nVersion %s\n\n", SUBREAD_VERSION); SUBREADputs("Usage:"); SUBREADputs(""); SUBREADputs(" ./exactSNP [options] -i input -g reference_genome -o output "); SUBREADputs(""); SUBREADputs("Required arguments:"); SUBREADputs(""); SUBREADputs(" -i Specify name of an input file including read mapping results. The"); SUBREADputs(" [-b if BAM] format of input file can be SAM or BAM (-b needs to be specified"); SUBREADputs(" if a BAM file is provided)."); SUBREADputs(""); SUBREADputs(" -g Specify name of the file including all reference sequences. Only"); SUBREADputs(" one single FASTA format file should be provided."); SUBREADputs(""); SUBREADputs(" -o Specify name of the output file. This program outputs a VCF format"); SUBREADputs(" file that includes discovered SNPs."); SUBREADputs(""); SUBREADputs("Optional arguments:"); SUBREADputs(""); SUBREADputs(" -a Provide a set of annotated SNPs (e.g. SNPs included in the dbSNP"); SUBREADputs(" database). The supplied file should be in VCF format (gzipped file"); SUBREADputs(" is accepted). Providing known SNPs to the program should improve"); SUBREADputs(" its SNP calling performance. Note that the provided SNPs may or"); SUBREADputs(" may not be called."); SUBREADputs(""); SUBREADputs(" -b Indicate the input file provided via -i is in BAM format."); SUBREADputs(""); SUBREADputs(" -Q Specify the q-value cutoff for SNP calling at sequencing depth of"); SUBREADputs(" 50X. 12 by default. The corresponding p-value cutoff is 10^(-1*Q)."); SUBREADputs(" Note that this program automatically adjusts the q-value cutoff"); SUBREADputs(" according to the sequencing depth at each chromosomal location."); SUBREADputs(""); SUBREADputs(" -f Specify the minimum fraction of mis-matched bases a SNP-containing"); SUBREADputs(" location must have. Its value must between 0 and 1. 0 by default."); SUBREADputs(""); SUBREADputs(" -n Specify the minimum number of mis-matched bases a SNP-containing"); SUBREADputs(" location must have. 1 by default."); SUBREADputs(""); SUBREADputs(" -r Specify the minimum number of mapped reads a SNP-containing"); SUBREADputs(" location must have (ie. the minimum coverage). 1 by default."); SUBREADputs(""); SUBREADputs(" -x Specify the maximum depth a SNP location is allowed to have."); SUBREADputs(" 1,000,000 reads by default. This option is useful for removing PCR"); SUBREADputs(" artefacts."); SUBREADputs(""); SUBREADputs(" -s Specify the minimum base quality scores (Phred scores) read bases"); SUBREADputs(" must satisfy to be used for SNP calling. 13 by default. Read bases"); SUBREADputs(" with quality scores less than 13 will be excluded from the"); SUBREADputs(" analysis."); SUBREADputs(""); SUBREADputs(" -t Specify the number of bases trimmed off from each end of the read."); SUBREADputs(" 3 by default."); SUBREADputs(""); SUBREADputs(" -T Specify the number of threads. 1 by default."); SUBREADputs(""); SUBREADputs(" -v output version of the program."); SUBREADputs(""); SUBREADputs(" -C Specify the path to save the temporary files."); SUBREADputs(""); SUBREADputs("Example:"); SUBREADputs(""); SUBREADputs(" ./exactSNP -i my-alignment.sam -g mm10.fa -o my-SNPs.txt"); SUBREADputs(""); } static struct option snp_long_options[]={ {"coverage-calc", no_argument ,0,'4'}, {0,0,0,0} }; #ifdef MAKE_STANDALONE int main(int argc,char ** argv) { snp_progress_report_callback = NULL; #else int main_snp_calling_test(int argc,char ** argv) { #endif int xk1 = 0; int ret = 0; for(xk1=0;xk1<1;xk1++){ int c; char in_SAM_file[5000]; char out_BED_file[MAX_FILE_NAME_LENGTH]; char temp_path[MAX_FILE_NAME_LENGTH]; char in_FASTA_file[MAX_FILE_NAME_LENGTH]; int threads, optindex=0; int t=0, k; struct SNP_Calling_Parameters parameters; unsigned int read_count; in_SAM_file [0] = 0; in_FASTA_file [0] = 0; out_BED_file[0] = 0; temp_path[0] = 0; read_count = 0; threads = 1; optind = 0; opterr = 1; optopt = 63; memset(¶meters, 0, sizeof(struct SNP_Calling_Parameters)); rebuild_command_line(¶meters . rebuilt_command_line, argc, argv); parameters.start_time = miltime(); parameters.empty_blocks = 0; parameters.reported_SNPs = 0; parameters.reported_indels = 0; parameters.supporting_read_rate = 0.; parameters.min_supporting_read_number = 1; parameters.min_alternative_read_number = 1; parameters.max_supporting_read_number = 1000000; parameters.neighbour_filter_testlen = -1; parameters.neighbour_filter_rate = 0.000000001; parameters.min_phred_score = 13; parameters.fisher_exact_testlen = 5; parameters.test_two_strands = 0; parameters.snp_interval = 0; parameters.excellent_phred_score = 39; parameters.bases_ignored_head_tail = 3; parameters.neighbour_abundance_test = 0; parameters.pile_file_name[0]=0; parameters.delete_piles = 1; parameters.subread_index[0]=0; parameters.subread_index_offsets=NULL; parameters.subread_index_array=NULL; parameters.is_phred_64 = 0; parameters.is_coverage_calculation = 0; parameters.is_BAM_file_input = 0; parameters.cutoff_multiplex = 12.f; parameters.cutoff_upper_bound = 5E-3f; parameters.all_mapped_bases = 0; parameters.fisher_normalisation_target = 50; parameters.known_SNP_vcf[0]=0; parameters.known_SNPs_number=0; parameters.background_input_file[0]=0; if(argc<2) { print_usage_snp(argv[0]); return 0; } while ((c = getopt_long (argc, argv, "S7:N:C:a:i:g:o:bQ:p:f:n:r:x:w:s:t:T:v4",snp_long_options, &optindex))!=-1) { switch (c) { case 'S': parameters.use_soft_clipping_bases = 1; break; case 'N': strcpy(parameters.background_input_file, optarg); break; case 'a': strcpy(parameters.known_SNP_vcf, optarg); break; case 'v': core_version_number("exactSNP"); return 0; case 'b': parameters.is_BAM_file_input = 1; break; case 'Q': parameters.cutoff_multiplex = 1.0*atoi(optarg); break; case 'p': parameters.cutoff_upper_bound = atof(optarg); break; case 'n': parameters.min_alternative_read_number = atoi(optarg); break; case '2': // UNUSED parameters.neighbour_abundance_test=0; break; case 't': parameters.bases_ignored_head_tail = atoi(optarg); break; case 'w': parameters.fisher_exact_testlen = atoi(optarg); break; case 's': parameters.min_phred_score = atoi(optarg); break; case '3': // UNUSED k=strlen(optarg); for(t=0;t optind){ SUBREADprintf("Invalid parameter '%s'\n", argv[optind]); return -1; } if(out_BED_file[0]==0 || in_FASTA_file[0]==0 || (parameters.pile_file_name [0] == 0 && in_SAM_file[0]==0)) { SUBREADprintf("The names of the input file, the output file and the reference sequence file must be specified using -i, -o and -g options.\n"); return -1; } FILE * tfp = f_subr_open(out_BED_file,"w"); if(!tfp) { SUBREADprintf("Cannot write the output file. Please check if the disk has space and you can create file in the current directory.\n"); return -1; } fclose(tfp); tfp = f_subr_open(in_FASTA_file,"r"); if(!tfp) { SUBREADprintf("Cannot open the reference sequence file: %s\n", in_FASTA_file); return -1; } fclose(tfp); if(in_SAM_file[0]) { tfp = f_subr_open(in_SAM_file,"r"); if(!tfp) { SUBREADprintf("Cannot open the input file: %s\n", in_SAM_file); return -1; } fclose(tfp); } SUBREADputs(""); print_subread_logo(); SUBREADputs(""); print_in_box(80,1,1,"exactSNP setting"); print_in_box(80,0,1,""); print_in_box(80,0,0," Input file : %s (%s)", get_short_fname(in_SAM_file), parameters.is_BAM_file_input?"BAM":"SAM"); print_in_box(80,0,0," Output file : %s", get_short_fname(out_BED_file)); print_in_box(80,0,0," Reference genome : %s", get_short_fname(in_FASTA_file)); print_in_box(80,0,0," Temp path : %s", temp_path); print_in_box(80,0,1,""); print_in_box(80,0,0," Threads : %d", threads); print_in_box(80,0,0," Min supporting reads : %d", parameters.min_supporting_read_number); print_in_box(81,0,0,"Min pct. of supporting reads : %.1f%%%%", 100* parameters.supporting_read_rate); print_in_box(80,0,0," Min base quality score : %d", parameters.min_phred_score); print_in_box(80,0,0," Number of trimmed bases : %d", parameters.bases_ignored_head_tail); print_in_box(80,0,1,""); print_in_box(80,0,0," Q value cutoff : 10E-%.1f", parameters.cutoff_multiplex); print_in_box(80,0,0," P value upper bound : %.5f", parameters.cutoff_upper_bound); print_in_box(80,0,0," Flanking windows size : %d", parameters.fisher_exact_testlen); if(parameters.known_SNP_vcf[0]) print_in_box(80,0,0," Known SNP annotations : %s", get_short_fname(parameters.known_SNP_vcf)); print_in_box(80,0,1,""); print_in_box(80,2,1,""); SUBREADputs(""); char tbuf[90]; char_strftime(tbuf); print_in_box(80,1,1,"Running (%s)", tbuf); print_in_box(80,0,1,""); warning_file_type(in_SAM_file, parameters.is_BAM_file_input?FILE_TYPE_BAM:FILE_TYPE_SAM); warning_file_type(in_FASTA_file, FILE_TYPE_FASTA); warning_file_limit(); int x1; if(temp_path[0]==0){ // temp path = out_dir for(x1 = strlen(out_BED_file); x1 >= 0; x1--){ if(out_BED_file[x1]=='/'){ memcpy(temp_path, out_BED_file, x1); temp_path[x1]=0; break; } } if(temp_path[0]==0)strcpy(temp_path, "./"); } ret = SNP_calling(in_SAM_file, out_BED_file, in_FASTA_file, temp_path, read_count, threads, ¶meters); if(ret != -1) { print_in_box(80,0,1,""); print_in_box(89,0,1,"%c[36mCompleted successfully.%c[0m",CHAR_ESC, CHAR_ESC); print_in_box(80,0,1,""); print_in_box(80,2,1,""); SUBREADputs(""); print_in_box(80,1,1,"Summary"); print_in_box(80,0,1,""); if(parameters.known_SNPs_number) print_in_box(80,0,0," Known SNPs : %u", parameters.known_SNPs_number); print_in_box(80,0,0," Processed reads : %u", parameters.real_read_count); print_in_box(80,0,0," Reported SNPs : %u", parameters.reported_SNPs); print_in_box(80,0,0," Reported indels : %u", parameters.reported_indels); print_in_box(80,0,1,""); print_in_box(80,0,0," Running time : %.1f minutes", (miltime() - parameters.start_time)/60); print_in_box(80,0,1,""); print_in_box(80,2,1,""); SUBREADputs(""); } EXSNP_SIGINT_hook(0); } // XK1 END return ret; } Rsubread/src/SNPCalling.h0000644000175200017520000000144614136047615016251 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #ifndef __SINGLE_NUCLEODITE_POLYMOREPHISM_H_ #define __SINGLE_NUCLEODITE_POLYMOREPHISM_H_ #endif Rsubread/src/aligner.c0000644000175200017520000000000614136047615015722 0ustar00biocbuildbiocbuild//SPA Rsubread/src/atgcContent.c0000644000175200017520000000655414136047615016570 0ustar00biocbuildbiocbuild/*************************************************************** The Subread and Rsubread software packages are free software packages: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #define _GNU_SOURCE #include #include #include #include #include long int total_app[5]; long int app[5][1000]; int get_index(size_t pt){ int index; char* a = (char *)pt; switch (*a) { case 'A': case 'a': index = 0; break; case 'T': case 't': index = 1; break; case 'G': case 'g': index = 2; break; case 'C': case 'c': index = 3; break; default: index = 4; break; } return index; } void initialise(){ int p,q; for(p=0; p<5; p++){ total_app[p] = 0; for(q=0;q<1000;q++){ app[p][q] = 0; } } } void retrieve_sequence(char ** input, char ** output_seq){ char * line = NULL; // size_t len = 0; // ssize_t z; int i; // int readlen; // int offset; int line_num = 0; // int data_ready=1; int MAX_LINE_LENGTH = 100000; FILE *fin,*fout; fin = fopen(*input,"r"); fout = fopen(*output_seq,"w"); line = (char*)calloc(MAX_LINE_LENGTH, 1); while (fgets(line, MAX_LINE_LENGTH, fin)){ line_num++; if(line[0] == '@'){ char * flin = fgets(line, MAX_LINE_LENGTH, fin); if(NULL == flin) continue; line_num++; i=0; while ((toascii(*(line+i))!=10) && (toascii(*(line+i))!=32)){ fprintf(fout, "%c",*(line+i)); i++; } fprintf(fout,"\n"); } } if (line) free(line); fclose(fin); fclose(fout); } void atgcContent(char ** input, char ** output, int *basewise){ FILE *fin, *fout; char * line = NULL; // size_t len = 0; // ssize_t z; int i,j; int readlen=1; int line_num=0; int index; double freq[5]; double total_freq[5]; int MAX_LINE_LENGTH = 100000; line = (char*)calloc(MAX_LINE_LENGTH, 1); initialise(); //corresponding index: // "A"-0 "T"-1 "G"-2 "C"-3 others-4 fin = fopen(*input,"r"); fout = fopen(*output, "w"); fprintf(fout,"A,T,G,C,N\n"); while (fgets(line, MAX_LINE_LENGTH, fin)){ line_num++; i=0; while ((toascii(*(line+i)) != 10) && (toascii(*(line+i)) != 32)){ index = get_index((size_t)(line+i)); app[index][i]++; total_app[index]++; i++; } readlen = i; } if (line) free(line); for(i=0; i<5; i++){ total_freq[i] =(((double)total_app[i]/readlen)/line_num); } fprintf(fout, "%2.4f,%2.4f,%2.4f,%2.4f,%2.4f\n",total_freq[0], total_freq[1], total_freq[2], total_freq[3], total_freq[4]); if (*basewise==1){ for(i=0; i #include #include #include #include #ifndef __MINGW32__ #include #endif #include #include #include #include "core.h" #include "core-bigtable.h" #include "gene-algorithms.h" #define TABLE_PRELOAD_SIZE 24 #define calc_offset(pair_NO) ((pair_NO) * (1 + global_context -> input_reads.is_paired_end_reads) + is_second_read) * (sizeof(short) * 3 * global_context -> config.big_margin_record_size + (sizeof(mapping_result_t) + global_context -> config.do_breakpoint_detection*sizeof(subjunc_result_t)) * global_context -> config.multi_best_reads) void bigtable_lock(global_context_t * global_context){ subread_lock_occupy(&global_context -> bigtable_lock); } void bigtable_unlock(global_context_t * global_context){ subread_lock_release(&global_context -> bigtable_lock); } unsigned int get_handling_thread_number(global_context_t * global_context , subread_read_number_t pair_number){ /* if(global_context -> config.all_threads<2) return 0; for(ret = 0; ret < global_context -> config.all_threads; ret++){ if(global_context -> input_reads.start_read_number_blocks[ret] > pair_number) return ret - 1; } return global_context -> config.all_threads - 1; */return 0; } #define get_inner_pair(a,b) (b) bigtable_cached_result_t * bigtable_retrieve_cache(global_context_t * global_context , thread_context_t * thread_context , subread_read_number_t pair_number, int is_second_read, int load_more); void bigtable_readonly_result(global_context_t * global_context , thread_context_t * thread_context , subread_read_number_t pair_number, int result_number, int is_second_read, mapping_result_t * return_ptr, subjunc_result_t * return_junction_ptr){ int rlen = -1; if(global_context -> bigtable_cache_file_fp){ int loadjunc; long long inner_pair_number = get_inner_pair(global_context, pair_number); if(global_context -> bigtable_cache_file_loaded_fragments_begin >=0){ bigtable_write_thread_cache(global_context); global_context -> bigtable_cache_file_loaded_fragments_begin = -1; } for(loadjunc = 0; loadjunc < 2; loadjunc++){ if(loadjunc){ if(!return_junction_ptr) continue; } else{ if(!return_ptr) continue; } unsigned long long offset = calc_offset(inner_pair_number); offset += sizeof(short) * 3 * global_context -> config.big_margin_record_size ; if(loadjunc) offset += sizeof(mapping_result_t) * global_context -> config.multi_best_reads + sizeof(subjunc_result_t) * result_number; else offset += sizeof(mapping_result_t) * result_number; fseeko(global_context -> bigtable_cache_file_fp , offset , SEEK_SET); void * write_ptr = return_ptr; if(loadjunc) write_ptr = return_junction_ptr; rlen = fread(write_ptr, loadjunc?sizeof(subjunc_result_t):sizeof(mapping_result_t), 1, global_context -> bigtable_cache_file_fp); if(rlen <1) SUBREADprintf("UNABLE TO READ RESULT\n"); } }else{ bigtable_cached_result_t * rett = bigtable_retrieve_cache(global_context , thread_context , pair_number, is_second_read,0); int best_offset = result_number; if(return_ptr)memcpy(return_ptr, rett -> alignment_res + best_offset, sizeof(mapping_result_t)); if(return_junction_ptr)memcpy(return_junction_ptr, rett -> subjunc_res + best_offset, sizeof(subjunc_result_t)); } } int init_bigtable_results(global_context_t * global_context, int is_rewinding) { if(global_context -> config.use_memory_buffer) { global_context -> bigtable_chunked_fragments = global_context -> config.reads_per_chunk+1; global_context -> bigtable_cache_size = global_context -> bigtable_chunked_fragments * (1+global_context -> input_reads.is_paired_end_reads); } else { global_context -> bigtable_chunked_fragments = 110llu*1024*1024; global_context -> bigtable_cache_size = (1+0*global_context -> config.all_threads) * global_context -> bigtable_chunked_fragments * (1+global_context -> input_reads.is_paired_end_reads); } //SUBREADprintf("reads_per_chunk = %u ; cached_single_reads = %u ; size of each read = %d + %d\n", global_context -> config.reads_per_chunk, global_context -> bigtable_cache_size, sizeof(mapping_result_t) , sizeof(subjunc_result_t)); // if(is_rewinding){ memset( global_context -> bigtable_cache_malloc_ptr, 0, sizeof(mapping_result_t) * global_context -> config.multi_best_reads * global_context -> bigtable_cache_size ); if(global_context -> config.do_breakpoint_detection) memset(global_context -> bigtable_cache_malloc_ptr_junc , 0, sizeof(subjunc_result_t) * global_context -> config.multi_best_reads * global_context -> bigtable_cache_size); }else{ global_context -> bigtable_cache = malloc(sizeof(bigtable_cached_result_t) * global_context -> bigtable_cache_size); global_context -> bigtable_cache_malloc_ptr = calloc( sizeof(mapping_result_t) , global_context -> config.multi_best_reads * global_context -> bigtable_cache_size ); if(global_context -> config.do_breakpoint_detection) global_context -> bigtable_cache_malloc_ptr_junc = calloc(sizeof(subjunc_result_t) , global_context -> config.multi_best_reads * global_context -> bigtable_cache_size); assert(NULL != global_context -> bigtable_cache_malloc_ptr ); } int xk1; if(!is_rewinding) for(xk1 = 0; xk1 < global_context -> bigtable_cache_size; xk1++){ global_context -> bigtable_cache [xk1].alignment_res = (mapping_result_t*) global_context -> bigtable_cache_malloc_ptr + global_context -> config.multi_best_reads * xk1; if(global_context -> config.do_breakpoint_detection) global_context -> bigtable_cache [xk1].subjunc_res = (subjunc_result_t*)global_context -> bigtable_cache_malloc_ptr_junc + global_context -> config.multi_best_reads * xk1; } if(global_context->config.do_big_margin_filtering_for_junctions)for(xk1 = 0; xk1 < global_context -> bigtable_cache_size; xk1++) memset(global_context -> bigtable_cache [xk1].big_margin_data, 0, sizeof(global_context -> bigtable_cache [xk1].big_margin_data)); subread_init_lock(&global_context -> bigtable_lock); assert(global_context -> config.use_memory_buffer); global_context -> bigtable_cache_file_fp = NULL; return 0; } mapping_result_t * _global_retrieve_alignment_ptr(global_context_t * global_context, subread_read_number_t pair_number, int is_second_read, int best_read_id){ mapping_result_t * ret; bigtable_retrieve_result(global_context, NULL, pair_number, best_read_id, is_second_read, &ret, NULL); return ret; } subjunc_result_t * _global_retrieve_subjunc_ptr(global_context_t * global_context, subread_read_number_t pair_number, int is_second_read, int best_read_id){ subjunc_result_t * ret; bigtable_retrieve_result(global_context, NULL, pair_number, best_read_id, is_second_read, NULL, &ret); return ret; } #define calc_file_location(pair_no) (pair_no)* (1 + global_context -> input_reads.is_paired_end_reads) * (sizeof(short) * 3 * global_context -> config.big_margin_record_size + (sizeof(mapping_result_t) + global_context -> config.do_breakpoint_detection*sizeof(subjunc_result_t)) * global_context -> config.multi_best_reads) void bigtable_write_thread_cache(global_context_t * global_context){ assert(global_context -> bigtable_cache_file_fp == NULL); return; } void wait_occupied(global_context_t * global_context , unsigned long long old_begin){ while(old_begin == global_context -> bigtable_cache_file_loaded_fragments_begin){ int i, all_released = 1; for(i=0; i< global_context -> bigtable_chunked_fragments ; i++) { if(global_context -> bigtable_cache[i].status == CACHE_STATUS_OCCUPIED) all_released = 0; } if(all_released) break; } } bigtable_cached_result_t * bigtable_retrieve_cache(global_context_t * global_context , thread_context_t * thread_context , subread_read_number_t pair_number, int is_second_read, int load_more) { long long inner_pair_number = get_inner_pair(global_context, pair_number); long long load_start_pair_no = inner_pair_number - inner_pair_number % global_context -> bigtable_chunked_fragments; bigtable_cached_result_t * ret_cache = global_context -> bigtable_cache + (inner_pair_number - load_start_pair_no)* (1+global_context -> input_reads.is_paired_end_reads) + is_second_read; return ret_cache; } int bigtable_retrieve_result(global_context_t * global_context , thread_context_t * thread_context , subread_read_number_t pair_number, int result_number, int is_second_read, mapping_result_t ** return_ptr, subjunc_result_t ** return_junction_ptr) { bigtable_cached_result_t * cache = bigtable_retrieve_cache(global_context, thread_context, pair_number, is_second_read, 0); int best_offset = result_number; if(return_ptr)(*return_ptr)= cache -> alignment_res + best_offset; if(return_junction_ptr)(*return_junction_ptr)= cache -> subjunc_res + best_offset; return 0; } unsigned short * _global_retrieve_big_margin_ptr(global_context_t * global_context, subread_read_number_t pair_number, subread_read_number_t is_second_read){ bigtable_cached_result_t * cache = bigtable_retrieve_cache(global_context, NULL, pair_number, is_second_read, 0); return cache -> big_margin_data; } void bigtable_release_result(global_context_t * global_context , thread_context_t * thread_context , subread_read_number_t pair_number, int commit_change){ assert(NULL == "File result cache is no longer used. Do not call this function."); } int finalise_bigtable_results(global_context_t * global_context){ assert(global_context -> bigtable_cache_file_fp == NULL); free(global_context -> bigtable_cache_malloc_ptr); if(global_context -> config.do_breakpoint_detection) free(global_context -> bigtable_cache_malloc_ptr_junc); free(global_context -> bigtable_cache); return 0; } void bktable_key_deallocator(void * key){ free(key); } void bktable_bucket_deallocator(void * vbuk){ bucketed_table_bucket_t * buk = vbuk; free(buk -> positions); free(buk -> details); free(buk); } void bktable_init(bucketed_table_t * tab, unsigned int maximum_interval_length, unsigned int expected_items){ memset(tab, 0, sizeof(bucketed_table_t)); tab -> expected_items = expected_items; tab -> maximum_interval_length = maximum_interval_length; tab -> entry_table = HashTableCreate(expected_items / 3); HashTableSetDeallocationFunctions(tab->entry_table, bktable_key_deallocator, bktable_bucket_deallocator); HashTableSetKeyComparisonFunction(tab->entry_table, fc_strcmp_chro); HashTableSetHashFunction(tab->entry_table, HashTableStringHashFunction); } void bktable_destroy(bucketed_table_t * tab){ HashTableDestroy(tab -> entry_table); } void bktable_append(bucketed_table_t * tab, char * chro, unsigned int pos, void * detail){ unsigned int twokeys[2], keyi; //assert(detail != NULL); if(detail == NULL) return; twokeys[0] = pos - pos % tab -> maximum_interval_length; if(twokeys[0] > tab -> maximum_interval_length) twokeys[1] = twokeys[0] - tab -> maximum_interval_length; else twokeys[1] = 0xffffffff; for(keyi = 0; keyi < 2 ; keyi++) { char static_key [20 + MAX_CHROMOSOME_NAME_LEN]; unsigned int curr_key_pos = twokeys[keyi]; if(curr_key_pos == 0xffffffff) continue; sprintf(static_key, "%s:%u", chro, curr_key_pos); bucketed_table_bucket_t * had_items = HashTableGet(tab -> entry_table, static_key); //SUBREADprintf("INSERT STATIC_KEY [%d] = %s ; FOUND = %p ; ITEMS=%d\n", keyi, static_key, had_items, had_items?had_items->items:-1); if(NULL == had_items) { had_items = malloc(sizeof(bucketed_table_bucket_t)); memset(had_items, 0, sizeof(bucketed_table_bucket_t)); had_items -> capacity = BUCKETED_TABLE_INIT_ITEMS; had_items -> positions = malloc(sizeof(int) * BUCKETED_TABLE_INIT_ITEMS); had_items -> details = malloc(sizeof(void *) *BUCKETED_TABLE_INIT_ITEMS); had_items -> keyed_bucket = curr_key_pos; had_items -> maximum_interval_length = tab -> maximum_interval_length; char * dynamic_key = malloc(strlen(static_key) + 1); strcpy(dynamic_key, static_key); HashTablePut(tab->entry_table, dynamic_key, had_items); } if(had_items -> capacity <= had_items -> items){ had_items -> capacity = max(had_items -> capacity + 5, had_items -> capacity * 1.3); had_items -> positions = realloc(had_items -> positions, had_items -> capacity * sizeof(int)); had_items -> details = realloc(had_items -> details, had_items -> capacity * sizeof(void *)); } had_items -> positions[ had_items -> items ] = pos; had_items -> details[ had_items -> items ] = detail; had_items -> items++; } tab -> fragments ++; } void bktable_free_ptrs(void * bukey, void * buckv, HashTable * tab){ int x1; bucketed_table_bucket_t * buck = buckv; for(x1 = 0; x1 < buck -> items; x1++) { if(buck->positions[x1] - buck->positions[x1] % buck -> maximum_interval_length == buck -> keyed_bucket) { //SUBREADprintf("FREE : %u ~ %u\n", buck->positions[x1] , buck -> keyed_bucket); free(buck->details[x1]); } } } int bktable_lookup(bucketed_table_t * tab, char * chro, unsigned int start_pos, unsigned int interval_length, unsigned int * hit_pos_list, void ** hit_ptr_list, int max_hits){ unsigned int my_key_pos; my_key_pos = start_pos - start_pos % tab -> maximum_interval_length; char static_key [20 + MAX_CHROMOSOME_NAME_LEN]; sprintf(static_key, "%s:%u", chro, my_key_pos); bucketed_table_bucket_t * had_items = HashTableGet(tab -> entry_table, static_key); //if(strcmp(chro, "X")==0 && abs(start_pos - 21067381 - 500) < 10){ // SUBREADprintf("LOOK STATIC_KEY = %s, BUCK=%p, ITEMS=%d\n", static_key, had_items, had_items?had_items->items:-1); //} if(!had_items) // no bucket at all. return 0; int item_i, ret = 0; for(item_i = 0 ; item_i < had_items->items; item_i++){ unsigned int potential_pos = had_items -> positions[item_i]; //if(strcmp(chro, "X")==0 && abs(start_pos - 21067381 - 500) < 10){ // SUBREADprintf("POTENTIAL_POS=%u, ACCEPT=%u ~ %u\n", potential_pos, start_pos, start_pos + interval_length); //} if(potential_pos >= start_pos && potential_pos < start_pos + interval_length) { hit_pos_list[ret] = potential_pos; hit_ptr_list[ret] = had_items -> details[item_i]; ret ++; if(ret >= max_hits) break; } } return ret; } void fraglist_init(fragment_list_t * list){ memset(list, 0, sizeof(fragment_list_t)); list -> capacity = FRAGMENT_LIST_INIT_ITEMS; list -> fragment_numbers = malloc(sizeof(subread_read_number_t) * list -> capacity); } void fraglist_destroy(fragment_list_t * list){ free(list -> fragment_numbers); } void fraglist_append(fragment_list_t * list, subread_read_number_t fragment_number){ if(list -> fragments >= list -> capacity){ //#warning "============== COMMENT DEBUG INFO =====================" //SUBREADprintf("REALLOC_PRT_IN = %d , %p\n",list -> capacity , list -> fragment_numbers ); list -> capacity = max(list -> capacity + 5, list -> capacity * 1.3); list -> fragment_numbers = realloc(list -> fragment_numbers, sizeof(subread_read_number_t) * list -> capacity); // SUBREADprintf("REALLOC_PRT_OUT = %d , %p\n",list -> capacity , list -> fragment_numbers ); } list -> fragment_numbers[ list -> fragments ++ ] = fragment_number; } Rsubread/src/core-bigtable.h0000644000175200017520000000462514136047615017020 0ustar00biocbuildbiocbuild#ifndef __CORE_BIGTABLE_H_ #define __CORE_BIGTABLE_H_ #include "subread.h" #include "core.h" #include "hashtable.h" #include "gene-algorithms.h" #define CACHE_STATUS_RELEASED 0 #define CACHE_STATUS_OCCUPIED 1 // This function creates an empty data structure for all results. // The number of reads is unknown at this stage. int init_bigtable_results(global_context_t * global_context, int is_rewind); // This function tries to retrieve the required result data structure into memory and set the return_ptr to the address of the data structure. // Junction ptr can be NULL. // This function returns ZERO if the record is available. It returns -1 if the record is unavailable. int bigtable_retrieve_result(global_context_t * global_context , thread_context_t * thread_context , subread_read_number_t pair_number, int result_number, int is_second_read, mapping_result_t ** return_ptr, subjunc_result_t ** return_junction_ptr); // This function notifies the bigtable subsystem to save changes and deallocate the memory block if necessary. // Junction ptr can be NULL. // If the data has been changed, commit_change must be set to an non-ZERO value void bigtable_release_result(global_context_t * global_context , thread_context_t * thread_context , subread_read_number_t pair_number, int commit_change); // This function destroies the buffers and deletes the temporary MMAP files. int finalise_bigtable_results(global_context_t * global_context); void bigtable_readonly_result(global_context_t * global_context , thread_context_t * thread_context , subread_read_number_t pair_number, int result_number, int is_second_read, mapping_result_t * return_ptr, subjunc_result_t * return_junction_ptr); void bktable_append(bucketed_table_t * tab, char * chro, unsigned int pos, void * detail); int bktable_lookup(bucketed_table_t * tab, char * chro, unsigned int start_pos, unsigned int interval_length, unsigned int * hit_pos_list, void ** hit_ptr_list, int max_hits); void bktable_init(bucketed_table_t * tab, unsigned int maximum_interval_length, unsigned int expected_items); void bktable_destroy(bucketed_table_t * tab); void bktable_free_ptrs(void * bkey, void * buckv, HashTable * tab); void fraglist_init(fragment_list_t * list); void fraglist_append(fragment_list_t * list, subread_read_number_t fragment_number); void fraglist_destroy(fragment_list_t * list); void bigtable_write_thread_cache(global_context_t * global_context); #endif Rsubread/src/core-indel.c0000644000175200017520000055426014136047615016342 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #include #include #include #include #include #include #include #include "hashtable.h" #include "HelperFunctions.h" #include "gene-value-index.h" #include "gene-algorithms.h" #include "input-files.h" #include "core.h" #include "core-indel.h" #include "core-junction.h" #include "core-bigtable.h" #include "sublog.h" //#define indel_debug //#define is_target_window_X(x) 0 int DIGIN_COUNTER=0; int FRESH_COUNTER=0; extern unsigned int BASE_BLOCK_LENGTH; void is_bad_read(char * r, int len, char * w) { } void merge_event_sides(void * arr, int start, int items, int items2) { void ** arrl = (void **)arr; chromosome_event_t * event_space = (chromosome_event_t*) arrl[1]; int * event_id_list = (int *)arrl[0]; int is_small_side = (arrl[2]!=NULL); int * merge_tmp = malloc(sizeof(int)* (items+items2)); int x1, first_cursor = start, second_cursor = start+items; for(x1=0;x1= start+items) choose_2 = 1; else if(second_cursor < start+items+items2) { chromosome_event_t * levent = event_space + event_id_list[first_cursor]; chromosome_event_t * revent = event_space + event_id_list[second_cursor]; if(is_small_side){ if(levent->event_small_side > revent ->event_small_side) choose_2 = 1; }else { if(levent->event_large_side > revent ->event_large_side) choose_2 = 1; } } if(choose_2) merge_tmp[x1] = event_id_list[second_cursor++]; else merge_tmp[x1] = event_id_list[first_cursor++]; } memcpy(event_id_list + start, merge_tmp, sizeof(int)* (items+items2)); free(merge_tmp); } void exchange_event_sides(void * arr, int l, int r) { void ** arrl = (void **)arr; int * event_id_list = (int *)arrl[0]; int tmp ; tmp = event_id_list[l]; event_id_list[l] = event_id_list[r]; event_id_list[r] = tmp; } int compare_event_inner(chromosome_event_t * event_space, int l, int r, int is_small_side) { chromosome_event_t * revent = event_space +r; chromosome_event_t * levent = event_space +l; if(is_small_side){ if(levent->event_small_side event_small_side) return -1; else if(levent->event_small_side ==revent ->event_small_side) return 0; else return 1; }else { if(levent->event_large_side event_large_side) return -1; else if(levent->event_large_side ==revent ->event_large_side) return 0; else return 1; } } int compare_event_sides(void * arr, int l, int r) { void ** arrl = (void **)arr; chromosome_event_t * event_space = (chromosome_event_t*) arrl[1]; int * event_id_list = (int *)arrl[0]; int is_small_side = (arrl[2]!=NULL); return compare_event_inner(event_space, event_id_list[l], event_id_list[r], is_small_side); } int BINsearch_event(chromosome_event_t * event_space, int * event_ids, int is_small_side, unsigned int pos, int events) { int imax, imin; assert(events > 0); imax = events - 1; imin = 0; while (1) { int imid = (imin+ imax)/2; unsigned tested_mid_pos; //assert(imaxevent_small_side:mid_event->event_large_side; if(tested_mid_pos == pos) return imid; else if(tested_mid_pos < pos) imin = imid +1; else imax = imid -1; if(imin > imax) return imax; } } typedef struct{ int thread_id; int block_start; int block_end; HashTable * result_tab; int * small_side_ordered_event_ids, * large_side_ordered_event_ids; chromosome_event_t * event_space; global_context_t * global_context; } AT_context_t; #define ANTI_SUPPORTING_READ_LIMIT 100 void * anti_support_thread_run(void * v_cont){ AT_context_t * atcont = v_cont; int * cancelled_event_list = malloc(sizeof(int)*ANTI_SUPPORTING_READ_LIMIT), x2; int * small_side_ordered_event_ids = atcont -> small_side_ordered_event_ids, * large_side_ordered_event_ids = atcont -> large_side_ordered_event_ids; global_context_t * global_context = atcont -> global_context; chromosome_event_t * event_space = atcont -> event_space; indel_context_t * indel_context = (indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]; if(indel_context->total_events<1)return NULL; int current_read_number, cancelled_events, x1; for(current_read_number = atcont -> block_start; current_read_number < atcont -> block_end; current_read_number++) { int is_second_read; for (is_second_read = 0; is_second_read < 1 + global_context -> input_reads.is_paired_end_reads; is_second_read ++) { int best_read_id; for(best_read_id = 0; best_read_id < global_context -> config.multi_best_reads; best_read_id++) { mapping_result_t *current_result = _global_retrieve_alignment_ptr(global_context, current_read_number, is_second_read, best_read_id); if(current_result -> selected_votes<1) break; if(!global_context->config.report_multi_mapping_reads)if(current_result -> result_flags & CORE_IS_BREAKEVEN) continue; //if(current_result -> result_flags & CORE_IS_GAPPED_READ) continue; if(current_result->selected_votes < global_context->config.minimum_subread_for_first_read) continue; cancelled_events=0; unsigned int mapping_head = current_result -> selected_position; unsigned int coverage_start = mapping_head + current_result -> confident_coverage_start; unsigned int coverage_end = mapping_head + current_result -> confident_coverage_end; int small_side_left_event = BINsearch_event(event_space, small_side_ordered_event_ids,1, coverage_start - 1, indel_context->total_events) + 1; int large_side_left_event = BINsearch_event(event_space, large_side_ordered_event_ids,0, coverage_start - 1, indel_context->total_events) + 1; int small_side_right_event = BINsearch_event(event_space, small_side_ordered_event_ids,1, coverage_end, indel_context->total_events)+20; int large_side_right_event = BINsearch_event(event_space, large_side_ordered_event_ids,0, coverage_end, indel_context->total_events)+20; for(x1 = small_side_left_event ; x1 <= small_side_right_event ; x1++) { if(x1 >= indel_context->total_events) break; if(ANTI_SUPPORTING_READ_LIMIT <= cancelled_events) break; chromosome_event_t * event_body = event_space+ small_side_ordered_event_ids[x1]; if(event_body -> event_small_side <= coverage_start + 5) continue; if(event_body -> event_small_side >= coverage_end - 5) continue; long long cur_count = HashTableGet(atcont -> result_tab , NULL+small_side_ordered_event_ids[x1]+1 ) - NULL; cur_count++; HashTablePut( atcont -> result_tab , NULL+small_side_ordered_event_ids[x1]+1 , NULL+cur_count ); cancelled_event_list[cancelled_events++] = small_side_ordered_event_ids[x1]; } for(x1 = large_side_left_event ; x1 <= large_side_right_event ; x1++) { if(x1 >= indel_context->total_events) break; chromosome_event_t * event_body = event_space+ large_side_ordered_event_ids[x1]; if(event_body -> event_large_side <= coverage_start + 5) continue; if(event_body -> event_large_side >= coverage_end- 5) continue; int to_be_add = 1; for(x2=0; x2 event_small_side, event_body -> event_large_side, event_body -> indel_length, event_body -> anti_supporting_reads); long long cur_count = HashTableGet(atcont -> result_tab , NULL+large_side_ordered_event_ids[x1]+1 ) - NULL; cur_count++; HashTablePut( atcont -> result_tab , NULL+large_side_ordered_event_ids[x1]+1 , NULL+cur_count ); } } } } } free(cancelled_event_list); return NULL; } void anti_support_add_count(void * ky, void * va, HashTable * tab){ chromosome_event_t * event_space = tab -> appendix1 ; int eno = ky-NULL-1; chromosome_event_t * event_body = event_space + eno; event_body -> anti_supporting_reads+= (va - NULL); } int anti_supporting_read_scan(global_context_t * global_context) { indel_context_t * indel_context = (indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]; if(indel_context->total_events<1)return 0; chromosome_event_t * event_space = indel_context -> event_space_dynamic; int x1, * small_side_ordered_event_ids, * large_side_ordered_event_ids; small_side_ordered_event_ids = malloc(sizeof(int)*indel_context -> total_events); large_side_ordered_event_ids = malloc(sizeof(int)*indel_context -> total_events); for(x1=0; x1total_events; x1++) { small_side_ordered_event_ids[x1]=x1; large_side_ordered_event_ids[x1]=x1; } void * sort_data[3]; sort_data[0] = small_side_ordered_event_ids; sort_data[1] = event_space; sort_data[2] = (void *)0xffff; // small_side_ordering merge_sort(sort_data, indel_context->total_events, compare_event_sides, exchange_event_sides, merge_event_sides); sort_data[0] = large_side_ordered_event_ids; sort_data[2] = NULL; // large_side_ordering merge_sort(sort_data, indel_context->total_events, compare_event_sides, exchange_event_sides, merge_event_sides); AT_context_t ATconts[64]; pthread_t AThreads[64]; int thread_no, last_end = 0; for(thread_no=0; thread_no< global_context -> config.all_threads; thread_no++){ AT_context_t * atc = ATconts + thread_no; atc -> thread_id = thread_no; atc -> block_start = last_end; atc -> block_end = last_end = global_context -> processed_reads_in_chunk / global_context -> config.all_threads * thread_no; if(thread_no == global_context -> config.all_threads-1) atc -> block_end = global_context -> processed_reads_in_chunk; atc -> global_context = global_context; atc -> result_tab = HashTableCreate(200000); atc -> small_side_ordered_event_ids = small_side_ordered_event_ids; atc -> large_side_ordered_event_ids = large_side_ordered_event_ids; atc -> event_space = event_space; pthread_create(AThreads + thread_no , NULL, anti_support_thread_run, atc); } for(thread_no=0; thread_no< global_context -> config.all_threads; thread_no++){ pthread_join(AThreads[thread_no], NULL); ATconts[thread_no].result_tab -> appendix1 = event_space; HashTableIteration( ATconts[thread_no].result_tab, anti_support_add_count ); HashTableDestroy(ATconts[thread_no].result_tab); } free(small_side_ordered_event_ids); free(large_side_ordered_event_ids); return 0; } chromosome_event_t * reallocate_event_space( global_context_t* global_context,thread_context_t * thread_context,int event_no) { int max_event_no; if(thread_context) { max_event_no = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> current_max_event_number; if(max_event_no<=event_no) { ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> current_max_event_number *= 1.6; ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> event_space_dynamic = realloc(((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> event_space_dynamic, sizeof(chromosome_event_t) * ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> current_max_event_number); } return ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; } else { max_event_no = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> current_max_event_number; if(max_event_no<=event_no) { ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> current_max_event_number *= 1.6; ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_space_dynamic = realloc(((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_space_dynamic, sizeof(chromosome_event_t) * ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> current_max_event_number); } return ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; } } int event_neighbour_sort_compare(void * arr, int l, int r){ unsigned int ** sort_data = (unsigned int **) arr; if(sort_data[1][l] > sort_data[1][r]) return 1; if(sort_data[1][l] < sort_data[1][r]) return -1; return 0; } void event_neighbour_sort_exchange(void * arr, int l, int r){ unsigned int ** sort_data = (unsigned int **) arr; unsigned int tmpi; tmpi = sort_data[0][l]; sort_data[0][l] = sort_data[0][r]; sort_data[0][r] = tmpi; tmpi = sort_data[1][l]; sort_data[1][l] = sort_data[1][r]; sort_data[1][r] = tmpi; } // smallFirst void event_neighbour_sort_merge(void * arr, int start, int items, int items2){ unsigned int ** sort_data = (unsigned int **) arr; unsigned int * tmp_global_id_list = malloc(sizeof(int) * (items+items2)); unsigned int * tmp_offset_list = malloc(sizeof(int) * (items+items2)); int i1_cursor = start, i2_cursor = items + start; int tmp_cursor = 0; while(1){ if(i1_cursor == items + start && i2_cursor == items + items2 + start )break; int select_items_1 = (i1_cursor < items + start && event_neighbour_sort_compare(arr, i1_cursor, i2_cursor) <= 0) || (i2_cursor == start + items + items2); if(select_items_1){ tmp_global_id_list[tmp_cursor] = sort_data[0][i1_cursor]; tmp_offset_list[tmp_cursor ++ ] = sort_data[1][i1_cursor ++]; }else{ tmp_global_id_list[tmp_cursor] = sort_data[0][i2_cursor]; tmp_offset_list[tmp_cursor ++ ] = sort_data[1][i2_cursor ++]; } } memcpy( sort_data[0] + start, tmp_global_id_list, sizeof(int) * (items+items2) ); memcpy( sort_data[1] + start, tmp_offset_list, sizeof(int) * (items+items2) ); free(tmp_global_id_list); free(tmp_offset_list); } int test_redundant_event( global_context_t * global_context, chromosome_event_t* testee, chromosome_event_t* neighbour ){ int length_delta = 3, is_same = 0; if(testee -> event_type == CHRO_EVENT_TYPE_INDEL) length_delta = 0; long long dist = testee -> event_large_side; dist -= neighbour -> event_large_side; if(abs(dist) <= length_delta){ if(testee -> event_type == CHRO_EVENT_TYPE_INDEL){ int indel_diff = testee -> indel_length; indel_diff -= neighbour -> indel_length; if(abs(indel_diff) <= length_delta) is_same = 1; }else is_same = 1; } int is_redund = 0; if(is_same){ if(testee -> indel_at_junction > neighbour -> indel_at_junction) is_redund = 1; else if(testee -> indel_at_junction == neighbour -> indel_at_junction){ if(testee -> supporting_reads < neighbour -> supporting_reads) is_redund = 1; else if( testee -> supporting_reads == neighbour -> supporting_reads ){ if(testee -> event_small_side < neighbour -> event_small_side) is_redund = 1; } } } return is_redund; } #define reallocate_to_be_removed_ids if(to_be_removed_number >= remove_id_size-1){\ remove_id_size = remove_id_size*3/2;\ to_be_removed_ids = realloc(to_be_removed_ids, remove_id_size);\ } #define MIN_EVENT_SUPPORT_NO 2 void remove_neighbour(global_context_t * global_context) { indel_context_t * indel_context = (indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]; HashTable * event_table = indel_context -> event_entry_table; chromosome_event_t * event_space = indel_context -> event_space_dynamic; int xk1,xk2,xk3; int * to_be_removed_ids; int to_be_removed_number = 0, all_junctions = 0; int remove_id_size = 999999; to_be_removed_ids = malloc(sizeof(int) * (1+remove_id_size)); for(xk1=0; xk1total_events; xk1++) { chromosome_event_t * event_body = &event_space[xk1]; if(CHRO_EVENT_TYPE_REMOVED == event_body->event_type) continue; if(global_context -> config.do_remove_neighbour_for_scRNA && global_context -> config.scRNA_input_mode && event_body -> supporting_reads < MIN_EVENT_SUPPORT_NO){ reallocate_to_be_removed_ids; to_be_removed_ids[to_be_removed_number++] = event_body -> global_event_id; continue; } all_junctions++; int event_type; for(event_type = 0; event_type < 2; event_type++) { if(event_type) // for INDELs { int neighbour_range = 3; if(event_body->event_type != CHRO_EVENT_TYPE_INDEL) continue; reallocate_to_be_removed_ids; if(is_ambiguous_indel_score(event_body))to_be_removed_ids[to_be_removed_number++] = event_body -> global_event_id; else for(xk2=-neighbour_range; xk2<=neighbour_range; xk2++) { //if(!xk2)continue; unsigned int test_pos_small = event_body -> event_small_side + xk2; chromosome_event_t * search_return [MAX_EVENT_ENTRIES_PER_SITE]; int found_events = search_event(global_context, event_table, event_space, test_pos_small , EVENT_SEARCH_BY_SMALL_SIDE, CHRO_EVENT_TYPE_INDEL, search_return); //if(test_pos_small > 284136 && test_pos_small < 284146) printf("POS=%u\tTRES=%d\n", test_pos_small, found_events); for(xk3 = 0; xk3 indel_length; length_diff -= event_body -> indel_length; if(length_diff == 0 && xk2==0) continue; if(is_ambiguous_indel_score(tested_neighbour)) to_be_removed_ids[to_be_removed_number++] = event_body -> global_event_id; else if(length_diff==0) { if(event_body -> event_small_side - event_body->connected_previous_event_distance + 1 == tested_neighbour-> event_large_side) continue; if(tested_neighbour -> event_small_side - tested_neighbour->connected_previous_event_distance + 1 == event_body-> event_large_side) continue; if(event_body -> event_large_side + event_body->connected_next_event_distance - 1 == tested_neighbour-> event_small_side) continue; if(tested_neighbour -> event_large_side + tested_neighbour->connected_next_event_distance - 1 == event_body-> event_small_side) continue; if(event_body->event_quality < tested_neighbour -> event_quality) to_be_removed_ids[to_be_removed_number++] = event_body -> global_event_id; else if(event_body->event_quality == tested_neighbour -> event_quality && (event_body -> supporting_reads < tested_neighbour -> supporting_reads||(event_body -> supporting_reads == tested_neighbour -> supporting_reads && xk2<0))) { to_be_removed_ids[to_be_removed_number++] = event_body -> global_event_id; } } } } } else // for junctions/fusions { int neighbour_range = 11; int indel_range = 4 , delta_small = 0;// max_range = 0; if(event_body -> event_type == CHRO_EVENT_TYPE_INDEL) continue; if(event_body -> is_donor_found_or_annotation & 64) continue; // do not remove known events //if(! global_context->config.check_donor_at_junctions )max_range = 10; //for(delta_small = - max_range; delta_small <= max_range ; delta_small ++) for(xk2=-neighbour_range; xk2<=neighbour_range; xk2++) { if(!xk2)continue; unsigned int test_pos_small = event_body -> event_small_side + xk2; chromosome_event_t * search_return [MAX_EVENT_ENTRIES_PER_SITE]; int found_events = search_event(global_context,event_table, event_space, test_pos_small + delta_small , EVENT_SEARCH_BY_SMALL_SIDE, CHRO_EVENT_TYPE_JUNCTION|CHRO_EVENT_TYPE_FUSION, search_return); for(xk3 = 0; xk3 indel_at_junction > event_body -> indel_at_junction) continue; if(event_body -> indel_at_junction > tested_neighbour -> indel_at_junction && abs(tested_neighbour -> event_large_side - tested_neighbour -> event_small_side + tested_neighbour -> indel_at_junction - event_body -> event_large_side + event_body -> event_small_side - event_body -> indel_at_junction) <= 16) to_be_removed_ids[to_be_removed_number++] = event_body -> global_event_id; else if(tested_neighbour -> event_large_side >= event_body -> event_large_side -indel_range + xk2 && tested_neighbour -> event_large_side <= event_body -> event_large_side + indel_range + xk2 && (event_body -> supporting_reads < tested_neighbour -> supporting_reads || (event_body -> supporting_reads == tested_neighbour -> supporting_reads && xk2<0))) to_be_removed_ids[to_be_removed_number++] = event_body -> global_event_id; } } if((global_context-> config.do_fusion_detection || global_context-> config.do_long_del_detection) && event_body -> event_type == CHRO_EVENT_TYPE_FUSION) { for(xk2=-10 ; xk2 < 10 ; xk2++) { if(!xk2)continue; reallocate_to_be_removed_ids; unsigned int test_pos_small = event_body -> event_small_side + xk2; chromosome_event_t * search_return [MAX_EVENT_ENTRIES_PER_SITE]; int found_events = search_event(global_context,event_table, event_space, test_pos_small + delta_small , EVENT_SEARCH_BY_BOTH_SIDES, CHRO_EVENT_TYPE_JUNCTION|CHRO_EVENT_TYPE_FUSION, search_return); for(xk3 = 0; xk3 supporting_reads < tested_neighbour -> supporting_reads) { to_be_removed_ids[to_be_removed_number++] = event_body -> global_event_id; break; } } } } } } } for(xk1=0; xk1event_large_side:deleted_event->event_small_side; unsigned int * res = HashTableGet(event_table, NULL+pos); if(res){ int current_size = res[0]&0x0fffffff; int wrt_ptr = 1; for(xk3 = 1 ; xk3< current_size+1 ; xk3++){ if(!res[xk3])break; if(res[xk3] -1 == event_no) continue; if(wrt_ptr != xk3) res[wrt_ptr]=res[xk3]; wrt_ptr++; } if(0 && wrt_ptr==1){ HashTableRemove(event_table, NULL+pos); free(res); }else if(wrt_ptr < current_size +1)res[wrt_ptr]=0; } } if(deleted_event -> event_type == CHRO_EVENT_TYPE_INDEL && deleted_event -> inserted_bases) free(deleted_event -> inserted_bases); deleted_event -> event_type = CHRO_EVENT_TYPE_REMOVED; // if(deleted_event -> event_small_side >= 232366259 -1 && deleted_event -> event_small_side <= 232366259 +61)SUBREADprintf("DELETED_THIS AT %u len=%d PTR %p\n", deleted_event -> event_small_side, deleted_event -> indel_length , deleted_event ); } free(to_be_removed_ids); } int localPointerCmp_forEventEntry(const void *pointer1, const void *pointer2) { unsigned int v1 = (unsigned int)(pointer1-NULL); unsigned int v2 = (unsigned int)(pointer2-NULL); return v1!=v2; } srUInt_64 localPointerHashFunction_forEventEntry(const void *pointer) { unsigned int v = (pointer-NULL); return (unsigned long) (v << 24) ^ (v>>8); } int init_indel_tables(global_context_t * context) { indel_context_t * indel_context = (indel_context_t*)malloc(sizeof(indel_context_t)); // event_entry_table is a table from an absolute chromosome location to a list of integers. // each integer is the index of event in the event array + 1. // unused values are set to 0. indel_context -> event_entry_table = NULL; context -> module_contexts[MODULE_INDEL_ID] = indel_context; indel_context -> total_events = 0; indel_context -> current_max_event_number = 0; //indel_context -> current_max_event_number = context->config.init_max_event_number; //indel_context -> event_space_dynamic = (chromosome_event_t *)malloc(sizeof(chromosome_event_t)*indel_context -> current_max_event_number); indel_context -> event_space_dynamic = NULL; if(context -> config.all_threads < 2) { indel_context -> event_entry_table = HashTableCreate(399997); indel_context -> event_entry_table -> appendix1=malloc(1024 * 1024 * 64); indel_context -> event_entry_table -> appendix2=malloc(1024 * 1024 * 64); memset(indel_context -> event_entry_table -> appendix1, 0, 1024 * 1024 * 64); memset(indel_context -> event_entry_table -> appendix2, 0, 1024 * 1024 * 64); HashTableSetKeyComparisonFunction(indel_context->event_entry_table, localPointerCmp_forEventEntry); HashTableSetHashFunction(indel_context->event_entry_table, localPointerHashFunction_forEventEntry); indel_context -> total_events = 0; indel_context -> current_max_event_number = context->config.init_max_event_number; indel_context -> event_space_dynamic = malloc(sizeof(chromosome_event_t)*indel_context -> current_max_event_number); if(!indel_context -> event_space_dynamic) { sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_FATAL, "Cannot allocate memory for threads. Please try to reduce the thread number."); return 1; } } if(context->config.is_third_iteration_running) { char * fns = malloc(200); fns[0]=0; exec_cmd("ulimit -n", fns, 200); int max_open_file = atoi(fns); //SUBREADprintf("SYS FILE LIMIT=%d\n", max_open_file); free(fns); max_open_file = max(100, max_open_file); max_open_file = min(3000, max_open_file); indel_context -> local_reassembly_pileup_files = HashTableCreate(100); indel_context -> local_reassembly_pileup_files -> appendix1 = NULL + max_open_file * 2/ 3; HashTableSetDeallocationFunctions(indel_context -> local_reassembly_pileup_files, NULL, NULL); HashTableSetKeyComparisonFunction(indel_context -> local_reassembly_pileup_files, my_strcmp); HashTableSetHashFunction(indel_context -> local_reassembly_pileup_files ,HashTableStringHashFunction); } indel_context -> dynamic_align_table = malloc(sizeof(short*)*MAX_READ_LENGTH); indel_context -> dynamic_align_table_mask = malloc(sizeof(char *)*MAX_READ_LENGTH); int xk1; for(xk1=0;xk1 dynamic_align_table[xk1] = malloc(sizeof(short)*MAX_READ_LENGTH); indel_context -> dynamic_align_table_mask[xk1] = malloc(sizeof(char)*MAX_READ_LENGTH); } for(xk1=0; xk1 event_body_locks+xk1); return 0; } int init_indel_thread_contexts(global_context_t * global_context, thread_context_t * thread_context, int task) { indel_thread_context_t * indel_thread_context = (indel_thread_context_t*)malloc(sizeof(indel_thread_context_t)); indel_context_t * indel_context = (indel_context_t *) global_context -> module_contexts[MODULE_INDEL_ID]; if(task == STEP_VOTING) { indel_thread_context -> event_entry_table = HashTableCreate(399997 * ( global_context -> config.scRNA_input_mode?18:2 )); indel_thread_context -> event_entry_table -> appendix1=NULL;//indel_context -> event_entry_table-> appendix1; indel_thread_context -> event_entry_table -> appendix2=NULL;//indel_context -> event_entry_table-> appendix2; HashTableSetKeyComparisonFunction(indel_thread_context->event_entry_table, localPointerCmp_forEventEntry); HashTableSetHashFunction(indel_thread_context->event_entry_table, localPointerHashFunction_forEventEntry); indel_thread_context -> total_events = 0; indel_thread_context -> current_max_event_number = global_context->config.init_max_event_number; indel_thread_context -> event_space_dynamic = malloc(sizeof(chromosome_event_t)*indel_thread_context -> current_max_event_number); if(!indel_thread_context -> event_space_dynamic) { sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_FATAL, "Cannot allocate memory for threads. Please try to reduce the thread number."); return 1; } indel_thread_context -> dynamic_align_table = malloc(sizeof(short*)*MAX_READ_LENGTH); indel_thread_context -> dynamic_align_table_mask = malloc(sizeof(char *)*MAX_READ_LENGTH); int xk1; for(xk1=0;xk1 dynamic_align_table[xk1] = malloc(sizeof(short)*MAX_READ_LENGTH); indel_thread_context -> dynamic_align_table_mask[xk1] = malloc(sizeof(char)*MAX_READ_LENGTH); } } else if(task == STEP_ITERATION_TWO) { indel_thread_context -> event_entry_table = indel_context -> event_entry_table; indel_thread_context -> total_events = indel_context -> total_events; indel_thread_context -> event_space_dynamic = indel_context -> event_space_dynamic; indel_thread_context -> final_reads_mismatches_array = (unsigned short*)malloc(sizeof(unsigned short)*indel_context -> total_events); indel_thread_context -> final_counted_reads_array = (unsigned short*)malloc(sizeof(unsigned short)*indel_context -> total_events); assert(indel_thread_context -> final_counted_reads_array); memset(indel_thread_context -> final_reads_mismatches_array , 0, sizeof(unsigned short)*indel_context -> total_events); memset(indel_thread_context -> final_counted_reads_array , 0, sizeof(unsigned short)*indel_context -> total_events); subread_init_lock(&thread_context -> output_lock); } thread_context -> module_thread_contexts[MODULE_INDEL_ID] = indel_thread_context; return 0; } void destory_event_entry_table(HashTable * old) { int bucket; KeyValuePair * cursor; for(bucket=0; bucket numOfBuckets; bucket++) { cursor = old -> bucketArray[bucket]; while(1) { if (!cursor) break; int * id_list = (int *) cursor ->value; //#warning "=============== UNCOMMENT THE NEXT LINE IN THE RELEASE!!! THIS IS FOR REMOVING A SEGFAULT ERROR ==================" free(id_list); cursor = cursor->next; } } } typedef struct { unsigned int scanning_positons; unsigned int thread_bodytable_number; } scanning_events_record_t; #define _get_global_body(iii) ( indel_context -> event_space_dynamic + records[iii].thread_bodytable_number) int scanning_events_compare(void * arr, int l, int r){ void ** arrr = (void **) arr; indel_context_t * indel_context = arrr[0]; scanning_events_record_t * records = arrr[1]; chromosome_event_t * body_l = _get_global_body(l); chromosome_event_t * body_r = _get_global_body(r); if(records[l].scanning_positons > records[r].scanning_positons)return 1; if(records[l].scanning_positons < records[r].scanning_positons)return -1; if((body_l -> is_donor_found_or_annotation & 64)!=0 && (body_r -> is_donor_found_or_annotation & 64)==0) return 1; // prefer known truth. if((body_l -> is_donor_found_or_annotation & 64)==0 && (body_r -> is_donor_found_or_annotation & 64)!=0) return -1; if(body_l -> supporting_reads > body_r -> supporting_reads) return -1; if(body_l -> supporting_reads < body_r -> supporting_reads) return 1; if(abs(body_l -> indel_length) < abs(body_r -> indel_length)) return 1; if(abs(body_l -> indel_length) > abs(body_r -> indel_length)) return -1; if(body_l -> indel_length > body_r -> indel_length) return -1; // same length, but L is del and R is ins -- prefer del than ins if(body_l -> indel_length < body_r -> indel_length) return 1; if(body_l -> event_small_side > body_r -> event_small_side)return 1; if(body_l -> event_small_side < body_r -> event_small_side)return -1; if(body_l -> event_large_side > body_r -> event_large_side)return 1; return -1; } void null_scanning_events_merge(void * arr, int start, int items, int items2){} void scanning_events_merge(void * arr, int start, int items, int items2){ void ** arrr = (void **) arr; scanning_events_record_t * records = arrr[1]; int read_1_ptr = start, read_2_ptr = start+items, write_ptr; scanning_events_record_t * merged_records = malloc(sizeof(scanning_events_record_t) * (items+items2)); for(write_ptr=0; write_ptr= start+items)||(read_2_ptr < start+items+items2 && scanning_events_compare(arr, read_1_ptr, read_2_ptr) > 0)) memcpy(merged_records+write_ptr, records+(read_2_ptr++), sizeof(scanning_events_record_t)); else memcpy(merged_records+write_ptr, records+(read_1_ptr++), sizeof(scanning_events_record_t)); } memcpy(records + start, merged_records, sizeof(scanning_events_record_t) * (items+items2)); free(merged_records); } void scanning_events_exchange(void * arr, int l, int r){ void ** arrr = (void **) arr; scanning_events_record_t * records = arrr[1]; unsigned long tmpi; tmpi = records[l].scanning_positons; records[l].scanning_positons = records[r].scanning_positons; records[r].scanning_positons = tmpi; tmpi = records[l].thread_bodytable_number; records[l].thread_bodytable_number = records[r].thread_bodytable_number; records[r].thread_bodytable_number = tmpi; } #define _test_record_size if(current_record_number >= current_record_size - 2){\ current_record_size *= 1.5;\ records=realloc(records, sizeof(scanning_events_record_t)*current_record_size);\ if(NULL == records) return -1;\ }\ #define _add_record records[current_record_number].scanning_positons = body -> event_small_side;\ records[current_record_number].thread_bodytable_number = xx1;\ current_record_number++;\ records[current_record_number].scanning_positons = body -> event_large_side;\ records[current_record_number].thread_bodytable_number = xx1;\ current_record_number++;\ int sort_junction_entry_table(global_context_t * global_context){ indel_context_t * indel_context = (indel_context_t*)global_context -> module_contexts[MODULE_INDEL_ID]; chromosome_event_t * event_space = indel_context -> event_space_dynamic; if(indel_context -> event_entry_table){ if(indel_context -> event_entry_table->appendix1) { free(indel_context -> event_entry_table -> appendix1); free(indel_context -> event_entry_table -> appendix2); } // free the entry table: the pointers. destory_event_entry_table(indel_context -> event_entry_table); HashTableDestroy(indel_context -> event_entry_table); } indel_context -> event_entry_table = HashTableCreate(399997); HashTableSetKeyComparisonFunction(indel_context->event_entry_table, localPointerCmp_forEventEntry); HashTableSetHashFunction(indel_context->event_entry_table, localPointerHashFunction_forEventEntry); if(global_context -> config.use_bitmap_event_table) { indel_context -> event_entry_table -> appendix1=malloc(1024 * 1024 * 64); indel_context -> event_entry_table -> appendix2=malloc(1024 * 1024 * 64); memset(indel_context -> event_entry_table -> appendix1, 0, 1024 * 1024 * 64); memset(indel_context -> event_entry_table -> appendix2, 0, 1024 * 1024 * 64); } else { indel_context -> event_entry_table -> appendix1=NULL; indel_context -> event_entry_table -> appendix2=NULL; } int xx1, current_record_number=0, current_record_size=10000; scanning_events_record_t * records = malloc(sizeof(scanning_events_record_t)*current_record_size); for(xx1 = 0; xx1 < indel_context -> total_events; xx1++){ chromosome_event_t * body = event_space + xx1; _test_record_size; _add_record; } void * sort_arr[2]; sort_arr[0] = indel_context; sort_arr[1] = records; // many repeated elements // do not use quick sort. merge_sort(sort_arr, current_record_number, scanning_events_compare, scanning_events_exchange, scanning_events_merge); unsigned int last_scannping_pos = records[0].scanning_positons; int merge_start = 0; HashTable * event_table = indel_context -> event_entry_table; for(xx1 =0; xx1 <= current_record_number; xx1++){ scanning_events_record_t * this_record = NULL; if(xx10){ if(xx1 == current_record_number || last_scannping_pos!= this_record -> scanning_positons){ int merge_end = xx1, merge_i; if( merge_end-merge_start > MAX_EVENT_ENTRIES_PER_SITE ) merge_end = merge_start + MAX_EVENT_ENTRIES_PER_SITE; unsigned int * id_list = malloc(sizeof(int) * (1+merge_end-merge_start)); assert(id_list); id_list[0] = merge_end-merge_start; for(merge_i = merge_start; merge_i < merge_end; merge_i++){ chromosome_event_t * body = _get_global_body(merge_i); id_list[merge_i - merge_start + 1] = records[merge_i].thread_bodytable_number + 1; mark_event_bitmap(event_table->appendix1, body -> event_small_side); mark_event_bitmap(event_table->appendix2, body -> event_large_side); } merge_start = xx1; //#warning "======= UNCOMMENT NEXT =======" HashTablePut(indel_context -> event_entry_table, NULL + last_scannping_pos, id_list); } } if(xx1 == current_record_number) break; last_scannping_pos = this_record -> scanning_positons; } free(records); return 0; } #define _get_event_body(iii) (records[iii].thread_no<0?( indel_context -> event_space_dynamic + records[iii].thread_bodytable_number ):(((indel_thread_context_t*)(thread_contexts[records[iii].thread_no].module_thread_contexts[MODULE_INDEL_ID]))-> event_space_dynamic + records[iii].thread_bodytable_number )) typedef struct { unsigned int thread_bodytable_number; short thread_no; } concatinating_events_record_t; #define _test_conc_size if(conc_rec_items >= conc_rec_size - 1){\ conc_rec_size *= 1.5;\ records = realloc(records, conc_rec_size * sizeof(concatinating_events_record_t));\ if(NULL == records) return -1;\ } int conc_sort_compare(void * arr, int l, int r){ void ** arrr = (void **)arr; concatinating_events_record_t * records = arrr[0]; indel_context_t * indel_context = arrr[1]; thread_context_t * thread_contexts = arrr[2]; chromosome_event_t * body_l = _get_event_body(l); chromosome_event_t * body_r = _get_event_body(r); if(body_l -> event_small_side > body_r -> event_small_side)return 3; if(body_l -> event_small_side < body_r -> event_small_side)return -3; if(body_l -> event_large_side > body_r -> event_large_side)return 3; if(body_l -> event_large_side < body_r -> event_large_side)return -3; if(abs(body_l -> indel_length) < abs(body_r -> indel_length)) return 2; if(abs(body_l -> indel_length) > abs(body_r -> indel_length)) return -2; if(body_l -> indel_length > body_r -> indel_length) return -2; // same length, but L is del and R is ins -- prefer del than ins if(body_l -> indel_length < body_r -> indel_length) return 2; if((body_l -> is_donor_found_or_annotation & 64)!=0 && (body_r -> is_donor_found_or_annotation & 64)==0) return 1; // prefer known truth. if((body_l -> is_donor_found_or_annotation & 64)==0 && (body_r -> is_donor_found_or_annotation & 64)!=0) return -1; if(body_l -> supporting_reads > body_r -> supporting_reads) return -1; if(body_l -> supporting_reads < body_r -> supporting_reads) return 1; return 0; } void conc_sort_merge(void * arr, int start, int items, int items2){ void ** arrr = (void **) arr; concatinating_events_record_t * records = arrr[0]; int read_1_ptr = start, read_2_ptr = start+items, write_ptr; concatinating_events_record_t * merged_records = malloc(sizeof(concatinating_events_record_t) * (items+items2)); for(write_ptr=0; write_ptr= start+items)||(read_2_ptr < start+items+items2 && conc_sort_compare(arr, read_1_ptr, read_2_ptr) > 0)) memcpy(merged_records+write_ptr, records+(read_2_ptr++), sizeof(concatinating_events_record_t)); else memcpy(merged_records+write_ptr, records+(read_1_ptr++), sizeof(concatinating_events_record_t)); } memcpy(records + start, merged_records, sizeof(concatinating_events_record_t) * (items+items2)); free(merged_records); } void conc_sort_exchange(void * arr, int l, int r){ void ** arrr = (void **)arr; concatinating_events_record_t * records = arrr[0]; unsigned int tmpi; tmpi = records[l].thread_bodytable_number; records[l].thread_bodytable_number = records[r].thread_bodytable_number; records[r].thread_bodytable_number = tmpi; tmpi = records[l].thread_no; records[l].thread_no = records[r].thread_no; records[r].thread_no = tmpi; } // sort and merge events from all threads and the global event space. int sort_global_event_table(global_context_t * global_context){ return finalise_indel_and_junction_thread(global_context, NULL, STEP_VOTING); } int finalise_indel_and_junction_thread(global_context_t * global_context, thread_context_t * thread_contexts, int task) { indel_context_t * indel_context = (indel_context_t*)global_context -> module_contexts[MODULE_INDEL_ID]; if(task == STEP_VOTING) { concatinating_events_record_t * records; int conc_rec_size = 10000, conc_rec_items = 0; records = malloc(sizeof(concatinating_events_record_t) * conc_rec_size); int xk1, thn; if(thread_contexts) for(thn =0; thn < global_context->config.all_threads; thn++){ thread_context_t * thread_context = thread_contexts+thn; indel_thread_context_t * indel_thread_context = (indel_thread_context_t*)thread_context -> module_thread_contexts[MODULE_INDEL_ID]; for(xk1 = 0; xk1 < indel_thread_context -> total_events; xk1++){ chromosome_event_t * old_body = indel_thread_context -> event_space_dynamic + xk1; if(old_body -> event_type == CHRO_EVENT_TYPE_REMOVED) continue; _test_conc_size; records[conc_rec_items].thread_no=thn; records[conc_rec_items++].thread_bodytable_number=xk1; } } for(xk1 = 0; xk1 < indel_context -> total_events; xk1++){ chromosome_event_t * old_body = indel_context -> event_space_dynamic + xk1; if(old_body -> event_type == CHRO_EVENT_TYPE_REMOVED) continue; _test_conc_size; records[conc_rec_items].thread_no=-1; records[conc_rec_items++].thread_bodytable_number=xk1; } void * sort_arr[3]; sort_arr[0] = records; sort_arr[1] = indel_context; sort_arr[2] = thread_contexts; // many repeated elements -- do not use quick sort. merge_sort(sort_arr, conc_rec_items, conc_sort_compare, conc_sort_exchange, conc_sort_merge); chromosome_event_t * prev_env = NULL; int merge_target_size = 10000; int merge_target_items = 0; chromosome_event_t * merge_target = malloc(sizeof(chromosome_event_t) * merge_target_size); int merge_start = 0; for(xk1 = 0; xk1 <= conc_rec_items; xk1++){ chromosome_event_t * this_event = (xk1 == conc_rec_items)?NULL:_get_event_body(xk1); if(xk1 > 0){ int compret = 0; if(xk1 < conc_rec_items) compret = conc_sort_compare(sort_arr, xk1-1, xk1); if(abs(compret)>1 || xk1 == conc_rec_items){// different events or last one -- merge [ prev_event_record_no , xk1 - 1 ] int xk_merge; // find a new slot in the target space if(merge_target_items >= merge_target_size - 1){ merge_target_size *= 1.5; merge_target = realloc(merge_target, sizeof(chromosome_event_t) * merge_target_size); } chromosome_event_t * merged_body = merge_target + merge_target_items; memcpy( merged_body, prev_env, sizeof(chromosome_event_t) ); merged_body -> global_event_id = merge_target_items++; for(xk_merge = merge_start; xk_merge < xk1 - 1; xk_merge++){ chromosome_event_t * old_body = _get_event_body(xk_merge); assert(merged_body -> event_type == old_body -> event_type); merged_body -> supporting_reads += old_body -> supporting_reads; merged_body -> anti_supporting_reads += old_body -> anti_supporting_reads; merged_body -> final_counted_reads += old_body -> final_counted_reads; merged_body -> final_reads_mismatches += old_body -> final_reads_mismatches; merged_body -> critical_supporting_reads += old_body -> critical_supporting_reads; merged_body -> junction_flanking_left = max(merged_body -> junction_flanking_left, old_body -> junction_flanking_left); merged_body -> junction_flanking_right = max(merged_body -> junction_flanking_right, old_body -> junction_flanking_right); merged_body -> is_donor_found_or_annotation |= old_body -> is_donor_found_or_annotation; if(merged_body -> connected_next_event_distance > 0 && old_body -> connected_next_event_distance > 0) merged_body -> connected_next_event_distance = min(merged_body -> connected_next_event_distance, old_body -> connected_next_event_distance); else merged_body -> connected_next_event_distance = max(merged_body -> connected_next_event_distance, old_body -> connected_next_event_distance); if(merged_body -> connected_previous_event_distance > 0 && old_body -> connected_previous_event_distance > 0) merged_body -> connected_previous_event_distance = min(merged_body -> connected_previous_event_distance, old_body -> connected_previous_event_distance); else merged_body -> connected_previous_event_distance = max(merged_body -> connected_previous_event_distance, old_body -> connected_previous_event_distance); if(old_body->inserted_bases && old_body -> event_type == CHRO_EVENT_TYPE_INDEL && old_body -> indel_length<0){ //printf("OCT27-FREEMEM-INS [%d] : thread-%d + %d %u-%p\n", xk_merge, records[xk_merge].thread_no, records[xk_merge].thread_bodytable_number, old_body->event_small_side, old_body->inserted_bases ); if(merged_body -> inserted_bases && merged_body -> inserted_bases != old_body -> inserted_bases){ // SUBREADprintf("FREE PTR=%p\n", old_body -> inserted_bases); free(old_body -> inserted_bases); } else merged_body -> inserted_bases = old_body -> inserted_bases; old_body -> inserted_bases = NULL; } } merge_start = xk1; } } if(xk1 == conc_rec_items) break; prev_env = this_event; } free(records); if(thread_contexts) for(thn =0; thn < global_context->config.all_threads; thn++){ thread_context_t * thread_context = thread_contexts+thn; indel_thread_context_t * indel_thread_context = (indel_thread_context_t*)thread_context -> module_thread_contexts[MODULE_INDEL_ID]; destory_event_entry_table(indel_thread_context -> event_entry_table); HashTableDestroy(indel_thread_context -> event_entry_table); free(indel_thread_context -> event_space_dynamic); for(xk1=0;xk1 dynamic_align_table[xk1]); free(indel_thread_context -> dynamic_align_table_mask[xk1]); } free(indel_thread_context->dynamic_align_table); free(indel_thread_context->dynamic_align_table_mask); free(indel_thread_context); } if(indel_context -> event_space_dynamic ) free(indel_context -> event_space_dynamic); indel_context -> event_space_dynamic = NULL; indel_context -> event_space_dynamic = merge_target; indel_context -> current_max_event_number = merge_target_size; indel_context -> total_events = merge_target_items; } else if(task == STEP_ITERATION_TWO) { int xk1, thn; if(thread_contexts) for(thn =0; thn < global_context->config.all_threads; thn++){ thread_context_t * thread_context = thread_contexts+thn; indel_thread_context_t * indel_thread_context = (indel_thread_context_t*)thread_context -> module_thread_contexts[MODULE_INDEL_ID]; for(xk1 = 0; xk1 < indel_thread_context -> total_events; xk1++) { indel_context -> event_space_dynamic [xk1] . final_counted_reads +=indel_thread_context -> final_counted_reads_array[xk1]; indel_context -> event_space_dynamic [xk1] . final_reads_mismatches +=indel_thread_context -> final_reads_mismatches_array[xk1]; } free(indel_thread_context -> final_counted_reads_array); free(indel_thread_context -> final_reads_mismatches_array); free(indel_thread_context); } } return 0; } void add_annotation_to_junctions(void * key, void * val, HashTable * tab){ global_context_t * global_context = tab->appendix1; indel_context_t * indel_context = (indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]; char * gene_chr = key; int * pos_strands = val; int current_lagre_end = -1; char chroname[MAX_CHROMOSOME_NAME_LEN]; int x1, ww=0, wi=0; for(x1 =0 ; ; x1++){ if(ww){ chroname[wi++]=gene_chr[x1]; chroname[wi]=0; } if(0==gene_chr[x1]) break; if(gene_chr[x1]==':') ww=1; } //#warning ">>>>>>> COMMENt NEXT <<<<<<<<<<<<<<" //if(1) SUBREADprintf("SEE LOCATION: %s has %d\n", gene_chr , pos_strands[0]+1 ); for(x1 = 1; x1 < pos_strands[0]+1; x1+=3){ if(pos_strands[x1]==0)break; //#warning ">>>>>>> COMMENt NEXT <<<<<<<<<<<<<<" //SUBREADprintf("SEE LOCATION ___ : %d >0 && %d < %d %d %d\n", current_lagre_end , current_lagre_end, pos_strands[x1], pos_strands[x1+1], pos_strands[x1+2]); if(current_lagre_end > 0 && current_lagre_end < pos_strands[x1]){ // add a junction between current_lagre_end and pos_strands[x1] if(indel_context -> total_events >= indel_context -> current_max_event_number -1){ indel_context -> current_max_event_number*=1.2; indel_context -> event_space_dynamic = realloc(indel_context -> event_space_dynamic , sizeof( chromosome_event_t )*indel_context -> current_max_event_number); } chromosome_event_t * newbody = indel_context -> event_space_dynamic + indel_context -> total_events ; memset(newbody, 0, sizeof(chromosome_event_t)); newbody -> global_event_id = indel_context -> total_events++; newbody -> event_type = CHRO_EVENT_TYPE_JUNCTION; newbody -> event_small_side = linear_gene_position(&global_context->chromosome_table , chroname, current_lagre_end); newbody -> event_large_side = linear_gene_position(&global_context->chromosome_table , chroname, pos_strands[x1]); //#warning ">>>>>>> COMMENt NEXT <<<<<<<<<<<<<<" if(0){ if ( 1 || newbody -> event_small_side == 625683723){ SUBREADprintf("Loaded event :"); debug_show_event(global_context, newbody); } } newbody -> is_negative_strand = pos_strands[x1+2]; newbody -> is_donor_found_or_annotation = 127; // printf("09NOV: add_annotation_to_junctions: %u -> %u '%s', %d,%d\n", newbody -> event_small_side , newbody -> event_large_side, chroname, current_lagre_end, pos_strands[x1]); } if(current_lagre_end < pos_strands[x1+1]) current_lagre_end = pos_strands[x1+1]; } } typedef struct { global_context_t * global_context; HashTable * feature_sorting_table; } do_load_juncs_context_t; int do_juncs_add_feature(char * gene_name, char * transcript_id, char * chro_name, unsigned int feature_start, unsigned int feature_end, int is_negative_strand, void * context){ //#warning ">>>>>>>>>>>>>>>>> COMMENT NEXT <<<<<<<<<<<<<<<<<<<<" //return 0; //#warning ">>>>>>>>>>>>>>>>> COMMENT NEXT <<<<<<<<<<<<<<<<<<<<" //SUBREADprintf("INJ LOCS: %s : %u, %u\n", chro_name, feature_start, feature_end); do_load_juncs_context_t * do_load_juncs_context = context; HashTable * feature_sorting_table = do_load_juncs_context -> feature_sorting_table; global_context_t * global_context = do_load_juncs_context -> global_context; char tmp_chro_name[MAX_CHROMOSOME_NAME_LEN]; if(global_context -> sam_chro_to_anno_chr_alias){ char * sam_chro = get_sam_chro_name_from_alias(global_context -> sam_chro_to_anno_chr_alias, chro_name); if(sam_chro!=NULL) chro_name = sam_chro; } int access_n = HashTableGet( global_context -> chromosome_table.read_name_to_index, chro_name ) - NULL; if(access_n < 1){ if(chro_name[0]=='c' && chro_name[1]=='h' && chro_name[2]=='r'){ chro_name += 3; }else{ strcpy(tmp_chro_name, "chr"); strcat(tmp_chro_name, chro_name); chro_name = tmp_chro_name; } } char sort_key[ MAX_CHROMOSOME_NAME_LEN * 3 + 2 ]; sprintf(sort_key, "%s:%s", gene_name, chro_name); int * old_features = HashTableGet(feature_sorting_table, sort_key), x1; int written_space = -1, written_last_item = 0; if(old_features){ int has_space = 0; for(x1= 1; x1 < old_features[0] + 1 ; x1+=3){ if(old_features[x1]==0){ has_space = 1; break; } } if(!has_space){ int old_size = old_features[0]; old_features[0] *= 1.5; old_features[0] -= old_features[0]%3; // I used malloc but not realloc here, because the old memory block will be freed by HashTablePutReplace. int * old_features2 = malloc( sizeof(int) * (old_features[0] +1)); memcpy(old_features2, old_features, (old_size + 1) * sizeof(int)); old_features2[old_size + 1] = 0; HashTablePutReplace(feature_sorting_table, sort_key, old_features2, 0); old_features = old_features2; } for(x1= 1; x1 < old_features[0] + 1 ; x1+=3){ if(old_features[x1]> feature_start || old_features[x1]==0){ written_space = x1; if(old_features[x1]> feature_start){ int x2; for(x2 = x1+3; x2 < old_features[0] + 1;x2+=3){ if(old_features[x2]==0)break; } x2 += 2; assert(x2 < old_features[0] + 1); if(x2 + 1 < old_features[0] + 1) old_features[x2+1]=0; for(; x2 >= x1 + 3; x2 --) old_features[x2] = old_features[x2-3]; } else written_last_item = 1; break; } } }else{ int init_space_sort = 15; old_features = malloc((init_space_sort + 1) * sizeof(int)); old_features[0]=init_space_sort; char * mm_sort_key = malloc(strlen(sort_key)+1); strcpy(mm_sort_key, sort_key); HashTablePut(feature_sorting_table, mm_sort_key, old_features); written_space = 1; written_last_item = 1; } assert(written_space >0); old_features[written_space]=feature_start - 1; old_features[written_space+1]=feature_end - 1; old_features[written_space+2]=is_negative_strand; if(written_last_item && written_space + 3 < old_features[0]+1) old_features[written_space+3]=0; return 0; } int load_known_junctions(global_context_t * global_context){ HashTable * feature_sorting_table = HashTableCreate(90239); HashTableSetKeyComparisonFunction(feature_sorting_table, my_strcmp); HashTableSetHashFunction(feature_sorting_table, HashTableStringHashFunction); HashTableSetDeallocationFunctions(feature_sorting_table, free, free); do_load_juncs_context_t do_load_juncs_context; memset(&do_load_juncs_context, 0, sizeof(do_load_juncs_context)); do_load_juncs_context.global_context = global_context; do_load_juncs_context.feature_sorting_table = feature_sorting_table; int features = load_features_annotation(global_context->config.exon_annotation_file , global_context->config.exon_annotation_file_type, global_context->config.exon_annotation_gene_id_column, NULL, global_context->config.exon_annotation_feature_name_column, &do_load_juncs_context, do_juncs_add_feature); feature_sorting_table -> appendix1 = global_context; HashTableIteration(feature_sorting_table, add_annotation_to_junctions); HashTableDestroy(feature_sorting_table); if(features <0)return -1; else{ return 0; } } int there_are_events_in_range(char * bitmap, unsigned int pos, int sec_len) { int ret = 0; if(!bitmap) return 1; unsigned int offset = pos >> 3; unsigned int offset_end = (pos + sec_len) >> 3; unsigned int offset_byte = (offset >> 3)&0x3ffffff; unsigned int offset_byte_end =1+((offset_end >> 3)&0x3ffffff); for(; offset_byte < offset_byte_end ; offset_byte++) { if(bitmap[offset_byte]){ ret = 1; break; } } // printf("TEST_JUMP: THERE ARE %d at %u\n" , ret , pos); return ret; } // bitmap has 64M bytes. int check_event_bitmap(unsigned char * bitmap, unsigned int pos) { unsigned int offset = pos >> 3; unsigned int offset_byte = (offset >> 3)&0x3ffffff; char keybyte = bitmap[offset_byte]; if(!keybyte) return 0; int offset_bit = offset & 7; return (keybyte & (1<> 3; unsigned int offset_byte = (offset >> 3)&0x3ffffff; int offset_bit = offset & 7; bitmap[offset_byte] |= (1<event_small_side; sides[1] = new_event->event_large_side; new_event -> global_event_id = event_no; int xk1, xk2; for(xk1=0; xk1<2; xk1++) { if(0 == sides[xk1])continue ; unsigned int * id_list = HashTableGet(event_table, NULL+sides[xk1]); if(!id_list) { id_list = malloc(sizeof(unsigned int)*(1+EVENT_ENTRIES_INIT_SIZE)); id_list[0]=EVENT_ENTRIES_INIT_SIZE; id_list[1]=0; HashTablePut(event_table , NULL+sides[xk1] , id_list); } for(xk2=1;xk2< id_list[0] ; xk2++) if(id_list[xk2]==0) break; if(xk2 < id_list[0]) id_list[xk2] = event_no+1; if(xk2 < id_list[0]) id_list[xk2+1] = 0; } if(event_table->appendix1) { mark_event_bitmap(event_table->appendix1, sides[0]); mark_event_bitmap(event_table->appendix2, sides[1]); } } int search_event(global_context_t * global_context, HashTable * event_table, chromosome_event_t * event_space, unsigned int pos, int search_type, unsigned char event_type, chromosome_event_t ** return_buffer) { int ret = 0; if(pos < 1 || pos > 0xffff0000)return 0; if(event_table->appendix1) { if(search_type == EVENT_SEARCH_BY_SMALL_SIDE) if(! check_event_bitmap(event_table -> appendix1, pos))return 0; if(search_type == EVENT_SEARCH_BY_LARGE_SIDE) if(! check_event_bitmap(event_table -> appendix2, pos))return 0; if(search_type == EVENT_SEARCH_BY_BOTH_SIDES) if(!(check_event_bitmap(event_table -> appendix1, pos) || check_event_bitmap(event_table -> appendix2, pos))) return 0; } unsigned int * res = HashTableGet(event_table, NULL+pos); if(res) { int xk2; int current_size = res[0]&0x0fffffff; for(xk2=1; xk2< current_size+1 ; xk2++) { if(!res[xk2])break; //if(res[xk2] - 1>= ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> current_max_event_number ) { SUBREADprintf("FATAL ERROR: Event id out-of-boundary: %u > %u.\n", res[xk2], ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> current_max_event_number ); continue;} chromosome_event_t * event_body = &event_space[res[xk2]-1]; if((event_body -> event_type & event_type) == 0)continue; //SUBREADprintf("VB1:%u\n", res[xk2]); if(search_type == EVENT_SEARCH_BY_SMALL_SIDE && event_body -> event_small_side != pos)continue; if(search_type == EVENT_SEARCH_BY_LARGE_SIDE && event_body -> event_large_side != pos)continue; if(search_type == EVENT_SEARCH_BY_BOTH_SIDES && event_body -> event_small_side != pos && event_body -> event_large_side != pos)continue; return_buffer[ret++] = event_body; } } return ret; } void get_insertion_sequence(global_context_t * global_context, thread_context_t * thread_context , char * binary_bases , char * read_text , int insertions) { int xk1; read_text[0]=0; for(xk1=0; xk1> bit_no))); } read_text[insertions]=0; } void set_insertion_sequence(global_context_t * global_context, thread_context_t * thread_context , char ** binary_bases , char * read_text , int insertions) { int xk1; (*binary_bases) = malloc((1+insertions)/4+2); //SUBREADprintf("ALLOC PTR=%p\n", (*binary_bases) ); assert(insertions <= MAX_INSERTION_LENGTH); memset((*binary_bases),0, (1+insertions)/4+2); for(xk1=0; xk1 module_thread_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; else event_space = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; int found_events = search_event(global_context, event_table, event_space, left_edge, EVENT_SEARCH_BY_SMALL_SIDE, CHRO_EVENT_TYPE_INDEL|CHRO_EVENT_TYPE_LONG_INDEL, search_return); //#warning ">>>>>>>>>>>>>>>>>> COMMENt THIS <<<<<<<<<<<<<<<<<<<<<<<" //printf("OCT27-STEPRR-TST LEFT=%u, INDEL=%d; FOUND=%d\n", left_edge , indels , found_events); if(found_events) { int kx1; for(kx1 = 0; kx1 < found_events ; kx1++) { if(search_return[kx1] -> indel_length == indels) { found = search_return[kx1]; break; } } } if(found){ if(old_event_id) (*old_event_id)=found->global_event_id; found -> supporting_reads += score_supporting_read_added; //found -> is_ambiguous = max(is_ambiguous , found -> is_ambiguous ); return NULL; } else { int event_no; //event_no =(thread_context)?((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> total_events: ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> total_events; if(thread_context) event_no = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> total_events ++; else event_no = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> total_events ++; event_space = reallocate_event_space(global_context, thread_context, event_no); chromosome_event_t * new_event = event_space+event_no; memset(new_event,0,sizeof(chromosome_event_t)); if(indels <0) // if it is an insertion: set_insertion_sequence(global_context, thread_context , &new_event -> inserted_bases , read_text, -indels); new_event -> event_small_side = left_edge; new_event -> event_large_side = left_edge + 1 + max(0, indels); //assert(new_event -> event_small_side && new_event -> event_large_side); new_event -> event_type = CHRO_EVENT_TYPE_INDEL; new_event -> indel_length = indels; new_event -> supporting_reads = 1; new_event -> event_quality = 1;//pow(0.5 , 3*mismatched_bases); //new_event -> is_ambiguous = is_ambiguous; //#warning ">>>>>>>>>>>>>>>>>> COMMENt THIS <<<<<<<<<<<<<<<<<<<<<<<" //printf("OCT27-STEPRR-NEW LEFT=%u RIGHT=%u, INDEL=%d\n", new_event -> event_small_side , new_event -> event_large_side, new_event -> indel_length ); put_new_event(event_table, new_event , event_no); return new_event; } } float EXON_RECOVER_MATCHING_RATE = 0.9; float EXON_INDEL_MATCHING_RATE_TAIL = 0.8; float EXON_INDEL_MATCHING_RATE_HEAD = 0.8; int core_extend_covered_region_15(global_context_t * global_context, gene_value_index_t *array_index, unsigned int read_start_pos, char * read, int read_len, int cover_start, int cover_end, int window_size, int req_match_5end , int req_match_3end, int indel_tolerance, int space_type, int tail_indel, short * head_indel_pos, int * head_indel_movement, short * tail_indel_pos, int * tail_indel_movement, int is_head_high_quality, char * qual_txt, int qual_format, float head_matching_rate, float tail_matching_rate){ int is_head; if(0){ char posout[100]; absoffset_to_posstr(global_context, read_start_pos, posout); SUBREADprintf("RTXT=%s, MAPP=%s\n", read, posout); } for(is_head = 0; is_head < 2; is_head ++){ int move_i; int best_match_n = -1, best_movement = 0; for(move_i = 0; move_i < 2* indel_tolerance-1; move_i ++){ int indel_move = (move_i+1)/2 * (move_i %2?1:-1); int indel_movement = indel_move + (is_head?0:tail_indel); int this_match_n; if(is_head) this_match_n = match_chro(read, array_index, read_start_pos - indel_movement, window_size, 0, space_type ); else this_match_n = match_chro(read + read_len - window_size, array_index, read_start_pos + read_len - window_size + indel_movement, window_size, 0, space_type ); // indel_movement : negativ = insertion, positive = deletion before or after the covered region //SUBREADprintf("MOVE: HEAD=%d, MATCH=%d, MOV=%d\n", is_head, this_match_n, indel_movement); if(this_match_n > best_match_n){ best_match_n = this_match_n; best_movement = indel_movement; } } int max_score=-1, best_splice_on_read=0; if(best_match_n >0 && best_movement!=0){ int test_start = is_head?window_size:(cover_end); int test_end = is_head?cover_start - max(0, -best_movement):(read_len - window_size - max(0, -best_movement)); int indel_first_piece_end_on_read, indel_second_piece_start_on_read; for(indel_first_piece_end_on_read = test_start ; indel_first_piece_end_on_read < test_end; indel_first_piece_end_on_read++){ unsigned int indel_first_piece_end_on_chro = read_start_pos + indel_first_piece_end_on_read + (is_head? -best_movement :tail_indel); indel_second_piece_start_on_read = indel_first_piece_end_on_read - min(0, best_movement); unsigned int indel_second_piece_start_on_chro = indel_first_piece_end_on_chro + max(0,best_movement); int first_piece_match = match_chro(read + indel_first_piece_end_on_read - window_size , array_index , indel_first_piece_end_on_chro - window_size, window_size, 0, space_type); int second_piece_match = match_chro(read + indel_second_piece_start_on_read , array_index , indel_second_piece_start_on_chro, window_size, 0, space_type); int score = first_piece_match + second_piece_match; if(score > max_score){ max_score = score; best_splice_on_read = indel_first_piece_end_on_read; } if(score == 2*window_size)break; //SUBREADprintf("FIND_SPLICE_POINT: IS_HEAD=%d, FIRST_PIECE_END=%d-%d, CHRO=%u-%u MATCHED=%d,%d\n", is_head, indel_first_piece_end_on_read, indel_second_piece_start_on_read, indel_first_piece_end_on_chro, indel_second_piece_start_on_chro, first_piece_match, second_piece_match); } } if(max_score >= 2*window_size - 1){ if(is_head){ (*head_indel_pos) = best_splice_on_read; (*head_indel_movement) = best_movement; } else { (*tail_indel_pos) = best_splice_on_read; (*tail_indel_movement) = best_movement; } } } return 0; } int core_extend_covered_region_13(gene_value_index_t *array_index, unsigned int read_start_pos, char * read, int read_len, int cover_start, int cover_end, int window_size, int req_match_5end , int req_match_3end, int indel_tolerance, int space_type, int tail_indel, short * head_indel_pos, int * head_indel_movement, short * tail_indel_pos, int * tail_indel_movement, int is_head_high_quality, char * qual_txt, int qual_format, float head_matching_rate, float tail_matching_rate) { int ret = 0; *head_indel_pos = -1; *tail_indel_pos = -1; if (cover_start >= window_size && head_matching_rate < 1.0001) { int head_test_len = cover_start; int roughly_mapped = match_chro(read, array_index, read_start_pos, head_test_len , 0, space_type); if (roughly_mapped >= head_test_len * EXON_RECOVER_MATCHING_RATE - 0.0001) { ret |= 1; } else { int window_end_pos = cover_start + window_size-1; int not_too_bad = 1; int right_match_number=0; while (1) { int matched_bases_in_window = match_chro_wronglen(read+ window_end_pos - window_size, array_index, read_start_pos + window_end_pos - window_size, window_size, space_type, NULL, &right_match_number); int best_indel_movement = -1000; int best_indel_pos = -1; if (matched_bases_in_window >= req_match_5end) // this window is not bad enough so that an indel is considered window_end_pos--; else { roughly_mapped = match_chro(read, array_index, read_start_pos, window_end_pos - right_match_number , 0, space_type); if (roughly_mapped < (int)(0.5+ (window_end_pos - right_match_number) * EXON_RECOVER_MATCHING_RATE)) { // the quality of this window is too low (at most 1 base is matched). I must consider if it is an indel. int indel_movement_i ; int best_matched_bases_after_indel = -1; not_too_bad = 0; for (indel_movement_i = 0; indel_movement_i < 2* indel_tolerance-1 ; indel_movement_i ++) { int indel_movement = (indel_movement_i+1)/2 * (indel_movement_i %2?1:-1); int test_length = window_end_pos /*- 1*/ - max(0, indel_movement) - right_match_number; if (test_length < window_size) continue; //if (test_length <= 1+ abs(indel_movement)/4) continue; //test_length = min(10, test_length); if (abs(indel_movement) > indel_tolerance) continue; int test_start =0;// window_end_pos - max(0, indel_movement) - right_match_number - test_length; float matched_bases_after_indel = match_chro_support(read +test_start, array_index, read_start_pos + indel_movement +test_start, test_length,0, space_type, qual_txt, qual_format); SUBREADprintf("HEAD : MATCHED_AFTER_INDEL = %f ; MVMT=%d ; WINDOW_END=%d\n", matched_bases_after_indel, indel_movement, window_end_pos); float test_rate = head_matching_rate; if(test_length < 3) test_rate = 1; if(best_matched_bases_after_indel = (int)(0.5+test_length * test_rate)) { not_too_bad = 1; best_matched_bases_after_indel = matched_bases_after_indel; best_indel_movement = indel_movement; best_indel_pos = window_end_pos - right_match_number; *head_indel_pos = best_indel_pos; *head_indel_movement = best_indel_movement; } } if(best_indel_pos<0) *head_indel_pos = window_end_pos - right_match_number; // break; } window_end_pos--; } if (window_end_pos - window_size <= 0) break; } if(not_too_bad) ret |=1; //else //*head_indel_pos = -1; } } else ret |= 1; if (cover_end <= read_len - window_size && tail_matching_rate < 1.0001) { int tail_test_len = read_len - cover_end; int roughly_mapped = match_chro(read + cover_end, array_index, read_start_pos + cover_end + tail_indel, tail_test_len , 0, space_type); if (roughly_mapped >= tail_test_len * EXON_RECOVER_MATCHING_RATE - 0.0001) { ret |= 2; } else { int window_start_pos = cover_end - window_size +1; int not_too_bad = 1; while (1) { int left_match_number = 0; int matched_bases_in_window = match_chro_wronglen(read+ window_start_pos, array_index, read_start_pos + window_start_pos + tail_indel, window_size, space_type, &left_match_number, NULL); int best_indel_movement = -1000; int best_indel_pos = -1; //printf("MOVE 4-bp WINDOW : %d ; matched = %d >=? %d\n", window_start_pos, matched_bases_in_window, req_match_3end); if (matched_bases_in_window >= req_match_3end) // this window is not bad enough so that an indel is considered window_start_pos++; else { roughly_mapped = match_chro(read+window_start_pos + left_match_number, array_index, read_start_pos + window_start_pos + tail_indel + left_match_number, read_len - window_start_pos - left_match_number , 0, space_type); if (roughly_mapped < (int)(0.5+(read_len - window_start_pos -left_match_number ) * EXON_RECOVER_MATCHING_RATE)) { // the quality of this window is too low (at most 1 base is matched). I must consider if it is an indel. int indel_movement_i ; int best_matched_bases_after_indel = -1; not_too_bad = 0; for (indel_movement_i = 0; indel_movement_i < 2* indel_tolerance; indel_movement_i ++) { int indel_adjustment = (indel_movement_i+1)/2 * (indel_movement_i %2?1:-1); int indel_movement = tail_indel + indel_adjustment; int test_length = read_len - window_start_pos - left_match_number + min(0, indel_adjustment); //test_length = min(10, test_length); if (test_length < window_size) continue; //if (test_length <= 1 + abs(indel_movement)/4) continue; if (abs(indel_movement) > indel_tolerance) continue; char * qual_txt_moved = qual_txt; if(qual_txt[0]) qual_txt_moved+=window_start_pos - min(0, indel_movement) + left_match_number; float matched_bases_after_indel = match_chro_support(read + window_start_pos - min(0, indel_movement) + left_match_number, array_index, read_start_pos + window_start_pos + max(0,indel_movement) +left_match_number , test_length,0, space_type, qual_txt_moved , qual_format); SUBREADprintf("TAIL : MATCHED_AFTER_INDEL = %f ; MVMT=%d ; WINDOW_END=%d\n", matched_bases_after_indel, indel_movement, window_start_pos - min(0, indel_movement) + left_match_number); float test_rate = tail_matching_rate; if(test_length < 3) test_rate = 1; //printf("%d =? %d\n", best_matched_bases_after_indel, matched_bases_after_indel, matched_bases_after_indel, (int)(0.5+test_length * test_rate)); if(best_matched_bases_after_indel = (int)(0.5+test_length * test_rate)) { not_too_bad = 1; best_matched_bases_after_indel = matched_bases_after_indel; best_indel_movement = indel_movement; best_indel_pos = window_start_pos + left_match_number ;//-1; *tail_indel_movement = best_indel_movement ; } } if(best_indel_pos<0) *tail_indel_pos = window_start_pos + left_match_number ; else *tail_indel_pos = best_indel_pos; } window_start_pos++; } if (window_start_pos + window_size >= read_len) break; } if(not_too_bad) ret |=2; //else // *tail_indel_pos =-1; } } else ret |= 2; return ret; } int core_dynamic_align(global_context_t * global_context, thread_context_t * thread_context, char * read, int read_len, unsigned int begin_position, char * movement_buffer, int expected_offset, char * read_name); int find_new_indels(global_context_t * global_context, thread_context_t * thread_context, int pair_number, char * read_name, char * read_text, char * qual_text, int read_len, int is_second_read, int best_read_id) { int i,j; int last_correct_subread = 0; int last_indel = 0; gene_vote_number_t * indel_recorder; unsigned int voting_position; HashTable * event_table = NULL; if(thread_context) event_table = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> event_entry_table; else event_table = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_entry_table; mapping_result_t *current_result = _global_retrieve_alignment_ptr(global_context, pair_number, is_second_read, best_read_id); if(global_context->config.do_big_margin_filtering_for_reads) if(is_ambiguous_voting(global_context, pair_number, is_second_read , current_result->selected_votes, current_result->confident_coverage_start, current_result->confident_coverage_end, read_len, (current_result->result_flags & CORE_IS_NEGATIVE_STRAND)?1:0))return 0; indel_recorder = current_result -> selected_indel_record; voting_position = current_result -> selected_position; if(!indel_recorder[0]) return 0; gene_value_index_t * current_value_index = thread_context?thread_context->current_value_index:global_context->current_value_index; if(0){ // test "shifting indels" -- not needed when "NEW" go_Q used int last_offset = 0; for(i=0; indel_recorder[i] && (iconfig.total_subreads , last_correct_subread) - 9; int first_correct_base = find_subread_end(read_len, global_context->config.total_subreads , next_correct_subread) - 16 + 9; last_correct_base = max(0, last_correct_base); last_correct_base = min(read_len-1, last_correct_base); first_correct_base = min(first_correct_base, read_len-1); first_correct_base = max(0, first_correct_base); first_correct_base = max(first_correct_base, last_correct_base); if(first_correct_base < last_correct_base || first_correct_base > last_correct_base + 3000) SUBREADprintf("WRONG ORDER: F=%u, L=%d\n", first_correct_base , last_correct_base); //printf("OCT27-STEPRR %s POS=%u, I=%d; INDELS=%d; COVER=%d -- %d (vote_no : %d - %d)\n", read_name, current_result->selected_position, i, indels, last_correct_base, first_correct_base, last_correct_subread, next_correct_subread); if(global_context -> config.use_dynamic_programming_indel || read_len > EXON_LONG_READ_LENGTH) { char movement_buffer[MAX_READ_LENGTH * 10 / 7]; //chromosome_event_t * last_event = NULL; int last_event_id = -1; first_correct_base = min(first_correct_base+10, read_len); int x1, dyna_steps; dyna_steps = core_dynamic_align(global_context, thread_context, read_text + last_correct_base, first_correct_base - last_correct_base, voting_position + last_correct_base + last_indel, movement_buffer, indels, read_name); movement_buffer[dyna_steps]=0; if(0 && FIXLENstrcmp("simulated.2467286", read_name) == 0){ SUBREADprintf("DYNAMIC: indels=%d, %s\n", indels, movement_buffer); } //#warning ">>>>>>> COMMENT NEXT BLOCK IN RELEASE <<<<<<<<" if(0) { char outstr[1000]; sprintf(outstr, "OCT27-STEPDD-IR %s %d %d~%d ", read_name, dyna_steps, last_correct_base, first_correct_base); for(x1=0; x1config.maximise_sensitivity_indel && total_mismatch <= 2 )) for(x1=0; x1current_value_index:global_context->current_value_index; int ambiguous_count=0; if(0){ int ambiguous_i, best_matched_bases = match_chro(read_text + cursor_on_read - 6, current_value_index, indel_left_boundary - 6, 6, 0, global_context->config.space_type) + match_chro(read_text + cursor_on_read - min(current_indel_len,0), current_value_index, indel_left_boundary + max(0, current_indel_len), 6, 0, global_context->config.space_type); for(ambiguous_i=-5; ambiguous_i<=5; ambiguous_i++) { int left_match = match_chro(read_text + cursor_on_read - 6, current_value_index, indel_left_boundary - 6, 6+ambiguous_i, 0, global_context->config.space_type); int right_match = match_chro(read_text + cursor_on_read + ambiguous_i - min(current_indel_len,0), current_value_index, indel_left_boundary + ambiguous_i + max(0, current_indel_len), 6-ambiguous_i, 0,global_context->config.space_type); if(left_match+right_match == best_matched_bases) ambiguous_count ++; } } if(abs(current_indel_len)<=global_context -> config.max_indel_length) { int old_event_id = -1; //if( indel_left_boundary >= 854782874 - 5 && indel_left_boundary <= 854782888 +5 ) // SUBREADprintf("PCCINDEL by %s at %u len=%d\n", read_name, indel_left_boundary , current_indel_len); chromosome_event_t * new_event = local_add_indel_event(global_context, thread_context, event_table, read_text + cursor_on_read + min(0,current_indel_len), indel_left_boundary - 1, current_indel_len, 1, ambiguous_count, 0, &old_event_id); mark_gapped_read(current_result); chromosome_event_t * event_space = NULL; if(thread_context) event_space = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; else event_space = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; if(last_event_id>=0){ chromosome_event_t * last_event = event_space + last_event_id; int dist = indel_left_boundary - last_event -> event_large_side ; if(last_event -> connected_next_event_distance<1) last_event -> connected_next_event_distance = dist; else last_event -> connected_next_event_distance = min(dist, last_event -> connected_next_event_distance); chromosome_event_t * old_new_event = new_event; if(old_event_id>=0){ assert(!new_event); old_new_event = event_space + old_event_id; } if(old_new_event -> connected_previous_event_distance < 1) old_new_event -> connected_previous_event_distance = dist; else old_new_event -> connected_previous_event_distance = min(dist, old_new_event -> connected_previous_event_distance); } if (new_event) last_event_id = new_event -> global_event_id; else last_event_id = old_event_id; } } if(mv == 0 || mv == 3) is_in_indel = 0; } if(is_in_indel && mv == 1) current_indel_len += 1; if(is_in_indel && mv == 2) current_indel_len -= 1; if(mv == 1 || mv == 3 || mv == 0) cursor_on_chromosome++; if(mv == 2 || mv == 3 || mv == 0) cursor_on_read++; last_mv = mv; } } else { if(first_correct_base - last_correct_base < -min(0,indels)) continue; int best_score=-99999, is_ambiguous_indel= 0; unsigned int best_pos = 0; int cutoff = first_correct_base-last_correct_base + min(0, indels) - global_context -> config.flanking_subread_indel_mismatch; int mid_j = (first_correct_base-last_correct_base )/2, min_j=99990; int mismatched_bases = 0; if(first_correct_base > last_correct_base + 4) for(j=0; j<(first_correct_base-last_correct_base + min(0, indels)); j++) { // j is the first UNWANTED base after the first matched part. int score , s2, s1; s1 = match_chro(read_text + last_correct_base, current_value_index, voting_position + last_correct_base + last_indel, j, 0, global_context->config.space_type); if(s1 >= j - global_context -> config.flanking_subread_indel_mismatch) { s2 = match_chro(read_text + last_correct_base + j - min(0, indels) , current_value_index, voting_position + last_correct_base + j + max(0,indels) + last_indel, first_correct_base-last_correct_base - j + min(0, indels), 0, global_context->config.space_type); score = s2+s1; if(score >= cutoff) { if(score == best_score) is_ambiguous_indel ++; if(score>best_score || (abs(mid_j-j)>min_j && best_score == score)) { mismatched_bases = first_correct_base-last_correct_base + min(0, indels) - score; best_score = score; // best_pos here is the absolute offset of the FIRST UNWANTED BASE. best_pos = voting_position + last_correct_base + j + last_indel; min_j = abs(mid_j-j); if(score>best_score) is_ambiguous_indel = 0; } } } } /* SUBREADprintf("\n%s\nRANGE:%d - %d ; BSCORE:%d @ %d ; AMB=%d ; INDELS=%d ; MM=%d\n", read_name, first_correct_base, last_correct_base, best_score, last_indel + last_correct_base + best_j, is_ambiguous_indel, indels, mismatched_bases); SUBREADprintf("%s\n", read_text); for(j=0;j< last_indel + last_correct_base + best_j; j++) putc(' ' , stderr); putc('\\' , stderr); for(j=0;j< indels; j++) putc(gvindex_get( current_value_index, best_pos + j) , stderr); putc('\n', stderr); */ if(best_score >0) { //#warning ">>>>>>>>>>>>>>>>>> COMMEN THIS <<<<<<<<<<<<<<<<<<<<<<" //printf("OCT27-STEPAD-%s POS=%u INDEL=%d\n", read_name, best_pos , indels); local_add_indel_event(global_context, thread_context, event_table, read_text + last_correct_base + last_indel, best_pos - 1, indels, 1, is_ambiguous_indel, mismatched_bases, NULL); mark_gapped_read(current_result); } } } last_correct_subread = indel_recorder[i+1]-1; last_indel = indel_recorder[i+2]; } if(global_context -> config.extending_search_indels && global_context -> config.max_indel_length>0) { // USING EXTENDING TO FIND INDELS // THIS IS UNSTABLE! int s_head = match_chro(read_text, current_value_index, voting_position, 8, 0, global_context->config.space_type); int s_tail = match_chro(read_text+read_len - 8, current_value_index, voting_position + read_len + current_result -> indels_in_confident_coverage - 8, 8, 0, global_context->config.space_type); //SUBREADprintf("EXT_START %d, %d\n", s_head, s_tail); //#warning "============ REMOVE THE FIRST '1' FROM THE NEXT LINE! ==========" if(s_head<=6 || s_tail<=6) { int is_head_high_quality = (current_result -> result_flags & CORE_IS_NEGATIVE_STRAND)?0:1; int k; int cover_start = find_subread_end(read_len, global_context -> config.total_subreads, current_result -> selected_indel_record[0]-1)- 15; for (k=0; current_result -> selected_indel_record[k]; k+=3); int cover_end = find_subread_end(read_len, global_context -> config.total_subreads, current_result -> selected_indel_record[k-2]-1) + max(0,-current_result -> selected_indel_record[k-1]); int head_qual = is_head_high_quality?0x7fffffff:0, tail_qual = is_head_high_quality?0:0x7fffffff; if(qual_text && qual_text[0]) { int j; head_qual = 0; tail_qual = 0; for (j = 0; j config.phred_score_format) { head_qual += (1000000-get_base_error_prob64i(qual_text[j])); } else { head_qual += (1000000-get_base_error_prob33i(qual_text[j])); } } for (j = 0; j config.phred_score_format) { tail_qual += (1000000-get_base_error_prob64i(qual_text[read_len - j -1])); } else { tail_qual += (1000000-get_base_error_prob33i(qual_text[read_len - j -1])); } } } float exon_tail_matching_rate=0.9, exon_head_matching_rate=0.9; int head_must_correct = 4; if(cover_start > 0) { if (head_qual / cover_start < 850000) { exon_head_matching_rate = 0.75; head_must_correct =2; } else if (head_qual / cover_start < 950000 ) { exon_head_matching_rate = 0.85; head_must_correct =3; } else exon_head_matching_rate = 0.92; }else exon_head_matching_rate = 9999; int tail_must_correct = 4; if(read_len-cover_end > 0) { if (tail_qual / (read_len-cover_end) < 850000) { exon_tail_matching_rate = 0.75; tail_must_correct =2; } else if (tail_qual / (read_len-cover_end) < 950000) { exon_tail_matching_rate = 0.85; tail_must_correct =3; } else exon_tail_matching_rate = 0.92; }else exon_tail_matching_rate = 9999; short head_indel_pos=-1 , tail_indel_pos=-1; int head_indel_movement=0, tail_indel_movement=0; if(0)SUBREADprintf("HQ=%.4f; TQ=%.4f; HM=%d; TM=%d; COVG = %d ~ %d\n", exon_head_matching_rate, exon_tail_matching_rate, head_must_correct, tail_must_correct, cover_start, cover_end); /* if(exon_head_matching_rate<1) exon_head_matching_rate = 0.97; if(exon_tail_matching_rate<1) exon_tail_matching_rate = 0.97; */ core_extend_covered_region_15(global_context, current_value_index, current_result->selected_position, read_text, read_len, cover_start, cover_end, 7, head_must_correct,tail_must_correct, global_context -> config.max_indel_length + 1, global_context -> config.space_type, current_result -> selected_indel_record[k-1], & head_indel_pos, & head_indel_movement, & tail_indel_pos, & tail_indel_movement, is_head_high_quality, qual_text, global_context -> config.phred_score_format, exon_head_matching_rate, exon_tail_matching_rate); //head_indel_movement = -head_indel_movement; if(0)SUBREADprintf("HMV=%d; TMV=%d; TPOS=%d\n", head_indel_movement, tail_indel_movement, tail_indel_pos); if(head_indel_movement && s_head < 7) { unsigned int head_indel_left_edge = head_indel_pos + current_result->selected_position - 1; //head_indel_left_edge -= max(0, head_indel_movement); if(abs(head_indel_movement)<=global_context -> config.max_indel_length) { local_add_indel_event(global_context, thread_context, event_table, read_text + head_indel_pos, head_indel_left_edge, head_indel_movement, 1, 1, 0,NULL); mark_gapped_read(current_result); } } if(tail_indel_movement && s_tail < 7) { unsigned int tail_indel_left_edge = tail_indel_pos + current_result->selected_position + current_result -> indels_in_confident_coverage - 1; if(abs(tail_indel_movement)<=global_context -> config.max_indel_length) { local_add_indel_event(global_context, thread_context, event_table, read_text + tail_indel_pos, tail_indel_left_edge, tail_indel_movement, 1, 1, 0,NULL); mark_gapped_read(current_result); } } } } return 0; } void print_indel_table(global_context_t * global_context){ } int write_indel_final_results(global_context_t * global_context) { int xk1, disk_is_full = 0; char * inserted_bases = NULL; indel_context_t * indel_context = (indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]; char * fn2, *ref_bases, *alt_bases; FILE * ofp = NULL; fn2 = malloc(MAX_FILE_NAME_LENGTH+30); sprintf(fn2, "%s.indel.vcf", global_context->config.output_prefix); ofp = f_subr_open(fn2, "wb"); //if(!ofp) // printf("HOW??? %s\n", fn2); inserted_bases = malloc(MAX_INSERTION_LENGTH + 2); ref_bases = malloc(1000); alt_bases = malloc(1000); fputs("##fileformat=VCFv4.0\n##INFO=\n##INFO=\n##INFO=\n", ofp); fputs("#CHROM\tPOS\tID\tREF\tALT\tQUAL\tFILTER\tINFO\n", ofp); for(xk1 = 0; xk1 < indel_context -> total_events ; xk1++) { char * chro_name; int chro_pos; chromosome_event_t * event_body = indel_context -> event_space_dynamic +xk1; // #warning " ================= REMOVE '- 1' from the next LINE!!! =========================" if((event_body -> event_type != CHRO_EVENT_TYPE_INDEL && event_body->event_type != CHRO_EVENT_TYPE_LONG_INDEL && event_body -> event_type != CHRO_EVENT_TYPE_POTENTIAL_INDEL)|| (event_body -> final_counted_reads < 1 && event_body -> event_type == CHRO_EVENT_TYPE_INDEL) ) continue; //assert((-event_body -> indel_length) < MAX_INSERTION_LENGTH); if(event_body -> indel_length<0) { get_insertion_sequence(global_context, NULL , event_body -> inserted_bases , inserted_bases , -event_body -> indel_length); free(event_body -> inserted_bases); } //assert(event_body -> event_small_side < event_body -> event_large_side ); locate_gene_position( event_body -> event_small_side , &global_context -> chromosome_table, &chro_name, &chro_pos); // VCF format: chr21 1000001 . AACC AAGGGGGCC 29 . INDEL;DP=20 if(event_body -> event_type == CHRO_EVENT_TYPE_INDEL || event_body -> event_type == CHRO_EVENT_TYPE_LONG_INDEL) { ref_bases[0]=0; alt_bases[0]=0; gene_value_index_t * current_index = find_current_value_index(global_context , event_body -> event_small_side-1 , max(0, event_body -> indel_length) + 2); if(current_index) { int rlen = max(0, event_body -> indel_length) + 2; assert(rlen<900); gvindex_get_string(ref_bases, current_index, event_body -> event_small_side-1 , rlen, 0); ref_bases[rlen] = 0; if(event_body -> indel_length > 0) // deletion { alt_bases[0] = ref_bases[0]; alt_bases[1] = ref_bases[event_body -> indel_length+1]; alt_bases[2] = 0; } else // insertion { alt_bases[0] = ref_bases[0]; strcpy(alt_bases+1,inserted_bases); strcat(alt_bases,ref_bases+1); } } if(CHRO_EVENT_TYPE_INDEL == event_body -> event_type) { if(event_body -> final_counted_reads<30) event_body -> event_quality = pow(0.5,(30-event_body -> final_counted_reads)); else event_body -> event_quality = 1; } int write_len = fprintf(ofp, "%s\t%u\t.\t%s\t%s\t%d\t.\tINDEL;DP=%d;SR=%d\n", chro_name, chro_pos, ref_bases, alt_bases, (int)(max(1, 250 + 10*log(event_body -> event_quality)/log(10))), event_body -> final_counted_reads + event_body -> anti_supporting_reads, event_body -> final_counted_reads); if(write_len < 10) disk_is_full = 1; } global_context->all_indels++; } fclose(ofp); free(ref_bases); free(alt_bases); free(inserted_bases); if(disk_is_full){ unlink(fn2); SUBREADprintf("ERROR: disk is full. Unable to write into the indel list.\n"); } free(fn2); return 0; } int destroy_indel_module(global_context_t * global_context) { int xk1; indel_context_t * indel_context = global_context -> module_contexts[MODULE_INDEL_ID]; if(indel_context -> event_entry_table->appendix1) { free(indel_context -> event_entry_table -> appendix1); free(indel_context -> event_entry_table -> appendix2); } // free the entry table: the pointers. destory_event_entry_table(indel_context -> event_entry_table); HashTableDestroy(indel_context -> event_entry_table); free(indel_context -> event_space_dynamic); for(xk1=0;xk1 dynamic_align_table[xk1]); free(indel_context -> dynamic_align_table_mask[xk1]); } for(xk1=0; xk1 event_body_locks+xk1); free(indel_context -> dynamic_align_table); free(indel_context -> dynamic_align_table_mask); return 0; } int trim_read(global_context_t *global_context , thread_context_t *thread_context, char * read_text, char * qual_text, int read_len, int * delta_position) { int xk1, bad_bases = 0; if(qual_text[0]==0) return read_len; int tail_trim_pos, last_ok = read_len /2; bad_bases=0; for(xk1=read_len/2 ; xk1< read_len ; xk1++) { if(qual_text[xk1] <= '#'+2) bad_bases++; else last_ok = xk1; if(bad_bases>=2) break; } tail_trim_pos = last_ok; int head_trim_pos; bad_bases=0; last_ok = read_len /2; for(xk1=read_len/2 ; xk1>=0 ; xk1--) { if(qual_text[xk1] <= '#'+2) bad_bases++; else last_ok = xk1; if(bad_bases>=2) break; } if(bad_bases>=2) head_trim_pos = last_ok; else head_trim_pos = 0; if(3*(tail_trim_pos - head_trim_pos )all_value_indexes[0] ; if(is_anchor_certain) { int head_match = match_chro(read_text, base_index , anchor_pos, READ_HEAD_TAIL_LEN, 0, global_context -> config.space_type); int tail_match = match_chro(read_text + read_len - READ_HEAD_TAIL_LEN, base_index , anchor_pos + read_len - READ_HEAD_TAIL_LEN, READ_HEAD_TAIL_LEN, 0, global_context -> config.space_type); if(head_match > READ_HEAD_TAIL_LEN-3 && tail_match> READ_HEAD_TAIL_LEN-3) return 0; } if(0 == locate_gene_position_max(anchor_pos, &global_context -> chromosome_table, &chro_name, &chro_offset, NULL, NULL, read_len)) { char temp_file_name[MAX_FILE_NAME_LENGTH+40]; int close_now = 0; sprintf(temp_file_name,"%s@%s-%04u.bin", global_context -> config.temp_file_prefix, chro_name , chro_offset / BASE_BLOCK_LENGTH ); FILE * pileup_fp = get_temp_file_pointer(temp_file_name, pileup_fp_table, &close_now); //assert(read_len == strlen(read_text) && read_len > 90); write_read_block_file(pileup_fp , 0, read_name, 0, chro_name , chro_offset, NULL, 0, read_text , qual_text, read_len , 1 , is_anchor_certain , anchor_pos, read_len, 0); if(close_now) fclose(pileup_fp); } return 0; } int build_local_reassembly(global_context_t *global_context , thread_context_t *thread_context, int pair_number , char * read_name , char * read_text ,char * qual_text , int read_len , int mate_read_len , int is_second_read, int best_read_id, int use_mate_pos, mapping_result_t *current_result, mapping_result_t *mate_result) { unsigned int read_anchor_position; if(!read_text) return 0; int is_position_certain = 1; indel_context_t * indel_context = (indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]; //mapping_result_t *current_result = _global_retrieve_alignment_ptr(global_context, pair_number, is_second_read, best_read_id); //mapping_result_t * mate_result = _global_retrieve_alignment_ptr(global_context, pair_number, !is_second_read, best_read_id); // No complex cigar strings are allowed. if(use_mate_pos) { read_anchor_position = mate_result -> selected_position; if(is_second_read + ((mate_result -> result_flags & CORE_IS_NEGATIVE_STRAND)?1:0) == 1) // I'm the second read so I'm after the first one. read_anchor_position += global_context -> config.expected_pair_distance; else // I'm the first read so I'm ahead the second one. read_anchor_position -= global_context -> config.expected_pair_distance; is_position_certain = 0; } else { read_anchor_position = current_result -> selected_position; } //gene_alue_index_t * value_index = &global_context->all_value_indexes[0] ; if(qual_text[0] &&global_context -> config.phred_score_format == FASTQ_PHRED64) fastq_64_to_33(qual_text); //int window_no = 2*(read_anchor_position / REASSEMBLY_WINDOW_LENGTH); write_local_reassembly(global_context, indel_context -> local_reassembly_pileup_files, read_anchor_position, read_name , read_text , qual_text , read_len, is_position_certain); return 0; } int insert_pileup_read(global_context_t * global_context , reassembly_block_context_t * block_context , unsigned int first_base_pos ,char * read_text , char * qual_text , int read_len, int is_read_head_aligned) { int window_i,xk1; for(window_i= -1; window_i<1; window_i++) { int de_brujin_grp_id = 2*(first_base_pos / REASSEMBLY_WINDOW_LENGTH) + window_i; int read_pos; if(de_brujin_grp_id<0)continue; HashTable * current_table = block_context -> de_bruijn_graphs [de_brujin_grp_id]; for(read_pos = 0; read_pos <= read_len - global_context -> config.k_mer_length; read_pos +=1) { unsigned long long int node_key = 0; for(xk1=0; xk1config.k_mer_length; xk1++) { node_key = (node_key << 2) | (base2int(read_text[xk1 + read_pos])); } unsigned long long int next_key_prefix = 0x8000000000000000ull | (node_key >> 2); if(!read_pos && is_read_head_aligned) { int start_offset = (first_base_pos - de_brujin_grp_id * REASSEMBLY_WINDOW_LENGTH/2); if(start_offset < block_context -> start_offsets [de_brujin_grp_id]) { block_context -> start_offsets [de_brujin_grp_id] = start_offset; block_context -> start_keys[1+10*de_brujin_grp_id]=0; block_context -> start_keys[10*de_brujin_grp_id]=next_key_prefix; } else if(start_offset == block_context -> start_offsets [de_brujin_grp_id]) { int start_key_id; for(start_key_id=0; start_key_id<10&&block_context -> start_keys[de_brujin_grp_id * 10 + start_key_id]; start_key_id++); if(start_key_id<10) block_context -> start_keys[start_key_id + 10 * de_brujin_grp_id] = next_key_prefix; } } int next_node_type = node_key&0x03; unsigned int next_jump_array = HashTableGet(current_table ,NULL+next_key_prefix) - NULL; if(next_jump_array) next_jump_array-=1; unsigned int current_read_count = 0xff&(next_jump_array >> (next_node_type*8)); if(current_read_count<254) current_read_count ++; next_jump_array = 1+((next_jump_array & ~(0xff << (next_node_type*8))) | ((current_read_count) << (next_node_type*8))); HashTablePut(current_table, NULL + next_key_prefix , NULL + next_jump_array); } } return 0; } int finalise_db_graph(global_context_t * global_context, reassembly_block_context_t * block_context, int de_brujin_graph_id, HashTable * de_brujin_graph, unsigned long long int start_key, short start_offset) { unsigned long long int current_key = start_key; char inserted_sequence[MAX_INSERTION_LENGTH+REASSEMBLY_WINDOW_LENGTH]; unsigned long long int used_keys[MAX_INSERTION_LENGTH+REASSEMBLY_WINDOW_LENGTH]; int inserted_length = 0; int is_looped = 0; int xk1; for(xk1=0; xk1 config.k_mer_length-1; xk1++) { int intbase = (start_key >> (2*(global_context -> config.k_mer_length-xk1-2))) & 3; inserted_sequence[xk1] = "AGCT"[intbase]; } inserted_length = global_context -> config.k_mer_length-1; while(1) { unsigned int next_moves; int max_move=-1; next_moves = HashTableGet(de_brujin_graph , NULL + current_key) - NULL; if(!next_moves) break; else { int test_next; int all_reads = 0; int ignored_moves=0; int max_move_reads=0; unsigned long long int previous_key = current_key<<2; next_moves-=1; for(test_next=0; test_next<4; test_next++) all_reads += 0xff&(next_moves>>(8*test_next)); while(1) { max_move = -1; max_move_reads = -1; for(test_next=0; test_next<4; test_next++) { if(ignored_moves & (1<>(8*test_next)); if(test_move_reads > max_move_reads) { max_move_reads = test_move_reads; max_move = test_next; } } if(max_move < 0) break; inserted_sequence[inserted_length] = "AGCT"[max_move]; int xk2, is_used_key = 0; current_key = (previous_key | max_move); for(xk2=0; xk2 config.k_mer_length + 1; xk2++) { if(current_key == used_keys[xk2]) { is_used_key = 1; is_looped = 1; ignored_moves |= (1< config.k_mer_length + 1] = current_key; current_key &= (0x3fffffffffffffffull >> (2*(32 - global_context -> config.k_mer_length))); current_key |= 0x8000000000000000ull; inserted_length++; } if(inserted_length > MAX_INSERTION_LENGTH + REASSEMBLY_WINDOW_LENGTH - 1 || max_move<0|| is_looped) { is_looped = 1; inserted_sequence[inserted_length]=0; break; } } if(!is_looped) { inserted_sequence[inserted_length]=0; assert(global_context -> index_block_number == 1); gene_value_index_t * base_index = &global_context->all_value_indexes[0] ; unsigned int window_start = block_context -> block_start_linear_pos + de_brujin_graph_id * REASSEMBLY_WINDOW_LENGTH / 2; int best_matched_bases = -1, best_end_pos = -1, best_start_pos = start_offset; #define END_POS_CONFIRM_LENGTH 24 for(xk1 = inserted_length + start_offset - END_POS_CONFIRM_LENGTH ; xk1>= start_offset+15 ; xk1--) //find the end of insertion { if(inserted_length - xk1 - END_POS_CONFIRM_LENGTH + best_start_pos >= MAX_INSERTION_LENGTH) break; int matched_bases = match_chro(inserted_sequence + inserted_length - END_POS_CONFIRM_LENGTH, base_index , window_start + xk1, END_POS_CONFIRM_LENGTH, 0, global_context -> config.space_type ); if(matched_bases>best_matched_bases) { best_matched_bases = matched_bases; best_end_pos = xk1 + END_POS_CONFIRM_LENGTH; } } int new_base_length = inserted_length - best_end_pos + best_start_pos; if(new_base_length>0 && best_matched_bases >= END_POS_CONFIRM_LENGTH - 1) { best_matched_bases = -1; int best_insertion_point = -1; for(xk1 = 15; xk1 config.space_type) + match_chro(inserted_sequence + xk1 + new_base_length , base_index , window_start + best_start_pos + xk1 , 15, 0 , global_context -> config.space_type ); if(insertion_matched>best_matched_bases && insertion_matched>=29) { best_insertion_point = xk1; best_matched_bases = insertion_matched; } } if(best_insertion_point>=0) { unsigned int best_pos = window_start + best_start_pos + best_insertion_point; int indels = -new_base_length; //inserted_sequence[best_insertion_point + new_base_length] = 0; indel_context_t * indel_context = (indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]; HashTable * event_table = indel_context -> event_entry_table; chromosome_event_t * event_space = indel_context -> event_space_dynamic; chromosome_event_t * found = NULL; chromosome_event_t * search_return [MAX_EVENT_ENTRIES_PER_SITE]; int found_events = search_event(global_context, event_table, event_space, best_pos - 1, EVENT_SEARCH_BY_SMALL_SIDE, CHRO_EVENT_TYPE_INDEL, search_return); if(found_events) { int kx1; for(kx1 = 0; kx1 < found_events ; kx1++) { if(search_return[kx1] -> indel_length == indels) { found = search_return[kx1]; break; } } } if(found){ found -> supporting_reads ++; } else { int event_no; event_no = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> total_events ++; event_space = reallocate_event_space(global_context, NULL, event_no); chromosome_event_t * new_event = event_space+event_no; memset(new_event,0,sizeof(chromosome_event_t)); set_insertion_sequence(global_context, NULL , &new_event -> inserted_bases , inserted_sequence+best_insertion_point, -indels); new_event -> event_small_side = best_pos - 1; new_event -> event_large_side = best_pos + max(0, indels); assert(new_event -> event_small_side && new_event -> event_large_side); new_event -> event_type = CHRO_EVENT_TYPE_INDEL; new_event -> indel_length = indels; new_event -> supporting_reads = 1; put_new_event(event_table, new_event , event_no); } fprintf(global_context -> long_insertion_FASTA_fp, ">BEGIN%u_%dM%dI%dM\n", window_start + best_start_pos, best_insertion_point , -indels , inserted_length + indels - best_insertion_point); int write_cursor; for(write_cursor = 0 ; write_cursor < inserted_length ; write_cursor += 70) { fwrite(inserted_sequence + write_cursor , min(70, inserted_length - write_cursor), 1, global_context -> long_insertion_FASTA_fp); fputs("\n",global_context -> long_insertion_FASTA_fp); } } } } return 0; } #define finalise_pileup_file_by_voting finalise_pileup_file #define REASSEMBLY_SUBREAD_GAP 1 int search_window_once(global_context_t * global_context, reassembly_by_voting_block_context_t * block_context, int window_no, int start_read_no, int search_direction, int anti_loop, float allele_quality); // search_direction = 1 : from 5' end to 3' end // search_direction = -1 : from 3'end to 5' end void search_window(global_context_t * global_context, reassembly_by_voting_block_context_t * block_context, int window_no, int start_read_no, int search_direction) { block_context->rebuilt_window[0] = 0; block_context->rebuilt_window_size = 0; memset(block_context->final_alleles, 0, sizeof(struct reassmebly_window_allele ) * global_context -> config.reassembly_window_alleles); block_context -> search_cost = 0; block_context -> used_read_number = 0; block_context -> total_matched_bases = 0; block_context -> max_matched_bases = 0; /*int ret = */search_window_once(global_context, block_context, window_no, start_read_no, search_direction, 0, 1); } // this function returns if the read is not rectifiable (i.e. likely to be misalignment) int rectify_read_text(global_context_t * global_context, reassembly_by_voting_block_context_t * block_context, int window_no, gehash_t * index, char * next_read_txt, int really_make_change) { // return 1; int read_offset,i,j; int read_len = strlen(next_read_txt); //int is_test_target = (strcmp("AATGGAACAACCGCCTCAAATGGAATGGAATGGCCTCCAAAGGAAGGGAGTGACATTTAATGAATGCATTCTAATGGAATGGCATGGAATGGACTGGAA", next_read_txt)==0); int is_test_target = 0; init_gene_vote(block_context -> vote_list_rectify); for(read_offset = 0; read_offset < read_len - global_context -> config.reassembly_subread_length+1; read_offset += 1) { unsigned int subread_int = 0, xk2; for(xk2=0; xk2< global_context -> config.reassembly_subread_length; xk2++) subread_int = (subread_int << 2) | base2int(next_read_txt[read_offset + xk2]); gehash_go_q(index, subread_int , read_offset, read_len, 0, block_context -> vote_list_rectify, 22, read_offset, 0, 0x7fffffff); } memset(block_context -> read_rectify_space, 0, MAX_READ_LENGTH * 4 * sizeof(short)); for (i=0; i vote_list_rectify -> items[i]; j++) { int xk1; int neighbour_read_no = (block_context -> vote_list_rectify -> pos[i][j] + MAX_READ_LENGTH+1)/10000; int neighbour_read_mapped_start, this_read_mapping_start, overlapped_length; unsigned long long int neighbour_read_key = (window_no * 1llu +1)<<32 ; neighbour_read_key |= (neighbour_read_no& 0xfffff)<<12; char * neighbour_read_text = HashTableGet(block_context->read_sequence_table,NULL+neighbour_read_key); assert(neighbour_read_text); int neighbour_read_len = strlen(neighbour_read_text); // neighbour_read_mapped_start is the location on the neigibour read of the first base of the current read. neighbour_read_mapped_start = - neighbour_read_no*10000 + block_context -> vote_list_rectify -> pos[i][j];// + block_context -> vote_list -> coverage_start[i][j]; if(neighbour_read_mapped_start>=0) { this_read_mapping_start = 0; overlapped_length = min(neighbour_read_len - neighbour_read_mapped_start, read_len); } else { // this_read_mapping_start is the location on this read of the first base of the neighbour read. this_read_mapping_start = -neighbour_read_mapped_start; neighbour_read_mapped_start = 0; overlapped_length = min(read_len - this_read_mapping_start, neighbour_read_len); if(is_test_target) { for(xk1 = 0; xk1 < 3+this_read_mapping_start; xk1++) fputc(' ',stdout ); printf("%s\n", neighbour_read_text); } } for(xk1 = 0; xk1 < overlapped_length; xk1++) { int this_read_pos = this_read_mapping_start + xk1; int nb_read_pos = neighbour_read_mapped_start + xk1; block_context -> read_rectify_space[this_read_pos * 4 +base2int(neighbour_read_text[nb_read_pos])] ++; } } if(is_test_target) printf(" %s\n", next_read_txt); char ori[2000]; strcpy(ori, next_read_txt); is_bad_read(ori, read_len, "RECT"); int rectified = 0; if(is_test_target) printf("R0=%s\n", next_read_txt); char changed[200]; // memset(changed, ' ', 200); for(i=0; i read_rectify_space[i*4+base2int(next_read_txt[i])]; int base_sel = -1; for(j=0; j<4; j++) { if(j!=base2int(next_read_txt[i]) && block_context -> read_rectify_space[i*4+j] > max_support) { base_sel = int2base(j); max_support = block_context -> read_rectify_space[i*4+j]; } } if(base_sel>0) { if(really_make_change) { next_read_txt[i]=base_sel; // changed[i] = '~'; } rectified ++; } } changed[i]=0; if(is_test_target) printf("R1=%s\n %s\n\n", next_read_txt, changed); is_bad_read(next_read_txt, read_len, "REC2"); return rectified<=2; } int search_window_once(global_context_t * global_context, reassembly_by_voting_block_context_t * block_context, int window_no, int start_read_no, int search_direction, int anti_loop, float allele_quality) { block_context -> search_cost++; if(block_context -> search_cost > 1000) return 0; int is_start_certain = 1, is_digged_in = 0; unsigned long long int starter_read_key = (window_no * 1llu +1)<<32 ; starter_read_key |= (start_read_no& 0xfffff)<<12; char * start_read_txt = HashTableGet(block_context->read_sequence_table,NULL+starter_read_key); assert(start_read_txt); int start_read_pos = HashTableGet(block_context->read_position_table, NULL+starter_read_key) - NULL ; if(start_read_pos == 0) is_start_certain = 0; else start_read_pos --; int read_len = strlen(start_read_txt), read_offset; init_gene_vote(block_context -> vote_list); int minimum_overlap_length = read_len /4; //if(!is_start_certain) minimum_overlap_length *= 1.5; //minimum_overlap_length = max(1,16-global_context ->config.reassembly_subread_length); for(read_offset = 0; read_offset < read_len - global_context -> config.reassembly_subread_length+1; read_offset += 1) { unsigned int subread_int = 0, xk2; for(xk2=0; xk2< global_context -> config.reassembly_subread_length; xk2++) subread_int = (subread_int << 2) | base2int(start_read_txt[read_offset + xk2]); if(global_context -> config.reassembly_tolerable_voting) gehash_go_q_tolerable(&block_context->voting_indexes[window_no], subread_int , read_offset, read_len, 0, block_context -> vote_list, 1, 22, 24, 0, read_offset, global_context -> config.reassembly_tolerable_voting, global_context -> config.reassembly_subread_length, 0, 0x7fffffff); else gehash_go_q(&block_context->voting_indexes[window_no], subread_int , read_offset, read_len, 0, block_context -> vote_list,0,read_offset, 0, 0x7fffffff); } int WINDOW_SEARCH_TREE_WIDTH = 1; int i,j; int max_read_scoring [8]; unsigned int max_read_ids [8]; int max_read_nonoverlap [8]; float max_allele_quality [8]; memset(max_read_scoring, 0, sizeof(int)* 8); memset(max_allele_quality, 0, sizeof(float)* 8); memset(max_read_ids, 0, sizeof(int)* 8); memset(max_read_nonoverlap, 0, sizeof(int)* 8); //intif(is_target_window_X(window_no)) //if(block_context -> window_start_pos > 1124357402 && block_context -> window_start_pos < 1124359402) // print_votes(block_context -> vote_list, global_context -> config.index_prefix); for (i=0; i vote_list -> items[i]; j++) { //if(is_digged_in)break; //if((block_context -> vote_list -> coverage_end[i][j] - block_context -> vote_list -> coverage_start[i][j]) * 6 > read_len ) // if(block_context -> window_start_pos > 1124357402 && block_context -> window_start_pos < 1124359402) // SUBREADprintf("LLLX PRINT_VOTE @%u : V=%d\n", block_context -> vote_list -> pos[i][j], block_context -> vote_list -> votes[i][j] ); if( block_context -> vote_list -> votes[i][j] > 4) { int next_read_no = (block_context -> vote_list -> pos[i][j] + MAX_READ_LENGTH +1 )/10000; if(next_read_no == start_read_no) continue; int next_read_nonoverlap = next_read_no * 10000 - block_context -> vote_list -> pos[i][j]; float next_read_allele_quality = allele_quality; int anchor_to_index = 0; if(abs(next_read_nonoverlap) < 2) anchor_to_index = 0; // PARALLEL or VALUELESS else if(next_read_nonoverlap < 0) anchor_to_index = -1; // INDEX -> ANCHOR else anchor_to_index = 1; // ANCHOR -> INDEX unsigned long long int test_next_read_key = (window_no * 1llu +1)<<32 ; test_next_read_key |= ( next_read_no& 0xfffff)<<12; int is_next_certain = 1; int next_read_pos = HashTableGet(block_context->read_position_table, NULL+test_next_read_key) - NULL; if(next_read_pos==0) is_next_certain =0; else next_read_pos--; /* if(!is_start_certain || !is_next_certain) { if(anchor_to_index == 1 && (block_context -> vote_list -> coverage_start[i][j] - next_read_nonoverlap) > 5) anchor_to_index = 0; // PARALLEL or VALUELESS if(anchor_to_index == -1 && block_context -> vote_list -> coverage_start[i][j] > 5) anchor_to_index = 0; // PARALLEL or VALUELESS }*/ char * next_read_txt = HashTableGet(block_context->read_sequence_table,NULL+test_next_read_key); assert(next_read_txt); int next_read_len = strlen(next_read_txt); int two_read_overlapped = next_read_len - next_read_nonoverlap; if(next_read_nonoverlap<0) two_read_overlapped = read_len + next_read_nonoverlap; if(two_read_overlapped< minimum_overlap_length) anchor_to_index = 0; if(search_direction>0) { if( next_read_nonoverlap + strlen(next_read_txt) <= read_len) anchor_to_index = 0; } if(anchor_to_index * search_direction > 0) { long long int pos_delta = next_read_pos; pos_delta -= start_read_pos; int xk3, next_read_scoring = 0, next_read_matching = 0; int order_reward = (is_next_certain && is_start_certain && search_direction * pos_delta >0)? 25000:0; if(is_start_certain * is_next_certain == 0) order_reward = 10000; int wrong_bases=0; if(anchor_to_index > 0) { if(next_read_len+ next_read_nonoverlap< read_len)continue;// no new bases are added by the new read. for(xk3 = next_read_nonoverlap ; xk3 < read_len ; xk3++) if(start_read_txt[xk3] == next_read_txt[xk3 - next_read_nonoverlap]) next_read_matching++; wrong_bases = read_len - next_read_nonoverlap - next_read_matching; if(wrong_bases>=2) { next_read_scoring = -1; } else { block_context -> total_matched_bases += next_read_matching; next_read_scoring = two_read_overlapped * 10000 + order_reward +(unsigned int)(HashTableGet(block_context -> read_quality_table, NULL+test_next_read_key)-NULL)/10 - wrong_bases * 20000; } } else { if(0< next_read_nonoverlap) continue; // no new bases are added by the new read. int should_match = min(next_read_len, read_len - next_read_nonoverlap); if(next_read_len > read_len - next_read_nonoverlap) continue; // the head of the next read is untested: bad! for(xk3 = - next_read_nonoverlap ; xk3 < should_match ; xk3++) if(next_read_txt[xk3] == start_read_txt[xk3 + next_read_nonoverlap]) next_read_matching++; wrong_bases = should_match + next_read_nonoverlap - next_read_matching; if(wrong_bases>=2) { next_read_scoring = -1; } else { block_context -> total_matched_bases += next_read_matching; next_read_scoring = two_read_overlapped * 10000 +order_reward + (unsigned int)(HashTableGet(block_context -> read_quality_table, NULL+test_next_read_key)-NULL)/10 - wrong_bases * 20000; } } if(wrong_bases) next_read_allele_quality *= 0.5; int is_rectifiable = rectify_read_text(global_context, block_context, window_no, &block_context->voting_indexes[window_no], next_read_txt , 0); /* if(block_context -> window_start_pos > 1124357402 && block_context -> window_start_pos < 1124359402) { SUBREADprintf("LLLX: NREAD #%d HAS QUAL %d ; is_rectifiable=%d ; WB=%d; ANCHOR_TO_I=%d\n", next_read_no, next_read_scoring, is_rectifiable, wrong_bases, anchor_to_index); SUBREADprintf("LLLX: NREAD OVL=%d, RLEN=%d, NRLEN=%d, NRMATCH=%d\n", next_read_nonoverlap, read_len, next_read_len, next_read_matching); SUBREADprintf("LLLX: THIS_READ=%s\n", start_read_txt); SUBREADprintf("LLLX: NEXT_READ=%s\n", next_read_txt); } */ if((next_read_scoring > max_read_scoring[ WINDOW_SEARCH_TREE_WIDTH-1]) && is_rectifiable) { int is_unique = 1, is_replace = 0; for(xk3=0; xk3 config.reassembly_window_alleles>1&&max_read_scoring[xk3]>0) { int position_diff = max_read_nonoverlap[xk3] - next_read_nonoverlap; int xk4; unsigned long long int test_prev_read_key = (window_no * 1llu +1)<<32 ; test_prev_read_key |= (max_read_ids[xk3] & 0xfffff)<<12; char *prev_read_txt = HashTableGet(block_context->read_sequence_table,NULL+test_prev_read_key); int mismatch=0; for(xk4 = abs(position_diff) ; ; xk4++) { int prev_read_pos = xk4-(position_diff>0?position_diff:0); int next_read_pos = xk4+(position_diff<0?position_diff:0); if(prev_read_txt[prev_read_pos]==0 || next_read_txt[next_read_pos]==0) break; mismatch += (prev_read_txt[prev_read_pos]!=next_read_txt[next_read_pos]); } if(mismatch<2) { is_unique = 0; if(position_diff > 0) is_replace = xk3+1; } } } if(is_replace) { max_read_scoring[is_replace-1] = next_read_scoring; max_read_ids[is_replace-1] = next_read_no; max_read_nonoverlap[is_replace-1] = next_read_nonoverlap; max_allele_quality[is_replace-1] = next_read_allele_quality; } else { if(is_unique) for(xk3=0; xk3 max_read_scoring[xk3]) { int xk4; for(xk4 = WINDOW_SEARCH_TREE_WIDTH-1; xk4>xk3; xk4--) { max_read_scoring[xk4] = max_read_scoring[xk4-1]; max_read_ids[xk4] = max_read_ids[xk4-1]; max_read_nonoverlap[xk4] = max_read_nonoverlap[xk4-1]; max_allele_quality[xk4] = max_allele_quality[xk4-1]; } max_read_scoring[xk3] = next_read_scoring; max_read_ids[xk3] = next_read_no; max_read_nonoverlap[xk3] = next_read_nonoverlap; max_allele_quality[xk3] = next_read_allele_quality; //break; } } } } } } } for(i = 0; i window_start_pos > 1124357402 && block_context -> window_start_pos < 1124359402) // SUBREADprintf("LLLX: TEST: %d\n", i); if(max_read_scoring[i]<=0) break; is_digged_in = 1; for(j=0; j< block_context -> used_read_number; j++) { if(block_context -> used_read_ids[j] == next_read_no) { is_fresh = 0; FRESH_COUNTER ++; break; } } if(!is_fresh)continue; block_context -> used_read_ids[block_context ->used_read_number++] = next_read_no; float next_allele_quality = max_allele_quality[i]; int next_read_nonoverlap = max_read_nonoverlap[i]; unsigned long long int test_next_read_key = (window_no * 1llu +1)<<32 ; test_next_read_key |= ( next_read_no& 0xfffff)<<12; char * next_read_txt = HashTableGet(block_context->read_sequence_table,NULL+test_next_read_key); assert(next_read_txt); int new_bases = (search_direction>0)?strlen(next_read_txt)-(read_len - next_read_nonoverlap):-next_read_nonoverlap; if (new_bases + block_context -> rebuilt_window_size > 1000){ return 1; } int basic_length = 1;//max(read_len, strlen(next_read_txt))/3; int max_length = 99999; //if(anti_loop) max_length = min(read_len, strlen(next_read_txt))/3; int current_rebuilt_size = block_context -> rebuilt_window_size; int current_matched_bases = block_context -> total_matched_bases; //int current_used_read_number = block_context -> used_read_number; if(new_bases>=basic_length && new_bases < max_length) { if(strlen(block_context -> rebuilt_window) != block_context ->rebuilt_window_size) return 1; //int is_rectifiable = rectify_read_text(global_context, block_context, window_no, &block_context->voting_indexes[window_no], next_read_txt , 1); //if(!is_rectifiable) continue; if(search_direction > 0) { int high_quality_offset = 0*min(2, strlen(next_read_txt) - new_bases); assert(high_quality_offset>=0); if(block_context ->rebuilt_window_size > high_quality_offset) { block_context -> rebuilt_window[ block_context ->rebuilt_window_size-high_quality_offset] =0; strcat(block_context -> rebuilt_window , next_read_txt + strlen(next_read_txt) - new_bases - high_quality_offset); } else strcat(block_context -> rebuilt_window , next_read_txt + strlen(next_read_txt) - new_bases); } else { int xk3; for(xk3= block_context -> rebuilt_window_size ; xk3>=0 ; xk3--) block_context -> rebuilt_window[new_bases + xk3] = block_context -> rebuilt_window[xk3]; int high_quality_offset = 0*min(2, strlen(next_read_txt) - new_bases); //high_quality_offset = 0; assert(high_quality_offset>=0); memcpy(block_context -> rebuilt_window , next_read_txt , new_bases + high_quality_offset); block_context -> rebuilt_window [block_context -> rebuilt_window_size +new_bases] = 0; } block_context -> rebuilt_window_size += new_bases; // if(block_context -> window_start_pos > 1124357402 && block_context -> window_start_pos < 1124359402) // SUBREADprintf("LLLX: DIG IN: %d -> %d (DIR=%d)\n", start_read_no, next_read_no, search_direction); search_window_once(global_context, block_context, window_no, next_read_no, search_direction, anti_loop, next_allele_quality); DIGIN_COUNTER ++; if(block_context -> search_cost > 1000) return 1; } if(search_direction > 0) // if the search is to the 3' end, the stack is simply dumpped. block_context -> rebuilt_window[current_rebuilt_size] = 0; else { // if the search is to the 5' end, we have to move the tail back to head. int xk4; int bytes_to_remove = block_context -> rebuilt_window_size - current_rebuilt_size; for(xk4 = bytes_to_remove; xk4 < block_context -> rebuilt_window_size; xk4++) block_context -> rebuilt_window[xk4-bytes_to_remove] = block_context -> rebuilt_window[xk4]; block_context -> rebuilt_window[current_rebuilt_size] = 0; } block_context -> rebuilt_window_size = current_rebuilt_size; block_context -> total_matched_bases = current_matched_bases; //block_context -> used_read_number = current_used_read_number; } // if(block_context -> window_start_pos > 1124357402 && block_context -> window_start_pos < 1124359402) // SUBREADprintf("LLLX: NOT DIG IN: %d :: %d DIR=%d\n", is_digged_in, block_context -> rebuilt_window_size, search_direction); if(!is_digged_in && block_context -> rebuilt_window_size > 0) { //if(block_context -> total_matched_bases * 10000 /block_context -> rebuilt_window_size > block_context -> max_matched_bases) int xk1, xk2; for(xk1 = 0; xk1 < global_context -> config.reassembly_window_alleles; xk1++) { //int is_same_allele = 0; // if(block_context -> window_start_pos > 1124357402 && block_context -> window_start_pos < 1124359402) // SUBREADprintf("LLLX [%d] CPY: %d<%d\n", xk1, block_context -> final_alleles[xk1].rebuilt_size , block_context -> rebuilt_window_size); if(block_context -> final_alleles[xk1].rebuilt_size < block_context -> rebuilt_window_size) { //int xk3=0; for(xk2= global_context -> config.reassembly_window_alleles - 1 ; xk2 > xk1; xk2--) memcpy(&block_context -> final_alleles[xk2], &block_context -> final_alleles[xk2 - 1], sizeof(struct reassmebly_window_allele)); strcpy(block_context -> final_alleles[xk1].rebuilt_window , block_context -> rebuilt_window); block_context -> max_matched_bases = block_context -> total_matched_bases * 10000 / block_context -> rebuilt_window_size; block_context -> final_alleles[xk1].rebuilt_size = block_context -> rebuilt_window_size; block_context -> final_alleles[xk1].allele_quality = allele_quality; break; } } } return 0; } #define MAX_INDELS_IN_WINDOW 10 #define PROBE_KEY_NUMBER 12 #define INDEL_FULL_ALIGN_MAX_MISMATCH 3 #define SINGLE_PROBE_MAX_MISMATCH 1 int full_indel_alignment(global_context_t * global_context, reassembly_by_voting_block_context_t * block_context, char * full_rebuilt_window, int full_rebuilt_window_size, gene_value_index_t * base_index, unsigned int window_start_pos, unsigned int * perfect_segment_start_pos, int * perfect_segment_lengths,int * indels_after_perfect_segments, short * indels_read_positions, float * indel_quality, unsigned int * contig_start_pos, unsigned int * contig_end_pos, int * head_removed_bases, int * tail_removed_bases, int * good_quality_indel) { int xk1; int is_unreliable = 0; unsigned int xk2 = window_start_pos; unsigned int probe_poses [PROBE_KEY_NUMBER]; int used_probe_in_rebuilt_window[PROBE_KEY_NUMBER]; int used_probes = 0; if(window_start_pos>300) xk2-=300; if(full_rebuilt_window_size<101)return 0; unsigned int last_best_xk2 = xk2; memset(probe_poses ,0 , sizeof(unsigned int)*PROBE_KEY_NUMBER); (*contig_start_pos) = 0; (*contig_end_pos) = 0xffffffff; for(xk1=0; xk1 config.reassembly_key_length) * xk1 /(PROBE_KEY_NUMBER-1); int max_probe_match = -1; unsigned int next_best_xk2 = 0; // xk2 is the first base of the location matching this probe. for(xk2 = last_best_xk2; xk2 < REASSEMBLY_WINDOW_LENGTH + window_start_pos + 300 + MAX_INSERTION_LENGTH; xk2++) { int probe_match = match_chro(probe_key, base_index, xk2, global_context -> config.reassembly_key_length, 0, global_context -> config.space_type); if(probe_match > max_probe_match ) { next_best_xk2 = xk2; max_probe_match = probe_match; } } if(max_probe_match >= global_context -> config.reassembly_key_length - SINGLE_PROBE_MAX_MISMATCH) { // calculate the contig start; if((*contig_start_pos)==0) { (*contig_start_pos) = next_best_xk2 + 1 - (probe_key-full_rebuilt_window); (*head_removed_bases) = probe_key-full_rebuilt_window; } (*contig_end_pos) = next_best_xk2 + 1 - (probe_key-full_rebuilt_window) + full_rebuilt_window_size; (*tail_removed_bases) = full_rebuilt_window_size - (probe_key-full_rebuilt_window) - global_context -> config.reassembly_key_length; probe_poses[used_probes] = next_best_xk2+1; used_probe_in_rebuilt_window[used_probes] = (full_rebuilt_window_size-global_context -> config.reassembly_key_length) * xk1 /(PROBE_KEY_NUMBER-1); //if(max_probe_match config.reassembly_key_length - 1) // is_unreliable += 2; used_probes +=1; } else { if(xk1 ==0 || xk1 == PROBE_KEY_NUMBER-1) { is_unreliable += 3; } } // if(next_best_xk2>0) } for(xk1=0; xk1= probe_poses[xk1+1]) return 0; } // find indels between every two mapped probes. // and we know the indel length already. int ret = 0, total_unmatched = 0; //int H0_matched = match_chro(full_rebuilt_window , base_index, probe_poses[0]-1, full_rebuilt_window_size, 0, global_context -> config.space_type); int total_delta = 0; for(xk1=0; xk1 < used_probes-1; xk1++) { // indels_in_section < 0 : insertion. // used_probe_in_rebuilt_window is the location of the probe on reads // probe_poses is the location of the probe on the chromosome. long long int indels_in_section = probe_poses[xk1+1]; indels_in_section -= probe_poses[xk1]; indels_in_section -= used_probe_in_rebuilt_window[xk1+1]; indels_in_section += used_probe_in_rebuilt_window[xk1]; total_delta += indels_in_section; if(indels_in_section) { // indels_in_section = -5: 5 insertion; indels_in_section = 3: 3 deletion // find the edge. int best_edge_score = -1; unsigned int section_best_edge = 0; // the smallest possible location of the second half is the location if the first half + deletion length xk2 = probe_poses[xk1] - 1 + max(0, indels_in_section); // find the exact indel point; xk2 is the tested point on the chromosome ( first WANTED base on the second half ). for(; xk2 < probe_poses[xk1+1] -1; xk2++) { int left_matched = match_chro(full_rebuilt_window + used_probe_in_rebuilt_window[xk1], base_index, probe_poses[xk1] - 1, (xk2 - probe_poses[xk1]+ 1 - max(0, indels_in_section)), 0, global_context -> config.space_type); int right_matched = match_chro(full_rebuilt_window + used_probe_in_rebuilt_window[xk1] + (xk2 - probe_poses[xk1] + 1) -indels_in_section , base_index, xk2, probe_poses[xk1+1] - 1 - xk2, 0, global_context -> config.space_type); if(left_matched+right_matched > best_edge_score) { best_edge_score = left_matched+right_matched; section_best_edge = xk2; } } int section_mismatch = probe_poses[xk1+1]-probe_poses[xk1]-max(0, indels_in_section) - best_edge_score; is_unreliable += section_mismatch; perfect_segment_start_pos[ret] = probe_poses[xk1] - 1; // perfect_segment_lengths is : first WANTED base on the second half - first base pos on the first half - deletions perfect_segment_lengths[ret] = section_best_edge - probe_poses[xk1] + 1 - max(0, indels_in_section); good_quality_indel[ret] = section_mismatch < INDEL_FULL_ALIGN_MAX_MISMATCH; indel_quality[ret] = pow(2,-is_unreliable); indels_after_perfect_segments[ret] = indels_in_section; indels_read_positions[ret] = used_probe_in_rebuilt_window[xk1] + perfect_segment_lengths[ret]; ret++; total_unmatched += section_mismatch; } else { int matched = match_chro(full_rebuilt_window + used_probe_in_rebuilt_window[xk1], base_index, probe_poses[xk1] - 1, (probe_poses[xk1+1] - probe_poses[xk1]), 0, global_context -> config.space_type); total_unmatched += ((probe_poses[xk1+1] - probe_poses[xk1]) - matched); } } if(ret>3|| total_unmatched>INDEL_FULL_ALIGN_MAX_MISMATCH || total_delta == 0) { return 0; } for(xk1=0; xk1 config.reassembly_key_length, 0, global_context->config.space_type); if(test_match>best_match) { best_match = test_match; best_location = xk2; } } * ret_location = best_location; return best_match; } int find_potential_ultralong_indels(global_context_t * global_context , reassembly_by_voting_block_context_t * block_context , int window_no, char * contig_1, char * contig_2, unsigned int * potential_ins_left, unsigned int * potential_ins_len) { int contig_len_1 = strlen(contig_1); int contig_len_2 = strlen(contig_2); unsigned int xk1, is_1_left; for(is_1_left = 0; is_1_left < 2; is_1_left ++) { int best_match = -1; char * test_key = is_1_left?contig_2:contig_1; char * testee = is_1_left?contig_1:contig_2; int testee_len = is_1_left?contig_len_1:contig_len_2; for(xk1=0; xk1< testee_len - global_context -> config.reassembly_key_length; xk1++) { int test_match = match_str(test_key, testee + xk1, global_context -> config.reassembly_key_length); if(test_match > best_match) best_match = test_match; } if(best_match >= global_context -> config.reassembly_key_length-1) return 0; } unsigned int window_left_pos = block_context->block_start_linear_pos + window_no * REASSEMBLY_WINDOW_LENGTH / 2 ; unsigned int search_start = window_left_pos; if(search_start>300)search_start-=300; else search_start=0; unsigned int search_end = 300 + global_context -> config.max_indel_length + window_left_pos; gene_value_index_t * base_index = &global_context->all_value_indexes[0] ; if(search_end > base_index -> length + base_index -> start_base_offset) search_end = base_index -> length + base_index -> start_base_offset; unsigned int contig_1_left_pos; int contig_1_left_match = find_best_location_for_probe(global_context, base_index, contig_1, search_start, search_end - search_start , &contig_1_left_pos); unsigned int contig_1_right_pos; int contig_1_right_match = find_best_location_for_probe(global_context, base_index, contig_1 + contig_len_1 - global_context -> config.reassembly_key_length, search_start, search_end - search_start , &contig_1_right_pos); unsigned int contig_2_left_pos; int contig_2_left_match = find_best_location_for_probe(global_context, base_index, contig_2, search_start, search_end - search_start , &contig_2_left_pos); unsigned int contig_2_right_pos; int contig_2_right_match = find_best_location_for_probe(global_context, base_index, contig_2 + contig_len_2 - global_context -> config.reassembly_key_length, search_start, search_end - search_start , &contig_2_right_pos); if(contig_1_left_match >= global_context -> config.reassembly_key_length -1 && contig_1_right_match >= global_context -> config.reassembly_key_length -1) return 0; if(contig_2_left_match >= global_context -> config.reassembly_key_length -1 && contig_2_right_match >= global_context -> config.reassembly_key_length -1) return 0; if(contig_1_left_match < global_context -> config.reassembly_key_length -1 && contig_1_right_match < global_context -> config.reassembly_key_length -1) return 0; if(contig_2_left_match < global_context -> config.reassembly_key_length -1 && contig_2_right_match < global_context -> config.reassembly_key_length -1) return 0; int ret = 0; unsigned int basic_ins_left = 0, basic_ins_right = 0; int is_1_at_left; if(contig_1_left_match >= global_context -> config.reassembly_key_length -1 && contig_2_right_match >= global_context -> config.reassembly_key_length -1 && contig_1_left_pos config.reassembly_key_length > contig_2_right_pos - contig_1_left_pos) { *potential_ins_len = contig_len_1 + contig_len_2 - global_context -> config.reassembly_key_length - (contig_2_right_pos - contig_1_left_pos); *potential_ins_left = contig_1_left_pos;// + global_context -> config.reassembly_key_length; basic_ins_left = contig_1_left_pos; basic_ins_right = contig_2_right_pos; is_1_at_left = 1; ret = 1; } } if(contig_1_right_match >= global_context -> config.reassembly_key_length -1 && contig_2_left_match >= global_context -> config.reassembly_key_length -1 && contig_1_right_pos>contig_2_left_pos) { if( contig_len_1 + contig_len_2 - global_context -> config.reassembly_key_length> contig_1_right_pos - contig_2_left_pos) { *potential_ins_len = contig_len_1 + contig_len_2 - global_context -> config.reassembly_key_length - (contig_1_right_pos - contig_2_left_pos); *potential_ins_left = contig_2_left_pos;// + global_context -> config.reassembly_key_length; basic_ins_right = contig_1_right_pos; basic_ins_left = contig_2_left_pos; is_1_at_left = 0; ret = 1; } } if(ret) { unsigned int x1=0,left_ins_edge = * potential_ins_left, best_edge = 0; char * left_contig = is_1_at_left ? contig_1: contig_2; char * right_contig = is_1_at_left ? contig_2: contig_1; char probe_window[global_context -> config.reassembly_key_length+1]; probe_window[global_context -> config.reassembly_key_length]=0; for(; left_ins_edge < (*potential_ins_left) + global_context -> config.reassembly_key_length; left_ins_edge++) probe_window[x1++]=gvindex_get(base_index, left_ins_edge); for(; left_ins_edge < basic_ins_right; left_ins_edge++) { int delta = left_ins_edge - global_context -> config.reassembly_key_length - (*potential_ins_left); char * test_key = left_contig + delta; if(probe_window[global_context -> config.reassembly_key_length-1] == test_key[global_context -> config.reassembly_key_length-1]) best_edge = left_ins_edge ; int matched = match_str(test_key, probe_window, global_context -> config.reassembly_key_length); if(matched< global_context -> config.reassembly_key_length-1) break; for(x1=0; x1 config.reassembly_key_length - 1; x1++) probe_window[x1] = probe_window[x1+1]; probe_window[global_context -> config.reassembly_key_length-1] = gvindex_get(base_index, left_ins_edge); } if(best_edge>0) *potential_ins_left = best_edge; if( ( basic_ins_right + global_context -> config.reassembly_key_length - best_edge) >strlen(right_contig) ||base_index -> length + base_index -> start_base_offset <= basic_ins_right + global_context -> config.reassembly_key_length || basic_ins_left < base_index -> start_base_offset || best_edge >= base_index -> length + base_index -> start_base_offset) ret = 0; else { int left_match = match_chro(left_contig, base_index, basic_ins_left, best_edge - basic_ins_left, 0, global_context->config.space_type); int right_match = match_chro(right_contig + strlen(right_contig)- ( basic_ins_right + global_context -> config.reassembly_key_length - best_edge), base_index, best_edge, basic_ins_right + global_context -> config.reassembly_key_length - best_edge, 0, global_context->config.space_type); if(left_match+right_match< basic_ins_right+global_context -> config.reassembly_key_length-basic_ins_left - 2) ret=0; } } return ret; } int put_long_indel_event(global_context_t * global_context, unsigned int best_pos, int indels, float indel_quality, char * inserted_bases, int event_type) { indel_context_t * indel_context = (indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]; HashTable * event_table = indel_context -> event_entry_table; chromosome_event_t * event_space = indel_context -> event_space_dynamic; chromosome_event_t * found = NULL; chromosome_event_t * search_return [MAX_EVENT_ENTRIES_PER_SITE]; int pos_delta; for(pos_delta = -10; pos_delta <= 10; pos_delta++) { int found_events = search_event(global_context, event_table, event_space, best_pos - 1 + pos_delta, EVENT_SEARCH_BY_SMALL_SIDE, CHRO_EVENT_TYPE_LONG_INDEL|CHRO_EVENT_TYPE_INDEL, search_return); if(found_events) { int kx1; for(kx1 = 0; kx1 < found_events ; kx1++) { if(search_return[kx1] -> indel_length == indels || event_type == CHRO_EVENT_TYPE_POTENTIAL_INDEL ) { found = search_return[kx1]; break; } } } if(found)break; } /* if(best_pos >= 1124357402&& best_pos < 1124359402) { fprintf(stderr, "LLLX-BB: %u :: FOUND=%p\n", best_pos, found); } */ if(found) { found -> supporting_reads ++; found -> event_quality = max(found ->event_quality, indel_quality); } else { int event_no; event_no = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> total_events ++; event_space = reallocate_event_space(global_context , NULL, event_no); chromosome_event_t * new_event = event_space+event_no; memset(new_event,0,sizeof(chromosome_event_t)); if(indels<0 && inserted_bases) set_insertion_sequence(global_context, NULL , &new_event -> inserted_bases , inserted_bases , -indels); new_event -> event_small_side = best_pos - 1; new_event -> event_large_side = best_pos + max(0, indels); assert(new_event -> event_small_side && new_event -> event_large_side); new_event -> event_type = event_type; new_event -> indel_length = indels; new_event -> supporting_reads = 1; new_event -> event_quality = indel_quality; put_new_event(event_table, new_event , event_no); } return 0; } #define BASE_NEEDED_PER_WINDOW 60 #define PILEUP_BOX_LEN_CERTAIN 12 #define MAX_PILEUP_BOX_COVERAGE_CERTAIN 12 #define PILEUP_BOX_LEN 50 #define MAX_PILEUP_BOX_COVERAGE 50 int finalise_pileup_file_by_voting(global_context_t * global_context , char * temp_file_name, char * chro_name, int block_no) { int global_read_no = 0; char * read_text, * qual_text; int xk1; int total_window_number =2 * (BASE_BLOCK_LENGTH / REASSEMBLY_WINDOW_LENGTH+1), window_adjust; unsigned int block_start_linear_pos = linear_gene_position(&global_context->chromosome_table, chro_name, block_no*BASE_BLOCK_LENGTH); //if(block_start_linear_pos> 150000000) return 0; FILE * tmp_fp = f_subr_open(temp_file_name,"rb"); if(!tmp_fp) return 0; print_in_box(80,0,0,"Processing %s\n", temp_file_name); unsigned int * window_center_read_no = (unsigned int *) malloc(sizeof(unsigned int)* total_window_number * global_context -> config.reassembly_start_read_number); unsigned int * window_base_number = (unsigned int *) malloc(sizeof(unsigned int)* total_window_number); unsigned int * window_center_read_qual = (unsigned int *) malloc(sizeof(unsigned int)* total_window_number * global_context -> config.reassembly_start_read_number); unsigned int * window_center_read_masks = (unsigned int *) malloc(sizeof(unsigned int)* total_window_number); struct reassmebly_window_allele *first_half_alleles = (struct reassmebly_window_allele *) malloc(sizeof(struct reassmebly_window_allele ) * global_context -> config.reassembly_window_alleles); struct reassmebly_window_allele *second_half_alleles = (struct reassmebly_window_allele *) malloc(sizeof(struct reassmebly_window_allele ) * global_context -> config.reassembly_window_alleles); unsigned char * mapquality_lower_bounds_certain = calloc(1, BASE_BLOCK_LENGTH / PILEUP_BOX_LEN_CERTAIN * MAX_PILEUP_BOX_COVERAGE_CERTAIN); unsigned char * box_read_number_certain = calloc(1, BASE_BLOCK_LENGTH / PILEUP_BOX_LEN_CERTAIN); unsigned char * mapquality_lower_bounds = calloc(1, BASE_BLOCK_LENGTH / PILEUP_BOX_LEN * MAX_PILEUP_BOX_COVERAGE); unsigned char * box_read_number = calloc(1, BASE_BLOCK_LENGTH / PILEUP_BOX_LEN); char * full_rebuilt_window = (char *)malloc(3000); reassembly_by_voting_block_context_t block_context; block_context.voting_indexes = (gehash_t*)malloc(sizeof(gehash_t)* total_window_number); block_context.block_start_linear_pos = block_start_linear_pos; block_context.start_keys = (unsigned long long int *) calloc(sizeof(unsigned long long int),10 * total_window_number); block_context.start_offsets = (short *)malloc(sizeof( short) * total_window_number); block_context.read_no_counter = (unsigned int *) calloc(sizeof(unsigned int),total_window_number); block_context.read_rectify_space = (short *) malloc(sizeof(unsigned short) * 4 * MAX_READ_LENGTH); block_context.final_alleles = malloc(sizeof(struct reassmebly_window_allele ) * global_context -> config.reassembly_window_alleles); read_text = (char *) malloc(MAX_READ_LENGTH); qual_text = (char *) malloc(MAX_READ_LENGTH); HashTable *read_sequence_table=HashTableCreate(300000); HashTable *read_position_table=HashTableCreate(300000); HashTable *read_quality_table=HashTableCreate(300000); block_context.read_sequence_table = read_sequence_table; block_context.read_quality_table = read_quality_table; block_context.read_position_table = read_position_table; memset(block_context.final_alleles, 0, sizeof(struct reassmebly_window_allele ) * global_context -> config.reassembly_window_alleles); memset(window_center_read_no, 0, sizeof(unsigned int)* total_window_number * global_context -> config.reassembly_start_read_number); memset(window_center_read_qual, 0, sizeof(unsigned int)* total_window_number*global_context -> config.reassembly_start_read_number); memset(window_center_read_masks, 0, sizeof(unsigned int)* total_window_number); memset(window_base_number, 0, sizeof(unsigned int)* total_window_number); if(!tmp_fp) return 1; while(!feof(tmp_fp)) { short read_len; long long int first_base_pos; //unsigned int qual_of_this_read = 1; base_block_temp_read_t read_rec; int rlen = fread(&read_rec, sizeof(read_rec), 1, tmp_fp); int is_position_certain = read_rec.strand; if(rlen<1) break; rlen = fread(&read_len, sizeof(short), 1, tmp_fp); if(rlen < 1){ SUBREADprintf("ERROR: Unable to parse piles\n"); return -1; } rlen = fread(read_text, sizeof(char), read_len, tmp_fp); if(rlen config.reassembly_window_multiplex ; window_adjust ++) { int window_no = 2*(first_base_pos / REASSEMBLY_WINDOW_LENGTH)+ window_adjust , xk2; if(window_no >=total_window_number) break; window_base_number[window_no]+=read_len; int qual_of_this_read = 127, read_offset; for(read_offset = 0; read_offset < read_len ; read_offset++) { int nch = qual_text[read_offset] - '!'; if(nch < 14) qual_of_this_read -=4; else if(nch < 22) qual_of_this_read -= 2; else if(nch < 33) qual_of_this_read --; } qual_of_this_read=max(1,qual_of_this_read); if(is_position_certain) { int box_no = first_base_pos / PILEUP_BOX_LEN_CERTAIN; for(xk1 = box_no * MAX_PILEUP_BOX_COVERAGE_CERTAIN ; xk1 < (box_no+1) * MAX_PILEUP_BOX_COVERAGE_CERTAIN; xk1++) { if(qual_of_this_read > mapquality_lower_bounds_certain[xk1]) { for(xk2 = (box_no+1) * MAX_PILEUP_BOX_COVERAGE_CERTAIN - 1; xk2 > xk1; xk2--) mapquality_lower_bounds_certain[xk2] = mapquality_lower_bounds_certain[xk2-1]; mapquality_lower_bounds_certain[xk1] = qual_of_this_read; break; } } } else { int box_no = first_base_pos / PILEUP_BOX_LEN; for(xk1 = box_no * MAX_PILEUP_BOX_COVERAGE ; xk1 < (box_no+1) * MAX_PILEUP_BOX_COVERAGE; xk1++) { if(qual_of_this_read > mapquality_lower_bounds[xk1]) { for(xk2 = (box_no+1) * MAX_PILEUP_BOX_COVERAGE - 1; xk2 > xk1; xk2--) mapquality_lower_bounds[xk2] = mapquality_lower_bounds[xk2-1]; mapquality_lower_bounds[xk1] = qual_of_this_read; break; } } } if(is_position_certain && window_adjust == 1) break; } } fclose(tmp_fp); for(xk1=0; xk1< total_window_number; xk1++) { if(window_base_number[xk1] >= BASE_NEEDED_PER_WINDOW) { gehash_create(&block_context.voting_indexes[xk1], max(4000,window_base_number[xk1]*4), 1); block_context . start_offsets[xk1] = 0x7fff; } } tmp_fp = f_subr_open(temp_file_name,"rb"); while(!feof(tmp_fp)) { short read_len; long long int first_base_pos; base_block_temp_read_t read_rec; int rlen = fread(&read_rec, sizeof(read_rec), 1, tmp_fp); int is_position_certain = read_rec.strand; if(rlen<1) break; rlen = fread(&read_len, sizeof(short), 1, tmp_fp); if(rlen <1){ SUBREADprintf("ERROR: cannot parse piles.\n"); return -1; } rlen = fread(read_text, sizeof(char), read_len, tmp_fp); if(rlen =0); read_text [read_len]=0; qual_text [read_len]=0; is_bad_read(read_text, read_len, "LOAD"); int read_offset; /* unsigned int qual_of_this_read = 1; for(read_offset = 0; read_offset < read_len ; read_offset++) qual_of_this_read += qual_text[read_offset]; qual_of_this_read = qual_of_this_read * 100 / read_len + (is_position_certain?200:0); if(qual_of_this_read<1)qual_of_this_read=1; */ int qual_of_this_read = 127; for(read_offset = 0; read_offset < read_len ; read_offset++) { int nch = qual_text[read_offset] - '!'; if(nch < 14) qual_of_this_read -=4; else if(nch < 22) qual_of_this_read -= 2; else if(nch < 33) qual_of_this_read --; } qual_of_this_read=max(1,qual_of_this_read); for(window_adjust = 0; window_adjust < global_context -> config.reassembly_window_multiplex; window_adjust++) { if(is_position_certain && window_adjust >= 2) break; int window_no = 2*(first_base_pos / REASSEMBLY_WINDOW_LENGTH) + window_adjust; if(window_no >= total_window_number) break; if(window_base_number[window_no]MAX_PILEUP_BOX_COVERAGE_CERTAIN)continue; box_read_number_certain[box_no]++; //qual_of_this_read += 200; } else { int box_no = first_base_pos / PILEUP_BOX_LEN; if(qual_of_this_read < mapquality_lower_bounds[box_no * MAX_PILEUP_BOX_COVERAGE + MAX_PILEUP_BOX_COVERAGE - 1]) continue; if(box_read_number[box_no]>MAX_PILEUP_BOX_COVERAGE)continue; box_read_number[box_no]++; } int read_no = block_context.read_no_counter[window_no]; block_context.read_no_counter[window_no]++; if(window_center_read_qual[window_no * global_context -> config.reassembly_start_read_number + global_context -> config.reassembly_start_read_number-1] < qual_of_this_read)//&& is_position_certain) { int xk44,xk3; for(xk44=0; xk44 config.reassembly_start_read_number; xk44++) if(window_center_read_qual[window_no * global_context -> config.reassembly_start_read_number + xk44] < qual_of_this_read) break; for(xk3=global_context -> config.reassembly_start_read_number -2; xk3>=xk44 ; xk3--) { window_center_read_no[window_no * global_context -> config.reassembly_start_read_number + xk3 +1] = window_center_read_no[window_no * global_context -> config.reassembly_start_read_number + xk3] ; window_center_read_qual[window_no * global_context -> config.reassembly_start_read_number + xk3 +1] = window_center_read_qual[window_no * global_context -> config.reassembly_start_read_number + xk3]; } window_center_read_no[window_no * global_context -> config.reassembly_start_read_number + xk44] = read_no + 1; window_center_read_qual[window_no * global_context -> config.reassembly_start_read_number + xk44] = qual_of_this_read; } for(read_offset = 0; read_offset < read_len - global_context -> config.reassembly_subread_length+1; read_offset += REASSEMBLY_SUBREAD_GAP) { unsigned int subread_int = 0; int xk2; for(xk2=0; xk2< global_context -> config.reassembly_subread_length; xk2++) subread_int = (subread_int << 2) | base2int(read_text[read_offset + xk2]); gehash_insert_limited(&block_context.voting_indexes[window_no], subread_int , read_offset +10000 *read_no, 400, 10000); } /* if(strstr(temp_file_name, "chr1-0005")) if(window_no == 63763 || window_no == 63762) { SUBREADprintf("LLLX-INSERT_WIN: [%d] : %s RN=%d RL=%d ITEMS=%llu\n",window_no, read_text, read_no, read_len, block_context.voting_indexes[window_no].current_items); } */ char * sequence_for_hash = (char *) malloc(read_len+1); assert(sequence_for_hash); strcpy(sequence_for_hash, read_text); unsigned long long int read_hashed_key = (window_no * 1llu +1)<<32 ; read_hashed_key |= (read_no & 0xfffff)<<12; if(is_position_certain) HashTablePut(read_position_table, NULL + read_hashed_key, NULL+first_base_pos+1); HashTablePut(read_sequence_table, NULL + read_hashed_key, sequence_for_hash); HashTablePut(read_quality_table, NULL + read_hashed_key, NULL + qual_of_this_read); } global_read_no ++; } fclose(tmp_fp); for(xk1=0; xk1< total_window_number; xk1++) { if(window_base_number[xk1]>=BASE_NEEDED_PER_WINDOW) gehash_sort(&block_context.voting_indexes[xk1]); } gene_vote_t *vote_list; vote_list = (gene_vote_t *) malloc(sizeof(gene_vote_t)); assert(vote_list); block_context.vote_list = vote_list; block_context.vote_list_rectify = (gene_vote_t *) malloc(sizeof(gene_vote_t)); indel_context_t * indel_context = (indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]; HashTable * event_table = indel_context -> event_entry_table; for(xk1 = 0; xk1 < total_window_number; xk1++) { // xk1 is the number of the current window. if(window_base_number[xk1] anchor_read -> 3'end //int IGNORED_BASES_IN_WINDOW = 10; //for(IGNORED_BASES_IN_WINDOW = 0; IGNORED_BASES_IN_WINDOW <=60; IGNORED_BASES_IN_WINDOW+=30) { int start_read_i; for(start_read_i = 0; start_read_i < global_context -> config.reassembly_start_read_number; start_read_i ++) { // from middle read to 5' end if(!window_center_read_no[xk1 * global_context -> config.reassembly_start_read_number + start_read_i ]) continue; unsigned int this_start_read_no = window_center_read_no[xk1 * global_context -> config.reassembly_start_read_number + start_read_i ]-1 ; unsigned int window_start_pos = block_context.block_start_linear_pos + xk1 * REASSEMBLY_WINDOW_LENGTH / 2; block_context.window_start_pos = window_start_pos; search_window(global_context, &block_context, xk1, this_start_read_no , -1); memcpy(first_half_alleles, block_context.final_alleles, global_context -> config.reassembly_window_alleles * sizeof(struct reassmebly_window_allele )); search_window(global_context, &block_context, xk1, this_start_read_no, 1); memcpy(second_half_alleles, block_context.final_alleles, global_context -> config.reassembly_window_alleles * sizeof(struct reassmebly_window_allele)); /* if(window_start_pos >= 1124357402&& window_start_pos < 1124359402) { fprintf(stderr, "LLLX: %u [%d] :: START READ = %u ; FN=%s ; BASES=%u\n", window_start_pos, xk1, this_start_read_no, temp_file_name , window_base_number[xk1]); char fname [100]; sprintf(fname, "DP-%d-%d", getpid(), xk1); gehash_dump(&block_context.voting_indexes[xk1], fname); }*/ unsigned long long int starter_read_key = (xk1 * 1llu +1)<<32 ; starter_read_key |= ( (this_start_read_no)& 0xfffff)<<12; char * start_read_txt = HashTableGet(block_context.read_sequence_table,NULL+starter_read_key); assert(start_read_txt); int first_allele_no, second_allele_no; for(first_allele_no = 0; first_allele_no < global_context -> config.reassembly_window_alleles && first_half_alleles[first_allele_no].rebuilt_size>0 ; first_allele_no++) for(second_allele_no = 0; second_allele_no < global_context -> config.reassembly_window_alleles && second_half_alleles[second_allele_no].rebuilt_size>0 ; second_allele_no++) { int all_indels_in_window = 0; strcpy(full_rebuilt_window, first_half_alleles [first_allele_no]. rebuilt_window); strcat(full_rebuilt_window, start_read_txt); strcat(full_rebuilt_window, second_half_alleles[second_allele_no].rebuilt_window); int xk2, full_rebuilt_window_size = strlen(full_rebuilt_window); gene_value_index_t * base_index = &global_context->all_value_indexes[0] ; unsigned int perfect_segment_start_pos[MAX_INDELS_IN_WINDOW], contig_start_pos, contig_end_pos, all_fresh = 0; int perfect_segment_lengths[MAX_INDELS_IN_WINDOW], head_removed_bases=0, tail_removed_bases=0; int indels_after_perfect_segments[MAX_INDELS_IN_WINDOW]; int good_quality_indels[MAX_INDELS_IN_WINDOW]; float quality_of_indels_aln[MAX_INDELS_IN_WINDOW]; short indels_read_positions[MAX_INDELS_IN_WINDOW]; char contig_CIGAR[200]; memset(good_quality_indels, 0, sizeof(int) * MAX_INDELS_IN_WINDOW); int indels_in_window = full_indel_alignment(global_context, &block_context, full_rebuilt_window, full_rebuilt_window_size, base_index, window_start_pos, perfect_segment_start_pos, perfect_segment_lengths, indels_after_perfect_segments, indels_read_positions, quality_of_indels_aln, &contig_start_pos, &contig_end_pos, &head_removed_bases, &tail_removed_bases, good_quality_indels); contig_CIGAR[0]=0; int read_position_cursor = head_removed_bases; int is_indel_contig = 0; for(xk2 = 0; xk2 < indels_in_window; xk2++) { unsigned int best_pos = perfect_segment_start_pos [xk2] + perfect_segment_lengths[xk2]; int indels = indels_after_perfect_segments[xk2], is_fresh = 1; float quality_of_this_indel = quality_of_indels_aln[xk2] * first_half_alleles [first_allele_no].allele_quality * second_half_alleles[second_allele_no].allele_quality; if(abs(indels) >= global_context -> config.max_indel_length){ //#warning "====================== REMOVE THE debug output ===============" continue; } /*if(abs(indels) <= 16 && 0){ continue; }*/ is_indel_contig=1; sprintf(contig_CIGAR+strlen(contig_CIGAR), "%dM%d%c", indels_read_positions[xk2] - read_position_cursor, abs(indels), indels<0?'I':'D'); read_position_cursor = indels_read_positions[xk2]; if(indels<0) read_position_cursor -= indels; all_indels_in_window++; if(indels >= -global_context -> config.max_indel_length && good_quality_indels[xk2]) { int neighbour_delta; for(neighbour_delta = - 30; neighbour_delta < 30 ; neighbour_delta++) { chromosome_event_t * search_return [MAX_EVENT_ENTRIES_PER_SITE]; chromosome_event_t * event_space = indel_context -> event_space_dynamic; int xk3, found_events = search_event(global_context, event_table, event_space, best_pos + neighbour_delta , EVENT_SEARCH_BY_SMALL_SIDE, CHRO_EVENT_TYPE_LONG_INDEL|CHRO_EVENT_TYPE_INDEL, search_return); for(xk3 = 0; xk3 event_quality >= quality_of_this_indel) is_fresh = 0; else tested_neighbour -> event_type = CHRO_EVENT_TYPE_REMOVED; } } if(is_fresh) put_long_indel_event(global_context, best_pos, indels, quality_of_this_indel, full_rebuilt_window + indels_read_positions[xk2], CHRO_EVENT_TYPE_LONG_INDEL); all_fresh += is_fresh; } } //#warning "====================== Make sure ' is_indel_contig ' in the next line is harmless before RELEASE ===============" if( is_indel_contig || all_fresh) { int write_cursor; char * chro_begin; int chro_offset_start = 0; int chro_offset_end = 0; locate_gene_position_max(contig_start_pos + head_removed_bases ,& global_context -> chromosome_table, &chro_begin, &chro_offset_start, NULL, NULL, 0); locate_gene_position_max(contig_end_pos - tail_removed_bases ,& global_context -> chromosome_table, &chro_begin, &chro_offset_end, NULL, NULL, 0); if(full_rebuilt_window_size - read_position_cursor - tail_removed_bases) fprintf(global_context -> long_insertion_FASTA_fp, ">%s-%u-%u-%s%dM\n", chro_name, chro_offset_start, chro_offset_end - 1, contig_CIGAR, full_rebuilt_window_size - read_position_cursor - tail_removed_bases); else fprintf(global_context -> long_insertion_FASTA_fp, ">%s-%u-%u-%s0M\n", chro_name, chro_offset_start, chro_offset_end - 1, contig_CIGAR); full_rebuilt_window[full_rebuilt_window_size - tail_removed_bases] = 0; for(write_cursor = head_removed_bases ; write_cursor < full_rebuilt_window_size - tail_removed_bases ; write_cursor += 70) { fwrite(full_rebuilt_window + write_cursor , min(70, full_rebuilt_window_size - write_cursor), 1, global_context -> long_insertion_FASTA_fp); fputs("\n",global_context -> long_insertion_FASTA_fp); } } } } } } free(vote_list); free(block_context.vote_list_rectify); for(xk1=0; xk1< total_window_number; xk1++) { if(window_base_number[xk1]>=BASE_NEEDED_PER_WINDOW) gehash_destory(&block_context.voting_indexes[xk1]); } free_values_destroy(read_sequence_table); free(mapquality_lower_bounds_certain); free(mapquality_lower_bounds); free(box_read_number); free(box_read_number_certain); free(full_rebuilt_window); free(block_context.read_no_counter); free(block_context.voting_indexes); free(block_context.start_keys); free(block_context.start_offsets); free(block_context.read_rectify_space); free(block_context.final_alleles); free(first_half_alleles); free(second_half_alleles); free(window_base_number); free(window_center_read_no); free(window_center_read_qual); free(window_center_read_masks); free(read_text); free(qual_text); return 0; } int finalise_pileup_file_by_debrujin(global_context_t * global_context , char * temp_file_name, char * chro_name, int block_no) { FILE * tmp_fp = f_subr_open(temp_file_name,"rb"); if(!tmp_fp) return 0; char * read_text, * qual_text; int xk1; unsigned int block_start_linear_pos = linear_gene_position(&global_context->chromosome_table, chro_name, block_no*BASE_BLOCK_LENGTH); int total_window_number =2 * (BASE_BLOCK_LENGTH / REASSEMBLY_WINDOW_LENGTH+1); reassembly_block_context_t * block_context = (reassembly_block_context_t*)malloc(sizeof(reassembly_block_context_t)); block_context -> block_start_linear_pos = block_start_linear_pos; block_context -> start_keys = (unsigned long long int *) calloc(sizeof(unsigned long long int),10 * total_window_number); block_context -> start_offsets = (short *)malloc(sizeof( short) * total_window_number); assert(block_context -> start_offsets); assert(block_context -> start_keys); block_context -> de_bruijn_graphs = ( HashTable ** )malloc(sizeof( HashTable *) * total_window_number); for(xk1=0; xk1< total_window_number; xk1++) { block_context -> de_bruijn_graphs[xk1] = HashTableCreate(10* REASSEMBLY_WINDOW_LENGTH); block_context -> start_offsets[xk1] = 0x7fff; } read_text = (char *) malloc(MAX_READ_LENGTH); qual_text = (char *) malloc(MAX_READ_LENGTH); while(!feof(tmp_fp)) { short read_len; unsigned int first_base_pos; base_block_temp_read_t read_rec; int rlen = fread(&read_rec, sizeof(read_rec), 1, tmp_fp); if(rlen<1) break; rlen = fread(&read_len, sizeof(short), 1, tmp_fp); if(rlen<1) return -1; rlen = fread(read_text, sizeof(char), read_len, tmp_fp); if(rlen0); } for(xk1=0; xk1< total_window_number; xk1++) { if(block_context -> start_offsets[xk1]<0x7fff && block_context -> de_bruijn_graphs[xk1] -> numOfElements >3) { finalise_db_graph(global_context, block_context, xk1, block_context -> de_bruijn_graphs[xk1] , block_context -> start_keys [xk1*10], block_context -> start_offsets [xk1]); } HashTableDestroy(block_context -> de_bruijn_graphs[xk1]); } free(block_context -> de_bruijn_graphs); free(block_context -> start_keys); free(block_context -> start_offsets); free(block_context); free(read_text); free(qual_text); fclose(tmp_fp); return 0; } int finalise_long_insertions_by_hashtable(global_context_t * global_context) { int chro_i; assert(global_context -> index_block_number == 1); unsigned int chro_start_pos = 0; char tmp_fname[MAX_FILE_NAME_LENGTH+40]; sprintf(tmp_fname,"%s.reassembly.fa", global_context->config.output_prefix); global_context->long_insertion_FASTA_fp = f_subr_open(tmp_fname ,"wb"); for(chro_i=0; chro_i < global_context -> chromosome_table.total_offsets ; chro_i++) { unsigned int chro_length = global_context -> chromosome_table.read_offsets[chro_i] - chro_start_pos; unsigned int chro_current_pos = 0; for(chro_current_pos = 0; chro_current_pos < chro_length ; chro_current_pos += BASE_BLOCK_LENGTH) { char temp_file_name[MAX_FILE_NAME_LENGTH + 50]; int block_no = chro_current_pos / BASE_BLOCK_LENGTH; sprintf(temp_file_name,"%s@%s-%04u.bin", global_context -> config.temp_file_prefix, global_context -> chromosome_table.read_names+chro_i*MAX_CHROMOSOME_NAME_LEN , block_no ); finalise_pileup_file(global_context , temp_file_name, global_context -> chromosome_table.read_names+MAX_CHROMOSOME_NAME_LEN*chro_i, block_no); unlink(temp_file_name); } chro_start_pos = global_context -> chromosome_table.read_offsets[chro_i]; } fclose(global_context -> long_insertion_FASTA_fp); /* for(xk1=0; xk1 < global_context -> config.long_indel_iterations ; xk1++) { explorer_insertions_in_unmapped(global_context); } */ return 0; } void destroy_pileup_table(HashTable* local_reassembly_pileup_files) { int bucket; KeyValuePair * cursor; for(bucket=0; bucket< local_reassembly_pileup_files -> numOfBuckets; bucket++) { cursor = local_reassembly_pileup_files -> bucketArray[bucket]; while (1) { if (!cursor) break; FILE * fp = (FILE *)cursor->value; if(fp != NULL+1) fclose(fp); free((void *)cursor->key); cursor = cursor->next; } } HashTableDestroy(local_reassembly_pileup_files); } char * _COREMAIN_delete_temp_prefix = NULL; void COREMAIN_SIGINT_hook(int param) { #ifdef MAKE_STANDALONE int xk1, last_slash = -1; if(_COREMAIN_delete_temp_prefix != NULL) { char del2[MAX_FILE_NAME_LENGTH], del_suffix[MAX_FILE_NAME_LENGTH], del_name[MAX_FILE_NAME_LENGTH]; SUBREADprintf("\n\nReceived a terminal signal. The temporary files were removed.\n"); for(xk1=0; _COREMAIN_delete_temp_prefix[xk1]; xk1++) { if(_COREMAIN_delete_temp_prefix[xk1]=='/') last_slash = xk1; else if(_COREMAIN_delete_temp_prefix[xk1]=='\\') { SUBREADprintf("The file name is unknown.\n"); return; } } if(last_slash>=0) { memcpy(del2, _COREMAIN_delete_temp_prefix, last_slash); del2[last_slash] = 0; strcpy(del_suffix , _COREMAIN_delete_temp_prefix + last_slash + 1); } else { strcpy(del2,"."); strcpy(del_suffix , _COREMAIN_delete_temp_prefix); } if(strlen(del_suffix)>8) { DIR *d; struct dirent *dir; d = opendir(del2); if (d) { while ((dir = readdir(d)) != NULL) { if(strstr(dir->d_name, del_suffix)) { //printf("%s\n", dir->d_name); strcpy(del_name, del2); strcat(del_name, "/"); strcat(del_name, dir->d_name); unlink(del_name); } } closedir(d); } } } exit(param); #endif } int finalise_long_insertions(global_context_t * global_context) { indel_context_t * indel_context = (indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]; HashTable * local_reassembly_pileup_files = indel_context -> local_reassembly_pileup_files; destroy_pileup_table(local_reassembly_pileup_files); return finalise_long_insertions_by_hashtable(global_context); } void init_global_context(global_context_t * context) { memset(context->module_contexts, 0, 5*sizeof(void *)); memset(&context->config, 0, sizeof(configuration_t)); context->config.fast_run = 0; context->config.memory_use_multiplex = 1; context->config.report_sam_file = 1; context->config.do_breakpoint_detection = 0; context->config.do_fusion_detection = 0; context->config.do_long_del_detection = 0; context->config.do_structural_variance_detection = 0; context->config.more_accurate_fusions = 1; context->config.report_multi_mapping_reads = 0; //#warning "============= best values for the SVs application: 8; 5; 32 ===============" context->config.top_scores = 8 - 5; context->config.max_vote_combinations = 5 - 3; context->config.max_vote_simples = 5; context->config.max_vote_number_cutoff = 1; context->config.experiment_type = 0; context->config.prefer_donor_receptor_junctions = 1; context->config.maximum_translocation_length = 10000; context->config.maximum_colocating_distance = 500; context->config.do_big_margin_filtering_for_reads = 0; context->config.do_big_margin_filtering_for_junctions = 0; context->config.maximum_intron_length = 500000; context->config.use_hamming_distance_in_exon = 0; context->config.is_third_iteration_running = 0; context->input_reads.is_paired_end_reads = 0; context->will_remove_input_file = 0; context->config.ignore_unmapped_reads = 0; context->config.report_unmapped_using_mate_pos = 1; context->config.downscale_mapping_quality=0; context->config.ambiguous_mapping_tolerance = 39; context->config.use_hamming_distance_break_ties = 0; context->config.use_quality_score_break_ties = 0; context->config.extending_search_indels = 0; context->config.PE_predominant_weight = 0; //#warning "============= best values for the SVs application: 3 =====================" context->config.multi_best_reads = 1; context->config.reported_multi_best_reads = 1; context->config.is_SAM_file_input=0; context->config.use_dynamic_programming_indel=0; context->config.use_bitmap_event_table = 1; context->config.convert_color_to_base = 0; context->config.is_gzip_fastq = 0; context->config.is_BAM_output = 1; context->config.is_BAM_input = 0; context->config.is_input_read_order_required = 0; context->config.read_trim_5 = 0; context->config.read_trim_3 = 0; context->config.minimum_exonic_subread_fraction = -1.0; context->config.is_first_read_reversed = 0; context->config.is_second_read_reversed = 1; context->config.space_type = GENE_SPACE_BASE; context->config.minimum_pair_distance = 50; context->config.maximum_pair_distance = 600; context->config.expected_pair_distance = 300; context->config.restrected_read_order = 1; context->config.k_mer_length = 28; context->config.reassembly_start_read_number = 2; context->config.reassembly_key_length = 28; context->config.reassembly_subread_length = 12; context->config.reassembly_window_multiplex = 3; context->config.reassembly_tolerable_voting = 1; context->config.reassembly_window_alleles = 1; context->config.do_superlong_indel_detection = 0; context->config.flanking_subread_indel_mismatch = 1; context->config.total_subreads = 10; context->config.minimum_subread_for_first_read = 3; context->config.minimum_subread_for_second_read = 1; context->config.min_mapped_fraction = 0; context->config.max_mismatch_exonic_reads = 5; context->config.max_mismatch_junction_reads = 2; context->config.all_threads = 1; context->config.is_first_iteration_running = 1; context->config.is_second_iteration_running = 1; context->config.reads_per_chunk = 20*1024*1024; //#warning "=========== 2*1024*1024 IS FOR TESTING BLOCKING AND SHOULD BE COMMENTED ===============" // context->config.reads_per_chunk = 2*1024*1024; context->config.use_memory_buffer = 1; context->config.is_methylation_reads = 0; context->config.report_no_unpaired_reads = 0; context->config.limited_tree_scan = 0; context->config.high_quality_base_threshold = 500000; context->config.report_multiple_best_in_pairs = 0; context->config.realignment_minimum_variant_distance = 16; context->config.init_max_event_number = 70000; context->config.show_soft_cliping = 1 ; context->config.big_margin_record_size = 9; context->config.read_group_id[0] = 0; context->config.read_group_txt[0] = 0; context->config.first_read_file[0] = 0; context->config.second_read_file[0] = 0; context->config.index_prefix[0] = 0; context->config.output_prefix[0] = 0; context->config.exon_annotation_file[0] = 0; context->config.SAM_extra_columns = 0; context->config.exon_annotation_file_type = FILE_TYPE_GTF; strcpy(context->config.exon_annotation_gene_id_column, "gene_id"); strcpy(context->config.exon_annotation_feature_name_column, "exon"); context->config.DP_penalty_create_gap = -1; context->config.DP_penalty_extend_gap = 0; context->config.DP_match_score = 2; context->config.DP_mismatch_penalty = 0; context->config.max_insertion_at_junctions=0; context->config.check_donor_at_junctions=1; memset(&context -> input_reads, 0, sizeof(read_input_t)); signal (SIGTERM, COREMAIN_SIGINT_hook); signal (SIGINT, COREMAIN_SIGINT_hook); int seed_rand[2]; double double_time = miltime(); memcpy(seed_rand, &double_time, 2*sizeof(int)); myrand_srand(seed_rand[0]^seed_rand[1]); // the seed is NOT used in R because myrand_srand will always takes four random numbers from R's RNG context->config.max_indel_length = 5; context->config.phred_score_format = FASTQ_PHRED33; context->start_time = miltime(); context->timecost_load_index = 0; context->timecost_voting = 0; context->timecost_before_realign = 0; context->timecost_for_realign = 0; } void init_core_temp_path(global_context_t * context){ int x1; char mac_rand[13]; mac_or_rand_str(mac_rand); context->config.temp_file_prefix[0] = 0; if(context->config.output_prefix[0]){ for(x1 = strlen(context->config.output_prefix); x1 >=0; x1--){ if(context->config.output_prefix[x1]=='/'){ memcpy(context->config.temp_file_prefix, context->config.output_prefix, x1); context->config.temp_file_prefix[x1]=0; break; } } } if(context->config.temp_file_prefix[0] == 0)strcpy(context->config.temp_file_prefix, "./"); sprintf(context->config.temp_file_prefix+strlen(context->config.temp_file_prefix), "/core-temp-sum-%06u-%s", getpid(), mac_rand ); _COREMAIN_delete_temp_prefix = context->config.temp_file_prefix; } #define INDEL_MASK_BY_INSERTION 1 #define INDEL_MASK_BY_DELETION 2 #define INDEL_MASK_BY_MATCH 0 #define INDEL_MASK_BY_MISMATCH 3 int CORE_DPALIGN_CREATEGAP_PENALTY = -1; int CORE_DPALIGN_EXTENDGAP_PENALTY = 0; int CORE_DPALIGN_MATCH_SCORE = 2; int CORE_DPALIGN_MISMATCH_PENALTY = 0; int core_dynamic_align(global_context_t * global_context, thread_context_t * thread_context, char * read, int read_len, unsigned int begin_position, char * movement_buffer, int expected_offset, char * read_name) // read must be converted to the positive strand. // movement buffer: 0:match, 1: read-insert, 2: gene-insert, 3:mismatch // the size of the movement buffer must be equal to the length of the read plus max_indel * 3. { int max_indel = min(16 , global_context->config.max_indel_length); int i,j; CORE_DPALIGN_CREATEGAP_PENALTY = global_context -> config.DP_penalty_create_gap; CORE_DPALIGN_EXTENDGAP_PENALTY = global_context -> config.DP_penalty_extend_gap; CORE_DPALIGN_MATCH_SCORE = global_context -> config.DP_match_score; CORE_DPALIGN_MISMATCH_PENALTY = global_context -> config.DP_mismatch_penalty; if(read_len < 3 || abs(expected_offset) > max_indel) return 0; if(expected_offset < 0 && read_len < (3-expected_offset)) return 0; gene_value_index_t * current_value_index = thread_context?thread_context->current_value_index:global_context->current_value_index; indel_context_t * indel_context = (indel_context_t*)global_context -> module_contexts[MODULE_INDEL_ID]; //unsigned long long table_ptr = (unsigned long long) indel_context -> dynamic_align_table; short ** table = indel_context -> dynamic_align_table; char ** table_mask = indel_context -> dynamic_align_table_mask; if(thread_context) { indel_thread_context_t * indel_thread_context = (indel_thread_context_t*)thread_context -> module_thread_contexts[MODULE_INDEL_ID]; table = indel_thread_context -> dynamic_align_table; table_mask = indel_thread_context -> dynamic_align_table_mask; } if(0 && strcmp(read_name, "MISEQ:13:000000000-A1H1M:1:1112:12194:5511") == 0) { int ii; for(ii = 0; ii thread_id, table_mask, read_len + expected_offset, read_len); for (i=0; i= read_len + expected_offset || j >= read_len)) SUBREADprintf("XXDM[%d]: %p %d,%d\n", thread_context -> thread_id, table_mask, read_len + expected_offset, read_len); table_mask[i][j]=0; if(0&&(i >= read_len + expected_offset || j >= read_len)) SUBREADprintf("YYDM[%d]: %p %d,%d\n", thread_context -> thread_id, table_mask, read_len + expected_offset, read_len); if (j < i - max_indel || j > max_indel + i) { table[i][j]=-9999; if(0 && strcmp(read_name, "MISEQ:13:000000000-A1H1M:1:1112:12194:5511") == 0) { putchar('\t'); } continue; } short from_upper; if (i>0) from_upper = table[i-1][j] + (table_mask[i-1][j] == INDEL_MASK_BY_DELETION?CORE_DPALIGN_EXTENDGAP_PENALTY:CORE_DPALIGN_CREATEGAP_PENALTY); else from_upper = -9999; short from_left; if (j>0) from_left = table[i][j-1] + (table_mask[i][j-1] == INDEL_MASK_BY_INSERTION?CORE_DPALIGN_EXTENDGAP_PENALTY:CORE_DPALIGN_CREATEGAP_PENALTY); else from_left = -9999; char chromo_ch = gvindex_get(current_value_index, begin_position + i); char is_matched_ij = (chromo_ch == read[j])?CORE_DPALIGN_MATCH_SCORE:CORE_DPALIGN_MISMATCH_PENALTY; short from_upperleft; if (i>0 && j>0) from_upperleft = table[i-1][j-1] + is_matched_ij; else if(i==0 && j==0) from_upperleft = is_matched_ij; else from_upperleft = -9999; if (from_upperleft == from_upper && from_upperleft > from_left) { table_mask[i][j]= INDEL_MASK_BY_DELETION; table[i][j] = from_upper; } else if(from_upperleft == from_left && from_upperleft > from_upper) { table_mask[i][j]= INDEL_MASK_BY_INSERTION; table[i][j] = from_left; } else if(from_upperleft > from_left && from_upperleft > from_upper) { table_mask[i][j]= (chromo_ch == read[j])?INDEL_MASK_BY_MATCH:INDEL_MASK_BY_MISMATCH; table[i][j] = from_upperleft; } else if(from_upperleft == from_left && from_upperleft == from_upper) { table_mask[i][j]= (chromo_ch == read[j])?INDEL_MASK_BY_MATCH:INDEL_MASK_BY_MISMATCH; table[i][j] = from_upperleft; } else if(from_left > from_upper) { table_mask[i][j]= INDEL_MASK_BY_INSERTION; table[i][j] = from_left; } else if(from_left <= from_upper) { table_mask[i][j]= INDEL_MASK_BY_DELETION; table[i][j] = from_upper; } if(0 && strcmp(read_name, "MISEQ:13:000000000-A1H1M:1:1112:12194:5511") == 0) SUBREADprintf("%c%c\t", chromo_ch, read[j]); } if(0 && strcmp(read_name, "MISEQ:13:000000000-A1H1M:1:1112:12194:5511") == 0) SUBREADputs(""); } #ifdef indel_debug //SUBREADputs(""); //SUBREADputs(""); #endif short path_i = read_len + expected_offset - 1; int out_pos = 0, delta=0; j = read_len - 1; if(0 && strcmp(read_name, "MISEQ:13:000000000-A1H1M:1:1112:12194:5511") == 0) { int ii,jj; for(ii=0;ii< path_i+1; ii++) { SUBREADprintf("%d\t", ii); for(jj=0; jj= (10734463 % BASE_BLOCK_LENGTH) && (x- 1) * REASSEMBLY_WINDOW_LENGTH /2-1 <= (10734463%BASE_BLOCK_LENGTH) ) #define is_target_window_X(x) 0 //#define MAXIMUM_EVENT_NUMBER 300000 typedef struct{ int is_precisely_called; unsigned int source_left_side; // the base BEFORE the translocated sequence. unsigned int target_left_side; // tge base BEFORE the inserted translocated sequence. unsigned int length; unsigned int event_P_number; unsigned int event_Q_number; unsigned int event_R_number; int is_inv; unsigned int all_sup_P; unsigned int max_sup_QR; } translocation_result_t; typedef struct{ int is_precisely_called; unsigned int event_Y_rough_small_abs; unsigned int event_Z_rough_large_abs; unsigned int small_side; // the base BEFORE the reversed sequence unsigned int length; unsigned int event_Y_number; // event_no in the event space. unsigned int event_Z_number; unsigned int all_sup_D; unsigned int max_sup_E; } inversion_result_t; struct reassmebly_window_allele { char rebuilt_window[8000]; float allele_quality; int rebuilt_size; }; typedef struct{ gehash_t * voting_indexes; char * chro_name; unsigned long long int * start_keys; short * start_offsets; unsigned int * read_no_counter; unsigned int block_start_linear_pos; HashTable * read_sequence_table; HashTable * read_position_table; HashTable * read_quality_table; gene_vote_t * vote_list; gene_vote_t * vote_list_rectify; short * read_rectify_space; char rebuilt_window[2500]; int rebuilt_window_size; struct reassmebly_window_allele * final_alleles; unsigned int used_read_ids[2000]; int used_read_number; int search_cost; int total_matched_bases; int max_matched_bases; unsigned int window_start_pos; } reassembly_by_voting_block_context_t; typedef struct{ HashTable ** de_bruijn_graphs; char * chro_name; unsigned long long int * start_keys; short * start_offsets; unsigned int block_start_linear_pos; } reassembly_block_context_t; #define EVENT_BODY_LOCK_BUCKETS 14929 typedef struct{ HashTable * event_entry_table; unsigned int total_events; unsigned int current_max_event_number; chromosome_event_t * event_space_dynamic; HashTable * local_reassembly_pileup_files; subread_lock_t event_body_locks[EVENT_BODY_LOCK_BUCKETS]; short ** dynamic_align_table; char ** dynamic_align_table_mask; } indel_context_t; typedef struct{ HashTable * event_entry_table; unsigned int total_events; unsigned int current_max_event_number; chromosome_event_t * event_space_dynamic; unsigned short * final_counted_reads_array; unsigned short * final_reads_mismatches_array; short ** dynamic_align_table; char ** dynamic_align_table_mask; } indel_thread_context_t; int init_indel_tables(global_context_t * context); int destroy_indel_module(global_context_t * context); int init_indel_thread_contexts(global_context_t * global_context, thread_context_t * thread_context, int task); int sort_global_event_table(global_context_t * global_context); int load_known_junctions(global_context_t * global_context); int finalise_indel_and_junction_thread(global_context_t * global_context, thread_context_t * thread_contexts, int task); int find_new_indels(global_context_t * global_context, thread_context_t * thread_context, int pair_number, char * read_name, char * read_text, char * qual_text, int read_len, int is_second_read, int best_read_id); int write_indel_final_results(global_context_t * context); int search_event(global_context_t * global_context,HashTable * event_table, chromosome_event_t * event_space, unsigned int pos, int search_type, unsigned char event_type, chromosome_event_t ** return_buffer); void set_alignment_result(global_context_t * global_context, int pair_number, int is_second_read, int best_read_id, unsigned int position, int votes, gene_vote_number_t * indel_record, short best_cover_start, short best_cover_end, int is_negative_strand, int is_PE, unsigned int minor_position, unsigned int minor_votes, unsigned int minor_coverage_start, unsigned int minor_coverage_end, unsigned int split_point, int inserted_bases, int is_strand_jumped, int is_GT_AG_donors, int used_subreads_in_vote, int noninformative_subreads_in_vote, int major_indel_offset, int minor_indel_offset, int main_hamming, int minor_hamming, int main_quality, int minor_quality); void put_new_event(HashTable * event_table, chromosome_event_t * new_event , int event_no); void remove_neighbour(global_context_t * global_context); int build_local_reassembly(global_context_t *global_context , thread_context_t *thread_context , int pair_number, char * read_name_1 , char * read_text_1 ,char * qual_text_1 , int read_len_1, int read_len_2, int is_second_read, int best_read_id, int is_paired_unmapped, mapping_result_t * current_res, mapping_result_t * mate_res); int finalise_long_insertions(global_context_t * global_context); // This function sets the global context with default values. void init_global_context(global_context_t * context); int write_local_reassembly(global_context_t *global_context, HashTable *pileup_fp_table, unsigned int anchor_pos, char * read_name , char * read_text ,char * qual_text , int read_len, int is_anchor_certain); int finalise_long_insertions_by_hashtable(global_context_t * global_context); void destroy_pileup_table(HashTable* local_reassembly_pileup_files); chromosome_event_t * reallocate_event_space(global_context_t* global_context,thread_context_t* thread_context,int event_no); int there_are_events_in_range(char * bitmap, unsigned int pos, int sec_len); int anti_supporting_read_scan(global_context_t * global_context); int core_dynamic_align(global_context_t * global_context, thread_context_t * thread_context, char * read, int read_len, unsigned int begin_position, char * movement_buffer, int expected_offset, char * read_name); void init_core_temp_path(global_context_t * context); chromosome_event_t * local_add_indel_event(global_context_t * global_context, thread_context_t * thread_context, HashTable * event_table, char * read_text, unsigned int left_edge, int indels, int score_supporting_read_added, int is_ambiguous, int mismatched_bases,int * old_event_id); void print_indel_table(global_context_t * global_context); int sort_junction_entry_table(global_context_t * global_context); void mark_event_bitmap(unsigned char * bitmap, unsigned int pos); int check_event_bitmap(unsigned char * bitmap, unsigned int pos); void exchange_event_sides(void * arr, int l, int r); void merge_event_sides(void * arr, int start, int items, int items2); int compare_event_sides(void * arr, int l, int r); srUInt_64 localPointerHashFunction_forEventEntry(const void *pointer); int localPointerCmp_forEventEntry(const void *pointer1, const void *pointer2); void anti_support_add_count(void * ky, void * va, HashTable * tab); int BINsearch_event(chromosome_event_t * event_space, int * event_ids, int is_small_side, unsigned int pos, int events); void destory_event_entry_table(HashTable * old); #define is_ambiguous_indel_score(e) (0) #endif Rsubread/src/core-interface-aligner.c0000644000175200017520000006650714136047615020630 0ustar00biocbuildbiocbuild#include #include #include #include #include #include "subread.h" #include "input-files.h" #include "core.h" #include "HelperFunctions.h" static struct option long_options[] = { {"index", required_argument, 0, 'i'}, {"read", required_argument, 0, 'r'}, {"read2", required_argument, 0, 'R'}, {"output", required_argument, 0, 'o'}, {"subreads", required_argument, 0, 'n'}, {"threads", required_argument, 0, 'T'}, {"indel", required_argument, 0, 'I'}, {"phred", required_argument, 0, 'P'}, {"minvotes", required_argument, 0, 'm'}, {"minvotes2", required_argument, 0, 'p'}, {"mindist", required_argument, 0, 'd'}, {"maxdist", required_argument, 0, 'D'}, {"order", required_argument, 0, 'S'}, {"DPMismatch", required_argument, 0, 'X'}, {"DPMatch", required_argument, 0, 'Y'}, {"DPGapOpen", required_argument, 0, 'G'}, {"DPGapExt", required_argument, 0, 'E'}, {"junction", no_argument, 0, 'J'}, {"unique", no_argument, 0, 'u'}, {"color-convert", no_argument, 0, 'b'}, {"multi", required_argument, 0, 'B'}, {"type", required_argument, 0, 't'}, {"quality", no_argument, 0, 'Q'}, {"trim5", required_argument, 0, '5'}, {"trim3", required_argument, 0, '3'}, {"exonAnnotation", required_argument, 0, 'a'}, {"exonAlias", required_argument, 0, 'A'}, {"exonFormat", required_argument, 0, 'F'}, {"memoryMultiplex", required_argument, 0, 0}, {"rg", required_argument, 0, 0}, {"gzFASTQinput", no_argument, 0, 0}, {"rg-id", required_argument, 0, 0}, {"SAMoutput", no_argument, 0, 0}, {"BAMinput", no_argument, 0, 0}, {"fast", no_argument, 0, 0}, {"SAMinput", no_argument, 0, 0}, {"reportPairedMultiBest", no_argument, 0, 0}, {"BCLinput", no_argument, 0, 0}, {"scRNA_FQinput", no_argument, 0, 0}, {"scRNA_BAMinput", no_argument, 0, 0}, {"sv", no_argument, 0, 0}, {"longDel", no_argument, 0, 0}, {"exonAnnotationScreenOut", required_argument, 0, 0}, {"gtfFeature", required_argument, 0, 0}, {"gtfAttr", required_argument, 0, 0}, {"extraColumns", no_argument, 0, 0}, {"forcedPE", no_argument, 0, 0}, {"ignoreUnmapped", no_argument, 0, 0}, {"accurateFusions", no_argument, 0, 0}, {"SVdetection", no_argument, 0, 0}, {"maxMismatches", required_argument, 0, 'M'}, {"minMappedLength", required_argument, 0, 0}, {"maxVoteSimples", required_argument, 0, 0}, {"complexIndels", no_argument, 0, 0}, {"minVoteCutoff", required_argument, 0, 0}, {"maxRealignLocations", required_argument, 0, 0}, {"multiMapping", no_argument, 0, 0}, {"keepReadOrder", no_argument, 0, 0}, {"sortReadsByCoordinates", no_argument, 0, 0}, {"noTLENpreference", no_argument, 0, 0}, {0, 0, 0, 0} }; void print_usage_core_aligner() { SUBREADprintf("\nVersion %s\n\n", SUBREAD_VERSION); SUBREADputs("Usage:"); SUBREADputs(""); SUBREADputs("./subread-align [options] -i -r -t -o "); SUBREADputs(""); SUBREADputs("## Mandatory arguments:"); SUBREADputs(" "); SUBREADputs(" -i Base name of the index."); SUBREADputs(""); SUBREADputs(" -r Name of an input read file. If paired-end, this should be"); SUBREADputs(" the first read file (typically containing \"R1\"in the file"); SUBREADputs(" name) and the second should be provided via \"-R\"."); SUBREADputs(" Acceptable formats include gzipped FASTQ, FASTQ, gzipped"); SUBREADputs(" FASTA and FASTA."); SUBREADputs(" These formats are identified automatically."); SUBREADputs(" "); SUBREADputs(" -t Type of input sequencing data. Its values include"); SUBREADputs(" 0: RNA-seq data"); SUBREADputs(" 1: genomic DNA-seq data."); SUBREADputs(" "); SUBREADputs("## Optional arguments:"); SUBREADputs("# input reads and output"); SUBREADputs(" "); SUBREADputs(" -o Name of an output file. By default, the output is in BAM"); SUBREADputs(" format. Omitting this option makes the output be written to"); SUBREADputs(" STDOUT."); SUBREADputs(""); SUBREADputs(" -R Name of the second read file in paired-end data (typically"); SUBREADputs(" containing \"R2\" the file name)."); SUBREADputs(""); SUBREADputs(" --SAMinput Input reads are in SAM format."); SUBREADputs(""); SUBREADputs(" --BAMinput Input reads are in BAM format."); SUBREADputs(""); SUBREADputs(" --SAMoutput Save mapping results in SAM format."); SUBREADputs(""); SUBREADputs("# Phred offset"); SUBREADputs(""); SUBREADputs(" -P <3:6> Offset value added to the Phred quality score of each read"); SUBREADputs(" base. '3' for phred+33 and '6' for phred+64. '3' by default."); SUBREADputs(""); SUBREADputs("# thresholds for mapping"); SUBREADputs(""); SUBREADputs(" -n Number of selected subreads, 10 by default."); SUBREADputs(""); SUBREADputs(" -m Consensus threshold for reporting a hit (minimal number of"); SUBREADputs(" subreads that map in consensus) . If paired-end, this gives"); SUBREADputs(" the consensus threshold for the anchor read (anchor read"); SUBREADputs(" receives more votes than the other read in the same pair)."); SUBREADputs(" 3 by default"); SUBREADputs(""); SUBREADputs(" -p Consensus threshold for the non- anchor read in a pair. 1 by"); SUBREADputs(" default."); SUBREADputs(""); SUBREADputs(" -M Maximum number of mis-matched bases allowed in each reported"); SUBREADputs(" alignment. 3 by default. Mis-matched bases found in soft-"); SUBREADputs(" clipped bases are not counted."); SUBREADputs(""); SUBREADputs("# unique mapping and multi-mapping"); SUBREADputs(""); SUBREADputs(" --multiMapping Report multi-mapping reads in addition to uniquely mapped"); SUBREADputs(" reads. Use \"-B\" to set the maximum number of equally-best"); SUBREADputs(" alignments to be reported."); SUBREADputs(""); SUBREADputs(" -B Maximum number of equally-best alignments to be reported for"); SUBREADputs(" a multi-mapping read. Equally-best alignments have the same"); SUBREADputs(" number of mis-matched bases. 1 by default."); SUBREADputs(""); SUBREADputs("# indel detection"); SUBREADputs(""); SUBREADputs(" -I Maximum length (in bp) of indels that can be detected. 5 by"); SUBREADputs(" default. Indels of up to 200bp long can be detected."); SUBREADputs(""); SUBREADputs(" --complexIndels Detect multiple short indels that are in close proximity"); SUBREADputs(" (they can be as close as 1bp apart from each other)."); SUBREADputs(""); SUBREADputs("# read trimming"); SUBREADputs(""); SUBREADputs(" --trim5 Trim off number of bases from 5' end of each read. 0"); SUBREADputs(" by default."); SUBREADputs(""); SUBREADputs(" --trim3 Trim off number of bases from 3' end of each read. 0"); SUBREADputs(" by default."); SUBREADputs(""); SUBREADputs("# distance and orientation of paired end reads"); SUBREADputs(""); SUBREADputs(" -d Minimum fragment/insert length, 50bp by default."); SUBREADputs(""); SUBREADputs(" -D Maximum fragment/insert length, 600bp by default."); SUBREADputs(""); SUBREADputs(" -S Orientation of first and second reads, 'fr' by default ("); SUBREADputs(" forward/reverse)."); SUBREADputs(""); SUBREADputs("# number of CPU threads"); SUBREADputs(""); SUBREADputs(" -T Number of CPU threads used, 1 by default."); SUBREADputs(""); SUBREADputs("# read group"); SUBREADputs(""); SUBREADputs(" --rg-id Add read group ID to the output."); SUBREADputs(""); SUBREADputs(" --rg Add to the read group (RG) header in the output."); SUBREADputs(""); SUBREADputs("# read order"); SUBREADputs(""); SUBREADputs(" --keepReadOrder Keep order of reads in BAM output the same as that in the"); SUBREADputs(" input file. Reads from the same pair are always placed next"); SUBREADputs(" to each other no matter this option is specified or not."); SUBREADputs(""); SUBREADputs(" --sortReadsByCoordinates Output location-sorted reads. This option is"); SUBREADputs(" applicable for BAM output only. A BAI index file is also"); SUBREADputs(" generated for each BAM file so the BAM files can be directly"); SUBREADputs(" loaded into a genome browser."); SUBREADputs(""); SUBREADputs("# color space reads"); SUBREADputs(""); SUBREADputs(" -b Convert color-space read bases to base-space read bases in"); SUBREADputs(" the mapping output. Note that read mapping is performed at"); SUBREADputs(" color-space."); SUBREADputs(""); SUBREADputs("# dynamic programming"); SUBREADputs(""); SUBREADputs(" --DPGapOpen Penalty for gap opening in short indel detection. -1 by"); SUBREADputs(" default."); SUBREADputs(""); SUBREADputs(" --DPGapExt Penalty for gap extension in short indel detection. 0 by"); SUBREADputs(" default."); SUBREADputs(""); SUBREADputs(" --DPMismatch Penalty for mismatches in short indel detection. 0 by"); SUBREADputs(" default."); SUBREADputs(""); SUBREADputs(" --DPMatch Score for matched bases in short indel detection. 2 by"); SUBREADputs(" default."); SUBREADputs(""); SUBREADputs("# detect structural variants"); SUBREADputs(""); SUBREADputs(" --sv Detect structural variants (eg. long indel, inversion,"); SUBREADputs(" duplication and translocation) and report breakpoints. Refer"); SUBREADputs(" to Users Guide for breakpoint reporting."); SUBREADputs(""); SUBREADputs("# gene annotation"); SUBREADputs(""); SUBREADputs(" -a Name of an annotation file (gzipped file is accepted)."); SUBREADputs(" GTF/GFF format by default. See -F option for more format"); SUBREADputs(" information."); SUBREADputs(""); SUBREADputs(" -F Specify format of the provided annotation file. Acceptable"); SUBREADputs(" formats include 'GTF' (or compatible GFF format) and"); SUBREADputs(" 'SAF'. 'GTF' by default. For SAF format, please refer to"); SUBREADputs(" Users Guide."); SUBREADputs(""); SUBREADputs(" -A Provide a chromosome name alias file to match chr names in"); SUBREADputs(" annotation with those in the reads. This should be a two-"); SUBREADputs(" column comma-delimited text file. Its first column should"); SUBREADputs(" include chr names in the annotation and its second column"); SUBREADputs(" should include chr names in the index. Chr names are case"); SUBREADputs(" sensitive. No column header should be included in the"); SUBREADputs(" file."); SUBREADputs(""); SUBREADputs(" --gtfFeature Specify feature type in GTF annotation. 'exon'"); SUBREADputs(" by default. Features used for read counting will be "); SUBREADputs(" extracted from annotation using the provided value."); SUBREADputs(""); SUBREADputs(" --gtfAttr Specify attribute type in GTF annotation. 'gene_id'"); SUBREADputs(" by default. Meta-features used for read counting will be "); SUBREADputs(" extracted from annotation using the provided value."); SUBREADputs(""); SUBREADputs("# others"); SUBREADputs(""); SUBREADputs(" -v Output version of the program."); SUBREADputs(""); SUBREADputs("Refer to Users Manual for detailed description to the arguments. "); SUBREADputs(""); } int parse_opts_aligner(int argc , char ** argv, global_context_t * global_context) { int c; int option_index = 0; int is_64_bit_computer = sizeof(char *)>4; optind = 0; opterr = 1; optopt = 63; subread_rebuild_cmd(argc, argv, global_context); global_context->config.entry_program_name = CORE_PROGRAM_SUBREAD; global_context->config.max_mismatch_exonic_reads = 3; global_context->config.max_mismatch_junction_reads = 3; global_context->config.use_dynamic_programming_indel = 1; // config.extending_search_indels is changed from 1 to 0 on 10/mar/2014 global_context->config.extending_search_indels = 0; global_context->config.limited_tree_scan = 0 ; //global_context->config.big_margin_record_size = 9; if(argc<2) { print_usage_core_aligner(); return -1; } /* for(c = 0; cconfig.DP_penalty_create_gap = atoi(optarg); break; case 'Y': if(!is_valid_digit(optarg, "Y")) STANDALONE_exit(-1); global_context->config.DP_match_score = atoi(optarg); break; case 'E': if(!is_valid_digit(optarg, "E")) STANDALONE_exit(-1); global_context->config.DP_penalty_extend_gap = atoi(optarg); break; case 'X': if(!is_valid_digit(optarg, "X")) STANDALONE_exit(-1); global_context->config.DP_mismatch_penalty = atoi(optarg); break; case '3': if(!is_valid_digit(optarg, "3")) STANDALONE_exit(-1); global_context->config.read_trim_3 = atoi(optarg); break; case '5': if(!is_valid_digit(optarg, "5")) STANDALONE_exit(-1); global_context->config.read_trim_5 = atoi(optarg); break; case 'J': global_context->config.show_soft_cliping = 0; break; case 'B': if(!is_valid_digit(optarg, "B")) STANDALONE_exit(-1); global_context->config.multi_best_reads = atoi(optarg); if(global_context->config.multi_best_reads<1) global_context->config.multi_best_reads=1; global_context->config.reported_multi_best_reads = global_context->config.multi_best_reads; global_context->config.max_vote_combinations = max(global_context->config.max_vote_combinations, global_context->config.reported_multi_best_reads + 1); global_context->config.max_vote_simples = max(global_context->config.max_vote_simples, global_context->config.reported_multi_best_reads + 1); break; case 'H': global_context->config.use_hamming_distance_break_ties = 1; break; case 's': global_context->config.downscale_mapping_quality = 1; break; case 'a': strncpy(global_context->config.exon_annotation_file, optarg, MAX_FILE_NAME_LENGTH-1); break; case 'A': strncpy(global_context->config.exon_annotation_alias_file, optarg, MAX_FILE_NAME_LENGTH-1); break; case 'F': if(strcmp(optarg,"GTF")==0){ global_context->config.exon_annotation_file_type = FILE_TYPE_GTF; } else if(strcmp(optarg,"SAF")==0){ global_context->config.exon_annotation_file_type = FILE_TYPE_RSUBREAD; } else { SUBREADprintf("Unknown annotation file format: %s.\nThe accepted formats are GTF and SAF only.\n", optarg); STANDALONE_exit(-1); } break; case 'S': global_context->config.is_first_read_reversed = optarg[0]=='r'?1:0; global_context->config.is_second_read_reversed = optarg[1]=='f'?0:1; break; case 'U': global_context->config.report_no_unpaired_reads = 1; break; case 'b': global_context->config.convert_color_to_base = 1; break; case 'D': if(!is_valid_digit(optarg, "D")) STANDALONE_exit(-1); global_context->config.maximum_pair_distance = atoi(optarg); break; case 'd': if(!is_valid_digit(optarg, "d")) STANDALONE_exit(-1); global_context->config.minimum_pair_distance = atoi(optarg); break; case 'n': if(!is_valid_digit(optarg, "n")) STANDALONE_exit(-1); global_context->config.total_subreads = atoi(optarg); //global_context->config.total_subreads = min(31,global_context->config.total_subreads ); break; case 'm': global_context->config.minimum_subread_for_first_read = atof(optarg); break; case 'T': if(!is_valid_digit_range(optarg, "T", 1, 64)) STANDALONE_exit(-1); global_context->config.all_threads = atoi(optarg); if(global_context->config.all_threads <1) global_context->config.all_threads = 1; if(global_context->config.all_threads > MAX_THREADS) global_context->config.all_threads = MAX_THREADS; break; case 'r': strncpy(global_context->config.first_read_file, optarg, MAX_FILE_NAME_LENGTH * 3 * MAX_SCRNA_FASTQ_FILES-1); break; case 'R': global_context->input_reads.is_paired_end_reads = 1; strncpy(global_context->config.second_read_file, optarg, MAX_FILE_NAME_LENGTH-1); break; case 'i': strncpy(global_context->config.index_prefix, optarg, MAX_FILE_NAME_LENGTH-1); break; case 'o': strncpy(global_context->config.output_prefix, optarg, MAX_FILE_NAME_LENGTH-1); break; case 'I': if(!is_valid_digit_range(optarg, "I", 0, 200)) STANDALONE_exit(-1); global_context->config.max_indel_length = atoi(optarg); if(!is_64_bit_computer) global_context->config.max_indel_length = min(global_context->config.max_indel_length , 16); if(global_context->config.max_indel_length <0)global_context->config.max_indel_length =0; if(global_context->config.max_indel_length > MAX_INSERTION_LENGTH)global_context->config.max_indel_length = MAX_INSERTION_LENGTH; if(global_context->config.max_indel_length > 16) { global_context->config.reassembly_subread_length = 16; global_context->config.reassembly_window_multiplex = 3; global_context->config.reassembly_start_read_number = 3; global_context->config.reassembly_tolerable_voting = 0; global_context->config.reassembly_window_alleles = 3; global_context->config.reassembly_key_length = 28; global_context->config.is_third_iteration_running = 1; // These options were removed for maximising the sensitivity. // They should be put back when we want a higher indel accuracy. //global_context->config.max_mismatch_exonic_reads = 1; //global_context->config.max_mismatch_junction_reads = 1; //global_context->config.total_subreads = 28; //global_context->config.minimum_subread_for_first_read = 3; //global_context->config.minimum_subread_for_second_read = 1; //global_context->config.do_big_margin_filtering_for_reads = 0; //global_context->config.extending_search_indels = 0; //global_context->config.use_dynamic_programming_indel = 1; //global_context->config.flanking_subread_indel_mismatch = 0; global_context->config.do_superlong_indel_detection = 0; } break; case 'M': if(!is_valid_digit(optarg, "M")) STANDALONE_exit(-1); global_context->config.max_mismatch_exonic_reads = atoi(optarg); break; case 'P': if (optarg[0]=='3') global_context->config.phred_score_format = FASTQ_PHRED33; else global_context->config.phred_score_format = FASTQ_PHRED64; break; case 'p': if(!is_valid_digit(optarg, "p")) STANDALONE_exit(-1); global_context->config.minimum_subread_for_second_read = atoi(optarg); break; case 't': if(strcmp(optarg, "1") == 0){ global_context->config.experiment_type = CORE_EXPERIMENT_DNASEQ; }else if(strcmp(optarg, "0") == 0){ global_context->config.experiment_type = CORE_EXPERIMENT_RNASEQ; }else{ SUBREADprintf("Error: unknown experiment type:%s (only 0 and 1 are allowed)\n", optarg); return -1; } break; case 'c': global_context->config.space_type = GENE_SPACE_COLOR; break; case 'Q': global_context->config.use_quality_score_break_ties = 1; break; case 0: if(strcmp("multiMapping", long_options[option_index].name)==0) { global_context->config.report_multi_mapping_reads = 1; } else if(strcmp("ignoreUnmapped", long_options[option_index].name)==0) { global_context->config.ignore_unmapped_reads = 1; } else if(strcmp("memoryMultiplex", long_options[option_index].name)==0) { global_context->config.memory_use_multiplex = atof(optarg); } else if(strcmp("BCLinput", long_options[option_index].name)==0) { global_context->config.scRNA_input_mode=GENE_INPUT_BCL; } else if(strcmp("scRNA_FQinput", long_options[option_index].name)==0) { global_context->config.scRNA_input_mode=GENE_INPUT_SCRNA_FASTQ; } else if(strcmp("scRNA_BAMinput", long_options[option_index].name)==0) { global_context->config.scRNA_input_mode=GENE_INPUT_SCRNA_BAM; } else if(strcmp("rg-id", long_options[option_index].name)==0) { strcpy(global_context->config.read_group_id, optarg); } else if(strcmp("rg", long_options[option_index].name)==0) { strcat(global_context->config.read_group_txt, "\t"); strcat(global_context->config.read_group_txt, optarg); } else if(strcmp("SAMoutput", long_options[option_index].name)==0) { global_context->config.is_BAM_output = 0; } else if(strcmp("BAMinput", long_options[option_index].name)==0) { if(global_context->config.is_SAM_file_input && global_context->config.is_BAM_input == 0){ SUBREADprintf("ERROR: you cannot specify both SAMinput and BAMinput\n"); return -1; } global_context->config.is_BAM_input = 1; global_context->config.is_SAM_file_input = 1; } else if(strcmp("keepReadOrder", long_options[option_index].name)==0) { global_context->config.is_input_read_order_required=1; } else if(strcmp("sortReadsByCoordinates", long_options[option_index].name)==0) { global_context->config.sort_reads_by_coordinates=1; } else if(strcmp("extraColumns", long_options[option_index].name)==0) { global_context->config.SAM_extra_columns=1; } else if(strcmp("SAMinput", long_options[option_index].name)==0) { if(global_context->config.is_SAM_file_input && global_context->config.is_BAM_input == 1){ SUBREADprintf("ERROR: you cannot specify both SAMinput and BAMinput\n"); return -1; } global_context->config.is_BAM_input = 0; global_context->config.is_SAM_file_input = 1; } else if(strcmp("reportPairedMultiBest", long_options[option_index].name)==0) { global_context->config.report_multiple_best_in_pairs = 1; } else if(strcmp("fast", long_options[option_index].name)==0) { global_context -> config.fast_run = 1; } else if(strcmp("sv", long_options[option_index].name)==0) { global_context->config.do_breakpoint_detection = 1; global_context->config.do_fusion_detection = 1; global_context->config.prefer_donor_receptor_junctions = 0; //global_context->config.do_big_margin_filtering_for_reads = 1; } else if(strcmp("longDel", long_options[option_index].name)==0) { global_context->config.do_breakpoint_detection = 1; global_context->config.do_long_del_detection = 1; global_context->config.prefer_donor_receptor_junctions = 0; } else if(strcmp("minMappedLength", long_options[option_index].name)==0) { if(!is_valid_digit(optarg, "minMappedLength")) STANDALONE_exit(-1); global_context->config.min_mapped_fraction = atoi(optarg); } else if(strcmp("accurateFusions", long_options[option_index].name)==0) { global_context->config.more_accurate_fusions = 1; } else if(strcmp("SVdetection", long_options[option_index].name)==0) { global_context -> config.do_structural_variance_detection = 1; global_context -> config.use_memory_buffer = 1; global_context -> config.reads_per_chunk = 600llu*1024*1024; } else if(strcmp("noTLENpreference", long_options[option_index].name) == 0) { global_context -> config.no_TLEN_preference = 1; } else if(strcmp("gtfFeature", long_options[option_index].name) == 0) { strncpy(global_context->config.exon_annotation_feature_name_column, optarg, MAX_READ_NAME_LEN - 1); } else if(strcmp("gtfAttr", long_options[option_index].name) == 0) { strncpy(global_context->config.exon_annotation_gene_id_column, optarg, MAX_READ_NAME_LEN - 1); } else if(strcmp("maxRealignLocations", long_options[option_index].name)==0) { global_context->config.max_vote_combinations = atoi(optarg); global_context->config.multi_best_reads = atoi(optarg); } else if(strcmp("maxVoteSimples", long_options[option_index].name)==0) { global_context->config.max_vote_simples = atoi(optarg); } else if(strcmp("complexIndels", long_options[option_index].name)==0) { global_context->config.maximise_sensitivity_indel = 1; global_context->config.realignment_minimum_variant_distance = 1; } else if(strcmp("exonAnnotationScreenOut", long_options[option_index].name)==0){ strcpy(global_context->config.exon_annotation_file_screen_out, optarg); } else if(strcmp("minVoteCutoff", long_options[option_index].name)==0) { global_context->config.max_vote_number_cutoff = atoi(optarg); } break; case '?': default: SUBREADprintf("Unknown option: -%c",c); print_usage_core_aligner(); return -1 ; } } if(argc > optind){ SUBREADprintf("Invalid parameter '%s'\n", argv[optind]); return -1; } if(global_context->config.reported_multi_best_reads > 1 && ! global_context->config.report_multi_mapping_reads) SUBREADprintf("WARNING: You required multi best alignments, but disallowed multi-mapping reads. You need to turn on the multi-mapping option.\n"); global_context->config.more_accurate_fusions = global_context->config.more_accurate_fusions && (global_context->config.do_fusion_detection || global_context->config.do_long_del_detection); if(global_context->config.more_accurate_fusions) { global_context->config.high_quality_base_threshold = 999999; //#warning "============ REMOVE THE NEXT LINE ======================" global_context->config.show_soft_cliping = 1; //#warning "============ REMOVE ' + 3' FROM NEXT LINE ==============" global_context->config.max_mismatch_junction_reads = 0 + 3; //#warning "============ REMOVE ' - 1' FROM NEXT LINE ==============" global_context->config.do_big_margin_filtering_for_junctions = 1 - 1; global_context->config.total_subreads = 28; //#warning "============ REMOVE THE NEXT LINE BEFORE RELEASE ===============" //global_context->config.multi_best_reads = 1; } if(global_context->config.is_SAM_file_input) global_context->config.phred_score_format = FASTQ_PHRED33; return 0; } #if defined MAKE_STANDALONE int main(int argc , char ** argv) { #elif defined RUNNING_ENV_JAVA int subread_aligner_main(int argc , char ** argv) { #else int main_align(int argc , char ** argv) { #endif // printf("SIZE_OF_ALN=%d\n", sizeof(mapping_result_t)); // printf("SIZE_OF_VOT=%d\n", sizeof(voting_context_t)); int ret = core_main(argc, argv, parse_opts_aligner); return ret; } Rsubread/src/core-interface-subjunc.c0000644000175200017520000007013614136047615020651 0ustar00biocbuildbiocbuild#include #include #include #include #include #include "subread.h" #include "input-files.h" #include "core.h" #include "HelperFunctions.h" static struct option long_options[] = { {"index", required_argument, 0, 'i'}, {"read", required_argument, 0, 'r'}, {"read2", required_argument, 0, 'R'}, {"output", required_argument, 0, 'o'}, {"subreads", required_argument, 0, 'n'}, {"minvotes", required_argument, 0, 'm'}, {"minvotes2", required_argument, 0, 'p'}, {"singleSAM", required_argument, 0, '1'}, {"pairedSAM", required_argument, 0, '2'}, {"threads", required_argument, 0, 'T'}, {"indel", required_argument, 0, 'I'}, {"phred", required_argument, 0, 'P'}, {"mindist", required_argument, 0, 'd'}, {"maxdist", required_argument, 0, 'D'}, {"order", required_argument, 0, 'S'}, {"trim5", required_argument, 0, '5'}, {"trim3", required_argument, 0, '3'}, {"color-convert", no_argument, 0, 'b'}, {"junctionIns", required_argument, 0, 0}, {"multi", required_argument, 0, 'B'}, {"exonAnnotation", required_argument, 0, 'a'}, {"exonAnnotationScreenOut", required_argument, 0, 0}, {"exonAlias", required_argument, 0, 'A'}, {"exonFormat", required_argument, 0, 'F'}, {"gtfAttr", required_argument, 0, 0}, {"gtfFeature", required_argument, 0, 0}, {"BCLinput", no_argument, 0, 0}, {"rg", required_argument, 0, 0}, {"rg-id", required_argument, 0, 0}, {"gzFASTQinput", no_argument, 0, 0}, {"unique", no_argument, 0, 'u'}, {"SAMoutput", no_argument, 0, 0}, {"BAMinput", no_argument, 0, 0}, {"SAMinput", no_argument, 0, 0}, {"hamming", no_argument, 0, 'H'}, {"quality", no_argument, 0, 'Q'}, {"fast", no_argument, 0, 0}, {"scRNA_FQinput", no_argument, 0, 0}, {"DPMismatch", required_argument, 0, 'X'}, {"DPMatch", required_argument, 0, 'Y'}, {"DPGapOpen", required_argument, 0, 'G'}, {"DPGapExt", required_argument, 0, 'E'}, {"extendIndelDetection", no_argument, 0, 0}, {"allJunctions", no_argument, 0, 0}, {"memoryMultiplex", required_argument, 0, 0}, {"ignoreUnmapped", no_argument, 0, 0}, {"extraColumns", no_argument, 0, 0}, {"disableBigMargin", no_argument, 0, 0}, {"relaxMismatchedBases", no_argument, 0, 0}, {"reportPairedMultiBest", no_argument, 0, 0}, {"maxMismatches", required_argument, 0, 'M'}, {"exonicSubreadFrac", required_argument, 0, 0}, {"SVdetection", no_argument, 0, 0}, {"maxVoteSimples", required_argument, 0, 0}, {"maxRealignLocations", required_argument, 0, 0}, {"minVoteCutoff", required_argument, 0, 0}, {"minMappedFraction", required_argument, 0, 0}, {"complexIndels", no_argument, 0, 0}, {"multiMapping", no_argument, 0, 0}, {"keepReadOrder", no_argument, 0, 0}, {"sortReadsByCoordinates", no_argument, 0, 0}, {0, 0, 0, 0} }; void print_usage_core_subjunc() { SUBREADprintf("\nVersion %s\n\n", SUBREAD_VERSION); SUBREADputs("Usage:"); SUBREADputs(""); SUBREADputs(" ./subjunc [options] -i -r -o "); SUBREADputs(""); SUBREADputs("## Mandatory arguments:"); SUBREADputs(""); SUBREADputs(" -i Base name of the index."); SUBREADputs(""); SUBREADputs(" -r Name of an input read file. If paired-end, this should be"); SUBREADputs(" the first read file (typically containing \"R1\"in the file"); SUBREADputs(" name) and the second should be provided via \"-R\"."); SUBREADputs(" Acceptable formats include gzipped FASTQ, FASTQ, gzipped"); SUBREADputs(" FASTA and FASTA."); SUBREADputs(" These formats are identified automatically."); SUBREADputs(""); SUBREADputs("## Optional arguments:"); SUBREADputs("# input reads and output"); SUBREADputs(" "); SUBREADputs(" -o Name of an output file. By default, the output is in BAM"); SUBREADputs(" format. Omitting this option makes the output be written to"); SUBREADputs(" STDOUT."); SUBREADputs(""); SUBREADputs(" -R Name of the second read file in paired-end data (typically"); SUBREADputs(" containing \"R2\" the file name)."); SUBREADputs(""); SUBREADputs(" --SAMinput Input reads are in SAM format."); SUBREADputs(""); SUBREADputs(" --BAMinput Input reads are in BAM format."); SUBREADputs(""); SUBREADputs(" --SAMoutput Save mapping results in SAM format."); SUBREADputs(""); SUBREADputs("# Phred offset"); SUBREADputs(""); SUBREADputs(" -P <3:6> Offset value added to the Phred quality score of each read"); SUBREADputs(" base. '3' for phred+33 and '6' for phred+64. '3' by default."); SUBREADputs(""); SUBREADputs("# thresholds for mapping"); SUBREADputs(""); SUBREADputs(" -n Number of selected subreads, 14 by default."); SUBREADputs(""); SUBREADputs(" -m Consensus threshold for reporting a hit (minimal number of"); SUBREADputs(" subreads that map in consensus) . If paired-end, this gives"); SUBREADputs(" the consensus threshold for the anchor read (anchor read"); SUBREADputs(" receives more votes than the other read in the same pair)."); SUBREADputs(" 1 by default."); SUBREADputs(""); SUBREADputs(" -p Consensus threshold for the non- anchor read in a pair. 1 by"); SUBREADputs(" default."); SUBREADputs(""); SUBREADputs(" -M Maximum number of mis-matched bases allowed in each reported"); SUBREADputs(" alignment. 3 by default. Mis-matched bases found in soft-"); SUBREADputs(" clipped bases are not counted."); SUBREADputs(""); SUBREADputs("# unique mapping and multi-mapping"); SUBREADputs(""); SUBREADputs(" --multiMapping Report multi-mapping reads in addition to uniquely mapped"); SUBREADputs(" reads. Use \"-B\" to set the maximum number of equally-best"); SUBREADputs(" alignments to be reported."); SUBREADputs(""); SUBREADputs(" -B Maximum number of equally-best alignments to be reported for"); SUBREADputs(" a multi-mapping read. Equally-best alignments have the same"); SUBREADputs(" number of mis-matched bases. 1 by default."); SUBREADputs(""); SUBREADputs("# indel detection"); SUBREADputs(""); SUBREADputs(" -I Maximum length (in bp) of indels that can be detected. 5 by"); SUBREADputs(" default. Indels of up to 200bp long can be detected."); SUBREADputs(""); SUBREADputs(" --complexIndels Detect multiple short indels that are in close proximity"); SUBREADputs(" (they can be as close as 1bp apart from each other)."); SUBREADputs(""); SUBREADputs("# read trimming"); SUBREADputs(""); SUBREADputs(" --trim5 Trim off number of bases from 5' end of each read. 0"); SUBREADputs(" by default."); SUBREADputs(""); SUBREADputs(" --trim3 Trim off number of bases from 3' end of each read. 0"); SUBREADputs(" by default."); SUBREADputs(""); SUBREADputs("# distance and orientation of paired end reads"); SUBREADputs(""); SUBREADputs(" -d Minimum fragment/insert length, 50bp by default."); SUBREADputs(""); SUBREADputs(" -D Maximum fragment/insert length, 600bp by default."); SUBREADputs(""); SUBREADputs(" -S Orientation of first and second reads, 'fr' by default ("); SUBREADputs(" forward/reverse)."); SUBREADputs(""); SUBREADputs("# number of CPU threads"); SUBREADputs(""); SUBREADputs(" -T Number of CPU threads used, 1 by default."); SUBREADputs(""); SUBREADputs("# read group"); SUBREADputs(""); SUBREADputs(" --rg-id Add read group ID to the output."); SUBREADputs(""); SUBREADputs(" --rg Add to the read group (RG) header in the output."); SUBREADputs(""); SUBREADputs("# read order"); SUBREADputs(""); SUBREADputs(" --keepReadOrder Keep order of reads in BAM output the same as that in the"); SUBREADputs(" input file. Reads from the same pair are always placed next"); SUBREADputs(" to each other no matter this option is specified or not."); SUBREADputs(""); SUBREADputs(" --sortReadsByCoordinates Output location-sorted reads. This option is"); SUBREADputs(" applicable for BAM output only. A BAI index file is also"); SUBREADputs(" generated for each BAM file so the BAM files can be directly"); SUBREADputs(" loaded into a genome browser."); SUBREADputs(""); SUBREADputs("# color space reads"); SUBREADputs(""); SUBREADputs(" -b Convert color-space read bases to base-space read bases in"); SUBREADputs(" the mapping output. Note that read mapping is performed at"); SUBREADputs(" color-space."); SUBREADputs(""); SUBREADputs("# dynamic programming"); SUBREADputs(""); SUBREADputs(" --DPGapOpen Penalty for gap opening in short indel detection. -1 by"); SUBREADputs(" default."); SUBREADputs(""); SUBREADputs(" --DPGapExt Penalty for gap extension in short indel detection. 0 by"); SUBREADputs(" default."); SUBREADputs(""); SUBREADputs(" --DPMismatch Penalty for mismatches in short indel detection. 0 by"); SUBREADputs(" default."); SUBREADputs(""); SUBREADputs(" --DPMatch Score for matched bases in short indel detection. 2 by"); SUBREADputs(" default."); SUBREADputs(""); SUBREADputs("# detect all junctions including gene fusions"); SUBREADputs(""); SUBREADputs(" --allJunctions Detect exon-exon junctions (both canonical and non-canonical"); SUBREADputs(" junctions) and structural variants in RNA-seq data. Refer to"); SUBREADputs(" Users Guide for reporting of junctions and fusions."); SUBREADputs(""); SUBREADputs("# gene annotation"); SUBREADputs(""); SUBREADputs(" -a Name of an annotation file (gzipped file is accepted)."); SUBREADputs(" GTF/GFF format by default. See -F option for more format"); SUBREADputs(" information."); SUBREADputs(""); SUBREADputs(" -F Specify format of the provided annotation file. Acceptable"); SUBREADputs(" formats include 'GTF' (or compatible GFF format) and"); SUBREADputs(" 'SAF'. 'GTF' by default. For SAF format, please refer to"); SUBREADputs(" Users Guide."); SUBREADputs(""); SUBREADputs(" -A Provide a chromosome name alias file to match chr names in"); SUBREADputs(" annotation with those in the reads. This should be a two-"); SUBREADputs(" column comma-delimited text file. Its first column should"); SUBREADputs(" include chr names in the annotation and its second column"); SUBREADputs(" should include chr names in the index. Chr names are case"); SUBREADputs(" sensitive. No column header should be included in the"); SUBREADputs(" file."); SUBREADputs(""); SUBREADputs(" --gtfFeature Specify feature type in GTF annotation. 'exon'"); SUBREADputs(" by default. Features used for read counting will be "); SUBREADputs(" extracted from annotation using the provided value."); SUBREADputs(""); SUBREADputs(" --gtfAttr Specify attribute type in GTF annotation. 'gene_id'"); SUBREADputs(" by default. Meta-features used for read counting will be "); SUBREADputs(" extracted from annotation using the provided value."); SUBREADputs(""); SUBREADputs("# others"); SUBREADputs(""); SUBREADputs(" -v Output version of the program."); SUBREADputs(""); SUBREADputs("Refer to Users Manual for detailed description to the arguments. "); SUBREADputs(""); } int parse_opts_subjunc(int argc , char ** argv, global_context_t * global_context) { int c; int option_index = 0; int is_64_bit_computer = sizeof(char *)>4; optind = 0; opterr = 1; optopt = 63; subread_rebuild_cmd(argc, argv, global_context); global_context->config.entry_program_name = CORE_PROGRAM_SUBJUNC; global_context->config.max_mismatch_exonic_reads = 3; global_context->config.max_mismatch_junction_reads = 3; global_context->config.ambiguous_mapping_tolerance = 39 - 20 ; global_context->config.extending_search_indels = 0; global_context->config.do_fusion_detection =0; global_context->config.use_dynamic_programming_indel = 1; global_context->config.do_breakpoint_detection = 1; global_context->config.total_subreads = 14; global_context->config.minimum_subread_for_first_read =1; global_context->config.minimum_subread_for_second_read = 1; global_context->config.minimum_exonic_subread_fraction = 0.3; global_context->config.high_quality_base_threshold = 990000; global_context->config.do_big_margin_filtering_for_junctions = 1; global_context->config.report_no_unpaired_reads = 0; global_context->config.experiment_type = CORE_EXPERIMENT_RNASEQ; //#warning " ========================= REMOVE ' + 1 ' FROM THE NEXT LINE !! ==========================" global_context->config.limited_tree_scan = 0; global_context->config.use_hamming_distance_in_exon = 0; //global_context->config.big_margin_record_size = 24; if(argc<2) { print_usage_core_subjunc(); return -1; } while ((c = getopt_long (argc, argv, "vxsJ1:2:S:L:A:a:Hd:D:n:m:p:P:R:r:i:l:o:G:Y:E:X:T:I:B:bQF:cuUfM:3:5:9:?", long_options, &option_index)) != -1) { switch(c) { case 'v': core_version_number("Subjunc"); return -1; case 'G': if(!is_valid_digit(optarg, "G")) STANDALONE_exit(-1); global_context->config.DP_penalty_create_gap = atoi(optarg); break; case 'Y': if(!is_valid_digit(optarg, "Y")) STANDALONE_exit(-1); global_context->config.DP_match_score = atoi(optarg); break; case 'E': if(!is_valid_digit(optarg, "E")) STANDALONE_exit(-1); global_context->config.DP_penalty_extend_gap = atoi(optarg); break; case 'X': if(!is_valid_digit(optarg, "X")) STANDALONE_exit(-1); global_context->config.DP_mismatch_penalty = atoi(optarg); break; case '3': if(!is_valid_digit(optarg, "3")) STANDALONE_exit(-1); global_context->config.read_trim_3 = atoi(optarg); break; case '5': if(!is_valid_digit(optarg, "5")) STANDALONE_exit(-1); global_context->config.read_trim_5 = atoi(optarg); break; case '1': case '2': case 'r': strncpy(global_context->config.first_read_file, optarg, MAX_FILE_NAME_LENGTH-1); break; case 'J': global_context->config.show_soft_cliping = 0; break; case 'Q': global_context->config.use_quality_score_break_ties = 1; break; case 'H': global_context->config.use_hamming_distance_break_ties = 1; break; case 's': global_context->config.downscale_mapping_quality = 1; break; case 'a': strncpy(global_context->config.exon_annotation_file, optarg, MAX_FILE_NAME_LENGTH-1); break; case 'A': strncpy(global_context->config.exon_annotation_alias_file, optarg, MAX_FILE_NAME_LENGTH-1); break; case 'F': if(strcmp(optarg,"GTF")==0){ global_context->config.exon_annotation_file_type = FILE_TYPE_GTF; } else if(strcmp(optarg,"SAF")==0){ global_context->config.exon_annotation_file_type = FILE_TYPE_RSUBREAD; } else { SUBREADprintf("Unknown annotation file format: %s.\nThe accepted formats are GTF and SAF only.\n", optarg); STANDALONE_exit(-1); } break; case 'S': global_context->config.is_first_read_reversed = optarg[0]=='r'?1:0; global_context->config.is_second_read_reversed = optarg[1]=='f'?0:1; break; case 'U': global_context->config.report_no_unpaired_reads = 1; break; case 'b': global_context->config.convert_color_to_base = 1; break; case 'D': if(!is_valid_digit(optarg, "D")) STANDALONE_exit(-1); global_context->config.maximum_pair_distance = atoi(optarg); break; case 'd': if(!is_valid_digit(optarg, "d")) STANDALONE_exit(-1); global_context->config.minimum_pair_distance = atoi(optarg); if(global_context->config.minimum_pair_distance <0) global_context->config.restrected_read_order = 0; break; case 'n': if(!is_valid_digit(optarg, "n")) STANDALONE_exit(-1); global_context->config.total_subreads = atoi(optarg); //global_context->config.total_subreads = min(31,global_context->config.total_subreads); break; case 'm': if(!is_valid_digit(optarg, "m")) STANDALONE_exit(-1); global_context->config.minimum_subread_for_first_read = atoi(optarg); break; case 'T': if(!is_valid_digit_range(optarg, "T", 1, 64)) STANDALONE_exit(-1); global_context->config.all_threads = atoi(optarg); if(global_context->config.all_threads <1) global_context->config.all_threads = 1; if(global_context->config.all_threads > MAX_THREADS) global_context->config.all_threads = MAX_THREADS; break; case 'M': if(!is_valid_digit(optarg, "M")) STANDALONE_exit(-1); global_context->config.max_mismatch_exonic_reads = atoi(optarg); global_context->config.max_mismatch_junction_reads = atoi(optarg); break; case 'R': global_context->input_reads.is_paired_end_reads = 1; strncpy(global_context->config.second_read_file, optarg, MAX_FILE_NAME_LENGTH-1); break; case 'i': strncpy(global_context->config.index_prefix, optarg, MAX_FILE_NAME_LENGTH-1); break; case 'o': strncpy(global_context->config.output_prefix, optarg, MAX_FILE_NAME_LENGTH-1); break; case 'I': if(!is_valid_digit(optarg, "I")) STANDALONE_exit(-1); global_context->config.max_indel_length = atoi(optarg); if(!is_64_bit_computer) global_context->config.max_indel_length = min(global_context->config.max_indel_length , 16); if(global_context->config.max_indel_length <0)global_context->config.max_indel_length =0; if(global_context->config.max_indel_length > MAX_INSERTION_LENGTH)global_context->config.max_indel_length = MAX_INSERTION_LENGTH; if(global_context->config.max_indel_length > 16) { global_context->config.reassembly_subread_length = 12; global_context->config.reassembly_window_multiplex = 3; global_context->config.reassembly_start_read_number = 5; global_context->config.reassembly_tolerable_voting = 0; global_context->config.reassembly_window_alleles = 2; global_context->config.reassembly_key_length = 28; global_context->config.flanking_subread_indel_mismatch = 0; global_context->config.is_third_iteration_running = 1; //global_context->config.max_mismatch_exonic_reads = 2; //global_context->config.max_mismatch_junction_reads = 2; //global_context->config.total_subreads = 28; //global_context->config.minimum_subread_for_first_read = 3; //global_context->config.minimum_subread_for_second_read = 1; //global_context->config.do_big_margin_filtering_for_reads = 1; global_context->config.do_superlong_indel_detection = 0; } break; case 'P': if (optarg[0]=='3') global_context->config.phred_score_format = FASTQ_PHRED33; else global_context->config.phred_score_format = FASTQ_PHRED64; break; case 'p': if(!is_valid_digit(optarg, "p")) STANDALONE_exit(-1); global_context->config.minimum_subread_for_second_read = atoi(optarg); break; case 'B': if(!is_valid_digit(optarg, "B")) STANDALONE_exit(-1); global_context->config.multi_best_reads = atoi(optarg); if(global_context->config.multi_best_reads<1) global_context->config.multi_best_reads=1; global_context->config.reported_multi_best_reads = global_context->config.multi_best_reads; global_context->config.max_vote_combinations = max(global_context->config.max_vote_combinations, global_context->config.reported_multi_best_reads + 1); global_context->config.max_vote_simples = max(global_context->config.max_vote_simples, global_context->config.reported_multi_best_reads + 1); break; case 'c': global_context->config.space_type = GENE_SPACE_COLOR; break; case 0: if(strcmp("multiMapping", long_options[option_index].name)==0) { global_context->config.report_multi_mapping_reads = 1; } else if(strcmp("memoryMultiplex", long_options[option_index].name)==0) { global_context->config.memory_use_multiplex = atof(optarg); } else if(strcmp("ignoreUnmapped", long_options[option_index].name)==0) { global_context->config.ignore_unmapped_reads = 1; } else if(strcmp("BCLinput", long_options[option_index].name)==0) { global_context->config.scRNA_input_mode=GENE_INPUT_BCL; } else if(strcmp("scRNA_FQinput", long_options[option_index].name)==0) { global_context->config.scRNA_input_mode=GENE_INPUT_SCRNA_FASTQ; } else if(strcmp("rg-id", long_options[option_index].name)==0) { strcpy(global_context->config.read_group_id, optarg); } else if(strcmp("rg", long_options[option_index].name)==0) { strcat(global_context->config.read_group_txt, "\t"); strcat(global_context->config.read_group_txt, optarg); } else if(strcmp("SAMoutput", long_options[option_index].name)==0) { global_context->config.is_BAM_output = 0; } else if(strcmp("BAMinput", long_options[option_index].name)==0) { if(global_context->config.is_SAM_file_input && global_context->config.is_BAM_input == 0){ SUBREADprintf("Error: you cannot specify both SAMinput and BAMinput\n"); return -1; } global_context->config.is_BAM_input = 1; global_context->config.is_SAM_file_input = 1; } else if(strcmp("SAMinput", long_options[option_index].name)==0) { if(global_context->config.is_SAM_file_input && global_context->config.is_BAM_input == 1){ SUBREADprintf("Error: you cannot specify both SAMinput and BAMinput\n"); return -1; } global_context->config.is_BAM_input = 0; global_context->config.is_SAM_file_input = 1; } else if(strcmp("junctionIns", long_options[option_index].name)==0) { if(!is_valid_digit(optarg, "junctionIns")) STANDALONE_exit(-1); global_context->config.check_donor_at_junctions=0; global_context->config.limited_tree_scan = 0; global_context->config.max_insertion_at_junctions = atoi(optarg); } else if(strcmp("keepReadOrder", long_options[option_index].name)==0) { global_context->config.is_input_read_order_required=1; } else if(strcmp("sortReadsByCoordinates", long_options[option_index].name)==0) { global_context->config.sort_reads_by_coordinates=1; } else if(strcmp("extraColumns", long_options[option_index].name)==0) { global_context->config.SAM_extra_columns=1; } else if(strcmp("minMappedFraction", long_options[option_index].name)==0) { if(!is_valid_digit(optarg, "minMappedFraction")) STANDALONE_exit(-1); global_context->config.min_mapped_fraction = atoi(optarg); } else if(strcmp("relaxMismatchedBases", long_options[option_index].name)==0) { global_context->config.max_mismatch_junction_reads = 999; global_context->config.max_mismatch_exonic_reads = 999; global_context->config.min_mapped_fraction = 61; } else if(strcmp("reportPairedMultiBest", long_options[option_index].name)==0) { global_context->config.report_multiple_best_in_pairs = 1; } else if(strcmp("exonicSubreadFrac", long_options[option_index].name)==0) { if(atof(optarg)>0) global_context->config.minimum_exonic_subread_fraction = atof(optarg); else SUBREADprintf("WARNING: unknown parameter: --exonicSubreadFrac '%s'\n", optarg); } else if(strcmp("fast", long_options[option_index].name)==0) { global_context -> config.fast_run = 1; } else if(strcmp("SVdetection", long_options[option_index].name)==0) { global_context -> config.do_structural_variance_detection = 1; global_context -> config.use_memory_buffer = 1; global_context -> config.reads_per_chunk = 300llu*1024*1024; } else if(strcmp("minDistanceBetweenVariants", long_options[option_index].name)==0) { int newdist = atoi(optarg); newdist = max(newdist, 1); newdist = min(newdist, MAX_READ_LENGTH); global_context->config.realignment_minimum_variant_distance = newdist; } else if(strcmp("disableBigMargin", long_options[option_index].name)==0) { global_context->config.do_big_margin_filtering_for_junctions = 0; global_context->config.limited_tree_scan = 0; } else if(strcmp("gtfFeature", long_options[option_index].name) == 0) { strncpy(global_context->config.exon_annotation_feature_name_column, optarg, MAX_READ_NAME_LEN - 1); } else if(strcmp("gtfAttr", long_options[option_index].name) == 0) { strncpy(global_context->config.exon_annotation_gene_id_column, optarg, MAX_READ_NAME_LEN - 1); } else if(strcmp("maxVoteSimples", long_options[option_index].name)==0) { global_context->config.max_vote_simples = atoi(optarg); } else if(strcmp("maxRealignLocations", long_options[option_index].name)==0) { global_context->config.max_vote_combinations = atoi(optarg); global_context->config.multi_best_reads = atoi(optarg); } else if(strcmp("complexIndels", long_options[option_index].name)==0) { global_context->config.maximise_sensitivity_indel = 1; global_context->config.realignment_minimum_variant_distance = 1; // global_context->config.max_indel_length = 16; } else if(strcmp("disableBigMargin", long_options[option_index].name)==0) { global_context->config.big_margin_record_size = 0; } else if(strcmp("extendIndelDetection", long_options[option_index].name)==0) { global_context->config.extending_search_indels = 1; } else if(strcmp("minVoteCutoff", long_options[option_index].name)==0) { global_context->config.max_vote_number_cutoff = atoi(optarg); } else if(strcmp("exonAnnotationScreenOut", long_options[option_index].name)==0){ strcpy(global_context->config.exon_annotation_file_screen_out, optarg); } else if(strcmp("allJunctions", long_options[option_index].name)==0) { global_context->config.do_fusion_detection = 1; } break; case '?': default: print_usage_core_subjunc(); return -1 ; } } if(argc > optind){ SUBREADprintf("Invalid parameter '%s'\n", argv[optind]); return -1; } if(global_context->config.reported_multi_best_reads > 1 && ! global_context->config.report_multi_mapping_reads) SUBREADprintf("WARNING: You required multi best alignments, but disallowed multi-mapping reads. You need to turn on the multi-mapping option.\n"); if(global_context->config.is_SAM_file_input) global_context->config.phred_score_format = FASTQ_PHRED33; global_context->config.more_accurate_fusions = global_context->config.more_accurate_fusions && global_context->config.do_fusion_detection; if(global_context->config.more_accurate_fusions) { global_context->config.high_quality_base_threshold = 999999; //#warning "============ REMOVE THE NEXT LINE ======================" global_context->config.show_soft_cliping = 1; //#warning "============ REMOVE ' + 3' FROM NEXT LINE ==============" global_context->config.max_mismatch_junction_reads = 0 + 3; //#warning "============ REMOVE ' - 1' FROM NEXT LINE ==============" global_context->config.do_big_margin_filtering_for_junctions = 1 - 1; global_context->config.total_subreads = 28; //#warning "============ REMOVE THE NEXT LINE BEFORE RELEASE ===============" //global_context->config.multi_best_reads = 1; } return 0; } #if defined MAKE_STANDALONE int main(int argc , char ** argv) { #elif defined RUNNING_ENV_JAVA int subread_subjunc_main(int argc , char ** argv) { #else int main_junction(int argc , char ** argv) { #endif int ret = core_main(argc, argv, parse_opts_subjunc); return ret; } Rsubread/src/core-junction.c0000644000175200017520000112732214136047615017074 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #include #include #include #include #include #include "subread.h" #include "sublog.h" #include "gene-value-index.h" #include "gene-algorithms.h" #include "input-files.h" #include "core.h" #include "core-indel.h" #include "core-junction.h" #include "core-bigtable.h" #define TTTSNAME "V0112_0155:7:1308:1308:136442" #define CLUSTER_ALIGNMENT_DONOR_R1_MAPPED 2 #define CLUSTER_ALIGNMENT_DONOR_R2_MAPPED 4 #define CLUSTER_ALIGNMENT_DONOR_NEGATIVE_STRAND 1 unsigned int abs32uint(unsigned int x){ if(x > 0x7fffffff) x = (0xffffffff - x) + 1; return x; } typedef struct{ unsigned int piece_main_abs_offset; unsigned int piece_minor_abs_offset; int piece_main_masks; short piece_main_coverage_start; short piece_main_coverage_end; short piece_main_hamming_match; short piece_main_read_quality; short piece_minor_hamming_match; short piece_minor_read_quality; int piece_minor_score; short intron_length; gene_vote_number_t *piece_main_indel_record; unsigned short piece_main_indels; unsigned short piece_minor_indel_offset; gene_vote_number_t piece_main_votes; gene_vote_number_t piece_minor_votes; short piece_minor_coverage_start; short piece_minor_coverage_end; short split_point; char inserted_bases; char is_GT_AG_donors; char is_donor_found_or_annotation; char is_strand_jumped; char is_break_even; //unsigned long long int Score_H; //unsigned long long int Score_L; } select_junction_record_t; void debug_show_event(global_context_t* global_context, chromosome_event_t * event){ char outpos1[100], outpos2[100]; absoffset_to_posstr(global_context, event -> event_small_side, outpos1); absoffset_to_posstr(global_context, event -> event_large_side, outpos2); SUBREADprintf("Event between %s and %s\n", outpos1, outpos2); } int get_offset_maximum_chro_pos(global_context_t * global_context, thread_context_t * thread_context, unsigned int linear){ gene_offset_t * chros =& global_context -> chromosome_table; int n = 0; int total_offsets = chros -> total_offsets; int LL = 0, RR = total_offsets-1; while(1){ if(LL >= RR-1) break; int MM = (LL+RR)/2; if( linear > chros->read_offsets[MM]) LL = MM; else if(linear < chros->read_offsets[MM]) RR = MM; else break; } n = max(0, LL - 2); for (; n < chros -> total_offsets; n++) { if (chros->read_offsets[n] > linear) { int ret; unsigned int last_linear = 0; if(n==0) ret = chros->read_offsets[0] - chros -> padding *2 +16; else{ ret = ( chros->read_offsets[n] - chros->read_offsets[n-1] ) - chros -> padding *2 +16; last_linear = chros->read_offsets[n-1]; } linear -= last_linear; if(linear < chros -> padding || linear >= chros -> padding + ret) return -1; return ret; } } return -2; } // read_head_abs_pos is the offset of the FIRST WANTED base. void search_events_to_front(global_context_t * global_context, thread_context_t * thread_context, explain_context_t * explain_context, char * read_text , char * qual_text, unsigned int read_head_abs_offset, short remainder_len, short sofar_matched, int suggested_movement, int do_not_jump) { short tested_read_pos; // #warning "SUBREAD_151 REMOVE THIS ASSERTION! " // if(remainder_len >= 102)SUBREADprintf("FATAL:%d\n", remainder_len ); // assert(remainder_len < 102); HashTable * event_table = NULL; chromosome_event_t * event_space = NULL; if(thread_context) { event_table = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> event_entry_table; event_space = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; } else { event_table = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_entry_table; event_space = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; } gene_value_index_t * value_index = thread_context?thread_context->current_value_index:global_context->current_value_index ; if((global_context -> config.do_fusion_detection || global_context -> config.do_long_del_detection)|| there_are_events_in_range(event_table->appendix1, read_head_abs_offset, remainder_len)) { int event_search_method; if((global_context -> config.do_fusion_detection || global_context -> config.do_long_del_detection)) event_search_method = EVENT_SEARCH_BY_BOTH_SIDES; else event_search_method = EVENT_SEARCH_BY_SMALL_SIDE; // tested_read_pos is the index of the first base unwanted! int move_start = do_not_jump?0:global_context -> config.realignment_minimum_variant_distance; if(suggested_movement) move_start = suggested_movement-1; int is_junction_scanned = 0; if(MAX_EVENTS_IN_READ - 1 > explain_context -> tmp_search_sections) for(tested_read_pos = move_start ; tested_read_pos <= remainder_len; tested_read_pos++) { int xk1, matched_bases_to_site; chromosome_event_t *site_events[MAX_EVENT_ENTRIES_PER_SITE+1]; int jump_penalty = 0; unsigned potential_event_pos; if(explain_context -> current_is_strand_jumped) potential_event_pos = read_head_abs_offset - tested_read_pos +1; else potential_event_pos = read_head_abs_offset + tested_read_pos -1; if(!check_event_bitmap( event_table->appendix1, potential_event_pos )) continue; int search_types = CHRO_EVENT_TYPE_INDEL | CHRO_EVENT_TYPE_JUNCTION | CHRO_EVENT_TYPE_FUSION; int site_events_no = search_event(global_context, event_table , event_space , potential_event_pos, event_search_method , search_types , site_events); if(0 && FIXLENstrcmp("R010442852", explain_context -> read_name) == 0) { SUBREADprintf("FOUND THE EVENT FRONT:%d at %u\n", site_events_no, potential_event_pos); if(site_events_no) SUBREADprintf("EVENT0_type = %d\n", site_events[0]->event_type); } if(!site_events_no)continue; unsigned int tested_chro_begin; if(explain_context -> current_is_strand_jumped) tested_chro_begin = read_head_abs_offset - tested_read_pos + 1; else tested_chro_begin = read_head_abs_offset; matched_bases_to_site = match_chro(read_text, value_index, tested_chro_begin, tested_read_pos, explain_context -> current_is_strand_jumped, global_context -> config.space_type); /* #warning "========= COMMENT TWO LINES ====================" SUBREADprintf("MBASETOSITE=%d, tested_read_pos=%d\n", matched_bases_to_site, tested_read_pos); SUBREADprintf("TXT=%s, tested_read_pos=%d\n", read_text, tested_chro_begin); */ int this_round_junction_scanned = 0; if(0 && FIXLENstrcmp("R010442852", explain_context -> read_name) == 0) SUBREADprintf("F_JUMP? match=%d / tested=%d\n", matched_bases_to_site , tested_read_pos); //#warning "========= remove - 2000 from next line =============" if(explain_context -> total_tries < REALIGN_TOTAL_TRIES && tested_read_pos >0 && ( matched_bases_to_site*10000/tested_read_pos > 9000 - 2000 || global_context->config.maximise_sensitivity_indel) ) for(xk1 = 0; xk1 < site_events_no ; xk1++) { chromosome_event_t * tested_event = site_events[xk1]; if(explain_context -> is_fully_covered && tested_event -> event_type == CHRO_EVENT_TYPE_FUSION && tested_event -> event_large_side - tested_event -> event_small_side > MAX_DELETION_LENGTH){ continue; } //if(explain_context -> pair_number == 23) if(0 && FIXLENstrcmp("R00000001230", explain_context -> read_name) ==0){ char outpos1[100]; absoffset_to_posstr(global_context, read_head_abs_offset , outpos1); SUBREADprintf("F_JUMP?%d > %d %s (%u) ; SEARCH_TAG=%u\n", (1+matched_bases_to_site)*10000 / tested_read_pos , 9000, read_text, tested_chro_begin, potential_event_pos); debug_show_event(global_context, tested_event); } // note that these two values are the index of the first wanted base. unsigned int new_read_head_abs_offset; if((global_context -> config.do_fusion_detection || global_context -> config.do_long_del_detection) && tested_event -> event_type == CHRO_EVENT_TYPE_INDEL) { if(explain_context ->current_is_strand_jumped){ if(potential_event_pos == tested_event-> event_small_side) continue; }else{ if(potential_event_pos == tested_event-> event_large_side) continue; } } if( tested_event -> event_type != CHRO_EVENT_TYPE_INDEL){ this_round_junction_scanned = 1; } if((global_context -> config.do_fusion_detection || global_context -> config.do_long_del_detection))// && tested_event->event_type == CHRO_EVENT_TYPE_FUSION) new_read_head_abs_offset = (potential_event_pos == tested_event -> event_large_side)?tested_event -> event_small_side:tested_event -> event_large_side; else new_read_head_abs_offset = tested_event -> event_large_side; short new_remainder_len = remainder_len - tested_read_pos + min(0, tested_event->indel_length) - tested_event -> indel_at_junction; // #warning "SUBREAD_151 REMOVE THIS ASSERTION! " // assert(new_remainder_len < 102); if(new_remainder_len>0) { //if(explain_context -> pair_number==2074) printf("JUMPPED IN.\n"); explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections].read_pos_end = explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections].read_pos_start + tested_read_pos; explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections].event_after_section = tested_event; explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections].is_connected_to_large_side = (potential_event_pos == tested_event -> event_large_side); explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections + 1].read_pos_start = tested_read_pos - min(0, tested_event -> indel_length) + tested_event -> indel_at_junction; explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections + 1].abs_offset_for_start = new_read_head_abs_offset; if(tested_event->event_type == CHRO_EVENT_TYPE_FUSION) jump_penalty = 2; int current_is_jumped = explain_context -> current_is_strand_jumped; int current_sup_as_complex = explain_context -> tmp_min_support_as_complex; int current_sup_as_simple = explain_context -> tmp_support_as_simple; //int current_unsup_as_simple = explain_context -> tmp_min_unsupport; int current_pure_donor_found = explain_context -> tmp_is_pure_donor_found_explain; explain_context -> tmp_support_as_simple = tested_event -> supporting_reads; explain_context -> tmp_min_support_as_complex = min((tested_event -> is_donor_found_or_annotation & 64)?0x7fffffff:tested_event -> supporting_reads,explain_context -> tmp_min_support_as_complex); explain_context -> tmp_min_unsupport = min(tested_event -> anti_supporting_reads,explain_context -> tmp_min_unsupport); explain_context -> tmp_is_pure_donor_found_explain = explain_context -> tmp_is_pure_donor_found_explain && tested_event -> is_donor_found_or_annotation; explain_context -> tmp_indel_penalty += ( tested_event -> event_type == CHRO_EVENT_TYPE_INDEL ); if(tested_event -> event_type == CHRO_EVENT_TYPE_FUSION && tested_event -> is_strand_jumped) explain_context -> current_is_strand_jumped = !explain_context -> current_is_strand_jumped; explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections + 1].is_strand_jumped = explain_context -> current_is_strand_jumped; explain_context -> tmp_search_sections ++; explain_context -> total_tries ++; search_events_to_front(global_context, thread_context, explain_context, read_text + tested_event -> indel_at_junction + tested_read_pos - min(0, tested_event->indel_length), qual_text + tested_read_pos - min(0, tested_event->indel_length), new_read_head_abs_offset, new_remainder_len, sofar_matched + matched_bases_to_site - jump_penalty, tested_event -> connected_next_event_distance, 0); explain_context -> tmp_search_sections --; explain_context -> current_is_strand_jumped = current_is_jumped; explain_context -> tmp_indel_penalty -= ( tested_event -> event_type == CHRO_EVENT_TYPE_INDEL ); explain_context -> tmp_min_support_as_complex = current_sup_as_complex; explain_context -> tmp_support_as_simple = current_sup_as_simple; //explain_context -> tmp_min_unsupport = current_unsup_as_simple; explain_context -> tmp_is_pure_donor_found_explain = current_pure_donor_found; } //if(global_context ->config.limited_tree_scan) break; } if( (global_context ->config.limited_tree_scan) && explain_context -> full_read_len <= EXON_LONG_READ_LENGTH) break; is_junction_scanned = max(is_junction_scanned, this_round_junction_scanned); } } //#warning "SUBREAD_151 REMOVE THE ASSERT! " //assert( remainder_len< 102 ); int whole_section_matched = match_chro(read_text , value_index, explain_context -> current_is_strand_jumped?read_head_abs_offset - remainder_len +1:read_head_abs_offset, remainder_len , explain_context -> current_is_strand_jumped, global_context -> config.space_type); explain_context -> tmp_total_matched_bases = whole_section_matched + sofar_matched ; new_explain_try_replace(global_context, thread_context, explain_context, remainder_len, 0); } void new_explain_try_replace(global_context_t* global_context, thread_context_t * thread_context, explain_context_t * explain_context, int remainder_len, int search_to_back) { int is_better_result = 0, is_same_best = 0; if(0 && FIXLENstrcmp("simulated.11420793", explain_context->read_name)==0){ SUBREADprintf("TRY_REPLACE : %s has best=%d, b_evn=%d, tscore=%d, t_evn=%d\n", explain_context->read_name, explain_context -> best_matching_bases , explain_context -> best_is_complex , explain_context-> tmp_total_matched_bases, explain_context -> tmp_search_sections ); } if(explain_context -> best_matching_bases - explain_context -> best_indel_penalty < explain_context-> tmp_total_matched_bases - explain_context -> tmp_indel_penalty) { is_better_result = 1; explain_context -> best_is_complex = explain_context -> tmp_search_sections ; explain_context -> is_currently_tie = 0; explain_context -> best_support_as_simple = explain_context -> tmp_support_as_simple; explain_context -> best_min_unsupport_as_simple = explain_context -> tmp_min_unsupport; explain_context -> best_min_support_as_complex = explain_context -> tmp_min_support_as_complex; explain_context -> best_is_pure_donor_found_explain = explain_context -> tmp_is_pure_donor_found_explain; explain_context -> second_best_matching_bases = max(explain_context -> second_best_matching_bases, explain_context -> best_matching_bases); explain_context -> best_matching_bases = explain_context-> tmp_total_matched_bases ; explain_context -> best_indel_penalty = explain_context -> tmp_indel_penalty; } else if(explain_context -> best_matching_bases - explain_context -> best_indel_penalty == explain_context-> tmp_total_matched_bases - explain_context -> tmp_indel_penalty) { // only gapped explainations are complex counted. explain_context -> best_is_complex += explain_context -> tmp_search_sections; explain_context -> second_best_matching_bases = explain_context -> best_matching_bases; explain_context -> best_indel_penalty = explain_context -> tmp_indel_penalty; if(0 && FIXLENstrcmp("R010442852", explain_context -> read_name) == 0){ SUBREADprintf("complexity: curr=%d, new=%d ; sections=%d\n", explain_context->best_min_support_as_complex, explain_context -> tmp_min_support_as_complex, explain_context -> tmp_search_sections ); } if(explain_context -> best_is_complex > 1) { // is complex now! if(explain_context -> tmp_search_sections == 0) { if(explain_context -> tmp_min_unsupport >explain_context->best_min_support_as_complex){ is_better_result = 1; explain_context->best_min_support_as_complex =explain_context -> tmp_min_unsupport; explain_context -> best_is_pure_donor_found_explain = explain_context -> tmp_is_pure_donor_found_explain; explain_context -> is_currently_tie = 0; } else if(explain_context -> tmp_min_unsupport == explain_context->best_min_support_as_complex) { explain_context -> is_currently_tie = 1; is_same_best = 1; } } else{ if(explain_context -> tmp_min_support_as_complex >explain_context->best_min_support_as_complex){ is_better_result = 1; explain_context -> best_min_support_as_complex =explain_context -> tmp_min_support_as_complex; explain_context -> best_is_pure_donor_found_explain = explain_context -> tmp_is_pure_donor_found_explain; explain_context -> is_currently_tie = 0; } else if(explain_context -> tmp_min_support_as_complex == explain_context->best_min_support_as_complex){ explain_context -> is_currently_tie = 1; is_same_best = 1; } } } else { // this branch is reached ONLY if the last best is ONE-gapped (50M3D50M) and the current best is ungapped (100M)! if(explain_context -> best_is_pure_donor_found_explain) { if(explain_context -> best_min_unsupport_as_simple >= explain_context -> best_support_as_simple+2) { is_better_result = 1; explain_context -> best_min_support_as_complex = explain_context -> best_min_unsupport_as_simple; explain_context -> best_is_pure_donor_found_explain = explain_context -> tmp_is_pure_donor_found_explain; explain_context -> is_currently_tie = 0; } } //#warning "======= MAKE if(0) IS CORRECT BEFORE RELEASE ======" else if(0) if(explain_context -> best_min_unsupport_as_simple >= explain_context -> best_support_as_simple) { is_better_result = 1; explain_context -> best_min_support_as_complex = explain_context -> best_min_unsupport_as_simple; explain_context -> best_is_pure_donor_found_explain = explain_context -> tmp_is_pure_donor_found_explain; explain_context -> is_currently_tie = 0; } } } else return; if(is_better_result || is_same_best){ if(search_to_back){ explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections].read_pos_start = 0; }else{ explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections].read_pos_end = explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections].read_pos_start + remainder_len; explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections].event_after_section = NULL; } } if(0 && FIXLENstrcmp("R010442852", explain_context -> read_name) == 0){ SUBREADprintf("TRY_REPLACE_DESICION TO %s: BETTER=%d, SAME=%d ; CURRENT : %d secs ; NEWBEST : %d secs\n", search_to_back?"BACK":"FRONT", is_better_result, is_same_best, search_to_back? explain_context -> result_back_junction_numbers[0]:explain_context -> result_front_junction_numbers[0] ,explain_context -> tmp_search_sections); int xx1; for(xx1 = 0; xx1 < explain_context -> tmp_search_sections;xx1++){ SUBREADprintf(" Event : %d ~ %d in read\n", explain_context -> tmp_search_junctions[xx1].read_pos_start, explain_context -> tmp_search_junctions[xx1].read_pos_end); if(explain_context -> tmp_search_junctions[xx1].event_after_section){ SUBREADprintf(" "); debug_show_event(global_context, explain_context -> tmp_search_junctions[xx1].event_after_section); } } } if(is_better_result) { if(search_to_back){ explain_context -> all_back_alignments = 1; explain_context -> result_back_junction_numbers[0] = explain_context -> tmp_search_sections +1; // checked: memory boundary memcpy(explain_context -> result_back_junctions[0], explain_context -> tmp_search_junctions , sizeof(perfect_section_in_read_t) * (explain_context -> tmp_search_sections +1)); }else{ explain_context -> all_front_alignments = 1; explain_context -> result_front_junction_numbers[0] = explain_context -> tmp_search_sections +1; // checked: memory boundary memcpy(explain_context -> result_front_junctions[0], explain_context -> tmp_search_junctions , sizeof(perfect_section_in_read_t) * (explain_context -> tmp_search_sections +1)); } }else if(is_same_best){ if(search_to_back && explain_context -> all_back_alignments < MAX_ALIGNMENT_PER_ANCHOR){ explain_context -> result_back_junction_numbers[explain_context -> all_back_alignments] = explain_context -> tmp_search_sections +1; // checked: memory boundary memcpy(explain_context -> result_back_junctions[explain_context -> all_back_alignments], explain_context -> tmp_search_junctions , sizeof(perfect_section_in_read_t) * (explain_context -> tmp_search_sections +1)); explain_context -> all_back_alignments ++; }else if((!search_to_back) && explain_context -> all_front_alignments < MAX_ALIGNMENT_PER_ANCHOR){ explain_context -> result_front_junction_numbers[explain_context -> all_front_alignments] = explain_context -> tmp_search_sections +1; // checked: memory boundary memcpy(explain_context -> result_front_junctions[explain_context -> all_front_alignments], explain_context -> tmp_search_junctions , sizeof(perfect_section_in_read_t) * (explain_context -> tmp_search_sections +1)); explain_context -> all_front_alignments ++; } } } void new_explain_try_replace_xe(global_context_t* global_context, thread_context_t * thread_context, explain_context_t * explain_context, int remainder_len, int search_to_back) { int is_better_result = 0, is_same_best = 0; //SUBREADprintf("TRYING SET %s %s : Matched_bases : %d -> %d ; SECS : %d -> %d\n", explain_context -> read_name, search_to_back?"BACK":"FRONT", explain_context -> best_matching_bases, explain_context-> tmp_total_matched_bases, search_to_back? explain_context -> result_back_junction_numbers[0]:explain_context -> result_front_junction_numbers[0] ,explain_context -> tmp_search_sections ); if(explain_context -> best_matching_bases < explain_context-> tmp_total_matched_bases) { is_better_result = 1; explain_context -> best_is_complex = explain_context -> tmp_search_sections ; explain_context -> is_currently_tie = 0; explain_context -> best_support_as_simple = explain_context -> tmp_support_as_simple; explain_context -> best_min_unsupport_as_simple = explain_context -> tmp_min_unsupport; explain_context -> best_min_support_as_complex = explain_context -> tmp_min_support_as_complex; explain_context -> best_is_pure_donor_found_explain = explain_context -> tmp_is_pure_donor_found_explain; explain_context -> second_best_matching_bases = max(explain_context -> second_best_matching_bases, explain_context -> best_matching_bases); explain_context -> best_matching_bases = explain_context-> tmp_total_matched_bases ; } else if(explain_context -> tmp_search_sections < search_to_back? explain_context -> result_back_junction_numbers[0]:explain_context -> result_front_junction_numbers[0] - 1 && explain_context -> best_matching_bases == explain_context-> tmp_total_matched_bases) { is_better_result = 1; explain_context -> best_is_complex = explain_context -> tmp_search_sections ; explain_context -> is_currently_tie = 0; explain_context -> best_support_as_simple = explain_context -> tmp_support_as_simple; explain_context -> best_min_unsupport_as_simple = explain_context -> tmp_min_unsupport; explain_context -> best_min_support_as_complex = explain_context -> tmp_min_support_as_complex; explain_context -> best_is_pure_donor_found_explain = explain_context -> tmp_is_pure_donor_found_explain; explain_context -> second_best_matching_bases = max(explain_context -> second_best_matching_bases, explain_context -> best_matching_bases); explain_context -> best_matching_bases = explain_context-> tmp_total_matched_bases ; } else if( 0 && explain_context -> best_matching_bases == explain_context-> tmp_total_matched_bases) { // only gapped explainations are complex counted. explain_context -> best_is_complex += explain_context -> tmp_search_sections; explain_context -> second_best_matching_bases = explain_context -> best_matching_bases; if(0 && FIXLENstrcmp("R010442852", explain_context -> read_name) == 0){ SUBREADprintf("complexity: curr=%d, new=%d ; sections=%d\n", explain_context->best_min_support_as_complex, explain_context -> tmp_min_support_as_complex, explain_context -> tmp_search_sections ); } if(explain_context -> best_is_complex > 1) { // is complex now! if(explain_context -> tmp_search_sections == 0) { if(explain_context -> tmp_min_unsupport >explain_context->best_min_support_as_complex){ is_better_result = 1; explain_context->best_min_support_as_complex =explain_context -> tmp_min_unsupport; explain_context -> best_is_pure_donor_found_explain = explain_context -> tmp_is_pure_donor_found_explain; explain_context -> is_currently_tie = 0; } else if(explain_context -> tmp_min_unsupport == explain_context->best_min_support_as_complex) { explain_context -> is_currently_tie = 1; is_same_best = 1; } } else { if(explain_context -> tmp_min_support_as_complex >explain_context->best_min_support_as_complex){ is_better_result = 1; explain_context -> best_min_support_as_complex =explain_context -> tmp_min_support_as_complex; explain_context -> best_is_pure_donor_found_explain = explain_context -> tmp_is_pure_donor_found_explain; explain_context -> is_currently_tie = 0; } else if(explain_context -> tmp_min_support_as_complex == explain_context->best_min_support_as_complex){ explain_context -> is_currently_tie = 1; is_same_best = 1; } } } else { // this branch is reached ONLY if the last best is ONE-gapped (50M3D50M) and the current best is ungapped (100M)! if(explain_context -> best_is_pure_donor_found_explain) { if(explain_context -> best_min_unsupport_as_simple >= explain_context -> best_support_as_simple+2) { is_better_result = 1; explain_context -> best_min_support_as_complex = explain_context -> best_min_unsupport_as_simple; explain_context -> best_is_pure_donor_found_explain = explain_context -> tmp_is_pure_donor_found_explain; explain_context -> is_currently_tie = 0; } } } } else return; if(is_better_result || is_same_best){ if(search_to_back){ explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections].read_pos_start = 0; }else{ explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections].read_pos_end = explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections].read_pos_start + remainder_len; explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections].event_after_section = NULL; } } if(0 && FIXLENstrcmp("R010442852", explain_context -> read_name) == 0){ SUBREADprintf("RNAME=%s TRY_REPLACE_DESICION TO %s: BETTER=%d, SAME=%d ; CURRENT : %d secs ; NEWBEST : %d secs MM_bases : %d -> %d\n", explain_context -> read_name, search_to_back?"BACK":"FRONT", is_better_result, is_same_best, search_to_back? explain_context -> result_back_junction_numbers[0]:explain_context -> result_front_junction_numbers[0] ,explain_context -> tmp_search_sections, explain_context -> best_matching_bases , explain_context-> tmp_total_matched_bases); int xx1; for(xx1 = 0; xx1 < explain_context -> tmp_search_sections;xx1++){ SUBREADprintf(" Event : %d ~ %d in read\n", explain_context -> tmp_search_junctions[xx1].read_pos_start, explain_context -> tmp_search_junctions[xx1].read_pos_end); if(explain_context -> tmp_search_junctions[xx1].event_after_section){ SUBREADprintf(" "); debug_show_event(global_context, explain_context -> tmp_search_junctions[xx1].event_after_section); } } } if(is_better_result) { if(search_to_back){ explain_context -> all_back_alignments = 1; explain_context -> result_back_junction_numbers[0] = explain_context -> tmp_search_sections +1; // checked: memory boundary memcpy(explain_context -> result_back_junctions[0], explain_context -> tmp_search_junctions , sizeof(perfect_section_in_read_t) * (explain_context -> tmp_search_sections +1)); }else{ explain_context -> all_front_alignments = 1; explain_context -> result_front_junction_numbers[0] = explain_context -> tmp_search_sections +1; // checked: memory boundary memcpy(explain_context -> result_front_junctions[0], explain_context -> tmp_search_junctions , sizeof(perfect_section_in_read_t) * (explain_context -> tmp_search_sections +1)); } }else if(is_same_best && 0){ if(search_to_back && explain_context -> all_back_alignments < MAX_ALIGNMENT_PER_ANCHOR){ explain_context -> result_back_junction_numbers[explain_context -> all_back_alignments] = explain_context -> tmp_search_sections +1; // checked: memory boundary memcpy(explain_context -> result_back_junctions[explain_context -> all_back_alignments], explain_context -> tmp_search_junctions , sizeof(perfect_section_in_read_t) * (explain_context -> tmp_search_sections +1)); explain_context -> all_back_alignments ++; }else if((!search_to_back) && explain_context -> all_front_alignments < MAX_ALIGNMENT_PER_ANCHOR){ explain_context -> result_front_junction_numbers[explain_context -> all_front_alignments] = explain_context -> tmp_search_sections +1; // checked: memory boundary memcpy(explain_context -> result_front_junctions[explain_context -> all_front_alignments], explain_context -> tmp_search_junctions , sizeof(perfect_section_in_read_t) * (explain_context -> tmp_search_sections +1)); explain_context -> all_front_alignments ++; } } } // read_tail_abs_offset is actually the offset of the base next to the last base in read tail. // read_tail_pos is the FIRST UNWANTED BASE, after the read. void search_events_to_back(global_context_t * global_context, thread_context_t * thread_context, explain_context_t * explain_context, char * read_text , char * qual_text, unsigned int read_tail_abs_offset, short read_tail_pos, short sofar_matched, int suggested_movement, int do_not_jump) { short tested_read_pos; HashTable * event_table = NULL; chromosome_event_t * event_space = NULL; if(thread_context) { event_table = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> event_entry_table; event_space = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; } else { event_table = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_entry_table; event_space = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; } gene_value_index_t * value_index = thread_context?thread_context->current_value_index:global_context->current_value_index ; if( there_are_events_in_range(event_table -> appendix2, read_tail_abs_offset - read_tail_pos, read_tail_pos) || global_context -> config.do_fusion_detection ||global_context -> config.do_long_del_detection ){ int event_search_method; if((global_context -> config.do_fusion_detection || global_context -> config.do_long_del_detection)) event_search_method = EVENT_SEARCH_BY_BOTH_SIDES; else event_search_method = EVENT_SEARCH_BY_LARGE_SIDE; int is_junction_scanned = 0; // minimum perfect section length is 1 // tested_read_pos is the first WANTED BASE in section. int move_start = read_tail_pos - (do_not_jump?0:global_context -> config.realignment_minimum_variant_distance); if(suggested_movement) move_start = read_tail_pos - suggested_movement + 1; //#warning ">>>>>>>>>>>>>> COMMENT THIS <<<<<<<<<<<<<<<<<<<<<" //printf("OCT27-STEP-BKIN : %s , STT=%d, %u, %d\n", explain_context -> read_name, move_start, read_tail_abs_offset, read_tail_pos); if(MAX_EVENTS_IN_READ - 1> explain_context -> tmp_search_sections) for(tested_read_pos = move_start; tested_read_pos >=0;tested_read_pos --) { int xk1, matched_bases_to_site; int jump_penalty = 0; chromosome_event_t *site_events[MAX_EVENT_ENTRIES_PER_SITE]; int potential_event_pos; if(explain_context -> current_is_strand_jumped) potential_event_pos = read_tail_abs_offset + ( read_tail_pos - tested_read_pos); else potential_event_pos = read_tail_abs_offset - ( read_tail_pos - tested_read_pos); if(!check_event_bitmap( event_table->appendix2, potential_event_pos )) continue; int search_types = CHRO_EVENT_TYPE_INDEL | CHRO_EVENT_TYPE_JUNCTION | CHRO_EVENT_TYPE_FUSION; int site_events_no = search_event(global_context, event_table , event_space , potential_event_pos, event_search_method , search_types, site_events); //#warning ">>>>>>>>>>>>>> COMMENT THIS <<<<<<<<<<<<<<<<<<<<<" //printf("OCT27-STEP-BKIN-SR: %s at %u, FOUND=%d\n" , explain_context -> read_name,potential_event_pos,site_events_no); if(!site_events_no)continue; unsigned int tested_chro_begin; if(explain_context -> current_is_strand_jumped) tested_chro_begin = read_tail_abs_offset + 1; else tested_chro_begin = read_tail_abs_offset - (read_tail_pos - tested_read_pos); matched_bases_to_site = match_chro(read_text + tested_read_pos, value_index, tested_chro_begin , read_tail_pos - tested_read_pos, explain_context -> current_is_strand_jumped, global_context -> config.space_type); int this_round_junction_scanned = 0; //#warning ">>>>>>>>>>>>>>>> REMOVE IT <<<<<<<<<<<<<<<<<<<<<<" //printf("OCT27-STEPSB-JB-%s: test %u = %d events; TEST=%d > 7000 : MA=%d; %s ; %u = %u - (%d - %d) ; LEV=%d\n", explain_context -> read_name, potential_event_pos, site_events_no, (read_tail_pos<=tested_read_pos)?(-1234):( matched_bases_to_site*10000/(read_tail_pos - tested_read_pos)) , matched_bases_to_site, read_text + tested_read_pos, potential_event_pos, read_tail_abs_offset, read_tail_pos, tested_read_pos, explain_context -> tmp_search_sections); //#warning "========= remove - 2000 from next line =============" if(explain_context -> total_tries < REALIGN_TOTAL_TRIES && (read_tail_pos>tested_read_pos) && ( matched_bases_to_site*10000/(read_tail_pos - tested_read_pos) > 9000 - 2000 || global_context->config.maximise_sensitivity_indel) ) for(xk1 = 0; xk1 < site_events_no ; xk1++) { chromosome_event_t * tested_event = site_events[xk1]; if(explain_context -> is_fully_covered && tested_event -> event_type == CHRO_EVENT_TYPE_FUSION && tested_event -> event_large_side - tested_event -> event_small_side > MAX_DELETION_LENGTH){ continue; } if((global_context -> config.do_fusion_detection || global_context -> config.do_long_del_detection) && tested_event -> event_type == CHRO_EVENT_TYPE_INDEL) { if(explain_context->current_is_strand_jumped){ if(potential_event_pos == tested_event-> event_large_side) continue; }else{ if(potential_event_pos == tested_event-> event_small_side) continue; } } if( tested_event -> event_type != CHRO_EVENT_TYPE_INDEL){ this_round_junction_scanned = 1; } if(0 && FIXLENstrcmp("R00000001230", explain_context -> read_name) == 0) SUBREADprintf("B_JUMP?%d LOC %u LEN %d EVT %p\n", (1+matched_bases_to_site)*10000 / (read_tail_pos - tested_read_pos) , tested_event-> event_large_side, tested_event-> indel_length, tested_event); // note that read_tail_pos is the first unwanted base. int new_read_tail_pos = tested_read_pos; if(tested_event->event_type == CHRO_EVENT_TYPE_INDEL) new_read_tail_pos += min(0, tested_event -> indel_length); // note that read_tail_abs_offset is the first unwanted base. unsigned int new_read_tail_abs_offset; if((global_context -> config.do_fusion_detection || global_context -> config.do_long_del_detection))// && tested_event->event_type == CHRO_EVENT_TYPE_FUSION) { new_read_tail_abs_offset = (potential_event_pos == tested_event -> event_small_side)? tested_event -> event_large_side : tested_event -> event_small_side; if(tested_event->is_strand_jumped + explain_context -> current_is_strand_jumped == 1) new_read_tail_abs_offset--; else new_read_tail_abs_offset++; } else new_read_tail_abs_offset = tested_event -> event_small_side + 1; new_read_tail_pos -= tested_event -> indel_at_junction; if(new_read_tail_pos>0) { explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections].read_pos_start = tested_read_pos; explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections + 1].event_after_section = tested_event; explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections + 1].is_connected_to_large_side = (potential_event_pos == tested_event -> event_small_side); explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections + 1].read_pos_end = tested_read_pos + min(0, tested_event->indel_length) - tested_event -> indel_at_junction; explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections + 1].abs_offset_for_start = new_read_tail_abs_offset; if(tested_event->event_type == CHRO_EVENT_TYPE_FUSION) jump_penalty = 2; int current_is_jumped = explain_context -> current_is_strand_jumped ; int current_sup_as_complex = explain_context -> tmp_min_support_as_complex; int current_sup_as_simple = explain_context -> tmp_support_as_simple; int current_pure_donor_found = explain_context -> tmp_is_pure_donor_found_explain; explain_context -> tmp_support_as_simple = tested_event -> supporting_reads; explain_context -> tmp_min_support_as_complex = min((tested_event -> is_donor_found_or_annotation & 64)?0x7fffffff:tested_event -> supporting_reads,explain_context -> tmp_min_support_as_complex); explain_context -> tmp_min_unsupport = min(tested_event -> anti_supporting_reads,explain_context -> tmp_min_unsupport); explain_context -> tmp_is_pure_donor_found_explain = explain_context -> tmp_is_pure_donor_found_explain && tested_event -> is_donor_found_or_annotation; explain_context -> tmp_indel_penalty += ( tested_event -> event_type == CHRO_EVENT_TYPE_INDEL ); if(tested_event -> event_type == CHRO_EVENT_TYPE_FUSION && tested_event -> is_strand_jumped) explain_context -> current_is_strand_jumped = !explain_context -> current_is_strand_jumped; explain_context -> tmp_search_junctions[explain_context -> tmp_search_sections + 1].is_strand_jumped = explain_context -> current_is_strand_jumped; explain_context -> tmp_search_sections ++; explain_context -> total_tries ++; search_events_to_back(global_context, thread_context, explain_context, read_text , qual_text, new_read_tail_abs_offset , new_read_tail_pos, sofar_matched + matched_bases_to_site - jump_penalty, tested_event -> connected_previous_event_distance, 0); explain_context -> tmp_search_sections --; explain_context -> tmp_indel_penalty -= ( tested_event -> event_type == CHRO_EVENT_TYPE_INDEL ); explain_context -> current_is_strand_jumped = current_is_jumped; explain_context -> tmp_min_support_as_complex = current_sup_as_complex; explain_context -> tmp_support_as_simple = current_sup_as_simple; explain_context -> tmp_is_pure_donor_found_explain = current_pure_donor_found; } } if(( global_context ->config.limited_tree_scan) && explain_context -> full_read_len <= EXON_LONG_READ_LENGTH) break; this_round_junction_scanned = max(this_round_junction_scanned, is_junction_scanned); } } int whole_section_matched = match_chro(read_text , value_index, read_tail_abs_offset - (explain_context -> current_is_strand_jumped?-1:read_tail_pos), read_tail_pos , explain_context -> current_is_strand_jumped, global_context -> config.space_type); explain_context -> tmp_total_matched_bases = whole_section_matched + sofar_matched ; new_explain_try_replace(global_context, thread_context, explain_context, 0, 1); } int init_junction_tables(global_context_t * context) { fraglist_init(&context -> funky_list_A); fraglist_init(&context -> funky_list_DE); bktable_init(&context -> funky_table_BC, FUNKY_COLOCATION_TOLERANCE * 2, 10000000); bktable_init(&context -> funky_table_DE, FUNKY_COLOCATION_TOLERANCE * 2, 10000000); bktable_init(&context -> breakpoint_table_P, 2 * context -> config.maximum_pair_distance, 1000000); bktable_init(&context -> breakpoint_table_QR, 2 * BREAK_POINT_MAXIMUM_TOLERANCE, 1000000); bktable_init(&context -> breakpoint_table_YZ, 2 * context -> config.maximum_pair_distance, 1000000); bktable_init(&context -> translocation_result_table, 2*BREAK_POINT_MAXIMUM_TOLERANCE, 1000000); bktable_init(&context -> inversion_result_table, 2*BREAK_POINT_MAXIMUM_TOLERANCE, 1000000); return 0; } int destroy_junction_tables(global_context_t * context) { fraglist_destroy(&context -> funky_list_A); fraglist_destroy(&context -> funky_list_DE); bktable_destroy(&context -> funky_table_BC); bktable_destroy(&context -> funky_table_DE); bktable_destroy(&context -> breakpoint_table_P); bktable_destroy(&context -> breakpoint_table_QR); bktable_destroy(&context -> breakpoint_table_YZ); HashTableIteration(context -> inversion_result_table.entry_table , bktable_free_ptrs); bktable_destroy(&context -> inversion_result_table); HashTableIteration(context -> translocation_result_table.entry_table , bktable_free_ptrs); bktable_destroy(&context -> translocation_result_table); return 0; } int init_junction_thread_contexts(global_context_t * global_context, thread_context_t * thread_context, int task) { return 0; } void insert_big_margin_record(global_context_t * global_context , unsigned short * big_margin_record, unsigned char votes, short read_pos_start, short read_pos_end, int read_len, int is_negative) { if( global_context->config.big_margin_record_size<3) return; unsigned short read_pos_start_2 = (is_negative?read_len -read_pos_end:read_pos_start) ; unsigned short read_pos_end_2 = (is_negative?read_len -read_pos_start:read_pos_end); int xk1; for(xk1=0; xk1< global_context->config.big_margin_record_size / 3; xk1++) { if( votes >= big_margin_record[xk1*3]) break; } if(xk1< global_context->config.big_margin_record_size / 3) { int xk2; for(xk2 = global_context->config.big_margin_record_size-4; xk2 >= xk1*3; xk2--) big_margin_record[xk2 + 3] = big_margin_record[xk2]; big_margin_record[xk1*3+0] = votes; big_margin_record[xk1*3+1] = read_pos_start_2; big_margin_record[xk1*3+2] = read_pos_end_2; } } int is_PE_distance(global_context_t * global_context, unsigned int pos1, unsigned int pos2, int rlen1, int rlen2, int is_negative_R1, int is_negative_R2) { long long int dist = pos2; dist -= pos1; is_negative_R1 = (is_negative_R1>0)?1:0; is_negative_R2 = (is_negative_R2>0)?1:0; if(pos1 > pos2) dist -= rlen1; else if(pos1 < pos2) dist += rlen2; else dist += max(rlen2, rlen1); if(abs(dist) > global_context->config.maximum_pair_distance || abs(dist)config.minimum_pair_distance) return 0; if(is_negative_R1 != is_negative_R2) return 0; if(pos1 > pos2 && !is_negative_R1) return 0; if(pos1 < pos2 && is_negative_R1) return 0; return 1; } #define MAX_VOTE_TOLERANCE 1 //returns 1 if the vote number is not significantly higher than the vote numbers in the vote list. int test_small_minor_votes(global_context_t * global_context, int minor_i, int minor_j, int major_i, int major_j , gene_vote_t * votes, int read_len) { int is_small_margin_minor = 0; long long dist = votes -> pos[minor_i][minor_j]; dist -= votes -> pos[major_i][major_j]; if(abs(dist)> global_context->config.maximum_intron_length) { int iii, jjj; for(iii=0; iiiitems[iii]; jjj++) { if(iii == minor_i && jjj == minor_j) continue; // "2" is the tolerance. if(votes -> votes[minor_i][minor_j] - votes -> votes[iii][jjj] >=1) continue; int minor_coverage_start = votes -> coverage_start[minor_i][minor_j] ; int minor_coverage_end = votes -> coverage_end[minor_i][minor_j] ; int other_coverage_start = votes -> coverage_start[iii][jjj]; int other_coverage_end = votes -> coverage_end[iii][jjj]; int minor_negative = votes -> masks[minor_i][minor_j] & IS_NEGATIVE_STRAND; int other_negative = votes -> masks[iii][jjj] & IS_NEGATIVE_STRAND; if(minor_negative) { int ttt = read_len - minor_coverage_end; minor_coverage_end = read_len - minor_coverage_start; minor_coverage_start = ttt; } if(other_negative){ int ttt = read_len - other_coverage_end; other_coverage_end = read_len - other_coverage_start; other_coverage_start = ttt; } if(abs(minor_coverage_end - other_coverage_end) < 7 && abs(minor_coverage_start - other_coverage_start)<7) is_small_margin_minor = 1; if(is_small_margin_minor) break; } if(is_small_margin_minor) break; } } return is_small_margin_minor; } // function test_junction_minor returns 1 if the current anchor and current_vote[i][j] are not good mates in terms of junction reads: // for example, if the distance is too far, if the coverered region overlapped or if the two mapped parts in the read are reversely arranged (expect in fusion detection) int test_junction_minor(global_context_t * global_context, thread_context_t * thread_context, gene_vote_t * votes, int vote_i, int vote_j, int i, int j, long long int dist) { if(abs(dist)> global_context->config.maximum_intron_length) return 1; if(votes -> coverage_start[vote_i][vote_j] == votes -> coverage_start[i][j])return 2; if(votes -> coverage_end[vote_i][vote_j] == votes -> coverage_end[i][j])return 3; //SUBREADprintf( " COV_IN_READ: %d ~ %d CHRO_POS: %u ~ %u \n", votes -> coverage_start[vote_i][vote_j] , votes -> coverage_start[i][j] , votes -> pos[vote_i][vote_j] , votes -> pos[i][j] ); if(votes -> coverage_start[vote_i][vote_j] > votes -> coverage_start[i][j]) { if(votes -> pos[vote_i][vote_j] < votes -> pos[i][j])return 4; } else { if(votes -> pos[vote_i][vote_j] > votes -> pos[i][j])return 5; } return 0; } void update_top_three(global_context_t * global_context, int * top_buffer_3i, int new_value){ if(new_value > top_buffer_3i[global_context -> config.top_scores - 1]){ int x1; for(x1 = 0;x1 < global_context -> config.top_scores ; x1++){ if(new_value > top_buffer_3i[x1]){ int x2; for(x2 = global_context -> config.top_scores - 1 ; x2 > x1 ; x2 --){ top_buffer_3i[x2] = top_buffer_3i[x2-1]; } top_buffer_3i[x1] = new_value; break; }else if(new_value == top_buffer_3i[x1]) break; } } } int comb_sort_compare(void * Vcomb_buffer, int i, int j){ vote_combination_t * comb_buffer = (vote_combination_t *)Vcomb_buffer; return comb_buffer[i].score_adj - comb_buffer[j].score_adj; } void comb_sort_exchange(void * Vcomb_buffer, int i, int j){ vote_combination_t * comb_buffer = (vote_combination_t *)Vcomb_buffer; vote_combination_t tmpv; memcpy(&tmpv, comb_buffer + i, sizeof(vote_combination_t)); memcpy(comb_buffer + i, comb_buffer + j, sizeof(vote_combination_t)); memcpy(comb_buffer + j, &tmpv, sizeof(vote_combination_t)); } void comb_sort_merge(void * Vcomb_buffer, int start, int items, int items2){ vote_combination_t * comb_buffer = (vote_combination_t *)Vcomb_buffer; vote_combination_t * merge_target = malloc(sizeof(vote_combination_t) * (items + items2)); int items1_cursor = start, items2_cursor = start + items, x1; for(x1=0; x1 < items+items2; x1++){ int select_items_1 = (items1_cursor < items + start && comb_sort_compare(comb_buffer, items1_cursor, items2_cursor) <=0) || (items2_cursor == start + items + items2); if(select_items_1){ memcpy(merge_target+x1, comb_buffer+items1_cursor, sizeof(vote_combination_t)); items1_cursor++; }else{ memcpy(merge_target+x1, comb_buffer+items2_cursor, sizeof(vote_combination_t)); items2_cursor++; } } memcpy(comb_buffer + start, merge_target, (items+items2) * sizeof(vote_combination_t)); free(merge_target); } int is_better_inner(global_context_t * global_context, thread_context_t * thread_context, subjunc_result_t * junc_res, int old_intron_length, gene_vote_number_t Vote_minor, int coverage_minor_length, int intron) { if( Vote_minor > junc_res -> minor_votes || (Vote_minor ==junc_res -> minor_votes && coverage_minor_length > junc_res -> minor_coverage_end - junc_res -> minor_coverage_start) || (Vote_minor ==junc_res -> minor_votes && coverage_minor_length ==junc_res -> minor_coverage_end - junc_res -> minor_coverage_start && intron < old_intron_length)) return 1; else return 0; } #define COVERAGE_STAB_NUMBER 100 int test_fully_covered(global_context_t * global_context, gene_vote_t * vote, int read_length){ int i,j,xk1,xk2; char local_strands[COVERAGE_STAB_NUMBER]; unsigned int local_locations[COVERAGE_STAB_NUMBER]; unsigned long long local_coverage[COVERAGE_STAB_NUMBER]; int used_stabs = 0; for (i=0; iitems[i]; j++) { if(vote -> votes[i][j]>2 && used_stabs < COVERAGE_STAB_NUMBER) { int is_fresh = 1; int is_negative = (vote -> masks[i][j] & IS_NEGATIVE_STRAND)?1:0; for(xk1=0; xk1 pos[i][j]; dist -= local_locations[xk1]; if(abs(dist) < MAX_DELETION_LENGTH) { is_fresh=0; break; } } } if(is_fresh){ local_strands[used_stabs]=is_negative; local_locations[used_stabs]= vote -> pos[i][j]; local_coverage[used_stabs] = 0; used_stabs++; } } } } if(!used_stabs) return 0; for (i=0; iitems[i]; j++) { if(vote -> votes[i][j]>=1) { int is_negative = (vote -> masks[i][j] & IS_NEGATIVE_STRAND)?1:0; for(xk1=0; xk1 pos[i][j]; dist -= local_locations[xk1]; if(abs(dist) < MAX_DELETION_LENGTH) { for(xk2 = vote -> coverage_start[i][j] * 64 / read_length; xk2 <= vote -> coverage_end[i][j] * 64 / read_length; xk2++){ local_coverage[xk1] |= 1llu< 54){ return 1; } } return 0; } int is_long_del_high_quality(global_context_t * global_context, thread_context_t * thread_context, int p1_start, int p1_end, int p2_start, int p2_end, int read_len, int p1_votes, int p2_votes){ if(p1_votes < 3 || p2_votes < 3) return 0; if( min( p1_start, p2_start ) > 10 ) return 0; if( read_len - max(p1_end, p2_end) > 10 ) return 0; return 1; } #define SE_READ_IN_KNOWN_EXON_REWARD 1 void copy_vote_to_alignment_res(global_context_t * global_context, thread_context_t * thread_context, mapping_result_t * align_res, subjunc_result_t * junc_res, gene_vote_t * current_vote, int vote_i, int vote_j, int curr_read_len, char * read_name, char * curr_read_text, int used_subreads_in_vote, int noninformative_subreads_in_vote, subread_read_number_t pair_number, int is_second_read, int * is_fully_covered) { int vv = current_vote -> votes[vote_i][vote_j]; //#warning "======== REMOVCE ==0 FROM NEXT ===========" if(global_context->config.scRNA_input_mode && !global_context -> input_reads.is_paired_end_reads) vv += SE_READ_IN_KNOWN_EXON_REWARD *is_pos_in_annotated_exon_regions(global_context, current_vote -> pos[vote_i][vote_j]); align_res -> selected_position = current_vote -> pos[vote_i][vote_j]; align_res -> selected_votes = vv; align_res -> indels_in_confident_coverage = indel_recorder_copy(align_res -> selected_indel_record, current_vote -> indel_recorder[vote_i][vote_j]); align_res -> confident_coverage_end = current_vote -> coverage_end[vote_i][vote_j]; align_res -> confident_coverage_start = current_vote -> coverage_start[vote_i][vote_j]; align_res -> result_flags = (current_vote -> masks[vote_i][vote_j] & IS_NEGATIVE_STRAND)?(CORE_IS_NEGATIVE_STRAND):0; align_res -> used_subreads_in_vote = used_subreads_in_vote; align_res -> noninformative_subreads_in_vote = noninformative_subreads_in_vote; align_res -> is_fully_covered = *is_fully_covered ; if(global_context -> config.do_breakpoint_detection) { int i,j; // iterate all the anchors we have found in step 1: for (i=0; iitems[i]; j++) { if(i == vote_i && j == vote_j) continue; if(align_res -> selected_votes < current_vote -> votes[i][j]) continue; // major half must be the anchor long long int dist = current_vote -> pos[vote_i][vote_j]; dist -= current_vote -> pos[i][j]; int is_strand_jumpped = (current_vote -> masks[vote_i][vote_j] & IS_NEGATIVE_STRAND)!=(current_vote -> masks[i][j] & IS_NEGATIVE_STRAND); if((global_context-> config.do_fusion_detection || global_context-> config.do_long_del_detection) && (*is_fully_covered) && (dist > MAX_DELETION_LENGTH || is_strand_jumpped)) continue; if((global_context-> config.do_fusion_detection || global_context-> config.do_long_del_detection)){ // function test_small_minor_votes returns 1 if the vote number is not significantly // higher than the vote numbers in the vote list. //#warning "SUBREAD_151 =========== THE TWO LINES SHOULD BE UNCOMMENTED IN RELEASED VERSION ==== WE COMMENT IT FOR A BETTER FUSION SENSITIVITY BUT ONLY FOR TEST ===================" if(1){ int small_minor_bigmargin = test_small_minor_votes(global_context , i, j, vote_i, vote_j, current_vote, curr_read_len); if(small_minor_bigmargin) continue; } }else{ // function test_junction_minor returns 1 if the current anchor and current_vote[i][j] // are not good mates in terms of junction reads: // // for example, if the distance is too far, if the coverered region overlapped or // if the covered region has a wrong arrangement to their relative positions. int test_minor_res = test_junction_minor(global_context, thread_context, current_vote, vote_i, vote_j, i, j, dist); //#warning " ============ DEBUG 1 ==================== " if(0 && FIXLENstrcmp("R002403247", read_name) == 0) { char posout2[100]; char posout1[100]; absoffset_to_posstr(global_context, current_vote -> pos[vote_i][vote_j], posout1); absoffset_to_posstr(global_context, current_vote -> pos[i][j], posout2); SUBREADprintf("SMALL_MARGIN=%d at %s ~ %s\n", test_minor_res, posout1, posout2); } // SUBREADprintf("TMR=%d (V=%d)\n", test_minor_res, current_vote -> votes[i][j]); if(test_minor_res)continue; } int is_better = is_better_inner(global_context, thread_context, junc_res, abs32uint(current_vote -> pos[vote_i][vote_j] - junc_res -> minor_position), current_vote -> votes[i][j], current_vote -> coverage_end[i][j] - current_vote -> coverage_start[i][j], abs32uint(current_vote -> pos[vote_i][vote_j] - current_vote -> pos[i][j])); int replace_minor = 0, minor_indel_offset = 0, inserted_bases = 0, is_GT_AG_donors = 0, is_donor_found_or_annotation = 0, final_split_point = 0, major_indels = 0, small_side_increasing_coordinate = 0, large_side_increasing_coordinate = 0; if(0 && FIXLENstrcmp("R002403247", read_name) == 0) { char posout[100]; absoffset_to_posstr(global_context, current_vote -> pos[i][j], posout); SUBREADprintf("IBT=%d (V=%d , OV=%d) at %s\n", is_better, current_vote -> votes[i][j], junc_res -> minor_votes, posout); SUBREADprintf("IBT OLD_INTRON=%d, INTRON=%d\n", abs32uint(current_vote -> pos[vote_i][vote_j] - junc_res -> minor_position), abs32uint(current_vote -> pos[vote_i][vote_j] - current_vote -> pos[i][j]) ); } if(is_better){ // Determine the splicing point of the fusion or the junction // If the splicing point is determined, then set replace_minor = 1 if(is_strand_jumpped){ if(!global_context -> config.do_fusion_detection) continue; int minor_cover_end_as_reversed = (current_vote -> masks[i][j] & IS_NEGATIVE_STRAND)? current_vote -> coverage_end[i][j]:(curr_read_len - current_vote -> coverage_start[i][j]); int minor_cover_start_as_reversed = (current_vote -> masks[i][j] & IS_NEGATIVE_STRAND)? current_vote -> coverage_start[i][j]:(curr_read_len - current_vote -> coverage_end[i][j]); int main_cover_end_as_reversed = (current_vote -> masks[vote_i][vote_j] & IS_NEGATIVE_STRAND)?current_vote -> coverage_end[vote_i][vote_j]:(curr_read_len - current_vote -> coverage_start[vote_i][vote_j]); int main_cover_start_as_reversed = (current_vote -> masks[vote_i][vote_j] & IS_NEGATIVE_STRAND)?current_vote -> coverage_start[vote_i][vote_j]:(curr_read_len - current_vote -> coverage_end[vote_i][vote_j]); int overlapped ; if(main_cover_start_as_reversed > minor_cover_start_as_reversed) overlapped = minor_cover_end_as_reversed - main_cover_start_as_reversed; else overlapped = main_cover_end_as_reversed - minor_cover_start_as_reversed; if(overlapped > 14) continue; int guess_start_as_reversed = (main_cover_start_as_reversed > minor_cover_start_as_reversed)? (minor_cover_end_as_reversed - 15): (main_cover_end_as_reversed - 15); int guess_end_as_reversed = (main_cover_start_as_reversed > minor_cover_start_as_reversed)? (main_cover_start_as_reversed + 15): (minor_cover_start_as_reversed + 15); int is_small_half_negative = 0 != ((current_vote -> pos[vote_i][vote_j]>current_vote -> pos[i][j]?current_vote -> masks[i][j]:current_vote -> masks[vote_i][vote_j])&IS_NEGATIVE_STRAND); int is_large_half_negative = !is_small_half_negative; int is_small_half_on_left_as_reversed = (main_cover_start_as_reversed > minor_cover_start_as_reversed) + (current_vote -> pos[vote_i][vote_j]> current_vote -> pos[i][j]) !=1; // small half on left(as reversed) === small half on right (as 'forward' form of the read, i.e., the raw FASTQ form for read_A and reversed FASTQ form for read_B) unsigned int small_half_abs_offset = min(current_vote -> pos[i][j], current_vote -> pos[vote_i][vote_j]); unsigned int large_half_abs_offset = max(current_vote -> pos[i][j], current_vote -> pos[vote_i][vote_j]); // curr_read_text is the 'reversed' form of the read. I.e., the reversed FASTQ form for read_A and the raw FASTQ form for read_B. replace_minor = donor_jumped_score(global_context, thread_context, small_half_abs_offset, large_half_abs_offset, max(0, guess_start_as_reversed) , min( guess_end_as_reversed, curr_read_len), curr_read_text, curr_read_len, is_small_half_negative, is_large_half_negative, is_small_half_on_left_as_reversed, & final_split_point, & is_GT_AG_donors, & is_donor_found_or_annotation, &small_side_increasing_coordinate, &large_side_increasing_coordinate); if( 0 && 1018082 == pair_number) { print_votes(current_vote, global_context -> config.index_prefix); SUBREADprintf("JUMP_001018082 NORMAL=%d SMALL_NEG=%d LARGE_NEG=%d, SMALL_ABS=%u LARGE_ABS=%u, REPLACE=%d, INCS=%d %d\n" , is_small_half_on_left_as_reversed, is_small_half_negative, is_large_half_negative, small_half_abs_offset, large_half_abs_offset, replace_minor, small_side_increasing_coordinate, large_side_increasing_coordinate); } // Now "final_split_point" is the read offset on the 'reversed' form of the read. It needs to be changed to (read_len - final_split_point) if the major half is on negative strand. if(replace_minor>0) replace_minor += current_vote -> votes[i][j] * 100000; } else { int overlapped ; if(current_vote -> coverage_start[vote_i][vote_j] > current_vote -> coverage_start[i][j]) overlapped = current_vote -> coverage_end[i][j] - current_vote -> coverage_start[vote_i][vote_j]; else overlapped = current_vote -> coverage_end[vote_i][vote_j] - current_vote -> coverage_start[i][j]; if(0 && FIXLENstrcmp("R000404427", read_name) == 0) { SUBREADprintf("OVL=%d, DIST=%u\n", overlapped, (unsigned int)abs(dist)); } if(overlapped > 14) continue; if(abs(dist)<6) continue; int guess_start = (current_vote -> coverage_start[vote_i][vote_j] > current_vote -> coverage_start[i][j])? (current_vote -> coverage_end[i][j] - 8): (current_vote -> coverage_end[vote_i][vote_j] - 8); int guess_end = (current_vote -> coverage_start[vote_i][vote_j] < current_vote -> coverage_start[i][j])? (current_vote -> coverage_start[i][j] + 8): (current_vote -> coverage_start[vote_i][vote_j] + 8); if((global_context -> config.do_fusion_detection || global_context -> config.do_long_del_detection) && !(current_vote -> masks[vote_i][vote_j] & IS_NEGATIVE_STRAND)) // if for fusion, the current read must have been reversed. // hence, it is now changed to "main half" view. reverse_read(curr_read_text, curr_read_len, global_context -> config.space_type); int left_indel_offset=0, right_indel_offset=0; int kx2; int normally_arranged = 1!=(current_vote -> coverage_start[vote_i][vote_j] > current_vote -> coverage_start[i][j]) + (current_vote -> pos[vote_i][vote_j] > current_vote -> pos[i][j]); if(curr_read_len > EXON_LONG_READ_LENGTH){ int kx1; gene_vote_number_t * indel_recorder = current_vote -> indel_recorder[vote_i][vote_j]; for(kx1=0; kx1 indel_recorder[i][j][kx2*3]) break; minor_indel_offset += (current_vote -> indel_recorder[i][j][kx2*3+2]); } if(current_vote -> pos[vote_i][vote_j] < current_vote -> pos[i][j]) { left_indel_offset=major_indels; right_indel_offset=minor_indel_offset; } else { right_indel_offset=major_indels; left_indel_offset=minor_indel_offset; } // the section having a smaller coordinate will have indel_offset !=0 // the section having a larger coordiname MUST HAVE indel_offset == 0 right_indel_offset=0; } if(is_long_del_high_quality( global_context, thread_context, current_vote -> coverage_start[i][j], current_vote -> coverage_end[i][j], current_vote -> coverage_start[vote_i][vote_j], current_vote -> coverage_end[vote_i][vote_j], curr_read_len, current_vote -> votes[i][j], current_vote -> votes[vote_i][vote_j])|| ! global_context -> config.do_long_del_detection) replace_minor = donor_score(global_context, thread_context, min(current_vote -> pos[vote_i][vote_j], current_vote -> pos[i][j]),max(current_vote -> pos[vote_i][vote_j] , current_vote -> pos[i][j]), left_indel_offset, right_indel_offset, normally_arranged, max(0, guess_start), min( guess_end, curr_read_len), curr_read_text, curr_read_len, & final_split_point, & is_GT_AG_donors, & is_donor_found_or_annotation, & inserted_bases, &small_side_increasing_coordinate, &large_side_increasing_coordinate, read_name); else replace_minor = 0; // Now "final_split_point" is the read offset on the 'reversed' form of the read (I.e., the reversed FASTQ form for read_A and the raw FASTQ form for read_B.) if do_fusion_detection AND if the main half is on negative strand. // However, because the final_split_point is ALWAYS on the form where the major half can be mapped, final_split_point will never be changed. if(replace_minor>0) replace_minor += current_vote -> votes[i][j] * 100000; if(0 && ( FIXLENstrcmp("R006232475", read_name) == 0 ) ) SUBREADprintf("NOJUMP_DONORs=%d LOC=%u\n", replace_minor , current_vote -> pos[i][j]); if((global_context -> config.do_fusion_detection || global_context -> config.do_long_del_detection) && !(current_vote -> masks[vote_i][vote_j] & IS_NEGATIVE_STRAND)) // changed back. reverse_read(curr_read_text, curr_read_len, global_context -> config.space_type); } } if(0 && ( FIXLENstrcmp("R006232475", read_name) == 0 ) ) { char posout[100]; absoffset_to_posstr(global_context, current_vote -> pos[i][j], posout); SUBREADprintf("TEST MINOR: POS=%s, REPLACE=%d\n", posout, replace_minor); } if(replace_minor){// && (replace_minor > current_piece_minor_score)){ junc_res -> minor_position = current_vote -> pos[i][j]; junc_res -> minor_votes = current_vote -> votes[i][j]; junc_res -> minor_coverage_start = current_vote -> coverage_start[i][j]; junc_res -> minor_coverage_end = current_vote -> coverage_end [i][j]; junc_res -> double_indel_offset = (minor_indel_offset & 0xf)|((major_indels & 0xf)<<4); junc_res -> split_point = final_split_point; if(0 && 1018082 == pair_number) { SUBREADprintf("REPLACED: LOC %u, INCS=%d %d\n", junc_res -> minor_position, small_side_increasing_coordinate, large_side_increasing_coordinate); } junc_res -> small_side_increasing_coordinate = small_side_increasing_coordinate; junc_res -> large_side_increasing_coordinate = large_side_increasing_coordinate; junc_res -> indel_at_junction = inserted_bases; align_res -> result_flags &=~0x3; if( (!is_donor_found_or_annotation) || is_GT_AG_donors > 2) align_res -> result_flags |= 3; else align_res -> result_flags = is_GT_AG_donors? (align_res -> result_flags|CORE_IS_GT_AG_DONORS):(align_res -> result_flags &~CORE_IS_GT_AG_DONORS); align_res -> result_flags = is_strand_jumpped? (align_res -> result_flags|CORE_IS_STRAND_JUMPED):(align_res -> result_flags &~CORE_IS_STRAND_JUMPED); } } } if(0 && memcmp("V0112_0155:7:1101:1173:2204", read_name, 26) == 0) { char leftpos[100], rightpos[100]; absoffset_to_posstr(global_context, current_vote -> pos[vote_i][vote_j] , leftpos); absoffset_to_posstr(global_context, junc_res -> minor_position, rightpos); SUBREADprintf("READ=%s, MAJOR=%s, MINOR=%s\n", read_name, leftpos, rightpos); } // This block runs after the minor half of this anchor is fully determined. // If the minor half is a fusion and there is a strand jump, move the minor half coverage to the major half strand. if(align_res -> result_flags & CORE_IS_STRAND_JUMPED) { // If "is_strand_jumped" is true, all coordinates so far are on the best voted strands (must be differnet strands, namely they're very likely to be overlapped). int tmpv = junc_res -> minor_coverage_start; junc_res -> minor_coverage_start = curr_read_len - junc_res -> minor_coverage_end; junc_res -> minor_coverage_end = curr_read_len - tmpv; // Split_point is now the "negative strand read" view. It has to be changed to "main piece" view junc_res -> split_point = (align_res -> result_flags & CORE_IS_NEGATIVE_STRAND)? junc_res -> split_point : (curr_read_len - junc_res -> split_point); } } } void simple_PE_and_same_chro(global_context_t * global_context , simple_mapping_t * r1, simple_mapping_t * r2 , int * is_PE_distance, int * is_same_chromosome , int rlen1, int rlen2){ test_PE_and_same_chro(global_context, r1 -> mapping_position, r2 -> mapping_position, is_PE_distance, is_same_chromosome, rlen1, rlen2); } #define MAX_CLUSTER_ELEMENTS 7 struct cluster_element{ unsigned int initial_position; char cluster_members; char from_second_read[MAX_CLUSTER_ELEMENTS]; int i_list[MAX_CLUSTER_ELEMENTS], j_list[MAX_CLUSTER_ELEMENTS]; }; int add_cluster_member(struct cluster_element * cl , int i, int j, int is_second_read){ if(cl->cluster_members < MAX_CLUSTER_ELEMENTS){ cl->i_list[(int)cl->cluster_members] = i; cl->j_list[(int)cl->cluster_members] = j; cl->from_second_read[(int)cl->cluster_members] = is_second_read; cl->cluster_members++; } return cl->cluster_members; } int is_same_cluster( global_context_t * global_context, struct cluster_element * cl , unsigned int pos){ long long int test_pos = pos; test_pos -= cl -> initial_position; if(abs(test_pos) < global_context -> config.maximum_intron_length) return 1; return 0; } int process_voting_junction_PE_topK(global_context_t * global_context, thread_context_t * thread_context, subread_read_number_t pair_number, gene_vote_t * vote_1, gene_vote_t * vote_2, char * read_name_1, char * read_name_2, char * read_text_1, char * read_text_2, int read_len_1, int read_len_2, int is_negative_strand, gene_vote_number_t v1_all_subreads, gene_vote_number_t v2_all_subreads); int align_cluster(global_context_t * global_context, thread_context_t * thread_context, struct cluster_element * this_cluster, char * read_name_1, char * read_name_2, char * read_text_1, char * read_text_2, int read_len_1, int read_len_2, int is_negative_strand, gene_vote_t * vote_1, gene_vote_t * vote_2, int * this_score, int * ii_path, int * jj_path, int * masks, int * path_len, int * R1R2_mapped); void simple_copy_vote_to_result( mapping_result_t * align_res, gene_vote_t * current_vote, int vote_i, int vote_j, int used_subreads_in_vote, int noninformative_subreads_in_vote, int score){ align_res -> selected_position = current_vote -> pos[vote_i][vote_j]; align_res -> selected_votes = score; align_res -> indels_in_confident_coverage = indel_recorder_copy(align_res -> selected_indel_record, current_vote -> indel_recorder[vote_i][vote_j]); align_res -> confident_coverage_end = current_vote -> coverage_end[vote_i][vote_j]; align_res -> confident_coverage_start = current_vote -> coverage_start[vote_i][vote_j]; align_res -> result_flags = (current_vote -> masks[vote_i][vote_j] & IS_NEGATIVE_STRAND)?(CORE_IS_NEGATIVE_STRAND):0; align_res -> used_subreads_in_vote = used_subreads_in_vote; align_res -> noninformative_subreads_in_vote = noninformative_subreads_in_vote; } int process_voting_junction_PE_juncs( global_context_t * global_context, thread_context_t * thread_context, subread_read_number_t pair_number, gene_vote_t * vote_1, gene_vote_t * vote_2, char * read_name_1, char * read_name_2, char * read_text_1, char * read_text_2, int read_len_1, int read_len_2, int is_negative_strand, gene_vote_number_t v1_all_subreads, gene_vote_number_t v2_all_subreads ){ int current_cluster_number = 0,max_clusters = global_context -> config.max_vote_simples * 2; int i,j, is_second_read, tested_votes, x1; struct cluster_element * cluster_buffer = malloc(max_clusters * sizeof(struct cluster_element)); int max_cluster_size_r1 = 0, max_cluster_size_r2 = 0; for( tested_votes = max(vote_1 -> max_vote, vote_2 -> max_vote); tested_votes > 0; tested_votes--) { for(is_second_read = 0 ; is_second_read < 1 + global_context -> input_reads.is_paired_end_reads; is_second_read ++) { gene_vote_t * current_vote = is_second_read?vote_2:vote_1; int * max_cluster_size = is_second_read?(&max_cluster_size_r2):(&max_cluster_size_r1); for (i=0; iitems[i]; j++) { if(current_vote->votes[i][j]!=tested_votes) continue; int is_added = 0; for(x1 = 0; x1 < current_cluster_number ; x1++){ if(is_same_cluster(global_context, cluster_buffer+x1, current_vote->pos[i][j])){ int new_size =add_cluster_member(cluster_buffer+x1, i, j, is_second_read); (*max_cluster_size) = max(*max_cluster_size, new_size); is_added = 1; } if(is_added)break; } if(current_cluster_number < max_clusters && !is_added){ cluster_buffer[current_cluster_number].initial_position = current_vote->pos[i][j]; cluster_buffer[current_cluster_number].i_list[0] = i; cluster_buffer[current_cluster_number].j_list[0] = j; cluster_buffer[current_cluster_number].from_second_read[0] = is_second_read; cluster_buffer[current_cluster_number].cluster_members = 1; current_cluster_number++; } } } } } if(1 || max_cluster_size_r1 == 3 || max_cluster_size_r2 == 3 ) // if there are 3-section clusters then parse it, else go to the regular procedure. There is a upper-limit to the sections to avoid fragile mapping. { for(x1 = 0 ; x1 < current_cluster_number ; x1++){ int this_score = -1, path_len = -1, R1R2_mapped = 0; int this_ii_path[ MAX_CLUSTER_ELEMENTS ], this_jj_path[ MAX_CLUSTER_ELEMENTS ], this_masks [ MAX_CLUSTER_ELEMENTS ]; align_cluster(global_context, thread_context, cluster_buffer + x1, read_name_1, read_name_2, read_text_1, read_text_2, read_len_1, read_len_2, is_negative_strand, vote_1, vote_2, &this_score, this_ii_path, this_jj_path, this_masks, &path_len, &R1R2_mapped); if(0 && FIXLENstrcmp("R00000003493", read_name_1)==0) SUBREADprintf("REAE_TEST : R12MAP=%d, PATHLEN=%d, SCORE=%d\n", R1R2_mapped, path_len, this_score); if(this_score > 0){ if(( R1R2_mapped & CLUSTER_ALIGNMENT_DONOR_R1_MAPPED) && ( R1R2_mapped & CLUSTER_ALIGNMENT_DONOR_R2_MAPPED)){ for(i = 0; i < global_context -> config.multi_best_reads; i++) { mapping_result_t * old_result_R1 = _global_retrieve_alignment_ptr(global_context, pair_number, 0, i); mapping_result_t * old_result_R2 = _global_retrieve_alignment_ptr(global_context, pair_number, 1, i); short old_score_R1 = old_result_R1 -> selected_votes; short old_score_R2 = old_result_R2 -> selected_votes; if( old_score_R1 < this_score || old_score_R2 < this_score ){ for(j = global_context -> config.multi_best_reads - 2; j>=i; j--){ mapping_result_t * shifted_result_R1 = _global_retrieve_alignment_ptr(global_context, pair_number, 0, j); mapping_result_t * shifted_result_R2 = _global_retrieve_alignment_ptr(global_context, pair_number, 1, j); mapping_result_t * target_result_R1 = _global_retrieve_alignment_ptr(global_context, pair_number, 0, j+1); mapping_result_t * target_result_R2 = _global_retrieve_alignment_ptr(global_context, pair_number, 1, j+1); memcpy( target_result_R1, shifted_result_R1 , sizeof(mapping_result_t)); memcpy( target_result_R2, shifted_result_R2 , sizeof(mapping_result_t) ); } int best_R1_i = -1, best_R1_j = - 1 , highest_vote_R1 = -1, highest_vote_R2 = -1, best_R2_i = -2, best_R2_j = -2; for(j = 0; j < path_len ; j++){ if( this_masks[j] & CLUSTER_ALIGNMENT_DONOR_R1_MAPPED ){ if( highest_vote_R1 < vote_1 -> votes [ this_ii_path [j] ] [ this_jj_path [j] ] ){ best_R1_i = this_ii_path [j] ; best_R1_j = this_jj_path [j] ; highest_vote_R1 = vote_1 -> votes [ this_ii_path [j] ] [ this_jj_path [j] ] ; } }else{ if( highest_vote_R2 < vote_2 -> votes [ this_ii_path [j] ] [ this_jj_path [j] ] ){ best_R2_i = this_ii_path [j] ; best_R2_j = this_jj_path [j] ; highest_vote_R2 = vote_2 -> votes [ this_ii_path [j] ] [ this_jj_path [j] ] ; } } //SUBREADprintf("MASK=%d\n", this_masks[j]); } //SUBREADprintf("IJ: R1=%d,%d R2=%d,%d MASK=%d\n", best_R1_i,best_R1_j,best_R2_i,best_R2_j); simple_copy_vote_to_result( old_result_R1, vote_1, best_R1_i, best_R1_j , v1_all_subreads, vote_1 -> noninformative_subreads, this_score); simple_copy_vote_to_result( old_result_R2, vote_2, best_R2_i, best_R2_j , v2_all_subreads, vote_2 -> noninformative_subreads, this_score); break; } } } else if( R1R2_mapped & ( CLUSTER_ALIGNMENT_DONOR_R1_MAPPED | CLUSTER_ALIGNMENT_DONOR_R2_MAPPED ) ) { int is_R2_mapped = ( R1R2_mapped & CLUSTER_ALIGNMENT_DONOR_R2_MAPPED)?1:0; for(i = 0; i < global_context -> config.multi_best_reads; i++) { mapping_result_t * old_result_R = _global_retrieve_alignment_ptr(global_context, pair_number, is_R2_mapped, i); short old_score_R = old_result_R -> selected_votes; if( old_score_R < this_score ){ for(j = global_context -> config.multi_best_reads - 2; j>=i; j--){ mapping_result_t * shifted_result_R = _global_retrieve_alignment_ptr(global_context, pair_number, is_R2_mapped, j); mapping_result_t * target_result_R = _global_retrieve_alignment_ptr(global_context, pair_number, is_R2_mapped, j+1); memcpy( target_result_R, shifted_result_R , sizeof(mapping_result_t)); } int best_R_i = -1, best_R_j = - 1 , highest_vote_R = -1; gene_vote_t * this_vote = is_R2_mapped?vote_2:vote_1; for(j = 0; j < path_len ; j++){ if( highest_vote_R < this_vote -> votes [ this_ii_path [j] ] [ this_jj_path [j] ] ){ best_R_i = this_ii_path [j] ; best_R_j = this_jj_path [j] ; highest_vote_R = this_vote -> votes [ this_ii_path [j] ] [ this_jj_path [j] ] ; } //SUBREADprintf("MASK=%d\n", this_masks[j]); } //SUBREADprintf("IJ: R1=%d,%d R2=%d,%d MASK=%d\n", best_R1_i,best_R1_j,best_R2_i,best_R2_j); simple_copy_vote_to_result( old_result_R, this_vote, best_R_i, best_R_j , v1_all_subreads, this_vote -> noninformative_subreads, this_score); break; } } } } /*if(highest_score > 0){ if(this_score >0){ if(this_score > highest_score){ highest_score = this_score; highest_occurance = 1; memcpy(best_ii_path, this_ii_path, sizeof(int)*path_len); memcpy(best_jj_path, this_jj_path, sizeof(int)*path_len); best_path_len = path_len; }else if(this_score == highest_score) highest_occurance ++; } }*/ } // call new junctions from the path // then put the alignment into the best list. }else{ return process_voting_junction_PE_topK(global_context, thread_context, pair_number, vote_1, vote_2, read_name_1, read_name_2, read_text_1, read_text_2, read_len_1, read_len_2, is_negative_strand, v1_all_subreads, v2_all_subreads); } free(cluster_buffer); return 0; } int compare_cluster_elements (void * arr, int l, int r){ int * ii_array = ((void **)arr)[0]; int * jj_array = ((void **)arr)[1]; int * second_vote = ((void **)arr)[2]; if(second_vote[l] != second_vote[r]) return second_vote[l] - second_vote[r]; gene_vote_t * vote_1 = ((void **)arr)[3]; gene_vote_t * vote_2 = ((void **)arr)[4]; gene_vote_t * this_vote_L = second_vote[l]?vote_2:vote_1; gene_vote_t * this_vote_R = second_vote[r]?vote_2:vote_1; return this_vote_L->coverage_start[ii_array[l]][jj_array[l]] - this_vote_R -> coverage_start[ii_array[r]][jj_array[r]]; } void exchange_cluster_elements (void * arr, int l, int r){ int * ii_array = ((void **)arr)[0]; int * jj_array = ((void **)arr)[1]; int * second_vote = ((void **)arr)[2]; int ti; ti = ii_array[l]; ii_array[l] = ii_array[r]; ii_array[r]=ti; ti = jj_array[l]; jj_array[l] = jj_array[r]; jj_array[r]=ti; ti = second_vote[l]; second_vote[l] = second_vote[r]; second_vote[r]=ti; } #define NEW_EXTEND_SCAN_INTRON_LONGEST 6000 #define NEW_EXTEND_SCAN_EXON_SHORTEST 14 int find_path(global_context_t * global_context, thread_context_t * thread_context, int start_element_i, int target_element_i, int * ii_array, int * jj_array, int * is_second_vote_array, gene_vote_t * vote_1, gene_vote_t * vote_2, char * read_name_1, char * read_name_2, char * read_text_1, char * read_text_2, int read_len_1, int read_len_2, int is_negative_strand, int * this_mask , int * exon_last_base); int find_donor_receptor(global_context_t * global_context, thread_context_t * thread_context, char * rname, char * rtext, int rlen, int start_coverage, int end_coverage, unsigned int start_pos, unsigned int end_pos, int indels_in_start, int v1, int v2, int * misma_bases, int * matched_bases, int * is_negative_donor); int extend_uncovered_region_juncs(global_context_t * global_context, thread_context_t * thread_context, char * rname, char * rtext, int rlen, int scan_to_tail, unsigned int scan_chro_start, int scan_read_start, unsigned short expect_donor, int * mismatched_bases_after_start, int * first_exon_last_base, unsigned int * first_exon_first_base, int * ret_mismatched_bases, int * is_negative_donor){ if( scan_to_tail ) assert( scan_read_start < rlen - NEW_EXTEND_SCAN_EXON_SHORTEST ); else assert( scan_read_start >= NEW_EXTEND_SCAN_EXON_SHORTEST); gene_value_index_t * value_index = thread_context?thread_context->current_value_index:global_context->current_value_index; int x1, best_best_score = -1, best_best_occurance = 0; unsigned long long matching_target = 0, rolling_bases = 0; for(x1 = 0 ; x1 < 8 ; x1++){ int nch = scan_to_tail? rtext[ rlen - 2 - x1 ] : rtext[ 10 - x1 ] ; matching_target = ( matching_target << 8 ) | nch; } if(0 && FIXLENstrcmp("V0112_0155:7:1101:13762:2349#ACTTGA", rname ) == 0 ) SUBREADprintf("TAG=%016llX\n",matching_target); for(x1 = 0; x1 < NEW_EXTEND_SCAN_INTRON_LONGEST ; x1++){ int best_last_exon_base = -1, matched_in_the_uncovered_gap = -1, mismatched_bases = -1, extended_should_mismatch = -1; unsigned int scan_cursor = scan_chro_start ; if(scan_to_tail) scan_cursor+=x1;else scan_cursor-=x1; unsigned long long nch = gvindex_get( value_index, scan_cursor ); if(scan_to_tail) rolling_bases = (rolling_bases >> 8) | nch << 56; else rolling_bases = nch | ( rolling_bases << 8 ); //SUBREADprintf("MATCH:%016llX,%016llX\n", rolling_bases, matching_target); if(rolling_bases == matching_target){ //SUBREADprintf("PNTT-M\n"); if(scan_to_tail) { best_last_exon_base = find_donor_receptor(global_context, thread_context, rname, rtext, rlen, scan_read_start, rlen - 2 - 7, scan_chro_start, scan_cursor - (rlen - 2) , 0, 0,0, &mismatched_bases , &matched_in_the_uncovered_gap, is_negative_donor); if(best_last_exon_base>0) extended_should_mismatch = match_chro( rtext + best_last_exon_base , value_index, scan_chro_start + best_last_exon_base, rlen - best_last_exon_base, 0, global_context->config.space_type); } else { best_last_exon_base = find_donor_receptor(global_context, thread_context, rname, rtext, rlen, 10, scan_read_start, scan_cursor - 3 , scan_chro_start, 0, 0,0, &mismatched_bases , &matched_in_the_uncovered_gap,is_negative_donor); if(best_last_exon_base>0) extended_should_mismatch = match_chro( rtext, value_index, scan_chro_start, best_last_exon_base , 0, global_context->config.space_type); } } if(best_last_exon_base >0 && extended_should_mismatch < ( scan_to_tail?( rlen - best_last_exon_base - 4 ):( best_last_exon_base - 4 )) && mismatched_bases < 2 ){ int this_score; if(scan_to_tail) this_score = rlen - scan_read_start - mismatched_bases; else this_score = scan_read_start - mismatched_bases; if(best_best_score < this_score){ best_best_score = this_score; (*mismatched_bases_after_start) = mismatched_bases; (*first_exon_last_base) = best_last_exon_base; (*first_exon_first_base) = scan_to_tail?( scan_cursor - (rlen - 2) ) : ( scan_cursor - 3 ); (*ret_mismatched_bases) = mismatched_bases; best_best_occurance = 1; }else if( best_best_score == this_score ) best_best_occurance ++; } if(0 && (!scan_to_tail) && best_last_exon_base >0 && extended_should_mismatch < best_last_exon_base - 4 && mismatched_bases < 2){ char out1pos[100], out2pos[100]; absoffset_to_posstr(global_context, scan_chro_start + best_last_exon_base, out1pos); absoffset_to_posstr(global_context, scan_cursor - (rlen - 2) + best_last_exon_base, out2pos); SUBREADprintf("LIMMISMA: %d < %d - 4\t\tfor %s\n" , extended_should_mismatch, best_last_exon_base ,rname); SUBREADprintf("HEAD MATCH: %s - %s : MM=%d ; SPLIT=%d\t%s\n",out1pos, out2pos, mismatched_bases, best_last_exon_base, rname); SUBREADprintf("R =%s\nS1=", rtext); int x2; for(x2 = 0; x2 < rlen; x2++){ if(x2 > best_last_exon_base + 16) SUBREADprintf(" "); else{ int nch = gvindex_get( value_index, scan_cursor - 3 + x2 ); SUBREADprintf("%c", nch); } } SUBREADprintf("\nS2="); for(x2 = 0; x2 < rlen; x2++){ if(x2 > best_last_exon_base + 16 ) SUBREADprintf(" "); else{ int nch = gvindex_get( value_index, scan_chro_start +x2); SUBREADprintf("%c", nch); } } SUBREADprintf("\n "); for(x2 = 0; x2 < rlen; x2++){ if(x2 < best_last_exon_base ) SUBREADprintf(" "); else if( x2 > best_last_exon_base + 1 ) SUBREADprintf(" "); else SUBREADprintf("|"); } SUBREADprintf("\n\n"); } if(0 && scan_to_tail && best_last_exon_base >0 && extended_should_mismatch < rlen - best_last_exon_base - 4 && mismatched_bases < 2){ SUBREADprintf("LIMMISMA: %d < %d - 4\t\tfor %s\n" , extended_should_mismatch, (rlen - best_last_exon_base ),rname); char out1pos[100], out2pos[100]; absoffset_to_posstr(global_context, scan_chro_start + best_last_exon_base, out1pos); absoffset_to_posstr(global_context, scan_cursor - (rlen - 2) + best_last_exon_base, out2pos); SUBREADprintf("TAIL MATCH: %s - %s : MM=%d ; SPLIT=%d\t%s\n",out1pos, out2pos, mismatched_bases, best_last_exon_base, rname); SUBREADprintf("R =%s\nS1=", rtext); int x2; for(x2 = 0; x2 < rlen; x2++){ if(x2 < scan_read_start - 16) SUBREADprintf(" "); else{ int nch = gvindex_get( value_index, x2 + scan_chro_start); SUBREADprintf("%c", nch); } } SUBREADprintf("\nS2="); for(x2 = 0; x2 < rlen; x2++){ if(x2 < best_last_exon_base - 16 ) SUBREADprintf(" "); else{ int nch = gvindex_get( value_index, scan_cursor - (rlen - 2)+x2); SUBREADprintf("%c", nch); } } SUBREADprintf("\n "); for(x2 = 0; x2 < rlen; x2++){ if(x2 < best_last_exon_base ) SUBREADprintf(" "); else if( x2 > best_last_exon_base + 1 ) SUBREADprintf(" "); else SUBREADprintf("|"); } SUBREADprintf("\n\n"); } } if(0&&best_best_occurance>0 && best_best_score>0) SUBREADprintf("OCCR=%d : SCR=%d\n", best_best_occurance, best_best_score); if(best_best_occurance == 1) return best_best_score; return -1; } void simple_add_junction( global_context_t * global_context, thread_context_t * thread_context, unsigned int left_edge_wanted, unsigned int right_edge_wanted, int indel_at_junction, int is_negative_donors ){ char * chro_name_left, *chro_name_right; int chro_pos_left,chro_pos_right; locate_gene_position( left_edge_wanted , &global_context -> chromosome_table, &chro_name_left, &chro_pos_left); locate_gene_position( right_edge_wanted , &global_context -> chromosome_table, &chro_name_right, &chro_pos_right); if((!( global_context-> config.do_fusion_detection || global_context-> config.do_long_del_detection) ) && chro_name_right!=chro_name_left) return; //insert event HashTable * event_table = NULL; chromosome_event_t * event_space = NULL; if(thread_context) { event_table = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> event_entry_table; event_space = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; } else { event_table = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_entry_table; event_space = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; } chromosome_event_t * search_return [MAX_EVENT_ENTRIES_PER_SITE]; chromosome_event_t * found = NULL; int found_events = search_event(global_context, event_table, event_space, left_edge_wanted , EVENT_SEARCH_BY_SMALL_SIDE, CHRO_EVENT_TYPE_INDEL | CHRO_EVENT_TYPE_JUNCTION | CHRO_EVENT_TYPE_FUSION, search_return); if(found_events) { int kx1; for(kx1 = 0; kx1 < found_events ; kx1++) { if(search_return[kx1] -> event_large_side == right_edge_wanted) { found = search_return[kx1]; break; } } } if(found) found -> supporting_reads ++; else { int event_no; if(thread_context) event_no = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> total_events ++; else event_no = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> total_events ++; chromosome_event_t * new_event = event_space+event_no; memset(new_event,0,sizeof(chromosome_event_t)); new_event -> event_small_side = left_edge_wanted; new_event -> event_large_side = right_edge_wanted; new_event -> is_negative_strand= is_negative_donors; new_event -> event_type = CHRO_EVENT_TYPE_JUNCTION; new_event -> supporting_reads = 1; new_event -> indel_length = 0; new_event -> indel_at_junction = indel_at_junction; new_event -> is_donor_found_or_annotation = 1; new_event -> small_side_increasing_coordinate = 0; new_event -> large_side_increasing_coordinate = 1; put_new_event(event_table, new_event , event_no); } } int align_cluster(global_context_t * global_context, thread_context_t * thread_context, struct cluster_element * this_cluster, char * read_name_1, char * read_name_2, char * read_text_1, char * read_text_2, int read_len_1, int read_len_2, int is_negative_strand, gene_vote_t * vote_1, gene_vote_t * vote_2, int * this_score, int * best_ii_path, int * best_jj_path, int * best_masks, int * best_path_length, int * R1R2_mapped){ //int cluster_x1; //SUBREADprintf("\n === Cluster %s %s === \n", is_negative_strand?"NEG":"POS", read_name_1); //unsigned int min_frag_start = 0xffffffff; int ii_array[MAX_CLUSTER_ELEMENTS], jj_array[MAX_CLUSTER_ELEMENTS], is_second_vote_array[MAX_CLUSTER_ELEMENTS], dynamic_highest_mask[MAX_CLUSTER_ELEMENTS], x1; void * sort_pointers [5]; for(x1 = 0 ; x1 < this_cluster->cluster_members; x1++){ ii_array[x1] = this_cluster -> i_list[x1]; jj_array[x1] = this_cluster -> j_list[x1]; is_second_vote_array[x1] = this_cluster -> from_second_read[x1]; } sort_pointers[0] = ii_array; sort_pointers[1] = jj_array; sort_pointers[2] = is_second_vote_array; sort_pointers[3] = vote_1; sort_pointers[4] = vote_2; basic_sort(sort_pointers, this_cluster->cluster_members, compare_cluster_elements, exchange_cluster_elements); if(0) for(x1 = 0 ; x1 < this_cluster->cluster_members; x1++){ gene_vote_t * this_vote = is_second_vote_array[x1]?vote_2:vote_1; int ii = ii_array[x1]; int jj = jj_array[x1]; SUBREADprintf(" R%d %d - %d POS=%u VOTES=%d\n", 1+is_second_vote_array[x1], this_vote->coverage_start[ii][jj], this_vote->coverage_end[ii][jj], this_vote -> pos[ii][jj], this_vote->votes[ii][jj]); } int dynamic_highest_scores[MAX_CLUSTER_ELEMENTS], dynamic_last_exon[MAX_CLUSTER_ELEMENTS]; char dynamic_highest_path[MAX_CLUSTER_ELEMENTS]; memset(dynamic_highest_scores,0,sizeof(int)*MAX_CLUSTER_ELEMENTS); int target_element_i; for(target_element_i = 0; target_element_i < this_cluster->cluster_members; target_element_i++){ gene_vote_t * this_vote = is_second_vote_array[target_element_i]?vote_2:vote_1; int ii = ii_array[target_element_i]; int jj = jj_array[target_element_i]; dynamic_highest_scores[target_element_i] = this_vote->coverage_end[ii][jj] - this_vote->coverage_start[ii][jj]; dynamic_highest_path[ target_element_i ] = -1; } int highest_score = -1; int highest_target_end = -1; for(target_element_i = 0; target_element_i < this_cluster->cluster_members; target_element_i++){ int start_element_i; for(start_element_i = 0; start_element_i < this_cluster->cluster_members; start_element_i++){ if(target_element_i <= start_element_i) continue; int this_mask = -1, breakpount_last_exon = -1; int increasing_score = find_path(global_context, thread_context, start_element_i, target_element_i, ii_array, jj_array, is_second_vote_array, vote_1, vote_2, read_name_1, read_name_2, read_text_1, read_text_2, read_len_1, read_len_2, is_negative_strand, &this_mask, &breakpount_last_exon); if(increasing_score >= 0 && increasing_score + dynamic_highest_scores[start_element_i] > dynamic_highest_scores[target_element_i]){ dynamic_highest_path[ target_element_i ] = start_element_i; dynamic_highest_scores[target_element_i] = increasing_score + dynamic_highest_scores[start_element_i]; dynamic_highest_mask[ target_element_i ] = this_mask; dynamic_last_exon[ target_element_i ] = breakpount_last_exon; if( dynamic_highest_scores[target_element_i] > highest_score ){ highest_score = dynamic_highest_scores[target_element_i] ; highest_target_end = target_element_i; } } } } if(highest_target_end >=0 && highest_score > 160 - 159){ int is_on_path [MAX_CLUSTER_ELEMENTS]; memset(is_on_path,0,sizeof(int)*MAX_CLUSTER_ELEMENTS); gene_vote_t * last_vote = is_second_vote_array[ highest_target_end ]?vote_2:vote_1; int last_section_read_end = last_vote -> coverage_end[ ii_array [highest_target_end] ] [ jj_array [highest_target_end] ]; int this_rlen = is_second_vote_array[ highest_target_end ]?read_len_2 : read_len_1; int this_votes = last_vote -> votes [ ii_array [highest_target_end] ] [ jj_array [highest_target_end] ]; int tail_first_exon_last_base_in_read=-1, tail_mismatched_bases=-1; unsigned int tail_first_exon_first_base_on_chro, tail_mapped_section_pos; int front_first_exon_last_base_in_read=-1, front_mismatched_bases=-1; unsigned int front_first_exon_first_base_on_chro, front_mapped_section_pos ; int front_score = 0, tail_score = 0, front_negative_donor = 0, tail_negative_donor = 0; if(0 && last_section_read_end < this_rlen - NEW_EXTEND_SCAN_EXON_SHORTEST && this_votes > 4) { char * this_rname = is_second_vote_array[ highest_target_end ]?read_name_2:read_name_1; char * this_rtext = is_second_vote_array[ highest_target_end ]?read_text_2:read_text_1; int scan_to_tail = 1, mismatched_bases_after_start; tail_mapped_section_pos = last_vote -> pos[ ii_array [highest_target_end] ] [ jj_array [highest_target_end] ] + last_vote -> current_indel_cursor[ ii_array [highest_target_end] ] [ jj_array [highest_target_end] ] ; if(0){ char out1pos[100]; absoffset_to_posstr(global_context, tail_mapped_section_pos, out1pos); SUBREADprintf("RN=%s\nSTART=%u, READ_START=%d, READ_FIRTS_BASE_POS=%s\n", this_rname, tail_mapped_section_pos , last_section_read_end, out1pos); } tail_score = extend_uncovered_region_juncs(global_context, thread_context, this_rname, this_rtext , this_rlen, scan_to_tail, tail_mapped_section_pos, last_section_read_end , -1, & mismatched_bases_after_start, &tail_first_exon_last_base_in_read, &tail_first_exon_first_base_on_chro, &tail_mismatched_bases, &tail_negative_donor); } (*best_path_length) = 0; if(highest_score>0) while(1){ best_ii_path[(*best_path_length)] = ii_array[highest_target_end]; best_jj_path[(*best_path_length)] = jj_array[highest_target_end]; best_masks[ (*best_path_length) ] = dynamic_highest_mask[highest_target_end]; if( dynamic_last_exon [ highest_target_end ] > 0 ) best_masks[ (*best_path_length) ] |= ( is_second_vote_array[ highest_target_end ]?CLUSTER_ALIGNMENT_DONOR_R2_MAPPED:CLUSTER_ALIGNMENT_DONOR_R1_MAPPED); if( is_second_vote_array[ highest_target_end ] ) (*R1R2_mapped) |= CLUSTER_ALIGNMENT_DONOR_R2_MAPPED; else (*R1R2_mapped) |= CLUSTER_ALIGNMENT_DONOR_R1_MAPPED; (*best_path_length)++; is_on_path[highest_target_end] = 1; if( dynamic_highest_path[highest_target_end] == -1 ) break; highest_target_end = dynamic_highest_path[highest_target_end]; } gene_vote_t * first_vote = is_second_vote_array[ highest_target_end ]?vote_2:vote_1; int first_section_read_start = first_vote -> coverage_start [ ii_array [highest_target_end] ] [ jj_array [highest_target_end] ] ; this_votes = first_vote -> votes [ ii_array [highest_target_end] ] [ jj_array [highest_target_end] ]; if(0 && first_section_read_start > NEW_EXTEND_SCAN_EXON_SHORTEST && this_votes > 4){ char * this_rname = is_second_vote_array[ highest_target_end ]?read_name_2:read_name_1; char * this_rtext = is_second_vote_array[ highest_target_end ]?read_text_2:read_text_1; int scan_to_tail = 0, mismatched_bases_after_start; front_mapped_section_pos = first_vote -> pos[ ii_array [highest_target_end] ] [ jj_array [highest_target_end] ]; front_score = extend_uncovered_region_juncs(global_context, thread_context, this_rname, this_rtext , this_rlen, scan_to_tail, front_mapped_section_pos, first_section_read_start , -1, & mismatched_bases_after_start, &front_first_exon_last_base_in_read, &front_first_exon_first_base_on_chro, &front_mismatched_bases, &front_negative_donor); } if(0 && front_score>0 && tail_score>0){ SUBREADprintf("\n>>> %s\n", read_name_1); for(target_element_i = 0; target_element_i < this_cluster->cluster_members; target_element_i++){ SUBREADprintf("R%d\t", is_second_vote_array[target_element_i]+1); } SUBREADprintf("\n"); for(target_element_i = 0; target_element_i < this_cluster->cluster_members; target_element_i++){ gene_vote_t * this_vote = is_second_vote_array[target_element_i]?vote_2:vote_1; int ii = ii_array[target_element_i]; int jj = jj_array[target_element_i]; SUBREADprintf("%d%c%d\t", this_vote->coverage_start[ii][jj], is_on_path[target_element_i]?'=':'-', this_vote->coverage_end[ii][jj]); } SUBREADprintf("\n"); for(target_element_i = 0; target_element_i < this_cluster->cluster_members; target_element_i++){ SUBREADprintf("%d\t", dynamic_highest_scores[target_element_i]); } SUBREADprintf("\n"); SUBREADprintf("Extra_scores = %d, %d\n", front_score, tail_score); } (*this_score) = highest_score + max(0, front_score) + max(0, tail_score); int applied_score_cut=0; if(((*R1R2_mapped) & CLUSTER_ALIGNMENT_DONOR_R1_MAPPED)&&( (*R1R2_mapped) & CLUSTER_ALIGNMENT_DONOR_R2_MAPPED ) ) applied_score_cut = read_len_2 + read_len_1 - 70; else if((*R1R2_mapped) & CLUSTER_ALIGNMENT_DONOR_R1_MAPPED) applied_score_cut = read_len_1 - 30; else if((*R1R2_mapped) & CLUSTER_ALIGNMENT_DONOR_R1_MAPPED) applied_score_cut = read_len_2 - 30; if( (*this_score) >= applied_score_cut){ for( x1 = 0; x1 < MAX_CLUSTER_ELEMENTS; x1++){ if(!is_on_path[x1]) continue; int x2, second_end = -1; for(x2 = x1 + 1; x2 < MAX_CLUSTER_ELEMENTS; x2++){ if(is_on_path[x2]){ second_end = x2; break; } } if(second_end > 0){ if( dynamic_last_exon[second_end] >0 ){ gene_vote_t * this_vote = is_second_vote_array[ x1 ]?vote_2:vote_1; unsigned int junction_small_side = this_vote -> pos[ ii_array[ x1 ]][ jj_array[ x1 ]] + this_vote -> current_indel_cursor[ ii_array[ x1 ]][ jj_array[ x1 ]] + dynamic_last_exon[second_end]; unsigned int junction_large_side = this_vote -> pos[ ii_array[ second_end ]][ jj_array[ second_end ]] + dynamic_last_exon[second_end] + 1; if(0){ char out1pos[100], out2pos[100]; absoffset_to_posstr(global_context, junction_small_side, out1pos); absoffset_to_posstr(global_context, junction_large_side, out2pos); SUBREADprintf("CLUSTER_JUNCTION %s %s\n%s\n%s\n\n", out1pos, out2pos, read_text_1, read_text_2); } //#warning "SUBREAD_151 ============= MAKE SURE: CHANGE '0' TO INSERTED BASES =================" simple_add_junction(global_context, thread_context, junction_small_side, junction_large_side, 0, (dynamic_highest_mask[ second_end ] & CLUSTER_ALIGNMENT_DONOR_NEGATIVE_STRAND)?1:0); } } } if(0 && front_mismatched_bases <1 && front_score >14){ unsigned int junction_small_side = front_first_exon_first_base_on_chro + front_first_exon_last_base_in_read; unsigned int junction_large_side = front_mapped_section_pos + front_first_exon_last_base_in_read + 1; char out1pos[100], out2pos[100]; absoffset_to_posstr(global_context, junction_small_side+1, out1pos); absoffset_to_posstr(global_context, junction_large_side+1, out2pos); //SUBREADprintf("FMB=%d\tFS=%d\nPOS=%s , %s\n\n", front_mismatched_bases, front_score, out1pos, out2pos); simple_add_junction(global_context, thread_context, junction_small_side, junction_large_side, 0, front_negative_donor); } if(0 && tail_mismatched_bases <1 && tail_score >14){ unsigned int junction_small_side = tail_mapped_section_pos + tail_first_exon_last_base_in_read; unsigned int junction_large_side = tail_first_exon_first_base_on_chro + tail_first_exon_last_base_in_read; char out1pos[100], out2pos[100]; absoffset_to_posstr(global_context, junction_small_side+1, out1pos); absoffset_to_posstr(global_context, junction_large_side+1, out2pos); //SUBREADprintf("BMB=%d\tBS=%d\nPOS=%s , %s\n\n", tail_mismatched_bases, tail_score, out1pos, out2pos); simple_add_junction(global_context, thread_context, junction_small_side, junction_large_side, 0, tail_negative_donor); } } } return 0; } #define paired_donor_receptor_m2(s, c1, c2 ) ( s[0] == c1 && s[1] == c2 ) int is_paired_donor_receptor( char * small_bases, char * large_bases ){ //SUBREADprintf("SITE1 = %c%c , SITE2 = %c%c\n", small_bases[0], small_bases[1], large_bases[0], large_bases[1]); // if ( paired_donor_receptor_m2( small_bases, 'G', 'T' ) && paired_donor_receptor_m2( large_bases, 'A', 'G' ) ) return 1; if ( paired_donor_receptor_m2( small_bases, 'C', 'T' ) && paired_donor_receptor_m2( large_bases, 'A', 'C' ) ) return 2; // http://www.ncbi.nlm.nih.gov/pmc/articles/PMC113136/ // the 99.24% of splice site pairs should be GT-AG, // 0.69% GC-AG, // 0.05% AT-AC // and finally only 0.02% could consist of other types of non-canonical splice sites. // non-canonical : GC-AG (+) or CT-GC (-) if ( paired_donor_receptor_m2( small_bases, 'G', 'C' ) && paired_donor_receptor_m2( large_bases, 'A', 'G' ) ) return 3; if ( paired_donor_receptor_m2( small_bases, 'C', 'T' ) && paired_donor_receptor_m2( large_bases, 'G', 'C' ) ) return 4; // non-canonical : AT-AC (+) or GT-AT (-) if ( paired_donor_receptor_m2( small_bases, 'A', 'T' ) && paired_donor_receptor_m2( large_bases, 'A', 'C' ) ) return 5; if ( paired_donor_receptor_m2( small_bases, 'G', 'T' ) && paired_donor_receptor_m2( large_bases, 'A', 'T' ) ) return 6; return 0; } int find_donor_receptor(global_context_t * global_context, thread_context_t * thread_context, char * rname, char * rtext, int rlen, int start_coverage, int end_coverage, unsigned int start_pos, unsigned int end_pos, int indels_in_start, int v1, int v2, int * misma_bases, int * matched_bases, int * is_negative_donor){ gene_value_index_t * value_index = thread_context?thread_context->current_value_index:global_context->current_value_index; int search_in_read_start = start_coverage - 8, search_in_read_end = end_coverage + 8; search_in_read_start = max(0, search_in_read_start); search_in_read_end = min( rlen, search_in_read_end ); unsigned int search_in_chro_start = start_pos + indels_in_start + search_in_read_start; char chro_bases_startside[ search_in_read_end - search_in_read_start ], chro_bases_endside[search_in_read_end - search_in_read_start]; int x1; for(x1 = 0; x1 < search_in_read_end - search_in_read_start; x1++){ chro_bases_startside[x1] = gvindex_get( value_index, search_in_chro_start + x1 ); chro_bases_endside[x1] = gvindex_get( value_index , end_pos + search_in_read_start + x1); } int insertion_in_between_i, best_testing_score = 500 * 1000; int best_insertion_in_between = -1, best_last_exon_base_in_start = -1; int applied_insertion_limit = global_context->config.max_insertion_at_junctions; for(insertion_in_between_i = 0; insertion_in_between_i <= applied_insertion_limit; insertion_in_between_i ++){ int start_site_match [ search_in_read_end - search_in_read_start ], end_site_match[ search_in_read_end - search_in_read_start ]; int start_last_exon_base, end_site_mismatches = 0, start_site_mismatches = 0; for(start_last_exon_base = 0 ; start_last_exon_base < search_in_read_end - search_in_read_start ; start_last_exon_base++){ start_site_match[start_last_exon_base] = ( rtext[ search_in_read_start + start_last_exon_base ] == chro_bases_startside[start_last_exon_base] ); int end_site_x = ( rtext[ search_in_read_start + start_last_exon_base] == chro_bases_endside[start_last_exon_base] ); end_site_match[start_last_exon_base] = end_site_x; if(start_last_exon_base >=insertion_in_between_i ) end_site_mismatches += !end_site_x; } for(start_last_exon_base = 0 ; start_last_exon_base < search_in_read_end - search_in_read_start - insertion_in_between_i ; start_last_exon_base++){ end_site_mismatches -= (! end_site_match[start_last_exon_base + insertion_in_between_i] ); start_site_mismatches += (! start_site_match[start_last_exon_base] ); if(start_last_exon_base >= 2 && start_last_exon_base < search_in_read_end - search_in_read_start -insertion_in_between_i -2){ if(0&& FIXLENstrcmp("V0112_0155:7:1101:12618:2466#ACTTGA", rname) == 0) SUBREADprintf("split=%d, ins=%d, MM=%d+%d \n", start_last_exon_base, insertion_in_between_i, start_site_mismatches, end_site_mismatches); if( (end_site_mismatches + start_site_mismatches) * 500 + insertion_in_between_i < best_testing_score ){ int donor_paired_ret=is_paired_donor_receptor( chro_bases_startside + start_last_exon_base + 1, chro_bases_endside + insertion_in_between_i + start_last_exon_base - 1 ); if( donor_paired_ret ) { best_insertion_in_between = insertion_in_between_i; best_last_exon_base_in_start = start_last_exon_base; best_testing_score = (end_site_mismatches + start_site_mismatches) * 500 + insertion_in_between_i; (*misma_bases) = (end_site_mismatches + start_site_mismatches); if(is_negative_donor) (*is_negative_donor) =(donor_paired_ret -1)%2; (*matched_bases) = end_coverage - start_coverage - insertion_in_between_i - (end_site_mismatches + start_site_mismatches); } } } } } if(0 && FIXLENstrcmp("V0112_0155:7:1101:12618:2466", rname)==0) { chro_bases_startside[x1] = 0; chro_bases_endside[x1] = 0; char sp1s[200]; for(x1 =0; x1<200; x1++) sp1s[x1]=' '; sp1s[search_in_read_start] =0; char spE[200]; for(x1 =0; x1<200; x1++) spE[x1]=' '; spE[search_in_read_start + best_last_exon_base_in_start] =0; char spBB[200]; for(x1 =0; x1<200; x1++) spBB[x1]=' '; spBB[ best_insertion_in_between] =0; char out1pos[100]; absoffset_to_posstr(global_context, search_in_chro_start, out1pos); if(0 && FIXLENstrcmp("chr14:105",out1pos)==0){ SUBREADprintf("POS=%s\t\tINS=%d\t\t%s\n", out1pos, best_insertion_in_between, rname); SUBREADprintf("R= %s\nS1=%s%s\nS2=%s%s\n %s|%s|\n\n", rtext, sp1s, chro_bases_startside, sp1s, chro_bases_endside, spE, spBB); } } if(best_last_exon_base_in_start>=0) return best_last_exon_base_in_start + search_in_read_start ; else return -1; } int find_path(global_context_t * global_context, thread_context_t * thread_context, int start_element_i, int target_element_i, int * ii_array, int * jj_array, int * is_second_vote_array, gene_vote_t * vote_1, gene_vote_t * vote_2, char * read_name_1, char * read_name_2, char * read_text_1, char * read_text_2, int read_len_1, int read_len_2, int is_negative_strand, int * this_mask , int * exon_last_base){ gene_vote_t * start_vote = is_second_vote_array[start_element_i]?vote_2:vote_1; gene_vote_t * end_vote = is_second_vote_array[target_element_i]?vote_2:vote_1; int start_coverage = start_vote->coverage_end[ ii_array[start_element_i] ][ jj_array[start_element_i] ]; int end_coverage = end_vote->coverage_start[ ii_array[target_element_i] ][ jj_array[target_element_i] ]; unsigned int start_pos = start_vote->pos[ ii_array[start_element_i] ][ jj_array[start_element_i] ]; unsigned int end_pos = end_vote->pos[ ii_array[target_element_i] ][ jj_array[target_element_i] ]; int ret = -1; long long dist = start_pos; dist -= end_pos; (*this_mask)=0; if( abs(dist)<50000 ) { if(start_vote == end_vote){ if(start_coverage < end_coverage + 9){ char * this_read_name = is_second_vote_array[start_element_i]?read_name_2:read_name_1; char * this_read_text = is_second_vote_array[start_element_i]?read_text_2:read_text_1; int this_read_len = is_second_vote_array[start_element_i]?read_len_2:read_len_1, mismatched_bases = 0, matched_in_the_uncovered_gap = 0; if(start_pos < end_pos){ int indels_in_start = start_vote -> current_indel_cursor [ ii_array[start_element_i]] [ jj_array[start_element_i] ] , donor_receptor_neg_strand = -1; int best_last_base_in_start_exon = find_donor_receptor(global_context, thread_context, this_read_name, this_read_text, this_read_len, start_coverage, end_coverage, start_pos, end_pos, indels_in_start, start_vote -> votes[ ii_array[start_element_i]] [ jj_array[start_element_i] ], start_vote -> votes[ ii_array[target_element_i]] [ jj_array[target_element_i] ], &mismatched_bases , &matched_in_the_uncovered_gap, &donor_receptor_neg_strand); if(best_last_base_in_start_exon > 0 && mismatched_bases<1){ ret = matched_in_the_uncovered_gap + end_vote->coverage_end[ ii_array[target_element_i] ][ jj_array[target_element_i] ] - end_coverage; (*this_mask) = donor_receptor_neg_strand? CLUSTER_ALIGNMENT_DONOR_NEGATIVE_STRAND : 0 ; if(0)SUBREADprintf("FROM %d-%d to %d-%d : INC=%d, UNCOV=%d/%d\n", start_vote->coverage_start[ ii_array[start_element_i] ][ jj_array[start_element_i] ], start_vote->coverage_end[ ii_array[start_element_i] ][ jj_array[start_element_i] ], end_vote -> coverage_start[ ii_array[target_element_i] ][ jj_array[target_element_i] ], end_vote -> coverage_end[ ii_array[target_element_i] ][ jj_array[target_element_i] ], ret, matched_in_the_uncovered_gap , end_coverage - start_coverage); // # of matched bases, from the end of the "start" section to the end of the end section. *exon_last_base = best_last_base_in_start_exon; } } } }else{ ret = end_vote->coverage_end[ ii_array[target_element_i] ][ jj_array[target_element_i] ] - end_vote->coverage_start[ ii_array[target_element_i] ][ jj_array[target_element_i] ] ; // if the two sections are on two reads, check the first base of the second read is after the first base of the first read. } } return ret; } int process_voting_junction_PE_topK(global_context_t * global_context, thread_context_t * thread_context, subread_read_number_t pair_number, gene_vote_t * vote_1, gene_vote_t * vote_2, char * read_name_1, char * read_name_2, char * read_text_1, char * read_text_2, int read_len_1, int read_len_2, int is_negative_strand, gene_vote_number_t v1_all_subreads, gene_vote_number_t v2_all_subreads) { topK_buffer_t * topbuf = thread_context?&thread_context->topKbuff:&global_context ->topKbuff ; vote_combination_t * comb_buffer = (vote_combination_t *) topbuf -> comb_buffer; simple_mapping_t * vote_simple_1_buffer, * vote_simple_2_buffer; vote_simple_1_buffer =(simple_mapping_t *) topbuf -> vote_simple_1_buffer; vote_simple_2_buffer =(simple_mapping_t *) topbuf -> vote_simple_2_buffer; //memset(comb_buffer, 0 , sizeof(vote_combination_t) * global_context -> config.max_vote_combinations); int is_second_read,i,j; int third_highest_votes[2][9]; int is_fully_covered_1 = 0; int is_fully_covered_2 = 0; for(is_second_read = 0 ; is_second_read < 1 + global_context -> input_reads.is_paired_end_reads; is_second_read ++) { gene_vote_t * current_vote = is_second_read?vote_2:vote_1; int *top_three_buff = third_highest_votes[is_second_read], i , j; int * is_fully_covered = is_second_read?&is_fully_covered_2:&is_fully_covered_1; int current_read_len = is_second_read?read_len_2:read_len_1; memset(top_three_buff, 0 , global_context -> config.top_scores * sizeof(int)); if((global_context-> config.do_fusion_detection || global_context-> config.do_long_del_detection)){ *is_fully_covered = test_fully_covered(global_context , current_vote, current_read_len); } for (i=0; iitems[i]; j++){ int vv = current_vote -> votes[i][j]; //#warning "======== REMOVCE ==0 FROM NEXT ===========" if(global_context->config.scRNA_input_mode && !global_context -> input_reads.is_paired_end_reads)vv += SE_READ_IN_KNOWN_EXON_REWARD*is_pos_in_annotated_exon_regions(global_context, current_vote -> pos[i][j]); update_top_three(global_context, top_three_buff, vv); } } if(0 && FIXLENstrcmp("R00000003493",read_name_1)==0)SUBREADprintf("3N [R %d] =%d,%d,%d\n", 1+is_second_read, top_three_buff[0], top_three_buff[1], top_three_buff[2]); for(i = 0; i < global_context -> config.multi_best_reads; i++) { mapping_result_t * old_result = _global_retrieve_alignment_ptr(global_context, pair_number, is_second_read, i); if(old_result -> selected_votes>0) { update_top_three(global_context, top_three_buff, old_result -> selected_votes); } } if(0 && FIXLENstrcmp("R00000003493",read_name_1)==0)SUBREADprintf("3Q [R %d] =%d,%d,%d\n", 1+is_second_read, top_three_buff[0], top_three_buff[1], top_three_buff[2]); } int simple_record_numbers[2], third_k; for(is_second_read = 0 ; is_second_read < 1 + global_context -> input_reads.is_paired_end_reads; is_second_read ++) { int current_simple_number = 0; int current_read_len = is_second_read?read_len_2:read_len_1; // populate the two simple read lists for(third_k = 0 ; third_k < global_context -> config.top_scores; third_k ++) { if(current_simple_number >= global_context -> config.max_vote_simples)break; int this_vote_N = third_highest_votes [is_second_read][third_k]; // only consider max_votes and max_votes - 1 if(this_vote_N<1 || (third_highest_votes[is_second_read][0] - this_vote_N > global_context -> config.max_vote_number_cutoff )) break; simple_mapping_t * current_simple = is_second_read ? vote_simple_2_buffer: vote_simple_1_buffer; gene_vote_t * current_vote = is_second_read?vote_2:vote_1; for (i=0; i= global_context -> config.max_vote_simples)break; for (j=0; j< current_vote->items[i]; j++) { if(current_simple_number >= global_context -> config.max_vote_simples)break; if(global_context->config.do_big_margin_filtering_for_junctions && third_k == 0 && current_vote->votes[i][j] >= third_highest_votes [is_second_read][global_context -> config.top_scores - 1]) insert_big_margin_record(global_context , _global_retrieve_big_margin_ptr(global_context,pair_number, is_second_read), current_vote -> votes[i][j], current_vote -> coverage_start[i][j], current_vote -> coverage_end[i][j] , current_read_len, (current_vote -> masks[i][j] & IS_NEGATIVE_STRAND)?1:0); int vv = current_vote->votes[i][j]; //#warning "======== REMOVCE ==0 FROM NEXT ===========" if(global_context->config.scRNA_input_mode && !global_context -> input_reads.is_paired_end_reads)vv += SE_READ_IN_KNOWN_EXON_REWARD*is_pos_in_annotated_exon_regions(global_context, current_vote -> pos[i][j]); if(vv == this_vote_N && current_vote->votes[i][j] >= global_context->config.minimum_subread_for_second_read) { current_simple[current_simple_number].is_vote_t_item = 1; current_simple[current_simple_number].item_index_i = i; current_simple[current_simple_number].item_index_j = j; current_simple[current_simple_number].read_start_base = current_vote -> coverage_start[i][j]; current_simple[current_simple_number].mapping_position = current_vote -> pos[i][j]; current_simple[current_simple_number].major_half_votes = vv; current_simple_number ++; } } } for(i = 0; i < global_context -> config.multi_best_reads; i++) { mapping_result_t * old_result = _global_retrieve_alignment_ptr(global_context, pair_number, is_second_read, i); if(current_simple_number >= global_context -> config.max_vote_simples)break; if(old_result -> selected_votes == this_vote_N) { current_simple[current_simple_number].is_vote_t_item = 0; current_simple[current_simple_number].item_index_i = i; current_simple[current_simple_number].mapping_position = old_result -> selected_position; current_simple[current_simple_number].major_half_votes = old_result -> selected_votes; current_simple[current_simple_number].read_start_base = old_result -> confident_coverage_start; current_simple_number ++; } } } simple_record_numbers[is_second_read] = current_simple_number; } int used_comb_buffer = 0; //calculate all combinations if(global_context -> input_reads.is_paired_end_reads){ for(i = 0; i < simple_record_numbers[0]; i++){ for(j = 0; j < simple_record_numbers[1]; j++){ int target_index; int is_PE_distance = 0, is_same_chromosome = 0, is_both_exonic_regions = 0; if(max(vote_simple_1_buffer[i].major_half_votes, vote_simple_2_buffer[j].major_half_votes) < global_context->config.minimum_subread_for_first_read)continue; simple_PE_and_same_chro(global_context , vote_simple_1_buffer+i, vote_simple_2_buffer+j , &is_PE_distance, &is_same_chromosome , read_len_1, read_len_2); if((!is_PE_distance) && min(vote_simple_1_buffer[i].major_half_votes, vote_simple_2_buffer[j].major_half_votes) < global_context->config.minimum_subread_for_first_read)continue; if( global_context -> exonic_region_bitmap && is_same_chromosome)is_both_exonic_regions = is_pos_in_annotated_exon_regions(global_context, vote_simple_1_buffer[i].mapping_position + vote_simple_1_buffer[i].read_start_base ) && is_pos_in_annotated_exon_regions(global_context, vote_simple_2_buffer[j].mapping_position + vote_simple_2_buffer[j].read_start_base ) ; int adjusted_weight; if(1){ if (is_both_exonic_regions && is_PE_distance) adjusted_weight = 1800; else if(is_both_exonic_regions) adjusted_weight = 1300; else if(is_PE_distance) adjusted_weight = 1300; else if(is_same_chromosome) adjusted_weight = 1000; else adjusted_weight = 800; }else{ if (is_both_exonic_regions) adjusted_weight = 1300; else if(is_PE_distance) adjusted_weight = 1300; else if(is_same_chromosome) adjusted_weight = 1000; else adjusted_weight = 800; } //int adjusted_weight = is_PE_distance?1600:(is_same_chromosome?1000:500); int adjusted_votes = (vote_simple_1_buffer[i].major_half_votes + vote_simple_2_buffer[j].major_half_votes) * adjusted_weight; for(target_index=0; target_index config.max_vote_combinations){ int move_i; for(move_i = min(used_comb_buffer, global_context -> config.max_vote_combinations - 1) ; move_i > target_index ; move_i --) //checked: memory boundary memcpy(comb_buffer + move_i, comb_buffer + move_i - 1 , sizeof(vote_combination_t) ); comb_buffer[target_index].r1_loc = vote_simple_1_buffer+i; comb_buffer[target_index].r2_loc = vote_simple_2_buffer+j; comb_buffer[target_index].score_adj = adjusted_votes; if(used_comb_buffer < global_context -> config.max_vote_combinations) used_comb_buffer ++; } } } } mapping_result_t * alignment_tmp_r1, * alignment_tmp_r2; alignment_tmp_r1 = (mapping_result_t *) topbuf -> alignment_tmp_r1; alignment_tmp_r2 = (mapping_result_t *) topbuf -> alignment_tmp_r2; subjunc_result_t * junction_tmp_r2 , * junction_tmp_r1; junction_tmp_r1 = (subjunc_result_t *) topbuf -> junction_tmp_r1; junction_tmp_r2 = (subjunc_result_t *) topbuf -> junction_tmp_r2; memset(junction_tmp_r1, 0, sizeof(subjunc_result_t) * global_context->config.multi_best_reads); memset(junction_tmp_r2, 0, sizeof(subjunc_result_t) * global_context->config.multi_best_reads); memset(alignment_tmp_r1, 0, sizeof(mapping_result_t) * global_context->config.multi_best_reads); memset(alignment_tmp_r2, 0, sizeof(mapping_result_t) * global_context->config.multi_best_reads); int alignment_res_r1_cursor = 0, alignment_res_r2_cursor = 0; if(used_comb_buffer > 0){ merge_sort(comb_buffer, used_comb_buffer, comb_sort_compare, comb_sort_exchange, comb_sort_merge); for(is_second_read = 0; is_second_read < 1 + global_context -> input_reads.is_paired_end_reads; is_second_read++){ int current_read_len = is_second_read ? read_len_2:read_len_1; char * current_read_text = is_second_read ? read_text_2:read_text_1; int current_all_subreads = is_second_read ? v2_all_subreads:v1_all_subreads; mapping_result_t * current_alignment_tmp = is_second_read?alignment_tmp_r2:alignment_tmp_r1; int * current_r_cursor = is_second_read ? &alignment_res_r2_cursor:&alignment_res_r1_cursor; int * is_fully_covered = is_second_read?&is_fully_covered_2:&is_fully_covered_1; gene_vote_t * current_vote = is_second_read?vote_2:vote_1; subjunc_result_t * current_junction_tmp = NULL; if(global_context -> config.do_breakpoint_detection) current_junction_tmp = is_second_read?junction_tmp_r2:junction_tmp_r1; for(i = used_comb_buffer - 1; i >=0; i--){ if((* current_r_cursor) >= global_context->config.multi_best_reads)break; // add the combination of comb_buffer[i] into the two mapping_result_t arrays simple_mapping_t * current_loc = is_second_read?comb_buffer[i].r2_loc:comb_buffer[i].r1_loc; assert(current_loc); unsigned int current_pos = current_loc->mapping_position; int is_exist = 0; for(j = 0; j < *current_r_cursor; j++) { if(current_alignment_tmp[j].selected_position == current_pos){ is_exist = 1; break; } } //SUBREADprintf("CLLL BUF %d R_%d : %u ; EXIST %d. Written into the %d-th best location\n", i, 1+is_second_read, current_loc->mapping_position, is_exist, *current_r_cursor); if(!is_exist){ if(current_loc -> is_vote_t_item) copy_vote_to_alignment_res(global_context, thread_context, current_alignment_tmp + (*current_r_cursor), current_junction_tmp ? current_junction_tmp + (*current_r_cursor) : NULL, current_vote, current_loc -> item_index_i, current_loc -> item_index_j, current_read_len, read_name_1, current_read_text, current_all_subreads , current_vote -> noninformative_subreads, pair_number, is_second_read, is_fully_covered); else{ //checked: memory boundary memcpy(current_alignment_tmp + (*current_r_cursor), _global_retrieve_alignment_ptr(global_context, pair_number, is_second_read, current_loc -> item_index_i), sizeof(mapping_result_t)); if(current_junction_tmp) //checked: memory boundary memcpy(current_junction_tmp + (*current_r_cursor), _global_retrieve_subjunc_ptr(global_context, pair_number, is_second_read, current_loc -> item_index_i), sizeof(subjunc_result_t)); } (*current_r_cursor)++; } } } }else{// if the one end is not mapped at all if(0 == simple_record_numbers[0]) _global_retrieve_alignment_ptr(global_context, pair_number, 0, 0) -> noninformative_subreads_in_vote = vote_1 -> noninformative_subreads; if(global_context -> input_reads.is_paired_end_reads && 0 == simple_record_numbers[1]) _global_retrieve_alignment_ptr(global_context, pair_number, 1, 0) -> noninformative_subreads_in_vote = vote_2 -> noninformative_subreads; if(simple_record_numbers[0]>0 || simple_record_numbers[1]>0) { // copy all the simple into the mapping_result_t for(is_second_read = 0; is_second_read < 1 + global_context -> input_reads.is_paired_end_reads; is_second_read++) { int * current_r_cursor = is_second_read ? &alignment_res_r2_cursor:&alignment_res_r1_cursor; int current_read_len = is_second_read ? read_len_2:read_len_1; char * current_read_text = is_second_read ? read_text_2:read_text_1; int current_all_subreads = is_second_read ? v2_all_subreads:v1_all_subreads; mapping_result_t * current_alignment_tmp = is_second_read?alignment_tmp_r2:alignment_tmp_r1; gene_vote_t * current_vote = is_second_read?vote_2:vote_1; int * is_fully_covered = is_second_read?&is_fully_covered_2:&is_fully_covered_1; subjunc_result_t * current_junction_tmp = NULL; if(global_context -> config.do_breakpoint_detection) current_junction_tmp = is_second_read?junction_tmp_r2:junction_tmp_r1; for(i = 0; i < simple_record_numbers[is_second_read]; i++){ if((*current_r_cursor) >= global_context->config.multi_best_reads)break; simple_mapping_t * current_loc = is_second_read?vote_simple_2_buffer+i:vote_simple_1_buffer+i; if(current_loc -> major_half_votes < global_context->config.minimum_subread_for_first_read) continue; unsigned int current_pos = current_loc->mapping_position; int is_exist = 0; for(j = 0; j < *current_r_cursor; j++) { if(current_alignment_tmp[j].selected_position == current_pos){ is_exist = 1; break; } } if(!is_exist){ if(current_loc -> is_vote_t_item) copy_vote_to_alignment_res(global_context, thread_context, current_alignment_tmp + (*current_r_cursor), current_junction_tmp ? current_junction_tmp + (*current_r_cursor): NULL, current_vote, current_loc -> item_index_i, current_loc -> item_index_j, current_read_len, read_name_1, current_read_text, current_all_subreads , current_vote -> noninformative_subreads, pair_number, is_second_read, is_fully_covered); else{ //checked:boundary memcpy(current_alignment_tmp + (*current_r_cursor), _global_retrieve_alignment_ptr(global_context, pair_number, is_second_read, current_loc -> item_index_i), sizeof(mapping_result_t)); if(current_junction_tmp) //checked:boundary memcpy(current_junction_tmp + (*current_r_cursor), _global_retrieve_subjunc_ptr(global_context, pair_number, is_second_read, current_loc -> item_index_i), sizeof(subjunc_result_t)); } (*current_r_cursor)++; } } } } } for(is_second_read = 0; is_second_read < 1 + global_context -> input_reads.is_paired_end_reads; is_second_read++){ int * current_r_cursor = is_second_read ? &alignment_res_r2_cursor:&alignment_res_r1_cursor; if((*current_r_cursor) > global_context->config.multi_best_reads){ SUBREADprintf("ERROR: multi_best_locations excessed the boundary: %d > %d\n", (*current_r_cursor), global_context->config.multi_best_reads); return -1; } } for(is_second_read = 0; is_second_read < 1 + global_context -> input_reads.is_paired_end_reads; is_second_read++) { int * current_r_cursor = is_second_read ? &alignment_res_r2_cursor:&alignment_res_r1_cursor; mapping_result_t * current_alignment_tmp = is_second_read?alignment_tmp_r2:alignment_tmp_r1; subjunc_result_t * current_junction_tmp = NULL; if(global_context -> config.do_breakpoint_detection) current_junction_tmp = is_second_read?junction_tmp_r2:junction_tmp_r1; for(i = 0; i < global_context->config.multi_best_reads ; i++){ mapping_result_t * cur_res = _global_retrieve_alignment_ptr(global_context, pair_number, is_second_read, i); if( i < (*current_r_cursor)) memcpy(cur_res, current_alignment_tmp + i, sizeof(mapping_result_t)); else cur_res -> selected_votes = 0; if(global_context -> config.do_breakpoint_detection) { subjunc_result_t * cur_junc = _global_retrieve_subjunc_ptr(global_context, pair_number, is_second_read, i); if(i < (*current_r_cursor)) memcpy(cur_junc, current_junction_tmp + i , sizeof(subjunc_result_t)); else cur_junc -> minor_votes = 0; } } } return 0; } // seq1 and seq2 must be on the same strand! // (seq2 is reversed) // The second half of seq1 MUST BE the same as the first half of seq2 if the two reads have an overlapping part. int is_gapped_as_funky(global_context_t * global_context, char * rname1, char * chr1, unsigned int pos1, int rlen1, int is_1_negative, char * cigar1, char * seq1, char * rname2, char * chr2, unsigned int pos2, int rlen2, int is_2_negative, char * cigar2, char * seq2, int tlen_removed_intron) { /* if(tlen_removed_intron >= rlen1 + rlen2) return 1; // may be gapped. int try_overlapping; int best_matched_bases = 0; int best_overlapping_len = -1; int assumed_overlapping = rlen1+rlen2-tlen_removed_intron; for(try_overlapping = 0; try_overlapping < min(rlen1, rlen2); try_overlapping++) { int r1_start = rlen1 - try_overlapping; int r2_end = try_overlapping; int xk1; int all_matched = 0, all_mismatched = 0; for(xk1 = 0; xk1 < r2_end; xk1++){ char r1ch = seq1[r1_start + xk1]; char r2ch = seq2[xk1]; if(r1ch==r2ch) all_matched++; else all_mismatched++; } if(all_mismatched <= 1 && try_overlapping == assumed_overlapping){ // the assumed overlapping length is good enough. return 0; } if(all_mismatched <= 1 && all_matched > best_matched_bases){ best_overlapping_len = try_overlapping; best_matched_bases = all_matched; } } if(best_overlapping_len <= 0)return 0; return assumed_overlapping */ return tlen_removed_intron > 600; } // the positions are not offset by adding the first soft clipping length. I.e., pos1 and pos2 may be smaller than those in the SAM files. // seq1 and seq2 must be on the same strand! // (seq2 is reversed) int is_funky_fragment(global_context_t * global_context, char * rname1, char * chr1, unsigned int pos1, int rlen1, int is_1_negative, char * cigar1, char * seq1, char * rname2, char * chr2, unsigned int pos2, int rlen2, int is_2_negative, char * cigar2, char * seq2, int tlen_removed_intron) { long long llraw_tlen = pos1; llraw_tlen -= pos2; if(llraw_tlen <0) llraw_tlen = -llraw_tlen; unsigned int raw_tlen = llraw_tlen; raw_tlen += max(rlen2, rlen1); //SUBREADprintf("CHRS=%p,%p, POS=%u,%u, RTLEN=%u\n", chr1, chr2, pos1, pos2, raw_tlen); if(chr1 != chr2) raw_tlen = 0; // note: the two pointers can be compared because they should be derived from the offset table. // Each chromosome name should have one and only one distinct char * pointer. if(chr1 == chr2 && raw_tlen <= global_context -> config.maximum_translocation_length && is_2_negative == is_1_negative) { if(is_gapped_as_funky(global_context, rname1, chr1, pos1, rlen1, is_1_negative, cigar1, seq1, rname2, chr2, pos2, rlen2, is_2_negative, cigar2, seq2, tlen_removed_intron)) return FUNKY_FRAGMENT_A; else return NOT_FUNKY; } else if( chr1 == chr2 && raw_tlen <= global_context -> config.maximum_translocation_length && is_2_negative != is_1_negative ) return FUNKY_FRAGMENT_DE; else if( chr1 != chr2 || raw_tlen > global_context -> config.maximum_translocation_length) return FUNKY_FRAGMENT_BC; return NOT_FUNKY; } int process_voting_junction(global_context_t * global_context, thread_context_t * thread_context, subread_read_number_t pair_number, gene_vote_t * vote_1, gene_vote_t * vote_2, char * read_name_1, char * read_name_2, char * read_text_1, char * read_text_2, int read_len_1, int read_len_2, int is_negative_strand, gene_vote_number_t v1_all_subreads, gene_vote_number_t v2_all_subreads){ //#warning "FOR TESTING CLUSTER_BASED JUNCTION DETECTION ONLY!!." //return process_voting_junction_PE_juncs(global_context, thread_context, pair_number, vote_1, vote_2, read_name_1, read_name_2, read_text_1, read_text_2, read_len_1, read_len_2, is_negative_strand, v1_all_subreads, v2_all_subreads); return process_voting_junction_PE_topK(global_context, thread_context, pair_number, vote_1, vote_2, read_name_1, read_name_2, read_text_1, read_text_2, read_len_1, read_len_2, is_negative_strand, v1_all_subreads, v2_all_subreads); } unsigned int explain_read(global_context_t * global_context, thread_context_t * thread_context, realignment_result_t * final_realignments, subread_read_number_t pair_number, int read_len, char * read_name , char *read_text, char *qual_text, int is_second_read, int best_read_id, int is_negative_strand) { explain_context_t explain_context; mapping_result_t *current_result = _global_retrieve_alignment_ptr(global_context, pair_number, is_second_read, best_read_id); if(global_context -> config.do_big_margin_filtering_for_reads) { int current_repeated_times = is_ambiguous_voting(global_context, pair_number, is_second_read, current_result->selected_votes, current_result->confident_coverage_start, current_result->confident_coverage_end, read_len, (current_result->result_flags & CORE_IS_NEGATIVE_STRAND)?1:0); if( global_context -> config.do_big_margin_filtering_for_reads && current_repeated_times>1) return 0; } memset(&explain_context,0, sizeof(explain_context_t)); explain_context.full_read_len = read_len; explain_context.is_fully_covered = current_result -> is_fully_covered ; explain_context.full_read_text = read_text; explain_context.full_qual_text = qual_text; explain_context.read_name = read_name; explain_context.is_confirmed_section_negative_strand = is_negative_strand ; explain_context.pair_number = pair_number; explain_context.is_second_read = is_second_read ; explain_context.best_read_id = best_read_id; explain_context.total_tries = 0; if(0 && FIXLENstrcmp("simulated.24700032", explain_context.read_name)==0)SUBREADprintf("BBFINAL %s SEL_POS=%u COV=%d - %d\n", explain_context.read_name, current_result -> selected_position, current_result -> confident_coverage_start, current_result -> confident_coverage_end); unsigned int back_search_tail_position,front_search_start_position; unsigned short back_search_read_tail, front_search_read_start; back_search_read_tail = min(explain_context.full_read_len , current_result -> confident_coverage_end );//- 5; back_search_tail_position = current_result -> selected_position + back_search_read_tail + current_result -> indels_in_confident_coverage; //if( back_search_read_tail > 102) //SUBREADprintf("MAX back_search_read_tail : MIN %d , %d\n", explain_context.full_read_len , current_result -> confident_coverage_end); explain_context.tmp_search_junctions[0].read_pos_end = back_search_read_tail; explain_context.tmp_search_junctions[0].abs_offset_for_start = back_search_tail_position; explain_context.all_back_alignments = 0; explain_context.tmp_search_sections = 0; explain_context.best_indel_penalty =0; explain_context.best_matching_bases = -9999; explain_context.second_best_matching_bases = -9999; explain_context.tmp_indel_penalty = 0; explain_context.tmp_total_matched_bases = 0; explain_context.is_currently_tie = 0; explain_context.best_is_complex = 0; explain_context.best_support_as_simple = 0; explain_context.best_min_unsupport_as_simple = 0; explain_context.tmp_support_as_simple = 0; explain_context.tmp_min_support_as_complex = 999999; explain_context.tmp_min_unsupport = 999999; explain_context.tmp_is_pure_donor_found_explain = 1; explain_context.best_is_pure_donor_found_explain = 0; if(1) { front_search_read_start = back_search_read_tail > 8? back_search_read_tail - 8:0; front_search_start_position = back_search_tail_position>8?back_search_tail_position - 8:0; } else { //front_search_read_start = current_result -> confident_coverage_start + 5; front_search_read_start = min(explain_context.full_read_len , current_result -> confident_coverage_end); if(front_search_read_start > 2*global_context -> config.realignment_minimum_variant_distance) front_search_read_start -= 2*global_context -> config.realignment_minimum_variant_distance; else front_search_read_start = 0; front_search_start_position = current_result -> selected_position + front_search_read_start; } search_events_to_back(global_context, thread_context, &explain_context, read_text , qual_text, back_search_tail_position , back_search_read_tail, 0, 0, 1); int back_penalty = explain_context.best_indel_penalty; //int is_backsearch_tie = explain_context.is_currently_tie; int back_search_matches_diff = -9999; /* if(explain_context.back_search_confirmed_sections>0) { short last_section_length = explain_context.back_search_junctions[0].read_pos_end - explain_context.back_search_junctions[0].read_pos_start; front_search_read_start = explain_context.back_search_junctions[0].read_pos_start; front_search_start_position = explain_context.back_search_junctions[0].abs_offset_for_start - last_section_length; int last_sec = explain_context.back_search_confirmed_sections-1; current_result -> selected_position = explain_context.back_search_junctions[last_sec].abs_offset_for_start - explain_context.back_search_junctions[last_sec].read_pos_end + explain_context.back_search_junctions[last_sec].read_pos_start; back_search_matches_diff = explain_context.best_matching_bases - explain_context.second_best_matching_bases; if(0 && memcmp(explain_context.read_name, TTTSNAME, 26)==0) { int xk1; for(xk1 = 0; xk1 < explain_context.back_search_confirmed_sections; xk1++) { short pr_section_length = explain_context.back_search_junctions[xk1].read_pos_end - explain_context.back_search_junctions[xk1].read_pos_start; if(explain_context.back_search_junctions[xk1].event_after_section) SUBREADprintf("BACK_SECTIONS [%d], START IS %u; RPSS=%d ; RPED=%d ; LEN=%d ; EVENT is %u %u INDEL=%d\n", xk1, explain_context.back_search_junctions[xk1].abs_offset_for_start, explain_context.back_search_junctions[xk1].read_pos_start, explain_context.back_search_junctions[last_sec].read_pos_end, pr_section_length, explain_context.back_search_junctions[xk1].event_after_section->event_small_side, explain_context.back_search_junctions[xk1].event_after_section->event_large_side, explain_context.back_search_junctions[xk1].event_after_section->indel_length); else SUBREADprintf("BACK_SECTIONS [%d], START IS %u; RPSS=%d ; RPED=%d ; LEN=%d\n", xk1, explain_context.back_search_junctions[xk1].abs_offset_for_start, explain_context.back_search_junctions[xk1].read_pos_start, explain_context.back_search_junctions[last_sec].read_pos_end, pr_section_length); } } //SUBREADprintf("DBI:%d - %d;\n", explain_context.best_matching_bases , explain_context.second_best_matching_bases); } else */ explain_context.all_front_alignments = 0; explain_context.tmp_search_sections = 0; explain_context.best_indel_penalty = 0; explain_context.best_matching_bases = -9999; explain_context.second_best_matching_bases = -9999; explain_context.tmp_total_matched_bases = 0; explain_context.tmp_indel_penalty = 0; explain_context.is_currently_tie = 0; explain_context.best_is_complex = 0; explain_context.best_support_as_simple = 0; explain_context.best_min_unsupport_as_simple = 0; explain_context.tmp_support_as_simple = 0; explain_context.tmp_min_support_as_complex = 999999; explain_context.tmp_min_unsupport = 999999; explain_context.tmp_is_pure_donor_found_explain = 1; explain_context.best_is_pure_donor_found_explain = 0; memset(explain_context.tmp_search_junctions, 0, sizeof(perfect_section_in_read_t ) * MAX_EVENTS_IN_READ); explain_context.tmp_search_junctions[0].read_pos_start = front_search_read_start; explain_context.tmp_search_junctions[0].abs_offset_for_start = front_search_start_position; if(0 && FIXLENstrcmp("R000002689",explain_context.read_name ) == 0) SUBREADprintf("Enter F_SEARCH: start=%u read_pos=%d REMAIN=%d\n", front_search_start_position, front_search_read_start, read_len - front_search_read_start ); short search_remain = read_len - front_search_read_start; //#warning "SUBREAD_151 REMOVE THE ASSERT! " //if(search_remain >= 102)SUBREADprintf("FATAL: RLEN=%d, SEARCH=%d\n", read_len, front_search_read_start); //assert( search_remain < 102 ); // if(0 && FIXLENstrcmp("R00000001230", explain_context.read_name) ==0){ char outpos1[100]; absoffset_to_posstr(global_context, front_search_start_position , outpos1); SUBREADprintf("QFPOS %s : %s , %d ==> \n", read_name, outpos1, front_search_read_start); } search_events_to_front(global_context, thread_context, &explain_context, read_text + front_search_read_start, qual_text + front_search_read_start, front_search_start_position, search_remain , 0, 0, 1); if(0 && FIXLENstrcmp("R_chr901_932716_91M1D9M",explain_context.read_name ) == 0) SUBREADprintf("F_SEARCH has found %d result sets\n", explain_context.all_front_alignments); explain_context.best_indel_penalty += back_penalty; //int is_frontsearch_tie = explain_context.is_currently_tie; //SUBREADprintf("DFI:%d - %d;\n", explain_context.best_matching_bases , explain_context.second_best_matching_bases); int front_search_matches_diff = explain_context.best_matching_bases - explain_context.second_best_matching_bases; explain_context.best_second_match_diff = front_search_matches_diff + back_search_matches_diff; int realignment_number = finalise_explain_CIGAR(global_context, thread_context, &explain_context, final_realignments); if(0 && FIXLENstrcmp("SRR3439488.572382", explain_context.read_name)==0) SUBREADprintf("TRYING_REALIGN:%s:%u\n", explain_context.read_name, explain_context.total_tries); return realignment_number; } void debug_clipping(global_context_t * global_context, thread_context_t * thread_context, gene_value_index_t * current_value_index, char * read_text, unsigned int mapped_pos, int test_len, int search_to_tail, int search_center, int number_of_clipped, char * read_name){ //if(test_len>100)return; int xk1; SUBREADprintf("\n %s CENTER=%d, CLIPPED=%d, TLEN=%d %s\n", read_name, search_center, number_of_clipped, test_len, search_to_tail?">>>>":"<<<<"); for(xk1 = 0 ; xk1 < test_len ; xk1++) { char reference_base = gvindex_get(current_value_index, xk1 + mapped_pos); SUBREADprintf("%c", reference_base == read_text[xk1] ? '-':'#'); } SUBREADprintf("\n"); for(xk1 = 0 ; xk1 < test_len ; xk1++) { if(xk1 == search_center) SUBREADprintf("%c", search_to_tail?'>':'<'); else SUBREADprintf(" "); } SUBREADprintf("\n"); for(xk1 = 0 ; xk1 < test_len ; xk1++) { if( search_to_tail && xk1 >= test_len - number_of_clipped) SUBREADprintf("R"); else if( (!search_to_tail) && xk1 <= number_of_clipped - 1) SUBREADprintf("L"); else SUBREADprintf(" "); } SUBREADprintf("\n"); } #define SOFT_CLIPPING_WINDOW_SIZE 5 #define SOFT_CLIPPING_MAX_ERROR 1 // it returns the number of bases to be clipped off. int find_soft_clipping(global_context_t * global_context, thread_context_t * thread_context, gene_value_index_t * current_value_index, char * read_text, unsigned int mapped_pos, int test_len, int search_to_tail, int search_center) { int base_in_window = 0; int added_base_index = 0, removed_base_index = 0; int search_start = 0; int matched_in_window = SOFT_CLIPPING_WINDOW_SIZE; int last_matched_base_index = -1, delta; if(search_to_tail) { if(search_center < 0) search_start = 0; else if(search_center >= test_len) // SHOULD NOT HAPPEN!!! search_start = test_len - 1; else search_start = search_center - 1; delta = 1; }else{ if(search_center < 0) // SHOULD NOT HAPPEN!!! search_start = 0; else if(search_center >= test_len) search_start = test_len - 1; else search_start = search_center + 1; delta = -1; } for(added_base_index = search_start; added_base_index >= 0 && added_base_index < test_len; added_base_index += delta) { // add the new base char reference_base = gvindex_get(current_value_index, added_base_index + mapped_pos); if(0){ char outpos1[100]; absoffset_to_posstr(global_context, added_base_index + mapped_pos, outpos1); SUBREADprintf("CHMAT [%s] %s (%u) ref:read = %c:%c\n", search_to_tail?"T":"H" ,outpos1, added_base_index + mapped_pos, reference_base, read_text[added_base_index]); } int added_is_matched = (reference_base == read_text[added_base_index]); matched_in_window += added_is_matched; if(added_is_matched) last_matched_base_index = added_base_index; base_in_window ++; if(base_in_window > SOFT_CLIPPING_WINDOW_SIZE){ removed_base_index = added_base_index - delta * SOFT_CLIPPING_WINDOW_SIZE; char removing_ref_base = gvindex_get(current_value_index, removed_base_index + mapped_pos); matched_in_window -= (removing_ref_base == read_text[removed_base_index]); }else{ matched_in_window --; } if(matched_in_window < SOFT_CLIPPING_WINDOW_SIZE - SOFT_CLIPPING_MAX_ERROR){ // clip, bondary is the last matched base. if(search_to_tail){ if(last_matched_base_index < 0) return test_len - search_start; else return test_len - last_matched_base_index - 1; }else{ if(last_matched_base_index >= 0) return last_matched_base_index; else return search_start - 1; } } } if(last_matched_base_index < 0) return test_len; if(search_to_tail){ if(last_matched_base_index < 0) return test_len - search_start; else return test_len - last_matched_base_index - 1; }else{ if(last_matched_base_index >= 0) return last_matched_base_index; else return search_start - 1; } } // read_head_abs_offset is the first WANTED base in read. // If the first section in read is reversed, read_head_abs_offset is the LAST WANTED bases in this section. (the abs offset of the first base in the section is actually larger than read_head_abs_offset) int final_CIGAR_quality(global_context_t * global_context, thread_context_t * thread_context, char * read_text, char * qual_text, int read_len, char * cigar_string, unsigned long read_head_abs_offset, int is_read_head_reversed, int * mismatched_bases, int covered_start, int covered_end, char * read_name, int * non_clipped_length, int *total_indel_length, int * matched_bases, int * chromosomal_length, int * full_section_clipped) { int cigar_cursor = 0; int read_cursor = 0; unsigned int current_perfect_section_abs = read_head_abs_offset; int rebuilt_read_len = 0, total_insertion_length = 0; float all_matched_bases = 0; gene_value_index_t * current_value_index = thread_context?thread_context->current_value_index:global_context->current_value_index; int current_reversed = is_read_head_reversed; int all_mismatched = 0; int is_First_M = 1, is_wrong_cigar = 0; int head_soft_clipped = -1, tail_soft_clipped = -1; unsigned int tmp_int = 0; //SUBREADprintf("Coverage : %d ~ %d\n", covered_start, covered_end); if(0){ char posout1[100]; int chro_max = get_offset_maximum_chro_pos(global_context,thread_context,read_head_abs_offset); absoffset_to_posstr(global_context, read_head_abs_offset, posout1); SUBREADprintf("READ %s : mapped to %s ; max_pos=%d\n", read_name, posout1, chro_max); } while(1) { char nch = cigar_string[cigar_cursor++]; if(!nch)break; if(isdigit(nch)) tmp_int = tmp_int*10+(nch-'0'); else{ if(tmp_int == 0)is_wrong_cigar = 1; if(is_wrong_cigar) break; if(nch == 'M' || nch == 'S') { char *qual_text_cur; if(qual_text[0])qual_text_cur = qual_text+read_cursor; else qual_text_cur = NULL; float section_qual; int is_Last_M = (cigar_string[cigar_cursor]==0); int has_clipping_this_section_head = 0, has_clipping_this_section_tail = 0; char * reversed_first_section_text = NULL; if(0){ int is_head_in_chro = get_offset_maximum_chro_pos(global_context,thread_context, current_perfect_section_abs ); int is_end_in_chro = get_offset_maximum_chro_pos(global_context,thread_context, current_perfect_section_abs + tmp_int ); char posout1[100]; char posout2[100]; int chro_max = get_offset_maximum_chro_pos(global_context,thread_context, current_perfect_section_abs ); absoffset_to_posstr(global_context, current_perfect_section_abs, posout1); absoffset_to_posstr(global_context, current_perfect_section_abs + tmp_int, posout2); SUBREADprintf(" %dM SECTION : mapped to %s ~ %s ; max_pos=%d ; Hin=%d, Ein=%d\n", tmp_int, posout1, posout2, chro_max, is_head_in_chro, is_end_in_chro); SUBREADprintf(" %dM SECTION : Hin=%d, Ein=%d\n", tmp_int, is_head_in_chro, is_end_in_chro); } // find "J" sections if it is the first M if(is_First_M && global_context -> config.show_soft_cliping) { int adj_coverage_start = covered_start - read_cursor; if(current_reversed) { reversed_first_section_text = malloc(MAX_READ_LENGTH); memcpy(reversed_first_section_text, read_text, tmp_int); reverse_read(reversed_first_section_text, tmp_int, global_context->config.space_type); head_soft_clipped = find_soft_clipping(global_context, thread_context, current_value_index, reversed_first_section_text, current_perfect_section_abs, tmp_int, 1, 0); } else head_soft_clipped = find_soft_clipping(global_context, thread_context, current_value_index, read_text, current_perfect_section_abs, tmp_int, 0, adj_coverage_start); //SUBREADprintf("SSHEAD:%d\n", head_soft_clipped); if(head_soft_clipped == tmp_int){ (*full_section_clipped) = 1; head_soft_clipped = 0; } else has_clipping_this_section_head = 1; if(has_clipping_this_section_head){ if( tmp_int - head_soft_clipped < 3 && head_soft_clipped > 1 ) (*full_section_clipped) = 1; } if(reversed_first_section_text) free(reversed_first_section_text); reversed_first_section_text = NULL; } if(is_Last_M && global_context -> config.show_soft_cliping) { int adj_coverage_end = covered_end - read_cursor; if(current_reversed) { reversed_first_section_text = malloc(MAX_READ_LENGTH); // checked: boundary memcpy(reversed_first_section_text, read_text + read_cursor, tmp_int); reverse_read(reversed_first_section_text, tmp_int, global_context->config.space_type); tail_soft_clipped = find_soft_clipping(global_context, thread_context, current_value_index, reversed_first_section_text, current_perfect_section_abs, tmp_int, 0, tmp_int); } else tail_soft_clipped = find_soft_clipping(global_context, thread_context, current_value_index, read_text + read_cursor, current_perfect_section_abs, tmp_int, 1, adj_coverage_end); if(1 && FIXLENstrcmp("NS500643:556:HGTMTBGXB:4:13403:18179:8012", read_name)==0) SUBREADprintf("SSTAIL:%d\n", tail_soft_clipped); if(1 && tail_soft_clipped == tmp_int){ tail_soft_clipped = 0; if(full_section_clipped)(*full_section_clipped) = 1; } else has_clipping_this_section_tail = 1; if( has_clipping_this_section_tail ){ if(tmp_int - tail_soft_clipped < 3 && tail_soft_clipped > 1) (*full_section_clipped) = 1; } if(reversed_first_section_text) free(reversed_first_section_text); } if(is_Last_M && is_First_M && tail_soft_clipped+head_soft_clipped >= tmp_int-1) { head_soft_clipped=0; tail_soft_clipped=0; } int mismatch_calculation_start = has_clipping_this_section_head?head_soft_clipped:0; int mismatch_calculation_end = has_clipping_this_section_tail?tail_soft_clipped:0; if(global_context -> config.space_type == GENE_SPACE_COLOR) section_qual = match_base_quality_cs(current_value_index, read_text+read_cursor, current_perfect_section_abs, qual_text_cur, tmp_int, global_context->config.phred_score_format , mismatched_bases, &all_mismatched, global_context -> config.high_quality_base_threshold, mismatch_calculation_start, mismatch_calculation_end); else section_qual = match_base_quality(current_value_index, read_text+read_cursor, current_perfect_section_abs, qual_text_cur, tmp_int, current_reversed, global_context->config.phred_score_format , mismatched_bases, &all_mismatched, global_context -> config.high_quality_base_threshold, mismatch_calculation_start, mismatch_calculation_end); all_matched_bases += section_qual; rebuilt_read_len += tmp_int; is_First_M=0; read_cursor += tmp_int; //move to the NEXT UNWANTED ABS OFFSET. if(current_reversed) current_perfect_section_abs --; else current_perfect_section_abs += tmp_int; } else if(nch == 'I') { rebuilt_read_len += tmp_int; read_cursor += tmp_int; all_matched_bases += tmp_int; total_indel_length += tmp_int; total_insertion_length += tmp_int; } else if(nch == 'D') { total_indel_length ++; if(!current_reversed) current_perfect_section_abs += tmp_int; } else if(tolower(nch) == 'n') { total_indel_length ++; current_perfect_section_abs += tmp_int; if(nch == 'n') current_reversed = !current_reversed; } else if(tolower(nch) == 'b') { total_indel_length ++; current_perfect_section_abs -= tmp_int; if(nch == 'b') current_reversed = !current_reversed; } if(read_cursor>MAX_READ_LENGTH){ SUBREADprintf("ERROR: Cigar section longer than read length: %d >= %d, '%s'\n", tmp_int , MAX_READ_LENGTH, cigar_string); is_wrong_cigar = 1; } tmp_int = 0; } } int my_non_clipped_length = read_len; my_non_clipped_length -= max(0,tail_soft_clipped); my_non_clipped_length -= max(0,head_soft_clipped); //#warning " ========== COMMENT THIS LINE !! =========" //printf("QCR ALL MM=%d, RBLEN=%d, MAPPED_LEN=%d ; CIGAR=%s\n", all_mismatched, rebuilt_read_len , my_non_clipped_length, cigar_string); if(is_wrong_cigar || rebuilt_read_len != read_len || my_non_clipped_length < global_context->config.min_mapped_fraction){ (*mismatched_bases)=99999; all_matched_bases = 0; sprintf(cigar_string, "%dM", read_len); } else if((head_soft_clipped>0 || tail_soft_clipped>0)) { char new_cigar_tmp[120]; is_First_M=1; new_cigar_tmp[0]=0; cigar_cursor = 0; while(1) { char nch = cigar_string[cigar_cursor++]; if(!nch)break; if(isdigit(nch)) tmp_int = tmp_int*10+(nch-'0'); else{ char cigar_piece [30]; cigar_piece[0]=0; if(nch == 'M') { char cigar_tiny [12]; int is_Last_M = (cigar_string[cigar_cursor]==0); if(is_First_M && head_soft_clipped>0) { tmp_int -= head_soft_clipped; sprintf(cigar_tiny,"%dS",head_soft_clipped); strcat(cigar_piece, cigar_tiny); } if(is_Last_M && tail_soft_clipped>0) { tmp_int -= tail_soft_clipped; } sprintf(cigar_tiny,"%dM",tmp_int); strcat(cigar_piece, cigar_tiny); if(is_Last_M && tail_soft_clipped>0) { sprintf(cigar_tiny,"%dS",tail_soft_clipped); strcat(cigar_piece, cigar_tiny); } is_First_M = 0; } else { sprintf(cigar_piece, "%u%c", tmp_int, nch); } strcat(new_cigar_tmp, cigar_piece); tmp_int = 0; } } if(1 && FIXLENstrcmp("NS500643:556:HGTMTBGXB:4:13403:18179:8012", read_name)==0) SUBREADprintf("NEW_CIGAR_2 : %s\n", new_cigar_tmp); strcpy(cigar_string, new_cigar_tmp); } if((*mismatched_bases) != 99999) (*mismatched_bases) = all_mismatched; (*non_clipped_length) = my_non_clipped_length; (*matched_bases) = my_non_clipped_length - all_mismatched - total_insertion_length; (*chromosomal_length) = current_perfect_section_abs - read_head_abs_offset + total_insertion_length; return max(0, (int)(all_matched_bases*60/my_non_clipped_length)); } // this function also adds final_counting_reads in chromosome_events. unsigned int finalise_explain_CIGAR(global_context_t * global_context, thread_context_t * thread_context, explain_context_t * explain_context, realignment_result_t * final_realignments) { int xk1, front_i, back_i; char tmp_cigar[120]; chromosome_event_t * to_be_supported [20]; short flanking_size_left[20], flanking_size_right[20]; int to_be_supported_count = 0; int is_junction_read = 0; int total_perfect_matched_sections = 0; mapping_result_t * result = _global_retrieve_alignment_ptr(global_context, explain_context->pair_number, explain_context->is_second_read, explain_context-> best_read_id); result -> result_flags &= ~CORE_IS_FULLY_EXPLAINED; result -> result_flags &= ~CORE_IS_PAIRED_END; //SUBREADprintf("FINAL_CIGAR R1 %d[%d] = %p, FLAGS=%d\n", explain_context -> pair_number , explain_context-> best_read_id , result , result -> result_flags); // reverse the back_search result for every equally best alignment // for(back_i = 0; back_i < explain_context -> all_back_alignments; back_i++){ if( explain_context -> result_back_junction_numbers[back_i] > MAX_EVENTS_IN_READ ){ SUBREADprintf("ERROR: Too many cigar sections: %d > %d\n", explain_context -> result_back_junction_numbers[back_i] , MAX_EVENTS_IN_READ); return 0; } for(xk1=0; xk1 result_back_junction_numbers[back_i]/2; xk1++) { perfect_section_in_read_t tmp_exp; // checked: boundary memcpy(&tmp_exp, &explain_context -> result_back_junctions[back_i][xk1], sizeof(perfect_section_in_read_t)); memcpy(&explain_context -> result_back_junctions[back_i][xk1], &explain_context -> result_back_junctions[back_i][explain_context -> result_back_junction_numbers[back_i] - xk1 - 1] , sizeof(perfect_section_in_read_t)); memcpy(&explain_context -> result_back_junctions[back_i][explain_context -> result_back_junction_numbers[back_i] - xk1 - 1] , &tmp_exp , sizeof(perfect_section_in_read_t)); } } // adding indel lengths in read lengths and relocate sections // note that the last section in back results has the same strand of the main piece. int is_cigar_overflow = 0, fusions_in_read = 0, final_alignment_number = 0; for(back_i = 0; back_i < explain_context -> all_back_alignments; back_i++){ if(final_alignment_number >= MAX_ALIGNMENT_PER_ANCHOR)break; int is_first_section_negative = (result ->result_flags & CORE_IS_NEGATIVE_STRAND)?1:0; for(xk1=0; xk1 result_back_junction_numbers[back_i]; xk1++) { int section_length = explain_context -> result_back_junctions[back_i][xk1].read_pos_end - explain_context -> result_back_junctions[back_i][xk1].read_pos_start; if(0 && FIXLENstrcmp("simulated.11420793", explain_context->read_name)==0)SUBREADprintf("FINAL_EXPLAIN %s BACK_%d SEC_%d OLD_START=%d SEC_LENG=%d\n", explain_context->read_name, back_i, xk1, explain_context -> result_back_junctions[back_i][xk1].abs_offset_for_start, section_length); unsigned int new_start_pos; if(explain_context -> result_back_junctions[back_i][xk1].is_strand_jumped) // the "strand_jumped" section do not need to move // however, the "abs_offset_for_start" is actually for the last base in this section. // this does not metter if we compare the reversed read to the chromosome. // "abs_offset_for_start" is the first UNWANTED base (smaller than the first WANTED base) new_start_pos = explain_context -> result_back_junctions[back_i][xk1].abs_offset_for_start +1; else // "abs_offset_for_start" is the first UNWANTED base. By subtracting the length, it becomes the first WANTED base. new_start_pos = explain_context -> result_back_junctions[back_i][xk1].abs_offset_for_start - section_length; explain_context -> result_back_junctions[back_i][xk1].abs_offset_for_start = new_start_pos; if(explain_context -> result_back_junctions[back_i][xk1].event_after_section && explain_context -> result_back_junctions[back_i][xk1].event_after_section->is_strand_jumped) is_first_section_negative=!is_first_section_negative; } // build CIGAR for(front_i = 0; front_i < explain_context -> all_front_alignments; front_i++){ if(final_alignment_number >= MAX_ALIGNMENT_PER_ANCHOR)break; to_be_supported_count = 0; tmp_cigar[0]=0; int known_junction_supp = 0; for(xk1 = 0; xk1 < explain_context -> result_back_junction_numbers[back_i] + explain_context -> result_front_junction_numbers[front_i] -1; xk1++) { char piece_cigar[25]; int read_pos_start, read_pos_end; perfect_section_in_read_t * current_section, *next_section = NULL; int is_front_search = 0; if(xk1 >= explain_context -> result_back_junction_numbers[back_i] - 1) { current_section = &explain_context -> result_front_junctions[front_i][xk1 - explain_context -> result_back_junction_numbers[back_i] +1]; if(xk1 - explain_context -> result_back_junction_numbers[back_i] +2 < explain_context -> result_front_junction_numbers[front_i]) next_section = &explain_context -> result_front_junctions[front_i][xk1 - explain_context -> result_back_junction_numbers[back_i] +2]; is_front_search = 1; } else { current_section = &explain_context -> result_back_junctions[back_i][xk1]; if(xk1+1 < explain_context -> result_back_junction_numbers[back_i]) next_section = &explain_context -> result_back_junctions[back_i][xk1+1]; } if(xk1 == explain_context -> result_back_junction_numbers[back_i] - 1) read_pos_start = explain_context -> result_back_junctions[back_i][xk1].read_pos_start; else read_pos_start = current_section -> read_pos_start; read_pos_end = current_section -> read_pos_end; chromosome_event_t *event_after = current_section -> event_after_section; sprintf(piece_cigar, "%dM", (read_pos_end - read_pos_start)); total_perfect_matched_sections += (read_pos_end - read_pos_start); flanking_size_left[xk1] = (read_pos_end - read_pos_start); if(xk1 result_back_junction_numbers[back_i] + explain_context -> result_front_junction_numbers[front_i] -2) assert(event_after); if(xk1>0) flanking_size_right[xk1-1] = (read_pos_end - read_pos_start); if(event_after) { if(event_after -> event_type == CHRO_EVENT_TYPE_INDEL) sprintf(piece_cigar+strlen(piece_cigar), "%d%c", abs(event_after->indel_length), event_after->indel_length>0?'D':'I'); else if(event_after -> event_type == CHRO_EVENT_TYPE_JUNCTION||event_after -> event_type == CHRO_EVENT_TYPE_FUSION) { // the distance in CIGAR is the NEXT UNWANTED BASE of piece#1 to the FIRST WANTED BASE in piece#2 int delta_one ; if(current_section -> is_strand_jumped + current_section -> is_connected_to_large_side == 1) delta_one = 1; else delta_one = -1; // if it is from front_search, the event side points to the first WANTED base of the next section; it should be moved to the last WANTED base the next section if the next section is jumped. if(next_section && (event_after -> is_strand_jumped + current_section -> is_strand_jumped==1)) { if(is_front_search) { if(current_section -> is_connected_to_large_side) delta_one += (next_section->read_pos_end - next_section-> read_pos_start - 1); else delta_one -= (next_section->read_pos_end - next_section-> read_pos_start - 1); } else { if(current_section -> is_connected_to_large_side) delta_one += (next_section->read_pos_end - next_section-> read_pos_start - 1); else delta_one -= (next_section->read_pos_end - next_section-> read_pos_start - 1); } } char jump_mode = current_section -> is_connected_to_large_side?'B':'N'; long long int movement = event_after -> event_large_side; movement -= event_after -> event_small_side - delta_one; if(1){ if(jump_mode == 'B' && movement < 0){ movement = - movement; jump_mode = 'N'; }else if(jump_mode == 'N' && movement < 0){ movement = - movement; jump_mode = 'B'; } } if(event_after -> is_strand_jumped) jump_mode = tolower(jump_mode); fusions_in_read += (event_after -> event_type == CHRO_EVENT_TYPE_FUSION); sprintf(piece_cigar+strlen(piece_cigar), "%u%c", (int)movement, jump_mode); if(event_after -> indel_at_junction) sprintf(piece_cigar+strlen(piece_cigar), "%dI", event_after -> indel_at_junction); is_junction_read ++; if(event_after -> is_donor_found_or_annotation & 64 ) known_junction_supp ++; } to_be_supported[to_be_supported_count++] = event_after; } strcat(tmp_cigar, piece_cigar); if(strlen(tmp_cigar) > CORE_MAX_CIGAR_STR_LEN - 14){ is_cigar_overflow=1; break; } } int mismatch_bases = 0; //#warning ">>>>>>>>>>>>>>>> COMMENT NEXT LINE <<<<<<<<<<<<<<<<<<<<<<<" //SUBREADprintf("ReadDebug:%s\t%s\n", explain_context -> read_name , tmp_cigar); if(is_cigar_overflow) sprintf(tmp_cigar, "%dM", explain_context -> full_read_len); unsigned int final_position; // #warning "'0 &&' is because there could be indels in the high-confident region but this indel is finally disused." if( 0 && explain_context -> result_back_junction_numbers[back_i] + explain_context -> result_front_junction_numbers[front_i] <= 2) final_position = result -> selected_position; else final_position = explain_context -> result_back_junctions[back_i][0].abs_offset_for_start; if(0 && FIXLENstrcmp("simulated.11420793", explain_context->read_name)==0)SUBREADprintf("FFFINAL %s : POS=%u, ABS=%u\n", explain_context->read_name, final_position, explain_context -> result_back_junctions[back_i][0].abs_offset_for_start); int is_exonic_read_fraction_OK = 1; if( global_context -> config.minimum_exonic_subread_fraction > 0.0000001 && (!is_junction_read) && result -> used_subreads_in_vote>0) { int min_subreads = global_context -> config.minimum_exonic_subread_fraction * result-> used_subreads_in_vote; if( result -> selected_votes < min_subreads ) is_exonic_read_fraction_OK = 0 ; } int final_qual = 0, applied_mismatch = 0, non_clipped_length = 0, total_indel_length = 0, final_MATCH = 0, chromosomal_length = 0, full_section_clipped = 0; if(is_exonic_read_fraction_OK) { if(0 && FIXLENstrcmp("R00000001230", explain_context -> read_name) ==0) SUBREADprintf("TSOFT_COVSTART %d\n", result -> confident_coverage_start); final_qual = final_CIGAR_quality(global_context, thread_context, explain_context -> full_read_text, explain_context -> full_qual_text, explain_context -> full_read_len , tmp_cigar, final_position, is_first_section_negative != ((result->result_flags & CORE_IS_NEGATIVE_STRAND)?1:0), &mismatch_bases, result -> confident_coverage_start, result -> confident_coverage_end, explain_context -> read_name, &non_clipped_length, &total_indel_length, & final_MATCH, & chromosomal_length, & full_section_clipped); //#warning ">>>>>>> COMMENT THIS <<<<<<<" //printf("OCT27-STEP2-%s:%d-POS%u-VOT%d-CIG-%s [ %d ]-INDELs=%llu; M/MM=%d,%d\n", explain_context -> read_name, explain_context -> is_second_read + 1, result -> selected_position, result -> selected_votes, tmp_cigar, is_cigar_overflow, ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> event_entry_table -> numOfElements, final_MATCH, mismatch_bases); applied_mismatch = is_junction_read? global_context->config.max_mismatch_junction_reads:global_context->config.max_mismatch_exonic_reads ; if(explain_context->full_read_len > EXON_LONG_READ_LENGTH) applied_mismatch = ((((explain_context->full_read_len+1)<<16) / 100) * applied_mismatch)>>16; if(global_context -> config.space_type == GENE_SPACE_COLOR) applied_mismatch += to_be_supported_count*2; } //#warning " ========== COMMENT THIS LINE !! =========" if(0 && FIXLENstrcmp("R00000001122", explain_context -> read_name) ==0){ char outpos1[100]; absoffset_to_posstr(global_context, final_position, outpos1); SUBREADprintf("FINALQUAL %s : FINAL_POS=%s ( %u )\tCIGAR=%s\tMM=%d / MAPLEN=%d > %d?\tVOTE=%d > %0.2f x %d ? MASK=%d\tQUAL=%d\tBRNO=%d\nKNOWN_JUNCS=%d PENALTY=%d\n\n", explain_context -> read_name, outpos1 , final_position , tmp_cigar, mismatch_bases, non_clipped_length, applied_mismatch, result -> selected_votes, global_context -> config.minimum_exonic_subread_fraction,result-> used_subreads_in_vote, result->result_flags, final_qual, explain_context -> best_read_id, known_junction_supp, explain_context -> best_indel_penalty); //exit(0); } if(mismatch_bases <= applied_mismatch && is_exonic_read_fraction_OK && fusions_in_read < 2 ){// && (0 == full_section_clipped || 0 == global_context -> config.do_breakpoint_detection)) { realignment_result_t * realign_res = final_realignments+final_alignment_number; final_alignment_number ++; realign_res -> realign_flags = result->result_flags; realign_res -> first_base_is_jumpped = 0; realign_res -> mapping_result = result; realign_res -> chromosomal_length = chromosomal_length; realign_res -> known_junction_supp = known_junction_supp; realign_res -> final_penalty = explain_context -> best_indel_penalty; if(mismatch_bases > applied_mismatch ) realign_res -> realign_flags |= CORE_TOO_MANY_MISMATCHES; else realign_res -> realign_flags &= ~CORE_TOO_MANY_MISMATCHES; if(((result -> result_flags & CORE_IS_NEGATIVE_STRAND)?1:0) != is_first_section_negative) { assert((global_context-> config.do_fusion_detection || global_context-> config.do_long_del_detection)); realign_res -> first_base_is_jumpped = 1; } strcpy(realign_res -> cigar_string, tmp_cigar); if(1) { int is_RNA_from_positive = -1; unsigned long long read_id = 2llu * explain_context -> pair_number + explain_context->is_second_read; for(xk1= 0; xk1 < to_be_supported_count; xk1++) { if(xk1 >= MAX_EVENTS_IN_READ) break; if(to_be_supported [xk1] -> event_type !=CHRO_EVENT_TYPE_INDEL && is_junction_read){ if(to_be_supported [xk1] -> event_type == CHRO_EVENT_TYPE_JUNCTION && to_be_supported [xk1] -> is_donor_found_or_annotation && is_RNA_from_positive == -1) is_RNA_from_positive = !(to_be_supported [xk1] -> is_negative_strand); } //final counts are added in function "add_realignment_event_support" in core.c realign_res -> supporting_chromosome_events[xk1] = to_be_supported[xk1]; realign_res -> flanking_size_left[xk1] = flanking_size_left[xk1]; realign_res -> flanking_size_right[xk1] = flanking_size_right[xk1]; realign_res -> crirical_support[xk1] += (read_id == to_be_supported [xk1] -> critical_read_id); } if(to_be_supported_count < MAX_EVENTS_IN_READ ) realign_res -> supporting_chromosome_events[to_be_supported_count] = NULL; result -> result_flags |= CORE_IS_FULLY_EXPLAINED; result -> read_length = explain_context->full_read_len; if(is_RNA_from_positive == -1) { realign_res -> realign_flags |= CORE_NOTFOUND_DONORS ; realign_res -> realign_flags &= ~(CORE_IS_GT_AG_DONORS); } else { realign_res -> realign_flags &= ~ (CORE_NOTFOUND_DONORS | CORE_IS_GT_AG_DONORS); if(is_RNA_from_positive) realign_res -> realign_flags |= CORE_IS_GT_AG_DONORS; } } realign_res -> first_base_position = final_position; realign_res -> final_quality = final_qual; realign_res -> final_mismatched_bases = mismatch_bases; realign_res -> final_matched_bases = (unsigned short)final_MATCH; realign_res -> best_second_diff_bases = (9 best_second_match_diff)?-1:explain_context -> best_second_match_diff; } } } return final_alignment_number; } #define ceq(c,t) ((c)[0]==(t)[0] && (c)[1]==(t)[1]) #define c2eq(ch1, ch2, tg1, tg2) ((ceq(ch1, tg1) && ceq(ch2, tg2)) || (ceq(ch1, tg2) && ceq(ch2, tg1)) ) int paired_chars_full_core(char * ch1, char * ch2, int is_reverse) { if (c2eq(ch1, ch2, "GT", "AG") || c2eq(ch1, ch2, "CT", "AC")) { if (is_reverse) if (ceq(ch1, "AG") || ceq(ch1, "AC")) return 2; if (!is_reverse) if (ceq(ch1, "CT") || ceq(ch1, "GT")) return 2; } else if ( c2eq(ch1, ch2,"GC","AG") || c2eq(ch1, ch2,"GC","CT") || c2eq(ch1, ch2,"AT","AC") || c2eq(ch1, ch2,"GT","AT")) { if (is_reverse) if (ceq(ch1, "GC") || ceq(ch1, "AT") || ceq(ch1, "AG") || ceq(ch1, "AC")) return 1; if (!is_reverse) if (ceq(ch1, "GC") || ceq(ch1, "AT") ||ceq(ch1, "GT") || ceq(ch1, "CT")) return 1; } return 0; } int paired_chars_part_core(char * ch1, char * ch2, int is_reverse) { if (c2eq(ch1, ch2, "GT", "AG") || c2eq(ch1, ch2, "CT", "AC")) { if (is_reverse){ if (ceq(ch1, "AG") || ceq(ch1, "AC")) return 1; } else { if (ceq(ch1, "CT") || ceq(ch1, "GT")) return 1; } } return 0; } #define is_donor_chars_full(cc) (((cc)[0]=='G' && (cc)[1]=='T') || \ ((cc)[0]=='A' && (cc)[1]=='G') || \ ((cc)[0]=='A' && (cc)[1]=='C') || \ ((cc)[0]=='C' && (cc)[1]=='T') || \ ((cc)[0]=='G' && (cc)[1]=='C') || \ ((cc)[0]=='A' && (cc)[1]=='T') || \ ((cc)[0]=='A' && (cc)[1]=='C') ) #define is_donor_chars_part(cc) (((cc)[0]=='G' && (cc)[1]=='T') || \ ((cc)[0]=='A' && (cc)[1]=='G') || \ ((cc)[0]=='A' && (cc)[1]=='C') || \ ((cc)[0]=='C' && (cc)[1]=='T')) //#warning "=============== NO DONOR-RECEPTOR NEEDED ==============" //#define is_donor_chars(x) 1 //#define paired_chars(x,y,z) 1 #define is_donor_chars is_donor_chars_part #define paired_chars paired_chars_part_core void print_big_margin(global_context_t * global_context, subread_read_number_t pair_number, int is_second_read){ unsigned short * big_margin_record = _global_retrieve_big_margin_ptr(global_context,pair_number, is_second_read); int x1; SUBREADprintf("\n >>> READ_NO=%u, SECOND=%d, MEM=%p <<< \n", (unsigned int)pair_number, is_second_read, big_margin_record); for(x1 = 0; x1 < global_context->config.big_margin_record_size/3 ; x1++) { SUBREADprintf("%d %d~%d ", big_margin_record[x1*3] , big_margin_record[x1*3+1] , big_margin_record[x1*3+2]); } SUBREADputs(""); } #define ABGIGUOUS_TOLERANCE 3 int is_ambiguous_voting(global_context_t * global_context, subread_read_number_t pair_number, int is_second_read, int selected_vote, int max_start,int max_end, int read_len, int is_negative) { // #warning "=========== THE NEXT LINE IS ONLY FOR COMPARING WITH STAR!! ============== " // return 0; if( global_context->config.big_margin_record_size<3) return 0; int xk1; int encounter = 0; if(is_negative) { int tmp = max_start; max_start = read_len - max_end; max_end = read_len - tmp; } unsigned short * big_margin_record = _global_retrieve_big_margin_ptr(global_context,pair_number, is_second_read); for(xk1 = 0; xk1 < global_context->config.big_margin_record_size/3 ; xk1++) { if(!big_margin_record[xk1*3])break; if(big_margin_record[xk1*3] >= selected_vote - 1) // actually, max-1 { if(0) { if ( max_start >= big_margin_record[xk1*3+1] - ABGIGUOUS_TOLERANCE && max_end <= big_margin_record[xk1*3+2] + ABGIGUOUS_TOLERANCE ) encounter++; else if ( big_margin_record[xk1*3+1] >= max_start - ABGIGUOUS_TOLERANCE && big_margin_record[xk1*3+2] <= max_end + ABGIGUOUS_TOLERANCE ) encounter++; } else { // 4 and 4 are the best setting for indel and fusion simulation. if(selected_vote >= big_margin_record[xk1*3]) { if(big_margin_record[xk1*3+1] >= max_start - 4 && big_margin_record[xk1*3+2] <= max_end + 4) encounter++; } else { if(big_margin_record[xk1*3+1] <= max_start + 4 && big_margin_record[xk1*3+2] >= max_end - 4) encounter++; } } } } if(encounter>1) return encounter; return 0; } #define JUNCTION_CONFIRM_WINDOW 17 // This function implements the same function of donor_score, except that the two halves are from different strands. // Both halves are forced to positive strand and the split point is found. // Note that the donor/receptor sides are still expected for distinguishing between Fusion Breaks and Fusion Junctions. // Note that the read_text is on reversed mode. The guess points are on reversed mode too. // "Left" and "Right" means the left/right half in the "reversed" read. int donor_jumped_score(global_context_t * global_context, thread_context_t * thread_context, unsigned int small_virtualHead_abs_offset, unsigned int large_virtualHead_abs_offset, int guess_start, int guess_end, char * read_text, int read_len, int is_small_half_negative, int is_large_half_negative, int small_half_on_left_reversed, int * final_split_point, int * is_GT_AG_strand, int * is_donor_found_or_annotation, int * small_side_increasing_coordinate, int * large_side_increasing_coordinate) { gene_value_index_t * value_index = thread_context?thread_context->current_value_index:global_context->current_value_index ; // guess_end is the index of the first UNWANTED BASE. int most_likely_point_as_reversed = (guess_start+guess_end)/2; int selected_real_split_point = -1, selected_junction_strand = -1; //char donor_left[2], donor_right[2]; int best_score = -111111; int real_split_point_i; int real_split_point_numbers = guess_end - guess_start; char positive_read[MAX_READ_LENGTH+1]; strcpy(positive_read, read_text) ; reverse_read(positive_read, read_len, global_context->config.space_type); //printf("TEST_JUMPED: %u - %u\n", small_virtualHead_abs_offset, large_virtualHead_abs_offset); (*small_side_increasing_coordinate) = (small_half_on_left_reversed != is_small_half_negative); (*large_side_increasing_coordinate) = (small_half_on_left_reversed == is_large_half_negative); for(real_split_point_i = 0 ; real_split_point_i < real_split_point_numbers; real_split_point_i++) { int left_should_match, right_should_match; int left_should_not_match, right_should_not_match; int real_split_point_as_reversed = (real_split_point_i % 2)?-((real_split_point_i+1)/2):((1+real_split_point_i)/2); real_split_point_as_reversed += most_likely_point_as_reversed; if(real_split_point_as_reversed > read_len-JUNCTION_CONFIRM_WINDOW)continue; if(real_split_point_as_reversed < JUNCTION_CONFIRM_WINDOW)continue; int is_donor_test_ok=0; if(small_half_on_left_reversed) { unsigned int small_pos_test_begin = small_virtualHead_abs_offset + (is_small_half_negative?real_split_point_as_reversed - JUNCTION_CONFIRM_WINDOW:(read_len - real_split_point_as_reversed)); char * small_pos_read_begin = (is_small_half_negative?read_text:positive_read) + (is_small_half_negative? (real_split_point_as_reversed - JUNCTION_CONFIRM_WINDOW) : (read_len - real_split_point_as_reversed) ); unsigned int large_pos_test_begin = large_virtualHead_abs_offset + (is_large_half_negative?real_split_point_as_reversed:(read_len - real_split_point_as_reversed - JUNCTION_CONFIRM_WINDOW)); char * large_pos_read_begin = (is_large_half_negative?read_text:positive_read) + (is_large_half_negative? (real_split_point_as_reversed) : (read_len - real_split_point_as_reversed - JUNCTION_CONFIRM_WINDOW)); left_should_match = match_chro(small_pos_read_begin , value_index , small_pos_test_begin , JUNCTION_CONFIRM_WINDOW , 0, global_context -> config.space_type); right_should_match = match_chro(large_pos_read_begin , value_index , large_pos_test_begin , JUNCTION_CONFIRM_WINDOW , 0, global_context -> config.space_type); left_should_not_match = right_should_not_match = 0; //match_chro(read_text + real_split_point - JUNCTION_CONFIRM_WINDOW, value_index, small_virtualHead_abs_offset + real_split_point - JUNCTION_CONFIRM_WINDOW , JUNCTION_CONFIRM_WINDOW , 0, global_context -> config.space_type); } else { unsigned int small_pos_test_begin = small_virtualHead_abs_offset + (is_small_half_negative?real_split_point_as_reversed:(read_len - real_split_point_as_reversed - JUNCTION_CONFIRM_WINDOW)); char * small_pos_read_begin = (is_small_half_negative?read_text:positive_read) + (is_small_half_negative? (real_split_point_as_reversed):(read_len - real_split_point_as_reversed - JUNCTION_CONFIRM_WINDOW)); unsigned int large_pos_test_begin = large_virtualHead_abs_offset + (is_large_half_negative?(real_split_point_as_reversed - JUNCTION_CONFIRM_WINDOW):(read_len - real_split_point_as_reversed)); char * large_pos_read_begin = (is_large_half_negative?read_text:positive_read) + (is_large_half_negative? (real_split_point_as_reversed - JUNCTION_CONFIRM_WINDOW):(read_len - real_split_point_as_reversed)); left_should_match = match_chro(small_pos_read_begin , value_index , small_pos_test_begin , JUNCTION_CONFIRM_WINDOW , 0, global_context -> config.space_type); right_should_match = match_chro(large_pos_read_begin , value_index , large_pos_test_begin , JUNCTION_CONFIRM_WINDOW , 0, global_context -> config.space_type); left_should_not_match = right_should_not_match = 0; } //#warning "============ REMOVE THE TWO '+ 1' FROM THE NEXT LINE =================" //#warning "============ ADD THE TWO '+ 1's IN THE BLANKETS FOR SVs GRANT APP =================" int mismatch_in_between_allowd = (global_context -> config.more_accurate_fusions)?(0):(1); if(left_should_match + right_should_match >= JUNCTION_CONFIRM_WINDOW*2 - mismatch_in_between_allowd && left_should_not_match <= JUNCTION_CONFIRM_WINDOW -3 && right_should_not_match <= JUNCTION_CONFIRM_WINDOW -3) { int test_score = is_donor_test_ok*500+left_should_match + right_should_match - left_should_not_match - right_should_not_match; if(test_score > best_score) { selected_real_split_point = real_split_point_as_reversed; best_score = test_score; } } } if(best_score>0) { //printf("TEST_JUMPED: BSCORE=%d SPLT=%d\n", best_score , selected_real_split_point); *final_split_point = selected_real_split_point; *is_donor_found_or_annotation = best_score>=500; *is_GT_AG_strand = selected_junction_strand; return best_score; } return 0; } int donor_score(global_context_t * global_context, thread_context_t * thread_context, unsigned int left_virtualHead_abs_offset, unsigned int right_virtualHead_abs_offset, int left_indel_offset, int right_indel_offset, int normally_arranged, int guess_start, int guess_end, char * read_text, int read_len, int * final_split_point, int * is_GT_AG_strand, int * is_donor_found_or_annotation, int * final_inserted_bases, int * small_side_increasing_coordinate, int * large_side_increasing_coordinate, char * read_name) { gene_value_index_t * value_index = thread_context?thread_context->current_value_index:global_context->current_value_index; int need_donor_test = global_context->config.do_breakpoint_detection && global_context -> config.check_donor_at_junctions && (!( global_context-> config.do_fusion_detection || global_context-> config.do_long_del_detection)); (*small_side_increasing_coordinate)=!normally_arranged; (*large_side_increasing_coordinate)= normally_arranged; // guess_end is the index of the first UNWANTED BASE. int most_likely_point = (guess_start+guess_end)/2; // "split_point" is the first base NOT IN piece 1; it is also the first base IN piece 2. int selected_real_split_point = -1, selected_junction_strand = -1 , selected_inserted_bases = 0; char donor_left[3], donor_right[3]; int best_score = -111111; int non_insertion_preferred = 0; int real_split_point_i; int real_split_point_numbers = guess_end - guess_start; if(0 && FIXLENstrcmp("R006856515", read_name) == 0) SUBREADprintf("TESTDON: LR=%d; RR=%d\n", left_indel_offset, right_indel_offset); for(real_split_point_i = 0 ; real_split_point_i < real_split_point_numbers; real_split_point_i++) { int left_should_match, right_should_match = 0; int left_should_not_match = 0, right_should_not_match = 0; int real_split_point = (real_split_point_i % 2)?-((real_split_point_i+1)/2):((1+real_split_point_i)/2); real_split_point += most_likely_point; int is_donor_test_ok = 0; if(real_split_point > read_len-JUNCTION_CONFIRM_WINDOW)continue; if(real_split_point < JUNCTION_CONFIRM_WINDOW)continue; if(global_context->config.prefer_donor_receptor_junctions) { if(normally_arranged) { gvindex_get_string (donor_left, value_index, left_virtualHead_abs_offset + real_split_point + left_indel_offset, 2, 0); if(is_donor_chars(donor_left)) { gvindex_get_string (donor_right, value_index, right_virtualHead_abs_offset + real_split_point + right_indel_offset - 2, 2, 0); if(is_donor_chars(donor_right)) { is_donor_test_ok = paired_chars(donor_left, donor_right,0); } } } else { gvindex_get_string (donor_left, value_index, right_virtualHead_abs_offset + real_split_point + left_indel_offset, 2, 0); gvindex_get_string (donor_right, value_index, left_virtualHead_abs_offset + real_split_point + right_indel_offset - 2, 2, 0); is_donor_test_ok = is_donor_chars(donor_left) && is_donor_chars(donor_right) && paired_chars(donor_left, donor_right,0); } } // donor_left[2]=0; donor_right[2]=0; if(0 && FIXLENstrcmp("R006856515", read_name) == 0) { donor_left[2]=0; donor_right[2]=0; SUBREADprintf("TESTDON: %s %s; OFFSET=%d; DON_OK=%d; NORMAL=%d; LEFT_OFF=%d; RIGHT_OFF=%d\n", donor_left, donor_right, real_split_point_i, is_donor_test_ok, normally_arranged, left_indel_offset, right_indel_offset); } //#warning "============ REMOVE THE TWO '+ 1' FROM THE NEXT LINE =================" //#warning "============ ADD TWO '+ 1' IN THE BLANKETS FOR SVs GRANT APP =================" int mismatch_in_between_allowd = (global_context -> config.more_accurate_fusions)?(0) : (1); if(is_donor_test_ok || !need_donor_test) { if(normally_arranged) { int inserted_bases=0; left_should_match = match_chro(read_text + real_split_point - JUNCTION_CONFIRM_WINDOW, value_index, left_virtualHead_abs_offset + real_split_point - JUNCTION_CONFIRM_WINDOW + left_indel_offset , JUNCTION_CONFIRM_WINDOW , 0, global_context -> config.space_type); //printf("INS=%d; LM=%d\t\tLOL=%u, LOR=%u, SP=%d\n", inserted_bases, left_should_match, left_virtualHead_abs_offset, right_virtualHead_abs_offset, real_split_point); if(left_should_match > JUNCTION_CONFIRM_WINDOW- (global_context->config.max_insertion_at_junctions?5:2)) { for(inserted_bases = 0; inserted_bases <= global_context->config.max_insertion_at_junctions; inserted_bases++) { right_should_match = match_chro(read_text + real_split_point + inserted_bases, value_index, right_virtualHead_abs_offset + real_split_point + right_indel_offset + inserted_bases, JUNCTION_CONFIRM_WINDOW , 0, global_context -> config.space_type); // printf("INS=%d; LM=%d; RM=%d\t\tLOL=%u, LOR=%u, SP=%d\n", inserted_bases, left_should_match, right_should_match, left_virtualHead_abs_offset, right_virtualHead_abs_offset, real_split_point); if(right_should_match >= 2*JUNCTION_CONFIRM_WINDOW - left_should_match - mismatch_in_between_allowd) { left_should_not_match = match_chro(read_text + real_split_point + inserted_bases, value_index, left_virtualHead_abs_offset + real_split_point + left_indel_offset, JUNCTION_CONFIRM_WINDOW , 0, global_context -> config.space_type); right_should_not_match = match_chro(read_text + real_split_point - JUNCTION_CONFIRM_WINDOW, value_index, right_virtualHead_abs_offset + real_split_point + right_indel_offset - JUNCTION_CONFIRM_WINDOW + inserted_bases, JUNCTION_CONFIRM_WINDOW , 0, global_context -> config.space_type); if(left_should_not_match <= JUNCTION_CONFIRM_WINDOW -5 && right_should_not_match <= JUNCTION_CONFIRM_WINDOW -5) { int test_score ; if(global_context->config.max_insertion_at_junctions) test_score = 100*(is_donor_test_ok*3000+left_should_match + right_should_match) - (left_should_not_match + right_should_not_match) - 20*inserted_bases; else test_score = 100*(is_donor_test_ok*3000+left_should_match + right_should_match - left_should_not_match - right_should_not_match); if(test_score > best_score) { //if(left_virtualHead_abs_offset > 2729745284 - 200 && left_virtualHead_abs_offset< 2729745284 + 200) // SUBREADprintf("INS=%d; BSS=%d; TSC=%d\n%s\n\n", inserted_bases , best_score, test_score, read_text); selected_junction_strand = (donor_left[0]=='G' || donor_right[1]=='G'); selected_inserted_bases = inserted_bases; selected_real_split_point = real_split_point; best_score = test_score; } } } if(global_context->config.max_insertion_at_junctions && 0 == inserted_bases && right_should_match >= 2*JUNCTION_CONFIRM_WINDOW - left_should_match - 5) non_insertion_preferred = 1; } } } else { right_should_match = match_chro(read_text + real_split_point - JUNCTION_CONFIRM_WINDOW, value_index, right_virtualHead_abs_offset + right_indel_offset + real_split_point - JUNCTION_CONFIRM_WINDOW , JUNCTION_CONFIRM_WINDOW , 0, global_context -> config.space_type); left_should_match = match_chro(read_text + real_split_point, value_index, left_virtualHead_abs_offset + real_split_point + left_indel_offset, JUNCTION_CONFIRM_WINDOW , 0, global_context -> config.space_type); right_should_not_match = match_chro(read_text + real_split_point, value_index, right_virtualHead_abs_offset + real_split_point + right_indel_offset, JUNCTION_CONFIRM_WINDOW , 0, global_context -> config.space_type); left_should_not_match = match_chro(read_text + real_split_point - JUNCTION_CONFIRM_WINDOW, value_index, left_virtualHead_abs_offset + left_indel_offset + real_split_point - JUNCTION_CONFIRM_WINDOW, JUNCTION_CONFIRM_WINDOW , 0, global_context -> config.space_type); //printf("LEFT:MA=%d UMA=%d RIGHT:MA=%d UMA=%d\n", left_should_match, left_should_not_match, right_should_match, right_should_not_match); if(left_should_match +right_should_match >= 2*JUNCTION_CONFIRM_WINDOW - mismatch_in_between_allowd && left_should_not_match <= JUNCTION_CONFIRM_WINDOW -5 && right_should_not_match <= JUNCTION_CONFIRM_WINDOW -5) { int test_score; test_score = 100*(is_donor_test_ok*3000+left_should_match + right_should_match - left_should_not_match - right_should_not_match); if(test_score > best_score) { selected_junction_strand = (donor_left[0]=='G' || donor_right[1]=='G'); selected_real_split_point = real_split_point; best_score = test_score; } } } } } if(best_score>0 && (0==non_insertion_preferred || 0==selected_inserted_bases)) { *final_split_point = selected_real_split_point; *is_donor_found_or_annotation = best_score>=290000; *is_GT_AG_strand = selected_junction_strand; *final_inserted_bases = selected_inserted_bases; if(0 && FIXLENstrcmp("R006856515", read_name)==0) SUBREADprintf("FINAL_INS_LEN=%d; BEST_SCORE=%d %s\n", selected_inserted_bases, best_score, read_name); return (1+best_score)/100; } return 0; } void find_new_junctions(global_context_t * global_context, thread_context_t * thread_context, subread_read_number_t pair_number, char * read_name, char * read_text, char * qual_text, int read_len, int is_second_read, int best_read_id) { mapping_result_t * result =_global_retrieve_alignment_ptr(global_context, pair_number, is_second_read, best_read_id); subjunc_result_t * subjunc_result =_global_retrieve_subjunc_ptr(global_context, pair_number, is_second_read, best_read_id); if(read_len > EXON_LONG_READ_LENGTH) { assert(result -> selected_position <= 0xffff0000); core_search_short_exons(global_context, thread_context, read_text, qual_text, read_len, result -> selected_position, (subjunc_result -> minor_votes < 1)? result -> selected_position:subjunc_result -> minor_position, result -> confident_coverage_start, result -> confident_coverage_end); } int selected_real_split_point = subjunc_result->split_point; //#warning " =============== remove "+ 2" FROM THE NEXT LINE (FOR A HIGHER ACCURACY FROM SubFusion on 19 JAN 2015) ==================" if((global_context -> config.do_fusion_detection || global_context -> config.do_long_del_detection) && subjunc_result -> minor_votes < 1)return; if((!(global_context -> config.do_fusion_detection || global_context -> config.do_long_del_detection)) && subjunc_result -> minor_votes < 1)return; //if(result -> selected_votes < global_context->config.minimum_subread_for_first_read)return; if(global_context->config.do_big_margin_filtering_for_junctions) if(is_ambiguous_voting(global_context, pair_number, is_second_read, result->selected_votes, result -> confident_coverage_start, result -> confident_coverage_end, read_len, (result->result_flags & CORE_IS_NEGATIVE_STRAND)?1:0))return; unsigned int left_virtualHead_abs_offset = min(result -> selected_position, subjunc_result -> minor_position); unsigned int right_virtualHead_abs_offset = max(result -> selected_position, subjunc_result -> minor_position); int is_GT_AG_donors = result->result_flags & 0x3; int is_donor_found_or_annotation = is_GT_AG_donors<3; int is_strand_jumped = (result->result_flags & CORE_IS_STRAND_JUMPED)?1:0; if(selected_real_split_point>0) { unsigned int left_edge_wanted, right_edge_wanted; if(is_strand_jumped) { if(0){ // note that splicing point and the coverage coordinates are "major negative" view. // recover the "negative view" splicing point location int S = (result->result_flags & CORE_IS_NEGATIVE_STRAND) ? selected_real_split_point : (read_len - selected_real_split_point); int Sbar = read_len - S; int is_abnormal_as_reversed = (subjunc_result->minor_coverage_start > result->confident_coverage_start) + (subjunc_result -> minor_position > result -> selected_position) == 1; if(!(result->result_flags & CORE_IS_NEGATIVE_STRAND)) is_abnormal_as_reversed = !is_abnormal_as_reversed; int is_small_half_negative = ((result->result_flags & CORE_IS_NEGATIVE_STRAND)?1:0) + (subjunc_result->minor_position < result->selected_position) ==1; if(is_abnormal_as_reversed && is_small_half_negative) { left_edge_wanted = left_virtualHead_abs_offset + S; right_edge_wanted = right_virtualHead_abs_offset + Sbar; } else if(is_abnormal_as_reversed && !is_small_half_negative) { left_edge_wanted = left_virtualHead_abs_offset + Sbar - 1; right_edge_wanted = right_virtualHead_abs_offset + S - 1; } else if(!is_abnormal_as_reversed && is_small_half_negative) { left_edge_wanted = left_virtualHead_abs_offset + S - 1; right_edge_wanted = right_virtualHead_abs_offset + Sbar - 1; } else // if(!is_abnormal_as_reversed && !is_small_half_negative) { left_edge_wanted = left_virtualHead_abs_offset + Sbar; right_edge_wanted = right_virtualHead_abs_offset + S; } if(left_edge_wanted >= right_edge_wanted){ SUBREADprintf("REVERSED NEW JUNC: %u ~ %u : ABN_REV=%d , SMALL_NEG=%d, LEFT_VH=%u, RIGHT_VH=%u, S/~S=%d/%d\n", left_edge_wanted, right_edge_wanted, is_abnormal_as_reversed, is_small_half_negative, left_virtualHead_abs_offset, right_virtualHead_abs_offset, S, Sbar); } }else{ unsigned int major_half_smallest_coordinate, minor_half_smallest_coordinate; major_half_smallest_coordinate = result -> selected_position + selected_real_split_point; minor_half_smallest_coordinate = subjunc_result->minor_position + read_len - selected_real_split_point; left_edge_wanted = min(major_half_smallest_coordinate, minor_half_smallest_coordinate); right_edge_wanted = max(major_half_smallest_coordinate, minor_half_smallest_coordinate); int is_abnormal_as_reversed = (subjunc_result->minor_coverage_start > result->confident_coverage_start) + (minor_half_smallest_coordinate > major_half_smallest_coordinate) == 1; int is_small_half_negative = ((result->result_flags & CORE_IS_NEGATIVE_STRAND)?1:0) + (minor_half_smallest_coordinate < major_half_smallest_coordinate) ==1; if(!(result->result_flags & CORE_IS_NEGATIVE_STRAND)) is_abnormal_as_reversed = !is_abnormal_as_reversed; if(is_small_half_negative != is_abnormal_as_reversed) { left_edge_wanted -=1; right_edge_wanted -=1; } } } else { int selected_real_split_point_for_left = selected_real_split_point; int selected_real_split_point_for_right = selected_real_split_point; if((subjunc_result->minor_coverage_start > result->confident_coverage_start) + (subjunc_result -> minor_position > result -> selected_position) == 1) //abnormally arranged halves selected_real_split_point_for_right --; else // normally arranged halves selected_real_split_point_for_left --; int minor_indel_offset = (subjunc_result->double_indel_offset & 0xf); int major_indel_offset = (subjunc_result->double_indel_offset >> 4) & 0xf; if(major_indel_offset>=8)major_indel_offset=-(16-major_indel_offset); //assert(minor_indel_offset==0); //assert(major_indel_offset==0); left_edge_wanted = left_virtualHead_abs_offset + selected_real_split_point_for_left + ((result -> selected_position > subjunc_result -> minor_position)?minor_indel_offset: major_indel_offset); right_edge_wanted = right_virtualHead_abs_offset + selected_real_split_point_for_right; } char * chro_name_left, *chro_name_right; int chro_pos_left,chro_pos_right; locate_gene_position( left_edge_wanted , &global_context -> chromosome_table, &chro_name_left, &chro_pos_left); locate_gene_position( right_edge_wanted , &global_context -> chromosome_table, &chro_name_right, &chro_pos_right); if((!( global_context-> config.do_fusion_detection || global_context-> config.do_long_del_detection) ) && chro_name_right!=chro_name_left) return; //insert event HashTable * event_table = NULL; chromosome_event_t * event_space = NULL; if(thread_context) { event_table = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> event_entry_table; event_space = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; } else { event_table = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_entry_table; event_space = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; } // note that selected_real_split_point is the first UNWANTED base after left half. //if(abs(left_edge_wanted-27286396) < 250 || abs(right_edge_wanted - 27286396)<250) if(0 && FIXLENstrcmp("R003738400", read_name) == 0) { char leftpos[100], rightpos[100]; absoffset_to_posstr(global_context, left_edge_wanted, leftpos); absoffset_to_posstr(global_context, right_edge_wanted, rightpos); SUBREADprintf("READ=%s, LEFT=%s, RIGHT=%s\n", read_name, leftpos, rightpos); } chromosome_event_t * found = NULL; chromosome_event_t * search_return [MAX_EVENT_ENTRIES_PER_SITE]; int found_events = search_event(global_context, event_table, event_space, left_edge_wanted , EVENT_SEARCH_BY_SMALL_SIDE, CHRO_EVENT_TYPE_INDEL | CHRO_EVENT_TYPE_JUNCTION | CHRO_EVENT_TYPE_FUSION, search_return); mark_gapped_read(result); if(found_events) { int kx1; for(kx1 = 0; kx1 < found_events ; kx1++) { if(search_return[kx1] -> event_large_side == right_edge_wanted) { found = search_return[kx1]; break; } } } //if( 1018082 == pair_number) // SUBREADprintf("NEW_CHIMERISM_HERE [%u:%d: R_%d] : %s , %s , %u , %u, %c ; INC=%d %d\n", pair_number, best_read_id, is_second_read+1, chro_name_left, chro_name_right, chro_pos_left, chro_pos_right, is_strand_jumped?'X':'=', subjunc_result -> small_side_increasing_coordinate, subjunc_result -> large_side_increasing_coordinate); int is_key_fusion = 0; if(0)if( ( 9566365 + 1210 - 200 <= left_edge_wanted && 9566365 + 1210 + 200 >= left_edge_wanted) && ( 36859887 + 1210 - 200 <= right_edge_wanted && 36859887 + 1210 + 200 >= right_edge_wanted) ){ SUBREADprintf("Read = %s, FOUND = %p in %d , %s:%u , %s:%u, INCs= %d, %d, JUMP=%d\n", read_name, found, found_events, chro_name_left, chro_pos_left, chro_name_right, chro_pos_right, subjunc_result -> small_side_increasing_coordinate, subjunc_result -> large_side_increasing_coordinate, is_strand_jumped); is_key_fusion = 1; } if(found) found -> supporting_reads ++; else { int event_no; if(thread_context) event_no = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> total_events ++; else event_no = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> total_events ++; event_space = reallocate_event_space(global_context, thread_context, event_no); chromosome_event_t * new_event = event_space+event_no; memset(new_event,0,sizeof(chromosome_event_t)); new_event -> event_small_side = left_edge_wanted; new_event -> event_large_side = right_edge_wanted + subjunc_result->indel_at_junction; new_event -> critical_read_id = 2llu * pair_number + is_second_read; int new_event_type =(((global_context -> config.entry_program_name == CORE_PROGRAM_SUBJUNC && global_context -> config.do_fusion_detection)||(global_context -> config.entry_program_name == CORE_PROGRAM_SUBJUNC && global_context -> config.do_long_del_detection))&& !global_context -> config.prefer_donor_receptor_junctions)?CHRO_EVENT_TYPE_FUSION:CHRO_EVENT_TYPE_JUNCTION; //#warning "=========================== DELETE NEXT LINE !!! ==================================" //new_event_type = CHRO_EVENT_TYPE_REMOVED; if(is_strand_jumped) new_event_type = CHRO_EVENT_TYPE_FUSION; if((subjunc_result->minor_coverage_start > result->confident_coverage_start) + (subjunc_result -> minor_position > result -> selected_position) ==1) new_event_type = CHRO_EVENT_TYPE_FUSION; if(chro_name_right!=chro_name_left) new_event_type = CHRO_EVENT_TYPE_FUSION; if(right_edge_wanted - left_edge_wanted > global_context -> config.maximum_intron_length) if(!(global_context -> config.do_fusion_detection || global_context -> config.do_long_del_detection)) new_event_type = CHRO_EVENT_TYPE_REMOVED; if(1) { unsigned int dist = new_event -> event_large_side - new_event -> event_small_side; int origin_type = new_event_type; int fusion_cover_len = -1; if(dist > MAX_INSERTION_LENGTH && new_event_type == CHRO_EVENT_TYPE_FUSION) { int cov_end, cover_start, major_cov; cov_end = max(subjunc_result->minor_coverage_end, result->confident_coverage_end ); cover_start = min(subjunc_result->minor_coverage_start, result->confident_coverage_start); major_cov = result->confident_coverage_end - result->confident_coverage_start; fusion_cover_len = cov_end - cover_start ; if(fusion_cover_len < read_len - 15 || major_cov > read_len - 15) new_event_type = CHRO_EVENT_TYPE_REMOVED; } if(dist > MAX_INSERTION_LENGTH && new_event_type == CHRO_EVENT_TYPE_FUSION && subjunc_result -> minor_votes < 2) new_event_type = CHRO_EVENT_TYPE_REMOVED; else if(new_event_type == CHRO_EVENT_TYPE_FUSION && subjunc_result -> minor_votes < 1) new_event_type = CHRO_EVENT_TYPE_REMOVED; if(dist > MAX_INSERTION_LENGTH && new_event_type == CHRO_EVENT_TYPE_FUSION && result -> selected_votes < 2) new_event_type = CHRO_EVENT_TYPE_REMOVED; else if(new_event_type == CHRO_EVENT_TYPE_FUSION && result -> selected_votes < 1) new_event_type = CHRO_EVENT_TYPE_REMOVED; if(0 && origin_type == CHRO_EVENT_TYPE_FUSION) { char leftpos[100], rightpos[100]; absoffset_to_posstr(global_context, new_event -> event_small_side, leftpos); absoffset_to_posstr(global_context, new_event -> event_large_side, rightpos); if(new_event_type == CHRO_EVENT_TYPE_REMOVED) SUBREADprintf("NEW_FUSION REMOVED %s SUGGEST %s ~ %s MAJOR COV=%d ~ %d, MINOR COV=%d ~ %d, RLEN=%d, COVED=%d, VOTES=%d, %d, %s, SPLIT=%d\n", read_name, leftpos, rightpos, result->confident_coverage_start, result->confident_coverage_end, subjunc_result->minor_coverage_start, subjunc_result->minor_coverage_end, read_len, fusion_cover_len, result -> selected_votes, subjunc_result -> minor_votes, is_strand_jumped?"JUMPED":"======", selected_real_split_point); else SUBREADprintf("NEW_FUSION WANTED %s SUGGEST %s ~ %s MAJOR COV=%d ~ %d, MINOR COV=%d ~ %d, RLEN=%d, COVED=%d, VOTES=%d, %d, %s, SPLIT=%d\n", read_name, leftpos, rightpos, result->confident_coverage_start, result->confident_coverage_end, subjunc_result->minor_coverage_start, subjunc_result->minor_coverage_end, read_len, fusion_cover_len, result -> selected_votes, subjunc_result -> minor_votes, is_strand_jumped?"JUMPED":"======", selected_real_split_point); } if(dist > MAX_INSERTION_LENGTH && new_event_type == CHRO_EVENT_TYPE_FUSION && (selected_real_split_point < read_len * 0.2 || selected_real_split_point >= read_len *0.8000) ) new_event_type = CHRO_EVENT_TYPE_REMOVED; } //if(pair_number == 13) //printf("MMMMX %d %u -- %u : TYPE %d\n" , event_no, left_edge_wanted, right_edge_wanted, new_event_type); // if((is_donor_found_or_annotation || !global_context -> config.check_donor_at_junctions) &&(!is_strand_jumped) && right_edge_wanted - left_edge_wanted <= global_context -> config.maximum_intron_length // && (subjunc_result->minor_coverage_start > result->confident_coverage_start) + (subjunc_result -> minor_position > result -> selected_position) !=1) if(is_key_fusion) SUBREADprintf(" INSERTED AS %d ( in %d or %d )\n", new_event_type , CHRO_EVENT_TYPE_JUNCTION, CHRO_EVENT_TYPE_FUSION); if(new_event_type == CHRO_EVENT_TYPE_JUNCTION) { new_event -> is_negative_strand= !is_GT_AG_donors; new_event -> event_type = CHRO_EVENT_TYPE_JUNCTION; new_event -> supporting_reads = 1; new_event -> indel_length = 0; new_event -> indel_at_junction = subjunc_result->indel_at_junction; new_event -> is_donor_found_or_annotation = is_donor_found_or_annotation; new_event -> small_side_increasing_coordinate = subjunc_result -> small_side_increasing_coordinate; new_event -> large_side_increasing_coordinate = subjunc_result -> large_side_increasing_coordinate; put_new_event(event_table, new_event , event_no); if(0 && FIXLENstrcmp("R000000052", read_name) == 0) SUBREADprintf("NEW_JUNCTION_HERE : %s , %u , %u (%u, %u)\n", chro_name_right, chro_pos_left, chro_pos_right, new_event -> event_small_side, new_event -> event_large_side); } else if(new_event_type == CHRO_EVENT_TYPE_FUSION) { if((global_context -> config.do_fusion_detection || global_context -> config.do_long_del_detection)) { new_event -> event_type = CHRO_EVENT_TYPE_FUSION; new_event -> is_strand_jumped = is_strand_jumped; new_event -> supporting_reads = 1; new_event -> indel_length = 0; new_event -> small_side_increasing_coordinate = subjunc_result -> small_side_increasing_coordinate; new_event -> large_side_increasing_coordinate = subjunc_result -> large_side_increasing_coordinate; put_new_event(event_table, new_event , event_no); //if( 1018082 == pair_number) // SUBREADprintf("NEW_CHIMERISM_HERE_FULL [%u:%d: R_%d] : %s , %s , %u , %u, %c ; INC=%d %d\n", pair_number, best_read_id, is_second_read+1, chro_name_left, chro_name_right, chro_pos_left, chro_pos_right, is_strand_jumped?'X':'=', new_event -> small_side_increasing_coordinate, new_event -> large_side_increasing_coordinate); } } } } } void write_translocation_results_final(void * key, void * buckv, HashTable * tab); void write_inversion_results_final(void * key, void * buckv, HashTable * tab); int write_fusion_final_results(global_context_t * global_context) { indel_context_t * indel_context = (indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]; char fn2 [MAX_FILE_NAME_LENGTH+30]; sprintf(fn2,"%s.breakpoints.vcf", global_context->config.output_prefix); FILE * ofp = f_subr_open(fn2, "wb"); fprintf(ofp,"##fileformat=VCFv4.1\n"); fprintf(ofp,"##INFO=\n"); fprintf(ofp,"##INFO=\n"); fprintf(ofp,"##INFO=\n"); fprintf(ofp,"#CHROM POS ID REF ALT QUAL FILTER INFO\n"); int xk1, disk_is_full = 0; unsigned int all_junctions = 0; int no_sup_juncs = 0; int all_juncs = 0; for(xk1 = 0; xk1 < indel_context -> total_events ; xk1++) { char * chro_name_left,* chro_name_right; int chro_pos_left, chro_pos_right; chromosome_event_t * event_body = indel_context -> event_space_dynamic +xk1; if(event_body -> event_type != CHRO_EVENT_TYPE_FUSION && (global_context->config.entry_program_name != CORE_PROGRAM_SUBREAD || event_body -> event_type != CHRO_EVENT_TYPE_JUNCTION)) continue; all_juncs++; if(event_body->final_counted_reads<1|| event_body->critical_supporting_reads < 1 - 1) { no_sup_juncs++; continue; } locate_gene_position( event_body -> event_small_side , &global_context -> chromosome_table, &chro_name_left, &chro_pos_left); locate_gene_position( event_body -> event_large_side , &global_context -> chromosome_table, &chro_name_right, &chro_pos_right); chro_pos_left+=1; chro_pos_right+=1; all_junctions ++; int wlen; char alt_base[500]; char ref_base; char bkt = event_body -> large_side_increasing_coordinate?'[':']'; gene_value_index_t * current_index = find_current_value_index(global_context , event_body -> event_small_side , 1); ref_base = gvindex_get( current_index, event_body -> event_small_side); if(event_body -> small_side_increasing_coordinate) sprintf(alt_base,"%c%s:%u%c%c", bkt, chro_name_right, chro_pos_right, bkt, ref_base); else sprintf(alt_base,"%c%c%s:%u%c", ref_base, bkt, chro_name_right, chro_pos_right, bkt); wlen = fprintf(ofp,"%s\t%u\tbnd_%d\t%c\t%s\t.\tPASS\tSVTYPE=BND;MATEID=bnd_%d;SR=%d\n", chro_name_left, chro_pos_left, all_junctions *2 -1, ref_base, alt_base, all_junctions*2, event_body -> final_counted_reads); current_index = find_current_value_index(global_context , event_body -> event_large_side , 1); ref_base = gvindex_get( current_index, event_body -> event_large_side ); bkt = event_body -> small_side_increasing_coordinate?'[':']'; if(event_body -> large_side_increasing_coordinate) sprintf(alt_base,"%c%s:%u%c%c", bkt, chro_name_left, chro_pos_left, bkt, ref_base); else sprintf(alt_base,"%c%c%s:%u%c", ref_base, bkt, chro_name_left, chro_pos_left, bkt); wlen += fprintf(ofp,"%s\t%u\tbnd_%d\t%c\t%s\t.\tPASS\tSVTYPE=BND;MATEID=bnd_%d;SR=%d\n", chro_name_right, chro_pos_right, all_junctions *2, ref_base, alt_base, all_junctions*2 -1, event_body -> final_counted_reads); if(wlen <18) disk_is_full = 1; // fprintf(ofp, "%s\t%u\t%s\t%u\t%s\t%d\t%s\t%s\n", chro_name_left, chro_pos_left, chro_name_right, chro_pos_right+1, event_body -> is_strand_jumped?"No":"Yes", event_body -> final_counted_reads, event_body -> small_side_increasing_coordinate?"Yes":"No", event_body -> large_side_increasing_coordinate?"Yes":"No"); } global_context -> all_fusions = all_junctions; if(global_context->config.do_structural_variance_detection){ global_context -> translocation_result_table.entry_table -> appendix1 = ofp; global_context -> translocation_result_table.entry_table -> appendix2 = global_context; HashTableIteration(global_context -> translocation_result_table.entry_table, write_translocation_results_final); global_context -> inversion_result_table.entry_table -> appendix1 = ofp; global_context -> inversion_result_table.entry_table -> appendix2 = global_context; HashTableIteration(global_context -> inversion_result_table.entry_table, write_inversion_results_final); } fclose(ofp); if(disk_is_full){ unlink(fn2); SUBREADprintf("ERROR: disk is full. No fusion table is generated.\n"); } return 0; } void write_inversion_results_final(void * bukey, void * buckv, HashTable * tab){ int x1; bucketed_table_bucket_t * buck = buckv; FILE * ofp = (FILE *)tab -> appendix1; global_context_t * global_context = (global_context_t * )tab -> appendix2; for(x1 = 0; x1 < buck -> items; x1++) { if(buck->positions[x1] - buck->positions[x1] % buck -> maximum_interval_length == buck -> keyed_bucket) { inversion_result_t * inv_res = buck -> details[x1]; char * src_chr; int src_pos; locate_gene_position(inv_res -> small_side, &global_context -> chromosome_table, &src_chr , &src_pos); fprintf(ofp, "INV\t%s\t%d\t%s\t%u\t%s\n", src_chr, src_pos + 1, src_chr, src_pos + 1 + inv_res -> length, inv_res -> is_precisely_called ? "PRECISE":"IMPRECISE"); fprintf(ofp, "INV\t%s\t%d\t%s\t%u\t%s\n", src_chr, src_pos + 2, src_chr, src_pos + inv_res -> length, inv_res -> is_precisely_called ? "PRECISE":"IMPRECISE"); //fprintf(ofp, "INVERSION\t%s\t%u\t%u\t%u\t%u\n", src_chr, src_pos, inv_res -> length, inv_res -> all_sup_D , inv_res -> max_sup_E); } } } void write_translocation_results_final(void * bukey, void * buckv, HashTable * tab){ int x1; bucketed_table_bucket_t * buck = buckv; FILE * ofp = (FILE *)tab -> appendix1; global_context_t * global_context = (global_context_t * )tab -> appendix2; for(x1 = 0; x1 < buck -> items; x1++) { if(buck->positions[x1] - buck->positions[x1] % buck -> maximum_interval_length == buck -> keyed_bucket) { char * src_chr, *targ_chr; int src_pos, targ_pos; translocation_result_t * trans_res = buck -> details[x1]; locate_gene_position(trans_res -> source_left_side, &global_context -> chromosome_table, &src_chr , &src_pos); locate_gene_position(trans_res -> target_left_side, &global_context -> chromosome_table, &targ_chr , &targ_pos); //fprintf(ofp, "TRANSLOCATION\t%s\t%u\t%u\t%s\t%u\t%s\t%u\t%u\n", src_chr, src_pos, trans_res -> length, targ_chr, targ_pos, trans_res -> is_inv?"INV":"STR", trans_res -> all_sup_P , trans_res -> max_sup_QR); /* SUBREADprintf("ABS=%u, %u, PRECISE=%d\n", trans_res -> source_left_side, trans_res -> target_left_side, trans_res -> is_precisely_called); SUBREADprintf("%u, %u\n", src_pos, targ_pos); SUBREADprintf("%s, %s\n", src_chr, targ_chr); */ fprintf(ofp, "%s\t%s\t%u\t%s\t%d\t%s\t%s\n", src_chr == targ_chr?"ITX":"CTX", src_chr, src_pos + 1, targ_chr, targ_pos + 1, trans_res -> is_inv?"X":"=", trans_res -> is_precisely_called ? "PRECISE":"IMPRECISE"); fprintf(ofp, "%s\t%s\t%u\t%s\t%d\t%s\t%s\n", src_chr == targ_chr?"ITX":"CTX", src_chr, src_pos + trans_res -> length + 1, targ_chr, targ_pos + 1, trans_res -> is_inv?"X":"=", trans_res -> is_precisely_called ? "PRECISE":"IMPRECISE"); fprintf(ofp, "DEL\t%s\t%d\t%u\t%s\n", src_chr, src_pos + 1, trans_res -> length , trans_res -> is_precisely_called ? "PRECISE":"IMPRECISE"); } } } int write_junction_final_results(global_context_t * global_context) { int no_sup_juncs = 0, disk_is_full = 0; indel_context_t * indel_context = (indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]; char fn2 [MAX_FILE_NAME_LENGTH+30]; sprintf(fn2,"%s.junction.bed", global_context->config.output_prefix); FILE * ofp = f_subr_open(fn2, "wb"); fprintf(ofp, "#Chr, StartLeftBlock, EndRightBlock, Junction_Name, nSupport, Strand, StartLeftBlock, EndRightBlock, Color, nBlocks, BlockSizes, BlockStarts\n"); int xk1; unsigned int all_junctions = 0; for(xk1 = 0; xk1 < indel_context -> total_events ; xk1++) { char * chro_name_left,* chro_name_right, indel_sect[10]; int chro_pos_left, chro_pos_right; chromosome_event_t * event_body = indel_context -> event_space_dynamic +xk1; if(event_body -> event_type != CHRO_EVENT_TYPE_JUNCTION) continue; //#warning " ================================== remove '- 1' from the next line!!! =================================" if(event_body->final_counted_reads < 1 || ( event_body->critical_supporting_reads < 1 - 1&& event_body->indel_at_junction)) { no_sup_juncs++; continue; } locate_gene_position( event_body -> event_small_side , &global_context -> chromosome_table, &chro_name_left, &chro_pos_left); locate_gene_position( event_body -> event_large_side , &global_context -> chromosome_table, &chro_name_right, &chro_pos_right); chro_pos_left++; unsigned int feature_start = chro_pos_left - event_body -> junction_flanking_left; if(chro_pos_left <= event_body -> junction_flanking_left){ feature_start = 1; event_body -> junction_flanking_left = chro_pos_left - 1; } unsigned int feature_end = chro_pos_right + event_body -> junction_flanking_right; all_junctions ++; indel_sect[0]=0; if(event_body->indel_at_junction) sprintf(indel_sect,"INS%d", event_body->indel_at_junction); if(event_body-> is_donor_found_or_annotation &64)strcat(indel_sect,"ANNO"); //else if(event_body->critical_supporting_reads < 1) // strcpy(indel_sect, "NOCRT"); int wlen = fprintf(ofp,"%s\t%u\t%u\tJUNC%08u%s\t%d\t%c\t%u\t%u\t%d,%d,%d\t2\t%d,%d\t0,%u\n", chro_name_left, feature_start, feature_end, all_junctions, indel_sect, event_body -> final_counted_reads, event_body->is_negative_strand?'-':'+', feature_start, feature_end, event_body->is_negative_strand?0:255, /*event_body -> anti_supporting_reads*/ event_body->is_negative_strand?255:0, event_body->is_negative_strand?255:0, event_body -> junction_flanking_left, event_body -> junction_flanking_right, feature_end-feature_start-event_body -> junction_flanking_right); if(wlen < 10) disk_is_full = 1; } fclose(ofp); if(disk_is_full){ unlink(fn2); SUBREADprintf("ERROR: disk is full; no junction table is created.\n"); } global_context -> all_junctions = all_junctions; //printf("Non-support juncs=%d; Final juncs = %d\n", no_sup_juncs, all_junctions); return 0; } void get_chro_2base(char *buf, gene_value_index_t * index, unsigned int pos, int is_negative_strand) { gvindex_get_string (buf, index, pos, 2, is_negative_strand); } int paired_chars_part(char * ch1, char * ch2, int is_reverse) { if (c2eq(ch1, ch2, "GT", "AG") || c2eq(ch1, ch2, "CT", "AC")) { if (is_reverse) if (ceq(ch1, "AG") || ceq(ch1, "AC")) return 1; if (!is_reverse) if (ceq(ch1, "CT") || ceq(ch1, "GT")) return 1; } return 0; } #define is_donar_chars_part(cc) (((cc)[0]=='G' && (cc)[1]=='T') || \ ((cc)[0]=='A' && (cc)[1]=='G') || \ ((cc)[0]=='A' && (cc)[1]=='C') || \ ((cc)[0]=='C' && (cc)[1]=='T')) #define SHORT_EXON_MIN_LENGTH 18 #define EXON_EXTENDING_SCAN 0 #define SHORT_EXON_WINDOW 6 #define SHORT_EXON_EXTEND 5000 void core_search_short_exons(global_context_t * global_context, thread_context_t * thread_context, char * read_text, char * qualityb0, int rl, unsigned int P1_Pos, unsigned int P2_Pos, short read_coverage_start, short read_coverage_end) { char inb[MAX_READ_LENGTH], qualityb[MAX_READ_LENGTH]; if ( (rl <= EXON_LONG_READ_LENGTH ) && (!EXON_EXTENDING_SCAN)) return; //return; gene_value_index_t * base_index = thread_context?thread_context->current_value_index:global_context->current_value_index ; //insert event HashTable * event_table = NULL; chromosome_event_t * event_space = NULL; if(thread_context) { event_table = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> event_entry_table; event_space = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; } else { event_table = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_entry_table; event_space = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; } strcpy(inb, read_text); strcpy(qualityb, qualityb0); unsigned int pos_small=min(P1_Pos, P2_Pos), pos_big = max(P1_Pos, P2_Pos); int max_score , test_score; unsigned int best_j1_edge=0 , best_j2_edge=0; int need_to_test = 0; ////////////////////////////////////////////////////////////////////////////////////////////// ////////////////////////////////////////////////////////////////////////////////////////////// // SCAN TO THE HEAD ///////////////////////////////////////////////////////////////////////// ////////////////////////////////////////////////////////////////////////////////////////////// ////////////////////////////////////////////////////////////////////////////////////////////// if (read_coverage_start > SHORT_EXON_MIN_LENGTH) { max_score = -1; int need_check2 = 1; if(qualityb[0]) { float head_quality = read_quality_score(qualityb , SHORT_EXON_MIN_LENGTH , global_context->config.phred_score_format); if(head_quality < 6 ) need_check2 = 0; } if(need_check2) if(SHORT_EXON_MIN_LENGTH *0.6 < match_chro(inb, base_index, pos_small, SHORT_EXON_MIN_LENGTH , 0, global_context->config.space_type)) need_check2 = 0; if(need_check2) { int delta_pos, is_indel = 0; for(delta_pos=-3; delta_pos <=3; delta_pos ++) { if(match_chro(inb, base_index, pos_small + delta_pos, SHORT_EXON_MIN_LENGTH , 0, global_context->config.space_type) >= SHORT_EXON_MIN_LENGTH*.7) { is_indel = 1; break; } } // The head of the read is incorrect. Do we need to search a long way? // See if there is a donor in the head area. int test_donor_pos; char cc[3]; cc[2]=0; if(!is_indel) for(test_donor_pos = SHORT_EXON_MIN_LENGTH ; test_donor_pos < read_coverage_start ; test_donor_pos ++) { get_chro_2base(cc, base_index, pos_small + test_donor_pos, 0); if(is_donar_chars_part(cc)) { need_to_test = 1; break; } } } } max_score = -999; int max_is_GTAG = 0; if(need_to_test && pos_small >= SHORT_EXON_MIN_LENGTH) { unsigned int test_end = pos_small - SHORT_EXON_EXTEND; if(SHORT_EXON_EXTEND > pos_small) test_end = 0; unsigned int new_pos = pos_small-SHORT_EXON_MIN_LENGTH; while(1) { new_pos = match_chro_range(inb, base_index, new_pos, 7 , new_pos - test_end , SEARCH_BACK); if(new_pos==0xffffffff) break; // There is an exact match. See if the donor/receptors are matched. // new_pos is the new head position of the read. int splice_point; for(splice_point = SHORT_EXON_MIN_LENGTH; splice_point < read_coverage_start ; splice_point ++) { char cc[3]; cc[2]=0; char cc2[3]; cc2[2]=0; get_chro_2base(cc, base_index, pos_small + splice_point -2, 0); if(is_donar_chars_part(cc)) { // <<< EXON---|CC2---INTRON---CC|---EXON get_chro_2base(cc2, base_index, new_pos + splice_point, 0); if(is_donar_chars_part(cc2) && paired_chars_part(cc2 , cc, 0)) { int matched_in_exon_old = match_chro(inb + splice_point, base_index, pos_small + splice_point , SHORT_EXON_WINDOW , 0, global_context->config.space_type); int matched_in_exon_new = match_chro(inb, base_index, new_pos , splice_point, 0, global_context->config.space_type); test_score = 1000000+ (matched_in_exon_new )*10000 + matched_in_exon_old * 1000 + new_pos - test_end; if(test_score <= max_score) continue; max_score = test_score; if(matched_in_exon_new < splice_point || matched_in_exon_old < SHORT_EXON_WINDOW ) continue; max_is_GTAG = (cc2[0]=='G' || cc2[1]=='G'); //printf("EX CC=%s\tCC2=%s\tis_GTAG=%d\n",cc,cc2,max_is_GTAG); best_j1_edge = new_pos + splice_point - 1; best_j2_edge = pos_small + splice_point; } } } } } if(best_j1_edge>0) { int event_no; chromosome_event_t * search_return [MAX_EVENT_ENTRIES_PER_SITE]; chromosome_event_t * found = NULL; int found_events = search_event(global_context, event_table, event_space, best_j1_edge , EVENT_SEARCH_BY_SMALL_SIDE, CHRO_EVENT_TYPE_JUNCTION|CHRO_EVENT_TYPE_FUSION, search_return); if(found_events) { int kx1; for(kx1 = 0; kx1 < found_events ; kx1++) { if(search_return[kx1] -> event_large_side == best_j2_edge) { found = search_return[kx1]; break; } } } if(found) found -> supporting_reads ++; else { if(thread_context) event_no = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> total_events ++; else event_no = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> total_events ++; event_space = reallocate_event_space(global_context, thread_context, event_no); chromosome_event_t * new_event = event_space+event_no; memset(new_event,0,sizeof(chromosome_event_t)); new_event -> event_small_side = best_j1_edge; new_event -> event_large_side = best_j2_edge; new_event -> is_negative_strand= !max_is_GTAG; new_event -> event_type = CHRO_EVENT_TYPE_JUNCTION; new_event -> supporting_reads = 1; new_event -> indel_length = 0; put_new_event(event_table, new_event , event_no); } //printf("FOUND NEW JUNCTION HEAD: %u - %u\n", best_j1_edge, best_j2_edge); } ////////////////////////////////////////////////////////////////////////////////////////////// ////////////////////////////////////////////////////////////////////////////////////////////// // SCAN TO THE TAIL ///////////////////////////////////////////////////////////////////////// ////////////////////////////////////////////////////////////////////////////////////////////// ////////////////////////////////////////////////////////////////////////////////////////////// need_to_test = 0; max_score = -999; if (read_coverage_end< rl - SHORT_EXON_MIN_LENGTH) { int need_check2 = 1; if(qualityb[0]) { float head_quality = read_quality_score(qualityb + rl - SHORT_EXON_MIN_LENGTH , SHORT_EXON_MIN_LENGTH , global_context->config.phred_score_format); if(head_quality < 6 ) need_check2 = 0; } if(SHORT_EXON_MIN_LENGTH *0.6 < match_chro(inb + rl - SHORT_EXON_MIN_LENGTH, base_index, pos_big + rl - SHORT_EXON_MIN_LENGTH , SHORT_EXON_MIN_LENGTH , 0, global_context->config.space_type)) need_check2 = 0; if(need_check2) { int delta_pos, is_indel = 0; for(delta_pos=-3; delta_pos <=3; delta_pos ++) { if(match_chro(inb + rl - SHORT_EXON_MIN_LENGTH, base_index, pos_big + rl - SHORT_EXON_MIN_LENGTH + delta_pos, SHORT_EXON_MIN_LENGTH , 0, global_context->config.space_type) >= SHORT_EXON_MIN_LENGTH*.7) { is_indel = 1; break; } } // The head of the read is incorrect. Do we need to search a long way? // See if there is a donor in the head area. int test_donor_pos; char cc[3]; cc[2]=0; if(!is_indel) for(test_donor_pos = read_coverage_end ; test_donor_pos < rl ; test_donor_pos ++) { get_chro_2base(cc, base_index, pos_big + test_donor_pos, 0); if(is_donar_chars_part(cc)) { need_to_test = 1; break; } } } } best_j1_edge = 0; max_is_GTAG = 0; if(need_to_test) { unsigned int test_end = pos_big + SHORT_EXON_EXTEND; if(test_end > base_index -> length + base_index -> start_point) test_end = base_index -> length + base_index -> start_point; unsigned int new_pos = pos_big +rl - SHORT_EXON_MIN_LENGTH +16; while(1) { if(new_pos + test_end - new_pos < base_index-> start_base_offset + base_index->length) { assert(new_pos<0xffff0000); new_pos = match_chro_range(inb + rl - SHORT_EXON_MIN_LENGTH, base_index, new_pos, 7 , test_end - new_pos , SEARCH_FRONT); } else break; if(new_pos==0xffffffff) break; // There is an exact match. See if the donor/receptors are matched. // (new_pos + SHORT_EXON_MIN_LENGTH -rl + splice_point) is the new exon start. int splice_point; for(splice_point = read_coverage_end ; splice_point < rl - SHORT_EXON_MIN_LENGTH; splice_point ++) { char cc[3]; cc[2]=0; char cc2[3]; cc2[2]=0; unsigned int new_pos_tail = (new_pos + SHORT_EXON_MIN_LENGTH -rl + splice_point); get_chro_2base(cc, base_index, pos_big + splice_point, 0); if(is_donar_chars_part(cc)) { get_chro_2base(cc2, base_index, new_pos_tail -2, 0); if(is_donar_chars_part(cc2) && paired_chars_part(cc , cc2, 0)) { int matched_in_exon_new = match_chro(inb + splice_point, base_index, new_pos_tail , rl - splice_point , 0, global_context->config.space_type); int matched_in_exon_old = match_chro(inb + splice_point - SHORT_EXON_WINDOW , base_index, pos_big + splice_point - SHORT_EXON_WINDOW , SHORT_EXON_WINDOW, 0, global_context->config.space_type); test_score = 1000000+ (matched_in_exon_new)*10000 + matched_in_exon_old * 1000 + test_end - new_pos; if(test_score <= max_score) continue; max_score = test_score; if(matched_in_exon_new < (rl - splice_point) || matched_in_exon_old < SHORT_EXON_WINDOW) continue; // EXON ---|CC---INTRON---CC2|--- EXON >>> max_is_GTAG = (cc[0]=='G'|| cc[1]=='G'); best_j1_edge = pos_big + splice_point - 1; best_j2_edge = new_pos_tail; } } } } } if(best_j1_edge>0) { int event_no; chromosome_event_t * search_return [MAX_EVENT_ENTRIES_PER_SITE]; chromosome_event_t * found = NULL; int found_events = search_event(global_context, event_table, event_space, best_j1_edge , EVENT_SEARCH_BY_SMALL_SIDE, CHRO_EVENT_TYPE_JUNCTION|CHRO_EVENT_TYPE_FUSION, search_return); if(found_events) { int kx1; for(kx1 = 0; kx1 < found_events ; kx1++) { if(search_return[kx1] -> event_large_side == best_j2_edge) { found = search_return[kx1]; break; } } } if(found) found -> supporting_reads ++; else { if(thread_context) event_no = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> total_events ++; else event_no = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> total_events ++; event_space = reallocate_event_space(global_context, thread_context, event_no); chromosome_event_t * new_event = event_space+event_no; memset(new_event,0,sizeof(chromosome_event_t)); new_event -> event_small_side = best_j1_edge; new_event -> event_large_side = best_j2_edge; new_event -> is_negative_strand= !max_is_GTAG; new_event -> event_type = CHRO_EVENT_TYPE_JUNCTION; new_event -> supporting_reads = 1; new_event -> indel_length = 0; put_new_event(event_table, new_event , event_no); //printf("FOUND NEW JUNCTION TAIL: %u - %u\n", best_j1_edge, best_j2_edge); } } } int core_select_best_matching_halves_maxone(global_context_t * global_context, gene_vote_t * vote, unsigned int * best_pos1, unsigned int * best_pos2, int * best_vote1, int * best_vote2, char * is_abnormal, short * half_marks, int * is_reversed_halves, float accept_rate, int read_len, long long int hint_pos, int tolerable_bases, short * read_coverage_start, short * read_coverage_end, gene_vote_number_t * indel_in_p1, gene_vote_number_t * indel_in_p2, gehash_data_t max_pos, gene_vote_number_t max_votes, short max_start, short max_end, short max_mask, gene_vote_number_t * max_indel_recorder, int* best_select_max_votes, int rl) { int best_splicing_point = -1, i,j; char * best_chro_name, is_reversed; int best_chro_pos; int selected_max_votes = -1; is_reversed = (max_mask & IS_NEGATIVE_STRAND)?1:0; for (i=0; iitems[i]; j++) { char * chro_name; char is_partner_reversed; int chro_pos; int overlapped_len, overlap_start, overlap_end; // All logical conditions //if( (vote->votes[i][j] < vote-> coverage_start[i][j]) < 12 && (vote-> coverage_end[i][j] > rl - 12 )) continue; is_partner_reversed = (vote->masks [i][j] & IS_NEGATIVE_STRAND) ? 1:0; overlap_start = max(max_start , vote->coverage_start[i][j]); overlap_end = min(max_end , vote->coverage_end[i][j]); overlapped_len =overlap_end - overlap_start; int coverage_len = max_end - max_start + vote->coverage_end[i][j] - vote->coverage_start[i][j]; if (overlapped_len >0)coverage_len -= overlapped_len; //SUBREADprintf("MAX: %d-%d OTHER %d-%d COV=%d OVLP=%d\n", max_start, max_end, vote->coverage_start[i][j], vote->coverage_end[i][j], coverage_len, overlapped_len); if(overlapped_len >=14) continue; long long int dist = vote->pos[i][j]; dist -= max_pos; //SUBREADprintf ("D=%lld\n", abs(dist)); if (abs(dist)<6) continue; int support_r1 = 1; int support_r2 = 1; if (max_votes < support_r1 || vote->votes[i][j]coverage_start[i][j] < max_start) + is_reversed == 1) { locate_gene_position(max_pos + read_len, &(global_context -> chromosome_table) , &best_chro_name, &best_chro_pos); locate_gene_position(vote->pos[i][j] , &(global_context -> chromosome_table), &chro_name, &chro_pos); }else { locate_gene_position(max_pos , &(global_context -> chromosome_table), &best_chro_name, &best_chro_pos); locate_gene_position(vote->pos[i][j] +read_len, &(global_context -> chromosome_table), &chro_name, &chro_pos); } if (chro_name != best_chro_name) // The pointers can be compared because they can be the same. continue; int is_fusion = 0; if(is_reversed != is_partner_reversed) is_fusion = 1; if( is_reversed && ((max_pos > vote->pos[i][j]) + (vote->coverage_start[i][j] < max_start) != 1))is_fusion = 1; if((! is_reversed) && ((max_pos > vote->pos[i][j]) + (vote->coverage_start[i][j] > max_start) != 1)) is_fusion = 1; if(abs(dist) > 500000 || chro_name != best_chro_name) continue; int test_vote_value ; test_vote_value = 8888888 + vote->votes[i][j]* 1000000 - abs(dist); if (hint_pos>=0) { long long int hint_dist = hint_pos; hint_dist -= vote->pos[i][j]; if (abs (hint_dist) < 100000) test_vote_value += 100; if (abs (hint_dist) < 5000) test_vote_value += 100; } if (test_vote_valuecoverage_start[i][j] < max_start && (((max_pos < vote->pos[i][j]) && !is_reversed) || ((max_pos > vote->pos[i][j]) && is_reversed) ) ) *half_marks |= IS_REVERSED_HALVES; if (vote->coverage_start[i][j] >= max_end && (((max_pos > vote->pos[i][j]) && !is_reversed) || ((max_pos < vote->pos[i][j]) && is_reversed) ) ) *half_marks |= IS_REVERSED_HALVES; if (vote->coverage_start[i][j] < max_start) { (*half_marks) = (*half_marks) & ~IS_R1_CLOSE_TO_5; } else { (*half_marks) |= IS_R1_CLOSE_TO_5; } if(max_mask & IS_NEGATIVE_STRAND) *half_marks = (*half_marks) | IS_NEGATIVE_STRAND_R1; else *half_marks = (*half_marks) & ~IS_NEGATIVE_STRAND_R1; if(vote->masks[i][j] & IS_NEGATIVE_STRAND) *half_marks = (*half_marks) | IS_NEGATIVE_STRAND_R2; else *half_marks = (*half_marks) & ~IS_NEGATIVE_STRAND_R2; best_splicing_point = ((vote->coverage_start[i][j] < max_start)? (vote->coverage_end[i][j]):(max_end)) + ((vote->coverage_start[i][j] < max_start)? (max_start):(vote->coverage_start[i][j])); best_splicing_point /=2; * best_pos1 = max_pos ; * best_pos2 = vote->pos[i][j] ; * best_vote1 = max_votes ; * best_vote2 = vote->votes[i][j] ; * read_coverage_start = min(vote->coverage_start[i][j] , max_start); * read_coverage_end = max(vote->coverage_end[i][j] , max_end); * read_coverage_start = max_start; * read_coverage_end = max_end; int k; for(k=0; kindel_recorder[i][j][k+3])break; * indel_in_p2 = vote->indel_recorder[i][j][k+2]; * is_reversed_halves = is_reversed; if (test_vote_value >=100) *half_marks = (*half_marks) | IS_PAIRED_HINTED; else *half_marks = (*half_marks) & ~(IS_PAIRED_HINTED); if (is_fusion) *half_marks = (*half_marks) | IS_FUSION; else *half_marks = (*half_marks) & ~( IS_FUSION); selected_max_votes = test_vote_value; } *best_select_max_votes = selected_max_votes ; return best_splicing_point; } int core_select_best_matching_halves(global_context_t * global_context , gene_vote_t * vote, unsigned int * best_pos1, unsigned int * best_pos2, int * best_vote1, int * best_vote2, char * is_abnormal, short * half_marks, int * is_reversed_halves, float accept_rate, int read_len, long long int hint_pos, int tolerable_bases, short * read_coverage_start, short * read_coverage_end, char * indel_in_p1, char * indel_in_p2 , int * max_cover_start, int * max_cover_end, int rl, int repeated_pos_base, int is_negative, char * repeat_record, unsigned int index_valid_range) { unsigned int tmp_best_pos1=0, tmp_best_pos2=0; int tmp_best_vote1=0, tmp_best_vote2=0, tmp_is_reversed_halves=0; char tmp_is_abnormal=0; gene_vote_number_t tmp_indel_in_p1=0, tmp_indel_in_p2=0; short tmp_half_marks=0, tmp_read_coverage_start=0, tmp_read_coverage_end=0; int ret = 0, best_ret = 0; int i,j; int test_select_votes=-1, best_select_votes = 1000000; //int max_minor = 0; /* for (i=0; iitems[i]; j++) { if(vote->votes[i][j] < vote->max_vote)continue; int ii,jj; for (ii=0; iiitems[ii]; jj++) { if(max_minor >= vote->votes[ii][jj]) continue; if(ii==i && jj==j)continue; long long int dist = vote->pos[ii][jj]; dist =abs(dist - vote->pos[i][j]); if(dist > 500000) continue; max_minor = vote->votes[ii][jj]; } } int encountered = 0; for (i=0; iitems[i]; j++) { if(vote->votes[i][j] < vote->max_vote)continue; int ii,jj; for (ii=0; iiitems[ii]; jj++) { if(max_minor != vote->votes[ii][jj]) continue; if(ii==i && jj==j)continue; long long int dist = vote->pos[ii][jj]; dist =abs(dist - vote->pos[i][j]); if(dist > 500000) continue; encountered++; } } */ int repeated_pos = repeated_pos_base; int offset_shifting = (rl > 220)?4:0; //int encounter = 0; for (i=0; iitems[i]; j++) { /*if((vote->votes[i][j] >= vote->max_vote -1) && (vote->max_coverage_start >= vote-> coverage_start[i][j] - EXON_MAX_BIGMARGIN_OVERLAPPING ) && (vote->max_coverage_end <= vote-> coverage_end[i][j] + EXON_MAX_BIGMARGIN_OVERLAPPING)) encounter++;*/ if(repeated_pos_base>=0 && vote->pos[i][j]<=index_valid_range) if(vote->votes[i][j] >= vote->max_vote && repeated_pos < repeated_pos_base+12) { repeat_record[repeated_pos] = (vote-> coverage_start[i][j] >> offset_shifting); repeat_record[repeated_pos+1] = (vote-> coverage_end[i][j] >> offset_shifting); repeat_record[repeated_pos+2] = (is_negative?0x80:0) | (vote->votes[i][j]&0x7f); repeated_pos+=3; } } for (i=0; iitems[i]; j++) { if(repeated_pos_base>=0 && vote->pos[i][j]<=index_valid_range) if(vote->votes[i][j] == vote->max_vote -1 && repeated_pos < repeated_pos_base+12) { repeat_record[repeated_pos] = (vote-> coverage_start[i][j] >> offset_shifting); repeat_record[repeated_pos+1] = (vote-> coverage_end[i][j] >> offset_shifting); repeat_record[repeated_pos+2] = (is_negative?0x80:0) | (vote->votes[i][j]&0x7f); repeated_pos+=3; } } /* if(encounter>=2) return 0; */ ret = core_select_best_matching_halves_maxone(global_context, vote, &tmp_best_pos1, &tmp_best_pos2, &tmp_best_vote1, &tmp_best_vote2, &tmp_is_abnormal,&tmp_half_marks, &tmp_is_reversed_halves, accept_rate, read_len, hint_pos, tolerable_bases, &tmp_read_coverage_start, &tmp_read_coverage_end, &tmp_indel_in_p1, &tmp_indel_in_p2, vote -> max_position, vote->max_vote, vote-> max_coverage_start, vote-> max_coverage_end, vote-> max_mask, vote->max_indel_recorder, &test_select_votes, rl); test_select_votes += vote->max_vote*1000000; //SUBREADprintf("TSV=%d\n",test_select_votes); if(test_select_votes > best_select_votes) { best_select_votes = test_select_votes; *best_pos1 = tmp_best_pos1; *best_pos2 = tmp_best_pos2; *is_reversed_halves= tmp_is_reversed_halves; *best_vote1 = tmp_best_vote1; *best_vote2 = tmp_best_vote2; *is_abnormal = tmp_is_abnormal; *indel_in_p1 = tmp_indel_in_p1; *indel_in_p2 = tmp_indel_in_p2; *half_marks = tmp_half_marks; *read_coverage_start = tmp_read_coverage_start; *read_coverage_end = tmp_read_coverage_end; * max_cover_start = vote-> max_coverage_start; * max_cover_end = vote-> max_coverage_end; best_ret = ret; } return best_ret; } #define EXON_DONOR_TEST_WINDOW 17 // pos1 must be small than pos2. int core13_test_donor(char *read, int read_len, unsigned int pos1, unsigned int pos2, int guess_break_point, char negative_strand, int test_range, char is_soft_condition, int EXON_INDEL_TOLERANCE, int* real_break_point, gene_value_index_t * my_value_array_index, int indel_offset1, int indel_offset2, int is_reversed, int space_type, int * best_donor_score, int * is_GTAG) { int bps_pos_x; int search_start = guess_break_point - test_range ; int search_end = guess_break_point + test_range ; char h1_2ch[3], h2_2ch[3]; h1_2ch[2] = h2_2ch[2]=0; search_start=max(10, search_start); search_end = min(read_len-10, search_end); int best_break = -1; int min_x = -9099; for (bps_pos_x = search_start; bps_pos_x < search_end ; bps_pos_x ++) { int paired_score = 0; get_chro_2base(h1_2ch, my_value_array_index, pos1 - indel_offset1+ bps_pos_x , is_reversed); get_chro_2base(h2_2ch, my_value_array_index, pos2 - 2 - indel_offset2 + bps_pos_x, is_reversed); //if(!is_reversed) //SUBREADprintf("C1=%s @%u, C2=%s @%u\n",h1_2ch, pos1 + bps_pos_x, h2_2ch,pos2 - 2 + indel_offset + bps_pos_x); if(h1_2ch[0]==h2_2ch[0] && h1_2ch[1]==h2_2ch[1]) continue; if(is_donar_chars_part(h1_2ch) && is_donar_chars_part(h2_2ch)) { paired_score = paired_chars_part(h1_2ch, h2_2ch, is_reversed); if(paired_score) { int m1, m2, x1, x2; int break_point_half = is_reversed?(read_len - bps_pos_x):bps_pos_x; int first_exon_end,second_half_start; int donar_conf_len = 0; donar_conf_len = min(break_point_half , EXON_DONOR_TEST_WINDOW); donar_conf_len = min(read_len - break_point_half, donar_conf_len); //SUBREADprintf("DONOR_CONF_LEN=%d\n", donar_conf_len); if (is_reversed) { first_exon_end = pos2 + bps_pos_x - indel_offset2; second_half_start = pos1 + bps_pos_x- indel_offset1; m1 = match_chro(read + break_point_half - donar_conf_len , my_value_array_index, first_exon_end, donar_conf_len, is_reversed, space_type); m2 = match_chro(read + break_point_half , my_value_array_index, second_half_start-donar_conf_len , donar_conf_len, is_reversed, space_type); x1 = match_chro(read + break_point_half , my_value_array_index, first_exon_end - donar_conf_len, donar_conf_len , is_reversed, space_type); x2 = match_chro(read + break_point_half - donar_conf_len , my_value_array_index, second_half_start , donar_conf_len, is_reversed, space_type); } else { first_exon_end = pos1 + bps_pos_x - indel_offset1; second_half_start = pos2 + bps_pos_x - indel_offset2; m1 = match_chro(read + break_point_half - donar_conf_len, my_value_array_index, first_exon_end-donar_conf_len , donar_conf_len, is_reversed, space_type); m2 = match_chro(read + break_point_half , my_value_array_index, second_half_start, donar_conf_len, is_reversed, space_type); x1 = match_chro(read + break_point_half , my_value_array_index, first_exon_end, donar_conf_len , is_reversed,space_type); x2 = match_chro(read + break_point_half - donar_conf_len, my_value_array_index, second_half_start - donar_conf_len, donar_conf_len , is_reversed,space_type); } #ifdef TEST_TARGET if(memcmp(read, TEST_TARGET, 15)==0) { SUBREADprintf("DONOR TEST STR=%s, %s ; pos=%d %d %d ; M=%d %d ; X=%d %d\n", h1_2ch, h2_2ch, bps_pos_x, indel_offset1, indel_offset2, m1, m2, x1, x2); } #endif int threshold = 3; if (paired_score == 1) threshold = 3; #ifdef QUALITY_KILL if (m1 >= donar_conf_len-1 && m2>=donar_conf_len-1 ) if(x1= donar_conf_len-1 && m2>=donar_conf_len -1) if(x10) { #ifdef TEST_TARGET if(memcmp(read, TEST_TARGET, 15)==0) SUBREADprintf("SELECRED!!!_BREAKPOINT=%d, RAW POS=%u,%u, R=%s\n", best_break, pos1 , pos2, read); #endif //SUBREADprintf ("FINAL BREAK: %d ; REV = %d\n ", best_break, is_reversed); *real_break_point = best_break; return 1; } else { #ifdef TEST_TARGET if(memcmp(read, TEST_TARGET, 15)==0) SUBREADprintf("KILLED!!!_BREAKPOINT=%d, R=%s\n", best_break+ pos1, read); #endif } return 0; } #define EXON_LARGE_WINDOW 60 #define ACCEPTED_SUPPORT_RATE 0.3 void core_fragile_junction_voting(global_context_t * global_context, thread_context_t * thread_context, char * rname, char * read, char * qual, unsigned int full_rl, int negative_strand, int color_space, unsigned int low_border, unsigned int high_border, gene_vote_t *vote_p1) { int windows = full_rl / EXON_LARGE_WINDOW +1; float overlap = (1.0*windows * EXON_LARGE_WINDOW - full_rl) / (windows-1); int ww; int window_cursor = 0; HashTable * event_table = NULL; chromosome_event_t * event_space = NULL; if(thread_context) { event_table = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> event_entry_table; event_space = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; } else { event_table = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_entry_table; event_space = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; } int GENE_SLIDING_STEP = global_context->current_index -> index_gap; for(ww=0; wwcurrent_index, subread_integer , subread_offset, read_len,negative_strand, vote_p1, 5, subread_no, low_border, high_border - read_len); } if(subread_offset1 >= read_len -16) break; } int ii, jj, kk; for(ii = 0; ii < GENE_VOTE_TABLE_SIZE; ii++) { for(jj = 0; jj < vote_p1 -> items[ii] ; jj++) { if(vote_p1 -> votes[ii][jj] < vote_p1 -> max_vote) continue; gene_vote_number_t * indel_recorder = vote_p1 -> indel_recorder[ii][jj]; unsigned int voting_position = vote_p1 -> pos[ii][jj]; int last_indel = 0, last_correct_subread=0; for(kk =0; indel_recorder[kk] && (kk < MAX_INDEL_SECTIONS); kk+=3){ char movement_buffer[MAX_READ_LENGTH * 10 / 7]; //chromosome_event_t * last_event = NULL; int last_event_id = -1; int indels = indel_recorder[kk+2] - last_indel; if(indels==0) continue; int next_correct_subread = indel_recorder[kk] -1; int last_correct_base = find_subread_end(read_len, global_context->config.total_subreads , last_correct_subread) - 9; int first_correct_base = find_subread_end(read_len, global_context->config.total_subreads , next_correct_subread) - 16 + 9; first_correct_base = min(first_correct_base+10, read_len); last_correct_base = max(0, last_correct_base); last_correct_base = min(read_len-1, last_correct_base); int x1, dyna_steps; dyna_steps = core_dynamic_align(global_context, thread_context, InBuff + last_correct_base, first_correct_base - last_correct_base, voting_position + last_correct_base + last_indel, movement_buffer, indels, rname); movement_buffer[dyna_steps]=0; if(0 && strcmp("MISEQ:13:000000000-A1H1M:1:1112:12194:5511", rname) == 0) { SUBREADprintf("IR= %d %d~%d\n", dyna_steps, last_correct_base, first_correct_base); for(x1=0; x1config.maximise_sensitivity_indel && total_mismatch <= 2 )) for(x1=0; x1current_value_index:global_context->current_value_index; int ambiguous_i, ambiguous_count=0; int best_matched_bases = match_chro(InBuff + cursor_on_read - 6, current_value_index, indel_left_boundary - 6, 6, 0, global_context->config.space_type) + match_chro(InBuff + cursor_on_read - min(current_indel_len,0), current_value_index, indel_left_boundary + max(0, current_indel_len), 6, 0, global_context->config.space_type); for(ambiguous_i=-5; ambiguous_i<=5; ambiguous_i++) { int left_match = match_chro(InBuff + cursor_on_read - 6, current_value_index, indel_left_boundary - 6, 6+ambiguous_i, 0, global_context->config.space_type); int right_match = match_chro(InBuff + cursor_on_read + ambiguous_i - min(current_indel_len,0), current_value_index, indel_left_boundary + ambiguous_i + max(0, current_indel_len), 6-ambiguous_i, 0,global_context->config.space_type); if(left_match+right_match == best_matched_bases) ambiguous_count ++; } if(0 && strcmp("MISEQ:13:000000000-A1H1M:1:1112:12194:5511", rname) == 0) SUBREADprintf("INDEL_DDADD: abs(I=%d); INDELS=%d; LOC=%u\n",i, current_indel_len, indel_left_boundary-1); if(abs(current_indel_len)<=global_context -> config.max_indel_length) { chromosome_event_t * new_event = local_add_indel_event(global_context, thread_context, event_table, InBuff + cursor_on_read + min(0,current_indel_len), indel_left_boundary - 1, current_indel_len, 1, ambiguous_count, 0, NULL); if(last_event_id >=0 && new_event){ // the event space can be changed when the new event is added. the location is updated everytime. chromosome_event_t * event_space = NULL; if(thread_context) event_space = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; else event_space = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; chromosome_event_t * last_event = event_space + last_event_id; int dist = new_event -> event_small_side - last_event -> event_large_side +1; new_event -> connected_previous_event_distance = dist; last_event -> connected_next_event_distance = dist; } if (new_event) last_event_id = new_event -> global_event_id; else last_event_id = -1; } } if(mv == 0 || mv == 3) is_in_indel = 0; } if(is_in_indel && mv == 1) current_indel_len += 1; if(is_in_indel && mv == 2) current_indel_len -= 1; if(mv == 1 || mv == 3 || mv == 0) cursor_on_chromosome++; if(mv == 2 || mv == 3 || mv == 0) cursor_on_read++; last_mv = mv; } last_correct_subread = indel_recorder[i+1]-1; } } } if(1) { finalise_vote(vote_p1); select_best_vote(vote_p1); //print_votes(vote_p1, global_context -> config.index_prefix); unsigned int best_pos1=0; unsigned int best_pos2=0; int best_vote1=0; int best_vote2=0; char is_abnormal=0; short half_marks=0; int is_reversed_halves=0, max_cover_start=0, max_cover_end=0; char indel_in_p1=0, indel_in_p2=0; short read_coverage_start =0, read_coverage_end=0; gene_value_index_t * base_index = thread_context?thread_context->current_value_index:global_context->current_value_index ; int splice_point = core_select_best_matching_halves(global_context, vote_p1, &best_pos1, &best_pos2, &best_vote1, &best_vote2, &is_abnormal ,&half_marks, &is_reversed_halves, ACCEPTED_SUPPORT_RATE, read_len, -1, 0, &read_coverage_start, &read_coverage_end, &indel_in_p1, &indel_in_p2, &max_cover_start, &max_cover_end, read_len, -1 , 0, NULL , 0xffffffff); //SUBREADprintf("RN=%s , WINDOW = %d ~ %d , SP=%d; BV=%d; BV2=%d\n", rname , window_cursor , window_cursor + read_len , splice_point, best_vote1, best_vote2); if (splice_point>0 && best_vote1 >= 1 && best_vote2>=1) { int test_real_break_point = -1, test_donor_score=-1; int is_GTAG = 0; int is_accepted = core13_test_donor(InBuff, read_len, min(best_pos1, best_pos2), max(best_pos1,best_pos2), splice_point, negative_strand, read_len/4, 0, 5, &test_real_break_point, base_index, 0, 0, negative_strand, color_space, &test_donor_score, &is_GTAG); if (is_accepted ){ unsigned int pos_small = min(test_real_break_point+ best_pos1, test_real_break_point+ best_pos2) - 1; unsigned int pos_big = max(test_real_break_point+ best_pos1, test_real_break_point+ best_pos2); int event_no; chromosome_event_t * search_return [MAX_EVENT_ENTRIES_PER_SITE]; chromosome_event_t * found = NULL; int found_events = search_event(global_context, event_table, event_space, pos_small , EVENT_SEARCH_BY_SMALL_SIDE, CHRO_EVENT_TYPE_JUNCTION|CHRO_EVENT_TYPE_FUSION, search_return); if(found_events) { int kx1; for(kx1 = 0; kx1 < found_events ; kx1++) { if(search_return[kx1] -> event_large_side == pos_big) { found = search_return[kx1]; break; } } } if(found) found -> supporting_reads ++; else { if(thread_context) event_no = ((indel_thread_context_t *)thread_context -> module_thread_contexts[MODULE_INDEL_ID]) -> total_events ++; else event_no = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> total_events ++; event_space = reallocate_event_space(global_context, thread_context, event_no); chromosome_event_t * new_event = event_space+event_no; memset(new_event,0,sizeof(chromosome_event_t)); new_event -> event_small_side = pos_small; new_event -> event_large_side = pos_big; new_event -> is_negative_strand= !is_GTAG; new_event -> event_type = CHRO_EVENT_TYPE_JUNCTION; new_event -> supporting_reads = 1; new_event -> indel_length = 0; put_new_event(event_table, new_event , event_no); // SUBREADprintf("ADD JUNCTION BY FRAGILE, %d-%d\n", pos_small, pos_big); } } } } InBuff[read_len] = tmp_char; } } void print_frags(global_context_t * global_context, fragment_list_t * fls){ int x1; for(x1 =0; x1 < fls -> fragments; x1++){ subread_read_number_t fno = fls -> fragment_numbers[x1] / 2; int f_is_B = fls -> fragment_numbers[x1] % 2; mapping_result_t * f_res = _global_retrieve_alignment_ptr(global_context, fno, f_is_B, 0); mapping_result_t * mate_res = _global_retrieve_alignment_ptr(global_context, fno, !f_is_B, 0); char outpos[100]; char outposm[100]; absoffset_to_posstr(global_context, f_res -> selected_position, outpos); absoffset_to_posstr(global_context, mate_res -> selected_position, outposm); int f_negative = (f_res -> result_flags & CORE_IS_NEGATIVE_STRAND)?1:0; int mate_negative = (mate_res -> result_flags & CORE_IS_NEGATIVE_STRAND)?1:0; if(f_is_B) f_negative=!f_negative; else mate_negative=!mate_negative; //SUBREADprintf("TRALOG: READ %09u %c AT %s (%c) ; MATE: %s (%c)\n", fno, f_is_B?'B':'A' , outpos, f_negative?'N':'P' , outposm, mate_negative?'N':'P'); } } // fragnos_paired_B = B_fragment_no * 2 + is_mate_b (is_mate_b points the mate that has the location in locations_mate_B) // fragnos_paired_C = C_fragment_no * 2 + is_mate_c (is_mate_c points the mate that has the location in locations_mate_C) // // locations_mate_B and locations_mate_C are the locations where the sequence is moved to. I.e., locations_mate_B and locations_mate_C are far far away from fragment A. // int find_translocation_BC_mates(global_context_t * global_context, mapping_result_t * res_A1, mapping_result_t * res_A2, fragment_list_t * listB, fragment_list_t * listC, int is_INV, unsigned long long * fragnos_paired_B, unsigned long long * fragnos_paired_C, unsigned int * locations_mate_B, unsigned int * locations_mate_C,unsigned int * guessed_brkP_small_sum, unsigned int * guessed_moved_length_sum , unsigned int * guessed_brkQ_small_sum){ int ret = 0, xk1, xk2; char * is_C_used = malloc(sizeof(char) * listC->fragments); memset(is_C_used, 0, sizeof(char) * listC->fragments); long long tmp_guessed_brkP_small_sum = 0, tmp_guessed_moved_length_sum = 0, tmp_guessed_brkQ_small_sum = 0; for(xk1 = 0; xk1 < listB->fragments; xk1++) { long long minimum_mate_distance = 0x7fffffff; int minimum_xk2 = -1; unsigned int mate_C_pos = 0; mapping_result_t * res_Ca = NULL, * res_Cc = NULL, * res_Ba = NULL, *res_Bb = NULL; mapping_result_t meta_C_res_body, res_Ca_body; res_Ca = &res_Ca_body; mapping_result_t * meta_C_res = &meta_C_res_body; subread_read_number_t B_read_no = listB->fragment_numbers[xk1]/2; int B_read_is_b = listB->fragment_numbers[xk1]%2; mapping_result_t meta_B_res_body, res_Ba_body; mapping_result_t * meta_B_res = &meta_B_res_body; res_Ba = &res_Ba_body; bigtable_readonly_result(global_context, NULL, B_read_no, 0, !B_read_is_b, meta_B_res, NULL); res_Bb = meta_B_res; bigtable_readonly_result(global_context, NULL, B_read_no, 0, B_read_is_b, res_Ba, NULL); for(xk2 = 0; xk2 < listC->fragments; xk2++) { if(is_C_used[xk2]) continue; subread_read_number_t C_read_no = listC->fragment_numbers[xk2]/2; int C_read_is_b = listC->fragment_numbers[xk2]%2; bigtable_readonly_result(global_context, NULL, C_read_no, 0, !C_read_is_b, meta_C_res, NULL); res_Cc = meta_C_res; bigtable_readonly_result(global_context, NULL, C_read_no, 0, C_read_is_b, res_Ca, NULL); int is_meta_B_negative = (meta_B_res -> result_flags & CORE_IS_NEGATIVE_STRAND)?1:0; if(!B_read_is_b) is_meta_B_negative = !is_meta_B_negative; int is_meta_C_negative = (meta_C_res -> result_flags & CORE_IS_NEGATIVE_STRAND)?1:0; if(!C_read_is_b) is_meta_C_negative = !is_meta_C_negative; //SUBREADprintf("TRALOG: MATES : B[%d] = %u (%c); C[%d] = %u (%c)\n", xk1, meta_B_res -> selected_position, is_meta_B_negative?'N':'P' , xk2, meta_C_res -> selected_position, is_meta_C_negative?'N':'P'); if(is_meta_B_negative != is_meta_C_negative && meta_B_res -> selected_position < meta_C_res -> selected_position && meta_C_res -> selected_position - meta_C_res -> selected_position < global_context -> config.maximum_translocation_length && meta_C_res -> selected_position - meta_B_res -> selected_position < minimum_mate_distance) { minimum_mate_distance = meta_C_res -> selected_position - meta_B_res -> selected_position; minimum_xk2 = xk2; mate_C_pos = meta_C_res -> selected_position; } } // read B has a mate of C[minimum xk2] if there is one. if(minimum_xk2>=0) { subread_read_number_t C_mate_fno = listC -> fragment_numbers[minimum_xk2] / 2; int C_mate_is_b = listC -> fragment_numbers[minimum_xk2] % 2; fragnos_paired_B[ret] = (B_read_no*2)+(!B_read_is_b); locations_mate_B[ret] = meta_B_res -> selected_position; fragnos_paired_C[ret] = (C_mate_fno*2)+(C_mate_is_b); locations_mate_C[ret] = mate_C_pos; is_C_used[minimum_xk2] = 1; int gapA, gapB, gapC; if(is_INV){ gapA = res_Ca -> selected_position - res_A1 -> selected_position - res_A1 -> read_length; gapB = res_A2 -> selected_position - res_Ba -> selected_position - res_Ba -> read_length; gapC = res_Cc -> selected_position - res_Bb -> selected_position - res_Bb -> read_length; }else{ gapA = res_Ba -> selected_position - res_A1 -> selected_position - res_A1 -> read_length; gapB = res_A2 -> selected_position - res_Ca -> selected_position - res_Ca -> read_length; gapC = res_Cc -> selected_position - res_Bb -> selected_position - res_Bb -> read_length; } tmp_guessed_brkP_small_sum += res_A1 -> selected_position + res_A1 -> read_length + gapA/2; tmp_guessed_moved_length_sum += res_A2 -> selected_position - res_A1 -> selected_position - res_A1 -> read_length - gapB/2 + gapA/2; tmp_guessed_brkQ_small_sum += res_Bb -> selected_position + res_Bb -> read_length + gapC/2; ret ++; } } free(is_C_used); if(ret>0){ *guessed_brkP_small_sum= tmp_guessed_brkP_small_sum / ret; *guessed_moved_length_sum = tmp_guessed_moved_length_sum/ ret; *guessed_brkQ_small_sum = tmp_guessed_brkQ_small_sum / ret; } return ret; } // This function sees if all the mates of read B_x and C_y are at the same location. // If mates of B_x and C_y spread on a large region, it is usually unreliable. // posesB and posesB are linear absolute positions of the mate reads. int find_translocation_BC_conformation(global_context_t * global_context, int PEmates, unsigned int * posesB, unsigned int * posesC){ unsigned int min_pos = 0xffffffff, max_pos = 0, xk1; if(PEmates<1) return 0; for(xk1 = 0; xk1 < PEmates; xk1++) { min_pos = min(min_pos, posesB[xk1]); min_pos = min(min_pos, posesC[xk1]); max_pos = max(max_pos, posesB[xk1]); max_pos = max(max_pos, posesC[xk1]); } if(max_pos - min_pos< 2*global_context -> config.maximum_pair_distance)return 1; return 0; } // fliB and fliB are : frag_[BC]_no * 2 + is_Read_b_close_to_BreakPoint_P int breakpoint_PQR_supported(global_context_t * global_context , unsigned int brkPno , unsigned int brkQno, unsigned int brkRno, fragment_list_t * fliB, fragment_list_t * fliC, int isInv){ int fli_i; int isFliB, nSupB=0, nSupC=0; for(isFliB = 0; isFliB < 2; isFliB++){ fragment_list_t * fli = isFliB?fliB:fliC; int * nSup = isFliB?&nSupB:&nSupC; // fliB => support source_small ~ target_large if inv, or source_small ~ target_small if !inv // fliC => support source_large ~ target_small if inv, or source_large ~ target_large if !inv // the read that is close to BreakPoint_P should support source, the other read should support target for(fli_i = 0; fli_i < fli -> fragments; fli_i ++){ subread_read_number_t frag_BC_no = fli -> fragment_numbers[fli_i]/2; int is_Read_b_close_to_BreakPoint_P = fli -> fragment_numbers[fli_i]%2; unsigned int source_small, source_large, target_smallQ, target_largeQ, target_smallR, target_largeR, target_large, target_small; get_event_two_coordinates(global_context, brkPno, NULL, NULL, &source_small, NULL, NULL, &source_large); get_event_two_coordinates(global_context, brkQno, NULL, NULL, &target_smallQ, NULL, NULL, &target_largeQ); get_event_two_coordinates(global_context, brkRno, NULL, NULL, &target_smallR, NULL, NULL, &target_largeR); if(target_smallQ <= target_smallR + BREAK_POINT_MAXIMUM_TOLERANCE && target_smallQ >= target_smallR - BREAK_POINT_MAXIMUM_TOLERANCE) { //target_smallQ is target, target_smallR is target target_large = target_smallR; target_small = target_smallQ; }else{ //target_largeQ is target, target_largeR is target target_large = target_largeQ; target_small = target_largeR; } mapping_result_t res_BC_close_P_body, res_BC_close_Q_body; mapping_result_t * res_BC_close_P = &res_BC_close_P_body, * res_BC_close_Q = & res_BC_close_Q_body; bigtable_readonly_result(global_context, NULL, frag_BC_no, 0, is_Read_b_close_to_BreakPoint_P, res_BC_close_P, NULL); bigtable_readonly_result(global_context, NULL, frag_BC_no, 0, !is_Read_b_close_to_BreakPoint_P, res_BC_close_Q, NULL); unsigned int P_pos = isInv?( isFliB?source_large:source_small ):( isFliB?source_small:source_large ); unsigned int Q_pos = isInv?( isFliB?target_large:target_small ):( isFliB?target_small:target_large ); SUBREADprintf("TRALOG: PQR_TARGET P=%u~%u; Q=%u~%u, R=%u~%u ; Ppos=%u, Qpos=%u, Pread=%u, Qread=%u on %s\n", source_small, source_large, target_smallQ, target_largeQ, target_smallR, target_largeR, P_pos, Q_pos, res_BC_close_P -> selected_position, res_BC_close_Q -> selected_position, isInv?"INV":"STR"); long long dist; dist = res_BC_close_P -> selected_position; dist -= P_pos; if(abs(dist) < global_context -> config.maximum_pair_distance){ dist = res_BC_close_Q -> selected_position; dist -= Q_pos; if(abs(dist) < global_context -> config.maximum_pair_distance) (*nSup)++; } } } //return nSupB + 1 >= fliB -> fragments/2 && nSupC + 1 >= fliC-> fragments/2 ; SUBREADprintf("TRALOG: PQR_NSUP: B=%d, C=%d on %s\n", nSupB, nSupC, isInv?"INV":"STR"); return nSupB > 0 && nSupC > 0 && nSupB + 2 >= fliB->fragments / 2 && nSupC + 2 >= fliC->fragments / 2; } // fragnoD1_mates and fragnoD2_mates are poteltial E reads 1/2. // D1: D's small read; D2: D's large read // E2 ~ D2 // E1 ~ D1 // E2.start > Y.large // E1.start > Y.small int breakpoint_YZ_supported(global_context_t * global_context, unsigned int brkYno, unsigned int brkZno, unsigned long long * fragnoD1_mates, int fragnoD1len, unsigned long long * fragnoD2_mates, int fragnoD2len){ int x1; int is_D2_mates; unsigned int inversion_small_edge, inversion_large_edge; get_event_two_coordinates(global_context, brkYno, NULL, NULL, &inversion_small_edge, NULL, NULL, &inversion_large_edge); int nSupD1mates = 0, nSupD2mates = 0; for(is_D2_mates = 0; is_D2_mates < 2; is_D2_mates ++){ unsigned long long * fragno_Dmates = is_D2_mates?fragnoD2_mates:fragnoD1_mates; int fragno_Dno = is_D2_mates?fragnoD2len:fragnoD1len; int * nSupMates = is_D2_mates?&nSupD2mates:&nSupD1mates; for(x1 = 0; x1 < fragno_Dno; x1++){ subread_read_number_t fragno_Dmate = fragno_Dmates[x1] / 2; int is_large_read_far_from_D = fragno_Dmates[x1] % 2; mapping_result_t frag_D_mate_a_body, frag_D_mate_b_body; mapping_result_t * frag_D_mate_a = &frag_D_mate_a_body, * frag_D_mate_b = & frag_D_mate_b_body; bigtable_readonly_result(global_context, NULL, fragno_Dmate, 0, 0, frag_D_mate_a, NULL); bigtable_readonly_result(global_context, NULL, fragno_Dmate, 0, 1, frag_D_mate_b, NULL); mapping_result_t * frag_D_mate_1 = (frag_D_mate_a -> selected_position > frag_D_mate_b -> selected_position)?frag_D_mate_b:frag_D_mate_a; mapping_result_t * frag_D_mate_2 = (frag_D_mate_a -> selected_position <=frag_D_mate_b -> selected_position)?frag_D_mate_b:frag_D_mate_a; mapping_result_t * res_to_support_small_edge = (is_D2_mates ^ is_large_read_far_from_D)?frag_D_mate_2:frag_D_mate_1; mapping_result_t * res_to_support_large_edge = (is_D2_mates ^ is_large_read_far_from_D)?frag_D_mate_1:frag_D_mate_2; long long distsm; distsm = res_to_support_small_edge -> selected_position; distsm -= inversion_small_edge; long long distla; distla = res_to_support_large_edge -> selected_position; distla -= inversion_large_edge; //SUBREADprintf("INVLOG: Dist_SM=%lld, Dist_LA=%lld\n", distsm, distla); if(distsm > -8 && distsm < global_context -> config.maximum_pair_distance){ if(distla > -8 && distla < global_context -> config.maximum_pair_distance) (*nSupMates) ++; } } } //SUBREADprintf("INVLOG: breakpoint_YZ_supported nSupD1=%d >= %d, nSupD2=%d >= %d\n", nSupD1mates, fragnoD1len, nSupD2mates, fragnoD2len); return nSupD1mates > 0 && nSupD2mates > 0 && nSupD1mates + 2 >= fragnoD1len / 2 && nSupD2mates + 2 >= fragnoD2len / 2; } #define _PQR_LIST_SIZE 48 int find_translocation_brk_PQR(global_context_t * global_context, mapping_result_t * resA1, mapping_result_t * resA2, fragment_list_t * fliB, fragment_list_t * fliC, unsigned int * brkPno, unsigned int * brkQno, unsigned int * brkRno, int isInv, unsigned int * is_cand_P_found) { unsigned int event_pos_list_A1[_PQR_LIST_SIZE]; void * event_ptr_list_A1[_PQR_LIST_SIZE]; char * chroA=NULL; int posA1=0; locate_gene_position(resA1 -> selected_position, &global_context -> chromosome_table, &chroA, &posA1); int candA1i, found_PQR = 0; int candA1Number = bktable_lookup(&global_context -> breakpoint_table_P, chroA, posA1, global_context -> config.maximum_pair_distance , event_pos_list_A1, event_ptr_list_A1, _PQR_LIST_SIZE); indel_context_t * indel_context = (indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]; int candBrkPi , candBrkPNumber=0; //SUBREADprintf("A FOUND %d P ", candA1Number); for(candA1i = 0; candA1i < candA1Number ; candA1i++){ int event_no = event_ptr_list_A1[candA1i] - NULL; chromosome_event_t * event_body = indel_context -> event_space_dynamic + event_no; long long small_dist = event_body -> event_small_side, large_dist = event_body -> event_large_side; small_dist -= resA1 -> selected_position; large_dist -= resA2 -> selected_position; } //SUBREADprintf(", (%d may be used)\n", candBrkPNumber); *is_cand_P_found = candBrkPNumber; for(candBrkPi = 0; candBrkPi < candBrkPNumber; candBrkPi++){ unsigned int event_no_P = event_ptr_list_A1[candBrkPi] - NULL; chromosome_event_t * event_body_P = indel_context -> event_space_dynamic + event_no_P; unsigned int anchor_for_brkQ = isInv?event_body_P -> event_large_side:event_body_P -> event_small_side; unsigned int anchor_for_brkR = isInv?event_body_P -> event_small_side:event_body_P -> event_large_side; unsigned int event_pos_list_Q[_PQR_LIST_SIZE]; void * event_ptr_list_Q[_PQR_LIST_SIZE]; unsigned int event_pos_list_R[_PQR_LIST_SIZE]; void * event_ptr_list_R[_PQR_LIST_SIZE]; char * charAncQ = NULL, * charAncR = NULL; int posAncQ=0, posAncR = 0; locate_gene_position(anchor_for_brkQ, &global_context -> chromosome_table, &charAncQ, &posAncQ); locate_gene_position(anchor_for_brkR, &global_context -> chromosome_table, &charAncR, &posAncR); int candQi, candQnumber = bktable_lookup(&global_context -> breakpoint_table_QR, charAncQ, posAncQ - BREAK_POINT_MAXIMUM_TOLERANCE , 2* BREAK_POINT_MAXIMUM_TOLERANCE , event_pos_list_Q, event_ptr_list_Q, _PQR_LIST_SIZE); int candRi, candRnumber = bktable_lookup(&global_context -> breakpoint_table_QR, charAncR, posAncR - BREAK_POINT_MAXIMUM_TOLERANCE , 2* BREAK_POINT_MAXIMUM_TOLERANCE , event_pos_list_R, event_ptr_list_R, _PQR_LIST_SIZE); SUBREADprintf("P [%s] FOUND %d Q AT %s:%u and %d R AT %s:%u\n", isInv?"INV":"STR", candQnumber, charAncQ, posAncQ, candRnumber, charAncR, posAncR); for(candQi = 0 ; candQi < candQnumber ; candQi++){ unsigned int event_no_Q = event_ptr_list_Q[candQi] - NULL; chromosome_event_t * event_body_Q = indel_context -> event_space_dynamic + event_no_Q; long long cand_Q_small_dist = event_body_Q -> event_small_side; cand_Q_small_dist -= isInv?event_body_P -> event_large_side:event_body_P -> event_small_side; int is_Q_small_side_close_to_P = abs(cand_Q_small_dist) <= BREAK_POINT_MAXIMUM_TOLERANCE; SUBREADprintf("Q: SMALL_CLOSE_P = %d, DIR = %c %c\n", is_Q_small_side_close_to_P, event_body_Q -> small_side_increasing_coordinate?'>':'<', event_body_Q -> large_side_increasing_coordinate?'>':'<'); if( is_Q_small_side_close_to_P && event_body_Q -> large_side_increasing_coordinate == 1) continue; // the large side is the target location. if((!is_Q_small_side_close_to_P) && event_body_Q -> small_side_increasing_coordinate == 1) continue; // the small side is the target location. if( isInv && event_body_Q -> large_side_increasing_coordinate != event_body_Q -> small_side_increasing_coordinate) continue; if((!isInv) && event_body_Q -> large_side_increasing_coordinate == event_body_Q -> small_side_increasing_coordinate) continue; for(candRi = 0 ; candRi < candRnumber ; candRi++){ unsigned int event_no_R = event_ptr_list_R[candRi] - NULL; chromosome_event_t * event_body_R = indel_context -> event_space_dynamic + event_no_R; srInt_64 cand_R_dist_to_Q = is_Q_small_side_close_to_P?event_body_Q -> event_large_side:event_body_Q -> event_small_side; cand_R_dist_to_Q -= is_Q_small_side_close_to_P?event_body_R -> event_large_side:event_body_R-> event_small_side; SUBREADprintf("R: candDist=%lld, DIR = %c %c\n", cand_R_dist_to_Q, event_body_Q -> small_side_increasing_coordinate?'>':'<', event_body_Q -> large_side_increasing_coordinate?'>':'<'); if(abs(cand_R_dist_to_Q) > BREAK_POINT_MAXIMUM_TOLERANCE) continue; int is_R_small_side_close_to_P = is_Q_small_side_close_to_P; if( is_R_small_side_close_to_P && !event_body_R -> large_side_increasing_coordinate) continue; if(!(is_R_small_side_close_to_P) && !event_body_R -> small_side_increasing_coordinate) continue; if( isInv && event_body_R -> large_side_increasing_coordinate != event_body_R -> small_side_increasing_coordinate) continue; if(!(isInv) && event_body_R -> large_side_increasing_coordinate == event_body_R -> small_side_increasing_coordinate) continue; (*brkPno) = event_no_P; (*brkQno) = event_no_Q; (*brkRno) = event_no_R; found_PQR++; return 1; } } } return found_PQR; } void get_event_two_coordinates(global_context_t * global_context, unsigned int event_no, char ** small_chro, int * small_pos, unsigned int * small_abs, char ** large_chro, int * large_pos, unsigned int * large_abs){ indel_context_t * indel_context = (indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]; chromosome_event_t * event_body = indel_context -> event_space_dynamic + event_no; if(small_abs)(*small_abs) = event_body -> event_small_side; if(large_abs)(*large_abs) = event_body -> event_large_side; if(small_chro && small_pos) locate_gene_position(event_body -> event_small_side, &global_context -> chromosome_table, small_chro, small_pos); if(large_chro && large_pos) locate_gene_position(event_body -> event_large_side, &global_context -> chromosome_table, large_chro, large_pos); } void create_or_update_translocation_imprecise_result(global_context_t * global_context , unsigned int guessed_P_small, unsigned int guessed_tra_len, unsigned int guessed_Q_small , int paired_BC_reads, int isInv){ char * brkPchr; int brkPsmall; void * trans_old_ptrs [_PQR_LIST_SIZE]; unsigned int trans_old_poses [_PQR_LIST_SIZE]; locate_gene_position(guessed_P_small, &global_context -> chromosome_table, &brkPchr, &brkPsmall); int is_trans_found = 0, old_res_i, old_res_number = bktable_lookup(&global_context -> translocation_result_table, brkPchr, brkPsmall - BREAK_POINT_MAXIMUM_TOLERANCE, 2*BREAK_POINT_MAXIMUM_TOLERANCE, trans_old_poses, trans_old_ptrs, _PQR_LIST_SIZE); for(old_res_i = 0; old_res_i < old_res_number; old_res_i++){ translocation_result_t * old_res = (translocation_result_t * )trans_old_ptrs[old_res_i]; long long target_dist = old_res -> target_left_side; target_dist -= guessed_Q_small; if(abs(target_dist) < BREAK_POINT_MAXIMUM_TOLERANCE && isInv == old_res -> is_inv){ target_dist = old_res -> length; target_dist -= guessed_tra_len; if(abs(target_dist) < BREAK_POINT_MAXIMUM_TOLERANCE){ old_res -> all_sup_P ++; old_res -> max_sup_QR = max(old_res -> max_sup_QR , paired_BC_reads); is_trans_found = 1; break; } } } if(0 == is_trans_found){ translocation_result_t * new_res = malloc(sizeof(translocation_result_t)); memset(new_res, 0, sizeof(translocation_result_t)); new_res -> target_left_side = guessed_Q_small; new_res -> length = guessed_tra_len; new_res -> source_left_side = guessed_P_small; new_res -> is_precisely_called = 0; new_res -> all_sup_P = 1; new_res -> max_sup_QR = paired_BC_reads; new_res -> is_inv = isInv; bktable_append(&global_context -> translocation_result_table,brkPchr, brkPsmall, new_res); } } void create_or_update_translocation_result(global_context_t * global_context , unsigned int brkPno, unsigned int brkQno, unsigned int brkRno , int paired_BC_reads, int isInv){ char *brkPchr, *brkQchr, *tmpchr; int brkPsmall, brkPlarge, brkQsmall, tmpint; unsigned int brkPabs_small, brkQabs_small, brkRabs_small, brkRabs_large, brkQabs_large; SUBREADprintf("\nTRALOG: FINALLY_CONFIRMED: %s ; %d PE_MATES\n", isInv?"INV":"STR", paired_BC_reads); get_event_two_coordinates(global_context, brkPno, &brkPchr, &brkPsmall, &brkPabs_small, &tmpchr, &brkPlarge, NULL); get_event_two_coordinates(global_context, brkQno, &brkQchr, &brkQsmall, &brkQabs_small, &tmpchr, &tmpint, &brkQabs_large); get_event_two_coordinates(global_context, brkRno, NULL, NULL, &brkRabs_small, NULL, NULL, &brkRabs_large); SUBREADprintf("TRARES: %s:%u (len=%d) => %s:%u (Coor: last_base_before)\n", brkPchr, brkPsmall, brkPlarge - brkPsmall - 1, brkQchr, brkQsmall); void * trans_old_ptrs [_PQR_LIST_SIZE]; unsigned int trans_old_poses [_PQR_LIST_SIZE]; unsigned int new_target_left_side, new_length; if(brkQabs_small >= brkRabs_small - BREAK_POINT_MAXIMUM_TOLERANCE && brkQabs_small <= brkRabs_small + BREAK_POINT_MAXIMUM_TOLERANCE) { // Q small and R large are target new_target_left_side = brkQabs_small; } else{ // Q large and R small are target new_target_left_side = brkQabs_large; } new_length = brkPlarge - brkPsmall - 1; int is_trans_found = 0, old_res_i, old_res_number = bktable_lookup(&global_context -> translocation_result_table, brkPchr, brkPsmall - BREAK_POINT_MAXIMUM_TOLERANCE, 2*BREAK_POINT_MAXIMUM_TOLERANCE, trans_old_poses, trans_old_ptrs, _PQR_LIST_SIZE); for(old_res_i = 0; old_res_i < old_res_number; old_res_i++){ translocation_result_t * old_res = (translocation_result_t * )trans_old_ptrs[old_res_i]; long long target_dist = old_res -> target_left_side; target_dist -= new_target_left_side; if(abs(target_dist) < BREAK_POINT_MAXIMUM_TOLERANCE && isInv == old_res -> is_inv){ target_dist = old_res -> length; target_dist -= new_length; if(abs(target_dist) < BREAK_POINT_MAXIMUM_TOLERANCE){ old_res -> all_sup_P ++; old_res -> max_sup_QR = max(old_res -> max_sup_QR , paired_BC_reads); is_trans_found = 1; break; } } } if(0 == is_trans_found){ translocation_result_t * new_res = malloc(sizeof(translocation_result_t)); memset(new_res, 0, sizeof(translocation_result_t)); new_res -> target_left_side = new_target_left_side; new_res -> length = new_length; new_res -> source_left_side = brkPabs_small; new_res -> is_precisely_called = 1; new_res -> event_P_number = brkPno; new_res -> event_Q_number = brkQno; new_res -> event_R_number = brkRno; new_res -> all_sup_P = 1; new_res -> max_sup_QR = paired_BC_reads; new_res -> is_inv = isInv; bktable_append(&global_context -> translocation_result_table,brkPchr, brkPsmall, new_res); } } void finalise_translocations(global_context_t * global_context){ void ** s1_ptrs, **s2_ptrs; unsigned int * s1_poses, * s2_poses; s1_ptrs = malloc(sizeof(void *) * S12_LIST_CAPACITY); s2_ptrs = malloc(sizeof(void *) * S12_LIST_CAPACITY); s1_poses = malloc(sizeof(int) * S12_LIST_CAPACITY); s2_poses = malloc(sizeof(int) * S12_LIST_CAPACITY); unsigned long long * s1_selected_list = malloc(sizeof(long long) * S12_LIST_CAPACITY); // fragment_no * 2 + is_second_read unsigned long long * s2_selected_list = malloc(sizeof(long long) * S12_LIST_CAPACITY); mapping_result_t ** s1_result_ptr_list = malloc(sizeof(mapping_result_t *) * S12_LIST_CAPACITY); mapping_result_t ** s2_result_ptr_list = malloc(sizeof(mapping_result_t *) * S12_LIST_CAPACITY); int frag_Q_larger_read; subread_read_number_t frag_A_i; for(frag_A_i = 0; frag_A_i < global_context -> funky_list_A.fragments; frag_A_i ++){ fragment_list_t fli_STR_B, fli_STR_C, fli_INV_B, fli_INV_C; fraglist_init(&fli_STR_B); fraglist_init(&fli_STR_C); fraglist_init(&fli_INV_B); fraglist_init(&fli_INV_C); subread_read_number_t frag_A_no = global_context -> funky_list_A.fragment_numbers[frag_A_i]; mapping_result_t q_res_A_body, q_res_B_body; mapping_result_t * q_res_A = &q_res_A_body; mapping_result_t * q_res_B = &q_res_B_body; bigtable_readonly_result(global_context, NULL, frag_A_no, 0, 0, q_res_A, NULL); bigtable_readonly_result(global_context, NULL, frag_A_no, 0, 1, q_res_B, NULL); mapping_result_t * q_res_1 = q_res_A -> selected_position > q_res_B -> selected_position?q_res_B:q_res_A; mapping_result_t * q_res_2 = q_res_A -> selected_position <= q_res_B -> selected_position?q_res_B:q_res_A; /*************************************************************************************************** * * is_q1_negative and is_q2_negative describes the strandness of the original FASTQ read sequence. * * For the very normal mappings, is_q1_negative must be 0 and is_q2_negative must be 1. * * If is_q1_negative != is_q2_negative, then there is a strand-jumpping fusion between the two reads. */ int is_q1_negative = (q_res_1 -> result_flags & CORE_IS_NEGATIVE_STRAND)?1:0; int is_q2_negative = (q_res_2 -> result_flags & CORE_IS_NEGATIVE_STRAND)?1:0; if(q_res_B == q_res_1)is_q1_negative=!is_q1_negative; if(q_res_B == q_res_2)is_q2_negative=!is_q2_negative; long long dist = q_res_A ->selected_position; dist -= q_res_B->selected_position; if( abs(dist) < 1000 && !(is_q1_negative == 0 && is_q2_negative == 1)) { SUBREADprintf("TRALOG: STRANDNESS_BUG %08llu\n", frag_A_no); } for(frag_Q_larger_read = 0; frag_Q_larger_read < 2; frag_Q_larger_read++){ void ** s_ptrs = frag_Q_larger_read?s2_ptrs:s1_ptrs; unsigned int * s_poses = frag_Q_larger_read?s2_poses:s1_poses; int q_res_offset = 0; mapping_result_t * q_res = frag_Q_larger_read?q_res_2:q_res_1; char * q_res_chro = NULL; locate_gene_position(q_res -> selected_position, &global_context -> chromosome_table, &q_res_chro, &q_res_offset); q_res_offset +=1 ; // all tables are one-based. unsigned int q_search_start = q_res_offset; if(q_search_start > FUNKY_COLOCATION_TOLERANCE) q_search_start -= FUNKY_COLOCATION_TOLERANCE; else q_search_start = 0; int cand_i, canidate_s_items = bktable_lookup(&global_context -> funky_table_BC, q_res_chro, q_search_start, 2*FUNKY_COLOCATION_TOLERANCE, s_poses, s_ptrs, S12_LIST_CAPACITY); if(0 && frag_A_no == 143736){ SUBREADprintf("TRALOG: SEARCH CLOSE TO %s READ: %s:%u ; HAD %d HITS\n", frag_Q_larger_read?"LARGE":"SMALL", q_res_chro, q_search_start, canidate_s_items); } // scan if candidate is reversed. // s_ptrs - NULL is the fragment no. for(cand_i = 0; cand_i < canidate_s_items; cand_i ++){ subread_read_number_t frag_S_no = (s_ptrs[cand_i] - NULL)/ 2; int frag_S_is_read_B = (s_ptrs[cand_i] - NULL) % 2; mapping_result_t read_S_res_body, mate_S_res_body; mapping_result_t * read_S_res = &read_S_res_body; mapping_result_t * mate_S_res = &mate_S_res_body; bigtable_readonly_result(global_context, NULL, frag_S_no, 0, frag_S_is_read_B, read_S_res, NULL); bigtable_readonly_result(global_context, NULL, frag_S_no, 0, !frag_S_is_read_B, mate_S_res, NULL); int is_read_S_negative = (read_S_res -> result_flags & CORE_IS_NEGATIVE_STRAND)?1:0; int is_mate_S_negative = (mate_S_res -> result_flags & CORE_IS_NEGATIVE_STRAND)?1:0; if(frag_S_is_read_B) is_read_S_negative = !is_read_S_negative; else is_mate_S_negative = !is_mate_S_negative; int is_INV_TRA = is_mate_S_negative == is_read_S_negative; if(is_INV_TRA && is_read_S_negative == !frag_Q_larger_read){ if(frag_Q_larger_read) fraglist_append(&fli_INV_B, frag_S_no * 2 + frag_S_is_read_B); else fraglist_append(&fli_INV_C, frag_S_no * 2 + frag_S_is_read_B); } if((!is_INV_TRA) && is_read_S_negative == !frag_Q_larger_read){ if(frag_Q_larger_read) fraglist_append(&fli_STR_C, frag_S_no * 2 + frag_S_is_read_B); else fraglist_append(&fli_STR_B, frag_S_no * 2 + frag_S_is_read_B); } } } unsigned int guesed_p_small, guessed_tra_length, guessed_q_small, is_brkP_cand_found = 0; if(fli_INV_B.fragments >= 1 && fli_INV_C.fragments >= 1){ int PEmates = find_translocation_BC_mates(global_context, q_res_1, q_res_2, &fli_INV_B, &fli_INV_C, 1, s1_selected_list, s2_selected_list, s1_poses, s2_poses, &guesed_p_small, &guessed_tra_length, &guessed_q_small); int ConformPE = find_translocation_BC_conformation(global_context, PEmates, s1_poses, s2_poses); int brkPQR_are_found = 0; unsigned int brkPno, brkQno, brkRno; char out1pos[100], out2pos[100]; absoffset_to_posstr(global_context, q_res_1 -> selected_position, out1pos); absoffset_to_posstr(global_context, q_res_2 -> selected_position, out2pos); SUBREADprintf("TRALOG: A_READ: %09llu: INV : %s ~ %s ; %d PE_MATES (%s)\n", frag_A_no, out1pos, out2pos, PEmates, ConformPE?"CONFORMABLE":"INCONSISTENT"); //SUBREADputs("TRALOG: INV_C:"); //print_frags(global_context,&fli_INV_C); //SUBREADputs("TRALOG: INV_B:"); //print_frags(global_context,&fli_INV_B); if(PEmates) brkPQR_are_found = find_translocation_brk_PQR(global_context, q_res_1, q_res_2, &fli_INV_B, &fli_INV_C, &brkPno, &brkQno, &brkRno, 1, &is_brkP_cand_found); if(brkPQR_are_found){ brkPQR_are_found = breakpoint_PQR_supported(global_context , brkPno , brkQno, brkRno, &fli_INV_B, &fli_INV_C, 1); SUBREADprintf("TRALOG: A_READ: INV BRK_PQR_SUPPED=%d\n", brkPQR_are_found); } if(brkPQR_are_found) create_or_update_translocation_result( global_context , brkPno, brkQno, brkRno , PEmates, 1); else if(ConformPE && fli_INV_B.fragments > 2 && fli_INV_C.fragments > 2 && is_brkP_cand_found) create_or_update_translocation_imprecise_result(global_context, guesed_p_small, guessed_tra_length, guessed_q_small, PEmates, 1); } if(fli_STR_B.fragments >= 1 && fli_STR_C.fragments >= 1){ int PEmates = find_translocation_BC_mates(global_context, q_res_1, q_res_2, &fli_STR_B, &fli_STR_C, 0, s1_selected_list, s2_selected_list, s1_poses, s2_poses, &guesed_p_small, &guessed_tra_length, &guessed_q_small); int ConformPE = find_translocation_BC_conformation(global_context, PEmates, s1_poses, s2_poses); char out1pos[100], out2pos[100]; absoffset_to_posstr(global_context, q_res_1 -> selected_position, out1pos); absoffset_to_posstr(global_context, q_res_2 -> selected_position, out2pos); SUBREADprintf("TRALOG: A_READ: %09llu: TRA : %s ~ %s ; %d PE_MATES (%s)\n", frag_A_no, out1pos, out2pos, PEmates, ConformPE?"CONFORMABLE":"INCONSISTENT"); //SUBREADputs("TRALOG: STR_B:"); //print_frags(global_context,&fli_STR_B); //SUBREADputs("TRALOG: STR_C:"); //print_frags(global_context,&fli_STR_C); int brkPQR_are_found = 0; unsigned int brkPno, brkQno, brkRno; if(PEmates) brkPQR_are_found = find_translocation_brk_PQR(global_context, q_res_1, q_res_2, &fli_STR_B, &fli_STR_C, &brkPno, &brkQno, &brkRno, 0, &is_brkP_cand_found); if(brkPQR_are_found){ brkPQR_are_found = breakpoint_PQR_supported(global_context , brkPno , brkQno, brkRno, &fli_STR_B, &fli_STR_C, 0); } if(brkPQR_are_found) create_or_update_translocation_result( global_context , brkPno, brkQno, brkRno , PEmates, 0); else if(ConformPE && fli_INV_B.fragments > 2 && fli_INV_C.fragments > 2 && is_brkP_cand_found) create_or_update_translocation_imprecise_result(global_context, guesed_p_small, guessed_tra_length, guessed_q_small, PEmates, 0); } fraglist_destroy(&fli_STR_B); fraglist_destroy(&fli_STR_C); fraglist_destroy(&fli_INV_B); fraglist_destroy(&fli_INV_C); } free(s1_result_ptr_list); free(s2_result_ptr_list); free(s1_ptrs); free(s2_ptrs); free(s1_poses); free(s2_poses); free(s1_selected_list); free(s2_selected_list); } void finalise_inversions(global_context_t * global_context){ subread_read_number_t frag_A_i; void ** s1_ptrs, **s2_ptrs; unsigned int * s1_poses, * s2_poses; s1_ptrs = malloc(sizeof(void *) * S12_LIST_CAPACITY); s2_ptrs = malloc(sizeof(void *) * S12_LIST_CAPACITY); s1_poses = malloc(sizeof(int) * S12_LIST_CAPACITY); s2_poses = malloc(sizeof(int) * S12_LIST_CAPACITY); unsigned long long * s1_selected_list = malloc(sizeof(long long) * S12_LIST_CAPACITY); // fragment_no * 2 + is_second_read unsigned long long * s2_selected_list = malloc(sizeof(long long) * S12_LIST_CAPACITY); mapping_result_t ** s1_result_ptr_list = malloc(sizeof(mapping_result_t *) * S12_LIST_CAPACITY); mapping_result_t ** s2_result_ptr_list = malloc(sizeof(mapping_result_t *) * S12_LIST_CAPACITY); int frag_Q_larger_read, xk1, xk2; for(frag_A_i = 0; frag_A_i < global_context -> funky_list_DE.fragments; frag_A_i ++){ int s1_list_items = 0, s2_list_items = 0; subread_read_number_t frag_A_no = global_context -> funky_list_DE.fragment_numbers[frag_A_i]; mapping_result_t q_res_A_body, q_res_B_body; mapping_result_t * q_res_A = &q_res_A_body, * q_res_B = &q_res_B_body; bigtable_readonly_result(global_context, NULL, frag_A_no, 0, 0, q_res_A, NULL); bigtable_readonly_result(global_context, NULL, frag_A_no, 0, 1, q_res_B, NULL); mapping_result_t * q_res_1 = q_res_A -> selected_position > q_res_B -> selected_position?q_res_B:q_res_A; mapping_result_t * q_res_2 = q_res_A -> selected_position <= q_res_B -> selected_position?q_res_B:q_res_A; /*************************************************************************************************** * * is_q1_negative and is_q2_negative describes the strandness of the original FASTQ read sequence. * * For the very normal mappings, is_q1_negative must be 0 and is_q2_negative must be 1. * * If is_q1_negative != is_q2_negative, then there is a strand-jumpping fusion between the two reads. */ int is_q1_negative = (q_res_1 -> result_flags & CORE_IS_NEGATIVE_STRAND)?1:0; int is_q2_negative = (q_res_2 -> result_flags & CORE_IS_NEGATIVE_STRAND)?1:0; if(q_res_B == q_res_1)is_q1_negative=!is_q1_negative; if(q_res_B == q_res_2)is_q2_negative=!is_q2_negative; if(is_q1_negative == 0 && is_q2_negative == 0) // D READ { for(frag_Q_larger_read = 0; frag_Q_larger_read < 2; frag_Q_larger_read++){ int * s_list_items = frag_Q_larger_read?&s2_list_items:&s1_list_items; void ** s_ptrs = frag_Q_larger_read?s2_ptrs:s1_ptrs; unsigned int * s_poses = frag_Q_larger_read?s2_poses:s1_poses; int q_res_offset = 0; mapping_result_t * q_res = frag_Q_larger_read?q_res_2:q_res_1; unsigned long long * s_selected_list = frag_Q_larger_read?s2_selected_list:s1_selected_list; mapping_result_t ** s_result_ptr_list = frag_Q_larger_read?s2_result_ptr_list:s1_result_ptr_list; char * q_res_chro = NULL; locate_gene_position(q_res -> selected_position, &global_context -> chromosome_table, &q_res_chro, &q_res_offset); q_res_offset +=1 ; // all tables are one-based. unsigned int q_search_start = q_res_offset; if(q_search_start > FUNKY_COLOCATION_TOLERANCE) q_search_start -= FUNKY_COLOCATION_TOLERANCE; else q_search_start = 0; int cand_i, canidate_s_items = bktable_lookup(&global_context -> funky_table_DE, q_res_chro, q_search_start, 2*FUNKY_COLOCATION_TOLERANCE, s_poses, s_ptrs, S12_LIST_CAPACITY); // scan if candidate is reversed. // s_ptrs - NULL is the fragment no. for(cand_i = 0; cand_i < canidate_s_items; cand_i ++){ subread_read_number_t frag_S_no = (s_ptrs[cand_i] - NULL)/2; int frag_S_larger_read = (s_ptrs[cand_i] - NULL)%2; if(frag_S_no == frag_A_no) continue; if(frag_S_larger_read == frag_Q_larger_read){ mapping_result_t res_S_A_body, res_S_B_body; mapping_result_t * res_S_A = &res_S_A_body , * res_S_B = &res_S_B_body; bigtable_readonly_result(global_context, NULL, frag_S_no, 0, 0, res_S_A, NULL); bigtable_readonly_result(global_context, NULL, frag_S_no, 0, 1, res_S_B, NULL); mapping_result_t * res_S_1 = res_S_A -> selected_position > res_S_B -> selected_position?res_S_B:res_S_A; mapping_result_t * res_S_2 = res_S_A -> selected_position <= res_S_B -> selected_position?res_S_B:res_S_A; mapping_result_t * co_locatted_S_res = frag_S_larger_read?res_S_2:res_S_1; int is_s1_negative = (res_S_1 -> result_flags & CORE_IS_NEGATIVE_STRAND)?1:0; int is_s2_negative = (res_S_2 -> result_flags & CORE_IS_NEGATIVE_STRAND)?1:0; if(res_S_B == res_S_1) is_s1_negative = !is_s1_negative; if(res_S_B == res_S_2) is_s2_negative = !is_s2_negative; if( is_s1_negative != 0 && is_s2_negative != 0 ){ // E READ s_selected_list[*s_list_items] = frag_S_no * 2 + frag_S_larger_read; s_result_ptr_list[*s_list_items] = co_locatted_S_res; (*s_list_items)++; } } } } } int found_INV_frags = 0; srInt_64 guessed_Z_large_abs_sum = 0, guessed_Y_small_abs_sum = 0; for(xk1 = 0; xk1 < s1_list_items; xk1++){ for(xk2 = 0; xk2 < s2_list_items ; xk2 ++){ if(s1_selected_list[xk1]/2 == s2_selected_list[xk2]/2) { found_INV_frags ++; // now there is only one D fragment. here we found the E fragment for it (E fragment is in s1[xk1] and s2[xk2]) // s1 is the E read that is close to D_1; s2 is the E read that is close to D_2; D_1 is the D read with smaller coordinate. // res_E1 is the read that is close to D_2; mapping location of E_1 should be larger than D_2 mapping_result_t * res_D1 = q_res_1; mapping_result_t * res_D2 = q_res_2; mapping_result_t * res_E1 = s2_result_ptr_list[xk2]; mapping_result_t * res_E2 = s1_result_ptr_list[xk1]; int Gap_a_length = res_E2 -> selected_position - res_D1 -> selected_position - res_D1 -> read_length; int Gap_b_length = res_E1 -> selected_position - res_D2 -> selected_position - res_D2 -> read_length; int average_gap_len = (Gap_b_length + Gap_a_length)/2; guessed_Y_small_abs_sum += res_D1 -> selected_position + res_D1 -> read_length - average_gap_len / 2; guessed_Z_large_abs_sum += res_E1 -> selected_position - average_gap_len / 2; SUBREADprintf("INVLOG: GUESSED_LEN = %d + %d / 2 = %d\n", Gap_a_length, Gap_b_length, average_gap_len); } } } unsigned int brkYno=0xffffffff, brkZno=0xffffffff; int cand_YZ_breakpoints = 0; if(found_INV_frags > 0) { char * q_small_chro = NULL; int q_small_pos = 0; guessed_Y_small_abs_sum /= found_INV_frags; guessed_Z_large_abs_sum /= found_INV_frags; SUBREADprintf("INVLOG: GUESSED_YZ=%lld, %lld\n", guessed_Y_small_abs_sum, guessed_Z_large_abs_sum); locate_gene_position(q_res_1 -> selected_position, &global_context -> chromosome_table, &q_small_chro, &q_small_pos); int cand_Y, cand_Z; cand_YZ_breakpoints = bktable_lookup(&global_context -> breakpoint_table_YZ, q_small_chro, q_small_pos, global_context -> config.maximum_pair_distance , s1_poses, s1_ptrs, S12_LIST_CAPACITY); //SUBREADprintf("INVLOG: %09u FOUND %d CANDIDATE BKs AT %s:%u\n", frag_A_no, cand_YZ_breakpoints, q_small_chro, q_small_pos); indel_context_t * indel_context = (indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]; for(cand_Y = 0; cand_Y < cand_YZ_breakpoints ; cand_Y ++){ if(brkYno < 0xffffffff) break; int event_no_Y = s1_ptrs[cand_Y] - NULL; chromosome_event_t * event_body_Y = indel_context -> event_space_dynamic + event_no_Y; if(event_body_Y -> small_side_increasing_coordinate) continue; if(event_body_Y -> small_side_increasing_coordinate != event_body_Y -> large_side_increasing_coordinate) assert(0); if(abs(event_body_Y -> event_large_side - q_res_2 -> selected_position) < global_context -> config.maximum_pair_distance){ for(cand_Z = 0; cand_Z < cand_YZ_breakpoints ; cand_Z ++){ int event_no_Z = s1_ptrs[cand_Z] - NULL; chromosome_event_t * event_body_Z = indel_context -> event_space_dynamic + event_no_Z; if(!event_body_Z -> small_side_increasing_coordinate) continue; if(event_body_Z -> small_side_increasing_coordinate != event_body_Z -> large_side_increasing_coordinate) assert(0); long long dist_small = event_body_Z -> event_small_side , dist_large = event_body_Z -> event_large_side; dist_small -= event_body_Y -> event_small_side; dist_large -= event_body_Y -> event_large_side; long long dist_small_large_diff = dist_small; dist_small_large_diff -= dist_large; if(abs(dist_small_large_diff) <= BREAK_POINT_MAXIMUM_TOLERANCE && abs(dist_large) <= BREAK_POINT_MAXIMUM_TOLERANCE && event_body_Z -> small_side_increasing_coordinate != event_body_Y -> small_side_increasing_coordinate){ brkYno = event_no_Y; brkZno = event_no_Z; break; } } if(1) { char outpos1[100], outpos2[100]; absoffset_to_posstr(global_context, event_body_Y -> event_small_side, outpos1); absoffset_to_posstr(global_context, event_body_Y -> event_large_side, outpos2); SUBREADprintf("INVLOG: %09llu FOUND BREAKPOINT YZ: %s ~ %s, INC_COR: %c %c , nSUP=%d\n", frag_A_no, outpos1, outpos2, event_body_Y -> small_side_increasing_coordinate?'>':'<', event_body_Y -> large_side_increasing_coordinate?'>':'<' , event_body_Y -> final_counted_reads); } } } } char *brkYchr = "NULL"; unsigned int brkYabs_small = 0, brkYabs_large = 0; int brkYsmall = 0, brkYlarge = 0; int is_precisely_called = 0, is_roughly_called = 0; if(brkYno < 0xffffffff){ // s1_selected_list : 2 * fragment_S_no + frag_S_larger_read int is_passed_YZ = breakpoint_YZ_supported(global_context, brkYno, brkZno, s1_selected_list, s1_list_items, s2_selected_list, s2_list_items); if(is_passed_YZ) { is_precisely_called = 1; get_event_two_coordinates(global_context, brkYno, &brkYchr, &brkYsmall, &brkYabs_small, &brkYchr, &brkYlarge, &brkYabs_large); } else is_roughly_called = 1; //SUBREADprintf("\nINVLOG: FINALLY_%sCONFIRMED: %09u %s:%u (len=%d) INVERSED.\n", is_passed_YZ?"":"NOT ", frag_A_no, brkYchr, brkYsmall, brkYlarge - brkYsmall); } //SUBREADprintf("\nINVLOG: FINALLY_GUESSED: %09u found_INV_frags=%d, s1_list_items=%d, s2_list_items=%d, cand_YZ_breakpoints=%d\n", frag_A_no, found_INV_frags, s1_list_items, s2_list_items, cand_YZ_breakpoints); //for(xk1 = 0; xk1 < s1_list_items; xk1++) SUBREADprintf("INVLOG: %09d S_1 MATES: %09llu\n" , frag_A_no , s1_selected_list[xk1]/2); //for(xk1 = 0; xk1 < s2_list_items; xk1++) SUBREADprintf("INVLOG: %09d S_2 MATES: %09llu\n" , frag_A_no , s2_selected_list[xk1]/2); /* if(found_INV_frags >= min(s1_list_items , s2_list_items) - 2 && found_INV_frags > 1 && !is_precisely_called && cand_YZ_breakpoints>0){ // guess brkYlarge, brkYsmall, brkZlarge, brkZsmall, brkYabsLarge, brkZabsLarge... locate_gene_position(guessed_Y_small_abs_sum, &global_context -> chromosome_table, &brkYchr, &brkYsmall); locate_gene_position(guessed_Z_large_abs_sum, &global_context -> chromosome_table, &brkYchr, &brkYlarge); //SUBREADprintf("\nINVLOG: FINALLY_GUESSED: %09u %s:%u (len=%llu) INVERSED.\n", frag_A_no, brkYchr, brkYsmall, guessed_Z_large_abs_sum - guessed_Y_small_abs_sum); is_roughly_called = 1; }*/ if( is_precisely_called || is_roughly_called ) { void * old_ptrs[_PQR_LIST_SIZE]; unsigned int old_poses[_PQR_LIST_SIZE]; int old_found = 0, old_i, old_inversions = bktable_lookup(&global_context -> inversion_result_table, brkYchr, brkYsmall - BREAK_POINT_MAXIMUM_TOLERANCE, 2*BREAK_POINT_MAXIMUM_TOLERANCE, old_poses, old_ptrs, _PQR_LIST_SIZE); for(old_i = 0; old_i < old_inversions; old_i ++){ inversion_result_t * inv_res_old = (inversion_result_t *) old_ptrs[old_i]; long long old_dist = inv_res_old -> length; old_dist -= brkYlarge - brkYsmall; // the difference on inversion length. if(abs(old_dist) < BREAK_POINT_MAXIMUM_TOLERANCE){ inv_res_old -> all_sup_D ++; inv_res_old -> max_sup_E = max(inv_res_old -> max_sup_E , found_INV_frags); old_found = 1; break; } } if(0 == old_found){ inversion_result_t * inv_res_new = malloc(sizeof(chromosome_event_t)); memset(inv_res_new, 0 , sizeof(chromosome_event_t)); inv_res_new -> length = brkYlarge - brkYsmall; inv_res_new -> is_precisely_called = is_precisely_called; if(is_precisely_called){ inv_res_new -> event_Y_number = brkYno; inv_res_new -> event_Z_number = brkZno; inv_res_new -> small_side = brkYabs_small; }else{ inv_res_new -> event_Y_rough_small_abs = guessed_Y_small_abs_sum; inv_res_new -> event_Z_rough_large_abs = guessed_Z_large_abs_sum; inv_res_new -> small_side = guessed_Y_small_abs_sum; } inv_res_new -> all_sup_D = 1; inv_res_new -> max_sup_E = found_INV_frags; bktable_append(&global_context -> inversion_result_table, brkYchr, brkYsmall, inv_res_new); } } } free(s1_result_ptr_list); free(s2_result_ptr_list); free(s1_ptrs); free(s2_ptrs); free(s1_poses); free(s2_poses); free(s1_selected_list); free(s2_selected_list); } void build_breakpoint_tables(global_context_t * global_context){ int xk1; indel_context_t * indel_context = (indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]; for(xk1 = 0; xk1 < indel_context -> total_events ; xk1++) { char * chro_name_left= NULL,* chro_name_right = NULL; int chro_pos_left= 0, chro_pos_right = 0; chromosome_event_t * event_body = indel_context -> event_space_dynamic + xk1; if(event_body -> event_type != CHRO_EVENT_TYPE_FUSION && event_body -> event_type != CHRO_EVENT_TYPE_JUNCTION) continue; locate_gene_position(event_body -> event_small_side, &global_context -> chromosome_table, &chro_name_left, &chro_pos_left); locate_gene_position(event_body -> event_large_side, &global_context -> chromosome_table, &chro_name_right, &chro_pos_right); long long dist = chro_pos_left; dist -= chro_pos_right; if(dist<0)dist=-dist; int breakpoint_group = -1; if(event_body -> is_strand_jumped){ // breakpoint QR or YZ if(chro_name_left != chro_name_right || dist > global_context -> config.maximum_translocation_length) breakpoint_group = 2; // QR else breakpoint_group = 3; // YZ }else{ // breakpoint QR or P if(chro_name_left != chro_name_right || dist > global_context -> config.maximum_translocation_length) breakpoint_group = 2; // QR else breakpoint_group = 1; // P } bucketed_table_t * index_table = breakpoint_group == 1? &global_context -> breakpoint_table_P : (breakpoint_group == 2? &global_context -> breakpoint_table_QR: (breakpoint_group == 3? &global_context -> breakpoint_table_YZ: NULL ) ); //SUBREADprintf("BPLOG: %s:%u ~ %s:%u (%c) GRP=%d (%p)\n", chro_name_left, chro_pos_left, chro_name_right, chro_pos_right, event_body -> is_strand_jumped?'X':'=', breakpoint_group, index_table); if(index_table) bktable_append(index_table, chro_name_left, chro_pos_left, NULL + xk1); if(index_table) bktable_append(index_table, chro_name_right, chro_pos_right, NULL + xk1); } } void finalise_structural_variances(global_context_t * global_context){ SUBREADprintf("Funky Tables: A:%u, BC:%u, DE:%u\n", (unsigned int) global_context -> funky_list_A.fragments, (unsigned int)global_context -> funky_table_BC.fragments / 2, (unsigned int)global_context -> funky_list_DE.fragments); build_breakpoint_tables(global_context); SUBREADprintf("Breakpoint Tables: P:%u, QR:%u, YZ:%u\n", (unsigned int)global_context -> breakpoint_table_P.fragments, (unsigned int)global_context -> breakpoint_table_QR.fragments, (unsigned int)global_context -> breakpoint_table_YZ.fragments); finalise_translocations(global_context); finalise_inversions(global_context); } Rsubread/src/core-junction.h0000644000175200017520000002177614136047615017106 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #ifndef SUBREAD_CORE_JUNCTION_H_ #define SUBREAD_CORE_JUNCTION_H_ #include "subread.h" #include "hashtable.h" #include "core.h" //#warning "======== REMOVE *2000 =============" #define REALIGN_TOTAL_TRIES (50) #define FUNKY_FRAGMENT_A 1 // same strand and gapped (0=tra_len) or chimeric. #define FUNKY_FRAGMENT_DE 4 // tlen < tra_len and strand jumpped #define NOT_FUNKY 0 // normal fragment #define FUNKY_COLOCATION_TOLERANCE 500 #define BREAK_POINT_MAXIMUM_TOLERANCE 80 #define S12_LIST_CAPACITY 100 // as the python sub-string rule: start is the first wanted base and end is the first unwanted base. typedef struct{ short read_pos_start; short read_pos_end; unsigned int abs_offset_for_start; // jumped from the "main piece" view char is_strand_jumped; char is_connected_to_large_side; chromosome_event_t * event_after_section; } perfect_section_in_read_t; typedef struct{ // result context //unsigned char back_search_confirmed_sections; //unsigned char front_search_confirmed_sections; // NOTE THAT EVERYTHING IN back_search_junctions IS BACKWARD. // 1, ORDER OF EXONS ARE BACKWARD // 2, "ABS_OFFSET_FOR_START" ARE ACTUALLY AT END OF SECTIONS //perfect_section_in_read_t back_search_junctions[MAX_EVENTS_IN_READ]; //perfect_section_in_read_t front_search_junctions[MAX_EVENTS_IN_READ]; // middle result context unsigned char tmp_search_sections; perfect_section_in_read_t tmp_search_junctions [MAX_EVENTS_IN_READ]; char current_is_strand_jumped; perfect_section_in_read_t result_back_junctions [MAX_ALIGNMENT_PER_ANCHOR][MAX_EVENTS_IN_READ]; perfect_section_in_read_t result_front_junctions [MAX_ALIGNMENT_PER_ANCHOR][MAX_EVENTS_IN_READ]; int result_back_junction_numbers[MAX_ALIGNMENT_PER_ANCHOR]; int result_front_junction_numbers[MAX_ALIGNMENT_PER_ANCHOR]; int all_back_alignments; int all_front_alignments; int known_junctions; unsigned int total_tries; // unsigned int tmp_jump_length; // unsigned int best_jump_length; // for the BEST record // they are not restored int best_matching_bases; int best_second_match_diff; int second_best_matching_bases; int best_indel_penalty; int tmp_total_matched_bases; int tmp_indel_penalty; int is_currently_tie; int best_is_complex; int best_support_as_simple; int best_min_unsupport_as_simple; int best_min_support_as_complex; int best_is_pure_donor_found_explain; // for the "current" stack (they are restored after poping from the stack) int tmp_support_as_simple; int tmp_min_unsupport; int tmp_min_support_as_complex; int tmp_is_pure_donor_found_explain; // input context int full_read_len; int is_fully_covered; char * full_read_text; char * full_qual_text; char * read_name; int is_confirmed_section_negative_strand; subread_read_number_t pair_number; int is_second_read; int best_read_id; }explain_context_t; typedef struct{ } junction_context_t; typedef struct { int read_len_1; int read_len_2; char * read_text_1; char * read_text_2; char is_negative_strand; } new_junction_context_t; void new_explain_try_replace(global_context_t* global_context, thread_context_t * thread_context, explain_context_t * explain_context, int remainder_len, int search_to_back); int init_junction_tables(global_context_t * context); int destroy_junction_tables(global_context_t * context); int process_voting_junction(global_context_t * global_context, thread_context_t * thread_context, subread_read_number_t pair_number, gene_vote_t * vote_1, gene_vote_t * vote_2, char * read_name_1, char * read_name_2, char * read_text_1, char * read_text_2, int read_len_1, int read_len_2, int is_negative_strand, gene_vote_number_t v1_all_subreads, gene_vote_number_t v2_all_subreads); int init_junction_thread_contexts(global_context_t * global_context, thread_context_t * thread_context, int task); int finalise_junction_thread(global_context_t * global_context, thread_context_t * thread_context, int task); unsigned int explain_read(global_context_t * global_context, thread_context_t * thread_context, realignment_result_t * realigns, subread_read_number_t pair_number,int read_len, char * read_name , char *read_text, char *qual, int is_second_read, int best_read_id, int is_negative_strand); int write_junction_final_results(global_context_t * global_context); // back_search_read_tail IS THE EXACT VERY SURE POSITION IN THE READ // back_search_tail_position IS THE EXACT VERY SURE POSITION ON CHROMOSOME CORRESPONDING TO back_search_read_tail int do_explain_back_search(global_context_t * global_context, thread_context_t * thread_context, explain_context_t * explain_context, char * read_text, char * read_qual, int back_search_read_tail, unsigned int back_search_tail_position); int do_explain_front_search(global_context_t * global_context, thread_context_t * thread_context, explain_context_t * explain_context, char * read_text, char * read_qual, int front_search_read_head, unsigned int front_search_head_position); unsigned int finalise_explain_CIGAR(global_context_t * global_context, thread_context_t * thread_context, explain_context_t * explain_context, realignment_result_t * realigns); void search_events_to_back(global_context_t * global_context, thread_context_t * thread_context, explain_context_t * explain_context, char * read_text , char * qual_text, unsigned int read_tail_abs_offset, short read_tail_pos, short sofar_matched, int suggested_movements, int do_not_jump); void search_events_to_back(global_context_t * global_context, thread_context_t * thread_context, explain_context_t * explain_context, char * read_text , char * qual_text, unsigned int read_tail_abs_offset, short read_tail_pos, short sofar_matched, int suggested_movements, int do_not_jump); void find_new_junctions(global_context_t * global_context, thread_context_t * thread_context, subread_read_number_t pair_number, char * read_name, char * read_text, char * qual_text, int read_len, int is_second_read, int best_read_id); int donor_score(global_context_t * global_context, thread_context_t * thread_context, unsigned int left_virtualHead_abs_offset, unsigned int right_virtualHead_abs_offset, int left_indels, int right_indels, int normally_arranged, int guess_start, int guess_end, char * read_text, int read_len, int * final_split_point, int * is_GT_AG_strand, int * is_donor_found, int * inserted_bases, int * small_side_inc_coor, int * large_side_inc_coor, char *read_name); int donor_jumped_score(global_context_t * global_context, thread_context_t * thread_context, unsigned int left_virtualHead_abs_offset, unsigned int right_virtualHead_abs_offset, int guess_start, int guess_end, char * read_text, int read_len, int is_left_half_negative, int is_right_half_negative, int is_left_part_on_left_as_reversed, int * final_split_point, int * is_GT_AG_strand, int * is_donor_found, int * small_side_inc_coor, int * large_side_inc_coor); int write_fusion_final_results(global_context_t * global_context); int is_ambiguous_voting(global_context_t * global_context, subread_read_number_t pair_number, int is_second_read, int max_vote, int max_start,int max_end, int read_len, int is_negative); void core_search_short_exons(global_context_t * global_context, thread_context_t * thread_context, char * read_text, char * qualityb0, int rl, unsigned int P1_Pos, unsigned int P2_Pos, short read_coverage_start, short read_coverage_end); void core_fragile_junction_voting(global_context_t * global_context, thread_context_t * thread_context, char * rname, char * read, char * qual, unsigned int full_rl, int negative_strand, int color_space, unsigned int low_border, unsigned int high_border, gene_vote_t *vote_p1); int is_funky_fragment(global_context_t * global_context, char * rname1, char * chr1, unsigned int pos1, int rlen1, int is_1_negative, char * cigar1, char * seq1, char * rname2, char * chr2, unsigned int pos2, int rlen2, int is_2_negative, char * cigar2, char * seq2, int tlen_removed_intron); void finalise_structural_variances(global_context_t * global_context); void debug_show_event(global_context_t* global_context, chromosome_event_t * event); void get_event_two_coordinates(global_context_t * global_context, unsigned int event_no, char ** small_chro, int * small_pos, unsigned int * small_abs, char ** large_chro, int * large_pos, unsigned int * large_abs); int get_offset_maximum_chro_pos(global_context_t * global_context, thread_context_t * thread_context, unsigned int linear); #endif Rsubread/src/core.c0000644000175200017520000060736314136047615015254 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ /*************************************************************** The ASCII Art used in this file was generated using FIGlet and the big font, contributed by Glenn Chappell to FIGlet. ***************************************************************/ #include #include #include #include #include #include #include #include #include #ifndef __MINGW32__ #include #endif #include #include #include #include #include "subread.h" #include "sublog.h" #include "core.h" #include "input-files.h" #include "input-blc.h" #include "sorted-hashtable.h" #include "HelperFunctions.h" #include "core-bigtable.h" #include "core-indel.h" #include "core-junction.h" static struct option long_options[] = { {"memory-optimisation", required_argument, 0, 0}, {0, 0, 0, 0} }; int (*progress_report_callback)(int, int, int); void core_version_number(char * program) { SUBREADprintf("\n%s v%s\n\n" , program, SUBREAD_VERSION); } int is_valid_float(char * optarg, char * optname){ int xk1=0; while(1){ int nch = optarg[xk1++]; if(!nch){ if(xk1 == 1){ SUBREADprintf("Value for argumant %s-%s is missing.\n", optname[1]?"-":"", optname); return 0; } break; } if(( nch!='-' || xk1 > 1 ) && nch != '.' && !isdigit(nch)){ SUBREADprintf("Value for argumant %s-%s is not a valid number: '%s'\n", optname[1]?"-":"", optname, optarg); return 0; } } return 1; } int is_valid_digit(char * optarg, char * optname){ int xk1=0; while(1){ int nch = optarg[xk1++]; if(!nch){ if(xk1 == 1){ SUBREADprintf("Value for argumant %s-%s is missing.\n", optname[1]?"-":"", optname); return 0; } break; } if(( nch!='-' || xk1 > 1 ) && !isdigit(nch)){ SUBREADprintf("Value for argumant %s-%s is not a valid number: '%s'\n", optname[1]?"-":"", optname, optarg); return 0; } } return 1; } int is_valid_digit_range(char * optarg, char * optname, int min, int max_inc){ if(!is_valid_digit( optarg, optname )) return 0; int tv = atoi(optarg); if(tv < min || tv > max_inc){ SUBREADprintf("Value for argumant %s-%s is out of range: %d to %d\n", optname[1]?"-":"", optname, min, max_inc); return 0; } return 1; } void warning_file_limit() { #ifndef __MINGW32__ struct rlimit limit_st; getrlimit(RLIMIT_NOFILE, & limit_st); { if(min(limit_st.rlim_cur , limit_st.rlim_max) < 400) { print_in_box(80,0,0,"WARNING This operation needs to open many files at same time,"); print_in_box(80,0,0," but your OS only allows to open %d files.", min(limit_st.rlim_cur , limit_st.rlim_max)); print_in_box(80,0,0," You can use command 'ulimit -n 500' to raise this limit"); print_in_box(80,0,0," to 500, or the program may become very slow."); print_in_box(80,0,0,""); } } #endif } int exec_cmd(char * cmd, char * outstr, int out_limit){ FILE* pipe = popen(cmd, "r"); if (!pipe) return -1; outstr[0]=0; char * linebuf = malloc(3000); int out_ptr = 0; while (!feof(pipe)) { if (fgets(linebuf, 128, pipe) != NULL){ if(out_ptr + strlen(linebuf) < out_limit){ strcat(outstr+out_ptr, linebuf); out_ptr += strlen(linebuf); } } } pclose(pipe); free(linebuf); return out_ptr; } void print_in_box(int line_width, int is_boundary, int options, char * pattern,...) { int put_color_for_colon, is_center, is_wrapped; va_list args; va_start(args , pattern); char * content, *out_line_buff; put_color_for_colon = (options & PRINT_BOX_NOCOLOR_FOR_COLON)?0:1; is_center = (options & PRINT_BOX_CENTER)?1:0; is_wrapped = (options & PRINT_BOX_WRAPPED)?1:0; content= malloc(1200); int content_len = vsprintf(content, pattern, args); out_line_buff= malloc(1200); out_line_buff[0]=0;; if(is_wrapped){ int seg_i = 0; for(seg_i=0; seg_i < content_len; seg_i += line_width-7){ strcpy(out_line_buff, content + seg_i); out_line_buff[line_width-7] = 0; print_in_box(line_width, is_boundary, options & (~PRINT_BOX_WRAPPED), out_line_buff); } }else{ int is_R_code,x1,content_len = strlen(content), state, txt_len, is_cut = 0, real_lenwidth; is_R_code = 1; #ifdef MAKE_STANDALONE is_R_code = 0; #endif if(content_len>0&&content[content_len-1]=='\r'){ content_len--; content[content_len] = 0; } if(content_len>0&&content[content_len-1]=='\n'){ content_len--; content[content_len] = 0; } state = 0; txt_len = 0; real_lenwidth = line_width; for(x1 = 0; content [x1]; x1++) { char nch = content [x1]; if(nch == CHAR_ESC) state = 1; if(state){ real_lenwidth --; }else{ txt_len++; if(txt_len == 80 - 6) { is_cut = 1; } } if(nch == 'm' && state) state = 0; } if(is_cut) { state = 0; txt_len = 0; for(x1 = 0; content [x1]; x1++) { char nch = content [x1]; if(nch == CHAR_ESC) state = 1; if(!state){ txt_len++; if(txt_len == 80 - 9) { strcpy(content+x1, "\x1b[0m ..."); content_len = line_width - 4; content_len = 80 - 4; line_width = 80; break; } } if(nch == 'm' && state) state = 0; } } if(content_len==0 && is_boundary) { strcat(out_line_buff,is_boundary==1?"//":"\\\\"); for(x1=0;x10 && col1w < content_len-1 && put_color_for_colon) { content[col1w+1]=0; strcat(out_line_buff,content); strcat(out_line_buff," "); sprintf(out_line_buff+strlen(out_line_buff),"%c[36m", CHAR_ESC); strcat(out_line_buff,content+col1w+2); sprintf(out_line_buff+strlen(out_line_buff),"%c[0m", CHAR_ESC); } else strcat(out_line_buff,content); } // for(x1=0;x1config.output_prefix); FILE * sumfp = fopen(sumname,"w"); #ifdef __MINGW32__ fprintf(sumfp, "Total_%s\t%I64u\n", global_context->input_reads.is_paired_end_reads?"fragments":"reads" , global_context -> all_processed_reads); #else fprintf(sumfp, "Total_%s\t%llu\n", global_context->input_reads.is_paired_end_reads?"fragments":"reads" , global_context -> all_processed_reads); #endif fprintf(sumfp, "Mapped_%s\t%u\n", global_context->input_reads.is_paired_end_reads?"fragments":"reads" , global_context -> all_mapped_reads); fprintf(sumfp, "Uniquely_mapped_%s\t%u\n", global_context->input_reads.is_paired_end_reads?"fragments":"reads" , global_context -> all_uniquely_mapped_reads); fprintf(sumfp, "Multi_mapping_%s\t%u\n", global_context->input_reads.is_paired_end_reads?"fragments":"reads" , global_context -> all_multimapping_reads); fprintf(sumfp, "Unmapped_%s\t%u\n", global_context->input_reads.is_paired_end_reads?"fragments":"reads" , global_context -> all_unmapped_reads); if(global_context->input_reads.is_paired_end_reads){ #ifdef __MINGW32__ fprintf(sumfp, "Properly_paired_fragments\t%I64u\n",global_context -> all_correct_PE_reads); #else fprintf(sumfp, "Properly_paired_fragments\t%llu\n",global_context -> all_correct_PE_reads); #endif fprintf(sumfp, "Singleton_fragments\t%u\n", global_context -> not_properly_pairs_only_one_end_mapped); fprintf(sumfp, "More_than_one_chr_fragments\t%u\n", global_context -> not_properly_pairs_different_chro); fprintf(sumfp, "Unexpected_strandness_fragments\t%u\n", global_context -> not_properly_different_strands); fprintf(sumfp, "Unexpected_template_length\t%u\n", global_context -> not_properly_pairs_TLEN_wrong); fprintf(sumfp, "Inversed_mapping\t%u\n", global_context -> not_properly_pairs_wrong_arrangement); } if(global_context->config.entry_program_name == CORE_PROGRAM_SUBJUNC && ( global_context -> config.prefer_donor_receptor_junctions || !(global_context -> config.do_fusion_detection || global_context -> config.do_long_del_detection))) fprintf(sumfp, "Junctions\t%u\n", global_context -> all_junctions); fprintf(sumfp, "Indels\t%u\n", global_context -> all_indels); fclose(sumfp); #endif if(progress_report_callback) { long long int all_reads_K = global_context -> all_processed_reads / 1000; float mapped_reads_percentage = global_context -> all_mapped_reads * 1./global_context -> all_processed_reads; if(global_context->input_reads.is_paired_end_reads) mapped_reads_percentage/=2; progress_report_callback(10, 900000, (int) (miltime()-global_context->start_time)); progress_report_callback(10, 900010, (int) all_reads_K); progress_report_callback(10, 900011, (int) (10000.*mapped_reads_percentage)); } print_in_box(80,0,1," "); print_in_box(89,0,1," %c[36mCompleted successfully.%c[0m", CHAR_ESC, CHAR_ESC); print_in_box(80,0,1," "); print_in_box(80,2,1," "); sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_INFO, ""); print_in_box(80, 1,1," Summary"); print_in_box(80, 0,1," "); #ifdef __MINGW32__ if(global_context->input_reads.is_paired_end_reads) print_in_box(80, 0,0," Total fragments : %I64d" , global_context -> all_processed_reads); else print_in_box(80, 0,0," Total reads : %I64d" , global_context -> all_processed_reads); print_in_box(81, 0,0," Mapped : %u (%.1f%%%%)", global_context -> all_mapped_reads, global_context -> all_mapped_reads*100.0 / global_context -> all_processed_reads); print_in_box(80, 0,0," Uniquely mapped : %u", global_context -> all_uniquely_mapped_reads); print_in_box(80, 0,0," Multi-mapping : %u", global_context -> all_multimapping_reads); print_in_box(80, 0,1," "); print_in_box(80, 0,0," Unmapped : %u", global_context -> all_unmapped_reads); if(global_context->input_reads.is_paired_end_reads){ print_in_box(80, 0,1," "); print_in_box(80, 0,0," Properly paired : %I64d", global_context -> all_correct_PE_reads); print_in_box(80, 0,0," Not properly paired : %I64d", global_context -> all_mapped_reads - global_context -> all_correct_PE_reads); print_in_box(80, 0,0," Singleton : %u", global_context -> not_properly_pairs_only_one_end_mapped); print_in_box(80, 0,0," Chimeric : %u", global_context -> not_properly_pairs_different_chro); print_in_box(80, 0,0," Unexpected strandness : %u", global_context -> not_properly_different_strands); print_in_box(80, 0,0," Unexpected fragment length : %u", global_context -> not_properly_pairs_TLEN_wrong); print_in_box(80, 0,0," Unexpected read order : %u", global_context -> not_properly_pairs_wrong_arrangement); } print_in_box(80, 0,1," "); if(global_context->config.output_prefix[0]) { if(global_context->config.entry_program_name == CORE_PROGRAM_SUBJUNC && ( global_context -> config.prefer_donor_receptor_junctions || !(global_context -> config.do_fusion_detection || global_context -> config.do_long_del_detection))) print_in_box(80, 0,0," Junctions : %u", global_context -> all_junctions); if((global_context-> config.do_fusion_detection || global_context-> config.do_long_del_detection)) print_in_box(80, 0,0," Fusions : %u", global_context -> all_fusions); print_in_box(80, 0,0," Indels : %u", global_context -> all_indels); } #else if(global_context->input_reads.is_paired_end_reads) print_in_box(80, 0,0," Total fragments : %'llu" , global_context -> all_processed_reads); else print_in_box(80, 0,0," Total reads : %'llu" , global_context -> all_processed_reads); print_in_box(81, 0,0," Mapped : %'u (%.1f%%%%)", global_context -> all_mapped_reads, global_context -> all_mapped_reads*100.0 / global_context -> all_processed_reads); print_in_box(80, 0,0," Uniquely mapped : %'u", global_context -> all_uniquely_mapped_reads); print_in_box(80, 0,0," Multi-mapping : %'u", global_context -> all_multimapping_reads); print_in_box(80, 0,1," "); print_in_box(80, 0,0," Unmapped : %'u", global_context -> all_unmapped_reads); if(global_context->input_reads.is_paired_end_reads){ print_in_box(80, 0,1," "); print_in_box(80, 0,0," Properly paired : %'llu", global_context -> all_correct_PE_reads); print_in_box(80, 0,0," Not properly paired : %'llu", global_context -> all_mapped_reads - global_context -> all_correct_PE_reads); print_in_box(80, 0,0," Singleton : %'u", global_context -> not_properly_pairs_only_one_end_mapped); print_in_box(80, 0,0," Chimeric : %'u", global_context -> not_properly_pairs_different_chro); print_in_box(80, 0,0," Unexpected strandness : %'u", global_context -> not_properly_different_strands); print_in_box(80, 0,0," Unexpected fragment length : %'u", global_context -> not_properly_pairs_TLEN_wrong); print_in_box(80, 0,0," Unexpected read order : %'u", global_context -> not_properly_pairs_wrong_arrangement); } print_in_box(80, 0,1," "); if(global_context->config.output_prefix[0]) { if(global_context->config.entry_program_name == CORE_PROGRAM_SUBJUNC && ( global_context -> config.prefer_donor_receptor_junctions || !(global_context -> config.do_fusion_detection || global_context -> config.do_long_del_detection))) print_in_box(80, 0,0," Junctions : %'u", global_context -> all_junctions); if((global_context-> config.do_fusion_detection || global_context-> config.do_long_del_detection)) print_in_box(80, 0,0," Fusions : %'u", global_context -> all_fusions); print_in_box(80, 0,0," Indels : %'u", global_context -> all_indels); } #endif if(global_context -> is_phred_warning) { print_in_box(80, 0,1," "); print_in_box(80,0,0," WARNING : Phred offset (%d) incorrect?", global_context->config.phred_score_format == FASTQ_PHRED33?33:64); } print_in_box(80, 0,1," "); print_in_box(80, 0,0," Running time : %.1f minutes", (miltime()-global_context->start_time)*1./60); if( global_context->input_reads.is_paired_end_reads && global_context -> config.reported_multi_best_reads<2 && global_context -> expected_TLEN_read_numbers < READPAIRS_FOR_CALC_EXPT_TLEN){ print_in_box(80, 0,1," "); print_in_box(80,0,0," NOTE : No enough read-pairs to derive expected fragment length."); } /* print_in_box(80, 0,0," Running time 0 : %.2f minutes", global_context->timecost_load_index/60); print_in_box(80, 0,0," Running time 1 : %.2f minutes", global_context->timecost_voting/60); print_in_box(80, 0,0," Running time 2 : %.2f minutes", global_context->timecost_before_realign/60); print_in_box(80, 0,0," Running time 3 : %.2f minutes", global_context->timecost_for_realign/60); */ print_in_box(80, 0,1,""); print_in_box(80, 2,1,""); sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_INFO, ""); return 0; } void show_progress(global_context_t * global_context, thread_context_t * thread_context, unsigned int current_read_no, int task) { // Read_chunk_start is the file_offset of the very first read in the entire file. // current_circle_start_position_file1 is the file_offset of the first read in this 5-million read chunk (or whatever the chunk size is) if(global_context->config.scRNA_input_mode){ if(task == 10){ char minchr[10]; float min_value = (miltime() - global_context -> start_time)*1./60; if(min_value < 9.91) sprintf(minchr, "%.01f", min_value); else sprintf(minchr, "% 3d", (int)min_value); print_in_box(80,0,0," processed % 3d million input reads in %s minutes", current_read_no/1000000, minchr); } //SUBREADprintf("TASK=%d, RNO=%u\n", task, current_read_no); return; } if(thread_context&&thread_context->thread_id) { SUBREADputs("show_progress can only be called by thread#0\n"); return; } gene_input_t * ginp1 = &global_context->input_reads.first_read_file; unsigned long long ginp1_file_pos = geinput_file_offset(ginp1); if(task == STEP_VOTING) { unsigned long long real_read_number = global_context -> all_processed_reads + current_read_no; if(real_read_number>1000) global_context -> input_reads . avg_read_length = (ginp1_file_pos - ginp1 -> read_chunk_start) * 1./real_read_number ; } unsigned long long total_file_size = global_context -> input_reads.first_read_file_size; unsigned long long guessed_all_reads = total_file_size / global_context -> input_reads . avg_read_length; unsigned long long current_block_start_file_offset = global_context -> current_circle_start_abs_offset_file1; unsigned long long guessed_this_chunk_reads = (total_file_size - current_block_start_file_offset) / global_context -> input_reads . avg_read_length ; if(guessed_this_chunk_reads > global_context ->config.reads_per_chunk) guessed_this_chunk_reads = global_context ->config.reads_per_chunk; unsigned long long guessed_all_reads_before_this_chunk = current_block_start_file_offset / global_context -> input_reads . avg_read_length ; unsigned long long reads_finished_in_this_chunk = (ginp1_file_pos - current_block_start_file_offset) / global_context -> input_reads . avg_read_length; int is_thred_step_running = global_context->config.is_third_iteration_running ? 1:0; if(task != STEP_VOTING) reads_finished_in_this_chunk = (ginp1_file_pos - current_block_start_file_offset) / global_context -> input_reads . avg_read_length; unsigned long long finished_steps = guessed_all_reads_before_this_chunk * (global_context -> index_block_number * 2 + 1 + is_thred_step_running); // add steps for voting if(task == STEP_VOTING) finished_steps += guessed_this_chunk_reads * global_context -> current_index_block_number * 2; else if(task == STEP_ITERATION_TWO) finished_steps += guessed_this_chunk_reads * global_context -> index_block_number * 2; else if(task > STEP_ITERATION_TWO) finished_steps += guessed_this_chunk_reads *(global_context -> index_block_number * 2+1); if(task == STEP_VOTING) finished_steps += reads_finished_in_this_chunk*2; else finished_steps += reads_finished_in_this_chunk; unsigned long long guessed_all_steps = guessed_all_reads * (global_context -> index_block_number *2 + 1 + is_thred_step_running); float finished_rate = finished_steps*1./guessed_all_steps; float reads_per_second = 0; if(task == STEP_VOTING) reads_per_second = finished_rate *1.*guessed_all_reads / (miltime() - global_context -> align_start_time); else reads_per_second = finished_rate *1.*guessed_all_reads / (miltime() - global_context -> start_time); if(current_read_no>1000 && !progress_report_callback) { char minchr[10]; float min_value = (miltime() - global_context -> start_time)*1./60; if(min_value < 9.91) sprintf(minchr, "%.01f", min_value); else sprintf(minchr, "% 3d", (int)min_value); // print_in_box(81,0,0, "%4d%%%% completed, %s mins elapsed, total=%dk %s, rate=%2.1fk/s\r", (int)(finished_rate*100), minchr,(int)(guessed_all_reads*1./1000), global_context -> input_reads.is_paired_end_reads?"frags":"reads", reads_per_second/1000); print_in_box(81,0,0, "%4d%%%% completed, %s mins elapsed, rate=%2.1fk %s per second\r", (int)(finished_rate*100), minchr, reads_per_second/1000, global_context -> input_reads.is_paired_end_reads?"fragments":"reads"); } if(progress_report_callback) { progress_report_callback(10 ,task,(int)(10000*finished_rate)); progress_report_callback(20 ,task,(int)(guessed_all_reads/1000)); } } /* int Xmain(int argc , char ** argv); int main(int argc, char ** argv) { int xk1=0; for(xk1=0; xk1<4; xk1++) Xmain(argc , argv); return 0; } */ int parse_opts_core(int argc , char ** argv, global_context_t * global_context) { int c; int option_index = 0; optind = 1; opterr = 1; optopt = 63; while ((c = getopt_long (argc, argv, "ExsS:L:AHd:D:n:m:p:P:R:r:i:l:o:T:Q:I:t:B:b:Q:FcuUfM?", long_options, &option_index)) != -1) { switch(c) { case 'Q': global_context->config.multi_best_reads = atoi(optarg); break; case 'H': global_context->config.use_hamming_distance_break_ties = 1; break; case 's': global_context->config.downscale_mapping_quality = 1; break; case 'M': global_context->config.do_big_margin_filtering_for_reads = 1; global_context->config.report_multi_mapping_reads = 0; break; case 'A': global_context->config.report_sam_file = 0; break; case 'E': global_context->config.max_mismatch_exonic_reads = 200; global_context->config.max_mismatch_junction_reads = 200; break; case 'f': global_context->config.max_mismatch_exonic_reads = 200; global_context->config.max_mismatch_junction_reads = 200; global_context->config.do_fusion_detection = 1; global_context->config.minimum_subread_for_first_read = 1; global_context->config.minimum_subread_for_second_read = 1; global_context->config.total_subreads = 28; global_context->config.report_no_unpaired_reads = 0; global_context->config.limited_tree_scan = 0; global_context->config.use_hamming_distance_in_exon = 1; break; case 'x': global_context->config.max_mismatch_exonic_reads = 10; global_context->config.max_mismatch_junction_reads = 1; global_context->config.ambiguous_mapping_tolerance = 39; global_context->config.extending_search_indels = 0; global_context->config.do_breakpoint_detection = 1; global_context->config.total_subreads = 14; global_context->config.minimum_subread_for_first_read = 3; global_context->config.minimum_subread_for_second_read = 1; global_context->config.high_quality_base_threshold = 990000; global_context->config.do_big_margin_filtering_for_junctions = 1; global_context->config.report_no_unpaired_reads = 0; global_context->config.limited_tree_scan = 1; global_context->config.use_hamming_distance_in_exon = 0; break; case 'S': global_context->config.is_first_read_reversed = optarg[0]=='r'?1:0; global_context->config.is_second_read_reversed = optarg[0]=='f'?0:1; break; case 'U': global_context->config.report_no_unpaired_reads = 1; break; case 'u': global_context->config.report_multi_mapping_reads = 0; break; case 'b': global_context->config.is_methylation_reads = 1; break; case 'D': global_context->config.maximum_pair_distance = atoi(optarg); break; case 'd': global_context->config.minimum_pair_distance = atoi(optarg); break; case 'n': global_context->config.total_subreads = atoi(optarg); break; case 'm': global_context->config.minimum_subread_for_first_read = atoi(optarg); break; case 'T': global_context->config.all_threads = atoi(optarg); if(global_context->config.all_threads <1) global_context->config.all_threads = 1; if(global_context->config.all_threads >32) global_context->config.all_threads = 32; break; case 'r': strncpy(global_context->config.first_read_file, optarg, MAX_FILE_NAME_LENGTH-1); break; case 'R': global_context->input_reads.is_paired_end_reads = 1; strncpy(global_context->config.second_read_file, optarg, MAX_FILE_NAME_LENGTH-1); break; case 'i': strncpy(global_context->config.index_prefix, optarg, MAX_FILE_NAME_LENGTH-1); break; case 'o': strncpy(global_context->config.output_prefix, optarg, MAX_FILE_NAME_LENGTH-1); break; case 'I': global_context->config.max_indel_length = atoi(optarg); if(global_context->config.max_indel_length <0)global_context->config.max_indel_length =0; if(global_context->config.max_indel_length > MAX_INSERTION_LENGTH)global_context->config.max_indel_length = MAX_INSERTION_LENGTH; if(global_context->config.max_indel_length > 16) { global_context->config.reassembly_subread_length = 12; global_context->config.reassembly_window_multiplex = 3; global_context->config.reassembly_start_read_number = 5; global_context->config.reassembly_tolerable_voting = 0; global_context->config.reassembly_window_alleles = 2; global_context->config.reassembly_key_length = 28; global_context->config.is_third_iteration_running = 1; global_context->config.max_mismatch_exonic_reads = 2; global_context->config.max_mismatch_junction_reads = 2; global_context->config.total_subreads = 28; global_context->config.do_big_margin_filtering_for_reads = 1; global_context->config.do_superlong_indel_detection = 0; } break; case 'P': if (optarg[0]=='3') global_context->config.phred_score_format = FASTQ_PHRED33; else global_context->config.phred_score_format = FASTQ_PHRED64; break; case 'p': global_context->config.minimum_subread_for_second_read = atoi(optarg); break; case 't': sprintf(global_context->config.temp_file_prefix, "%s/core-temp-sum-%06u-%05u", optarg, getpid(), myrand_rand() ); break; case 'F': global_context->config.is_second_iteration_running = 0; global_context->config.report_sam_file = 0; break; case 'B': strcpy(global_context->config.exon_annotation_file, optarg); break; case 'c': global_context->config.space_type = GENE_SPACE_COLOR; break; case 0: //if(strcmp("memory-optimisation",long_options[option_index].name)==0) // memory_optimisation = atoi(optarg); break; case '?': default: return -1 ; } } return 0; } #ifdef MAKE_STANDALONE int main_back(int argc , char ** argv) { progress_report_callback = NULL; #else int subread_core_main(int argc , char ** argv) { if(progress_report_callback) progress_report_callback(0,0,1); #endif return core_main(argc, argv, parse_opts_core); } int check_configuration(global_context_t * global_context) { int expected_type = FILE_TYPE_FAST_; if(global_context -> config.is_SAM_file_input && global_context -> config.is_BAM_input) expected_type = FILE_TYPE_BAM; else if(global_context -> config.is_SAM_file_input && !global_context -> config.is_BAM_input) expected_type = FILE_TYPE_SAM; else if(global_context -> config.is_gzip_fastq) expected_type = FILE_TYPE_GZIP_FAST_; if(global_context -> config.max_indel_length > 16) warning_file_limit(); int wret = 0, wret2 = 0; if( global_context ->config.scRNA_input_mode == 0 ) warning_file_type(global_context -> config.first_read_file, expected_type); if(global_context -> config.second_read_file[0]) { if(expected_type==FILE_TYPE_FAST_ || expected_type==FILE_TYPE_GZIP_FAST_) wret2 = warning_file_type(global_context -> config.second_read_file, expected_type); else print_in_box(80,0,0,"Only one input SAM or BAM file is needed. The second input file is ignored."); } if(wret == -1 || wret2 == -1){ return -1; } if(4 == sizeof(void *) && global_context->config.sort_reads_by_coordinates){ SUBREADputs("ERROR: the sort by coordinate function only works on 64-bit computers."); return -1; } if(global_context->config.is_input_read_order_required && global_context->config.sort_reads_by_coordinates){ SUBREADputs("ERROR: you shouldn't specify keep input order and sort by coordinate at same time."); return -1; } if((global_context -> config.is_BAM_output == 0 || global_context->config.output_prefix[0]==0) && global_context->config.sort_reads_by_coordinates){ if(global_context -> config.is_BAM_output==0)SUBREADputs("ERROR: SAM output doesn't support read sorting by coordinates."); else SUBREADputs("ERROR: STDOUT output doesn't support read sorting by coordinates."); return -1; } return 0; } unsigned long long myrand_seed = 0; unsigned long long myrand_seed2 = 0; void myrand_srand(unsigned long long seed){ // when it is in R, the seed is NOT used but the R random numbers are used as the seed. myrand_seed = 0; myrand_seed2 = 0; #ifndef MAKE_STANDALONE GetRNGstate(); seed = 0; seed = (seed << 16)+ (int)(unif_rand()*65521); seed = (seed << 16)+ (int)(unif_rand()*65521); seed = (seed << 16)+ (int)(unif_rand()*65521); seed = (seed << 16)+ (int)(unif_rand()*65521); PutRNGstate(); #endif myrand_seed = seed; myrand_rand(); myrand_rand(); myrand_rand(); myrand_rand(); myrand_rand(); myrand_rand(); myrand_rand(); myrand_rand(); myrand_rand(); } #define THE_966666773323RD_PRIME 28962406029617llu int myrand_rand(){ //if(myrand_seed % 3133LLU == 0) myrand_srand(0); myrand_seed ^= (myrand_seed % 858173 + myrand_seed % 104729); myrand_seed2 += myrand_seed; myrand_seed ^= myrand_seed2 << 13; return (int)(((myrand_seed^myrand_seed2) %THE_966666773323RD_PRIME) % (1LLU+RAND_MAX)); } int core_main(int argc , char ** argv, int (parse_opts (int , char **, global_context_t * ))) { struct timeval xtime; gettimeofday(&xtime,NULL); myrand_srand(time(NULL)^xtime.tv_usec); global_context_t * global_context; global_context = (global_context_t*)malloc(sizeof(global_context_t)); memset(global_context, 0, sizeof(global_context_t)); init_global_context(global_context); int ret = parse_opts(argc , argv, global_context); init_core_temp_path(global_context); //global_context->config.reads_per_chunk = 200*1024; if(global_context->config.max_indel_length > 20 && !global_context->input_reads.is_paired_end_reads) { global_context->config.total_subreads = 28; global_context->config.reassembly_start_read_number = 3; global_context->config.do_superlong_indel_detection = 1; } if(global_context->config.fast_run){ global_context -> config.top_scores = 1; global_context -> config.max_vote_combinations = 1; global_context -> config.max_vote_simples = 1; global_context -> config.multi_best_reads = 1; } ret = ret || print_configuration(global_context); ret = ret || check_configuration(global_context); ret = ret || load_global_context(global_context); ret = ret || init_modules(global_context); ret = ret || read_chunk_circles(global_context); ret = ret || write_final_results(global_context); ret = ret || destroy_modules(global_context); ret = ret || destroy_global_context(global_context); ret = ret || show_summary(global_context); free(global_context); return ret; } // the new file name is written into fname then. int convert_BAM_to_SAM(global_context_t * global_context, char * fname, int is_bam) { char temp_file_name[MAX_FILE_NAME_LENGTH+80], *fline=malloc(3000), tmp_readname[MAX_READ_NAME_LEN]; short tmp_flags; SamBam_FILE * sambam_reader; char * env_no_sort = getenv("SUBREAD_DO_NOT_CHECK_INPUT"); if(env_no_sort){ global_context->input_reads.is_paired_end_reads = 1; SUBREADprintf("\nWARNING: The SAM input file was assumed to be sorted by name.\nENV: SUBREAD_DO_NOT_CHECK_INPUT\n"); return 0; } int is_file_sorted = 1; unsigned long long int read_no = 0; sambam_reader = SamBam_fopen(fname, is_bam?SAMBAM_FILE_BAM:SAMBAM_FILE_SAM); if(!sambam_reader) { SUBREADprintf("ERROR: Unable to open file '%s'. The file is not in the specified format.\n", fname); free(fline); return -1; } while(1) { char * is_ret = SamBam_fgets(sambam_reader, fline, 2999, 1); if(!is_ret) break; if(sambam_reader ->is_bam_broken ) { SUBREADputs("ERROR: the BAM format is broken."); return -1; } if(fline[0]=='@')continue; if(is_SAM_unsorted(fline, tmp_readname, &tmp_flags , read_no)){ if(tmp_flags & 1) global_context->input_reads.is_paired_end_reads = 1; is_file_sorted = 0; read_no++; break; } read_no++; if(tmp_flags & 1) global_context->input_reads.is_paired_end_reads = 1; else global_context->input_reads.is_paired_end_reads = 0; if(! global_context->input_reads.is_paired_end_reads) break; } if(read_no<1) { SUBREADprintf("ERROR: unable to find any reads from file '%s'. The file is not in the specified format or is empty.\n", fname); return -1; } SamBam_fclose(sambam_reader); print_in_box(80,0,0,"The input %s file contains %s-end reads.", is_bam?"BAM":"SAM", global_context->input_reads.is_paired_end_reads?"paired":"single"); if(!is_file_sorted) print_in_box(80,0,0,"The input %s file is unsorted. Reorder it...", is_bam?"BAM":"SAM"); else if(is_bam) print_in_box(80,0,0,"Convert the input BAM file..."); int disk_is_full = 0; if(is_bam || (global_context->input_reads.is_paired_end_reads && !is_file_sorted)) { sprintf(temp_file_name, "%s.sam", global_context->config.temp_file_prefix); sambam_reader = SamBam_fopen(fname, is_bam?SAMBAM_FILE_BAM: SAMBAM_FILE_SAM); if(!sambam_reader){ SUBREADprintf("Unable to open %s.\n", fname); return -1; } FILE * sam_fp = NULL; SAM_sort_writer writer; int writer_opened = 0; if(is_file_sorted) sam_fp = f_subr_open(temp_file_name,"w"); else writer_opened = sort_SAM_create(&writer, temp_file_name, NULL); if((is_file_sorted && !sam_fp) || (writer_opened && !is_file_sorted)){ SUBREADprintf("Failed to write to the directory. You may not have permission to write to this directory or the disk is full.\n"); return -1; } while(1) { char * is_ret = SamBam_fgets(sambam_reader, fline, 2999, 1); if(!is_ret) break; if(is_file_sorted){ int wlen = fputs(fline, sam_fp); if(wlen <0){ SUBREADprintf("ERROR: unable to write into the temporary SAM file. Please check the disk space in the output directory.\n"); disk_is_full = 1; break; } }else{ int ret = sort_SAM_add_line(&writer, fline, strlen(fline)); if(ret== -1) { print_in_box(80,0,0,"ERROR: read name is too long; check input format."); break; } if(ret== -2) { SUBREADprintf("ERROR: unable to write into the temporary SAM file. Please check the disk space in the output directory.\n"); disk_is_full = 1; break; } } } if(is_file_sorted) fclose(sam_fp); else{ int ret = sort_SAM_finalise(&writer); if(writer.unpaired_reads) #ifdef __MINGW32__ print_in_box(80,0,0,"%I64d single-end mapped reads in reordering.", writer.unpaired_reads); #else print_in_box(80,0,0,"%lld single-end mapped reads in reordering.", writer.unpaired_reads); #endif if(ret) { disk_is_full = 1; SUBREADprintf("ERROR: unable to create the temporary file. Please check the disk space in the output directory.\n"); } } SamBam_fclose(sambam_reader); strcpy(fname, temp_file_name); global_context -> will_remove_input_file = 1; } free(fline); return disk_is_full; } int convert_GZ_to_FQ(global_context_t * global_context, char * fname, int half_n) { int is_OK = 0; char temp_file_name[MAX_FILE_NAME_LENGTH+30]; char * linebuff=malloc(3001); gzFile rawfp = gzopen(fname, "r"); if(rawfp) { print_in_box(80,0,0,"Decompress %s...", fname); sprintf(temp_file_name, "%s-%d.fq", global_context->config.temp_file_prefix, half_n); FILE * outfp = fopen(temp_file_name, "w"); if(outfp) { while(1) { char * bufr =gzgets(rawfp, linebuff, 3000); if(!bufr) break; fputs(bufr, outfp); } is_OK = 1; fclose(outfp); } else{ SUBREADprintf("Unable to create temporary file '%s'\nPlease run the program in a directory where you have the privilege to create files.\n", temp_file_name); } gzclose(rawfp); } strcpy(fname, temp_file_name); global_context -> will_remove_input_file |= (1<< (half_n-1)); return !is_OK; } int core_geinput_open(global_context_t * global_context, gene_input_t * fp, int half_number) { char *fname; if(global_context->config.is_SAM_file_input) { fname = global_context ->config.first_read_file; if(half_number == 1) if(convert_BAM_to_SAM(global_context, global_context ->config.first_read_file, global_context ->config.is_BAM_input)) return -1; if(!global_context->input_reads.is_paired_end_reads) half_number=0; return geinput_open_sam(fname, fp, half_number); } else { int rv = -1; //SUBREADprintf("SCRNA_MODE=%d\n", global_context->config.scRNA_input_mode ); if(global_context -> config.is_gzip_fastq) if(convert_GZ_to_FQ(global_context, (half_number==2)? global_context ->config.second_read_file : global_context ->config.first_read_file, half_number)) return -1; fname = (half_number == 2)?global_context -> config.second_read_file:global_context -> config.first_read_file; if(global_context->config.scRNA_input_mode == GENE_INPUT_BCL) rv = geinput_open_bcl(fname , fp, global_context -> config.reads_per_chunk, global_context -> config.all_threads ); else if(global_context->config.scRNA_input_mode == GENE_INPUT_SCRNA_FASTQ) rv = geinput_open_scRNA_fqs(fname , fp, global_context -> config.reads_per_chunk, global_context -> config.all_threads ); else if(global_context->config.scRNA_input_mode == GENE_INPUT_SCRNA_BAM) rv = geinput_open_scRNA_BAM(fname , fp, global_context -> config.reads_per_chunk, global_context -> config.all_threads ); else rv = geinput_open(fname, fp); if(global_context->input_reads.is_paired_end_reads && global_context->config. scRNA_input_mode){ SUBREADprintf("ERROR: No paired-end input is allowed on scRNA mode.\n"); return -1; } return rv; } } int fetch_next_read_pair(global_context_t * global_context, thread_context_t * thread_context, gene_input_t* ginp1, gene_input_t* ginp2, int *read_len_1, int *read_len_2, char * read_name_1, char * read_name_2, char * read_text_1, char * read_text_2, char * qual_text_1, char *qual_text_2, int remove_color_head, subread_read_number_t * read_no_in_chunk) { int rl1=0, rl2=0; int is_second_R1, is_second_R2; subread_read_number_t this_number = -1; geinput_preload_buffer(ginp1, &global_context -> input_reads.input_lock); if(ginp2) geinput_preload_buffer(ginp2, &global_context -> input_reads.input_lock); subread_lock_occupy(&global_context -> input_reads.input_lock); if(global_context -> running_processed_reads_in_chunk < global_context -> config.reads_per_chunk) { do { is_second_R1 = 0; is_second_R2 = 0; rl1 = geinput_next_read_trim(ginp1, read_name_1, read_text_1 , qual_text_1, global_context->config.read_trim_5, global_context->config.read_trim_3, &is_second_R1); //if(global_context -> running_processed_reads_in_chunk < 100)SUBREADprintf("GETRNAMES %s LEN=%d\n", read_name_1, rl1); if(global_context->config.space_type == GENE_SPACE_COLOR && remove_color_head) { if(isalpha(read_text_1[0])) { int xk1; for(xk1=2; read_text_1[xk1]; xk1++) read_text_1[xk1-2]=read_text_1[xk1]; read_text_1[xk1-2]=0; } } if(ginp2) { rl2 = geinput_next_read_trim(ginp2, read_name_2, read_text_2 , qual_text_2, global_context->config.read_trim_5, global_context->config.read_trim_3, &is_second_R2); if(global_context->config.space_type == GENE_SPACE_COLOR && remove_color_head) { if(isalpha(read_text_2[0])) { int xk1; for(xk1=2; read_text_2[xk1]; xk1++) read_text_2[xk1-2]=read_text_2[xk1]; read_text_2[xk1-2]=0; } } } if(rl1 <= 0 || (rl2 <= 0 && ginp2)) break; } while(is_second_R1||is_second_R2) ; //SUBREADprintf("IMBRL1=%d\tRL2=%d\n", rl1, rl2); // if(rl1 >0 || (rl2 > 0 && ginp2)){ this_number = global_context -> running_processed_reads_in_chunk; global_context -> running_processed_reads_in_chunk ++; } } subread_lock_release(&global_context -> input_reads.input_lock); if( global_context->config.space_type == GENE_SPACE_COLOR) { rl1-=1;rl2-=1; } if(ginp2 && rl1 * rl2 <=0 && (rl1>0 || rl2>0)){ if(!global_context-> input_reads.is_internal_error) SUBREADprintf("\nERROR: two input files have different amounts of reads.\n\n"); global_context-> input_reads.is_internal_error = 1; *read_no_in_chunk = -1; return 1; } else if(rl1>0 && (rl2>0 || !ginp2) && this_number>=0) { if(global_context->config.is_first_read_reversed) { reverse_read(read_text_1, rl1, global_context->config.space_type); if(qual_text_1) reverse_quality(qual_text_1, rl1); } if(ginp2 && global_context->config.is_second_read_reversed) { reverse_read(read_text_2, rl2, global_context->config.space_type); if(qual_text_2) reverse_quality(qual_text_2, rl2); } *read_no_in_chunk = this_number; *read_len_1 = rl1; if(ginp2) *read_len_2 = rl2; return 0; } else { // normally finished *read_no_in_chunk = -1; return 1; } } int write_final_results(global_context_t * context) { if((context -> config.do_fusion_detection || context -> config.do_long_del_detection) && context -> config.do_structural_variance_detection) finalise_structural_variances(context); if(context -> config.output_prefix[0] && 0==context -> input_reads.is_internal_error && !(context -> config.is_BAM_output && context -> output_bam_writer -> is_internal_error) ) { write_indel_final_results(context); if(context -> config.entry_program_name == CORE_PROGRAM_SUBJUNC && (context -> config.prefer_donor_receptor_junctions||!(context -> config.do_fusion_detection || context -> config.do_long_del_detection))) write_junction_final_results(context); if((context -> config.do_fusion_detection || context -> config.do_long_del_detection)) write_fusion_final_results(context); } return 0; } int get_soft_clipping_length(char* CIGAR) { int nch; int cigar_cursor, tmp_int = 0; for(cigar_cursor = 0; (nch = CIGAR[cigar_cursor]) > 0;cigar_cursor++) { if(isdigit(nch)) tmp_int = tmp_int*10+(nch-'0'); else { if(nch=='S') return tmp_int; return 0; } } return 0; } #define CIGAR_PERFECT_SECTIONS 12 typedef struct{ char current_cigar_decompress[CORE_MAX_CIGAR_STR_LEN + 1]; char cigar [CORE_MAX_CIGAR_STR_LEN]; unsigned short chimeric_sections; unsigned int out_poses[CIGAR_PERFECT_SECTIONS]; short out_lens[CIGAR_PERFECT_SECTIONS]; char out_cigars[CIGAR_PERFECT_SECTIONS][60]; char out_strands[CIGAR_PERFECT_SECTIONS]; char additional_information[CORE_ADDITIONAL_INFO_LENGTH + 1]; mapping_result_t * raw_result; unsigned int linear_position; short soft_clipping_movements; char * chro; int offset; int strand; int is_first_section_jumpped; int mapping_quality; int is_NM_appied; }subread_output_tmp_t; typedef struct{ long long int *PE_distance; char * out_cigar_buffer[CIGAR_PERFECT_SECTIONS]; subread_output_tmp_t *r1; subread_output_tmp_t *r2; subread_output_tmp_t ** out_pairs; mapping_result_t ** out_raws; } subread_output_context_t; void init_output_context(global_context_t * global_context, subread_output_context_t * out_context) { int xk1; memset(out_context, 0, sizeof(subread_output_context_t)); out_context -> r1 = malloc(sizeof(subread_output_tmp_t)); for(xk1=0;xk1 out_cigar_buffer[xk1] = malloc(60); out_context -> out_pairs = malloc(sizeof( subread_output_context_t *) * global_context->config.multi_best_reads * 2); out_context -> out_raws = malloc(sizeof( mapping_result_t * *) * global_context->config.multi_best_reads * 2); if(global_context -> input_reads.is_paired_end_reads) { out_context -> PE_distance = malloc(sizeof(long long) * global_context->config.multi_best_reads); out_context -> r2 = malloc(sizeof(subread_output_tmp_t)); } else { out_context -> PE_distance = NULL; out_context -> r2 = NULL; } } void destroy_output_context(global_context_t * global_context , subread_output_context_t * out_context) { int xk1; for(xk1=0;xk1 out_cigar_buffer[xk1]); free(out_context -> out_raws); free(out_context -> out_pairs); free(out_context -> r1 ); if(global_context -> input_reads.is_paired_end_reads) { free(out_context -> r2); free(out_context -> PE_distance); } } int locate_current_value_index(global_context_t * global_context, thread_context_t * thread_context, mapping_result_t * result, int rlen); int calc_edit_dist(global_context_t * global_context, mapping_result_t * current_result, char * cigar , unsigned int pos , char * read_text, int all_mm) { unsigned int cigar_cursor = 0, tmpi=0; while(1) { char nch = cigar[cigar_cursor++]; if(!nch)break; if('0'<=nch && '9' >=nch) { tmpi = tmpi*10+nch-'0'; } else{ if(nch == 'I' || nch == 'D') { all_mm+=tmpi; } tmpi = 0; } } return all_mm; } unsigned int move_to_read_head(unsigned int tailpos, char * cigar){ int cigar_i = 0, nch; unsigned int tmpi = 0; while(0<(nch = cigar[cigar_i++])){ if(isdigit(nch)){ tmpi = tmpi * 10 + nch - '0'; }else{ if(nch == 'N' || nch == 'M' || nch == 'D') tailpos -= tmpi; tmpi = 0; } } return tailpos; } // This function returns 1 if the cut was added. // It returns 0 if the head or tail cut is not able to be added (e.g., the cigar ends up like "50M4I10S" or "10S30N90M") int add_head_tail_cut_softclipping(global_context_t * global_context, unsigned int linear, char * cigar, int rlen, int head_cut, int tail_cut){ //SUBREADprintf("ADD_SOFT: %s , %d, %d\n", cigar,head_cut,tail_cut); char cigar_added [CORE_MAX_CIGAR_STR_LEN]; int cigar_cursor = 0, read_cursor = 0, next_read_cursor = 0; int tmpi = 0, nch, has_M = 0; unsigned int linear_cursor = linear; cigar_added[0]=0; while(1){ nch = cigar[cigar_cursor++]; if(nch == 0) break; if(isdigit(nch)){ tmpi = 10 * tmpi + (nch - '0'); }else{ if('M' == nch || 'S' == nch || 'I' == nch) next_read_cursor = read_cursor + tmpi; if('M' == nch || 'D' == nch || 'S' == nch || 'N' == nch){ linear_cursor += tmpi; } int head_S = 0, tail_S =0, remainder_tmpi = tmpi, is_skip = 0; if(next_read_cursor <= head_cut) is_skip = 1; if(read_cursor >= rlen - tail_cut) is_skip = 1; if(!is_skip){ if(nch != 'S'){ if(read_cursor <= head_cut)head_S = head_cut; if(next_read_cursor >= rlen - tail_cut) tail_S = tail_cut; remainder_tmpi = tmpi; if(head_S) remainder_tmpi-= (head_S - read_cursor); if(tail_S) remainder_tmpi-= next_read_cursor - (rlen - tail_S); } //SUBREADprintf("OPTR=%c, THREE-LENS=%d,%d,%d\n", nch, head_S, remainder_tmpi, tail_S); if(head_S >0 || tail_S > 0) if(nch !='M') return 0; if(head_S > 0) sprintf(cigar_added + strlen(cigar_added), "%dS", head_S ); if(remainder_tmpi > 0){ sprintf(cigar_added + strlen(cigar_added), "%d%c", remainder_tmpi , nch ); if( nch == 'M' ) has_M = 1; } if(tail_S > 0) sprintf(cigar_added + strlen(cigar_added), "%dS", tail_S ); } read_cursor = next_read_cursor; tmpi = 0; } } //SUBREADprintf("RPCIGAR: %s => %s\n", cigar, cigar_added); strcpy(cigar, cigar_added); return has_M; } int convert_read_to_tmp(global_context_t * global_context , subread_output_context_t * output_context, int read_number, int is_second_read, int read_len, char * read_text, char * qual_text, realignment_result_t * current_result, subread_output_tmp_t * r, char * read_name) { int is_r_OK; r -> raw_result = current_result -> mapping_result; r -> additional_information[0]=0; is_r_OK = (current_result -> mapping_result -> result_flags & CORE_IS_FULLY_EXPLAINED) > 0; if(0 && FIXLENstrcmp("V0112_0155:7:1101:5279:29143#ATCACG", read_name) == 0) SUBREADprintf("%s R_%d CPOINT1 : is_OK=%d\n", read_name , is_second_read + 1 , is_r_OK); if(is_r_OK) { int current_strand = (current_result -> mapping_result -> result_flags & CORE_IS_NEGATIVE_STRAND)?1:0; int is_first_section_jumped = current_result -> first_base_is_jumpped; if(is_first_section_jumped) assert((global_context -> config.do_fusion_detection || global_context -> config.do_long_del_detection)); strcpy(r-> current_cigar_decompress, current_result -> cigar_string); int chimeric_sections = 0; r->is_first_section_jumpped = is_first_section_jumped; r->linear_position = current_result -> first_base_position; //r->mapping_quality = current_result -> final_quality; r->mapping_quality = 40; // changed on 10-JUN-2016 if(current_result -> realign_flags & CORE_IS_BREAKEVEN) r -> mapping_quality = 0; else r -> mapping_quality /= current_result->MAPQ_adjustment; //SUBREADprintf("REP=%d\n", current_result->MAPQ_adjustment); strcpy(r->cigar, r -> current_cigar_decompress); r->strand = (current_result -> mapping_result -> result_flags & CORE_IS_NEGATIVE_STRAND)?1:0; //sprintf(r->additional_information, "\tSM:i:%d",current_result -> final_mismatched_bases); //printf("SM='%s'\n", r->additional_information); if((global_context -> config.do_fusion_detection || global_context -> config.do_long_del_detection)) { chimeric_sections = chimeric_cigar_parts(global_context, r->linear_position, is_first_section_jumped ^ current_strand, is_first_section_jumped, r->current_cigar_decompress, r->out_poses, output_context->out_cigar_buffer, r->out_strands, read_len, r->out_lens, read_name); if(chimeric_sections > 0){ int xk1; r->chimeric_sections = chimeric_sections; strcpy(r->out_cigars[0], output_context->out_cigar_buffer[0]); for(xk1=1; xk1out_cigars[xk1], output_context->out_cigar_buffer[xk1]); unsigned int vitual_head_pos = r->out_poses[xk1]; char strand_xor = (r->out_strands[xk1] == '-') != is_second_read;//!= (r->out_strands[0]=='-') ; //if(( r->out_strands[xk1] == '-' ) != (r->out_strands[0]=='-' )) vitual_head_pos = move_to_read_head(vitual_head_pos, r->out_cigars[xk1]); if(0==locate_gene_position_max(vitual_head_pos ,& global_context -> chromosome_table, & chimaric_chr, & chimeric_pos, NULL, NULL, 0+r->out_lens[xk1])) { int soft_clipping_movement = 0; soft_clipping_movement = get_soft_clipping_length(r->out_cigars[xk1]); assert(chimaric_chr); sprintf(r->additional_information + strlen(r->additional_information), "\tCG:Z:%s\tCP:i:%u\tCT:Z:%c\tCC:Z:%s", r->out_cigars[xk1] , max(1,chimeric_pos + soft_clipping_movement + 1), strand_xor?'-':'+' , chimaric_chr ); } else is_r_OK = 0; } r->linear_position = r->out_poses[0]; r->strand = r->out_strands[0]=='-'; strcpy(r->cigar , r->out_cigars[0]); }else is_r_OK = 0; } if(is_r_OK) r->soft_clipping_movements = get_soft_clipping_length(r->cigar); } if(is_r_OK) { int head_cut = 0 , tail_cut = 0; if(locate_gene_position_max(r->linear_position + r->soft_clipping_movements,& global_context -> chromosome_table, &r-> chro , &r -> offset, &head_cut, &tail_cut,(global_context-> config.do_fusion_detection || global_context-> config.do_long_del_detection)?read_len:(current_result->chromosomal_length - r->soft_clipping_movements))){ is_r_OK = 0; } else { int is_added_OK = 1; if(head_cut!=0 || tail_cut!=0) is_added_OK = add_head_tail_cut_softclipping(global_context, r->linear_position, r->cigar , read_len, head_cut, tail_cut); if(is_added_OK){ r -> offset++; assert(r-> chro); }else{ is_r_OK = 0; } } if(global_context -> config.do_breakpoint_detection && !(current_result -> realign_flags & CORE_NOTFOUND_DONORS)) { sprintf(r->additional_information + strlen(r->additional_information), "\tXS:A:%c", (current_result -> realign_flags & CORE_IS_GT_AG_DONORS)?'+':'-'); } /* if(global_context -> config.more_accurate_fusions) { sprintf(r->additional_information + strlen(r->additional_information), "\tDF:i:%d", current_result -> best_second_diff_bases >0?current_result -> best_second_diff_bases:9999 ); }*/ } return is_r_OK; } int add_event_detected_from_cigar(global_context_t * global_context, unsigned int left_last_base, unsigned int right_first_base, int is_indel, int indel_len, int left_extend, int right_extend, int read_length, char * read_name) { HashTable * event_table = NULL; chromosome_event_t * event_space = NULL; event_table = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_entry_table; event_space = ((indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]) -> event_space_dynamic; chromosome_event_t *site_events[MAX_EVENT_ENTRIES_PER_SITE]; int search_types ; if(is_indel) search_types = CHRO_EVENT_TYPE_INDEL; else search_types = CHRO_EVENT_TYPE_JUNCTION | CHRO_EVENT_TYPE_FUSION; if(left_last_base > right_first_base) { unsigned int ttt; ttt = right_first_base; right_first_base = left_last_base; left_last_base = ttt; } int xk1, site_events_no = search_event(global_context, event_table , event_space , left_last_base, EVENT_SEARCH_BY_BOTH_SIDES , search_types , site_events); int is_found = 0; for(xk1 = 0; xk1 < site_events_no; xk1++){ chromosome_event_t * this_event = site_events[xk1]; if(is_indel && this_event -> event_type != CHRO_EVENT_TYPE_INDEL) continue; if((!is_indel) && this_event -> event_type == CHRO_EVENT_TYPE_INDEL) continue; #ifndef __MINGW32__ if(0 && ( memcmp(read_name, "V0112_0155:7:1104:17648:117432",28)==0 || ( this_event -> event_small_side > 2613701363 - 200 && this_event -> event_small_side < 2613701363 + 100) )) SUBREADprintf("EVENT: L=%u, R=%u (TABLE L=%u , R=%u), LEXT=%d, REXT=%d -- %s\n", left_last_base, right_first_base, this_event -> event_small_side, this_event -> event_large_side, left_extend, right_extend, read_name); #endif if(this_event -> event_small_side == left_last_base && this_event -> event_large_side == right_first_base){ if(is_indel && this_event -> indel_length == indel_len){ this_event -> final_counted_reads ++; is_found = 1; break; } if(!is_indel){ this_event -> final_counted_reads ++; // if(read_length >= 80) { if(left_extend > 30 && right_extend > 30) this_event -> critical_supporting_reads ++; }else{ if(left_extend > 18 && right_extend > 18) this_event -> critical_supporting_reads ++; } this_event -> junction_flanking_left = max(this_event -> junction_flanking_left, left_extend); this_event -> junction_flanking_right = max(this_event -> junction_flanking_right, right_extend); is_found = 1; break; } } } if(!is_found){ //SUBREADprintf("\nEVENT NOT FOUND.\n\n"); return 1; } return 0; } int get_junction_right_extension(char * remainder_cigar){ int ret = 0; unsigned int tmpi = 0; int cigar_cursor, nch; for(cigar_cursor = 0;;cigar_cursor++){ nch = remainder_cigar[cigar_cursor]; if(0 == nch)break; if(isdigit(nch))tmpi = tmpi*10 + nch - '0'; else{ if(nch == 'M' || nch == 'D') ret += tmpi; if(nch == 'N' || nch == 'n' || nch == 'B' || nch == 'b') break; tmpi = 0; } } return ret; } void remove_soft_clipping(char * dst, char * src){ unsigned int tmpi = 0, head_clip = 0, tail_clip = 0, last_M = 0; int cigar_cursor, is_first_s = 1, nch, nch2; dst[0] = 0; for(cigar_cursor = 0;;cigar_cursor++){ nch = src[cigar_cursor]; nch2 = src[cigar_cursor+1]; if(0 == nch)break; if(isdigit(nch))tmpi = tmpi*10 + nch - '0'; else{ if('S' == nch){ if(is_first_s) head_clip = tmpi; if(nch2 == 0) tail_clip = tmpi; } else if('M' == nch){ last_M = tmpi; } else{ if(last_M){ sprintf(dst + strlen(dst), "%uM", last_M + head_clip); last_M = 0; head_clip = 0; } sprintf(dst + strlen(dst), "%u%c", tmpi, nch); } tmpi=0; is_first_s = 0; } } if(last_M){ sprintf(dst + strlen(dst), "%uM" , last_M + tail_clip + head_clip); } } int getFirstM(char * cig){ int tmpi = 0, nch, x1; for(x1=0; 0!=(nch = cig[x1]) ; x1++){ if(isdigit(nch)){ tmpi=tmpi*10 + nch - '0'; }else break; } return tmpi; } int calc_tlen(global_context_t * global_context, subread_output_tmp_t * rec1 , subread_output_tmp_t * rec2, int read_len_1, int read_len_2); int calc_flags(global_context_t * global_context, thread_context_t * thread_context, subread_output_tmp_t * rec1 , subread_output_tmp_t * rec2, int is_second_read, int read_len_1, int read_len_2, int current_location_no , int tlen, int this_OK, int mate_OK) { int ret, is_TLEN_wrong = 0; if(global_context->input_reads.is_paired_end_reads) { ret = SAM_FLAG_PAIRED_TASK; ret |= is_second_read?SAM_FLAG_SECOND_READ_IN_PAIR:SAM_FLAG_FIRST_READ_IN_PAIR; subread_output_tmp_t * this_rec = is_second_read?rec2:rec1; subread_output_tmp_t * mate_rec = is_second_read?rec1:rec2; if(this_OK == 0) ret |= SAM_FLAG_UNMAPPED; else if(this_rec->strand + is_second_read == 1) ret |= SAM_FLAG_REVERSE_STRAND_MATCHED; if(mate_OK == 0) ret |= SAM_FLAG_MATE_UNMATCHED; else if(mate_rec->strand + is_second_read != 1) ret |= SAM_FLAG_MATE_REVERSE_STRAND_MATCHED; if(rec1 && rec2) { int TLEN = tlen;//calc_tlen(global_context , rec1, rec2, read_len_1, read_len_2); int is_PEM = 0; if( rec1 -> chro == rec2 -> chro /* two pointers can be directly compared. */ && TLEN >= global_context->config.minimum_pair_distance && TLEN <= global_context-> config.maximum_pair_distance && this_rec->strand == mate_rec->strand ) { if(global_context -> config.is_first_read_reversed && !(global_context -> config.is_second_read_reversed)) { if(this_rec -> strand == 0) { if((is_second_read + (mate_rec-> offset > this_rec -> offset) == 1) || mate_rec-> offset == this_rec -> offset) is_PEM = 1; else is_TLEN_wrong = 1; } } else { if(this_rec -> strand) { if((is_second_read + (mate_rec-> offset < this_rec -> offset) == 1) || mate_rec-> offset == this_rec -> offset) is_PEM = 1; else is_TLEN_wrong = 1; }else { if((is_second_read + (mate_rec-> offset > this_rec -> offset) == 1) || mate_rec-> offset == this_rec -> offset) is_PEM = 1; else is_TLEN_wrong = 1; } } } if(is_PEM) ret |= SAM_FLAG_MATCHED_IN_PAIR; else if(is_second_read){ if(rec1 -> chro != rec2 -> chro){ if(thread_context) thread_context -> not_properly_pairs_different_chro ++; else global_context -> not_properly_pairs_different_chro++; }else if(this_rec->strand != mate_rec->strand){ if(thread_context) thread_context -> not_properly_different_strands ++; else global_context -> not_properly_different_strands ++; }else if(TLEN < global_context->config.minimum_pair_distance || TLEN > global_context-> config.maximum_pair_distance){ if(thread_context) thread_context -> not_properly_pairs_TLEN_wrong ++; else global_context -> not_properly_pairs_TLEN_wrong ++; }else if(is_TLEN_wrong){ if(thread_context) thread_context -> not_properly_pairs_wrong_arrangement ++; else global_context -> not_properly_pairs_wrong_arrangement ++; } } } } else { ret = 0; if(this_OK == 0) ret |= SAM_FLAG_UNMAPPED; else if(rec1->strand) ret |= SAM_FLAG_REVERSE_STRAND_MATCHED; } if(current_location_no>0){ if((rec1 && !is_second_read)||(rec2 && is_second_read)) ret |= SAM_FLAG_SECONDARY_MAPPING; } return ret; } int calc_tlen(global_context_t * global_context, subread_output_tmp_t * rec1 , subread_output_tmp_t * rec2, int read_len_1, int read_len_2) { int ret = -1; unsigned int r1_head_pos = rec1 -> offset;// - rec1 -> soft_clipping_movements; unsigned int r2_head_pos = rec2 -> offset;// - rec2 -> soft_clipping_movements; if(r1_head_pos == r2_head_pos) ret = max(read_len_1, read_len_2); // the two reads are fully overlapping else{ int is_r2_smaller = r2_head_pos < r1_head_pos; subread_output_tmp_t * smaller_rec = is_r2_smaller?rec2:rec1; unsigned int smaller_head_pos = is_r2_smaller?r2_head_pos:r1_head_pos; unsigned int larger_head_pos = is_r2_smaller?r1_head_pos:r2_head_pos; int len_larger_rec = is_r2_smaller?read_len_1:read_len_2; int len_smaller_rec = is_r2_smaller?read_len_2:read_len_1; unsigned int tmpi = 0; unsigned int chro_cursor = smaller_head_pos; unsigned int section_end = 0; unsigned int read_cursor = 0; int cigar_cursor, nch, nch2; for(cigar_cursor = 0; ; cigar_cursor++){ nch = smaller_rec -> cigar[cigar_cursor]; nch2 = smaller_rec -> cigar[cigar_cursor+1]; if(nch > 0){ if(isdigit(nch)) tmpi = tmpi * 10 + nch - '0'; else{ if(nch == 'M' || nch == 'S'){ chro_cursor += tmpi; read_cursor += tmpi; section_end = chro_cursor; } if(nch == 'N' || nch == 'B' || nch == 'b' || nch == 'n' || nch == 'I' || nch == 'D' || nch2 == 0){ if(nch == 'N' || nch == 'D') chro_cursor += tmpi; //SUBREADprintf("NCH=%c, NCH2=%d, SEC_END=%u, LARGE_HEAD=%u\n", nch, nch2, section_end, larger_head_pos); if(section_end >= larger_head_pos) { ret = read_cursor + larger_head_pos - section_end + len_larger_rec; break; } } if(nch == 'I'){ read_cursor += tmpi; } if(nch == 'B' || nch == 'b' || nch == 'n') break; // fusion block, unable to determine the TLEN after the fusion point. tmpi = 0; } }else{ break; } } if(ret < 0) ret = larger_head_pos - section_end + len_larger_rec + len_smaller_rec; } //SUBREADprintf("TLEN = %d :: \tREC1 : %s:%u (%s - %d) ; REC2: %s:%u (%s - %d )\n", ret, rec1 -> chro, rec1 -> offset, rec1 -> cigar, rec1 -> soft_clipping_movements, // rec2 -> chro, rec2 -> offset, rec2 -> cigar, rec2 -> soft_clipping_movements); return ret; } int calc_should_reverse(global_context_t * global_context, subread_output_tmp_t * rec1 , subread_output_tmp_t * rec2, int is_second_read) { subread_output_tmp_t * cur_rec = is_second_read?rec2:rec1; if(cur_rec) return cur_rec->strand; else { return is_second_read; //subread_output_tmp_t * mate_rec = is_second_read?rec2:rec1; //return mate_rec?mate_rec->strand:is_second_read; } } void remove_nm_i(char * st) { char * st_nm = strstr(st, "\tNM:i:"); if(st_nm) { char * write_cursor = st_nm; int started = 0; st_nm++; while(1) { char nch = *st_nm; if(!nch) { *write_cursor=0; break; } if(nch == '\t') started = 1; if(started) { *write_cursor=*st_nm; write_cursor++; } st_nm++; } } } double last_t1 = 0; int for_other_thread = 0; int for_one_threads = 0; void add_buffered_fragment(global_context_t * global_context, thread_context_t * thread_context, subread_read_number_t pair_number , char * read_name1, unsigned int flags1, char * chro_name1, unsigned int chro_position1, int mapping_quality1, char * cigar1, char * next_chro_name1, unsigned int next_chro_pos1, int temp_len1, int read_len1, char * read_text1, char * qual_text1, char * additional_columns1, char * read_name2, unsigned int flags2, char * chro_name2, unsigned int chro_position2, int mapping_quality2, char * cigar2, char * next_chro_name2, unsigned int next_chro_pos2, int temp_len2, int read_len2, char * read_text2, char * qual_text2, char * additional_columns2, int all_locations, int this_location ){ assert(global_context -> config.all_threads > 1); if( global_context -> config.is_BAM_output && !global_context -> config.is_input_read_order_required){ //SUBREADprintf("MTBAM : THR_%d CORE: %s\n", thread_context -> thread_id, read_name1); SamBam_writer_add_read(global_context -> output_bam_writer, thread_context -> thread_id, read_name1, flags1, chro_name1, chro_position1, mapping_quality1, cigar1, next_chro_name1, next_chro_pos1, temp_len1, read_len1, read_text1, qual_text1, additional_columns1, !global_context->input_reads.is_paired_end_reads); if(global_context->input_reads.is_paired_end_reads) SamBam_writer_add_read(global_context -> output_bam_writer, thread_context -> thread_id, read_name2, flags2, chro_name2, chro_position2, mapping_quality2, cigar2, next_chro_name2, next_chro_pos2, temp_len2, read_len2, read_text2, qual_text2, additional_columns2, 1); }else{ while(1){ int is_fin = 0; subread_lock_occupy(&global_context -> output_lock); //SUBREADprintf("THWT : TH_%d IS AFTER %lld ; LAST=%lld (%d/%d)\n", thread_context -> thread_id, pair_number -1, global_context -> last_written_fragment_number, this_location, all_locations); if(global_context -> last_written_fragment_number == pair_number-1){ if(global_context -> config.is_BAM_output){ SamBam_writer_add_read(global_context -> output_bam_writer, -1, read_name1, flags1, chro_name1 , chro_position1, mapping_quality1, cigar1, next_chro_name1 , next_chro_pos1, temp_len1, read_len1, read_text1, qual_text1, additional_columns1, !global_context->input_reads.is_paired_end_reads); if(global_context->input_reads.is_paired_end_reads) SamBam_writer_add_read(global_context -> output_bam_writer, -2, read_name2, flags2, chro_name2 , chro_position2, mapping_quality2, cigar2, next_chro_name2 , next_chro_pos2, temp_len2, read_len2, read_text2, qual_text2, additional_columns2, 1); }else{ int write_len_2 = 100, write_len = sambamout_fprintf(global_context -> output_sam_fp , "%s\t%d\t%s\t%u\t%d\t%s\t%s\t%u\t%d\t%s\t%s%s%s\n", read_name1, flags1, chro_name1, chro_position1, mapping_quality1, cigar1, next_chro_name1, next_chro_pos1, temp_len1, read_text1, qual_text1, additional_columns1[0]?"\t":"", additional_columns1); if(global_context->input_reads.is_paired_end_reads) write_len_2 = sambamout_fprintf(global_context -> output_sam_fp , "%s\t%d\t%s\t%u\t%d\t%s\t%s\t%u\t%d\t%s\t%s%s%s\n", read_name2, flags2, chro_name2, chro_position2, mapping_quality2, cigar2, next_chro_name2, next_chro_pos2, temp_len2, read_text2, qual_text2, additional_columns2[0]?"\t":"", additional_columns2); if( write_len < 10 || write_len_2 < 10 ){ global_context -> output_sam_is_full = 1; } } if(all_locations <= this_location +1)global_context -> last_written_fragment_number = pair_number; is_fin = 1; } subread_lock_release(&global_context -> output_lock); if(is_fin)break; usleep(2); } } } #define write_chunk_results_145 write_chunk_results // rec1 or rec2 is OK if they are not NULL. void write_single_fragment(global_context_t * global_context, thread_context_t * thread_context, subread_output_tmp_t * rec1, realignment_result_t * raw_r1, subread_output_tmp_t * rec2, realignment_result_t * raw_r2, int all_locations , int current_location , char * read_name_1, char * read_name_2, int read_len_1, int read_len_2, char * read_text_1, char * read_text_2, char * qual_text_1, char * qual_text_2, subread_read_number_t pair_number, int non_informative_subread_r1, int non_informative_subread_r2, int is_R1_OK, int is_R2_OK) { //assert(all_locations <= global_context -> config.reported_multi_best_reads); int tlen = 0; if(is_R1_OK && is_R2_OK && rec1->chro == rec2->chro)tlen = calc_tlen(global_context , rec1, rec2, read_len_1, read_len_2); //#warning "SUBREAD_151 ============= REMOVE '+1' when not doing structure variance detection for the proposal ==========" if(0) if((global_context -> config.do_fusion_detection || global_context -> config.do_long_del_detection) && rec1 && rec2 && current_location == 0 && rec1 -> chimeric_sections == 1 && rec2 -> chimeric_sections == 1){ // when current_location == 0, both r1 and r2 were on the same strand. int is_funky = is_funky_fragment(global_context, read_name_1, rec1 -> chro, rec1 -> offset, read_len_1, rec1 -> strand, rec1 -> out_cigars[0], read_text_1, read_name_2, rec2 -> chro, rec2 -> offset, read_len_2, rec2 -> strand, rec2 -> out_cigars[0] , read_text_2, tlen); if (0 &&is_funky) SUBREADprintf("RNAME %s is %d, CHRO = '%s' %p '%s' %p, POS=%u, %u\n", read_name_1, is_funky, rec1 -> chro,rec1 -> chro,rec2 -> chro,rec2 -> chro, rec1 -> offset, rec2 -> offset); if(is_funky & FUNKY_FRAGMENT_A){ fraglist_append(&global_context -> funky_list_A, pair_number); } if(1)if(is_funky & FUNKY_FRAGMENT_BC){ //#warning "LOGIC WRONG: R1 AND R2 SHOULD BE DECIDED BY THEIR MAPPING POSITIONS" bktable_append(&global_context -> funky_table_BC, rec1 -> chro, rec1 -> offset , NULL + (2*pair_number)); bktable_append(&global_context -> funky_table_BC, rec2 -> chro, rec2 -> offset , NULL + (2*pair_number+1)); } if(1)if(is_funky & FUNKY_FRAGMENT_DE){ fraglist_append(&global_context -> funky_list_DE, pair_number); bktable_append(&global_context -> funky_table_DE, rec1 -> chro, rec1 -> offset , NULL + (2*pair_number + (rec1 -> offset > rec2 -> offset ? 1:0))); bktable_append(&global_context -> funky_table_DE, rec2 -> chro, rec2 -> offset , NULL + (2*pair_number + (rec1 -> offset < rec2 -> offset ? 1:0))); } } int flag1 = calc_flags( global_context, thread_context , rec1, rec2, 0, read_len_1, read_len_2, current_location, tlen, is_R1_OK, is_R2_OK); int flag2 = -1; if(global_context->input_reads.is_paired_end_reads) { flag2 = calc_flags( global_context , thread_context, rec1, rec2, 1, read_len_1, read_len_2, current_location, tlen, is_R2_OK, is_R1_OK); if((0 == current_location) && (flag2 & SAM_FLAG_MATCHED_IN_PAIR)){ if(thread_context)thread_context->all_correct_PE_reads ++; else global_context->all_correct_PE_reads ++; } } int applied_reverse_space; applied_reverse_space = global_context->config.space_type; if(global_context -> config.convert_color_to_base) { //SUBREADprintf("BEST_READ_NO = %d / %d\n", current_location, all_locations); //SUBREADprintf("ORGI: %s\n", read_text_1); colorread2base(read_text_1, read_len_1+1); //SUBREADprintf("CONV: %s\n\n", read_text_1); if(global_context->input_reads.is_paired_end_reads) colorread2base(read_text_2, read_len_2+1); applied_reverse_space = GENE_SPACE_BASE; } int should_1_reverse = calc_should_reverse( global_context , rec1, rec2, 0); if(should_1_reverse) { reverse_read(read_text_1, read_len_1 + global_context->config.convert_color_to_base, applied_reverse_space); reverse_quality(qual_text_1, read_len_1); } if(global_context->input_reads.is_paired_end_reads) { int should_2_reverse = calc_should_reverse( global_context , rec1, rec2, 1); if(should_2_reverse) { reverse_read(read_text_2, read_len_2 + global_context->config.convert_color_to_base, applied_reverse_space); reverse_quality(qual_text_2, read_len_2); } } remove_backslash(read_name_1); if(global_context->input_reads.is_paired_end_reads) remove_backslash(read_name_2); int display_offset1 = 0, display_tailgate1 = 0; int display_offset2 = 0, display_tailgate2 = 0; if(global_context -> config.space_type == GENE_SPACE_COLOR) { if(rec1 && rec1 -> strand) { display_offset1 = 0; display_tailgate1 = 1; } else { if(!global_context -> config.convert_color_to_base) { // the first base was a fake prime base; the second base is the first meaningful base. char second_char = read_text_1[1]; read_text_1[1] = color2char(second_char, read_text_1[0]); } display_offset1 = 1; display_tailgate1 = 0; } if(rec2 && rec2 -> strand) { if(!global_context -> config.convert_color_to_base) { // the first base was a fake prime base; the second base is the first meaningful base. char second_char = read_text_2[1]; read_text_2[1] = color2char(second_char, read_text_2[0]); } display_offset2 = 1; display_tailgate2 = 0; } else { display_offset2 = 0; display_tailgate2 = 1; } assert(display_offset1 + display_tailgate1 == 1); assert(display_offset2 + display_tailgate2 == 1); } if(!qual_text_1[0]) { int xi2; for(xi2=display_offset1;read_text_1[xi2 + display_tailgate1];xi2++) qual_text_1[xi2 - display_offset1] = 'I'; qual_text_1[xi2 - display_offset1]=0; for(xi2=display_offset2;read_text_2[xi2 + display_tailgate2];xi2++) qual_text_2[xi2 - display_offset2] = 'I'; qual_text_2[xi2 - display_offset2]=0; } if(display_tailgate1) read_text_1[strlen(read_text_1)-1]=0; if(display_tailgate2) read_text_2[strlen(read_text_2)-1]=0; if(global_context -> config.space_type == GENE_SPACE_BASE){ if(is_R1_OK) {// && !strstr(rec1->additional_information, "\tNM:i:")){ short rec1_edit = calc_edit_dist(global_context, rec1->raw_result, rec1->cigar , rec1->linear_position, read_text_1, raw_r1 -> final_mismatched_bases); sprintf(rec1->additional_information + strlen( rec1->additional_information), "\tNM:i:%d", rec1_edit ); } if(global_context->input_reads.is_paired_end_reads && is_R2_OK) //&& !strstr(rec2->additional_information, "\tNM:i:")) { short rec2_edit = calc_edit_dist(global_context, rec2->raw_result, rec2->cigar , rec2->linear_position, read_text_2, raw_r2 -> final_mismatched_bases); sprintf(rec2->additional_information + strlen( rec2->additional_information), "\tNM:i:%d", rec2_edit ); } } char extra_additional_1 [1000+CORE_ADDITIONAL_INFO_LENGTH], extra_additional_2[1000+CORE_ADDITIONAL_INFO_LENGTH]; int extra_additional_1_ptr=0, extra_additional_2_ptr=0; extra_additional_1[0] = extra_additional_2[0]=0; if(is_R1_OK || is_R2_OK){ // no NH and HI only if both ends are unmapped. extra_additional_1_ptr +=snprintf(extra_additional_1, 310, "HI:i:%d\tNH:i:%d", current_location+1, all_locations); extra_additional_2_ptr +=snprintf(extra_additional_2, 310, "HI:i:%d\tNH:i:%d", current_location+1, all_locations); } if(global_context->config.read_group_id[0]) { extra_additional_1_ptr += snprintf(extra_additional_1+ extra_additional_1_ptr, 310, "\tRG:Z:%s", global_context->config.read_group_id); extra_additional_2_ptr += snprintf(extra_additional_2+ extra_additional_2_ptr, 310, "\tRG:Z:%s", global_context->config.read_group_id); } char * out_chro1, * out_chro2, *out_cigar1, *out_cigar2; if(is_R1_OK) { strcpy(extra_additional_1 + extra_additional_1_ptr, rec1->additional_information); out_chro1 = rec1->chro; out_cigar1 = rec1->cigar; } else { out_chro1 = "*"; out_cigar1 = "*"; } if(is_R2_OK) { strcpy(extra_additional_2 + extra_additional_2_ptr, rec2->additional_information); out_chro2 = rec2->chro; out_cigar2 = rec2->cigar; } else { out_chro2 = "*"; out_cigar2 = "*"; } int out_offset1=0, out_offset2=0; long long int out_tlen1, out_tlen2; int out_mapping_quality1 = 0, out_mapping_quality2 = 0; out_tlen1 = tlen; out_tlen2 = tlen; if(is_R1_OK && is_R2_OK) { if( rec1->offset > rec2->offset) out_tlen1 = - out_tlen1; else if(rec2->offset > rec1->offset) out_tlen2 = -out_tlen2; else{ if( rec1 -> strand ) out_tlen1 = - out_tlen1; else out_tlen2 = -out_tlen2; } } if(0==current_location) { if(global_context -> input_reads.is_paired_end_reads) { if(is_R1_OK || is_R2_OK){ //SUBREADprintf("MAPTOLEN\n"); if(thread_context) thread_context -> all_mapped_reads ++; else global_context -> all_mapped_reads ++; } } else if(is_R1_OK){ if(thread_context) thread_context -> all_mapped_reads ++; else global_context -> all_mapped_reads ++; } } if(is_R1_OK){ out_offset1 = max(1, rec1->offset); out_mapping_quality1 = rec1->mapping_quality; } if(is_R2_OK){ out_offset2 = max(1, rec2->offset); out_mapping_quality2 = rec2->mapping_quality; } char * mate_chro_for_1 = out_chro2; char * mate_chro_for_2 = out_chro1; if(out_chro1 == out_chro2 && out_chro1 && out_chro1[0]!='*'){ mate_chro_for_1="="; mate_chro_for_2="="; } //if(pair_number<10)SUBREADprintf("FIN_ADD #%d: %s in %p \n", pair_number, read_name_1, thread_context); if(thread_context) add_buffered_fragment(global_context, thread_context, pair_number, read_name_1, flag1, out_chro1 , out_offset1, out_mapping_quality1, out_cigar1, mate_chro_for_1 , out_offset2, out_tlen1, read_len_1, read_text_1 + display_offset1, qual_text_1, extra_additional_1, read_name_2, flag2, out_chro2 , out_offset2, out_mapping_quality2, out_cigar2, mate_chro_for_2 , out_offset1, out_tlen2, read_len_2, read_text_2 + display_offset2, qual_text_2, extra_additional_2, all_locations, current_location); else{ //subread_lock_occupy(&global_context -> output_lock); if(global_context -> config.is_BAM_output){ SamBam_writer_add_read(global_context -> output_bam_writer, -1, read_name_1, flag1, out_chro1 , out_offset1, out_mapping_quality1, out_cigar1, mate_chro_for_1 , out_offset2, out_tlen1, read_len_1, read_text_1 + display_offset1, qual_text_1, extra_additional_1, !global_context->input_reads.is_paired_end_reads); if(global_context->input_reads.is_paired_end_reads) SamBam_writer_add_read(global_context -> output_bam_writer, -1, read_name_2, flag2, out_chro2 , out_offset2, out_mapping_quality2, out_cigar2, mate_chro_for_2 , out_offset1, out_tlen2, read_len_2, read_text_2 + display_offset2, qual_text_2, extra_additional_2, 1); } else { int write_len_2 = 100, #ifdef __MINGW32__ write_len = sambamout_fprintf(global_context -> output_sam_fp , "%s\t%d\t%s\t%u\t%d\t%s\t%s\t%u\t%I64d\t%s\t%s%s%s\n", read_name_1, flag1, out_chro1, out_offset1, out_mapping_quality1, out_cigar1, mate_chro_for_1, out_offset2, out_tlen1, read_text_1 + display_offset1, qual_text_1, extra_additional_1[0]?"\t":"", extra_additional_1); #else write_len = sambamout_fprintf(global_context -> output_sam_fp , "%s\t%d\t%s\t%u\t%d\t%s\t%s\t%u\t%lld\t%s\t%s%s%s\n", read_name_1, flag1, out_chro1, out_offset1, out_mapping_quality1, out_cigar1, mate_chro_for_1, out_offset2, out_tlen1, read_text_1 + display_offset1, qual_text_1, extra_additional_1[0]?"\t":"", extra_additional_1); #endif if(global_context->input_reads.is_paired_end_reads) #ifdef __MINGW32__ write_len_2 = sambamout_fprintf(global_context -> output_sam_fp , "%s\t%d\t%s\t%u\t%d\t%s\t%s\t%u\t%I64d\t%s\t%s%s%s\n", read_name_2, flag2, out_chro2, out_offset2, out_mapping_quality2, out_cigar2, mate_chro_for_2, out_offset1, out_tlen2, read_text_2 + display_offset2, qual_text_2, extra_additional_2[0]?"\t":"", extra_additional_2); #else write_len_2 = sambamout_fprintf(global_context -> output_sam_fp , "%s\t%d\t%s\t%u\t%d\t%s\t%s\t%u\t%lld\t%s\t%s%s%s\n", read_name_2, flag2, out_chro2, out_offset2, out_mapping_quality2, out_cigar2, mate_chro_for_2, out_offset1, out_tlen2, read_text_2 + display_offset2, qual_text_2, extra_additional_2[0]?"\t":"", extra_additional_2); #endif if( write_len < 10 || write_len_2 < 10 ){ global_context -> output_sam_is_full = 1; } } //subread_lock_release(&global_context -> output_lock); } if(is_R1_OK) rec1->raw_result->selected_position += rec1->soft_clipping_movements; if(is_R2_OK) rec2->raw_result->selected_position += rec2->soft_clipping_movements; } void init_chunk_scanning_parameters(global_context_t * global_context, thread_context_t * thread_context, gene_input_t ** ginp1, gene_input_t ** ginp2) { *ginp2 = NULL; *ginp1 = &global_context->input_reads.first_read_file; if(global_context->input_reads.is_paired_end_reads) *ginp2 = &global_context->input_reads.second_read_file; } gene_value_index_t * find_current_value_index(global_context_t * global_context, unsigned int pos, int len) { int block_no; if(global_context->index_block_number<2) { unsigned index_begin = global_context -> all_value_indexes [0] . start_base_offset; unsigned index_end = global_context -> all_value_indexes [0] . start_base_offset + global_context -> all_value_indexes [0] . length; if(pos>=index_begin && pos + lenall_value_indexes [0]; else return NULL; } else for(block_no=0; block_noindex_block_number; block_no++) { unsigned index_begin = global_context -> all_value_indexes [block_no] . start_base_offset; unsigned index_end = global_context -> all_value_indexes [block_no] . start_base_offset + global_context -> all_value_indexes [block_no] . length; if((block_no == 0 && pos >= index_begin && pos < index_end - 1000000) || (block_no>0 && block_noindex_block_number-1 && pos >= index_begin+ 1000000 && pos < index_end - 1000000) || (block_no == global_context->index_block_number-1 && pos >= index_begin + 1000000 && pos < index_end )) { return & global_context -> all_value_indexes [block_no]; } } return NULL; } //this function selects the correct all_value_indexes from global_context and put it to global_context or thread_context if thread_context is not NULL. int locate_current_value_index(global_context_t * global_context, thread_context_t * thread_context, mapping_result_t * result, int rlen) { int block_no; if(global_context->index_block_number<2) { unsigned index_begin = global_context -> all_value_indexes [0] . start_base_offset; unsigned index_end = global_context -> all_value_indexes [0] . start_base_offset + global_context -> all_value_indexes [0] . length; //SUBREADprintf("RESET2 : %u should <= %u\n", result->selected_position + rlen, index_end); if(result->selected_position>=index_begin && result->selected_position + rlen<=index_end) { if(thread_context)thread_context->current_value_index = & global_context->all_value_indexes [0]; else global_context->current_value_index =& global_context->all_value_indexes [0]; return 0; } else return 1; } for(block_no=0; block_noindex_block_number; block_no++) { unsigned index_begin = global_context -> all_value_indexes [block_no] . start_base_offset; unsigned index_end = global_context -> all_value_indexes [block_no] . start_base_offset + global_context -> all_value_indexes [block_no] . length; if((block_no == 0 && result->selected_position >= index_begin && result->selected_position < index_end - 1000000) || (block_no>0 && block_noindex_block_number-1 && result->selected_position >= index_begin+ 1000000 && result->selected_position < index_end - 1000000) || (block_no == global_context->index_block_number-1 && result->selected_position >= index_begin + 1000000 && result->selected_position < index_end )) { if(thread_context)thread_context->current_value_index =& global_context -> all_value_indexes [block_no]; else global_context->current_value_index =& global_context -> all_value_indexes [block_no]; return 0; } } return 1; } int do_iteration_one(global_context_t * global_context, thread_context_t * thread_context) { assert(0); // not needed anymore return 0; } int finish_iteration_three(global_context_t * global_context, thread_context_t * thread_context) { return 0; } int do_iteration_three(global_context_t * global_context, thread_context_t * thread_context) { int ret; gene_input_t * ginp1 = NULL , * ginp2 = NULL; subread_read_number_t current_read_number=0; char read_text_1[MAX_READ_LENGTH+1], read_text_2[MAX_READ_LENGTH+1]; char qual_text_1[MAX_READ_LENGTH+1], qual_text_2[MAX_READ_LENGTH+1]; char read_name_1[MAX_READ_NAME_LEN+1], read_name_2[MAX_READ_NAME_LEN+1]; int read_len_1, read_len_2=0, sqr_interval, sqr_read_number = 0; //unsigned int low_index_border = global_context -> current_value_index -> start_base_offset; //unsigned int high_index_border = global_context -> current_value_index -> start_base_offset + global_context -> current_value_index -> length; print_in_box(80,0,0,"Prepare for long indel deleteion..."); init_chunk_scanning_parameters(global_context,thread_context, & ginp1, & ginp2); sqr_interval = max(5000,global_context -> processed_reads_in_chunk/10/ global_context -> config.all_threads); while(1) { int is_second_read; sqr_read_number++; ret = fetch_next_read_pair(global_context, thread_context, ginp1, ginp2, &read_len_1, &read_len_2, read_name_1, read_name_2, read_text_1, read_text_2, qual_text_1, qual_text_2, 1, ¤t_read_number); if(ret) break; int best_read_id = 0; for (is_second_read = 0; is_second_read < 1 + global_context -> input_reads.is_paired_end_reads; is_second_read ++) { int is_reversed_already = 0; for(best_read_id = 0; best_read_id < global_context -> config.multi_best_reads; best_read_id++) { mapping_result_t current_result_body; mapping_result_t mate_result_body; mapping_result_t * current_result = ¤t_result_body, *mate_result = NULL; bigtable_readonly_result(global_context, NULL, current_read_number, best_read_id, is_second_read, current_result, NULL); if(global_context -> input_reads.is_paired_end_reads){ mate_result = &mate_result_body; bigtable_readonly_result(global_context, NULL, current_read_number, best_read_id,!is_second_read, mate_result, NULL); } //if(best_read_id && (current_result->selected_votes <1)) break; char * current_read_name = is_second_read?read_name_2 : read_name_1; char * current_read = is_second_read?read_text_2 : read_text_1; char * current_qual = is_second_read?qual_text_2 : qual_text_1; int current_rlen = is_second_read?read_len_2:read_len_1; int mate_rlen = is_second_read?read_len_1:read_len_2; if((current_result->result_flags & CORE_IS_FULLY_EXPLAINED) || (global_context -> input_reads.is_paired_end_reads &&(mate_result -> result_flags & CORE_IS_FULLY_EXPLAINED))) { //SUBREADprintf("BUILD BLOCK FOR READ_%d '%s' BEST=%d\n", is_second_read + 1, current_read_name , best_read_id); if(current_result->result_flags & CORE_IS_FULLY_EXPLAINED) { int is_negative_strand = ((current_result -> result_flags & CORE_IS_NEGATIVE_STRAND)?1:0); if(is_negative_strand + is_reversed_already ==1) { reverse_read(current_read, current_rlen, global_context->config.space_type); reverse_quality(current_qual, current_rlen); is_reversed_already=!is_reversed_already; } build_local_reassembly(global_context , thread_context , current_read_number, current_read_name , current_read , current_qual , current_rlen, 0 , is_second_read, best_read_id, 0, current_result, mate_result); } else if(global_context -> input_reads.is_paired_end_reads && (mate_result -> result_flags & CORE_IS_FULLY_EXPLAINED)) { int is_negative_strand = ((mate_result -> result_flags & CORE_IS_NEGATIVE_STRAND)?1:0); if(is_negative_strand+is_reversed_already==1) { reverse_read(current_read, current_rlen, global_context->config.space_type); reverse_quality(current_qual, current_rlen); is_reversed_already=!is_reversed_already; } build_local_reassembly(global_context , thread_context , current_read_number , current_read_name , current_read , current_qual , current_rlen, mate_rlen , is_second_read, best_read_id, 1, current_result, mate_result); } } } } if(!thread_context || thread_context->thread_id == 0) { if(sqr_read_number>sqr_interval) { show_progress(global_context, thread_context, current_read_number, STEP_ITERATION_THREE); sqr_read_number = 0; } } } return 0; } void add_realignment_event_support(global_context_t * global_context , realignment_result_t * res){ int xk1; indel_context_t * indel_context = (indel_context_t *)global_context -> module_contexts[MODULE_INDEL_ID]; for(xk1 = 0; xk1 < MAX_EVENTS_IN_READ ; xk1++){ chromosome_event_t *sup = res -> supporting_chromosome_events[xk1]; if(!sup)break; int lock_hash = sup -> global_event_id % EVENT_BODY_LOCK_BUCKETS; subread_lock_occupy(indel_context -> event_body_locks+lock_hash); sup -> final_counted_reads ++; sup -> junction_flanking_left = max(sup -> junction_flanking_left, res -> flanking_size_left[xk1]); sup -> junction_flanking_right = max(sup -> junction_flanking_right, res -> flanking_size_right[xk1]); subread_lock_release(indel_context -> event_body_locks+lock_hash); } } unsigned int calc_end_pos(unsigned int p, char * cigar, unsigned int * all_skipped_len, int * is_exonic_regions, global_context_t * global_context); void test_PE_and_same_chro_align(global_context_t * global_context , realignment_result_t * res1, realignment_result_t * res2, int * is_exonic_regions, int * is_PE_distance, int * is_same_chromosome, int read_len_1, int read_len_2, char * rname, int * res_tlen); void write_realignments_for_fragment(global_context_t * global_context, thread_context_t * thread_context, subread_output_context_t * out_context, unsigned int read_number, realignment_result_t * res1, realignment_result_t * res2, char * read_name_1, char * read_name_2, char * read_text_1, char * read_text_2, char * qual_text_1, char * qual_text_2 , int rlen1 , int rlen2, int multi_mapping_number, int this_multi_mapping_i, int non_informative_subreads_r1, int non_informative_subreads_r2){ int is_2_OK = 0, is_1_OK = 0; if(res1){ is_1_OK = convert_read_to_tmp(global_context , out_context, read_number, 0, rlen1, read_text_1, qual_text_1, res1, out_context -> r1, read_name_1); if(is_1_OK) add_realignment_event_support(global_context, res1); } if(res2){ is_2_OK = convert_read_to_tmp(global_context , out_context, read_number, 1, rlen2, read_text_2, qual_text_2, res2, out_context -> r2, read_name_2); if(is_2_OK) add_realignment_event_support(global_context, res2); } subread_output_tmp_t * r1_output = NULL; subread_output_tmp_t * r2_output = NULL; if(res1){ r1_output = out_context -> r1; } if(res2){ r2_output = out_context -> r2; } if(this_multi_mapping_i < 1){ if( is_1_OK == 0 && is_2_OK == 0) if(thread_context) thread_context -> all_unmapped_reads ++; else global_context -> all_unmapped_reads ++; else if( is_1_OK == 0 || is_2_OK == 0){ if(thread_context) thread_context -> not_properly_pairs_only_one_end_mapped ++; else global_context -> not_properly_pairs_only_one_end_mapped ++; if((is_1_OK && (res1 -> realign_flags & CORE_IS_BREAKEVEN )) || (is_2_OK && (res2 -> realign_flags & CORE_IS_BREAKEVEN))) { if(thread_context) thread_context -> all_multimapping_reads ++; else global_context -> all_multimapping_reads ++; }else{ if(thread_context) thread_context -> all_uniquely_mapped_reads ++; else global_context -> all_uniquely_mapped_reads ++; } }else{ if(res1 -> realign_flags & CORE_IS_BREAKEVEN){ if(thread_context) thread_context -> all_multimapping_reads ++; else global_context -> all_multimapping_reads ++; }else{ if(thread_context) thread_context -> all_uniquely_mapped_reads ++; else global_context -> all_uniquely_mapped_reads ++; } } } if((!global_context->config.ignore_unmapped_reads) || (is_2_OK || is_1_OK)) write_single_fragment(global_context, thread_context, r1_output, res1, r2_output, res2, multi_mapping_number , this_multi_mapping_i , read_name_1, read_name_2, rlen1, rlen2, read_text_1, read_text_2, qual_text_1, qual_text_2, read_number, non_informative_subreads_r1, non_informative_subreads_r2, is_1_OK, is_2_OK); } void clear_repeated_buffer(global_context_t * global_context, unsigned int * repeated_buffer_position, char ** repeated_buffer_cigar, int * repeated_count){ (*repeated_count) = 0; } int add_repeated_buffer(global_context_t * global_context, unsigned int * repeated_buffer_position, char ** repeated_buffer_cigar, int * repeated_count, realignment_result_t * res1, realignment_result_t * res2){ int x1, is_repeated = 0; char * r1_cigar = "*"; unsigned int r1_pos = 0; if(res1){ r1_cigar = res1 -> cigar_string; r1_pos = res1 -> first_base_position; } char * r2_cigar = "*"; unsigned int r2_pos = 0; if(res2){ r2_cigar = res2 -> cigar_string; r2_pos = res2 -> first_base_position; } for(x1 = 0; x1 < (*repeated_count); x1 += 2){ //if(strcmp(r1_cigar, "48M2769N53M")==0) // SUBREADprintf("KYKY %u=%u %s=%s\n" , r1_pos, repeated_buffer_position[x1], r1_cigar , repeated_buffer_cigar[x1]); if(repeated_buffer_position[x1] == r1_pos && repeated_buffer_position[x1+1] == r2_pos) if(strcmp(repeated_buffer_cigar[x1], r1_cigar) == 0 && strcmp(repeated_buffer_cigar[x1+1], r2_cigar) == 0) { is_repeated = 1; break; } } if( (!is_repeated) && (*repeated_count) < MAX_ALIGNMENT_PER_ANCHOR * 2 * global_context -> config.reported_multi_best_reads){ //if(strcmp(r1_cigar, "48M2769N53M")==0) // SUBREADprintf("CPCP %u=%u %s=%s\n" , r1_pos, repeated_buffer_position[x1], r1_cigar , repeated_buffer_cigar[x1]); repeated_buffer_position[*repeated_count] = r1_pos; repeated_buffer_position[1 + *repeated_count] = r2_pos; strcpy(repeated_buffer_cigar[*repeated_count], r1_cigar); strcpy(repeated_buffer_cigar[1 + *repeated_count], r2_cigar); (*repeated_count) +=2; } return is_repeated; } int do_iteration_two(global_context_t * global_context, thread_context_t * thread_context) { gene_input_t * ginp1 = NULL , * ginp2 = NULL; subread_read_number_t current_read_number=0; char read_text_1[MAX_READ_LENGTH+1], read_text_2[MAX_READ_LENGTH+1]; char qual_text_1[MAX_READ_LENGTH+1], qual_text_2[MAX_READ_LENGTH+1]; char raw_read_text_1[MAX_READ_LENGTH+1], raw_read_text_2[MAX_READ_LENGTH+1]; char raw_qual_text_1[MAX_READ_LENGTH+1], raw_qual_text_2[MAX_READ_LENGTH+1]; char read_name_1[MAX_READ_NAME_LEN+1], read_name_2[MAX_READ_NAME_LEN+1]; char * repeated_buffer_cigars[MAX_ALIGNMENT_PER_ANCHOR * 2 * global_context -> config.reported_multi_best_reads]; unsigned int repeated_buffer_pos[MAX_ALIGNMENT_PER_ANCHOR * 2 * global_context -> config.reported_multi_best_reads]; int repeated_count; int read_len_1=0, read_len_2=0; int sqr_interval, sqr_read_number=0; int * final_MATCH_buffer1, *final_MISMATCH_buffer1, *final_PENALTY_buffer1; int * final_MATCH_buffer2, *final_MISMATCH_buffer2, *final_PENALTY_buffer2, non_informative_subreads_r1=0, non_informative_subreads_r2=0; int * final_realignment_index1, *final_realignment_index2; unsigned int *final_realignment_number; unsigned long long * final_SCORE_buffer; unsigned int * final_TLEN_buffer; realignment_result_t * final_realignments; subread_output_context_t out_context; //SUBREADprintf("THREAD_OPT2_start\n"); init_output_context(global_context, &out_context); for(repeated_count = 0;repeated_count < MAX_ALIGNMENT_PER_ANCHOR * 2 * global_context -> config.reported_multi_best_reads ; repeated_count ++ ){ repeated_buffer_cigars[repeated_count] = malloc(2*CORE_MAX_CIGAR_STR_LEN); } final_MATCH_buffer1 = malloc(sizeof(int) * 2 * global_context -> config.multi_best_reads * MAX_ALIGNMENT_PER_ANCHOR); final_MISMATCH_buffer1 = malloc(sizeof(int) * 2 * global_context -> config.multi_best_reads * MAX_ALIGNMENT_PER_ANCHOR); final_PENALTY_buffer1 = malloc(sizeof(int) * 2 * global_context -> config.multi_best_reads * MAX_ALIGNMENT_PER_ANCHOR); final_realignment_index1 = malloc(sizeof(int) * global_context -> config.multi_best_reads * MAX_ALIGNMENT_PER_ANCHOR); final_MATCH_buffer2 = malloc(sizeof(int) * 2 * global_context -> config.multi_best_reads * MAX_ALIGNMENT_PER_ANCHOR); final_MISMATCH_buffer2 = malloc(sizeof(int) * 2 * global_context -> config.multi_best_reads * MAX_ALIGNMENT_PER_ANCHOR); final_PENALTY_buffer2 = malloc(sizeof(int) * 2 * global_context -> config.multi_best_reads * MAX_ALIGNMENT_PER_ANCHOR); final_realignment_index2 = malloc(sizeof(int) * global_context -> config.multi_best_reads * MAX_ALIGNMENT_PER_ANCHOR); final_SCORE_buffer = malloc(sizeof(long long) * global_context -> config.multi_best_reads * global_context -> config.multi_best_reads * MAX_ALIGNMENT_PER_ANCHOR*MAX_ALIGNMENT_PER_ANCHOR); final_TLEN_buffer = malloc(sizeof(int) * global_context -> config.multi_best_reads * global_context -> config.multi_best_reads * MAX_ALIGNMENT_PER_ANCHOR*MAX_ALIGNMENT_PER_ANCHOR); final_realignments = malloc(sizeof(realignment_result_t) * global_context -> config.multi_best_reads * 2 * MAX_ALIGNMENT_PER_ANCHOR); final_realignment_number = malloc(sizeof(int) * global_context -> config.multi_best_reads * 2); mapping_result_t * r1_align_result_buffer, * r2_align_result_buffer; subjunc_result_t * r1_subjunc_result_buffer, * r2_subjunc_result_buffer; r1_align_result_buffer = malloc(sizeof(mapping_result_t) * global_context -> config.multi_best_reads); r2_align_result_buffer = malloc(sizeof(mapping_result_t) * global_context -> config.multi_best_reads); r1_subjunc_result_buffer = malloc(sizeof(subjunc_result_t) * global_context -> config.multi_best_reads); r2_subjunc_result_buffer = malloc(sizeof(subjunc_result_t) * global_context -> config.multi_best_reads); //unsigned int low_index_border = global_context -> current_value_index -> start_base_offset; //unsigned int high_index_border = global_context -> current_value_index -> start_base_offset + global_context -> current_value_index -> length; init_chunk_scanning_parameters(global_context,thread_context, & ginp1, & ginp2); sqr_interval = max(5000,global_context -> processed_reads_in_chunk/10/ global_context -> config.all_threads); while(1) { int is_second_read; int max_votes; sqr_read_number++; fetch_next_read_pair(global_context, thread_context, ginp1, ginp2, &read_len_1, &read_len_2, read_name_1, read_name_2, read_text_1, read_text_2, qual_text_1, qual_text_2, 0, ¤t_read_number); //if(current_read_number%500000==0)SUBREADprintf("THREAD_OPT2_fetch %s %s ; RNO=%lld\n", read_name_1, read_text_1, current_read_number); strcpy(raw_read_text_1, read_text_1); strcpy(raw_qual_text_1, qual_text_1); //printf("OCT27-STEPB - %s\n", read_name_1); if(global_context -> input_reads.is_paired_end_reads){ strcpy(raw_read_text_2, read_text_2); strcpy(raw_qual_text_2, qual_text_2); } if(global_context->config.space_type == GENE_SPACE_COLOR){ if(isalpha(read_text_1[0])){ //SUBREADprintf("FIRST : %s\n\n", read_text_1); int xk1; for(xk1=2; read_text_1[xk1]; xk1++){ read_text_1[xk1-2] = read_text_1[xk1]; } read_text_1[xk1-2] = 0; } if(global_context -> input_reads.is_paired_end_reads){ //SUBREADprintf("SECOND: %s\n\n", read_text_2); if(isalpha(read_text_2[0])){ int xk1; for(xk1=1; read_text_2[xk1]; xk1++){ read_text_2[xk1-1] = read_text_2[xk1]; } read_text_2[xk1-1] = 0; } } } if(raw_qual_text_1[0] && global_context -> config.phred_score_format == FASTQ_PHRED64){ fastq_64_to_33(raw_qual_text_1); if(global_context->input_reads.is_paired_end_reads) fastq_64_to_33(raw_qual_text_2); } if((global_context -> config.is_BAM_output && global_context -> output_bam_writer -> is_internal_error) || global_context -> input_reads.is_internal_error || (global_context -> output_sam_is_full))break; if(current_read_number < 0) break; // if no more reads if( global_context -> input_reads.is_paired_end_reads) max_votes = max(_global_retrieve_alignment_ptr(global_context, current_read_number, 0, 0)->selected_votes, _global_retrieve_alignment_ptr(global_context, current_read_number, 1, 0)->selected_votes); else max_votes = _global_retrieve_alignment_ptr(global_context, current_read_number, 0, 0)->selected_votes; //if( current_read_number < 10 ) SUBREADprintf("BSSS2 : %s : %d\n", read_name_1, max_votes); int best_read_id=0; //memset(final_MATCH_buffer, 0, sizeof(int) * 2 * global_context -> config.multi_best_reads); //memset(final_MISMATCH_buffer, 0, sizeof(int) * 2 * global_context -> config.multi_best_reads); int r1_candidate_locations = 0, r2_candidate_locations = 0; int r1_step2_locations = 0, r2_step2_locations = 0; clear_repeated_buffer(global_context, repeated_buffer_pos, repeated_buffer_cigars, &repeated_count); for (is_second_read = 0; is_second_read < 1 + global_context -> input_reads.is_paired_end_reads; is_second_read ++) { char * current_read_name = is_second_read?read_name_2 : read_name_1; int is_reversed_already = 0, realignment_i; int * current_candidate_locations = is_second_read?&r2_candidate_locations:&r1_candidate_locations; int * current_MATCH_buffer = is_second_read?final_MATCH_buffer2:final_MATCH_buffer1; int * current_MISMATCH_buffer = is_second_read?final_MISMATCH_buffer2:final_MISMATCH_buffer1; int * current_PENALTY_buffer = is_second_read?final_PENALTY_buffer2:final_PENALTY_buffer1; int * current_realignment_index = is_second_read?final_realignment_index2:final_realignment_index1; for(best_read_id = 0; best_read_id < global_context -> config.multi_best_reads; best_read_id++) { mapping_result_t *current_result = _global_retrieve_alignment_ptr(global_context, current_read_number, is_second_read, best_read_id); if(best_read_id == 0){ if(is_second_read) non_informative_subreads_r2 = current_result -> noninformative_subreads_in_vote; else non_informative_subreads_r1 = current_result -> noninformative_subreads_in_vote; } char * current_read = is_second_read?read_text_2 : read_text_1; char * current_qual = is_second_read?qual_text_2 : qual_text_1; int current_rlen = is_second_read?read_len_2:read_len_1; if(current_result->selected_votes < global_context->config.minimum_subread_for_second_read || max_votes < global_context->config.minimum_subread_for_first_read) { // if(current_read_number == 111 || current_read_number == 112) //SUBREADprintf("RESET0 [%lld] R_%d SEL=%d MAX=%d\n", current_read_number, is_second_read + 1, current_result->selected_votes, max_votes); current_result -> selected_votes = 0; continue; } if(locate_current_value_index(global_context, thread_context, current_result, current_rlen)) { current_result -> selected_votes = 0; //SUBREADprintf("RESET1 Read position excesses index boundary.\n"); continue; } //#warning ">>>>>>> COMMENT THIS <<<<<<<" //printf("OCT27-STEP21-%s:%d-ALN%02d-THRE %d in MM=%d\n", current_read_name, is_second_read + 1, best_read_id, thread_context -> thread_id, global_context -> config.multi_best_reads); int is_negative_strand = (current_result -> result_flags & CORE_IS_NEGATIVE_STRAND)?1:0; if(is_negative_strand + is_reversed_already == 1) { reverse_read(current_read, current_rlen, global_context->config.space_type); reverse_quality(current_qual, current_rlen); is_reversed_already = !is_reversed_already; } current_result -> result_flags &= ~CORE_IS_FULLY_EXPLAINED; if(is_second_read) r2_step2_locations = best_read_id + 1; else r1_step2_locations = best_read_id + 1; unsigned int final_alignments = explain_read(global_context, thread_context , final_realignments + (is_second_read + 2 * best_read_id) * MAX_ALIGNMENT_PER_ANCHOR, current_read_number, current_rlen, current_read_name, current_read, current_qual, is_second_read, best_read_id, is_negative_strand); final_realignment_number[ best_read_id * 2 + is_second_read ] = final_alignments; for(realignment_i = 0 ; realignment_i < final_alignments ; realignment_i ++){ if((* current_candidate_locations) >= global_context -> config.multi_best_reads * MAX_ALIGNMENT_PER_ANCHOR) break; int realign_index = (is_second_read + 2 * best_read_id)* MAX_ALIGNMENT_PER_ANCHOR + realignment_i; int final_MATCH = final_realignments[realign_index].final_matched_bases; int final_PENALTY = final_realignments[realign_index].final_penalty; if((current_result -> result_flags & CORE_IS_FULLY_EXPLAINED) && final_MATCH>0) { int final_MISMATCH = final_realignments[realign_index].final_mismatched_bases; //#warning ">>>>>>>>>>>>>>>>>>>>>>>>>>>>> REMOVE THIS <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<" //printf("OCT27-STEPMSM-MMM %s M=%d MM=%d\n", read_name_1, final_MATCH ,final_MISMATCH); current_PENALTY_buffer[*current_candidate_locations] = final_PENALTY; current_MATCH_buffer[*current_candidate_locations] = final_MATCH; current_MISMATCH_buffer[*current_candidate_locations] = final_MISMATCH; current_realignment_index[*current_candidate_locations] = realign_index; (*current_candidate_locations) ++; } } //#warning ">>>>>>> COMMENT THIS <<<<<<<" //printf("OCT27-FI-%s:%d-ALN%02d-THRE %d\n", current_read_name, is_second_read + 1, best_read_id, thread_context -> thread_id); } } //if(161430 <= current_read_number) SUBREADprintf("LOC1=%d, LOC2=%d\n", r1_candidate_locations, r2_candidate_locations); int need_expect_TLEN = r2_candidate_locations && r1_candidate_locations && global_context -> config.reported_multi_best_reads<2 && global_context -> expected_TLEN_read_numbers < READPAIRS_FOR_CALC_EXPT_TLEN; int output_cursor = 0; if(r2_candidate_locations == 0 || r1_candidate_locations == 0) { int is_second_read, highest_score_occurence =0; for(is_second_read = 0; is_second_read < 1+ global_context -> input_reads.is_paired_end_reads; is_second_read++) { int current_candidate_locations = is_second_read?r2_candidate_locations:r1_candidate_locations; if(current_candidate_locations > 0){ int * current_MATCH_buffer = is_second_read?final_MATCH_buffer2:final_MATCH_buffer1; int * current_PENALTY_buffer = is_second_read?final_PENALTY_buffer2:final_PENALTY_buffer1; int * current_MISMATCH_buffer = is_second_read?final_MISMATCH_buffer2:final_MISMATCH_buffer1; int * current_realignment_index = is_second_read?final_realignment_index2:final_realignment_index1; int read_record_i; unsigned long long int best_score_highest = 0; unsigned long long int scores_array [global_context -> config.multi_best_reads * MAX_ALIGNMENT_PER_ANCHOR]; for(read_record_i = 0; read_record_i < current_candidate_locations; read_record_i++){ realignment_result_t * current_realignment_result = final_realignments + current_realignment_index[read_record_i]; unsigned int this_MATCH = current_MATCH_buffer[read_record_i]; unsigned int this_MISMATCH = current_MISMATCH_buffer[read_record_i]; unsigned int this_PENALTY = current_PENALTY_buffer[read_record_i]; unsigned long long this_SCORE; if(global_context -> config.experiment_type == CORE_EXPERIMENT_DNASEQ){ this_SCORE = this_MATCH * 100000llu + (10000 - this_MISMATCH); }else{ unsigned int skip = 0; int is_exonic_regions = 1; if(global_context -> exonic_region_bitmap)calc_end_pos(current_realignment_result -> first_base_position, current_realignment_result -> cigar_string, &skip, &is_exonic_regions, global_context ); if(global_context -> config.scRNA_input_mode) this_SCORE =((100000llu * (10000 - this_MISMATCH + 2*is_exonic_regions) + this_MATCH)*50llu - this_PENALTY)*20llu+ current_realignment_result -> known_junction_supp; else this_SCORE =((100000llu * (10000 - this_MISMATCH) + this_MATCH)*50llu - this_PENALTY)*20llu+ current_realignment_result -> known_junction_supp; } best_score_highest = max(best_score_highest, this_SCORE); scores_array[read_record_i] = this_SCORE; } for(read_record_i = 0; read_record_i < current_candidate_locations ; read_record_i++){ realignment_result_t * current_realignment_result = final_realignments + current_realignment_index[read_record_i]; if( scores_array[read_record_i] >= best_score_highest && (current_realignment_result -> realign_flags & CORE_TOO_MANY_MISMATCHES)==0) { int is_repeated = 0; is_repeated = add_repeated_buffer(global_context, repeated_buffer_pos, repeated_buffer_cigars, &repeated_count, is_second_read?NULL:current_realignment_result , is_second_read?current_realignment_result:NULL); if(is_repeated) scores_array[read_record_i] = 0; else highest_score_occurence ++; } } if(highest_score_occurence<2 || global_context -> config.report_multi_mapping_reads){ int is_break_even = 0; if(highest_score_occurence>1) is_break_even = 1; highest_score_occurence = min(highest_score_occurence, global_context -> config.reported_multi_best_reads); for(read_record_i = 0; read_record_i < current_candidate_locations ; read_record_i++){ realignment_result_t * current_realignment_result = final_realignments + current_realignment_index[read_record_i]; if( scores_array[read_record_i] >= best_score_highest && (current_realignment_result -> realign_flags & CORE_TOO_MANY_MISMATCHES)==0 && output_cursor < global_context -> config.reported_multi_best_reads){ strcpy(read_text_1, raw_read_text_1); strcpy(read_text_2, raw_read_text_2); strcpy(qual_text_1, raw_qual_text_1); strcpy(qual_text_2, raw_qual_text_2); if(is_break_even) current_realignment_result -> realign_flags |= CORE_IS_BREAKEVEN; current_realignment_result -> MAPQ_adjustment = current_MISMATCH_buffer [read_record_i] + ( is_second_read?(r2_step2_locations): (r1_step2_locations)); // SUBREADprintf("THREAD_OPT1_write %s\n", read_name_1); if(is_second_read) write_realignments_for_fragment(global_context, thread_context, &out_context, current_read_number, NULL, current_realignment_result, read_name_1, read_name_2, read_text_1, read_text_2, qual_text_1, qual_text_2, read_len_1, read_len_2, highest_score_occurence, output_cursor, non_informative_subreads_r1, non_informative_subreads_r2); else write_realignments_for_fragment(global_context, thread_context, &out_context , current_read_number, current_realignment_result, NULL, read_name_1, read_name_2, read_text_1, read_text_2, qual_text_1, qual_text_2, read_len_1, read_len_2, highest_score_occurence, output_cursor, non_informative_subreads_r1, non_informative_subreads_r2); output_cursor ++; } } assert(output_cursor == highest_score_occurence); } } } } else { int r1_best_id, r2_best_id, highest_score_occurence = 0; int expected_TLEN; if(global_context -> expected_TLEN_read_numbers >= READPAIRS_FOR_CALC_EXPT_TLEN) expected_TLEN = global_context -> expected_TLEN_sum / global_context -> expected_TLEN_read_numbers; else expected_TLEN = (global_context -> config.minimum_pair_distance + global_context -> config.maximum_pair_distance)/2; unsigned long long highest_score = 0; if(need_expect_TLEN) memset(final_TLEN_buffer, 0 , sizeof(int) * global_context -> config.multi_best_reads * global_context -> config.multi_best_reads * MAX_ALIGNMENT_PER_ANCHOR*MAX_ALIGNMENT_PER_ANCHOR ); memset(final_SCORE_buffer, 0 , sizeof(long long) * global_context -> config.multi_best_reads * global_context -> config.multi_best_reads * MAX_ALIGNMENT_PER_ANCHOR*MAX_ALIGNMENT_PER_ANCHOR ); for(r1_best_id = 0; r1_best_id < r1_candidate_locations; r1_best_id ++) { int r1_matched = final_MATCH_buffer1[r1_best_id]; if(r1_matched < 1) continue; realignment_result_t * realignment_result_R1 = final_realignments + final_realignment_index1[r1_best_id]; for(r2_best_id = 0; r2_best_id < r2_candidate_locations; r2_best_id ++) { int r2_matched = final_MATCH_buffer2[r2_best_id]; if(r2_matched < 1) continue; realignment_result_t * realignment_result_R2 = final_realignments + final_realignment_index2[r2_best_id]; int is_PE = 0, tlen=0; int is_same_chro = 0, is_exonic_regions = 0; unsigned long long final_SCORE = 0; test_PE_and_same_chro_align(global_context , realignment_result_R1 , realignment_result_R2, &is_exonic_regions, &is_PE, &is_same_chro , read_len_1, read_len_2, read_name_1, &tlen); unsigned long long TLEN_exp_score = 0; if(is_PE && global_context -> config.no_TLEN_preference == 0 && global_context -> config.reported_multi_best_reads<2){ TLEN_exp_score = (tlen > expected_TLEN)?(tlen - expected_TLEN):(expected_TLEN - tlen); if(TLEN_exp_score>999) TLEN_exp_score=0; else TLEN_exp_score = 999-TLEN_exp_score; } if(global_context -> config.experiment_type == CORE_EXPERIMENT_DNASEQ){ int weight; //#warning " ============ USE THE FIRST THREE WEIGHTS! ======== " if(is_PE) weight = 120; //weight = 300; else if(is_same_chro) weight = 100; else weight = 80; //weight = 30; final_SCORE = weight * (final_MATCH_buffer1[r1_best_id] + final_MATCH_buffer2[r2_best_id]); //#warning "=========== ADD BY YANG LIAO FOR MORE MAPPED READS WITH '-u' OPTION ================" final_SCORE = final_SCORE * 1000llu - final_MISMATCH_buffer1[r1_best_id] - final_MISMATCH_buffer2[r2_best_id]; final_SCORE = final_SCORE * 20llu - final_PENALTY_buffer1[r1_best_id] - final_PENALTY_buffer2[r2_best_id]; final_SCORE = final_SCORE * 1000llu + TLEN_exp_score; if(is_PE && need_expect_TLEN) final_TLEN_buffer[r1_best_id * global_context -> config.multi_best_reads * MAX_ALIGNMENT_PER_ANCHOR + r2_best_id] = tlen; //SUBREADprintf("%s FINAL_SCORE=%llu TLEN_SCORE=%llu DIST=%d EXP_TLEN=%d\n", read_name_1, final_SCORE, TLEN_exp_score, tlen, expected_TLEN); } else if (global_context -> config.experiment_type == CORE_EXPERIMENT_RNASEQ) { int weight; if(1){ if(is_exonic_regions && is_PE) weight = 5000; else if(is_PE || is_exonic_regions) weight = 3000; else if(is_same_chro) weight = 1000; else weight = 300; }else{ if(is_PE) weight = 3000; else if(is_same_chro) weight = 1000; else weight = 300; } //#warning "=========== ADD BY YANG LIAO ' + 2' ====================" final_SCORE = weight / (final_MISMATCH_buffer1[r1_best_id] + final_MISMATCH_buffer2[r2_best_id] + 1 + 2); //#warning "=========== ADD BY YANG LIAO FOR MORE MAPPED READS WITH '-u' OPTION ================" final_SCORE = final_SCORE * 3000llu + (final_MATCH_buffer1[r1_best_id] + final_MATCH_buffer2[r2_best_id]); final_SCORE = final_SCORE * 20 + realignment_result_R2 -> known_junction_supp + realignment_result_R1 -> known_junction_supp; final_SCORE = final_SCORE * 20 - final_PENALTY_buffer1[r1_best_id] - final_PENALTY_buffer2[r2_best_id]; final_SCORE = final_SCORE * 1000 + TLEN_exp_score; //#warning ">>>>>>>>>>>>>>>>>>>>>>>>>>>>> REMOVE THIS <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<" //printf("OCT27-STEPMSM-FNSC %s M=%d,%d MM=%d,%d CHROSAME=%d FSCR=%llu\n", read_name_1, final_MATCH_buffer1[r1_best_id], final_MATCH_buffer2[r2_best_id], final_MISMATCH_buffer1[r1_best_id] , final_MISMATCH_buffer2[r2_best_id], is_same_chro, final_SCORE ); if(is_PE && need_expect_TLEN) final_TLEN_buffer[r1_best_id * global_context -> config.multi_best_reads * MAX_ALIGNMENT_PER_ANCHOR + r2_best_id] = tlen; } else assert(0); assert(final_SCORE > 0); final_SCORE_buffer[r1_best_id * global_context -> config.multi_best_reads * MAX_ALIGNMENT_PER_ANCHOR + r2_best_id] = final_SCORE; if(final_SCORE > highest_score) { //#warning ">>>>>>>>>>>> COMMENT THIS <<<<<<<<<<<<<<<<<<<" //printf("OCT27-STEPMSM-REPL %s : SCORE %llu -> %llu, pos=%u,%u\n", read_name_1, final_SCORE, highest_score , realignment_result_R1->mapping_result->selected_position, realignment_result_R2->mapping_result->selected_position ); highest_score_occurence = 1; highest_score = final_SCORE; //SUBREADprintf("29NOV2016-FIRSTBEST %s [%d : score %llu] LOC = %d,%d\n", read_name_1, highest_score_occurence, final_SCORE, r1_best_id, r2_best_id); clear_repeated_buffer(global_context, repeated_buffer_pos, repeated_buffer_cigars, &repeated_count); add_repeated_buffer(global_context, repeated_buffer_pos, repeated_buffer_cigars, &repeated_count, realignment_result_R1, realignment_result_R2); } else if(final_SCORE == highest_score) { int is_repeat = 0; if(global_context -> config.reported_multi_best_reads) is_repeat = add_repeated_buffer(global_context, repeated_buffer_pos, repeated_buffer_cigars, &repeated_count, realignment_result_R1, realignment_result_R2); if(is_repeat) final_SCORE_buffer[r1_best_id * global_context -> config.multi_best_reads * MAX_ALIGNMENT_PER_ANCHOR + r2_best_id] = 0; else{ highest_score_occurence ++; // SUBREADprintf("29NOV2016-BEST %s [%d : score %llu] LOC = %d,%d\n", read_name_1, highest_score_occurence, final_SCORE, r1_best_id, r2_best_id); } } } } //SUBREADprintf("Highest score = %llu, Occurance = %d\n", highest_score , highest_score_occurence); // Then, copy the (R1, R2) that have the highest score into the align_res buffer. //#warning ">>>>>>> COMMENT THIS <<<<<<<" //printf("OCT27-WRITE-BOTHMAOOED-%s-THRE %d ; occu=%d\n", read_name_1, thread_context -> thread_id, highest_score_occurence); if(highest_score_occurence <= 1 || global_context -> config.report_multi_mapping_reads){ int is_break_even = 0; if(highest_score_occurence>1)is_break_even = 1; highest_score_occurence = min(highest_score_occurence, global_context -> config.reported_multi_best_reads); for(r1_best_id = 0; r1_best_id < r1_candidate_locations; r1_best_id ++) { int r1_matched = final_MATCH_buffer1[r1_best_id]; if(r1_matched < 1) continue; for(r2_best_id = 0; r2_best_id < r2_candidate_locations; r2_best_id ++) { int r2_matched = final_MATCH_buffer2[r2_best_id]; if(r2_matched < 1) continue; if(final_SCORE_buffer[r1_best_id * global_context -> config.multi_best_reads * MAX_ALIGNMENT_PER_ANCHOR + r2_best_id] == highest_score && output_cursor < global_context -> config.reported_multi_best_reads){ if(need_expect_TLEN){ int this_tlen = final_TLEN_buffer[r1_best_id * global_context -> config.multi_best_reads * MAX_ALIGNMENT_PER_ANCHOR + r2_best_id]; if(this_tlen>0){ subread_lock_occupy(&global_context -> output_lock); global_context -> expected_TLEN_read_numbers++; global_context -> expected_TLEN_sum += this_tlen; if(global_context -> expected_TLEN_read_numbers == READPAIRS_FOR_CALC_EXPT_TLEN) print_in_box(80,0,0," Estimated fragment length : %d bp\n", (int)(global_context -> expected_TLEN_sum / global_context -> expected_TLEN_read_numbers )); subread_lock_release(&global_context -> output_lock); } } realignment_result_t * r1_realign = final_realignments + final_realignment_index1[r1_best_id]; realignment_result_t * r2_realign = final_realignments + final_realignment_index2[r2_best_id]; strcpy(read_text_1, raw_read_text_1); strcpy(read_text_2, raw_read_text_2); strcpy(qual_text_1, raw_qual_text_1); strcpy(qual_text_2, raw_qual_text_2); if(is_break_even){ r1_realign -> realign_flags |= CORE_IS_BREAKEVEN; r2_realign -> realign_flags |= CORE_IS_BREAKEVEN; } r1_realign -> MAPQ_adjustment = r1_step2_locations + final_MISMATCH_buffer1[r1_best_id]; r2_realign -> MAPQ_adjustment = r2_step2_locations + final_MISMATCH_buffer2[r2_best_id]; // SUBREADprintf("THREAD_OPT2_write %s\n", read_name_1); write_realignments_for_fragment(global_context, thread_context, &out_context, current_read_number, r1_realign, r2_realign, read_name_1, read_name_2, read_text_1, read_text_2, qual_text_1, qual_text_2, read_len_1, read_len_2, highest_score_occurence, output_cursor, non_informative_subreads_r1, non_informative_subreads_r2); output_cursor ++; } } } assert(output_cursor == highest_score_occurence ); } } //if( current_read_number < 10 ) SUBREADprintf("BEEE2 : %s : %d\n", read_name_1, max_votes); //#warning ">>>>>>> COMMENT THIS <<<<<<<" //printf("OCT27-WRITE-UNMAP?-%s-THRE %d\n", read_name_1, thread_context -> thread_id); if(output_cursor<1) { strcpy(read_text_1, raw_read_text_1); strcpy(read_text_2, raw_read_text_2); strcpy(qual_text_1, raw_qual_text_1); strcpy(qual_text_2, raw_qual_text_2); // SUBREADprintf("THREAD_OPT3_write %s\n", read_name_1); write_realignments_for_fragment(global_context, thread_context, &out_context, current_read_number, NULL, NULL, read_name_1, read_name_2, read_text_1, read_text_2, raw_qual_text_1, raw_qual_text_2, read_len_1, read_len_2, 0, 0, non_informative_subreads_r1, non_informative_subreads_r2); } if(!thread_context || thread_context->thread_id == 0) { if(sqr_read_number>sqr_interval) { show_progress(global_context, thread_context, current_read_number, STEP_ITERATION_TWO); sqr_read_number=0; } } //#warning ">>>>>>> COMMENT THIS <<<<<<<" //printf("OCT27-FIN-%s-THRE %d\n", read_name_1, thread_context -> thread_id); //bigtable_release_result(global_context, thread_context, current_read_number, 1); } free(final_realignments); free(final_realignment_number); free(final_MATCH_buffer1); free(final_MISMATCH_buffer1); free(final_PENALTY_buffer1); free(final_realignment_index1); free(final_MATCH_buffer2); free(final_MISMATCH_buffer2); free(final_PENALTY_buffer2); free(final_realignment_index2); free(final_SCORE_buffer); free(final_TLEN_buffer); free(r1_align_result_buffer); free(r1_subjunc_result_buffer); free(r2_align_result_buffer); free(r2_subjunc_result_buffer); for(repeated_count = 0;repeated_count < MAX_ALIGNMENT_PER_ANCHOR * 2 * global_context -> config.reported_multi_best_reads ; repeated_count ++ ){ free(repeated_buffer_cigars[repeated_count]); } destroy_output_context(global_context, &out_context); //SUBREADprintf("OPT2-FINISHED\n"); if(global_context ->config.all_threads >1 && global_context ->config.is_BAM_output && !global_context ->config.is_input_read_order_required)SamBam_writer_finalise_thread(global_context -> output_bam_writer, thread_context -> thread_id); if(global_context ->config.all_threads <=1 && global_context ->config.is_BAM_output && global_context ->config.sort_reads_by_coordinates)SamBam_writer_finalise_one_thread(global_context -> output_bam_writer); return 0; } int core_get_subread_quality(global_context_t * global_context, thread_context_t * thread_context, char * qual, int qual_len) { int x1, ret=1; if(!qual)return 1; if(!qual[0])return 1; int offset = (global_context->config.phred_score_format == FASTQ_PHRED33)?33:64; for(x1=0; (x1 < qual_len) && qual[x1]; x1++) ret += (qual[x1] - offset); return ret; } int has_better_mapping(global_context_t * global_context, thread_context_t * thread_context, subread_read_number_t current_read_number, int is_second_read, int this_aln_id){ int better_read_id; mapping_result_t * this_r = _global_retrieve_alignment_ptr(global_context, current_read_number, is_second_read, this_aln_id); for(better_read_id = 0; better_read_id < this_aln_id; better_read_id++){ mapping_result_t * better_r = _global_retrieve_alignment_ptr(global_context, current_read_number, is_second_read, better_read_id); if( this_r -> selected_position >= better_r -> selected_position - global_context -> config.max_indel_length - 1 && this_r -> selected_position <= better_r -> selected_position + global_context -> config.max_indel_length +1 ) if( this_r -> confident_coverage_start >= better_r -> confident_coverage_start && this_r -> confident_coverage_end <= better_r -> confident_coverage_end) return 1; } return 0; } int do_voting(global_context_t * global_context, thread_context_t * thread_context) { int xk1; gene_input_t * ginp1 = NULL , * ginp2 = NULL; subread_read_number_t current_read_number=0, last_shown_curr = 0; char * read_text_1, * read_text_2; char * qual_text_1, * qual_text_2; char read_name_1[MAX_READ_NAME_LEN+1], read_name_2[MAX_READ_NAME_LEN+1]; int read_len_1, read_len_2=0; int min_first_read_votes = global_context -> config.minimum_subread_for_first_read; int voting_max_indel_length = min(16, global_context->config.max_indel_length); int sqr_interval=10000, sqr_read_number = 0; read_text_1 = malloc(MAX_READ_LENGTH+1); read_text_2 = malloc(MAX_READ_LENGTH+1); qual_text_1 = malloc(MAX_READ_LENGTH+1); qual_text_2 = malloc(MAX_READ_LENGTH+1); gene_vote_t * vote_1, * vote_2, * vote_fg; vote_1 = (gene_vote_t *) malloc(sizeof(gene_vote_t)); vote_2 = (gene_vote_t *) malloc(sizeof(gene_vote_t)); vote_fg = (gene_vote_t *) malloc(sizeof(gene_vote_t)); if(!(vote_1&&vote_2)) { sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_FATAL,"Cannot allocate voting memory."); return 1; } init_chunk_scanning_parameters(global_context,thread_context, & ginp1, & ginp2); unsigned int low_index_border = global_context -> current_value_index -> start_base_offset; unsigned int high_index_border = global_context -> current_value_index -> start_base_offset + global_context -> current_value_index -> length; int has_second_read = 1 + global_context -> input_reads.is_paired_end_reads; if(thread_context) thread_context -> current_value_index = global_context -> current_value_index; int GENE_SLIDING_STEP = global_context -> current_index -> index_gap; int need_junction_step = global_context -> config.do_breakpoint_detection || global_context -> config.do_fusion_detection ||global_context -> config.do_long_del_detection; while(1) { int is_second_read; int subread_no; int is_reversed, applied_subreads = 0, v1_all_subreads=0, v2_all_subreads=0; fetch_next_read_pair(global_context, thread_context, ginp1, ginp2, &read_len_1, &read_len_2, read_name_1, read_name_2, read_text_1, read_text_2, qual_text_1, qual_text_2,1, ¤t_read_number); //SUBREADprintf("VOTING READ # %lld: %s %s %d\n", current_read_number, read_name_1, read_text_1, read_len_1); if(current_read_number < 0){ // #warning ">>>>>>>>>>>>> COMMENT THIS <<<<<<<<<<<<<<<<<<<" // printf("OCT27-STEPB-QUIT :T%d\n", thread_context -> thread_id); break; } for(is_reversed = 0; is_reversed<2; is_reversed++) { //printf("MAP_REA = %s / %s\n", read_text_1, read_text_2); for (is_second_read = 0; is_second_read < has_second_read; is_second_read ++) { gene_vote_t * current_vote = is_second_read?vote_2: vote_1; char * current_read = is_second_read?read_text_2 : read_text_1; int current_rlen = is_second_read?read_len_2:read_len_1; int subread_step; if(current_rlen< 16) continue; int CR15GLS = (current_rlen - 15 - GENE_SLIDING_STEP)<<16; if(current_rlen<= EXON_LONG_READ_LENGTH) { subread_step = CR15GLS /(global_context -> config.total_subreads -1); if(subread_step<(GENE_SLIDING_STEP<<16))subread_step = GENE_SLIDING_STEP<<16; }else{ subread_step = 6<<16; if( CR15GLS / subread_step > 62) subread_step = CR15GLS/62; } int noninformative_subreads_for_each_gap[GENE_SLIDING_STEP]; applied_subreads = 1 + CR15GLS / subread_step; if(is_second_read) v2_all_subreads = applied_subreads; else v1_all_subreads = applied_subreads; //SUBREADprintf("NSUBR=%d\tAPPLIED_SUBR=%d\tSTEP=%d\n", global_context -> config.total_subreads, applied_subreads, subread_step); unsigned int current_high_border = high_index_border - current_rlen; if(global_context->config.do_breakpoint_detection && current_rlen > EXON_LONG_READ_LENGTH)//&& global_context->config.all_threads<2) { char * current_qual = is_second_read?qual_text_2 : qual_text_1; core_fragile_junction_voting(global_context, thread_context, read_name_1, current_read, current_qual, current_rlen, is_reversed, global_context->config.space_type, low_index_border, current_high_border, vote_fg); } if(global_context->config.SAM_extra_columns) { for(xk1=0;xk1 config.do_fusion_detection || global_context-> config.do_long_del_detection)){ if(is_second_read == 0)init_gene_vote(vote_1); if(is_second_read == 1)init_gene_vote(vote_2); } for(subread_no=0; subread_no < applied_subreads ; subread_no++) { for(xk1=0; xk1config.SAM_extra_columns) { current_vote -> noninformative_subreads = noninformative_subreads_for_each_gap[xk1]; } int subread_offset = ((subread_step * subread_no) >> 16); if(GENE_SLIDING_STEP > 1) subread_offset -= subread_offset%(GENE_SLIDING_STEP) - xk1; char * subread_string = current_read + subread_offset; gehash_key_t subread_integer = genekey2int(subread_string, global_context->config.space_type); if(global_context->config.is_methylation_reads) gehash_go_q_CtoT(global_context->current_index, subread_integer , subread_offset, current_rlen, is_reversed, current_vote, 1, 0xffffff, voting_max_indel_length, subread_no, 1, low_index_border, high_index_border - current_rlen); else gehash_go_X(global_context->current_index, subread_integer , subread_offset, current_rlen, is_reversed, current_vote, voting_max_indel_length, subread_no, low_index_border, current_high_border, allow_indel_i, shift_indel_locs, &shift_indel_NO); if(global_context->config.SAM_extra_columns) noninformative_subreads_for_each_gap[xk1] = current_vote -> noninformative_subreads; } } if(shift_indel_NO == 0 || global_context-> config.do_fusion_detection || global_context-> config.do_long_del_detection)break; } if(global_context->config.SAM_extra_columns) { short max_noninformative_subreads = -1; for(xk1=0;xk1 max_noninformative_subreads) max_noninformative_subreads = noninformative_subreads_for_each_gap[xk1]; current_vote -> noninformative_subreads = max_noninformative_subreads; } } if(is_reversed==1 || !(global_context-> config.do_fusion_detection || global_context-> config.do_long_del_detection)) { //#warning "====== CHECK PRINTING !!! ==========" if(0 && FIXLENstrcmp("R00000000181", read_name_1) ==0){ SUBREADprintf(">>>%llu<<<\n%s [%d] %s\n%s [%d] %s VOTE1_MAX=%d >= %d\n", current_read_number, read_name_1, read_len_1, read_text_1, read_name_2, read_len_2, read_text_2, vote_1->max_vote, min_first_read_votes); SUBREADprintf(" ======= PAIR %s = %llu ; NON_INFORMATIVE = %d, %d =======\n", read_name_1, current_read_number, vote_1 -> noninformative_subreads, vote_2 -> noninformative_subreads); print_votes(vote_1, global_context -> config.index_prefix); print_votes(vote_2, global_context -> config.index_prefix); } if(global_context -> input_reads.is_paired_end_reads) process_voting_junction(global_context, thread_context, current_read_number, vote_1, vote_2, read_name_1, read_name_2, read_text_1, read_text_2, read_len_1, read_len_2, is_reversed, v1_all_subreads, v2_all_subreads); else{ if(vote_1->max_vote >= min_first_read_votes) process_voting_junction(global_context, thread_context, current_read_number, vote_1, vote_2, read_name_1, NULL , read_text_1, NULL, read_len_1, 0, is_reversed, v1_all_subreads, 0); else if(_global_retrieve_alignment_ptr(global_context, current_read_number, 0,0) -> selected_votes < 1) { mapping_result_t * allll = _global_retrieve_alignment_ptr(global_context, current_read_number, 0,0); allll -> noninformative_subreads_in_vote = 0; _global_retrieve_alignment_ptr(global_context, current_read_number, 0,0)->used_subreads_in_vote = max(_global_retrieve_alignment_ptr(global_context, current_read_number, 0,0)->used_subreads_in_vote, applied_subreads); _global_retrieve_alignment_ptr(global_context, current_read_number, 0,0)->noninformative_subreads_in_vote = max(_global_retrieve_alignment_ptr(global_context, current_read_number, 0,0)->noninformative_subreads_in_vote, vote_1 -> noninformative_subreads); } } } if(is_reversed == 0) { reverse_read(read_text_1, read_len_1, global_context->config.space_type); if(global_context -> input_reads.is_paired_end_reads) reverse_read(read_text_2, read_len_2, global_context->config.space_type); } } int read_1_reversed = 1; int read_2_reversed = 1; int best_read_id; if(global_context -> is_final_voting_run){ for(is_second_read = 0; is_second_read < 1 + global_context -> input_reads.is_paired_end_reads; is_second_read ++) { int * this_read_has_reversed = is_second_read ? &read_2_reversed:&read_1_reversed; char * current_read_name = is_second_read?read_name_2 : read_name_1; char * current_read = is_second_read?read_text_2 : read_text_1; char * current_qual = is_second_read?qual_text_2 : qual_text_1; int current_rlen = is_second_read?read_len_2:read_len_1; for(best_read_id = 0; best_read_id < global_context -> config.multi_best_reads; best_read_id++) { mapping_result_t * current_r = _global_retrieve_alignment_ptr(global_context, current_read_number, is_second_read,best_read_id); if(current_r -> selected_votes < 1) continue; int this_read_should_be_reversed = (current_r -> result_flags & CORE_IS_NEGATIVE_STRAND) ? 1:0; //SUBREADprintf("DETECT INDEL: should_reverse = %d, this_has_reversed = %d\n", this_read_should_be_reversed, *this_read_has_reversed); if(this_read_should_be_reversed != (*this_read_has_reversed)) { (*this_read_has_reversed) = !(*this_read_has_reversed); reverse_read(current_read, current_rlen, global_context->config.space_type); if(current_qual) reverse_quality(current_qual , current_rlen); } gene_value_index_t * curr_val_index = thread_context? thread_context -> current_value_index: global_context -> current_value_index; locate_current_value_index(global_context, thread_context, current_r, current_rlen); // #warning "==== UNCOMMENT THE NEXT LINE ====" if(0 && FIXLENstrcmp("R00000003493", current_read_name)==0) SUBREADprintf("TEST_BETTER: better_id = %d, votes = %d, has=%d\n", best_read_id, current_r -> selected_votes, has_better_mapping(global_context, thread_context, current_read_number, is_second_read,best_read_id)); if(!has_better_mapping(global_context, thread_context, current_read_number, is_second_read,best_read_id)) find_new_indels(global_context, thread_context, current_read_number, current_read_name, current_read, current_qual, current_rlen, is_second_read, best_read_id); if(need_junction_step) find_new_junctions(global_context, thread_context, current_read_number, current_read_name, current_read, current_qual, current_rlen, is_second_read, best_read_id); // #warning "==== UNCOMMENT THE NEXT LINE ====" if(0 && FIXLENstrcmp("R00000003493", current_read_name)==0){ SUBREADprintf("TEST_BETTER_END: better_id = %d, votes = %d, has=%d\n", best_read_id, current_r -> selected_votes, has_better_mapping(global_context, thread_context, current_read_number, is_second_read,best_read_id)); exit(1); } if(thread_context) thread_context -> current_value_index = curr_val_index; else global_context -> current_value_index = curr_val_index; } } } if(!thread_context || thread_context->thread_id == 0) { if(0 == global_context -> config.scRNA_input_mode && sqr_read_number > sqr_interval) { show_progress(global_context, thread_context, current_read_number, STEP_VOTING); sqr_read_number = 0; unsigned long long total_file_size = global_context -> input_reads.first_read_file_size; unsigned long long guessed_all_reads = total_file_size / global_context -> input_reads . avg_read_length;// / (1+global_context -> config.is_SAM_file_input); sqr_interval = max(10000,guessed_all_reads / global_context -> config.all_threads/10); } if(global_context -> config.scRNA_input_mode && current_read_number - last_shown_curr >= 1000000){ show_progress(global_context, thread_context, current_read_number + global_context -> all_processed_reads, STEP_VOTING); last_shown_curr = current_read_number; } } //bigtable_release_result(global_context, thread_context, current_read_number, 1); sqr_read_number++; } free(vote_1); free(vote_2); free(vote_fg); free(read_text_1); free(read_text_2); free(qual_text_1); free(qual_text_2); return 0; } void subread_init_topKbuff(global_context_t * global_context, topK_buffer_t * topKbuff){ topKbuff -> vote_simple_1_buffer = malloc(global_context -> config.max_vote_simples * sizeof(simple_mapping_t)); topKbuff -> vote_simple_2_buffer = malloc(global_context -> config.max_vote_simples * sizeof(simple_mapping_t)); topKbuff -> junction_tmp_r1 = malloc(sizeof(subjunc_result_t) * global_context->config.multi_best_reads); topKbuff -> junction_tmp_r2 = malloc(sizeof(subjunc_result_t) * global_context->config.multi_best_reads); topKbuff -> alignment_tmp_r1 = malloc(sizeof(mapping_result_t) * global_context->config.multi_best_reads); topKbuff -> alignment_tmp_r2 = malloc(sizeof(mapping_result_t) * global_context->config.multi_best_reads); topKbuff -> comb_buffer = malloc(global_context -> config.max_vote_combinations * sizeof(vote_combination_t)); } void subread_free_topKbuff(global_context_t * global_context, topK_buffer_t * topKbuff){ free(topKbuff ->junction_tmp_r1); free(topKbuff ->junction_tmp_r2); free(topKbuff ->alignment_tmp_r1); free(topKbuff ->alignment_tmp_r2); free(topKbuff ->comb_buffer); free(topKbuff ->vote_simple_1_buffer); free(topKbuff ->vote_simple_2_buffer); } void * run_in_thread(void * pthread_param) { void ** parameters = (void **)pthread_param; global_context_t * global_context = (global_context_t * ) parameters[0]; thread_context_t * thread_context = (thread_context_t *) parameters[1]; int task = *((int *)(parameters[2])); subread_lock_t * thread_init_lock = (subread_lock_t * ) parameters[3]; int * ret_value_pointer = (int *)parameters[4]; if(thread_init_lock) subread_lock_release(thread_init_lock); switch(task) { case STEP_VOTING: *ret_value_pointer = do_voting(global_context, thread_context); break; case STEP_ITERATION_TWO: *ret_value_pointer = do_iteration_two(global_context, thread_context); break; } //sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_DETAILS, "finished running %d", task); return NULL; } int run_maybe_threads(global_context_t *global_context, int task) { void * thr_parameters [5]; int ret_value =0; if(task == STEP_ITERATION_TWO){ global_context -> last_written_fragment_number = -1; } if(global_context->config.all_threads<2) { thr_parameters[0] = global_context; thr_parameters[1] = NULL; thr_parameters[2] = &task; thr_parameters[3] = NULL; thr_parameters[4] = &ret_value; run_in_thread(thr_parameters); if(STEP_VOTING == task){ // sort and merge events from all threads and the global event space. sort_global_event_table(global_context); // sort the event entry table at each location. //#warning "==== UNCOMMENT NEXT LINE ======" sort_junction_entry_table(global_context); } } else { int current_thread_no ; thread_context_t thread_contexts[64]; int ret_values[64]; memset(thread_contexts, 0, sizeof(thread_context_t)*64); global_context -> all_thread_contexts = thread_contexts; for(current_thread_no = 0 ; current_thread_no < global_context->config.all_threads ; current_thread_no ++) { thread_contexts[current_thread_no].thread_id = current_thread_no; init_indel_thread_contexts(global_context, thread_contexts+current_thread_no, task); if((global_context->config.do_breakpoint_detection || global_context-> config.do_fusion_detection || global_context->config.do_breakpoint_detection || global_context-> config.do_long_del_detection)) init_junction_thread_contexts(global_context, thread_contexts+current_thread_no, task); if(STEP_VOTING == task) subread_init_topKbuff(global_context,&thread_contexts[current_thread_no].topKbuff); subread_lock_occupy(&global_context -> thread_initial_lock); thr_parameters[0] = global_context; thr_parameters[1] = thread_contexts+current_thread_no; thr_parameters[2] = &task; thr_parameters[3] = (void *)&global_context -> thread_initial_lock; thr_parameters[4] = ret_values + current_thread_no; pthread_create(&thread_contexts[current_thread_no].thread, NULL, run_in_thread, &thr_parameters); } for(current_thread_no = 0 ; current_thread_no < global_context->config.all_threads ; current_thread_no ++) { pthread_join(thread_contexts[current_thread_no].thread, NULL); if(STEP_ITERATION_TWO == task){ global_context -> all_mapped_reads += thread_contexts[current_thread_no].all_mapped_reads; global_context -> all_correct_PE_reads += thread_contexts[current_thread_no].all_correct_PE_reads; global_context -> not_properly_pairs_wrong_arrangement += thread_contexts[current_thread_no].not_properly_pairs_wrong_arrangement; global_context -> not_properly_pairs_different_chro += thread_contexts[current_thread_no].not_properly_pairs_different_chro; global_context -> not_properly_different_strands += thread_contexts[current_thread_no].not_properly_different_strands; global_context -> not_properly_pairs_TLEN_wrong += thread_contexts[current_thread_no].not_properly_pairs_TLEN_wrong; global_context -> all_unmapped_reads += thread_contexts[current_thread_no].all_unmapped_reads; global_context -> not_properly_pairs_only_one_end_mapped += thread_contexts[current_thread_no].not_properly_pairs_only_one_end_mapped; global_context -> all_multimapping_reads += thread_contexts[current_thread_no].all_multimapping_reads; global_context -> all_uniquely_mapped_reads += thread_contexts[current_thread_no].all_uniquely_mapped_reads; } if(STEP_VOTING == task) subread_free_topKbuff(global_context,&thread_contexts[current_thread_no].topKbuff); ret_value += *(ret_values + current_thread_no); if(ret_value)break; } // sort and merge events from all threads and the global event space. finalise_indel_and_junction_thread(global_context, thread_contexts, task); if(STEP_VOTING == task) // sort the event entry table at each location. sort_junction_entry_table(global_context); } if(CORE_SOFT_BR_CHAR == '\r') sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_INFO, ""); return ret_value; } void clean_context_after_chunk(global_context_t * context) { context -> running_processed_reads_in_chunk = 0; context -> processed_reads_in_chunk = 0; init_bigtable_results(context, 1); indel_context_t * indel_context = (indel_context_t *)context -> module_contexts[MODULE_INDEL_ID]; chromosome_event_t * event_space = indel_context -> event_space_dynamic; int event_id; //memset(context -> big_margin_record , 0 , sizeof(*context -> big_margin_record) * context ->config.reads_per_chunk * (context->input_reads.is_paired_end_reads?2:1) * context->config.big_margin_record_size); for(event_id = 0; event_id < indel_context->total_events; event_id++){ chromosome_event_t * event_body = event_space + event_id; event_body -> critical_read_id = 0xffffffffffffffffllu; } } #define SKIP_CORE_NOEMPTY(fp_loc, buf_loc) { while(1){char *ret_loc = fgets(buf_loc, 3000, fp_loc); if(buf_loc[0]!='\n' || !ret_loc) break; } } void locate_read_files(global_context_t * global_context, int type) { // The BCL input module uses its own chunking algorithm. if(global_context -> input_reads.first_read_file.file_type == GENE_INPUT_BCL) return; if(type==SEEK_SET) { geinput_tell(&global_context -> input_reads.first_read_file, &global_context -> current_circle_start_position_file1); if(global_context ->input_reads.is_paired_end_reads) geinput_tell(&global_context -> input_reads.second_read_file, &global_context -> current_circle_start_position_file2); } else { geinput_tell(&global_context -> input_reads.first_read_file, &global_context -> current_circle_end_position_file1); if(global_context ->input_reads.is_paired_end_reads) geinput_tell(&global_context -> input_reads.second_read_file, &global_context -> current_circle_end_position_file2); } if(global_context -> input_reads.first_read_file.file_type == GENE_INPUT_SCRNA_FASTQ) return; if(global_context -> input_reads.first_read_file.file_type == GENE_INPUT_SCRNA_BAM) return; // This variable is only used to display the running progress. // That is why the scRNA-seq mode doesn't need to keep the start pos. if(type==SEEK_SET) global_context -> current_circle_start_abs_offset_file1 = geinput_file_offset(&(global_context -> input_reads.first_read_file)); } void rewind_read_files(global_context_t * global_context, int type) { if(type==SEEK_SET) { geinput_seek(&global_context -> input_reads.first_read_file, & global_context -> current_circle_start_position_file1); if(global_context ->input_reads.is_paired_end_reads) geinput_seek(&global_context -> input_reads.second_read_file, & global_context -> current_circle_start_position_file2); } else { geinput_seek(&global_context -> input_reads.first_read_file, & global_context -> current_circle_end_position_file1); if(global_context ->input_reads.is_paired_end_reads) geinput_seek(&global_context -> input_reads.second_read_file, & global_context -> current_circle_end_position_file2); } } void go_chunk_start( global_context_t * global_context ){ if(global_context -> input_reads.first_read_file.file_type == GENE_INPUT_BCL) cacheBCL_go_chunk_start(&global_context -> input_reads.first_read_file.bcl_input); else rewind_read_files(global_context, SEEK_SET); global_context -> running_processed_reads_in_chunk=0; } void go_chunk_nextchunk( global_context_t * global_context ){ if(global_context -> input_reads.first_read_file.file_type == GENE_INPUT_BCL) cacheBCL_go_chunk_end(&global_context -> input_reads.first_read_file.bcl_input); else rewind_read_files(global_context, SEEK_END); global_context -> running_processed_reads_in_chunk=0; } int read_chunk_circles(global_context_t *global_context) { int block_no; // printf("GINP1 AT %llu\n", ftello(global_context -> input_reads.first_read_file.input_fp)); unsigned int chunk_no = 0; global_context -> current_index = (gehash_t*) malloc(sizeof(gehash_t)); global_context -> current_value_index = global_context -> all_value_indexes; global_context -> running_processed_reads_in_chunk=0; global_context -> processed_reads_in_chunk=0; double time_load_index = miltime(); for(block_no = 0; block_no< global_context->index_block_number; block_no++) { char tmp_fname[MAX_FILE_NAME_LENGTH+ 30]; sprintf(tmp_fname, "%s.%02d.%c.array", global_context->config.index_prefix, block_no, global_context->config.space_type == GENE_SPACE_COLOR?'c':'b'); if(gvindex_load(&global_context -> all_value_indexes[block_no], tmp_fname)) return -1; } double period_load_index = miltime() - time_load_index; global_context -> timecost_load_index += period_load_index; while(1) { int ret; locate_read_files(global_context, SEEK_SET); for(global_context->current_index_block_number = 0; global_context->current_index_block_number < global_context->index_block_number; global_context->current_index_block_number++) { char tmp_fname[MAX_FILE_NAME_LENGTH+30]; time_load_index = miltime(); if(global_context->index_block_number > 1 || chunk_no == 0) { sprintf(tmp_fname, "%s.%02d.%c.tab", global_context->config.index_prefix, global_context->current_index_block_number, global_context->config.space_type == GENE_SPACE_COLOR?'c':'b'); print_in_box(80,0,0, "Load the %d-th index block...",1+ global_context->current_index_block_number); if(gehash_load(global_context -> current_index, tmp_fname)) return -1; print_in_box(80,0,0, "The index block has been loaded."); sprintf(tmp_fname, "%s.%02d.%c.array", global_context->config.index_prefix, global_context->current_index_block_number, global_context->config.space_type == GENE_SPACE_COLOR?'c':'b'); } period_load_index = miltime() - time_load_index; global_context -> timecost_load_index += period_load_index; global_context -> current_value_index = global_context -> all_value_indexes + global_context->current_index_block_number; if(global_context->current_index_block_number ==0 && global_context -> all_processed_reads==0) global_context->align_start_time = miltime(); if(global_context->index_block_number == global_context->current_index_block_number + 1) global_context -> is_final_voting_run = 1; else global_context -> is_final_voting_run = 0; print_in_box(80,0,0, "Start read mapping in chunk."); double time_start_voting = miltime(); ret = run_maybe_threads(global_context, STEP_VOTING); double period_voting = miltime() - time_start_voting; global_context -> timecost_voting += period_voting; if(0 == global_context->current_index_block_number){ locate_read_files(global_context, SEEK_END); global_context -> processed_reads_in_chunk = global_context -> running_processed_reads_in_chunk; } if(global_context->current_index_block_number < global_context->index_block_number -1) go_chunk_start(global_context); // rewind_read_files(global_context, SEEK_SET); int is_last_chunk = global_context -> processed_reads_in_chunk < global_context->config.reads_per_chunk; if(global_context->index_block_number > 1 || is_last_chunk) gehash_destory_fast(global_context -> current_index); if(ret) break; if(!global_context -> processed_reads_in_chunk) break; } // after the voting step, all subread index blocks are released and all base index blocks are loaded at once. double time_before_realign = miltime(); if(0 == chunk_no && global_context->config.exon_annotation_file[0] && global_context->config.do_breakpoint_detection){ ret = ret || load_known_junctions(global_context); if(!ret){ // sort and merge events from all threads and the global event space. sort_global_event_table(global_context); // sort the event entry table at each location. sort_junction_entry_table(global_context); } } ret = ret || anti_supporting_read_scan(global_context); remove_neighbour(global_context); go_chunk_start(global_context); // rewind_read_files(global_context, SEEK_SET); double period_before_realign = miltime() - time_before_realign; global_context -> timecost_before_realign += period_before_realign; double time_realign = miltime(); ret = ret || run_maybe_threads(global_context, STEP_ITERATION_TWO); double period_realign = miltime() - time_realign; global_context -> timecost_for_realign += period_realign; if(global_context -> config.is_third_iteration_running) { go_chunk_start(global_context); //rewind_read_files(global_context, SEEK_SET); ret = ret || do_iteration_three(global_context, NULL); } go_chunk_nextchunk(global_context); //rewind_read_files(global_context, SEEK_END); global_context -> all_processed_reads+= global_context ->processed_reads_in_chunk; if(ret) return ret; if(global_context -> processed_reads_in_chunk < global_context->config.reads_per_chunk || (global_context -> config.is_BAM_output && global_context -> output_bam_writer -> is_internal_error) || global_context -> input_reads.is_internal_error || (global_context -> output_sam_is_full)) // base value indexes loaded in the last circle are not destroyed and are used in writting the indel VCF. // the indexes will be destroyed in destroy_global_context break; if(0){ SUBREADprintf("WARNINGqqq: EARLY BREAK!\n"); SUBREADprintf("WARNINGqqq: EARLY BREAK!\n"); SUBREADprintf("WARNINGqqq: EARLY BREAK!\n"); SUBREADprintf("WARNINGqqq: EARLY BREAK!\n"); SUBREADprintf("WARNINGqqq: EARLY BREAK!\n"); SUBREADprintf("WARNINGqqq: EARLY BREAK!\n"); SUBREADprintf("WARNINGqqq: EARLY BREAK!\n"); SUBREADprintf("WARNINGqqq: EARLY BREAK!\n"); break; } clean_context_after_chunk(global_context); chunk_no++; } free(global_context -> current_index); if(global_context -> config.is_third_iteration_running) finalise_long_insertions(global_context); return 0; } void char_strftime(char * tbuf){ time_t rawtime; struct tm * timeinfo; time (&rawtime); timeinfo = localtime (&rawtime); strftime (tbuf,80,"%d-%b-%Y %H:%M:%S",timeinfo); } void print_subread_logo() { sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_INFO ," %c[44;37m ========== %c[0m%c[36m _____ _ _ ____ _____ ______ _____ ", CHAR_ESC, CHAR_ESC, CHAR_ESC); sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_INFO ," %c[44;37m ===== %c[0m%c[36m / ____| | | | _ \\| __ \\| ____| /\\ | __ \\ ", CHAR_ESC, CHAR_ESC, CHAR_ESC); sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_INFO ," %c[44;37m ===== %c[0m%c[36m | (___ | | | | |_) | |__) | |__ / \\ | | | |", CHAR_ESC, CHAR_ESC, CHAR_ESC); sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_INFO ," %c[44;37m ==== %c[0m%c[36m \\___ \\| | | | _ <| _ /| __| / /\\ \\ | | | |", CHAR_ESC, CHAR_ESC, CHAR_ESC); sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_INFO ," %c[44;37m ==== %c[0m%c[36m ____) | |__| | |_) | | \\ \\| |____ / ____ \\| |__| |", CHAR_ESC, CHAR_ESC, CHAR_ESC); sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_INFO ," %c[44;37m ========== %c[0m%c[36m |_____/ \\____/|____/|_| \\_\\______/_/ \\_\\_____/%c[0m", CHAR_ESC, CHAR_ESC, CHAR_ESC, CHAR_ESC); #ifdef MAKE_STANDALONE char * spaces = ""; if(strlen(SUBREAD_VERSION) == 8) spaces = ""; else if(strlen(SUBREAD_VERSION) == 5) spaces = " "; sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_INFO ," %sv%s",spaces,SUBREAD_VERSION); #else sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_INFO ," %s",SUBREAD_VERSION); #endif } int print_configuration(global_context_t * context) { setlocale(LC_NUMERIC, ""); sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_ERROR,""); print_subread_logo(); sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_ERROR,""); print_in_box(80, 1, 1, context->config.entry_program_name == CORE_PROGRAM_SUBJUNC?"setting":"setting"); print_in_box(80, 0, 1, ""); if(context->config.do_breakpoint_detection) { if(context-> config.do_fusion_detection) print_in_box(80, 0, 0, "Function : Read alignment + Junction/Fusion detection%s", context->config.experiment_type == CORE_EXPERIMENT_DNASEQ?" (DNA-Seq)":" (RNA-Seq)"); else if(context-> config.do_long_del_detection) print_in_box(80, 0, 0, "Function : Read alignment + Long Deletion detection%s", context->config.experiment_type == CORE_EXPERIMENT_DNASEQ?" (DNA-Seq)":" (RNA-Seq)"); else print_in_box(80, 0, 0, "Function : Read alignment + Junction detection (%s)", context->config.experiment_type == CORE_EXPERIMENT_DNASEQ?"DNA-Seq":"RNA-Seq"); } else print_in_box(80, 0, 0, "Function : Read alignment%s", context->config.experiment_type == CORE_EXPERIMENT_DNASEQ?" (DNA-Seq)":" (RNA-Seq)"); if( context->config.second_read_file[0]){ print_in_box(80, 0, 0, "Input file 1 : %s", get_short_fname(context->config.first_read_file)); print_in_box(80, 0, 0, "Input file 2 : %s", get_short_fname(context->config.second_read_file)); }else if(context->config.scRNA_input_mode == GENE_INPUT_SCRNA_FASTQ){ const char *strmm_tmp = context->config.first_read_file; int sample_no = 1; while((strmm_tmp = strstr(strmm_tmp, SCRNA_FASTA_SPLIT1))!=NULL) { sample_no++; strmm_tmp++; } print_in_box(80, 0, 0, "Input file : %d samples from scRNA-seq", sample_no); }else if(context->config.scRNA_input_mode == GENE_INPUT_BCL){ print_in_box(80, 0, 0, "Input file : %s%s", get_short_fname(context->config.first_read_file), " (scRNA)"); }else if(context->config.scRNA_input_mode == GENE_INPUT_SCRNA_BAM){ print_in_box(80, 0, 0, "Input file : %s%s", get_short_fname(context->config.first_read_file), " (10X BAM)"); }else{ print_in_box(80, 0, 0, "Input file : %s%s", get_short_fname(context->config.first_read_file), context->config.is_SAM_file_input?(context->config.is_BAM_input?" (BAM)":" (SAM)"):("")); } if(context->config.output_prefix [0]) print_in_box(80, 0, 0, "Output file : %s (%s)%s", get_short_fname(context->config.output_prefix), context->config.is_BAM_output?"BAM":"SAM", context->config.is_input_read_order_required?", Keep Order":(context->config.sort_reads_by_coordinates?", Sorted":"")); else print_in_box(80, 0, 0, "Output method : STDOUT (%s)" , context->config.is_BAM_output?"BAM":"SAM"); print_in_box(80, 0, 0, "Index name : %s", get_short_fname(context->config.index_prefix)); print_in_box(80, 0, 0, ""); print_in_box(80, 0, 1, "------------------------------------"); print_in_box(80, 0, 0, ""); print_in_box(80, 0, 0, " Threads : %d", context->config.all_threads); print_in_box(80, 0, 0, " Phred offset : %d", (context->config.phred_score_format == FASTQ_PHRED33)?33:64); if( context->config.second_read_file[0]) { print_in_box(80, 0, 0, " # of extracted subreads : %d", context->config.total_subreads); print_in_box(80, 0, 0, " Min read1 vote : %d", context->config.minimum_subread_for_first_read); print_in_box(80, 0, 0, " Min read2 vote : %d", context->config.minimum_subread_for_second_read); print_in_box(80, 0, 0, " Max fragment size : %d", context->config.maximum_pair_distance); print_in_box(80, 0, 0, " Min fragment size : %d", context->config.minimum_pair_distance); } else print_in_box(80, 0, 0, " Min votes : %d / %d", context->config.minimum_subread_for_first_read, context->config.total_subreads); print_in_box(80, 0, 0, " Max mismatches : %d", context->config.max_mismatch_exonic_reads); print_in_box(80, 0, 0, " Max indel length : %d", context->config.max_indel_length); print_in_box(80, 0, 0, " Report multi-mapping reads : %s", context->config.report_multi_mapping_reads?"yes":"no"); print_in_box(80, 0, 0, "Max alignments per multi-mapping read : %d", context->config.multi_best_reads); if(context->config.exon_annotation_file[0]){ if(context->config.exon_annotation_file_screen_out[0]) print_in_box(80, 0, 0, " Annotations : %s", context->config.exon_annotation_file_screen_out); else print_in_box(80, 0, 0, " Annotations : %s (%s)",get_short_fname( context->config.exon_annotation_file), context->config.exon_annotation_file_type==FILE_TYPE_GTF?"GTF":"SAF"); } if(context->config.max_insertion_at_junctions) print_in_box(80, 0, 0, " Insertions at junc : %d", context->config.max_insertion_at_junctions); if(context->config.read_group_id[0]) print_in_box(80, 0, 0, " Read group name : %s", context->config.read_group_id); print_in_box(80, 0, 1, ""); print_in_box(80, 2, 1, ""); sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_ERROR,""); if(!context->config.experiment_type){ sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_ERROR,"You have to specify the experiment type by using the '-t' option.\n"); return -1; } if(!context->config.first_read_file[0]) { sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_ERROR,"You have to specify at least one input file in the FASTQ/FASTA/PLAIN format using the '-r' option.\n"); return -1; } if(0 && !context->config.output_prefix[0]) { sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_ERROR,"You have to specify the path of output using the '-o' option.\n"); return -1; } if(!context->config.index_prefix[0]) { sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_ERROR,"You have to specify the prefix of the index files using the '-i' option.\n"); return -1; } char tbuf[90]; char_strftime(tbuf); print_in_box(80,1,1,"Running (%s, pid=%d)", tbuf, getpid()); print_in_box(80,0,1,""); return 0; } int init_paired_votes(global_context_t *context) { init_bigtable_results(context, 0); return 0; } #define FETCH_SEQ_LEN(x1) (context->chromosome_table.read_offsets[xk1] - last_offset+16 - ( 2 * context->chromosome_table.padding )) void write_sam_headers(global_context_t * context) { char * sorting_str = context -> config.sort_reads_by_coordinates?"coordinate":"unsorted"; if(context -> config.is_BAM_output) { char header_buff[100]; sprintf(header_buff, "@HD\tVN:1.0\tSO:%s", sorting_str); SamBam_writer_add_header(context -> output_bam_writer, header_buff, 0); int xk1; int last_offset = 0; char * obuf = malloc(10000+5000); for(xk1=0; xk1< context->chromosome_table.total_offsets; xk1++) { int seq_len = FETCH_SEQ_LEN(x1); //context->chromosome_table.read_offsets[xk1] - last_offset+16 SamBam_writer_add_chromosome(context -> output_bam_writer, context->chromosome_table.read_names+ xk1 * MAX_CHROMOSOME_NAME_LEN, seq_len, 1); last_offset = context->chromosome_table.read_offsets[xk1]; } if(context->config.read_group_id[0]) { snprintf(obuf,10000, "@RG\tID:%s%s",context->config.read_group_id, context->config.read_group_txt); SamBam_writer_add_header(context -> output_bam_writer,obuf, 0); } snprintf(obuf,9999+4900, "@PG\tID:subread\tPN:subread\tVN:%s\tCL:%s", SUBREAD_VERSION, context->rebuilt_command_line); SamBam_writer_add_header(context -> output_bam_writer,obuf, 0); SamBam_writer_finish_header(context -> output_bam_writer); free(obuf); } else { sambamout_fprintf(context -> output_sam_fp, "@HD\tVN:1.0\tSO:%s\n", sorting_str); int xk1; int last_offset = 0; for(xk1=0; xk1< context->chromosome_table.total_offsets; xk1++) { int seq_len = FETCH_SEQ_LEN(x1); sambamout_fprintf(context -> output_sam_fp, "@SQ\tSN:%s\tLN:%u\n", context->chromosome_table.read_names+ xk1 * MAX_CHROMOSOME_NAME_LEN, seq_len); last_offset = context->chromosome_table.read_offsets[xk1]; } if(context->config.read_group_id[0]) sambamout_fprintf(context -> output_sam_fp, "@RG\tID:%s%s\n",context->config.read_group_id, context->config.read_group_txt); sambamout_fprintf(context -> output_sam_fp, "@PG\tID:subread\tPN:subread\tVN:%s\tCL:%s\n", SUBREAD_VERSION, context->rebuilt_command_line); } } #define EXONIC_REGION_RESOLUTION 16 int is_pos_in_annotated_exon_regions(global_context_t * global_context, unsigned int pos){ int exonic_map_byte = pos / EXONIC_REGION_RESOLUTION / 8; int exonic_map_bit = (pos / EXONIC_REGION_RESOLUTION) % 8; return (global_context ->exonic_region_bitmap [exonic_map_byte] & (1< numOfBuckets; x1 ++){ cursor = tab -> bucketArray[x1]; while(1){ if(NULL == cursor)break; char * tab_anno_chro = cursor -> value; if(strcmp(tab_anno_chro, anno_chro) == 0) return (char *)cursor -> key; cursor = cursor -> next; } } return NULL; } int do_anno_bitmap_add_feature(char * gene_name, char * tracnscript_id, char * chro_name, unsigned int feature_start, unsigned int feature_end, int is_negative_strand, void * context){ global_context_t * global_context = context; if(global_context -> sam_chro_to_anno_chr_alias){ char * sam_chro = get_sam_chro_name_from_alias(global_context -> sam_chro_to_anno_chr_alias, chro_name); if(sam_chro!=NULL) chro_name = sam_chro; } if(!HashTableGet(global_context -> annotation_chro_table,chro_name)) HashTablePut(global_context -> annotation_chro_table, memstrcpy( chro_name ), NULL+1); char tmp_chro_name[MAX_CHROMOSOME_NAME_LEN]; int access_n = HashTableGet( global_context -> chromosome_table.read_name_to_index, chro_name ) - NULL; if(access_n < 1){ if(chro_name[0]=='c' && chro_name[1]=='h' && chro_name[2]=='r'){ chro_name += 3; }else{ strcpy(tmp_chro_name, "chr"); strcat(tmp_chro_name, chro_name); chro_name = tmp_chro_name; } } unsigned int exonic_map_start = linear_gene_position(&global_context->chromosome_table , chro_name, feature_start); unsigned int exonic_map_stop = linear_gene_position(&global_context->chromosome_table , chro_name, feature_end); if(exonic_map_start > 0xffffff00 || exonic_map_stop > 0xffffff00){ return -1; } exonic_map_start -= exonic_map_start%EXONIC_REGION_RESOLUTION; exonic_map_stop -= exonic_map_stop%EXONIC_REGION_RESOLUTION; for(; exonic_map_start <= exonic_map_stop; exonic_map_start+=EXONIC_REGION_RESOLUTION){ int exonic_map_byte = exonic_map_start / EXONIC_REGION_RESOLUTION / 8; int exonic_map_bit = (exonic_map_start / EXONIC_REGION_RESOLUTION) % 8; global_context ->exonic_region_bitmap[exonic_map_byte] |= (1< appendix2; HashTable * result_annot_chros = tab -> appendix1; char * index_name = ky; char * annot_name = HashTableGet(alias_index_to_annot, index_name); if(annot_name==NULL) annot_name = index_name; HashTablePut( result_annot_chros, annot_name, va ); } void warning_anno_vs_index(HashTable * anno_chros_tab, gene_offset_t * index_chros_offset, HashTable * alias_index_to_annot){ HashTable * index_chros_tab = index_chros_offset -> read_name_to_index; HashTable * chros_in_annot_using_index_names = anno_chros_tab ; HashTable * chros_in_index_using_annot_names = index_chros_tab ; HashTable * alias_annot_to_index = NULL; if(alias_index_to_annot){ chros_in_annot_using_index_names = StringTableCreate(1000); chros_in_index_using_annot_names = StringTableCreate(1000); alias_annot_to_index = StringTableReverse(alias_index_to_annot); anno_chros_tab -> appendix1 = chros_in_annot_using_index_names; anno_chros_tab -> appendix2 = alias_annot_to_index; HashTableIteration(anno_chros_tab, convert_table_key_to_alias); index_chros_tab -> appendix1 = chros_in_index_using_annot_names; index_chros_tab -> appendix2 = alias_index_to_annot; HashTableIteration(index_chros_tab, convert_table_key_to_alias); } warning_hash_hash(anno_chros_tab, chros_in_index_using_annot_names, "Chromosomes/contigs in annotation but not in index :"); warning_hash_hash(index_chros_tab, chros_in_annot_using_index_names, "Chromosomes/contigs in index but not in annotation :"); if(alias_index_to_annot){ HashTableDestroy(alias_annot_to_index); HashTableDestroy(chros_in_annot_using_index_names); HashTableDestroy(chros_in_index_using_annot_names); } } int load_annotated_exon_regions(global_context_t * global_context){ int bitmap_size = (4096 / EXONIC_REGION_RESOLUTION / 8)*1024*1024; global_context ->exonic_region_bitmap = malloc(bitmap_size); memset( global_context ->exonic_region_bitmap , 0, bitmap_size ); global_context -> annotation_chro_table = HashTableCreate(1003); HashTableSetDeallocationFunctions( global_context -> annotation_chro_table, free, NULL); HashTableSetKeyComparisonFunction( global_context -> annotation_chro_table, my_strcmp); HashTableSetHashFunction( global_context -> annotation_chro_table, fc_chro_hash); int loaded_features = load_features_annotation(global_context->config.exon_annotation_file, global_context->config.exon_annotation_file_type, global_context->config.exon_annotation_gene_id_column, NULL, global_context->config.exon_annotation_feature_name_column, global_context, do_anno_bitmap_add_feature); if(loaded_features < 0)return -1; else print_in_box(80,0,0,"%d annotation records were loaded.\n", loaded_features); return 0; } int load_global_context(global_context_t * context) { char tmp_fname [MAX_FILE_NAME_LENGTH + 50]; int min_phred_score = -1 , max_phred_score = -1; context -> is_phred_warning = 0; if(context->config.multi_best_reads>1 && ! context->config.report_multi_mapping_reads){ print_in_box(80,0,0,"WARNING: Multi-mapping reads are reported."); context->config.report_multi_mapping_reads = 1; } if(context->config.scRNA_input_mode){ context -> config.multi_best_reads = 3; context -> config.do_remove_neighbour_for_scRNA = 1; context -> config.reads_per_chunk = 30000000*context -> config.multi_best_reads; context -> config.multi_best_reads = max(context -> config.multi_best_reads , context -> config.reported_multi_best_reads); // opening a BCL input needs the exact chunk size. if(context->config.multi_best_reads>1) context -> config.reads_per_chunk /= context->config.multi_best_reads; char * reads_per_chunk = getenv("CC_READS_PER_CHUNK"); char * remove_beighbour = getenv("CC_REMOVE_NEIGHBOUR"); if(remove_beighbour) context -> config.do_remove_neighbour_for_scRNA = remove_beighbour[0]-'0'; if(reads_per_chunk) context -> config.reads_per_chunk = atoi(reads_per_chunk); if(0){ SUBREADprintf("WARNINGqqq: small-chunk!\n"); SUBREADprintf("WARNINGqqq: small-chunk!\n"); SUBREADprintf("WARNINGqqq: small-chunk!\n"); SUBREADprintf("WARNINGqqq: small-chunk!\n"); SUBREADprintf("WARNINGqqq: small-chunk!\n"); SUBREADprintf("WARNINGqqq: small-chunk!\n"); context -> config.reads_per_chunk /= 15; } } print_in_box(80,0,0,"Check the input reads."); subread_init_lock(&context->input_reads.input_lock); if(core_geinput_open(context, &context->input_reads.first_read_file, 1)) { sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_ERROR,"Unable to open the input file '%s'\n", context->config.first_read_file); return -1; } context->config.space_type = context->input_reads.first_read_file.space_type; print_in_box(89,0,0,"The input file contains %c[36m%s%c[0m space reads.", CHAR_ESC, context->config.space_type == GENE_SPACE_COLOR?"color":"base", CHAR_ESC); if(context->config.space_type == GENE_SPACE_COLOR && context->config.is_BAM_output && !context->config.convert_color_to_base) { print_in_box(80,0,0,"The color-space bases will be converted into base space in the BAM output."); context->config.convert_color_to_base=1; } else if(context->config.space_type == GENE_SPACE_BASE && context->config.convert_color_to_base) { print_in_box(80,0,0,"The reads will not be converted into base space."); context->config.convert_color_to_base=0; } if(context->input_reads.is_paired_end_reads) { if(core_geinput_open(context, &context->input_reads.second_read_file, 2)) { //sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_ERROR,"Unable to open '%s' as input. Please check if it exists, you have the permission to read it, and it is in the correct format.\n", context->config.second_read_file); return -1; } context -> config.max_vote_combinations = 3; context -> config.multi_best_reads = 3; context -> config.max_vote_simples = 64; context -> config.max_vote_number_cutoff = 2; }else{ context -> config.max_vote_combinations = 3; context -> config.multi_best_reads = 3; context -> config.max_vote_simples = 3; context -> config.max_vote_number_cutoff = 2; } print_in_box(80,0,0,"Initialise the memory objects."); context -> config.max_vote_simples = max(context -> config.max_vote_simples , context -> config.reported_multi_best_reads); context -> config.max_vote_combinations = max(context -> config.max_vote_combinations , context -> config.reported_multi_best_reads); if(!context->config.scRNA_input_mode){ // if it is BCL input then these two parameters have been decided . if(context->config.multi_best_reads>1) context->config.reads_per_chunk /= context->config.multi_best_reads; if(context->input_reads.is_paired_end_reads) context->config.reads_per_chunk /= 2; } struct stat ginp1_stat; int guess_tested_reads = 0; stat(context->config.first_read_file , &ginp1_stat); context->input_reads.first_read_file_size = ginp1_stat.st_size; print_in_box(80,0,0,"Estimate the mean read length."); context -> input_reads.avg_read_length = 100; if(context->config.scRNA_input_mode != GENE_INPUT_SCRNA_BAM && context->config.scRNA_input_mode != GENE_INPUT_SCRNA_FASTQ) context -> input_reads.avg_read_length = guess_reads_density_format(context->config.first_read_file , context->config.is_SAM_file_input?1:0, &min_phred_score, &max_phred_score , &guess_tested_reads); if(context -> input_reads.avg_read_length<0 )context -> input_reads.avg_read_length = 250; // SUBREADprintf("QR=[%d,%d]; ALEN=%f\n", min_phred_score, max_phred_score, context -> input_reads.avg_read_length); if(max_phred_score>=0) { int inferred_offset; if(abs(min_phred_score - 33) < abs(min_phred_score - 64)) inferred_offset = 33; else inferred_offset = 64; if(context -> input_reads.first_read_file.file_type != GENE_INPUT_GZIP_FASTA){ if((context->config.phred_score_format == FASTQ_PHRED64 && inferred_offset == 33) || (context->config.phred_score_format == FASTQ_PHRED33 && inferred_offset == 64)) { print_in_box(80,0,0, "WARNING - The specified Phred score offset (%d) seems incorrect.", context->config.phred_score_format == FASTQ_PHRED33?33:64); print_in_box(80,0,0, " ASCII values of the quality scores of read bases included in"); print_in_box(80,0,0, " the first %d reads were found to be within the range of",guess_tested_reads); print_in_box(80,0,0, " [%d,%d].", min_phred_score, max_phred_score); print_in_box(80,0,0, ""); context -> is_phred_warning = 1; } else{ print_in_box(80,0,0, "The range of Phred scores observed in the data is [%d,%d]", min_phred_score - inferred_offset, max_phred_score - inferred_offset); } } } subread_init_lock(&context -> output_lock); if(context->config.report_sam_file && context -> config.output_prefix[0]) { // ====== open output files ====== // Only the sam file is opened here; other files like bed, indel and etc are opened in init_modules() sprintf(tmp_fname,"%s", context->config.output_prefix); print_in_box(80,0,0,"Create the output %s file.", context -> config.is_BAM_output?"BAM":"SAM"); if(context -> config.is_BAM_output) { context -> output_bam_writer = malloc(sizeof(SamBam_Writer)); SamBam_writer_create(context -> output_bam_writer , tmp_fname, context -> config.is_input_read_order_required?1:context -> config.all_threads, context -> config.sort_reads_by_coordinates, context->config.scRNA_input_mode, context -> config.temp_file_prefix); context -> output_sam_fp = NULL; } else { context -> output_sam_fp = f_subr_open(tmp_fname,"wb"); //context -> output_sam_inner_buffer = malloc(OUTPUT_BUFFER_SIZE); //setvbuf (context -> output_sam_fp, context -> output_sam_inner_buffer, _IOFBF, OUTPUT_BUFFER_SIZE); context -> output_bam_writer = NULL; } if((!context -> output_bam_writer) && (!context->output_sam_fp)) { sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_ERROR,"Unable to open '%s' to write. Please check if the path exists and you have the permission to create/write this file", tmp_fname); return -1; } } else { if(context -> config.is_BAM_output) { context -> output_bam_writer = malloc(sizeof(SamBam_Writer)); SamBam_writer_create(context -> output_bam_writer ,NULL, context -> config.is_input_read_order_required?1:context -> config.all_threads, context -> config.sort_reads_by_coordinates, 0, context -> config.temp_file_prefix); } context->output_sam_fp = NULL; } // ====== check index files, count blocks and load chro table ====== print_in_box(80,0,0,"Check the index."); sprintf(tmp_fname, "%s.reads", context->config.index_prefix); if(!does_file_exist(tmp_fname)) { sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_ERROR,"Unable top open index '%s'. Please make sure that the correct prefix is specified and you have the permission to read these files. For example, if there are files '/opt/my_index.reads', '/opt/my_index.files' and etc, the index prefix should be specified as '/opt/my_index' without any suffix. \n", context->config.index_prefix); return -1; } context->current_index_block_number = 0; if(load_offsets(&context->chromosome_table, context->config.index_prefix)){ sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_ERROR,"\nThe index was built by using an old version of Subread; its format is no longer supported. Please use the current version of the index builder to rebuild it.\n"); return 1; } if(context->config.space_type == GENE_SPACE_COLOR) sprintf(tmp_fname, "%s.00.c.tab", context->config.index_prefix); else sprintf(tmp_fname, "%s.00.b.tab", context->config.index_prefix); if(!does_file_exist(tmp_fname)) { sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_ERROR,"Your reads are in the %s space but the index was not built in the same space. Unable to precess the reads.\n", context->config.space_type == GENE_SPACE_COLOR?"color":"base"); return -1; } context->index_block_number = 0; while(1) { sprintf(tmp_fname, "%s.%02d.%c.tab", context->config.index_prefix, context->index_block_number, context->config.space_type == GENE_SPACE_COLOR?'c':'b'); if(!does_file_exist(tmp_fname))break; context->index_block_number ++; if(context->index_block_number>=2 && context->config.max_indel_length > 16) { print_in_box(80,0,0,"ERROR You cannot use multi-block index for very-long indel detection."); print_in_box(80,0,0,"Please set the maximum indel length <= 16."); return -1; } } if(context->config.report_sam_file) write_sam_headers(context); print_in_box(80,0,0,"Init the voting space."); // ====== init other variables ====== if(context -> config.all_threads>1) subread_init_lock(&context -> thread_initial_lock); else subread_init_topKbuff(context, &context -> topKbuff); if(init_paired_votes(context)) { sublog_printf(SUBLOG_STAGE_RELEASED, SUBLOG_LEVEL_ERROR,"Cannot initialise the voting space. You need at least 2GB of empty physical memory to run this program.\n"); return 1; } sublog_printf(SUBLOG_STAGE_DEV1, SUBLOG_LEVEL_DEBUG, "load_global_context: finished"); memset( context->all_value_indexes , 0 , 100 * sizeof(gene_value_index_t)); context -> sam_chro_to_anno_chr_alias = NULL; if(context->config.exon_annotation_file[0]){ print_in_box(80,0,0,"Load the annotation file."); if( load_annotated_exon_regions( context ) ) return -1; if(context->config.exon_annotation_alias_file[0]) context -> sam_chro_to_anno_chr_alias = load_alias_table(context->config.exon_annotation_alias_file); warning_anno_vs_index(context -> annotation_chro_table, &context -> chromosome_table, context -> sam_chro_to_anno_chr_alias); HashTableDestroy(context -> annotation_chro_table); } else context -> exonic_region_bitmap = NULL; print_in_box(80,0,0,"Global environment is initialised."); return 0; } int init_modules(global_context_t * context) { sublog_printf(SUBLOG_STAGE_DEV1, SUBLOG_LEVEL_DEBUG, "init_modules: begin"); int ret = init_indel_tables(context); if((context->config.do_breakpoint_detection || context-> config.do_fusion_detection || context->config.do_breakpoint_detection || context-> config.do_long_del_detection)) ret = ret || init_junction_tables(context); sublog_printf(SUBLOG_STAGE_DEV1, SUBLOG_LEVEL_DEBUG, "init_modules: finished: %d",ret); return ret; } int destroy_modules(global_context_t * context) { destroy_indel_module(context); if((context->config.do_breakpoint_detection || context-> config.do_fusion_detection || context->config.do_breakpoint_detection || context-> config.do_long_del_detection)) destroy_junction_tables(context); return 0; } int destroy_global_context(global_context_t * context) { int xk1, block_no, ret = 0; if(context -> exonic_region_bitmap) free(context -> exonic_region_bitmap); for(block_no = 0; block_no< context->index_block_number; block_no++) gvindex_destory(&context -> all_value_indexes[block_no]); if(context->config.all_threads<2){ subread_free_topKbuff( context,&context -> topKbuff ); } if(context->output_sam_fp) { if(context -> output_sam_is_full){ unlink(context->config.output_prefix); SUBREADprintf("\nERROR: cannot finish the SAM file. Please check the disk space in the output directory.\nNo output file was generated.\n"); ret = 1; } fclose(context -> output_sam_fp); } if(context->input_reads.is_internal_error){ unlink(context->config.output_prefix); return 1; } if(context->output_bam_writer) { SamBam_writer_close(context->output_bam_writer); if(context->output_bam_writer -> is_internal_error){ unlink(context->config.output_prefix); SUBREADprintf("\nERROR: cannot finish the BAM file. Please check the disk space in the output directory.\nNo output file was generated.\n"); ret = 1; } free(context->output_bam_writer); context->output_bam_writer=NULL; } //free(context->big_margin_record); for(xk1=0; xk1<5; xk1++) if(context->module_contexts[xk1])free(context->module_contexts[xk1]); geinput_close(&context -> input_reads.first_read_file); if(context->input_reads.is_paired_end_reads) geinput_close(&context -> input_reads.second_read_file); destroy_offsets(&context->chromosome_table); finalise_bigtable_results(context); if((context -> will_remove_input_file & 1) && strstr(context ->config.first_read_file, "/core-temp")) unlink(context ->config.first_read_file); if((context -> will_remove_input_file & 2) && strstr(context ->config.second_read_file, "/core-temp")) unlink(context ->config.second_read_file); free(context -> rebuilt_command_line); return ret; } void subread_rebuild_cmd(int argc, char ** argv, global_context_t * global_context){ global_context -> rebuilt_command_line_size = rebuild_command_line(&global_context -> rebuilt_command_line , argc, argv); } int write_bincigar_part(char * bincigar, int chropt, unsigned int optlen, int bincigar_len) { int binopt, binbytes, x1; if(optlen<1) return -1; if(optlen < 4) binbytes=1; else if(optlen < 1024) binbytes=2; else if(optlen < 262144) binbytes=3; else if(optlen < 67108864) binbytes=4; else binbytes=5; if(bincigar_len>= 2; for(x1=1;x1>=8; } return binbytes; } // function returns the actual length of bincigar, or -1 if anything is wrong, e.g., bincigar_len is too short or unrecognized operations. int OLD_cigar2bincigar(char *cigar, char *bincigar, int bincigar_len) { int xk1=0; unsigned int tmpv=0, bincigar_cursor=0; while(1) { int nch = cigar[xk1]; if(!nch) break; xk1++; if(isdigit(nch)) tmpv=tmpv*10+(nch-'0'); else { int bincigar_sec_len = write_bincigar_part(bincigar+bincigar_cursor, nch, tmpv, bincigar_len-bincigar_cursor); if(bincigar_sec_len<0){ bincigar[0]=0; return -1; } bincigar_cursor += bincigar_sec_len; tmpv=0; } } if(bincigar_cursor> 3); binopt = 7 & bincigar[0]; switch(binopt) { case CORE_CIGAR_OPT_D: charopt='D'; break; case CORE_CIGAR_OPT_I: charopt='I'; break; case CORE_CIGAR_OPT_M: charopt='M'; break; case CORE_CIGAR_OPT_S: charopt='S'; break; case CORE_CIGAR_OPT_B: charopt='B'; break; case CORE_CIGAR_OPT_BB: charopt='b'; break; case CORE_CIGAR_OPT_N: charopt='N'; break; default: charopt='n'; break; } tmpv = (bincigar[0]>>6) & 3; for(x1 = 1; x1 < binbytes; x1++) { unsigned int dtmpv = 0xff & bincigar[x1]; dtmpv <<= (x1*8 - 6); tmpv += dtmpv; } int added_len = sprintf(sec_buf, "%u%c", tmpv, charopt); if(added_len > cigar_len) return -1; memcpy(cigar, sec_buf, added_len); (*bincigar_move) = binbytes; return added_len; } int OLD_bincigar2cigar(char * cigar, int cigar_len, char * bincigar, int bincigar_max_len, int read_len) { int cigar_cursor = 0, bincigar_cursor = 0; while(1) { int bincigar_move = 0; int cigar_sec_len = write_cigar_part(bincigar + bincigar_cursor, cigar+cigar_cursor, cigar_len-cigar_cursor-1, &bincigar_move); if(cigar_sec_len<0){ sprintf(cigar,"%dM", read_len); return -1; } //printf("NPC=%s\n", cigar); cigar_cursor += cigar_sec_len; bincigar_cursor += bincigar_move; if(bincigar_cursor>=bincigar_max_len) break; if(bincigar[bincigar_cursor] == 0) break; } cigar[cigar_cursor] = 0; return cigar_cursor; } int term_strncpy(char * dst, char * src, int max_dst_mem) { int i; for(i=0; i= max_dst_mem) i = max_dst_mem-1; dst[i] = 0; return 0; } void absoffset_to_posstr(global_context_t * global_context, unsigned int pos, char * res){ char * ch; int off; locate_gene_position(pos, &global_context -> chromosome_table, & ch, &off); sprintf(res, "%s:%u", ch, off); } int chimeric_cigar_parts(global_context_t * global_context, unsigned int sel_pos, int is_first_section_negative_strand, int is_first_section_reversed, char * in_cigar, unsigned int * out_poses, char ** out_cigars, char * out_strands, int read_len, short * perfect_lens, char * read_name) { unsigned int current_perfect_map_start = sel_pos; int current_perfect_section_no = 0; unsigned int current_perfect_cursor = sel_pos; int is_reversed = is_first_section_reversed; int is_negative = is_first_section_negative_strand; int read_cursor = 0; int out_cigar_writer_ptr = 0; unsigned int tmpi = 0; short perfect_len = 0; int cigar_cursor; out_poses[0] = current_perfect_map_start - (is_reversed?1:0); out_strands[0] = is_negative?'-':'+'; char main_piece_strand = (is_first_section_negative_strand == is_first_section_reversed)?'+':'-'; for(cigar_cursor=0;;cigar_cursor++) { char ncch = in_cigar[cigar_cursor]; int is_chimeric_section_end = 0; if(!ncch){ perfect_lens [current_perfect_section_no] = perfect_len ; current_perfect_section_no++; break; } if(toupper(ncch)=='N'||toupper(ncch)=='B') { unsigned int jummped_location; int is_chro_jump = 0, is_long_jump = 0; if(is_reversed) { if(toupper(ncch)=='N') jummped_location = current_perfect_map_start - 1 + tmpi; else jummped_location = current_perfect_map_start - 1 - tmpi; } else { if(toupper(ncch)=='N') jummped_location = current_perfect_cursor + tmpi; else jummped_location = current_perfect_cursor - tmpi; } if(ncch == 'N') { char * curr_chr, * new_chr; int curr_offset, new_offset; locate_gene_position_max(current_perfect_cursor, &global_context -> chromosome_table, & curr_chr, & curr_offset, NULL, NULL, 1); locate_gene_position_max(jummped_location , &global_context -> chromosome_table, & new_chr, & new_offset, NULL, NULL, 1); if( curr_chr == NULL || new_chr == NULL ){ /* char outpos[100]; absoffset_to_posstr(global_context, sel_pos + 1, outpos); SUBREADprintf("Wrong CIGAR: mapped to %s, CIGAR=%s\n", outpos , in_cigar); */ return -1; } assert(curr_chr); assert(new_chr); is_chro_jump = (curr_chr != new_chr); long long int dist = (long long int)current_perfect_cursor; dist -= (long long int)jummped_location; if(abs(dist) >= global_context -> config.maximum_intron_length) is_long_jump = 1; //#warning ">>>>>> COMMENT WHEN RELEASE <<<<<<" if(0 && FIXLENstrcmp("R000000007", read_name)==0) SUBREADprintf("dist=%lld, abs=%lld, %u, %u\n",dist,abs(dist), current_perfect_cursor, jummped_location); // A long jump is the jump longer than 2^27. // Picard does not like it!! } // is_long_jump is true only if the two sections are on different chromosomes. //#warning ">>>>>> COMMENT WHEN RELEASE <<<<<<" if(0 && FIXLENstrcmp("R000000007", read_name)==0) SUBREADprintf("CHR_JMP=%d, NCCH=%c, LONG_JMP=%d\n", is_chro_jump, ncch, is_long_jump); if(is_chro_jump || islower(ncch) || ncch == 'B' || is_long_jump) { current_perfect_cursor = jummped_location; if(islower(ncch)){ is_reversed = !is_reversed; is_negative = !is_negative; if(is_reversed) current_perfect_cursor --; } current_perfect_map_start = current_perfect_cursor; tmpi = 0; if(read_cursor=CIGAR_PERFECT_SECTIONS)break; out_poses[current_perfect_section_no] = current_perfect_map_start - read_cursor; out_strands[current_perfect_section_no] = is_negative?'-':'+'; out_cigar_writer_ptr = sprintf(out_cigars[current_perfect_section_no],"%dS", read_cursor); is_chimeric_section_end = 1; } } if(!is_chimeric_section_end) { if(isalpha(ncch)) { out_cigar_writer_ptr+=sprintf(out_cigars[current_perfect_section_no]+out_cigar_writer_ptr, "%u%c", tmpi, ncch); } if(ncch == 'M'|| ncch == 'S') { read_cursor += tmpi; if(ncch == 'M') perfect_len += tmpi; if(is_reversed) out_poses[current_perfect_section_no] += tmpi; else current_perfect_cursor += tmpi; tmpi = 0; } else if(ncch == 'D' || ncch == 'N') { if(is_reversed) out_poses[current_perfect_section_no] += tmpi; else current_perfect_cursor += tmpi; tmpi = 0; } else if(ncch == 'I') { read_cursor += tmpi; tmpi = 0; } else if(isdigit(ncch)) tmpi = tmpi*10+(ncch-'0'); } } int xk1 = 0, best_match = -9999; for(xk1=0; xk1perfect_lens[best_match])) best_match = xk1; } if(best_match>0) { unsigned int tmpv; char cigar_sec[100]; tmpv = out_poses[0]; out_poses[0]=out_poses[best_match]; out_poses[best_match] = tmpv; tmpv = out_strands[0]; out_strands[0] = out_strands[best_match]; out_strands[best_match] = tmpv; strcpy(cigar_sec, out_cigars[0]); strcpy(out_cigars[0], out_cigars[best_match]); strcpy(out_cigars[best_match] , cigar_sec); tmpv = perfect_lens[0]; perfect_lens[0] = perfect_lens[best_match]; perfect_lens[best_match] = tmpv; } return current_perfect_section_no; } void quick_sort_run(void * arr, int spot_low,int spot_high, int compare (void * arr, int l, int r), void exchange(void * arr, int l, int r)); void quick_sort(void * arr, int arr_size, int compare (void * arr, int l, int r), void exchange(void * arr, int l, int r)) { quick_sort_run(arr, 0, arr_size-1, compare, exchange); } void quick_sort_run(void * arr, int spot_low,int spot_high, int compare (void * arr, int l, int r), void exchange(void * arr, int l, int r)) { // https://en.wikipedia.org/wiki/Quicksort // Lomuto partition scheme int pivot,j,i; pivot = spot_high; i = spot_low-1; for(j = spot_low; j spot_low) quick_sort_run(arr, spot_low, i, compare, exchange); if(spot_high>i+2) quick_sort_run(arr, i+2, spot_high, compare, exchange); } void basic_sort_run(void * arr, int start, int items, int compare (void * arr, int l, int r), void exchange(void * arr, int l, int r)){ int i, j; for(i=start; i< start + items - 1; i++) { int min_j = i; for(j=i + 1; j< start + items; j++) { if(compare(arr, min_j, j) > 0) min_j = j; } if(i!=min_j) exchange(arr, i, min_j); } } void basic_sort(void * arr, int items, int compare (void * arr, int l, int r), void exchange(void * arr, int l, int r)){ basic_sort_run(arr, 0, items, compare, exchange); } void merge_sort_run(void * arr, int start, int items, int compare (void * arr, int l, int r), void exchange(void * arr, int l, int r), void merge(void * arr, int start, int items, int items2)) { if(items > 11) { int half_point = items/2; merge_sort_run(arr, start, half_point, compare, exchange, merge); merge_sort_run(arr, start + half_point, items - half_point, compare, exchange, merge); merge(arr, start, half_point, items - half_point); } else { basic_sort_run(arr, start, items, compare, exchange); } } void merge_sort(void * arr, int arr_size, int compare (void * arr, int l, int r), void exchange(void * arr, int l, int r), void merge(void * arr, int start, int items, int items2)) { merge_sort_run(arr, 0, arr_size, compare, exchange, merge); } unsigned int calc_end_pos(unsigned int p, char * cigar, unsigned int * all_skipped_len, int * is_exonic_regions, global_context_t * global_context){ unsigned int cursor = p, tmpi=0; int nch, cigar_cursor; for(cigar_cursor = 0; 0!=(nch = cigar[cigar_cursor]); cigar_cursor++){ if(isdigit(nch)){ tmpi = tmpi * 10 + (nch - '0'); }else{ if((nch == 'S' && cursor == p) || nch == 'M' || nch == 'N' || nch == 'D'){ if(nch == 'M' && global_context -> exonic_region_bitmap){ if(global_context -> config.do_breakpoint_detection){ if(is_pos_in_annotated_exon_regions(global_context, cursor) == 0 || is_pos_in_annotated_exon_regions(global_context, cursor + tmpi - 1) == 0) ( * is_exonic_regions) = 0; } else { if(is_pos_in_annotated_exon_regions(global_context, cursor + tmpi/2) == 0) ( * is_exonic_regions) = 0; } } cursor += tmpi; if(nch == 'N' || nch == 'D')(*all_skipped_len) += tmpi; } tmpi = 0; } } return cursor; } void test_PE_and_same_chro_cigars(global_context_t * global_context , unsigned int pos1, unsigned int pos2, int * is_exonic_regions, int * is_PE_distance, int * is_same_chromosome, int read_len_1, int read_len_2, char * cigar1, char * cigar2, char *read_name, int * res_tlen){ char * r1_chr = NULL, * r2_chr = NULL; int r1_pos, r2_pos; (*is_same_chromosome) = 0; (*is_PE_distance) = 0; (*is_exonic_regions) = 1; locate_gene_position(pos1, &global_context -> chromosome_table, & r1_chr, & r1_pos); locate_gene_position(pos2, &global_context -> chromosome_table, & r2_chr, & r2_pos); if(r1_chr == r2_chr){ unsigned int skip_1 = 0; unsigned int skip_2 = 0; unsigned int r1_end_pos = calc_end_pos(pos1, cigar1, &skip_1, is_exonic_regions, global_context ); unsigned int r2_end_pos = calc_end_pos(pos2, cigar2, &skip_2, is_exonic_regions, global_context ); unsigned int tlen = max(r1_end_pos, r2_end_pos) - min(pos1, pos2); if(tlen > skip_1) tlen -= skip_1; if(tlen > skip_2) tlen -= skip_2; (*is_same_chromosome) = 1; if(tlen >= global_context -> config.minimum_pair_distance && tlen <= global_context -> config.maximum_pair_distance) (* is_PE_distance) = 1; *res_tlen = tlen; }else *res_tlen = 0x7fffffff; } void test_PE_and_same_chro_align(global_context_t * global_context , realignment_result_t * res1, realignment_result_t * res2, int * is_exonic_regions, int * is_PE_distance, int * is_same_chromosome, int read_len_1, int read_len_2, char * read_name, int *res_tlen){ return test_PE_and_same_chro_cigars(global_context, res1 -> first_base_position, res2 -> first_base_position, is_exonic_regions, is_PE_distance, is_same_chromosome , read_len_1 , read_len_2, res1 -> cigar_string, res2 -> cigar_string, read_name, res_tlen); } void test_PE_and_same_chro(global_context_t * global_context , unsigned int pos1, unsigned int pos2, int * is_PE_distance, int * is_same_chromosome, int read_len_1, int read_len_2) { char * r1_chr, * r2_chr; int r1_pos, r2_pos; (*is_same_chromosome) = 0; (*is_PE_distance) = 0; int re1 = locate_gene_position(pos1, &global_context -> chromosome_table, & r1_chr, & r1_pos); int re2 = locate_gene_position(pos2, &global_context -> chromosome_table, & r2_chr, & r2_pos); if(re1 ==0 && 0 ==re2) { long long tlen = r1_pos; tlen -= r2_pos; tlen = abs(tlen); tlen += (r1_pos > r2_pos)?read_len_1:read_len_2; unsigned int tlenI = (unsigned int) tlen; //SUBREADprintf("TEST PE: %p == %p , TLEN=%u\n", r1_chr, r2_chr, tlenI); if(r1_chr == r2_chr){ (*is_same_chromosome) = 1; if(tlenI >= global_context -> config.minimum_pair_distance && tlenI <= global_context -> config.maximum_pair_distance) (* is_PE_distance) = 1; } } } int FIXLENstrcmp(char * flen, char * rname){ char * tt=NULL; char * fixed_len = malloc(strlen(flen)+1); strcpy(fixed_len, flen); char * s1 = strtok_r(fixed_len, "\n", &tt); int found = 0; while(1){ int this_ok = 1,x=0; for(; s1[x]; x++){ if(rname[x]!=s1[x]) { this_ok = 0; break; } } if(this_ok){ found = 1; break; } s1 = strtok_r(NULL, "\n", &tt); if(!s1)break; } free(fixed_len); return !found; } Rsubread/src/core.h0000644000175200017520000006044514136047615015253 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ //Core.c is the totally refactored body of the current subread, subjunc, subfusion and subindel programs. //This program runs in this way: // 1, it loads reads from FASTQ, FASTA, PLAIN or SAM files // 2, it change the read and the quality strings according to FF/FR/RR/RF orders, such that every read is in the "F" manner. // Namely, the second read in a pair is reversed. // 3, it builds voting table for each read. // 4, it performs the first iteration. // 5, it performs the second iteration. // 6, it reports results regarding the reads. // 7, if the input files contain too many reads (e.g., greater than 14M in total), step 1 ~ 6 are repeated until all reads are finalised. // 8, it reports global results. //Core.c maintains the following context: // 1, the global context. // 2, the overall context for each module. // 3, the local context for voting each read for each module. // The structure of each context is named as // Context_ModuleName_Scope //Thread context is maintained for each thread. // There should be a function for each module to store/load its global context. #ifndef _SUBREAD_CORE_H_ #define _SUBREAD_CORE_H_ #include #include "subread.h" #include "sambam-file.h" //#define is_target(s) (memcmp((s),"chr901_242237_242737_0:0:0_0:0:0_3761", 22)==0) #define is_target(s) 0 //#define _global_retrieve_voting_context(global_context, pair_number) (global_context->chunk_vote_records[pair_number]) //#define _global_retrieve_alignment_ptr(global_context, pair_number, is_second_read, best_read_id) ((global_context->input_reads.is_paired_end_reads?(global_context -> chunk_alignment_records + (2*pair_number+is_second_read)* global_context->config.multi_best_reads + best_read_id):(global_context -> chunk_alignment_records+pair_number*global_context->config.multi_best_reads + best_read_id))) //#define _global_retrieve_subjunc_ptr(global_context, pair_number, is_second_read, best_read_id) ((global_context->input_reads.is_paired_end_reads?(global_context -> chunk_subjunc_records + (2*pair_number+is_second_read)* global_context->config.multi_best_reads + best_read_id):(global_context -> chunk_subjunc_records+pair_number*global_context->config.multi_best_reads + best_read_id))) //#define _global_retrieve_big_margin_ptr(global_context,pair_number, is_second_read) (is_second_read?(global_context->big_margin_record + (2*pair_number+is_second_read)* global_context->config.big_margin_record_size):(global_context->big_margin_record + pair_number * global_context->config.big_margin_record_size)) #define mark_gapped_read(res) (res)-> result_flags|= CORE_IS_GAPPED_READ; #define CORE_MAX_CIGAR_STR_LEN 110 #define CORE_ADDITIONAL_INFO_LENGTH 400 #define READPAIRS_FOR_CALC_EXPT_TLEN 1000 typedef struct{ subread_read_number_t fragments; unsigned int maximum_interval_length; unsigned int expected_items; HashTable * entry_table; } bucketed_table_t; typedef struct{ int capacity; int items; unsigned int keyed_bucket; unsigned int maximum_interval_length; unsigned int * positions; void ** details; } bucketed_table_bucket_t; typedef struct{ unsigned long long capacity; subread_read_number_t fragments; subread_read_number_t * fragment_numbers; } fragment_list_t; typedef struct{ int is_paired_end_reads; int is_internal_error; gene_input_t first_read_file; gene_input_t second_read_file; unsigned long long first_read_file_size; subread_lock_t input_lock; double avg_read_length; } read_input_t; typedef struct{ char read_name[MAX_READ_NAME_LEN]; unsigned short flags; char chro_name[MAX_CHROMOSOME_NAME_LEN]; unsigned int location; unsigned short map_quality; char cigar[CORE_MAX_CIGAR_STR_LEN]; char other_chro_name[MAX_CHROMOSOME_NAME_LEN]; unsigned int other_location; long long int tlen; int rlen; char read_text[MAX_READ_LENGTH]; char qual_text[MAX_READ_LENGTH]; char additional_columns[CORE_ADDITIONAL_INFO_LENGTH]; } output_read_buffer_t; typedef struct{ int fragment_number_in_chunk; int multi_mapping_locations; int this_mapping_location; output_read_buffer_t r1; output_read_buffer_t r2; } output_fragment_buffer_t; typedef struct{ // running_scheme int all_threads; int is_first_iteration_running; int is_second_iteration_running; int is_third_iteration_running; int use_memory_buffer; float memory_use_multiplex; char temp_file_prefix[MAX_FILE_NAME_LENGTH]; subread_read_number_t reads_per_chunk; int fast_run; // input_scheme char first_read_file[MAX_FILE_NAME_LENGTH * MAX_SCRNA_FASTQ_FILES * 3]; char second_read_file[MAX_FILE_NAME_LENGTH]; char exon_annotation_file[MAX_FILE_NAME_LENGTH]; char exon_annotation_file_screen_out[MAX_FILE_NAME_LENGTH]; char exon_annotation_alias_file[MAX_FILE_NAME_LENGTH]; int exon_annotation_file_type; char exon_annotation_gene_id_column[MAX_READ_NAME_LEN]; char exon_annotation_feature_name_column[MAX_READ_NAME_LEN]; int do_remove_neighbour_for_scRNA; int scRNA_input_mode; short read_trim_5; short read_trim_3; int is_first_read_reversed; int is_second_read_reversed; int space_type; int is_methylation_reads; int phred_score_format; int is_SAM_file_input; int is_gzip_fastq; // reporting scheme char read_group_id[MAX_FILE_NAME_LENGTH]; char read_group_txt[MAX_FILE_NAME_LENGTH]; char output_prefix[MAX_FILE_NAME_LENGTH]; int report_sam_file; int report_no_unpaired_reads; int min_mapped_fraction; int max_mismatch_exonic_reads; int max_mismatch_junction_reads; int ignore_unmapped_reads; int report_unmapped_using_mate_pos; int report_multi_mapping_reads; int downscale_mapping_quality; int is_BAM_input; int is_BAM_output; int is_input_read_order_required; int sort_reads_by_coordinates; int convert_color_to_base; int SAM_extra_columns; int report_multiple_best_in_pairs; unsigned int multi_best_reads; unsigned int reported_multi_best_reads; // basic voting char index_prefix[MAX_FILE_NAME_LENGTH]; int top_scores; int max_vote_combinations; int max_vote_simples; int max_vote_number_cutoff; int total_subreads; int minimum_subread_for_first_read; int minimum_subread_for_second_read; float minimum_exonic_subread_fraction; int minimum_pair_distance; int maximum_pair_distance; int restrected_read_order; int show_soft_cliping; int max_indel_length; int expected_pair_distance; int ambiguous_mapping_tolerance; int use_hamming_distance_break_ties; int use_quality_score_break_ties; int big_margin_record_size; int PE_predominant_weight; int no_TLEN_preference; // subjunc int entry_program_name; int experiment_type; int do_breakpoint_detection; int do_big_margin_filtering_for_junctions; int do_big_margin_filtering_for_reads; int limited_tree_scan; int use_hamming_distance_in_exon; unsigned int maximum_intron_length; int high_quality_base_threshold; int max_insertion_at_junctions; int check_donor_at_junctions; // subfusion int do_fusion_detection; int do_long_del_detection; int do_structural_variance_detection; int prefer_donor_receptor_junctions; int more_accurate_fusions; int maximum_translocation_length; int maximum_colocating_distance; // indel char do_superlong_indel_detection; char extending_search_indels; int k_mer_length; int realignment_minimum_variant_distance; int reassembly_start_read_number; int reassembly_key_length; int reassembly_subread_length; int reassembly_window_multiplex; int reassembly_tolerable_voting; int reassembly_window_alleles; int init_max_event_number; int use_dynamic_programming_indel; int use_bitmap_event_table; int maximise_sensitivity_indel; int flanking_subread_indel_mismatch; int DP_penalty_create_gap; int DP_penalty_extend_gap; int DP_match_score; int DP_mismatch_penalty; } configuration_t; #define CORE_IS_GT_AG_DONORS 1 #define CORE_NOTFOUND_DONORS 2 #define CORE_IS_STRAND_JUMPED 4 #define CORE_IS_NEGATIVE_STRAND 8 #define CORE_IS_FULLY_EXPLAINED 16 #define CORE_IS_BREAKEVEN 32 #define CORE_IS_GAPPED_READ 64 #define CORE_IS_PAIRED_END 128 #define CORE_TOO_MANY_MISMATCHES 256 #define CORE_CIGAR_OPT_M 0 #define CORE_CIGAR_OPT_S 1 #define CORE_CIGAR_OPT_D 2 #define CORE_CIGAR_OPT_I 3 #define CORE_CIGAR_OPT_B 4 #define CORE_CIGAR_OPT_N 5 #define CORE_CIGAR_OPT_BB 6 #define CORE_CIGAR_OPT_NN 7 #define CORE_PROGRAM_SUBREAD 100 #define CORE_PROGRAM_SUBJUNC 200 #define CORE_PROGRAM_SUBINDEL 1000 #define CORE_EXPERIMENT_DNASEQ 1000 #define CORE_EXPERIMENT_RNASEQ 2000 #define PRINT_BOX_WRAPPED 4 #define PRINT_BOX_NOCOLOR_FOR_COLON 2 #define PRINT_BOX_CENTER 1 typedef struct{ unsigned int event_small_side; // the last base before the event unsigned int event_large_side; // the first base after the event // for exon-exon junctions, these two numbers are the last and first base in the exons. (ZERO-BASED INDEX) // for indels, the smaller side is the base before in inserted/deleted bases; the larger side is the base after them. // for a fusion, the two sides are the base before the fusion point (the fusion point is not a base, but a vertical bar between two bases -- two sides.) // // // The 'V' marked bases are the two sides. // V V // Exon-exon junction: 100 .......s|gt........ag|s....... 300 chrX // ^ ^ These two bars are the junction point. // // // // DNA-fusions: // // small_side ===> .|. <=== large_side normal arrangement (N/Y) // // small_side ===> .| // large_side ===> .| strand jumpped (N/N) // // |. <=== small_side strand jumpped (Y/Y) // |. <=== large_side // // ^ This is fusion point. It is not a base, but a vertical bar between two sides. // // // // note: the fusion point is larger than event_small_side if small_side_increasing_coordinate == N; it is smaller than event_small_side if small_side_increasing_coordinate == Y; // the fusion point is smaller than event_large_side if large_side_increasing_coordinate == Y; it is larger than event_large_side if large_side_increasing_coordinate == N. short indel_length; short junction_flanking_left; short junction_flanking_right; char indel_at_junction; char is_negative_strand; // this only works to junction detection, according to 'GT/AG' or 'CT/AC' donors. This only applys to junctions. char is_strand_jumped; // "strand jumped" means that the left and right sides are on different strands. This only applys to fusions. char is_donor_found_or_annotation; // only for junctions: GT/AG is found at the location. 1: found, 0:not found: 64: from annotation (thus unknown) // Also, if "is_strand_jumped" is true, all coordinates (e.g., splicing points, cover_start, cover_end, etc) are on "reversed read" view. char small_side_increasing_coordinate; char large_side_increasing_coordinate; // normal exon-exon junctions must have N and Y on small/large increasing coordinates. //char is_ambiguous; char connected_next_event_distance; // the distance (negative or positive) to the next event in the table. For example, if the cigar string is 10M3I1M1I10M, event "3I" will have 1 here . char connected_previous_event_distance; // the distance (negative or positive) to the next event in the table. For example, if the cigar string is 10M3I1M1I10M, event "1I" will have 1 here. //char inserted_bases[(1+MAX_INSERTION_LENGTH) / 4 + 1]; char * inserted_bases; unsigned short supporting_reads; unsigned short anti_supporting_reads; unsigned short final_counted_reads; unsigned short final_reads_mismatches; unsigned char event_type; unsigned int global_event_id; float event_quality; unsigned long long critical_read_id; int critical_supporting_reads; } chromosome_event_t; typedef struct { unsigned int selected_position; short result_flags; short read_length; // 4 bytes gene_vote_number_t selected_votes; gene_vote_number_t used_subreads_in_vote; unsigned char noninformative_subreads_in_vote; // this coverage is the range on reads, in point of view of "main piece" strand (i.e., "is_negative_strand") char indels_in_confident_coverage; char is_fully_covered; gene_vote_number_t selected_indel_record [MAX_INDEL_SECTIONS*3 + 1]; unsigned short confident_coverage_start; unsigned short confident_coverage_end; short subread_quality; } mapping_result_t; typedef struct{ mapping_result_t * mapping_result; unsigned int first_base_position; char cigar_string[CORE_MAX_CIGAR_STR_LEN]; chromosome_event_t * supporting_chromosome_events[MAX_EVENTS_IN_READ]; short flanking_size_left[MAX_EVENTS_IN_READ]; short flanking_size_right[MAX_EVENTS_IN_READ]; char crirical_support[MAX_EVENTS_IN_READ]; char first_base_is_jumpped; short final_mismatched_bases; short final_matched_bases; short best_second_diff_bases; short realign_flags; short final_quality; short chromosomal_length; short MAPQ_adjustment; int known_junction_supp; int final_penalty; } realignment_result_t; #define BUCKETED_TABLE_INIT_ITEMS 3 #define FRAGMENT_LIST_INIT_ITEMS 3 typedef struct { short split_point; gene_vote_number_t minor_votes; char double_indel_offset; char indel_at_junction; char small_side_increasing_coordinate; char large_side_increasing_coordinate; unsigned int minor_position; // this coverage is the range on reads, in point of view of "main piece" strand unsigned short minor_coverage_start; unsigned short minor_coverage_end; } subjunc_result_t; // THREE_TOP_UPDATE dictates the number of top-votes. typedef struct { union{ int is_vote_t_item; int is_exon_item; }; int item_index_i; int item_index_j; unsigned int mapping_position; int major_half_votes; unsigned short read_start_base; }simple_mapping_t; typedef struct{ simple_mapping_t * r1_loc; simple_mapping_t * r2_loc; unsigned long long score_adj; } vote_combination_t; typedef struct{ unsigned long long maxinum_read_number; int result_chunk_fd; int junction_chunk_fd; int big_margin_chunk_fd; mapping_result_t * result_chunk_addr; subjunc_result_t * junction_chunk_addr; unsigned short * big_margin_chunk_addr; subread_lock_t resize_lock; } bigtable_t; // this cached item is for an entire read (not read pair, not one best result) typedef struct { int status; subread_read_number_t pair_number; int is_second_read; unsigned short big_margin_data[9*3]; mapping_result_t * alignment_res; subjunc_result_t * subjunc_res; } bigtable_cached_result_t; typedef struct{ void * junction_tmp_r1; void * junction_tmp_r2; void * alignment_tmp_r1; void * alignment_tmp_r2; void * vote_simple_1_buffer; void * vote_simple_2_buffer; void * comb_buffer; } topK_buffer_t; typedef struct{ unsigned long long all_correct_PE_reads; int thread_id; pthread_t thread; // module_thread_context is different to module_context, though some contents in module_thread_context could be also in module context (e.g., junction tables) // modules functions may deside to use objects in which context. void * module_thread_contexts[5]; gene_value_index_t * current_value_index; subread_lock_t output_lock; unsigned int all_mapped_reads; unsigned int not_properly_pairs_wrong_arrangement; unsigned int not_properly_pairs_different_chro; unsigned int not_properly_different_strands; unsigned int not_properly_pairs_TLEN_wrong; unsigned int all_unmapped_reads; unsigned int not_properly_pairs_only_one_end_mapped; unsigned int all_multimapping_reads; unsigned int all_uniquely_mapped_reads; topK_buffer_t topKbuff; } thread_context_t; typedef struct{ // basic running configuration configuration_t config; // for the index gehash_t * current_index; gene_value_index_t * current_value_index; gene_value_index_t all_value_indexes[100]; int index_block_number; int current_index_block_number; int will_remove_input_file; int is_phred_warning; int is_final_voting_run; // global locks subread_lock_t thread_initial_lock; // global FILE* SamBam_Writer * output_bam_writer; FILE * output_sam_fp; FILE * long_insertion_FASTA_fp; char * output_sam_inner_buffer; int output_sam_is_full; // running contexts void * module_contexts[5]; thread_context_t * all_thread_contexts; subread_read_number_t last_written_fragment_number; int need_merge_buffer_now; read_input_t input_reads; bigtable_t bigtable; int rebuilt_command_line_size; char * rebuilt_command_line; subread_lock_t bigtable_lock; subread_lock_t output_lock; int bigtable_cache_size; FILE * bigtable_cache_file_fp; long long bigtable_cache_file_loaded_fragments_begin; long long bigtable_cache_file_fragments; bigtable_cached_result_t * bigtable_cache; char *bigtable_cache_malloc_ptr, *bigtable_cache_malloc_ptr_junc; unsigned int bigtable_chunked_fragments; int bigtable_dirty_data; gene_offset_t chromosome_table; double start_time; double align_start_time; double timecost_load_index; double timecost_voting; double timecost_before_realign; double timecost_for_realign; unsigned long long expected_TLEN_sum; unsigned int expected_TLEN_read_numbers; unsigned long long all_processed_reads; unsigned long long all_correct_PE_reads; unsigned int all_junctions; unsigned int all_fusions; unsigned int all_indels; unsigned int all_mapped_reads; unsigned int not_properly_pairs_wrong_arrangement; unsigned int not_properly_pairs_different_chro; unsigned int not_properly_different_strands; unsigned int not_properly_pairs_TLEN_wrong; unsigned int all_unmapped_reads; unsigned int not_properly_pairs_only_one_end_mapped; unsigned int all_multimapping_reads; unsigned int all_uniquely_mapped_reads; unsigned long long current_circle_start_abs_offset_file1; gene_inputfile_position_t current_circle_start_position_file1; gene_inputfile_position_t current_circle_start_position_file2; gene_inputfile_position_t current_circle_end_position_file1; gene_inputfile_position_t current_circle_end_position_file2; subread_read_number_t processed_reads_in_chunk; subread_read_number_t running_processed_reads_in_chunk; // sunfusion structural variance bucketed_table_t funky_table_BC; bucketed_table_t funky_table_DE; fragment_list_t funky_list_A; fragment_list_t funky_list_DE; bucketed_table_t breakpoint_table_P; bucketed_table_t breakpoint_table_QR; bucketed_table_t breakpoint_table_YZ; bucketed_table_t translocation_result_table; bucketed_table_t inversion_result_table; topK_buffer_t topKbuff; // per chunk parameters subread_read_number_t read_block_start; char * exonic_region_bitmap; HashTable * sam_chro_to_anno_chr_alias; HashTable * annotation_chro_table; } global_context_t; #define MODULE_INDEL_ID 0 #define MODULE_JUNCTION_ID 1 #define STEP_VOTING 10 #define STEP_ITERATION_ONE 20 #define STEP_ITERATION_TWO 30 #define STEP_ITERATION_THREE 40 #define STEP_WRITE_CHUNK_RESULTS 90 #define MEMORY_OPTIMISATION_LAPTOP 10 #define MEMORY_OPTIMISATION_DESKTOP 20 #define MEMORY_OPTIMISATION_SUPERCOMPUTER 100 // This function initialise the module contexts that are needed according to the running configuration. int init_modules(global_context_t * context); // This function undo any memory allocation and close opened files in init_modules. int destroy_modules(global_context_t * context); // This function shows welcome messages and shows the current configuration to stdout. // This function also checks the legality of the parameters in context->config int print_configuration(global_context_t * context); // This function opens all related files except the index. It however counts the piece number of the index. int load_global_context(global_context_t * context); // This function deallocates memory and closes files that were allowcated and opened in load_global_context. int destroy_global_context(global_context_t * context); // This function processes the read chunks (24M reads each), including three steps: 1, voting; 2, iteration one; 3, iteration two. int read_chunk_circles(global_context_t * context); // write the final results in each module. Note that the SAM file is writen in read_chunk_circles, after each read chunk is processed. int write_final_results(global_context_t * context); // this function is called by an interface to start the main process with a customized opt parser. int core_main(int argc , char ** argv, int (parse_opts (int , char **, global_context_t * ))); // decompress a cigar string to cigar // cigar_len is the maximum length of decompressed cigar // bincigar is the maximum length of compressed cigar // it returns the length of decompressed cigar, or -1 if buffer is too short. int bincigar2cigar(char * cigar, int cigar_len, char * bincigar, int bincigar_max_len, int read_len); // compress a cigar string to bincigar // bincigar_len is the maximum length of compressed cigar // it returns the length of compressed cigar, or -1 if buffer is too short. int cigar2bincigar(char *cigar, char *bincigar, int bincigar_len); // print the logo of our program void print_subread_logo(); // print a line in the box void print_in_box(int line_width, int is_boundary, int options, char * pattern,...); // find the value index covering this read // it returns NULL if no index is found. gene_value_index_t * find_current_value_index(global_context_t * global_context, unsigned int pos, int len); // generate the time string void char_strftime(char * tbuf); int term_strncpy(char * dst, char * src, int max_dst_memory); int is_result_in_PE(mapping_result_t * aln); void core_version_number(char * program); mapping_result_t * _global_retrieve_alignment_ptr(global_context_t * global_context, subread_read_number_t pair_number, int is_second_read, int best_read_id); subjunc_result_t * _global_retrieve_subjunc_ptr(global_context_t * global_context, subread_read_number_t pair_number, int is_second_read, int best_read_id); unsigned short * _global_retrieve_big_margin_ptr(global_context_t * global_context, subread_read_number_t pair_number, subread_read_number_t is_second_read); // This assumes the first part of Cigar has differet strandness to the main part of the cigar. // Pos is the LAST WANTED BASE location before the first strand jump (split by 'b' or 'n'). // The first base in the read actually has a larger coordinate than Pos. unsigned int reverse_cigar(unsigned int pos, char * cigar, char * new_cigar); int chimeric_cigar_parts(global_context_t * global_context , unsigned int sel_pos, int is_first_section_negative_strand, int is_first_section_reversed, char * in_cigar, unsigned int * out_poses, char ** out_cigars, char * out_strands, int read_len, short * out_read_lens, char * read_name); void warning_file_limit(); void quick_sort(void * arr,int arr_size, int compare (void * arr, int l, int r), void exchange(void * arr, int l, int r)); void basic_sort(void * arr,int arr_size, int compare (void * arr, int l, int r), void exchange(void * arr, int l, int r)); // L_Minus_R should return -1, 0 or 1 when LR. // The result is from Small to Large. void merge_sort(void * arr, int arr_size, int L_Minus_R (void * arr, int l, int r), void exchange(void * arr, int l, int r), void merge_SmallFirst(void * arr, int start, int items, int items2)); void absoffset_to_posstr(global_context_t * global_context, unsigned int pos, char * res); void test_PE_and_same_chro(global_context_t * global_context , unsigned int pos1, unsigned int pos2, int * is_PE_distance, int * is_same_chromosome , int rlen1, int rlen2); int FIXLENstrcmp(char * fixed_len, char * rname); int is_valid_digit(char * optarg, char * optname); int is_valid_digit_range(char * optarg, char * optname, int min, int max_inc); int is_valid_float(char * optarg, char * optname); int exec_cmd(char * cmd, char * outstr, int out_limit); int is_pos_in_annotated_exon_regions(global_context_t * global_context, unsigned int pos); char * get_sam_chro_name_from_alias(HashTable * tab, char * anno_chro); void subread_rebuild_cmd(int argc, char ** argv, global_context_t * global_context); int get_soft_clipping_length(char* CIGAR); #endif Rsubread/src/detection-calls.c0000644000175200017520000004650614136047615017372 0ustar00biocbuildbiocbuild#include #include #include #include #include #include #include #include "subread.h" #include "HelperFunctions.h" #include "hashtable.h" #include "input-files.h" #include "gene-algorithms.h" typedef struct{ char gene_name[FEATURE_NAME_LENGTH+2]; char chro_name[MAX_CHROMOSOME_NAME_LEN+2]; unsigned int start, end; char is_negative_strand; } DTCexon_t; typedef struct { ArrayList * exon_table; ArrayList * chro_list; HashTable * chro_4bit_table; HashTable * chro_length_table; HashTable * gene_to_GC_TOTAL_table; HashTable * sam_chro_to_anno_chr_alias; FILE * out_FP_genes, * out_FP_bins; char out_file_name[MAX_FILE_NAME_LENGTH]; char anno_file_name[MAX_FILE_NAME_LENGTH]; char alias_file_name[MAX_FILE_NAME_LENGTH]; int anno_file_format; char gene_id_column_name[MAX_FILE_NAME_LENGTH]; char transcript_id_column_name[MAX_FILE_NAME_LENGTH]; char used_feature_type[MAX_FILE_NAME_LENGTH]; char fasta_file_name[MAX_FILE_NAME_LENGTH]; int use_intron_bins; int filled_bins; } DTCcontext_t; int DTCdo_add_feature(char * gene_name, char * transcript_name, char * chro_name, unsigned int start, unsigned int end, int is_negative_strand, void * vcontext){ DTCcontext_t * context = vcontext; DTCexon_t * new_exon = malloc(sizeof(DTCexon_t)); if(context -> sam_chro_to_anno_chr_alias){ char * sam_chro = get_sam_chro_name_from_alias(context -> sam_chro_to_anno_chr_alias, chro_name); if(sam_chro!=NULL) chro_name = sam_chro; } strncpy(new_exon -> gene_name, gene_name, FEATURE_NAME_LENGTH); strncpy(new_exon -> chro_name, chro_name, MAX_CHROMOSOME_NAME_LEN); new_exon -> start = start; new_exon -> end = end; new_exon -> is_negative_strand = is_negative_strand; ArrayListPush(context -> exon_table , new_exon); return 0; } int DTCcompare_merge_genes(void * L_elem, void * R_elem, ArrayList *me){ unsigned int * le = L_elem, * re = R_elem; if(le[1] > re[1]) return 1; if(le[1] < re[1]) return -1; return 0; } int DTCcompare_exons(void * L_elem, void * R_elem, ArrayList *me){ DTCexon_t * le = L_elem, * re = R_elem; int chname_cmp = strcmp(le -> chro_name, re -> chro_name); if(chname_cmp) return chname_cmp; long long lll = le -> start; lll -= re -> start; if(lll > 0) return 1; if(lll < 0) return -1; return 0; } #define BIT2_START_SIZE 1024 void DTCload_fasta(DTCcontext_t * context){ FILE * fp = fopen(context -> fasta_file_name, "r"); assert(fp); char * base_2bits = NULL;//malloc(BIT2_START_SIZE); unsigned int base_2bits_capasity = BIT2_START_SIZE*2; unsigned int base_current_chro_len = 0; char * curr_chro_mem = NULL; while(!feof(fp)){ char fasta_line[501]; char * fl = fgets(fasta_line, 501, fp); if(!fl) break; if(fl[0]=='>'){ if(curr_chro_mem){ ArrayListPush(context -> chro_list, curr_chro_mem); HashTablePut(context -> chro_length_table, curr_chro_mem , NULL + base_current_chro_len); HashTablePut(context -> chro_4bit_table, curr_chro_mem , base_2bits); } curr_chro_mem = malloc(strlen(fl)); int ii; for(ii = 1; fl[ii] != '\n' && fl[ii] != '\r' && fl[ii] != 0 ; ii++){ curr_chro_mem[ii-1] = fl[ii]; } curr_chro_mem[ii-1] = 0; base_current_chro_len = 0; base_2bits = malloc(BIT2_START_SIZE); base_2bits_capasity = BIT2_START_SIZE*2; }else{ int ii; for(ii=0; fl[ii] != '\n' && fl[ii] != '\r' && fl[ii] != 0 ; ii++){ if(base_2bits_capasity <= base_current_chro_len){ if(base_2bits_capasity < 1000000) base_2bits_capasity = base_2bits_capasity*2; else base_2bits_capasity = base_2bits_capasity*3/2; base_2bits = realloc(base_2bits, base_2bits_capasity/2 ); HashTablePutReplaceEx(context -> chro_4bit_table, curr_chro_mem , base_2bits,0,0,0); } int ubase = toupper(fl[ii]), b2v=15; if(ubase == 'A')b2v = 0; else if(ubase == 'C')b2v = 1; else if(ubase == 'G')b2v = 2; else if(ubase == 'T')b2v = 3; int new_bch = base_2bits[ base_current_chro_len/2 ]; if( base_current_chro_len%2 ) new_bch = ( b2v << 4 ) | (new_bch&15); else new_bch = b2v; base_2bits[ base_current_chro_len/2 ] = new_bch; base_current_chro_len ++; } } } if(curr_chro_mem){ ArrayListPush(context -> chro_list, curr_chro_mem); HashTablePut(context -> chro_length_table, curr_chro_mem , NULL + base_current_chro_len); HashTablePut(context -> chro_4bit_table, curr_chro_mem , base_2bits); } SUBREADprintf("%lld items loaded from FASTA.\n", context -> chro_4bit_table -> numOfElements); fclose(fp); } void DTCadd_annotation(DTCcontext_t * context, char * gene_name, char * chro_name, unsigned int start, unsigned int stop, int is_negative_strand, unsigned int GCs, unsigned int totals, int is_bins){ float fc = NAN; if(totals>0) fc =(float)GCs/totals; fprintf(is_bins? context -> out_FP_bins :context -> out_FP_genes , "%s\t%s\t%u\t%u\t%c\t%.5f\n", gene_name, chro_name, start, stop, is_negative_strand?'-':'+', fc); } int DTCwrite_annotations(char * gene_name, char * transcript_name, char * chro_name, unsigned int start, unsigned int end, int is_negative_strand, void * vcontext){ DTCcontext_t * context = vcontext; unsigned int * gc_2int = HashTableGet(context ->gene_to_GC_TOTAL_table, gene_name); if(!gc_2int) gc_2int = (unsigned int[]){0,0,0}; DTCadd_annotation(context, gene_name, chro_name, start, end, is_negative_strand, gc_2int[0], gc_2int[1], 0); return 0; } void DTCprint_lentab_items(void * key, void * hashed_obj, HashTable * tab){ SUBREADprintf("%s => %ld\n", (char*)key, (long)(hashed_obj - NULL)); } // start and end are 1-based // chro 4bit bin are 0-based unsigned int DTCtotal_GC_bases(DTCcontext_t * context, char * chro, unsigned int start, unsigned int end, unsigned int * total_bases){ unsigned int ret = 0; char * bin2 = HashTableGet(context -> chro_4bit_table, chro); unsigned int chro_len = HashTableGet(context -> chro_length_table, chro) - NULL; //SUBREADprintf("TCHO: %s : %p , len = %u , range = %u ~ %u\n", chro, bin2, chro_len, start, end); if(NULL == bin2 && strlen(chro) <3){ char tna [MAX_CHROMOSOME_NAME_LEN]; sprintf(tna, "chr%s", chro); bin2 = HashTableGet(context -> chro_4bit_table, tna); chro_len = HashTableGet(context -> chro_length_table, tna) - NULL; //HashTableIteration(context -> chro_length_table, DTCprint_lentab_items); } if(NULL == bin2 && strlen(chro) >3 && memcmp("chr", chro, 3)==0){ char *tna = chro + 3; bin2 = HashTableGet(context -> chro_4bit_table, tna); chro_len = HashTableGet(context -> chro_length_table, tna) - NULL; } if(chro_len < end) return 0; unsigned int ii; for(ii = start - 1; ii < end; ii++){ int byte_no = ii / 2; int baseint = (ii%2)?( (bin2[byte_no]>> 4) & 0xf ):( bin2[byte_no] & 0xf ); ret += (baseint == 1 || baseint == 2); (*total_bases) += (baseint<4); } return ret; } #define DTC_GAP_BIN_MIN_LENGTH 2000 void DTCanno_fill_gap(DTCcontext_t * context, char * chro_name, unsigned int start, unsigned int stop){ //SUBREADprintf("FILL: %s - %u ~ %u\n", chro_name, start, stop); unsigned int cur; for(cur = start; ; cur += DTC_GAP_BIN_MIN_LENGTH){ unsigned int bin_end = cur + DTC_GAP_BIN_MIN_LENGTH; if(bin_end > stop + 1 - DTC_GAP_BIN_MIN_LENGTH) bin_end = stop +1; unsigned int binlens = 0; unsigned int GCs = DTCtotal_GC_bases(context, chro_name, cur, bin_end - 1, &binlens); char bin_name[40]; sprintf(bin_name, "_fill_bin_%07d", context -> filled_bins++); DTCadd_annotation(context, bin_name, chro_name, cur, bin_end - 1, 0, GCs, binlens, 1); if(bin_end >= stop) break; } } void DTCprocess_gene_tab(void * key, void * hashed_obj, HashTable * tab){ ArrayList * merged_gene_list = tab -> appendix1; ArrayListPush(merged_gene_list , hashed_obj); } typedef struct{ ArrayList * gene_3int_ptr_list; unsigned int start, end; } MergedExon_t; void DTC_free_ME_list(void * mel){ MergedExon_t * me = mel; ArrayListDestroy(me -> gene_3int_ptr_list); free(mel); } void DTC_arrayListDestroy(void * l){ ArrayListDestroy(( ArrayList *)l); } int DTCparse_GTF_and_Genome(DTCcontext_t * context){ DTCload_fasta(context); load_features_annotation(context -> anno_file_name, context -> anno_file_format, context -> gene_id_column_name, context -> transcript_id_column_name, context -> used_feature_type, context, DTCdo_add_feature); SUBREADprintf("%lld items loaded from annotation file.\n", context -> exon_table -> numOfElements); long chri, ii; HashTable * chro_to_exons_table = HashTableCreate(1000); HashTableSetDeallocationFunctions(chro_to_exons_table, free, DTC_arrayListDestroy); HashTableSetKeyComparisonFunction(chro_to_exons_table, my_strcmp); HashTableSetHashFunction(chro_to_exons_table, fc_chro_hash); HashTable * gene_to_GC_TOTAL_table = HashTableCreate(10000); HashTableSetDeallocationFunctions(gene_to_GC_TOTAL_table, free, free); HashTableSetKeyComparisonFunction(gene_to_GC_TOTAL_table, my_strcmp); HashTableSetHashFunction(gene_to_GC_TOTAL_table, fc_chro_hash); for(ii = 0; ii < context -> exon_table -> numOfElements; ii++){ DTCexon_t * tmpexon = ArrayListGet(context -> exon_table , ii); ArrayList * in_chro_exons = HashTableGet(chro_to_exons_table, tmpexon -> chro_name); if(NULL == in_chro_exons){ in_chro_exons = ArrayListCreate(2000); char * memky = strdup(tmpexon -> chro_name); HashTablePut(chro_to_exons_table, memky, in_chro_exons); } ArrayListPush(in_chro_exons, tmpexon); } for(chri = 0; chri < context -> chro_list -> numOfElements ; chri++){ char * current_chro = ArrayListGet(context -> chro_list, chri); ArrayList * in_chro_exons = HashTableGet(chro_to_exons_table , current_chro); if(NULL == in_chro_exons && memcmp("chr", current_chro, 3) == 0){ char * tchro = current_chro + 3; in_chro_exons = HashTableGet(chro_to_exons_table , tchro); } if(NULL == in_chro_exons && strlen(current_chro) < 3){ char tchro[MAX_CHROMOSOME_NAME_LEN]; sprintf(tchro, "chr%s", current_chro); in_chro_exons = HashTableGet(chro_to_exons_table , tchro); } if(NULL == in_chro_exons) continue; HashTable * genename_to_3int_table = HashTableCreate(10000); HashTableSetDeallocationFunctions(genename_to_3int_table, free, free); HashTableSetKeyComparisonFunction(genename_to_3int_table, my_strcmp); HashTableSetHashFunction(genename_to_3int_table, fc_chro_hash); ArrayListSort(in_chro_exons, DTCcompare_exons); assert(in_chro_exons -> numOfElements > 0); SUBREADprintf("Annotations for %s (%lld) were sorted.\n", current_chro, in_chro_exons -> numOfElements); unsigned int gene_number = 1; char * current_chro_name_in_anno = NULL; for(ii = 0; ii < in_chro_exons -> numOfElements; ii++){ DTCexon_t * tmpexon = ArrayListGet(in_chro_exons , ii); if(ii==0) current_chro_name_in_anno = tmpexon -> chro_name; char * tmpky = tmpexon -> gene_name; unsigned int * tmp_gene_info = HashTableGet(genename_to_3int_table, tmpky); unsigned int * gc_total_2int = HashTableGet(gene_to_GC_TOTAL_table, tmpky); if(tmp_gene_info == NULL){ tmp_gene_info = malloc(sizeof(unsigned int) * 3); tmp_gene_info[0] = gene_number++; tmp_gene_info[1] = tmpexon -> start; tmp_gene_info[2] = tmpexon -> end; if(NULL == gc_total_2int){ gc_total_2int = malloc(sizeof(int)*2); gc_total_2int[0] = gc_total_2int[1] = 0; HashTablePut(gene_to_GC_TOTAL_table, strdup(tmpky), gc_total_2int); } gc_total_2int[0] += DTCtotal_GC_bases(context, current_chro, tmpexon -> start, tmpexon -> end, &gc_total_2int[1]); char * memky = strdup(tmpky); HashTablePut(genename_to_3int_table , memky , tmp_gene_info); } else { assert(tmpexon -> start >= tmp_gene_info[1]); unsigned int new_start = max(tmpexon -> start, tmp_gene_info[2] +1); if(new_start <= tmpexon -> end){ gc_total_2int[0] += DTCtotal_GC_bases(context, current_chro, new_start, tmpexon -> end, &gc_total_2int[1]); tmp_gene_info[2] = max(tmp_gene_info[2], tmpexon -> end); } } } /*for(ii = 0; ii < in_chro_exons -> numOfElements; ii++){ DTCexon_t * tmpexon = ArrayListGet(in_chro_exons , ii); unsigned int * tmp_gene_info = HashTableGet(genename_to_3int_table , tmpexon -> gene_name); DTCadd_annotation(context, tmpexon -> gene_name, current_chro, tmpexon -> start, tmpexon -> end, tmpexon -> is_negative_strand, tmp_gene_info[3], tmp_gene_info[4]); }*/ if(context -> use_intron_bins){ DTCexon_t * tmpexon = ArrayListGet(in_chro_exons , 0); unsigned int top_start = tmpexon -> start; unsigned int top_end = tmpexon -> end; ArrayList * current_ME_3int_ptrs = ArrayListCreate(5); ArrayList * merged_exon_list = ArrayListCreate(5000); ArrayListSetDeallocationFunction(merged_exon_list, DTC_free_ME_list); ArrayListPush(current_ME_3int_ptrs, HashTableGet(genename_to_3int_table, tmpexon -> gene_name)); for(ii = 1; ii < in_chro_exons -> numOfElements; ii++){ tmpexon = ArrayListGet(in_chro_exons , ii); if(top_end < tmpexon -> start){ MergedExon_t * new_ME = malloc(sizeof(MergedExon_t)); new_ME -> gene_3int_ptr_list = current_ME_3int_ptrs; new_ME -> start = top_start; new_ME -> end = top_end; ArrayListPush(merged_exon_list, new_ME); top_start = tmpexon -> start; top_end = tmpexon -> end; current_ME_3int_ptrs = ArrayListCreate(5); ArrayListPush(current_ME_3int_ptrs, HashTableGet(genename_to_3int_table, tmpexon -> gene_name)); }else if(top_end < tmpexon -> end){ top_end = tmpexon -> end; ArrayListPush_NoRepeatedPtr(current_ME_3int_ptrs, HashTableGet(genename_to_3int_table, tmpexon -> gene_name)); }else{ ArrayListPush_NoRepeatedPtr(current_ME_3int_ptrs, HashTableGet(genename_to_3int_table, tmpexon -> gene_name)); } } if(1){ MergedExon_t * new_ME = malloc(sizeof(MergedExon_t)); new_ME -> gene_3int_ptr_list = current_ME_3int_ptrs; new_ME -> start = top_start; new_ME -> end = top_end; ArrayListPush(merged_exon_list, new_ME); } unsigned int last_exon_end = 0; unsigned int max_exon_end = 0; for(ii = 0; ii < merged_exon_list -> numOfElements; ii++){ long ingene; MergedExon_t * current_ME = ArrayListGet(merged_exon_list, ii); if(last_exon_end>0){ assert(last_exon_end < current_ME -> start); DTCanno_fill_gap(context, current_chro_name_in_anno, last_exon_end + 1, current_ME -> start -1); } for(ingene = 0; ingene < current_ME -> gene_3int_ptr_list -> numOfElements; ingene++){ unsigned int *int3_gene = ArrayListGet(current_ME -> gene_3int_ptr_list, ingene); max_exon_end = max(max_exon_end, int3_gene[2]); } //SUBREADprintf("MAX_EXON_END = %u , THIS = %u - %u\n" , max_exon_end, current_ME -> start, current_ME -> end); if( max_exon_end > current_ME -> end ){ last_exon_end = current_ME -> end; } else last_exon_end = 0; } ArrayListDestroy(merged_exon_list); }else{ ArrayList * merged_gene_list = ArrayListCreate(10000); genename_to_3int_table -> appendix1 = merged_gene_list; HashTableIteration(genename_to_3int_table ,DTCprocess_gene_tab); ArrayListSort(merged_gene_list, DTCcompare_merge_genes); SUBREADprintf("%s has %lld merged genes from %lld item hashtable\n", current_chro, merged_gene_list -> numOfElements, genename_to_3int_table -> numOfElements); assert(merged_gene_list -> numOfElements>0); unsigned int * geneints = ArrayListGet(merged_gene_list, 0); unsigned int top_start = geneints[1]; unsigned int top_end = geneints[2]; DTCanno_fill_gap(context, current_chro_name_in_anno, 1, top_start); for(ii = 1; ii < merged_gene_list -> numOfElements; ii++){ geneints = ArrayListGet(merged_gene_list, ii); if(top_end < geneints[1]){ DTCanno_fill_gap(context, current_chro_name_in_anno, top_end + 1, geneints[1] - 1); top_start = geneints[1]; top_end = geneints[2]; }else if(top_end < geneints[2]) top_end = geneints[2]; } DTCanno_fill_gap(context, current_chro_name_in_anno, top_end + 1, HashTableGet(context -> chro_length_table, current_chro) - NULL - 1); } HashTableDestroy(genename_to_3int_table); } context -> gene_to_GC_TOTAL_table = gene_to_GC_TOTAL_table; load_features_annotation(context -> anno_file_name, context -> anno_file_format, context -> gene_id_column_name, context -> transcript_id_column_name, context -> used_feature_type, context, DTCwrite_annotations); HashTableDestroy(chro_to_exons_table); HashTableDestroy(gene_to_GC_TOTAL_table); return 0; } int DTCinit_context(DTCcontext_t ** context, int argc, char ** argv){ DTCcontext_t * ret = malloc(sizeof(DTCcontext_t)); memset(ret, 0, sizeof(DTCcontext_t)); ret -> filled_bins = 0; ret -> anno_file_format = FILE_TYPE_GTF; ret -> alias_file_name [0] = 0; strcpy(ret -> gene_id_column_name, "gene_id"); strcpy(ret -> transcript_id_column_name, "transcript_id"); strcpy(ret -> used_feature_type, "exon"); ret -> chro_list = ArrayListCreate(100); ret -> exon_table = ArrayListCreate(100); ArrayListSetDeallocationFunction(ret -> exon_table, free); ret -> chro_4bit_table = HashTableCreate(100); HashTableSetDeallocationFunctions(ret -> chro_4bit_table, free, free); HashTableSetKeyComparisonFunction(ret -> chro_4bit_table, my_strcmp); HashTableSetHashFunction(ret -> chro_4bit_table, fc_chro_hash); ret -> chro_length_table = HashTableCreate(100); // chro_length_table is deallocated when chro_4bit_table is deallocated. HashTableSetKeyComparisonFunction(ret -> chro_length_table, my_strcmp); HashTableSetHashFunction(ret -> chro_length_table, fc_chro_hash); int c = 0; optind = 0; opterr = 1; optopt = 63; while ((c = getopt (argc, argv, "a:G:o:F:A:g:e:I"))!=-1){ switch(c){ case 'e': strcpy(ret -> used_feature_type, optarg); break; case 'g': strcpy(ret -> gene_id_column_name, optarg); break; case 'A': strcpy(ret -> alias_file_name, optarg); break; case 'F': if(strcmp("GTF", optarg)==0) ret -> anno_file_format = FILE_TYPE_GTF; else if(strcmp("SAF", optarg)==0) ret -> anno_file_format = FILE_TYPE_RSUBREAD; else{ SUBREADprintf("ERROR: unknown annotation format: %s\n", optarg); return 1; } break; case 'o': strcpy(ret -> out_file_name, optarg); break; case 'a': strcpy(ret -> anno_file_name, optarg); break; case 'G': strcpy(ret -> fasta_file_name, optarg); break; case 'I': ret -> use_intron_bins = 1; break; } } if(ret -> alias_file_name[0]) ret -> sam_chro_to_anno_chr_alias = load_alias_table(ret -> alias_file_name); else ret -> sam_chro_to_anno_chr_alias = NULL; ret -> out_FP_genes = fopen(ret -> out_file_name,"w"); char binfn[MAX_FILE_NAME_LENGTH+12]; sprintf(binfn,"%s-bins", ret -> out_file_name); ret -> out_FP_bins = fopen(binfn,"w"); fprintf(ret -> out_FP_genes, "GeneID\tChr\tStart\tEnd\tStrand\tGCfraction\n"); fprintf(ret -> out_FP_bins, "GeneID\tChr\tStart\tEnd\tStrand\tGCfraction\n"); *context = ret; return 0; } int DTCdestroy_context(DTCcontext_t *context){ ArrayListDestroy(context -> exon_table); ArrayListDestroy(context -> chro_list); if(context -> sam_chro_to_anno_chr_alias)HashTableDestroy(context -> sam_chro_to_anno_chr_alias); HashTableDestroy(context -> chro_length_table); HashTableDestroy(context -> chro_4bit_table); fclose(context -> out_FP_genes); fclose(context -> out_FP_bins); free(context); return 0; } #ifdef MAKE_STANDALONE int main(int argc, char ** argv) #else int detectionCall_Main(int argc, char ** argv) #endif { DTCcontext_t * context; int ret = DTCinit_context(&context, argc, argv); ret = ret || DTCparse_GTF_and_Genome(context); ret = ret || DTCdestroy_context(context); return ret; } Rsubread/src/detectionCall.c0000644000175200017520000002610414136047615017062 0ustar00biocbuildbiocbuild/*************************************************************** The Subread and Rsubread software packages are free software packages: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #include #include #include #include #include #include //typedef int int; /* INPUT: SAM FILE * GENE(EXON) ANNOTATION FILE BINSIZE=2000 WITH GC CONTENT * BG ANNOTATION FILE BINSIZE=2000 WITH GC CONTENT * OUTPUT: MAPPING RESULT FOR TWO ANNOTATION FILES * * PURPOSE: THE OUTPUT OF THIS FUNCTION IS THEN USED BY R FUNCTION * TO CALCULATE THE P-VALUE FOR EACH GENE * */ /* 1. build up data structure for exon and integenic region * 2. simplify SAM, save just chr_id and read_position * 3. read_mapping procedure, map this SAM file reads to exon/ir * */ /* Constants*/ #define BINSIZE 2000 #define STR 300 #define MAXCHRNUM 24 #define MAXREADSPERCHR 2000000 /* chromosome names are in alphabic order that */ char *chrs_map[] = {"chr1", "chr10", "chr11","chr12", "chr13", "chr14","chr15","chr16", "chr17","chr18", "chr19", "chr2","chr20","chr21","chr22","chr3", "chr4", "chr5","chr6", "chr7", "chr8","chr9", "chrX","chrY"}; /* Variables*/ char *SAM_file; char *simplified_SAM_file; char *sorted_simplified_SAM_file; char *annotation_exon_file; char *annotation_ir_file; char *mapping_result_exon; char *mapping_result_ir; /* Time Variables*/ time_t start_time; time_t timer; double timediff; /* Data Structure */ typedef struct an_node{ int start, end, gene; int read, nnum, gcnum, atnum; struct an_node *next; }node; typedef struct a_chr{ char *id; node *list; }chr; chr exon[MAXCHRNUM]; chr ir[MAXCHRNUM]; int chr_num; char current_chr_id[STR]; void generate_filenames(char *dataset, char *exon_file, char *ir_file, char *temp_header){ SAM_file = (char *)malloc(STR); simplified_SAM_file = (char *)malloc(STR); sorted_simplified_SAM_file = (char *)malloc(STR); annotation_exon_file = (char *)malloc(STR); annotation_ir_file = (char *)malloc(STR); mapping_result_exon = (char *)malloc(STR); mapping_result_ir = (char *)malloc(STR); strcpy(SAM_file, dataset); strcpy(simplified_SAM_file, temp_header); strcat(simplified_SAM_file,"_simplified.txt"); strcpy(sorted_simplified_SAM_file, temp_header); strcat(sorted_simplified_SAM_file,"_simplified_sorted.txt"); strcpy(annotation_exon_file, exon_file); strcpy(annotation_ir_file, ir_file); strcpy(mapping_result_exon, temp_header); strcat(mapping_result_exon,"_mapping_exon_GC.txt"); strcpy(mapping_result_ir, temp_header); strcat(mapping_result_ir,"_mapping_binned_integenic_region_GC.txt"); } void * make_empty_node_map(void){ node *new; new = (node *) malloc(sizeof(node)); new->next = NULL; new->start = 0; new->end = 0; new->gene = 0; new->read = 0; new->nnum = 0; new->gcnum = 0; new->atnum = 0; return new; } void build_exon_data_structure_map(void){ FILE *fin; int read_start, read_end, read_entrezid, read_n, read_gc, read_at; char chr_id[STR]; node *cnode=NULL; char current_chr_id[STR]; chr_num = 0; fin = fopen(annotation_exon_file,"r"); while (fscanf(fin, "%d %s %d %d %d %d %d",&read_entrezid, chr_id, &read_start, &read_end, &read_n, &read_gc, &read_at) != -1){ if (strcmp(chr_id, current_chr_id) != 0){ strcpy(current_chr_id, chr_id); chr_num++; exon[chr_num-1].id = (char *)malloc(STR); strcpy(exon[chr_num-1].id, chr_id); exon[chr_num-1].list = (node *)make_empty_node_map(); cnode = exon[chr_num-1].list; } node *new_node; new_node = (node *)make_empty_node_map(); new_node->start = read_start; new_node->end = read_end; new_node->gene = read_entrezid; new_node->nnum = read_n; new_node->gcnum = read_gc; new_node->atnum = read_at; cnode->next = new_node; cnode = cnode->next; } fclose(fin); } void build_ir_data_structure_map(void){ FILE *fin; int read_start, read_end, read_n, read_gc, read_at; char chr_id[STR]; node *cnode=NULL; char current_chr_id[STR]; chr_num = 0; fin = fopen(annotation_ir_file,"r"); while (fscanf(fin, "%s %d %d %d %d %d",chr_id, &read_start, &read_end, &read_n, &read_gc, &read_at) != -1){ if (strcmp(chr_id, current_chr_id) != 0){ strcpy(current_chr_id, chr_id); chr_num++; ir[chr_num-1].id = (char *)malloc(STR); strcpy(ir[chr_num-1].id, chr_id); ir[chr_num-1].list = (node *)make_empty_node_map(); cnode = ir[chr_num-1].list; } node *new_node; new_node = (node *)make_empty_node_map(); new_node->start = read_start; new_node->end = read_end; new_node->nnum = read_n; new_node->gcnum = read_gc; new_node->atnum = read_at; cnode->next = new_node; cnode = cnode->next; } fclose(fin); } void simplify_SAM_file(void){ FILE *fin; FILE *fout; char * line = NULL; char * read_chr; size_t len = 1000; // size_t z; int read_pos; char *readline; fin = fopen(SAM_file,"r"); fout = fopen(simplified_SAM_file,"w"); line = (char *)malloc(len+1); while ((readline = fgets(line, len, fin)) != NULL){ if(line[0] == '@') continue; strtok(line,"\t"); strtok(NULL,"\t"); read_chr = strtok(NULL,"\t"); if(*read_chr == '*') continue; read_pos = atoi(strtok(NULL,"\t")); fprintf(fout, "%s\t%d\n", read_chr, read_pos); } fclose(fin); fclose(fout); } void read_mapping(void){ /* local variables */ FILE *fread; char read_chr[STR]; int read_pos, count; int chr_pos; node *cnode; /* read_mapping_exon*/ chr_pos = 0; // count is the counter, every 2,000,000 for a chromosome, // we reset the current node to the head of list count = 0; cnode = exon[chr_pos].list->next; fread = fopen(sorted_simplified_SAM_file,"r"); while ((fscanf(fread, "%s %d", read_chr, &read_pos)) != -1){ // firstly find the chromosome if (strcmp(read_chr, exon[chr_pos].id) != 0){ chr_pos++; cnode = exon[chr_pos].list->next; count = 1; } else { count++; } if (chr_pos == chr_num){ // This read has an unknown chromosome id // do nothing } else { // go through the list, find the exon position. // increment read by 1 if ((read_pos >= cnode->start) && (read_pos <= cnode->end)){ cnode->read++; } else { // search the rest of list while ((cnode->next != NULL) && (cnode->next->start < read_pos)){ cnode = cnode->next; } if ((cnode->start <= read_pos) && (cnode->end >= read_pos)){ cnode->read++; } } } if (count == MAXREADSPERCHR){ // reset the current node cnode = exon[chr_pos].list->next; } } fclose(fread); /* read_mapping_integenic_region*/ chr_pos = 0; count = 0; cnode = ir[chr_pos].list->next; fread = fopen(sorted_simplified_SAM_file,"r"); while ((fscanf(fread, "%s %d", read_chr, &read_pos)) != -1){ // firstly find the chromosome if (strcmp(read_chr, ir[chr_pos].id) != 0){ chr_pos++; cnode = ir[chr_pos].list->next; count = 1; } else { count++; } if (chr_pos == chr_num){ // This read has an unknown chromosome id // do nothing } else { // go through the list, find the exon position. // increment read by 1 if ((read_pos >= cnode->start) && (read_pos <= cnode->end)){ cnode->read++; } else { // search the rest of list while ((cnode->next != NULL) && (cnode->next->start < read_pos)){ cnode = cnode->next; } if ((cnode->start <= read_pos) && (cnode->end >= read_pos)){ cnode->read++; } } } if (count == MAXREADSPERCHR){ // reset the current node cnode = exon[chr_pos].list->next; } } fclose(fread); } void output_mapping_result(void){ /* Local Variables */ int i; node *dh; /* Output exon result to file*/ FILE *fout_exon = fopen(mapping_result_exon, "w"); fprintf(fout_exon,"entrezid\tchr\tchr_start\tchr_stop\tnnum\tgcnum\tatnum\tnreads\n"); for(i=0; inext != NULL){ dh = dh->next; fprintf(fout_exon, "%d\t%s\t%d\t%d\t%d\t%d\t%d\t%d\n", dh->gene, exon[i].id, dh->start, dh->end, dh->nnum, dh->gcnum, dh->atnum, dh->read); } } fclose(fout_exon); /* Output integenic result to file*/ FILE *fout_ir = fopen(mapping_result_ir, "w"); fprintf(fout_ir,"chr\tchr_start\tchr_stop\tnnum\tgcnum\tatnum\tnreads\n"); for(i=0; inext != NULL){ dh = dh->next; fprintf(fout_ir, "%s\t%d\t%d\t%d\t%d\t%d\t%d\n", ir[i].id, dh->start, dh->end, dh->nnum, dh->gcnum, dh->atnum, dh->read); } } fclose(fout_ir); } void q_sort(int numbers[], int left, int right) { int pivot, l_hold, r_hold; l_hold = left; r_hold = right; pivot = numbers[left]; while (left < right) { while ((numbers[right] >= pivot) && (left < right)) right--; if (left != right) { numbers[left] = numbers[right]; left++; } while ((numbers[left] <= pivot) && (left < right)) left++; if (left != right) { numbers[right] = numbers[left]; right--; } } numbers[left] = pivot; pivot = left; left = l_hold; right = r_hold; if (left < pivot) q_sort(numbers, left, pivot-1); if (right > pivot) q_sort(numbers, pivot+1, right); } /* From the simplified SAM file generated above * for each chromosome id, find all reads on that chromosome * sort the read positions and then output to a sorted_SAM_simplified file*/ void sort_reads(void){ FILE *freadin; FILE *freadout; int pos[MAXREADSPERCHR]; int i; char read_chr[STR]; int read_pos; int count; int j; freadout = fopen(sorted_simplified_SAM_file,"w"); for (i=0; i<24; i++){ count = 0; // extracting reads with chromosome id == chrs_map[i] freadin = fopen(simplified_SAM_file,"r"); while ((fscanf(freadin, "%s %d", read_chr, &read_pos)) != -1){ if (strcmp(read_chr, chrs_map[i]) == 0){ // this read is on the target chromosome pos[count] = read_pos; count++; if (count == MAXREADSPERCHR){ // sort these reads from the particular chromosome // then write them to sorted file q_sort(pos, 0, count-1); // export sorted list to file. for (j=0; j #include #include #include #include //typedef int int32_t; /* Constants */ #define MAXCHRNUM 24 #define STR 100 //maximum string length #define NPERCENTAGE 0.3 /* Data Structure */ typedef struct an_node{ int start, end, gene; int read, nnum, gcnum, atnum; struct an_node *next; }node; typedef struct a_chr{ char *id; node *list; }chr; /* Variables */ int binsize; chr DCC_exon[MAXCHRNUM]; chr DCC_ir[MAXCHRNUM]; int DCC_chr_num; char DCC_current_chr_id[STR]; char *utr_cds_file; char *DCC_exon_file; char *DCC_ir_file; char *binned_DCC_ir_file; char *fa_header; void * make_empty_node(void){ node *new; new = (node *) malloc(sizeof(node)); new->next = NULL; new->start = 0; new->end = 0; new->gene = 0; new->read = 0; new->nnum = 0; new->gcnum = 0; new->atnum = 0; return new; } /*cheating function, build DCC_exon data structure from DCC_exon file*/ void build_DCC_exon_data_structure(void){ FILE *fin; int read_start, read_end, read_entrezid; char chr_id[STR]; node *cnode=NULL; char DCC_current_chr_id[STR]; DCC_chr_num = 0; fin = fopen(DCC_exon_file,"r"); while (fscanf(fin, "%d %s %d %d",&read_entrezid, chr_id, &read_start, &read_end) != -1){ if (strcmp(chr_id, DCC_current_chr_id) != 0){ strcpy(DCC_current_chr_id, chr_id); DCC_chr_num++; //printf("new chromosome is: %s\n", chr_id); DCC_exon[DCC_chr_num-1].id = (char *)malloc(STR); strcpy(DCC_exon[DCC_chr_num-1].id, chr_id); DCC_exon[DCC_chr_num-1].list = (node *)make_empty_node(); cnode = DCC_exon[DCC_chr_num-1].list; } node *new_node; new_node = (node *)make_empty_node(); new_node->start = read_start; new_node->end = read_end; new_node->gene = read_entrezid; cnode->next = new_node; cnode = cnode->next; } fclose(fin); } /* Build from binned integenic region */ void build_DCC_ir_data_structure(void){ FILE *fin; int read_start, read_end; char chr_id[STR]; node *cnode=NULL; char DCC_current_chr_id[STR]; DCC_chr_num = 0; fin = fopen(binned_DCC_ir_file,"r"); while (fscanf(fin, "%s %d %d",chr_id, &read_start, &read_end) != -1){ if (strcmp(chr_id, DCC_current_chr_id) != 0){ strcpy(DCC_current_chr_id, chr_id); DCC_chr_num++; DCC_ir[DCC_chr_num-1].id = (char *)malloc(STR); strcpy(DCC_ir[DCC_chr_num-1].id, chr_id); DCC_ir[DCC_chr_num-1].list = (node *)make_empty_node(); cnode = DCC_ir[DCC_chr_num-1].list; } node *new_node; new_node = (node *)make_empty_node(); new_node->start = read_start; new_node->end = read_end; cnode->next = new_node; cnode = cnode->next; } fclose(fin); } /* BREAK INTEGENIC REGION INTO BINSIZE BINS * IN THIS APPROACH, WE MAKE SURE ALL BINS ARE IN BINSIZE, * WE IGNORE BINS THAT ARE SMALLER THAN BINSIZE FOR INTEGENIC REGION*/ void breakIntegenicRegion(){ FILE *fin; FILE *fout; int read_start, read_end; char chr_id[STR]; int binnum = 0; fin = fopen(DCC_ir_file, "r"); fout = fopen(binned_DCC_ir_file, "w"); while (fscanf(fin, "%s %d %d", chr_id, &read_start, &read_end) != -1){ if ((read_end - read_start +1) >= binsize){ // need to break this integenic region // break it down into bins with bins being BINSIZE length and last being [binsize, 2*binsize) while ((read_end - read_start + 1) >= binsize){ fprintf(fout, "%s\t%d\t%d\n",chr_id, read_start, (read_start+binsize-1)); read_start = read_start + binsize; binnum++; } } } fclose(fin); fclose(fout); } void calculateExonGCContent(void){ int i,j; FILE *fseq; int pos; char line[200]; int len; node *bin; int linenum; char *filename; for (i=0; inext; pos = 0; linenum = 0; while ( fgets ( line, sizeof(line), fseq) != NULL ){ linenum++; len = strlen(line)-1; for (j=0;j= bin->start) && (pos <= bin->end)){ switch (toupper(line[j])) { case 'N': bin->nnum++; break; case 'G': bin->gcnum++; break; case 'C': bin->gcnum++; break; case 'A': bin->atnum++; break; case 'T': bin->atnum++; break; default: break; } } else { if (pos > bin->end){ bin = bin->next; } } // end-else } // end-if } // end-for } // end-while } // end-for /*remove heavyN DCC_exons*/ node *to_remove; for (i=0; inext != NULL){ if ((1.0*bin->next->nnum/(bin->next->end - bin->next->start + 1)) > NPERCENTAGE){ // this bin contains too much N, remove it to_remove = bin->next; bin->next = bin->next->next; free(to_remove); } else { bin = bin->next; } } } /* Output result to file*/ char *DCC_exon_GC_file = "exon_GC.txt"; FILE *f_annotation_DCC_exon = fopen(DCC_exon_GC_file, "w"); node *dh; //fprintf(f_annotation_DCC_exon,"entrezid\tchr\tstart\tend\tnnum\tgcnum\tatnum\n"); for(i=0; inext != NULL){ dh = dh->next; fprintf(f_annotation_DCC_exon, "%d\t%s\t%d\t%d\t%d\t%d\t%d\n", dh->gene, DCC_exon[i].id, dh->start, dh->end, dh->nnum, dh->gcnum, dh->atnum); } } fclose(f_annotation_DCC_exon); } void calculateIRGCContent(void){ int i,j; FILE *fseq; int pos; char line[200]; int len; node *bin; int linenum; char *filename; for (i=0; inext; pos = 0; linenum = 0; while ( fgets ( line, sizeof(line), fseq) != NULL ){ linenum++; len = strlen(line)-1; for (j=0;j= bin->start) && (pos <= bin->end)){ switch (toupper(line[j])) { case 'N': bin->nnum++; break; case 'G': bin->gcnum++; break; case 'C': bin->gcnum++; break; case 'A': bin->atnum++; break; case 'T': bin->atnum++; break; default: break; } } else { if (pos > bin->end){ bin = bin->next; } } // end-else } // end-if } // end-for } // end-while } // end-for /*remove heavyN integenic region bins*/ node *to_remove; for (i=0; inext != NULL){ if ((1.0*bin->next->nnum/(bin->next->end - bin->next->start + 1)) > NPERCENTAGE){ // this bin contains too much N, remove it to_remove = bin->next; bin->next = bin->next->next; free(to_remove); } else { bin = bin->next; } } } /* Output result to file*/ char *DCC_ir_GC_file = "binned_integenic_region_GC.txt"; FILE *f_annotation_DCC_ir = fopen(DCC_ir_GC_file, "w"); node *dh; //fprintf(f_annotation_DCC_ir,"chr\tstart\tend\tnnum\tgcnum\tatnum\n"); for(i=0; inext != NULL){ dh = dh->next; fprintf(f_annotation_DCC_ir, "%s\t%d\t%d\t%d\t%d\t%d\n", DCC_ir[i].id, dh->start, dh->end, dh->nnum, dh->gcnum, dh->atnum); } } fclose(f_annotation_DCC_ir); } void DetectionCallAnnotationBody(void){ breakIntegenicRegion(); // printf("finsihed break into binss\n"); build_DCC_ir_data_structure(); //printf("The chr number after building DCC_ir structure is : %d\n", DCC_chr_num); calculateIRGCContent(); build_DCC_exon_data_structure(); //printf("The chr number after building DCC_exon structure is : %d\n", DCC_chr_num); calculateExonGCContent(); } void detectionCallAnnotation(char **DCC_exonfile, char **DCC_irfile, char **species, int *user_binsize){ // initialisation /* DCC_exon and DCC_ir files are pre-processed without unwanted chromosome data */ DCC_exon_file = (char *)malloc(STR); strcpy(DCC_exon_file, *DCC_exonfile); DCC_ir_file = (char *)malloc(STR); strcpy(DCC_ir_file, *DCC_irfile); fa_header = (char *)malloc(STR); binned_DCC_ir_file = (char *)malloc(STR); if (strcmp(*species, "hg") == 0){ strcpy(binned_DCC_ir_file, "hg19_binned_integenic_region.txt"); strcpy(fa_header,"human_sequence_data/hs_ref_GRCh37_"); } if (strcmp(*species, "mm") == 0){ strcpy(binned_DCC_ir_file, "mm9_binned_integenic_region.txt"); strcpy(fa_header, "mouse_sequence_data/"); } binsize = *user_binsize; // printf("going to main body of function\n"); DetectionCallAnnotationBody(); /* printf("\nUSAGE: DetectionCallAnnotation(DCC_exonFilename, integenicRegionFilename, binsizse)\n\n"); printf("BY DEFAULT: DCC_exonFilename = DCC_exon.txt\n\t\tintegenicRegionFilename = integenic_region.txt\n\t\tbinsize = 2000\n\n"); */ } Rsubread/src/exon-algorithms.c0000644000175200017520000000000614136047615017421 0ustar00biocbuildbiocbuild//SPA Rsubread/src/exon-algorithms.h0000644000175200017520000000000614136047615017426 0ustar00biocbuildbiocbuild//SPA Rsubread/src/exon-align-indel.c0000644000175200017520000000000614136047615017433 0ustar00biocbuildbiocbuild//SPA Rsubread/src/exon-align.c0000644000175200017520000000000614136047615016342 0ustar00biocbuildbiocbuild//SPA Rsubread/src/flattenAnnotations.c0000644000175200017520000003202014136047615020155 0ustar00biocbuildbiocbuild/*************************************************************** The Subread and Rsubread software packages are free software packages: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #define _GNU_SOURCE #include #include #include #include #include #include #include #ifndef MAKE_STANDALONE #include #endif #include #include #include #include #include "subread.h" #include "interval_merge.h" #include "core.h" #include "gene-algorithms.h" #include "sambam-file.h" #include "input-files.h" #include "hashtable.h" #include "seek-zlib.h" #include "HelperFunctions.h" #define MERGING_MODE_CHOPPING 100 // keep all the edges : [100,199] + [150,249] = [100,149], [150,199], [200,249] #define MERGING_MODE_MERGING 0 // just merge into one: [100,199] + [150,249] = [100, 249] typedef struct{ char GTF_gene_id_column[MAX_READ_NAME_LEN]; char GTF_wanted_feature_type[MAX_READ_NAME_LEN]; char GTF_file_name[MAX_FILE_NAME_LENGTH]; char output_file_name[MAX_FILE_NAME_LENGTH]; FILE * output_FP; int merging_mode; HashTable * gene_to_chro_strand_table; HashTable * gene_chro_strand_to_exons_table; } flatAnno_context_t; static struct option long_options[] = { {0, 0, 0, 0} }; void flatAnno_print_usage(){ SUBREADprintf("flattenGTF Version %s\n\n", SUBREAD_VERSION); SUBREADputs(" Flatten features included in a GTF annotation and save the modified annotation"); SUBREADputs(" to a SAF format file."); SUBREADputs(""); SUBREADputs("Usage:"); SUBREADputs(" ./flattenGTF [options] -a -o "); SUBREADputs(""); SUBREADputs("## Mandatory arguments: "); SUBREADputs(""); SUBREADputs(" -a Name of an annotation file in GTF/GFF format."); SUBREADputs(""); SUBREADputs(" -o Name of output file."); SUBREADputs(""); SUBREADputs("## Optional arguments: "); SUBREADputs(""); SUBREADputs(" -t Specify feature type in a GTF annotation. 'exon' by default."); SUBREADputs(" Features with the specified feature type are extracted from the"); SUBREADputs(" annotation for processing."); SUBREADputs(""); SUBREADputs(" -g Specify attribute type in GTF annotation. 'gene_id' by default."); SUBREADputs(" This attribute type is used to group features into meta-"); SUBREADputs(" features."); SUBREADputs(""); SUBREADputs(" -C Merging overlapping exons into multiple non-overlapping exons but"); SUBREADputs(" all the edges are kept."); SUBREADputs(""); } int flatAnno_finalise(flatAnno_context_t * context){ HashTableDestroy(context -> gene_to_chro_strand_table); HashTableDestroy(context -> gene_chro_strand_to_exons_table); fclose(context -> output_FP ); SUBREADputs("Finished.\n"); return 0; } int flatAnno_do_anno_1R(char * gene_name, char * transcript_name, char * chro_name, unsigned int start, unsigned int end, int is_negative_strand, void * Vcontext){ flatAnno_context_t * context = Vcontext; ArrayList * chro_strand_list_for_gene = HashTableGet(context -> gene_to_chro_strand_table, gene_name); if(NULL == chro_strand_list_for_gene){ char * mem_gene = malloc(strlen(gene_name)+1); strcpy(mem_gene, gene_name); chro_strand_list_for_gene = ArrayListCreate(3); ArrayListSetDeallocationFunction(chro_strand_list_for_gene, free); HashTablePut(context -> gene_to_chro_strand_table, mem_gene, chro_strand_list_for_gene); } int i, found = 0; char chro_strand[MAX_CHROMOSOME_NAME_LEN+10+FEATURE_NAME_LENGTH]; sprintf( chro_strand, "%s\t%s\t%c", gene_name,chro_name,is_negative_strand?'-':'+'); for(i=0; inumOfElements; i++){ char * old_ch_st = ArrayListGet(chro_strand_list_for_gene,i); if(strcmp(old_ch_st, chro_strand) == 0){ found=1; break; } } if(!found){ char * mem_ch_st = strdup(chro_strand); ArrayListPush(chro_strand_list_for_gene, mem_ch_st); } ArrayList * ge_ch_st_exon_list = HashTableGet(context -> gene_chro_strand_to_exons_table, chro_strand); if(NULL == ge_ch_st_exon_list){ ge_ch_st_exon_list = ArrayListCreate(3); ArrayListSetDeallocationFunction(ge_ch_st_exon_list,free); HashTablePut(context -> gene_chro_strand_to_exons_table, strdup(chro_strand), ge_ch_st_exon_list); } int * mem_start_end = malloc(sizeof(int)*2); mem_start_end[0] = (int)start; mem_start_end[1] = (int)end; ArrayListPush(ge_ch_st_exon_list, mem_start_end); return 0; } int flatAnno_do_anno_merge_one_array_compare(void * vL, void * vR, ArrayList *me){ int * iL = vL, *iR = vR; if((*iL)>(*iR))return 1; if((*iL)<(*iR))return -1; return 0; } void flatAnno_do_anno_chop_one_array(void * key, void * hashed_obj, HashTable * tab){ ArrayList * this_list = hashed_obj; // each element is a pair of integers defining start and end of an exon. ArrayList * edge_before_me_List = ArrayListCreate( this_list->numOfElements * 2 ); int i; for(i=0; i numOfElements; i++){ int * curr_2i = this_list -> elementList[ i ]; int findi, endi; for(endi = 0; endi < 2; endi++){ assert(curr_2i[endi]>0); int search_tag = curr_2i[endi] + endi, found=0; for(findi = 0; findi < edge_before_me_List->numOfElements; findi++){ if(ArrayListGet(edge_before_me_List,findi)-NULL == search_tag){ found = 1; break; } } if(0==found) ArrayListPush(edge_before_me_List, NULL+search_tag); } } ArrayListSort(edge_before_me_List, NULL); char * continue_after_an_edge = malloc(edge_before_me_List -> numOfElements -1); memset(continue_after_an_edge,0, edge_before_me_List -> numOfElements -1); int missed_gaps = edge_before_me_List->numOfElements -1; for(i=0; inumOfElements -1; i++){ int an_edge_before_me = edge_before_me_List-> elementList[i]-NULL; int j; for(j=0; jnumOfElements; j++){ int * curr_2i = this_list -> elementList[ j ]; if( curr_2i[0] <= an_edge_before_me && curr_2i[1] >= an_edge_before_me ) { continue_after_an_edge[i]=1; missed_gaps --; break; } } } long old_capacity = this_list -> capacityOfElements, old_items = this_list -> numOfElements; if(edge_before_me_List->numOfElements -1 - missed_gaps > old_capacity){ this_list -> elementList = realloc( this_list -> elementList, sizeof(void*) * edge_before_me_List->numOfElements -1 -missed_gaps ); this_list -> capacityOfElements = edge_before_me_List->numOfElements -1 -missed_gaps ; } int written_i = 0; for(i=0; inumOfElements -1; i++){ int * curr_2i = NULL; //if(edge_before_me_List->elementList[i] - NULL == 108395906)SUBREADprintf("CV_I %d = %ld ~ %ld ; CTN=%d\n", i, edge_before_me_List->elementList[i] - NULL, edge_before_me_List -> elementList[i+1] -NULL-1, continue_after_an_edge[i]); if(0==continue_after_an_edge[i]){ // SUBREADprintf("NC_I %d ~ [ 0 , %ld ]\n", i, edge_before_me_List->numOfElements -2); assert(i>0 && inumOfElements -2); assert(continue_after_an_edge[i+1] && continue_after_an_edge[i-1] ); // otherwise it is a singleton edge. continue; } if(written_i < old_items){ curr_2i = this_list -> elementList[ written_i ]; }else{ curr_2i = malloc(sizeof(int)*2); this_list -> elementList[ written_i ] = curr_2i; } curr_2i[0] = edge_before_me_List -> elementList[i] -NULL; curr_2i[1] = edge_before_me_List -> elementList[i+1] -NULL-1; written_i++; } assert(written_i == edge_before_me_List->numOfElements -1 - missed_gaps ); for(i=written_i; i elementList[i]); this_list -> numOfElements = written_i; ArrayListDestroy(edge_before_me_List); free(continue_after_an_edge); } void flatAnno_do_anno_merge_one_array(void * key, void * hashed_obj, HashTable * tab){ ArrayList * this_list = hashed_obj; ArrayListSort(this_list, flatAnno_do_anno_merge_one_array_compare); int i, n1_items = 0; for(i=1; i numOfElements; i++){ int * last_2i = this_list -> elementList[ n1_items ]; int * curr_2i = this_list -> elementList[ i ]; if(last_2i[1] >= curr_2i[1]) continue; if(last_2i[1] >= curr_2i[0] -1){ last_2i[1]=curr_2i[1]; continue; } n1_items++; if(n1_items< i){ last_2i = this_list -> elementList[ n1_items ]; last_2i[0] = curr_2i[0]; last_2i[1] = curr_2i[1]; } } for(i=n1_items+1; i numOfElements; i++)free(this_list -> elementList[i]); this_list -> numOfElements = n1_items+1; } int flatme_strcmp(void * L, void * R, ArrayList * me){ return strcmp(L, R); } int flatAnno_do_anno_merge_and_write(flatAnno_context_t * context){ context -> gene_chro_strand_to_exons_table -> appendix1 = context; HashTableIteration(context -> gene_chro_strand_to_exons_table, context -> merging_mode == MERGING_MODE_CHOPPING? flatAnno_do_anno_chop_one_array :flatAnno_do_anno_merge_one_array); ArrayList * all_chro_st_list = HashTableKeyArray(context -> gene_chro_strand_to_exons_table); ArrayListSort(all_chro_st_list, flatme_strcmp); fprintf( context -> output_FP , "GeneID\tChr\tStart\tEnd\tStrand\n"); int i,j; for(i = 0; i< all_chro_st_list -> numOfElements; i++){ char * ge_chro_strand = ArrayListGet(all_chro_st_list,i); char * local_ge_chro_strand = strdup(ge_chro_strand); char * strand_ptr = local_ge_chro_strand; for(j=0; j<2; strand_ptr++) if(*strand_ptr=='\t')j++; strand_ptr[-1] = 0; ArrayList * exon_in_chro_strand = HashTableGet(context -> gene_chro_strand_to_exons_table, ge_chro_strand); for(j=0; j< exon_in_chro_strand -> numOfElements; j++){ int * start_end_2i = ArrayListGet(exon_in_chro_strand,j); fprintf( context -> output_FP ,"%s\t%d\t%d\t%s\n", local_ge_chro_strand, start_end_2i[0], start_end_2i[1], strand_ptr); } free(local_ge_chro_strand); } ArrayListDestroy(all_chro_st_list ); return 0; } int flatAnno_do_anno(flatAnno_context_t * context){ int loaded_features = load_features_annotation(context -> GTF_file_name, FILE_TYPE_GTF, context -> GTF_gene_id_column, NULL, context -> GTF_wanted_feature_type, context, flatAnno_do_anno_1R); if(loaded_features<0)SUBREADputs("ERROR: Unable to open the GTF file."); if(loaded_features==0)SUBREADprintf("ERROR: No '%s' feature was found in the GTF file. (the '%s' attribute is required)\n", context -> GTF_wanted_feature_type, context -> GTF_gene_id_column); if(loaded_features<=0) return -1; return flatAnno_do_anno_merge_and_write(context); } int flatAnno_start(flatAnno_context_t * context){ SUBREADputs(""); if(!context -> GTF_file_name[0]){ flatAnno_print_usage(); if(context -> output_file_name[0]) SUBREADprintf("Error: no input file is specified.\n"); return -1; } if(!context -> output_file_name[0]){ flatAnno_print_usage(); SUBREADprintf("Error: no output file is specified.\n"); return -1; } SUBREADprintf("Flattening GTF file: %s\n", context -> GTF_file_name); SUBREADprintf("Output SAF file: %s\n", context -> output_file_name); context -> output_FP = fopen(context -> output_file_name, "w"); if(NULL == context -> output_FP ){ SUBREADprintf("Error: unable to open the output file.\n"); return -1; } SUBREADprintf("\nLooking for '%s' features... (grouped by '%s')\n\n", context -> GTF_wanted_feature_type, context -> GTF_gene_id_column); context -> gene_to_chro_strand_table = StringTableCreate(30000); HashTableSetDeallocationFunctions(context -> gene_to_chro_strand_table, free, (void(*)(void *))ArrayListDestroy); context -> gene_chro_strand_to_exons_table = StringTableCreate(30000); HashTableSetDeallocationFunctions(context -> gene_chro_strand_to_exons_table, free, (void(*)(void *))ArrayListDestroy); return 0; } #ifdef MAKE_STANDALONE int main(int argc, char ** argv) #else int R_flattenAnnotations(int argc, char ** argv) #endif { flatAnno_context_t context; memset(&context, 0, sizeof(context)); strcpy(context.GTF_gene_id_column, "gene_id"); strcpy(context.GTF_wanted_feature_type, "exon"); int option_index = 0,c; optind=0; opterr=1; optopt=63; while ((c = getopt_long (argc, argv, "Ct:g:a:o:v?", long_options, &option_index)) != -1) switch(c) { case 'C': context.merging_mode = MERGING_MODE_CHOPPING; break; case 'o': strcpy(context.output_file_name, optarg); break; case 'a': strcpy(context.GTF_file_name, optarg); break; case 'g': strcpy(context.GTF_gene_id_column, optarg); break; case 't': strcpy(context.GTF_wanted_feature_type, optarg); break; case '?': default : flatAnno_print_usage(); return -1; } int ret = 0; ret = ret || flatAnno_start(&context); ret = ret || flatAnno_do_anno(&context); ret = ret || flatAnno_finalise(&context); return ret; } Rsubread/src/fullscan.c0000644000175200017520000001237414136047615016123 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #include #include #include #include #include #include #include "subread.h" #include "gene-value-index.h" #include "input-files.h" float MIN_REPORTING_RATIO = 0.8; gene_offset_t _global_offsets; unsigned int SCAN_TOTAL_BASES=0; char * only_chro = NULL; void fullscan_usage() { SUBREADprintf("\nsubread-fullscan Version %s\n\n", SUBREAD_VERSION); SUBREADputs(" This program scans the entire genome to find all high-similarity locations to"); SUBREADputs("a specified read."); SUBREADputs(""); SUBREADputs("Usage:"); SUBREADputs(""); SUBREADputs(" ./subread-fullscan [options] -i "); SUBREADputs(""); SUBREADputs("Required arguments:"); SUBREADputs(""); SUBREADputs(" -i Base name of the index."); SUBREADputs(""); SUBREADputs(" read_string The read bases."); SUBREADputs(""); SUBREADputs("Optional arguments:"); SUBREADputs(""); SUBREADputs(" -m The minimum fraction of matched bases in the read, 0.8 by default"); SUBREADputs(""); } void report_pos(unsigned int pos) { char * chro_name; int chro_pos; locate_gene_position(pos, &_global_offsets, &chro_name, &chro_pos); SUBREADprintf ("%s,%u\n", chro_name,chro_pos); } int str_match_count(char * c1, char * c2, int rl, int th) { int i,ret =0; for (i=0; i th) return 0; } return rl-ret; } void scan_test_match(char * read, char * read_rev, char * chro, int rl, unsigned int pos) { int threshold = (int)(MIN_REPORTING_RATIO * rl - 0.001); int m = str_match_count(read, chro, rl, rl- threshold); int mr = str_match_count(read_rev, chro, rl,rl- threshold); if (m>=threshold) { SUBREADprintf("\nFound on positive strand (%0.2f%%): ", m*100./rl); report_pos(pos); } if (mr>=threshold) { SUBREADprintf("\nFound on negative strand (%0.2f%%): ", mr*100./rl); report_pos(pos); } //if (pos > 19999999) //SUBREADprintf ("m=%d mr=%d T=%d\n", m, mr, threshold); } void full_scan_read(char * index_name, char * read_str) { int read_len = strlen(read_str); char read_rev_str[1208]; char chro_str[1208]; gene_value_index_t index; int tabno = 0; unsigned int current_pos =0xffffffffu; strcpy(read_rev_str, read_str); reverse_read(read_rev_str, read_len, GENE_SPACE_BASE); while (1){ char table_fn[1250]; struct stat filestat; sprintf(table_fn, "%s.%02d.b.array", index_name, tabno); int stat_ret = stat(table_fn, &filestat); if (stat_ret !=0 ) { if(!tabno) SUBREADprintf("The index does not contain any raw base data which is required in scanning. Please use the -b option while building the index.\n"); return ; } if (tabno>0)gvindex_destory(&index); gvindex_load(&index,table_fn); if (tabno==0) { gvindex_get_string (chro_str, &index, 0, read_len, 0); current_pos = 0; } for(; current_pos + read_len < index.start_point + index.length ; current_pos++) { if(only_chro){ char * chro_name; int chro_pos; locate_gene_position(current_pos, &_global_offsets, &chro_name, &chro_pos); if(strcmp(chro_name, only_chro)!=0)continue; } scan_test_match(read_str, read_rev_str, chro_str, read_len, current_pos); char nch = gvindex_get(&index, current_pos + read_len); int i; for (i=0; i0)gvindex_destory(&index); } int main (int argc , char ** argv) { char index_name [MAX_FILE_NAME_LENGTH]; char read_str [1208]; char c; int i; index_name[0]=0; while ((c = getopt (argc, argv, "i:m:c:?")) != -1) switch(c) { case 'i': strncpy(index_name, optarg, MAX_FILE_NAME_LENGTH-1); break; case 'm': MIN_REPORTING_RATIO = atof(optarg); break; case 'c': only_chro = optarg; break; case '?': return -1 ; } if (!index_name[0] || optind == argc) { fullscan_usage(); return -1; } i=0; while(argv[optind][i]) { argv[optind][i] = toupper(argv[optind][i]); i++; } strncpy(read_str, argv[optind], 1199); load_offsets (&_global_offsets, index_name); SUBREADprintf ("Reporting threshold=%0.2f%%\n", MIN_REPORTING_RATIO*100); /* for(i=0;i<1000;i++) { if (!_global_offsets.read_offset[i])break; SCAN_TOTAL_BASES = _global_offsets.read_offset[i]; }*/ SUBREADprintf ("All bases =%u\n", SCAN_TOTAL_BASES); SUBREADprintf ("Scanning the full index for %s...\n\n", read_str); full_scan_read(index_name, read_str); SUBREADprintf ("\nFinished.\n"); return 0; } Rsubread/src/fusion-align.c0000644000175200017520000000000614136047615016674 0ustar00biocbuildbiocbuild//SPA Rsubread/src/gen_rand_reads.c0000644000175200017520000010714114136047615017244 0ustar00biocbuildbiocbuild#include #include #include #include #include #include #include #include #include #include #include #ifndef __MINGW32__ #include #endif #include #include #include #include #ifndef MAKE_STANDALONE #include #endif #include "subread.h" #include "core.h" #include "HelperFunctions.h" #include "seek-zlib.h" #include "input-files.h" #include "gene-algorithms.h" #define MAX_SIMULATION_READ_LEN 250 #define STRATEGY_LESS_THAN_N 10 #define STRATEGY_RANDOM_ASSIGN 20 #define STRATEGY_ITERATIVE_M 30 int print_usage_gen_reads(char * pgname) { SUBREADputs(""); SUBREADputs("Usage:"); SUBREADputs(""); SUBREADputs(" For scanning a FASTA/gz file:"); SUBREADprintf(" %s --summarizeFasta \\\n", pgname); SUBREADputs(" --transcriptFasta --outputPrefix [--simpleTranscriptId]"); SUBREADputs(""); SUBREADputs(" For generating read/pairs:"); SUBREADprintf(" %s --transcriptFasta \\\n", pgname); SUBREADputs(" --outputPrefix --expressionLevels [other options]"); SUBREADputs(""); SUBREADputs(" --summarizeFasta Only output the transcript names and lengths."); SUBREADputs(""); SUBREADputs(" --transcriptFasta The transcript database in FASTA/gz format."); SUBREADputs(""); SUBREADputs(" --outputPrefix The prefix of the output files."); SUBREADputs(""); SUBREADputs(" --totalReads Total read/pairs in output."); SUBREADputs(""); SUBREADputs(" --expressionLevels Two column table delimited by , giving the"); SUBREADputs(" wanted TPM values. Columns: TranscriptID and TPM"); SUBREADputs(""); SUBREADputs(" --readLen The length of the output reads. 100 by default."); SUBREADputs(""); SUBREADputs(" --totalReads Total read/pairs in the output."); SUBREADputs(""); SUBREADputs(" --randSeed Seed to generate random numbers. UNIXTIME is used"); SUBREADputs(" as the random seed by default."); SUBREADputs(""); SUBREADputs(" --qualityRefFile A textual file containing Phred+33 quanlity strings"); SUBREADputs(" for simulating sequencing errors. The quality"); SUBREADputs(" strings have to have the same length as the output"); SUBREADputs(" reads. No sequencing errors are simulated when this"); SUBREADputs(" option is omitted."); SUBREADputs(""); SUBREADputs(" --floorStrategy How to deal with round-up errors. 'FLOOR': generate"); SUBREADputs(" less than wanted reads; 'RANDOM': randomly assign"); SUBREADputs(" margin reads to transcripts; 'ITERATIVE': find the"); SUBREADputs(" best M value to have ~N reads."); SUBREADputs(""); SUBREADputs(" --pairedEnd Generate paired-end reads."); SUBREADputs(""); SUBREADputs(" --insertionLenMean ,--insertionLenSigma ,--insertionLenMin ,"); SUBREADputs(" --insertionLenMax Parameters of a truncated normal distribution for"); SUBREADputs(" deciding insertion lengths of paired-end reads."); SUBREADputs(" Default values: mean=160, sigma=30, min=110, max=400"); SUBREADputs(""); SUBREADputs(" --simpleTranscriptId Truncate transcript names to the first '|' or space."); SUBREADputs(""); SUBREADputs(" --truthInReadNames Encode the true locations of reads in read names."); SUBREADputs(""); SUBREADputs(" --noActualReads Do not actually generate reads in fastq.gz files."); SUBREADputs(""); return 1; } static struct option long_options[] = { {"quiet", no_argument, 0, 'Q'}, {"truthInReadNames", no_argument, 0, 'T'}, {"simpleTranscriptId", no_argument, 0, 'C'}, {"summarizeFasta", no_argument, 0, 'M'}, {"transcriptFasta", required_argument, 0, 't'}, {"totalReads", required_argument, 0, 'r'}, {"pairedEnd", no_argument, 0, 'p'}, {"expressionLevels", required_argument, 0, 'e'}, {"qualityRefFile", required_argument, 0, 'q'}, {"outputPrefix", required_argument, 0, 'o'}, {"randSeed", required_argument, 0, 'S'}, {"readLen", required_argument, 0, 'L'}, {"floorStrategy", required_argument, 0, 'O'}, {"noActualReads", no_argument, 0, 'x'}, {"insertionLenMean", required_argument, 0, 'F'}, {"insertionLenMin", required_argument, 0, 'N'}, {"insertionLenMax", required_argument, 0, 'X'}, {"insertionLenSigma", required_argument, 0, 'V'}, {0, 0, 0, 0} }; typedef struct { char random_seeds[16]; char transcript_fasta_file[MAX_FILE_NAME_LENGTH]; char output_prefix[MAX_FILE_NAME_LENGTH]; char expression_level_file[MAX_FILE_NAME_LENGTH]; char quality_string_file[MAX_FILE_NAME_LENGTH]; unsigned long long output_sample_size; unsigned long long applied_M; int floor_strategy; int is_paired_end; int simple_transcript_names; int truth_in_read_names; float insertion_length_mean; int insertion_length_max; int insertion_length_min; float insertion_length_sigma; int quiet; int read_length; int no_actual_reads; ArrayList * quality_strings; ArrayList * transcript_hitting_space; ArrayList * transcript_names; HashTable * transcript_sequences; HashTable * transcript_lengths; HashTable * expression_levels; char fake_quality_string[MAX_SIMULATION_READ_LEN+3]; char * cmd_line; gzFile out_fps[2]; FILE * counts_out_fp; } genRand_context_t; void grc_incrand(genRand_context_t * grc){ unsigned long long round_rand =0; memcpy(&round_rand, grc->random_seeds+8, sizeof(round_rand)); round_rand++; memcpy(grc->random_seeds+8, &round_rand, sizeof(round_rand)); } void grc_sequencing_error_read(char * seq, int qlen, char * qua){ int b; for(b=0; b read_length+1]; memcpy(read_seq, seq, grc -> read_length); read_seq[grc -> read_length]=0; if(trans_negative) reverse_read(read_seq , grc -> read_length , GENE_SPACE_BASE); char * qual_str = NULL; if(grc->quality_strings->numOfElements>0){ qual_str = ArrayListRandom(grc->quality_strings); //SUBREADprintf("TESTQUAL\t%p\n", qual_str); grc_sequencing_error_read(read_seq, grc -> read_length, qual_str); }else{ if(!grc->fake_quality_string[0]){ int xx; for(xx = 0; xx < grc -> read_length; xx++) grc->fake_quality_string[xx]='H'; grc->fake_quality_string[xx]=0; } qual_str = grc->fake_quality_string; } gzFile thisfp = (is_PE_second==1) ? grc -> out_fps[1] : grc -> out_fps[0]; int R1_pos = is_PE_second?mate_pos:my_pos; int R2_pos = is_PE_second?my_pos:mate_pos; if(grc->truth_in_read_names){ if(is_PE_second<0) gzprintf(thisfp, "@R%09llu:%s:%d\n%s\n+\n%s\n", rno, seq_name, 1+my_pos, read_seq, qual_str); else gzprintf(thisfp, "@R%09llu:%s:%d:%d\n%s\n+\n%s\n", rno, seq_name, 1+R1_pos, 1+R2_pos, read_seq, qual_str); } else gzprintf(thisfp, "@R%09llu\n%s\n+\n%s\n", rno, read_seq, qual_str); } void gen_a_read_from_one_transcript(genRand_context_t * grc, long this_transcript_no, unsigned long long rno){ char * trans_name = ArrayListGet(grc->transcript_names, this_transcript_no); char * trans_seq = HashTableGet(grc->transcript_sequences, trans_name); int actual_transcript_len = HashTableGet(grc->transcript_lengths, trans_name) - NULL; int applied_insertion_maxlen = min(grc -> insertion_length_max, actual_transcript_len); double rand_01 = plain_txt_to_long_rand(grc->random_seeds, 16)*1./0xffffffffffffffffllu; int rand_01_int = (int)(rand_01*901267351); myrand_srand(rand_01_int); // for generating sequencing errors. NOTE: the argument is unused in R. MYRAND module uses R's RNG for seeding. grc_incrand(grc); if(grc -> is_paired_end){ float insertion_len = inverse_sample_normal(rand_01) * grc-> insertion_length_sigma + grc -> insertion_length_mean; int fraglen = (int)(min(max(insertion_len, grc -> insertion_length_min), applied_insertion_maxlen)); rand_01 = plain_txt_to_long_rand(grc->random_seeds, 16)*1./0xffffffffffffffffllu; grc_incrand(grc); int start_pos = (actual_transcript_len - fraglen) * rand_01; int is_first_end_negative = rand_01_int % 2; if(is_first_end_negative){ gen_one_read_here(grc, trans_seq + start_pos + fraglen - grc -> read_length, 0, 1, rno, trans_name, start_pos + fraglen - grc -> read_length, start_pos); gen_one_read_here(grc, trans_seq + start_pos, 1, 0, rno, trans_name, start_pos, start_pos + fraglen - grc -> read_length); }else{ gen_one_read_here(grc, trans_seq + start_pos, 0, 0, rno, trans_name, start_pos, start_pos + fraglen - grc -> read_length); gen_one_read_here(grc, trans_seq + start_pos + fraglen - grc -> read_length, 1, 1, rno, trans_name, start_pos + fraglen - grc -> read_length, start_pos); } }else{ int start_pos = (actual_transcript_len - grc -> read_length)*rand_01; int is_negative = rand_01_int % 2; gen_one_read_here(grc, trans_seq + start_pos, -1, is_negative, rno, trans_name, start_pos, -1); } } int grc_check_parameters(genRand_context_t * grc){ int ret = 0; if(grc->read_length > MAX_SIMULATION_READ_LEN){ SUBREADprintf("Error: the read length cannot be higher than %d.\n", MAX_SIMULATION_READ_LEN); ret=1; } if(grc->is_paired_end){ if(grc->insertion_length_min>grc->insertion_length_max){ SUBREADprintf("Error: the minimum insertion length must be equal or higher than the maximum insertion length.\n"); ret=1; } if(grc->insertion_length_minread_length){ SUBREADprintf("Error: the minimum insertion length must be equal or higher than read length.\n"); ret=1; } if(grc->insertion_length_max<1){ SUBREADprintf("Error: the maximum insertion length must be a positive number.\n"); ret=1; } } if(grc->read_length<1){ SUBREADprintf("Error: the read length must be a positive number.\n"); ret=1; } if(!grc->transcript_fasta_file[0]){ SUBREADprintf("Error: a transcript file must be provide.\n"); ret=1; } if(!grc->output_prefix[0]){ SUBREADprintf("Error: the output prefix must be provide.\n"); ret=1; }else{ char outname[MAX_FILE_NAME_LENGTH+30]; sprintf(outname, "%s.for_test.log",grc->output_prefix); FILE * test_out = fopen(outname, "w"); if(test_out){ fclose(test_out); unlink(outname); }else{ SUBREADprintf("Error: cannot create the output file.\n"); ret=1; } } if(!grc->expression_level_file[0]){ SUBREADprintf("Error: the wanted expression levels must be provided.\n"); ret=1; } if(grc->output_sample_size < 1){ SUBREADprintf("Warning: no read number is specified. Generating one million read%s.\n", grc->is_paired_end?"-pairs":"s"); grc->output_sample_size = 1000000; } return ret; } unsigned long long calc_N_from_M(genRand_context_t *grc , unsigned long long Mval){ long ti; unsigned long long space_end = ArrayListGet(grc->transcript_hitting_space, grc->transcript_hitting_space->numOfElements -1)-NULL; unsigned long long ret =0, lastv=0; for(ti = 0; ti < grc->transcript_hitting_space->numOfElements; ti++){ unsigned long long thisv = ArrayListGet(grc->transcript_hitting_space, ti) - NULL; unsigned long long this_space_span = thisv - lastv; unsigned long long expected_reads =(unsigned long long )(this_space_span *1.0/space_end * Mval); // expected_reads = exp_level * seq_length / sum(Q*L) * M ret += expected_reads; lastv = thisv; } return ret; } unsigned long long itr_find_M(genRand_context_t *grc){ long transcripts= grc->transcript_hitting_space->numOfElements; unsigned long long wanted_reads = grc->output_sample_size; unsigned long long LL = wanted_reads, HH = wanted_reads + transcripts; unsigned long long MM = 0; while(1){ MM = (LL+HH)/2; unsigned long long thisN_at_M = calc_N_from_M(grc, MM); if(thisN_at_M < wanted_reads) LL = MM+1; else if(thisN_at_M > wanted_reads) HH = MM-1; else break; if(LL >=HH){ MM = max(LL,HH); break; } } return MM; } unsigned long long convert_hitting_space_to_num_of_reads(genRand_context_t *grc , ArrayList * num_of_frags_per_transcript, int min_seq_len){ unsigned long long lastv = 0, current_total =0; ArrayList * rescure_hitting_space = ArrayListCreate(100000); unsigned long long to_rescure_read_top=0; unsigned long long read_i = 0; unsigned long long space_end = ArrayListGet(grc->transcript_hitting_space, grc->transcript_hitting_space->numOfElements -1)-NULL; if(grc -> floor_strategy == STRATEGY_ITERATIVE_M){ grc->applied_M = itr_find_M(grc); }else grc->applied_M = grc->output_sample_size; // after M is here, the reminder is just like "LESS_THAN_N. { for(read_i = 0; read_i < grc->transcript_hitting_space->numOfElements ; read_i++){ char *seq_name = ArrayListGet(grc->transcript_names, read_i); int seq_len = HashTableGet(grc-> transcript_lengths, seq_name)-NULL; unsigned long long thisv = ArrayListGet(grc->transcript_hitting_space, read_i) - NULL; unsigned long long this_space_span = thisv - lastv; // the many 9's is to aovid the sum of read numbers larger than wanted. double floor_chopping = grc -> floor_strategy == STRATEGY_RANDOM_ASSIGN ?0.999999999:1; unsigned long long expected_reads =(unsigned long long )((this_space_span *1.0/space_end) * grc->applied_M*floor_chopping); unsigned long long to_rescure_reads = 0; if(grc -> floor_strategy == STRATEGY_RANDOM_ASSIGN) to_rescure_reads = (unsigned long long)((this_space_span *1.0/space_end * grc->applied_M- 1.*expected_reads)*100000.); if(this_space_span < 1) to_rescure_reads=0; if( seq_len < min_seq_len ){ to_rescure_reads = 0; expected_reads = 0; } to_rescure_read_top+= to_rescure_reads; assert(to_rescure_read_top < 0x5fffffffffffffffllu); ArrayListPush(rescure_hitting_space, NULL+to_rescure_read_top); ArrayListPush(num_of_frags_per_transcript, NULL+expected_reads); current_total += expected_reads; lastv = thisv; } // I'm not sure why this is so important but let it be. assert(current_total<=grc->applied_M); if(grc -> floor_strategy == STRATEGY_RANDOM_ASSIGN)for(read_i = current_total; read_i < grc->applied_M; read_i++){ unsigned long long longrand = plain_txt_to_long_rand(grc->random_seeds, 16); grc_incrand(grc); longrand = longrand % to_rescure_read_top; long this_transcript_no = ArrayListFindNextDent(rescure_hitting_space, longrand); unsigned long long expected_reads = ArrayListGet(num_of_frags_per_transcript, this_transcript_no)-NULL; expected_reads++; num_of_frags_per_transcript->elementList[this_transcript_no] = NULL+expected_reads; current_total ++; } } ArrayListDestroy(rescure_hitting_space); return current_total; } // The 823,532,653,200th prime is 24,537,224,085,139. // -- https://primes.utm.edu/nthprime/index.php #define A_LARGE_PRIME_FOR_MOD 24537224085139llu int grc_gen( genRand_context_t *grc ){ int ret = 0; unsigned long long read_i = 0; ArrayList * num_of_frags_per_transcript = ArrayListCreate(100000); int min_seq_len = grc->is_paired_end?grc->insertion_length_min:grc->read_length; convert_hitting_space_to_num_of_reads(grc, num_of_frags_per_transcript, min_seq_len); ArrayList * per_transcript_reads_hitting_space = ArrayListCreate(100000); unsigned long long total_read_top =0; for(read_i =0; read_i < num_of_frags_per_transcript -> numOfElements; read_i++) { char *seq_name = ArrayListGet(grc->transcript_names, read_i); int seq_len = HashTableGet(grc-> transcript_lengths, seq_name)-NULL; unsigned long long expected_reads = ArrayListGet(num_of_frags_per_transcript, read_i)-NULL; if(seq_len >= min_seq_len) #ifdef __MINGW32__ fprintf(grc->counts_out_fp, "%s\t%d\t%I64u\n", seq_name, seq_len, expected_reads); #else fprintf(grc->counts_out_fp, "%s\t%d\t%llu\n", seq_name, seq_len, expected_reads); #endif else fprintf(grc->counts_out_fp, "%s\t%d\tNA\n", seq_name, seq_len); total_read_top+=expected_reads; ArrayListPush(per_transcript_reads_hitting_space, NULL+total_read_top); } if(grc -> floor_strategy == STRATEGY_RANDOM_ASSIGN) assert(total_read_top == grc->applied_M); else grc->applied_M = total_read_top; unsigned long long longrand = plain_txt_to_long_rand(grc->random_seeds, 16); grc_incrand(grc); if(grc->no_actual_reads == 0){ unsigned long long mod_class = longrand % grc->applied_M; // an arbitratry starting point. for(read_i =0; read_i < grc->applied_M; read_i++) { mod_class += A_LARGE_PRIME_FOR_MOD; mod_class = mod_class % grc->applied_M; long this_transcript_no = ArrayListFindNextDent(per_transcript_reads_hitting_space, mod_class); //char * trans_name = ArrayListGet(grc->transcript_names, this_transcript_no); //SUBREADprintf("TESTGEN\t%s\n", trans_name); gen_a_read_from_one_transcript(grc, this_transcript_no, read_i); } } ArrayListDestroy(num_of_frags_per_transcript); ArrayListDestroy(per_transcript_reads_hitting_space); return ret; } int grc_finalize(genRand_context_t *grc){ HashTableDestroy(grc->expression_levels); HashTableDestroy(grc->transcript_sequences); HashTableDestroy(grc->transcript_lengths); ArrayListDestroy(grc->quality_strings); ArrayListDestroy(grc->transcript_hitting_space); ArrayListDestroy(grc->transcript_names); gzclose(grc->out_fps[0]); if(grc->out_fps[1]) gzclose(grc->out_fps[1]); fclose(grc->counts_out_fp); free(grc->cmd_line); #ifdef __MINGW32__ SUBREADprintf("Finished. Actual sample size : I64u\n", grc->applied_M); #else SUBREADprintf("Finished. Actual sample size : %llu\n", grc->applied_M); #endif return 0; } #define TRANSCRIPT_FASTA_LINE_INIT 800 #define TRANSCRIPT_MAX_EXPRESSION_LEVEL 1000001.0 #define TRANSCRIPT_FASTA_LINE_WIDTH 1000 int grc_summary_fasta(genRand_context_t * grc){ char outname[MAX_FILE_NAME_LENGTH+30]; autozip_fp auto_FP; if(!grc->output_prefix[0]){ SUBREADprintf("Error: the output prefix must be provide.\n"); return -1; } sprintf(outname,"%s.faSummary", grc->output_prefix); int ret = autozip_open(grc->transcript_fasta_file, &auto_FP); if(ret<0){ SUBREADprintf("Error: cannot open the fasta file as input\n"); return -1; }else ret = 0; FILE * sumfp = fopen(outname, "w"); if(sumfp == NULL){ SUBREADprintf("Error: cannot open the putput file\n"); return -1; } fprintf(sumfp, "TranscriptID\tLength\tMD5\tUnique\tOccurrence\tDuplicated\n"); char * seq_name = NULL; unsigned char md5res[16]; int seq_len = 0, total_rep_seq = 0; HelpFuncMD5_CTX md5ctx; HelpFuncMD5_Init(&md5ctx); HashTable * seq_duplicate_tab = StringTableCreate(100000); HashTable * name_duplicate_tab = StringTableCreate(100000); HashTable * seq_length_tab = StringTableCreate(100000); HashTable * seq_md5_tab = StringTableCreate(100000); ArrayList * seq_name_list = ArrayListCreate(100000); HashTableSetDeallocationFunctions(seq_duplicate_tab, free,NULL); ArrayListSetDeallocationFunction(seq_name_list, free); while(1){ char clinebuf[TRANSCRIPT_FASTA_LINE_WIDTH]; int rlength = autozip_gets(&auto_FP, clinebuf, TRANSCRIPT_FASTA_LINE_WIDTH -1); if(rlength < 1)break; if(rlength >= TRANSCRIPT_FASTA_LINE_WIDTH -1 || clinebuf[rlength]!='\0' || clinebuf[rlength-1]!='\n'){ SUBREADprintf("Error: The line width of the fasta file excessed %d bytes.\n", TRANSCRIPT_FASTA_LINE_WIDTH); ret = 1; break; } if(clinebuf[0]=='>'){ if(seq_name){ if(seq_len<1){ SUBREADprintf("Error: a transcript has no sequence: '%s'\n", seq_name); return -1; } HelpFuncMD5_Final(md5res, &md5ctx); //SUBREADprintf("%s\t",seq_name);int md5i;for(md5i=0;md5i<16;md5i++)SUBREADprintf("%02X",0xff&(int)md5res[md5i]);SUBREADputs(""); char * md5mem = malloc(33); int md5i;for(md5i=0;md5i<16;md5i++)sprintf(md5mem+2*md5i, "%02X", 0xff&(int)md5res[md5i]); long dupval = HashTableGet(seq_duplicate_tab,md5mem)-NULL; dupval++; HashTablePutReplace( seq_duplicate_tab, md5mem, NULL+dupval , 0); if(dupval>1){ char * newmd5mem =HashTableGetKey(seq_duplicate_tab, md5mem); free(md5mem); md5mem = newmd5mem; } ArrayListPush( seq_name_list, seq_name); HashTablePut( seq_length_tab, seq_name, NULL+seq_len); HashTablePut( seq_md5_tab, seq_name, md5mem); //if(strcmp("ENST00000410691.1",seq_name)==0)SUBREADprintf("MD5CHECK: %s has %s (%p)\n", seq_name, md5mem, md5mem); //if(strcmp("ENST00000634833.2",seq_name)==0)SUBREADprintf("MD5CHECK: %s has %s (%p), not %s (%p)\n", seq_name, md5mem, md5mem, HashTableGet(seq_md5_tab, "ENST00000410691.1"), HashTableGet(seq_md5_tab, "ENST00000410691.1")); seq_len = 0; } clinebuf[rlength-1]=0; if(grc->simple_transcript_names){ int xx; for(xx=1; xx %p\n", clinebuf+1, had_tab); if(had_tab){ SUBREADprintf("Error: duplicate sequence name was found : '%s'.\n", clinebuf+1); return -1; } seq_name=malloc(rlength); strcpy(seq_name, clinebuf+1); HashTablePut(name_duplicate_tab, seq_name, NULL+1); HelpFuncMD5_Init(&md5ctx); }else{ int xx; for(xx=0; xx1){ char * newmd5mem =HashTableGetKey(seq_duplicate_tab, md5mem); free(md5mem); md5mem = newmd5mem; } ArrayListPush( seq_name_list, seq_name); HashTablePut( seq_length_tab, seq_name, NULL+seq_len); HashTablePut( seq_md5_tab, seq_name, md5mem); } int seqi; HashTable * reprs_tab = StringTableCreate(100000); for(seqi = 0 ; seqi < seq_name_list->numOfElements; seqi++){ char * seqnam = ArrayListGet(seq_name_list,seqi); char * md5str = HashTableGet(seq_md5_tab,seqnam); long md5repeated = HashTableGet(seq_duplicate_tab, md5str) - NULL; if(md5repeated>1) total_rep_seq++; long seqlen = HashTableGet(seq_length_tab,seqnam)-NULL; int is_reprs = HashTableGet(reprs_tab,md5str)==NULL; HashTablePut(reprs_tab,md5str,NULL+1); fprintf(sumfp, "%s\t%ld\t%s\t%s\t%ld\t%s\n", seqnam, seqlen, md5str, md5repeated>1?"FALSE":"TRUE" /*"Unique"*/, md5repeated, is_reprs?"FALSE":"TRUE" /*"Duplicated"*/); } HashTableDestroy(reprs_tab); if(total_rep_seq && grc->quiet==0)SUBREADprintf("Warning: %d duplicate sequences were found in the input. Please check the summary table.\n",total_rep_seq); ArrayListDestroy(seq_name_list); HashTableDestroy(seq_length_tab); HashTableDestroy(seq_md5_tab); HashTableDestroy(seq_duplicate_tab); HashTableDestroy(name_duplicate_tab); autozip_close(&auto_FP); fclose(sumfp); return ret; } void grc_put_new_trans(genRand_context_t *grc, char * seq_name, char * seq_str, unsigned int seq_len, unsigned long long * linear_space_top){ if(seq_len<1){ SUBREADprintf("Warning: a transcript, '%s', has a zero length. No read is generated from it.\n", seq_name); } HashTablePut(grc-> transcript_sequences,seq_name, seq_str); HashTablePut(grc-> transcript_lengths, seq_name, NULL+ seq_len); unsigned long long this_seq_exp_10000 = HashTableGet(grc->expression_levels, seq_name)-NULL; if(this_seq_exp_10000<1){ SUBREADprintf("Warning: a transcript, '%s', has no wanted expression level. No read is generated from it.\n", seq_name); this_seq_exp_10000=0; }else this_seq_exp_10000-=1; //SUBREADprintf("TESTLEN\t%s\t%d\n", seq_name, seq_len); (*linear_space_top) += this_seq_exp_10000 * seq_len; ArrayListPush(grc->transcript_names, seq_name); ArrayListPush(grc->transcript_hitting_space, NULL+*linear_space_top); } int grc_load_env(genRand_context_t *grc){ grc->expression_levels = HashTableCreate(100000); HashTableSetDeallocationFunctions(grc->expression_levels, free, NULL); HashTableSetKeyComparisonFunction(grc->expression_levels, fc_strcmp_chro); HashTableSetHashFunction(grc->expression_levels, fc_chro_hash); grc->transcript_sequences = HashTableCreate(100000); HashTableSetDeallocationFunctions(grc->transcript_sequences, free, free); HashTableSetKeyComparisonFunction(grc->transcript_sequences, fc_strcmp_chro); HashTableSetHashFunction(grc->transcript_sequences, fc_chro_hash); grc->transcript_lengths = HashTableCreate(100000); HashTableSetKeyComparisonFunction(grc->transcript_lengths, fc_strcmp_chro); HashTableSetHashFunction(grc->transcript_lengths, fc_chro_hash); grc -> quality_strings = ArrayListCreate(100000); ArrayListSetDeallocationFunction(grc -> quality_strings, free); grc -> transcript_hitting_space = ArrayListCreate(100000); grc -> transcript_names = ArrayListCreate(100000); // the names are destroyed by destroying grc->transcript_sequences autozip_fp auto_FP; int xk1; int ret = autozip_open(grc->expression_level_file, &auto_FP); if(ret<0){ ret = 1; SUBREADprintf("Error: unable to open the expression level file.\n"); }else ret = 0; if(ret) return ret; unsigned long long total_tpm = 0; while(1){ char linebuf[400], * tokbuf=NULL; int rline = autozip_gets(&auto_FP, linebuf, 399); if(rline<1) break; if(strstr(linebuf, "ID\tTPM"))continue; char * seqname = strtok_r(linebuf, "\t", &tokbuf); char * seqexp_str = tokbuf; if(NULL == seqexp_str){ SUBREADprintf("Error: expression level file format error.\n"); ret = 1; } double seqexp = atof(seqexp_str); if(seqexp > TRANSCRIPT_MAX_EXPRESSION_LEVEL){ SUBREADprintf("Error: The transcript expression level shouldn't excess %.0f\n", TRANSCRIPT_MAX_EXPRESSION_LEVEL); } unsigned long long seqexp_int = (unsigned long long )(seqexp*10000.); total_tpm += seqexp_int; char * seqname_buf = malloc(strlen(seqname)+1); strcpy(seqname_buf, seqname); void * had_tab = HashTableGet(grc->expression_levels, seqname_buf); if(had_tab){ SUBREADprintf("Error: duplicate transcript name was found in the TMP table: '%s'.\n", seqname_buf); return -1; } HashTablePut(grc->expression_levels, seqname_buf, NULL+seqexp_int+1); } autozip_close(&auto_FP); if(ret) return ret; #define ROUNDUP_TOLERANCE ( 1000llu * 10000llu ) if(total_tpm > 1000000llu * 10000llu + ROUNDUP_TOLERANCE || total_tpm < 1000000llu * 10000llu - ROUNDUP_TOLERANCE) { SUBREADprintf("Error: total TPM is not 1,000,000\n"); return 1; } if(grc->quality_string_file[0]){ ret = autozip_open(grc->quality_string_file, &auto_FP); if(ret<0){ ret = 1; SUBREADprintf("Error: unable to open the quality string file.\n"); }else ret = 0; if(ret) return ret; while(1){ char linebuf[400]; int rline = autozip_gets(&auto_FP, linebuf, 399); if(rline<1) break; if(rline==grc->read_length +1 || ( rline==grc->read_length && linebuf[rline-1]!='\n' ) ) { // is OK. }else{ SUBREADprintf("Error: all your quality strings must be %d-byte long.\n", grc->read_length); ret = 1; break; } char * qstr = malloc(rline); memcpy(qstr, linebuf, rline); if(qstr[rline-1]=='\n') qstr[rline-1]=0; ArrayListPush(grc -> quality_strings, qstr); } autozip_close(&auto_FP); } if(ret) return ret; ret = autozip_open(grc->transcript_fasta_file, &auto_FP); if(ret<0){ ret = 1; SUBREADprintf("Error: unable to open the transcript file.\n"); } else ret = 0; if(ret) return ret; HelpFuncMD5_CTX md5ctx; HelpFuncMD5_Init(&md5ctx); HashTable * seq_duplicate_tab = StringTableCreate(100000); HashTableSetDeallocationFunctions(seq_duplicate_tab, free, NULL); unsigned long long linear_space_top = 0; char * lbuf = NULL, * seq_name = NULL; unsigned int lbuf_cap = 0, lbuf_used = 0, this_seq_len = 0, total_dup=0; while(1){ char clinebuf[TRANSCRIPT_FASTA_LINE_WIDTH]; int rlength = autozip_gets(&auto_FP, clinebuf, TRANSCRIPT_FASTA_LINE_WIDTH -1); if(rlength < 1)break; if(rlength >= TRANSCRIPT_FASTA_LINE_WIDTH -1 || clinebuf[rlength]!='\0' || clinebuf[rlength-1]!='\n'){ SUBREADprintf("Error: The line width of the fasta file excessed %d bytes.\n", TRANSCRIPT_FASTA_LINE_WIDTH); ret = 1; break; } if(clinebuf[0]=='>'){ if(NULL != seq_name){ char * md5mem = malloc(33); unsigned char md5res[16]; HelpFuncMD5_Final(md5res, &md5ctx); int md5i;for(md5i=0;md5i<16;md5i++)sprintf(md5mem+2*md5i, "%02X", 0xff&(int)md5res[md5i]); char * had_tab = HashTableGet(seq_duplicate_tab, md5mem); long seq_exp = HashTableGet(grc->expression_levels, seq_name)-NULL; if(had_tab && seq_exp>1) total_dup++;//SUBREADprintf("Warning: duplicate sequence was found in '%s' and '%s'.\n", seq_name, had_tab); if(seq_exp>1)HashTablePut(seq_duplicate_tab, md5mem, 1+NULL); else free(md5mem); had_tab = HashTableGet(grc-> transcript_sequences, seq_name); if(had_tab){ SUBREADprintf("Error: duplicate sequence names were found in the input: '%s'.\n", seq_name); return -1; } grc_put_new_trans(grc, seq_name, lbuf, this_seq_len, &linear_space_top); } clinebuf[rlength-1]=0; if(grc->simple_transcript_names) for(xk1=1; xk1 0 lbuf_used += rlength -1; this_seq_len = lbuf_used; } } if(lbuf_used<1){ SUBREADprintf("Error: The fasta file did not end correctly! \n"); ret = 1; } if(NULL != seq_name && lbuf_used >0){ char * md5mem = malloc(33); unsigned char md5res[16]; HelpFuncMD5_Final(md5res, &md5ctx); int md5i;for(md5i=0;md5i<16;md5i++)sprintf(md5mem+2*md5i, "%02X", 0xff&(int)md5res[md5i]); long seq_exp = HashTableGet(grc->expression_levels, seq_name)-NULL; char * had_tab = HashTableGet(seq_duplicate_tab, md5mem); if(had_tab && seq_exp>1)total_dup++;// SUBREADprintf("Warning: duplicate sequence was found in '%s' and '%s'.\n", seq_name, had_tab); free(md5mem); had_tab = HashTableGet(grc-> transcript_sequences, seq_name); if(had_tab){ SUBREADprintf("Error: duplicate sequence names were found in the input: '%s'.\n", seq_name); return -1; } grc_put_new_trans(grc, seq_name, lbuf, this_seq_len, &linear_space_top); } if(total_dup)SUBREADprintf("Warning: there are %d transcripts that have replicate sequences and the wanted expression levels are non-zero. You may use scanFasta() to find their names.\n", total_dup); autozip_close(&auto_FP); HashTableDestroy(seq_duplicate_tab); if(linear_space_top<1){ SUBREADprintf("Error: no valid transcript found in the input. No reads can be generated.\n"); return -1; } char outname[MAX_FILE_NAME_LENGTH+30]; sprintf(outname,"%s.truthCounts", grc->output_prefix); grc->counts_out_fp = fopen(outname,"w"); fprintf(grc->counts_out_fp, "## CMD :%s\nTranscriptID\tLength\tCount\n", grc->cmd_line); sprintf(outname,"%s_R1.fastq.gz", grc->output_prefix); grc->out_fps[0] = gzopen(outname, "wb"); if(grc->is_paired_end){ sprintf(outname,"%s_R2.fastq.gz", grc->output_prefix); grc->out_fps[1] = gzopen(outname, "wb"); }else grc->out_fps[1]=NULL; return ret; } #ifdef MAKE_STANDALONE int main(int argc, char ** argv) #else int gen_rnaseq_reads_main(int argc, char ** argv) #endif { int do_fasta_summary = 0; int c; int option_index = 0; genRand_context_t grc; memset(&grc,0,sizeof(grc)); optind = 0; opterr = 1; optopt = 63; rebuild_command_line(&grc.cmd_line, argc, argv); // default settings of the read/insertion length: 100bp, a general illumina feeling grc.insertion_length_sigma = 30.; grc.insertion_length_min = 110; grc.insertion_length_max = 400; grc.insertion_length_mean = 160.; grc.read_length = 100; grc.floor_strategy = STRATEGY_RANDOM_ASSIGN; long long seed = -1; while ((c = getopt_long (argc, argv, "QO:TCxS:V:N:X:F:L:q:r:t:e:o:pM?", long_options, &option_index)) != -1) { switch(c){ case 'Q': grc.quiet=1; break; case 'x': grc.no_actual_reads = 1; break; case 'O': if(strcmp(optarg,"FLOOR")==0) grc.floor_strategy = STRATEGY_LESS_THAN_N; if(strcmp(optarg,"ITERATIVE")==0) grc.floor_strategy = STRATEGY_ITERATIVE_M; if(strcmp(optarg,"RANDOM")==0) grc.floor_strategy = STRATEGY_RANDOM_ASSIGN; break; case 'M': do_fasta_summary = 1; break; case 'V': grc.insertion_length_sigma = atof(optarg); break; case 'N': grc.insertion_length_min = atoi(optarg); break; case 'X': grc.insertion_length_max = atoi(optarg); break; case 'F': grc.insertion_length_mean = atof(optarg); break; case 'L': grc.read_length = atoi(optarg); break; case 'p': grc.is_paired_end = 1; break; case 'o': strcpy(grc.output_prefix, optarg); break; case 'e': strcpy(grc.expression_level_file, optarg); break; case 't': strcpy(grc.transcript_fasta_file, optarg); break; case 'T': grc.truth_in_read_names=1; break; case 'C': grc.simple_transcript_names = 1; break; case 'r': grc.output_sample_size = atoll(optarg); break; case 'S': seed = atoll(optarg); assert(seed>=0); break; case 'q': strcpy(grc.quality_string_file, optarg); break; default: case '?': print_usage_gen_reads(argv[0]); return 0; } } #ifdef MAKE_STANDALONE if(seed<0){ double timemil = miltime(); memcpy(&seed, &timemil, sizeof(seed)); } #else { // in R, we use the random seed generated by R's RNG to initialize the myrand module, then use four random numbers from it to initialize the random_seed inside this program. // This guarantees that when R's RNG was seeded by the same seed, the behaviour of the read generator is fixed. myrand_srand(0);// the seed is ignored in R so give it zero. seed = 0; seed = (seed<<16) | (myrand_rand()&0xffff); seed = (seed<<16) | (myrand_rand()&0xffff); seed = (seed<<16) | (myrand_rand()&0xffff); seed = (seed<<16) | (myrand_rand()&0xffff); } #endif memcpy(grc.random_seeds, &seed, sizeof(seed)); int ret = 0; if(do_fasta_summary){ ret = grc_summary_fasta(&grc); if(ret && strlen(grc.output_prefix)>0){ char delfn[30+MAX_FILE_NAME_LENGTH]; sprintf(delfn, "%s.faSummary", grc.output_prefix); //SUBREADprintf("UNLINK: %s\n", delfn); unlink(delfn); } free(grc.cmd_line); }else{ int ret = grc_check_parameters(&grc) && print_usage_gen_reads(argv[0]); ret = ret || grc_load_env(&grc); ret = ret || grc_gen(&grc); ret = ret || grc_finalize(&grc); } return ret; } Rsubread/src/gene-algorithms.c0000644000175200017520000024123614136047615017402 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #define _GNU_SOURCE #include #include #include #include #include #include #include #include #include #include #ifndef FREEBSD #include #endif #include "subread.h" #include "input-files.h" #include "gene-algorithms.h" #include "sorted-hashtable.h" void non_func(const char * fmt, ...) { } void subread_lock_release(subread_lock_t * lock) { #ifdef MACOS pthread_mutex_unlock(lock); #else pthread_spin_unlock(lock); #endif } void subread_lock_occupy(subread_lock_t * lock) { #ifdef MACOS pthread_mutex_lock(lock); #else pthread_spin_lock(lock); #endif } void subread_destroy_lock(subread_lock_t * lock) { #ifdef MACOS pthread_mutex_destroy(lock); #else pthread_spin_destroy(lock); #endif } void subread_init_lock(subread_lock_t * lock) { #ifdef MACOS pthread_mutex_init(lock, NULL); #else pthread_spin_init(lock, PTHREAD_PROCESS_PRIVATE); #endif } //int EXON_MAX_CIGAR_LEN = 26; //#define indel_debug #ifdef indel_debug #define ddprintf SUBREADprintf #define ddfflush fflush #else #define ddprintf non_func #define ddfflush(a) #endif #define get_base_quality_score(quality_chr, quality_scale) ((quality_scale)==QUALITY_SCALE_NONE?-0.01: ((quality_scale) == QUALITY_SCALE_LINEAR?( (quality_chr) - '@')*0.01-0.03: correct_rate_table[(quality_chr) - '@'])) double begin_ftime; unsigned char get_next_char(FILE * fp) { int find_br = 0; while (!feof(fp)) { char nch; nch = fgetc(fp); if (find_br) { if (nch == '\n') find_br=0; } else if (nch == '>') find_br = 1; else if (nch > 32) return nch; } return 0; } int is_valid_subread(const char * read_str) { return 1; int i; for (i=0; i<16; i++) if(read_str[i] == 'N' || read_str[i] == '.') return 0; return 1; } float read_quality_score(char * qualityb, int rl , int format) { int i; int qual = 0; int testlen = 0; char base; if(format==FASTQ_PHRED64) base = 'B'; else base = '#'; for(i=0; i 1) { qual += testv; testlen ++; } } return qual*1./testlen; } int min_matched_bases(char * qualityb, int rl , int format, float match_score) { if(!(qualityb) || !qualityb[0]) return 0; int i; char base; int ret = 0; if(format==FASTQ_PHRED64) base = 'B'; else base = '#'; for(i=0; i"); for(i = 0; i< space_width; i++) strcat(out_buf," "); strcat(out_buf,"]"); print_in_box(80,0,0,"%s%c",out_buf,CORE_SOFT_BR_CHAR); */ print_in_box(81,0,0," [ %.1f%%%% finished ] %c",percentage*100, CORE_SOFT_BR_CHAR); } void print_text_scrolling_bar(char * hint, float percentage, int width, int * internal_counter) { char fan = '-'; int bar_width = width - 7 - strlen(hint) , i; int dash_width = (int)(bar_width * percentage+0.5); dash_width = min(dash_width, bar_width - 1); int space_width = bar_width - 1 - dash_width; /* for (i=0; i"); for(i = 0; i< space_width; i++) strcat(lbuf, " "); strcat(lbuf, "]"); SUBREADprintf("%s%c",lbuf,CORE_SOFT_BR_CHAR); } void add_repeated_numbers(int qid, gene_vote_t * vote, unsigned char * repeated_regions) { int i,j; for (i=0; iitems[i]; j++) { int v_2 ; if(vote-> votes[i][j] >=2) { v_2 = vote-> votes[i][j] - 2; if(repeated_regions[v_2 + qid * 16] < 255) repeated_regions[v_2 + qid * 16]++; } } } int remove_repeated_reads(gehash_t * table, gehash_t * huge_table, int index_threshold) { int i; int vals[200000]; int val_len[200000]; int scroll_count = 0; unsigned int all_removed = 0; for (i=0; i buckets_number ; i++) { struct gehash_bucket * cb = table->buckets + i; int j; int val_c = 0; if(i % 300 == 0) print_text_scrolling_bar ("Removing non-informative subreads", 1.0*i/table->buckets_number, 80, &scroll_count); for(j=0; jcurrent_items; j++) { int k, found = 0; for(k=0;kitem_keys[j]) { val_len[k]++; found = 1; break; } } if(!found) { if(val_c>199999) { SUBREADprintf("\nThere are too many items in a bucket; you may reduce the threshold of non-informative subreads to eliminate this problem.\n"); #ifdef MAKE_STANDALONE exit(-1); #else return -1; #endif } else { vals[val_c] = cb->item_keys[j]; val_len[val_c] = 1; val_c++; } } } // SUBREADprintf ("F3\n"); for(j=0; j index_threshold) { gehash_remove(table, vals[j]); gehash_insert(huge_table, vals[j], 1, NULL); all_removed += val_len[j]; } } } if(IS_DEBUG) SUBREADprintf ("@LOG There are %u subreads removed from the index.\n", all_removed); return all_removed; } unsigned int linear_gene_position(const gene_offset_t* offsets , char *chro_name, unsigned int chro_pos) { unsigned int ret = 0 ; int n; n = HashTableGet(offsets -> read_name_to_index, chro_name)-NULL; //printf("09NOV: GET '%s' = %d\n", chro_name, n); if(n<1) return 0xffffffff; if(n>1) ret = offsets->read_offsets[n-2]; ret += offsets -> padding ; return ret + chro_pos; } unsigned int get_gene_linear(int chrono, int offset, const unsigned int offsets []) { if (chrono>1)return offsets[chrono-1]+offset; return offset; } int locate_gene_position_max(unsigned int linear, const gene_offset_t* offsets , char ** chro_name, int * pos, int * head_cut_length, int * tail_cut_length, int rl) { int n = 0; (*chro_name) = NULL; (*pos) = -1; int low_idx=0, high_idx= offsets->total_offsets; while(1){ if(high_idx <= low_idx+1) { n = max(low_idx-2,0); break; } int mid_idx = (low_idx+high_idx)/2; unsigned int mid_val = offsets->read_offsets[mid_idx]; if(mid_val > linear) high_idx = mid_idx; else low_idx = mid_idx+1; } for (; ntotal_offsets; n++) { if (offsets->read_offsets[n] > linear) { //#warning "======== COMMENT THIS LINE !! ========" //SUBREADprintf("max=%u <= lim=%u : ACCEPTED.\n", rl + linear , offsets->read_offsets[n] + 16); // the end of the read should not excess the end of the chromosome if (n==0) *pos = linear; else *pos = linear - offsets->read_offsets[n-1]; if(tail_cut_length == NULL){ if(rl + linear > offsets->read_offsets[n] + 15 - offsets -> padding){ return 1; } } else { unsigned int posn1 = 0; if(n>0) posn1 = offsets->read_offsets[n-1]; long long int tct = ( linear + rl - posn1 - offsets -> padding ); if(tct < rl)tct = rl; long long int chro_leng = (offsets->read_offsets[n] - posn1 - 2*offsets -> padding + 16); //SUBREADprintf("CHRO_LEN : %lld, READ_TAIL %lld , RL=%d\n", chro_leng, tct, rl); tct -= chro_leng; if( tct >= rl ){ return 1; } if( tct <0 )tct=0; (*tail_cut_length) = tct; } if( (*pos) < offsets -> padding ) { if(head_cut_length == NULL || (*pos) + rl <= offsets -> padding){ return 1; }else{ (*head_cut_length) = offsets -> padding - (*pos); (*pos) = offsets -> padding; } } (*pos) -= offsets -> padding; *chro_name = (char *)offsets->read_names+n*MAX_CHROMOSOME_NAME_LEN; return 0; } } return -1; } int locate_gene_position(unsigned int linear, const gene_offset_t* offsets , char ** chro_name, int * pos) { return locate_gene_position_max(linear, offsets, chro_name, pos, NULL, NULL, 0); } #define _index_vote(key) (((unsigned int)key)%GENE_VOTE_TABLE_SIZE) #ifdef USE_SLOW_HASHTABLE_INDEX int vv=0; void add_gene_vote(gene_vote_t* vote, int key , int add_new) { add_gene_vote_weighted(vote, key, add_new, 1); } void add_gene_vote_weighted(gene_vote_t* vote, int key , int add_new, int w) { int offset = _index_vote(key); int datalen = vote -> items[offset]; unsigned int * dat = vote -> pos[offset]; int i; for (i=0;ivotes[offset][i]); test_max += w; vote->votes[offset][i] = test_max; if(test_max > vote->max_vote){ vote->max_vote = test_max; vote->max_position = key; } return; } } if(!add_new || datalen >=GENE_VOTE_SPACE) return; // w += 16; vote -> items[offset] ++; dat[i] = key; vote->votes[offset][i]=w; } #endif int max_gene_vote(gene_vote_t* vote, int * position_result, int query) { int n, i, max_index=0; int max_votes = -1; for (n=0; nitems[n]; gene_vote_number_t * vots = vote->votes[n]; for (i=0; i max_votes) { max_index = n<<16|i; max_votes = vots[i]; } } } if(max_votes == -1) { *position_result = -1; return 0; } else { *position_result = vote->pos[max_index>>16][max_index&0xffff]; return max_votes; } } void clear_allvote(gene_allvote_t* allvote) { memset(allvote -> max_votes,0, allvote -> max_len* sizeof(*allvote -> max_votes)); memset(allvote -> masks,0, allvote -> max_len* sizeof(*allvote -> masks)); memset(allvote -> span_coverage ,0, allvote -> max_len* sizeof(char)); } void destory_allvote(gene_allvote_t* allvote) { free(allvote -> max_positions); free(allvote -> max_votes); free(allvote -> max_quality); free(allvote -> max_final_quality); free(allvote -> masks); #ifdef REPORT_ALL_THE_BEST free(allvote -> best_records); #endif free(allvote -> is_counterpart); free(allvote -> span_coverage); if(allvote -> max_indel_recorder) free(allvote -> max_indel_recorder); } int init_allvote(gene_allvote_t* allvote, int expected_len, int allowed_indels) { int is_OK = 0; allvote -> max_len = expected_len; allvote -> max_positions = (unsigned int *) malloc(sizeof(int)*expected_len); allvote -> max_votes = (gene_vote_number_t *) calloc(sizeof(gene_vote_number_t), expected_len); allvote -> max_quality = (gene_quality_score_t *) calloc(sizeof(gene_quality_score_t), expected_len); allvote -> max_final_quality = (gene_quality_score_t *) calloc(sizeof(gene_quality_score_t), expected_len); allvote -> masks = (short *) calloc(sizeof(short), expected_len); #ifdef REPORT_ALL_THE_BEST allvote -> best_records = (gene_best_record_t *) malloc(sizeof(gene_best_record_t)* expected_len); #endif allvote -> is_counterpart = (unsigned char *) malloc(expected_len); allvote -> max_indel_tolerance = allowed_indels; allvote -> indel_recorder_length = max(3*(allowed_indels+1)+1, 28); allvote -> span_coverage = (char *)calloc(sizeof(char), expected_len); allvote -> repeated_regions = (unsigned char *)calloc(sizeof(unsigned char), 16 * expected_len); if((allvote -> max_quality && allvote -> max_positions && allvote -> max_votes && allvote -> max_final_quality && allvote -> masks && allvote -> is_counterpart && allvote -> span_coverage)) is_OK = 1; if(allowed_indels && is_OK) { allvote -> max_indel_recorder = (char *)malloc( allvote -> indel_recorder_length *expected_len); is_OK = (allvote -> max_indel_recorder!=NULL); } else allvote -> max_indel_recorder = NULL; if(is_OK) return 0; else { SUBREADputs(MESSAGE_OUT_OF_MEMORY); return 1; } } // rl_adjust >0: "D" in read; rl_adjust<0: "I" in read void compress_cigar(char *cigar, int total_length, char * read, int * pos_offset, int *rl_adjust) { char tmp[200]; char cigar_piece [10]; int cigar_len = strlen(cigar); cigar_len = min(cigar_len, EXON_MAX_CIGAR_LEN); cigar[cigar_len]=0; int i; srInt_64 tmpv = 0; char last_operation = 'X'; srInt_64 last_tmpv = 0; int is_first_M = 1; int delta_i = 0; int delta_d = 0; int delta_rl = 0; tmp[0]=0; int cigar_length=0; for(i=0; i < cigar_len; i++) { char cc = cigar[i]; if(isdigit(cc)) { tmpv=tmpv*10+(cc-'0'); } else if(cc=='-') { last_tmpv = 0; cigar_length = 0; break; } else { if(is_first_M && pos_offset && cc=='S') *pos_offset = tmpv; else if(cc=='M') is_first_M = 0; if((cc!=last_operation) && (last_operation!='X')) { if(last_operation == 'M' || last_operation == 'S' || last_operation == 'N' || last_operation == 'B' || last_operation == 'J' || last_operation == 'j' || last_operation == 'b') { if(delta_i) { sprintf(cigar_piece,"%dI", delta_i); strcat(tmp, cigar_piece); } delta_i = 0; if(delta_d) { sprintf(cigar_piece,"%dD", delta_d); strcat(tmp, cigar_piece); } delta_d = 0; #ifdef __MINGW32__ sprintf(cigar_piece,"%I64d%c", last_tmpv, last_operation); #else sprintf(cigar_piece,"%lld%c", last_tmpv, last_operation); #endif strcat(tmp, cigar_piece); } if(last_operation == 'M' || last_operation == 'S' || last_operation == 'I') cigar_length += last_tmpv; last_tmpv = 0; } if(cc=='I' ) { delta_i += tmpv; delta_rl -= tmpv; } if(cc=='D' ) { delta_d += tmpv; delta_rl += tmpv; } last_tmpv += tmpv; tmpv = 0; last_operation = cc; } } if(last_tmpv) { if(delta_i) { sprintf(cigar_piece,"%dI", delta_i); strcat(tmp, cigar_piece); } if(delta_d) { sprintf(cigar_piece,"%dD", delta_d); strcat(tmp, cigar_piece); } if(last_operation =='M' || last_operation =='S') { #ifdef __MINGW32__ sprintf(cigar_piece,"%I64d%c", tmpv+last_tmpv, last_operation); #else sprintf(cigar_piece,"%lld%c", tmpv+last_tmpv, last_operation); #endif strcat(tmp, cigar_piece); } if(last_operation == 'M' || last_operation == 'S' || last_operation == 'I') cigar_length += tmpv+last_tmpv; } if(cigar_length == total_length) { if(rl_adjust)(*rl_adjust)=delta_rl; strcpy(cigar, tmp); } else { sprintf(cigar, "%dM", total_length); } } // adjust_len <0: "I" in read; adjust_len>0: "D" in read void show_cigar(char * info, int len, int is_reversed_map, char * buf, int indel_tolerance, int total_subreads, char *read, int * pos_offset, int * adjust_len) { int i; int last_offset = 0, cursor = 0; if(info[0]==-1){ sprintf(buf, "%dM", len); return; } else if(info[0]==-2){ if (strchr( info+1, '-')) sprintf(buf, "%dM", len); else { strncpy(buf, info+1, 98); compress_cigar(buf, len, read, pos_offset, adjust_len); } return; } else if(info[0]==-3){ info ++; } for(i=0; i0?offset:0) <0) { buf[0]=0; cursor = 0; break; } if (i >0) { sprintf(buf+strlen(buf), "%d%c%dM", abs(offset), offset>0?'I':'D', base_end - cursor - (offset>0?offset:0)); if(adjust_len) (*adjust_len) -= offset; } else sprintf(buf+strlen(buf), "%dM", base_end); last_offset = dist; cursor = base_end; } compress_cigar(buf, len, read, pos_offset, adjust_len); } void add_allvote_q(gene_allvote_t* allvote,int qid , int pos, gene_vote_number_t votes, gene_quality_score_t quality, int is_counterpart, short mask, char * max_indel_recorder, gene_value_index_t * array_index, char * read_txt, int read_len, int max_indel, int total_subreads, int space_type, int report_junction, int is_head_high_quality, char * qual_txt, int phred_version, char span_coverage, short **dynamic_programming_short , char ** dynamic_programming_char) { // SUBREADprintf("Ospan=%d Nspan=%d\n", allvote -> span_coverage[qid], span_coverage); } float CORE_RECOVER_MATCHING_RATE = 0.9; float CORE_INDEL_MATCHING_RATE_TAIL = 0.8; float CORE_INDEL_MATCHING_RATE_HEAD = 0.8; void find_and_explain_indel(gene_allvote_t* allvote,int qid , int pos, gene_vote_number_t votes, gene_quality_score_t quality, int is_counterpart, char mask, char * max_indel_recorder, gene_value_index_t * array_index, char * read_txt, int read_len, int max_indel, int total_subreads, int space_type, int report_junction, int is_head_high_quality, char * qual_txt, int phred_version , short ** dynamic_programming_short, char **dynamic_programming_char) { //unsigned int pos0 = pos; if(allvote -> max_indel_recorder) { //PNT111 short head_indel_pos=-1 , tail_indel_pos=-1; int head_indel_movement=0, tail_indel_movement=0; if (array_index) { int k; if (max_indel_recorder[0]) { int cover_start = find_subread_end(read_len, total_subreads, max_indel_recorder[0]-1)- 15; for (k=0; max_indel_recorder[k]; k+=3); int cover_end = find_subread_end(read_len, total_subreads, max_indel_recorder[k-2]-1) + max(0,-max_indel_recorder[k-1]); int head_qual = is_head_high_quality?0x7fffffff:0, tail_qual = is_head_high_quality?0:0x7fffffff; if(qual_txt && qual_txt[0]) { int j; head_qual = 0; tail_qual = 0; for (j = 0; j 0) { if (head_qual / cover_start < 850000) { CORE_INDEL_MATCHING_RATE_HEAD = 0.75; head_must_correct =2; } else if (head_qual / cover_start < 950000 ) { CORE_INDEL_MATCHING_RATE_HEAD = 0.85; head_must_correct =3; } else CORE_INDEL_MATCHING_RATE_HEAD = 0.92; }else CORE_INDEL_MATCHING_RATE_HEAD = 9999; int tail_must_correct = 4; if(read_len-cover_end > 0) { if (tail_qual / (read_len-cover_end) < 850000) { CORE_INDEL_MATCHING_RATE_TAIL = 0.75; tail_must_correct =2; } else if (tail_qual / (read_len-cover_end) < 950000) { CORE_INDEL_MATCHING_RATE_TAIL = 0.85; tail_must_correct =3; } else CORE_INDEL_MATCHING_RATE_TAIL = 0.92; }else CORE_INDEL_MATCHING_RATE_TAIL = 9999; int is_full_covered = 0; is_full_covered = extend_covered_region(array_index, pos, read_txt, read_len, cover_start, cover_end, 4, head_must_correct, tail_must_correct, max_indel, space_type, max_indel_recorder[k-1], &head_indel_pos, &head_indel_movement, &tail_indel_pos, &tail_indel_movement, is_head_high_quality, qual_txt, phred_version, CORE_INDEL_MATCHING_RATE_HEAD, CORE_INDEL_MATCHING_RATE_TAIL); if(head_indel_movement) { pos += head_indel_movement; allvote -> max_positions[qid] = pos; } if(is_full_covered == 3) { allvote -> masks[qid] &= ~IS_RECOVERED_JUNCTION_READ; } else allvote -> masks[qid] |= IS_RECOVERED_JUNCTION_READ; } } *(allvote -> max_indel_recorder + qid * allvote -> indel_recorder_length) = 0xff ; if ((max_indel_recorder[3] || head_indel_pos>=0 || tail_indel_pos>0)){ int head_pos = 0; if((head_indel_pos>=0 && report_junction) || head_indel_movement)head_pos = head_indel_pos ;//- max(0,head_indel_movement); int tail_pos = read_len; if((tail_indel_pos>0 && report_junction) || tail_indel_movement)tail_pos = tail_indel_pos ;//- min(0, tail_indel_movement); explain_indel_in_middle(allvote, qid , pos, max_indel_recorder, array_index, read_txt, read_len, max_indel, total_subreads, head_pos, tail_pos , head_indel_movement, tail_indel_movement, report_junction, dynamic_programming_short, dynamic_programming_char); //if(*(allvote -> max_indel_recorder + qid * allvote -> indel_recorder_length) != 0xfd ) allvote -> max_positions[qid] = pos0; } } } void explain_indel_in_middle(gene_allvote_t* allvote, int qid , int pos, char * max_indel_recorder, gene_value_index_t * array_index, char * read_txt, int read_len, int max_indel, int total_subreads, int head_start_point, int tail_end_point, int head_indel_movement, int tail_indel_movement, int report_junction, short ** dynamic_programming_short , char ** dynamic_programming_char) { char indel_operations[1500]; int i,xx; char tmp_cigar [EXON_MAX_CIGAR_LEN+1]; int current_pos = head_start_point; int explain_cursor = head_start_point; char last_operation = 0; int del_number = 0; int dynamic_delta = 0; tmp_cigar[0]=0; if(current_pos >0) { if (head_indel_movement != 0) sprintf(tmp_cigar, "%dM%d%c", head_start_point - max(head_indel_movement, 0), abs(head_indel_movement), head_indel_movement>0?'I':'D'); else if (report_junction) sprintf(tmp_cigar, "%dS", head_start_point); } //ddprintf ("R=%s; REC[8]=%d [9]=%d\n", read_txt, max_indel_recorder[8], max_indel_recorder[9]); for (i=3; max_indel_recorder[i]; i+=3) { if (i >= max_indel*3) break; int last_dist = max_indel_recorder[i-1]; int black_subread_start = max_indel_recorder[i-2]-1; int black_subread_end = max_indel_recorder[i]-1; if (black_subread_end < black_subread_start+1) black_subread_end = black_subread_start+1; while(max_indel_recorder[i+3]) { if (max_indel_recorder[i+3]- black_subread_end>2 || i >= max_indel*3-3) break; black_subread_end = max_indel_recorder[i+3]-1; i+=3; } int black_base_start = find_subread_end(read_len, total_subreads,black_subread_start )- 3 + max(0,-last_dist); int black_base_end = find_subread_end(read_len, total_subreads, black_subread_end)-15 + 3 + max(0,-max_indel_recorder[i+2]); int blackref_base_start = black_base_start - max(0,-last_dist) + max(0,last_dist); // SUBREADprintf("baseref_offset=%d\n", max(0,last_dist)); int gap_end_read = black_base_end + max(0,max_indel_recorder[i+2]); blackref_base_start = max(0,blackref_base_start); black_base_start = max(0,black_base_start); black_base_end = min(read_len, black_base_end); int vpos = strlen(tmp_cigar); if (vpos > EXON_MAX_CIGAR_LEN -6) break; int exp_indel = last_dist - max_indel_recorder[i+2]; int moves = 0; if(black_base_end > black_base_start) moves = dynamic_align (read_txt + black_base_start, black_base_end-black_base_start , array_index, pos + blackref_base_start, max_indel, indel_operations, exp_indel, -10, black_base_end - black_base_start+5, dynamic_programming_short, dynamic_programming_char); last_operation = 0; if(moves)// < (black_base_end-black_base_start) + 6 + 1.5 * exp_indel) { int pre_read_len = 0; current_pos = black_base_start ; xx=0; while (pre_read_len < 0 && xx < moves) { if(indel_operations[xx]!=2)pre_read_len++; xx++; } for (; xxEXON_MAX_CIGAR_LEN-6) break; sprintf(tmp_cigar + vpos, "%d%c", last_operation==1?del_number:(current_pos - explain_cursor), last_operation==0?'M':(last_operation==1?'D':'I')); explain_cursor = current_pos ; del_number = 0; } if(current_pos >= gap_end_read-1 || xx == moves - 1){ if(current_operation != 0 && current_pos!=explain_cursor) { int vpos = strlen(tmp_cigar); sprintf(tmp_cigar + vpos, "%d%c", current_operation==1?del_number:(current_pos - explain_cursor), current_operation==1?'D':'I'); explain_cursor = current_pos; del_number=0; } break; } if (indel_operations[xx]==0) current_pos ++; else if (indel_operations[xx]==1) {del_number++; dynamic_delta++;}//"D" else if (indel_operations[xx]==2) {current_pos ++; dynamic_delta--;} //"I" else if (indel_operations[xx]==3) current_pos ++; last_operation = current_operation; } } else { int movement = last_dist - max_indel_recorder[i+2]; int vpos = strlen(tmp_cigar); if (vpos>EXON_MAX_CIGAR_LEN-6) break; current_pos = find_subread_end(read_len, total_subreads,max_indel_recorder[i]-1)-15 + (max_indel_recorder[i+2] >0?max_indel_recorder[i+2]:-max_indel_recorder[i+2]); current_pos -= (black_base_end - black_base_start)/2 -3; if(movement) sprintf(tmp_cigar + vpos, "%dM%d%c", current_pos - explain_cursor , abs(movement), movement<0?'D':'I' ); else if (current_pos - explain_cursor>0) sprintf(tmp_cigar + vpos, "%dM", current_pos - explain_cursor ); explain_cursor = current_pos + (movement >0?movement:0); current_pos = find_subread_end(read_len, total_subreads,max_indel_recorder[i+1]-1); last_operation = 0; del_number = 0; moves = 0; } last_operation = 0; current_pos = black_base_end; } int vpos = strlen(tmp_cigar); if (vpos > EXON_MAX_CIGAR_LEN-9 ) { *(allvote -> max_indel_recorder + qid * allvote -> indel_recorder_length) = 0xfd; memcpy(allvote -> max_indel_recorder + qid * allvote -> indel_recorder_length+1, max_indel_recorder, 3 * max_indel); } else { if (explain_cursor explain_cursor) sprintf(tmp_cigar+vpos,"%dM", tail_end_point - explain_cursor); vpos = strlen(tmp_cigar); if(tail_end_point< read_len) { if (tail_indel_movement!=0) { int tail_len_m = read_len -(tail_end_point - min(tail_indel_movement, 0)); if(tail_len_m) sprintf(tmp_cigar+vpos, "%d%c%dM", abs(tail_indel_movement), tail_indel_movement<0?'I':'D', tail_len_m); else sprintf(tmp_cigar+vpos, "%d%c", abs(tail_indel_movement), tail_indel_movement<0?'I':'D'); } else if (report_junction) sprintf(tmp_cigar+vpos, "%dS",read_len - tail_end_point ); } } // if (head_start_point >0 || tail_end_point < read_len) // strcat(tmp_cigar,"X"); *(allvote -> max_indel_recorder + qid * allvote -> indel_recorder_length) = 0xfe ; strncpy(allvote -> max_indel_recorder + qid * allvote -> indel_recorder_length+1, tmp_cigar, allvote -> indel_recorder_length - 2); } } int evaluate_piece(char * piece_str, int chron, int offset, int is_counterpart, int start_pos, int end_pos) { char fname[MAX_FILE_NAME_LENGTH]; int inner_pos = 0, i; FILE * fp; char next_char=0; int ret = 0; if (chron == 0) sprintf(fname, "/opt/Work2001/Gene-Search/src/GENE-LIB/%02da.fa", chron); else sprintf(fname, "/opt/Work2001/Gene-Search/src/GENE-LIB/%02d.fa", chron); inner_pos = offset + offset / 70; fp = f_subr_open(fname,"r"); while(next_char!='\n') next_char=fgetc(fp); fseek(fp, inner_pos, SEEK_CUR); for(i=0 ; i start_base_offset + index -> length && pos > -i) ret += match_chro(read, index, pos + i, test_len, is_negative_strand, space_type); } return ret; } void mark_votes_array_index(char * read_str, int read_len, gene_vote_t * dest, gene_vote_t * src, gene_value_index_t * my_array_index, int color_space, int indel_tolerance, int min_minor, const char quality_str [], int quality_scale, int is_negative_strand) { int i, j; dest -> max_vote = 0; dest -> max_quality = 0; for (i=0; i items[i] = src -> items[i]; for(j=0; j< src->items[i]; j++) { unsigned int potential_position = src-> pos[i][j]; float matchingness_count = 0; if (src -> votes [i][j]>= min_minor) //matchingness_count = match_read(read_str, read_len, potential_position, my_array_index, color_space, indel_tolerance, quality_str, quality_scale); matchingness_count = 1.*match_chro_indel(read_str, my_array_index, potential_position, read_len, 0, color_space, indel_tolerance, NULL, 0); dest -> pos[i][j] = potential_position; dest -> quality[i][j] = matchingness_count; dest -> votes [i][j] =src -> votes [i][j]; dest -> masks[i][j] = src -> masks[i][j]; dest -> coverage_start[i][j] = src -> coverage_start[i][j]; dest -> coverage_end[i][j] = src -> coverage_end[i][j]; memcpy(dest -> indel_recorder[i][j], src -> indel_recorder[i][j], MAX_INDEL_TOLERANCE*3*sizeof(char)); // find for the `best positions'; only the best positions are replaced by the base machingness scores. if((matchingness_count > dest -> max_quality && src -> votes [i][j] == dest -> max_vote) || (src -> votes [i][j] > dest -> max_vote)) { memcpy(dest -> max_indel_recorder, src -> indel_recorder[i][j], MAX_INDEL_TOLERANCE*3*sizeof(char)); dest -> max_vote = src -> votes [i][j]; dest -> max_mask = src -> masks[i][j]; dest -> max_quality = matchingness_count; dest -> max_position = potential_position; dest -> max_coverage_start = src -> coverage_start[i][j]; dest -> max_coverage_end = src -> coverage_end[i][j]; } } } } int select_positions_array(char * read1_str, int read1_len, char * read2_str, int read2_len, gene_vote_t * vote_read1, gene_vote_t * vote_read2, gene_vote_number_t * numvote_read1, gene_vote_number_t * numvote_read2, gene_quality_score_t * sum_quality, gene_quality_score_t * qual_r1, gene_quality_score_t * qual_r2, gene_vote_number_t * r1_recorder, gene_vote_number_t * r2_recorder, gehash_data_t * pos_read1, gehash_data_t * pos_read2, unsigned int max_pair_dest, unsigned int min_pair_dest, int min_major, int min_minor,int is_negative_strand, gene_value_index_t * my_array_index, int color_space, int indel_tolerance, int number_of_anchors_quality, const char quality_str1 [], const char quality_str2 [], int quality_scale, int max_indel_len, int * is_break_even) { gene_vote_t base_vote_1 , base_vote_2; mark_votes_array_index(read1_str, read1_len, &base_vote_1, vote_read1, my_array_index, color_space, indel_tolerance, min_minor, quality_str1, quality_scale, is_negative_strand); mark_votes_array_index(read2_str, read2_len, &base_vote_2, vote_read2, my_array_index, color_space, indel_tolerance, min_minor, quality_str2, quality_scale, is_negative_strand); // SUBREADprintf("Q=%.3f\n", *qual_r2); return select_positions(&base_vote_1, &base_vote_2, numvote_read1, numvote_read2, sum_quality, qual_r1, qual_r2, pos_read1, pos_read2, r1_recorder , r2_recorder ,max_pair_dest, min_pair_dest, min_major, min_minor, is_negative_strand, number_of_anchors_quality,max_indel_len, is_break_even); } int select_positions(gene_vote_t * vote_read1, gene_vote_t * vote_read2, gene_vote_number_t * numvote_read1, gene_vote_number_t * numvote_read2, gene_quality_score_t * sum_quality, gene_quality_score_t * qual_r1, gene_quality_score_t * qual_r2 , gehash_data_t * pos_read1, gehash_data_t * pos_read2, gene_vote_number_t * read1_indel_recorder, gene_vote_number_t * read2_indel_recorder, unsigned int max_pair_dest, unsigned int min_pair_dest, int min_major, int min_minor,int is_negative_strand, int number_of_anchors_quality, int max_indel_len, int * is_breakeven) { int k, i, j, anchors = 0; gehash_data_t anchors_position [ANCHORS_NUMBER]; unsigned char anchor_read [ANCHORS_NUMBER]; gene_vote_number_t anchor_votes = max(vote_read1->max_vote, vote_read2->max_vote); gene_vote_number_t anchors_votes [ANCHORS_NUMBER]; gene_quality_score_t anchors_quality [ANCHORS_NUMBER]; int anchors_buckets [ANCHORS_NUMBER]; int anchors_index [ANCHORS_NUMBER]; gene_vote_number_t minor_votes [ANCHORS_NUMBER]; gene_quality_score_t minor_quality [ANCHORS_NUMBER]; gehash_data_t minor_position [ANCHORS_NUMBER]; int minor_buckets [ANCHORS_NUMBER]; int minor_index [ANCHORS_NUMBER]; char is_minor_breakeven [ANCHORS_NUMBER]; if(anchor_votes < min_major) return 0; for (k=0; k<2; k++) { gene_vote_t * current_vote = k?vote_read2:vote_read1; if (current_vote->max_vote < anchor_votes) continue; for (i=0; iitems[i]; j++) { if(current_vote->votes[i][j] >= anchor_votes - 0) { anchors_position[anchors] = current_vote->pos[i][j]; anchors_votes [anchors] = current_vote->votes[i][j]; anchors_quality [anchors] = current_vote->quality[i][j]; anchor_read [anchors] = k; anchors_buckets [anchors] = i; anchors_index [anchors] = j; anchors++; if(anchors >= ANCHORS_NUMBER) { // SUBREADprintf("Abnormally too many anchors [%d, %d] %f\n", i, j, anchor_votes); anchors --; } } } } memset(minor_votes,0, ANCHORS_NUMBER*sizeof(gene_vote_number_t)); memset(minor_quality,0, ANCHORS_NUMBER*sizeof(gene_quality_score_t)); memset(is_minor_breakeven,0, ANCHORS_NUMBER); for (k=0; k<2; k++) { gene_vote_t * current_vote = k?vote_read2:vote_read1; gene_vote_t * current_vote2 = k?vote_read1:vote_read2; if (current_vote2->max_vote < anchor_votes) continue; for (i=0; iitems[i]; j++) { if(current_vote->votes[i][j] >= min_minor) { int l; for (l=0; lpos[i][j]; abdist -= anchors_position[l]; if((anchor_read[l] && !is_negative_strand) || ((!anchor_read[l]) && is_negative_strand)) abdist = -abdist; if (abdist < 0) //#warning WGSIM generates paired-end data in a wrong way; abdist is given its absolute value in such a case. abdist = -abdist; // continue; long long int abdist_old = minor_position[l]; abdist_old -= anchors_position[l]; if(abdist_old <0) abdist_old=-abdist_old; if ( (minor_votes[l] < current_vote->votes[i][j] || (minor_votes[l] == current_vote->votes[i][j] && current_vote->quality [i][j] > minor_quality[l]) || (minor_votes[l] == current_vote->votes[i][j] && abs(current_vote->quality [i][j] - minor_quality[l])<0.0001 && abdist < abdist_old))&& (abdist <= max_pair_dest) && (abdist >= min_pair_dest) ) { minor_votes[l] = current_vote->votes[i][j]; minor_position[l] = current_vote->pos[i][j]; minor_quality[l] = current_vote->quality [i][j]; minor_buckets[l] = i; minor_index[l] = j; } } } } } gene_vote_number_t selection_minor_vote = 0; gene_vote_number_t selection_major_vote = 0; gehash_data_t selection_minor_pos = 0; gehash_data_t selection_major_pos = 0; int selected_major_index=0, selected_major_bucket=0, selected_minor_bucket=0, selected_minor_index=0; *sum_quality = 0; unsigned char selection_major_read_no = 0; //int reason = 0; for (k=0; k (selection_minor_vote+selection_major_vote) || ((minor_votes[k]+anchors_votes[k]) == (selection_minor_vote+selection_major_vote) && minor_quality[k] + anchors_quality[k] > *sum_quality)) { need_replace = 1; } else if (minor_votes[k]+anchors_votes[k] == selection_minor_vote+selection_major_vote && minor_quality[k] + anchors_quality[k] == *sum_quality) { if (selection_minor_pos != minor_position[k] && minor_position[k] != selection_major_pos) { * is_breakeven = 1; if (selection_major_pos > anchors_position[k]) need_replace = 1; } } if(need_replace){ selection_minor_pos = minor_position[k]; selection_major_pos = anchors_position[k]; selection_minor_vote = minor_votes[k]; selection_major_vote = anchors_votes[k]; selection_major_read_no = anchor_read[k]; selected_major_bucket = anchors_buckets[k]; selected_minor_bucket = minor_buckets[k]; selected_major_index = anchors_index[k]; selected_minor_index = minor_index[k]; * is_breakeven = is_minor_breakeven[k]; * qual_r1 = anchor_read[k] ? minor_quality[k]:anchors_quality[k]; * qual_r2 = anchor_read[k] ? anchors_quality[k]: minor_quality[k]; * sum_quality = minor_quality[k] + anchors_quality[k]; } } if(selection_minor_vote > 0) { if(selection_major_read_no) // major is on read 2 { *numvote_read1 = selection_minor_vote; *numvote_read2 = selection_major_vote; *pos_read1 = selection_minor_pos; *pos_read2 = selection_major_pos ; indel_recorder_copy(read1_indel_recorder, vote_read1 -> indel_recorder[selected_minor_bucket][selected_minor_index]); indel_recorder_copy(read2_indel_recorder, vote_read2 -> indel_recorder[selected_major_bucket][selected_major_index]); } else { *numvote_read1 = selection_major_vote; *numvote_read2 = selection_minor_vote; *pos_read1 = selection_major_pos; *pos_read2 = selection_minor_pos; indel_recorder_copy(read1_indel_recorder, vote_read1 -> indel_recorder[selected_major_bucket][selected_major_index]); indel_recorder_copy(read2_indel_recorder, vote_read2 -> indel_recorder[selected_minor_bucket][selected_minor_index]); } return 1; } return 0; } void destroy_offsets(gene_offset_t* offsets) { free(offsets->read_names); free(offsets->read_offsets); HashTableDestroy(offsets->read_name_to_index); } int load_offsets(gene_offset_t* offsets , const char index_prefix []) { char fn[MAX_FILE_NAME_LENGTH]; FILE * fp; int n=0; int padding = 0; int is_V3_index = gehash_load_option(index_prefix, SUBREAD_INDEX_OPTION_INDEX_PADDING , &padding); if(!is_V3_index) return 1; memset(offsets, 0, sizeof( gene_offset_t)); sprintf(fn, "%s.reads", index_prefix); fp = f_subr_open(fn, "r"); if(!fp) { SUBREADprintf("file not found :%s\n", fn); return 1; } int current_max_n = 100; offsets->read_names = malloc(current_max_n * MAX_READ_NAME_LEN); offsets->read_offsets= malloc(current_max_n * sizeof(int)); offsets->read_name_to_index = HashTableCreate(5000); offsets->padding = padding; HashTableSetKeyComparisonFunction(offsets->read_name_to_index, my_strcmp); HashTableSetHashFunction(offsets->read_name_to_index ,HashTableStringHashFunction); HashTableSetDeallocationFunctions(offsets->read_name_to_index ,free, NULL); while (!feof(fp)) { int i=0, step = 0, j=0; read_line(MAX_FILE_NAME_LENGTH-1,fp, fn, 0); if (strlen(fn)<2)continue; while (fn[i]) { if (fn[i] == '\t') { fn[i]=0; offsets->read_offsets[n] = (unsigned int)atoll(fn); step = 1; } else if (step) { if(jread_names+MAX_CHROMOSOME_NAME_LEN*n + (j++)) = fn[i]; *(offsets->read_names+MAX_CHROMOSOME_NAME_LEN*n + j) = 0; } } i++; } char * read_name_mem = malloc(MAX_CHROMOSOME_NAME_LEN); strcpy(read_name_mem, offsets->read_names + n*MAX_CHROMOSOME_NAME_LEN); //printf("09NOV: add_annotation_to_junctions: '%s',%u\n", read_name_mem , n); HashTablePut(offsets->read_name_to_index, read_name_mem , NULL + 1 + n); n++; if(n >= current_max_n) { offsets->read_names = realloc(offsets->read_names, 2*current_max_n * MAX_CHROMOSOME_NAME_LEN); offsets->read_offsets = realloc(offsets->read_offsets, 2*current_max_n * sizeof(int)); current_max_n*=2; } offsets->read_offsets[n] = 0; } offsets->total_offsets=n; fclose(fp); return 0; } #ifndef MAKE_STANDALONE #define CLOCK_USE_GETTIME #endif double miltime(){ double ret; #ifdef FREEBSD struct timeval tp; struct timezone tz; tz.tz_minuteswest=0; tz.tz_dsttime=0; gettimeofday(&tp,&tz); ret = tp.tv_sec+ 0.001*0.001* tp.tv_usec; #else #ifdef CLOCK_USE_GETTIME struct timespec tsc; clock_gettime(CLOCK_REALTIME, &tsc); ret = tsc.tv_sec*1. + tsc.tv_nsec*1./1000/1000/1000; #else struct timeb trp; ftime(&trp); ret = trp.time*1.0+(trp.millitm*1.0/1000.0); #endif #endif return ret; } #define front2(str, bias) (*((str)+(bias))+*((str)+1+(bias))) #define front4(str, bias) (front2(str, bias)+front2(str, bias+2)) #define front8(str, bias) (front4(str, bias)+front4(str, bias+4)) #define front16(str, bias) (front8(str, bias)+front8(str, bias+8)) float match_read(const char read_str[], int read_len, unsigned int potential_position, gene_value_index_t * my_array_index, int space_type, int indel_tolerance, const char quality_str [], int quality_scale) { float ret = 0; int i, bias; char read_matchingness [7][1250]; if(indel_tolerance>3) indel_tolerance = 3; for(bias=-indel_tolerance; bias<=indel_tolerance;bias++) { for(i=0;i max_matchness) { max_matchness = m*1.; best_movement = j; } } // SUBREADprintf("best-match=%f\n", max_matchness); // if (quality_str[0]) { max_matchness = 0; // float qs0 = max_matchness; for (j=0; j<4; j++) { if(read_matchingness[best_movement][j+i]) { // penalty max_matchness += 1.03+get_base_quality_score(quality_str[j+i], quality_scale); } else { // relief //max_matchness += 0.2-get_base_phred(quality_str[j+i])*0.01; } } // SUBREADprintf("Delta matchingness = %f\n", qs0 - max_matchness); } ret += max_matchness; //front8(read_matchingness[movement], i); // (front4(read_matchingness[movement], i)==4?4:0); } return ret; } void final_matchingness_scoring(const char read_str[], const char quality_str[], int read_len, gene_vote_t * vote, gehash_data_t * max_position, gene_vote_number_t * max_vote, short *max_mask, gene_quality_score_t * max_quality, gene_value_index_t * my_array_index, int space_type, int indel_tolerance, int quality_scale, gene_vote_number_t * max_indel_recorder, int * max_coverage_start, int * max_coverage_end) { int i, j; gene_quality_score_t max_matching = -1.0; *max_vote = vote -> max_vote; for (i=0; iitems[i]; j++) { if( vote->votes[i][j] < vote -> max_vote -1 ) continue; unsigned int potential_position = vote -> pos[i][j]; gene_quality_score_t matchingness_count = 1.*match_chro_indel((char *)read_str, my_array_index, potential_position, read_len, 0, space_type, indel_tolerance, NULL, 0); if(matchingness_count > max_matching) { max_matching = matchingness_count; *max_position = potential_position; *max_mask = vote -> masks[i][j]; *max_coverage_start = vote-> coverage_start[i][j]; *max_coverage_end = vote -> coverage_end[i][j]; indel_recorder_copy(max_indel_recorder, vote-> indel_recorder[i][j]); * max_quality = max_matching; } else if (matchingness_count == max_matching) { // SUBREADprintf("\nBREAK EVEN DETECTED AT SORTED TABLE: %u (kept) and %u\n", vote->max_position, vote -> pos[i][j]); (*max_mask) |= IS_BREAKEVEN_READ ; } } } int DPALIGN_CREATEGAP_PENALTY = -2 ; int DPALIGN_EXTENDGAP_PENALTY = 0 ; int DPALIGN_MISMATCH_PENALTY = 0; int DPALIGN_MATCH_SCORE = 2; int search_DP_branch(char * read, int read_len, gene_value_index_t * index, unsigned int begin_position, int path_i, int path_j, short ** table,char ** table_mask, int max_indel, char * movement_buffer, int expected_offset, int current_score, int out_pos, int current_offset, int init_read_offset, int shutdown_read_offset, int * all_steps) { if (1499 - out_pos > (read_len <<2) || (*all_steps) > 3000 + (read_len << 3) || (*all_steps) > 33000){ /*ddprintf("\nTOO MANY STEPS: rl = %d len=%d steps=%d\n", read_len, 1499 - out_pos, *all_steps); ddfflush(stdout) ;*/ return 0; } if(path_j < 0 || path_i < 0) { int i; //ddprintf("\nFINAL TEST exp %d real-offset %d\n", expected_offset, current_offset); if (expected_offset !=current_offset)return 0; for (i=0; i<=path_j; i++) movement_buffer[out_pos--] = 1; for (i=0; i<=path_i; i++) movement_buffer[out_pos--] = 2; return out_pos ; } short upper_score; if (path_i>0) upper_score = table[path_i-1][path_j]; else upper_score = 0; short left_score; if (path_j>0) left_score = table[path_i][path_j-1]; else left_score = 0; short upperleft_score; if (path_j>0 && path_i>0) upperleft_score = table[path_i-1][path_j-1]; else upperleft_score = 0; char is_matched_ij = gvindex_get(index, begin_position + path_j) == read[path_i]?DPALIGN_MATCH_SCORE :DPALIGN_MISMATCH_PENALTY; int found = 0; int table_mask_j_1 = 0, table_mask_i_1 = 0; int table_mask2_i_1 = 0; if(path_j >0 && path_i >=0) { table_mask_j_1 = left_score + (table_mask[path_i][path_j-1]? DPALIGN_EXTENDGAP_PENALTY: DPALIGN_CREATEGAP_PENALTY) == current_score; } if(path_i >0 && path_j >=0) { table_mask_i_1 = upper_score + (table_mask[path_i-1][path_j]? DPALIGN_EXTENDGAP_PENALTY: DPALIGN_CREATEGAP_PENALTY) == current_score; table_mask2_i_1 = upper_score + (table_mask[path_i-1][path_j]? DPALIGN_EXTENDGAP_PENALTY: DPALIGN_CREATEGAP_PENALTY) < 0 ; } if ((!found ) && ( table_mask_j_1 || (current_score == 0 && table_mask_i_1))) { movement_buffer[out_pos] = 1; (*all_steps) ++; //ddprintf ("Path_i = %d ; Path_j = %d ; Offset = %d => 1\n", path_i, path_j, current_offset); //ddfflush(stdout) ; found =search_DP_branch (read, read_len, index, begin_position, path_i , path_j -1, table , table_mask, max_indel, movement_buffer, expected_offset, left_score, out_pos -1, current_offset - ((path_i >= init_read_offset && path_i <= shutdown_read_offset)?1:0), init_read_offset, shutdown_read_offset, all_steps); } if ((!found )&& ( table_mask_i_1 || (current_score == 0 && table_mask2_i_1))) { movement_buffer[out_pos] = 2; (*all_steps) ++; //ddprintf ("Path_i = %d ; Path_j = %d ; Offset = %d => 2\n", path_i, path_j, current_offset); //ddfflush(stdout) ; found =search_DP_branch (read, read_len, index, begin_position, path_i -1 , path_j , table , table_mask , max_indel, movement_buffer, expected_offset, upper_score, out_pos -1, current_offset + ((path_i >= init_read_offset && path_i <= shutdown_read_offset)?1:0), init_read_offset, shutdown_read_offset, all_steps); } if((!found )&& (is_matched_ij + (upperleft_score - current_score) == 0)) { movement_buffer[out_pos] = is_matched_ij==2?0:3; //ddprintf ("Path_i = %d ; Path_j = %d ; Offset = %d => 0\n", path_i, path_j, current_offset); (*all_steps) ++; found = search_DP_branch (read, read_len, index, begin_position, path_i -1 , path_j -1, table , table_mask, max_indel, movement_buffer, expected_offset, upperleft_score, out_pos -1, current_offset, init_read_offset, shutdown_read_offset, all_steps); } return found; } #define INDEL_WINDOW_WIDTH 4 int window_indel_align(char * read, int read_len, gene_value_index_t * index, unsigned int begin_position, int max_indel, char * movement_buffer, int expected_offset, int init_read_offset, int shutdown_read_offset) { short indel_windows[32]; int scores [32][MAX_READ_LENGTH]; int i,j,windows_number; char chro_str[200]; memset(indel_windows,0, 32*sizeof(short)); windows_number = abs(expected_offset)+1; //ddprintf ("\nWindow size = %d ; ExpOffset = %d\n", windows_number , expected_offset); ddfflush(stdout) ; for (i=0; i< read_len; i++) for (j=0; j< windows_number; j++) { int matchingness ; if (j==0) chro_str[i] = gvindex_get(index, begin_position+i); if (expected_offset<0) matchingness = *(read + i) == gvindex_get(index, begin_position + j + i) ; else matchingness = *(read + i) == gvindex_get(index, begin_position - windows_number + j + 1 + i) ; indel_windows [j] += matchingness; if (i >= INDEL_WINDOW_WIDTH)// The result for i - INDEL_WINDOW_WIDTH is OK { scores[j][i - INDEL_WINDOW_WIDTH] = indel_windows [j] ; } if (i >= INDEL_WINDOW_WIDTH)// The result for i - INDEL_WINDOW_WIDTH is OK { if (expected_offset<0) matchingness = *(read - INDEL_WINDOW_WIDTH + i) == gvindex_get(index, begin_position + j + i - INDEL_WINDOW_WIDTH) ; else matchingness = *(read - INDEL_WINDOW_WIDTH + i) == gvindex_get(index, begin_position -windows_number + j +1 + i - INDEL_WINDOW_WIDTH) ; indel_windows [j] -= matchingness; } } chro_str[i]=0; j = read[read_len]; read[read_len]=0; ddprintf ("CHRO=%s\nREAD=%s\n", chro_str, read); /* float contingency_list [MAX_READ_LENGTH]; for (i=0; i< read_len - windows_number-INDEL_WINDOW_WIDTH; i++) { float contingency; if (expected_offset >0) contingency = ((scores[0][i]*1. + 1)-(scores[windows_number-1][i]*1.+1)); else contingency = ((scores[windows_number-1][i]*1. + 1)-(scores[0][i]*1.+1)); contingency_list[i]=contingency; }*/ int max_score=-1; int max_pos = -1; if (expected_offset>0) { //insertion for (i=read_len-INDEL_WINDOW_WIDTH-1; i>=0; i--) { if (scores[windows_number - 1][i - expected_offset]>=2 && scores[0][i] >= max_score) { max_pos = i - windows_number+1; max_score = scores[0][i]; } } } else { //deletion for (i=read_len-INDEL_WINDOW_WIDTH-1; i>=0; i--) { if (scores[windows_number -1][i] >= max_score && scores[0][i + expected_offset] >=2) { max_score = scores[windows_number -1][i]; max_pos = i ; } } } max_pos = min(read_len, max(0, max_pos)); int move_number = 0; for (i=0; i< read_len -INDEL_WINDOW_WIDTH; i++) { if (i==max_pos) for (j = 0; j < windows_number-1; j++) movement_buffer[move_number++] = 1+(expected_offset>0); // 1=del; 2=ins if (i!=max_pos || expected_offset <0) movement_buffer [move_number++] = 0; // matched #ifdef indel_debug for (j=0; j< windows_number; j++) SUBREADprintf("%d ",scores[j][i]); SUBREADprintf (" %c\n", (i==max_pos)?'*':' ') ; #endif } for(; i< read_len; i++) movement_buffer [move_number++] = 0; // matched read[read_len] = j; return move_number; } int dynamic_align(char * read, int read_len, gene_value_index_t * index, unsigned int begin_position, int max_indel, char * movement_buffer, int expected_offset, int init_read_offset, int shutdown_read_offset, short **table , char ** table_mask) // read must be converted to the positive strand. // movement buffer: 0:match, 1: read-insert, 2: gene-insert, 3:mismatch // the size of the movement buffer must be equal to the length of the read plus max_indel * 3. { int i,j; #ifdef indel_debug int ii, jj; for(ii = 0; ii max_indel + i) { table[i][j]=-9999; #ifdef indel_debug putchar('\t'); #endif continue; } short from_upper; if (i>0) from_upper = table[i-1][j] + (table_mask[i-1][j]?DPALIGN_EXTENDGAP_PENALTY:DPALIGN_CREATEGAP_PENALTY); else from_upper = DPALIGN_CREATEGAP_PENALTY; short from_left; if (j>0) from_left = table[i][j-1] + (table_mask[i][j-1]?DPALIGN_EXTENDGAP_PENALTY:DPALIGN_CREATEGAP_PENALTY); else from_left = DPALIGN_CREATEGAP_PENALTY; char chromo_ch = gvindex_get(index, begin_position + j); char is_matched_ij = (chromo_ch == read[i])?DPALIGN_MATCH_SCORE:DPALIGN_MISMATCH_PENALTY; short from_upperleft; if (i>0 && j>0) from_upperleft = table[i-1][j-1] + is_matched_ij; else from_upperleft = is_matched_ij; if ((i ==0 || j ==0) && (i+j>0)) from_upperleft += DPALIGN_CREATEGAP_PENALTY; if (from_upperleft <=from_left || from_upperleft <= from_upper) table_mask[i][j]=1; table[i][j]=max(0,max(from_upper, max(from_left, from_upperleft))); #ifdef indel_debug SUBREADprintf("%c%c\t", chromo_ch, read[i]); #endif } #ifdef indel_debug SUBREADputs(""); #endif } #ifdef indel_debug //SUBREADputs(""); //SUBREADputs(""); #endif short current_score = table[read_len-1] [read_len - expected_offset-1]; short path_i = read_len-1 ; int out_pos = 1499; char out_tmp [1500]; int all_steps = 0; j = read_len-expected_offset -1; #ifdef indel_debug for(ii=0;ii< 20; ii++) { for(jj=0; jj<20; jj++) SUBREADprintf("%d\t",table[ii][jj]); SUBREADputs(""); } SUBREADputs(""); SUBREADputs(""); for(ii=0;ii< 20; ii++) { for(jj=0; jj<20; jj++) SUBREADprintf("%d\t",table_mask[ii][jj]); SUBREADputs(""); } #endif out_pos = search_DP_branch(read, read_len, index, begin_position, path_i, j, table, table_mask, max_indel, out_tmp , expected_offset, current_score , out_pos, 0, init_read_offset,shutdown_read_offset, &all_steps); if (out_pos) { memcpy(movement_buffer, out_tmp + out_pos +1, 1499 - out_pos); return 1499 - out_pos; } else return 0; } int extend_covered_region(gene_value_index_t *array_index, unsigned int read_start_pos, char * read, int read_len, int cover_start, int cover_end, int window_size, int req_match_5end , int req_match_3end, int indel_tolerance, int space_type, int tail_indel, short * head_indel_pos, int * head_indel_movement, short * tail_indel_pos, int * tail_indel_movement, int is_head_high_quality, char * qual_txt, int qual_format, float head_matching_rate, float tail_matching_rate) { int ret = 0; *head_indel_pos = -1; *tail_indel_pos = -1; if (cover_start >= window_size && head_matching_rate < 1.0001) { int head_test_len = cover_start; int roughly_mapped = match_chro(read, array_index, read_start_pos, head_test_len , 0, space_type); if (roughly_mapped >= head_test_len * CORE_RECOVER_MATCHING_RATE - 0.0001) { ret |= 1; } else { int window_end_pos = cover_start + window_size-1; int not_too_bad = 1; int right_match_number=0; while (1) { int matched_bases_in_window = match_chro_wronglen(read+ window_end_pos - window_size, array_index, read_start_pos + window_end_pos - window_size, window_size, space_type, NULL, &right_match_number); int best_indel_movement = -1000; int best_indel_pos = -1; if (matched_bases_in_window >= req_match_5end) // this window is not bad enough so that an indel is considered window_end_pos--; else { roughly_mapped = match_chro(read, array_index, read_start_pos, window_end_pos - right_match_number , 0, space_type); if (roughly_mapped < (int)(0.5+ (window_end_pos - right_match_number) * CORE_RECOVER_MATCHING_RATE)) { // the quality of this window is too low (at most 1 base is matched). I must consider if it is an indel. int indel_movement_i ; int best_matched_bases_after_indel = -1; not_too_bad = 0; for (indel_movement_i = 0; indel_movement_i < 2* indel_tolerance-1 ; indel_movement_i ++) { int indel_movement = (indel_movement_i+1)/2 * (indel_movement_i %2?1:-1); int test_length = window_end_pos /*- 1*/ - max(0, indel_movement) - right_match_number; if (test_length < window_size) continue; //if (test_length <= 1+ abs(indel_movement)/4) continue; //test_length = min(10, test_length); if (abs(indel_movement) > indel_tolerance) continue; int test_start =0;// window_end_pos - max(0, indel_movement) - right_match_number - test_length; int matched_bases_after_indel = match_chro_support(read +test_start, array_index, read_start_pos + indel_movement +test_start, test_length,0, space_type, qual_txt, qual_format); float test_rate = head_matching_rate; if(test_length < 3) test_rate = 1; if(best_matched_bases_after_indel = (int)(0.5+test_length * test_rate)) { not_too_bad = 1; best_matched_bases_after_indel = matched_bases_after_indel; best_indel_movement = indel_movement; best_indel_pos = window_end_pos - right_match_number; *head_indel_pos = best_indel_pos; *head_indel_movement = best_indel_movement; } } if(best_indel_pos<0) *head_indel_pos = window_end_pos - right_match_number; break; }else window_end_pos--; } if (window_end_pos - window_size <= 0) break; } if(not_too_bad) ret |=1; //else //*head_indel_pos = -1; } } else ret |= 1; if (cover_end <= read_len - window_size && tail_matching_rate < 1.0001) { int tail_test_len = read_len - cover_end; int roughly_mapped = match_chro(read + cover_end, array_index, read_start_pos + cover_end + tail_indel, tail_test_len , 0, space_type); if (roughly_mapped >= tail_test_len * CORE_RECOVER_MATCHING_RATE - 0.0001) { ret |= 2; } else { int window_start_pos = cover_end - window_size +1; int not_too_bad = 1; while (1) { int left_match_number = 0; int matched_bases_in_window = match_chro_wronglen(read+ window_start_pos, array_index, read_start_pos + window_start_pos + tail_indel, window_size, space_type, &left_match_number, NULL); int best_indel_movement = -1000; int best_indel_pos = -1; if (matched_bases_in_window >= req_match_3end) // this window is not bad enough so that an indel is considered window_start_pos++; else { roughly_mapped = match_chro(read+window_start_pos + left_match_number, array_index, read_start_pos + window_start_pos + tail_indel + left_match_number, read_len - window_start_pos - left_match_number , 0, space_type); if (roughly_mapped < (int)(0.5+(read_len - window_start_pos -left_match_number ) * CORE_RECOVER_MATCHING_RATE)) { // the quality of this window is too low (at most 1 base is matched). I must consider if it is an indel. int indel_movement_i ; int best_matched_bases_after_indel = -1; not_too_bad = 0; for (indel_movement_i = 0; indel_movement_i < 2* indel_tolerance; indel_movement_i ++) { int indel_adjustment = (indel_movement_i+1)/2 * (indel_movement_i %2?1:-1); int indel_movement = tail_indel + indel_adjustment; int test_length = read_len - window_start_pos - left_match_number + min(0, indel_adjustment); //test_length = min(10, test_length); if (test_length < window_size) continue; //if (test_length <= 1 + abs(indel_movement)/4) continue; if (abs(indel_movement) > indel_tolerance) continue; int matched_bases_after_indel = match_chro_support(read + window_start_pos - min(0, indel_adjustment) + left_match_number, array_index, read_start_pos + window_start_pos + max(0,indel_movement) +left_match_number , test_length,0, space_type, qual_txt + window_start_pos - min(0, indel_adjustment) + left_match_number , qual_format); float test_rate = tail_matching_rate; if(test_length < 3) test_rate = 1; if(best_matched_bases_after_indel = (int)(0.5+test_length * test_rate)) { not_too_bad = 1; best_matched_bases_after_indel = matched_bases_after_indel; best_indel_movement = indel_movement; best_indel_pos = window_start_pos + left_match_number ;//-1; *tail_indel_movement = best_indel_movement ; } } if(best_indel_pos<0) *tail_indel_pos = window_start_pos + left_match_number ; else *tail_indel_pos = best_indel_pos; break; }else window_start_pos++; } if (window_start_pos + window_size >= read_len) break; } if(not_too_bad) ret |=2; //else // *tail_indel_pos =-1; } } else ret |= 2; return ret; } float match_base_quality_cs(gene_value_index_t *array_index, char * read_txt, unsigned int pos, char * qual_txt, int read_len, int phred_version, int * high_qual_unmatch, int * all_mismatched, int ql_kill, int head_clipped, int tail_clipped) { int i; int ret =0; char lastch; if(pos < array_index -> start_base_offset || pos + read_len >= array_index -> start_base_offset + array_index -> length){ //SUBREADprintf("WARNING: BASE INDEX OUT OF LIMIT: %u < %u < %u\n%s\n", array_index -> start_base_offset , pos, array_index -> start_base_offset + array_index -> length, read_txt); // exit(-1); return (read_len - tail_clipped - head_clipped); } lastch = gvindex_get(array_index, pos); for(i=head_clipped; i start_base_offset || pos + read_len >= array_index -> start_base_offset + array_index -> length){ //SUBREADprintf("WARNING: BASE INDEX OUT OF LIMIT: %u < %u < %u\n%s\n", array_index -> start_base_offset , pos, array_index -> start_base_offset + array_index -> length, read_txt); // exit(-1); return (read_len - tail_clipped - head_clipped); } for(i=head_clipped; i 196 #define QL_MIN 999000 #endif #endif #ifndef QL_MIN #define QL_MIN 200000 #endif*/ if( ql < ql_kill) (*high_qual_unmatch)++; ret += ql-1000000; } } } //printf ("SECTION QUAL = %d LEN = %d\n", ret, read_len); return ret/1000000.; } float final_mapping_quality(gene_value_index_t *array_index, unsigned int pos, char * read_txt, char * qual_txt, char * cigar_txt, int phred_version, int * mismatch, int rl, char * refined_cigar, unsigned int * new_pos) { int cigar_cursor = 0; int read_cursor = 0; long long int chromosome_cursor = pos, x; int cigar_length = strlen(cigar_txt); int i; int ret = 0; x= 0; while(cigar_cursor < cigar_length) { if(cigar_txt[cigar_cursor] =='X') { cigar_cursor++; continue; } if(cigar_txt[cigar_cursor]>='0' && cigar_txt[cigar_cursor]<='9') x = x*10+ (cigar_txt[cigar_cursor]-'0'); else { if(cigar_txt[cigar_cursor] == 'M' || cigar_txt[cigar_cursor] == 'S') { int all_MM=0; float nret = match_base_quality(array_index, read_txt + read_cursor, chromosome_cursor , (qual_txt && qual_txt[0])?qual_txt + read_cursor:NULL, x, 0, phred_version, mismatch, &all_MM, 200000,0,0); //printf ("%s: Q=%.6f; L=%d ; POS=%u\n", read_txt + read_cursor, nret, x, chromosome_cursor); ret += (int)(nret*1000000); chromosome_cursor +=x; read_cursor +=x; } else if(cigar_txt[cigar_cursor] == 'I') { if(!qual_txt) { ret += x*1000000; read_cursor +=x; } else if(FASTQ_PHRED64 == phred_version) { for(i = 0; i='0' && cigar_txt[cigar_cursor]<='9') x = x*10+ (cigar_txt[cigar_cursor]-'0'); else { if(cigar_txt[cigar_cursor] == 'M' || cigar_txt[cigar_cursor] == 'S') { gvindex_get_string(read_data + read_cursor , array_index , chromosome_cursor , x , 0); chromosome_cursor +=x; read_cursor +=x; } else if(cigar_txt[cigar_cursor] == 'I') { int xk; for(xk=read_cursor;xk=0) { if(window_matched >= REFINE_WINDOW_SIZE -1) if(read_txt[read_cursor+REFINE_WINDOW_SIZE] == read_data[read_cursor+REFINE_WINDOW_SIZE]) last_confirmed_read_pos = read_cursor+REFINE_WINDOW_SIZE+1; window_matched -= (read_txt[read_cursor] == read_data[read_cursor] || read_txt[read_cursor] == 'I'); } } window_matched = 0; int first_confirmed_read_pos = 99999; for(read_cursor = rl -1 ; read_cursor >=0 ; read_cursor--) { window_matched += (read_txt[read_cursor] == read_data[read_cursor]|| read_txt[read_cursor] == 'I'); if(read_cursor <= rl- REFINE_WINDOW_SIZE - 1) { if(window_matched >= REFINE_WINDOW_SIZE -1) if(read_txt[read_cursor] == read_data[read_cursor]) first_confirmed_read_pos = read_cursor; window_matched -= (read_txt[read_cursor+REFINE_WINDOW_SIZE] == read_data[read_cursor+REFINE_WINDOW_SIZE] || read_txt[read_cursor + REFINE_WINDOW_SIZE] == 'I'); } } cigar_cursor = 0; read_cursor = 0; refined_cigar[0]=0; x=0; int begin_copy =0; chromosome_cursor = pos; if(first_confirmed_read_pos<99990 && last_confirmed_read_pos<99990) { if(first_confirmed_read_pos>0) sprintf(refined_cigar,"%dS",first_confirmed_read_pos); while(cigar_cursor < cigar_length) { if(cigar_txt[cigar_cursor] =='X') { cigar_cursor++; continue; } if(cigar_txt[cigar_cursor]>='0' && cigar_txt[cigar_cursor]<='9') x = x*10+ (cigar_txt[cigar_cursor]-'0'); else { if(cigar_txt[cigar_cursor] == 'M' || cigar_txt[cigar_cursor] == 'S' || cigar_txt[cigar_cursor] == 'I') { int out_len = 0; if(read_cursor < first_confirmed_read_pos && read_cursor + x >= first_confirmed_read_pos) out_len = read_cursor + x - first_confirmed_read_pos; else if(first_confirmed_read_pos <= read_cursor) out_len = x; if(out_len) { if(!begin_copy) *new_pos = chromosome_cursor - read_cursor; if(read_cursor >= last_confirmed_read_pos) out_len=0; else if(read_cursor < last_confirmed_read_pos && read_cursor +x>last_confirmed_read_pos) out_len -= (read_cursor +x - last_confirmed_read_pos); } if(out_len) { sprintf(refined_cigar+strlen(refined_cigar), "%d%c", out_len, cigar_txt[cigar_cursor]); begin_copy=1; } if(cigar_txt[cigar_cursor] == 'M' || cigar_txt[cigar_cursor] == 'S') chromosome_cursor +=x; read_cursor +=x; if(read_cursor >= last_confirmed_read_pos) break; } else { if(begin_copy) #ifdef __MINGW32__ sprintf(refined_cigar+strlen(refined_cigar), "%I64d%c", x, cigar_txt[cigar_cursor]); #else sprintf(refined_cigar+strlen(refined_cigar), "%lld%c", x, cigar_txt[cigar_cursor]); #endif if(cigar_txt[cigar_cursor] == 'D' || cigar_txt[cigar_cursor] == 'N'|| cigar_txt[cigar_cursor] == 'j' || cigar_txt[cigar_cursor] == 'J') chromosome_cursor +=x; else if(cigar_txt[cigar_cursor] == 'B' || cigar_txt[cigar_cursor] == 'b') chromosome_cursor -=x; } x= 0; } cigar_cursor++; } if(last_confirmed_read_pos<=rl-1) sprintf(refined_cigar+strlen(refined_cigar),"%dS",rl-last_confirmed_read_pos); } else if(new_pos)*new_pos=pos; } else if(new_pos)*new_pos=pos; return match_rate; } void print_votes(gene_vote_t * vote, char *index_prefix) { gene_offset_t offsets; int i,j; char * chrname = NULL; int chrpos = 0; load_offsets (&offsets, index_prefix); //locate_gene_position(vote -> max_position, &offsets, &chrname, &chrpos); SUBREADprintf(" ========== Max votes = %d ==========\n", vote->max_vote);// , Position is %s,%u\n", vote->max_vote, chrname, chrpos ); for (i=0; iitems[i]; j++) { locate_gene_position(vote -> pos[i][j] +1, &offsets, &chrname, &chrpos); int toli = vote->toli [i][j]; int last_offset = vote -> indel_recorder[i][j][toli+2]; SUBREADprintf(" %s\tVote = %d , Position is (1-base):%s,%d (+%u) Coverage is (%d, %d) Indel %d %s (%d)\n", vote->votes[i][j] == vote->max_vote?"***":" ", vote->votes[i][j] , chrname, chrpos, vote -> pos[i][j], vote -> coverage_start[i][j], vote -> coverage_end[i][j],last_offset, (vote -> masks[i][j])?"NEG":"POS", vote -> masks[i][j]); if(1){ int k; for(k=0; k indel_recorder[i][j][k], vote -> indel_recorder[i][j][k+1], vote -> indel_recorder[i][j][k+2]); } SUBREADputs(""); } } } static float PROB_QUAL_TABLE[] = {1.000000000 , 0.794328235 , 0.630957344 , 0.501187234 , 0.398107171 , 0.316227766 , 0.251188643 , 0.199526231 , 0.158489319 , 0.125892541 , 0.100000000 , 0.079432823 , 0.063095734 , 0.050118723 , 0.039810717 , 0.031622777 , 0.025118864 , 0.019952623 , 0.015848932 , 0.012589254 , 0.010000000 , 0.007943282 , 0.006309573 , 0.005011872 , 0.003981072 , 0.003162278 , 0.002511886 , 0.001995262 , 0.001584893 , 0.001258925 , 0.001000000 , 0.000794328 , 0.000630957 , 0.000501187 , 0.000398107 , 0.000316228 , 0.000251189 , 0.000199526 , 0.000158489 , 0.000125893 , 0.000100000 , 0.000079433 , 0.000063096 , 0.000050119 , 0.000039811 , 0.000031623 , 0.000025119 , 0.000019953 , 0.000015849 , 0.000012589 , 0.000010000 , 0.000007943 , 0.000006310 , 0.000005012 , 0.000003981 , 0.000003162 , 0.000002512 , 0.000001995 , 0.000001585 , 0.000001259 , 0.000001000 , 0.000000794 , 0.000000631 , 0.000000501}; float get_base_error_prob33(char v) { return PROB_QUAL_TABLE[v-'!']; } float get_base_error_prob64(char v) { return PROB_QUAL_TABLE[v-'@']; } static int PROB_QUAL_INT_TABLE[] = { 1000000 , 794328 , 630957 , 501187 , 398107 , 316227 , 251188 , 199526 , 158489 , 125892 , 100000 , 79432 , 63095 , 50118 , 39810 , 31622 , 25118 , 19952 , 15848 , 12589 , 10000 , 7943 , 6309 , 5011 , 3981 , 3162 , 2511 , 1995 , 1584 , 1258 , 1000 , 794 , 630 , 501 , 398 , 316 , 251 , 199 , 158 , 125 , 100 , 79 , 63 , 50 , 39 , 31 , 25 , 19 , 15 , 12 , 10 , 7 , 6 , 5 , 3 , 3 , 2 , 1 , 1 , 1 , 1 , 0 , 0 , 0 , }; int old_get_base_error_prob33i(char v) { return PROB_QUAL_INT_TABLE[v-'!']; } int get_base_error_prob64i(char v) { return PROB_QUAL_INT_TABLE[v-'@']; } #ifdef SKIP_THIS_PART void bad_reverse_cigar(char * cigar) { int cigar_cursor = 0; srInt_64 tmpv=0; char ncg[100]; ncg[0]=0; while(cigar[cigar_cursor]) { char cc = cigar[cigar_cursor]; if(isdigit(cc)) { tmpv=tmpv*10+(cc-'0'); } else if((cc>='A'&&cc<='Z')||(cc>='a'&&cc<='z')) { char ncg2[103]; #ifdef __MINGW32__ sprintf(ncg2, "%I64d%c", tmpv, cc); #else sprintf(ncg2, "%lld%c", tmpv, cc); #endif strncat(ncg2, ncg, 99); strncpy(ncg, ncg2, 99); tmpv=0; } else { char ncg2[103]; sprintf(ncg2, "%c%s",cc,ncg); strncpy(ncg, ncg2, 99); tmpv=0; } cigar_cursor++; } strcpy(cigar, ncg); } #ifdef TEST_WHATCIGARREVERSE int main() #else int debug_main() #endif { char cg[100]; sprintf(cg,"10M8H20M9I100N30M"); bad_reverse_cigar(cg); SUBREADprintf("%s\n",cg); return 0; } #endif void remove_indel_neighbours(HashTable * indel_table) { unsigned int * to_delete; char * to_delete_len; int xi; KeyValuePair * cursor; return; to_delete=(unsigned int* )malloc(sizeof(int)*400000); to_delete_len=(char* )malloc(sizeof(char)*400000); int num_delete=0, bucket; for(bucket=0; bucket numOfBuckets; bucket++) { cursor = indel_table -> bucketArray[bucket]; while (1) { if(!cursor) break; indel_record_t * p = (indel_record_t *) cursor -> key; indel_result_t * pv = (indel_result_t *)cursor -> value; indel_record_t p_nb; p_nb.len = p->len; for(xi=-5;xi<=5;xi++) { if(!xi)continue; p_nb.pos = p->pos+xi; indel_result_t * p_nbv = (indel_result_t *)HashTableGet(indel_table, &p_nb); if(p_nbv && (p_nbv->support > pv->support || (p_nbv->support == pv->support && xi>0))) { if(num_delete<399999) { to_delete_len[num_delete]=p->len; to_delete[num_delete++]= p->pos+xi; } break; } } cursor = cursor->next; } } for(xi=0;xiitems, 0, GENE_VOTE_TABLE_SIZE*sizeof( *((a)->items))); (a)->max_vote = 0; (a) -> max_indel_recorder[0]=0; (a)->max_tmp_indel_recorder = NULL; (a)->max_mask = 0; (a) -> noninformative_subreads = 0; } // return current votes for a given position // if create_new_pos == 0 then do not take this position if it does not exist in the vote array //void add_gene_vote(gene_vote_t* vote, int position, int create_new_pos); //void add_gene_vote_weighted(gene_vote_t* vote, int position, int create_new_pos, int w); // return the votes; the position is put into position_result int max_gene_vote(gene_vote_t* vote, int * position_result, int query_id); // evaluate the number of matched characters in the piece_str int evaluate_piece(char * piece_str, int chromosome, int offset, int is_counterpart, int start_p, int end_p); // These two functions maintain the global vote for pieces int init_allvote(gene_allvote_t* allvote, int expected_len, int allowed_indels); void clear_allvote(gene_allvote_t* allvote); void add_allvote_q(gene_allvote_t* allvote,int qid , int pos, gene_vote_number_t votes, gene_quality_score_t quality, int is_counterpart, short mask, char * max_indel_recorder, gene_value_index_t * array_index, char * read_txt, int read_len, int max_indel, int total_subreads, int space_type, int report_junction, int is_head_high_quality, char * qual_txt, int phred_version, char span_coverage, short **dynamic_programming_short , char** dynamic_programming_char); unsigned char get_next_char(FILE * fp); extern double begin_ftime; void print_text_scrolling_bar(char * hint, float percentage, int width, int * internal_counter); void print_window_scrolling_bar(char * hint, float percentage, int width, int * internal_counter); void print_running_log(double finished_rate, double read_per_second, double expected_seconds, unsigned long long int total_reads, int is_pair) ; int select_positions(gene_vote_t * vote_read1, gene_vote_t * vote_read2, gene_vote_number_t * numvote_read1, gene_vote_number_t * numvote_read2, gene_quality_score_t * sum_quality, gene_quality_score_t * qual_r1, gene_quality_score_t * qual_r2, gehash_data_t * pos_read1, gehash_data_t * pos_read2, gene_vote_number_t * read1_indel_recorder, gene_vote_number_t* read2_indel_recorder, unsigned int max_pair_dist, unsigned int min_pair_dist, int min_major, int min_minor, int is_negatve, int number_of_anchors_quality, int max_indel_len, int * is_breakeven); int select_positions_array(char * read1_str, int read1_len, char * read2_str, int read2_len, gene_vote_t * vote_read1, gene_vote_t * vote_read2, gene_vote_number_t * numvote_read1, gene_vote_number_t * numvote_read2, gene_quality_score_t * sum_quality, gene_quality_score_t * q_r1, gene_quality_score_t *q_r2, gene_vote_number_t * indel_rec_r1, gene_vote_number_t * indel_rec_r2, gehash_data_t * pos_read1, gehash_data_t * pos_read2, unsigned int max_pair_dest, unsigned int min_pair_dest, int min_major, int min_minor,int is_negative_strand, gene_value_index_t * my_array_index, int color_space, int indel_tolerance, int number_of_anchors_quality, const char quality_str1 [], const char quality_str2 [], int quality_scale, int max_indel_len, int * is_breakeven); #define QUALITY_SCALE_NONE 0 #define QUALITY_SCALE_LINEAR 1 #define QUALITY_SCALE_LOG 2 // This function returns the lowest score of phred in the given 16-byte quality string // Assuming that the score = char - '@' gene_quality_score_t get_subread_quality(const char * quality_str, const char * read_str, int quality_scale, int phred_version); gene_vote_number_t calculate_penalty_score(const char * quality_str, const char * read_str, int quality_scale, int base_matchness); int is_valid_subread(const char * read_str); void final_matchingness_scoring(const char read_str[], const char quality_str[], int read_len, gene_vote_t * vote, gehash_data_t * max_position, gene_vote_number_t * max_vote, short *max_mask, gene_quality_score_t * max_quality, gene_value_index_t * my_array_index, int space_type, int indel_tolerance, int quality_scale, gene_vote_number_t * max_indel_recorder, int * max_coverage_start, int * max_coverage_end); float match_read(const char read_str[], int read_len, unsigned int potential_position, gene_value_index_t * my_array_index, int space_type, int indel_tolerance, const char quality_str [], int quality_scale) ; void show_cigar(char * info, int len, int is_reversed_map, char * buf, int max_indel, int total_subreads, char * read, int * pos_offset, int * adjust_rl); int dynamic_align(char * read, int read_len, gene_value_index_t * index, unsigned int begin_position, int max_indel, char * movement_buffer, int expected_offset,int begin_read_offset, int end_read_offset, short **dynamic_programming_short , char** dynamic_programming_char ); int window_indel_align(char * read, int read_len, gene_value_index_t * index, unsigned int begin_position, int max_indel, char * movement_buffer, int expected_offset,int begin_read_offset, int end_read_offset); void explain_indel_in_middle(gene_allvote_t* allvote, int qid , int pos, char * max_indel_recorder, gene_value_index_t * array_index, char * read_txt, int read_len, int max_indel, int total_subreads, int head_start_point, int tail_end_point, int head_indel_movement, int tail_indel_movement, int find_junctions, short **dynamic_programming_short , char** dynamic_programming_char); void find_and_explain_indel(gene_allvote_t* allvote,int qid , int pos, gene_vote_number_t votes, gene_quality_score_t quality, int is_counterpart, char mask, char * max_indel_recorder, gene_value_index_t * array_index, char * read_txt, int read_len, int max_indel, int total_subreads, int space_type,int find_junctions, int is_head_high_quality, char * qual_txt, int phred_version, short** dynamic_programming_short , char **dynamic_programming_char ); int extend_covered_region(gene_value_index_t *array_index, unsigned int read_start_pos, char * read, int read_len, int cover_start, int cover_end, int window_size, int match_req_5end, int match_req_3end, int indel_tolerance, int space_type, int tail_indel, short * head_indel_pos, int * head_indel_movement, short * tail_indel_pos, int * tail_indel_movement, int is_head_high_quality, char * qual_str, int qual_format, float head_matching_rate, float tail_matching_rate); float final_mapping_quality(gene_value_index_t *array_index, unsigned int pos, char * read_txt, char * qual_txt, char * cigar_txt, int phred_version, int * mismatch, int rl, char * refined_cigar, unsigned int * new_pos); int bad_quality_base_number(char * qualityb, int rl , int format); int min_matched_bases(char * qualityb, int rl , int format, float match_score); float read_quality_score(char * qualityb, int rl , int format); void compress_cigar(char * cigar, int read_len, char *read, int * position_offset, int * adjust_rl); void print_votes(gene_vote_t * vote, char *index_prefix); void destory_allvote(gene_allvote_t* allvote); float get_base_error_prob33(char v); float get_base_error_prob64(char v); #define qv33s { 1000000 , 794328 , 630957 , 501187 , 398107 , 316227 , 251188 , 199526 , 158489 , 125892 , 100000 , 79432 , 63095 , 50118 , 39810 , 31622 , 25118 , 19952 , 15848 , 12589 , 10000 , 7943 , 6309 , 5011 , 3981 , 3162 , 2511 , 1995 , 1584 , 1258 , 1000 , 794 , 630 , 501 , 398 , 316 , 251 , 199 , 158 , 125 , 100 , 79 , 63 , 50 , 39 , 31 , 25 , 19 , 15 , 12 , 10 , 7 , 6 , 5 , 3 , 3 , 2 , 1 , 1 , 1 , 1 , 0 , 0 , 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 } #define get_base_error_prob33i(v) (((int [])qv33s)[(v) -33]) //int get_base_error_prob33i(char v); int get_base_error_prob64i(char v); void add_repeated_numbers(int qid, gene_vote_t * vote, unsigned char * repeated_regions); void bad_reverse_cigar(char * cigar); void subread_lock_occupy(subread_lock_t * lock); void subread_init_lock(subread_lock_t * lock); void subread_destroy_lock(subread_lock_t * lock); void subread_lock_release(subread_lock_t * lock); void remove_indel_neighbours(HashTable * indel_table); float match_base_quality(gene_value_index_t *array_index, char * read_txt, unsigned int pos, char * qual_txt, int read_len, int is_negative, int phred_version, int * high_qual_unmatch, int * all_MM, int ql_kill, int head_clipped, int tail_clipped); int match_chro_indel(char * read, gene_value_index_t * index, unsigned int pos, int test_len, int is_negative_strand, int space_type, int indel_size, gene_vote_number_t * indel_recorder, int total_subreads); float match_base_quality_cs(gene_value_index_t *array_index, char * read_txt, unsigned int pos, char * qual_txt, int read_len, int phred_version, int * high_qual_unmatch, int * all_MM, int ql_kill, int head_clipped, int tail_clipped); void print_version_info(); int fc_strcmp_chro(const void * s1, const void * s2); srUInt_64 fc_chro_hash(const void *key) ; #endif Rsubread/src/gene-value-index.c0000644000175200017520000010036314136047615017445 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #include #include #include #include #include #include "gene-value-index.h" #include "input-files.h" int gvindex_init(gene_value_index_t * index, unsigned int start_point, unsigned int total_bases_estimated) { index->start_point = start_point; index->memory_block_size = total_bases_estimated/4+10; index->values = calloc(index->memory_block_size,1); if(!index->values) { SUBREADputs(MESSAGE_OUT_OF_MEMORY); return 1; } index -> start_base_offset = index -> start_point - index -> start_point%4; return 0; } #define gvindex_baseno2offset_m(base_number, index, offset_byte, offset_bit) {offset_byte = (base_number - index -> start_base_offset) >>2; offset_bit = base_number % 4 * 2;} void gvindex_baseno2offset(unsigned int base_number, gene_value_index_t * index, unsigned int * offset_byte, unsigned int * offset_bit) { // the base number corrsponding to the 0-th bit in the whole value array; unsigned int offset = (base_number - index -> start_base_offset); * offset_byte = offset >>2 ; * offset_bit = base_number % 4 * 2; } int is_offset_in_chro(gene_value_index_t * offsets, gehash_data_t linear){ int ret = 1; if(offsets -> appendix1 && offsets -> appendix2){ gene_offset_t * chros = offsets -> appendix1; int n = 0; int total_offsets = chros -> total_offsets; int LL = 0, RR = total_offsets-1; while(1){ if(LL >= RR-1) break; int MM = (LL+RR)/2; if( linear > chros->read_offsets[MM]) LL = MM; else if(linear < chros->read_offsets[MM]) RR = MM; else break; } n = max(0, LL - 2); for (; n < chros -> total_offsets; n++) { if (chros->read_offsets[n] > linear) { unsigned int pos; if (n==0) pos = linear; else pos = linear - chros->read_offsets[n-1]; if( pos < chros -> padding || pos >= ( chros->read_offsets[n] - chros->read_offsets[n-1] - chros -> padding )) ret = 0; SUBREADprintf("INCHRO:%d ; POS:%d\n", ret, pos); break; } } } return ret; } // return 'A', 'G', 'T' and 'C' int gvindex_get(gene_value_index_t * index, gehash_data_t offset) { unsigned int offset_byte, offset_bit; //if(!is_offset_in_chro( index, offset ))return -1; gvindex_baseno2offset_m(offset, index , offset_byte, offset_bit); if(offset_byte >= index-> values_bytes -1)return 'N'; unsigned int one_base_value = (index->values [offset_byte]) >> (offset_bit); //SUBREADprintf("RECV_BASE=%d (%d - %d)\n",one_base_value & 3, offset_byte , offset_bit); return int2base(one_base_value & 3); } int gvindex_match(gene_value_index_t * index, gehash_data_t offset, gehash_key_t base_values) { unsigned int offset_byte, offset_bit; gvindex_baseno2offset_m(offset, index , offset_byte, offset_bit); int i, ret = 0; for (i=0; i<16; i++) { unsigned char mask = 0x3 << (offset_bit); unsigned char one_base_value = (index->values [offset_byte] & mask) >> (8-offset_bit); if ( ((base_values >> (30 - i*2)) & 0x3) == one_base_value) ret |= 1 << i; offset_bit +=2; if(offset_bit >=8) { offset_bit = 0; offset_byte ++; } } return ret; } void gvindex_set(gene_value_index_t * index, gehash_data_t offset, gehash_key_t base_values, int padding) { unsigned int offset_byte, offset_bit; gvindex_baseno2offset(offset, index , &offset_byte, &offset_bit); int i; unsigned int offset_byte_margin = offset_byte + (padding / 8 +1); if(index -> memory_block_size <= offset_byte_margin + 2) { assert(index -> memory_block_size> offset_byte_margin+2); index -> memory_block_size *= 1.5; index->values = realloc(index->values, index -> memory_block_size); } for (i=0; i<16; i++) { // 11110011 // ^^ base unsigned char mask = 0xff << (offset_bit+2) | 0xff >> (8-offset_bit); index->values [offset_byte] &= mask; index->values [offset_byte] |= ((base_values >> (30 - i*2))&0x03) << (offset_bit); //SUBREADprintf("PUT_BASE=%d (%d - %d)\n", (base_values >> (30 - i*2))&0x03, offset_byte, offset_bit); offset_bit +=2; if(offset_bit >=8) { offset_bit = 0; offset_byte ++; } } index -> length = offset + 16 - index -> start_point + padding; } int gvindex_dump(gene_value_index_t * index, const char filename []) { FILE * fp = f_subr_open(filename, "wb"); int is_full = 0; int write_len = fwrite(&index->start_point,4,1, fp); write_len += fwrite(&index->length, 4, 1, fp); if(write_len < 2){ is_full = 1; } unsigned int useful_bytes, useful_bits; gvindex_baseno2offset (index -> length+ index -> start_point, index,&useful_bytes,&useful_bits); write_len = fwrite(index->values, 1, useful_bytes+1, fp); if(write_len <= useful_bytes) is_full = 1; fclose(fp); if(is_full) SUBREADprintf("ERROR: unable to writeinto the output file. Please check the disk space in the output directory.\n"); return is_full; } int gvindex_load(gene_value_index_t * index, const char filename []) { memset(index,0, sizeof(gene_value_index_t)); FILE * fp = f_subr_open(filename, "rb"); int read_length; read_length = fread(&index->start_point,4,1, fp); if(read_length<1){ SUBREADprintf("ERROR: the array index is incomplete : %d\n", read_length ); return 1; } read_length = fread(&index->length,4,1, fp); if(read_length<1){ SUBREADputs("ERROR: the index is incomplete."); return 1; } //SUBREADprintf ("\nBINDEX %s : %u ~ +%u\n",filename, index->start_point, index->length ); unsigned int useful_bytes, useful_bits; index -> start_base_offset = index -> start_point - index -> start_point%4; gvindex_baseno2offset (index -> length+ index -> start_point, index ,&useful_bytes,&useful_bits); index -> values = malloc(useful_bytes+1); index -> values_bytes = useful_bytes+1; if(!index->values) { SUBREADputs(MESSAGE_OUT_OF_MEMORY); return 1; } read_length =fread(index->values, 1, useful_bytes+1, fp); if(read_length < useful_bytes){ SUBREADprintf("ERROR: the array index is incomplete : %d < %d.\n", read_length, useful_bytes+1 ); return 1; } fclose(fp); return 0; } int match_chro_wronglen(char * read, gene_value_index_t * index, unsigned int pos, int test_len, int space_type, int * left_match_bases, int * right_match_bases) { int ret = 0; int i; char last_char='A'; int left_correct_end = 0; if(left_match_bases) *left_match_bases=0; if(right_match_bases) *right_match_bases=0; if (space_type == GENE_SPACE_COLOR) last_char = (pos <= index -> start_point)?'A': gvindex_get(index,pos-1); for (i=0;i= max_test_length)break; // printf("Xi=%d POS=%u INDEL=%d MATCH=%d TT vs R = %c , %c ; IPOS=%d\n", i, npos, offset, read[i] ==tt, tt, read[i], *indel_point); if(read[i]==tt)ret++; else if((i + offset < test_len - INDEL_TEST_WINDOW - 3 && i >0)) { // if there is a base unmatched, it is potentially an indel from here. int window_match = match_chro(read+i, index, pos+i+ offset, INDEL_TEST_WINDOW ,0,GENE_SPACE_BASE); if(window_match < INDEL_TEST_WINDOW -1) { // if the window is badly matched, it is very likely to be an indel from this base. int indel_test_i; for(indel_test_i =0; indel_test_i < 7; indel_test_i++) { int indel_test = (indel_test_i+1)/2*(indel_test_i%2?1:-1); if(abs(indel_test)>max_indel_number) continue; if(indel_test < 0) //insertion { int matched_tail = match_chro(read+i-indel_test, index, pos+i, test_len - i+indel_test,0,GENE_SPACE_BASE); int matched_score = matched_tail * 10000 / ( test_len - i + indel_test); if(matched_score > bast_match_score_remailing && matched_score > MIN_INDEL_SEARCH_MATCH_SCORE) { offset = indel_test; bast_match_score_remailing = matched_score; } }else // deletion { int matched_tail = match_chro(read+i, index, pos+i+indel_test, test_len - i ,0,GENE_SPACE_BASE); int matched_score = matched_tail * 10000 / (test_len - i); if(matched_score > bast_match_score_remailing && matched_score > MIN_INDEL_SEARCH_MATCH_SCORE) { offset = indel_test; bast_match_score_remailing = matched_score; } } } } if(bast_match_score_remailing>0) { if(offset > 0)//deletion { *indel_point = i; npos = pos + i + offset; tt = gvindex_get (index, npos); ret += read[i] == tt; } else if(offset<0) { *indel_point = i; i-=offset+1; } } } } *indels = offset; return ret; } int find_all_indels(HashTable *indel_table, unsigned long pos, indel_record_t * ret, int search_to_back) { int max_test_indels = 16; int xi, iret=0; if(!search_to_back) { int mask_pos = (pos>>3)& 0x1fffffff; if(!((char *)indel_table->appendix1)[mask_pos]) return 0; int mask_off = pos %8; if(!(((char *)indel_table->appendix1)[mask_pos] & (1<0)search_pos-= xi; search_pos--; } if(search_to_back) { int mask_pos = (search_pos>>3)& 0x1fffffff; if(!((char *)indel_table->appendix1)[mask_pos]) continue; int mask_off = search_pos %8; if(!(((char *)indel_table->appendix1)[mask_pos] & (1<9)break; } } //printf("%u has %d indel for %d\n", pos, iret, search_to_back); return iret; } void match_indel_table_to_front_in(HashTable * indel_table , char * read, int read_pos_first_base, gene_value_index_t * index, unsigned int first_base_pos, int test_len, short * total_indels, short * indel_point, int max_indel_number, int min_test_offset, short * indels, short * indel_poses, int sofar_matched_bases, short * best_indels, short * best_indel_poses, int * best_matching_bases, int level); // This function iterates all possible combinations of indels, and select the one that has the highest matched base number as the final result. // The final result will be written in *sec int match_indel_table_to_front(HashTable * indel_table , char * read, gene_value_index_t * index, unsigned int first_base_pos, int test_len, short * indels, short * indel_point, int max_indel_number, int min_test_offset, struct explorer_section_t * sec) { short tmp_indel_rec[10], tmp_indel_pos_rec[10]; memset(tmp_indel_rec, 0, sizeof(short)*10); memset(tmp_indel_pos_rec, 0, sizeof(short)*10); short *best_indel_rec=NULL, *best_indel_pos_rec=NULL; if(sec) { best_indel_rec = sec -> all_indels ; best_indel_pos_rec = sec -> all_indel_poses; memset(best_indel_rec, 0, sizeof(short)*10); memset(best_indel_pos_rec, 0, sizeof(short)*10); } int best_matching_bases = -1; match_indel_table_to_front_in(indel_table, read, 0, index, first_base_pos, test_len , indels , indel_point , max_indel_number , min_test_offset , tmp_indel_rec , tmp_indel_pos_rec , 0, best_indel_rec, best_indel_pos_rec , &best_matching_bases , 0); return best_matching_bases; } #define TEST_TARGET "AGGAAAGTGAGACCATGCTCAGGAAAACAGAAGGCAACAGAAACTATGACAAATTCCAGACATCGGATTAACAGACAAAGACAG" void match_indel_table_to_front_in(HashTable * indel_table , char * read, int read_pos_first_base, gene_value_index_t * index, unsigned int first_base_pos, int test_len, short * total_indels, short * indel_point, int max_indel_number, int min_test_offset, short * indels, short * indel_poses, int sofar_matched_bases, short * best_indels, short * best_indel_poses, int * best_matching_bases, int level) { unsigned int xi; int xj, xk; unsigned int index_low_boundary = index -> start_base_offset; unsigned int index_up_boundary = index -> start_base_offset + index -> length; if(first_base_pos <= index_low_boundary)return; if(first_base_pos + test_len >= index_up_boundary)return; if(level >9)return; for(xi = 1; xi < test_len; xi++) { indel_record_t found_records[20]; int section_matched_bases = 0 , num_records = find_all_indels(indel_table , xi + first_base_pos, found_records, 0); if(num_records >0) section_matched_bases = match_chro(read, index, first_base_pos, xi ,0,GENE_SPACE_BASE); // if(num_records) printf("XI %d FOUND %d INDELS AT %u ; SECMATCH=%d\n",xi, num_records , first_base_pos + xi, section_matched_bases); for(xj=0; xj len >0) next_first_base_pos += this_record -> len; // if this is a deletion, jump the gap. char * next_read = read + xi; if(this_record -> len <0) next_read -= this_record -> len ; int remainder_testlen = test_len - xi; if(this_record -> len <0) remainder_testlen += this_record -> len ; indels[level] = this_record -> len; indel_poses[level] = read_pos_first_base + xi; int next_read_pos_first_base = read_pos_first_base + xi; if(this_record -> len <0) next_read_pos_first_base -= this_record -> len; if(remainder_testlen>0 && (remainder_testlen *best_matching_bases) { if(best_indels) { memcpy(best_indels, indels, sizeof(short)*10); memcpy(best_indel_poses, indel_poses, sizeof(short)*10); } *best_matching_bases = onepiece_matched_bases + sofar_matched_bases; xk=0; int ret_indels=0; while(indels[xk]) ret_indels += indels[xk++]; *total_indels = ret_indels; if(indels[0]) *indel_point = indel_poses[0]; else *indel_point = 0; } } void match_indel_table_to_back_in(HashTable * indel_table , char * read, gene_value_index_t * index, unsigned int last_base_pos, int test_len, short * total_indels, short * indel_point, int max_indel_number, int min_test_offset, short * indels, short * indel_poses, int sofar_matched_bases, short * best_indels, short * best_indel_poses, int * best_matching_bases, int level); int match_indel_table_to_back(HashTable * indel_table, char * read, gene_value_index_t * index, unsigned int last_base_pos, int test_len, short * indels, short * indel_point, int max_indel_number, int min_test_offset, struct explorer_section_t * sec) { short tmp_indel_rec[10], tmp_indel_pos_rec[10]; memset(tmp_indel_rec, 0, sizeof(short)*10); memset(tmp_indel_pos_rec, 0, sizeof(short)*10); short best_indel_rec [10] , best_indel_pos_rec [10]; memset(best_indel_rec, 0, sizeof(short)*10); memset(best_indel_pos_rec, 0, sizeof(short)*10); int best_matching_bases = -1; match_indel_table_to_back_in(indel_table, read, index, last_base_pos, test_len , indels , indel_point , max_indel_number , min_test_offset , tmp_indel_rec , tmp_indel_pos_rec , 0, best_indel_rec, best_indel_pos_rec , &best_matching_bases , 0); int xi; if(sec) { int retv=0; while(best_indel_rec[retv])retv++; //printf("BACK-OUT NO.INDEL=%d\n", retv); for(xi=0;xiall_indels[xi] = best_indel_rec[retv-xi-1]; sec->all_indel_poses[xi] = best_indel_pos_rec[retv-xi-1]; } for(xi=retv;xi<10;xi++) sec->all_indels[xi] = 0; } return best_matching_bases; } void match_indel_table_to_back_in(HashTable * indel_table , char * read, gene_value_index_t * index, unsigned int last_base_pos, int test_len, short * total_indels, short * indel_point, int max_indel_number, int min_test_offset, short * indels, short * indel_poses, int sofar_matched_bases, short * best_indels, short * best_indel_poses, int * best_matching_bases, int level) { unsigned int xi; int xj, xk; unsigned int index_low_boundary = index -> start_base_offset; unsigned int index_up_boundary = index -> start_base_offset + index -> length; if(last_base_pos - test_len <= index_low_boundary)return; if(last_base_pos >= index_up_boundary)return; if(level >9)return; if(0&&memcmp(read, TEST_TARGET, 60)==0) printf("BACK-OUT: %s, LEV=%d, LEN=%d, TAILPOS=%u\n",read, level, test_len, last_base_pos); for(xi = 1 ; xi < test_len-1 ; xi++) { indel_record_t found_records[20]; int section_matched_bases = 0, num_records = find_all_indels(indel_table , last_base_pos - xi, found_records, 1); if(num_records>0) section_matched_bases = match_chro(read + test_len - xi, index, last_base_pos - xi, xi ,0,GENE_SPACE_BASE); // if(num_records) printf("XI %d FOUND %d INDELS AT %u ; SECMATCH=%d\n",xi, num_records , last_base_pos - xi, section_matched_bases); for(xj=0; xj len >0) next_last_base_pos -= this_record -> len; // if this is a deletion, jump the gap. int remainder_testlen = test_len - xi -1; if(this_record -> len <0) remainder_testlen += this_record -> len ; indels[level] = this_record -> len; indel_poses[level] = test_len - xi -1; if(this_record -> len <0) indel_poses[level] += this_record -> len ; if(remainder_testlen> 0 &&remainder_testlen *best_matching_bases) { if(0&&memcmp(read, TEST_TARGET, 60)==0) { for(xk=0; xk 0xffff0000 || pos + test_len>= index-> length + index->start_point) { *indels = 0; return 0; } // i is the distance from the tail. for(i=1 ; i < test_len /*max(0,offset)*/; i++) { unsigned int npos = tail_pos - i - 1 - offset; char tt; tt = gvindex_get (index, npos); //printf("POS=%u INDEL=%d MATCH=%d\n", npos, offset, read[test_len - i - 1] ==tt); if(read[test_len - i - 1] ==tt)ret++; else if(test_len - i - 1 > INDEL_TEST_WINDOW +1 && i > 1) { // if there is a base unmatched, it is potentially an indel from here. int window_match = match_chro(read + test_len - i - INDEL_TEST_WINDOW, index, tail_pos - i - offset - INDEL_TEST_WINDOW, INDEL_TEST_WINDOW ,0,GENE_SPACE_BASE); if(window_match < INDEL_TEST_WINDOW - 1) { // if the window is badly matched, it is very likely to be an indel from this base. int indel_test_i; for(indel_test_i =0; indel_test_i < 7; indel_test_i++) { int indel_test = (indel_test_i+1)/2*(indel_test_i%2?1:-1); if(abs(indel_test)>max_indel_number) continue; if(indel_test > 0) //deletion { int matched_tail = match_chro(read , index, tail_pos - test_len - indel_test , test_len - i ,0,GENE_SPACE_BASE); int matched_score = matched_tail * 10000 / ( test_len - i ); if(matched_score > bast_match_score_remailing && matched_score > MIN_INDEL_SEARCH_MATCH_SCORE) { offset = indel_test; bast_match_score_remailing = matched_score; } }else //insertion { int matched_tail = match_chro(read, index, tail_pos - test_len - indel_test , test_len - i + indel_test ,0,GENE_SPACE_BASE); int matched_score = matched_tail * 10000 / (test_len - i + indel_test); if(matched_score > bast_match_score_remailing && matched_score > MIN_INDEL_SEARCH_MATCH_SCORE) { offset = indel_test; bast_match_score_remailing = matched_score; } } } } if(bast_match_score_remailing>0) { if(offset < 0)//insertion { //ret += read[i+offset] == tt; *indel_point = test_len - i + offset ; i -= offset + 1; } else if(offset >0) //deletion { npos = (tail_pos - i - offset) ; tt = gvindex_get (index, npos); ret += read[test_len - i - 1] == tt; *indel_point = test_len - i ; } } } } //if(memcmp(read, "AACCCCTTGCAGAAAA", 15)==0) // SUBREADprintf("\nINDEL_SPOT %d\n", offset); *indels = offset; return ret; } float match_chro_support(char * read, gene_value_index_t * index, unsigned int pos, int test_len, int is_negative_strand, int space_type, char * qual_txt, int qual_format) { int i; int all_qual = 0; int supported_qual = 0; if (is_negative_strand) { for (i=test_len -1;i>=0;i--) { char tt = gvindex_get(index, pos+test_len-1-i); int is_correct = 0; switch(tt) { case 'A': is_correct = read[i] == 'T'; break; case 'T': is_correct = read[i] == 'A'; break; case 'G': is_correct = read[i] == 'C'; break; case 'C': is_correct = read[i] == 'G'; break; } int base_p = 0.; if(qual_txt[0]) { if(FASTQ_PHRED64 == qual_format) { base_p = get_base_error_prob64i(qual_txt[i]); } else { base_p = get_base_error_prob33i(qual_txt[i]); } } if(base_p > 300000) continue; base_p=0; all_qual += (1000000-base_p); if(is_correct) supported_qual += (1000000-base_p); } } else { if(qual_txt[0]) { if(FASTQ_PHRED33 == qual_format) { for (i=0;i 300000) continue; base_p=0; all_qual += (1000000-base_p); if(is_correct) supported_qual += (1000000-base_p); } } else { for (i=0;i 300000) continue; base_p=0; all_qual += (1000000-base_p); if(is_correct) supported_qual += (1000000-base_p); } } } else for (i=0;i= index -> length + index -> start_point) return 0; if (pos > 0xffff0000) return 0; if (is_negative_strand) { if (space_type == GENE_SPACE_COLOR) { pos++; last_char = (pos+test_len>= index -> length + index -> start_point)?'A': gvindex_get(index,pos+test_len); for (i=test_len -1;i>=0;i--) { char tt = gvindex_get (index, pos+test_len-1-i); ret += read[i] == '0'+chars2color(tt, last_char); last_char = tt; } } else { for (i=test_len -1;i>=0;i--) { char tt = gvindex_get (index, pos+test_len-1-i); switch(tt) { case 'A': ret += read[i] == 'T'; break; case 'T': ret += read[i] == 'A'; break; case 'G': ret += read[i] == 'C'; break; case 'C': ret += read[i] == 'G'; break; } } } } else { if(space_type == GENE_SPACE_BASE) { unsigned int offset_byte, offset_bit; //puts("P1"); gvindex_baseno2offset_m(pos, index , offset_byte, offset_bit); //printf("OB=%d; V=%016llX\n", offset_byte, (long long )index->values); if(offset_byte >= index-> values_bytes)return 0; char int_value = index->values [offset_byte]; for (i=0;i> offset_bit) & 3; char tv = read[i]; switch(tv){ case 'A': ret += tt==0; break; case 'G': ret += tt==1; break; case 'C': ret += tt==2; break; case 0: //SUBREADprintf("NON-ATGC-CHAR:%d\n", tv); //assert(0); break; default: ret += tt==3; } offset_bit+=2; if(offset_bit==8) { offset_byte++; if(offset_byte == index-> values_bytes)return 0; int_value = index->values [offset_byte]; offset_bit = 0; } } //puts("P3"); } else { last_char = (pos <= index -> start_point)?'A': gvindex_get(index,pos); for (i=0;istart_base_offset; int ret = 0, offset, bits; for(i=(pos - index->start_base_offset); i< test_end ; i++) { offset = i/4; bits = i%4*2; ret += ((index -> values[offset] >> bits) & 0x3 )== base2int(*read); read++; } return ret; } unsigned int match_chro_range(char * read, gene_value_index_t * index, unsigned int pos, int read_len, int search_length, int search_to_back) { short key[4]; int i, j; for(i = 0; i < 4; i++) { key[i]=0; for(j = i+7; j >= i; j--) { key[i] = key[i] << 2 | base2int(read[j]); } } unsigned int offset_byte, offset_bit, search_dist; gvindex_baseno2offset(pos, index , &offset_byte, &offset_bit); /* if(offset_byte > 0x35000000) SUBREADprintf("POS=%u, OFFBYTE=%u, index->start_base_off=%u\n" , pos, offset_byte, index -> start_base_offset); */ search_dist = search_length /4; if(search_to_back == SEARCH_BACK) { if(search_dist > offset_byte - 500)search_dist = offset_byte- 500; }else if(search_dist + offset_byte >= index -> values_bytes - 500) search_dist = index -> values_bytes - offset_byte -501; for (i=2; ivalues +test_offset); for(j=0; j<4; j++) { if(tv == key[j]) { //test the whole read unsigned int hit_pos = test_offset*4; hit_pos += index -> start_base_offset - j;// + index -> start_point; int retv = match_chro_maxerror(read, index, hit_pos, read_len, 0, 0, 0); if(retv >0) { //SUBREADprintf("POS=%u, TOFF=%u, STARTBASE=%d, j=%d , RETV=%d\n" , test_offset*4 + index -> start_base_offset - j , test_offset , index -> start_base_offset , j , retv); return hit_pos; } } } } return 0xffffffff; } int match_chro_maxerror(char * read, gene_value_index_t * index, unsigned int pos, int test_len, int is_negative_strand, int space_type, int max_error) { int ret = 0; int i; char last_char='A'; if (is_negative_strand) { if (space_type == GENE_SPACE_COLOR) { pos++; last_char = (pos+test_len>= index -> length + index -> start_point)?'A': gvindex_get(index,pos+test_len); } for (i=test_len -1;i>=0;i--) { char tt = gvindex_get (index, pos+test_len-1-i); if(space_type == GENE_SPACE_COLOR) { ret += read[i] != '0'+chars2color(tt, last_char); last_char = tt; } else switch(tt) { case 'A': ret += read[i] != 'T'; break; case 'T': ret += read[i] != 'A'; break; case 'G': ret += read[i] != 'C'; break; case 'C': ret += read[i] != 'G'; break; } if(ret>max_error)return 0; } } else { if (space_type == GENE_SPACE_COLOR) last_char = (pos <= index -> start_point)?'A': gvindex_get(index,pos-1); for (i=0;imax_error)return 0; } } return test_len - ret; } int gvindex_match_base(gene_value_index_t * index, gehash_data_t offset, const char base_int_value) { unsigned int offset_byte, offset_bit; gvindex_baseno2offset(offset, index, &offset_byte, &offset_bit); unsigned char mask = 0x3 << (offset_bit); if(offset_byte >= index->values_bytes) return 0; // SUBREADprintf("\nERROR: %u > %u\n", offset_byte, index->values_bytes); char reference_base = ((index->values [offset_byte] & mask) >> offset_bit); return (reference_base == base_int_value)?1:0 ; } void gvindex_destory(gene_value_index_t * index) { free(index -> values); } void gvindex_get_string(char *buf, gene_value_index_t * index, unsigned int pos, int len, int is_negative_strand) { int i; if (is_negative_strand) for (i=len-1;i>=0;i--) { buf[i] = gvindex_get (index, pos + len - 1 - i); switch(buf[i]) { case 'A': buf[i] = 'T'; break; case 'T': buf[i] = 'A'; break; case 'G': buf[i] = 'C'; break; case 'C': buf[i] = 'G'; break; } } else for (i=0;i #include #include #include #include #include "hashtable.h" #include "core.h" #include "input-files.h" static int pointercmp(const void *pointer1, const void *pointer2); static srUInt_64 pointerHashFunction(const void *pointer); static int isProbablePrime(srInt_64 number); static srInt_64 calculateIdealNumOfBuckets(HashTable *hashTable); srInt_64 long_random_val(){ srInt_64 ret = 0; if(RAND_MAX<255){ SUBREADprintf("Is this a embedded computer????\n"); return -1; } int i; for(i=0;i<8;i++){ if(i>0)ret = (ret << 8) ^ (myrand_rand() & 0xff); else ret = (ret << 8) ^ (myrand_rand() & 0x7f); } return ret; } HashTable * ArrayListToLookupTable_Int(ArrayList * arr){ srInt_64 x1; HashTable * ret = HashTableCreate( max(1,arr -> numOfElements / 6) ); for(x1 = 0; x1 < arr->numOfElements; x1++) HashTablePut(ret, ArrayListGet(arr, x1)+1, NULL+x1+1); return ret; } int ArrayListContainsPtr(ArrayList * list, void * who){ srInt_64 x1; for(x1 = 0; x1 < list->numOfElements; x1++) if(list->elementList [ x1 ]==who) return 1; return 0; } void * ArrayListShift(ArrayList * list){ if(list->numOfElements<1) return NULL; void *ret = list->elementList [0]; srInt_64 xx; list->numOfElements -- ; for(xx=0; xxnumOfElements; xx++) list->elementList [ xx ] = list->elementList [ xx+1 ]; return ret; } void * ArrayListPop(ArrayList * list){ if(list->numOfElements<1) return NULL; return list->elementList [ -- list->numOfElements]; } void * ArrayListRandom(ArrayList * list){ srInt_64 ii = long_random_val() % list -> numOfElements; return list -> elementList[ii]; } ArrayList * ArrayListCreate(int init_capacity){ ArrayList * ret = malloc(sizeof(ArrayList)); memset(ret,0,sizeof(ArrayList)); ret -> capacityOfElements = init_capacity; ret -> elementList = malloc(sizeof(void *)*init_capacity); return ret; } ArrayList * ArrayListDuplicate(ArrayList * ori){ ArrayList * ret = malloc(sizeof(ArrayList)); memset(ret,0,sizeof(ArrayList)); ret -> capacityOfElements = ori -> numOfElements; ret -> numOfElements = ori -> numOfElements; ret -> elementList = malloc(sizeof(void *)*ret -> capacityOfElements); memcpy(ret -> elementList, ori -> elementList, sizeof(void *)*ret -> capacityOfElements); ret -> elemDeallocator = ori -> elemDeallocator; return ret; } void ArrayListDestroy(ArrayList * list){ srInt_64 x1; if(list -> elemDeallocator) for(x1 = 0;x1 < list->numOfElements; x1++) list -> elemDeallocator(list -> elementList[x1]); free(list -> elementList); free(list); } void * ArrayListGet(ArrayList * list, srInt_64 n){ if(n<0 || n >= list->numOfElements)return NULL; return list -> elementList[n]; } int ArrayListPush_NoRepeatedPtr(ArrayList * list, void * new_elem){ srInt_64 ii; for(ii = 0; ii < list->numOfElements; ii++){ if(list -> elementList[ii] == new_elem) return -1; } return ArrayListPush(list,new_elem); } int ArrayListPush(ArrayList * list, void * new_elem){ if(list -> capacityOfElements <= list->numOfElements){ if(list -> capacityOfElements *1.3 > list -> capacityOfElements + 10) list -> capacityOfElements = list -> capacityOfElements *1.3; else list -> capacityOfElements = list -> capacityOfElements + 10; list -> elementList=realloc(list -> elementList, sizeof(void *) * list -> capacityOfElements); assert(list -> elementList); } list->elementList[list->numOfElements++] = new_elem; return list->numOfElements; } void ArrayListSetDeallocationFunction(ArrayList * list, void (*elem_deallocator)(void *elem)){ list -> elemDeallocator = elem_deallocator; } int ArrayListSort_comp_pntr( void * L_elem, void * R_elem, ArrayList * me ){ if( L_elem > R_elem )return 1; if( L_elem < R_elem )return -1; return 0; } int ArrayListSort_compare(void * sortdata0, int L, int R){ void ** sortdata = sortdata0; ArrayList * list = sortdata[0]; int (*comp_elems)(void * L_elem, void * R_elem, ArrayList * me) = sortdata[1]; void * L_elem = list -> elementList[L]; void * R_elem = list -> elementList[R]; return comp_elems(L_elem, R_elem, list); } void ArrayListSort_exchange(void * sortdata0, int L, int R){ void ** sortdata = sortdata0; ArrayList * list = sortdata[0]; void * tmpp = list -> elementList[L]; list -> elementList[L] = list -> elementList[R]; list -> elementList[R] = tmpp; } void ArrayListSort_merge(void * sortdata0, int start, int items, int items2){ void ** sortdata = sortdata0; ArrayList * list = sortdata[0]; int (*comp_elems)(void * L_elem, void * R_elem, ArrayList * me) = sortdata[1]; void ** merged = malloc(sizeof(void *)*(items + items2)); int write_cursor, read1=start, read2=start+items; for(write_cursor = 0; write_cursor < items + items2; write_cursor++){ void * Elm1 = (read1 == start + items)? NULL:list -> elementList[read1]; void * Elm2 = (read2 == start + items + items2)?NULL:list -> elementList[read2]; int select_1 = (read1 == start + items)?0:( read2 == start + items + items2 || comp_elems(Elm1, Elm2, list) < 0 ); if(select_1) merged[write_cursor] = list -> elementList[read1++]; else merged[write_cursor] = list -> elementList[read2++]; if(read2 > start + items + items2){ SUBREADprintf("R1: %d < %d ; R2: %d < %d\n", read1, start + items, read2, start + items + items2); } assert(read2 <=start + items + items2); assert(read1 <=start + items); } memcpy(list -> elementList + start, merged, sizeof(void *) * (items + items2)); free(merged); } void ArrayListSort(ArrayList * list, int compare_L_minus_R(void * L_elem, void * R_elem, ArrayList * me)){ void * sortdata[2]; sortdata[0] = list; sortdata[1] = compare_L_minus_R?compare_L_minus_R:ArrayListSort_comp_pntr; if(list -> numOfElements > 20) merge_sort(sortdata, list -> numOfElements, ArrayListSort_compare, ArrayListSort_exchange, ArrayListSort_merge); else basic_sort(sortdata, list -> numOfElements, ArrayListSort_compare, ArrayListSort_exchange); } int ArrayListLLUComparison(void * L_elem, void * R_elem){ srUInt_64 lint = L_elem-NULL; srUInt_64 rint = R_elem-NULL; if(lintrint)return 1; return 0; } srInt_64 ArrayListFindNextDent(ArrayList * list, srUInt_64 value_less_than_dent){ srInt_64 h=list->numOfElements-1,l=0,m=-1l; if( list -> elementList[h]- NULL <= value_less_than_dent )return -1l; while(h>l){ m=(h+l)/2; srUInt_64 mv = list -> elementList[m] - NULL; if(mv < value_less_than_dent) l=m+1; else if(mv > value_less_than_dent) h=m-1; else break; } if(m<2)m=0; else m-=2; for(; m>=0 && list -> elementList[m] - NULL >= value_less_than_dent; m--); for(m = max(0, m); m < list->numOfElements ; m++){ if( list -> elementList[m] - NULL > value_less_than_dent )return m; } SUBREADprintf("ALGORITHM ERROR!! DID YOU SORT THE LIST???\n"); return -2l; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableCreate() - creates a new HashTable * DESCRIPTION: * Creates a new HashTable. When finished with this HashTable, it * should be explicitly destroyed by calling the HashTableDestroy() * function. * EFFICIENCY: * O(1) * ARGUMENTS: * numOfBuckets - the number of buckets to start the HashTable out with. * Must be greater than zero, and should be prime. * Ideally, the number of buckets should between 1/5 * and 1 times the expected number of elements in the * HashTable. Values much more or less than this will * result in wasted memory or decreased performance * respectively. The number of buckets in a HashTable * can be re-calculated to an appropriate number by * calling the HashTableRehash() function once the * HashTable has been populated. The number of buckets * in a HashTable may also be re-calculated * automatically if the ratio of elements to buckets * passes the thresholds set by HashTableSetIdealRatio(). * RETURNS: * HashTable - a new Hashtable, or NULL on error \*--------------------------------------------------------------------------*/ HashTable * StringTableCreate(srInt_64 numOfBuckets){ HashTable * ret = HashTableCreate(numOfBuckets); HashTableSetHashFunction(ret, HashTableStringHashFunction); HashTableSetKeyComparisonFunction(ret , my_strcmp); return ret; } void StringTableReverse_Run(void * ky, void * va, HashTable * tab){ HashTable * res = tab -> appendix1; HashTablePut(res, va, ky); } HashTable * StringTableReverse(HashTable * ori){ HashTable * res = StringTableCreate(ori -> numOfBuckets); void * oriap1 = ori -> appendix1; ori -> appendix1 = res; HashTableIteration(ori, StringTableReverse_Run); ori -> appendix1 = oriap1; return res; } ArrayList * HashTableKeys(HashTable * tab){ int i; ArrayList * ret = ArrayListCreate(tab -> numOfElements); for (i=0; i< tab ->numOfBuckets; i++) { KeyValuePair *pair = tab ->bucketArray[i]; while (pair != NULL) { ArrayListPush(ret, pair -> key); KeyValuePair *nextPair = pair->next; pair = nextPair; } } return ret; } HashTable *HashTableCreate(srInt_64 numOfBuckets) { HashTable *hashTable; int i; assert(numOfBuckets > 0); hashTable = (HashTable *) malloc(sizeof(HashTable)); if (hashTable == NULL) return NULL; hashTable->appendix1=NULL; hashTable->appendix2=NULL; hashTable->appendix3=NULL; hashTable->counter1=0; hashTable->counter2=0; hashTable->counter3=0; hashTable->bucketArray = (KeyValuePair **) malloc(numOfBuckets * sizeof(KeyValuePair *)); if (hashTable->bucketArray == NULL) { free(hashTable); return NULL; } hashTable->numOfBuckets = numOfBuckets; hashTable->numOfElements = 0; for (i=0; ibucketArray[i] = NULL; hashTable->idealRatio = 3.0; hashTable->lowerRehashThreshold = 0.0; hashTable->upperRehashThreshold = 15.0; hashTable->keycmp = pointercmp; hashTable->valuecmp = pointercmp; hashTable->hashFunction = pointerHashFunction; hashTable->keyDeallocator = NULL; hashTable->valueDeallocator = NULL; return hashTable; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableDestroy() - destroys an existing HashTable * DESCRIPTION: * Destroys an existing HashTable. * EFFICIENCY: * O(n) * ARGUMENTS: * hashTable - the HashTable to destroy * RETURNS: * \*--------------------------------------------------------------------------*/ void HashTableDestroy(HashTable *hashTable) { int i; for (i=0; inumOfBuckets; i++) { KeyValuePair *pair = hashTable->bucketArray[i]; while (pair != NULL) { KeyValuePair *nextPair = pair->next; if (hashTable->keyDeallocator != NULL) hashTable->keyDeallocator((void *) pair->key); if (hashTable->valueDeallocator != NULL){ // fprintf(stderr,"FREE %p\n", pair->value); hashTable->valueDeallocator(pair->value); } free(pair); pair = nextPair; } } free(hashTable->bucketArray); free(hashTable); } /*--------------------------------------------------------------------------*\ * NAME: * HashTableContainsKey() - checks the existence of a key in a HashTable * DESCRIPTION: * Determines whether or not the specified HashTable contains the * specified key. Uses the comparison function specified by * HashTableSetKeyComparisonFunction(). * EFFICIENCY: * O(1), assuming a good hash function and element-to-bucket ratio * ARGUMENTS: * hashTable - the HashTable to search * key - the key to search for * RETURNS: * bool - whether or not the specified HashTable contains the * specified key. \*--------------------------------------------------------------------------*/ int HashTableContainsKey(const HashTable *hashTable, const void *key) { return (HashTableGet(hashTable, key) != NULL); } /*--------------------------------------------------------------------------*\ * NAME: * HashTableContainsValue() * - checks the existence of a value in a HashTable * DESCRIPTION: * Determines whether or not the specified HashTable contains the * specified value. Unlike HashTableContainsKey(), this function is * not very efficient, since it has to scan linearly looking for a * match. Uses the comparison function specified by * HashTableSetValueComparisonFunction(). * EFFICIENCY: * O(n) * ARGUMENTS: * hashTable - the HashTable to search * value - the value to search for * RETURNS: * bool - whether or not the specified HashTable contains the * specified value. \*--------------------------------------------------------------------------*/ int HashTableContainsValue(const HashTable *hashTable, const void *value) { int i; for (i=0; inumOfBuckets; i++) { KeyValuePair *pair = hashTable->bucketArray[i]; while (pair != NULL) { if (hashTable->valuecmp(value, pair->value) == 0) return 1; pair = pair->next; } } return 0; } /*--------------------------------------------------------------------------*\ * NAME: * HashTablePut() - adds a key/value pair to a HashTable * DESCRIPTION: * Adds the specified key/value pair to the specified HashTable. If * the key already exists in the HashTable (determined by the comparison * function specified by HashTableSetKeyComparisonFunction()), its value * is replaced by the new value. May trigger an auto-rehash (see * HashTableSetIdealRatio()). It is illegal to specify NULL as the * key or value. * EFFICIENCY: * O(1), assuming a good hash function and element-to-bucket ratio * ARGUMENTS: * hashTable - the HashTable to add to * key - the key to add or whose value to replace * value - the value associated with the key * RETURNS: * err - 0 if successful, -1 if an error was encountered \*--------------------------------------------------------------------------*/ int HashTablePut(HashTable *hashTable, const void *key, void *value) { return HashTablePutReplace(hashTable, key, value, 1); } int HashTablePutReplaceEx(HashTable *hashTable, const void *key, void *value, int replace_key, int dealloc_key, int dealloc_value) { srInt_64 hashValue; KeyValuePair *pair; assert(key != NULL); assert(value != NULL); hashValue = hashTable->hashFunction(key) % hashTable->numOfBuckets; pair = hashTable->bucketArray[hashValue]; while (pair != NULL && hashTable->keycmp(key, pair->key) != 0) pair = pair->next; if (pair) { if (pair->key != key) { if(replace_key) { if(hashTable->keyDeallocator && dealloc_key) hashTable->keyDeallocator((void *) pair->key); pair->key = (void *)key; } } if (pair->value != value) { if (hashTable->valueDeallocator != NULL && dealloc_value) hashTable->valueDeallocator(pair->value); pair->value = (void *)value; } } else { KeyValuePair *newPair = (KeyValuePair *) malloc(sizeof(KeyValuePair)); if (newPair == NULL) { return -1; } else { newPair->key = (void *)key; newPair->value = (void *)value; newPair->next = hashTable->bucketArray[hashValue]; hashTable->bucketArray[hashValue] = newPair; hashTable->numOfElements++; if (hashTable->upperRehashThreshold > hashTable->idealRatio) { float elementToBucketRatio = (float) hashTable->numOfElements / (float) hashTable->numOfBuckets; if (elementToBucketRatio > hashTable->upperRehashThreshold) HashTableRehash(hashTable, 0); } } } return 0; } int HashTablePutReplace(HashTable *hashTable, const void *key, void *value, int replace_key) { return HashTablePutReplaceEx(hashTable, key, value, replace_key, 1, 1); } /*--------------------------------------------------------------------------*\ * NAME: * HashTableGet() - retrieves the value of a key in a HashTable * DESCRIPTION: * Retrieves the value of the specified key in the specified HashTable. * Uses the comparison function specified by * HashTableSetKeyComparisonFunction(). * EFFICIENCY: * O(1), assuming a good hash function and element-to-bucket ratio * ARGUMENTS: * hashTable - the HashTable to search * key - the key whose value is desired * RETURNS: * void * - the value of the specified key, or NULL if the key * doesn't exist in the HashTable \*--------------------------------------------------------------------------*/ void *HashTableGetKey(const HashTable *hashTable, const void *key) { srInt_64 hashValue = hashTable->hashFunction(key) % hashTable->numOfBuckets; KeyValuePair *pair = hashTable->bucketArray[hashValue]; while (pair != NULL && hashTable->keycmp(key, pair->key) != 0) pair = pair->next; return (pair == NULL)? NULL : pair->key; } void *HashTableGet(const HashTable *hashTable, const void *key) { srInt_64 hashValue = hashTable->hashFunction(key) % hashTable->numOfBuckets; KeyValuePair *pair = hashTable->bucketArray[hashValue]; while (pair != NULL && hashTable->keycmp(key, pair->key) != 0) pair = pair->next; return (pair == NULL)? NULL : pair->value; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableRemove() - removes a key/value pair from a HashTable * DESCRIPTION: * Removes the key/value pair identified by the specified key from the * specified HashTable if the key exists in the HashTable. May trigger * an auto-rehash (see HashTableSetIdealRatio()). * EFFICIENCY: * O(1), assuming a good hash function and element-to-bucket ratio * ARGUMENTS: * hashTable - the HashTable to remove the key/value pair from * key - the key specifying the key/value pair to be removed * RETURNS: * \*--------------------------------------------------------------------------*/ void HashTableRemove(HashTable *hashTable, const void *key) { srInt_64 hashValue = hashTable->hashFunction(key) % hashTable->numOfBuckets; KeyValuePair *pair = hashTable->bucketArray[hashValue]; KeyValuePair *previousPair = NULL; while (pair != NULL && hashTable->keycmp(key, pair->key) != 0) { previousPair = pair; pair = pair->next; } if (pair != NULL) { if (hashTable->keyDeallocator != NULL) hashTable->keyDeallocator((void *) pair->key); if (hashTable->valueDeallocator != NULL) hashTable->valueDeallocator(pair->value); if (previousPair != NULL) previousPair->next = pair->next; else hashTable->bucketArray[hashValue] = pair->next; free(pair); hashTable->numOfElements--; if (hashTable->lowerRehashThreshold > 0.0) { float elementToBucketRatio = (float) hashTable->numOfElements / (float) hashTable->numOfBuckets; if (elementToBucketRatio < hashTable->lowerRehashThreshold) HashTableRehash(hashTable, 0); } } } /*--------------------------------------------------------------------------*\ * NAME: * HashTableRemoveAll() - removes all key/value pairs from a HashTable * DESCRIPTION: * Removes all key/value pairs from the specified HashTable. May trigger * an auto-rehash (see HashTableSetIdealRatio()). * EFFICIENCY: * O(n) * ARGUMENTS: * hashTable - the HashTable to remove all key/value pairs from * RETURNS: * \*--------------------------------------------------------------------------*/ void HashTableRemoveAll(HashTable *hashTable) { int i; for (i=0; inumOfBuckets; i++) { KeyValuePair *pair = hashTable->bucketArray[i]; while (pair != NULL) { KeyValuePair *nextPair = pair->next; if (hashTable->keyDeallocator != NULL) hashTable->keyDeallocator((void *) pair->key); if (hashTable->valueDeallocator != NULL) hashTable->valueDeallocator(pair->value); free(pair); pair = nextPair; } hashTable->bucketArray[i] = NULL; } hashTable->numOfElements = 0; HashTableRehash(hashTable, 5); } /*--------------------------------------------------------------------------*\ * NAME: * HashTableIsEmpty() - determines if a HashTable is empty * DESCRIPTION: * Determines whether or not the specified HashTable contains any * key/value pairs. * EFFICIENCY: * O(1) * ARGUMENTS: * hashTable - the HashTable to check * RETURNS: * bool - whether or not the specified HashTable contains any * key/value pairs \*--------------------------------------------------------------------------*/ int HashTableIsEmpty(const HashTable *hashTable) { return (hashTable->numOfElements == 0); } /*--------------------------------------------------------------------------*\ * NAME: * HashTableSize() - returns the number of elements in a HashTable * DESCRIPTION: * Returns the number of key/value pairs that are present in the * specified HashTable. * EFFICIENCY: * O(1) * ARGUMENTS: * hashTable - the HashTable whose size is requested * RETURNS: * long - the number of key/value pairs that are present in * the specified HashTable \*--------------------------------------------------------------------------*/ srInt_64 HashTableSize(const HashTable *hashTable) { return hashTable->numOfElements; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableGetNumBuckets() - returns the number of buckets in a HashTable * DESCRIPTION: * Returns the number of buckets that are in the specified HashTable. * This may change dynamically throughout the life of a HashTable if * automatic or manual rehashing is performed. * EFFICIENCY: * O(1) * ARGUMENTS: * hashTable - the HashTable whose number of buckets is requested * RETURNS: * long - the number of buckets that are in the specified * HashTable \*--------------------------------------------------------------------------*/ srInt_64 HashTableGetNumBuckets(const HashTable *hashTable) { return hashTable->numOfBuckets; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableSetKeyComparisonFunction() * - specifies the function used to compare keys in a HashTable * DESCRIPTION: * Specifies the function used to compare keys in the specified * HashTable. The specified function should return zero if the two * keys are considered equal, and non-zero otherwise. The default * function is one that simply compares pointers. * ARGUMENTS: * hashTable - the HashTable whose key comparison function is being * specified * keycmp - a function which returns zero if the two arguments * passed to it are considered "equal" keys and non-zero * otherwise * RETURNS: * \*--------------------------------------------------------------------------*/ void HashTableSetKeyComparisonFunction(HashTable *hashTable, int (*keycmp)(const void *key1, const void *key2)) { assert(keycmp != NULL); hashTable->keycmp = keycmp; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableSetValueComparisonFunction() * - specifies the function used to compare values in a HashTable * DESCRIPTION: * Specifies the function used to compare values in the specified * HashTable. The specified function should return zero if the two * values are considered equal, and non-zero otherwise. The default * function is one that simply compares pointers. * ARGUMENTS: * hashTable - the HashTable whose value comparison function is being * specified * valuecmp - a function which returns zero if the two arguments * passed to it are considered "equal" values and non-zero * otherwise * RETURNS: * \*--------------------------------------------------------------------------*/ void HashTableSetValueComparisonFunction(HashTable *hashTable, int (*valuecmp)(const void *value1, const void *value2)) { assert(valuecmp != NULL); hashTable->valuecmp = valuecmp; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableSetHashFunction() * - specifies the hash function used by a HashTable * DESCRIPTION: * Specifies the function used to determine the hash value for a key * in the specified HashTable (before modulation). An ideal hash * function is one which is easy to compute and approximates a * "random" function. The default function is one that works * relatively well for pointers. If the HashTable keys are to be * strings (which is probably the case), then this default function * will not suffice, in which case consider using the provided * HashTableStringHashFunction() function. * ARGUMENTS: * hashTable - the HashTable whose hash function is being specified * hashFunction - a function which returns an appropriate hash code * for a given key * RETURNS: * \*--------------------------------------------------------------------------*/ void HashTableSetHashFunction(HashTable *hashTable, srUInt_64 (*hashFunction)(const void *key)) { assert(hashFunction != NULL); hashTable->hashFunction = hashFunction; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableRehash() - reorganizes a HashTable to be more efficient * DESCRIPTION: * Reorganizes a HashTable to be more efficient. If a number of * buckets is specified, the HashTable is rehashed to that number of * buckets. If 0 is specified, the HashTable is rehashed to a number * of buckets which is automatically calculated to be a prime number * that achieves (as closely as possible) the ideal element-to-bucket * ratio specified by the HashTableSetIdealRatio() function. * EFFICIENCY: * O(n) * ARGUMENTS: * hashTable - the HashTable to be reorganized * numOfBuckets - the number of buckets to rehash the HashTable to. * Should be prime. Ideally, the number of buckets * should be between 1/5 and 1 times the expected * number of elements in the HashTable. Values much * more or less than this will result in wasted memory * or decreased performance respectively. If 0 is * specified, an appropriate number of buckets is * automatically calculated. * RETURNS: * \*--------------------------------------------------------------------------*/ void HashTableRehash(HashTable *hashTable, srInt_64 numOfBuckets) { KeyValuePair **newBucketArray; int i; assert(numOfBuckets >= 0); if (numOfBuckets == 0) numOfBuckets = calculateIdealNumOfBuckets(hashTable); if (numOfBuckets == hashTable->numOfBuckets) return; /* already the right size! */ newBucketArray = (KeyValuePair **) malloc(numOfBuckets * sizeof(KeyValuePair *)); if (newBucketArray == NULL) { /* Couldn't allocate memory for the new array. This isn't a fatal * error; we just can't perform the rehash. */ return; } for (i=0; inumOfBuckets; i++) { KeyValuePair *pair = hashTable->bucketArray[i]; while (pair != NULL) { KeyValuePair *nextPair = pair->next; srInt_64 hashValue = hashTable->hashFunction(pair->key) % numOfBuckets; pair->next = newBucketArray[hashValue]; newBucketArray[hashValue] = pair; pair = nextPair; } } free(hashTable->bucketArray); hashTable->bucketArray = newBucketArray; hashTable->numOfBuckets = numOfBuckets; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableSetIdealRatio() * - sets the ideal element-to-bucket ratio of a HashTable * DESCRIPTION: * Sets the ideal element-to-bucket ratio, as well as the lower and * upper auto-rehash thresholds, of the specified HashTable. Note * that this function doesn't actually perform a rehash. * * The default values for these properties are 3.0, 0.0 and 15.0 * respectively. This is likely fine for most situations, so there * is probably no need to call this function. * ARGUMENTS: * hashTable - a HashTable * idealRatio - the ideal element-to-bucket ratio. When a rehash * occurs (either manually via a call to the * HashTableRehash() function or automatically due the * the triggering of one of the thresholds below), the * number of buckets in the HashTable will be * recalculated to be a prime number that achieves (as * closely as possible) this ideal ratio. Must be a * positive number. * lowerRehashThreshold * - the element-to-bucket ratio that is considered * unacceptably low (i.e., too few elements per bucket). * If the actual ratio falls below this number, a * rehash will automatically be performed. Must be * lower than the value of idealRatio. If no ratio * is considered unacceptably low, a value of 0.0 can * be specified. * upperRehashThreshold * - the element-to-bucket ratio that is considered * unacceptably high (i.e., too many elements per bucket). * If the actual ratio rises above this number, a * rehash will automatically be performed. Must be * higher than idealRatio. However, if no ratio * is considered unacceptably high, a value of 0.0 can * be specified. * RETURNS: * \*--------------------------------------------------------------------------*/ void HashTableSetIdealRatio(HashTable *hashTable, float idealRatio, float lowerRehashThreshold, float upperRehashThreshold) { assert(idealRatio > 0.0); assert(lowerRehashThreshold < idealRatio); assert(upperRehashThreshold == 0.0 || upperRehashThreshold > idealRatio); hashTable->idealRatio = idealRatio; hashTable->lowerRehashThreshold = lowerRehashThreshold; hashTable->upperRehashThreshold = upperRehashThreshold; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableSetDeallocationFunctions() * - sets the key and value deallocation functions of a HashTable * DESCRIPTION: * Sets the key and value deallocation functions of the specified * HashTable. This determines what happens to a key or a value when it * is removed from the HashTable. If the deallocation function is NULL * (the default if this function is never called), its reference is * simply dropped and it is up to the calling program to perform the * proper memory management. If the deallocation function is non-NULL, * it is called to free the memory used by the object. E.g., for simple * objects, an appropriate deallocation function may be free(). * * This affects the behaviour of the HashTableDestroy(), HashTablePut(), * HashTableRemove() and HashTableRemoveAll() functions. * ARGUMENTS: * hashTable - a HashTable * keyDeallocator * - if non-NULL, the function to be called when a key is * removed from the HashTable. * valueDeallocator * - if non-NULL, the function to be called when a value is * removed from the HashTable. * RETURNS: * \*--------------------------------------------------------------------------*/ void HashTableSetDeallocationFunctions(HashTable *hashTable, void (*keyDeallocator)(void *key), void (*valueDeallocator)(void *value)) { hashTable->keyDeallocator = keyDeallocator; hashTable->valueDeallocator = valueDeallocator; } /*--------------------------------------------------------------------------*\ * NAME: * HashTableStringHashFunction() - a good hash function for strings * DESCRIPTION: * A hash function that is appropriate for hashing strings. Note that * this is not the default hash function. To make it the default hash * function, call HashTableSetHashFunction(hashTable, * HashTableStringHashFunction). * ARGUMENTS: * key - the key to be hashed * RETURNS: * unsigned long - the unmodulated hash value of the key \*--------------------------------------------------------------------------*/ srUInt_64 HashTableStringHashFunction(const void *key) { const unsigned char *str = (const unsigned char *) key; srUInt_64 hashValue = 0; int i; for (i=0; str[i] != '\0'; i++) hashValue = hashValue * 37 + str[i]; return hashValue; } static int pointercmp(const void *pointer1, const void *pointer2) { return (pointer1 != pointer2); } static srUInt_64 pointerHashFunction(const void *pointer) { return (srUInt_64)(pointer - NULL); } static int isProbablePrime(srInt_64 oddNumber) { srInt_64 i; for (i=3; i<51; i+=2) if (oddNumber == i) return 1; else if (oddNumber%i == 0) return 0; return 1; /* maybe */ } static srInt_64 calculateIdealNumOfBuckets(HashTable *hashTable) { srInt_64 idealNumOfBuckets = hashTable->numOfElements / hashTable->idealRatio; if (idealNumOfBuckets < 5) idealNumOfBuckets = 5; else idealNumOfBuckets |= 0x01; /* make it an odd number */ while (!isProbablePrime(idealNumOfBuckets)) idealNumOfBuckets += 2; return idealNumOfBuckets; } void free_values_destroy(HashTable * tab) { KeyValuePair * cursor; int bucket; for(bucket=0; bucket< tab -> numOfBuckets; bucket++) { cursor = tab -> bucketArray[bucket]; while (1) { if(!cursor) break; char * read_txt = (char *) cursor ->value; free(read_txt); cursor = cursor->next; } } HashTableDestroy(tab); } ArrayList * HashTableKeyArray(HashTable * tab){ ArrayList *ret = ArrayListCreate(20); int i; for (i=0; i< tab ->numOfBuckets; i++) { KeyValuePair *pair = tab ->bucketArray[i]; while (pair != NULL) { ArrayListPush(ret, (void *)pair -> key); KeyValuePair *nextPair = pair->next; pair = nextPair; } } return ret; } void HashTableIteration(HashTable * tab, void process_item(void * key, void * hashed_obj, HashTable * tab) ) { int i; for (i=0; i< tab ->numOfBuckets; i++) { KeyValuePair *pair = tab ->bucketArray[i]; while (pair != NULL) { process_item(( void * )pair -> key, pair -> value, tab); KeyValuePair *nextPair = pair->next; pair = nextPair; } } } void HashTableSortedIndexes_copy_idx( void *k, void *v, HashTable * k2int_tab ){ ArrayList * dsta = k2int_tab -> appendix1; ArrayListPush(dsta, k); } int HashTableSortedIndexes_cmp_idx( void * Lv, void * Rv, ArrayList * me ){ void ** appdx = me -> appendix1; HashTable * k2int_tab = appdx[0]; void * Lhash = HashTableGet(k2int_tab, Lv); void * Rhash = HashTableGet(k2int_tab, Rv); void * large_first = appdx[1]; if(large_first){ if(Lhash > Rhash) return -1; if(Lhash < Rhash) return 1; }else{ if(Lhash > Rhash) return 1; if(Lhash < Rhash) return -1; } return 0; } ArrayList * HashTableSortedIndexes(HashTable * k2int_tab, int larger_value_first){ ArrayList *ret = HashTableKeys(k2int_tab); void * appx[2]; ret -> appendix1 = appx; appx[0]=k2int_tab; appx[1]=NULL+larger_value_first; ArrayListSort(ret, HashTableSortedIndexes_cmp_idx); return ret; } Rsubread/src/hashtable.h0000644000175200017520000005470614136047615016261 0ustar00biocbuildbiocbuild/*--------------------------------------------------------------------------*\ * -----===== HashTable =====----- * * Author: Keith Pomakis (pomakis@pobox.com) * Date: August, 1998 * Released to the public domain. * *-------------------------------------------------------------------------- * $Id: hashtable.h,v 9999.30 2019/12/05 03:05:37 cvs Exp $ \*--------------------------------------------------------------------------*/ #ifndef _HASHTABLE_H #define _HASHTABLE_H #ifndef SRINT_64_DEFINED #define SRINT_64_DEFINED typedef long long srInt_64; typedef unsigned long long srUInt_64; #endif /* These structs should not be accessed directly from user code. * All access should be via the public functions declared below. */ typedef struct KeyValuePair_struct { void *key; void *value; struct KeyValuePair_struct *next; } KeyValuePair; typedef struct { srInt_64 numOfBuckets; srInt_64 numOfElements; KeyValuePair **bucketArray; float idealRatio, lowerRehashThreshold, upperRehashThreshold; int (*keycmp)(const void *key1, const void *key2); int (*valuecmp)(const void *value1, const void *value2); srUInt_64 (*hashFunction)(const void *key); void (*keyDeallocator)(void *key); void (*valueDeallocator)(void *value); void * appendix1; void * appendix2; void * appendix3; srInt_64 counter1; srInt_64 counter2; srInt_64 counter3; } HashTable; typedef struct { void ** elementList; srInt_64 numOfElements; srInt_64 capacityOfElements; void (*elemDeallocator)(void *elem); void * appendix1; } ArrayList; int ArrayListContainsPtr(ArrayList * list, void * who); ArrayList * ArrayListCreate(int init_capacity); ArrayList * ArrayListDuplicate(ArrayList * ori); void ArrayListDestroy(ArrayList * list); void * ArrayListGet(ArrayList * list, srInt_64 n); void * ArrayListRandom(ArrayList * list); int ArrayListPush(ArrayList * list, void * new_elem); int ArrayListPush_NoRepeatedPtr(ArrayList * list, void * new_elem); // Shift is the other direction of Pop: // Say, a list has elements 0,1,2,3,4 // Pop removes 4 from the list and returns it. // Shift repoves 0 from the list and returns it. // // Caller has to be sure that at least one element is in the list or a NULL is returned. // Due to its nature, the caller has to deallocate the returned object. The internal deallocater is NOT called on the returned object. void * ArrayListShift(ArrayList * list); void * ArrayListPop(ArrayList * list); void ArrayListSetDeallocationFunction(ArrayList * list, void (*elem_deallocator)(void *elem)); void ArrayListSort(ArrayList * list, int compare_L_minus_R(void * L_elem, void * R_elem, ArrayList * me)); // A simple comparison function if you want to sort unsigned long long ints. int ArrayListLLUComparison(void * L_elem, void * R_elem); // This function returns the index of the value in the list that is the minumum value greater than "value_less_than_dent". // The elements in the list are all zero-based unsigned long long ints. The list must be sorted (small -> large) before calling this function. // If the "value_less_than_dent" is larger than all the values in the list, "-1" is returned; // If there are repeated numbers in the list, this function will always return the index of the first of them when this repeated number is the next dent. // srInt_64 ArrayListFindNextDent(ArrayList * list, srUInt_64 value_less_than_dent); ArrayList * HashTableKeys(HashTable * tab); void HashTableIteration(HashTable * tab, void process_item(void * key, void * hashed_obj, HashTable * tab) ); ArrayList * HashTableKeyArray(HashTable * tab); /*--------------------------------------------------------------------------*\ * NAME: * HashTableCreate() - creates a new HashTable * DESCRIPTION: * Creates a new HashTable. When finished with this HashTable, it * should be explicitly destroyed by calling the HashTableDestroy() * function. * EFFICIENCY: * O(1) * ARGUMENTS: * numOfBuckets - the number of buckets to start the HashTable out with. * Must be greater than zero, and should be prime. * Ideally, the number of buckets should between 1/5 * and 1 times the expected number of elements in the * HashTable. Values much more or less than this will * result in wasted memory or decreased performance * respectively. The number of buckets in a HashTable * can be re-calculated to an appropriate number by * calling the HashTableRehash() function once the * HashTable has been populated. The number of buckets * in a HashTable may also be re-calculated * automatically if the ratio of elements to buckets * passes the thresholds set by HashTableSetIdealRatio(). * RETURNS: * HashTable - a new Hashtable, or NULL on error \*--------------------------------------------------------------------------*/ HashTable * StringTableCreate(srInt_64 numOfBuckets); HashTable * StringTableReverse(HashTable * ori); HashTable *HashTableCreate(srInt_64 numOfBuckets); /*--------------------------------------------------------------------------*\ * NAME: * HashTableDestroy() - destroys an existing HashTable * DESCRIPTION: * Destroys an existing HashTable. * EFFICIENCY: * O(n) * ARGUMENTS: * hashTable - the HashTable to destroy * RETURNS: * \*--------------------------------------------------------------------------*/ void HashTableDestroy(HashTable *hashTable); /*--------------------------------------------------------------------------*\ * NAME: * HashTableContainsKey() - checks the existence of a key in a HashTable * DESCRIPTION: * Determines whether or not the specified HashTable contains the * specified key. Uses the comparison function specified by * HashTableSetKeyComparisonFunction(). * EFFICIENCY: * O(1), assuming a good hash function and element-to-bucket ratio * ARGUMENTS: * hashTable - the HashTable to search * key - the key to search for * RETURNS: * bool - whether or not the specified HashTable contains the * specified key. \*--------------------------------------------------------------------------*/ int HashTableContainsKey(const HashTable *hashTable, const void *key); /*--------------------------------------------------------------------------*\ * NAME: * HashTableContainsValue() * - checks the existence of a value in a HashTable * DESCRIPTION: * Determines whether or not the specified HashTable contains the * specified value. Unlike HashTableContainsKey(), this function is * not very efficient, since it has to scan linearly looking for a * match. Uses the comparison function specified by * HashTableSetValueComparisonFunction(). * EFFICIENCY: * O(n) * ARGUMENTS: * hashTable - the HashTable to search * value - the value to search for * RETURNS: * bool - whether or not the specified HashTable contains the * specified value. \*--------------------------------------------------------------------------*/ int HashTableContainsValue(const HashTable *hashTable, const void *value); /*--------------------------------------------------------------------------*\ * NAME: * HashTablePut() - adds a key/value pair to a HashTable * DESCRIPTION: * Adds the specified key/value pair to the specified HashTable. If * the key already exists in the HashTable (determined by the comparison * function specified by HashTableSetKeyComparisonFunction()), its value * is replaced by the new value. May trigger an auto-rehash (see * HashTableSetIdealRatio()). It is illegal to specify NULL as the * key or value. * EFFICIENCY: * O(1), assuming a good hash function and element-to-bucket ratio * ARGUMENTS: * hashTable - the HashTable to add to * key - the key to add or whose value to replace * value - the value associated with the key * RETURNS: * err - 0 if successful, -1 if an error was encountered \*--------------------------------------------------------------------------*/ int HashTablePut(HashTable *hashTable, const void *key, void *value); int HashTablePutReplace(HashTable *hashTable, const void *key, void *value, int replace_key); /*--------------------------------------------------------------------------*\ * NAME: * HashTableGet() - retrieves the value of a key in a HashTable * DESCRIPTION: * Retrieves the value of the specified key in the specified HashTable. * Uses the comparison function specified by * HashTableSetKeyComparisonFunction(). * EFFICIENCY: * O(1), assuming a good hash function and element-to-bucket ratio * ARGUMENTS: * hashTable - the HashTable to search * key - the key whose value is desired * RETURNS: * void * - the value of the specified key, or NULL if the key * doesn't exist in the HashTable \*--------------------------------------------------------------------------*/ void * HashTableGetKey(const HashTable *hashTable, const void *key); void * HashTableGet(const HashTable *hashTable, const void *key); /*--------------------------------------------------------------------------*\ * NAME: * HashTableRemove() - removes a key/value pair from a HashTable * DESCRIPTION: * Removes the key/value pair identified by the specified key from the * specified HashTable if the key exists in the HashTable. May trigger * an auto-rehash (see HashTableSetIdealRatio()). * EFFICIENCY: * O(1), assuming a good hash function and element-to-bucket ratio * ARGUMENTS: * hashTable - the HashTable to remove the key/value pair from * key - the key specifying the key/value pair to be removed * RETURNS: * \*--------------------------------------------------------------------------*/ void HashTableRemove(HashTable *hashTable, const void *key); /*--------------------------------------------------------------------------*\ * NAME: * HashTableRemoveAll() - removes all key/value pairs from a HashTable * DESCRIPTION: * Removes all key/value pairs from the specified HashTable. May trigger * an auto-rehash (see HashTableSetIdealRatio()). * EFFICIENCY: * O(n) * ARGUMENTS: * hashTable - the HashTable to remove all key/value pairs from * RETURNS: * \*--------------------------------------------------------------------------*/ void HashTableRemoveAll(HashTable *hashTable); /*--------------------------------------------------------------------------*\ * NAME: * HashTableIsEmpty() - determines if a HashTable is empty * DESCRIPTION: * Determines whether or not the specified HashTable contains any * key/value pairs. * EFFICIENCY: * O(1) * ARGUMENTS: * hashTable - the HashTable to check * RETURNS: * bool - whether or not the specified HashTable contains any * key/value pairs \*--------------------------------------------------------------------------*/ int HashTableIsEmpty(const HashTable *hashTable); /*--------------------------------------------------------------------------*\ * NAME: * HashTableSize() - returns the number of elements in a HashTable * DESCRIPTION: * Returns the number of key/value pairs that are present in the * specified HashTable. * EFFICIENCY: * O(1) * ARGUMENTS: * hashTable - the HashTable whose size is requested * RETURNS: * long - the number of key/value pairs that are present in * the specified HashTable \*--------------------------------------------------------------------------*/ srInt_64 HashTableSize(const HashTable *hashTable); /*--------------------------------------------------------------------------*\ * NAME: * HashTableGetNumBuckets() - returns the number of buckets in a HashTable * DESCRIPTION: * Returns the number of buckets that are in the specified HashTable. * This may change dynamically throughout the life of a HashTable if * automatic or manual rehashing is performed. * EFFICIENCY: * O(1) * ARGUMENTS: * hashTable - the HashTable whose number of buckets is requested * RETURNS: * long - the number of buckets that are in the specified * HashTable \*--------------------------------------------------------------------------*/ srInt_64 HashTableGetNumBuckets(const HashTable *hashTable); /*--------------------------------------------------------------------------*\ * NAME: * HashTableSetKeyComparisonFunction() * - specifies the function used to compare keys in a HashTable * DESCRIPTION: * Specifies the function used to compare keys in the specified * HashTable. The specified function should return zero if the two * keys are considered equal, and non-zero otherwise. The default * function is one that simply compares pointers. * ARGUMENTS: * hashTable - the HashTable whose key comparison function is being * specified * keycmp - a function which returns zero if the two arguments * passed to it are considered "equal" keys and non-zero * otherwise * RETURNS: * \*--------------------------------------------------------------------------*/ void HashTableSetKeyComparisonFunction(HashTable *hashTable, int (*keycmp)(const void *key1, const void *key2)); /*--------------------------------------------------------------------------*\ * NAME: * HashTableSetValueComparisonFunction() * - specifies the function used to compare values in a HashTable * DESCRIPTION: * Specifies the function used to compare values in the specified * HashTable. The specified function should return zero if the two * values are considered equal, and non-zero otherwise. The default * function is one that simply compares pointers. * ARGUMENTS: * hashTable - the HashTable whose value comparison function is being * specified * valuecmp - a function which returns zero if the two arguments * passed to it are considered "equal" values and non-zero * otherwise * RETURNS: * \*--------------------------------------------------------------------------*/ void HashTableSetValueComparisonFunction(HashTable *hashTable, int (*valuecmp)(const void *value1, const void *value2)); /*--------------------------------------------------------------------------*\ * NAME: * HashTableSetHashFunction() * - specifies the hash function used by a HashTable * DESCRIPTION: * Specifies the function used to determine the hash value for a key * in the specified HashTable (before modulation). An ideal hash * function is one which is easy to compute and approximates a * "random" function. The default function is one that works * relatively well for pointers. If the HashTable keys are to be * strings (which is probably the case), then this default function * will not suffice, in which case consider using the provided * HashTableStringHashFunction() function. * ARGUMENTS: * hashTable - the HashTable whose hash function is being specified * hashFunction - a function which returns an appropriate hash code * for a given key * RETURNS: * \*--------------------------------------------------------------------------*/ void HashTableSetHashFunction(HashTable *hashTable, srUInt_64 (*hashFunction)(const void *key)); /*--------------------------------------------------------------------------*\ * NAME: * HashTableRehash() - reorganizes a HashTable to be more efficient * DESCRIPTION: * Reorganizes a HashTable to be more efficient. If a number of * buckets is specified, the HashTable is rehashed to that number of * buckets. If 0 is specified, the HashTable is rehashed to a number * of buckets which is automatically calculated to be a prime number * that achieves (as closely as possible) the ideal element-to-bucket * ratio specified by the HashTableSetIdealRatio() function. * EFFICIENCY: * O(n) * ARGUMENTS: * hashTable - the HashTable to be reorganized * numOfBuckets - the number of buckets to rehash the HashTable to. * Should be prime. Ideally, the number of buckets * should be between 1/5 and 1 times the expected * number of elements in the HashTable. Values much * more or less than this will result in wasted memory * or decreased performance respectively. If 0 is * specified, an appropriate number of buckets is * automatically calculated. * RETURNS: * \*--------------------------------------------------------------------------*/ void HashTableRehash(HashTable *hashTable, srInt_64 numOfBuckets); /*--------------------------------------------------------------------------*\ * NAME: * HashTableSetIdealRatio() * - sets the ideal element-to-bucket ratio of a HashTable * DESCRIPTION: * Sets the ideal element-to-bucket ratio, as well as the lower and * upper auto-rehash thresholds, of the specified HashTable. Note * that this function doesn't actually perform a rehash. * * The default values for these properties are 3.0, 0.0 and 15.0 * respectively. This is likely fine for most situations, so there * is probably no need to call this function. * ARGUMENTS: * hashTable - a HashTable * idealRatio - the ideal element-to-bucket ratio. When a rehash * occurs (either manually via a call to the * HashTableRehash() function or automatically due the * the triggering of one of the thresholds below), the * number of buckets in the HashTable will be * recalculated to be a prime number that achieves (as * closely as possible) this ideal ratio. Must be a * positive number. * lowerRehashThreshold * - the element-to-bucket ratio that is considered * unacceptably low (i.e., too few elements per bucket). * If the actual ratio falls below this number, a * rehash will automatically be performed. Must be * lower than the value of idealRatio. If no ratio * is considered unacceptably low, a value of 0.0 can * be specified. * upperRehashThreshold * - the element-to-bucket ratio that is considered * unacceptably high (i.e., too many elements per bucket). * If the actual ratio rises above this number, a * rehash will automatically be performed. Must be * higher than idealRatio. However, if no ratio * is considered unacceptably high, a value of 0.0 can * be specified. * RETURNS: * \*--------------------------------------------------------------------------*/ void HashTableSetIdealRatio(HashTable *hashTable, float idealRatio, float lowerRehashThreshold, float upperRehashThreshold); /*--------------------------------------------------------------------------*\ * NAME: * HashTableSetDeallocationFunctions() * - sets the key and value deallocation functions of a HashTable * DESCRIPTION: * Sets the key and value deallocation functions of the specified * HashTable. This determines what happens to a key or a value when it * is removed from the HashTable. If the deallocation function is NULL * (the default if this function is never called), its reference is * simply dropped and it is up to the calling program to perform the * proper memory management. If the deallocation function is non-NULL, * it is called to free the memory used by the object. E.g., for simple * objects, an appropriate deallocation function may be free(). * * This affects the behaviour of the HashTableDestroy(), HashTablePut(), * HashTableRemove() and HashTableRemoveAll() functions. * ARGUMENTS: * hashTable - a HashTable * keyDeallocator * - if non-NULL, the function to be called when a key is * removed from the HashTable. * valueDeallocator * - if non-NULL, the function to be called when a value is * removed from the HashTable. * RETURNS: * \*--------------------------------------------------------------------------*/ void HashTableSetDeallocationFunctions(HashTable *hashTable, void (*keyDeallocator)(void *key), void (*valueDeallocator)(void *value)); /*--------------------------------------------------------------------------*\ * NAME: * HashTableStringHashFunction() - a good hash function for strings * DESCRIPTION: * A hash function that is appropriate for hashing strings. Note that * this is not the default hash function. To make it the default hash * function, call HashTableSetHashFunction(HashTableStringHashFunction). * ARGUMENTS: * key - the key to be hashed * RETURNS: * long - the unmodulated hash value of the key \*--------------------------------------------------------------------------*/ srUInt_64 HashTableStringHashFunction(const void *key); void free_values_destroy(HashTable * tab); int HashTablePutReplaceEx(HashTable *hashTable, const void *key, void *value, int replace_key, int dealloc_key, int dealloc_value); ArrayList * HashTableSortedIndexes(HashTable * k2int_tab, int larger_value_first); // NOTE: values are all added by 1. HashTable * ArrayListToLookupTable_Int(ArrayList * arr); #endif /* _HASHTABLE_H */ Rsubread/src/index-builder.c0000644000175200017520000011302714136047615017044 0ustar00biocbuildbiocbuild/*************************************************************** The Subread and Rsubread software packages are free software packages: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #include #include #include #include #include #include #include #include #include "hashtable.h" #include "gene-value-index.h" #include "HelperFunctions.h" #include "gene-algorithms.h" #include "sorted-hashtable.h" #include "seek-zlib.h" #include "hashtable.h" #include "input-files.h" #define NO_GENE_DEBUG_ #define _GENE_DEBUG_SIZE_ 40000 #define MIN_READ_SPLICING 2000000 #define MAX_KEY_MATCH GENE_VOTE_SPACE int GENE_SLIDING_STEP = 3; int IS_COLOR_SPACE = 0; int MARK_NONINFORMATIVE_SUBREADS = 0; int IS_FORCED_ONE_BLOCK = 0; int ignore_bar_in_seqnames = 0; double begin00_ftime = 0.0; #define NEXT_READ 1 #define NEXT_FILE 2 #define FULL_PARTITION 4 long long estimate_memory_peak(unsigned int * bucket_sizes, unsigned int bucket_no, unsigned int tables ){ long long peakuse = 0; int tn; for(tn=0; tn 199) { SUBREADprintf("ERROR: There are too many FASTA files. You may merge them into one FASTA file.\n"); return -1; } if (strlen (index_prefix) > 290) { SUBREADprintf("ERROR: The path is too long. It should not be longer than 290 chars.\n"); return -1; } if(all_bases<1) { SUBREADprintf("ERROR: File '%s' is inaccessible.\n", chro_files[-all_bases-1]); return -1; } current_tab_items = 0; table_no = 0; int padding_around_contigs = MAX_READ_LENGTH; if(0 == for_measure_buckets){ if(gehash_create_ex(& table, expected_hash_items, 0, SUBINDEX_VER2, GENE_SLIDING_STEP, padding_around_contigs)) return 1; if(gvindex_init(&value_array_index, 0, (unsigned int) actual_bases))return 1; } if(1==for_measure_buckets){ *bucket_sizes = realloc(*bucket_sizes, sizeof(int) * (table_no+1) * bucket_no); memset((*bucket_sizes) + table_no * bucket_no, 0, sizeof(int) * bucket_no); memset(&table,0 , sizeof(gehash_t)); memset(&value_array_index, 0, sizeof(gene_value_index_t)); table.padding = padding_around_contigs; table.index_gap = GENE_SLIDING_STEP; } file_number = 0; offset = table.padding; read_no = 0; char * fn = malloc(3100); memset(read_offsets,0, chro_table_maxsize*sizeof(int)); sprintf(fn, "%s.files", index_prefix); unlink(fn); status = NEXT_FILE; if(1==for_measure_buckets) print_in_box(80,0,0,"Estimate the index size..."); else print_in_box(80,0,0,"Build the index..."); { char window [16], last_color_base=-1, last_last_color_base=-1; int i, read_len = 0; unsigned int int_key = 0, array_int_key = 0; int skips=0, all_skips = 0; //Pre-fill while(1) { //Subread Cycle char next_char; if (status == NEXT_FILE) { if(file_number == chro_file_number) { FILE * fp; geinput_close(ginp); //SUBREADprintf ("Processing chromosome files ...\n"); if(0==for_measure_buckets){ sprintf (fn, "%s.%02d.%c.tab", index_prefix, table_no, IS_COLOR_SPACE?'c':'b'); SUBREADfflush(stdout); if(!dump_res)dump_res |= gehash_dump(&table, fn); sprintf (fn, "%s.%02d.%c.array", index_prefix, table_no, IS_COLOR_SPACE?'c':'b'); if(!dump_res)dump_res |= gvindex_dump(&value_array_index, fn); gvindex_destory(&value_array_index) ; gehash_destory(&table); } read_offsets[read_no-1] = offset + table.padding; for(i=table_no+1; i<100; i++) { sprintf(fn, "%s.%02d.%c.tab", index_prefix, i, IS_COLOR_SPACE?'c':'b'); unlink(fn); sprintf(fn, "%s.%02d.%c.array", index_prefix, i, IS_COLOR_SPACE?'c':'b'); unlink(fn); } sprintf (fn, "%s.reads", index_prefix); fp = f_subr_open(fn, "wb"); for (i=0; i0){ read_offsets[read_no-1] = offset + table.padding; offset += 2*table.padding; } //printf("TTTXT FN=%s\n",fn); for(i=0;(fn[i+1] != ' ' && fn[i+1] != '\0' && fn[i+1] != '\t' && i filename, ftell(ginp -> input_fp)); fclose(fname_fp); for (i=0; i<16; i++) { char nch = geinput_next_char(ginp); if (nch == 'N') skips = 16; else if (skips>0) skips--; window[i] = nch; } read_len = 16; read_no ++; if(read_no >= chro_table_maxsize) { read_offsets = realloc(read_offsets, 2* chro_table_maxsize * sizeof(unsigned int)); read_names = realloc(read_names, 2* chro_table_maxsize * MAX_READ_NAME_LEN); chro_table_maxsize *= 2; } if(IS_COLOR_SPACE) { int_key = genekey2color('A',window); array_int_key = genekey2int(window,GENE_SPACE_BASE); last_last_color_base = -1; last_color_base = window[15]; } else array_int_key = int_key = genekey2int(window,GENE_SPACE_BASE); } if (status == FULL_PARTITION) { int seek_back_reads ; if (read_len < MIN_READ_SPLICING) { seek_back_reads = read_len; offset -= seek_back_reads; offset += 16; } else { seek_back_reads = MIN_READ_SPLICING - 10; seek_back_reads -= seek_back_reads%3; seek_back_reads += 1; offset -= seek_back_reads; offset += 16; } if(0==for_measure_buckets){ sprintf(fn, "%s.%02d.%c.tab", index_prefix, table_no, IS_COLOR_SPACE?'c':'b'); SUBREADfflush(stdout); if(!dump_res)dump_res |= gehash_dump(&table, fn); sprintf(fn, "%s.%02d.%c.array", index_prefix, table_no, IS_COLOR_SPACE?'c':'b'); if(!dump_res)dump_res |= gvindex_dump(&value_array_index, fn); } table_no ++; if(0==for_measure_buckets){ gehash_destory(&table); gvindex_destory(&value_array_index); } read_len -= seek_back_reads; read_len += 16; while(seek_back_reads) { fseek(ginp -> input_fp, -1, SEEK_CUR); char bnch = fgetc(ginp -> input_fp); if ((bnch >='A' && bnch <= 'Z' ) || (bnch >='a' && bnch <= 'z' ) || bnch == '-' || bnch == 'N' || bnch=='.')seek_back_reads--; fseek(ginp -> input_fp, -1, SEEK_CUR); } for (i=0; i<16; i++) { char nch = geinput_next_char(ginp); if (nch == 'N' ) skips = 16; else if (skips>0) skips--; window[i] = nch; } if(IS_COLOR_SPACE) { int_key = genekey2color('A',window); array_int_key = genekey2int(window,GENE_SPACE_BASE); last_last_color_base = -1; last_color_base = window[15]; } else array_int_key = int_key = genekey2int(window, GENE_SPACE_BASE); current_tab_items = 0; if(0==for_measure_buckets){ if(gehash_create_ex(&table, expected_hash_items, 0, SUBINDEX_VER2, GENE_SLIDING_STEP, padding_around_contigs)) return 1; if(gvindex_init(&value_array_index, offset, (unsigned int) actual_bases)) return 1; } if(1==for_measure_buckets){ //unsigned int * tmpp = *bucket_sizes; *bucket_sizes = realloc(*bucket_sizes, sizeof(int) * (table_no+1) * bucket_no); assert(NULL!= *bucket_sizes); memset((*bucket_sizes) + table_no * bucket_no, 0, sizeof(int) * bucket_no); } } status = 0; if(skips || (IS_COLOR_SPACE && last_last_color_base<0)) all_skips ++; else { int is_no_info = HashTableGet(huge_table, NULL+1+int_key)-NULL; if(is_no_info){ // SUBREADprintf("NOINFO KEY=%u AT %u\n", int_key, offset); }else { if(0 == for_measure_buckets)if(gehash_insert(&table, int_key, offset - (IS_COLOR_SPACE?1:0), (*bucket_sizes)+ table_no * bucket_no))return 1; if(1 == for_measure_buckets) gehash_try_insert_measure((*bucket_sizes)+ table_no * bucket_no , bucket_no, int_key); current_tab_items++; } if( 0 == for_measure_buckets ) gvindex_set(&value_array_index, offset - (IS_COLOR_SPACE?0:0), array_int_key, padding_around_contigs); } if((!IS_FORCED_ONE_BLOCK) && current_tab_items >= expected_hash_items && (read_len > MIN_READ_SPLICING || read_len < 32)) { status = FULL_PARTITION; continue; } for (i=0; i0){ skips--; last_color_base = -1; } int_key = int_key << 2; if (IS_COLOR_SPACE) { if(last_color_base>0) int_key += chars2color(last_color_base, next_char); array_int_key = array_int_key << 2; array_int_key += base2int (next_char); last_last_color_base = last_color_base; last_color_base = next_char; } else { int_key += base2int (next_char); array_int_key = int_key; } if(actual_bases > 12){ if (offset>0 && offset % (actual_bases / 12) == 0) { double finished_rate = offset*1.0/actual_bases; double base_per_second = offset / (miltime()-begin_ftime); double ETA = (actual_bases-offset) / base_per_second; print_build_log(finished_rate,base_per_second,ETA, actual_bases); SUBREADfflush(stdout) ; } } offset ++; read_len ++; if(offset > 0xFFFFFFFDU) { SUBREADprintf("ERROR: the provided reference sequences include more than 4 billion bases.\n") ; return -1; } } } } free(read_names); free(read_offsets); if(dump_res){ SUBREADprintf("No index was built.\n"); sprintf(fn, "%s.files", index_prefix); unlink(fn); sprintf(fn, "%s.reads", index_prefix); unlink(fn); int index_i; for(index_i = 0; index_i <= 99; index_i++){ sprintf(fn, "%s.%02d.b.tab", index_prefix, index_i); unlink(fn); sprintf(fn, "%s.%02d.c.tab", index_prefix, index_i); unlink(fn); sprintf(fn, "%s.%02d.b.array", index_prefix, index_i); unlink(fn); sprintf(fn, "%s.%02d.c.array", index_prefix, index_i); unlink(fn); } } free(fn); free(ginp); *total_tables = table_no+1; return 0; } int add_repeated_subread(gehash_t * tab , unsigned int subr, unsigned char ** huge_index) { int huge_byte = (subr>>1); int huge_offset = (subr % 2) * 4; int ind_id = (huge_byte >> 24) & 127; unsigned int byte_value = huge_index[ ind_id ][huge_byte&0xffffff] ; int huge_value = (byte_value>> huge_offset) & 0xf; if(huge_value <15){ huge_value ++; byte_value = (byte_value & (~(0xf << huge_offset))) | (huge_value << huge_offset); huge_index[ ind_id ][huge_byte&0xffffff] = (unsigned char)(byte_value&0xff); return 0; } unsigned int times = 0; int matched = gehash_get(tab, subr, ×, 1); if(matched) gehash_update(tab, subr, times+1); else if(gehash_insert(tab, subr, 16, NULL)) return 1; return 0; } int scan_gene_index(const char index_prefix [], char ** chro_files, int chro_file_number, int threshold, HashTable *huge_table, long long * actual_total_bases_inc_marging) { int file_number, i ,j; int status = NEXT_FILE; unsigned int offset, read_no; long long int guess_all_bases = guess_gene_bases(chro_files,chro_file_number); int padding_around_contigs = MAX_READ_LENGTH; *actual_total_bases_inc_marging = padding_around_contigs; gehash_t occurrence_table; unsigned char * huge_index[128]; for(i=0;i<128;i++) { huge_index[i] = calloc(1024*1024*16,1); if(NULL == huge_index[i]){ SUBREADprintf("ERROR: No memory can be allocated.\n"); return -1; } } if(gehash_create_ex(&occurrence_table, 500000, 0, SUBINDEX_VER0, 1, 0)) return 1; gene_input_t * ginp = malloc(sizeof(gene_input_t)); print_in_box(80,0,0,"Scan uninformative subreads in reference sequences ..."); if (chro_file_number > 199) { SUBREADprintf("ERROR: There are too many FASTA files. You may merge them into one FASTA file.\n"); return -1; } if (strlen (index_prefix) > 290) { SUBREADprintf("ERROR: The path is too long. It should not be longer than 290 chars.\n"); return -1; } if(guess_all_bases<1) { SUBREADprintf("ERROR: File '%s' is inaccessible.\n", chro_files[-guess_all_bases-1]); return -1; } file_number = 0; offset = 0; read_no = 0; char * fn = malloc(3100); sprintf(fn, "%s.files", index_prefix); unlink(fn); status = NEXT_FILE; { char window [16], last_color_base=-1, last_last_color_base=-1; int i, read_len = 0; unsigned int int_key = 0, array_int_key = 0; int skips=0, all_skips = 0; //Pre-fill while(1) { //Subread Cycle char next_char; //if(status > 0)SUBREADprintf("BDSSTATUS = %d\n", status); if (status == NEXT_FILE) { (*actual_total_bases_inc_marging)+=padding_around_contigs; if(file_number == chro_file_number) { geinput_close(ginp); break; } else { if (file_number) geinput_close(ginp); geinput_open(chro_files[file_number++], ginp); status = NEXT_READ; } } if (status == NEXT_READ) { (*actual_total_bases_inc_marging)+=2*padding_around_contigs; geinput_readline(ginp, fn, 0); //SUBREADprintf("HEADER_SCAN '''%s'''\n", fn); for (i=0; i<16; i++) { char nch = geinput_next_char(ginp); if (nch == 'N') skips = 16; else if (skips>0) skips--; window[i] = nch; } read_len = 16; read_no ++; if(IS_COLOR_SPACE) { int_key = genekey2color('A',window); array_int_key = genekey2int(window,GENE_SPACE_BASE); last_last_color_base = -1; last_color_base = window[15]; } else array_int_key = int_key = genekey2int(window,GENE_SPACE_BASE); } status = 0; if(skips || (IS_COLOR_SPACE && last_last_color_base<0)) all_skips ++; else { int rv = add_repeated_subread(&occurrence_table, int_key, huge_index); if(rv<0) { return -1; } } for (i=0; i0){ skips--; last_color_base = -1; } int_key = int_key << 2; if (IS_COLOR_SPACE) { if(last_color_base>0) int_key += chars2color(last_color_base, next_char); array_int_key = array_int_key << 2; array_int_key += base2int (next_char); last_last_color_base = last_color_base; last_color_base = next_char; } else { int_key += base2int (next_char); array_int_key = int_key; } offset ++; //if(offset % 100000 == 0) SUBREADprintf("SCANNEDOF %d BASES; LLBASE %d\n", offset, next_char); (*actual_total_bases_inc_marging)++; read_len ++; if(offset > 0xFFFFFFFDU) { SUBREADprintf("ERROR: the provided reference sequences include more than 4 billion bases.\n") ; return -1; } } } } free(fn); for (i=0; i current_items>=1) { for (j=0; j current_items; j++) { if(current_bucket -> item_values [j] > threshold) { HashTablePut(huge_table,NULL+1+ current_bucket -> item_keys[j], NULL+current_bucket -> item_values [j]); } } } } for(i=0;i<128;i++) if(huge_index[i]) free(huge_index[i]); free(ginp); gehash_destory(&occurrence_table); if(huge_table -> numOfElements) { print_in_box(80,0,0,"%llu uninformative subreads were found.", huge_table -> numOfElements); print_in_box(80,0,0,"These subreads were excluded from index building."); } return 0; } char *rtrim(char *s) { char* back = s + strlen(s); while(isspace(*--back)); *(back+1) = '\0'; return s; } typedef struct{ char * filename; unsigned int line_no; int byte_in_line; } format_check_context_t; int ERROR_FOUND_IN_FASTA = 0; void check_and_convert_warn(format_check_context_t * fcc, char * msg, FILE * log_fp){ ERROR_FOUND_IN_FASTA += 1; fprintf(log_fp, "A format issue is found in the %u-th line in file '%s': %s.\n", fcc->line_no, fcc->filename, msg); } void check_and_convert_warnOLD(char * FN, long long int fpos_line_head, unsigned line_no, int line_pos, char * msg, FILE * log_fp) { #define CHAR_ESC 27 int x1,brs=0; long long int back_search_ptr; char * line_buf = malloc(MAX_READ_LENGTH+1); ERROR_FOUND_IN_FASTA += 1; fprintf(log_fp,"\n"); //fprintf(log_fp,"%c[33m", CHAR_ESC); for(x1=0;x1<81;x1++) fprintf(log_fp,"="); fprintf(log_fp,"\n"); //fprintf(log_fp,"%c[32m", CHAR_ESC); fprintf(log_fp,"Input file '%s':\n", FN); //fprintf(log_fp,"%c[31m", CHAR_ESC); fprintf(log_fp,"%s\n", msg); //fprintf(log_fp,"%c[33m", CHAR_ESC); for(x1=0;x1<81;x1++) fprintf(log_fp,"."); fprintf(log_fp,"\n"); //fprintf(log_fp,"%c[37m", CHAR_ESC); FILE * warn_fp = f_subr_open(FN, "r"); for(back_search_ptr = fpos_line_head - 1; back_search_ptr>=0; back_search_ptr--) { int nch; fseeko(warn_fp, back_search_ptr, SEEK_SET); nch = fgetc(warn_fp); if(nch == '\n') brs++; if(brs >2) break; fseeko(warn_fp, back_search_ptr, SEEK_SET); } if(back_search_ptr<=0)brs++; int print_line_no = line_no - brs + 1; while(1) { char * ret = fgets(line_buf, MAX_READ_LENGTH, warn_fp); if(!ret)break; /* if(ftello(warn_fp) > fpos_line_head) fprintf(log_fp,"%c[9m%c[31m", CHAR_ESC, CHAR_ESC); else fprintf(log_fp,"%c[29m%c[37m", CHAR_ESC, CHAR_ESC); */ fprintf(log_fp," % 9d ", print_line_no++); rtrim(line_buf); fprintf(log_fp,"%s%s\n",line_buf,strlen(line_buf)<16?" ":""); if(ftello(warn_fp) > fpos_line_head) break; } for(x1=0;x1= MAX_READ_LENGTH-2){ SUBREADprintf("ERROR: A fasta file cannot have a line longer than 1000 bytes. You need to split a very long line into many lines.\n"); return -1; } line_no ++; fcc.line_no = line_no; int line_buf_len = strlen(line_buf); for(; line_buf[line_buf_len-1] == '\r' || line_buf[line_buf_len-1]=='\n' ;line_buf_len--) { if(line_buf[line_buf_len-1]=='\r') { if(!is_R_warnned) { is_R_warnned=1; fcc.byte_in_line = line_buf_len-1; check_and_convert_warn(&fcc ,"This line ends with '\\r\\n'. It is not a problem for building the index but we suggest to use Unix-styled line breaks.", log_fp); } } line_buf[line_buf_len-1] =0; } //if(strchr(line_buf,'>') || strchr(line_buf,'\r') || strchr(line_buf,'\n'))SUBREADprintf("TESTING: string contains '''%s''' at %I64u\n", line_buf, ftello(fafp.plain_fp)); if(line_buf_len<1) { fcc.byte_in_line = -1; check_and_convert_warn(&fcc ,"This line is empty. This is not allowed in the FASTA file.", log_fp); continue; } if(line_buf[0]=='>') { if(line_no>1 &&!is_head_written) { fcc.byte_in_line = 0; check_and_convert_warn(&fcc,"This sequence has less than 16 bases. It is ignored in the index because no subreads can be extracted.", log_fp); } is_head_written = 0; read_len = 0; read_head_buf[0]=0; strcat(read_head_buf, line_buf); strcat(read_head_buf, "\n"); int chro_name_end=1; while(line_buf[chro_name_end]) { if(line_buf[chro_name_end]==' '||line_buf[chro_name_end]=='|' || line_buf[chro_name_end]=='\t')break; chro_name_end++; } line_buf[chro_name_end] = 0; int is_exist = HashTableGet(rep_name_table , line_buf+1) - NULL; if(is_exist) { SUBREADprintf("ERROR: repeated chromosome name '%s' is observed in the FASTA file(s).\nThe index was NOT built.\n", line_buf+1); is_repeated_chro=1; break; } char * keymem = malloc(chro_name_end); strcpy(keymem, line_buf+1); HashTablePut(rep_name_table, keymem, NULL+1); } else if(line_no<1) { fcc.byte_in_line=0; check_and_convert_warn(&fcc ,"This file is not started with a header line. It seems not to be a FASTA file.", log_fp); } else { int xk2; for(xk2=0; xk2 < line_buf_len; xk2++) { int nextch = line_buf[xk2]; int lowerch = tolower(nextch); if(!( lowerch == 'a' || lowerch == 't' || lowerch == 'g' || lowerch == 'c' || nextch == '.' || nextch=='-' || lowerch=='n')) { fcc.byte_in_line=xk2; check_and_convert_warn(&fcc, "The non-ACGT base was converted to an 'A'.", log_fp); line_buf[xk2] = 'A'; } else if(nextch == '.' || nextch=='-' || lowerch=='n') line_buf[xk2] = 'A'; else line_buf[xk2] = toupper(nextch); } read_len += line_buf_len; if(read_len > 16 && !is_head_written) { fputs(read_head_buf, out_fp); written_chrs++; is_head_written = 1; chrom_lens_len++; if((chrom_lens_max_len-1) <= chrom_lens_len) { (*chrom_lens) = realloc((*chrom_lens), 2*chrom_lens_max_len*sizeof(unsigned int)); chrom_lens_max_len*=2; } } if(is_head_written) { int line_buf_len = strlen(line_buf); int writen_len = fprintf(out_fp,"%s\n", line_buf); if(writen_len < line_buf_len){ SUBREADprintf("ERROR: unable to write into the temporary file. Please check the free space of the output directory.\n"); is_disk_full = 1; break; } (*chrom_lens)[chrom_lens_len-1] = read_len; (*chrom_lens)[chrom_lens_len] = 0; } else { strcat(read_head_buf, line_buf); strcat(read_head_buf, "\n"); } } } autozip_close(&fafp); if(is_disk_full) break; } HashTableDestroy(rep_name_table); free(line_buf); free(read_head_buf); fclose(out_fp); if(!written_chrs){ SUBREADprintf("ERROR: No index was built because there were no subreads extracted. A chromosome needs at least 16 bases to be indexed."); return 1; } if(is_repeated_chro|| is_disk_full) return 1; if(ERROR_FOUND_IN_FASTA) { print_in_box( 80,0,0,"There were %d notes for reference sequences.", ERROR_FOUND_IN_FASTA); print_in_box( 89,0,0,"The notes can be found in the log file, %c[36m'%s'%c[0m.", CHAR_ESC, log_fn, CHAR_ESC); } else print_in_box( 80,0,0,"No format issues were found"); return 0; } char * tmp_file_for_signal; void SIGINT_hook(int param) { #ifdef MAKE_STANDALONE if(tmp_file_for_signal[0]) { unlink(tmp_file_for_signal); SUBREADprintf("\n\nReceived a terminal signal. The temporary file was removed. The index was NOT built successfully. Please DO NOT use the new index until they are rebuilt.\n\n"); } exit(param); #endif } static struct option ib_long_options[]={ {0, 0, 0, 0} }; #ifdef MAKE_STANDALONE int main(int argc,char ** argv) #else int main_buildindex(int argc,char ** argv) #endif { int threshold = 100, optindex=0; int memory_limit; // 8000 MBytes char output_file[MAX_FILE_NAME_LENGTH], c, tmp_fa_file[MAX_FILE_NAME_LENGTH], log_file_name[MAX_FILE_NAME_LENGTH+20]; char *ptr_tmp_fa_file[1]; unsigned int * chromosome_lengths; begin00_ftime = miltime(); if(sizeof(char *)>4) memory_limit=8000; else memory_limit=3000; ptr_tmp_fa_file[0]=tmp_fa_file; output_file[0] = 0; tmp_fa_file[0] = 0; tmp_file_for_signal = tmp_fa_file; IS_FORCED_ONE_BLOCK = 0; GENE_SLIDING_STEP = 3; IS_COLOR_SPACE = 0; SUBREADprintf("\n"); optind = 0; while (1){ c = getopt_long (argc, argv, "kvcBFM:o:f:Db?", ib_long_options, &optindex); if(c==-1) break; switch(c) { case 'b': ignore_bar_in_seqnames= 1; break; case 'B': IS_FORCED_ONE_BLOCK =1; break; case 'F': GENE_SLIDING_STEP =1; break; case 'v': core_version_number("Subread-buildindex"); return 0; case 'c': IS_COLOR_SPACE = 1; break; case 'M': memory_limit = atoi(optarg); break; case 'f': threshold = atoi(optarg); break; case 'o': strncpy(output_file, optarg, MAX_FILE_NAME_LENGTH-1); break; case 'k': if(memcmp(SUBREAD_VERSION , "1.3.",4)==0) { SUBREADprintf("The \"-k\" option is not supported in version 1.3.x. "); return -1; } MARK_NONINFORMATIVE_SUBREADS = 1; break; case '?': default : return -1; } } if (argc == optind || !output_file[0]) { SUBREADprintf("Version %s\n\n", SUBREAD_VERSION); /* SUBREADputs("Usage:"); SUBREADputs(""); SUBREADputs(" ./subread-buildindex [options] -o {FASTA file1} [FASTA file2] ..."); SUBREADputs(""); SUBREADputs("Required arguments:"); SUBREADputs(""); SUBREADputs(" -o base name of the index to be created"); SUBREADputs(""); SUBREADputs("Optional arguments:"); SUBREADputs(""); SUBREADputs(" -G build a gapped index for the reference genome. 16bp subreads"); SUBREADputs(""); SUBREADputs(" -M size of requested memory(RAM) in megabytes. Index is split into blocks if necessary."); SUBREADputs(""); SUBREADputs(" -f specify the threshold for removing uninformative subreads"); SUBREADputs(" (highly repetitive 16mers in the reference). 24 by default."); SUBREADputs(""); SUBREADputs(" -c build a color-space index."); SUBREADputs(""); SUBREADputs(" -v output version of the program."); SUBREADputs(""); SUBREADputs("For more information about these arguments, please refer to the User Manual.\n"); */ SUBREADputs("Usage:"); SUBREADputs(""); SUBREADputs(" ./subread-buildindex [options] -o {FASTA[.gz] file1}\\"); SUBREADputs(" [FASTA[.gz] file2] ..."); SUBREADputs(""); SUBREADputs("Required arguments:"); SUBREADputs(""); SUBREADputs(" -o base name of the index to be created"); SUBREADputs(""); SUBREADputs("Optional arguments:"); SUBREADputs(""); SUBREADputs(" -F build a full index for the reference genome. 16bp subreads"); SUBREADputs(" will be extracted from every position of the reference"); SUBREADputs(" genome. Size of the index is typically 3 times the size of"); SUBREADputs(" index built from using the default setting."); SUBREADputs(""); SUBREADputs(" -B create one block of index. The built index will not be split"); SUBREADputs(" into multiple pieces. This makes the largest amount of"); SUBREADputs(" memory be requested when running alignments, but it enables"); SUBREADputs(" the maximum mapping speed to be achieved. This option"); SUBREADputs(" overrides -M when it is provided as well."); SUBREADputs(""); SUBREADputs(" -M size of requested memory(RAM) in megabytes, 8000 by default."); SUBREADputs(""); SUBREADputs(" -f specify the threshold for removing uninformative subreads"); SUBREADputs(" (highly repetitive 16mers in the reference). 100 by default."); SUBREADputs(""); SUBREADputs(" -c build a color-space index."); SUBREADputs(""); SUBREADputs(" -v output version of the program."); SUBREADputs(""); SUBREADputs("For more information about these arguments, please refer to the User Manual.\n"); return -1 ; } if(threshold < 5) { SUBREADprintf("ERROR: The threshold of non-informative reads cannot be less than 5.\n"); return -1; } // ********************************************** // print config summary // ********************************************** print_subread_logo(); size_t free_mem=-1, total_mem=-1; if(get_free_total_mem( &total_mem, &free_mem )){ free_mem=-1; total_mem=-1; } SUBREADputs(""); print_in_box(80, 1, 1, "setting"); print_in_box(80, 0, 1, ""); print_in_box(80, 0, 0, " Index name : %s", get_short_fname(output_file)); print_in_box(80, 0, 0, " Index space : %s", IS_COLOR_SPACE?"color space":"base space"); if(IS_FORCED_ONE_BLOCK) { print_in_box(80, 0, 0, " Index split : no-split"); memory_limit = GENE_SLIDING_STEP==1?22000:11500; } else { if(memory_limit > 12000 && GENE_SLIDING_STEP>2) { print_in_box(80, 0, 0, " Memory : %u -> %u Mbytes", memory_limit, 12000); memory_limit = 12000; } else print_in_box(80, 0, 0, " Memory : %u Mbytes", memory_limit); } print_in_box(80, 0, 0, " Repeat threshold : %d repeats", threshold); print_in_box(80, 0, 0, " Gapped index : %s", GENE_SLIDING_STEP>1?"yes":"no"); print_in_box(80, 0, 0, ""); if(free_mem>0)print_in_box(80, 0, 0, " Free / total memory : %.1fGB / %.1fGB", free_mem*1./1024/1024/1024, total_mem*1./1024/1024/1024); print_in_box(80, 0, 0, ""); print_in_box(80, 0, 0, " Input files : %d file%s in total", argc - optind, (argc - optind>1)?"s":""); int x1; for(x1=0;x1< argc - optind; x1++) { char * fasta_fn = *(argv+optind+x1); int f_type = probe_file_type_fast(fasta_fn); char o_char = '?'; if(f_type == FILE_TYPE_FASTA){ o_char = 'o'; } if(f_type == FILE_TYPE_GZIP_FASTA){ o_char = 'o'; } print_in_box(94, 0, 0, " %c[32m%c%c[36m %s%c[0m", CHAR_ESC, o_char, CHAR_ESC, get_short_fname(fasta_fn) , CHAR_ESC); } print_in_box(80, 0, 0, ""); #ifndef __MINGW32__ if(free_mem>0 && free_mem < 3*1024ll*1024*1024){ print_in_box(80, 0, 0, ""); print_in_box( 80, 0, 0, " WARNING: the free memory is lower than 3.0GB." ); print_in_box( 80, 0, 0, " the program may run very slow or crash." ); print_in_box(80, 0, 0, ""); } #endif print_in_box(80, 2, 1, ""); SUBREADputs(""); print_in_box(80, 1, 1, "Running"); print_in_box(80, 0, 0, ""); for(x1=0;x1< argc - optind; x1++) { char * fasta_fn = *(argv+optind+x1); int f_type = probe_file_type_fast(fasta_fn); if(f_type != FILE_TYPE_GZIP_FASTA && f_type != FILE_TYPE_FASTA && f_type != FILE_TYPE_NONEXIST){ SUBREADprintf("ERROR: '%s' is not a Fasta file.\n", fasta_fn); STANDALONE_exit(-1); } } for(x1 = strlen(output_file); x1 >=0; x1--){ if(output_file[x1]=='/'){ memcpy(tmp_fa_file, output_file, x1); tmp_fa_file[x1]=0; break; } } if(tmp_fa_file[0]==0)strcpy(tmp_fa_file, "./"); #ifdef __MINGW32__ sprintf(tmp_fa_file+strlen(tmp_fa_file), "/subread-index-sam-%06u-%06d", getpid(),(int)(time(NULL) % 1000000)); #else sprintf(tmp_fa_file+strlen(tmp_fa_file), "/subread-index-sam-%06u-XXXXXX", getpid()); int tmpfdd = mkstemp(tmp_fa_file); if(tmpfdd == -1){ SUBREADprintf("ERROR: cannot create temp file\n"); return -1; } #endif sprintf(log_file_name, "%s.log", output_file); FILE * log_fp = f_subr_open(log_file_name,"wb"); signal (SIGTERM, SIGINT_hook); signal (SIGINT, SIGINT_hook); int ret = check_and_convert_FastA(argv+optind , argc - optind, tmp_fa_file, &chromosome_lengths, log_fp, log_file_name); if(log_fp) fclose(log_fp); if(!ret) { long long actual_bases=0; HashTable * huge_table; huge_table = HashTableCreate(200000); unsigned int expected_hash_items = (unsigned int)(memory_limit * 1024.0 / 8.) * 1024 ; unsigned int * bucket_sizes = NULL, total_tables=0; unsigned int bucket_no = calculate_buckets_by_size(expected_hash_items, SUBINDEX_VER2, 0, GENE_SLIDING_STEP); ret = ret || scan_gene_index(output_file, ptr_tmp_fa_file , 1, threshold, huge_table, &actual_bases); ret = ret || build_gene_index(output_file, ptr_tmp_fa_file , 1, threshold, huge_table, chromosome_lengths, actual_bases, 1, &bucket_sizes, expected_hash_items, bucket_no, &total_tables); long long estm_need_memory = estimate_memory_peak( bucket_sizes, bucket_no, total_tables ); long long needed_mem = estm_need_memory + huge_table->numOfElements * sizeof(KeyValuePair) + 560ll*1024*1024 + actual_bases / ((GENE_SLIDING_STEP > 1 ) ? 7:5); if(free_mem>0 && free_mem < needed_mem){ print_in_box(80, 0, 1, ""); print_in_box(80, 0, 1, "WARNING: available memory is lower than %.1f GB." ,needed_mem*1./1024l/1024/1024); print_in_box(80, 0, 1, " The program may run very slow."); print_in_box(80, 0, 1, "Build a gapped index and/or split index into blocks to reduce memory use."); print_in_box(80, 0, 1, ""); }else print_in_box(80, 0, 0, "%.1f GB of memory is needed for index building." ,needed_mem*1./1024l/1024/1024); ret = ret || build_gene_index(output_file, ptr_tmp_fa_file , 1, threshold, huge_table, chromosome_lengths, actual_bases, 0, &bucket_sizes, expected_hash_items, bucket_no, &total_tables); if(!ret){ print_in_box(80, 0, 1, "Total running time: %.1f minutes.", (miltime()-begin00_ftime)/60); print_in_box(89, 0, 1, "Index %c[36m%s%c[0m was successfully built.", CHAR_ESC, output_file, CHAR_ESC); } HashTableDestroy(huge_table); free(chromosome_lengths); free(bucket_sizes); } unlink(tmp_fa_file); tmp_fa_file[0]=0; print_in_box(80, 0, 0, ""); print_in_box(80, 2, 1, ""); SUBREADputs(""); return ret; } Rsubread/src/input-blc.c0000644000175200017520000015522214136047615016211 0ustar00biocbuildbiocbuild#include #include #include #include #include #include "subread.h" #include "HelperFunctions.h" #include "seek-zlib.h" #include "gene-algorithms.h" #include "input-files.h" #include "input-blc.h" #include "sambam-file.h" #ifdef __MINGW32__ #define SR_PATH_SPLIT_STR "\\" #else #define SR_PATH_SPLIT_STR "/" #endif struct iBLC_scan_t{ char out_format_string[MAX_FILE_NAME_LENGTH]; char filter_format_string[MAX_FILE_NAME_LENGTH]; int found_answer; int filter_is_gzipped; int bcl_is_gzipped; int reads_per_cluster; int read_lengths[INPUT_BLC_MAX_READS]; int read_is_index[INPUT_BLC_MAX_READS]; }; int iBLC_guess_scan(struct iBLC_scan_t * scancon, char * data_dir ){ DIR * this_level = opendir(data_dir); if(this_level == NULL) return -1; struct dirent *dp; int filter_found = 0, bcl_found = 0; char testfile_name[MAX_FILE_NAME_LENGTH]; while ((dp = readdir (this_level)) != NULL) { #ifdef __MINGW32__ if(1){ #else if(dp -> d_type == DT_DIR && dp->d_name[0]!='.'){ #endif strcpy(testfile_name,data_dir); strcat(testfile_name, SR_PATH_SPLIT_STR); strcat(testfile_name, dp->d_name); //SUBREADprintf("DIG: %s\n", testfile_name); #ifdef __MINGW32__ if(strcmp( dp->d_name, ".") && strcmp( dp->d_name,"..")) if(0==iBLC_guess_scan( scancon, testfile_name)) continue; #else if(iBLC_guess_scan( scancon, testfile_name))return -1; }else if(dp -> d_type == DT_REG){ #endif //SUBREADprintf( "%s %s %p %p\n" , data_dir, dp->d_name , strstr( dp->d_name , "0001.bcl." ) , strstr( dp->d_name , ".bci") ); if(0==strcmp(dp->d_name, "RunInfo.xml")){ if(scancon->reads_per_cluster > 0){ SUBREADprintf("ERROR: the root directory contains multiple scRNA data sets.\n"); return -1; } strcpy(testfile_name, data_dir); strcat(testfile_name, SR_PATH_SPLIT_STR); strcat(testfile_name, dp->d_name); FILE *fp = fopen(testfile_name,"rb"); if(NULL == fp){ SUBREADprintf("ERROR: cannot open the run info file: %s\n", testfile_name); } while(1){ char inbuf[MAX_READ_LENGTH]; if(!fgets( inbuf, MAX_READ_LENGTH-1, fp))break; if(strstr( inbuf, "my_index); if(my_index >0 && is_idx >=0 && rlen>0){ scancon -> read_lengths[my_index-1]=rlen; scancon -> read_is_index[my_index-1]=is_idx; scancon -> reads_per_cluster = max(scancon -> reads_per_cluster, my_index); }else assert( my_index >0 && is_idx >=0 && rlen>0 ); //SUBREADprintf("LOAD CLUSTER %d = %d\n", my_index, rlen); } } fclose(fp); if(scancon -> reads_per_cluster <1){ SUBREADprintf("ERROR: the format of RunInfo.xml is unknown\n"); return -1; } } if(0==memcmp(data_dir+ strlen(data_dir)-5, SR_PATH_SPLIT_STR "L001",5 ) && strstr( dp->d_name , "s_1.filter")){ autozip_fp tfp; strcpy(testfile_name, data_dir); strcat(testfile_name, SR_PATH_SPLIT_STR); strcat(testfile_name, dp->d_name); int resop = autozip_open(testfile_name, &tfp); if(0 <= resop){ autozip_close(&tfp); char * gen_fmt = str_replace(dp->d_name , "s_1.filter", "s_%d.filter"); char * gen_fmt2 = str_replace(data_dir , SR_PATH_SPLIT_STR "L001", SR_PATH_SPLIT_STR "L%03d"); strcpy(scancon -> filter_format_string, gen_fmt2); strcat(scancon -> filter_format_string, SR_PATH_SPLIT_STR); strcat(scancon -> filter_format_string, gen_fmt); free(gen_fmt2); free(gen_fmt); filter_found = resop + 1; } } if(0==memcmp(data_dir+ strlen(data_dir)-5, SR_PATH_SPLIT_STR "L001",5 ) && strstr( dp->d_name , "0001.bcl." ) && !strstr( dp->d_name , ".bci") ){ int tti; bcl_found = 1; char * gen_fmt = str_replace(dp->d_name , "0001.bcl.", "%04d.bcl."); for(tti = 0; tti<22; tti++){ strcpy(testfile_name, data_dir); strcat(testfile_name, SR_PATH_SPLIT_STR); sprintf(testfile_name+strlen(testfile_name), gen_fmt, 1, 2+tti); autozip_fp tfp; int resop = autozip_open(testfile_name, &tfp); //printf("%d === %s %s\n", resop, gen_fmt, testfile_name); if(0<=resop){ scancon -> bcl_is_gzipped = resop; autozip_close(&tfp); }else bcl_found=0; } if(bcl_found){ char * gen_fmt2 = str_replace(data_dir , SR_PATH_SPLIT_STR "L001", SR_PATH_SPLIT_STR "L%03d"); strcpy(scancon -> out_format_string, gen_fmt2); free(gen_fmt2); strcat(scancon -> out_format_string, SR_PATH_SPLIT_STR); strcat(scancon -> out_format_string, gen_fmt); } free(gen_fmt); } } } if(bcl_found && filter_found){ scancon -> found_answer = 1; scancon -> filter_is_gzipped = filter_found - 1; } closedir(this_level); return 0; } int iBLC_guess_format_string(char * data_dir, int * cluster_bases, char * format_string, char * filter_format, int * bcl_is_gzipped, int * filter_is_gzipped, int * read_lens, int * is_index){ struct iBLC_scan_t sct; memset(&sct, 0, sizeof(sct)); int tii = iBLC_guess_scan(&sct, data_dir); if(tii || ! sct.found_answer) return -1; strcpy(format_string, sct.out_format_string); strcpy(filter_format, sct.filter_format_string); *filter_is_gzipped = sct.filter_is_gzipped; *bcl_is_gzipped = sct.bcl_is_gzipped; *cluster_bases=0; for(tii=0; tiiis_EOF) return; if(NULL == blc_input -> bcl_gzip_fps && blc_input -> bcl_is_gzipped)return; if(NULL == blc_input -> bcl_fps && !blc_input -> bcl_is_gzipped)return; for(ii=0; ii < blc_input->total_bases_in_each_cluster; ii++){ if(blc_input -> bcl_is_gzipped){ seekgz_close(blc_input -> bcl_gzip_fps[ii]); free(blc_input -> bcl_gzip_fps[ii]); blc_input -> bcl_gzip_fps[ii] = NULL; }else{ fclose(blc_input -> bcl_fps[ii]); blc_input -> bcl_fps[ii] = NULL; } } if(blc_input -> filter_is_gzipped){ seekgz_close(blc_input -> filter_gzip_fp); free(blc_input -> filter_gzip_fp); blc_input -> filter_gzip_fp = NULL; }else{ fclose(blc_input -> filter_fp); blc_input -> filter_fp = NULL; } if( blc_input -> bcl_is_gzipped ){ free(blc_input -> bcl_gzip_fps); blc_input -> bcl_gzip_fps = NULL; }else{ free(blc_input -> bcl_fps); blc_input -> bcl_fps = NULL; } } int iBLC_open_batch(input_BLC_t * blc_input ){ char fname[MAX_FILE_NAME_LENGTH]; iBLC_close_batch(blc_input); int fii, xx; blc_input -> is_EOF=1; if(blc_input -> bcl_gzip_fps == NULL) blc_input -> bcl_gzip_fps = calloc( sizeof(void *), blc_input -> total_bases_in_each_cluster ); // for both FILE** and seekgz ** for(fii = 0; fii < blc_input -> total_bases_in_each_cluster; fii++){ sprintf(fname, blc_input -> bcl_format_string, blc_input -> current_lane, fii+1); if(blc_input -> bcl_is_gzipped){ blc_input -> bcl_gzip_fps[fii] = calloc( sizeof(seekable_zfile_t), 1); int rv = seekgz_open(fname, blc_input -> bcl_gzip_fps[fii], NULL); if(rv){ SUBREADprintf("ERROR: Unable to open %s\n", fname); return -1; } for(xx = 0; xx < 4; xx++) seekgz_next_int8(blc_input -> bcl_gzip_fps[fii]); // skip the first 32-b integer }else{ blc_input -> bcl_fps[fii] = fopen(fname, "rb"); if(NULL == blc_input -> bcl_fps[fii]){ SUBREADprintf("ERROR: Unable to open %s\n", fname); return -1; } for(xx = 0; xx < 4; xx++) fgetc(blc_input -> bcl_fps[fii]); // skip the first 32-b integer } } sprintf(fname, blc_input -> filter_format_string, blc_input -> current_lane,blc_input -> current_lane); if(blc_input -> filter_is_gzipped){ blc_input -> filter_gzip_fp = calloc( sizeof(seekable_zfile_t), 1); int rv = seekgz_open(fname, blc_input -> filter_gzip_fp, NULL); if(rv){ SUBREADprintf("ERROR: Unable to open %s\n", fname); return -1; } for(xx = 0; xx < 12; xx++) seekgz_next_int8(blc_input -> filter_gzip_fp); // skip the 12-byte header }else{ blc_input -> filter_fp = fopen(fname, "rb"); if(NULL == blc_input -> filter_fp){ SUBREADprintf("ERROR: Unable to open %s\n", fname); return -1; } for(xx = 0; xx < 12; xx++) fgetc(blc_input -> filter_fp); // skip the 12-byte header } blc_input -> is_EOF=0; return 0; } int iCache_open_batch( cache_BCL_t * cache_input){ cache_input -> bcl_gzip_fps = calloc(sizeof(autozip_fp), cache_input -> total_bases_in_each_cluster); return 0; } int cacheBCL_go_chunk_end( cache_BCL_t * cache_input ){ cache_input -> read_no_in_chunk= cache_input -> reads_available_in_chunk; return 0; } int cacheBCL_go_chunk_start( cache_BCL_t * cache_input ){ cache_input -> read_no_in_chunk=0; return 0; } void cacheBCL_close(cache_BCL_t * cache_input){ int x1; for(x1=0;x1 total_bases_in_each_cluster;x1++){ if( cache_input -> bcl_gzip_fps[x1]. plain_fp || cache_input -> bcl_gzip_fps[x1].gz_fp.gz_fp )autozip_close(&cache_input -> bcl_gzip_fps[x1]); free(cache_input -> bcl_bin_cache[x1]); } free(cache_input -> bcl_gzip_fps); if(cache_input -> filter_fp. plain_fp || cache_input -> filter_fp.gz_fp.gz_fp )autozip_close(&cache_input -> filter_fp); free(cache_input -> lane_no_in_chunk); free(cache_input -> flt_bin_cache); } int cacheBCL_init( cache_BCL_t * cache_input, char * data_dir, int reads_in_chunk, int all_threads ){ memset(cache_input, 0, sizeof( cache_BCL_t)); subread_init_lock(&cache_input -> read_lock); int rv = iBLC_guess_format_string(data_dir, &cache_input -> total_bases_in_each_cluster, cache_input -> bcl_format_string, cache_input -> filter_format_string, &cache_input -> bcl_is_gzipped, &cache_input -> filter_is_gzipped, cache_input -> single_read_lengths, cache_input -> single_read_is_index); if(rv) return -1; cache_input -> current_lane = 1; cache_input -> reads_per_chunk = reads_in_chunk; cache_input -> bcl_bin_cache = malloc(sizeof(char*) * cache_input -> total_bases_in_each_cluster); int x1; for(x1 = 0; x1 < cache_input -> total_bases_in_each_cluster; x1++) cache_input -> bcl_bin_cache[x1] = malloc(reads_in_chunk); cache_input -> flt_bin_cache = malloc(reads_in_chunk*2); cache_input -> flt_bin_cache_size = reads_in_chunk*2; cache_input -> lane_no_in_chunk = malloc(reads_in_chunk); cache_input -> chunk_end_lane = 1; // no "0th lane" cache_input -> all_threads = all_threads; return iCache_open_batch(cache_input)?1:0; } void iCache_close_one_fp( cache_BCL_t * cache_input, int bcl_no){ autozip_fp * tfp = bcl_no<0?&cache_input-> filter_fp :(cache_input -> bcl_gzip_fps + bcl_no); // SUBREADprintf("CLOSE_AUTO: %d\n", bcl_no); autozip_close(tfp); memset(tfp,0,sizeof(autozip_fp)); } int iCache_open_one_fp( cache_BCL_t * cache_input, int bcl_no, int lane_no){ autozip_fp * tfp = bcl_no<0?&cache_input-> filter_fp :(cache_input -> bcl_gzip_fps + bcl_no); char fname[MAX_FILE_NAME_LENGTH+1]; if(bcl_no <0) sprintf(fname, cache_input -> filter_format_string, lane_no, lane_no); else sprintf(fname, cache_input -> bcl_format_string, lane_no, bcl_no+1); int rv = autozip_open(fname, tfp); // SUBREADprintf("OPEN_AUTO %s = %d\n", fname, rv); if(rv>=0){ int sk = bcl_no<0?12:4; for(; sk>0; sk--){ autozip_getch(tfp); //SUBREADprintf("SKEP=%d\n", nch); } }else{ memset(tfp,0,sizeof(autozip_fp)); } return rv < 0; } // it returns the number of bytes loaded. 0 if no bytes are available. int iCache_continuous_read_lanes( cache_BCL_t * cache_input, int bcl_no){ autozip_fp * tfp = bcl_no<0?&cache_input-> filter_fp :(cache_input -> bcl_gzip_fps + bcl_no); int my_lane = cache_input -> chunk_start_lane ,wptr=0; char * wpt = bcl_no<0?cache_input-> flt_bin_cache:cache_input -> bcl_bin_cache[bcl_no]; int total_valid_reads = 0; int raw_ptr = 0; while(1){ if(!(tfp -> plain_fp || tfp -> gz_fp.gz_fp)){ int fpr = iCache_open_one_fp( cache_input, bcl_no, my_lane ); if(fpr){ if(bcl_no<0)cache_input -> last_chunk_in_cache = 1; break; } } while(1){ int nch = autozip_getch( tfp ); //if(bcl_no>=0)SUBREADprintf("NCH=%d\n", nch); if(nch>=0){ if( bcl_no < 0 || cache_input -> flt_bin_cache [raw_ptr] ){ if(bcl_no <0){ if(nch>0) cache_input -> lane_no_in_chunk[total_valid_reads ++] = my_lane; if(wptr == cache_input -> flt_bin_cache_size){ cache_input -> flt_bin_cache_size *= 1.6; wpt = cache_input-> flt_bin_cache = realloc( wpt, cache_input -> flt_bin_cache_size ); } }else total_valid_reads++; //if(wptr == 1000000)SUBREADprintf("BaseNo=%d; Nch=%d\n", bcl_no, nch); wpt[wptr++] = nch&0xff; if(total_valid_reads == cache_input -> reads_per_chunk) break; } }else{ // SUBREADprintf("CONTINUOUSLY [%d-th base] BREAK : %d\n", bcl_no, nch); break; } raw_ptr ++; } if(total_valid_reads == cache_input -> reads_per_chunk) break; iCache_close_one_fp(cache_input, bcl_no); my_lane++; } if(bcl_no <0){ cache_input -> reads_available_in_chunk = total_valid_reads; cache_input -> chunk_end_lane = my_lane; } //if(bcl_no<0) SUBREADprintf("CONTINUOUSLY [%d-th base] READ %d reads into the %d-th lanel fpntr = %ld\n", bcl_no, total_valid_reads, my_lane, tfp -> plain_fp ? ftello(tfp -> plain_fp ): -1); return total_valid_reads; } void * iCache_decompress_chunk_1T(void * arg){ cache_BCL_t * cache_input = arg; while (1){ int my_bcl_no ; subread_lock_occupy(&cache_input -> read_lock); for(my_bcl_no =0; my_bcl_no total_bases_in_each_cluster; my_bcl_no++){ if(!cache_input -> bcl_no_is_used[my_bcl_no]) { cache_input -> bcl_no_is_used[my_bcl_no]=1; break; } } subread_lock_release(&cache_input -> read_lock); if(my_bcl_no>= cache_input -> total_bases_in_each_cluster) return NULL; iCache_continuous_read_lanes( cache_input, my_bcl_no ); } return NULL; } int cacheBCL_next_chunk(cache_BCL_t * cache_input){ int x1; //SUBREADprintf("BCL: READ_CHUNK_NEXT ( %d )\n", cache_input -> chunk_no+1); cache_input -> chunk_start_lane = cache_input -> chunk_end_lane; memset(cache_input -> bcl_no_is_used, 0 , sizeof(int)* MAX_READ_LENGTH); pthread_t * threads = malloc(sizeof(pthread_t)*cache_input -> all_threads); iCache_continuous_read_lanes( cache_input, -1 ); // read filtering binary for(x1=0; x1 all_threads; x1++) pthread_create(threads+x1, NULL, iCache_decompress_chunk_1T, cache_input); for(x1=0; x1 all_threads; x1++) pthread_join(threads[x1],NULL); free(threads); cache_input -> read_no_in_chunk = 0; cache_input -> chunk_no ++; return 0; } int iCache_copy_read(cache_BCL_t * cache_input, char * read_name, char * seq, char * qual, srInt_64 rno){ int bii, idx_offset, base_offset; int * srii = cache_input -> single_read_lengths; #ifdef __MINGW32__ sprintf(read_name, "R%011I64u:", rno); #else sprintf(read_name, "R%011llu:", rno); #endif int is_dual_index = srii[3]>0; idx_offset = srii[0]; base_offset = srii[1] + ( is_dual_index?srii[2]:0) + idx_offset; read_name[13+idx_offset]='|'; read_name[14+2*idx_offset]='|'; read_name[15+base_offset+idx_offset]='|'; sprintf(read_name +16 +2*base_offset, "|@RgLater@L%03d" , cache_input -> lane_no_in_chunk[cache_input -> read_no_in_chunk]); for(bii = 0; bii < cache_input -> total_bases_in_each_cluster; bii++){ int nch = cache_input -> bcl_bin_cache[bii][cache_input -> read_no_in_chunk]; //if(rno == 1000000){ // SUBREADprintf("COPY: %d base NCH=%d\n", bii, nch); //} if(nch<0) nch+=256; int nbase = 'N'; int nqual = '#'; if(nch > 0){ nbase="ACGT"[nch%4]; nqual=33+(nch>>2); } if(nqual >= '/' && bii < base_offset ) nqual++; if(bii < srii[0]){ read_name[13+bii] = nbase; read_name[14+idx_offset+bii]= nqual; }else if(bii < base_offset ){ read_name[15+idx_offset+bii] = nbase; read_name[16+base_offset+bii]= nqual; }else{ seq[bii - base_offset ] = nbase; qual[bii - base_offset ] = nqual; } } cache_input -> read_no_in_chunk++; //SUBREADprintf("GOT READ #%d ; ret=%d\n" , cache_input -> read_no_in_chunk, srii[2]); return srii[2+is_dual_index]; } int cacheBCL_next_read(cache_BCL_t * cache_input, char * read_name, char * seq, char * qual, srInt_64 * read_number_in_all){ if(cache_input -> read_no_in_chunk >= cache_input -> reads_available_in_chunk){ if(cache_input -> last_chunk_in_cache) return 0; cacheBCL_next_chunk(cache_input); if(cache_input -> read_no_in_chunk >= cache_input -> reads_available_in_chunk) // no reads are loaded in the previous step return 0; } srInt_64 rnumb =(cache_input -> chunk_no -1)*1ll * cache_input -> reads_per_chunk +(cache_input -> read_no_in_chunk); if(read_number_in_all) *read_number_in_all = rnumb; return iCache_copy_read(cache_input, read_name, seq, qual, rnumb); } int input_BLC_init( input_BLC_t * blc_input , char * data_dir ){ memset(blc_input, 0, sizeof(input_BLC_t)); subread_init_lock(&blc_input -> read_lock); int rv = iBLC_guess_format_string(data_dir, &blc_input -> total_bases_in_each_cluster, blc_input -> bcl_format_string, blc_input -> filter_format_string, &blc_input -> bcl_is_gzipped, &blc_input -> filter_is_gzipped, blc_input -> single_read_lengths, blc_input -> single_read_is_index); if(rv) return -1; blc_input -> current_lane = 1; return iBLC_open_batch(blc_input)?1:0; } // load the next read W/O switch lane. // Return 0 if EOF, -1 if error or bases if loaded correctly. int iBLC_current_lane_next_read(input_BLC_t * blc_input, char * readname , char * read, char * qual){ int bii, idx_offset, base_offset; #ifdef __MINGW32__ sprintf(readname, "R%011I64u:", blc_input -> read_number +1); #else sprintf(readname, "R%011llu:", blc_input -> read_number +1); #endif { idx_offset = blc_input -> single_read_lengths[0]; base_offset = idx_offset + blc_input -> single_read_lengths[1]; } readname[13+idx_offset]='|'; readname[14+2*idx_offset]='|'; readname[15+base_offset+idx_offset]='|'; sprintf(readname +16 +2*base_offset, "|L%03d" , blc_input -> current_lane); while(1){ int fch = blc_input -> filter_is_gzipped? seekgz_next_int8(blc_input -> filter_gzip_fp) :fgetc(blc_input -> filter_fp); if(fch < 0) return 0; int baseii =0; for(bii =0; bii< blc_input -> total_bases_in_each_cluster; bii++){ int nch = blc_input -> bcl_is_gzipped?seekgz_next_int8(blc_input -> bcl_gzip_fps[bii]):fgetc(blc_input -> bcl_fps[bii]), bv, qv; if(fch == 1){ if(0==nch){ bv='N'; qv='#'; }else{ bv="ACGT"[nch%4]; qv=33+(nch>>2); if(qv >= '/') qv++; } if(bii < idx_offset){ assert(bv !=0 && qv !=0); readname[13+ bii]=bv; readname[14+ bii+idx_offset]=qv; }else if(bii < base_offset){ assert(bv !=0 && qv !=0); readname[15+ bii+ idx_offset]=bv; readname[16+ bii+ base_offset]=qv; }else{ read[baseii] = bv; qual[baseii] = qv; baseii++; } } } if(fch==1){ blc_input -> read_number ++; return baseii; } } } int iBLC_inc_lane(input_BLC_t * blc_input){ blc_input -> current_lane ++; return iBLC_open_batch(blc_input); // this function automatically closes BCL fps and FILTER fp. } // return : -1: error, 0: end of all files and all lanes, >0: actual read is loaded (return the read len). The read name is the combination of the short-end and the index-end. // NOTE: this only works with scRNA protocol!! int input_BLC_next_read(input_BLC_t * blc_input , char * readname, char * read, char * qual){ int nextlane, rrv=0; if(blc_input->is_EOF) return 0; subread_lock_occupy(&blc_input -> read_lock); for(nextlane = 0; nextlane <2; nextlane++){ int rv = iBLC_current_lane_next_read(blc_input, readname, read, qual); if(rv >0 || rv <0){ rrv = rv; break; } if(rv ==0 && nextlane){ rrv = 0; break; } if(nextlane>0){ rrv = -1; break; } rv = iBLC_inc_lane(blc_input); if(rv){ rrv = 0; break; } } subread_lock_release(&blc_input -> read_lock); return rrv; } int input_BLC_tell ( input_BLC_t * blc_input , input_BLC_pos_t * pos ){ int xx1; memset(pos,0, sizeof(*pos)); pos -> lane_id = blc_input -> current_lane; pos -> read_number = blc_input -> read_number; pos -> is_EOF = blc_input -> is_EOF; if(pos->is_EOF) return 0; if(blc_input->bcl_is_gzipped){ pos -> pos_of_bclgzs = calloc(sizeof(void *) , blc_input -> total_bases_in_each_cluster); for(xx1=0; xx1 total_bases_in_each_cluster; xx1++){ pos -> pos_of_bclgzs[xx1] = malloc(sizeof(seekable_position_t)); seekgz_tell(blc_input->bcl_gzip_fps[xx1], pos -> pos_of_bclgzs[xx1]); } }else{ pos -> pos_of_bcls = calloc(sizeof(srInt_64) , blc_input -> total_bases_in_each_cluster); for(xx1=0; xx1 total_bases_in_each_cluster; xx1++) pos -> pos_of_bcls[xx1] = ftello(blc_input->bcl_fps[xx1]); } if(blc_input->filter_is_gzipped){ pos -> pos_of_filtergz = malloc(sizeof(seekable_position_t)); seekgz_tell(blc_input->filter_gzip_fp, pos -> pos_of_filtergz); }else pos -> pos_of_filter = ftello(blc_input->filter_fp); return 0; } int input_BLC_seek( input_BLC_t * blc_input , input_BLC_pos_t * pos ){ int xx1; blc_input -> read_number = pos -> read_number; if(pos -> is_EOF){ iBLC_close_batch(blc_input); blc_input -> is_EOF = pos -> is_EOF; blc_input -> current_lane = pos -> lane_id; return 0; } if(pos -> lane_id != blc_input -> current_lane){ blc_input -> current_lane = pos -> lane_id; iBLC_open_batch(blc_input); } for(xx1=0; xx1 total_bases_in_each_cluster; xx1++) if(blc_input->bcl_is_gzipped) seekgz_seek(blc_input->bcl_gzip_fps[xx1], pos -> pos_of_bclgzs[xx1]); else fseeko(blc_input->bcl_fps[xx1], pos -> pos_of_bcls[xx1], SEEK_SET); if(blc_input->filter_is_gzipped) seekgz_seek(blc_input->filter_gzip_fp, pos -> pos_of_filtergz); else fseeko(blc_input->filter_fp, pos -> pos_of_filter, SEEK_SET); return 0; } void input_BLC_destroy_pos(input_BLC_t * blc_input , input_BLC_pos_t *pos){ int xx1; for(xx1=0; xx1 total_bases_in_each_cluster; xx1++){ if(blc_input->bcl_is_gzipped) free(pos -> pos_of_bclgzs[xx1]); } free((blc_input->bcl_is_gzipped? (void*)pos -> pos_of_bclgzs:(void*)pos -> pos_of_bcls)); } void input_BLC_close(input_BLC_t * blc_input){ iBLC_close_batch(blc_input); subread_destroy_lock(&blc_input -> read_lock); } void iBLC_free_sample_items(void * sample_arr){ ArrayList * ar = (ArrayList * ) sample_arr; ArrayListDestroy(ar); } void iBLC_free_3tp(void * t){ char **tt = (char**)t; free(tt[1]); free(tt); } #define SHEET_FORMAT_RAWDIR_INPUT 10 #define SHEET_FORMAT_FASTQ_INPUT 20 #define SHEET_FORMAT_BAM_INPUT 30 HashTable * input_BLC_parse_SampleSheet(char * fname){ HashTable * ret = StringTableCreate(30); HashTableSetDeallocationFunctions(ret, free, iBLC_free_sample_items); FILE * fp = fopen(fname, "rb"); if(fp==NULL) return NULL; char linebuf[MAX_FILE_NAME_LENGTH]; int state = -1, file_format=-1, stat1_line = 0; while(!feof(fp)){ char * gret = fgets(linebuf, MAX_FILE_NAME_LENGTH-1, fp); if(gret == NULL) break; int linelen = strlen(linebuf); if(linelen<5)continue; if(linebuf[linelen -1] == '\r' || linebuf[linelen -1] == '\n') linebuf[linelen -1] =0; if(linebuf[linelen -2] == '\r' || linebuf[linelen -2] == '\n') linebuf[linelen -2] =0; if(state < 0 && strstr(linebuf,"EMFileVersion,4")) state = 0; if(state == 1 && linebuf[0]=='[') state = 99999; if(state == 1){ if(0==stat1_line){ if(strstr( linebuf, "Lane")){ file_format = SHEET_FORMAT_RAWDIR_INPUT; continue; } if(strstr( linebuf, "BAMFile")){ file_format = SHEET_FORMAT_BAM_INPUT; continue; } if(strstr( linebuf, "BarcodeUMIFile")){ file_format = SHEET_FORMAT_FASTQ_INPUT; continue; } } if(strlen(linebuf)>10)stat1_line ++; char * tokp=NULL; int lane_no = 1; char * sample_name = NULL; char * sample_index = NULL; if(file_format == SHEET_FORMAT_RAWDIR_INPUT){ lane_no = atoi(strtok_r(linebuf, ",", &tokp)); strtok_r(NULL, ",", &tokp); sample_name = strtok_r(NULL, ",", &tokp); sample_index = strdup(strtok_r(NULL, ",", &tokp)); }else if(file_format == SHEET_FORMAT_FASTQ_INPUT){ strtok_r(linebuf, ",", &tokp); // fastq file 1 strtok_r(NULL, ",", &tokp); // fastq file 2 sample_name = strtok_r(NULL, ",", &tokp); // sample name }else{ strtok_r(linebuf, ",", &tokp); // bam file sample_name = strtok_r(NULL, ",", &tokp); // sample name } char ** entry = malloc(sizeof(void*)*3); entry[0] = NULL + lane_no; entry[1] = sample_index; entry[2] = NULL + stat1_line; // the N-th line in the file , 1 based ArrayList * arr = HashTableGet(ret, sample_name); if(NULL == arr){ arr = ArrayListCreate(16); ArrayListSetDeallocationFunction(arr, iBLC_free_3tp); HashTablePut( ret, strdup(sample_name), arr ); } //SUBREADprintf("PUT_SAMPLE=%s of lane_no %d\n", sample_name, lane_no); ArrayListPush(arr,entry); } if(state == 0 && strstr(linebuf,"ata]")){ state = 1; stat1_line = 0; } } if(state <1){ SUBREADprintf("ERROR: the sample sheet doesn't contain any sample.\n"); return NULL; } return ret; } ArrayList * input_BLC_parse_CellBarcodes(char * fname){ autozip_fp fp; int resop = autozip_open(fname, &fp); if(resop<0) return NULL; ArrayList * ret = ArrayListCreate(10000000); ArrayListSetDeallocationFunction(ret, free); while(1){ char tmp_fl[MAX_BARCODE_LEN+1]; int skr = autozip_gets(&fp, tmp_fl, MAX_BARCODE_LEN); if(skr<1) break; //if(ret -> numOfElements <40) SUBREADprintf("LOAD_CELL_BAR : %s , %d\n", tmp_fl, skr); int x1; if(tmp_fl[skr-1]=='\n') tmp_fl[skr-1]=0; for(x1=0; tmp_fl[x1]; x1++) if(!isalpha(tmp_fl[x1])){ tmp_fl[x1]=0; break; } ArrayListPush(ret, strdup(tmp_fl)); } autozip_close(&fp); return ret; } int is_ATGC(char c){ return c=='A'||c=='C'||c=='G'||c=='T'||c=='N'; } int hamming_dist_ATGC_max3(char* s1, char* s2 ){ int xx,ret=0; for(xx=0;;xx++){ char nch1 = s1[xx]; char nch2 = s2[xx]; if(is_ATGC(nch1) && is_ATGC(nch2)){ ret += nch1==nch2; if(xx -ret >3) return 999; }else break; } return xx-ret; } int hamming_dist_ATGC_max1_2p(char* s1, char* s2 ){ int xx,sl=0; while(is_ATGC(s1[sl]) && is_ATGC(s2[sl])) sl++; sl /=2; int p1_mm=0, p2_mm=0; for(xx=0;;xx++){ char nch1 = s1[xx]; char nch2 = s2[xx]; if(is_ATGC(nch1) && is_ATGC(nch2)){ if(nch1!=nch2){ if(xx1 || p2_mm>1 ) return 999; return p1_mm+p2_mm; } int hamming_dist_ATGC_max1(char* s1, char* s2 ){ int xx,ret=0; for(xx=0;;xx++){ char nch1 = s1[xx]; char nch2 = s2[xx]; if(is_ATGC(nch1) && is_ATGC(nch2)){ ret += nch1==nch2; if(xx -ret >1) return 999; }else break; } return xx-ret; } int hamming_dist_ATGC_max2(char* s1, char* s2 ){ int xx,ret=0; for(xx=0;;xx++){ char nch1 = s1[xx]; char nch2 = s2[xx]; if(is_ATGC(nch1) && is_ATGC(nch2)){ ret += nch1==nch2; if(xx -ret >2) return 999; }else break; } return xx-ret; } int iCache_get_sample_id(ArrayList * sample_sheet_list, char * sbc, int read_laneno){ int x1; //SUBREADprintf("TOTAL_SBC=%ld\n", global_context -> scRNA_sample_barcode_list -> numOfElements); for(x1=0; x1 < sample_sheet_list -> numOfElements ; x1++ ){ char ** lane_and_barcode = ArrayListGet(sample_sheet_list, x1); int lane_no = lane_and_barcode[0]-(char*)NULL; if(read_laneno == lane_no){ int sample_no = lane_and_barcode[1]-(char*)NULL; char * knownbar = lane_and_barcode[2]; int hd = hamming_dist_ATGC_max2( sbc, knownbar ); if(hd<=2) return sample_no; } } return -1; } void iCache_copy_sample_table_2_list(void* ky, void* va, HashTable* tab){ ArrayList * cbclist = tab -> appendix1; ArrayList * hashed_arr = va ; srInt_64 xx1; for(xx1 =0; xx1< hashed_arr -> numOfElements; xx1++){ char ** push_arr = malloc(sizeof(char*)*3); char ** sbc_lane_sample = ArrayListGet(hashed_arr, xx1); srInt_64 lane_sample_int = sbc_lane_sample[0]-(char*)NULL; ArrayListPush(cbclist, push_arr); push_arr[0] = NULL + lane_sample_int; push_arr[1] = NULL + cbclist -> numOfElements+1; push_arr[2] = sbc_lane_sample[1]; // Sample Barcode } } #define IMPOSSIBLE_MEMORY_ADDRESS 0x5CAFEBABE0000000 int iCache_get_cell_no(HashTable * cell_barcode_table, ArrayList * cell_barcode_list, char * cell_barcode, int cell_barcode_length){ char tmpc [MAX_READ_NAME_LEN]; int xx1; ArrayList * ret=NULL; for(xx1=0;xx1<3;xx1++){ int xx2; if(xx1==1) ret = ArrayListCreate(100); if(xx1>0){ tmpc[0] = (xx1==2)?'S':'F'; for(xx2=0; xx2numOfElements; xx2++){ int bcno = ArrayListGet(rawarr, xx2)-NULL; found=0; for(xx3=0;xx3 numOfElements;xx3++){ if(ArrayListGet(ret, xx3)==NULL+bcno){ found=1; break; } } if(!found)ArrayListPush(ret, NULL+bcno); } } } } int tb1=-1; for(xx1=0; xx1 numOfElements; xx1++){ int tbcn = ArrayListGet(ret,xx1)-NULL; char * known_cbc = ArrayListGet(cell_barcode_list, tbcn); int hc = hamming_dist_ATGC_max2( known_cbc, cell_barcode ); // cbc[16]=0; if(hc <=3)SUBREADprintf("TEST_CBC %s ~ %s = %d\n", known_cbc, cbc, hc); if(hc==1){ tb1 = tbcn; break; } } //SUBREADprintf("CANDIDATE CELL BARCODES=%ld ; hit = %d\n", ret->numOfElements, tb1); ArrayListDestroy(ret); return tb1; } void iCache_delete_bcb_key(void *k){ if(((k-NULL) & 0xFFFFFFFFF0000000llu)!= IMPOSSIBLE_MEMORY_ADDRESS) ArrayListDestroy(k); } int cacheBCL_quality_test(int input_mode, char * datadir, HashTable * sample_sheet_table, ArrayList * cell_barcode_list, int testing_reads, int * tested_reads, int * valid_sample_index, int * valid_cell_barcode); #ifdef MAKE_TEST_QUALITY_TEST int main(int argc,char ** argv){ #else int do_R_try_cell_barcode_files(int argc, char ** argv){ #endif assert(argc>=6); int input_mode = GENE_INPUT_BCL; if(strcmp("fastq", argv[5])==0) input_mode = GENE_INPUT_SCRNA_FASTQ; if(strcmp("bam", argv[5])==0) input_mode = GENE_INPUT_SCRNA_BAM; int testing_reads = atoi(argv[4]); ArrayList * cell_barcode_list = input_BLC_parse_CellBarcodes(argv[3]); SUBREADprintf("Loaded %lld cell barcodes from %s\n", cell_barcode_list -> numOfElements, argv[3]); HashTable * sample_sheet = NULL; if(input_mode==GENE_INPUT_BCL) input_BLC_parse_SampleSheet(argv[2]); char * input_BCL_raw_dir = argv[1]; int tested_reads=0, valid_sample_index=0, valid_cell_barcode=0; int rv = cacheBCL_quality_test(input_mode, input_BCL_raw_dir, sample_sheet, cell_barcode_list, testing_reads, &tested_reads, &valid_sample_index, &valid_cell_barcode ); #ifdef MAKE_TEST_QUALITY_TEST printf("Samples = %ld loaded\n", sample_sheet -> numOfElements); printf("Cell barcodes = %ld loaded\n", cell_barcode_list -> numOfElements); printf("QUALITY-TEST : rv=%d , tested = %d , good-sample = %d , good-cell = %d\n", rv, tested_reads, valid_sample_index, valid_cell_barcode); #else argv[6]=NULL+rv; argv[7]=NULL+tested_reads; argv[8]=NULL+valid_sample_index; argv[9]=NULL+valid_cell_barcode; #endif ArrayListDestroy(cell_barcode_list); return 0; } int cacheBCL_qualTest_BAMmode(char * datadir, int testing_reads, int known_cell_barcode_length, ArrayList * sample_sheet_list, ArrayList * cell_barcode_list, HashTable * cell_barcode_table, int * tested_reads, int * valid_sample_index, int * valid_cell_barcode){ input_scBAM_t * scBAM_input = malloc(sizeof(input_scBAM_t)); int ret = input_scBAM_init(scBAM_input, datadir); SUBREADprintf("cacheBCL_qualTest_RET_BAMmode = %d for %s\n", ret, datadir); if(ret)return ret; while(1){ char base[MAX_READ_LENGTH], qual[MAX_READ_LENGTH], rname[MAX_READ_NAME_LEN]; base[0]=qual[0]=rname[0]=0; ret = scBAM_next_read(scBAM_input, rname , base, qual); if(ret<=0)break; char *cell_barcode = NULL, *sample_barcode=NULL, *lane_str=NULL; int xx=0; char *testi; for(testi = rname+1; * testi; testi ++){ if( * testi=='|'){ xx++; if(xx == 1) { cell_barcode = testi +1; }else if(xx == 2) { sample_barcode = testi +1; }else if(xx == 4){ lane_str = testi+1; break; } } } int cell_no = iCache_get_cell_no(cell_barcode_table, cell_barcode_list, cell_barcode, known_cell_barcode_length); if(cell_no>0) (*valid_cell_barcode)++; (*tested_reads)++; if((*tested_reads) >= testing_reads)break; } input_scBAM_close(scBAM_input); free(scBAM_input); return 0; } int cacheBCL_qualTest_FQmode(char * datadir, int testing_reads, int known_cell_barcode_length, ArrayList * sample_sheet_list, ArrayList * cell_barcode_list, HashTable * cell_barcode_table, int * tested_reads, int * valid_sample_index, int * valid_cell_barcode){ input_mFQ_t fqs_input; int ret = input_mFQ_init_by_one_string(&fqs_input, datadir); if(ret)return ret; while(1){ char base[MAX_READ_LENGTH], qual[MAX_READ_LENGTH], rname[MAX_READ_NAME_LEN]; base[0]=qual[0]=rname[0]=0; ret = input_mFQ_next_read(&fqs_input, rname , base, qual); if(ret<=0)break; char *cell_barcode = NULL; //SUBREADprintf("RETV=%d, RN=%s\n" ,ret, rname); int xx=0; char *testi; for(testi = rname+1; * testi; testi ++){ if( * testi=='|'){ xx++; if(xx == 1) { cell_barcode = testi+1; break; } } } int cell_no = iCache_get_cell_no(cell_barcode_table, cell_barcode_list, cell_barcode, known_cell_barcode_length); if(cell_no>0) (*valid_cell_barcode)++; (*tested_reads)++; if((*tested_reads) >= testing_reads)break; } input_mFQ_close(&fqs_input); return 0; } int cacheBCL_qualTest_BCLmode(char * datadir, int testing_reads, int known_cell_barcode_length, ArrayList * sample_sheet_list, ArrayList * cell_barcode_list, HashTable * cell_barcode_table, int * tested_reads, int * valid_sample_index, int * valid_cell_barcode){ cache_BCL_t blc_input; int orv = cacheBCL_init(&blc_input, datadir,testing_reads+1, 1); if(orv)return -1; while(1){ char base[MAX_READ_LENGTH], qual[MAX_READ_LENGTH], rname[MAX_READ_NAME_LEN]; srInt_64 readno = 0; base[0]=qual[0]=rname[0]=0; orv = cacheBCL_next_read(&blc_input, rname, base, qual, &readno); if(0==orv) break; char * testi, *lane_str = NULL, * sample_barcode = NULL, * cell_barcode, * umi_barcode; // cell_barcode MUST be 16-bp long, see https://community.10xgenomics.com/t5/Data-Sharing/Cell-barcode-and-UMI-with-linked-reads/td-p/68376 cell_barcode = rname + 13; umi_barcode = cell_barcode + known_cell_barcode_length; int xx=0, laneno=0; for(testi = umi_barcode+1; * testi; testi ++){ if( * testi=='|'){ xx++; if(xx == 2) { sample_barcode = testi +1; }else if(xx == 4){ lane_str = testi+1; break; } } } assert(xx ==4 && (*lane_str)=='L'); for(testi = lane_str+1; *testi; testi++){ assert(isdigit(*testi)); laneno = laneno*10 + (*testi)-'0'; } int sample_no = -1; if(sample_sheet_list->numOfElements)iCache_get_sample_id(sample_sheet_list, sample_barcode, laneno); int cell_no = iCache_get_cell_no(cell_barcode_table, cell_barcode_list, cell_barcode, known_cell_barcode_length); //printf("CELL_CALL %d = %s\n", cell_no, cell_barcode); if(sample_no>0) (*valid_sample_index)++; if(cell_no>0) (*valid_cell_barcode)++; (*tested_reads)++; if((*tested_reads) >= testing_reads)break; } cacheBCL_close(&blc_input); return 0; } int cacheBCL_quality_test(int input_mode, char * datadir, HashTable * sample_sheet_table, ArrayList * cell_barcode_list, int testing_reads, int * tested_reads, int * valid_sample_index, int * valid_cell_barcode){ ArrayList * sample_sheet_list = ArrayListCreate(100); ArrayListSetDeallocationFunction(sample_sheet_list, free); if(sample_sheet_table){ sample_sheet_table -> appendix1 = sample_sheet_list; HashTableIteration(sample_sheet_table, iCache_copy_sample_table_2_list); } HashTable * cell_barcode_table = StringTableCreate(1000000); HashTableSetDeallocationFunctions(cell_barcode_table, free, iCache_delete_bcb_key); char bctmp[MAX_READ_NAME_LEN]; int xx1,xx2, known_cell_barcode_length=-1; for(xx1=0; xx1< cell_barcode_list -> numOfElements ; xx1++){ char * bcbstr = ArrayListGet(cell_barcode_list, xx1); if(-1==known_cell_barcode_length) known_cell_barcode_length = strlen(bcbstr); else if(known_cell_barcode_length != strlen(bcbstr)){ SUBREADprintf("ERROR: the cell barcodes have different lengths (%d!=%ld at %d). The program cannot process the cell barcodes.\n", known_cell_barcode_length, strlen(bcbstr),xx1); return -1; } HashTablePut(cell_barcode_table, strdup(bcbstr), NULL+IMPOSSIBLE_MEMORY_ADDRESS+xx1); //if(cell_barcode_table -> numOfElements % 1000000 < 10)printf("size=%ld\n", cell_barcode_table -> numOfElements); for(xx2=0; xx2<2; xx2++){ bctmp[0] = xx2?'S':'F'; int xx3; for(xx3 = 0; xx3< known_cell_barcode_length/2; xx3++) bctmp[xx3+1] = bcbstr[ xx3*2+xx2 ]; bctmp[known_cell_barcode_length/2+1]=0; ArrayList * array_of_codes = HashTableGet(cell_barcode_table, bctmp); if(!array_of_codes){ array_of_codes = ArrayListCreate(4); HashTablePut(cell_barcode_table, strdup(bctmp), array_of_codes); } ArrayListPush(array_of_codes, NULL+xx1); } } if(known_cell_barcode_length<0){ SUBREADprintf("ERROR: cannot load any cell barcode from database\n"); return -1; } int ret = -1; if(input_mode == GENE_INPUT_SCRNA_FASTQ) ret=cacheBCL_qualTest_FQmode(datadir, testing_reads, known_cell_barcode_length, sample_sheet_list, cell_barcode_list, cell_barcode_table, tested_reads, valid_sample_index, valid_cell_barcode); else if(input_mode == GENE_INPUT_SCRNA_BAM) ret=cacheBCL_qualTest_BAMmode(datadir, testing_reads, known_cell_barcode_length, sample_sheet_list, cell_barcode_list, cell_barcode_table, tested_reads, valid_sample_index, valid_cell_barcode); else ret=cacheBCL_qualTest_BCLmode(datadir, testing_reads, known_cell_barcode_length, sample_sheet_list, cell_barcode_list, cell_barcode_table, tested_reads, valid_sample_index, valid_cell_barcode); ArrayListDestroy(sample_sheet_list); HashTableDestroy(cell_barcode_table); return ret; } #ifdef MAKE_TEST_SAMPLESHEET int main(int argc, char ** argv){ assert(argc>1); char * samplesheet = argv[1]; HashTable * st = input_BLC_parse_SampleSheet(samplesheet); printf("P=%p\n", st); } #endif #ifdef MAKE_TEST_ICACHE int main(int argc, char ** argv){ assert(argc>1); cache_BCL_t blc_input; int orv = cacheBCL_init(&blc_input, argv[1],5000000, 5), total_poses = 0; printf("orv=%d, bases=%d, filter_gzip=%d, data_gzip=%d\nBCL-pattern = %s\nFLT-pattern=%s\n", orv, blc_input.total_bases_in_each_cluster, blc_input.filter_is_gzipped, blc_input.bcl_is_gzipped, blc_input.bcl_format_string, blc_input.filter_format_string); while(1){ char base[1000], qual[1000], rname[200]; srInt_64 readno = 0; base[0]=qual[0]=rname[0]=0; orv = cacheBCL_next_read(&blc_input, rname, base, qual, &readno); assert(orv>=0); if(0==orv) break; if(1||readno%1000000==0)printf("%s %s %s\n", rname, base, qual); } cacheBCL_close(&blc_input); } #endif #ifdef MAKE_TEST_IBLC int main(int argc, char ** argv){ assert(argc>1); input_BLC_t blc_input; input_BLC_pos_t poses[12]; int orv = input_BLC_init(&blc_input, argv[1]), total_poses = 0; printf("orv=%d, bases=%d, filter_gzip=%d, data_gzip=%d\n", orv, blc_input.total_bases_in_each_cluster, blc_input.filter_is_gzipped, blc_input.bcl_is_gzipped); while(1){ char base[1000], qual[1000], rname[200]; base[0]=qual[0]=rname[0]=0; orv = input_BLC_next_read(&blc_input, rname, base, qual); assert(orv>=0); if(0==orv) break; if(blc_input.read_number%1000000==1)printf("%s %s %s\n", rname, base, qual); if(blc_input.read_number % 20000000 == 1){ input_BLC_tell(&blc_input, poses + total_poses); total_poses++; } if(blc_input.read_number > 180000010) break; } int ii; for(ii = 0; ii < total_poses; ii++){ srInt_64 jj; printf("\n\n========== SEEKING %d OF %d ================\n", ii,total_poses); input_BLC_seek( &blc_input, poses+ii ); input_BLC_destroy_pos( &blc_input, poses+ii ); for(jj = 0 ; jj < 3000011; jj++){ char base[1000], qual[1000], rname[200]; orv = input_BLC_next_read(&blc_input, rname, base, qual); assert(orv>=0); if(blc_input.read_number%1000000==2)printf("%s %s %s\n", rname, base, qual); } } printf("END CORRECTLY R=%llu\n", blc_input.read_number); input_BLC_close(&blc_input); return 0; } #endif void scBAM_tell(input_scBAM_t * bam_input, input_scBAM_pos_t * pos){ pos -> current_BAM_file_no = bam_input -> current_BAM_file_no; pos -> section_start_pos = bam_input -> section_start_pos; pos -> current_read_no = bam_input -> current_read_no; pos -> in_section_offset = bam_input -> in_section_offset; } int scBAM_next_char(input_scBAM_t * bam_input); int scBAM_next_string(input_scBAM_t * bam_input, char * strbuff, int lenstr); int scBAM_next_int(input_scBAM_t * bam_input, int *ret){ *ret = 0; int xk1; for(xk1=0;xk1<4;xk1++){ int nbyte = scBAM_next_char(bam_input); if(nbyte<0) return -1; (*ret) += nbyte <<(8*xk1); } return 0; } int scBAM_skip_bam_header(input_scBAM_t * bam_input){ int ret = 0, tmpi = 0, nref=0, x1; ret = scBAM_next_int(bam_input, &tmpi); if(ret < 0 || tmpi != 0x014d4142)return -1; // 0x014d4142 = 'BAM\1' little-endian scBAM_next_int(bam_input, &tmpi); // header txt length while(tmpi--) scBAM_next_char(bam_input); // skip header txt scBAM_next_int(bam_input, &nref); // n_ref bam_input -> chro_table = calloc(sizeof(SamBam_Reference_Info), nref); SUBREADprintf("OPEN '%s' : %d refs\n",bam_input -> BAM_file_names[bam_input -> current_BAM_file_no], nref); for(x1=0;x1 chro_table [x1].chro_name, tmpi); ret = scBAM_next_int(bam_input, (int*)&bam_input -> chro_table [x1].chro_length); if(ret < 0) return -1; } return 0; } int scBAM_inner_fopen(input_scBAM_t * bam_input){ bam_input -> os_file = f_subr_open(bam_input -> BAM_file_names[bam_input -> current_BAM_file_no], "rb"); if(!bam_input -> os_file)return -1; return scBAM_skip_bam_header(bam_input); } void scBAM_inner_fclose(input_scBAM_t * bam_input){ free(bam_input -> chro_table); fclose(bam_input -> os_file); } int scBAM_rebuffer(input_scBAM_t * bam_input){ int bin_len=0; while(1){ if(bam_input -> current_BAM_file_no== bam_input -> total_BAM_files) return -1; if(feof(bam_input -> os_file)){ scBAM_inner_fclose(bam_input); bam_input -> current_BAM_file_no++; if(bam_input -> current_BAM_file_no== bam_input -> total_BAM_files){ return -1; } scBAM_inner_fopen(bam_input); } char zipped_bam_buf[66000]; bam_input -> section_start_pos = ftello(bam_input -> os_file); int ziplen = PBam_get_next_zchunk(bam_input -> os_file, zipped_bam_buf, 66000, (unsigned int*)&bin_len); if(ziplen<1) return -1; bin_len = SamBam_unzip(bam_input -> section_buff, 65536, zipped_bam_buf, ziplen, 0); if(bin_len>0){ bam_input -> section_bin_bytes = bin_len; bam_input -> in_section_offset = 0; break; }else if(bin_len<0) return -1; } return bin_len; } // negative : EOF int scBAM_next_char(input_scBAM_t * bam_input){ if(bam_input -> current_BAM_file_no== bam_input -> total_BAM_files) return -1; if(bam_input -> in_section_offset == bam_input -> section_bin_bytes) if(0>scBAM_rebuffer(bam_input))return -1; int ret = bam_input -> section_buff[bam_input -> in_section_offset++]; if(ret<0)ret+=256; return ret; } void scBAM_seek(input_scBAM_t * bam_input, input_scBAM_pos_t * pos){ if(bam_input -> current_BAM_file_no!= pos -> current_BAM_file_no){ if(bam_input -> current_BAM_file_no < bam_input -> total_BAM_files) scBAM_inner_fclose(bam_input); bam_input -> current_BAM_file_no = pos -> current_BAM_file_no; if(bam_input -> current_BAM_file_no < bam_input -> total_BAM_files) scBAM_inner_fopen(bam_input); } if(bam_input -> current_BAM_file_no < bam_input -> total_BAM_files){ bam_input -> section_start_pos = pos -> section_start_pos; fseeko(bam_input -> os_file, bam_input -> section_start_pos, SEEK_SET); scBAM_rebuffer(bam_input); bam_input -> current_read_no = pos -> current_read_no; bam_input -> in_section_offset = pos -> in_section_offset; } } int scBAM_next_string(input_scBAM_t * bam_input, char * strbuff, int lenstr){ int x1=0; while(lenstr--){ int nbyte = scBAM_next_char(bam_input); if(nbyte<0) return -1; strbuff[x1++]=nbyte; } return x1; } // binbuf >= FC_LONG_READ_RECORD_HARDLIMIT // the alignment block with binlen will be written into binbuf. // negative : EOF int scBAM_next_alignment_bin(input_scBAM_t * bam_input, char * binbuf){ int reclen = 0; int ret = scBAM_next_int(bam_input, &reclen); if(ret<0)return -1; if(reclen < 36 || reclen > FC_LONG_READ_RECORD_HARDLIMIT -4)return -1; memcpy(binbuf, &reclen, 4); return scBAM_next_string(bam_input, binbuf +4, reclen); } int scBAM_next_read(input_scBAM_t * bam_input, char * read_name, char * seq, char * qual){ int ret = scBAM_next_alignment_bin(bam_input, bam_input -> align_buff); if(ret<0)return -1; int binlen=0, l_read_name = 0, flag=0, n_cigar_op=0, l_seq=0, x1; memcpy(&binlen, bam_input -> align_buff, 4); memcpy(&l_read_name, bam_input -> align_buff+12,1); memcpy(&n_cigar_op, bam_input -> align_buff+16,2); memcpy(&flag, bam_input -> align_buff+18,2); memcpy(&l_seq, bam_input -> align_buff+20,4); int rname_build_ptr = l_read_name-1; strcpy(read_name, bam_input -> align_buff+36); char * seq_start = bam_input -> align_buff+36+l_read_name+4*n_cigar_op; for(x1=0;x1> ( 4*!(x1%2) ) ) & 15]; char * qual_start = seq_start + (l_seq+1)/2; memcpy(qual, qual_start, l_seq); for(x1=0;x1 align_buff + 4 + binlen - extag_start, tagname, &tag_type, &tag_str_val); if(tag_type!='Z')return -1; int tag_str_len = strlen(tag_str_val); if(x1==0||x1==2||x1==4)read_name[rname_build_ptr++] = '|'; memcpy( read_name + rname_build_ptr, tag_str_val, tag_str_len ); rname_build_ptr += tag_str_len; } read_name[rname_build_ptr] = 0; return l_seq; } int input_scBAM_init(input_scBAM_t * bam_input, char * bam_fnames){ char * ubamnames = strdup(bam_fnames); char * tpl1 = NULL; char * fnl1 = strtokmm(ubamnames, SCRNA_FASTA_SPLIT1, &tpl1); int no_file = 0; memset(bam_input, 0, sizeof(input_scBAM_t)); while(fnl1){ bam_input -> BAM_file_names[no_file++] = strdup(fnl1); fnl1 = strtokmm(NULL, SCRNA_FASTA_SPLIT1, &tpl1); } bam_input -> total_BAM_files = no_file; free(ubamnames); return scBAM_inner_fopen(bam_input); } void input_scBAM_close(input_scBAM_t * bam_input){ int x1; for(x1=0; x1 total_BAM_files; x1++)free(bam_input ->BAM_file_names[x1]); if(bam_input -> current_BAM_file_no < bam_input -> total_BAM_files)scBAM_inner_fclose(bam_input); } void input_mFQ_fp_close(input_mFQ_t * fqs_input){ if(fqs_input -> autofp1.filename[0]){ autozip_close(&fqs_input -> autofp1); if(fqs_input->files2)autozip_close(&fqs_input -> autofp2); autozip_close(&fqs_input -> autofp3); } fqs_input -> autofp1.filename[0] = 0; } int input_mFQ_guess_lane_no(char * f1_name){ int fnamelen = strlen(f1_name); char * lst = strstr(f1_name + fnamelen - 24, "_L0"); if(lst){ int lno = 0; lno += lst[3]-'0'; lno = lno*10+( lst[4]-'0' ); } return 999; } int input_mFQ_open_files(input_mFQ_t * fqs_input){ fqs_input -> current_guessed_lane_no = input_mFQ_guess_lane_no(fqs_input->files1[fqs_input-> current_file_no]); int gzipped_ret = autozip_open(fqs_input->files1[fqs_input-> current_file_no],&fqs_input -> autofp1); if(fqs_input -> files2)gzipped_ret = gzipped_ret < 0 ?gzipped_ret:autozip_open(fqs_input->files2[fqs_input-> current_file_no],&fqs_input -> autofp2); gzipped_ret = gzipped_ret < 0?gzipped_ret:autozip_open(fqs_input->files3[fqs_input-> current_file_no],&fqs_input -> autofp3); return gzipped_ret < 0; } int input_mFQ_next_file(input_mFQ_t * fqs_input){ input_mFQ_fp_close(fqs_input); fqs_input-> current_file_no ++; if(fqs_input-> current_file_no >= fqs_input-> total_files)return -1; return input_mFQ_open_files(fqs_input); } int input_mFQ_init_by_one_string(input_mFQ_t * fqs_input, char * three_paired_fqnames){ int total_files = 0; char * fnames = strdup(three_paired_fqnames); char ** files1 = malloc(sizeof(char*) * MAX_SCRNA_FASTQ_FILES); char ** files2 = malloc(sizeof(char*) * MAX_SCRNA_FASTQ_FILES); char ** files3 = malloc(sizeof(char*) * MAX_SCRNA_FASTQ_FILES); char * tpl1 = NULL, * tpl2 = NULL; char * fnl1 = strtokmm(fnames, SCRNA_FASTA_SPLIT1, &tpl1); int no_file2 = 0; while(fnl1){ char * fnl2 = strtokmm(fnl1, SCRNA_FASTA_SPLIT2, &tpl2); files1[total_files] = fnl2; fnl2 = strtokmm(NULL, SCRNA_FASTA_SPLIT2, &tpl2); files2[total_files] = fnl2; no_file2 = no_file2 || strlen(fnl2)<2; fnl2 = strtokmm(NULL, SCRNA_FASTA_SPLIT2, &tpl2); files3[total_files] = fnl2; fnl1 = strtokmm(NULL, SCRNA_FASTA_SPLIT1, &tpl1); total_files++; } int rv = input_mFQ_init(fqs_input, files1, no_file2?NULL:files2, files3, total_files); free(fnames); free(files1); free(files2); free(files3); return rv; } int input_mFQ_init( input_mFQ_t * fqs_input, char ** files1, char ** files2, char** files3, int total_files ){ int x1; memset(fqs_input, 0, sizeof(input_mFQ_t)); fqs_input->files1 = malloc(sizeof(char*)*total_files); fqs_input->files2 = files2?malloc(sizeof(char*)*total_files):NULL; fqs_input->files3 = malloc(sizeof(char*)*total_files); fqs_input->total_files = total_files; for(x1=0; x1 total_files; x1++){ fqs_input->files1[x1] = strdup(files1[x1]); if(files2)fqs_input->files2[x1] = strdup(files2[x1]); fqs_input->files3[x1] = strdup(files3[x1]); } fqs_input->current_file_no = 0; fqs_input->current_read_no = 0; return input_mFQ_open_files(fqs_input); } int input_mFQ_next_read(input_mFQ_t * fqs_input, char * readname , char * read, char * qual ){ char tmpline [MAX_READ_NAME_LEN+1]; int ret = -1,x1; if(fqs_input -> current_file_no == fqs_input -> total_files) return -1; while(1){ ret = autozip_gets(&fqs_input -> autofp1, tmpline, MAX_READ_NAME_LEN); int write_ptr=0; if(ret==0){ ret = input_mFQ_next_file(fqs_input); if(ret >=0) continue; return -1; } else if(ret<0) return -1; #ifdef __MINGW32__ sprintf(readname, "R%011I64d", fqs_input -> current_read_no); #else sprintf(readname, "R%011lld", fqs_input -> current_read_no); #endif readname[12]='|'; ret = autozip_gets(&fqs_input -> autofp1, readname+13, MAX_READ_NAME_LEN); readname[13+ret-1]='|'; write_ptr = 13+ret; autozip_gets(&fqs_input -> autofp1, tmpline, MAX_READ_NAME_LEN); ret = autozip_gets(&fqs_input -> autofp1, readname+write_ptr, MAX_READ_NAME_LEN); ret --; for(x1=write_ptr; x1='/') readname[x1]++; readname[write_ptr+ret]='|'; write_ptr += ret; if(fqs_input->files2){ ret = autozip_gets(&fqs_input -> autofp2, tmpline, MAX_READ_NAME_LEN); if(ret<=0) return -1; ret = autozip_gets(&fqs_input -> autofp2, readname+write_ptr, MAX_READ_NAME_LEN); ret --; readname[write_ptr+ret]='|'; write_ptr += ret; autozip_gets(&fqs_input -> autofp2, tmpline, MAX_READ_NAME_LEN); ret = autozip_gets(&fqs_input -> autofp2, readname+write_ptr, MAX_READ_NAME_LEN); for(x1=write_ptr; x1='/') readname[x1]++; ret --; readname[write_ptr+ret]=0; }else write_ptr+=sprintf(readname+write_ptr,"|input#%04d@L%03d", fqs_input -> current_file_no, fqs_input -> current_guessed_lane_no); ret = autozip_gets(&fqs_input -> autofp3, tmpline, MAX_READ_NAME_LEN); if(ret<=0) return -1; ret = autozip_gets(&fqs_input -> autofp3, read, MAX_READ_LENGTH); ret --; // read length excludes "\n" read[ret]=0; autozip_gets(&fqs_input -> autofp3, tmpline, MAX_READ_NAME_LEN); autozip_gets(&fqs_input -> autofp3, qual, MAX_READ_LENGTH); qual[ret]=0; break; } fqs_input->current_read_no ++; return ret; } void input_mFQ_close(input_mFQ_t * fqs_input){ int x1; input_mFQ_fp_close(fqs_input); for(x1=0; x1 total_files; x1++){ free(fqs_input->files1[x1]); if(fqs_input->files2)free(fqs_input->files2[x1]); free(fqs_input->files3[x1]); } free(fqs_input->files1); if(fqs_input->files2)free(fqs_input->files2); free(fqs_input->files3); } int input_mFQ_seek(input_mFQ_t * fqs_input, input_mFQ_pos_t * pos ){ if(fqs_input -> current_file_no != pos -> current_file_no){ if(fqs_input -> current_file_no < fqs_input -> total_files)input_mFQ_fp_close(fqs_input); fqs_input -> current_file_no = pos -> current_file_no; if(fqs_input -> current_file_no < fqs_input -> total_files)input_mFQ_open_files(fqs_input); } if(fqs_input -> current_file_no < fqs_input -> total_files){ fqs_input -> current_read_no = pos -> current_read_no; if(fqs_input -> autofp1.is_plain){ fseeko(fqs_input -> autofp1.plain_fp, pos -> pos_file1, SEEK_SET); if(fqs_input -> files2)fseeko(fqs_input -> autofp2.plain_fp, pos -> pos_file2, SEEK_SET); fseeko(fqs_input -> autofp3.plain_fp, pos -> pos_file3, SEEK_SET); }else{ seekgz_seek(&fqs_input -> autofp1.gz_fp,&pos -> zpos_file1); if(fqs_input -> files2)seekgz_seek(&fqs_input -> autofp2.gz_fp,&pos -> zpos_file2); seekgz_seek(&fqs_input -> autofp3.gz_fp,&pos -> zpos_file3); } } return 0; } int input_mFQ_tell(input_mFQ_t * fqs_input, input_mFQ_pos_t * pos ){ memset(pos, 0, sizeof(input_mFQ_pos_t)); pos -> current_file_no = fqs_input -> current_file_no; pos -> current_read_no = fqs_input -> current_read_no; if(fqs_input -> current_file_no < fqs_input -> total_files){ if(fqs_input -> autofp1.is_plain){ pos -> pos_file1 = ftello(fqs_input -> autofp1.plain_fp); if(fqs_input -> files2)pos -> pos_file2 = ftello(fqs_input -> autofp2.plain_fp); pos -> pos_file3 = ftello(fqs_input -> autofp3.plain_fp); }else{ seekgz_tell(&fqs_input -> autofp1.gz_fp,&pos -> zpos_file1); if(fqs_input -> files2)seekgz_tell(&fqs_input -> autofp2.gz_fp,&pos -> zpos_file2); seekgz_tell(&fqs_input -> autofp3.gz_fp,&pos -> zpos_file3); } } return 0; } Rsubread/src/input-blc.h0000644000175200017520000000534014136047615016211 0ustar00biocbuildbiocbuild#ifndef _INPUT_BLC_H #define _INPUT_BLC_H #include #include #include "subread.h" #include "seek-zlib.h" #include "hashtable.h" int input_BLC_init( input_BLC_t * blc_input , char * data_dir ); int input_BLC_next_read( input_BLC_t * blc_input, char * readname , char * read, char * qual ); int input_BLC_tell ( input_BLC_t * blc_input , input_BLC_pos_t * pos ); int input_BLC_seek ( input_BLC_t * blc_input , input_BLC_pos_t * pos ); void input_BLC_close (input_BLC_t * blc_input); int input_scBAM_init(input_scBAM_t * bam_input, char * bam_fname); void input_scBAM_close(input_scBAM_t * bam_input); void scBAM_seek(input_scBAM_t * bam_input, input_scBAM_pos_t * pos); void scBAM_tell(input_scBAM_t * bam_input, input_scBAM_pos_t * pos); int scBAM_next_read(input_scBAM_t * bam_input, char * readname , char * read, char * qual ); int input_mFQ_init( input_mFQ_t * fqs_input, char ** files1, char ** files2, char** files3, int total_files ); int input_mFQ_init_by_one_string(input_mFQ_t * fqs_input, char * three_paired_fqnames); int input_mFQ_next_read(input_mFQ_t * fqs_input, char * readname , char * read, char * qual ); int input_mFQ_tell(input_mFQ_t * fqs_input, input_mFQ_pos_t * pos ); int input_mFQ_seek(input_mFQ_t * fqs_input, input_mFQ_pos_t * pos ); void input_mFQ_close(input_mFQ_t * fqs_input); // "cached BCL" maintains a chunk of reads; it decompresses int cacheBCL_init( cache_BCL_t * cache_input, char * data_dir, int reads_in_chunk, int all_threads ); int cacheBCL_next_read( cache_BCL_t * cache_input, char * read_name, char * seq, char * qual, srInt_64 * read_number_in_all); int cacheBCL_go_chunk_start( cache_BCL_t * blc_input ); int cacheBCL_go_chunk_end( cache_BCL_t * blc_input ); void cacheBCL_close ( cache_BCL_t * blc_input ); // it returns a hashtable : sample name -> [ ( "ACGTAATT", 1 ), ( "CGTTATGG", 2 ), ... ] // The hashtable can be simply destroyed and all contents are deallocated automatically // It returns NULL if no sample sheet is found HashTable * input_BLC_parse_SampleSheet(char * fname); // It returns a list of barcodes (not the barcode conversation table) // It returns NULL if no list is found // The arraylist can be simply destroyed and all contents are deallocated automatically ArrayList * input_BLC_parse_CellBarcodes(char * fname); int hamming_dist_ATGC_max1_2p(char* s1, char* s2 ); int hamming_dist_ATGC_max1(char* s1, char* s2 ); int hamming_dist_ATGC_max2(char* s1, char* s2 ); int hamming_dist_ATGC_max3(char* s1, char* s2 ); // returns -1 if error, or 0 if no error. int cacheBCL_quality_test(int is_FASTQ_input, char * datadir, HashTable * sample_sheet_table, ArrayList * cell_barcode_list, int testing_reads, int * tested_reads, int * valid_sample_index, int * valid_cell_barcode); #endif Rsubread/src/input-files.c0000644000175200017520000062710014141550405016542 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #include #include #include #include #include #include #include #ifndef __MINGW32__ #include #endif #include #include #include #include #include #include "input-files.h" #include "input-blc.h" #include "sambam-file.h" #include "HelperFunctions.h" #include "hashtable.h" #include "seek-zlib.h" #include "gene-algorithms.h" #include "sublog.h" unsigned int BASE_BLOCK_LENGTH = 15000000; FILE * f_subr_open(const char * fname, const char * mode) { #ifdef __MINGW32__ return fopen64(fname, mode); #else #if defined(__LP64__) || defined(_LP64) || defined(MACOS) return fopen(fname, mode); #else return fopen64(fname, mode); #endif #endif } void fastq_64_to_33(char * qs) { int i=0; while(qs[i]) qs[i++] -= 31; } void * delay_run(void * ptr){ usleep(100000); free(ptr); return NULL; } void * delay_realloc(void * old_pntr, size_t old_size, size_t new_size){ pthread_t thread; void * new_ret = malloc(new_size); memcpy(new_ret, old_pntr, old_size); pthread_create(&thread, NULL, delay_run, old_pntr); return new_ret; } // the caller is in charge of deallocation char * memstrcpy(char * in){ int ilen = strlen(in); char * ret = malloc(ilen+1); memcpy(ret, in, ilen); ret[ilen]=0; return ret; } double guess_reads_density(char * fname, int is_sam) { return guess_reads_density_format(fname, is_sam, NULL, NULL, NULL); } srInt_64 geinput_file_offset( gene_input_t * input){ if(input -> file_type == GENE_INPUT_GZIP_FASTQ || input -> file_type == GENE_INPUT_GZIP_FASTA){ if(((seekable_zfile_t*)input -> input_fp) -> blocks_in_chain<1)return 0; seekable_decompressed_block_t * ct = ((seekable_zfile_t*)input -> input_fp) -> block_rolling_chain+((seekable_zfile_t*)input -> input_fp) -> block_chain_current_no; return ct -> block_start_in_file_offset + ((seekable_zfile_t*)input -> input_fp) -> current_block_txt_read_ptr * 5/16; // compressed text ~= plain text * 28% }else{ return ftello((FILE*)input ->input_fp); } } double guess_reads_density_format(char * fname, int is_sam, int * min_phred_score, int * max_phred_score, int * tested_reads) { gene_input_t *ginp = malloc(sizeof(gene_input_t)); srInt_64 fpos =0, fpos2 = 0; int i; int max_qual_chr = -1, min_qual_chr = 127; char buff[MAX_READ_LENGTH] , qbuf[MAX_READ_LENGTH]; float retv = 0; if(is_sam == 0) { if(geinput_open(fname, ginp))retv= -1.0; }else if(is_sam == 1) { if(geinput_open_sam(fname, ginp,0))retv= -1.0; }else if(is_sam == 2) { if(geinput_open_sam(fname, ginp,1))retv= -1.0; } if(retv > -0.1){ geinput_next_read(ginp, NULL, buff, NULL); fpos = geinput_file_offset(ginp); for(i=0; i<3000; i++) { if(geinput_next_read(ginp, NULL, buff, qbuf)<0) break; if(qbuf[0]) { int xk=0; while(qbuf[xk]) { min_qual_chr = min(min_qual_chr,qbuf[xk]); max_qual_chr = max(max_qual_chr,qbuf[xk++]); } } if(tested_reads) (*tested_reads) ++; } if(min_phred_score) { (*min_phred_score) = min_qual_chr; (*max_phred_score) = max_qual_chr; } fpos2 = geinput_file_offset(ginp) - fpos; geinput_close(ginp); retv= fpos2*1.0/i; } free(ginp); return retv; } int is_gene_char(char c) { //if(c== 'M' || c == 'm' || c == 'U' || c == 'u' || c == 'A' || c=='a' || c=='G' || c=='g' || c=='C' || c=='c' || c=='T' || c=='t' || c=='N' || c=='n') if(c=='-' || c == '.' || c == 'N') return GENE_SPACE_BASE; if((c>='A' && c<'Z') || (c>='a' && c<='z')) return GENE_SPACE_BASE; if(c>='0' && c<'9') return GENE_SPACE_COLOR; return 0; } srInt_64 guess_gene_bases(char ** files, int file_number) { int i; srInt_64 ret = 0; for(i=0; i file_type == GENE_INPUT_GZIP_FASTQ? (seekgz_next_char((seekable_zfile_t*)input -> input_fp)):(fgetc((FILE*)input -> input_fp)) ) int geinput_preload_buffer(gene_input_t * input, subread_lock_t * read_lock){ if(input -> file_type == GENE_INPUT_GZIP_FASTQ) return seekgz_preload_buffer((seekable_zfile_t*)input -> input_fp, read_lock); return 0; } // read the line EXCLUDE last \n // returns bytes WITHOUT \n int read_line_noempty(int max_read_len, gene_input_t * input, char * buff, int must_upper) { int ret =0; if(input -> file_type == GENE_INPUT_GZIP_FASTQ || input -> file_type == GENE_INPUT_GZIP_FASTA){ seekgz_preload_buffer((seekable_zfile_t*)input -> input_fp, NULL); ret = seekgz_gets((seekable_zfile_t*)input->input_fp, buff, MAX_READ_LENGTH-1); if(ret > 0){ if(must_upper){ int ii; for(ii=0; ii input_fp, buff, conv_to_upper); } int is_read(char * in_buff) { int p=0; char c; int space_type = GENE_SPACE_BASE; while((c=in_buff[p++])!='\0') { if(c!='\r' && c!='\n'){ int x = is_gene_char(c); if (x == GENE_SPACE_COLOR) space_type = GENE_SPACE_COLOR; else if(!x) return 0; } } return space_type; } char *strtokmm(char *str, const char *delim, char ** next) { char *tok; char *m; if (delim == NULL) return NULL; tok = (str) ? str : (*next); if (tok == NULL) return NULL; m = strstr(tok, delim); if (m) { (*next) = m + strlen(delim); *m = '\0'; } else { (*next) = NULL; } return tok; } int geinput_open_scRNA_BAM(char * rfnames, gene_input_t * input, int reads_per_chunk, int threads ){ strcpy(input->filename,rfnames); int rv = input_scBAM_init(&input -> scBAM_input, rfnames); input -> file_type = GENE_INPUT_SCRNA_BAM; input -> space_type = GENE_SPACE_BASE; return rv; } int geinput_open_scRNA_fqs(char * rfnames, gene_input_t * input, int reads_per_chunk, int threads){ strcpy(input->filename,rfnames); int rv = input_mFQ_init_by_one_string(&input -> scRNA_fq_input, rfnames); input -> file_type = GENE_INPUT_SCRNA_FASTQ; input -> space_type = GENE_SPACE_BASE; return rv; } int geinput_open_bcl( const char * dir_name, gene_input_t * input, int reads_per_chunk, int threads){ int rv = cacheBCL_init(&input -> bcl_input , (char*) dir_name, reads_per_chunk, threads ); strcpy(input->filename, dir_name); if(rv) return -1; input -> file_type = GENE_INPUT_BCL; input -> space_type = GENE_SPACE_BASE; return 0; } int geinput_open_sam(const char * filename, gene_input_t * input, int half_number) { input->input_fp = f_subr_open(filename, "rb"); strcpy(input->filename, filename); if(input->input_fp == NULL) return 1; input -> file_type = half_number + GENE_INPUT_SAM_SINGLE; while(1){ char in_buff[3001]; srInt_64 current_pos = ftello(input -> input_fp); int rlen = read_line(3000, input->input_fp, in_buff, 0); if(rlen < 1) return 1; if(in_buff[0] != '@') { int x, tab_no = 0; char *read_buf=NULL; for(x=0; xspace_type = is_read(read_buf); if (GENE_INPUT_SAM_PAIR_2 != input -> file_type) fseeko(input -> input_fp , current_pos, SEEK_SET); input -> read_chunk_start = ftell(input -> input_fp); break; } } return 0; } int geinput_open(const char * filename, gene_input_t * input) { char in_buff[MAX_READ_LENGTH]; int line_no = 0, ret = 0; if(strlen(filename)>298) return 1; input -> gzfa_last_name[0]=0; strcpy(input->filename, filename); FILE * TMP_FP = f_subr_open(filename, "rb"); if(TMP_FP == NULL) return 1; int id1, id2; id1 = fgetc(TMP_FP); id2 = fgetc(TMP_FP); if(id1 == 31 && id2 == 139) { fclose(TMP_FP); input->input_fp = malloc(sizeof(seekable_zfile_t)); ret = seekgz_open(filename, input->input_fp, NULL ); if(ret == 0){ int fq_stat = 0; for(line_no = 0; line_no < 1000; line_no++){ int fl = seekgz_gets(input->input_fp, in_buff, 1000); if(fl < 1)break; // EOF else if(fl == 1)continue; // empty line else{ // text line if(line_no==0)input->file_type = in_buff[0]=='@'? GENE_INPUT_GZIP_FASTQ: GENE_INPUT_GZIP_FASTA; if(fq_stat%4 == 1) // read text { input->space_type = is_read(in_buff); break; } fq_stat ++; } } seekgz_close(input->input_fp); seekgz_open(filename, input->input_fp, NULL); } //SUBREADprintf("ZFAtest: type=%d\n", input->file_type); }else{ input->file_type = GENE_INPUT_FASTQ; input->input_fp = TMP_FP; fseeko(input->input_fp, 0, SEEK_SET); while (1){ srInt_64 last_pos = ftello(input->input_fp); int rlen = read_line_noempty(MAX_READ_LENGTH, input, in_buff, 0); if (rlen<=0){ ret = 1; break; }else{ if(line_no==0 && is_read(in_buff)) { input->file_type = GENE_INPUT_PLAIN; input->space_type = is_read(in_buff); fseeko(input->input_fp,last_pos,SEEK_SET); break; } if(in_buff[0]=='>') { input->file_type = GENE_INPUT_FASTA; // printf("FILE %s OPENED AS FATSA.\n", filename); rlen += read_line(MAX_READ_LENGTH, input->input_fp, in_buff, 0); input->space_type = is_read(in_buff); fseeko(input->input_fp,last_pos,SEEK_SET); break; } if(in_buff[0]=='@') { input->file_type = GENE_INPUT_FASTQ; rlen += read_line_noempty(MAX_READ_LENGTH, input, in_buff, 0); input->space_type = is_read(in_buff); fseeko(input->input_fp, last_pos,SEEK_SET); break; } line_no++; } } } input -> read_chunk_start = geinput_file_offset(input); if(0 == input->space_type)input->space_type = GENE_SPACE_BASE; return ret; } int geinput_next_char(gene_input_t * input) { if(input->file_type == GENE_INPUT_FASTA) { int last_br = 0; while (1) { char nch = fgetc((FILE *)input->input_fp); if ((nch <0) && feof((FILE *)input->input_fp)) return -2; else if (nch < 0 || nch > 126)SUBREADprintf("\nUnrecognised char = #%d\n", nch); if (nch == '\r') { #ifndef __MINGW32__ SUBREADprintf("The input FASTA file contains \\r characters. This should not result in any problem but we suggest to use UNIX-style line breaks.\n"); #endif last_br ++; continue; } if (nch == '\n') { last_br ++; continue; } if (nch == ' ' || nch == '\t') continue; if (nch == '>' && last_br) { // if this is a new segment fseeko(input->input_fp, -last_br , SEEK_CUR); return -1; } if (is_gene_char(nch)) return toupper(nch); else { srInt_64 fpos = ftello(input->input_fp); int back_search_len =2; int is_empty_seq = 0; char *out_buf = malloc(2000); while( fpos >= back_search_len ) { fseeko(input->input_fp, fpos - back_search_len, SEEK_SET); int bc_nch = fgetc(input->input_fp); //SUBREADprintf("SEEKINGBACK : %d : ch=%d '%c' ; bch=%d '%c'\n", back_search_len, nch, nch, bc_nch, bc_nch); if(bc_nch=='\n') { if(nch == '>' && back_search_len==2) is_empty_seq=1; break; } back_search_len++; } char * fgin = fgets(out_buf, 1999,input->input_fp); if(NULL == fgin) out_buf[0]=0; if(is_empty_seq) { if(strlen(out_buf)>0) out_buf[strlen(out_buf)-1]=0; SUBREADprintf ("\nEmpty chromosome sequence before '%s'. The file offset is %llu\n",out_buf, fpos); free(out_buf); return -1; } else { #ifdef __MINGW32__ SUBREADprintf ("\nUnknown character in the chromosome data: '%c' (ASCII:%02X), ignored. The file offset is %lu\n", nch, nch, fpos); #else SUBREADprintf ("\nUnknown character in the chromosome data: '%c' (ASCII:%02X), ignored. The file offset is %llu\n", nch, nch, fpos); #endif SUBREADprintf("%s", out_buf); for(; back_search_len>2; back_search_len--) SUBREADprintf(" "); SUBREADprintf("^\n"); fseeko(input->input_fp, fpos, SEEK_SET); free(out_buf); return 'N'; } } if(nch !='\r' && nch != '\n')last_br = 0; } } else { SUBREADprintf("Only the FASTA format is accepted for input chromosome data.\n"); return -3; } } int geinput_readline_back(gene_input_t * input, char * linebuffer_3000) { srInt_64 last_pos = ftello(input -> input_fp); int ret = read_line(3000, input->input_fp, linebuffer_3000, 0); if(ret<1) return -1; fseeko(input -> input_fp, last_pos, SEEK_SET); return ret; } #define SKIP_LINE { nch=' '; while(nch != EOF && nch != '\n') nch = geinput_getc(input); } #define SKIP_LINE_NOEMPTY {int content_line_l = 0; nch=' '; while(nch != EOF && (nch != '\n' ||! content_line_l)){nch = geinput_getc(input); content_line_l += (nch != '\n');} } //#define SKIP_LINE { nch=' '; while(nch != EOF && nch != '\n') nch = geinput_getc(input); } unsigned int read_numbers(gene_input_t * input) { unsigned int ret = 0; char nch; srInt_64 fpos = ftello(input->input_fp); if(input->file_type >= GENE_INPUT_SAM_SINGLE) { while(1) { nch = fgetc(input->input_fp); if(nch=='@') SKIP_LINE else break; } } while(1) { SKIP_LINE if(nch==EOF) break; ret ++; } fseeko(input->input_fp, fpos, SEEK_SET); if (input->file_type == GENE_INPUT_FASTQ) return ret/4; if (input->file_type == GENE_INPUT_FASTA) return ret/2; return ret; } void geinput_tell(gene_input_t * input, gene_inputfile_position_t * pos){ if(input -> file_type == GENE_INPUT_SCRNA_BAM){ scBAM_tell(&input -> scBAM_input, &pos -> scBAM_position); }else if(input -> file_type == GENE_INPUT_SCRNA_FASTQ){ input_mFQ_tell(&input -> scRNA_fq_input, &pos -> mFQ_position); }else if(input -> file_type == GENE_INPUT_BCL){ assert(input -> file_type != GENE_INPUT_BCL); }else if(input -> file_type == GENE_INPUT_GZIP_FASTQ || input -> file_type == GENE_INPUT_GZIP_FASTA){ seekgz_tell(( seekable_zfile_t *)input -> input_fp, &pos -> seekable_gzip_position); if(input -> gzfa_last_name[0]) strcpy(pos -> gzfa_last_name, input -> gzfa_last_name); else pos -> gzfa_last_name[0]=0; }else{ pos -> simple_file_position = ftello((FILE *)input -> input_fp); } } void geinput_seek(gene_input_t * input, gene_inputfile_position_t * pos){ if(input -> file_type == GENE_INPUT_SCRNA_BAM){ scBAM_seek(&input -> scBAM_input, &pos -> scBAM_position); }else if(input -> file_type == GENE_INPUT_SCRNA_FASTQ){ input_mFQ_seek(&input -> scRNA_fq_input, &pos -> mFQ_position); }else if(input -> file_type == GENE_INPUT_BCL){ assert(input -> file_type != GENE_INPUT_BCL); }else if(input -> file_type == GENE_INPUT_GZIP_FASTQ || input -> file_type == GENE_INPUT_GZIP_FASTA){ seekgz_seek(( seekable_zfile_t *)input -> input_fp, &pos -> seekable_gzip_position); if(pos -> gzfa_last_name[0]) strcpy(input -> gzfa_last_name, pos -> gzfa_last_name); else input -> gzfa_last_name[0]=0; }else{ fseeko((FILE *)input -> input_fp, pos -> simple_file_position, SEEK_SET); } } int trim_read_inner(char * read_text, char * qual_text, int rlen, short t_5, short t_3) { if(rlen > t_5) { int xk1; for(xk1 = 0; xk1 < rlen - t_5 ; xk1++) read_text[xk1] = read_text[xk1+t_5]; if(qual_text) for(xk1 = 0; xk1 < rlen - t_5 ; xk1++) qual_text[xk1] = qual_text[xk1+t_5]; } else{ read_text[0]=0; if(qual_text)qual_text[0]=0; return 0; } if(rlen - t_5 > t_3) { read_text[rlen - t_5 - t_3]=0; if(qual_text)qual_text[rlen - t_5 - t_3]=0; } else{ read_text[0]=0; if(qual_text)qual_text[0]=0; return 0; } return max(0, rlen - t_5 - t_3); } srInt_64 tell_current_line_no(gene_input_t * input){ srInt_64 fpos = ftello(input->input_fp); fseeko(input->input_fp,0,SEEK_SET); srInt_64 ret = 0, fscanpos = 0; while(1) { char nch = fgetc(input->input_fp); if(nch == EOF) return -1; if(nch == '\n') ret ++; fscanpos ++; if(fscanpos >= fpos){ fseeko(input->input_fp, fpos, SEEK_SET); return ret; } } } int geinput_next_read(gene_input_t * input, char * read_name, char * read_string, char * quality_string) { return geinput_next_read_trim( input, read_name, read_string, quality_string, 0, 0, NULL); } // returns read length if OK int geinput_next_read_with_lock(gene_input_t * input, char * read_name, char * read_string, char * quality_string, short trim_5, short trim_3, int * is_secondary, cellCounts_lock_t * lock){ if(input -> file_type == GENE_INPUT_BCL) { int rv = cacheBCL_next_read(&input -> bcl_input, read_name, read_string, quality_string, NULL); if(rv<=0) return -1; if(trim_5 || trim_3) rv = trim_read_inner(read_string, quality_string, rv, trim_5, trim_3); return rv; } else if(input -> file_type == GENE_INPUT_SCRNA_FASTQ) { int rv = input_mFQ_next_read(&input -> scRNA_fq_input, read_name, read_string, quality_string); if(rv<=0) return -1; if(trim_5 || trim_3) rv = trim_read_inner(read_string, quality_string, rv, trim_5, trim_3); return rv; } return 0; } // returns read length if OK int geinput_next_read_trim(gene_input_t * input, char * read_name, char * read_string, char * quality_string, short trim_5, short trim_3, int * is_secondary) { if(input -> file_type == GENE_INPUT_BCL) { int rv = cacheBCL_next_read(&input -> bcl_input, read_name, read_string, quality_string, NULL); if(rv<=0) return -1; if(trim_5 || trim_3) rv = trim_read_inner(read_string, quality_string, rv, trim_5, trim_3); return rv; } else if(input -> file_type == GENE_INPUT_SCRNA_FASTQ) { int rv = input_mFQ_next_read(&input -> scRNA_fq_input, read_name, read_string, quality_string); if(rv<=0) return -1; if(trim_5 || trim_3) rv = trim_read_inner(read_string, quality_string, rv, trim_5, trim_3); return rv; } else if(input -> file_type == GENE_INPUT_SCRNA_BAM) { int rv = scBAM_next_read(&input -> scBAM_input, read_name, read_string, quality_string); if(rv<=0) return -1; if(trim_5 || trim_3) rv = trim_read_inner(read_string, quality_string, rv, trim_5, trim_3); return rv; } else if(input -> file_type == GENE_INPUT_PLAIN) { int ret = read_line(MAX_READ_LENGTH, input->input_fp, read_string, 0); if(quality_string) *quality_string=0; if(ret <3)return -1; if(trim_5 || trim_3) ret = trim_read_inner(read_string, NULL, ret, trim_5, trim_3); return ret; } else if(input->file_type >= GENE_INPUT_SAM_SINGLE) { char in_buff [3001]; int tabs; int current_str_pos; int i; int ret = -1; int need_reverse; char mask_buf[5]; while(1) { // int is_second_map = 0; int linelen = read_line(3000, input->input_fp, in_buff, 0); if(linelen <1)return -1; if(read_name) *read_name = 0; if(quality_string) *quality_string = 0; *read_string = 0; need_reverse = 0; current_str_pos = 0; ret = -1; tabs=0; for(i=0; ifile_type > GENE_INPUT_SAM_SINGLE) // skip a line if not single-end read_line(1, input->input_fp, in_buff, 0); break; //printf("Repeated read skipped : %s\n", read_name); } if(need_reverse) { if(quality_string) reverse_quality(quality_string, ret); reverse_read(read_string, ret, input->space_type); } if(trim_5 || trim_3) ret = trim_read_inner(read_string, quality_string, ret, trim_5, trim_3); return ret; } else if(input->file_type == GENE_INPUT_GZIP_FASTA) { // it is currently at ">" int tr = 0, ret = 0; char rbuf [MAX_READ_LENGTH+2]; if(input -> gzfa_last_name [0] == 0){ ret = read_line_noempty(MAX_READ_NAME_LEN, input, rbuf, 0); if(ret <1) return -1; if(read_name)strcpy(read_name, rbuf+1); } else if(read_name)strcpy(read_name, input -> gzfa_last_name); ret=0; while(1){ tr = read_line_noempty(MAX_READ_LENGTH, input, rbuf, 0); if(tr<1) { if(ret<1) return -1; break; }else{ if(rbuf[0]=='>'){ strcpy(input -> gzfa_last_name, rbuf+1); break; }else{ strcpy(read_string+ret, rbuf); ret += tr; // read_line_noempty have no \n } read_string[ret]=0; } } if(trim_5 || trim_3) ret = trim_read_inner(read_string, quality_string, ret, trim_5, trim_3); return ret; } else if(input->file_type == GENE_INPUT_FASTA) { int ret; if(quality_string) (*quality_string)=0; #ifdef __MINGW32__ assert(0); #endif while(1) // fetch read name { ret = read_line(MAX_READ_LENGTH, input->input_fp, read_string, 0); if(ret <1) { sublog_printf(SUBLOG_STAGE_RELEASED,SUBLOG_LEVEL_DEBUG, "The input file normally exhausted."); return -1; } int cursor = 0; while(read_string[cursor]) { if(cursor >=2 &&(read_string[cursor] == ' ' || read_string[cursor] == '\t')) { read_string [cursor] = 0; break; } cursor++; } if(read_string[0]=='>'){ if (read_name != NULL) strncpy(read_name, read_string+1, MAX_READ_NAME_LEN); break; } else sublog_printf(SUBLOG_STAGE_RELEASED,SUBLOG_LEVEL_FATAL,"The input file may be broken."); } ret = 0; while(1) // fetch read text { char nch = 0; ret += read_line(MAX_READ_LENGTH-ret, input->input_fp, read_string+ret, 1); nch = fgetc(input->input_fp); if(nch!=EOF) fseeko(input->input_fp, -1, SEEK_CUR); if(nch == '>'||nch<1 || nch == EOF) break; } // printf("LOAD R=|%s|\nRETV=%d\n", read_string, ret); if(ret <1)return -1; if(trim_5 || trim_3) ret = trim_read_inner(read_string, quality_string, ret, trim_5, trim_3); return ret; } else if(input->file_type == GENE_INPUT_FASTQ || input->file_type == GENE_INPUT_GZIP_FASTQ) { char nch = 0; int ret; //if(input->file_type == GENE_INPUT_GZIP_FASTQ)seekgz_preload_buffer(input, NULL); //READ NAME if (read_name == NULL) { SKIP_LINE_NOEMPTY; if(nch == EOF) return -1; } else { do{ nch = geinput_getc(input); //SUBREADprintf("B4_READ_NAME: %d '%c'\n", nch,nch); } while (nch == '\n'); if(nch==EOF) return -1; if(nch != '@') { if(input->file_type == GENE_INPUT_FASTQ){ srInt_64 lineno = tell_current_line_no(input); SUBREADprintf("ERROR: a format issue %d is found on the %lld-th line in input file '%s'.\nProgram aborted.\n", nch, lineno, input -> filename); } else { SUBREADprintf("ERROR: a format issue %d is found on the input file '%s'.\nProgram aborted.\n", nch, input -> filename); SUBREADprintf("The lines after the error point:\n"); read_line_noempty(MAX_READ_LENGTH, input, read_string, 0); SUBREADprintf("%s\n", read_string); read_line_noempty(MAX_READ_LENGTH, input, read_string, 0); SUBREADprintf("%s\n", read_string); read_line_noempty(MAX_READ_LENGTH, input, read_string, 0); SUBREADprintf("%s\n", read_string); read_line_noempty(MAX_READ_LENGTH, input, read_string, 0); SUBREADprintf("%s\n", read_string); } return -1; } read_line_noempty(MAX_READ_NAME_LEN, input, read_name, 0); int cursor = 1; while(read_name[cursor]) { if(read_name[cursor] == ' ' || read_name[cursor] == '\t') { read_name [cursor] = 0; break; } cursor++; } } //if(input->file_type == GENE_INPUT_GZIP_FASTQ)seekgz_preload_buffer(input, NULL); // READ LINE ret = read_line_noempty(MAX_READ_LENGTH, input, read_string, 0); //SUBREADprintf("READ_SHOULD_ATGC [len=%d] : '''%s'''\n", ret, read_string); // SKIP "+" do{ nch = geinput_getc(input); } while( nch == '\n' ); if(nch != '+'){ if(input->file_type == GENE_INPUT_FASTQ){ srInt_64 lineno = tell_current_line_no(input); SUBREADprintf("ERROR: a format issue %c is found on the %lld-th line in input file '%s'.\nProgram aborted.\n", nch, lineno, input -> filename); }else{ SUBREADprintf("ERROR: a format issue %d (should be +) is found on the input file '%s'.\nProgram aborted.\n", nch, input -> filename); read_line_noempty(MAX_READ_LENGTH, input, read_string, 0); SUBREADprintf("%s\n", read_string); read_line_noempty(MAX_READ_LENGTH, input, read_string, 0); SUBREADprintf("%s\n", read_string); read_line_noempty(MAX_READ_LENGTH, input, read_string, 0); SUBREADprintf("%s\n", read_string); read_line_noempty(MAX_READ_LENGTH, input, read_string, 0); SUBREADprintf("%s\n", read_string); } return -1; } SKIP_LINE; // QUAL LINE if (quality_string) read_line_noempty(MAX_READ_LENGTH, input, quality_string, 0); else SKIP_LINE_NOEMPTY; #ifdef MODIFIED_READ_LEN { int modified_start = 0; if(modified_start) { int i; for(i=0;i file_type == GENE_INPUT_SCRNA_BAM) input_scBAM_close(&input -> scBAM_input); else if(input -> file_type == GENE_INPUT_SCRNA_FASTQ) input_mFQ_close(&input -> scRNA_fq_input); else if(input -> file_type == GENE_INPUT_BCL) cacheBCL_close(&input -> bcl_input); else if(input -> file_type == GENE_INPUT_GZIP_FASTQ || input -> file_type == GENE_INPUT_GZIP_FASTA) seekgz_close((seekable_zfile_t * ) input->input_fp); else fclose((FILE*)input->input_fp); } char * __converting_char_table = "NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTNGNNNCNNNNNNNNNNNNAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTNGNNNCNNNNNNNNNNNNAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN "; void reverse_read(char * InBuff, int read_len, int space_type) { int i; if(space_type == GENE_SPACE_COLOR) { int start_pos = 0; char last_base = InBuff[0]; //printf("CLRLEN0=%d\nS0=%s\n", read_len, InBuff); if(isalpha(last_base)) { read_len ++; for (i=1; i=0; i-=2) { char kv = *(key++); ret |= base2int(kv)<=0; i-=2) { char c1 = *(key++); ret |= base2int(c1)<>16)+15; } else { int subread_step; subread_step = 6<<16; if(((len - 18)<<16) / subread_step > 62) subread_step = ((len - 18)<<16)/62; return ((subread_step*(subread))>>16)+15; } } void fix_cigar_SAM14(char * cig){ int tmpi = 0, ci = 0, tmpM = 0, wi = 0; char ncig[EXON_MAX_CIGAR_LEN]; if(cig[0]=='*'){ return; } while(1){ int nch = cig[ci]; if(isdigit(nch)) tmpi = tmpi * 10 + nch - '0'; else{ if(nch == '=' || nch == 'X' || nch == 'M'){ tmpM += tmpi; }else{ if(tmpM > 0){ wi += sprintf(ncig + wi, "%dM", tmpM); tmpM = 0; } if(0 == nch) break; else wi += sprintf(ncig + wi, "%d%c", tmpi, nch); } tmpi = 0; } ci++; } memcpy(cig, ncig, wi+1); } //This function returns 0 if the line is a mapped read; -1 if the line is in a wrong format and 1 if the read is unmapped. int parse_SAM_line(char * sam_line, char * read_name, int * flags, char * chro, unsigned int * pos, char * cigar, int * mapping_quality, unsigned int * pair_dist, char * sequence, char * quality_string, int * rl, int * repeated) { char cc; int ci = 0, k=0, field=0, ret_quality = 0, ret_flag = 0, ret_pairdist=0; unsigned int ret_pos = 0; int is_rep = 0; while( (cc = sam_line[k]) ) { if(cc=='\t') { field++; k++; if(field == 1)read_name[ci]=0; else if(field == 3)chro[ci]=0; else if(field == 6)cigar[ci]=0; else if(field == 10) { sequence[ci]=0; (*rl) = ci; } else if(field == 11)quality_string[ci]=0; ci=0; is_rep = 0; continue; } if(field == 9) sequence[ci++] = cc; else if(field == 10) quality_string[ci++] = cc; else if(field == 0) read_name[ci++] = cc; else if(field == 1) ret_flag = ret_flag*10 + (cc-'0'); else if(field == 8) { if(cc!='-') ret_pairdist = ret_pairdist*10 + (cc-'0'); } else if(field == 2) { //if(ci == 0 && cc == '*') return 1; chro[ci++] = cc; } else if(field == 3) ret_pos = ret_pos * 10 + (cc-'0'); else if(field == 4) ret_quality = ret_quality * 10 + (cc-'0'); else if(field == 5) cigar[ci++] = cc; else if(field > 10) { if(cc == 'I' && ci==0) is_rep = 1; if(cc != 'H' && ci==1 ) is_rep = 0; if(is_rep && ci == 4) *repeated = 0; if(is_rep && ci>4) (*repeated)=(*repeated)*10+(cc-'0'); ci++; } k++; } //printf("REP=%d\n", *repeated); if(field == 10 && ci>0)quality_string[ci]=0; else if(field < 10) return -1; if(ret_flag & 4) (*mapping_quality) = 0; else (*mapping_quality) = ret_quality; (*pos) = ret_pos; (*flags) = ret_flag; (*pair_dist) = ret_pairdist; //printf("FLAG=%d\n", (*flags)); if(((*flags) & 4) == 4) return 1; fix_cigar_SAM14(cigar); return 0; } // This function returns 0 if the block is determined. // The block is undeterminable if the chromosome name is not in known_chromosomes, or the position is larger than the known length. // Pos is in terms of [1, ... , max_length] int get_read_block(char *chro, unsigned int pos, char *temp_file_suffix, chromosome_t *known_chromosomes, unsigned int * max_base_position) { int chro_no; unsigned int max_known_chromosome=0; for(chro_no=0;known_chromosomes[chro_no].chromosome_name[0]; chro_no++) { if(strcmp(chro , known_chromosomes[chro_no].chromosome_name) == 0) { max_known_chromosome = known_chromosomes[chro_no].known_length; break; } //if(chro_no > 1) // printf("TOO MANY CHROS:%d\n", chro_no); } if(!known_chromosomes[chro_no].chromosome_name[0]) return 1; if(pos >= known_chromosomes[chro_no].known_length) return 1; int block_no = (pos-1) / BASE_BLOCK_LENGTH; sprintf(temp_file_suffix , "%s-%04u.bin", chro, block_no); if(max_base_position)*max_base_position=min((block_no+1)*BASE_BLOCK_LENGTH, max_known_chromosome); return 0; } FILE * get_temp_file_pointer(char *temp_file_name, HashTable* fp_table, int * close_immediately) { FILE * temp_file_pointer = (FILE *) HashTableGet(fp_table, temp_file_name); *close_immediately = 0; if(temp_file_pointer == NULL || temp_file_pointer == NULL + 1) { int need_put = (temp_file_pointer == NULL ); char *key_name; key_name = (char *)SUBREAD_malloc(300); if(!key_name) return NULL; strcpy(key_name, temp_file_name); temp_file_pointer = f_subr_open(key_name,"ab"); if(!temp_file_pointer){ SUBREADprintf("File cannot be opened: '%s'.\nPlease increase the maximum open files by command 'ulimit -n'.\nThis number should be set to at least 500 for human genome, and more chromosomes require more opened files.\n\n", key_name); return NULL; } int maximum_open_file = fp_table -> appendix1 - NULL; if( fp_table -> numOfElements < maximum_open_file && need_put) HashTablePut(fp_table, key_name ,temp_file_pointer); else{ if(need_put) HashTablePut(fp_table, key_name , NULL + 1); *close_immediately = 1; } } return temp_file_pointer; } void my_fclose(void * fp) { if(fp && fp != NULL+1) fclose((FILE *)fp); } int my_strcmp(const void * s1, const void * s2) { int ret = strcmp((char*)s1, (char*)s2); //SUBREADprintf("SCM:%s %s = %d\n", s1, s2, ret); return ret; } int write_read_block_file(FILE *temp_fp , unsigned int read_number, char *read_name, int flags, char * chro, unsigned int pos, char *cigar, int mapping_quality, char *sequence , char *quality_string, int rl , int is_sequence_needed, char strand, unsigned short read_pos, unsigned short read_len, unsigned short mapped_seg) { base_block_temp_read_t datum; memset(&datum,0,sizeof(datum)); datum.record_type = 100; datum.read_number = read_number; datum.pos = pos; datum.flags = flags; datum.strand = strand; datum.read_pos = read_pos; datum.read_len = read_len; datum.mapping_quality = mapping_quality; datum.mapped_segment_in_read = mapped_seg; if(rl < 1|| rl > MAX_READ_LENGTH) { SUBREADprintf("READ IS TOO LONG:%d\n", rl); return -1; } fwrite(&datum, sizeof(datum), 1, temp_fp); if(is_sequence_needed) { unsigned short srl = rl&0xffff; int wlen = fwrite(&srl, sizeof(short),1, temp_fp); if(wlen != 1) return -1; wlen = fwrite(sequence , 1, rl,temp_fp ); if(wlen != rl) return -1; wlen = fwrite(quality_string , 1, rl,temp_fp ); if(wlen != rl) return -1; } return 0; } int get_known_chromosomes(char * in_SAM_file, chromosome_t * known_chromosomes) { int i, is_first_read_PE; int is_BAM = is_certainly_bam_file(in_SAM_file, &is_first_read_PE, NULL); SamBam_FILE * fp = SamBam_fopen(in_SAM_file,is_BAM?SAMBAM_FILE_BAM:SAMBAM_FILE_SAM); while(1) { char line_buffer [3000]; char * is_ret = SamBam_fgets(fp, line_buffer, 2999, 0); if(!is_ret) break; int linelen = strlen(line_buffer); if(line_buffer[0]=='@') { int chro_numb=0, field = 0, ci=0, ciw = 0; if(line_buffer[1]!='S' || line_buffer[2]!='Q' || line_buffer[3]!='\t' ) continue; while(known_chromosomes[chro_numb].chromosome_name[0]!=0) chro_numb++; if(chro_numb > XOFFSET_TABLE_SIZE-1) { SUBREADprintf("FATAL ERROR: the number of chromosomes excessed %d\n", XOFFSET_TABLE_SIZE); return -1; } known_chromosomes[chro_numb].known_length = 0; for(i=0; i< linelen; i++) { char cc = line_buffer[i]; if(cc == '\r' || cc=='\n') continue; if(cc == '\t') { if(field == 1) known_chromosomes[chro_numb].chromosome_name[ciw]=0; ci = 0; ciw = 0; field ++; } else if(field == 1) { if(ci >2) known_chromosomes[chro_numb].chromosome_name[ciw++]=cc; ci++; } else if(field == 2) { if(ci >2) known_chromosomes[chro_numb].known_length = known_chromosomes[chro_numb].known_length * 10 + (cc - '0'); ci++; } } } else break; } SamBam_fclose(fp); return 0; } void add_cigar_indel_event(HashTable * event_table_ptr, char * chro, unsigned int chro_pos, int indels , char * ins_seq) { if(abs(indels)>100) return; char event_token[100]; snprintf(event_token, 99,"%s\t%u", chro, chro_pos); int x1; unsigned int indel_event_id = 0xffffffff, token_len; int exist_indel_count = HashTableGet(event_table_ptr, event_token) - NULL; unsigned short * app2_ptr = event_table_ptr->appendix2; if(exist_indel_count) for(x1 = 0; x1< exist_indel_count; x1++) { snprintf(event_token, 99,"%s\t%u\t%d", chro, chro_pos, x1); srInt_64 t64v = (HashTableGet(event_table_ptr, event_token)-NULL); srInt_64 indel_len = (t64v&0xff) - 0x80; if(indel_len == indels){ indel_event_id = 0xffffff&(t64v >> 8) ; if(app2_ptr[indel_event_id]<65000) app2_ptr[indel_event_id] +=1; return; } } if(event_table_ptr->counter2<0xffff00) { unsigned int event_space_max_size = event_table_ptr-> counter1; indel_event_id = event_table_ptr->counter2 ++; if(indel_event_id >= event_space_max_size) { event_table_ptr->appendix1 = realloc(event_table_ptr->appendix1 , sizeof(char *) * event_space_max_size*2); event_table_ptr->appendix2 = realloc(event_table_ptr->appendix2 , sizeof(short) * event_space_max_size*2); memset(event_table_ptr->appendix2 + event_space_max_size * sizeof(short), 0, sizeof(short) * event_space_max_size); event_table_ptr-> counter1 = event_space_max_size*2; app2_ptr = event_table_ptr->appendix2; } token_len=snprintf(event_token, 99,"%s\t%u", chro, chro_pos); if(exist_indel_count<1) { char * token_1 = malloc(token_len+1); strcpy(token_1, event_token); HashTablePut(event_table_ptr, token_1, NULL+1); } else { HashTablePutReplace(event_table_ptr, event_token, NULL+exist_indel_count+1, 0); } token_len=snprintf(event_token, 99,"%s\t%u\t%d", chro, chro_pos, exist_indel_count); char * token_2 = malloc(token_len+1); strcpy(token_2, event_token); srInt_64 indel_event_id_long = indel_event_id; app2_ptr[indel_event_id] +=1; HashTablePut(event_table_ptr, token_2, NULL + ((0xff & (0x80 + indels)) | ((indel_event_id_long&0xffffff) << 8))); if(indels<0) { char * ins_seq_2 = malloc(-indels), ** app1_ptrptr = event_table_ptr->appendix1; memcpy(ins_seq_2, ins_seq, -indels); app1_ptrptr[indel_event_id] = ins_seq_2; } } } void destroy_cigar_event_table(HashTable * event_table) { int bucket; KeyValuePair * cursor; char ** seq_tab = event_table->appendix1; for(bucket=0; bucket numOfBuckets; bucket++) { cursor = event_table -> bucketArray[bucket]; while (1) { int xk1, tabs; if (!cursor) break; char * token = (char *)cursor -> key; tabs = 0; for(xk1=0; token[xk1]; xk1++) if(token[xk1]=='\t') tabs++; srInt_64 tmpv = cursor -> value - NULL; //printf("%s\t%lld\n", token, tmpv); if(tabs==3) { unsigned int event_id = (tmpv>>8)&0xffffff; free(seq_tab[event_id]); } free(token); cursor = cursor->next; } } free(event_table->appendix1); free(event_table->appendix2); HashTableDestroy(event_table); } void break_VCF_file(char * vcf_file, HashTable * fp_table, char * temp_file_prefix, chromosome_t* known_chromosomes) { autozip_fp vzfp; int vret = autozip_open(vcf_file, &vzfp); char temp_file_suffix[MAX_CHROMOSOME_NAME_LEN+20]; int close_now = 0; if(vret < 0) { SUBREADprintf("The specified VCF does not exist.\n"); return; } char * linebuf = malloc(3000); char * tmpfname = malloc(MAX_FILE_NAME_LENGTH); while(1) { char * tok_tmp; int aretc = autozip_gets(&vzfp, linebuf, 2999); if(aretc < 1) break; if(linebuf[0]=='#') continue; if(strstr(linebuf, "INDEL")) continue; //SUBREADprintf("VLINE:%s\n", linebuf); char * chro = strtok_r(linebuf, "\t", &tok_tmp); if(!tok_tmp) continue; char * pos_str = strtok_r(NULL, "\t", &tok_tmp); if(!tok_tmp) continue; strtok_r(NULL, "\t", &tok_tmp);// name if(!tok_tmp) continue; char * ref_seq = strtok_r(NULL, "\t", &tok_tmp); if(!tok_tmp) continue; char * alt_seq = strtok_r(NULL, "\t", &tok_tmp); if(!tok_tmp) continue; int is_snp = 0; if(strstr(alt_seq,",")) { char * com_tmp = NULL; char * com_sec = strtok_r(alt_seq, ",", &com_tmp); while(com_sec) { if(strlen(com_sec)==strlen(ref_seq)) { is_snp=1; break; } com_sec = strtok_r(NULL, ",", &com_tmp); } }else if(strlen(ref_seq) == strlen(alt_seq)) is_snp=1; if(!is_snp)continue; unsigned int max_section_pos; if(get_read_block(chro, atoi(pos_str) , temp_file_suffix, known_chromosomes, &max_section_pos))continue; sprintf(tmpfname, "%s%s", temp_file_prefix , temp_file_suffix); FILE * temp_fp = get_temp_file_pointer(tmpfname, fp_table, &close_now); if(temp_fp) { VCF_temp_read_t datum; datum.record_type = 200; datum.pos = atoi(pos_str); datum.type = CHRO_EVENT_TYPE_SNP; fwrite(&datum, sizeof(VCF_temp_read_t), 1, temp_fp); if(close_now) fclose(temp_fp); } } free(linebuf); free(tmpfname); autozip_close(&vzfp); } int break_SAM_file(char * in_SAM_file, int is_BAM_file, char * temp_file_prefix, unsigned int * real_read_count, int * block_count, chromosome_t * known_chromosomes, int is_sequence_needed, int base_ignored_head_tail, gene_value_index_t *array_index, gene_offset_t * offsets, srInt_64 * all_mapped_bases, HashTable * event_table, char * VCF_file, srInt_64 * all_mapped_reads, int do_fragment_filtering, int push_to_read_head, int use_softclipped_bases ) { int i, is_first_read=1, is_error = 0; HashTable * fp_table; unsigned int read_number = 0; char line_buffer [3000]; SamBam_FILE * sambam_reader; sambam_reader = SamBam_fopen(in_SAM_file, is_BAM_file?SAMBAM_FILE_BAM:SAMBAM_FILE_SAM); if(!sambam_reader){ SUBREADprintf("SAM file does not exist or is not accessible: '%s'\n", in_SAM_file); return 1; } if(push_to_read_head)assert(is_sequence_needed==0); fp_table = HashTableCreate( 11011 ); HashTableSetDeallocationFunctions(fp_table, free, my_fclose); HashTableSetKeyComparisonFunction(fp_table, my_strcmp); HashTableSetHashFunction(fp_table,HashTableStringHashFunction); char * fns = malloc(200); fns[0]=0; exec_cmd("ulimit -n", fns, 200); int max_open_file = atoi(fns); //SUBREADprintf("SYS FILE LIMIT=%d\n", max_open_file); free(fns); max_open_file = max(100, max_open_file); max_open_file = min(3000, max_open_file); fp_table -> appendix1 = NULL + max_open_file * 2/ 3; if(event_table!=NULL && event_table->appendix1==NULL) { event_table->appendix1 = malloc(sizeof(char *) * 100); event_table->appendix2 = malloc(sizeof(unsigned short) * 100); memset(event_table->appendix2, 0, sizeof(unsigned short) * 100); event_table->counter1 = 100; event_table->counter2 = 0; } while(1) { //srInt_64 file_position = ftello(fp); //int linelen = read_line(2999, fp, line_buffer, 0); char * is_ret = SamBam_fgets(sambam_reader, line_buffer, 2999, 1); if(!is_ret) break; if(line_buffer[0]=='@') { int chro_numb=0, field = 0, ci=0, ciw = 0; if(line_buffer[1]!='S' || line_buffer[2]!='Q' || line_buffer[3]!='\t' ) continue; while(known_chromosomes[chro_numb].chromosome_name[0]!=0) chro_numb++; if(chro_numb > XOFFSET_TABLE_SIZE-1) { SUBREADprintf("FATAL ERROR: the number of chromosomes excessed %d\n", XOFFSET_TABLE_SIZE); return -1; } known_chromosomes[chro_numb].known_length = 0; for(i=0; ; i++) { char cc = line_buffer[i]; if(!cc) break; if(cc == '\r' || cc=='\n') continue; if(cc == '\t') { if(field == 1) known_chromosomes[chro_numb].chromosome_name[ciw]=0; ci = 0; ciw = 0; field ++; } else if(field == 1) { if(ci >2) known_chromosomes[chro_numb].chromosome_name[ciw++]=cc; ci++; } else if(field == 2) { if(ci >2) known_chromosomes[chro_numb].known_length = known_chromosomes[chro_numb].known_length * 10 + (cc - '0'); ci++; } } if(chro_numb < XOFFSET_TABLE_SIZE-1) known_chromosomes[chro_numb+1].chromosome_name[0]=0; } else { char read_name[MAX_READ_NAME_LEN], chro[MAX_CHROMOSOME_NAME_LEN], cigar[EXON_MAX_CIGAR_LEN], sequence[MAX_READ_LENGTH+1], quality_string[MAX_READ_LENGTH+1]; int flags = 0, mapping_quality = 0, rl=0; char is_negative_strand = 0; unsigned int pos = 0, pairdist = 0; char temp_file_suffix[MAX_FILE_NAME_LENGTH]; char temp_file_name[MAX_FILE_NAME_LENGTH]; FILE * temp_fp; int repeated = -1, close_now = 0; if(is_first_read) { is_first_read=0; if(VCF_file && VCF_file[0]) break_VCF_file(VCF_file, fp_table, temp_file_prefix, known_chromosomes); } //SUBREADprintf("ARRI_0=%p ; OFFS=%p ; EVT=%p\n%s\n",array_index, offsets, event_table, line_buffer); int line_parse_result = parse_SAM_line(line_buffer, read_name, &flags, chro, &pos, cigar, & mapping_quality, &pairdist, sequence, quality_string, &rl, &repeated); if(line_parse_result<0)SUBREADprintf("WRONG LINE FORMAT: %s\n", line_buffer); if(strlen(quality_string)<2) { int xk1; for(xk1=0; xk1 500000)){ read_number ++; continue; } if(do_fragment_filtering && array_index) { int mismatch = 0; unsigned int linear_pos = linear_gene_position(offsets , chro, pos)-1; float match_rate = final_mapping_quality(array_index, linear_pos, sequence, quality_string, cigar, FASTQ_PHRED33, & mismatch, rl, NULL, NULL); if(mismatch>8 || match_rate < 160) { read_number ++; continue; } } is_negative_strand = (flags & SAM_FLAG_REVERSE_STRAND_MATCHED)?1:0; if((flags & 4) ==0 && all_mapped_reads)(*all_mapped_reads)++; if(is_sequence_needed == 2) { } else if(is_sequence_needed == 1) { int read_cursor = 0; int is_first_S = 1; unsigned int chromosome_cursor = pos; int j, tmpv=0; char cc; unsigned short M_parts=0; for(j=0; cigar[j]; j++) { cc = cigar[j]; if(cc>='0' && cc<='9') tmpv= tmpv*10+(cc-'0'); else if(cc == 'S'||cc == 'M') { if(cc == 'M') is_first_S = 0; if(cc == 'M' || use_softclipped_bases) { unsigned int insertion_cursor = chromosome_cursor - ((cc=='S' && is_first_S)?tmpv:0); unsigned int insertion_end = chromosome_cursor + ((cc=='S' && is_first_S)?0:tmpv); // DO INSERTION while(insertion_cursor < insertion_end && read_cursor < (rl - base_ignored_head_tail)) { unsigned int max_section_pos, insert_length; int need_write = 1; if(get_read_block(chro, insertion_cursor , temp_file_suffix, known_chromosomes, &max_section_pos))break; insert_length = min(max_section_pos + 1, insertion_end) - insertion_cursor; if(insert_length<1) break; if(base_ignored_head_tail) { if(read_cursor+insert_length < base_ignored_head_tail) need_write = 0; else if(read_cursor < base_ignored_head_tail) { int ignored_length = base_ignored_head_tail - read_cursor; insert_length = read_cursor + insert_length - base_ignored_head_tail; read_cursor = base_ignored_head_tail; insertion_cursor += ignored_length; } if(read_cursor >= (rl - base_ignored_head_tail)) need_write = 0; else if(read_cursor +insert_length >= (rl - base_ignored_head_tail)) insert_length = (rl - base_ignored_head_tail) - read_cursor; } // printf("INST: RL=%d; INSL=%d; READ_CUR=%d; IGNORE=%d\n", rl, insert_length, read_cursor , base_ignored_head_tail); //#warning " ======= DEBUG OUT =========" if(0 && FIXLENstrcmp("SRR768163.14829906", read_name) == 0) SUBREADprintf("INST: RL=%d; NEED=%d; INSL=%d; READ_CUR=%d; IGNORE=%d; RN=%s\nWRT AT %u (one-based): %s\n\n", rl, need_write, insert_length, read_cursor , base_ignored_head_tail, read_name, insertion_cursor, sequence + read_cursor); if(0 && strcmp(chro, "chr12") == 0 && insertion_cursor <= 114788620 && insertion_cursor + insert_length > 114788620){ int read_pos0 = 114788620 - insertion_cursor + read_cursor; SUBREADprintf("INST_114788620 : %s : val=%c ; NEED=%d\n", read_name, sequence[read_pos0], need_write); } if(need_write && insert_length > 0 && sequence[0]!='*') { sprintf(temp_file_name, "%s%s", temp_file_prefix , temp_file_suffix); temp_fp = get_temp_file_pointer(temp_file_name, fp_table, &close_now); if(!temp_fp) return -1; if(all_mapped_bases) (*all_mapped_bases) += insert_length; is_error |= write_read_block_file(temp_fp , read_number, read_name, flags, chro, insertion_cursor, cigar, mapping_quality, sequence + read_cursor , quality_string + read_cursor, insert_length , 1, is_negative_strand, read_cursor, rl, M_parts); if(close_now) fclose(temp_fp); } insertion_cursor += insert_length; read_cursor += insert_length; } if(M_parts < 65535)M_parts ++; } else read_cursor += tmpv; if(!is_first_S) chromosome_cursor += tmpv; tmpv=0; } else if(cc == 'D' || cc == 'N') { // the left edge ( last WANTED base ) is chromosome_cursor-1 // the indel length is tmpv; // now we add this into the event table. if(event_table && cc=='D') add_cigar_indel_event(event_table, chro, chromosome_cursor-1, tmpv, NULL); chromosome_cursor += tmpv; tmpv = 0; } else if(cc == 'I' ) { // the left edge ( last WANTED base ) is chromosome_cursor-1 // the indel length is -tmpv; // now we add this into the event table. if(event_table && sequence[0]!='*') add_cigar_indel_event(event_table, chro, chromosome_cursor-1, -tmpv, sequence + read_cursor); read_cursor += tmpv; tmpv = 0; } else tmpv = 0; } }else{ // NO sequence is needed : no CIGAR is parsed. int cgi, cc; int pushback = 0; for(cgi=0; cigar[cgi]; cgi++){ cc = cigar[cgi]; if(cc >='0' && cc<='9') pushback = pushback*10 + cc-'0'; else{ if(cc!='S') pushback=0; break; } } assert(pos>=pushback); pos -= pushback; if(get_read_block(chro, pos, temp_file_suffix, known_chromosomes, NULL)) { read_number ++; continue; } sprintf(temp_file_name, "%s%s", temp_file_prefix , temp_file_suffix); temp_fp = get_temp_file_pointer(temp_file_name, fp_table, &close_now); is_error |= write_read_block_file(temp_fp , read_number, read_name, flags, chro, pos, cigar, mapping_quality, sequence , quality_string, rl , is_sequence_needed, is_negative_strand, 0,rl, 0); if(close_now)fclose(temp_fp); } read_number ++; } } if(block_count) (*block_count) = fp_table->numOfElements; HashTableDestroy(fp_table); SamBam_fclose(sambam_reader); if(real_read_count) (*real_read_count) = read_number; if(is_error){ SUBREADprintf("ERROR: cannot write into the temporary files. Please check the disk space in the temp directory.\n"); } return is_error; } int is_in_exon_annotations(gene_t *output_genes, unsigned int offset, int is_start) { int i,j; for(i=0; i< MAX_ANNOTATION_EXONS; i++) { if(!output_genes[i].end_offset) break; if(output_genes[i].end_offset >= offset && output_genes[i].start_offset <= offset) { for(j=0; j< MAX_EXONS_PER_GENE; j++) { if(output_genes[i].exon_ends[j] >= offset && output_genes[i].exon_starts[j] <= offset) { if(output_genes[i].exon_starts[j] == offset && is_start) return 2; // 2==exactly matched if(output_genes[i].exon_ends[j] == offset && !is_start) return 2; return 1; // 1==enclosed } } } } return 0; //0==exon not found } int load_exon_annotation(char * annotation_file_name, gene_t ** output_genes, gene_offset_t* offsets) { int line_len, gene_number = 0, exons = 0; char old_gene_name[MAX_GENE_NAME_LEN]; FILE * fp = f_subr_open(annotation_file_name, "rb"); if(!fp) { SUBREADprintf("Cannot open the exon annotation file: %s\n", annotation_file_name); return -1; } (*output_genes) = malloc(sizeof(gene_t)*MAX_ANNOTATION_EXONS); if(!*output_genes) { SUBREADprintf("Cannot allocate memory for the exon table. \n"); return -1; } old_gene_name[0]=0; (*output_genes)[0].end_offset = 0; (*output_genes)[0].start_offset = 0xffffffff; while(gene_number < MAX_ANNOTATION_EXONS) { char buff[1200], this_gene_name[MAX_GENE_NAME_LEN], chromosome_name[MAX_CHROMOSOME_NAME_LEN]; int i = 0, j=0; unsigned int exon_location; line_len = read_line(1200, fp, buff, 0); if(line_len>0) //Not EOF { if(!isdigit(buff[0])) // it is a title line or something else continue; for(i=0; buff[i] != '\t' && buff[i] != '\n' && i < 1200; i++) this_gene_name[i] = buff[i]; this_gene_name[i] = 0; } if(line_len<=0 || (exons && old_gene_name[0] && strcmp(this_gene_name , old_gene_name))) // it is a new gene { strncpy((*output_genes)[gene_number].gene_name , old_gene_name, MAX_GENE_NAME_LEN); (*output_genes)[gene_number].exon_ends[exons] = 0; gene_number++; exons = 0; (*output_genes)[gene_number].end_offset = 0; (*output_genes)[gene_number].start_offset = 0xffffffff; } if(line_len<=0) break; // copy chromosome name for(i++; buff[i] != '\t' && buff[i] != '\n' && i < 1200; i++) chromosome_name[j++] = buff[i]; chromosome_name[j] = 0; // start location exon_location = 0; for(i++; buff[i] != '\t' && buff[i] != '\n' && i < 1200; i++) if(isdigit(buff[i])) exon_location = exon_location*10 + buff[i] - '0'; (*output_genes)[gene_number].exon_starts[exons] = linear_gene_position(offsets, chromosome_name , exon_location-1); if( (*output_genes)[gene_number].exon_starts[exons] == 0xffffffff) continue; if((*output_genes)[gene_number].start_offset > (*output_genes)[gene_number].exon_starts[exons]) (*output_genes)[gene_number].start_offset = (*output_genes)[gene_number].exon_starts[exons]; // end location exon_location = 0; for(i++; buff[i] != '\t' && buff[i] != '\n' && buff[i] && i < 1200; i++) if(isdigit(buff[i])) exon_location = exon_location*10 + buff[i] - '0'; (*output_genes)[gene_number].exon_ends[exons] = linear_gene_position(offsets, chromosome_name , exon_location); if((*output_genes)[gene_number].end_offset < (*output_genes)[gene_number].exon_ends[exons]) (*output_genes)[gene_number].end_offset = (*output_genes)[gene_number].exon_ends[exons]; exons ++; if(exons >= MAX_EXONS_PER_GENE) { SUBREADprintf("The number of exons excesses the limit. Please increase the value of MAX_EXONS_PER_GENE in subread.h.\n"); return -1; } strncpy(old_gene_name, this_gene_name , MAX_GENE_NAME_LEN); } fclose(fp); return 0; } int does_file_exist(char * path) { int ret ; FILE * fp = f_subr_open(path, "rb"); ret = fp!=NULL; if(fp)fclose(fp); return ret; } srUInt_64 sort_SAM_hash(char * str) { srUInt_64 hash = 5381; int c, xk1=0; while (1) { c = str[xk1++]; if(!c)break; hash = ((hash << 5) + hash) + c; /* hash * 33 + c */ } return hash; } void do_SIGINT_remove(char * prefix, int param); char * _SAMSORT_SNP_delete_temp_prefix = NULL; char * _REPAIRER_delete_temp_prefix = NULL; void SAM_SORT_SIGINT_hook(int param) { do_SIGINT_remove(_SAMSORT_SNP_delete_temp_prefix, param); } void REPAIR_SIGINT_hook(int param) { do_SIGINT_remove(_REPAIRER_delete_temp_prefix, param); } void delete_with_prefix(char * prefix){ if(prefix != NULL) { int xk1, last_slash = -1; char del2[MAX_FILE_NAME_LENGTH], del_suffix[MAX_FILE_NAME_LENGTH], del_name[MAX_FILE_NAME_LENGTH]; for(xk1=0; prefix[xk1]; xk1++) { if(prefix[xk1]=='/') last_slash = xk1; else if(prefix[xk1]=='\\') { SUBREADprintf("The file name is unknown.\n"); return; } } if(last_slash>=0) { memcpy(del2, prefix, last_slash); del2[last_slash] = 0; strcpy(del_suffix , prefix + last_slash + 1); } else { strcpy(del2,"."); strcpy(del_suffix , prefix); } //#warning ">>>>>>>> COMMENT THIS OUT <<<<<<<<<<<<<<<<<<<<<" //SUBREADprintf("SCANDEL: %s, PREFIX %s, SUFFIX %s\n", del2, prefix, del_suffix); if(strlen(del_suffix)>8) { DIR *d; struct dirent *dir; d = opendir(del2); if (d) { while ((dir = readdir(d)) != NULL) { if(strstr(dir->d_name, del_suffix)) { strcpy(del_name, del2); strcat(del_name, "/"); strcat(del_name, dir->d_name); unlink(del_name); // #warning ">>>>>>>> COMMENT THIS OUT <<<<<<<<<<<<<<<<<<<<<" // SUBREADprintf("DEL: %s\n", del_name); //test fix } } closedir(d); } } } } void do_SIGINT_remove(char * prefix, int param) { #ifdef MAKE_STANDALONE delete_with_prefix(prefix); SUBREADprintf("\n\nReceived a terminal signal. The temporary files were removed.\n"); exit(param); #endif } void * old_sig_TERM = NULL, * old_sig_INT = NULL; int SAM_pairer_writer_create( SAM_pairer_writer_main_t * bam_main , int all_threads , int has_dummy, int BAM_input, int c_level, char * out_file){ int x1; memset(bam_main, 0, sizeof(SAM_pairer_writer_main_t)); bam_main -> bam_fp = f_subr_open(out_file, "wb"); if(NULL == bam_main -> bam_fp) return 1; strcpy(bam_main -> bam_name, out_file); bam_main -> threads = malloc(all_threads * sizeof(SAM_pairer_writer_thread_t)); bam_main -> all_threads = all_threads; bam_main -> has_dummy = has_dummy; bam_main -> compression_level = c_level; subread_init_lock(&bam_main -> output_fp_lock); for(x1 = 0; x1 < all_threads ; x1 ++){ bam_main -> threads[x1].BIN_buffer_ptr = 0; bam_main -> threads[x1].strm.zalloc = Z_NULL; bam_main -> threads[x1].strm.zfree = Z_NULL; bam_main -> threads[x1].strm.opaque = Z_NULL; bam_main -> threads[x1].strm.avail_in = 0; bam_main -> threads[x1].strm.next_in = Z_NULL; deflateInit2(&bam_main -> threads[x1].strm, bam_main -> compression_level, Z_DEFLATED, PAIRER_GZIP_WINDOW_BITS, PAIRER_DEFAULT_MEM_LEVEL, Z_DEFAULT_STRATEGY); } return 0; } void SAM_pairer_write_BAM_header(FILE * writer, int compressed_size) { // the four magic characters fputc(31, writer); fputc(139, writer); fputc(8, writer); fputc(4, writer); time_t time_now = 0; fwrite(&time_now,4,1, writer); int tmp_i; // Extra flags and OS fputc(0, writer); fputc(0xff, writer); // Extra length tmp_i = 6; fwrite(&tmp_i,2,1, writer); // SI1 and SI2 magic numbers, and SLEN fputc(66, writer); fputc(67, writer); tmp_i = 2; fwrite(&tmp_i,2,1, writer); tmp_i = compressed_size + 19 + 6; fwrite(&tmp_i,2,1, writer); } int SAM_pairer_multi_thread_compress(SAM_pairer_writer_main_t * bam_main , SAM_pairer_writer_thread_t * bam_thread) { #define BAM_compressed_space 65536 char * BAM_compressed = malloc(BAM_compressed_space); int ret, have; if(bam_thread -> BIN_buffer_ptr>0){ deflateReset(&bam_thread -> strm); bam_thread -> strm.avail_in = bam_thread -> BIN_buffer_ptr; bam_thread -> strm.next_in = bam_thread -> BIN_buffer; bam_thread -> strm.avail_out = BAM_compressed_space; bam_thread -> strm.next_out = (unsigned char *)BAM_compressed; ret = deflate( &bam_thread -> strm , Z_FINISH); have = BAM_compressed_space - bam_thread -> strm.avail_out; assert(bam_thread -> strm.avail_in == 0); }else{ z_stream nstrm; nstrm.zalloc = Z_NULL; nstrm.zfree = Z_NULL; nstrm.opaque = Z_NULL; nstrm.avail_in = 0; nstrm.next_in = Z_NULL; deflateInit2(&nstrm, SAMBAM_COMPRESS_LEVEL_NORMAL, Z_DEFLATED, PAIRER_GZIP_WINDOW_BITS, PAIRER_DEFAULT_MEM_LEVEL, Z_DEFAULT_STRATEGY); nstrm.avail_in = 0; nstrm.next_in = bam_thread -> BIN_buffer; nstrm.avail_out = BAM_compressed_space; nstrm.next_out = (unsigned char *)BAM_compressed; ret = deflate(&nstrm, Z_FINISH); deflateEnd(&nstrm); have = BAM_compressed_space - nstrm.avail_out; } if(ret == Z_OK || 1){ //SUBREADprintf("Compress: %d -> %d %p\n", bam_thread -> BIN_buffer_ptr, have, bam_main -> bam_fp); //if(bam_thread -> BIN_buffer_ptr == 0) have = 0; unsigned int crc0 = crc32(0, NULL, 0); unsigned int CRC32 = crc32(crc0, (unsigned char *) bam_thread -> BIN_buffer ,bam_thread -> BIN_buffer_ptr); subread_lock_occupy( &bam_main -> output_fp_lock ); SAM_pairer_write_BAM_header( bam_main -> bam_fp , have); fwrite(BAM_compressed,1, have, bam_main -> bam_fp ); fwrite(&CRC32 , 4, 1, bam_main -> bam_fp); fwrite( &bam_thread -> BIN_buffer_ptr , 4, 1, bam_main -> bam_fp); subread_lock_release( &bam_main -> output_fp_lock ); bam_thread -> BIN_buffer_ptr = 0; } else { SUBREADprintf("ERROR: Cannot compress a BAM block : %d\n", ret); return 1; } free(BAM_compressed); return 0; } void SAM_pairer_writer_destroy( SAM_pairer_writer_main_t * bam_main ) { int x1; for(x1 = 0; x1 < bam_main -> all_threads ; x1 ++){ if(bam_main -> threads[x1].BIN_buffer_ptr>0){ SAM_pairer_multi_thread_compress(bam_main, bam_main->threads+x1); } if(x1 == bam_main -> all_threads - 1){ assert(0 == bam_main -> threads[x1].BIN_buffer_ptr); SAM_pairer_multi_thread_compress(bam_main, bam_main->threads+x1); } deflateEnd(&bam_main -> threads[x1].strm); } subread_destroy_lock(&bam_main -> output_fp_lock); fclose(bam_main -> bam_fp); free(bam_main -> threads); } void SAM_pairer_set_unsorted_notification(SAM_pairer_context_t * pairer, void (* unsorted_notification) (void * pairer, char * bin1, char * bin2)){ pairer -> unsorted_notification = unsorted_notification; } int SAM_pairer_warning_file_open_limit(){ #ifndef __MINGW32__ struct rlimit limit_st; getrlimit(RLIMIT_NOFILE, & limit_st); if(min(limit_st.rlim_cur, limit_st.rlim_max ) < MIN_FILE_POINTERS_ALLOWED){ SUBREADprintf(" ERROR: the maximum file open number (%d) is too low. Please increase this number to a number larger than 50 by using the 'ulimit -n' command.\n\n",(int)(min(limit_st.rlim_cur, limit_st.rlim_max))); return 1; } #endif return 0; } #define BAM_EOF_BLOCK_BIN "\x1f\x8b\x08\x04\x00\x00\x00\x00\x00\xff\x06\x00\x42\x43\x02\x00\x1b\x00\x03\x00\x00\x00\x00\x00\x00\x00\x00\x00" #define BAM_EOF_BLOCK_LEN 28 int BAM_check_EOF_block(char * in_file){ char testingbin [BAM_EOF_BLOCK_LEN]; FILE * fp = f_subr_open(in_file, "rb"); if(!fp) return 0; // this fuction doesn't care whether the file exists. fseeko(fp, -BAM_EOF_BLOCK_LEN, SEEK_END); int frl = fread(testingbin, BAM_EOF_BLOCK_LEN,1, fp); fclose(fp); if(frl<1) return -1; if(memcmp(testingbin, BAM_EOF_BLOCK_BIN, BAM_EOF_BLOCK_LEN)!=0) return -1; return 0; } // Tiny_Mode only write the following information: // Name Flag Chro Pos Mapq Cigar MateChro MatePos Tlen N I NH:i:xx HI:i:xx // Tiny_Mode does not work when output and input are both in BAM format // in_format can be either // bin_buff_size_per_thread is in Mega-Bytes. // It returns 0 if no error int SAM_pairer_create(SAM_pairer_context_t * pairer, int all_threads, int bin_buff_size_per_thread, int BAM_input, int is_Tiny_Mode, int is_single_end_mode, int force_do_not_sort, int need_read_group_tag, int display_progress, char * in_file, void (* reset_output_function) (void * pairer), int (* output_header_function) (void * pairer, int thread_no, int is_text, unsigned int items, char * bin, unsigned int bin_len), int (* output_function) (void * pairer, int thread_no, char * bin1, char * bin2), char * tmp_path, void * appendix1, int long_read_minimum_length) { memset(pairer, 0, sizeof(SAM_pairer_context_t)); if(in_file[0]=='<'){ in_file++; strncpy(pairer -> in_file_name, "", MAX_FILE_NAME_LENGTH); }else strncpy(pairer -> in_file_name, in_file, MAX_FILE_NAME_LENGTH); if(BAM_input && in_file[0]!='<' && BAM_check_EOF_block(in_file)){ SUBREADprintf("ERROR: the BAM input file, '%s', doesn't have a valid EOF block.\n", in_file); return 1; } pairer -> input_fp = f_subr_open(in_file, "rb"); if(NULL == pairer -> input_fp) return 1; SAM_pairer_warning_file_open_limit(); pairer -> input_is_BAM = BAM_input; pairer -> tiny_mode = is_Tiny_Mode; pairer -> reset_output_function = reset_output_function; pairer -> output_function = output_function; pairer -> output_header = output_header_function; pairer -> display_progress = display_progress; pairer -> is_single_end_mode = is_single_end_mode; pairer -> force_do_not_sort = force_do_not_sort; pairer -> need_read_group_tag = need_read_group_tag; pairer -> long_read_minimum_length = long_read_minimum_length; subread_init_lock(&pairer -> unsorted_notification_lock); subread_init_lock(&pairer -> input_fp_lock); subread_init_lock(&pairer -> SAM_BAM_table_lock); pairer -> total_threads = all_threads; if(pairer ->input_is_BAM){ pairer -> input_buff_SBAM_size = bin_buff_size_per_thread * 1024 * 1024; }else{ pairer -> input_buff_SBAM_size = max(bin_buff_size_per_thread * 1024 * 1024 + FC_LONG_READ_RECORD_HARDLIMIT , 3*FC_LONG_READ_RECORD_HARDLIMIT/2); } pairer -> input_buff_BIN_size = max(1024*1024, pairer -> input_buff_SBAM_size ); pairer -> appendix1 = appendix1; old_sig_TERM = signal (SIGTERM, REPAIR_SIGINT_hook); old_sig_INT = signal (SIGINT, REPAIR_SIGINT_hook); strcpy(pairer -> tmp_file_prefix, tmp_path); _REPAIRER_delete_temp_prefix = pairer -> tmp_file_prefix; pairer -> threads = malloc(all_threads * sizeof(SAM_pairer_thread_t)); memset(pairer -> threads, 0, all_threads * sizeof(SAM_pairer_thread_t)); if(pairer ->input_is_BAM){ pairer ->bam_margin_table = HashTableCreate(2191); HashTableSetHashFunction(pairer -> bam_margin_table, fc_chro_hash); HashTableSetKeyComparisonFunction(pairer -> bam_margin_table, fc_strcmp_chro); HashTableSetDeallocationFunctions(pairer -> bam_margin_table, free, free); }else{ pairer -> sam_contig_number_table = HashTableCreate(21907); HashTableSetHashFunction(pairer -> sam_contig_number_table, fc_chro_hash); HashTableSetKeyComparisonFunction(pairer -> sam_contig_number_table, fc_strcmp_chro); HashTableSetDeallocationFunctions(pairer -> sam_contig_number_table, free, NULL); } pairer -> unsorted_notification_table = HashTableCreate(2191); HashTableSetHashFunction(pairer -> unsorted_notification_table, fc_chro_hash); HashTableSetKeyComparisonFunction(pairer -> unsorted_notification_table, fc_strcmp_chro); HashTableSetDeallocationFunctions(pairer -> unsorted_notification_table, free, free); int x1; for(x1 = 0; x1 < all_threads ; x1++){ pairer -> threads[x1].thread_id = x1; pairer -> threads[x1].reads_in_SBAM = 0; pairer -> threads[x1].input_buff_SBAM = malloc(pairer -> input_buff_SBAM_size); pairer -> threads[x1].input_buff_BIN_capacity = pairer -> input_buff_BIN_size; pairer -> threads[x1].input_buff_BIN = malloc(pairer -> threads[x1].input_buff_BIN_capacity ); pairer -> threads[x1].input_buff_BIN_used = 0; pairer -> threads[x1].orphant_table = HashTableCreate(pairer -> input_buff_SBAM_size / 100); HashTableSetHashFunction(pairer -> threads[x1].orphant_table, fc_chro_hash); HashTableSetKeyComparisonFunction(pairer -> threads[x1].orphant_table, fc_strcmp_chro); HashTableSetDeallocationFunctions(pairer -> threads[x1].orphant_table, free, free); pairer -> threads[x1].strm.zalloc = Z_NULL; pairer -> threads[x1].strm.zfree = Z_NULL; pairer -> threads[x1].strm.opaque = Z_NULL; pairer -> threads[x1].strm.avail_in = 0; pairer -> threads[x1].strm.next_in = Z_NULL; inflateInit2(&pairer -> threads[x1].strm, PAIRER_GZIP_WINDOW_BITS); if(force_do_not_sort) subread_init_lock(&pairer -> threads[x1].SBAM_lock); } return 0; } void SAM_pairer_print_keys(void * key, void * hashed_obj, HashTable * tab){ int dlen =0; memcpy(&dlen, hashed_obj,4); SUBREADprintf("ESKY = %s LEN = %d\n",(char*)key,dlen); } void SAM_pairer_destroy(SAM_pairer_context_t * pairer){ int x1; srInt_64 all_orphants = 0; for(x1 = 0; x1 < pairer -> total_threads ; x1++){ inflateEnd(&pairer -> threads[x1].strm); free(pairer -> threads[x1].input_buff_BIN); free(pairer -> threads[x1].input_buff_SBAM); if(pairer -> force_do_not_sort) subread_destroy_lock(&pairer -> threads[x1].SBAM_lock); all_orphants += pairer -> threads[x1].orphant_table->numOfElements; HashTableDestroy(pairer -> threads[x1].orphant_table); } if(pairer->input_is_BAM){ //HashTableIteration(pairer -> bam_margin_table, SAM_pairer_print_keys); HashTableDestroy(pairer -> bam_margin_table); } else HashTableDestroy(pairer -> sam_contig_number_table); HashTableDestroy(pairer -> unsorted_notification_table); subread_destroy_lock(&pairer -> unsorted_notification_lock); subread_destroy_lock(&pairer -> input_fp_lock); subread_destroy_lock(&pairer -> SAM_BAM_table_lock); //#warning "==== NOT DELETING TEMP ====" // if(0) delete_with_prefix(pairer -> tmp_file_prefix); fclose(pairer -> input_fp); free(pairer -> threads); signal (SIGTERM, old_sig_TERM); signal (SIGINT, old_sig_INT); //SUBREADprintf("All orphans=%llu frags\n", all_orphants); } // always assume that fp is at the start of a BAM GZ block. int SAM_pairer_read_BAM_block(FILE * fp, int max_read_len, char * inbuff) { unsigned char gz_header_12 [12]; //SUBREADprintf("STAT GZ POS=%llu\n", ftello(fp)); int read_len = fread(gz_header_12, 1, 12, fp ); if(read_len < 12){ return -1; } if(gz_header_12[0]!=31 || gz_header_12[1]!=139){ //SUBREADprintf("Unrecognized Gzip headers: %u, %u\nPlease make sure if the input file is in the BAM format.\n", gz_header_12[0], gz_header_12[1]); return -1; } unsigned short xlen = 0, bsize = 0; memcpy(&xlen, gz_header_12 + 10, 2); int xlen_read = 0; while( xlen_read < xlen ){ unsigned char x_header_4[4]; unsigned short slen = 0; read_len = fread(x_header_4, 1, 4, fp); if(read_len < 4){ SUBREADprintf("BAD GZ BAM 6LEN\n"); return -1; } memcpy(&slen, x_header_4+2 , 2); xlen_read += 4; if(x_header_4[0]==66 && x_header_4[1]==67 && slen == 2){ read_len = fread(&bsize, 2, 1, fp); if(read_len < 1){ SUBREADprintf("BAD GZ BAM XLEN\n"); return -1; } }else{ fseeko(fp, slen, SEEK_CUR); } xlen_read += slen; } if(bsize < 1 || bsize < xlen + 19){ SUBREADprintf("BAD GZ BAM BSIZE\n"); return -1; } read_len = fread(inbuff, 1, bsize - xlen - 19, fp); //SUBREADprintf("ABBO : GOOD GZ , LEN=%d , POS=%llu\n", read_len, ftello(fp)); // seek over CRC and ISIZE fseeko(fp, 8, SEEK_CUR); if(read_len < bsize - xlen - 19) return -1; return read_len; } #define MIN_BAM_BLOCK_SIZE 66000 int SAM_pairer_read_SAM_MB( FILE * fp, int max_read_len, char * inbuff ){ int ret = 0; if(feof(fp)) return 0; while(1){ if(ret >= max_read_len - FC_LONG_READ_RECORD_HARDLIMIT || feof(fp))break; int rlen = fread(inbuff +ret , 1, max_read_len - FC_LONG_READ_RECORD_HARDLIMIT - ret , fp); if(rlen > 0){ int x1; for(x1 = 0; x1 < min(200, rlen); x1++) if(*(inbuff+ret+x1)<8 || *(inbuff+ret+x1)> 127){ SUBREADprintf("NOT_SAM_ACTUALLY\n"); return -1; } ret += rlen; } } if(!feof(fp)){ int nch; while(1){ nch = fgetc(fp); if(nch < 0 || nch == '\n'){ break; }else{ inbuff[ret++]=nch; } } } if(inbuff[ret-1] != '\n') inbuff[ret++]='\n'; inbuff[ret] = 0; return ret; } void SAM_pairer_fill_BIN_buff(SAM_pairer_context_t * pairer , SAM_pairer_thread_t * thread_context , int * is_finished){ // load continuous 64MB of data into the SBAM buffer of the current thread // For BAM files: must be the entire blocks. // For SAM files: must be the full lines. int current_buffer_used = 0; int current_blocks = 0; int last_read_len = -1, this_size; if(pairer -> input_is_BAM){ thread_context -> input_buff_SBAM_file_start = ftello(pairer -> input_fp); while(1){ if( feof(pairer -> input_fp)){ *is_finished = 1; break; } if(pairer -> input_buff_SBAM_size - current_buffer_used < MIN_BAM_BLOCK_SIZE) { break; } this_size = SAM_pairer_read_BAM_block( pairer -> input_fp , pairer -> input_buff_SBAM_size - current_buffer_used , thread_context -> input_buff_SBAM + current_buffer_used); current_blocks ++; if(this_size >= 0) { current_buffer_used += this_size; } else { if(feof(pairer -> input_fp) && last_read_len != -1 ){ pairer -> is_bad_format |= (last_read_len > 2); pairer -> is_incomplete_BAM |= (last_read_len > 2); if(pairer -> is_incomplete_BAM)SUBREADprintf("ERROR: the BAM file seems incomplete : this %d, last %d.\n", this_size , last_read_len ); } *is_finished = 1; break; } last_read_len = this_size; } thread_context -> input_buff_SBAM_file_end = ftello(pairer -> input_fp); }else{ // is_SAM current_buffer_used = SAM_pairer_read_SAM_MB(pairer -> input_fp , pairer -> input_buff_SBAM_size , thread_context -> input_buff_SBAM); if(current_buffer_used < 1) *is_finished = 1; } //SUBREADprintf("PAPA:READ=%d by %d blocks %p, PTRS=%p %p\n", current_buffer_used, current_blocks, thread_context, thread_context -> input_buff_SBAM, thread_context -> input_buff_BIN); thread_context -> input_buff_SBAM_used = current_buffer_used; thread_context -> input_buff_SBAM_ptr = 0; thread_context -> input_buff_BIN_used = 0; thread_context -> input_buff_BIN_ptr = 0; thread_context -> readno_in_chunk = 0; } int SAM_pairer_find_start(SAM_pairer_context_t * pairer , SAM_pairer_thread_t * thread_context ); #define BAM_next_nch { \ int retXX = 0; while(thread_context -> input_buff_BIN_ptr >= thread_context -> input_buff_BIN_used){retXX = SAM_pairer_fetch_BAM_block(pairer, thread_context); if(retXX) break;}\ if(retXX) nch=-1; else nch = thread_context -> input_buff_BIN[thread_context -> input_buff_BIN_ptr++];} #define SAM_next_line {\ if( thread_context -> input_buff_SBAM_used <= thread_context -> input_buff_SBAM_ptr ){ line_ptr = NULL;}else{\ line_ptr = thread_context -> input_buff_SBAM + thread_context -> input_buff_SBAM_ptr;line_len = 0;\ while(line_len + thread_context -> input_buff_SBAM_ptr < thread_context -> input_buff_SBAM_used){ int ccch = thread_context -> input_buff_SBAM[ thread_context -> input_buff_SBAM_ptr + line_len ]; if(ccch == '\n')break; line_len ++;}\ thread_context -> input_buff_SBAM_ptr += line_len+1;}} int SAM_pairer_fetch_BAM_block(SAM_pairer_context_t * pairer , SAM_pairer_thread_t * thread_context){ if(thread_context -> input_buff_SBAM_used <= thread_context -> input_buff_SBAM_ptr){ return 1; } int remained_BIN = thread_context -> input_buff_BIN_used - thread_context -> input_buff_BIN_ptr; if( remained_BIN > 0) { int x1; for(x1 = 0 ; x1 < thread_context -> input_buff_BIN_used - thread_context -> input_buff_BIN_ptr; x1++) thread_context -> input_buff_BIN[x1] = thread_context -> input_buff_BIN[x1+thread_context -> input_buff_BIN_ptr]; thread_context -> input_buff_BIN_used -= thread_context -> input_buff_BIN_ptr; } else thread_context -> input_buff_BIN_used = 0; thread_context -> input_buff_BIN_ptr = 0; inflateReset(&thread_context -> strm); int lin, lout; lin=thread_context -> strm.avail_in = (unsigned int)(thread_context -> input_buff_SBAM_used - thread_context -> input_buff_SBAM_ptr); thread_context -> strm.next_in = (unsigned char *)thread_context -> input_buff_SBAM + thread_context -> input_buff_SBAM_ptr; if( thread_context -> input_buff_BIN_capacity < thread_context -> input_buff_BIN_used + 128*1024){ thread_context -> input_buff_BIN_capacity = max(thread_context -> input_buff_BIN_used, thread_context -> input_buff_BIN_capacity )*1.5; if(thread_context -> input_buff_BIN_capacity > 1024*1024*1024){ SUBREADprintf("ERROR: buffer size larger than 1GB\n"); return 1; }else{ //SUBREADprintf("Resize Buffer of Th_%d to %d (used %d); In_ava=%d - %d\n", thread_context -> thread_id, thread_context -> input_buff_BIN_capacity, thread_context -> input_buff_BIN_used, thread_context -> input_buff_SBAM_used , thread_context -> input_buff_SBAM_ptr); } thread_context -> input_buff_BIN = realloc( thread_context -> input_buff_BIN , thread_context -> input_buff_BIN_capacity); //SUBREADprintf(" PTR=%p\n",thread_context -> input_buff_BIN); assert( thread_context -> input_buff_BIN ); } lout=thread_context -> strm.avail_out = thread_context -> input_buff_BIN_capacity - thread_context -> input_buff_BIN_used; thread_context -> strm.next_out = (unsigned char *)thread_context -> input_buff_BIN + thread_context -> input_buff_BIN_used; int ret = inflate(&thread_context ->strm, Z_FINISH); if(ret == Z_OK || ret == Z_STREAM_END) { int have = lout - thread_context ->strm.avail_out; int used_BAM = lin - thread_context -> strm.avail_in; //SUBREADprintf("ABBO TH %d : INFLATED BAM_CONSUMED: %d BIN_USED: %d => %d NEED_FIND_START=%d\n", thread_context -> thread_id, used_BAM, thread_context -> input_buff_BIN_used , thread_context -> input_buff_BIN_used+have, thread_context -> need_find_start); thread_context -> input_buff_BIN_used += have; thread_context -> input_buff_SBAM_ptr += used_BAM; if(thread_context -> need_find_start){ int test_read_bin = SAM_pairer_find_start(pairer, thread_context); if(test_read_bin<1 && thread_context -> input_buff_BIN_used >= 32 ){ pairer -> is_bad_format = 1; //SUBREADprintf("ERROR: cannot find the start of the next BAM block.\n"); print_in_box(80,0,0," Switch to the safe mode."); } } //SUBREADprintf("FETCHED BLOCK DECOMP=%d FROM COMP=%d\n", have, used_BAM); } else { if(ret == -5){ SUBREADprintf("Cannot parse the input BAM file.\n"); }else{ SUBREADprintf("GZIP ERROR:%d\n", ret); } pairer -> is_bad_format = 1; pairer -> is_internal_error = 1; return 1; } return 0; } #define BAM_next_u32(v) {\ (v) = 0; unsigned int poww = 1 ; \ BAM_next_nch; (v) += nch*poww; poww *= 256;\ BAM_next_nch; (v) += nch*poww; poww *= 256;\ BAM_next_nch; (v) += nch*poww; poww *= 256;\ BAM_next_nch; (v) += nch*poww;\ } void SAM_pairer_reduce_BAM_bin(SAM_pairer_context_t * pairer, SAM_pairer_thread_t * thread_context, unsigned char * bin_where, int * bin_len){ unsigned int seq_len, name_len, cigar_ops; memcpy(&seq_len, bin_where + 20, 4); if(seq_len<=1) return; memcpy(&name_len, bin_where + 12, 4); name_len = name_len & 0xff; memcpy(&cigar_ops, bin_where + 16, 4); cigar_ops = cigar_ops & 0xffff; int targ_pos = 36+name_len+4*cigar_ops + 2; int src_pos = 36+name_len+4*cigar_ops + (1+seq_len) / 2 + seq_len; bin_where[targ_pos-2]=0xff; bin_where[targ_pos-1]=0xff; //SUBREADprintf("REDUCE by Thread %d : %d -> %d in %d ; seq_len = %u\n", thread_context -> thread_id, src_pos, targ_pos, *bin_len, seq_len); seq_len = 1; memcpy(bin_where + 20, &seq_len, 4); while(src_pos < (*bin_len)){ bin_where[targ_pos++]=bin_where[src_pos++]; } (* bin_len) = targ_pos - 4; memcpy(bin_where, bin_len, 4); (* bin_len) += 4; } #define MAX_BIN_RECORD_LENGTH ( 20*1024*1024) int reduce_SAM_to_BAM(SAM_pairer_context_t * pairer , SAM_pairer_thread_t * thread_context, int include_sequence); int is_read_bin(char * bin, int bin_len, int max_refID); int SAM_pairer_get_next_read_BIN( SAM_pairer_context_t * pairer , SAM_pairer_thread_t * thread_context , unsigned char ** bin_where, int * bin_len ) { if( pairer -> input_is_BAM ){ int nch = 0; while(1){ if(!pairer -> BAM_header_parsed){ int x1; unsigned int bam_signature; BAM_next_u32(bam_signature); BAM_next_u32(pairer -> BAM_l_text); char * header_txt = NULL; int header_txt_dynamic_length = -1; if(pairer->BAM_l_text>0) header_txt = malloc(max(1000000,pairer->BAM_l_text)); for(x1 = 0 ; x1 < pairer -> BAM_l_text; x1++){ BAM_next_nch; header_txt [x1] = nch; } int is_OK = 0; if(pairer -> output_header)pairer -> output_header(pairer, thread_context -> thread_id, 1, pairer -> BAM_l_text , header_txt , pairer -> BAM_l_text ); BAM_next_u32(pairer -> BAM_n_ref); unsigned int ref_bin_len = 0; for(x1 = 0; x1 < pairer -> BAM_n_ref; x1++) { unsigned int l_name, l_ref, x2; BAM_next_u32(l_name); assert(l_name < 256); if(header_txt == NULL){ header_txt = malloc(3000000); header_txt_dynamic_length = 3000000; } if( header_txt_dynamic_length>0 && ref_bin_len > header_txt_dynamic_length - 1000000 ){ header_txt_dynamic_length *= 2; header_txt = realloc( header_txt, header_txt_dynamic_length); } memcpy(header_txt + ref_bin_len, &l_name, 4); ref_bin_len += 4; for(x2 = 0; x2 < l_name; x2++){ BAM_next_nch; header_txt[ref_bin_len++] = nch; } BAM_next_u32(l_ref); memcpy(header_txt + ref_bin_len, &l_ref, 4); ref_bin_len += 4; } is_OK = is_OK || (pairer -> output_header?pairer -> output_header(pairer, thread_context -> thread_id, 0, pairer -> BAM_n_ref , header_txt , ref_bin_len ):0); //SUBREADprintf("TFMT:HEADER REFS=%d TXTS=%d SIGN=%u\n", pairer -> BAM_n_ref, pairer->BAM_l_text, bam_signature); if(header_txt) free(header_txt); if(is_OK){ pairer -> is_incomplete_BAM = 1; return 0; } pairer -> BAM_header_parsed = 1; SAM_pairer_fetch_BAM_block(pairer, thread_context); } if(pairer -> is_bad_format) return 0; unsigned int record_len = 0xffffffff; while(1){ if(thread_context -> input_buff_BIN_ptr <= thread_context -> input_buff_BIN_used - 4) memcpy(&record_len, thread_context -> input_buff_BIN + thread_context -> input_buff_BIN_ptr, 4); if(record_len < 0xfffffff0 && thread_context -> input_buff_BIN_ptr +4 + record_len <= thread_context -> input_buff_BIN_used){ break; } int ret_fetch = SAM_pairer_fetch_BAM_block(pairer, thread_context); // if ret != 0 then load next big chunk of BAM. if(ret_fetch){ if(thread_context -> input_buff_BIN_used > thread_context -> input_buff_BIN_ptr){ char * margin_key = malloc(40); char * margin_data = malloc(thread_context -> input_buff_BIN_used - thread_context -> input_buff_BIN_ptr+4); int margin_size = thread_context -> input_buff_BIN_used - thread_context -> input_buff_BIN_ptr; memcpy(margin_data, &margin_size, 4); memcpy(margin_data+4, thread_context -> input_buff_BIN + thread_context -> input_buff_BIN_ptr, thread_context -> input_buff_BIN_used - thread_context -> input_buff_BIN_ptr); #ifdef __MINGW32__ sprintf(margin_key,"E%lu", (unsigned long)thread_context -> input_buff_SBAM_file_end); #else sprintf(margin_key,"E%llu", thread_context -> input_buff_SBAM_file_end); #endif subread_lock_occupy(&pairer -> SAM_BAM_table_lock); HashTablePut(pairer -> bam_margin_table, margin_key, margin_data); subread_lock_release(&pairer -> SAM_BAM_table_lock); } return 0; } } //SUBREADprintf("TFMT:RLEN=%d\n", record_len); if(!pairer -> is_bad_format){ unsigned int seq_len = 0; thread_context -> input_buff_BIN_ptr += 4; memcpy(&seq_len, thread_context -> input_buff_BIN + thread_context -> input_buff_BIN_ptr + 16, 4); //SUBREADprintf("REDUCE_2: record %u, %u\n", record_len, seq_len); // #warning "=========== CHECK IF '0 && ' IS CORRECT ===========" if(record_len < 32 || (0 && record_len > min(MAX_BIN_RECORD_LENGTH,60000))|| seq_len >= pairer -> long_read_minimum_length){ if(seq_len >= pairer -> long_read_minimum_length) pairer -> is_single_end_mode = 1; SUBREADprintf("ERROR: sequence length in the BAM record is out of the expected region: %d, %d\n", record_len , seq_len ); pairer -> is_bad_format = 1; return 0; } (* bin_where) = thread_context -> input_buff_BIN + thread_context -> input_buff_BIN_ptr - 4; (* bin_len) = record_len + 4; thread_context -> input_buff_BIN_ptr += record_len; } return 1; } } else { // if input is SAM char *line_ptr; int line_len=0, passed_read_SBAM_ptr = -1; if(!pairer -> BAM_header_parsed){ char * header_start = NULL; int header_len = 0; while(1){ SAM_next_line; //SUBREADprintf("LINE_PTR[%d][used bytes=%d]='''%s'''\n", thread_context -> thread_id, thread_context -> input_buff_SBAM_used, line_ptr); if(NULL == header_start && line_ptr[0] == '@') header_start = line_ptr; if(NULL == line_ptr){ passed_read_SBAM_ptr = line_ptr - thread_context -> input_buff_SBAM; //SUBREADprintf("FATAL: the header is too large to the buffer.\n"); break; }else{ //SUBREADprintf("LINELEN=%d, PTR=%d, FIRST=%c\n", line_len, thread_context -> input_buff_SBAM_ptr , line_ptr[0]); } if(line_ptr[0]=='@'){ header_len += 1+line_len; }else{ passed_read_SBAM_ptr = line_ptr - thread_context -> input_buff_SBAM; break; } } int is_OK = pairer -> output_header(pairer, thread_context -> thread_id, 1, header_len , header_start , header_len); thread_context -> input_buff_SBAM_ptr = 0; int header_bin_ptr = 0, header_contigs = 0; while(1){ SAM_next_line; if(line_ptr == NULL || line_ptr[0]!='@') break; if(memcmp(line_ptr, "@SQ\t",4)==0){ unsigned int ct_len = 0, ctptr = 4, status = 0, sqname_len = 0; char * sqname = NULL; while(1){ char ctnch = line_ptr[ctptr++]; if( status == 0){ if(ctnch=='S' && line_ptr[ctptr] == 'N' && line_ptr[ctptr+1] == ':'){ ctptr += 2; status = 10; sqname = line_ptr + ctptr; }else if(ctnch=='L' && line_ptr[ctptr] == 'N' && line_ptr[ctptr+1] == ':'){ ctptr += 2; status = 20; }else status = 30; }else if(status == 10 || status == 20 || status == 30){ if(ctnch == '\t' || ctnch == '\n'){ status = 0; if(ctnch == '\n') break; //break; } if(status == 10) sqname_len ++; else if(status == 20) ct_len = ct_len * 10 + ctnch - '0'; } } sqname_len += 1; memcpy(header_start + header_bin_ptr, &sqname_len, 4); header_bin_ptr += 4; memcpy(header_start + header_bin_ptr, sqname, sqname_len-1); *(header_start + header_bin_ptr + sqname_len - 1) = 0; char * mem_contig_name = malloc(sqname_len); strcpy(mem_contig_name , header_start + header_bin_ptr); //SUBREADprintf("CONTIG %d : %s (len=%d = %d)\n", header_contigs, header_start + header_bin_ptr , sqname_len, strlen(mem_contig_name)); HashTablePut(pairer -> sam_contig_number_table , mem_contig_name, NULL + 1 + header_contigs); header_bin_ptr += sqname_len; memcpy(header_start + header_bin_ptr, &ct_len, 4); header_bin_ptr += 4; header_contigs++; } } is_OK = is_OK || pairer -> output_header(pairer, thread_context -> thread_id, 0, header_contigs , header_start , header_bin_ptr); pairer -> BAM_header_parsed = 1; if(is_OK){ pairer -> is_incomplete_BAM = 1; return 0; } } if(passed_read_SBAM_ptr >=0) thread_context -> input_buff_SBAM_ptr = passed_read_SBAM_ptr; if( thread_context -> input_buff_SBAM_ptr < thread_context -> input_buff_SBAM_used ){ thread_context -> input_buff_BIN_ptr = 0; //SUBREADprintf("reduce_SAM_to_BAM_0 \n"); *bin_len = reduce_SAM_to_BAM(pairer, thread_context,!pairer -> tiny_mode); *bin_where = (unsigned char *)thread_context -> input_buff_BIN; return ((*bin_len) > 0 && !pairer->is_bad_format)?1:0; } return 0; } return 0; } int online_register_contig(SAM_pairer_context_t * pairer , SAM_pairer_thread_t * thread_context, char * ref){ SUBREADprintf("ERROR: Unable to find chromosome '%s' in the SAM header.\n", ref); assert(0); int reflen = strlen(ref); char * header_sec = malloc(reflen + 20); reflen++; memcpy(header_sec, &reflen, 4); memcpy(header_sec + 4, ref, reflen); memset(header_sec + 4+reflen, 0, 4); subread_lock_occupy(&pairer -> SAM_BAM_table_lock); int refId = HashTableGet(pairer->sam_contig_number_table, ref) - NULL - 1; if(refId < 0){ refId = pairer->sam_contig_number_table->numOfElements; pairer -> output_header(pairer, thread_context -> thread_id, 0, 1 , header_sec , 8+reflen); char * mem_ref = malloc(reflen+1); memcpy(mem_ref, ref, reflen); mem_ref[reflen]=0; HashTablePut(pairer->sam_contig_number_table, mem_ref, NULL + refId + 1); } subread_lock_release(&pairer -> SAM_BAM_table_lock); free(header_sec); return refId; } #define set_memory_int(ptr, iii) { *(ptr) = (iii)&0xff; *(ptr+1) = (iii>>8)&0xff; *(ptr+2) = (iii>>16)&0xff;*(ptr+3) = (iii>>24); } int reduce_SAM_to_BAM(SAM_pairer_context_t * pairer , SAM_pairer_thread_t * thread_context, int include_sequence){ int column_no = 0, in_ptr = 0; char * in_str = thread_context -> input_buff_SBAM + thread_context -> input_buff_SBAM_ptr; char * read_name = NULL, * ref = NULL, * mate_ref = NULL, * cigar = NULL, * seq = NULL, * qual = NULL; int flag = 0, pos = 0, mapq = 0, old_read_pos = 0, tlen = 0, l_read_name = 0, tlen_sign = 1, l_seq = 0; read_name = in_str; while(1){ int nch = in_str[in_ptr]; if(nch == '\n' || nch == '\0') { break; }else if(nch == '\t'){ if(column_no == 0 || column_no == 2 || column_no == 5 || column_no == 6 || column_no == 9) in_str[in_ptr] = 0; column_no ++; if(column_no == 2) ref = in_str + in_ptr + 1; else if(column_no == 5) cigar = in_str + in_ptr + 1; else if(column_no == 6) mate_ref = in_str + in_ptr + 1; else if(column_no == 9) seq = in_str + in_ptr + 1; else if(column_no == 10) qual = in_str + in_ptr + 1; else if(column_no == 11) break; }else{ if(column_no == 0) l_read_name ++; else if(column_no == 1) flag = flag *10 + nch - '0'; else if(column_no == 3) pos = pos *10 + nch - '0'; else if(column_no == 4) mapq = mapq *10 + nch - '0'; else if(column_no == 7) old_read_pos = old_read_pos *10 + nch - '0'; else if(column_no == 9) l_seq ++; else if(column_no == 8){ if(nch == '-') tlen_sign = -1; else tlen = tlen *10 + nch - '0'; } } in_ptr++; } if(column_no < 10){ //SUBREADprintf("RETURN_LESS:%d\n", column_no); return -1; } l_read_name++; char * bin_tmp = (char *)thread_context -> input_buff_BIN + thread_context -> input_buff_BIN_ptr; int refID = HashTableGet(pairer->sam_contig_number_table, ref) - NULL - 1; if(refID < 0 && ref[0]!='*') refID = online_register_contig(pairer, thread_context, ref); set_memory_int(bin_tmp + 4, refID); pos -= 1; set_memory_int(bin_tmp + 8, pos); int mapq_nl = mapq << 8 | l_read_name; set_memory_int(bin_tmp + 12, mapq_nl); int coverage; int cigar_ops = SamBam_compress_cigar(cigar, (int *)(bin_tmp + 36 + l_read_name), &coverage, 65535); int flag_nc = flag << 16 | cigar_ops; set_memory_int(bin_tmp + 16, flag_nc); int seq_len = qual - seq - 1; if(seq_len >=pairer -> long_read_minimum_length ){ pairer -> is_single_end_mode = 1; include_sequence = 0; pairer -> tiny_mode = 1; pairer -> long_cigar_mode = 1; } if(include_sequence){ set_memory_int(bin_tmp + 20, l_seq); // SEQ_LEN }else set_memory_int(bin_tmp + 20, 1); int mate_refID = refID; if(mate_ref[0]!='=' || mate_ref[1]!=0) mate_refID = HashTableGet(pairer->sam_contig_number_table, mate_ref) - NULL - 1; if(mate_refID < 0 && mate_ref[0]!='*') mate_refID = online_register_contig(pairer, thread_context, mate_ref); set_memory_int(bin_tmp + 24, mate_refID); old_read_pos -= 1; set_memory_int(bin_tmp + 28, old_read_pos); tlen = tlen * tlen_sign; set_memory_int(bin_tmp + 32, tlen); memcpy(bin_tmp + 36, read_name, l_read_name); int bin_ptr = 36 + l_read_name + 4 * cigar_ops; if(include_sequence){ int xk1, nch; //SUBREADprintf("SEQ (%d = %d) = %s\n", strlen(seq), l_seq, seq); //SUBREADprintf("QUA (%d = %d) = %s\n\n", strlen(qual), l_seq, qual); SamBam_read2bin(seq , bin_tmp + bin_ptr); bin_ptr += (l_seq + 1) / 2; for(xk1=0; xk1 < l_seq; xk1++){ nch = qual[xk1]; bin_tmp[bin_ptr++] = nch - 33; } }else{ bin_tmp[bin_ptr ++] = 0xff; bin_tmp[bin_ptr ++] = 0xff; } if(column_no == 11) // has extra tags { while(in_str[in_ptr] == '\t'){ if((!isalpha(in_str[in_ptr+1])) || (!isalpha(in_str[in_ptr+4]))){ while(in_str[in_ptr] !='\n')in_ptr++; break; } in_ptr ++; //SUBREADprintf("EXTRA_TAGS : %c%c : %c\n",in_str[in_ptr+0], in_str[in_ptr+1], in_str[in_ptr+3]); int is_important_tag = (in_str[in_ptr+0] == 'N' && in_str[in_ptr+1] == 'H') || (in_str[in_ptr+0] == 'H' && in_str[in_ptr+1] == 'I') || (in_str[in_ptr+0] == 'R' && in_str[in_ptr+1] == 'G') || (in_str[in_ptr+0] == 'N' && in_str[in_ptr+1] == 'M') ; int xxnch; if(in_str[in_ptr + 3] == 'Z' || in_str[in_ptr + 3] == 'H'){ if(is_important_tag||!pairer -> tiny_mode){ bin_tmp[bin_ptr+0] = in_str[in_ptr+0]; bin_tmp[bin_ptr+1] = in_str[in_ptr+1]; bin_tmp[bin_ptr+2] = in_str[in_ptr + 3]; bin_ptr += 3; } in_ptr += 5; while(1){ xxnch = *(in_str + in_ptr); if(xxnch == '\n' || xxnch == '\t' || xxnch == 0) break; if(is_important_tag||!pairer -> tiny_mode) *(bin_tmp + (bin_ptr++)) = xxnch; in_ptr ++; } if(is_important_tag||!pairer -> tiny_mode){ *(bin_tmp + (bin_ptr++)) = 0; } }else if(in_str[in_ptr + 3] == 'i'){ int tmpi = 0, tmpi_sign = 1; if(is_important_tag || !pairer -> tiny_mode){ bin_tmp[bin_ptr+0] = in_str[in_ptr+0]; bin_tmp[bin_ptr+1] = in_str[in_ptr+1]; bin_tmp[bin_ptr+2] = 'i'; bin_ptr += 3; } in_ptr += 5; while(1){ xxnch = *(in_str + in_ptr); if(xxnch == '\n' || xxnch == '\t' || xxnch == 0) break; else if(xxnch == '-') tmpi_sign = -1; else tmpi = tmpi * 10 + xxnch - '0'; in_ptr ++; } tmpi *= tmpi_sign; if(is_important_tag || !pairer -> tiny_mode){ set_memory_int(bin_tmp+bin_ptr, tmpi); bin_ptr += 4; } }else if(in_str[in_ptr + 3] == 'f'){ char ftxt[30]; int fi=0; while(1){ xxnch = *(in_str + in_ptr + 5 + fi); if(xxnch== '\n' || xxnch == '\t'|| xxnch == 0) break; ftxt[fi++]=xxnch; ftxt[fi]=0; } if(!pairer -> tiny_mode){ float fv = atof(ftxt); bin_tmp[bin_ptr+0] = in_str[in_ptr+0]; bin_tmp[bin_ptr+1] = in_str[in_ptr+1]; bin_tmp[bin_ptr+2] = 'f'; memcpy( bin_tmp + bin_ptr + 3, &fv, 4); bin_ptr += 7; } in_ptr += 5 + fi; }else if(in_str[in_ptr + 3] == 'B'){ char elemtype = in_str[in_ptr + 5]; int txi=0, eles=0; char ttxt[30], *elen_ptr = NULL;; if(!pairer -> tiny_mode){ bin_tmp[bin_ptr+0] = in_str[in_ptr+0]; bin_tmp[bin_ptr+1] = in_str[in_ptr+1]; bin_tmp[bin_ptr+2] = 'B'; bin_tmp[bin_ptr+3] = elemtype; elen_ptr = bin_tmp+4 + bin_ptr; bin_ptr += 8; } in_ptr += 6; int elembytes_no = 4; if(elemtype == 'C' || elemtype == 'c') elembytes_no = 1; if(elemtype == 'S' || elemtype == 's') elembytes_no = 2; while(1){ xxnch = *(in_str + in_ptr); if((!pairer -> tiny_mode)){ if((xxnch ==',' || xxnch =='\n' || xxnch == '\t' || xxnch == 0) && txi > 0){ //SUBREADprintf("ADD VAL : `%s`\n", ttxt); if(elemtype == 'f'){ float fv = atof(ttxt); memcpy( bin_tmp + bin_ptr, &fv, 4); }else{ int iv = atoi(ttxt); memcpy( bin_tmp + bin_ptr, &iv, elembytes_no); } bin_ptr+=elembytes_no; txi=0; eles++; }else{ if(xxnch!=','){ ttxt[txi++] = xxnch; ttxt[txi] = 0; } } } if(xxnch =='\n' || xxnch == '\t' || xxnch == 0)break; in_ptr ++; } if((!pairer -> tiny_mode)) memcpy(elen_ptr, & eles, 4); }else if(in_str[in_ptr + 3] == 'A'){ if(!pairer -> tiny_mode){ bin_tmp[bin_ptr+0] = in_str[in_ptr+0]; bin_tmp[bin_ptr+1] = in_str[in_ptr+1]; bin_tmp[bin_ptr+2] = 'A'; bin_tmp[bin_ptr+3] = in_str[in_ptr+5]; bin_ptr += 4; } in_ptr += 6; }else{ in_ptr += 5; while(1){ xxnch = *(in_str + in_ptr); if(xxnch == '\n' || xxnch == '\t' || xxnch == 0) break; in_ptr++; } } // #warning "=============== COMMENT NEXT =====================" // SUBREADprintf("Z_len PTR = %d + %d\n", bin_ptr, thread_context -> input_buff_BIN_ptr); } } thread_context -> input_buff_SBAM_ptr += in_ptr + 1; bin_ptr -= 4; set_memory_int(bin_tmp, bin_ptr); bin_ptr += 4; //memcpy(buf, bin_tmp, bin_ptr); return bin_ptr; } int SAP_pairer_skip_tag_body_len(char *bin){ int skip_content = 0; if(bin[2]=='i' || bin[2]=='I' || bin[2]=='f') skip_content = 4; else if(bin[2]=='s' || bin[2]=='S') skip_content = 2; else if(bin[2]=='c' || bin[2]=='C' || bin[2]=='A') skip_content = 1; else if(bin[2]=='Z' || bin[2]=='H'){ while(bin[skip_content + 3]) skip_content++; skip_content ++; } else if(bin[2]=='B'){ char cell_type = tolower(bin[3]); memcpy(&skip_content, bin + 4, 4); if(cell_type == 's')skip_content *=2; else if(cell_type == 'i' || cell_type == 'f')skip_content *= 4; skip_content += 4+1; // 32-bit count, 1 byte type }else{ SUBREADprintf("UnknownTag=%c\n\n", bin[2]); assert(0); } return skip_content+3; } int SAM_pairer_iterate_tags(unsigned char * bin, int bin_len, char * tag_name, char * data_type, char ** saved_value){ int found = 0; int bin_cursor = 0; while(bin_cursor < bin_len){ if(0){ char outc[3]; outc[0] = bin[bin_cursor]; outc[1] = bin[bin_cursor+1]; outc[2]=0; SUBREADprintf("TAG=%s, TYP=%c %d %c\n", outc, bin[bin_cursor+2], bin[bin_cursor+3], bin[bin_cursor+4]); } if(bin[bin_cursor] == tag_name[0] && bin[bin_cursor+1] == tag_name[1]){ (* data_type) = bin[bin_cursor+2]; (* saved_value) = (char *)bin+bin_cursor+3; found = 1; break; } int skip_content = SAP_pairer_skip_tag_body_len((char*)bin+bin_cursor); bin_cursor += skip_content ; } return found; } int SAM_pairer_iterate_int_tags(unsigned char * bin, int bin_len, char * tag_name, int * saved_value){ char * data_ptr = NULL; char data_type = 0; (*saved_value) = 0; int ret = SAM_pairer_iterate_tags(bin, bin_len, tag_name, &data_type, &data_ptr); //SUBREADprintf(" NEED %s , FOUND %d, TYPE %c\n", tag_name, ret, data_type); if(ret){ if(data_type == 'i' || data_type == 'I') memcpy(saved_value, data_ptr, 4); else if(data_type == 's' || data_type == 'S') memcpy(saved_value, data_ptr, 2); else if(data_type == 'c' || data_type == 'C') memcpy(saved_value, data_ptr, 1); else return 0; } return ret; } int SAM_pairer_get_read_full_name( SAM_pairer_context_t * pairer , SAM_pairer_thread_t * thread_context , unsigned char * bin, int bin_len , char * full_name, int * this_flag){ full_name[0]=0; int rlen = 0; unsigned int l_read_name = 0; unsigned int refID = 0; unsigned int next_refID = 0; unsigned int pos = 0, l_seq = 0, cigar_opts; unsigned int next_pos = 0, tmpi = 0; int FLAG; int HItag = -1; memcpy(&refID, bin + 4, 4); memcpy(&pos, bin + 8, 4); memcpy(&tmpi, bin + 12, 4); l_read_name = tmpi & 0xff; memcpy(&tmpi, bin + 16, 4); FLAG = (tmpi >> 16)&0xffff; (*this_flag) = FLAG; cigar_opts = tmpi & 0xffff; memcpy(&next_refID, bin + 24, 4); memcpy(&next_pos, bin + 28, 4); memcpy(full_name, bin+36, l_read_name); assert(l_read_name > 0); unsigned int r1_refID, old_read_pos, r2_refID, new_dummy_pos; if(FLAG & 4){ refID = -1; pos = 0; } if(FLAG & 8){ next_refID = -1; next_pos = 0; } if((FLAG & 0x40) == 0x40){ r1_refID = refID; old_read_pos = pos; r2_refID = next_refID; new_dummy_pos = next_pos; } else { r2_refID = refID; new_dummy_pos = pos; r1_refID = next_refID; old_read_pos = next_pos; } memcpy(&l_seq, bin + 20, 4); //SUBREADprintf("LQ=%d, RL=%d, CIGAR_OPT=%d\n", l_seq, (l_seq+1)/2, cigar_opts); unsigned int tags_start = 36+l_read_name+4*cigar_opts+(l_seq+1)/2+l_seq; unsigned int tags_len = bin_len - tags_start; if(tags_len > 2){ int found = SAM_pairer_iterate_int_tags(bin + tags_start, tags_len, "HI", &HItag); if(!found) HItag = -1; } int slash_pos = 0; for(; slash_pos < l_read_name - 1; slash_pos++){ if(full_name[slash_pos] == '/') break; } rlen = slash_pos + sprintf(full_name+slash_pos, "\027%d\027%u\027%d\027%u\027%d", r1_refID, old_read_pos, r2_refID, new_dummy_pos, HItag); return rlen; } int SAM_pairer_multi_thread_header (void * pairer_vp, int thread_no, int is_text, unsigned int items, char * bin, unsigned int bin_len){ SAM_pairer_context_t * pairer = (SAM_pairer_context_t *) pairer_vp; SAM_pairer_writer_main_t * bam_main = (SAM_pairer_writer_main_t * )pairer -> appendix1; SAM_pairer_writer_thread_t * bam_thread = bam_main -> threads + thread_no; unsigned int BIN_block_cursor = 0, bin_cursor = 0; //SUBREADprintf("WRITE HEADER TYPE=%d; ITEMS=%d\n", is_text, items); if(is_text){ memcpy( bam_thread -> BIN_buffer, "BAM\1", 4 ); memcpy( bam_thread -> BIN_buffer + 4 , & items , 4 ); BIN_block_cursor = 8; }else{ memcpy( bam_thread -> BIN_buffer , & items , 4 ); BIN_block_cursor = 4; } while( bin_cursor < bin_len ){ int write_text_len = min(SAM_PAIRER_WRITE_BUFFER - BIN_block_cursor, bin_len - bin_cursor); // SUBREADprintf("WRITE TLEN=%d\n", write_text_len); memcpy(bam_thread -> BIN_buffer + BIN_block_cursor , bin + bin_cursor, write_text_len); bam_thread -> BIN_buffer_ptr = write_text_len + BIN_block_cursor; SAM_pairer_multi_thread_compress(bam_main, bam_thread); bin_cursor += write_text_len; BIN_block_cursor = 0; } bam_thread -> BIN_buffer_ptr = 0; return 0; } int SAM_pairer_get_tag_bin_start(char * bin1){ int seq_len = 0; int cigar_opts = 0; int len_name = (unsigned char)bin1[12]; memcpy(&seq_len, bin1 + 20,4); memcpy(&cigar_opts, bin1 + 16, 2); return 36 + len_name + seq_len + (seq_len+1)/2 + 4 * cigar_opts; } void SAM_pairer_make_dummy(char * rname, char * bin1, char * out_bin2, int need_RG_tag){ char * realname = bin1 + 36; int block1len =-1; int len_name = (unsigned char)bin1[12] -1; int old_read_chro =-1; int old_read_pos =-1; int new_dummy_chro =-1; int new_dummy_pos =-1; memcpy(&block1len, bin1, 4); memcpy(&old_read_chro, bin1 + 4, 4); memcpy(&old_read_pos, bin1 + 8, 4); memcpy(&new_dummy_chro, bin1 + 24, 4); memcpy(&new_dummy_pos, bin1 + 28, 4); int HItag =-1; int NHtag =-1; int seq_len = -1; int cigar_opts = -1; memcpy(&seq_len, bin1 + 20,4); int old_read_FLAG = -1; memcpy(&old_read_FLAG, bin1 + 16, 4); cigar_opts = old_read_FLAG & 0xffff; char * RG_tag_val = NULL; int bin1ptr = 36 + len_name +1 + seq_len + (seq_len+1)/2 + 4 * cigar_opts; //SUBREADprintf("MAKE_DUMMY: %s ; need_RG=%d, %d > %d\n", realname, need_RG_tag, block1len + 4 ,bin1ptr + 3); if( block1len + 4 > bin1ptr + 3 ){ SAM_pairer_iterate_int_tags((unsigned char *)(bin1+bin1ptr),block1len + 4 - bin1ptr, "NH", &NHtag); SAM_pairer_iterate_int_tags((unsigned char *)(bin1+bin1ptr),block1len + 4 - bin1ptr, "HI", &HItag); if( need_RG_tag ){ char RG_type=0; SAM_pairer_iterate_tags((unsigned char *)(bin1+bin1ptr),block1len + 4 - bin1ptr, "RG", &RG_type, &RG_tag_val); if(RG_type != 'Z') RG_tag_val = NULL; //SUBREADprintf("type=%c\tval=%s\n", RG_type, RG_tag_val); } } old_read_FLAG = 0xffff&(old_read_FLAG >>16); int mate_tlen = 0; memcpy(&mate_tlen, bin1 + 32, 4); if(old_read_chro<0) old_read_pos=-1; if(new_dummy_chro<0) new_dummy_pos=-1; int bin_mq_nl = (len_name+1); int new_dummy_FLAG = (old_read_FLAG&0x40)? 0x80:0x40; new_dummy_FLAG |= 1; // Dummy reads should always be unmapped! //if(old_read_FLAG & 8)new_dummy_FLAG |=4; if(old_read_FLAG & 4)new_dummy_FLAG |=8; if(old_read_FLAG & 8)new_dummy_FLAG |=4; if(old_read_FLAG & 0x10) new_dummy_FLAG |= 0x20; if(old_read_FLAG & 0x20) new_dummy_FLAG |= 0x10; new_dummy_FLAG = new_dummy_FLAG << 16; memcpy(out_bin2+4, &new_dummy_chro,4); memcpy(out_bin2+8, &new_dummy_pos,4); memcpy(out_bin2+12, &bin_mq_nl, 4); memcpy(out_bin2+16, &new_dummy_FLAG, 4); new_dummy_FLAG = 1; memcpy(out_bin2+20, &new_dummy_FLAG, 4); memcpy(out_bin2+24, &old_read_chro, 4); memcpy(out_bin2+28, &old_read_pos, 4); mate_tlen = -mate_tlen; memcpy(out_bin2+32, &mate_tlen, 4); memcpy(out_bin2+36, realname, len_name+1); out_bin2[36 + len_name+1] = 0xff; out_bin2[36 + len_name+2] = 0x20; int all_len = 36 + len_name + 3 - 4; int tag_ptr = 36 + len_name + 3; //SUBREADprintf("HI=%d\n", HItag); if(HItag>0){ out_bin2[tag_ptr++]='H'; out_bin2[tag_ptr++]='I'; if(HItag<128){ out_bin2[tag_ptr++]='C'; memcpy(out_bin2 + (tag_ptr++), &HItag, 1); all_len += 4; }else if(HItag<32767){ out_bin2[(tag_ptr+=2)]='S'; memcpy(out_bin2 + 36 + len_name+6, &HItag, 2); all_len += 5; }else { out_bin2[(tag_ptr+=4)]='I'; memcpy(out_bin2 + 36 + len_name+6, &HItag, 4); all_len += 7; } } if(NHtag>0){ out_bin2[tag_ptr++]='N'; out_bin2[tag_ptr++]='H'; if(NHtag<128){ out_bin2[tag_ptr++]='C'; memcpy(out_bin2 + (tag_ptr++), &NHtag, 1); all_len += 4; }else if(NHtag<32767){ out_bin2[(tag_ptr+=2)]='S'; memcpy(out_bin2 + 36 + len_name+6, &NHtag, 2); all_len += 5; }else { out_bin2[(tag_ptr+=4)]='I'; memcpy(out_bin2 + 36 + len_name+6, &NHtag, 4); all_len += 7; } } if(RG_tag_val){ out_bin2[tag_ptr++]='R'; out_bin2[tag_ptr++]='G'; out_bin2[tag_ptr++]='Z'; all_len +=3; while(*RG_tag_val){ out_bin2[tag_ptr++]=*(RG_tag_val++); all_len ++; } out_bin2[tag_ptr++]=0; all_len ++; } memcpy(out_bin2,&all_len,4); } void SAM_pairer_reset( SAM_pairer_context_t * pairer ) { int x1; pairer -> is_finished = 0; pairer -> BAM_header_parsed = 0; pairer -> total_input_reads = 0; pairer -> input_chunk_no = 0; pairer -> merge_level_finished = 0; for(x1 = 0; x1 < pairer -> total_threads ; x1 ++){ pairer -> threads[x1].reads_in_SBAM = 0; pairer -> threads[x1].input_buff_BIN_used = 0; pairer -> threads[x1].input_buff_BIN_ptr = 0; pairer -> threads[x1].input_buff_SBAM_used = 0; pairer -> threads[x1].input_buff_SBAM_ptr = 0; pairer -> threads[x1].orphant_block_no = 0; pairer -> threads[x1].readno_in_chunk = 0; pairer -> threads[x1].immediate_last_read_full_name[0]=0; HashTableDestroy(pairer -> threads[x1].orphant_table); pairer -> threads[x1].orphant_table = HashTableCreate(pairer -> input_buff_SBAM_size / 100); HashTableSetHashFunction(pairer -> threads[x1].orphant_table, fc_chro_hash); HashTableSetKeyComparisonFunction(pairer -> threads[x1].orphant_table, fc_strcmp_chro); HashTableSetDeallocationFunctions(pairer -> threads[x1].orphant_table, free, free); inflateReset(&pairer -> threads[x1].strm); } HashTableDestroy(pairer -> unsorted_notification_table); pairer -> unsorted_notification_table = HashTableCreate(2191); HashTableSetHashFunction(pairer -> unsorted_notification_table, fc_chro_hash); HashTableSetKeyComparisonFunction(pairer -> unsorted_notification_table, fc_strcmp_chro); HashTableSetDeallocationFunctions(pairer -> unsorted_notification_table, free, free); HashTableDestroy(pairer ->bam_margin_table); pairer ->bam_margin_table = HashTableCreate(2191); HashTableSetHashFunction(pairer -> bam_margin_table, fc_chro_hash); HashTableSetKeyComparisonFunction(pairer -> bam_margin_table, fc_strcmp_chro); HashTableSetDeallocationFunctions(pairer -> bam_margin_table, free, free); } void SAM_pairer_writer_reset( void * pairer_vp ) { SAM_pairer_context_t * pairer = (SAM_pairer_context_t *) pairer_vp; SAM_pairer_writer_main_t * bam_main = (SAM_pairer_writer_main_t * )pairer -> appendix1; int rlen = ftruncate(fileno(bam_main -> bam_fp), 0); if(rlen != 0)SUBREADprintf("ERROR: Cannot reset the output file."); fclose(bam_main -> bam_fp); bam_main -> bam_fp = f_subr_open(bam_main -> bam_name, "wb"); int x1; for(x1 = 0; x1 < pairer -> total_threads ; x1 ++){ bam_main -> threads[x1].BIN_buffer_ptr = 0; deflateReset(&bam_main -> threads[x1].strm); } } int SAM_pairer_multi_thread_output(void * pairer_vp, int thread_no, char * bin1, char * bin2 ){ SAM_pairer_context_t * pairer = (SAM_pairer_context_t *) pairer_vp; SAM_pairer_writer_main_t * bam_main = (SAM_pairer_writer_main_t * )pairer -> appendix1; SAM_pairer_writer_thread_t * bam_thread = bam_main -> threads + thread_no; char dummy_bin2 [MAX_READ_NAME_LEN*2 + 180 ]; if(bin2==NULL && bam_main -> has_dummy){ SAM_pairer_make_dummy( "DUMMY", bin1, dummy_bin2, pairer -> need_read_group_tag ); bin2 = dummy_bin2; } int bin_len1, bin_len2 = 0; memcpy(&bin_len1, bin1, 4); bin_len1 +=4; if(bin2) { memcpy(&bin_len2, bin2, 4); bin_len2 +=4; } if( bin_len1 + bin_len2 >= SAM_PAIRER_WRITE_BUFFER){ SUBREADprintf("ERROR: BAM Record larger than a BAM block.\n"); return 1; } if(bin_len1 + bin_len2 + bam_thread -> BIN_buffer_ptr >= SAM_PAIRER_WRITE_BUFFER){ int ret = SAM_pairer_multi_thread_compress(bam_main, bam_thread); if(ret)return 1; } memcpy( bam_thread -> BIN_buffer + bam_thread -> BIN_buffer_ptr, bin1, bin_len1 ); if(bin2) memcpy( bam_thread -> BIN_buffer + bam_thread -> BIN_buffer_ptr + bin_len1, bin2, bin_len2 ); bam_thread -> BIN_buffer_ptr += bin_len1 + bin_len2; return 0; } void SAM_pairer_do_read_test( SAM_pairer_context_t * pairer , SAM_pairer_thread_t * thread_context , int read_name_len, char * read_full_name, int bin_len, char * bin , int flags){ unsigned char * mate_bin = HashTableGet(thread_context -> orphant_table, read_full_name); if(mate_bin){ if(pairer -> output_function) pairer -> output_function(pairer, thread_context -> thread_id, bin, (char*)mate_bin); HashTableRemove(thread_context -> orphant_table, read_full_name); if(thread_context -> orphant_space > bin_len) thread_context -> orphant_space -= bin_len; else thread_context -> orphant_space = 0; //SUBREADprintf("Mate_found: %s\n", read_full_name); } else { char * mem_name = malloc(read_name_len + 1); memcpy(mem_name, read_full_name, read_name_len); mem_name[read_name_len] = 0; char * mem_bin = malloc(bin_len); memcpy(mem_bin, bin , bin_len); HashTablePut(thread_context -> orphant_table, mem_name, mem_bin); thread_context -> orphant_space += bin_len; //#warning "============= COMMENT NEXT ==================" //SUBREADprintf("Orphant_created [%d]: %s ; BINLEN=%d, OPSIZE=%d\n", thread_context -> thread_id, read_full_name, bin_len, thread_context -> orphant_space); } } void SAM_pairer_register_matcher(SAM_pairer_context_t * pairer , unsigned int chunk_number, unsigned int readno_in_chunk, char * read_full_name , char * bin, int bin_len , int this_flags){ char * mem_bin = malloc(bin_len); memcpy(mem_bin, bin , bin_len); subread_lock_occupy(&pairer -> unsorted_notification_lock); char * mem_name = malloc(24); sprintf(mem_name, "B:%u:%d", chunk_number , (readno_in_chunk>0)?1:0); HashTablePut(pairer -> unsorted_notification_table, mem_name, mem_bin); mem_bin = malloc(bin_len); sprintf(mem_bin,"%010u %d", chunk_number, (readno_in_chunk>0)?1:0); mem_name = malloc(strlen(read_full_name) + 5); sprintf(mem_name, "C:%s:%d", read_full_name , (this_flags & 0x80)?1:0); HashTablePut(pairer -> unsorted_notification_table, mem_name, mem_bin); subread_lock_release(&pairer -> unsorted_notification_lock); } void SAM_pairer_do_one_BIN(SAM_pairer_context_t * pairer , SAM_pairer_thread_t * thread_context , char * bin, int bin_len){ char read_full_name[ MAX_READ_NAME_LEN*2 +80 ]; // rname:chr_r1:pos_r1:chr_r2:pos_r2:HI_tag int this_flags=0; int name_len = SAM_pairer_get_read_full_name(pairer, thread_context, (unsigned char *)bin, bin_len, read_full_name, & this_flags); if(pairer -> is_single_end_mode == 0 && ( this_flags & 1 ) == 1){ // if the reads are PE if(strcmp(read_full_name , thread_context -> immediate_last_read_full_name) == 0){ if(pairer -> output_function) pairer -> output_function(pairer, thread_context -> thread_id, (char*) bin, (char*)thread_context -> immediate_last_read_bin); thread_context -> immediate_last_read_full_name[0] = 0; }else{ if(thread_context -> immediate_last_read_full_name[0]){ if(thread_context -> readno_in_chunk>2){ if(pairer -> is_unsorted_notified == 0){ if(pairer -> unsorted_notification){ //SUBREADprintf("READ_%d : UNSORT1 : %s != %s\n", thread_context -> readno_in_chunk, thread_context -> immediate_last_read_full_name , read_full_name); pairer -> unsorted_notification(pairer , thread_context -> immediate_last_read_bin, (char *) bin); } pairer -> is_unsorted_notified = 1; } }else if(thread_context -> readno_in_chunk == 1 && !pairer -> is_unsorted_notified ) { SAM_pairer_register_matcher(pairer, thread_context -> chunk_number, thread_context -> readno_in_chunk - 1, thread_context -> immediate_last_read_full_name, thread_context -> immediate_last_read_bin, thread_context -> immediate_last_read_bin_len , thread_context -> immediate_last_read_flags ); } SAM_pairer_do_read_test(pairer , thread_context , thread_context -> immediate_last_read_name_len , thread_context -> immediate_last_read_full_name , thread_context -> immediate_last_read_bin_len , thread_context -> immediate_last_read_bin, thread_context -> immediate_last_read_flags); } thread_context -> immediate_last_read_bin_len = bin_len; thread_context -> immediate_last_read_name_len = name_len; thread_context -> immediate_last_read_flags = this_flags; strcpy(thread_context -> immediate_last_read_full_name, read_full_name); memcpy(thread_context -> immediate_last_read_bin, bin, bin_len); } }else{ // else just write. if(pairer -> output_function) pairer -> output_function(pairer, thread_context -> thread_id, (char*) bin, NULL); } thread_context -> readno_in_chunk ++; } int SAM_pairer_do_next_read( SAM_pairer_context_t * pairer , SAM_pairer_thread_t * thread_context ){ unsigned char * bin = NULL; int bin_len = 0; int has_next_read = SAM_pairer_get_next_read_BIN(pairer, thread_context, &bin, &bin_len); if(has_next_read && !pairer -> is_bad_format){ SAM_pairer_do_one_BIN( pairer, thread_context,(char *)bin, bin_len ); return 0; } else pairer -> BAM_header_parsed = 1; return 1; } // all orphants are written into files, each has a size of buffer size. // when the orphants are longer than buffer_size, then sort and save to disk. void SAM_pairer_sort_exchange(void * arr, int l, int r){ unsigned char *** sort_data = (unsigned char ***) arr; unsigned char * tmpc; tmpc = sort_data[0][r]; sort_data[0][r] = sort_data[0][l]; sort_data[0][l] = tmpc; tmpc = sort_data[1][r]; sort_data[1][r] = sort_data[1][l]; sort_data[1][l] = tmpc; } int SAM_pairer_sort_compare(void * arr, int l, int r){ char *** sort_data = (char ***) arr; return strcmp(sort_data[0][l], sort_data[0][r]); } void SAM_pairer_sort_merge( void * arr, int start, int items, int items2 ){ unsigned char *** sort_data = (unsigned char ***) arr; unsigned char ** tmp_name_list = malloc(sizeof(char *) * (items+items2)); unsigned char ** tmp_bin_list = malloc(sizeof(char *) * (items+items2)); int i1_cursor = start, i2_cursor = items + start; int tmp_cursor = 0; while(1){ if(i1_cursor == items + start && i2_cursor == items + items2 + start )break; int select_items_1 = (i2_cursor == start + items + items2) || (i1_cursor < items + start && SAM_pairer_sort_compare(arr, i1_cursor, i2_cursor) <= 0); if(select_items_1){ tmp_name_list[tmp_cursor] = sort_data[0][i1_cursor]; tmp_bin_list[tmp_cursor ++] = sort_data[1][i1_cursor++]; }else{ tmp_name_list[tmp_cursor] = sort_data[0][i2_cursor]; tmp_bin_list[tmp_cursor ++] = sort_data[1][i2_cursor++]; } } assert(tmp_cursor == items + items2); memcpy( sort_data[0] + start, tmp_name_list, sizeof(char *) * (items+items2) ); memcpy( sort_data[1] + start, tmp_bin_list, sizeof(char *) * (items+items2) ); free(tmp_name_list); free(tmp_bin_list); } unsigned int SAM_pairer_osr_hash(char * st){ int x1 = 0, nch; unsigned int ret = 0, ret2=0; while((nch = st[x1++])!=0){ ret = (ret << 2) ^ nch; ret2 = (ret << 3) ^ nch; } return (ret^ret2) % 39846617; } int SAM_pairer_osr_next_name(FILE * fp , char * name, int thread_no, int all_threads){ while(1){ if(feof(fp)) return 0; int rlen =0; int retv = fread(&rlen, 1, 2, fp); if(retv < 2) return 0; if(rlen < 1) return 0; assert(rlen < 1024); int rlen2 = fread(name, 1, rlen, fp); if(rlen2 != rlen) return 0; name[rlen]=0; if(all_threads < 0 || SAM_pairer_osr_hash(name)% all_threads == thread_no ) { fseeko(fp, -2-rlen, SEEK_CUR); return 1; } retv = fread(&rlen, 1, 4, fp); if(retv!=4) return -1; rlen +=4; fseeko(fp, rlen, SEEK_CUR); } return 0; } void SAM_pairer_osr_next_bin(FILE * fp, char * bin){ int rlen =0; int retv = fread(&rlen, 1, 2, fp); if(retv <2) *((int*)bin)=0; assert(rlen < 1024); fseeko(fp, rlen, SEEK_CUR); rlen =0; retv = fread(&rlen, 1, 4, fp); if(retv <4) *((int*)bin)=0; rlen +=4; retv = fread(bin, 1, rlen, fp); if(retv tmp_file_prefix); char * names = malloc( fps_no * max_name_len ); bin_tmp1 = malloc(FC_LONG_READ_RECORD_HARDLIMIT); bin_tmp2 = malloc(FC_LONG_READ_RECORD_HARDLIMIT); FILE * out_fp = fopen(tmp_fname, "wb"); // initialize the "current_first_name" for each orphan file for(x1 = 0 ; x1 < fps_no; x1++) { int has = SAM_pairer_osr_next_name( fps[x1] , names + max_name_len*x1 , -1 , -1); if(!has) *(names + max_name_len*x1)=0; } while(1){ int min_name_fileno = -1; int min2_name_fileno = -1; // find the min_name in all FPs // and find the same min_name if there is any for(x1 = 0 ; x1 < fps_no; x1++){ int has = *(names + max_name_len*x1); if(has){ int strcv_12 = 1; if(min_name_fileno >=0) strcv_12 = strcmp(names+(min_name_fileno * max_name_len), names+(x1 * max_name_len)); if(strcv_12 > 0){ min_name_fileno = x1; min2_name_fileno = -1; }else if( strcv_12 == 0){ min2_name_fileno = x1; } } } if(min_name_fileno >= 0 && !is_disk_full){ SAM_pairer_osr_next_bin( fps[ min_name_fileno ] , bin_tmp1); if(min2_name_fileno>=0){ SAM_pairer_osr_next_bin( fps[ min2_name_fileno ] , bin_tmp2); pairer -> output_function(pairer, 0, (char*) bin_tmp1, (char*)bin_tmp2); if(0 && 0 == pairer -> is_unsorted_notified){ char * name_tmp_1 = malloc(strlen(names+(min_name_fileno * max_name_len))+5), *name_tmp_2 = malloc(strlen(names+(min_name_fileno * max_name_len))+5); char * min1_chunk_info, * min2_chunk_info; sprintf(name_tmp_1, "C:%s:%d", names+(min_name_fileno * max_name_len), 0); sprintf(name_tmp_2, "C:%s:%d", names+(min2_name_fileno * max_name_len), 1); min1_chunk_info = HashTableGet( pairer -> unsorted_notification_table , name_tmp_1); min2_chunk_info = HashTableGet( pairer -> unsorted_notification_table , name_tmp_2); if(min1_chunk_info == NULL || min2_chunk_info == NULL || !SAM_pairer_is_matched_chunks(min1_chunk_info, min2_chunk_info)){ sprintf(name_tmp_1, "B:%s:%d", names+(min_name_fileno * max_name_len), 0); if( pairer -> unsorted_notification ){ //SUBREADprintf("FINAL STEP\n"); //SUBREADprintf("UNSORT2\n"); pairer -> unsorted_notification(pairer , HashTableGet( pairer -> unsorted_notification_table , name_tmp_1), NULL); } pairer -> is_unsorted_notified = 1; } free(name_tmp_1); } int read_has = SAM_pairer_osr_next_name( fps[min2_name_fileno], names + max_name_len*min2_name_fileno, -1, -1); if(!read_has) *(names + max_name_len*min2_name_fileno)=0; }else{ unsigned short wlen; unsigned int rbinlen = 0; wlen = strlen( names+(min_name_fileno * max_name_len) ); fwrite( &wlen, 2, 1,out_fp ); fwrite( names+(min_name_fileno * max_name_len), 1, wlen, out_fp ); memcpy( &rbinlen, bin_tmp1 , 4); rbinlen += 4; fwrite( bin_tmp1, 4, 1, out_fp ); int write_len = fwrite( bin_tmp1, 1, rbinlen, out_fp ); if(write_len < rbinlen)is_disk_full = 1; } int read_has = SAM_pairer_osr_next_name( fps[min_name_fileno], names + max_name_len*min_name_fileno, -1, -1); if(!read_has) *(names + max_name_len*min_name_fileno)=0; } else break; } fclose(out_fp); unlink(fname); rename(tmp_fname, fname); free(names); free(bin_tmp1); free(bin_tmp2); return is_disk_full; } #define PAIRER_WAIT_TICK_TIME 10000 int SAM_pairer_get_merge_max_fp(SAM_pairer_context_t * pairer){ return pairer -> max_file_open_number; } void SAM_pairer_set_merge_max_fp(SAM_pairer_context_t * pairer, int fon){ pairer -> max_file_open_number = fon; } int SAM_pairer_probe_maxfp( SAM_pairer_context_t * pairer){ int orphant_fp_no=0, is_disk_full = 0; int thno, bkno, x1; int thread_fps [ pairer -> total_threads ]; char tmp_fname[MAX_FILE_NAME_LENGTH+50]; memset(thread_fps, 0, sizeof(int) * pairer -> total_threads); for( thno = 0 ; thno < pairer -> total_threads ; thno ++ ){ for( bkno = 0 ; ; bkno++){ sprintf(tmp_fname, "%s-TH%02d-BK%06d.tmp", pairer->tmp_file_prefix, thno, bkno); FILE * in_fp = fopen(tmp_fname, "rb"); if(NULL == in_fp) break; thread_fps[thno] = bkno; fclose(in_fp); orphant_fp_no ++; } } int max_open_fps = 0, has_limit = 0; int orphant_fp_size = 50; FILE ** orphant_fps = malloc(sizeof(FILE *) * orphant_fp_size); for( bkno = 0 ; bkno < 5; bkno++){ sprintf(tmp_fname, "%s-FTEST-%d.tmp", pairer->tmp_file_prefix, bkno); FILE * tfp = fopen(tmp_fname, "w"); if(NULL == tfp){ has_limit = 1; break; } orphant_fps[max_open_fps++] = tfp; } //#warning ">>>>>>> COMMENT NEXT LINE <<<<<<<<" for( thno = 0 ; thno < pairer -> total_threads ; thno ++ ){ if(has_limit) break; for( bkno = 0 ; ; bkno++){ sprintf(tmp_fname, "%s-TH%02d-BK%06d.tmp", pairer->tmp_file_prefix, thno, bkno); FILE * in_fp = fopen(tmp_fname, "rb"); if(NULL == in_fp){ if( bkno <= thread_fps[thno] ) has_limit = 1; break; } orphant_fps[max_open_fps++] = in_fp; if(max_open_fps >= orphant_fp_size - 1){ orphant_fp_size *= 2; orphant_fps = realloc(orphant_fps, orphant_fp_size * sizeof(FILE *)); } } } for( bkno = 0 ;bkno < max_open_fps; bkno ++) fclose(orphant_fps[bkno]); SAM_pairer_set_merge_max_fp(pairer, max_open_fps - 5); //#warning ">>>>>>> COMMENT NEXT LINE <<<<<<<<" //SUBREADprintf("Needed FPS = %d, Ulimit FPS = %d, Has_Limit = %d \n", orphant_fp_no, max_open_fps, has_limit); if( SAM_pairer_get_merge_max_fp(pairer) < orphant_fp_no * pairer -> total_threads){ int processed_orphant = 0; int current_opened_fp_no = 0 ; FILE * level_merge_fps [ SAM_pairer_get_merge_max_fp(pairer) ]; for( thno = 0 ; thno < pairer -> total_threads ; thno ++ ){ for( bkno = 0 ; ; bkno++){ char tmp_fname[MAX_FILE_NAME_LENGTH+50]; sprintf(tmp_fname, "%s-TH%02d-BK%06d.tmp", pairer->tmp_file_prefix, thno, bkno); FILE * in_fp = fopen(tmp_fname, "rb"); if(NULL == in_fp) break; // #warning ">>>> COMMENT DEBUG OUTPUT <<<<" // SUBREADprintf("Adding temp file:%s\n", tmp_fname); level_merge_fps[current_opened_fp_no ++] = in_fp; processed_orphant ++; if(current_opened_fp_no >= SAM_pairer_get_merge_max_fp(pairer) || processed_orphant == orphant_fp_no){ sprintf(tmp_fname, "%s-LEVELMERGE.tmp", pairer->tmp_file_prefix); // #warning ">>>> COMMENT DEBUG OUTPUT <<<<" // SUBREADprintf("Merging temp files\n"); is_disk_full |= merge_level_fps(pairer , tmp_fname, level_merge_fps, current_opened_fp_no); for(x1 = 0; x1 < current_opened_fp_no; x1++) fclose(level_merge_fps[x1]); if(processed_orphant < orphant_fp_no){ level_merge_fps[0] = fopen(tmp_fname, "rb"); current_opened_fp_no = 1; } if(is_disk_full) break; } } } pairer -> merge_level_finished = 1; } free(orphant_fps); return is_disk_full; } void * SAM_pairer_rescure_orphants_max_FP(void * params){ void ** param_ptr = (void **) params; SAM_pairer_context_t * pairer = param_ptr[0]; int thread_no = (int)(param_ptr[1]-NULL); free(params); srInt_64 died=0; int orphant_fp_no=0; int thno, bkno, x1; char tmp_fname[MAX_FILE_NAME_LENGTH+60]; int max_name_len = MAX_READ_NAME_LEN*2 +80, orphant_fp_size = 50; FILE ** orphant_fps = malloc(sizeof(FILE *) * orphant_fp_size); if(0 == thread_no && pairer -> display_progress) SUBREADprintf("Finished scanning the input file. Processing unpaired reads.\n"); //SUBREADprintf("merged = %d\n", pairer -> merge_level_finished); if(pairer -> merge_level_finished){ sprintf(tmp_fname, "%s-LEVELMERGE.tmp", pairer->tmp_file_prefix); FILE * in_fp = fopen(tmp_fname, "rb"); orphant_fps[0] = in_fp; orphant_fp_no=1; }else{ orphant_fp_no = 0; for( thno = 0 ; thno < pairer -> total_threads ; thno ++ ){ for( bkno = 0 ; ; bkno++){ sprintf(tmp_fname, "%s-TH%02d-BK%06d.tmp", pairer->tmp_file_prefix, thno, bkno); FILE * in_fp = fopen(tmp_fname, "rb"); if(NULL == in_fp) break; if(orphant_fp_no >= orphant_fp_size){ orphant_fp_size *= 1.5; orphant_fps = realloc(orphant_fps, orphant_fp_size * sizeof(FILE *)); } orphant_fps[orphant_fp_no++]=in_fp; } } } char * names = malloc( orphant_fp_no * max_name_len ); memset(names, 0, orphant_fp_no * max_name_len ); char * bin_tmp1 , * bin_tmp2; bin_tmp1 = malloc(66000); bin_tmp2 = malloc(66000); for(x1 = 0 ; x1 < orphant_fp_no; x1++) { int has = SAM_pairer_osr_next_name( orphant_fps[x1] , names + max_name_len*x1 , thread_no , pairer-> total_threads); if(!has) *(names + max_name_len*x1)=0; } while(1){ int min_name_fileno = -1; int min2_name_fileno = -1; for(x1 = 0 ; x1 < orphant_fp_no; x1++){ int has = *(names + max_name_len*x1); if(has){ int strcv_12 = 1; if(min_name_fileno >=0) strcv_12 = strcmp(names+(min_name_fileno * max_name_len), names+(x1 * max_name_len)); if(strcv_12 > 0){ min_name_fileno = x1; min2_name_fileno = -1; }else if( strcv_12 == 0){ min2_name_fileno = x1; } } } if(min_name_fileno >= 0){ SAM_pairer_osr_next_bin( orphant_fps[ min_name_fileno ] , bin_tmp1); if( min2_name_fileno >=0){ SAM_pairer_osr_next_bin( orphant_fps[ min2_name_fileno ] , bin_tmp2); pairer -> output_function(pairer, thread_no, (char*) bin_tmp1, (char*)bin_tmp2); if(0 && 0 == pairer -> is_unsorted_notified){ char *name_tmp_1 = malloc(strlen(names+(min_name_fileno * max_name_len))+5), *name_tmp_2 = malloc(strlen(names+(min_name_fileno * max_name_len))+5); char * min1_chunk_info, * min2_chunk_info; sprintf(name_tmp_1, "C:%s:%d", names+(min_name_fileno * max_name_len), 0); sprintf(name_tmp_2, "C:%s:%d", names+(min2_name_fileno * max_name_len), 1); min1_chunk_info = HashTableGet( pairer -> unsorted_notification_table , name_tmp_1); min2_chunk_info = HashTableGet( pairer -> unsorted_notification_table , name_tmp_2); //#warning ">>>>>>> COMMENT NEXT LINE <<<<<<<<" //SUBREADprintf("RESCURE MATCHER: %s , %s == %s , %s, %s\n", name_tmp_1, name_tmp_2, min1_chunk_info, min2_chunk_info, // SAM_pairer_is_matched_chunks(min1_chunk_info, min2_chunk_info)?"MATCH":"XXXXX"); if(min1_chunk_info == NULL || min2_chunk_info == NULL || !SAM_pairer_is_matched_chunks(min1_chunk_info, min2_chunk_info)){ sprintf(name_tmp_1, "B:%s:%d", names+(min_name_fileno * max_name_len), 0); if( pairer -> unsorted_notification ){ SUBREADprintf("UNSORT3\n"); //SUBREADprintf("FINAL STEP\n"); pairer -> unsorted_notification(pairer , HashTableGet( pairer -> unsorted_notification_table , name_tmp_1), NULL); } pairer -> is_unsorted_notified = 1; } } int read_has = SAM_pairer_osr_next_name( orphant_fps[min2_name_fileno], names + max_name_len*min2_name_fileno, thread_no, pairer-> total_threads); if(!read_has) *(names + max_name_len*min2_name_fileno)=0; }else{ //#warning ">>>>>>> COMMENT NEXT LINE <<<<<<<<" //SUBREADprintf("FINAL_ORPHAN:%s\n" , names + max_name_len*min_name_fileno); pairer -> output_function(pairer, thread_no, (char*) bin_tmp1, NULL); died++; } int read_has = SAM_pairer_osr_next_name( orphant_fps[min_name_fileno], names + max_name_len*min_name_fileno, thread_no, pairer-> total_threads); //#warning ">>>>>>> COMMENT NEXT BLOCK <<<<<<<<" if(0){ if(!read_has) SUBREADprintf("FP %d FINISHED\n", min_name_fileno); } if(!read_has) *(names + max_name_len*min_name_fileno)=0; } else break; } free(names); //#warning ">>>>>>> COMMENT NEXT LINE <<<<<<<<" //SUBREADprintf("finished_fps= %d\n", orphant_fp_no); for(x1 = 0 ; x1 < orphant_fp_no; x1++) { fclose ( orphant_fps[x1] ); } free( bin_tmp1 ); free( bin_tmp2 ); free(orphant_fps); pairer -> total_orphan_reads += died; return NULL; } int SAM_pairer_update_orphant_table(SAM_pairer_context_t * pairer , SAM_pairer_thread_t * thread_context){ unsigned int x2 = 0; unsigned char ** name_list, ** bin_list; //SUBREADprintf("ELES=%lu\n", thread_context->orphant_table->numOfElements); name_list = malloc(sizeof(char*) * thread_context->orphant_table->numOfElements); bin_list = malloc(sizeof(char*) * thread_context->orphant_table->numOfElements); int x1, is_error = 0; for(x1 = 0; x1 < thread_context->orphant_table->numOfBuckets; x1 ++){ KeyValuePair *pair = thread_context->orphant_table->bucketArray[x1]; while (pair != NULL) { KeyValuePair *nextPair = pair->next; name_list [x2] = (unsigned char *)pair -> key; bin_list [x2] = pair -> value; x2++; pair = nextPair; } } assert(x2 == thread_context->orphant_table->numOfElements); unsigned char ** sort_data[2]; sort_data[0]=name_list; sort_data[1]=bin_list; merge_sort(sort_data, thread_context->orphant_table->numOfElements, SAM_pairer_sort_compare, SAM_pairer_sort_exchange, SAM_pairer_sort_merge); char tmp_fname[MAX_FILE_NAME_LENGTH+40]; sprintf(tmp_fname, "%s-TH%02d-BK%06d.tmp", pairer->tmp_file_prefix, thread_context -> thread_id, thread_context -> orphant_block_no++); FILE * tmp_fp = fopen(tmp_fname, "wb"); if(tmp_fp){ for(x1 = 0; x1 < x2; x1 ++){ unsigned int bin_len; memcpy(&bin_len, bin_list[x1] , 4); int namelen = strlen((char *)name_list[x1]); int write_len = fwrite(&namelen,2,1,tmp_fp); is_error |= (write_len <1); write_len = fwrite(name_list[x1], 1, namelen, tmp_fp); is_error |= (write_len orphant_table , name_list[x1]); } fclose(tmp_fp); }else is_error =1; assert(thread_context -> orphant_table-> numOfElements == 0); free(name_list); free(bin_list); thread_context -> orphant_space = 0; if(is_error) SUBREADprintf("ERROR: unable to write into the temporary file. Please check the disk space in the output directory.\n"); return is_error; } int is_read_bin_ONE(char * bin, int bin_len, int max_refID, int * block_len){ memcpy(block_len, bin, 4); if((*block_len) > MAX_BIN_RECORD_LENGTH - 4 || (*block_len) < 32) return -1; if((*block_len) > bin_len - 4) return -2; int refID, mate_refID; memcpy(&refID, bin + 4, 4); memcpy(&mate_refID, bin + 24, 4); if(refID != -1 && (refID< 0 || refID >=max_refID)) return -3; if(mate_refID != -1 && (mate_refID< 0 || mate_refID >=max_refID)) return -4; int l_seq; memcpy(&l_seq, bin + 20, 4); if(l_seq > bin_len*2 || l_seq > MAX_BIN_RECORD_LENGTH || l_seq < 0) return -5; int min_mq_nl; memcpy(&min_mq_nl, bin + 12, 4); int name_len = min_mq_nl & 0xff; if(name_len < 1) return -20; int flag_nc; memcpy(&flag_nc, bin + 16, 4); int cigar_opts = flag_nc & 0xffff; // int flag = flag_nc >> 16; if(cigar_opts > 100) return -6; int rname_cursor = 36; if(bin[rname_cursor] == '@') return -7; for(; rname_cursor< 36 + name_len - 1; rname_cursor ++){ int nch = bin[rname_cursor]; if(nch < 0x20 || nch >=0x7f) return -9; if(nch == '\t') return -8; } if(bin[rname_cursor]!=0)return -10; if((*block_len) < 32 + name_len + 4*cigar_opts + l_seq + (l_seq+1)/2) return -11; int cigar_i; for(cigar_i = 0; cigar_i < cigar_opts ; cigar_i++){ int cigar_v; memcpy(&cigar_v , bin + 36 + name_len + 4*cigar_i, 4); int cigar_op = cigar_v & 0xf; int cigar_value = cigar_v & 0xfffffff; if(cigar_op > 8) return -12; if((cigar_op == 0 || cigar_op == 1 || cigar_op > 6) && (cigar_value < 1 || cigar_value > MAX_BIN_RECORD_LENGTH)){ //#warning ">>>>>> COMMENT NEXT LINE IN RELEASE <<<<<<" if(0){ char * rname = bin + 36; SUBREADprintf("OP=%d, VAL=%d [%s]\n", cigar_op, cigar_value, rname); } return -13; } } int ext_cursor = 36 + name_len + 4*cigar_opts + l_seq + (l_seq+1)/2; if(ext_cursor < (*block_len) + 4){ if(ext_cursor > (*block_len) + 4 - 4) return -17; if((!isalpha(bin[ext_cursor])) || bin[ext_cursor+1]>122 || bin[ext_cursor+1]<48 ||!isalpha(bin[ext_cursor+2])){ // SUBREADprintf("TAGERR: %c%c%c\n", bin[ext_cursor], bin[ext_cursor+1], bin[ext_cursor+2]); return -16; } } return 1; } #define TESTING_READS_FOR_START 3 int tchecks=0; // A block MUST have at least three reads as evidence; otherwise the BAM file is converted into the conservative format. int is_read_bin(char * bin, int bin_len, int max_refID){ int testing_i; int bin_cursor = 0; for(testing_i = 0; testing_i < TESTING_READS_FOR_START; testing_i++){ int block_len = 0; int rr = is_read_bin_ONE(bin + bin_cursor, bin_len - bin_cursor, max_refID, &block_len); if(0) SUBREADprintf("CHECK_START # %d: RET=%d\n", ++tchecks, rr); if(rr!=1) return rr; bin_cursor += block_len +4; if(bin_cursor == bin_len) return 1; } return 1; } int tfinds = 0; int SAM_pairer_find_start(SAM_pairer_context_t * pairer , SAM_pairer_thread_t * thread_context ){ thread_context -> need_find_start = 0; int start_pos = 0; for(start_pos = 0; start_pos < min(MAX_BIN_RECORD_LENGTH, thread_context -> input_buff_BIN_used); start_pos++){ if(1==is_read_bin((char *)thread_context -> input_buff_BIN + start_pos, thread_context -> input_buff_BIN_used - start_pos , pairer -> BAM_n_ref)){ //if(1) SUBREADprintf("STFIND # %d : start = %d\n", ++tfinds, start_pos); if(start_pos>0){ char * margin_key = malloc(22); char * margin_data = malloc(start_pos+4); memcpy(margin_data, &start_pos, 4); memcpy(margin_data+4, thread_context -> input_buff_BIN, start_pos); #ifdef __MINGW32__ sprintf(margin_key,"S%lu", (unsigned long) thread_context -> input_buff_SBAM_file_start); #else sprintf(margin_key,"S%llu", thread_context -> input_buff_SBAM_file_start); #endif subread_lock_occupy(&pairer -> SAM_BAM_table_lock); HashTablePut(pairer -> bam_margin_table, margin_key, margin_data); subread_lock_release(&pairer -> SAM_BAM_table_lock); } break; } } thread_context -> input_buff_BIN_ptr = start_pos; // SUBREADprintf("ABBO TH %d : FOUND START AT %d in %d\n", thread_context -> thread_id , start_pos, thread_context -> input_buff_BIN_used); return start_pos < min(MAX_BIN_RECORD_LENGTH, thread_context -> input_buff_BIN_used); } void * SAM_pairer_thread_run( void * params ){ void ** param_ptr = (void **) params; SAM_pairer_context_t * pairer = param_ptr[0]; int thread_no = (int)(param_ptr[1]-NULL), is_disk_full = 0; free(params); SAM_pairer_thread_t * thread_context = pairer -> threads + thread_no; int is_finished = 0; while(1){ subread_lock_occupy(&pairer -> input_fp_lock); if(pairer -> BAM_header_parsed || thread_no == 0){ thread_context -> need_find_start = pairer -> BAM_header_parsed; //SUBREADprintf("ABBO TH %d : FILL_BIN AT FILE %lld\n", thread_context -> thread_id, ftello(pairer -> input_fp )); SAM_pairer_fill_BIN_buff(pairer, thread_context, &is_finished); thread_context -> chunk_number = pairer -> input_chunk_no; pairer -> input_chunk_no ++; } subread_lock_release(&pairer -> input_fp_lock); if(!pairer -> BAM_header_parsed && thread_no > 0) { usleep(PAIRER_WAIT_TICK_TIME); } else if(thread_context -> input_buff_SBAM_used>0) { unsigned int processed_reads = 0; while(1){ int has_no_more = SAM_pairer_do_next_read(pairer, thread_context); if(has_no_more)break; processed_reads++; } pairer -> total_input_reads += processed_reads; } if(pairer -> is_bad_format) break; if(thread_context -> immediate_last_read_full_name[0]){ SAM_pairer_register_matcher(pairer, thread_context -> chunk_number, thread_context -> readno_in_chunk - 1, thread_context -> immediate_last_read_full_name, thread_context -> immediate_last_read_bin, thread_context -> immediate_last_read_bin_len , thread_context -> immediate_last_read_flags); SAM_pairer_do_read_test(pairer , thread_context , thread_context -> immediate_last_read_name_len , thread_context -> immediate_last_read_full_name , thread_context -> immediate_last_read_bin_len , thread_context -> immediate_last_read_bin, thread_context -> immediate_last_read_flags); thread_context -> immediate_last_read_full_name[0] = 0; } if(thread_context -> orphant_space > pairer -> input_buff_SBAM_size) if(!is_disk_full)is_disk_full |= SAM_pairer_update_orphant_table(pairer, thread_context); if(is_finished){ pairer -> BAM_header_parsed = 1; break; } } if(thread_context -> orphant_table -> numOfElements > 0) if(!is_disk_full)is_disk_full |= SAM_pairer_update_orphant_table(pairer, thread_context); pairer -> is_internal_error |= is_disk_full; return NULL; } // This function returns 1 if the bin is EXACTLY a whole read. int SAM_pairer_verify_read_bin_ONE(SAM_pairer_context_t * pairer, SAM_pairer_thread_t * thread_context , char * bin, int binlen){ int block_len = 9; int ret = is_read_bin_ONE(bin, binlen, pairer -> BAM_n_ref, &block_len); if(ret != 1 || block_len+4 != binlen){ SUBREADprintf("ERROR: cannot retrieve a read from the BAM file: %d, %d\n", block_len+4, ret); ret = -1; } //SUBREADprintf("FINAL_BIN_MATCH VERIFY : %d\n", ret); return ret; } void SAM_pairer_finish_margins(void * kv, void * val , HashTable * tab){ char * key = kv; if(key[0]=='E'){ char keyS [40]; strcpy(keyS, key); keyS[0]='S'; char * Sbin = HashTableGet(tab, keyS); assert(Sbin); char * Ebin = val; tab -> appendix2 ++; // SUBREADprintf("PAIRED_BINS: %s %s\n", key, keyS); SAM_pairer_context_t * pairer = tab -> appendix1; SAM_pairer_thread_t * thread_context = pairer -> threads+0; thread_context -> readno_in_chunk = 0; int Elen = 0, Slen = 0; memcpy(&Elen, Ebin, 4); memcpy(&Slen, Sbin, 4); char * tb = malloc(Elen + Slen); memcpy(tb, Ebin+4, Elen); memcpy(tb+ Elen, Sbin+4, Slen); if(SAM_pairer_verify_read_bin_ONE(pairer , thread_context, tb, Elen + Slen)==1) SAM_pairer_do_one_BIN( pairer, thread_context, tb, Elen + Slen); else{ pairer -> is_bad_format = 1; } free(tb); }else tab -> appendix2 --; } void SAM_pairer_finish_margin_table( SAM_pairer_context_t * pairer){ pairer -> bam_margin_table -> appendix1 = pairer; pairer -> bam_margin_table -> appendix2 = NULL; SAM_pairer_thread_t * thread_context = pairer -> threads+0; thread_context -> immediate_last_read_full_name[0] = 0; HashTableIteration(pairer -> bam_margin_table, SAM_pairer_finish_margins); if(thread_context -> immediate_last_read_full_name[0]){ SAM_pairer_register_matcher(pairer, thread_context -> chunk_number, thread_context -> readno_in_chunk - 1, thread_context -> immediate_last_read_full_name, thread_context -> immediate_last_read_bin, thread_context -> immediate_last_read_bin_len , thread_context -> immediate_last_read_flags); SAM_pairer_do_read_test(pairer , thread_context , thread_context -> immediate_last_read_name_len , thread_context -> immediate_last_read_full_name , thread_context -> immediate_last_read_bin_len , thread_context -> immediate_last_read_bin, thread_context -> immediate_last_read_flags); thread_context -> immediate_last_read_full_name[0] = 0; } pairer -> is_internal_error |= SAM_pairer_update_orphant_table(pairer, pairer -> threads+0); assert(NULL == pairer -> bam_margin_table -> appendix2); } // not only run, but also finalise. // It returns 0 if no error. int SAM_pairer_run_once( SAM_pairer_context_t * pairer){ int x1; for(x1 = 0; x1 < pairer -> total_threads ; x1++){ // this 16-byte memory block is freed in the thread worker. void ** init_params = malloc(sizeof(void *) * 2); init_params[0] = pairer; init_params[1] = (void *)(NULL+x1); pthread_create(&(pairer -> threads[x1].thread_stab), NULL, SAM_pairer_thread_run, init_params); } for(x1 = 0; x1 < pairer -> total_threads ; x1++){ pthread_join(pairer -> threads[x1].thread_stab, NULL); } if(0 == pairer -> is_bad_format){ if(pairer -> input_is_BAM) SAM_pairer_finish_margin_table(pairer); int is_disk_full = SAM_pairer_probe_maxfp( pairer ); if(is_disk_full){ SUBREADprintf("ERROR: cannot write into the temporary file. Please check the disk space in the output directory.\n"); pairer -> is_internal_error = 1; }else{ for(x1 = 0; x1 < pairer -> total_threads ; x1++){ // this 16-byte memory block is freed in the thread worker. void ** init_params = malloc(sizeof(void *) * 2); init_params[0] = pairer; init_params[1] = (void *)(NULL+x1); pthread_create(&(pairer -> threads[x1].thread_stab), NULL, SAM_pairer_rescure_orphants_max_FP, init_params); } for(x1 = 0; x1 < pairer -> total_threads ; x1++){ pthread_join(pairer -> threads[x1].thread_stab, NULL); } } } return 0; } int fix_load_next_block(FILE * in, char * binbuf, z_stream * strm){ char * bam_buf = malloc(70000); int x1, ret = 0; x1 = fgetc(in); if(x1 != 31) ret = -1; x1 = fgetc(in); if(x1 != 139) ret = -1; x1 = fgetc(in); if(x1 != 8) ret = -1; x1 = fgetc(in); if(x1 != 4) ret = -1; if(ret == 0){ x1 = fgetc(in); x1 = fgetc(in); x1 = fgetc(in); x1 = fgetc(in); x1 = fgetc(in);//XFL x1 = fgetc(in);//OS int xlen; xlen = fgetc(in); xlen += fgetc(in) * 256; int bsize = -1, xlen_ptr = 0; while(xlen_ptr < xlen){ int si1 = fgetc(in); int si2 = fgetc(in); int slen = fgetc(in); slen += fgetc(in) * 256; if(si1 == 66 && si2==67){ bsize = fgetc(in); bsize += 256*fgetc(in); }else{ fseeko(in , slen, SEEK_CUR); } xlen_ptr += 4 + slen; } if(bsize > 0){ int rlenv = fread(bam_buf, 1, bsize - xlen - 19, in); if(rlenv < bsize - xlen - 19) return -1; } fseeko(in, 8, SEEK_CUR); strm -> avail_in = bsize - xlen - 19; strm -> next_in = (unsigned char*)bam_buf; strm -> avail_out = 70000; strm -> next_out = (unsigned char*)binbuf; int ret_inf = inflate(strm, Z_FINISH); if(ret_inf == Z_STREAM_END) ret = 70000 - strm -> avail_out; else ret = -2; inflateReset(strm); } free(bam_buf); return ret; } int fix_write_block(FILE * out, char * bin, int binlen, z_stream * strm){ int is_end_mode = binlen == 0, written=0; // SUBREADprintf("FIX_WRTR : %d\n", binlen); while(1){ if(binlen - written<1 && !is_end_mode) return 0; char * bam_buf = malloc(70000); int x1, bam_len = 0, this_sec_len = 0, old_start = written; if(binlen - written > 0){ this_sec_len = binlen - written; if(this_sec_len>61000) this_sec_len=61000; strm -> avail_in = this_sec_len; strm -> next_in = (unsigned char*)bin + written; strm -> avail_out = 70000; strm -> next_out = (unsigned char*)bam_buf; deflate(strm , Z_FINISH); bam_len = 70000 - strm -> avail_out; written += this_sec_len; deflateReset(strm); }else{ z_stream nstrm; nstrm.zalloc = Z_NULL; nstrm.zfree = Z_NULL; nstrm.opaque = Z_NULL; nstrm.avail_in = 0; nstrm.next_in = Z_NULL; deflateInit2(&nstrm, SAMBAM_COMPRESS_LEVEL_NORMAL, Z_DEFLATED, PAIRER_GZIP_WINDOW_BITS, PAIRER_DEFAULT_MEM_LEVEL, Z_DEFAULT_STRATEGY); nstrm.avail_in = 0; nstrm.next_in = (unsigned char*)bin; nstrm.avail_out = 70000; nstrm.next_out = (unsigned char*)bam_buf; deflate(&nstrm, Z_FINISH); bam_len = 70000 - nstrm.avail_out; deflateEnd(&nstrm); } // SUBREADprintf(" FIX_COMPR: %d -> %d\n", this_sec_len , bam_len); unsigned int crc0 = crc32(0, NULL, 0); unsigned int crc = crc32(crc0, (unsigned char *) bin + old_start, this_sec_len); fputc(31, out); fputc(139, out); fputc(8, out); fputc(4, out); fputc(0, out); fputc(0, out); fputc(0, out); fputc(0, out); fputc(0, out);//XFL fputc(0xff, out);//OS x1 = 6; fwrite( &x1, 2, 1 , out ); fputc( 66, out ); fputc( 67, out ); x1 = 2; fwrite( &x1, 2, 1 , out ); x1 = bam_len + 19 + 6; fwrite( &x1, 2, 1 , out ); int write_len = fwrite( bam_buf , 1,bam_len, out ); fwrite( &crc, 4, 1, out ); fwrite( &this_sec_len, 4, 1, out ); free(bam_buf); if(write_len < bam_len)return 1; if(binlen<1) return 0; } return 0; } #define FIX_GET_NEXT_NCH { while(in_bin_ptr == in_bin_size){ \ in_bin_ptr = 0; in_bin_size = 0;\ int newsize = fix_load_next_block(old_fp, in_bin, &in_strm);\ if(newsize < 0){ in_bin_size = -1; if(newsize<-1)SUBREADprintf("ERROR: failed to decompress the BAM file %s\n", pairer -> in_file_name) ;break;}else{in_bin_size = newsize;}\ } if(in_bin_size>0){nch = in_bin[in_bin_ptr++]; if(nch < 0)nch += 256; } else nch = -1; } #define FIX_FLASH_OUT { if(out_bin_ptr > 0)disk_is_full |= fix_write_block(new_fp, out_bin, out_bin_ptr, &out_strm); out_bin_ptr = 0; } #define FIX_APPEND_OUT(p, c) { if(out_bin_ptr > 60002){FIX_FLASH_OUT} ; memcpy(out_bin + out_bin_ptr, p, c); out_bin_ptr +=c ; } #define FIX_APPEND_READ(p, c){ memcpy(out_bin + out_bin_ptr, p, c); out_bin_ptr +=c ; } int SAM_pairer_fix_format(SAM_pairer_context_t * pairer){ FILE * old_fp = pairer -> input_fp; fseeko(old_fp, 0, SEEK_SET); char tmpfname [MAX_FILE_NAME_LENGTH+14], readname[256]; sprintf(tmpfname, "%s.fixbam", pairer -> tmp_file_prefix); FILE * new_fp = f_subr_open(tmpfname, "wb"); char * in_bin = malloc(1024*70); char * out_bin = malloc(20*1024*1024); z_stream in_strm; z_stream out_strm; in_strm.zalloc = Z_NULL; in_strm.zfree = Z_NULL; in_strm.opaque = Z_NULL; in_strm.avail_in = 0; in_strm.next_in = Z_NULL; inflateInit2(&in_strm, PAIRER_GZIP_WINDOW_BITS); out_strm.zalloc = Z_NULL; out_strm.zfree = Z_NULL; out_strm.opaque = Z_NULL; out_strm.avail_in = 0; out_strm.next_in = Z_NULL; deflateInit2(&out_strm, Z_NO_COMPRESSION, Z_DEFLATED, PAIRER_GZIP_WINDOW_BITS, PAIRER_DEFAULT_MEM_LEVEL, Z_DEFAULT_STRATEGY); int disk_is_full = 0; int in_bin_ptr = 0; int out_bin_ptr = 0; int in_bin_size = 0; int content_count = 0; int content_size = 0; int x1, nch = 0, is_longcigar = 0; for(x1 = 0; x1 < 4; x1++){ FIX_GET_NEXT_NCH; // BAM1 if(nch < 0) return -1; FIX_APPEND_OUT(&nch, 1); } // ====== The header texts content_size = 0; for(x1 = 0; x1 < 4; x1++){ FIX_GET_NEXT_NCH; if(nch < 0) return -1; // SUBREADprintf("FIX: TLEN: %d\n", nch); content_size += (nch << (8 * x1)); } FIX_APPEND_OUT(&content_size, 4); //SUBREADprintf("FIX: TXTLEN=%d\n", content_size); for(content_count = 0; content_count < content_size; content_count++){ FIX_GET_NEXT_NCH; if(nch < 0) return -1; FIX_APPEND_OUT(&nch, 1); // fputc(nch, stderr); } FIX_FLASH_OUT; // ====== The chromosome table content_size = 0; for(x1 = 0; x1 < 4; x1++){ FIX_GET_NEXT_NCH; if(nch < 0) return -1; content_size += (nch << (8 * x1)); } FIX_APPEND_OUT(&content_size, 4); //SUBREADprintf("LONGFIX: CHROLEN=%d\n", content_size); for(content_count = 0; content_count < content_size; content_count++){ int namelen = 0; for(x1 = 0; x1 < 4; x1++){ FIX_GET_NEXT_NCH; if(nch < 0) return -1; namelen+= (nch << (8 * x1)); } FIX_APPEND_READ(&namelen, 4); for(x1 = 0; x1 < namelen + 4; x1++){ // inc. length FIX_GET_NEXT_NCH; if(nch < 0) return -1; FIX_APPEND_READ(&nch, 1); } if(out_bin_ptr > 60003){ FIX_FLASH_OUT; } } FIX_FLASH_OUT; // ===== The reads int seq_len = 0, name_len = 0, cigar_opts = 0, block_no=0; srInt_64 reads =0; pairer -> is_bad_format = 0; while(! is_longcigar){ int block_size = 0, new_block_size; char * block_size_ptr = out_bin + out_bin_ptr; char * sqlen_ptr = NULL; seq_len = 0, name_len = 0, cigar_opts = 0; // block_length FIX_GET_NEXT_NCH; if(nch<0) break; block_size = nch; for(x1 = 1; x1 < 4; x1++){ FIX_GET_NEXT_NCH; if(nch < 0) return -1; block_size += (nch << (8 * x1)); } FIX_APPEND_READ(&block_size, 4); if(pairer -> tiny_mode){ // block_remainder int extag_new_len = 0; // if(block_no>280000)SUBREADprintf("DOING BLOCK %d\n", block_no); block_no ++ ; for(x1 = 0; x1 < block_size; x1++){ FIX_GET_NEXT_NCH; if(nch < 0) return -1; if(x1 == 8) name_len = nch; else if(x1 >= 16 && x1 < 20){ seq_len += ( nch << (8 * (x1 - 16))); if(x1 == 16) sqlen_ptr = out_bin + out_bin_ptr; }else if(x1 == 12 || x1 == 13){ cigar_opts += ( nch << (8 * (x1 - 12))); }else if(seq_len > 1){ if(x1 == 32 + name_len + 4 * cigar_opts || x1 == 32 + name_len + 4 * cigar_opts + 1){ nch = 0xff; }else if(x1 > 32 + name_len + 4 * cigar_opts + 1 && x1 < 32 + name_len + 4 * cigar_opts + seq_len + (seq_len+1)/2){ continue; } } if( x1 == 32 && seq_len >= pairer -> long_read_minimum_length){ is_longcigar = 1; int x2; for(x2 = 0; x2 < name_len; x2++){ FIX_GET_NEXT_NCH; readname[x2] = nch; } break; } char etag_name0 = -1, etag_name1, etag_type; if(x1 == 32 + name_len + 4 * cigar_opts + seq_len + (seq_len+1)/2){ while(x1 < block_size){ int this_tag_output = 0; if(etag_name0 > 0){ FIX_GET_NEXT_NCH; if(nch < 0) return -1; } etag_name0 = nch; FIX_GET_NEXT_NCH; if(nch < 0) return -1; etag_name1 = nch; FIX_GET_NEXT_NCH; if(nch < 0) return -1; etag_type = nch; x1 += 3; if((( etag_name0 == 'H' && etag_name1 == 'I' ) || ( etag_name0 == 'N' && etag_name1 == 'H' ) || ( etag_name0 == 'R' && etag_name1 == 'G' ) || ( etag_name0 == 'N' && etag_name1 == 'M' ) ) && ( etag_type == 'c' || etag_type=='Z' || etag_type == 'C'||etag_type == 's'||etag_type == 'S'||etag_type == 'i'||etag_type == 'I') ){ FIX_APPEND_READ(&etag_name0,1); FIX_APPEND_READ(&etag_name1,1); FIX_APPEND_READ(&etag_type,1); this_tag_output = 1; } if(etag_type == 'Z'||etag_type =='H'){ if(this_tag_output) extag_new_len +=3; while(1){ FIX_GET_NEXT_NCH; if(nch < 0) return -1; if(this_tag_output){ assert(x1 < 20000); FIX_APPEND_READ(&nch, 1); extag_new_len++; } x1++; if(nch == 0)break; } }else if(etag_type == 'A'){ FIX_GET_NEXT_NCH; if(nch < 0) return -1; x1++; }else if(etag_type =='B'){ FIX_GET_NEXT_NCH; if(nch < 0) return -1; char array_type = nch; int x2, adlen = 1, aditems = 0; if(array_type == 's'||array_type == 'S')adlen = 2; if(array_type == 'i'||array_type == 'I'||array_type == 'f')adlen = 4; for(x2=0;x2<4; x2++) { FIX_GET_NEXT_NCH; if(nch < 0) return -1; aditems += nch << (8*x2); } x1 += 5 + aditems * adlen; for(x2 = 0; x2 < aditems * adlen; x2++){ FIX_GET_NEXT_NCH; if(nch < 0) return -1; } }else{ int dlen = 1; if(etag_type == 's'||etag_type == 'S') dlen = 2; if(etag_type == 'i'||etag_type == 'I' || etag_type == 'f') dlen = 4; if(this_tag_output) extag_new_len += dlen + 3; x1 += dlen; while(dlen > 0){ FIX_GET_NEXT_NCH; if(nch < 0) return -1; if(this_tag_output) FIX_APPEND_READ(&nch, 1); dlen--; } } } break; } FIX_APPEND_READ(&nch, 1); } if(!is_longcigar){ seq_len = min(1, seq_len); sqlen_ptr[0]=seq_len; sqlen_ptr[1]=0, sqlen_ptr[2]=0; sqlen_ptr[3]=0; new_block_size = 32 + name_len + 4 * cigar_opts + seq_len + (seq_len+1)/2 + extag_new_len; memcpy(block_size_ptr, &new_block_size, 4); } }else{ for(x1 = 0; x1 < block_size; x1++){ FIX_GET_NEXT_NCH; if(nch < 0) return -1; if(x1 == 8) name_len = nch; else if(x1 >= 16 && x1 < 20){ seq_len += ( nch << (8 * (x1 - 16))); if(x1 == 16) sqlen_ptr = out_bin + out_bin_ptr; }else if(x1 == 12 || x1 == 13){ cigar_opts += ( nch << (8 * (x1 - 12))); } if(x1 == 32 && seq_len >= pairer -> long_read_minimum_length){ is_longcigar = 1; int x2; for(x2 = 0; x2 < name_len; x2++){ FIX_GET_NEXT_NCH; readname[x2] = nch; } break; } FIX_APPEND_READ(&nch, 1); } } reads ++; if(out_bin_ptr > 60000){ FIX_FLASH_OUT; } } FIX_FLASH_OUT; disk_is_full |= fix_write_block(new_fp, out_bin, 0, &out_strm); deflateEnd(&out_strm); inflateEnd(&in_strm); fclose(new_fp); free(in_bin); free(out_bin); if(is_longcigar){ unlink(tmpfname); pairer -> long_cigar_mode = 1; pairer -> tiny_mode = 1; if(0 && ! pairer -> is_single_end_mode){ print_in_box(80,0,0," Switch to long-read mode; reads, not read-pairs, will be counted."); print_in_box(80,0,0," Read name: %s", readname); print_in_box(80,0,0," It had %d cigar opts and %d bases, more than %d.", cigar_opts, seq_len, pairer -> long_read_minimum_length); } }else{ fclose(old_fp); pairer -> input_fp = f_subr_open(tmpfname, "rb"); } if(disk_is_full)SUBREADprintf("ERROR: cannot write into the temporary file. Please check the empty space in the output directory.\n"); return disk_is_full; } unsigned int nosort_tick_time = 100; #define NOSORT_SBAM_BUFF_SIZE 5000000 #define NOSORT_BIN_BUFF_SIZE (2*5010000) void * SAM_nosort_thread_run( void * params ){ void ** param_ptr = (void **) params; SAM_pairer_context_t * pairer = param_ptr[0]; int thread_no = (int)(param_ptr[1]-NULL); free(params); SAM_pairer_thread_t * thread_context = pairer -> threads + thread_no; char * read_ptr_1 = (char *)thread_context -> input_buff_BIN; char * read_ptr_2 = (char *)thread_context -> input_buff_BIN + NOSORT_BIN_BUFF_SIZE / 2; while(1){ int has_found = 0, to_quit = 0; subread_lock_occupy(&thread_context -> SBAM_lock); // SUBREADprintf("CONSUME:RINS=%d, PTR=%d\n", thread_context -> reads_in_SBAM, thread_context -> input_buff_BIN_ptr ); if(thread_context -> reads_in_SBAM > 1){ if(pairer -> input_is_BAM){ int record_len, seq_len1 = 0, seq_len2 = 0; // SUBREADprintf("LOAD BY THREAD %d:", thread_no); memcpy(&record_len, thread_context -> input_buff_SBAM + thread_context -> input_buff_SBAM_ptr, 4); // SUBREADprintf("RLEN=%d\n", record_len); assert(record_len > 32 &&record_len < NOSORT_SBAM_BUFF_SIZE); memcpy(read_ptr_1 , thread_context -> input_buff_SBAM + thread_context -> input_buff_SBAM_ptr, 4 + record_len); memcpy(&seq_len1, thread_context -> input_buff_SBAM + thread_context -> input_buff_SBAM_ptr + 20, 4); thread_context -> input_buff_SBAM_ptr += record_len + 4; memcpy(&record_len, thread_context -> input_buff_SBAM + thread_context -> input_buff_SBAM_ptr, 4); assert(record_len > 32 &&record_len < NOSORT_SBAM_BUFF_SIZE); memcpy(read_ptr_2 , thread_context -> input_buff_SBAM + thread_context -> input_buff_SBAM_ptr, 4 + record_len); memcpy(&seq_len2, thread_context -> input_buff_SBAM + thread_context -> input_buff_SBAM_ptr + 20, 4); thread_context -> input_buff_SBAM_ptr += record_len + 4; has_found = 1; thread_context -> reads_in_SBAM -= 2; if(seq_len1 >= pairer -> long_read_minimum_length || seq_len2 >= pairer -> long_read_minimum_length) pairer -> long_cigar_mode = 1; }else{ thread_context -> input_buff_BIN_ptr = 0; int rret = reduce_SAM_to_BAM(pairer, thread_context , 0); thread_context -> reads_in_SBAM -- ; if(rret > 0){ thread_context -> input_buff_BIN_ptr = NOSORT_BIN_BUFF_SIZE/2; rret = reduce_SAM_to_BAM(pairer, thread_context, 0); thread_context -> reads_in_SBAM -- ; if(rret > 0){ has_found = 1; } } } } if(pairer -> is_finished) to_quit = 1; subread_lock_release(&thread_context -> SBAM_lock); if(has_found) pairer -> output_function(pairer, thread_no, (char*) read_ptr_1,(char*) read_ptr_2); else{ if(to_quit) break; usleep(nosort_tick_time); } } return NULL; } int SAM_nosort_decompress_next_block(SAM_pairer_context_t * pairer){ int SBAM_used; unsigned int decompressed_len; char * SBAM_buff = pairer -> appendix2; char * BIN_buff = pairer -> appendix3; int * BIN_buff_used = pairer -> appendix4; int * BIN_buff_ptr = pairer -> appendix5; SBAM_used = PBam_get_next_zchunk(pairer -> input_fp, SBAM_buff, NOSORT_SBAM_BUFF_SIZE, &decompressed_len); if(SBAM_used<0){ if(SBAM_used == -2){ SUBREADputs("ERROR: the BAM format is broken."); pairer->is_internal_error = 1; } return -1; } //SUBREADprintf("PRE-LOAD BAM: USED %d, PTR %d\n", * BIN_buff_used , * BIN_buff_ptr); if((* BIN_buff_ptr) < (* BIN_buff_used)){ int diff = (* BIN_buff_used) - (* BIN_buff_ptr); int x1; for(x1 = 0; x1 < diff; x1++){ BIN_buff[x1] = BIN_buff[x1 + (* BIN_buff_ptr)]; } (* BIN_buff_used) = diff; } else (* BIN_buff_used) = 0; (* BIN_buff_ptr) = 0; int binlen = SamBam_unzip(BIN_buff + (* BIN_buff_used), 65536, SBAM_buff , SBAM_used, 0); //assert(binlen == decompressed_len); if(binlen < 0) return -1; (* BIN_buff_used) += binlen; return binlen; } #define NOSORT_BAM_next_nch { while( BIN_buff_used == BIN_buff_ptr ){int rlen = SAM_nosort_decompress_next_block(pairer); if(rlen < 0) { BIN_buff_used = -1 ; break;}} if(BIN_buff_used < 0) nch = -1; else nch = BIN_buff[BIN_buff_ptr++]; } #define NOSORT_BAM_next_u32(v){ NOSORT_BAM_next_nch; if(nch < 0)v=-1;else{; v= nch; NOSORT_BAM_next_nch; v+=nch*256; NOSORT_BAM_next_nch; v+=nch*65536; NOSORT_BAM_next_nch; v+=nch*16777216;} } #define NOSORT_SAM_next_line {NOSORT_SAM_eof = fgets(line_ptr, NOSORT_SBAM_BUFF_SIZE, pairer -> input_fp);} #if FEATURECOUNTS_BUFFER_SIZE < ( 12*1024*1024 ) #error "FEATURECOUNTS_BUFFER_SIZE MUST BE GREATER THAN 12MB!." #endif #define NOSORT_REFILL_LOWBAR ( 3 * 1024 * 1024 ) #define NOSORT_REFILL_HIGHBAR ( 6 * 1024 * 1024 ) void SAM_nosort_run_once(SAM_pairer_context_t * pairer){ int x1; for(x1 = 0; x1 < pairer -> total_threads ; x1++){ // this 16-byte memory block is freed in the thread worker. void ** init_params = malloc(sizeof(void *) * 2); init_params[0] = pairer; init_params[1] = (void *)(NULL+x1); pthread_create(&(pairer -> threads[x1].thread_stab), NULL, SAM_nosort_thread_run, init_params); } char * SBAM_buff = malloc(NOSORT_SBAM_BUFF_SIZE); int nch; unsigned char * BIN_buff = malloc(NOSORT_BIN_BUFF_SIZE); char *NOSORT_SAM_eof=NULL; int BIN_buff_used = 0; int BIN_buff_ptr = 0; pairer -> appendix2 = SBAM_buff; pairer -> appendix3 = BIN_buff; pairer -> appendix4 = &BIN_buff_used; pairer -> appendix5 = &BIN_buff_ptr; if(pairer -> input_is_BAM){ int x1; unsigned int bam_signature; NOSORT_BAM_next_u32(bam_signature); NOSORT_BAM_next_u32(pairer -> BAM_l_text); char * header_txt = malloc(max(1000000,pairer->BAM_l_text)); for(x1 = 0 ; x1 < pairer -> BAM_l_text; x1++){ NOSORT_BAM_next_nch; header_txt [x1] = nch; } int is_OK = pairer -> output_header(pairer, 0, 1, pairer -> BAM_l_text , header_txt , pairer -> BAM_l_text ); NOSORT_BAM_next_u32(pairer -> BAM_n_ref); unsigned int ref_bin_len = 0; for(x1 = 0; x1 < pairer -> BAM_n_ref; x1++) { unsigned int l_name, l_ref, x2; NOSORT_BAM_next_u32(l_name); assert(l_name < 256); memcpy(header_txt + ref_bin_len, &l_name, 4); ref_bin_len += 4; for(x2 = 0; x2 < l_name; x2++){ NOSORT_BAM_next_nch; header_txt[ref_bin_len++] = nch; } NOSORT_BAM_next_u32(l_ref); memcpy(header_txt + ref_bin_len, &l_ref, 4); ref_bin_len += 4; assert(ref_bin_len < pairer -> BAM_l_text); } is_OK = is_OK || pairer -> output_header(pairer, 0, 0, pairer -> BAM_n_ref , header_txt , ref_bin_len ); free(header_txt); if(is_OK){ pairer -> is_incomplete_BAM = 1; return; } while(1){ if(pairer -> is_finished) break; int need_sleep = 1; for(x1 = 0; x1 < pairer -> total_threads ; x1++){ if(pairer -> is_finished) break; SAM_pairer_thread_t * this_thread = pairer -> threads + x1; if(this_thread -> input_buff_SBAM_used - this_thread -> input_buff_SBAM_ptr < NOSORT_REFILL_LOWBAR && (this_thread -> input_buff_SBAM_used == 0 || this_thread -> input_buff_SBAM_ptr > 0)){ subread_lock_occupy(&this_thread -> SBAM_lock); int to_be_add = NOSORT_REFILL_HIGHBAR - (this_thread -> input_buff_SBAM_used - this_thread -> input_buff_SBAM_ptr); int x2, x3; if(this_thread -> input_buff_SBAM_ptr < this_thread -> input_buff_SBAM_used){ for(x2 = 0; x2 < this_thread -> input_buff_SBAM_used - this_thread -> input_buff_SBAM_ptr; x2++) this_thread -> input_buff_SBAM[x2] = this_thread -> input_buff_SBAM[x2 + this_thread -> input_buff_SBAM_ptr]; this_thread -> input_buff_SBAM_used -= this_thread -> input_buff_SBAM_ptr; }else this_thread -> input_buff_SBAM_used =0; this_thread -> input_buff_SBAM_ptr = 0; for(x2 = 0 ; ; x2++){ int record_len; NOSORT_BAM_next_u32(record_len); if(record_len < 32 || record_len > 500000){ if(record_len!=-1) SUBREADprintf("Unexpected record length: %d.\n", record_len); pairer -> is_finished = 1; break; } memcpy(this_thread -> input_buff_SBAM + this_thread -> input_buff_SBAM_used , &record_len, 4); this_thread -> input_buff_SBAM_used += 4; for(x3 =0; x3 < record_len; x3++){ NOSORT_BAM_next_nch; this_thread -> input_buff_SBAM[this_thread -> input_buff_SBAM_used++] = nch; } this_thread -> reads_in_SBAM ++; if(x2 % 2 == 1 && to_be_add <= this_thread -> input_buff_SBAM_used + 20000 )break; } need_sleep = 0; subread_lock_release(&this_thread -> SBAM_lock); } } if(need_sleep) usleep(nosort_tick_time); } }else{ // if input is SAM char * line_ptr = SBAM_buff; char * header_start = NULL; int passed_read_SBAM_ptr = -1; unsigned int header_buffer_safe_size = 0; while(1){ passed_read_SBAM_ptr = ftello(pairer -> input_fp); NOSORT_SAM_next_line; if(NOSORT_SAM_eof == NULL)break; header_buffer_safe_size += strlen(line_ptr); if(NULL== header_start && line_ptr[0] == '@') header_start = line_ptr; if(NULL == line_ptr){ SUBREADprintf("FATAL: the header is too large to the buffer.\n"); break; }else{ //SUBREADprintf("LINELEN=%d, PTR=%d, FIRST=%c\n", line_len, thread_context -> input_buff_SBAM_ptr , line_ptr[0]); } if(line_ptr[0]!='@'){ break; } } fseeko(pairer -> input_fp, 0 , SEEK_SET); int header_bin_ptr = 0, header_contigs = 0; char * header_bin = malloc(header_buffer_safe_size); while(1){ NOSORT_SAM_next_line; if(NOSORT_SAM_eof == NULL)break; if(line_ptr[0]!='@') break; if(memcmp(line_ptr, "@SQ\t",4)==0){ unsigned int ct_len = 0, ctptr = 4, status = 0, sqname_len = 0; char * sqname = NULL; while(1){ char ctnch = line_ptr[ctptr++]; if( status == 0){ if(ctnch=='S' && line_ptr[ctptr] == 'N' && line_ptr[ctptr+1] == ':'){ ctptr += 2; status = 10; sqname = line_ptr + ctptr; }else if(ctnch=='L' && line_ptr[ctptr] == 'N' && line_ptr[ctptr+1] == ':'){ ctptr += 2; status = 20; }else status = 30; }else if(status == 10 || status == 20 || status == 30){ if(ctnch == '\t' || ctnch == '\n'){ status = 0; if(ctnch == '\n') break; //break; } if(status == 10) sqname_len ++; else if(status == 20) ct_len = ct_len * 10 + ctnch - '0'; } } sqname_len += 1; memcpy(header_bin + header_bin_ptr, &sqname_len, 4); header_bin_ptr += 4; memcpy(header_bin + header_bin_ptr, sqname, sqname_len-1); *(header_bin + header_bin_ptr + sqname_len - 1) = 0; char * mem_contig_name = malloc(sqname_len); strcpy(mem_contig_name , header_bin + header_bin_ptr); // SUBREADprintf("CONTIG %d : %s (len=%d = %d)\n", header_contigs, header_bin + header_bin_ptr , sqname_len, strlen(mem_contig_name)); HashTablePut(pairer -> sam_contig_number_table , mem_contig_name, NULL + 1 + header_contigs); header_bin_ptr += sqname_len; memcpy(header_bin + header_bin_ptr, &ct_len, 4); header_bin_ptr += 4; header_contigs++; } } pairer -> BAM_header_parsed = 1; int is_OK = pairer -> output_header(pairer, 0, 0, header_contigs , header_bin , header_bin_ptr); free(header_bin); if(is_OK){ pairer -> is_incomplete_BAM = 1; return; } fseeko(pairer -> input_fp, passed_read_SBAM_ptr, SEEK_SET); line_ptr = SBAM_buff; while(1){ if(pairer -> is_finished) break; int need_sleep = 1; for(x1 = 0; x1 < pairer -> total_threads ; x1++){ if(pairer -> is_finished) break; SAM_pairer_thread_t * this_thread = pairer -> threads + x1; if(this_thread -> input_buff_SBAM_used - this_thread -> input_buff_SBAM_ptr < NOSORT_REFILL_LOWBAR && (this_thread -> input_buff_SBAM_used == 0 || this_thread -> input_buff_SBAM_ptr > 0)){ subread_lock_occupy(&this_thread -> SBAM_lock); int to_be_add = NOSORT_REFILL_HIGHBAR - (this_thread -> input_buff_SBAM_used - this_thread -> input_buff_SBAM_ptr); int x2; if(this_thread -> input_buff_SBAM_ptr < this_thread -> input_buff_SBAM_used){ for(x2 = 0; x2 < this_thread -> input_buff_SBAM_used - this_thread -> input_buff_SBAM_ptr; x2++) this_thread -> input_buff_SBAM[x2] = this_thread -> input_buff_SBAM[x2 + this_thread -> input_buff_SBAM_ptr]; this_thread -> input_buff_SBAM_used -= this_thread -> input_buff_SBAM_ptr; }else this_thread -> input_buff_SBAM_used =0; this_thread -> input_buff_SBAM_ptr = 0; for(x2 = 0 ; ; x2++){ int record_len; NOSORT_SAM_next_line; if(NULL==NOSORT_SAM_eof || line_ptr[0]==0){ pairer -> is_finished = 1; break; } record_len = strlen(line_ptr); // SUBREADprintf("1CHR=%c, ECHR=%d , RL=%d, RINS=%d, USED=%d, SIZE=%d\n", line_ptr[0], line_ptr[record_len - 1], record_len, this_thread -> reads_in_SBAM, this_thread -> input_buff_SBAM_used, pairer -> input_buff_SBAM_size); memcpy(this_thread -> input_buff_SBAM + this_thread -> input_buff_SBAM_used , line_ptr, record_len); this_thread -> input_buff_SBAM_used += record_len; this_thread -> reads_in_SBAM ++; if(x2 % 2 == 1 && to_be_add <= this_thread -> input_buff_SBAM_used + 20000 )break; } need_sleep = 0; subread_lock_release(&this_thread -> SBAM_lock); } } if(need_sleep) usleep(nosort_tick_time); } } free(SBAM_buff); free(BIN_buff); for(x1 = 0; x1 < pairer -> total_threads ; x1++){ pthread_join(pairer -> threads[x1].thread_stab, NULL); } } #define BINADD_NCHAR { if(binptr >= bin_buff_capacity - 10){\ bin_buff_capacity = bin_buff_capacity * 14 / 10;\ bin_buffer = realloc(bin_buffer, bin_buff_capacity);\ } bin_buffer[binptr++] = nch;} // only one thread; very large buffer size. int SAM_pairer_long_cigar_run(SAM_pairer_context_t * pairer){ char *bin_buffer, *bam_buffer; FILE * old_fp = pairer -> input_fp; int bin_buff_capacity = 1000000, block_size = 0; char * in_bin = malloc(140000); bin_buffer = malloc(bin_buff_capacity); bam_buffer = malloc(70000); z_stream in_strm; in_strm.zalloc = Z_NULL; in_strm.zfree = Z_NULL; in_strm.opaque = Z_NULL; in_strm.avail_in = 0; in_strm.next_in = Z_NULL; inflateInit2(&in_strm, PAIRER_GZIP_WINDOW_BITS); fseeko(old_fp, 0, SEEK_SET); if(1){ int disk_is_full = 0; int in_bin_ptr = 0; int out_bin_ptr = 0; int in_bin_size = 0; int content_count = 0; int content_size = 0; int is_finished = 0; int x1, nch = 0, binptr = 0; disk_is_full = disk_is_full?out_bin_ptr:out_bin_ptr; // stupid code to avoid warning messages from GCC 7 for(x1 = 0; x1 < 4; x1++){ FIX_GET_NEXT_NCH; // BAM1 if(nch < 0) return -1; } // ====== The header texts content_size = 0; binptr = 0; for(x1 = 0; x1 < 4; x1++){ FIX_GET_NEXT_NCH; if(nch < 0) return -1; content_size += (nch << (8 * x1)); } for(content_count = 0; content_count < content_size; content_count++){ FIX_GET_NEXT_NCH; BINADD_NCHAR; if(nch < 0) return -1; } pairer -> output_header (pairer , 0, 1, binptr, bin_buffer, binptr); // ====== The chromosome table binptr = 0; content_size = 0; for(x1 = 0; x1 < 4; x1++){ FIX_GET_NEXT_NCH; if(nch < 0) return -1; content_size += (nch << (8 * x1)); } for(content_count = 0; content_count < content_size; content_count++){ block_size = 0; for(x1 = 0; x1 < 4; x1++){ FIX_GET_NEXT_NCH; if(nch < 0) return -1; BINADD_NCHAR; block_size += (nch << (8 * x1)); } for(x1 = 0; x1 < block_size + 4; x1++){ FIX_GET_NEXT_NCH; if(nch < 0) return -1; BINADD_NCHAR; } } pairer -> output_header (pairer , 0, 0, content_size, bin_buffer, binptr); // go through the reads int reads = 0; while(1){ binptr = 0; block_size = 0; for(x1 = 0; x1 < 4; x1++){ FIX_GET_NEXT_NCH; if(x1 == 0 && nch < 0){ is_finished=1; break; } if(nch < 0) return -1; BINADD_NCHAR; block_size += (nch << (8 * x1)); } if(is_finished)break; for(x1 = 0; x1 < block_size; x1 ++){ FIX_GET_NEXT_NCH; if(nch < 0) return -1; BINADD_NCHAR; } pairer -> output_function(pairer, 0, bin_buffer, NULL); reads++; } } free(bam_buffer); free(bin_buffer); free(in_bin); return 0; } void pairer_increase_SAMBAM_buffer(SAM_pairer_context_t * pairer){ pairer -> input_buff_SBAM_size *= 5; pairer -> input_buff_BIN_size = max(1024*1024, pairer -> input_buff_SBAM_size ); int x1; for(x1 = 0; x1< pairer -> total_threads; x1++){ pairer -> threads[x1].input_buff_SBAM = realloc( pairer -> threads[x1].input_buff_SBAM, pairer -> input_buff_SBAM_size); pairer -> threads[x1].input_buff_BIN = realloc( pairer -> threads[x1].input_buff_BIN, pairer -> input_buff_BIN_size); } } int SAM_pairer_run( SAM_pairer_context_t * pairer){ int corrected_run; if(pairer -> force_do_not_sort){ SAM_nosort_run_once(pairer); }else for(corrected_run = 0; corrected_run < 2 ; corrected_run ++){ pairer -> is_final_run = corrected_run; SAM_pairer_run_once(pairer); //SUBREADprintf("RUN %d : BAD_format %d\n", corrected_run, pairer -> is_bad_format); if(pairer -> is_bad_format && pairer->input_is_BAM && ( ! pairer -> is_internal_error ) && ( ! pairer -> is_incomplete_BAM )){ //#warning ">>>>>> REMOVE '+ 1' FROM NEXT LINE IN RELEASE <<<<<<" assert(1 != corrected_run); delete_with_prefix(pairer -> tmp_file_prefix); pairer -> is_internal_error |= SAM_pairer_fix_format(pairer); if(pairer -> is_bad_format || pairer -> is_internal_error) return -1; SAM_pairer_reset(pairer); if(pairer -> reset_output_function)pairer -> reset_output_function(pairer); pairer_increase_SAMBAM_buffer(pairer); if(pairer -> long_cigar_mode) return SAM_pairer_long_cigar_run(pairer); }else break; } return pairer -> is_bad_format || pairer -> is_internal_error || pairer -> is_incomplete_BAM; } int sort_SAM_create(SAM_sort_writer * writer, char * output_file, char * tmp_path) { char tmp_fname[MAX_FILE_NAME_LENGTH+40], mac_rand[13]; memset(writer, 0, sizeof(SAM_sort_writer)); old_sig_TERM = signal (SIGTERM, SAM_SORT_SIGINT_hook); old_sig_INT = signal (SIGINT, SAM_SORT_SIGINT_hook); mac_or_rand_str(mac_rand); if(tmp_path == NULL){ int slash_pos = 0; for(slash_pos = strlen(output_file); slash_pos >=0; slash_pos--){ if(output_file[slash_pos]=='/')break; } if(slash_pos >= 0){ memcpy(writer -> tmp_path, output_file, slash_pos+1); sprintf(writer -> tmp_path + slash_pos+1, "temp-sort-%06u-%s-", getpid(), mac_rand); }else sprintf(writer -> tmp_path, "./temp-sort-%06u-%s-", getpid(), mac_rand); }else sprintf(writer -> tmp_path, "%s/temp-sort-%06u-%s-", tmp_path, getpid(), mac_rand); //#warning " >>>>>>>>>>>>>>>> REMOVE THE NEXT LINE <<<<<<<<<<<<<<<<<<<< " //SUBREADprintf("TMP_SORT=%s FROM %s\n", writer -> tmp_path, output_file); _SAMSORT_SNP_delete_temp_prefix = writer -> tmp_path; sprintf(tmp_fname, "%s%s", writer -> tmp_path, "headers.txt"); writer -> all_chunks_header_fp = f_subr_open(tmp_fname,"w"); if(!writer -> all_chunks_header_fp) return -1; fclose(writer -> all_chunks_header_fp); unlink(tmp_fname); writer -> out_fp = f_subr_open(output_file,"w"); if(!writer -> out_fp) return -1; return 0; } void find_tag_out(char * read_line_buf, char * tag, char * hi_tag_out) { int hi_tag = -1; char tag_str[10]; sprintf(tag_str , "\t%s:i:", tag); char * hi_tag_str = strstr(read_line_buf, tag_str); if(hi_tag_str) { hi_tag = 0; int line_cursor; for(line_cursor=6; ; line_cursor++) { char nch = hi_tag_str[line_cursor]; // printf("HI:i=%s; nch [%d] ='%c'\n", hi_tag_str, line_cursor, nch); if(!isdigit(nch)) break; hi_tag = hi_tag*10 + (nch-'0'); } } if(hi_tag >=0) { sprintf(hi_tag_out,"\t%s:i:%d", tag, hi_tag); }else hi_tag_out[0] = 0; } int sort_SAM_finalise(SAM_sort_writer * writer) { int x1_chunk, x1_block, is_disk_full = 0; int xk1; for(xk1=0;xk1 current_block_fp_array[xk1]) fclose(writer -> current_block_fp_array[xk1]); } memset(writer -> current_block_fp_array, 0, sizeof(FILE *)*SAM_SORT_BLOCKS); writer -> current_chunk_size = 0; writer -> current_chunk++; for(x1_block = 0; x1_block current_chunk; x1_chunk++) { char tmpfname[MAX_FILE_NAME_LENGTH+40]; sprintf(tmpfname, "%sCHK%08d-BLK%03d.bin", writer -> tmp_path, x1_chunk , x1_block); FILE * bbfp = f_subr_open(tmpfname,"rb"); if(!bbfp) continue; while(!feof(bbfp)) { char * read_name = NULL; short flags; short read_name_len; short read_len; int ret = fread(&flags, 2,1 , bbfp); if(ret<1) break; ret = fread(&read_name_len, 2,1 , bbfp); if(ret<1) break; if(flags & SAM_FLAG_SECOND_READ_IN_PAIR) fseeko(bbfp, read_name_len, SEEK_CUR); else { read_name = malloc(read_name_len+1); ret = fread(read_name, 1, read_name_len, bbfp); if(ret< read_name_len) break; read_name[read_name_len] = 0; } ret =fread(&read_len,2,1,bbfp); if(ret<1) break; if(flags & SAM_FLAG_SECOND_READ_IN_PAIR) fseeko(bbfp, read_len, SEEK_CUR); else { char * new_line_mem = malloc(read_len+1); ret = fread(new_line_mem, 1, read_len, bbfp); if(ret numOfElements<4)printf("RV=%s\n", read_name); } } fclose(bbfp); } //printf("BLK=%d; CKS=%d; READS=%llu\n", x1_block, x1_chunk, first_read_name_table -> numOfElements); srInt_64 finished_second_reads = 0; for(x1_chunk = 0; x1_chunk < writer -> current_chunk; x1_chunk++) { char tmpfname[MAX_FILE_NAME_LENGTH+40]; sprintf(tmpfname, "%sCHK%08d-BLK%03d.bin", writer -> tmp_path, x1_chunk , x1_block); // printf("START_BLOCK: %s\n", tmpfname); FILE * bbfp = f_subr_open(tmpfname,"rb"); if(!bbfp) continue; char * read_line_buf = malloc(3000); char * read_name_buf = malloc(MAX_READ_NAME_LEN + MAX_CHROMOSOME_NAME_LEN * 2 + 26); while(!feof(bbfp)) { short flags; short read_name_len; short read_len; int ret = fread(&flags, 2,1 , bbfp); if(ret<1) break; ret = fread(&read_name_len, 2,1 , bbfp); if(ret < 1) break; if(read_name_len>=MAX_READ_NAME_LEN + MAX_CHROMOSOME_NAME_LEN * 2 + 26) SUBREADprintf("VERY_LONG_NAME(%d)\n", read_name_len); if(flags & SAM_FLAG_SECOND_READ_IN_PAIR) { ret = fread(read_name_buf, 1, read_name_len, bbfp); if(ret < read_name_len) break; read_name_buf[read_name_len] = 0; } else fseeko(bbfp, read_name_len, SEEK_CUR); ret = fread(&read_len, 2,1 , bbfp); if(ret < 1) break; if(flags & SAM_FLAG_SECOND_READ_IN_PAIR) { ret = fread(read_line_buf, 1, read_len, bbfp); if(ret < 1) break; read_line_buf[read_len] = 0; } else fseeko(bbfp, read_len, SEEK_CUR); if(flags & SAM_FLAG_SECOND_READ_IN_PAIR) { // printf("RRNAME:%s\n", read_name_buf); char * first_read_text = HashTableGet(first_read_name_table, read_name_buf); strtok(read_name_buf,"\t"); if(first_read_text && first_read_text[0]!=(char)0xff) { fputs(read_name_buf, writer->out_fp); putc('\t', writer->out_fp); fputs(first_read_text, writer->out_fp); fputs(read_name_buf, writer->out_fp); putc('\t', writer->out_fp); int write_len = fputs(read_line_buf, writer->out_fp); if(write_len < 0) is_disk_full = 1; read_name_buf[strlen(read_name_buf)]='\t'; HashTableRemove(first_read_name_table, read_name_buf); finished_second_reads ++; } else{ int dummy_flags = 4 | 1, mate_flags = 0; char * dummy_mate_chr = NULL; char dummy_mate_chr_buf[120]; unsigned int dummy_old_read_pos = 0, tmpi=0,dummy_char_strpos = 0; int tabs = 0; int read_cursor = 0; for(read_cursor = 0;; read_cursor++) { char nch = read_line_buf[read_cursor]; if(!nch) break; if(nch == '\t') { if(tabs == 0){ mate_flags = tmpi; dummy_mate_chr = read_line_buf+read_cursor+1; } else if(tabs == 1) dummy_char_strpos = read_cursor; else if(tabs == 2) { dummy_old_read_pos = tmpi; break; } tmpi=0; tabs++; }else{ if(tabs==0 || tabs == 2) tmpi = tmpi * 10 + (nch - '0'); } } dummy_flags |= SAM_FLAG_FIRST_READ_IN_PAIR; if(mate_flags & SAM_FLAG_UNMAPPED) dummy_flags |= SAM_FLAG_MATE_UNMATCHED; if(mate_flags & SAM_FLAG_REVERSE_STRAND_MATCHED) dummy_flags |= SAM_FLAG_MATE_REVERSE_STRAND_MATCHED; if(mate_flags & SAM_FLAG_MATE_REVERSE_STRAND_MATCHED) dummy_flags |= SAM_FLAG_REVERSE_STRAND_MATCHED; memcpy(dummy_mate_chr_buf, dummy_mate_chr, read_line_buf +dummy_char_strpos - dummy_mate_chr); dummy_mate_chr_buf[read_line_buf +dummy_char_strpos - dummy_mate_chr]=0; char hi_tag_out[18]; char nh_tag_out[18]; find_tag_out(read_line_buf, "HI", hi_tag_out); find_tag_out(read_line_buf, "NH", nh_tag_out); // build a fake FIRST read for the mapped SECOND read. // note that the TLEN, MATE_POS and MATE_CHAR are incorrect for general use. fprintf(writer->out_fp, "%s\t%d\t*\t0\t0\t*\t%s\t%d\t0\tN\tI%s%s\n", read_name_buf, dummy_flags, dummy_mate_chr_buf, dummy_old_read_pos, nh_tag_out, hi_tag_out); fputs(read_name_buf, writer->out_fp); putc('\t', writer->out_fp); int write_len = fputs(read_line_buf, writer->out_fp); if(write_len < 0) is_disk_full = 1; writer -> unpaired_reads +=1; } //else SUBREADprintf("WARNING: Unpaired read found in file:%s\n", read_name_buf); } } fclose(bbfp); unlink(tmpfname); free(read_name_buf); free(read_line_buf); } if(1) { writer -> unpaired_reads += first_read_name_table -> numOfElements; KeyValuePair * cursor; int bucket; // go through the hash table and write correct FIRST lines and dummy SECOND lines. for(bucket=0; bucket< first_read_name_table -> numOfBuckets; bucket++) { cursor = first_read_name_table -> bucketArray[bucket]; while(1) { if (!cursor) break; char * first_read_text = (char *)cursor -> value; char * first_read_name = (char *)cursor -> key; if(first_read_text[0]!=(char)0xff) { int dummy_flags = 4 | 1, mate_flags = 0; char * dummy_mate_chr = NULL; unsigned int dummy_old_read_pos = 0, tmpi=0, dummy_char_strpos = 0; int tabs = 0; int read_cursor = 0; for(read_cursor = 0;; read_cursor++) { char nch = first_read_text[read_cursor]; if(!nch) break; if(nch == '\t') { if(tabs == 0){ mate_flags = tmpi; dummy_mate_chr = first_read_text+read_cursor+1; } else if(tabs == 1) dummy_char_strpos = read_cursor; else if(tabs == 2) { dummy_old_read_pos = tmpi; break; } tmpi=0; tabs++; }else{ if(tabs==0 || tabs == 2) tmpi = tmpi * 10 + (nch - '0'); } } dummy_flags |= SAM_FLAG_SECOND_READ_IN_PAIR; if(mate_flags & SAM_FLAG_UNMAPPED) dummy_flags |= SAM_FLAG_MATE_UNMATCHED; if(mate_flags & SAM_FLAG_REVERSE_STRAND_MATCHED) dummy_flags |= SAM_FLAG_MATE_REVERSE_STRAND_MATCHED; if(mate_flags & SAM_FLAG_MATE_REVERSE_STRAND_MATCHED) dummy_flags |= SAM_FLAG_REVERSE_STRAND_MATCHED; if((!first_read_text[0])||(!first_read_text[1])) { SUBREADprintf("unable to recover the first read : '%s' , flags = %d\n", first_read_name, mate_flags); assert(0); } char nh_tag_out[18]; char hi_tag_out[18]; find_tag_out(first_read_text, "NH", nh_tag_out); find_tag_out(first_read_text, "HI", hi_tag_out); strtok(first_read_name, "\t"); fputs(first_read_name, writer->out_fp); putc('\t', writer->out_fp); fputs(first_read_text, writer->out_fp); first_read_text[dummy_char_strpos] = 0; fprintf(writer->out_fp, "%s\t%d\t*\t0\t0\t*\t%s\t%d\t0\tN\tI%s%s\n", first_read_name, dummy_flags, dummy_mate_chr, dummy_old_read_pos, nh_tag_out,hi_tag_out); } cursor = cursor->next; } } } HashTableDestroy(first_read_name_table); } fclose(writer -> out_fp); signal (SIGTERM, old_sig_TERM); signal (SIGINT, old_sig_INT); return is_disk_full; } void sort_SAM_check_chunk(SAM_sort_writer * writer) { if(writer -> current_chunk_size > SAM_SORT_BLOCK_SIZE * SAM_SORT_BLOCKS) { int xk1; for(xk1=0;xk1 current_block_fp_array[xk1]) fclose(writer -> current_block_fp_array[xk1]); } memset(writer -> current_block_fp_array, 0, sizeof(FILE *)*SAM_SORT_BLOCKS); writer -> current_chunk_size = 0; writer -> current_chunk++; } } // the SAM_line includes "\n" at the tail! // line_len = strlen(SAM_line) int sort_SAM_add_line(SAM_sort_writer * writer, char * SAM_line, int line_len) { int is_disk_full = 0; assert(writer -> all_chunks_header_fp); if(line_len<3) return 0; if(SAM_line[0]=='@'){ int wlen = fputs(SAM_line, writer -> out_fp); if(wlen < 0){ return -2; } } else { char read_name[MAX_READ_NAME_LEN + MAX_CHROMOSOME_NAME_LEN * 2 + 26]; char chromosome_1_name[MAX_CHROMOSOME_NAME_LEN]; char chromosome_2_name[MAX_CHROMOSOME_NAME_LEN]; unsigned int pos_1, pos_2; int hi_tag,flags = 0, line_cursor = 0, field_cursor = 0, tabs=0; char * second_col_pos = NULL; chromosome_1_name[0]=0; chromosome_2_name[0]=0; pos_1 = 0; pos_2 = 0; hi_tag = -1; while(line_cursor < line_len) { char nch = SAM_line[line_cursor++]; if(!nch)break; if(nch == '\t') { field_cursor = 0; tabs++; if(tabs == 1) second_col_pos = SAM_line + line_cursor; if(tabs>7) break; } else if(tabs == 0) { read_name[field_cursor++] = nch; if(MAX_READ_NAME_LEN<=field_cursor){ return -1; } read_name[field_cursor] = 0; } else if(tabs == 1) flags = flags*10+(nch-'0'); else if(tabs == 2) { chromosome_1_name[field_cursor++] = nch; chromosome_1_name[field_cursor]=0; if(MAX_CHROMOSOME_NAME_LEN - 1 <= field_cursor) return -1; } else if(tabs == 3) pos_1 = pos_1 * 10 + (nch-'0'); else if(tabs == 6) { chromosome_2_name[field_cursor++] = nch; chromosome_2_name[field_cursor] = 0; if(MAX_CHROMOSOME_NAME_LEN - 1 <= field_cursor) return -1; } else if(tabs == 7) pos_2 = pos_2 * 10 + (nch-'0'); } if(tabs <= 7) return -1; //if(memcmp("V0112_0155:7:1101:4561:132881", read_name, 27)==0) char * hi_tag_str = strstr(SAM_line,"\tHI:i:"); if(hi_tag_str) { hi_tag = 0; for(line_cursor=6; ; line_cursor++) { char nch = hi_tag_str[line_cursor]; if(!isdigit(nch)) break; hi_tag = hi_tag*10 + (nch-'0'); } } line_len = strlen(second_col_pos); sort_SAM_check_chunk(writer); for(field_cursor = 0; read_name[field_cursor] ; field_cursor++) if(read_name[field_cursor] == '/') read_name[field_cursor] = 0; if(chromosome_2_name[0]=='=') strcpy(chromosome_2_name, chromosome_1_name); // new read name format: OLD_READ_NAME\tCHR_R1:POS_R1:CHR_R2:POS_R2 if(flags & SAM_FLAG_MATE_UNMATCHED) { if(chromosome_2_name[0] != '*') strcpy(chromosome_2_name , "*"); pos_2 = 0; } if(flags & SAM_FLAG_UNMAPPED) { if(chromosome_1_name[0] != '*') strcpy(chromosome_1_name , "*"); pos_1 = 0; } char hi_key [13]; if(hi_tag >=0)// && pos_1 && pos_2) sprintf(hi_key, ":%d", hi_tag); else hi_key[0]=0; if(flags & SAM_FLAG_SECOND_READ_IN_PAIR) sprintf(read_name+strlen(read_name), "\t%s:%u:%s:%u%s",chromosome_2_name, pos_2, chromosome_1_name, pos_1, hi_key); else sprintf(read_name+strlen(read_name), "\t%s:%u:%s:%u%s",chromosome_1_name, pos_1, chromosome_2_name, pos_2, hi_key); //if(memcmp("V0112_0155:7:1101:4561:132881", read_name, 27)==0) // printf("RRN=%s\n", read_name); int read_name_len = strlen(read_name); srUInt_64 read_line_hash = sort_SAM_hash(read_name); int block_id = read_line_hash % SAM_SORT_BLOCKS; if(!writer -> current_block_fp_array[block_id]) { char tmpfname[MAX_FILE_NAME_LENGTH+40]; sprintf(tmpfname,"%sCHK%08d-BLK%03d.bin", writer -> tmp_path , writer -> current_chunk , block_id); writer -> current_block_fp_array[block_id] = f_subr_open(tmpfname, "wb"); } if(line_len < 2) { SUBREADprintf("unable to put the first read.\n"); assert(0); } if(second_col_pos[0]==0 || second_col_pos[1]==0) { SUBREADprintf("unable to put the first read TEXT.\n"); assert(0); } // printf("WRNAME:%s\n", read_name); fwrite(&flags, 2, 1, writer -> current_block_fp_array[block_id]); fwrite(&read_name_len, 2, 1, writer -> current_block_fp_array[block_id]); fwrite(read_name, 1, read_name_len, writer -> current_block_fp_array[block_id]); fwrite(&line_len, 2, 1, writer -> current_block_fp_array[block_id]); int write_len = fwrite(second_col_pos, 1, line_len, writer -> current_block_fp_array[block_id]); if(write_len < line_len)is_disk_full = -2; writer -> output_file_size += line_len; writer -> current_chunk_size += line_len; writer -> written_reads ++; } return is_disk_full; } int is_SAM_unsorted(char * SAM_line, char * tmp_read_name, short * tmp_flag, srInt_64 read_no) { char read_name[MAX_READ_NAME_LEN]; int flags = 0, line_cursor = 0, field_cursor = 0, tabs=0; read_name[0] =0; while(1) { char nch = SAM_line[line_cursor++]; if(!nch)break; if(nch == '\t') { field_cursor = 0; tabs++; if(tabs>1) break; } else if(tabs == 0) { read_name[field_cursor++] = nch; assert(MAX_READ_NAME_LEN>field_cursor); read_name[field_cursor] = 0; } else if(tabs == 1) flags = flags*10+(nch-'0'); } //int is_second_read = (flags & 0x80) ? 1:0; for(field_cursor = 0; read_name[field_cursor] ; field_cursor++) if(read_name[field_cursor] == '/') read_name[field_cursor] = 0; (*tmp_flag) = flags; if(!(flags &1)) return 0; if(read_no % 2 == 0) { if(flags & SAM_FLAG_SECOND_READ_IN_PAIR)return 1; strcpy(tmp_read_name , read_name); } else { if(flags & SAM_FLAG_FIRST_READ_IN_PAIR) return 1; if(strcmp(tmp_read_name, read_name))return 1; } return 0; } int is_certainly_bam_file(char * fname, int * is_first_read_PE, srInt_64 * SAMBAM_header_size) { int read_type = probe_file_type_EX(fname, is_first_read_PE, SAMBAM_header_size); if(read_type == FILE_TYPE_NONEXIST || read_type == FILE_TYPE_EMPTY || read_type == FILE_TYPE_UNKNOWN) return -1; if(read_type == FILE_TYPE_BAM) return 1; return 0; } int is_pipe_file(char * fname) { FILE * fp = fopen(fname,"r"); if(!fp) return 0; int seeked = fseeko(fp, 0, SEEK_SET); fclose(fp); return (seeked != 0); } int warning_file_type(char * fname, int expected_type) { int ret_pipe_file = is_pipe_file(fname); if(ret_pipe_file) { print_in_box(80,0,0,"WARNING file '%s' is not a regular file.", fname); print_in_box(80,0,0," No alignment can be done on a pipe file."); print_in_box(80,0,0," If the FASTQ file is gzipped, please use gzFASTQinput option."); print_in_box(80,0,0,""); return 1; } int read_type = probe_file_type(fname, NULL); if(read_type == FILE_TYPE_NONEXIST) { SUBREADprintf("ERROR: unable to open file '%s'. File name might be incorrect, or you do not have the permission to read the file.\n", fname); return -1; } else if(read_type == FILE_TYPE_EMPTY) { SUBREADprintf("\nERROR: file '%s' is empty.\n\n", fname); return -1; } else if((expected_type == FILE_TYPE_FAST_ && (read_type!= FILE_TYPE_FASTQ && read_type!= FILE_TYPE_FASTA && read_type!= FILE_TYPE_GZIP_FASTQ && read_type!= FILE_TYPE_GZIP_FASTA))|| (expected_type == FILE_TYPE_GZIP_FAST_ && read_type!= FILE_TYPE_GZIP_FASTA) || (( expected_type != FILE_TYPE_GZIP_FAST_ && expected_type != FILE_TYPE_FAST_) && expected_type != read_type)) { char * req_fmt = "SAM"; if(expected_type==FILE_TYPE_BAM) req_fmt = "BAM"; else if(expected_type==FILE_TYPE_FAST_) req_fmt = "FASTQ or FASTA"; else if(expected_type==FILE_TYPE_GZIP_FAST_) req_fmt = "gzip FASTQ or FASTA"; char * real_fmt = "SAM"; if(read_type==FILE_TYPE_BAM) real_fmt = "BAM"; else if(read_type==FILE_TYPE_FASTA) real_fmt = "FASTA"; else if(read_type==FILE_TYPE_FASTQ) real_fmt = "FASTQ"; else if(read_type==FILE_TYPE_GZIP_FASTQ) real_fmt = "gzip FASTQ"; else if(read_type==FILE_TYPE_GZIP_FASTA) real_fmt = "gzip FASTA"; print_in_box(80,0,0,"WARNING format issue in file '%s':", fname); print_in_box(80,0,0," The required format is : %s", req_fmt); if(read_type == FILE_TYPE_UNKNOWN) print_in_box(80,0,0," The file format is unknown."); else print_in_box(80,0,0," The real format seems to be : %s", real_fmt); print_in_box(80,0,0,"A wrong format may result in wrong results or crash the program."); print_in_box(80,0,0,"Please refer to the manual for file format options."); print_in_box(80,0,0,"If the file is in the correct format, please ignore this message."); print_in_box(80,0,0,""); return 1; } return 0; } char * gzgets_noempty(void * fp, char * buf, int maxlen) { char * ret; while(1) { ret = gzgets(fp,buf, maxlen); if(!ret)return NULL; if(ret[0]!='\n') return ret; } } char * fgets_noempty(char * buf, int maxlen, FILE * fp) { char * ret; while(1) { ret = fgets(buf, maxlen, fp); if(!ret)return NULL; if(ret[0]!='\n') return ret; } } int is_comment_line(const char * l, int file_type, unsigned int lineno) { int tabs = 0, xk1 = 0; if(l[0]=='#') return 1; if(isalpha(l[0]) && file_type == FILE_TYPE_RSUBREAD) { char target_chr[16]; memcpy(target_chr, l, 16); for(xk1=0; xk1<16; xk1++) target_chr[xk1] = tolower(target_chr[xk1]); if(memcmp(target_chr, "geneid\tchr\tstart",16)==0) return 1; } xk1=0; while(l[xk1]) tabs += (l[xk1++] == '\t'); return tabs < ((file_type == FILE_TYPE_GTF)?8:4); } int probe_file_type_fast(char * fname){ FILE * fp = f_subr_open(fname, "rb"); if(!fp) return FILE_TYPE_NONEXIST; int ret = FILE_TYPE_UNKNOWN; int nch; char *test_buf=malloc(5000); nch = fgetc(fp); if(feof(fp)) ret = FILE_TYPE_EMPTY; else { if(nch == '@') // FASTQ OR SAM { char * rptr = fgets_noempty(test_buf, 4999, fp); int second_line_len = 0; if(rptr) { rptr = fgets_noempty(test_buf, 4999, fp); if(rptr) { second_line_len = strlen(test_buf); int tabs = 0, x1; for(x1=0;x1<4999;x1++) { if(test_buf[x1]=='\n' || !test_buf[x1]) break; if(test_buf[x1]=='\t'){ tabs++; continue; } if(tabs == 1) if(!isdigit(test_buf[x1]))break; } if(rptr[0]=='@' || tabs>7) ret = FILE_TYPE_SAM; } } if(ret == FILE_TYPE_UNKNOWN) { rptr = fgets_noempty(test_buf, 4999, fp); if(rptr[0] == '+') { rptr = fgets_noempty(test_buf, 4999, fp); if(rptr && second_line_len == strlen(test_buf)) ret = FILE_TYPE_FASTQ; } } } else if(nch == '>') // FASTA { ret = FILE_TYPE_FASTA; } else if(nch == 31) // BAM OR GZ_FASTQ { nch = fgetc(fp); if(nch == 139) { fclose(fp); fp=NULL; gzFile zfp = gzopen(fname, "rb"); if(zfp) { int rlen = gzread(zfp, test_buf,4); if(rlen == 4 && memcmp(test_buf,"BAM\1",4)==0) ret = FILE_TYPE_BAM; if(rlen == 4 && test_buf[0]=='@') ret = FILE_TYPE_GZIP_FASTQ; if(rlen == 4 && test_buf[0]=='>') ret = FILE_TYPE_GZIP_FASTA; gzclose(zfp); } } } else if(nch >= 0x20 && nch <= 0x7f) // SAM without headers { int tabs = 0, x1; char * rptr = fgets(test_buf, 4999, fp); if(rptr) for(x1=0;x1<4999;x1++) { if(test_buf[x1]=='\n' || !test_buf[x1]) break; if(test_buf[x1]=='\t'){ tabs++; continue; } if(tabs == 1) if(!isdigit(test_buf[x1]))break; } if(tabs>7) ret = FILE_TYPE_SAM; } } if(fp)fclose(fp); free(test_buf); return ret; } int probe_file_type(char * fname, int * is_first_read_PE) { return probe_file_type_EX(fname, is_first_read_PE, NULL); } int probe_file_type_EX(char * fname, int * is_first_read_PE, srInt_64 * SAMBAM_header_length) { FILE * fp = f_subr_open(fname, "rb"); if(!fp) return FILE_TYPE_NONEXIST; int ret = FILE_TYPE_UNKNOWN; int nch; char *test_buf=malloc(5000); nch = fgetc(fp); if(feof(fp)) ret = FILE_TYPE_EMPTY; else { if(nch == '@') // FASTQ OR SAM { char * rptr = fgets_noempty(test_buf, 4999, fp); int second_line_len = 0; if(rptr) { rptr = fgets_noempty(test_buf, 4999, fp); if(rptr) { second_line_len = strlen(test_buf); int tabs = 0, x1; for(x1=0;x1<4999;x1++) { if(test_buf[x1]=='\n' || !test_buf[x1]) break; if(test_buf[x1]=='\t'){ tabs++; continue; } if(tabs == 1) if(!isdigit(test_buf[x1]))break; } if(rptr[0]=='@' || tabs>7) ret = FILE_TYPE_SAM; } } if(ret == FILE_TYPE_UNKNOWN) { rptr = fgets_noempty(test_buf, 4999, fp); if(rptr[0] == '+') { rptr = fgets_noempty(test_buf, 4999, fp); if(rptr && second_line_len == strlen(test_buf)) ret = FILE_TYPE_FASTQ; } } } else if(nch == '>') // FASTA { char * rptr = fgets(test_buf, 4999, fp); int x1; if(rptr) { ret = FILE_TYPE_FASTA; for(x1=0;x1<4999;x1++) { if(test_buf[x1]=='\n' || !test_buf[x1]) break; nch = toupper(test_buf[x1]); if(nch < ' ' || nch>127) { ret = FILE_TYPE_UNKNOWN; break; } } rptr = fgets(test_buf, 4999, fp); if(rptr && ret == FILE_TYPE_FASTA) { for(x1=0;x1<4999;x1++) { if(test_buf[x1]=='\n' || !test_buf[x1]) break; nch = toupper(test_buf[x1]); if(nch == 'A' || nch == 'T' || nch == 'G' || nch == 'C' || nch == 'N' || nch == '.' || (nch >='0' && nch <= '3')) ; else { ret = FILE_TYPE_UNKNOWN; break; } } if(x1==0) ret = FILE_TYPE_UNKNOWN; } } } else if(nch == 31) // BAM OR GZ_FASTQ { nch = fgetc(fp); if(nch == 139) { fclose(fp); fp=NULL; gzFile zfp = gzopen(fname, "rb"); if(zfp) { int rlen = gzread(zfp, test_buf,4); if(rlen == 4 && memcmp(test_buf,"BAM\1",4)==0) ret = FILE_TYPE_BAM; if(rlen == 4 && test_buf[0]=='@') ret = FILE_TYPE_GZIP_FASTQ; if(rlen == 4 && test_buf[0]=='>') ret = FILE_TYPE_GZIP_FASTA; gzclose(zfp); } } } else if(nch >= 0x20 && nch <= 0x7f) // SAM without headers { int tabs = 0, x1; char * rptr = fgets(test_buf, 4999, fp); if(rptr) for(x1=0;x1<4999;x1++) { if(test_buf[x1]=='\n' || !test_buf[x1]) break; if(test_buf[x1]=='\t'){ tabs++; continue; } if(tabs == 1) if(!isdigit(test_buf[x1]))break; } if(tabs>7) ret = FILE_TYPE_SAM; } } if(fp)fclose(fp); //SUBREADprintf("RET=%d, FIRSTPE=%p, SAMLEN=%p\n" , ret, is_first_read_PE, SAMBAM_header_length); if(FILE_TYPE_BAM == ret || FILE_TYPE_SAM == ret) if(is_first_read_PE || SAMBAM_header_length) { SamBam_FILE * tpfp = SamBam_fopen(fname, (FILE_TYPE_BAM == ret)?SAMBAM_FILE_BAM:SAMBAM_FILE_SAM); while(1) { char * tbr = SamBam_fgets(tpfp, test_buf, 4999, 0); if( is_first_read_PE && tpfp -> is_paired_end >= 10) (*is_first_read_PE) = tpfp -> is_paired_end - 10; if(tbr == NULL)break; if(tbr[0]=='@') continue; break; } if( SAMBAM_header_length) (*SAMBAM_header_length) = tpfp -> header_length; SamBam_fclose(tpfp); } free(test_buf); //if(is_first_read_PE)assert(0); return ret; } void warning_hash_hash(HashTable * t1, HashTable * t2, char * msg){ int buck_i, shown = 0; for(buck_i = 0; buck_i < t1 -> numOfBuckets; buck_i++){ KeyValuePair * cursor = t1 -> bucketArray[buck_i]; while(cursor){ char * t1chro = (char *) cursor -> key; int found = HashTableGet(t2, t1chro) != NULL; if(!found) if(strlen(t1chro)>3 && t1chro[0]=='c'&&t1chro[1]=='h'&&t1chro[2]=='r' ) found = HashTableGet(t2, t1chro+3) != NULL; if(!found) { char tmp_t1chro [MAX_CHROMOSOME_NAME_LEN+1]; sprintf(tmp_t1chro, "chr%s", t1chro); found = HashTableGet(t2, tmp_t1chro) != NULL; } if(!found){ if(!shown){ print_in_box(80,0,0,""); print_in_box(80,0,0,msg); shown = 1; } print_in_box(80,0,0," %s", t1chro); } cursor = cursor -> next; } } if(shown) print_in_box(80,0,0,""); } #ifdef MAKE_INPUTTEST int main(int argc, char ** argv) { FILE * ifp; srInt_64 rno=0; short tmp_flags, is_sorted = 1; char buff[3000], tmp_rname[MAX_FILE_NAME_LENGTH]; ifp = f_subr_open(argv[1],"r"); while(1) { char * rr = fgets(buff,2999, ifp); if(!rr) break; if(buff[0]=='@')continue; if(is_SAM_unsorted(buff, tmp_rname, &tmp_flags, rno)) { printf("The input file is unsorted.\n"); is_sorted = 0; break; } rno++; } fclose(ifp); //if(is_sorted) return 0; ifp = f_subr_open(argv[1],"r"); SAM_sort_writer writer; if(sort_SAM_create(&writer, argv[2], ".")){ printf("ERROR: unable to create the writer.\n"); return -1; } while(1) { char * rr = fgets(buff,2999, ifp); if(!rr) break; int line_len = strlen(buff); sort_SAM_add_line(&writer, buff, line_len); } fclose(ifp); sort_SAM_finalise(&writer); printf("WRITTEN=%llu\nUNPAIR=%llu\n", writer.written_reads, writer.unpaired_reads); } #endif #ifdef MAKE_TYPETEST int main(int argc, char ** argv) { char * fn = argv[1]; int type = probe_file_type(fn, NULL); switch(type) { case FILE_TYPE_FASTQ: printf("Type: FASTQ\n"); break; case FILE_TYPE_FASTA: printf("Type: FASTA\n"); break; case FILE_TYPE_SAM : printf("Type: SAM\n"); break; case FILE_TYPE_BAM : printf("Type: BAM\n"); break; default: printf("Unknown type.\n"); } } #endif Rsubread/src/input-files.h0000644000175200017520000003161314136047615016555 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #ifndef __INPUT_FILES_H_ #define __INPUT_FILES_H_ #include "subread.h" #include "hashtable.h" #define GENE_SPACE_BASE 1 #define GENE_SPACE_COLOR 2 #define GENE_INPUT_PLAIN 0 #define GENE_INPUT_FASTQ 1 #define GENE_INPUT_FASTA 2 #define GENE_INPUT_BCL 3 #define GENE_INPUT_SCRNA_FASTQ 4 #define GENE_INPUT_SCRNA_BAM 5 #define GENE_INPUT_GZIP_FASTQ 51 #define GENE_INPUT_GZIP_FASTA 52 #define GENE_INPUT_SAM_SINGLE 93 #define GENE_INPUT_SAM_PAIR_1 94 #define GENE_INPUT_SAM_PAIR_2 95 #define MAX_LINE_LENGTH 3000 #define MIN_FILE_POINTERS_ALLOWED 50 #define FILE_TYPE_SAM 50 #define FILE_TYPE_BAM 500 #define FILE_TYPE_FAST_ 100 #define FILE_TYPE_FASTQ 105 #define FILE_TYPE_FASTA 110 #define FILE_TYPE_GZIP_FAST_ 1000 #define FILE_TYPE_GZIP_FASTQ 1105 #define FILE_TYPE_GZIP_FASTA 1110 #define FILE_TYPE_UNKNOWN 999 #define FILE_TYPE_EMPTY 999990 #define FILE_TYPE_NONEXIST 999999 #define FILE_TYPE_RSUBREAD 10 #define FILE_TYPE_GTF 100 #define FEATURECOUNTS_BUFFER_SIZE (1024*1024*12) #include #include #include "core-indel.h" #include "hashtable.h" #define SAM_SORT_BLOCKS 229 #define SAM_SORT_BLOCK_SIZE 512333303LLU //#define SAM_SORT_BLOCK_SIZE 11123333LLU // typedef struct { srInt_64 output_file_size; srInt_64 current_chunk_size; unsigned int current_chunk; srInt_64 written_reads; srInt_64 unpaired_reads; FILE * current_block_fp_array [SAM_SORT_BLOCKS]; FILE * all_chunks_header_fp; FILE * out_fp; char tmp_path[MAX_FILE_NAME_LENGTH]; } SAM_sort_writer; typedef struct { int thread_id; char * input_buff_SBAM; int input_buff_SBAM_used; int input_buff_SBAM_ptr; int reads_in_SBAM; subread_lock_t SBAM_lock; srInt_64 input_buff_SBAM_file_start; srInt_64 input_buff_SBAM_file_end; unsigned int chunk_number; unsigned int readno_in_chunk; unsigned char * input_buff_BIN; int input_buff_BIN_used; int input_buff_BIN_ptr; int input_buff_BIN_capacity; int orphant_block_no; int need_find_start; srInt_64 orphant_space; z_stream strm; char immediate_last_read_bin[FC_LONG_READ_RECORD_HARDLIMIT]; char immediate_last_read_full_name[MAX_READ_NAME_LEN*2 +80 ]; int immediate_last_read_flags; int immediate_last_read_bin_len; int immediate_last_read_name_len; HashTable * orphant_table; pthread_t thread_stab; } SAM_pairer_thread_t; typedef struct { FILE * input_fp; int input_is_BAM; int tiny_mode; int display_progress; int is_bad_format; int is_single_end_mode; int force_do_not_sort; int long_cigar_mode; int long_read_minimum_length; int is_finished; int merge_level_finished; int max_file_open_number; subread_lock_t input_fp_lock; subread_lock_t SAM_BAM_table_lock; subread_lock_t unsorted_notification_lock; srInt_64 total_input_reads; srInt_64 total_orphan_reads; HashTable * unsorted_notification_table; HashTable * sam_contig_number_table; HashTable * bam_margin_table; int total_threads; int input_chunk_no; int input_buff_SBAM_size; int input_buff_BIN_size; char tmp_file_prefix[MAX_FILE_NAME_LENGTH+1]; char in_file_name[MAX_FILE_NAME_LENGTH+1]; SAM_pairer_thread_t * threads; int BAM_header_parsed; unsigned int BAM_l_text; unsigned int BAM_n_ref; int is_unsorted_notified; int is_incomplete_BAM; int need_read_group_tag; int is_internal_error; int is_final_run; void (* reset_output_function) (void * pairer); int (* output_function) (void * pairer, int thread_no, char * bin1, char * bin2); int (* output_header) (void * pairer, int thread_no, int is_text, unsigned int items, char * bin, unsigned int bin_len); void (* unsorted_notification) (void * pairer, char * bin1, char * bin2); // it is called only once // reserved for the application passing its own data to the output function. void * appendix1; void * appendix2; void * appendix3; void * appendix4; void * appendix5; } SAM_pairer_context_t; #define SAM_PAIRER_WRITE_BUFFER ( 64000 ) typedef struct { unsigned char BIN_buffer[SAM_PAIRER_WRITE_BUFFER]; int BIN_buffer_ptr; z_stream strm; } SAM_pairer_writer_thread_t; typedef struct { SAM_pairer_writer_thread_t * threads; int all_threads; int compression_level; int has_dummy; FILE * bam_fp; char bam_name[MAX_FILE_NAME_LENGTH]; subread_lock_t output_fp_lock; } SAM_pairer_writer_main_t; void fastq_64_to_33(char * qs); // the caller is in charge of deallocation char * memstrcpy(char * in); int chars2color(char c1, char c2); int genekey2color(char last_base,char * key); // Convert a string key into an integer key int genekey2int(char * key, int space_type); // Open a read file. This function automatically decides its type. // Return 0 if successfully opened, or 1 if error occurred int geinput_open(const char * filename, gene_input_t * input); // Open a sam file. Parameter half_no indicates which read of the pair is concerned. // half_no = 1: the first read; half_no = 2: the last read; half_no = 0: single-end // Return 0 if successfully opened, or 1 if error occurred int geinput_open_sam(const char * filename, gene_input_t * input, int half_no); // Read a line from the input and reward the pointer to the last position. int geinput_readline_back(gene_input_t * input, char * linebuffer) ; // Get the next read from the input file // Return the length of this read or -1 if EOF. // The memory space for read_string must be at least 512 bytes. int geinput_next_read(gene_input_t * input, char * read_name, char * read_string, char * quality_string); int geinput_next_read_trim(gene_input_t * input, char * read_name, char * read_string, char * quality_string, short trim_5, short trim_3, int * is_secondary); int geinput_next_read_with_lock(gene_input_t * input, char * read_name, char * read_string, char * quality_string, short trim_5, short trim_3, int * is_secondary, cellCounts_lock_t * lock); void geinput_jump_read(gene_input_t * input); // Close the input file void geinput_close(gene_input_t * input); // return the next ATGC char from a input file. // return 0 if this read segment reaches the end; return -1 if EOF; return -2 if error int geinput_next_char(gene_input_t * input); // line buffer has to be at least 300 bytes // it returns the length of reading int geinput_readline(gene_input_t * input, char * linebuffer, int conv_to_upper) ; // read a line into the buff, // the line should not be longer than 300 chars or the remaining part will be discarded. // therefore the buff has to be at least 300 chars. int read_line(int max_len, FILE * fp, char * buff, int conv_to_upper); // count the number of reads in a flie double guess_reads_density(char * fname, int is_sam) ; // guess the size of the chromosome lib // return the number of bases, or (-index-1) if the file at the index is not found. srInt_64 guess_gene_bases(char ** files, int file_number); void reverse_read(char * ReadString, int Length, int space_type); void reverse_quality(char * QualtyString, int Length); unsigned int read_numbers(gene_input_t * input); //This function returns 0 if the line is a mapped read; -1 if the line is in a wrong format and 1 if the read is unmapped. int parse_SAM_line(char * sam_line, char * read_name, int * flags, char * chro, unsigned int * pos, char * cigar, int * mapping_quality, unsigned int * pair_dist, char * sequence , char * quality_string, int * rl, int * repeated); #define reverse_char(c) ((c)=='A'?'T':((c)=='G'?'C':((c)=='C'?'G':'A'))) int find_subread_end(int len, int TOTAL_SUBREADS,int subread) ; int break_SAM_file(char * in_SAM_file, int is_BAM, char * temp_file_prefix, unsigned int * real_read_count, int * block_no, chromosome_t * known_chromosomes, int is_sequence_needed, int base_ignored_head_tail, gene_value_index_t *array_index, gene_offset_t * offsets, srInt_64 * all_Mapped_bases , HashTable * event_table_ptr, char * VCF_file, srInt_64 * all_mapped_reads, int do_fragment_filtering, int push_to_read_head, int use_soft_clipped_bases); int get_known_chromosomes(char * in_SAM_file, chromosome_t * known_chromosomes); int load_exon_annotation(char * annotation_file_name, gene_t ** output_genes, gene_offset_t* offsets ); int is_in_exon_annotations(gene_t *output_genes, unsigned int offset, int is_start); int does_file_exist (char * filename); double guess_reads_density_format(char * fname, int is_sam, int * min_phred, int * max_phred, int * tested_reads); FILE * get_temp_file_pointer(char *temp_file_name, HashTable* fp_table, int * close_immediately); int write_read_block_file(FILE *temp_fp , unsigned int read_number, char *read_name, int flags, char * chro, unsigned int pos, char *cigar, int mapping_quality, char *sequence , char *quality_string, int rl , int is_sequence_needed, char strand, unsigned short read_pos, unsigned short read_len, unsigned short M_seg); int get_read_block(char *chro, unsigned int pos, char *temp_file_suffix, chromosome_t *known_chromosomes, unsigned int * max_base_position); int my_strcmp(const void * s1, const void * s2); void destroy_cigar_event_table(HashTable * event_table); int is_SAM_unsorted(char * SAM_line, char * tmp_read_name, short * tmp_flag, srInt_64 read_no); int sort_SAM_add_line(SAM_sort_writer * writer, char * SAM_line, int line_len); int sort_SAM_finalise(SAM_sort_writer * writer); int sort_SAM_create(SAM_sort_writer * writer, char * output_file, char * tmp_path); void colorread2base(char * read_buffer, int read_len); int warning_file_type(char * fname, int expected_type); char color2char(char clr, char c1); int is_certainly_bam_file(char * fname, int * is_firstread_PE, srInt_64 * SAMBAM_header_length); srUInt_64 sort_SAM_hash(char * str); char * fgets_noempty(char * buf, int maxlen, FILE * fp); char * gzgets_noempty(void * fp, char * buf, int maxlen); int probe_file_type(char * fname, int * is_first_PE); int probe_file_type_fast(char * fname); void geinput_seek(gene_input_t * input, gene_inputfile_position_t * pos); void geinput_tell(gene_input_t * input, gene_inputfile_position_t * pos); srInt_64 geinput_file_offset( gene_input_t * input); int probe_file_type_EX(char * fname, int * is_first_read_PE, srInt_64 * SAMBAM_header_length); int SAM_pairer_create(SAM_pairer_context_t * pairer, int all_threads, int bin_buff_size_per_thread, int BAM_input, int is_Tiny_Mode, int is_single_end_mode, int force_do_not_sort, int need_read_group_tag, int display_progress, char * in_file, void (* reset_output_function) (void * pairer), int (* output_header_function) (void * pairer, int thread_no, int is_text, unsigned int items, char * bin, unsigned int bin_len), int (* output_function) (void * pairer, int thread_no, char * bin1, char * bin2), char * tmp_path, void * appendix1, int long_read_minimum_length) ; int SAM_pairer_run( SAM_pairer_context_t * pairer); void SAM_pairer_destroy(SAM_pairer_context_t * pairer); void SAM_pairer_writer_reset(void * pairer); void SAM_pairer_set_unsorted_notification(SAM_pairer_context_t * pairer, void (* unsorted_notification) (void * pairer, char * bin1, char * bin2)); int SAM_pairer_multi_thread_output( void * pairer, int thread_no, char * bin1, char * bin2 ); int SAM_pairer_multi_thread_header (void * pairer_vp, int thread_no, int is_text, unsigned int items, char * bin, unsigned int bin_len); int SAP_pairer_skip_tag_body_len(char * tag_start); int SAM_pairer_writer_create( SAM_pairer_writer_main_t * bam_main , int all_threads, int has_dummy , int BAM_output, int BAM_compression_level, char * out_file); void SAM_pairer_writer_destroy( SAM_pairer_writer_main_t * bam_main ) ; int SAM_pairer_iterate_int_tags(unsigned char * bin, int bin_len, char * tag_name, int * saved_value); int SAM_pairer_iterate_tags(unsigned char * bin, int bin_len, char * tag_name, char * data_type, char ** saved_value); int SAM_pairer_warning_file_open_limit(); int SAM_pairer_get_tag_bin_start(char * bin); void *delay_realloc(void * old_pntr, size_t old_size, size_t new_size); int is_comment_line(const char * l, int file_type, unsigned int lineno); void warning_hash_hash(HashTable * t1, HashTable * t2, char * msg); int geinput_preload_buffer(gene_input_t * input, subread_lock_t* read_lock); int geinput_open_scRNA_fqs(char * fnames, gene_input_t * input, int reads_per_chunk, int threads); int geinput_open_scRNA_BAM(char * fnames, gene_input_t * input, int reads_per_chunk, int threads); int geinput_open_bcl( const char * dir_name, gene_input_t * input, int reads_in_chunk, int threads); char *strtokmm(char *str, const char *delim, char ** next); #endif Rsubread/src/interval_merge.c0000644000175200017520000000655514136047615017323 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #include #include #include #define NORMAL_INTS 35 int mergeIntervals(unsigned int * intervals, unsigned int * result_ints, int nints) { unsigned int * stack_buffer_mem; int stock_pointer = 0; stack_buffer_mem = result_ints; int ii,jj; for(ii=0; ii intervals[jj*2]) { small_jj_value = intervals[jj*2]; small_jj_id = jj; } } if(small_jj_value < intervals[ii*2]) { unsigned int tmpv; tmpv = intervals[ii*2]; intervals[ii*2] = small_jj_value; intervals[small_jj_id * 2] = tmpv; tmpv = intervals[ii*2 + 1]; intervals[ii*2 + 1] = intervals[small_jj_id * 2 + 1]; intervals[small_jj_id * 2 + 1] = tmpv; } } stack_buffer_mem[stock_pointer*2] = intervals[0]; stack_buffer_mem[stock_pointer*2+1] = intervals[1]; stock_pointer++; for (ii = 1 ; ii < nints; ii++) { //int top_start = stack_buffer_mem[stock_pointer*2-2]; int top_stop = stack_buffer_mem[stock_pointer*2-1]; if (top_stop < intervals[ii*2]) { stack_buffer_mem[stock_pointer * 2] = intervals[ii*2]; stack_buffer_mem[stock_pointer * 2+1] = intervals[ii*2+1]; stock_pointer++; } else if (top_stop < intervals[ii*2+1]) { top_stop = intervals[ii*2+1]; stack_buffer_mem[stock_pointer*2-1] = top_stop; } // Otherwise the ii-th interval is useless because it is enclosed in the top item } return stock_pointer; } #ifdef MAKE_TEST_INTERVL_MERGE void print_gaps(unsigned int * gaps, int gapn) { int ii; for(ii=0; ii #include #include #ifndef MACOS #ifndef FREEBSD #include #endif #endif #include #include"core.h" #include"long-hashtable.h" int lnhash_create(lnhash_t * the_table, unsigned int num_buckets) { int x1; the_table -> num_elements = 0; the_table -> num_buckets = num_buckets; the_table -> is_sorted = 0; the_table -> subread_repeat_max = 25600; the_table -> buckets = malloc(num_buckets * sizeof(lnhash_buckct_t)); if(sizeof(void*) == 4) the_table -> key_repeated_numbers = malloc(sizeof(short) * 0xfffffff); else the_table -> key_repeated_numbers = malloc(sizeof(short) * 0x100000000llu); for(x1=0; x1 buckets[x1].num_elements = 0; the_table -> buckets[x1].space_elements = 0; the_table -> buckets[x1].key_array = NULL; the_table -> buckets[x1].value_array = NULL; } return 0; } void lnhash_destroy(lnhash_t * the_table) { int x1; free(the_table -> key_repeated_numbers); for(x1=0; x1 num_buckets; x1++) { if(the_table -> buckets[x1].key_array) free(the_table -> buckets[x1].key_array); if(the_table -> buckets[x1].value_array) free(the_table -> buckets[x1].value_array); } free(the_table -> buckets); } unsigned int lnhash_locate_bucket(lnhash_t * the_table, gehash_key_t key) { return key % the_table -> num_buckets; } void lnhash_resize_bucket(lnhash_buckct_t * buck, unsigned int new_size) { if(buck -> space_elements < new_size) { if(buck -> space_elements>0) { buck -> space_elements *= LNHASH_BUCKET_SIZE_INCREMENT; buck -> key_array = realloc(buck -> key_array, sizeof(gehash_key_t) * buck -> space_elements); buck -> value_array = realloc(buck -> value_array, sizeof(lnhash_data_t) * buck -> space_elements); } else { buck -> space_elements = LNHASH_INIT_BUCKET_SIZE; buck -> key_array = malloc(sizeof(gehash_key_t) * buck -> space_elements); buck -> value_array = malloc(sizeof(lnhash_data_t) * buck -> space_elements); } } } int lnhash_insert(lnhash_t * the_table, gehash_key_t key, lnhash_data_t data) { int bucket_no = lnhash_locate_bucket(the_table, key); lnhash_buckct_t * buck = &(the_table -> buckets[bucket_no]); if(the_table -> key_repeated_numbers[key] < the_table -> subread_repeat_max) { the_table -> key_repeated_numbers[key]++; lnhash_resize_bucket(buck, buck -> num_elements +1); buck -> key_array[buck -> num_elements] = key; buck -> value_array[buck -> num_elements] = data; buck -> num_elements++; return 0; } else if(the_table -> key_repeated_numbers[key] == the_table -> subread_repeat_max) { return 1; int read_c, write_c=0; for(read_c =0; read_c < buck -> num_elements; read_c++) { if(buck -> key_array[read_c] != key) { if(write_c!=read_c) { buck -> key_array[write_c] = buck -> key_array[read_c]; buck -> value_array[write_c] = buck -> value_array[read_c]; } write_c++; } } buck -> num_elements = write_c; the_table -> key_repeated_numbers[key] = 65535; } return 1; } //void merge_sort(void * arr, int arr_size, int compare (void * arr, int l, int r), void exchange(void * arr, int l, int r), void merge(void * arr, int start, int items, int items2)); // int lnhash_mergesort_compare(void * arr, int l, int r) { lnhash_buckct_t * buck = arr; gehash_key_t lk = buck->key_array[l]; gehash_key_t rk = buck->key_array[r]; if(lk==rk)return 0; if(lk> rk)return 1; return -1; } void lnhash_mergesort_exchange(void * arr, int l, int r) { lnhash_buckct_t * buck = arr; gehash_key_t tmpk; lnhash_data_t tmpd; tmpk = buck->key_array[l]; tmpd = buck->value_array[l]; buck->value_array[l] = buck->value_array[r]; buck->key_array[l] = buck->key_array[r]; buck->key_array[r] = tmpk; buck->value_array[r] = tmpd; } void lnhash_mergesort_merge(void * arr, int start, int items, int items2) { lnhash_buckct_t * buck = arr; int i1_cursor = start, i2_cursor = start + items, out_cursor = 0; int end_point = start + items + items2; gehash_key_t * tmpk_arr = malloc(sizeof(gehash_key_t) * (items + items2)); lnhash_data_t * tmpd_arr = malloc(sizeof(lnhash_data_t) * (items + items2)); while(1) { if(i1_cursor == start + items && i2_cursor == end_point) break; assert(out_cursor = start+items) choose_2 = 1; if(i2_cursor < end_point) if(buck->key_array[i1_cursor] > buck->key_array[i2_cursor]) choose_2 = 1; if(choose_2) { tmpk_arr[out_cursor] = buck->key_array[i2_cursor]; tmpd_arr[out_cursor] = buck->value_array[i2_cursor]; i2_cursor++; } else { tmpk_arr[out_cursor] = buck->key_array[i1_cursor]; tmpd_arr[out_cursor] = buck->value_array[i1_cursor]; i1_cursor++; } out_cursor++; } memcpy(buck->key_array + start, tmpk_arr, sizeof(gehash_key_t ) * (items + items2)); memcpy(buck->value_array + start, tmpd_arr, sizeof(lnhash_data_t) * (items + items2)); free(tmpk_arr); free(tmpd_arr); } void lnhash_sort_bucket(lnhash_buckct_t * buck) { merge_sort(buck, buck->num_elements, lnhash_mergesort_compare, lnhash_mergesort_exchange, lnhash_mergesort_merge); } void lnhash_resort(lnhash_t * the_table) { int x1; for(x1 = 0;x1 < the_table -> num_buckets; x1++) { lnhash_buckct_t * buck = &(the_table -> buckets[x1]); lnhash_sort_bucket(buck); } the_table -> is_sorted = 1; } int lnhash_get_record_column(lnhash_data_t pos) { return (int)(pos % LNHASH_VOTE_ARRAY_HEIGHT); } int lnhash_update_votes(lnhash_vote_t * votes, int record_column, lnhash_data_t test_pos, short dist_to_head) { int row_cursor; for(row_cursor=0; row_cursorvote_record_items[record_column]; row_cursor++) { if(votes -> vote_records[record_column][row_cursor].head_position == test_pos) { votes -> vote_records[record_column][row_cursor].num_votes++; votes -> vote_records[record_column][row_cursor].coverage_start = min( votes -> vote_records[record_column][row_cursor].coverage_start, dist_to_head); votes -> vote_records[record_column][row_cursor].coverage_end = max( votes -> vote_records[record_column][row_cursor].coverage_end, dist_to_head + 16); return 1; } } return 0; } void lnhash_add_votes(lnhash_vote_t * votes, int record_column, lnhash_data_t test_pos, short dist_to_head) { if(votes->vote_record_items[record_column]vote_record_items[record_column]; votes -> vote_records[record_column][row_cursor].head_position = test_pos; votes -> vote_records[record_column][row_cursor].num_votes = 1; votes -> vote_records[record_column][row_cursor].coverage_start = dist_to_head; votes -> vote_records[record_column][row_cursor].coverage_end = dist_to_head + 16; votes->vote_record_items[record_column]++; } } // no indels are considered. int lnhash_query(lnhash_t * the_table, lnhash_vote_t * votes, gehash_key_t queried_key, short dist_to_head) { // assert(the_table -> is_sorted); unsigned int bucket_no = lnhash_locate_bucket(the_table , queried_key); int lower_cursor, higher_cursor, match_cursor = 0, hits=0; lnhash_buckct_t * buck = &(the_table -> buckets[bucket_no]); if(the_table -> is_sorted) { lower_cursor = 0; higher_cursor = buck -> num_elements; if(higher_cursor<1) return 0; while(1) { if(lower_cursor> higher_cursor) return 0; int middle_no = (lower_cursor+higher_cursor)/2; if(buck->key_array[middle_no] == queried_key) { match_cursor = middle_no; break; } else if(buck->key_array[middle_no] > queried_key) higher_cursor = middle_no-1; else lower_cursor = middle_no+1; } while( match_cursor > 0 ) { if(buck->key_array[match_cursor-1] != queried_key) break; match_cursor--; } } for(; match_cursor < buck -> num_elements; match_cursor++) { if(buck->key_array[match_cursor]!=queried_key) { if(the_table -> is_sorted) break; else continue; } lnhash_data_t test_pos = buck -> value_array[match_cursor]; test_pos -= dist_to_head; //if((test_pos & 0x3fff) > 1300) continue; int record_column = lnhash_get_record_column(test_pos); int found = lnhash_update_votes(votes, record_column, test_pos, dist_to_head); if(!found) lnhash_add_votes (votes, record_column, test_pos, dist_to_head); hits ++; } return hits; } int compare_voting_items(void * arrV, int i, int j) { lnhash_vote_record_t * arr = (lnhash_vote_record_t*) arrV; if(arr[i].head_position > arr[j].head_position) return 1; else if(arr[i].head_position < arr[j].head_position) return -1; return 0; } void exchange_voting_items(void * arrV, int i, int j) { lnhash_vote_record_t * arr = (lnhash_vote_record_t*) arrV; lnhash_vote_record_t tmp; memcpy(&tmp, arr + i, sizeof(lnhash_vote_record_t)); memcpy(arr + i, arr + j, sizeof(lnhash_vote_record_t)); memcpy(arr + j, &tmp, sizeof(lnhash_vote_record_t)); } void merge_vorting_items(void * arrV, int start, int items1, int items2) { lnhash_vote_record_t * arr = (lnhash_vote_record_t*) arrV; lnhash_vote_record_t * arrtmp = malloc( sizeof(lnhash_vote_record_t) * (items1 + items2)); int cursor_1 = 0, cursor_2 = 0, xk1; for(xk1=0; xk1 head_position >= curr_pnt2_item -> head_position) use_item1 = 0; if(use_item1){ memcpy(arrtmp+xk1, arr+start+cursor_1, sizeof(lnhash_vote_record_t)); cursor_1++; } else{ memcpy(arrtmp+xk1, arr+start+items1+cursor_2, sizeof(lnhash_vote_record_t)); cursor_2++; } } memcpy(arr + start, arrtmp, sizeof(lnhash_vote_record_t) * (items1 + items2)); free(arrtmp); } int sorted_voting_table_EX(lnhash_vote_t * vote, lnhash_vote_record_t ** result_buffer, int minimum_votes, int offsetted) { int ret_size = LNHASH_VOTE_ARRAY_HEIGHT * 3; int ret_items = 0; lnhash_vote_record_t * ret = malloc(sizeof(lnhash_vote_record_t) * ret_size); int vote_X, vote_Y; for(vote_X = 0; vote_X < LNHASH_VOTE_ARRAY_HEIGHT; vote_X++) for(vote_Y = 0; vote_Y < vote -> vote_record_items[vote_X]; vote_Y++) { lnhash_vote_record_t * rec = &(vote -> vote_records[vote_X][vote_Y]); if(rec -> num_votes >= minimum_votes) { if(ret_size <= ret_items) { ret_size *= 1.3; ret = realloc(ret, sizeof(lnhash_vote_record_t) * ret_size); } if(offsetted) rec -> head_position += rec -> coverage_start; memcpy(ret + ret_items, rec, sizeof(lnhash_vote_record_t)); ret_items ++; } } merge_sort(ret, ret_items, compare_voting_items , exchange_voting_items, merge_vorting_items); (*result_buffer) = ret; return ret_items; } int sorted_voting_table_offset(lnhash_vote_t * vote, lnhash_vote_record_t ** result_buffer, int minimum_votes){ return sorted_voting_table_EX(vote, result_buffer, minimum_votes, 1); } int sorted_voting_table(lnhash_vote_t * vote, lnhash_vote_record_t ** result_buffer, int minimum_votes) { return sorted_voting_table_EX(vote, result_buffer, minimum_votes, 0); } Rsubread/src/long-hashtable.h0000644000175200017520000000603114136047615017202 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #ifndef _LONG_HASHTABLE_H_ #define _LONG_HASHTABLE_H_ #include #include #include "subread.h" #define LNHASH_BUCKET_SIZE_INCREMENT 1.4 #define LNHASH_INIT_BUCKET_SIZE 300 typedef unsigned long long lnhash_data_t; typedef struct { unsigned int num_elements; unsigned int space_elements; gehash_key_t * key_array; lnhash_data_t * value_array; } lnhash_buckct_t; typedef struct { int is_sorted; unsigned long long int num_elements; unsigned int num_buckets; unsigned short * key_repeated_numbers; unsigned short subread_repeat_max; lnhash_buckct_t * buckets; } lnhash_t; #define LNHASH_VOTE_ARRAY_WIDTH 240 #define LNHASH_VOTE_ARRAY_HEIGHT 233 //#define LNHASH_VOTE_ARRAY_WIDTH 48 //#define LNHASH_VOTE_ARRAY_HEIGHT 31 typedef struct{ lnhash_data_t head_position; short coverage_start; short coverage_end; short num_votes; } lnhash_vote_record_t; typedef struct{ int max_votes; int vote_record_items [LNHASH_VOTE_ARRAY_HEIGHT]; lnhash_vote_record_t vote_records [LNHASH_VOTE_ARRAY_HEIGHT][LNHASH_VOTE_ARRAY_WIDTH]; } lnhash_vote_t; #define lnhash_init_votes(v) memset(v->vote_record_items, 0, sizeof(int) * LNHASH_VOTE_ARRAY_HEIGHT) // The EX version creates a hashtable with the given version number int lnhash_create(lnhash_t * the_table, unsigned int num_buckets); // This function puts a data item into the table. If there is duplication, it insert another copy into the table but do not overlap on the old one. int lnhash_insert(lnhash_t * the_table, gehash_key_t key, lnhash_data_t data); // this function must be called before querying or dumpping. void lnhash_resort(lnhash_t * the_table); // this function returns the number of items being hit int lnhash_query(lnhash_t * the_table, lnhash_vote_t * vote, gehash_key_t queried_key, short dist_to_head); // Free all memory that is allocated for the table. Only the table structure itself is not freed. void lnhash_destroy(lnhash_t * the_table); // This function returns the number of votes that has at least "minimum_votes" votes; the vote records are sorted by their head_position. int sorted_voting_table(lnhash_vote_t * vote, lnhash_vote_record_t ** result_buffer, int minimum_votes); // This function adds rec -> coverage_start to rec -> head_position. int sorted_voting_table_offset(lnhash_vote_t * vote, lnhash_vote_record_t ** result_buffer, int minimum_votes); #endif Rsubread/src/longread-mapping.c0000644000175200017520000016031614136047615017540 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #include #include #include #include #include #include #include #include #include #include #include #ifndef __MINGW32__ #include #endif #include #include #include #include #include #include #include "LRMconfig.h" #include "LRMhelper.h" #include "LRMsorted-hashtable.h" #include "LRMbase-index.h" #include "LRMchro-event.h" #include "LRMfile-io.h" int LRMvalidate_and_init_context(LRMcontext_t ** context, int argc, char ** argv); #ifdef MAKE_STANDALONE int main(int argc, char ** argv){ #else int longread_mapping_R(int argc, char ** argv){ #endif int retv=0; LRMcontext_t *context = NULL; retv = retv || LRMvalidate_and_init_context(&context, argc, argv); retv = retv || LRMshow_conf(context); retv = retv || LRMrun_task(context); retv = retv || LRMfinalise(context); retv = retv || LRMprint_mapping_summary(context); retv = retv || LRMdestroy_context(context); context = NULL; return retv; } int LRMprint_mapping_summary(LRMcontext_t * context){ LRMprintf("\n\nAll finished.\n\nTotal processed reads : %d\n", context -> all_processed_reads); LRMprintf("Mapped reads: %u (%.1f%%)\n", context->mapped_reads, context->mapped_reads*100./context->all_processed_reads); LRMprintf("Time: %.1f minutes\n\n" , (LRMmiltime() - context->start_running_time)/60); return 0; } void LRMprint_version(){ LRMprintf("\nSublong v%s\n", SUBREAD_VERSION); LRMputs(""); } void LRMprint_usage(){ LRMprint_version(); LRMputs("Usage:"); LRMputs(""); LRMputs("./sublong [options] -i -r -o "); LRMputs(""); LRMputs("## Mandatory arguments:"); LRMputs(""); LRMputs(" -i Base name of the index. The index must be built as a full index"); LRMputs(" and has only one block."); LRMputs(""); LRMputs(" -r Name of an input read file. Acceptable formats include gzipped"); LRMputs(" FASTQ and FASTQ (automatically identified)."); LRMputs(""); LRMputs(" -o Name of an output file in BAM format."); LRMputs(""); LRMputs("## Optional arguments:"); LRMputs("# input reads and output"); LRMputs(""); LRMputs(" --SAMoutput Save mapping results in SAM format."); LRMputs(""); LRMputs("# number of CPU threads"); LRMputs(""); LRMputs(" -T Number of CPU threads used. 1 by default."); LRMputs(""); LRMputs("# others"); LRMputs(""); LRMputs(" -v Output version of the program."); LRMputs(""); LRMputs(" -X Turn on the RNA-seq mode."); LRMputs(""); LRMputs("Refer to Users Manual for detailed description to the arguments."); LRMputs(""); } static struct option long_options[] ={ {"SAMoutput", no_argument, 0, 's'}, {0, 0, 0, 0} }; int LRMvalidate_and_init_context(LRMcontext_t ** context, int argc, char ** argv){ int c; (*context) = malloc(sizeof(LRMcontext_t)); memset((*context), 0, sizeof(LRMcontext_t)); LRMset_default_values_context(*context); (*context) -> input_file_name[0] = 0; (*context) -> output_file_name[0] = 0; (*context) -> index_prefix[0] = 0; optind = 0; opterr = 1; optopt = 63; int option_index = 0; while ((c = getopt_long (argc, argv, "Xr:i:o:B:T:v", long_options, &option_index))!=-1){ switch(c){ case 'B': (*context)->multi_best_read_alignments = min(max(1,atoi(optarg)),20); break; case 'X': (*context) -> is_RNAseq_mode = 1; (*context)->dynamic_programming_score_match = 6; (*context)->dynamic_programming_score_mismatch = 0; (*context)->dynamic_programming_score_create_gap = -6; (*context)->dynamic_programming_score_extend_gap = -1; break; case 'r': strcpy((*context) -> input_file_name, optarg); break; case 'i': strcpy((*context) -> index_prefix, optarg); break; case 's': (*context) -> max_cigars_in_read = 99900; (*context) -> is_SAM_output = 1; break; case 'o': strcpy((*context) -> output_file_name, optarg); break; case 'T': (*context) -> threads = min(max(1,atoi(optarg)),LRMMAX_THREADS); break; case 'm': (*context) -> min_voting_number = atoi(optarg); break; case 'v': LRMprint_version(); return 1; case '?': default: LRMprint_usage(); return 1; } } if((*context) -> input_file_name[0] ==0 || (*context) -> output_file_name[0]==0 || (*context) -> index_prefix[0]==0){ LRMprintf("Please specify the input, output files and the index.\n"); LRMprint_usage(); return 1; } (*context) -> user_command_line[0]=0; for(c = 0; c user_command_line+strlen( (*context) -> user_command_line), "\"%s\" ", argv[c]); LRMthread_lockinit(&(*context) -> input_lock); LRMthread_lockinit(&(*context) -> sam_bam_file_lock); (*context)-> sam_bam_chromosome_table = LRMHashTableCreate(199); LRMHashTableSetKeyComparisonFunction((*context)-> sam_bam_chromosome_table, LRMhash_strcmp); LRMHashTableSetHashFunction((*context)-> sam_bam_chromosome_table, LRMhash_strhash); LRMHashTableSetDeallocationFunctions((*context)-> sam_bam_chromosome_table, NULL, NULL); (*context)-> chromosome_size_list = LRMArrayListCreate(29); (*context)-> chromosome_size_table = LRMHashTableCreate(199); LRMHashTableSetKeyComparisonFunction((*context)-> chromosome_size_table, LRMhash_strcmp); LRMHashTableSetHashFunction((*context)-> chromosome_size_table, LRMhash_strhash); LRMHashTableSetDeallocationFunctions((*context)-> chromosome_size_table, free, NULL); (*context) -> sam_bam_chromosome_list = LRMArrayListCreate(29); LRMload_offsets(*context); int retv = LRMgeinput_open((*context)->input_file_name,&(*context) -> input_file); (*context)->sam_bam_file = fopen( (*context) -> output_file_name, "w"); if(NULL == (*context)->sam_bam_file) retv = 1; (*context)->event_space = malloc(sizeof(LRMevent_t)*20000); (*context)->event_space_size = 20000; LRMthread_lockinit(&(*context) -> event_space_lock); (*context)->events_realignment = LRMHashTableCreate(320000); return retv; } #ifndef MAKE_STANDALONE #define LRM_CLOCK_USE_GETTIME #endif double LRMmiltime(){ double ret; #ifdef LRM_CLOCK_USE_GETTIME struct timespec tsc; clock_gettime(CLOCK_REALTIME, &tsc); ret = tsc.tv_sec*1. + tsc.tv_nsec*1./1000/1000/1000; #else struct timeb trp; ftime(&trp); ret = trp.time*1.0+(trp.millitm*1.0/1000.0); #endif return ret; } void LRMset_default_values_context(LRMcontext_t * context){ context->threads = 1; context->start_running_time = LRMmiltime(); context->multi_best_read_alignments = 1; context->max_dynamic_indel_length = 5000; context->max_read_indel_length = 0; context->max_junction_distance = 100000; context->min_matched_bases_in_alignment =40; context->max_mismatched_bases_in_subread = 0; context->min_voting_number = 1; context->result_merge_tolerance = LRMDYNAMIC_MAXIMUM_GAP_LENGTH; context->max_cigars_in_read = 65000; context->dynamic_programming_score_match = 6; context->dynamic_programming_score_mismatch = 0; context->dynamic_programming_score_create_gap = -6; context->dynamic_programming_score_extend_gap = -1; } int LRMshow_conf(LRMcontext_t * context){ LRMprintf("\n ====== Subread long read mapping %s======\n\n", context -> is_RNAseq_mode? "(RNA-seq) ": ""); LRMprintf("Threads: %d\n" , context->threads); LRMprintf("Input file: %s\n" , context->input_file_name); LRMprintf("Output file: %s (%s)\n" , context->output_file_name, context->is_SAM_output?"SAM":"BAM"); LRMprintf("Index: %s\n\n" , context->index_prefix); return 0; } int LRMinit_chunk(LRMcontext_t * context){ return 0; } int LRMrun_task(LRMcontext_t * context){ int retv = 0; retv = LRMload_index( context ); LRMprintf("Index was loaded; the gap bewteen subreads is %d bases\n", context -> current_index.index_gap ); while(!(retv ||LRMinput_has_finished( context ))){ retv=retv || LRMinit_chunk(context); retv=retv || LRMsave_input_pos(context); retv=retv || LRMiterate_reads( context , LRMRUNNING_STEP_VOTING); retv=retv || LRMfinalise_chunk_reads(context); } return retv; } int LRMfinalise(LRMcontext_t * context){ return 0; } int LRMdestroy_context(LRMcontext_t * context){ LRMgehash_destory(&(context -> current_index)); LRMgvindex_destory(&(context -> current_base_index)); LRMHashTableDestroy(context -> chromosome_size_table); LRMArrayListDestroy(context -> chromosome_size_list); LRMHashTableDestroy(context -> sam_bam_chromosome_table); LRMArrayListDestroy(context -> sam_bam_chromosome_list); LRMHashTableSetDeallocationFunctions(context -> events_realignment, NULL, free); LRMHashTableDestroy(context -> events_realignment); free(context -> event_space); if(!context -> is_SAM_output){ fwrite(context -> bam_file_tail_binary,1, context -> bam_file_tail_length, context->sam_bam_file); } LRMgeinput_close(&context->input_file); fclose(context->sam_bam_file); //free(context->user_command_line); free(context); return 0; } int LRMinput_has_finished( LRMcontext_t * context ){ return context -> input_exhausted ; } int LRMload_index(LRMcontext_t * context){ int retv = 0; char indextab_fname[LRMMAX_FILENAME_LENGTH + 20]; sprintf(indextab_fname, "%s.00.b.tab", context -> index_prefix); retv = retv || LRMgehash_load(&(context -> current_index), indextab_fname); sprintf(indextab_fname, "%s.00.b.array", context -> index_prefix); retv = retv || LRMgvindex_load(&(context -> current_base_index), indextab_fname); return retv; } int LRMiterate_reads( LRMcontext_t * context, int task ){ int retv = 0; retv = retv || LRMstart_thread( context , task ); retv = retv || LRMwait_threads( context ); retv = retv || LRMmerge_threads( context, task ); return retv; } void * LRM_thread_runner (void * args){ void ** argv = args; LRMcontext_t * context = argv[0]; int thid = argv[1]-NULL; int task = argv[2]-NULL; free(args); LRMchunk_read_iteration(context, thid, task); return NULL; } int LRMstart_thread_init_context(LRMcontext_t * context, int thread_id, int step){ LRMthread_context_t * thread_context = context -> thread_contexts+thread_id; memset(thread_context, 0, sizeof(LRMthread_context_t)); thread_context->thread_id = thread_id; if( thread_context -> thread_id == 0 )LRMsambam_write_header(context, thread_context); thread_context -> mapped_reads = 0; thread_context -> out_SAMBAM_buffer = malloc(3000000); if(thread_context -> out_SAMBAM_buffer == NULL) return 1; thread_context -> out_buff_used = 0; thread_context -> out_buff_capacity = 3000000; thread_context -> dynamic_programming_movement_buffer = malloc(( 2* LRMINDEL_DYNAMIC_CHANNEL_TOLERANCE + 1) * LRMDYNAMIC_MAXIMUM_GAP_LENGTH); thread_context -> dynamic_programming_score_buffer = malloc(sizeof(int) * ( 2 * LRMINDEL_DYNAMIC_CHANNEL_TOLERANCE + 1) *( LRMDYNAMIC_MAXIMUM_GAP_LENGTH+1)); thread_context -> dynamic_programming_indel_movement_buf = malloc( LRMMAX_READ_LENGTH + 1 ); thread_context -> final_cigar_string = malloc( LRMMAX_READ_LENGTH + 1 ); return 0; } int LRMstart_thread(LRMcontext_t * context, int task ){ int th_id, retv=0; for(th_id=0; th_id threads; th_id++){ retv = retv || LRMstart_thread_init_context(context,th_id,task); if(retv) break; else { void ** th_args=malloc(sizeof(void *)*3); // to be freed in the thread. th_args[0] = context; th_args[1] = NULL + th_id; th_args[2] = NULL + task; LRMpthread_create(context -> running_threads+th_id, NULL, LRM_thread_runner, th_args); } } return retv; } int LRMwait_threads( LRMcontext_t * context ){ int th_id; for(th_id=0; th_id threads; th_id++) LRMpthread_join(context -> running_threads[th_id], NULL); return 0; } void LRMmerge_threads_destroy_context(LRMcontext_t * context, LRMthread_context_t * thread_context, int task){ free(thread_context -> dynamic_programming_movement_buffer); free(thread_context -> dynamic_programming_score_buffer); free(thread_context -> dynamic_programming_indel_movement_buf); free(thread_context -> final_cigar_string); } int LRMmerge_threads( LRMcontext_t * context , int step){ int retv = 0; int th_id; for(th_id=0; th_id threads; th_id++){ LRMwrite_chunk_check_buffer_write(context, context -> thread_contexts+th_id, 1); LRMmerge_threads_destroy_context(context, context -> thread_contexts+th_id, step); if(th_id == context -> threads-1)LRMbam_generate_tail_binary(context, context -> thread_contexts+th_id); context -> mapped_reads += context -> thread_contexts[th_id].mapped_reads; } return retv; } int LRMrewind_input_pos(LRMcontext_t * context){ context -> processed_reads_in_chunk = 0; if(context->input_file.file_type == LRMGENE_INPUT_GZIP_FASTQ) LRMseekgz_seek(context->input_file.input_fp, &context->last_saved_zlib_pos); else fseeko(context->input_file.input_fp, context->last_saved_raw_pos, SEEK_SET); return 0; } int LRMsave_input_pos( LRMcontext_t * context){ context -> processed_reads_in_chunk = 0; if(context->input_file.file_type == LRMGENE_INPUT_GZIP_FASTQ) LRMseekgz_tell(context->input_file.input_fp, &context->last_saved_zlib_pos); else context -> last_saved_raw_pos = ftello(context->input_file.input_fp); return 0; } int LRMdo_one_voting_read_process_samechro(LRMcontext_t * context, unsigned int p1, unsigned int p2){ char * chro_name1, *chro_name2; int chro_pos1, chro_pos2; LRMlocate_gene_position(context, p1, &chro_name1, & chro_pos1); LRMlocate_gene_position(context, p2, &chro_name2, & chro_pos2); return chro_name1 == chro_name2; // they can be compared in this way because they are pointers in the sam_bam_chromosome_list. } void LRMreverse_read_and_qual(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context){ LRMreverse_read(iteration_context -> read_text, iteration_context -> read_length); LRMreverse_quality(iteration_context -> qual_text, iteration_context -> read_length); } void LRMcalc_total_subreads(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context){ if(iteration_context->read_length < 16){ iteration_context -> subread_total = 0; return; } int maximum_subreads = iteration_context->read_length - 16 + 1; maximum_subreads = maximum_subreads/3; //LRMprintf("TOTAL SUBREADS=%d\n", min(maximum_subreads, LRMMAX_SUBREAD_PER_READ_HARDLIMIT)); iteration_context -> subread_total = min(maximum_subreads, LRMMAX_SUBREAD_PER_READ_HARDLIMIT); int subread_last = iteration_context -> subread_total -1; iteration_context -> subread_extract_gap = (iteration_context->read_length - 16)*1.0 / ( subread_last ) + 0.000001; } int LRMcalc_subread_start(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context, int subread_no){ if(iteration_context->read_length < 16)return 0; int subread_last = iteration_context -> subread_total -1; if(subread_no < subread_last){ int ret = (int)(iteration_context -> subread_extract_gap * subread_no); assert(ret <= iteration_context->read_length - 16); return ret; } return iteration_context->read_length - 16; } void LRMdo_one_voting_read_single(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context){ int subread_i; LRMcalc_total_subreads(context,thread_context,iteration_context); for(subread_i=0; subread_i< iteration_context -> subread_total; subread_i++){ int subread_start = LRMcalc_subread_start(context,thread_context,iteration_context,subread_i); char * subread_text = iteration_context->read_text + subread_start; unsigned int subread_int = LRMgenekey2int(subread_text); LRMgehash_go_QQ(context, thread_context, iteration_context,& context->current_index, subread_int , subread_start, iteration_context->read_length, iteration_context -> is_reversed, & iteration_context-> vote_table, context -> max_read_indel_length, subread_i); } } void LRMdo_one_voting_read(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context){ LRMinit_gene_vote((& iteration_context-> vote_table)); for(iteration_context->is_reversed = 0; iteration_context->is_reversed<2; iteration_context->is_reversed++){ LRMdo_one_voting_read_single(context, thread_context, iteration_context); if(0 == iteration_context->is_reversed) LRMreverse_read_and_qual(context, thread_context, iteration_context); } iteration_context->is_reversed=1; } int LRM_longvote_location_compare(void * arr, int l, int r){ LRMread_iteration_context_t * iteration_context = arr; if(iteration_context -> sorting_vote_locations[l] > iteration_context -> sorting_vote_locations[r])return 1; if(iteration_context -> sorting_vote_locations[l] < iteration_context -> sorting_vote_locations[r])return -1; return 0; } void LRM_longvote_location_exchange(void * arr, int l, int r){ LRMread_iteration_context_t * iteration_context = arr; unsigned int t; unsigned short ts; t = iteration_context -> sorting_vote_locations[l]; iteration_context -> sorting_vote_locations[l] = iteration_context -> sorting_vote_locations[r]; iteration_context -> sorting_vote_locations[r] = t; t = iteration_context -> sorting_subread_nos[l]; iteration_context -> sorting_subread_nos[l] = iteration_context -> sorting_subread_nos[r]; iteration_context -> sorting_subread_nos[r] = t; ts = iteration_context -> sorting_subread_votes[l]; iteration_context -> sorting_subread_votes[l] = iteration_context -> sorting_subread_votes[r]; iteration_context -> sorting_subread_votes[r] = ts; ts = iteration_context -> sorting_is_negative_strand[l]; iteration_context -> sorting_is_negative_strand[l] = iteration_context -> sorting_is_negative_strand[r]; iteration_context -> sorting_is_negative_strand[r] = ts; } void LRM_longvote_location_merge(void * arr, int start, int items, int items2){ LRMread_iteration_context_t * iteration_context = arr; unsigned int * tmp_locs = malloc(sizeof(unsigned int) * (items + items2)); unsigned int * tmp_subread_nos = malloc(sizeof(unsigned int) * (items + items2)); unsigned int * tmp_negative = malloc(sizeof(unsigned int) * (items + items2)); unsigned short * tmp_votes = malloc(sizeof(unsigned short) * (items + items2)); int rcur_1 = start, rcur_2 = start + items, wcur = 0; for(wcur=0; wcur < items + items2 ; wcur++){ int select_from_1 = rcur_1 < start + items; if(select_from_1 && rcur_2 < start + items + items2)select_from_1 = (LRM_longvote_location_compare(arr, rcur_1, rcur_2)<0); if(select_from_1){ tmp_locs[wcur] = iteration_context -> sorting_vote_locations[rcur_1]; tmp_subread_nos[wcur] = iteration_context -> sorting_subread_nos[rcur_1]; tmp_votes[wcur] = iteration_context -> sorting_subread_votes[rcur_1]; tmp_negative[wcur] = iteration_context -> sorting_is_negative_strand[rcur_1++]; }else{ tmp_locs[wcur] = iteration_context -> sorting_vote_locations[rcur_2]; tmp_subread_nos[wcur] = iteration_context -> sorting_subread_nos[rcur_2]; tmp_votes[wcur] = iteration_context -> sorting_subread_votes[rcur_2]; tmp_negative[wcur] = iteration_context -> sorting_is_negative_strand[rcur_2++]; } } memcpy(iteration_context -> sorting_vote_locations + start , tmp_locs, sizeof(unsigned int) * (items + items2)); memcpy(iteration_context -> sorting_subread_nos + start , tmp_subread_nos, sizeof(unsigned int) * (items + items2)); memcpy(iteration_context -> sorting_subread_votes + start , tmp_votes, sizeof(unsigned short) * (items + items2)); memcpy(iteration_context -> sorting_is_negative_strand + start , tmp_negative, sizeof(unsigned int) * (items + items2)); free(tmp_locs); free(tmp_subread_nos); free(tmp_votes); free(tmp_negative); } void LRMprint_longvote( LRMcontext_t * context, LRMthread_context_t * thread_context,LRMread_iteration_context_t * iteration_context){ int ii; LRMprintf("\n+++ READ %s ++++++++++++++++++++\n", iteration_context -> read_name); if(0) for(ii = 0; ii< iteration_context -> sorting_total_votes; ii++){ char postxt[55]; LRMgene_vote_t * vtab = &(iteration_context->vote_table); if( iteration_context -> sorting_subread_votes[ii] >0){ int bb = iteration_context -> sorting_subread_nos[ii] >> 16; int tt = iteration_context -> sorting_subread_nos[ii] & 0xffff; LRMpos2txt(context, iteration_context -> sorting_vote_locations[ii], postxt); LRMprintf(" %c %d ~ %d %u (%s) : %d votes Array %d %d\n", iteration_context -> sorting_is_negative_strand[ii]?'-':'+', vtab -> coverage_start[bb][tt], vtab -> coverage_end[bb][tt], iteration_context -> sorting_vote_locations[ii], postxt, iteration_context -> sorting_subread_votes[ii], bb, tt); } } LRMprintf("===========================================================\n"); for(ii=0; ii sorted_window_vote_stop[ii]<1)break; int sorted_no = iteration_context -> sorted_window_vote_start[ii]; char postxt[55]; LRMpos2txt(context, iteration_context -> sorting_vote_locations[sorted_no], postxt); LRMprintf(" Window %s subread no: %d ~ %d : Votes = %d ; Pos = %u (%s)\n", iteration_context -> sorted_window_is_negative_strand[ii]?"NEG":"POS", iteration_context -> sorted_window_vote_start[ii], iteration_context -> sorted_window_vote_stop[ii], iteration_context -> sorted_window_total_votes[ii], iteration_context -> sorting_vote_locations[sorted_no], postxt); } LRMprintf(":::::::::::::::::::::::::::::::::::::::::::::::::::::::::::\n"); unsigned int prev_end_chro = 0; int prev_end_read = 0; for(ii = 0; ii < iteration_context -> chain_total_items ; ii++){ char postxt[55]; long long to_prev_delta = prev_end_chro + iteration_context -> chain_cov_start[ii] - prev_end_read; to_prev_delta -= iteration_context -> chain_chro_at_cov_start[ii]; if(ii < 1) to_prev_delta = 0; LRMpos2txt(context, iteration_context -> chain_chro_at_cov_start[ii], postxt); #ifndef __MINGW32__ LRMprintf(" >> COV = %d ~ %d : Pos = %u (%s) DELTA_PREV=%lld\n", iteration_context -> chain_cov_start[ii], iteration_context -> chain_cov_end[ii], iteration_context -> chain_chro_at_cov_start[ii], postxt, to_prev_delta); #endif prev_end_chro = iteration_context -> chain_chro_at_cov_start[ii] + (iteration_context -> chain_cov_end[ii] - iteration_context -> chain_cov_start[ii]); prev_end_read = iteration_context -> chain_cov_end[ii]; } LRMprintf("\n"); } void LRMcopy_longvotes_to_itr( LRMcontext_t * context, LRMthread_context_t * thread_context,LRMread_iteration_context_t * iteration_context){ int bb, ii; iteration_context -> sorting_total_votes = 0; LRMgene_vote_t * vtab = &(iteration_context->vote_table); for(bb =0; bb< LRMGENE_VOTE_TABLE_SIZE; bb++){ for(ii = 0; ii< vtab -> items[bb]; ii++){ // if( vtab -> votes[bb][ii]<2)continue; iteration_context -> sorting_vote_locations[iteration_context -> sorting_total_votes] = vtab -> pos[bb][ii] + vtab -> coverage_start[bb][ii]; iteration_context -> sorting_is_negative_strand[iteration_context -> sorting_total_votes] = (vtab ->masks[bb][ii] & LRMIS_NEGATIVE_STRAND)? 1:0; iteration_context -> sorting_subread_nos[iteration_context -> sorting_total_votes] = ii | (bb<<16); iteration_context -> sorting_subread_votes[iteration_context -> sorting_total_votes] = vtab -> votes[bb][ii]; iteration_context -> sorting_total_votes ++; } } } int LRMis_gap_in_used_gap(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context, unsigned int vote_start_pos, unsigned int vote_cov_len){ unsigned int vote_end_pos = vote_start_pos + vote_cov_len; int xxi; for(xxi = 0; xxi < iteration_context -> chain_used_gaps -> numOfElements; xxi+=2){ unsigned int used_start = LRMArrayListGet(iteration_context -> chain_used_gaps, xxi) - NULL; unsigned int used_end = LRMArrayListGet(iteration_context -> chain_used_gaps, xxi+1) - NULL; //LRMprintf("ADD_VOTE? OLD: %u ~ %u ; NEXT: %u ~ %u\n", used_start, used_end, vote_start_pos, vote_end_pos); if(used_start <= vote_start_pos && used_end >= vote_start_pos) return 1; if(vote_start_pos <= used_start && vote_end_pos >= used_start) return 1; } return 0; } #define LRM_SORTED_VOTE_WINDOW_SIZE 1500000 void LRMfind_top_windows( LRMcontext_t * context, LRMthread_context_t * thread_context,LRMread_iteration_context_t * iteration_context){ int ii, max_window_no = LRMMAX_SORTED_WINDOW_NO_HARD_LIMIT, is_reversed; int testing_window_width = min(LRM_SORTED_VOTE_WINDOW_SIZE, iteration_context -> read_length *4/3); if(iteration_context -> read_length < 10000) testing_window_width = 20000; if(context -> is_RNAseq_mode ) testing_window_width = min(1000000, testing_window_width); if(iteration_context -> sorting_total_votes<1){ iteration_context -> sorted_window_total_votes[0]=0; return; } memset(iteration_context -> sorted_window_total_votes, 0, sizeof(int) * LRMMAX_SORTED_WINDOW_NO_HARD_LIMIT); for(is_reversed = 0; is_reversed < 2; is_reversed++){ unsigned int votes_in_win = 0, win_start_ii = 0; for(win_start_ii = 0; win_start_ii < iteration_context -> sorting_total_votes && iteration_context -> sorting_is_negative_strand[win_start_ii] != is_reversed; win_start_ii++) win_start_ii ++; if(win_start_ii == iteration_context -> sorting_total_votes) continue; for(ii = win_start_ii; ii< iteration_context -> sorting_total_votes; ii++){ if( iteration_context -> sorting_is_negative_strand[ii] != is_reversed ) continue; if(0 && LRMFIXLENstrcmp("R4", iteration_context->read_name) == 0) LRMprintf(" PASSING %d += %d : Vote in win = %d\n", ii, iteration_context -> sorting_subread_votes[ii], votes_in_win); votes_in_win += iteration_context -> sorting_subread_votes[ii]; while(win_start_ii < iteration_context -> sorting_total_votes && win_start_ii < ii){ while(iteration_context -> sorting_is_negative_strand[win_start_ii] != is_reversed) win_start_ii ++; if(iteration_context -> sorting_vote_locations[ii] - iteration_context -> sorting_vote_locations[win_start_ii] < testing_window_width && !LRMis_gap_in_used_gap(context, thread_context, iteration_context,iteration_context -> sorting_vote_locations[win_start_ii], 15))break; votes_in_win -= iteration_context -> sorting_subread_votes[win_start_ii]; win_start_ii ++; while(iteration_context -> sorting_is_negative_strand[win_start_ii] != is_reversed){ if(win_start_ii >= iteration_context -> sorting_total_votes) break; win_start_ii ++; } } if(0 && LRMFIXLENstrcmp("R4", iteration_context->read_name) == 0) LRMprintf(" PASSING2 %d += %d : Vote in win = %d\n", ii, iteration_context -> sorting_subread_votes[ii], votes_in_win); if(0 && LRMFIXLENstrcmp("R4", iteration_context->read_name) == 0 && votes_in_win > 8200){ LRMprintf(" EXCHANGE: %d > %d ; window = %d ~ %d\n=================================\n", votes_in_win , iteration_context -> sorted_window_total_votes[max_window_no-1], win_start_ii, ii); } //LRMprintf("LRMtest_window_in : %u ~ %u\n", iteration_context->sorting_vote_locations[win_start_ii], iteration_context->sorting_vote_locations[ii] - iteration_context -> sorting_vote_locations[win_start_ii] + 16); if(votes_in_win > iteration_context -> sorted_window_total_votes[max_window_no-1] && ! LRMis_gap_in_used_gap(context, thread_context, iteration_context, iteration_context->sorting_vote_locations[win_start_ii], iteration_context->sorting_vote_locations[ii] - iteration_context -> sorting_vote_locations[win_start_ii] + 15)){ int tins = -1; int tt; int has_in_window = 0; for(tt = max_window_no - 1; tt >= 0 ; tt --){ if( is_reversed == iteration_context -> sorted_window_is_negative_strand[tt] && ( (iteration_context -> sorted_window_vote_start[tt] <=win_start_ii && iteration_context -> sorted_window_vote_stop[tt] > win_start_ii)|| ( win_start_ii <= iteration_context -> sorted_window_vote_start[tt] && ii > iteration_context -> sorted_window_vote_start[tt] ))){ if(votes_in_win >iteration_context -> sorted_window_total_votes[tt]){ int mm; for(mm = tt; mm < max_window_no - 1; mm++){ iteration_context -> sorted_window_total_votes[mm] = iteration_context -> sorted_window_total_votes[mm+1]; iteration_context -> sorted_window_vote_start[mm] = iteration_context -> sorted_window_vote_start[mm+1]; iteration_context -> sorted_window_vote_stop[mm] = iteration_context -> sorted_window_vote_stop[mm+1]; iteration_context -> sorted_window_is_negative_strand[mm] = iteration_context -> sorted_window_is_negative_strand[mm+1]; } iteration_context -> sorted_window_total_votes[max_window_no - 1] = 0; }else{ has_in_window = 1; break; } } } if(!has_in_window){ for(tt = max_window_no - 1; tt >= 0 ; tt --){ if(votes_in_win > iteration_context -> sorted_window_total_votes[tt]) tins = tt; } for(tt = max_window_no-1; tt > tins; tt--){ iteration_context -> sorted_window_total_votes[tt] = iteration_context -> sorted_window_total_votes[tt -1] ; iteration_context -> sorted_window_vote_start[tt] = iteration_context -> sorted_window_vote_start[tt -1] ; iteration_context -> sorted_window_vote_stop[tt] = iteration_context -> sorted_window_vote_stop[tt -1] ; iteration_context -> sorted_window_is_negative_strand[tt] = iteration_context -> sorted_window_is_negative_strand[tt -1]; } iteration_context -> sorted_window_total_votes[tins] = votes_in_win; iteration_context -> sorted_window_vote_start[tins] = win_start_ii; iteration_context -> sorted_window_vote_stop[tins] = ii; iteration_context -> sorted_window_is_negative_strand[tins] = iteration_context -> sorting_is_negative_strand[ii]; } } } } } #define MAX_INTRON_LENGTH 700000 int LRM_test_chain_extension(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context, int window_no, int go_larger, long long N2W_read_dist, long long N2W_chro_dist, unsigned int last_chro_pos, unsigned int this_chro_pos, unsigned int this_cov_len){ //LRMprintf("TESTING CHAIN: N2W_READ: %lld ; N2W_CHRO: %lld %s\n", N2W_read_dist, N2W_chro_dist, go_larger?"GO_LARGE":"GO_SMALL"); if(LRMis_gap_in_used_gap( context, thread_context, iteration_context, this_chro_pos, this_cov_len )) return 0; if(N2W_read_dist == 0 || N2W_chro_dist == 0) return 0; if(go_larger){ if(N2W_read_dist < 0 || N2W_chro_dist < 0) return 0; }else{ if(N2W_read_dist > 0 || N2W_chro_dist > 0) return 0; } if(!context -> is_RNAseq_mode)if( abs(N2W_chro_dist) > 50 || abs(N2W_read_dist) > 50 )if(abs(N2W_chro_dist) >= abs(N2W_read_dist)* 14/10 || abs(N2W_chro_dist) <= abs(N2W_read_dist) * 6 / 10) return 0; if(context -> is_RNAseq_mode)if( abs(N2W_chro_dist) > 50 || abs(N2W_read_dist) > 50 )if( abs(N2W_chro_dist) >= abs(N2W_read_dist) + MAX_INTRON_LENGTH || abs(N2W_chro_dist) <= abs(N2W_read_dist) * 6 / 10) return 0; if(abs(N2W_read_dist) > context -> result_merge_tolerance) return 0; if(!LRMdo_one_voting_read_process_samechro(context , last_chro_pos, this_chro_pos) ) return 0; return 1; } void LRMfix_extension_overlapping(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context, int window_no ){ int ii; int last_end_read = iteration_context -> chain_cov_end[0]; unsigned int last_chro_end = iteration_context -> chain_chro_at_cov_start[0] + iteration_context -> chain_cov_end[0] - iteration_context -> chain_cov_start[0]; if(0){ for(ii = 1; ii < iteration_context -> chain_total_items ; ii++){ LRMprintf("BEFORE_FIX READ COV: %d ~ %d\n", iteration_context -> chain_cov_start[ii], iteration_context -> chain_cov_end[ii]); } } for(ii = 1; ii < iteration_context -> chain_total_items ; ii++){ int this_start_read = iteration_context -> chain_cov_start[ii]; int add_to_this = last_end_read - this_start_read; if(add_to_this >= 0){ iteration_context -> chain_cov_start[ii] += add_to_this+1; iteration_context -> chain_chro_at_cov_start[ii] += add_to_this+1; //LRMprintf("FIXING OVERLAPPING MOVE : %d\n", add_to_this); } //LRMprintf("ITEM %d / %d ; LAST_END %u <= THIS_START %u ; READ START END = %d, %d ; LAST_READ_END = %d\n", ii, iteration_context -> chain_total_items , last_chro_end, iteration_context -> chain_chro_at_cov_start[ii], iteration_context -> chain_cov_start[ii], iteration_context -> chain_cov_end[ii] , last_end_read ); if(iteration_context -> chain_cov_start[ii]>=iteration_context -> chain_cov_end[ii] || iteration_context -> chain_cov_start[ii] < last_end_read || iteration_context -> chain_chro_at_cov_start[ii] < last_chro_end){ // LRMprintf("DELETED COV\n"); //assert( iteration_context -> chain_cov_start[ii] < iteration_context -> chain_cov_end[ii] ); int ti; for(ti=ii; ti chain_total_items-1; ti++){ iteration_context -> chain_cov_start[ti] = iteration_context -> chain_cov_start[ti+1]; iteration_context -> chain_cov_end[ti] = iteration_context -> chain_cov_end[ti+1]; iteration_context -> chain_chro_at_cov_start [ti] = iteration_context -> chain_chro_at_cov_start [ti+1]; } iteration_context -> chain_total_items--; last_end_read = iteration_context -> chain_cov_end[ii-1]; last_chro_end = iteration_context -> chain_chro_at_cov_start [ii-1] + iteration_context -> chain_cov_end[ii-1] - iteration_context -> chain_cov_start[ii-1]; if(ii < iteration_context -> chain_total_items) ii--; }else{ last_end_read = iteration_context -> chain_cov_end[ii]; last_chro_end = iteration_context -> chain_chro_at_cov_start [ii] + iteration_context -> chain_cov_end[ii] - iteration_context -> chain_cov_start[ii]; } } } void LRMbuild_chains(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context, int window_no ){ int testing_window_width = min(LRM_SORTED_VOTE_WINDOW_SIZE, iteration_context -> read_length *4/3); if(iteration_context -> read_length < 10000) testing_window_width = 20000; if(iteration_context -> sorted_window_total_votes[window_no] < 1){ iteration_context -> chain_total_items = iteration_context -> chain_tosmall_items = iteration_context -> chain_tolarge_items = 0; return; } int seed_subread_no = iteration_context -> sorted_window_vote_start[window_no]; int test_subread_no, go_large, has_overlapping = 0; for(test_subread_no = iteration_context -> sorted_window_vote_start[window_no] ; test_subread_no < iteration_context -> sorted_window_vote_stop[window_no] ; test_subread_no++){ if(iteration_context -> sorted_window_is_negative_strand[window_no] != iteration_context -> sorting_is_negative_strand[test_subread_no]) continue; if(iteration_context -> sorting_subread_votes[test_subread_no] > iteration_context -> sorting_subread_votes[seed_subread_no]) seed_subread_no = test_subread_no; } LRMgene_vote_t * vtab = &(iteration_context->vote_table); for(go_large = 0; go_large < 2; go_large ++){ int last_added_tt, last_added_ii; last_added_tt = iteration_context -> sorting_subread_nos[seed_subread_no] >> 16; last_added_ii = iteration_context -> sorting_subread_nos[seed_subread_no] & 0xffff; unsigned int last_added_read_pos = vtab -> coverage_start[last_added_tt][last_added_ii]; long long last_added_chro_pos = vtab -> pos[last_added_tt][last_added_ii] + vtab -> coverage_start[last_added_tt][last_added_ii]; //LRMprintf("SEED %s : [%d:%d] ; pos=%u OR %u\n", iteration_context -> sorted_window_is_negative_strand[window_no]?"NEG":"POS", last_added_tt, last_added_ii, last_added_read_pos, iteration_context -> sorting_vote_locations[seed_subread_no]); int last_added_read_edge = go_large?vtab -> coverage_end[last_added_tt][last_added_ii]: vtab -> coverage_start[last_added_tt][last_added_ii]; if(!go_large){ iteration_context ->chain_tosmall_items = 1; iteration_context ->chain_tolarge_items = 0; iteration_context -> chain_cov_start[0] = last_added_read_pos; iteration_context -> chain_cov_end[0] = vtab -> coverage_end[last_added_tt][last_added_ii]; iteration_context -> chain_chro_at_cov_start[0] = last_added_chro_pos; } int delta = go_large?1:-1; int next_test_subread_no = -1; for(test_subread_no = seed_subread_no ; test_subread_no >= 0 && test_subread_no < iteration_context -> sorting_total_votes; test_subread_no+= delta){ int test_tt, test_ii; test_tt = iteration_context -> sorting_subread_nos[test_subread_no] >> 16; test_ii = iteration_context -> sorting_subread_nos[test_subread_no] &0xffff; if( test_ii >= LRMGENE_VOTE_SPACE || test_tt >= LRMGENE_VOTE_TABLE_SIZE) LRMprintf("Error: Table oversize %s , Subr: %d/%d : %d %d\n", iteration_context -> read_name, test_subread_no, iteration_context -> sorting_total_votes, test_tt, test_ii); assert(test_ii < LRMGENE_VOTE_SPACE); assert(test_tt < LRMGENE_VOTE_TABLE_SIZE); int this_read_pos = vtab -> coverage_start[test_tt][test_ii]; int this_read_end = vtab -> coverage_end[test_tt][test_ii]; long long this_chro_pos = iteration_context -> sorting_vote_locations[test_subread_no]; int same_min_subread_test_no=-1; long long min_same_DELTA = 99999999999; for(same_min_subread_test_no = test_subread_no; same_min_subread_test_no sorting_total_votes && same_min_subread_test_no >=0; same_min_subread_test_no+= delta){ int same_min_tt = iteration_context -> sorting_subread_nos[same_min_subread_test_no] >> 16; int same_min_ii = iteration_context -> sorting_subread_nos[same_min_subread_test_no] & 0xffff; int same_min_read_pos = vtab -> coverage_start[same_min_tt][same_min_ii]; if(same_min_read_pos!= this_read_pos){ next_test_subread_no = same_min_subread_test_no - delta; break; } long long same_min_chro_pos = iteration_context -> sorting_vote_locations[same_min_subread_test_no]; long long int same_read_dist = same_min_read_pos; same_read_dist -= last_added_read_pos; long long same_chro_dist = same_min_chro_pos; same_chro_dist -= last_added_chro_pos; long long same_DELTA = same_chro_dist - same_read_dist; if(abs(same_DELTA) < min_same_DELTA){ min_same_DELTA = abs(same_DELTA); test_subread_no = same_min_subread_test_no; } if(0){ //int Asame_min_tt = iteration_context -> sorting_subread_nos[same_min_subread_test_no+delta] >> 16; //int Asame_min_ii = iteration_context -> sorting_subread_nos[same_min_subread_test_no+delta] & 0xffff; //int Asame_min_read_pos = vtab -> coverage_start[Asame_min_tt][Asame_min_ii]; //if(Asame_min_read_pos == same_min_read_pos || same_min_subread_test_no > test_subread_no) // LRMprintf("SAME_MIN %s TO %d - D=%lld ; LOC # %lld = %u\n", go_large?"LARGE":"SMALL" , same_min_read_pos, same_DELTA, same_min_subread_test_no, same_min_chro_pos); } if(same_min_subread_test_no == iteration_context -> sorting_total_votes -1 || same_min_subread_test_no == 0) next_test_subread_no = same_min_subread_test_no; } test_tt = iteration_context -> sorting_subread_nos[test_subread_no] >> 16; test_ii = iteration_context -> sorting_subread_nos[test_subread_no] &0xffff; this_read_pos = vtab -> coverage_start[test_tt][test_ii]; this_read_end = vtab -> coverage_end[test_tt][test_ii]; this_chro_pos = iteration_context -> sorting_vote_locations[test_subread_no]; if( test_ii >= LRMGENE_VOTE_SPACE || test_tt >= LRMGENE_VOTE_TABLE_SIZE) LRMprintf("Error: Table oversize %s , Subr: %d/%d : %d %d\n", iteration_context -> read_name, test_subread_no, iteration_context -> sorting_total_votes, test_tt, test_ii); int N2W_read_dist = this_read_pos; N2W_read_dist -= last_added_read_pos; long long N2W_chro_dist = this_chro_pos; N2W_chro_dist -= last_added_chro_pos; if(abs(N2W_chro_dist) > testing_window_width) break; //LRMprintf("CALC N2W_chro_dist : %lld = %lld - %lld \n", N2W_chro_dist, last_added_chro_pos, this_chro_pos); int can_add_this = iteration_context -> sorted_window_is_negative_strand[window_no] == iteration_context -> sorting_is_negative_strand[test_subread_no] && LRM_test_chain_extension(context, thread_context, iteration_context, window_no, go_large, N2W_read_dist, N2W_chro_dist, last_added_chro_pos , this_chro_pos, this_read_end - this_read_pos); if(can_add_this){ int idx = iteration_context ->chain_tosmall_items; if(go_large) idx += iteration_context ->chain_tolarge_items; int this_added_read_inner = go_large? this_read_pos:this_read_end; if( go_large && this_added_read_inner <= last_added_read_edge ){ //LRMprintf("OVERLAPPING_LAGRE %s : %d <= %d\n", iteration_context -> read_name, this_added_read_inner, last_added_read_edge); has_overlapping = 1; }else if( (!go_large) && this_added_read_inner >= last_added_read_edge ){ //LRMprintf("OVERLAPPING_SMALL %s : %d >= %d\n", iteration_context -> read_name, this_added_read_inner, last_added_read_edge); has_overlapping = 1; } iteration_context -> chain_cov_start[idx] = this_read_pos; iteration_context -> chain_cov_end[idx] = this_read_end; iteration_context -> chain_chro_at_cov_start[idx] = this_chro_pos; if(go_large) iteration_context ->chain_tolarge_items++; else iteration_context ->chain_tosmall_items++; last_added_read_pos = this_read_pos; last_added_chro_pos = this_chro_pos; last_added_read_edge = go_large? this_read_end : this_read_pos ; } //assert(next_test_subread_no >= 0); test_subread_no = next_test_subread_no; } } int ii; for(ii = 0; ii < iteration_context -> chain_tosmall_items/2 ; ii++){ unsigned int tv; tv = iteration_context -> chain_cov_start[ii]; iteration_context -> chain_cov_start[ii] = iteration_context -> chain_cov_start[iteration_context -> chain_tosmall_items - 1 - ii]; iteration_context -> chain_cov_start[iteration_context -> chain_tosmall_items - 1 - ii] = tv; tv = iteration_context -> chain_cov_end[ii]; iteration_context -> chain_cov_end[ii] = iteration_context -> chain_cov_end[iteration_context -> chain_tosmall_items - 1 - ii]; iteration_context -> chain_cov_end[iteration_context -> chain_tosmall_items - 1 - ii] = tv; tv = iteration_context -> chain_chro_at_cov_start[ii]; iteration_context -> chain_chro_at_cov_start[ii] = iteration_context -> chain_chro_at_cov_start[iteration_context -> chain_tosmall_items - 1 - ii]; iteration_context -> chain_chro_at_cov_start[iteration_context -> chain_tosmall_items - 1 - ii] = tv; } iteration_context -> chain_total_items = iteration_context -> chain_tosmall_items + iteration_context -> chain_tolarge_items; if(has_overlapping) LRMfix_extension_overlapping(context, thread_context, iteration_context, window_no); unsigned int this_gap_start_corr = iteration_context -> chain_chro_at_cov_start[0]; unsigned int this_gap_end_corr = iteration_context -> chain_chro_at_cov_start[iteration_context -> chain_total_items -1] + iteration_context -> chain_cov_end[iteration_context -> chain_total_items -1] - iteration_context -> chain_cov_start[ iteration_context -> chain_total_items -1 ]; if(iteration_context -> chain_total_items>0){ LRMArrayListPush(iteration_context -> chain_used_gaps, NULL+this_gap_start_corr); LRMArrayListPush(iteration_context -> chain_used_gaps, NULL+this_gap_end_corr); //LRMprintf("USED_CHAIN_RANGE ITEMS=%d : %u ~ %u\n", iteration_context -> chain_total_items, this_gap_start_corr , this_gap_end_corr); }else{ //LRMprintf("NO_CHAIN_IS_BUILT\n"); } } void LRMfill_gaps_addNM(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context, int window_no, int subread_no){ int middle_delta = subread_no?1:1; thread_context -> dynamic_programming_indel_movement_start += sprintf( thread_context -> dynamic_programming_indel_movement_buf + thread_context -> dynamic_programming_indel_movement_start, "%dM/", iteration_context -> chain_cov_end[subread_no] - iteration_context -> chain_cov_start[subread_no] - middle_delta); //LRMprintf("NORMAL MS: %dM\n", iteration_context -> chain_cov_end[subread_no] - iteration_context -> chain_cov_start[subread_no] - middle_delta); } void LRMsave_mapping_result(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context, int window_no){ int flags = 4; //if(iteration_context -> total_matched_bases >= context -> min_matched_bases_in_alignment) char * chro_name; int chro_pos, map_quality = 10, mis_matched = 0, corr_report = 0; if( iteration_context -> chain_total_items > 0 ){ flags = iteration_context -> sorted_window_is_negative_strand[window_no]?16:0; LRMlocate_gene_position(context, iteration_context -> final_mapping_location, &chro_name, &chro_pos); if(NULL != chro_name){ corr_report = 1; thread_context -> mapped_reads++; } } if(!corr_report){ strcpy(thread_context -> final_cigar_string, "*"); chro_name = "*"; chro_pos = -1; map_quality = 0; } //LRMprintf("Writing [%d] : %u => %s , %d\n", loc_ret, iteration_context -> final_mapping_location, chro_name , chro_pos); LRMwrite_chunk_add_buffered_output(context, thread_context, iteration_context, flags, chro_name, chro_pos +1, map_quality, thread_context -> final_cigar_string, mis_matched); } void LRMfill_gaps_reduce_Cigar(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context, int * PTRmapped_length, int window_no){ //LRMtest_move_buff( context, thread_context, iteration_context, thread_context -> dynamic_programming_indel_movement_buf, strlen(thread_context -> dynamic_programming_indel_movement_buf), iteration_context -> read_length); int tmpi = -1; int ci, nch, repeat_i = 0, old_opt = 0, wcur=0, r_rebuilt_len=0, mapped_length = 0; for(ci = 0; ; ci++){ nch = thread_context -> dynamic_programming_indel_movement_buf[ci]; if(!nch) break; if(nch == '/') continue; if(nch == '.') continue; if(nch == 'X') nch='M'; if(isdigit(nch)){ if(tmpi<0) tmpi = 0; tmpi = tmpi*10 + (nch-'0'); }else{ if(tmpi<0) tmpi = 1; if(old_opt != nch && repeat_i>0){ wcur += sprintf( thread_context -> final_cigar_string + wcur, "%d%c", repeat_i, old_opt ); if( old_opt == 'M' || old_opt == 'I' || old_opt == 'S' ) r_rebuilt_len += repeat_i; if( old_opt == 'M' ) mapped_length += repeat_i; repeat_i = 0; } repeat_i += tmpi; tmpi = -1; old_opt = nch; } } if(repeat_i>0){ if( old_opt == 'M' || old_opt == 'I' || old_opt == 'S' ) r_rebuilt_len += repeat_i; if( old_opt == 'M' ) mapped_length += repeat_i; sprintf( thread_context -> final_cigar_string + wcur, "%d%c", repeat_i, old_opt ); } //LRMprintf("Rebuild Rlen of %s = %d\n", iteration_context -> read_name, r_rebuilt_len); if(r_rebuilt_len != iteration_context -> read_length) LRMprintf("WRONG_REBUILD : %s : %d != %d ; %s\n", iteration_context -> read_name, r_rebuilt_len , iteration_context -> read_length, thread_context -> dynamic_programming_indel_movement_buf); *PTRmapped_length = mapped_length; } unsigned int LRMfill_gaps_find_final_mapping_loc(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context, int window_no){ int ii; int rollback = 0; for(ii = thread_context -> dynamic_programming_indel_movement_start ; ii >=0; ii--){ int nch = thread_context -> dynamic_programming_indel_movement_buf[ii]; if( nch == 'I' || nch == '/' || nch == '.' ) continue; if( nch == 'S') break; rollback ++; } return iteration_context -> chain_chro_at_cov_start[0] - rollback; } void LRMfill_gaps(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context, int window_no){ int ii; int total_read_len = 0; //LRMprintf("READ_REVERSING: %d == %d ?\n", iteration_context -> is_reversed , iteration_context -> sorted_window_is_negative_strand[window_no]); if(iteration_context -> is_reversed != iteration_context -> sorted_window_is_negative_strand[window_no] ){ LRMreverse_read(iteration_context -> read_text, iteration_context -> read_length); iteration_context -> is_reversed=!iteration_context -> is_reversed; } int first_mapped_subread_start = iteration_context -> chain_cov_start[0]; int new_moves = LRMdynamic_to_ends(context, thread_context, iteration_context, first_mapped_subread_start, iteration_context -> chain_chro_at_cov_start[0], 0); //LRMprintf("MOVES_START %s: moves=%d ; read_len = %d ; %s\n", iteration_context -> read_name, new_moves, first_mapped_subread_start, thread_context -> dynamic_programming_indel_movement_buf); if(new_moves<0){ LRMprintf("MINUS MOVES : %d\n", new_moves); new_moves = 0; } total_read_len += first_mapped_subread_start; thread_context -> dynamic_programming_indel_movement_start += new_moves; iteration_context -> final_mapping_location = LRMfill_gaps_find_final_mapping_loc(context, thread_context, iteration_context, window_no); LRMfill_gaps_addNM(context, thread_context, iteration_context, window_no, 0); total_read_len += iteration_context -> chain_cov_end[0] - iteration_context -> chain_cov_start[0]; int last_subread_end = iteration_context -> chain_cov_end[0]; unsigned int last_subread_chro_end = iteration_context -> chain_chro_at_cov_start[0] + (last_subread_end - first_mapped_subread_start); for(ii = 1; ii < iteration_context -> chain_total_items; ii++){ unsigned int this_subread_chro_start = iteration_context -> chain_chro_at_cov_start[ii]; int this_subread_start = iteration_context -> chain_cov_start[ii]; int read_minus_chro_delta = (this_subread_start - last_subread_end) - ( this_subread_chro_start - last_subread_chro_end ); //LRMprintf("read_minus_chro_delta== %d = (%d - %d) - (%d - %d)\n", read_minus_chro_delta, this_subread_start, last_subread_end, this_subread_chro_start, last_subread_chro_end); if(this_subread_start <=last_subread_end -1) LRMprintf("Error: Reversed order of %s : %d < %d\n", iteration_context -> read_name, this_subread_start, last_subread_end -1 ); assert( this_subread_start > last_subread_end -1 ); if( this_subread_start - last_subread_end >= LRMDYNAMIC_MAXIMUM_GAP_LENGTH ){ int indel_after_M = -read_minus_chro_delta; int gap_read_M = this_subread_start - last_subread_end +1 - max( 0, -indel_after_M ); int gap_read_M_L = gap_read_M/2; int gap_read_M_R = gap_read_M - gap_read_M_L; int indel_move = indel_after_M<0?'I':'D'; thread_context -> dynamic_programming_indel_movement_start += sprintf( thread_context -> dynamic_programming_indel_movement_buf + thread_context -> dynamic_programming_indel_movement_start, "%dM%d%c%dM/", gap_read_M_L,abs(indel_after_M), indel_move, gap_read_M_R); LRMprintf("LONG GAP %s : %d\n", iteration_context -> read_name, this_subread_start - last_subread_end ); LRMprintf("LONG GAP CIGAR : %dM%d%c\n", gap_read_M,abs(indel_after_M), indel_move); } else{ new_moves = LRMdynamic_in_middle(context, thread_context, iteration_context, last_subread_end -1, this_subread_start, last_subread_chro_end - 1, read_minus_chro_delta); if(new_moves<0){ LRMprintf("MINUS MOVES : %d\n", new_moves); new_moves = 0; } thread_context -> dynamic_programming_indel_movement_start += new_moves; } total_read_len += iteration_context -> chain_cov_end[ii] - last_subread_end ; LRMfill_gaps_addNM(context, thread_context, iteration_context, window_no, ii); last_subread_end = iteration_context -> chain_cov_end[ii]; last_subread_chro_end = this_subread_chro_start + (last_subread_end - this_subread_start); } unsigned int last_end_chro_pos = iteration_context -> chain_chro_at_cov_start[ii-1]; last_end_chro_pos += ( iteration_context -> chain_cov_end[ii-1] - iteration_context -> chain_cov_start[ii-1] ); new_moves = LRMdynamic_to_ends(context, thread_context, iteration_context, last_subread_end -1, last_end_chro_pos -1, 1 ); if(new_moves<0){ LRMprintf("MINUS MOVES : %d\n", new_moves); new_moves = 0; } total_read_len += iteration_context -> read_length - last_subread_end; if( total_read_len!=iteration_context -> read_length )LRMprintf("WRONG LENGTH %s: %d (mapping) != %d (fastq length) \n", iteration_context -> read_name, total_read_len, iteration_context -> read_length); thread_context -> dynamic_programming_indel_movement_start += new_moves; int mapped_length = 0; LRMfill_gaps_reduce_Cigar(context, thread_context, iteration_context, &mapped_length, window_no); if(mapped_length < context->min_matched_bases_in_alignment) iteration_context -> chain_total_items=0; } void LRMreset_iteration_context_before_read_one_alignment(LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context){ iteration_context -> total_matched_bases = 0; iteration_context -> chain_tolarge_items = 0; iteration_context -> chain_tosmall_items = 0; iteration_context -> chain_total_items = 0; memset(iteration_context -> sorted_window_total_votes, 0, sizeof(int) * LRMMAX_SORTED_WINDOW_NO_HARD_LIMIT); thread_context -> dynamic_programming_indel_movement_start = 1; thread_context -> dynamic_programming_indel_movement_buf[0]='/'; } void LRMreset_iteration_context_before_read( LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context ){ if(iteration_context -> chain_used_gaps) LRMArrayListDestroy(iteration_context -> chain_used_gaps); iteration_context -> chain_used_gaps = LRMArrayListCreate(40); } void LRMdo_dynamic_programming_read( LRMcontext_t * context, LRMthread_context_t * thread_context, LRMread_iteration_context_t * iteration_context ){ LRMreset_iteration_context_before_read(context , thread_context , iteration_context); LRMcopy_longvotes_to_itr(context, thread_context, iteration_context); LRMmerge_sort(iteration_context, iteration_context -> sorting_total_votes, LRM_longvote_location_compare, LRM_longvote_location_exchange, LRM_longvote_location_merge); for(iteration_context -> current_alignment_no = 0; iteration_context -> current_alignment_no < context -> multi_best_read_alignments; iteration_context -> current_alignment_no++){ LRMreset_iteration_context_before_read_one_alignment(context , thread_context , iteration_context); LRMfind_top_windows(context, thread_context, iteration_context); int ww = 0; if(iteration_context -> sorted_window_total_votes [ww] >0){ LRMbuild_chains(context, thread_context, iteration_context, ww); if(0&& LRMFIXLENstrcmp("R4", iteration_context->read_name) == 0 ) LRMprint_longvote(context, thread_context, iteration_context); LRMfill_gaps(context, thread_context, iteration_context, ww); }else iteration_context -> chain_total_items = iteration_context -> chain_tosmall_items = iteration_context -> chain_tolarge_items = 0; LRMsave_mapping_result(context, thread_context, iteration_context, ww); } } int LRMchunk_read_iteration(LRMcontext_t * context, int thread_id, int task){ LRMthread_context_t * thread_context = context -> thread_contexts+ thread_id; LRMread_iteration_context_t * iteration_context; iteration_context = malloc(sizeof(LRMread_iteration_context_t)); memset(iteration_context, 0, sizeof(LRMread_iteration_context_t)); while(1){ int retv = LRMfetch_next_read(context, thread_context, &iteration_context-> read_length, iteration_context->read_name, iteration_context->read_text, iteration_context->qual_text, &iteration_context -> read_no_in_chunk); if(retv) break; LRMdo_one_voting_read(context, thread_context, iteration_context); LRMdo_dynamic_programming_read(context, thread_context, iteration_context); if(iteration_context -> read_no_in_chunk % 2000 == 0) LRMprintf("Processing %d-th read for task %d; used %.1f minutes\n", context -> all_processed_reads + iteration_context -> read_no_in_chunk, task, (LRMmiltime() - context -> start_running_time)/60); } if(iteration_context -> chain_used_gaps) LRMArrayListDestroy(iteration_context -> chain_used_gaps); iteration_context -> chain_used_gaps = NULL; free(iteration_context); return 0; } int LRMfinalise_chunk_reads(LRMcontext_t* context){ context -> all_processed_reads += context -> processed_reads_in_chunk; return 0; } int LRMFIXLENstrcmp(char * fixed_len, char * rname){ int x=0; for(; fixed_len[x]; x++){ if(rname[x]!=fixed_len[x]) return 1; } return 0; } Rsubread/src/mergeVCF.c0000644000175200017520000002532714136047615015754 0ustar00biocbuildbiocbuild#include #include #include #include #include #include #include "subread.h" #include "core.h" #include "hashtable.h" #include "gene-algorithms.h" #include "HelperFunctions.h" char output_file_name[MAX_FILE_NAME_LENGTH]; FILE * output_file_pointer; static struct option propm_long_options[] = { {"out", required_argument, 0, 'o'}, {0, 0, 0, 0} }; typedef struct{ char ** keys; } VCF_sort_context; void VCF_sort_getv(char * ky, char * chro, int * pos) { int cursor, nch, tabs =0, chro_cursor=0; (*pos)= 0; for(cursor = 0;0!=(nch=ky[cursor]); cursor++){ if(nch == '\t') { tabs ++; if(tabs == 3)break; } else if(tabs == 1) chro[chro_cursor++]=nch; else if(tabs == 2) (*pos) = (*pos)*10 + (nch - '0'); } chro[chro_cursor]=0; } int compare_VCF_rows(void * arri, int l, int r) { VCF_sort_context * arr = (VCF_sort_context*) arri; char * lk = arr -> keys[l]; char * rk = arr -> keys[r]; char chro_1[MAX_CHROMOSOME_NAME_LEN+1]; char chro_2[MAX_CHROMOSOME_NAME_LEN+1]; int pos1 =0, pos2 = 0; VCF_sort_getv(lk,chro_1, &pos1); VCF_sort_getv(rk,chro_2, &pos2); // printf("LK=%s; CHRO=%s; POS=%d\n", lk, chro_1, pos1); int ret = strcmp_number(chro_1, chro_2); if(ret==0) { if(pos1>pos2) ret = 1; if(pos1 keys[l]; char * rk = arr -> keys[r]; arr -> keys[l] = rk; arr -> keys[r] = lk; } void merge_VCF_rows(void * arri, int start, int items, int items2) { VCF_sort_context * arr = (VCF_sort_context*) arri; int x1, first_cursor = start, second_cursor = start+items; char ** merge_tmp = malloc(sizeof(char *)* (items+items2)); for(x1=0;x1= start+items) choose_2 = 1; else if(second_cursor < start+items+items2) choose_2 = (0 < compare_VCF_rows(arr, first_cursor, second_cursor)); if(choose_2) merge_tmp[x1] = arr -> keys[second_cursor++]; else merge_tmp[x1] = arr -> keys[first_cursor++]; } memcpy(arr -> keys + start, merge_tmp, sizeof(char *) * (items+items2)); free(merge_tmp); } int endswith(const char *str, const char *suffix) { if (!str || !suffix) return 0; size_t lenstr = strlen(str); size_t lensuffix = strlen(suffix); if (lensuffix > lenstr) return 0; return strncmp(str + lenstr - lensuffix, suffix, lensuffix) == 0; } void set_hash_funcs(HashTable * tab, int is_key_freed, int is_value_string) { HashTableSetDeallocationFunctions(tab,is_key_freed?free:NULL,is_value_string?free:NULL); HashTableSetHashFunction(tab , fc_chro_hash); HashTableSetKeyComparisonFunction(tab,fc_strcmp_chro); } int warning_reported_repeated; void do_find_common(char ** file_names, int files) { int badfiles = 0; char * file_line = malloc(3000); int file_no; HashTable * extra_count_table = HashTableCreate(200000); set_hash_funcs(extra_count_table,1,0); HashTable * extra_qual_table = HashTableCreate(200000); set_hash_funcs(extra_qual_table,0,0); HashTable * extra_info_table = HashTableCreate(200000); set_hash_funcs(extra_info_table,0,1); HashTable * extra_id_qual_filter_table = HashTableCreate(200000); set_hash_funcs(extra_id_qual_filter_table,0,1); for(file_no=0; file_no < files; file_no++) { FILE * ifp = fopen(file_names[file_no],"r"); if(NULL == ifp) { SUBREADprintf("Error: unable to open file '%s'\n", file_names[file_no]); badfiles ++; continue; } else SUBREADprintf("Process file '%s' ...\n", file_names[file_no]); int lines = 0; while(1) { char * tmp_pnt; char * ret = fgets(file_line, 3000, ifp); if(!ret)break; if(ret[0]=='#') continue; char * chro = strtok_r(ret,"\t",&tmp_pnt); if(chro == NULL|| tmp_pnt == NULL) { SUBREADprintf("Unknown format in the file! Only the VCF format is allowed for input files.\n"); break; } char * pos_str = strtok_r(NULL,"\t",&tmp_pnt); if(pos_str == NULL|| strlen(pos_str)>10|| tmp_pnt == NULL) { SUBREADprintf("Unknown format in the file! Only the VCF format is allowed for input files.\n"); break; } char * id_str = strtok_r(NULL,"\t",&tmp_pnt); // snp_id char * ref = strtok_r(NULL,"\t",&tmp_pnt); char * alt_str = strtok_r(NULL,"\t",&tmp_pnt); // alt char * qual_str = strtok_r(NULL,"\t",&tmp_pnt); //qual char * filter_str = strtok_r(NULL,"\t",&tmp_pnt); //filter char * info = strtok_r(NULL,"\t",&tmp_pnt); //addition columns are discarded if(info == NULL) { SUBREADprintf("Unknown format in the file! Only the VCF format is allowed for input files.\n"); break; } int is_indel = 0; if(strstr(info, "INDEL;")) is_indel = 1; if(endswith(info, "INDEL")) is_indel = 1; char *tmp_pnt_alt = NULL; //SUBREADprintf("GO_INTO_STR: %s\n", alt_str); while(1) { char * alt_one = strtok_r(tmp_pnt_alt?NULL:alt_str,",", &tmp_pnt_alt); if(!alt_one) break; lines ++; char key_type = is_indel?'I':'S'; char * ky = malloc(strlen(chro)+strlen(alt_one)+strlen(ref)+40); sprintf(ky, "%c\t%s\t%s\t.\t%s\t%s\t.", key_type, chro, pos_str, ref, alt_one); int qual = atoi(qual_str); if(file_no - badfiles == 0) { char * IQF_buf = malloc(strlen(qual_str)+strlen(filter_str)+strlen(id_str)+4); char * info_buf = malloc(strlen(info)+1); assert(info_buf); strcpy(info_buf, info); sprintf(IQF_buf,"%s\t%s\t%s", id_str,qual_str,filter_str); int repeat_times = HashTableGet(extra_count_table, ky) - NULL; if(0 == repeat_times) { HashTablePut(extra_count_table, ky, NULL+1); HashTablePut(extra_id_qual_filter_table, ky, IQF_buf); HashTablePut(extra_info_table, ky, info_buf); HashTablePut(extra_qual_table, ky, NULL + qual + 1); } else { if(!warning_reported_repeated)SUBREADprintf("Warning: repeated rows are found in the first input file.\n"); warning_reported_repeated=1; free(ky); } } else{ int repeat_times = HashTableGet(extra_count_table, ky) - NULL; if(repeat_times >0) { int old_qual = HashTableGet(extra_qual_table, ky) - NULL - 1; repeat_times ++; HashTablePutReplace(extra_count_table, ky, NULL + repeat_times, 0); if(old_qual > qual) { char * info_buf = malloc(strlen(info)+1); char * IQF_buf = malloc(strlen(qual_str)+strlen(filter_str)+strlen(id_str)+4); assert(info_buf); strcpy(info_buf, info); sprintf(IQF_buf,"%s\t%s\t%s", id_str,qual_str,filter_str); HashTablePutReplace(extra_info_table, ky, info_buf, 0); HashTablePutReplace(extra_id_qual_filter_table, ky, IQF_buf, 0); HashTablePutReplace(extra_qual_table, ky, NULL + 1 + qual, 0); } } free(ky); } if(NULL == tmp_pnt_alt) break; } } SUBREADprintf("There are %d variants found in this file.\n\n", lines); fclose(ifp); } KeyValuePair *cursor; int bucket, x1; char ** output_list; int out_list_size = 300, out_list_items = 0; output_list = malloc(out_list_size * sizeof(char *)); for(bucket=0; bucket < extra_count_table -> numOfBuckets; bucket++) { cursor = extra_count_table -> bucketArray[bucket]; while (1) { if (!cursor) break; char * ky = (char *)cursor -> key; int counts = cursor -> value - NULL; assert(counts <= files); if(counts == files - badfiles) { if(out_list_items >= out_list_size) { out_list_size *= 1.5; output_list = realloc(output_list, out_list_size * sizeof(char *)); } output_list[out_list_items] = ky; out_list_items ++; } cursor = cursor->next; } } VCF_sort_context vcf_context; vcf_context.keys = output_list; merge_sort(&vcf_context, out_list_items,compare_VCF_rows,exchange_VCF_rows,merge_VCF_rows); fprintf(output_file_pointer, "#CHROM POS ID REF ALT QUAL FILTER INFO"); for(x1 =0 ; x1< out_list_items; x1++) { char * ky = output_list[x1]; char * info = HashTableGet(extra_info_table, ky); char * IQF_buffer = HashTableGet(extra_id_qual_filter_table, ky); char * tok_buf_tab = NULL; char * id_str = strtok_r(IQF_buffer,"\t", &tok_buf_tab); char * qual_str = strtok_r(NULL,"\t", &tok_buf_tab); char * filter_str = strtok_r(NULL,"\t", &tok_buf_tab); char * chro_str = strtok_r(ky+2,"\t",&tok_buf_tab); char * pos_str = strtok_r(NULL,"\t",&tok_buf_tab); strtok_r(NULL,"\t",&tok_buf_tab);// fake_id char * ref_str = strtok_r(NULL,"\t",&tok_buf_tab); char * alt_str = strtok_r(NULL,"\t",&tok_buf_tab); char *brk = "\n"; if(info[strlen(info)-1]=='\n') brk=""; fprintf(output_file_pointer, "%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s%s", chro_str, pos_str, id_str, ref_str , alt_str, qual_str , filter_str , info, brk); } SUBREADprintf("Finished! There are %d common variants from the %d input file%s.\n\n", out_list_items, files - badfiles, (files - badfiles>1)?"s":""); free(output_list); HashTableDestroy(extra_qual_table); HashTableDestroy(extra_info_table); HashTableDestroy(extra_id_qual_filter_table); HashTableDestroy(extra_count_table); free(file_line); } void common_var_usage() { SUBREADprintf("\nmergeVCF v%s\n", SUBREAD_VERSION); SUBREADputs(""); SUBREADputs(" Calculating the intersection between multiple VCF files containing indels"); SUBREADputs(" and/or SNPs."); SUBREADputs(""); SUBREADputs(" The supported VCF files include VCF files generated by subread, subjunc,"); SUBREADputs(" ExactSNP, subindel and other tools."); SUBREADputs(""); SUBREADputs("Usage:"); SUBREADputs(""); SUBREADputs(" ./mergeVCF -o input_vcf_1 {input_vcf_2} {input_vcf_3} ..."); SUBREADputs(""); SUBREADputs("Required arguments:"); SUBREADputs(""); SUBREADputs(" -o : Name of the file containing the variants in all input VCF files."); SUBREADputs(""); } #ifdef MAKE_STANDALONE int main(int argc, char ** argv) #else int findCommonVariants(int argc, char ** argv) #endif { int c; int option_index = 0; optind = 0; opterr = 1; optopt = 63; output_file_name[0]=0; warning_reported_repeated=0; while((c = getopt_long (argc, argv, "o:h", propm_long_options, &option_index)) != -1) { switch(c){ case 'o': strcpy(output_file_name, optarg); break; default: common_var_usage(); return -1; } } if(!output_file_name[0]) { common_var_usage(); return -1; } else{ output_file_pointer = f_subr_open(output_file_name,"w"); if(output_file_pointer) { if( argc-optind ) do_find_common(argv + optind, argc-optind); else SUBREADprintf("At least one input file is needed.\n\n"); fclose(output_file_pointer); } else { SUBREADprintf("Unable to open the output file: '%s'\n", output_file_name); return -1; } } return 0; } Rsubread/src/processExons.c0000644000175200017520000001602014136047615016777 0ustar00biocbuildbiocbuild/*************************************************************** The Subread and Rsubread software packages are free software packages: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #define _GNU_SOURCE #include #include #include #include #include #define MAX_GENE 1000000 #define MAX_CHR 200 #define STR 100 //typedef int int; typedef struct an_exon{ int start, end; char orientation; struct an_exon *next; }exon; typedef struct a_chr{ char *id; exon *exon_list; }chr; typedef struct a_gene{ int id; int chr_index; int chr_num; char *current_chr; chr chr_array[MAX_CHR]; }gene; // global variables int exon_num; int gene_index; int gene_num; //gene gene_array[MAX_GENE]; gene * gene_array; int current_gene_id; char *PEE_exon_file; char *gene_file; void initialise_process_exons(){ gene_array = (gene *) calloc(MAX_GENE,sizeof(gene)); gene_num = 0; gene_index = 0; current_gene_id = 0; exon_num = 0; PEE_exon_file = (char *)malloc(STR); gene_file = (char *)malloc(STR); } // returns a pointer to a dummy head node to start off the linked list // usage: exon *dh = (exon *)make_empty(); void * make_empty(void) { exon *head; head = (exon *) malloc(sizeof(*head)); head->next = NULL; // next = NULL implies end of list return head; } void print_list(FILE *fout, FILE *fgene, int x, int y){ char *chromosome = gene_array[x].chr_array[y].id; int gene = gene_array[x].id; exon *list = gene_array[x].chr_array[y].exon_list;; list = list->next; int gene_s, gene_e; gene_s = list->start; while (list !=NULL){ fprintf(fout, "%d\t%s\t%d\t%d\n",gene, chromosome, list->start, list->end); exon_num++; gene_e = list->end; list = list->next; } fprintf(fgene, "%d\t%s\t%d\t%d\n", gene, chromosome, gene_s, gene_e); } void insert_new_exon(void *pt, int start, int end){ exon *node = (exon *)pt; exon *new_node; new_node = (exon *)(malloc(sizeof(exon))); new_node->start = start; new_node->end = end; new_node->next = node->next; node->next = new_node; } void insert_exon(void *pt, int start, int end){ exon *node = (exon *)pt; while (node ->next != NULL){ //node = node->next; if ((start >= node-> next->start) && (start <= node->next->end)){ if (end > node->next->end){ node->next->end = end; return; } else { return; } } if ((end >= node->next->start) && (end <= node->next->end)){ if (start < node->next->start){ node->next->start = start; return; } else { return; } } if (start == ((node->next->end)+1)){ node->next->end = end; return; } if (end == ((node->next->start)-1)){ node->next->start = start; return; } if((node->end < start) && (node->next->start > end)){ insert_new_exon(node, start, end); return; } node = node->next; } if (start == ((node->end)+1)){ node->end = end; return; } if (end == ((node->start)-1)){ node->start = start; return; } insert_new_exon(node,start,end); } void output_exons_to_file(){ FILE *fexon; FILE *fgene; fexon = fopen(PEE_exon_file,"w"); fgene = fopen(gene_file,"w"); //fprintf(fexon, "entrezid\tchromosome\tchr_start\tchr_stop\n"); //fprintf(fgene, "entrezid\tchromosome\tchr_start\tchr_stop\n"); int j,i; int total_chr_num; for(j=0; j1){ Rprintf("GeneID: %d appears on chromosomes: ", gene_array[j].id); for(i=0; i=0) && (gene_array[gene_index].id != target_gene_id)){ gene_index--; } // new gene_id, build a new gene record if ((gene_index < 0) && (gene_num < MAX_GENE)) { gene_num++; gene_index = gene_num - 1; gene_array[gene_index].id = target_gene_id; gene_array[gene_index].chr_index = 0; gene_array[gene_index].chr_num = 0; gene_array[gene_index].current_chr = ""; } current_gene_id = target_gene_id; return gene_index; } int find_chr(int gene_pos, char *chr_id){ int current_chr_index; if (strcmp(chr_id, gene_array[gene_pos].current_chr) != 0){ // current chromosome id doesn't match what we are processing. current_chr_index = gene_array[gene_pos].chr_num - 1; while ((current_chr_index >=0) && (strcmp(chr_id, gene_array[gene_pos].chr_array[current_chr_index].id) != 0)){ current_chr_index--; } if ((current_chr_index < 0) && (gene_array[gene_pos].chr_num < MAX_CHR)){ //build a new chromosome gene_array[gene_pos].chr_num++; gene_array[gene_pos].chr_index = gene_array[gene_pos].chr_num - 1; current_chr_index = gene_array[gene_pos].chr_index; gene_array[gene_pos].chr_array[current_chr_index].id = (char *)malloc(STR); // char *pt = strcpy(gene_array[gene_pos].chr_array[current_chr_index].id,chr_id); strcpy(gene_array[gene_pos].chr_array[current_chr_index].id,chr_id); gene_array[gene_pos].current_chr = gene_array[gene_pos].chr_array[current_chr_index].id; gene_array[gene_pos].chr_array[current_chr_index].exon_list =(exon *)make_empty(); } } else { current_chr_index = gene_array[gene_pos].chr_index; } return(current_chr_index); } void * find_list(int gene_id, char *chr_id){ int p_gene=0; int p_chr=0; // locate gene array index if (current_gene_id == gene_id){ p_gene = gene_index; } else { p_gene = find_gene(gene_id); } if (p_gene < 0){ // printf("exceeding max number of genes that can be processed.\n"); return(NULL); } // locate chromosome array index p_chr = find_chr(p_gene, chr_id); if (p_chr < 0) { // printf("exceed the maximum number of chromosomes for parallel gene with id:%d\n",gene_id); return(NULL); } // now, we locate the linked list head. exon *dh = gene_array[p_gene].chr_array[p_chr].exon_list; return dh; } void processExons(char **utr_cds, char **fexon, char** fgene){ FILE *fin; int start, end, gene_id; char chr_id[20]; char ori; exon *dh; initialise_process_exons(); strcpy(PEE_exon_file, *fexon); strcpy(gene_file, *fgene); fin = fopen(*utr_cds,"r"); while (fscanf(fin, "%s %d %d %c %d ",chr_id, &start, &end, &ori, &gene_id) != -1){ dh = (exon *)find_list(gene_id, chr_id); if (dh != NULL){ insert_exon(dh, start, end); } } fclose(fin); output_exons_to_file(); if(gene_array) free(gene_array); } Rsubread/src/propmapped.c0000644000175200017520000003342214136047615016460 0ustar00biocbuildbiocbuild/*************************************************************** The Subread and Rsubread software packages are free software packages: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #define _GNU_SOURCE #include #include #include #include #include #include #include #include #ifndef __MINGW32__ #include #endif #include #include #include "subread.h" #include "core.h" #include "hashtable.h" #include "sambam-file.h" #include "input-files.h" #include "gene-algorithms.h" #include "HelperFunctions.h" typedef struct { char input_file_name [MAX_FILE_NAME_LENGTH]; char output_file_name [MAX_FILE_NAME_LENGTH]; char temp_file_prefix [MAX_FILE_NAME_LENGTH]; int is_BAM_input; unsigned long long all_reads; unsigned long long all_records; unsigned long long mapped_reads; int is_fragments_counted; int is_proppair_needed; int sort_buckets; int verbose; HashTable * split_fp_table; } propMapped_context; char * _PROPMAPPED_delete_tmp_prefix = NULL; void PROPMAPPED_SIGINT_hook(int param) { #ifdef MAKE_STANDALONE int xk1, last_slash = -1; if(_PROPMAPPED_delete_tmp_prefix != NULL) { char del2[MAX_FILE_NAME_LENGTH], del_suffix[MAX_FILE_NAME_LENGTH], del_name[MAX_FILE_NAME_LENGTH]; SUBREADprintf("\n\nReceived a terminal signal. The temporary files were removed.\n"); for(xk1=0; _PROPMAPPED_delete_tmp_prefix[xk1]; xk1++) { if(_PROPMAPPED_delete_tmp_prefix[xk1]=='/') last_slash = xk1; else if(_PROPMAPPED_delete_tmp_prefix[xk1]=='\\') { SUBREADprintf("The file name is unknown.\n"); return; } } if(last_slash>=0) { memcpy(del2, _PROPMAPPED_delete_tmp_prefix, last_slash); del2[last_slash] = 0; strcpy(del_suffix , _PROPMAPPED_delete_tmp_prefix + last_slash + 1); } else { strcpy(del2,"."); strcpy(del_suffix , _PROPMAPPED_delete_tmp_prefix); } if(strlen(del_suffix)>8) { DIR *d; struct dirent *dir; d = opendir(del2); if (d) { while ((dir = readdir(d)) != NULL) { if(strstr(dir->d_name, del_suffix)) { //printf("%s\n", dir->d_name); strcpy(del_name, del2); strcat(del_name, "/"); strcat(del_name, dir->d_name); unlink(del_name); } } closedir(d); } } } exit(param); #endif } int propMapped(propMapped_context * context) { SamBam_FILE * in_fp = SamBam_fopen(context -> input_file_name ,context -> is_BAM_input?SAMBAM_FILE_BAM:SAMBAM_FILE_SAM); if(!in_fp) { SUBREADprintf("Unable to open file '%s'.\n", context -> input_file_name); return -1; } char * line_buffer = malloc(3000); while(1) { char * is_ret = SamBam_fgets(in_fp, line_buffer, 2999, 1); if(!is_ret) break; if(line_buffer[0]=='@') continue; char * tok_tmp; strtok_r (line_buffer,"\t", &tok_tmp); // read name char * flags_str = strtok_r (NULL ,"\t", &tok_tmp); int flags = atoi(flags_str); context -> all_reads++; if( (flags & 4 ) == 0) context -> mapped_reads ++; } SamBam_fclose(in_fp); return 0; } int write_result(propMapped_context * context) { if(context -> output_file_name[0]) { FILE * outfp = f_subr_open(context -> output_file_name, "a"); #ifdef __MINGW32__ fprintf(outfp, "%s,%I64u,%I64u,%f\n", context -> input_file_name, context -> all_reads, context -> mapped_reads, context -> mapped_reads*1./context -> all_reads); #else fprintf(outfp, "%s,%llu,%llu,%f\n", context -> input_file_name, context -> all_reads, context -> mapped_reads, context -> mapped_reads*1./context -> all_reads); #endif fclose(outfp); } char * objname = context -> is_fragments_counted? "fragment":"read"; #ifdef __MINGW32__ if(context -> verbose) SUBREADprintf("Finished. All records: %I64u; all %ss: %I64u; mapped %ss: %I64u; the mappability is %.2f%%\n", context->all_records, objname, context -> all_reads, objname, context -> mapped_reads, context -> mapped_reads*100./context -> all_reads); #else if(context -> verbose) SUBREADprintf("Finished. All records: %llu; all %ss: %llu; mapped %ss: %llu; the mappability is %.2f%%\n", context->all_records, objname, context -> all_reads, objname, context -> mapped_reads, context -> mapped_reads*100./context -> all_reads); #endif return 0; } static struct option propm_long_options[] = { {"in", required_argument, 0, 'i'}, {"out", required_argument, 0, 'o'}, {0, 0, 0, 0} }; void propMapped_usage() { SUBREADprintf("\npropMapped v%s\n", SUBREAD_VERSION); SUBREADputs(""); SUBREADputs(" Calculate the proportion of mapped reads/fragments."); SUBREADputs(""); SUBREADputs("Usage:"); SUBREADputs(""); SUBREADputs(" ./prommapped [options] -i "); SUBREADputs(""); SUBREADputs("Required arguments:"); SUBREADputs(""); SUBREADputs(" -i An input file containing read mapping results. Both SAM or BAM"); SUBREADputs(" formats are supported."); SUBREADputs(""); SUBREADputs("Optional arguments:"); SUBREADputs(""); SUBREADputs(" -o Name of output file including mapping statistics."); SUBREADputs(""); SUBREADputs(" -f If specified, fragments (read pairs) will be counted instead of"); SUBREADputs(" individual reads. This option is only applicable for paired-end"); SUBREADputs(" reads."); SUBREADputs(""); SUBREADputs(" -p If specified, only properly paired reads will be counted. This"); SUBREADputs(" option is only applicable for paired-end reads."); SUBREADputs(""); } int finalise_PE_split(propMapped_context * context) { int bucket; KeyValuePair *cursor; for(bucket=0; bucket < context -> split_fp_table -> numOfBuckets; bucket++) { cursor = context -> split_fp_table -> bucketArray[bucket]; while (1) { if (!cursor) break; FILE * fp = (FILE *) cursor -> value; fclose(fp); cursor = cursor->next; } } HashTableDestroy(context -> split_fp_table); return 0; } FILE * get_FP_by_read_name(propMapped_context * context, char * read_name) { unsigned long long int hash_key = sort_SAM_hash(read_name); hash_key = hash_key % context -> sort_buckets; FILE * ret = HashTableGet(context -> split_fp_table, NULL+1+(int)(hash_key)); if(!ret) { char fname [MAX_FILE_NAME_LENGTH+40]; #ifdef __MINGW32__ sprintf(fname, "%s-%I64u.bin", context->temp_file_prefix, hash_key); #else sprintf(fname, "%s-%llu.bin", context->temp_file_prefix, hash_key); #endif ret = f_subr_open(fname, "wb"); HashTablePut(context -> split_fp_table, NULL+1+(int)(hash_key), ret); } return ret; } int add_read_flags(propMapped_context * context, FILE * fp, char * read_name, unsigned short flags) { int x1; int rname_len = strlen(read_name); for(x1=rname_len-1; x1>0;x1--) { if(read_name[x1]=='/'){ read_name[x1]=0; break; } } if(1&flags) if(!context->is_fragments_counted) { strcat(read_name, (flags&0x40)?"!!_1":"!!_2"); } rname_len = strlen(read_name); if(rname_len>250) return -1; unsigned char rname_len_char = (unsigned char)rname_len; int wlen = fwrite(&rname_len_char,1,1,fp); if(wlen < 1) return -1; wlen = fwrite(read_name,rname_len,1,fp); if(wlen < 1) return -1; wlen = fwrite(&flags, sizeof(short), 1, fp); if(wlen < 1) return -1; return 0; } int init_PE_sambam(propMapped_context * context) { char mac_rand[13]; mac_or_rand_str(mac_rand); #ifdef MAKE_STANDALONE myrand_srand(time(NULL)); #endif int x1; context->temp_file_prefix[0]=0; for(x1 = strlen(context->output_file_name) ; x1 >=0; x1--){ if(context->output_file_name[x1]=='/'){ memcpy(context->temp_file_prefix, context->output_file_name, x1); context->temp_file_prefix[x1] = 0; } } if(context->temp_file_prefix[0]==0) strcpy(context->temp_file_prefix, "./"); sprintf(context->temp_file_prefix+strlen(context->temp_file_prefix), "/prpm-temp-sum-%06u-%s", getpid(), mac_rand); _PROPMAPPED_delete_tmp_prefix = context -> temp_file_prefix; signal (SIGTERM, PROPMAPPED_SIGINT_hook); signal (SIGINT, PROPMAPPED_SIGINT_hook); context -> split_fp_table = HashTableCreate(500); return 0; } int split_PE_sambam(propMapped_context * context) { SamBam_FILE * in_fp = SamBam_fopen(context -> input_file_name ,context -> is_BAM_input?SAMBAM_FILE_BAM:SAMBAM_FILE_SAM); if(!in_fp) { SUBREADprintf("Unable to open file '%s'.\n", context -> input_file_name); return -1; } char * line_buffer = malloc(3000); int is_error = 0; while(1) { char * is_ret = SamBam_fgets(in_fp, line_buffer, 2999, 1); if(!is_ret) break; if(line_buffer[0]=='@') continue; //SUBREADprintf("TELLO: %ld\n", ftello(in_fp -> os_file)); char * tok_tmp; char * read_name = strtok_r (line_buffer,"\t", &tok_tmp); // read name char * flags_str = strtok_r (NULL ,"\t", &tok_tmp); unsigned flags = atoi(flags_str); FILE * fp = get_FP_by_read_name(context , read_name); is_error = add_read_flags(context, fp, read_name, flags); if(is_error) break; context -> all_records++; } free(line_buffer); SamBam_fclose(in_fp); if(is_error) SUBREADprintf("ERROR: Unable to write into the temporary file. Please check the disk space in the output directory."); return is_error; } int prop_PE(propMapped_context * context) { int bini; for(bini = 0; bini < context -> sort_buckets; bini++) { char fname[MAX_FILE_NAME_LENGTH+25]; HashTable * rname_table = HashTableCreate(100000); HashTableSetKeyComparisonFunction(rname_table , fc_strcmp_chro); HashTableSetHashFunction(rname_table, fc_chro_hash); HashTableSetDeallocationFunctions(rname_table , free, NULL); sprintf(fname, "%s-%d.bin", context->temp_file_prefix, bini); FILE * fp = f_subr_open(fname, "rb"); if(fp) { while(!feof(fp)) { unsigned char read_len; unsigned short flags; char * read_name; int retvv = fread(&read_len,1,1,fp); if(retvv<1) break; if(feof(fp))break; read_name = malloc((int)read_len+1); retvv = fread(read_name,read_len,1,fp); if(retvv<1) break; read_name[read_len]=0; retvv = fread(&flags,1, sizeof(short), fp); if(retvv<1) break; int new_OK; if(context -> is_fragments_counted){ if(context -> is_proppair_needed ) new_OK = (flags & 2)>0; else new_OK=1; if(new_OK) new_OK = (!(flags & 4)) || ( (flags & 1) && !(flags&8) ); } else { new_OK = ((flags & 4) == 0); } int old_OK = HashTableGet(rname_table, read_name) - NULL; if(old_OK) { old_OK--; // if the old read is unmapped or the old read is not properly mapped pair. if(new_OK && !old_OK ) HashTablePut(rname_table, read_name, NULL+2); } else HashTablePut(rname_table, read_name, NULL + new_OK + 1); } fclose(fp); unlink(fname); int bucket; KeyValuePair *cursor; for(bucket=0; bucket < rname_table -> numOfBuckets; bucket++) { cursor = rname_table -> bucketArray[bucket]; while (1) { if (!cursor) break; if(cursor -> value == NULL+2) context -> mapped_reads++; context -> all_reads++; cursor = cursor->next; } } } //SUBREADprintf("BIN %d ALLR %llu MAPPED %llu ELES=%ld\n", bini, context -> all_reads, context -> mapped_reads, rname_table -> numOfElements); HashTableDestroy(rname_table); } return 0; } void ppm_warning_file_limit() { #ifndef __MINGW32__ struct rlimit limit_st; getrlimit(RLIMIT_NOFILE, & limit_st); { if(min(limit_st.rlim_cur , limit_st.rlim_max) < 400) { SUBREADprintf("Your operation system does not allow a single process to open more then 400 files. You may need to change this setting by using a 'ulimit -n 500' command, or the program may crash.\n"); } } #endif } #ifdef MAKE_STANDALONE int main(int argc, char ** argv) #else int propmapped(int argc, char ** argv) #endif { int ret = 0; int c; int option_index = 0; _PROPMAPPED_delete_tmp_prefix = NULL; propMapped_context * context = malloc(sizeof(propMapped_context)); memset(context, 0 , sizeof(propMapped_context)); optind = 0; opterr = 1; optopt = 63; context -> sort_buckets = 253; while((c = getopt_long (argc, argv, "Vi:o:bfph", propm_long_options, &option_index)) != -1) { switch(c){ case 'i': strcpy(context -> input_file_name, optarg); break; case 'o': strcpy(context -> output_file_name, optarg); break; case 'f': context -> is_fragments_counted = 1; break; case 'V': context -> verbose = 1; case 'p': context -> is_proppair_needed = 1; break; case 0: // long names break; default: propMapped_usage(); return 0; } } if(!context -> input_file_name[0]) { propMapped_usage(); return 0; } int is_bam = is_certainly_bam_file(context -> input_file_name, NULL, NULL); if(1==is_bam){ context -> is_BAM_input = 1; } else if(is_bam < 0) { ret = -1; SUBREADprintf("Unable to open input file '%s' or the input file is empty.\n", context -> input_file_name); } if(context -> verbose) SUBREADprintf("The input file is opened as a %cAM file.\nThe %ss in the input file are being counted.\n", context -> is_BAM_input?'B':'S', context -> is_fragments_counted?"fragment":"read"); ppm_warning_file_limit (); ret = ret || init_PE_sambam(context); ret = ret || split_PE_sambam(context); ret = ret || finalise_PE_split(context); ret = ret || prop_PE(context); ret = ret || write_result(context); free(context); return ret; } Rsubread/src/qualityScores.c0000644000175200017520000004167514136047615017171 0ustar00biocbuildbiocbuild/*************************************************************** The Subread and Rsubread software packages are free software packages: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #define _GNU_SOURCE #include #include #include #include #include #include #include "subread.h" #include "core.h" #include "sambam-file.h" #include "input-files.h" //static int ERROR_PROB_INT_TABLE[] = { 1000000 , 794328 , 630957 , 501187 , 398107 , 316227 , 251188 , 199526 , 158489 , 125892 , 100000 , 79432 , 63095 , 50118 , 39810 , 31622 , 25118 , 19952 , 15848 , 12589 , 10000 , 7943 , 6309 , 5011 , 3981 , 3162 , 2511 , 1995 , 1584 , 1258 , 1000 , 794 , 630 , 501 , 398 , 316 , 251 , 199 , 158 , 125 , 100 , 79 , 63 , 50 , 39 , 31 , 25 , 19 , 15 , 12 , 10 , 7 , 6 , 5 , 3 , 3 , 2 , 1 , 1 , 1 , 1 , 0 , 0 , 0 ,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0}; typedef struct { char * input_name; char * output_name; int input_file_type; int phred_offset; long long needed_reads; int sam_end; int max_read_length; char * IO_line_buff; FILE * result_fp; union{ SamBam_FILE * sambam_reader; gzFile gzfq_reader; FILE * fq_reader; }; unsigned long long scored_reads; unsigned long long line_counter; unsigned long long scored_bases; unsigned long long total_phred_sum; unsigned long long total_errorprob_sum; unsigned long long * base_phred_sum; unsigned long long * base_errorprob_sum; int quality_offset_warning_shown; } qualscore_context; int finalise_qs_context(qualscore_context * qs_context, int prev_ret) { fclose(qs_context -> result_fp); free(qs_context-> IO_line_buff); SUBREADprintf("\n"); if(prev_ret) { SUBREADprintf("No results were generated.\n"); } else { if(qs_context -> input_file_type == FILE_TYPE_FASTQ) fclose(qs_context -> fq_reader); else if(qs_context -> input_file_type == FILE_TYPE_SAM || qs_context -> input_file_type == FILE_TYPE_BAM) SamBam_fclose(qs_context -> sambam_reader); else if(qs_context -> input_file_type == FILE_TYPE_GZIP_FASTQ) gzclose(qs_context -> gzfq_reader); else return 1; //double avg_phred = qs_context -> total_phred_sum*1./ qs_context->scored_bases; //double avg_prob = qs_context -> total_errorprob_sum*1./1000000/qs_context->scored_bases; SUBREADprintf("Completed successfully. Quality scores for %llu reads (equally spaced in the file) are returned.\n", qs_context -> scored_reads); if(qs_context-> quality_offset_warning_shown) SUBREADprintf("However, the Phred score offset (%d) seemed to be wrong. The quality scores can be meaningless.\n", qs_context -> phred_offset); // else // SUBREADprintf("Average Phred score is %.1f. Average wrong sequence probability is %.2f%%.\n", avg_phred, avg_prob*100); } SUBREADprintf("\n"); return 0; } int add_read_scores(qualscore_context * qs_context, char * qual_text, unsigned int total_cols) { int xk1, nch, out_cursor = 0, aval = 1; qs_context -> IO_line_buff[0]=0; for(xk1=0; xk1 < total_cols; xk1++){ nch = -1; if(aval) nch = qual_text[xk1]; if(nch == 0){ aval = 0; nch=-1; } if(nch>0){ nch -= qs_context -> phred_offset; if(nch <=0 || nch >= 65) { if(!qs_context-> quality_offset_warning_shown) { SUBREADprintf("Warning: the Phred score offset (%d) seems wrong : it ended up with a phred score of %d.\n", qs_context -> phred_offset, nch); qs_context-> quality_offset_warning_shown=1; } } out_cursor+=sprintf(qs_context->IO_line_buff+out_cursor,"%d,", nch); } else { out_cursor+=sprintf(qs_context->IO_line_buff+out_cursor,"NA,"); } } if(out_cursor>0) qs_context->IO_line_buff[out_cursor-1]='\n';//remove the last comma and change it to a linebreak. fwrite(qs_context->IO_line_buff, 1, out_cursor, qs_context -> result_fp); qs_context -> scored_reads ++; return 0; } int qs_next_qual(qualscore_context * qs_context, char * qual_buff) { int ret = 0, need_reverse=0; if(qs_context -> input_file_type == FILE_TYPE_FASTQ || qs_context -> input_file_type == FILE_TYPE_GZIP_FASTQ) { char * retstr; if(qs_context -> input_file_type == FILE_TYPE_FASTQ) retstr = fgets_noempty(qual_buff, 2999, qs_context -> fq_reader); else retstr = gzgets_noempty(qs_context -> fq_reader, qual_buff, 2999); if(!retstr) ret = 1; else { if(retstr[0]!='@') { SUBREADprintf("ERROR: The input fastq file has a wrong format.\n"); return -1; } qs_context -> line_counter ++; if(qs_context -> input_file_type == FILE_TYPE_FASTQ) retstr = fgets_noempty(qual_buff, 2999, qs_context -> fq_reader); else retstr = gzgets_noempty(qs_context -> fq_reader, qual_buff, 2999); if(qs_context -> input_file_type == FILE_TYPE_FASTQ) retstr = fgets_noempty(qual_buff, 2999, qs_context -> fq_reader); else retstr = gzgets_noempty(qs_context -> fq_reader, qual_buff, 2999); if(!retstr) ret = 1; else { if(retstr[0]!='+') { SUBREADprintf("ERROR: The input fastq file has a wrong format.\n"); return -1; } qs_context -> line_counter += 2; } if(qs_context -> input_file_type == FILE_TYPE_FASTQ) retstr = fgets_noempty(qual_buff, 2999, qs_context -> fq_reader); else retstr = gzgets_noempty(qs_context -> fq_reader, qual_buff, 2999); if(!retstr) ret = 1; } } else// sam or bam { while(1) { char * retstr = SamBam_fgets(qs_context -> sambam_reader , qs_context -> IO_line_buff, 5999, qual_buff?1:0); if(!retstr) ret = 1; else { if(qs_context -> IO_line_buff[0]!='@') { char * tmpptr; int flags = 0; char * nstr = strtok_r(qs_context -> IO_line_buff, "\t", &tmpptr); // name if(!nstr) continue; nstr = strtok_r(NULL, "\t", &tmpptr); // name if(nstr) flags = atoi(nstr); else continue; if(0&& qs_context -> sam_end>0 && !(flags &SAM_FLAG_PAIRED_TASK )) { SUBREADprintf("ERROR: The SAM/BAM input file contains single-end reads, please do not specify paired-end specific arguments (e.g., first-end or second-end).\n"); ret = 1; break; } nstr = strtok_r(NULL, "\t", &tmpptr); // chro if(!nstr) continue; nstr = strtok_r(NULL, "\t", &tmpptr); // pos if(!nstr) continue; nstr = strtok_r(NULL, "\t", &tmpptr); // mapq if(!nstr) continue; nstr = strtok_r(NULL, "\t", &tmpptr); // cigar if(!nstr) continue; nstr = strtok_r(NULL, "\t", &tmpptr); // matechr if(!nstr) continue; nstr = strtok_r(NULL, "\t", &tmpptr); // matepos if(!nstr) continue; nstr = strtok_r(NULL, "\t", &tmpptr); // tlen if(!nstr) continue; nstr = strtok_r(NULL, "\t", &tmpptr); // read_text if(!nstr) continue; nstr = strtok_r(NULL, "\t", &tmpptr); // qual_text if(!nstr) continue; if((qs_context -> sam_end == 1 && (!(flags & SAM_FLAG_SECOND_READ_IN_PAIR))) || (qs_context -> sam_end == 2 && (flags & SAM_FLAG_SECOND_READ_IN_PAIR)) || (qs_context -> sam_end == 0)) { if((flags & 256)==0 ) { if(flags & SAM_FLAG_REVERSE_STRAND_MATCHED) need_reverse=1; strcpy(qual_buff, nstr); break; } } } } if(ret) break; } } int qblen = strlen(qual_buff); if(qual_buff[qblen-1]=='\n'){ qual_buff[qblen-1]=0; qblen--; } if(need_reverse) reverse_quality(qual_buff, qblen); return ret; } int rewind_qs_file(qualscore_context * qs_context) { if(qs_context -> input_file_type == FILE_TYPE_FASTQ) fclose(qs_context -> fq_reader); else if(qs_context -> input_file_type == FILE_TYPE_SAM || qs_context -> input_file_type == FILE_TYPE_BAM) SamBam_fclose(qs_context -> sambam_reader); else if(qs_context -> input_file_type == FILE_TYPE_GZIP_FASTQ) gzclose(qs_context -> gzfq_reader); if(qs_context -> input_file_type == FILE_TYPE_FASTQ) qs_context -> fq_reader = f_subr_open(qs_context->input_name, "r"); else if(qs_context -> input_file_type == FILE_TYPE_SAM || qs_context -> input_file_type == FILE_TYPE_BAM) qs_context -> sambam_reader = SamBam_fopen(qs_context->input_name, (qs_context -> input_file_type == FILE_TYPE_BAM)?SAMBAM_FILE_BAM:SAMBAM_FILE_SAM); else if(qs_context -> input_file_type == FILE_TYPE_GZIP_FASTQ) qs_context -> gzfq_reader = gzopen(qs_context->input_name, "rb"); return 0; } int qs_str_input_type(char * strtype) { if(strcmp(strtype,"SAM")==0) return FILE_TYPE_SAM; else if(strcmp(strtype, "BAM")==0) return FILE_TYPE_BAM; else if(strcmp(strtype, "FASTQ")==0) return FILE_TYPE_FASTQ; else if(strcmp(strtype, "GZFASTQ")==0) return FILE_TYPE_GZIP_FASTQ; return -1; } int start_qs_context(qualscore_context * qs_context) { int ret =0; SUBREADprintf("\nqualityScores %s\n\n", SUBREAD_VERSION); if(qs_context -> input_file_type == FILE_TYPE_FASTQ) qs_context -> fq_reader = f_subr_open(qs_context->input_name, "r"); else if(qs_context -> input_file_type == FILE_TYPE_SAM || qs_context -> input_file_type == FILE_TYPE_BAM) qs_context -> sambam_reader = SamBam_fopen(qs_context->input_name, (qs_context -> input_file_type == FILE_TYPE_BAM)?SAMBAM_FILE_BAM:SAMBAM_FILE_SAM); else if(qs_context -> input_file_type == FILE_TYPE_GZIP_FASTQ) qs_context -> gzfq_reader = gzopen(qs_context->input_name, "rb"); else ret = 1; qs_context -> result_fp = f_subr_open(qs_context->output_name, "w"); if(!qs_context -> result_fp) { ret = 1; SUBREADprintf("ERROR: cannot open output file '%s'.\n", qs_context->output_name); } else { if(ret || !qs_context -> fq_reader) { SUBREADprintf("ERROR: cannot open input file '%s'.\n", qs_context->input_name); SUBREADprintf(" The file does not exist, or it is not in the %s format.\n", qs_context -> input_file_type == FILE_TYPE_FASTQ?"FASTQ":(qs_context -> input_file_type == FILE_TYPE_SAM?"SAM":(qs_context -> input_file_type == FILE_TYPE_BAM?"BAM":"gzipped FASTQ"))); ret=1; } else { qs_context -> IO_line_buff = malloc(6000); if(!qs_context -> IO_line_buff){ SUBREADprintf("ERROR: cannot allocate 3KB memory??\n"); ret=1; } } } if(qs_context->phred_offset!=33 && (qs_context -> input_file_type == FILE_TYPE_SAM || qs_context -> input_file_type == FILE_TYPE_BAM)) SUBREADprintf("WARNING: your input file is in SAM or BAM format. In most cases, the Phred score offset in SAM or BAM is 33.\n"); if(!ret) SUBREADprintf("Scan the input file...\n"); return ret; } int retrieve_scores(char ** input, int *offset_pt, long long *size, int * sam_whichend, char **input_file_type, char ** output_sc){ qualscore_context qs_context; memset(&qs_context, 0, sizeof(qualscore_context)); // input_name is a FastQ file, a GZ FastQ file, a SAM file or a BAM file. qs_context.input_name = input[0]; // output_sc_name is the final result file used in donwstream analysis. // the output_sc file has a format like // 40,40,40,40,40,....,30,30,10,3,3,3,3 { elements = read length } // 40,35,35,30,30,....,30,30,10,3,3,3,3 { elements = read length } // .... {total lines = needed_reads +- 1} qs_context.output_name = output_sc[0]; // input_file_type can be "BAM", "SAM", "FASTQ" or "GZFASTQ" char *input_file_type_str = *input_file_type; qs_context.input_file_type = qs_str_input_type(input_file_type_str); // if the input file is SAM or BAM, sam_end=0 : both ends; sam_end=1 : first end; sam_end=2 : second end. qs_context.sam_end = *sam_whichend; // offset is 33 or 64 qs_context.phred_offset = *offset_pt; // total number of sampled reads qs_context.needed_reads = *size; double start_time = miltime(); int ret = start_qs_context(&qs_context); if(!ret) { unsigned long long read_number = 0; unsigned int all_read_max_len = 0; char *linebuff = malloc(3000); while (1) { int reti = qs_next_qual(&qs_context, linebuff); if(reti<0) ret = 1; if(reti) break; read_number++; int rlen = strlen(linebuff); if(linebuff[rlen-1]=='\n') rlen--; all_read_max_len = max(all_read_max_len, rlen); if(read_number % 1000000 == 0) SUBREADprintf(" %llu reads have been scanned in %.1f seconds.\n", read_number, miltime() - start_time); } if(!ret) { if(read_number<1) SUBREADprintf("Warning: the input file is empty%s.\n", (qs_context.sam_end ==2)?", or it does not contain any second-end read":""); else { rewind_qs_file(&qs_context); double sampling_rate = (read_number-0.1)/qs_context.needed_reads; double next_sample_number = 0; sampling_rate = max(1.0, sampling_rate); SUBREADprintf("Totally %llu reads were scanned; the sampling interval is %d.\nNow extract read quality information...\n", read_number, (int)(sampling_rate)); read_number = 0; while(1) { int reti = qs_next_qual(&qs_context, linebuff); if(reti) break; if(read_number >= next_sample_number - 0.0000000001) { add_read_scores(&qs_context, linebuff, all_read_max_len); next_sample_number += sampling_rate; } read_number++; if(read_number % 1000000 == 0) SUBREADprintf(" %llu reads have been scanned in %.1f seconds.\n", read_number, miltime() - start_time); } } } free(linebuff); } finalise_qs_context(&qs_context, ret); return ret; } static struct option qs_long_options[] = { {"BAMinput", no_argument, 0, '9'}, {"SAMinput", no_argument, 0, '8'}, {"gzFASTQinput", no_argument, 0, '7'}, {"FASTQinput", no_argument, 0, '6'}, {"first-end", no_argument, 0, '1'}, {"second-end", no_argument, 0, '2'}, {"both-ends", no_argument, 0, '0'}, {"counted-reads", required_argument, 0, 'n'}, {"phred-offset", required_argument, 0, 'P'}, {0, 0, 0, 0} }; void qualscore_usage() { SUBREADprintf("\nqualityScore Version %s\n\n", SUBREAD_VERSION); SUBREADputs(" Retrieve Phred score for read bases\n"); SUBREADputs("Usage:"); SUBREADputs(""); SUBREADputs(" ./qualityScores [options] -i -o "); SUBREADputs(""); SUBREADputs("Required arguments:"); SUBREADputs(""); SUBREADputs(" -i Name of input file including read data. The default format is"); SUBREADputs(" Fastq."); SUBREADputs(""); SUBREADputs(" -o Name of output file that is a text file including Phred scores"); SUBREADputs(" for each read base."); SUBREADputs(""); SUBREADputs("Optional arguments:"); SUBREADputs(""); SUBREADputs(" --gzFASTQinput Input file is in gzipped Fastq format."); SUBREADputs(""); SUBREADputs(" --BAMinput Input file is in BAM format."); SUBREADputs(""); SUBREADputs(" --SAMinput Input file is in SAM format."); SUBREADputs(""); SUBREADputs(" --first-end Use only first reads in paired-end data. Only applicable for"); SUBREADputs(" paired-end BAM/SAM input."); SUBREADputs(""); SUBREADputs(" --second-end Use only second reads in paired-end data. Only applicable for"); SUBREADputs(" paired-end BAM/SAM input."); SUBREADputs(""); SUBREADputs(" --counted-reads Total number of reads to be extracted from the input"); SUBREADputs(" file. 10,000 by default."); SUBREADputs(""); SUBREADputs(" --phred-offset <33|64> refer to subread aligner."); SUBREADputs(""); } #ifdef MAKE_STANDALONE int main(int argc, char ** argv) #else int main_qualityScores(int argc, char ** argv) #endif { int ret; int c; long long needed_reads = 10000ll; int option_index = 0 , offset_pt = 33, sam_end = 0; char in_name[MAX_FILE_NAME_LENGTH]; char out_name[MAX_FILE_NAME_LENGTH]; char * input_format = "FASTQ"; char * in_nameptr = in_name; char * out_nameptr = out_name; in_name[0]=out_name[0]=0; sam_end = 1; // default: first-end only optind = 0; opterr = 1; optopt = 63; while((c = getopt_long (argc, argv, "n:i:o:P:12987", qs_long_options, &option_index)) != -1) { switch(c){ case 'n': needed_reads = atoll(optarg); break; case 'P': offset_pt = optarg[0]=='6'?64:33; break; case '9': input_format="BAM"; break; case '8': input_format="SAM"; break; case '7': input_format="GZFASTQ"; break; case '6': input_format="FASTQ"; break; case '0': sam_end = 0; break; case '1': sam_end = 1; break; case '2': sam_end = 2; break; case 'i': strcpy(in_name, optarg); break; case 'o': strcpy(out_name, optarg); break; case 0: // long names break; default: qualscore_usage(); return 0; } } if(in_name[0]==0 || out_name[0]==0) { qualscore_usage(); return 0; } ret = retrieve_scores(&in_nameptr, &offset_pt, &needed_reads, &sam_end, &input_format, &out_nameptr); return ret; } Rsubread/src/read-repair.c0000644000175200017520000001060314136047615016500 0ustar00biocbuildbiocbuild#include #include #include #include #include #include "subread.h" #include "core.h" #include "input-files.h" #include "HelperFunctions.h" void print_usage_pairer(char * cmd){ SUBREADprintf("\nrepair Version %s\n\n", SUBREAD_VERSION); SUBREADputs(" Find reads that are from the same pair in the input and then place them next"); SUBREADputs("to each other in the output. A dummy read is added for each singleton read"); SUBREADputs("that does not have a pair. The output file is compatible with featureCounts"); SUBREADputs("program."); SUBREADputs(""); SUBREADputs("Usage:"); SUBREADputs(""); SUBREADputs(" ./repair [options] -i -o \n"); SUBREADputs(""); SUBREADputs("Required arguments:"); SUBREADputs(""); SUBREADputs(" -i Name of input file. BAM format by default."); SUBREADputs(""); SUBREADputs(" -o Name of output file. The output file is in BAM format."); SUBREADputs(""); SUBREADputs("Optional arguments:"); SUBREADputs(""); SUBREADputs(" -S The input file is in SAM format."); SUBREADputs(""); SUBREADputs(" -c Compress the output BAM file. This will reduce the size of BAM"); SUBREADputs(" file, but will increase the time of retrieving reads from BAM"); SUBREADputs(" file."); SUBREADputs(""); SUBREADputs(" -T Number of CPU threads. 8 by default."); SUBREADputs(""); SUBREADputs(" -d Do not add dummy reads for singleton reads."); SUBREADputs(""); SUBREADputs(" -t Do not include sequences and quality scores of reads in the"); SUBREADputs(" output file."); SUBREADputs(""); } #ifdef MAKE_STANDALONE int main(int argc, char ** argv) #else int main_read_repair(int argc, char ** argv) #endif { double t0 = miltime(); int threads = 8, is_BAM = 1; char c; char in_BAM_file[MAX_FILE_NAME_LENGTH+1]; char out_BAM_file[MAX_FILE_NAME_LENGTH+1]; char rand_prefix[40]; int no_compression = 1; int has_dummy = 1; int tiny_mode = 0; optind = 1; opterr = 1; optopt = 63; int memory = 64; in_BAM_file[0] = out_BAM_file[0] = 0; while ((c = getopt(argc, argv, "i:T:M:o:vtdcS?")) != -1) { switch(c) { case '?': case 'v': print_usage_pairer(argv[0]); STANDALONE_exit(0); break; case 'S': is_BAM = 0; break; case 't': tiny_mode = 1; break; case 'd': has_dummy = 0; break; case 'o': strcpy(out_BAM_file, optarg); break; case 'M': memory = atoi(optarg); if(memory < 1) memory = 1; break; case 'T': threads = atoi(optarg); if(threads < 1) threads = 1; if(threads > MAX_THREADS) threads = MAX_THREADS; break; case 'c': no_compression = 0; break; case 'i': strcpy(in_BAM_file, optarg); break; } } if(in_BAM_file[0]==0 || out_BAM_file[0]==0){ print_usage_pairer(argv[0]); //SUBREADprintf("\nNo input or output files are specified.\n"); STANDALONE_exit(-1); } if(!is_paired_end_BAM(in_BAM_file)){ SUBREADprintf("Error: the input file contains single-end reads. Repair can only process paired-end reads.\n"); STANDALONE_exit(-1); } char mac_rand[13]; mac_or_rand_str(mac_rand); sprintf(rand_prefix, "fsbm-p%06d-%s", getpid(), mac_rand); SAM_pairer_context_t pairer; SAM_pairer_writer_main_t writer_main; int ret = SAM_pairer_writer_create(&writer_main, threads, has_dummy,1, no_compression?Z_NO_COMPRESSION:Z_DEFAULT_COMPRESSION, out_BAM_file); if(ret){ SUBREADprintf("Unable to open the output file. Program terminated.\n"); return -1; }else{ ret = SAM_pairer_create(&pairer, threads, memory, is_BAM, tiny_mode,0,0 , 0, 1, in_BAM_file, SAM_pairer_writer_reset, SAM_pairer_multi_thread_header, SAM_pairer_multi_thread_output, rand_prefix, &writer_main, 99999999); if(ret){ SUBREADprintf("Unable to open the input file. Program terminated.\n"); return -1; }else{ SAM_pairer_run(&pairer); int has_error = pairer.is_bad_format; SAM_pairer_destroy(&pairer); SAM_pairer_writer_destroy(&writer_main); SUBREADprintf("\n%s %.2f minutes\nTotal input reads: %llu ; Unpaired reads: %llu\n\n", has_error?"Program terminated WITH ERRORS!!! Used":"All finished in", (miltime()-t0)/60, pairer.total_input_reads, pairer.total_orphan_reads); if(has_error){ SUBREADprintf("No output file was generated.\n"); unlink(out_BAM_file); return -1; } return 0; } } } Rsubread/src/readSummary.c0000644000175200017520000143560114136047615016610 0ustar00biocbuildbiocbuild/*************************************************************** The Subread and Rsubread software packages are free software packages: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #define _GNU_SOURCE #include #include #include #include #include #include #include #include #ifndef MAKE_STANDALONE #include #endif #include #include #include #include #include "subread.h" #include "interval_merge.h" #include "core.h" #include "gene-algorithms.h" #include "sambam-file.h" #include "input-files.h" #include "input-blc.h" #include "hashtable.h" #include "seek-zlib.h" #include "HelperFunctions.h" /********************************************************************/ /********************************************************************/ /********************************************************************/ // NEW FUNCTION FOR MULTI-THREADING /********************************************************************/ /********************************************************************/ /********************************************************************/ #define CHROMOSOME_NAME_LENGTH 256 #define MAX_FC_READ_LENGTH 10001 #define MAX_HIT_NUMBER (1000*1000*1000) #define MAX_EXTRA_COLS 15 #define MAX_UMI_LEN 14 #define FC_FLIST_SPLITOR "\026" typedef struct{ char * gene_name; unsigned int pos_first_base; unsigned int pos_last_base; } fc_junction_gene_t; #define MAXIMUM_INSERTION_IN_SECTION 8 typedef struct { char * chro; unsigned int start_pos; unsigned int chromosomal_length; short insertions; unsigned int insertion_start_pos[ MAXIMUM_INSERTION_IN_SECTION ]; unsigned short insertion_lengths[ MAXIMUM_INSERTION_IN_SECTION ]; } CIGAR_interval_t; typedef struct { int space; int used; fc_junction_gene_t ** genes; } gene_info_list_t; typedef struct { char chromosome_name_left[CHROMOSOME_NAME_LENGTH + 1]; char chromosome_name_right[CHROMOSOME_NAME_LENGTH + 1]; unsigned int last_exon_base_left; unsigned int first_exon_base_right; } fc_junction_info_t; typedef struct { srInt_64 feature_name_pos; unsigned int start; unsigned int end; unsigned int sorted_order; unsigned short chro_name_pos_delta; char is_negative_strand; char * extra_columns; } fc_feature_info_t; typedef struct { srInt_64 assigned_reads; srInt_64 unassigned_unmapped; srInt_64 unassigned_read_type; srInt_64 unassigned_singleton; srInt_64 unassigned_mappingquality; srInt_64 unassigned_chimericreads; srInt_64 unassigned_fragmentlength; srInt_64 unassigned_duplicate; srInt_64 unassigned_multimapping; srInt_64 unassigned_secondary; srInt_64 unassigned_junction_condition; srInt_64 unassigned_nofeatures; srInt_64 unassigned_overlapping_length; srInt_64 unassigned_ambiguous; } fc_read_counters; typedef srInt_64 read_count_type_t; typedef struct { unsigned short thread_id; srInt_64 nreads_mapped_to_exon; srInt_64 all_reads; //unsigned short current_read_length1; //unsigned short current_read_length2; unsigned int count_table_size; read_count_type_t * count_table; unsigned int chunk_read_ptr; pthread_t thread_object; int hits_number_capacity; unsigned int * hits_start_pos1; unsigned int * hits_start_pos2; unsigned short * hits_length1; unsigned short * hits_length2; char ** hits_chro1; char ** hits_chro2; srInt_64 * hits_indices1; srInt_64 * hits_indices2; unsigned int proc_Starting_Chro_Points_1BASE[65536]; unsigned short proc_Starting_Read_Points[65536]; unsigned short proc_Section_Read_Lengths[65536]; char * proc_ChroNames[65536]; char proc_Event_After_Section[65536]; CIGAR_interval_t proc_CIGAR_intervals_R1[65536], proc_CIGAR_intervals_R2[65536]; char ** scoring_buff_gap_chros; unsigned int * scoring_buff_gap_starts; unsigned short * scoring_buff_gap_lengths; char * read_details_buff; char * bam_compressed_buff; int read_details_buff_used; unsigned int * scoring_buff_numbers; unsigned int * scoring_buff_flags; unsigned int * scoring_buff_overlappings; srInt_64 * scoring_buff_exon_ids; srInt_64 del4_added_reads; char * chro_name_buff; z_stream bam_file_output_stream; HashTable ** scRNA_sample_bc_tables; // sample_ID ==> int64s: cell barcode id <<32 | umi barcode id HashTable * scRNA_registered_UMI_table; // UMI bases ==> UMI_no +1 in this thread HashTable * junction_counting_table; // key: string chro_name \t last_base_previous_exont \t first_base_next_exon HashTable * splicing_point_table; HashTable * RG_table; // rg_name -> [ count_table, sum_fc_read_counters, junction_counting_table, splicing_point_table] // NOTE: some reads have no RG tag. These reads are put into the tables in this object but not in the RG_table -> tables. srInt_64 scRNA_pooled_reads; srInt_64 *scRNA_reads_per_sample; srInt_64 *scRNA_mapped_reads_per_sample; srInt_64 *scRNA_assigned_reads_per_sample; srInt_64 scRNA_has_valid_sample_index; srInt_64 scRNA_has_valid_cell_barcode; fc_read_counters read_counters; } fc_thread_thread_context_t; #define READ_SHIFT_UPSTREAM 10 #define READ_SHIFT_DOWNSTREAM 20 #define READ_SHIFT_LEFT 30 #define READ_SHIFT_RIGHT 40 #define REVERSE_TABLE_BUCKET_LENGTH 131072 #define REDUCE_TO_5_PRIME_END 5 #define REDUCE_TO_3_PRIME_END 3 typedef struct { unsigned int chro_number; unsigned int chro_features; unsigned int chro_feature_table_start; unsigned int chro_block_table_start; unsigned int chro_block_table_end; unsigned int chro_possible_length; unsigned short chro_reverse_table_current_size; unsigned int * reverse_table_start_index; int reverse_table_start_index_size; //unsigned int * reverse_table_end_index; } fc_chromosome_index_info; typedef struct { int is_gene_level; int is_paired_end_mode_assign; int is_paired_end_reads_expected; int is_multi_overlap_allowed; int restricted_no_multi_overlap; char * strand_check_mode; int is_strand_checked; int is_both_end_required; int is_chimertc_disallowed; int is_PE_distance_checked; int is_multi_mapping_allowed; int is_primary_alignment_only; int is_SAM_file; int is_read_details_out; int is_junction_no_chro_shown; int is_unpaired_warning_shown; int is_stake_warning_shown; int is_read_too_long_to_SAM_BAM_shown; int is_split_or_exonic_only; int is_duplicate_ignored; int is_first_read_reversed; int is_second_read_straight; int is_verbose; int long_read_minimum_length; int assign_reads_to_RG; int use_stdin_file; int is_mixed_PE_SE; int disk_is_full; int do_not_sort; int reduce_5_3_ends_to_one; int isCVersion; int use_fraction_multi_mapping; int do_junction_counting; int do_detection_call; int this_input_number; int need_calculate_overlap_len; int need_calculate_fragment_len; int min_mapping_quality_score; int min_paired_end_distance; int max_paired_end_distance; int max_M; int feature_block_size; int read_length; int line_length; int longest_chro_name; int five_end_extension; int three_end_extension; int read_shift_type; int read_shift_size; int fragment_minimum_overlapping; int do_scRNA_table; float fractional_minimum_overlapping; float fractional_minimum_feature_overlapping; int use_overlapping_break_tie; int max_missing_bases_in_read, max_missing_bases_in_feature; srInt_64 all_reads; unsigned short thread_number; fc_thread_thread_context_t * thread_contexts; int sambam_chro_table_items; int is_input_bad_format; int any_reads_are_PE; SamBam_Reference_Info * sambam_chro_table; pthread_spinlock_t read_details_out_lock; SAM_pairer_context_t read_pairer; SAM_pairer_context_t scRNA_read_pairer; char * debug_command; char * unistr_buffer_space; srInt_64 max_BAM_header_size; srInt_64 unistr_buffer_size; srInt_64 unistr_buffer_used; int is_scRNA_BAM_FQ_out_generated; int scRNA_input_mode; int scRNA_dual_index; float scRNA_umi_cutoff; int scRNA_rerun_on_persample_BAM; int * scRNA_applied_umi_cut; HashTable * scRNA_sample_sheet_table; ArrayList * scRNA_sample_barcode_list; ArrayList * scRNA_cell_barcodes_array; HashTable * scRNA_cell_barcode_head_tail_table; HashTable * scRNA_lineno1B_to_sampleno1B_tab; ArrayList * scRNA_sample_id_to_name; HashTable * scRNA_sample_BAM_writers; // sample_ID(1-base) ==> SamBam_writer HashTable * lineno_2_sortedno_tab; int known_cell_barcode_length; HashTable * junction_features_table; HashTable * junction_bucket_table; fasta_contigs_t * fasta_contigs; HashTable * gene_name_table; // gene_name -> gene_number HashTable * BAM_chros_to_anno_table; // name in annotation file -> alias name HashTable * GCcontent_table; // gene_name -> "qc_content_frac" int scRNA_do_one_batch_runner_current; pthread_spinlock_t scRNA_do_one_batch_runner_lock; FILE ** scRNA_barcode_batched_bins; pthread_spinlock_t * scRNA_barcode_batched_locks; int scRNA_barcode_batched_bin_no; int scRNA_barcode_batched_max_Rbin_len; int scRNA_barcode_batched_max_genes; int scRNA_UMI_length; parallel_gzip_writer_t scRNA_fastq_unassigned_writer[4]; pthread_spinlock_t scRNA_fastq_unassigned_lock; char * RGnames_set; int RGnames_capacity; int RGnames_ptr; char alias_file_name[MAX_FILE_NAME_LENGTH]; char input_file_name[MAX_FILE_NAME_LENGTH]; char * input_file_short_name; char raw_input_file_name[MAX_FILE_NAME_LENGTH]; char output_file_name[MAX_FILE_NAME_LENGTH]; char output_file_path[MAX_FILE_NAME_LENGTH]; char temp_file_dir[MAX_FILE_NAME_LENGTH]; char read_details_path[MAX_FILE_NAME_LENGTH]; char annotation_file_screen_output[MAX_FILE_NAME_LENGTH]; char scRNA_sample_sheet[MAX_FILE_NAME_LENGTH]; char scRNA_cell_barcode_list[MAX_FILE_NAME_LENGTH]; unsigned char ** gene_name_array; // gene_internal_number -> gene_name int input_file_unique; char * reported_extra_columns; HashTable * exontable_chro_table; // gene_name -> fc_chromosome_index_info structure (contains chro_number, feature_number, block_start, block_end, etc) int exontable_nchrs; int exontable_exons; int * exontable_geneid; char * exontable_strand; char ** exontable_chr; srInt_64 * exontable_start; srInt_64 * exontable_stop; char feature_name_column[2000]; char gene_id_column[100]; srInt_64 * exontable_block_end_index; srInt_64 * exontable_block_max_end; srInt_64 * exontable_block_min_start; char ** exontable_anno_chrs; char * exontable_anno_chr_2ch; srInt_64 * exontable_anno_chr_heads; FILE * read_details_out_FP; double start_time; char * cmd_rebuilt; char redo; fc_read_counters read_counters; } fc_thread_global_context_t; unsigned int tick_time = 1000; int fetch_boundaries(char * chroname,char * cigar, unsigned int pos, char strand, int *has_left, unsigned short *left_on_read, unsigned int *left_pos, int *has_right, unsigned short *right_on_read, unsigned int *right_pos, fc_junction_info_t * result_junctions, int junction_space){ int cigar_cursor = 0, nch, read_len = 0, ret = 0; unsigned int chro_cursor = pos, tmpi = 0; unsigned int right_boundary = 0; unsigned short left_clipped = 0; unsigned short right_clipped = 0; *has_right = 0; *has_left = 0; for(; (nch = cigar[cigar_cursor])!=0 ; cigar_cursor++){ if(isdigit(nch)){ tmpi = tmpi*10 + (nch - '0'); } else { if (nch == 'S'){ if(chro_cursor == pos) left_clipped = tmpi;else right_clipped=tmpi; read_len += tmpi; } else if(nch == 'M' || nch == 'D'){ if(nch == 'M')read_len += tmpi; chro_cursor += tmpi; right_boundary = chro_cursor -1; } else if(nch == 'N'){ unsigned int last_exon_last_base = chro_cursor - 1; unsigned int next_exon_first_base = chro_cursor + tmpi; chro_cursor += tmpi; if(ret < junction_space){ result_junctions[ret].last_exon_base_left = last_exon_last_base; result_junctions[ret].first_exon_base_right = next_exon_first_base; strcpy(result_junctions[ret].chromosome_name_left, chroname); strcpy(result_junctions[ret].chromosome_name_right, chroname); ret ++; } } else if(nch == 'I') read_len += tmpi; tmpi = 0; } } if(left_clipped){ *has_left = 1; *left_on_read = left_clipped; *left_pos = pos; } if(right_clipped){ *has_right = 1; *right_on_read = read_len - right_clipped - 1; *right_pos = right_boundary; } return ret; } // This function parses the cigar string and returns the number of exon-exon junctions found in the cigar. // It returns 0 if no junctions are found. int calc_junctions_from_cigar(fc_thread_global_context_t * global_context, int flag, char * chroname, unsigned int pos, char * cigar , char * extra_tags, fc_junction_info_t * result_junctions){ unsigned short boundaries_inclusive_base_on_read[global_context -> max_M]; unsigned int boundaries_inclusive_base_pos[global_context -> max_M]; char boundaries_chromosomes[global_context -> max_M][MAX_CHROMOSOME_NAME_LEN]; char boundaries_extend_to_left_on_read[global_context -> max_M]; int boundaries = 0; int cigar_cursor = 0, nch, ret = 0, read_len = 0, x1, x2; unsigned int chro_cursor = pos, tmpi = 0; unsigned short left_clipped = 0; unsigned short right_clipped = 0; for(; (nch = cigar[cigar_cursor])!=0 ; cigar_cursor++){ if(isdigit(nch)){ tmpi = tmpi*10 + (nch - '0'); } else { if (nch == 'S'){ if(chro_cursor == pos) left_clipped = tmpi;else right_clipped=tmpi; read_len += tmpi; } else if(nch == 'M' || nch == 'D'){ if(nch == 'M')read_len += tmpi; chro_cursor += tmpi; } else if(nch == 'N'){ unsigned int last_exon_last_base = chro_cursor - 1; unsigned int next_exon_first_base = chro_cursor + tmpi; if(ret <= global_context -> max_M - 1){ result_junctions[ret].last_exon_base_left = last_exon_last_base; result_junctions[ret].first_exon_base_right = next_exon_first_base; strcpy(result_junctions[ret].chromosome_name_left, chroname); strcpy(result_junctions[ret].chromosome_name_right, chroname); ret ++; } chro_cursor += tmpi; } else if(nch == 'I') read_len += tmpi; tmpi = 0; } } if(left_clipped){ strcpy(boundaries_chromosomes[boundaries] , chroname); boundaries_extend_to_left_on_read[boundaries] = 0; boundaries_inclusive_base_pos[boundaries] = pos; boundaries_inclusive_base_on_read[boundaries++] = left_clipped; } if(right_clipped){ strcpy(boundaries_chromosomes[boundaries] , chroname); boundaries_extend_to_left_on_read[boundaries] = 1; boundaries_inclusive_base_pos[boundaries] = chro_cursor - 1; boundaries_inclusive_base_on_read[boundaries++] = read_len - right_clipped - 1; } int tag_cursor=0; //if(strstr(extra_tags, "CG:Z")) { // SUBREADprintf("CIGAR=%s, EXTRA=%s\n", cigar, extra_tags); //} int status = PARSE_STATUS_TAGNAME; char tag_name[2], typechar=0; int tag_inner_cursor=0; char read_main_strand = (((flag & 0x10) == 0x10) == ((flag & 0x40)==0x40))?'-':'+'; char current_fusion_char[MAX_CHROMOSOME_NAME_LEN]; unsigned int current_fusion_pos = 0; char current_fusion_strand = 0; char current_fusion_cigar[global_context -> max_M * 15]; current_fusion_cigar [0] =0; current_fusion_char [0]=0; while(1){ int nch = extra_tags[tag_cursor]; if(status == PARSE_STATUS_TAGNAME){ tag_name[tag_inner_cursor++] = nch; if(tag_inner_cursor == 2){ status = PARSE_STATUS_TAGTYPE; tag_cursor += 1; assert(extra_tags[tag_cursor] == ':'); } }else if(status == PARSE_STATUS_TAGTYPE){ typechar = nch; tag_cursor +=1; assert(extra_tags[tag_cursor] == ':'); tag_inner_cursor = 0; status = PARSE_STATUS_TAGVALUE; }else if(status == PARSE_STATUS_TAGVALUE){ if(nch == '\t' || nch == 0){ if(current_fusion_cigar[0] && current_fusion_char[0] && current_fusion_pos && current_fusion_strand){ unsigned int left_pos = 0, right_pos = 0; unsigned short left_on_read = 0, right_on_read = 0; int has_left = 0, has_right = 0; unsigned int start_pos = current_fusion_pos; if(current_fusion_strand!=read_main_strand) start_pos = find_left_end_cigar(current_fusion_pos, current_fusion_cigar); ret += fetch_boundaries(current_fusion_char, current_fusion_cigar, start_pos, current_fusion_strand, &has_left, &left_on_read, &left_pos, &has_right, &right_on_read, &right_pos, result_junctions + ret, global_context -> max_M - ret ); if(has_left){ strcpy(boundaries_chromosomes[boundaries] , current_fusion_char); boundaries_extend_to_left_on_read[boundaries] = 0; boundaries_inclusive_base_pos[boundaries] = left_pos; boundaries_inclusive_base_on_read[boundaries++] = left_on_read; } if(has_right){ strcpy(boundaries_chromosomes[boundaries] , current_fusion_char); boundaries_extend_to_left_on_read[boundaries] = 1; boundaries_inclusive_base_pos[boundaries] = right_pos; boundaries_inclusive_base_on_read[boundaries++] = right_on_read; } // SUBREADprintf("BOUND_EXT: %s:%u (at %u) (%c) ~ %s:%u (at %u) (%c)\n", current_fusion_char, left_pos, left_on_read, has_left?'Y':'X' , current_fusion_char, right_pos, right_on_read, has_right?'Y':'X'); current_fusion_pos = 0; current_fusion_strand = 0; current_fusion_cigar [0] =0; current_fusion_char [0]=0; } tag_inner_cursor = 0; status = PARSE_STATUS_TAGNAME; }else{ if(tag_name[0]=='C' && tag_name[1]=='C' && typechar == 'Z'){ current_fusion_char[tag_inner_cursor++]=nch; current_fusion_char[tag_inner_cursor]=0; }else if(tag_name[0]=='C' && tag_name[1]=='G' && typechar == 'Z'){ current_fusion_cigar[tag_inner_cursor++]=nch; current_fusion_cigar[tag_inner_cursor]=0; }else if(tag_name[0]=='C' && tag_name[1]=='P' && typechar == 'i'){ current_fusion_pos = current_fusion_pos * 10 + (nch - '0'); }else if(tag_name[0]=='C' && tag_name[1]=='T' && typechar == 'Z'){ current_fusion_strand = nch; } } } if(nch == 0){ assert(status == PARSE_STATUS_TAGNAME); break; } tag_cursor++; } //for(x1 = 0; x1 < boundaries; x1++) // SUBREADprintf("HAS: LR:%d, READ:%d\n", boundaries_extend_to_left_on_read[x1], boundaries_inclusive_base_on_read[x1]); for(x1 = 0; x1 < boundaries; x1++) for(x2 = 0; x2 < boundaries; x2++){ if(x1==x2) continue; if(boundaries_chromosomes[x1][0]==0 || boundaries_chromosomes[x2][0]==0) continue; if(boundaries_extend_to_left_on_read[x1] == 1 && boundaries_extend_to_left_on_read[x2] == 0){ if( boundaries_inclusive_base_on_read[x1] == boundaries_inclusive_base_on_read[x2]-1 ){ if(ret <= global_context -> max_M - 1){ result_junctions[ret].last_exon_base_left = boundaries_inclusive_base_pos[x1]; result_junctions[ret].first_exon_base_right = boundaries_inclusive_base_pos[x2]; strcpy(result_junctions[ret].chromosome_name_left, boundaries_chromosomes[x1]); strcpy(result_junctions[ret].chromosome_name_right, boundaries_chromosomes[x2]); ret++; } // SUBREADprintf("MATCH: %d ~ %d\n", boundaries_inclusive_base_on_read[x1], boundaries_inclusive_base_on_read[x2]); boundaries_chromosomes[x1][0]=0; boundaries_chromosomes[x2][0]=0; } } } //for(x1 = 0; x1 < boundaries; x1++) // if(boundaries_chromosomes[x1][0]) // SUBREADprintf("LEFT: LR:%d, READ:%d\n", boundaries_extend_to_left_on_read[x1], boundaries_inclusive_base_on_read[x1]); return ret; } srInt_64 unistr_cpy(fc_thread_global_context_t * global_context, char * str, int strl) { srInt_64 ret; if(global_context->unistr_buffer_used + strl >= global_context->unistr_buffer_size-1) { if( global_context->unistr_buffer_size < (1000llu*1000u*1000u*32)) // 32GB { global_context -> unistr_buffer_size = global_context->unistr_buffer_size /2 *3; global_context -> unistr_buffer_space = realloc(global_context -> unistr_buffer_space, global_context->unistr_buffer_size); } else { SUBREADprintf("Error: exceed memory limit (32GB) for storing feature names.\n"); return 0xffffffffu; } } strcpy(global_context -> unistr_buffer_space + global_context->unistr_buffer_used, str); ret = global_context->unistr_buffer_used; global_context->unistr_buffer_used += strl +1; return ret; } int print_FC_configuration(fc_thread_global_context_t * global_context, char * annot, char * sam, char * out, int is_sam, int is_GTF, int *n_input_files, int isReadSummaryReport, char * PE_exp, char * PE_ass) { char * tmp_ptr1 = NULL , * next_fn, *sam_used = malloc(strlen(sam)+MAX_FILE_NAME_LENGTH), sam_ntxt[30],bam_ntxt[30], next_ntxt[50]; int nfiles=1, nBAMfiles = 0, nNonExistFiles = 0, x1; char MAC_or_random[13]; mac_or_rand_str(MAC_or_random); /* if(global_context -> max_missing_bases_in_read >= 0 && global_context -> fractional_minimum_overlapping > 0.000001){ SUBREADprintf("\nERROR: multiple filtering conditions on overlapping bases in reads\n"); return 1; } if(global_context -> max_missing_bases_in_feature >= 0 && global_context -> fractional_minimum_feature_overlapping > 0.000001){ SUBREADprintf("\nERROR: multiple filtering conditions on overlapping bases in features\n"); return 1; }*/ sprintf(sam_used, "%s/featureCounts_test_file_writable-%06d-%s.tmp", global_context -> temp_file_dir, getpid(), MAC_or_random); FILE * fp = fopen(sam_used,"w"); if(fp){ fclose(fp); unlink(sam_used); }else{ SUBREADprintf("\nERROR: temporary directory is not writable: '%s'\n\n", global_context -> temp_file_dir); return 1; } strcpy(sam_used, sam); nfiles = 0; while(1) { next_fn = strtok_r(nfiles==0?sam_used:NULL, FC_FLIST_SPLITOR, &tmp_ptr1); if(next_fn == NULL || strlen(next_fn)<1) break; nfiles++; srInt_64 BAM_header_size = -1; int file_probe = is_certainly_bam_file(next_fn, NULL, &BAM_header_size); if(BAM_header_size>0) global_context -> max_BAM_header_size = max( global_context -> max_BAM_header_size , BAM_header_size + 180000); if(file_probe==-1){ nNonExistFiles++; if(global_context -> use_stdin_file){ SUBREADprintf("\nERROR: no valid SAM or BAM file is received from \n\n"); }else{ SUBREADprintf("\nERROR: invalid parameter: '%s'\n\n", next_fn); } return 1; } if(file_probe == 1) nBAMfiles++; } SUBREADputs(""); print_subread_logo(); SUBREADputs(""); print_in_box(80,1,1,"featureCounts setting"); print_in_box(80,0,0,""); sam_ntxt[0]=0; bam_ntxt[0]=0; next_ntxt[0]=0; if(nNonExistFiles) sprintf(next_ntxt, "%d unknown file%s", nNonExistFiles, nNonExistFiles>1?"s":""); if(nBAMfiles) sprintf(bam_ntxt, "%d BAM file%s ", nBAMfiles, nBAMfiles>1?"s":""); if(nfiles-nNonExistFiles-nBAMfiles) sprintf(sam_ntxt, "%d SAM file%s ", nfiles-nNonExistFiles-nBAMfiles , (nfiles-nNonExistFiles-nBAMfiles)>1?"s":""); strcpy(sam_used, sam); print_in_box(80,0,0," Input files : %s%s%s", sam_ntxt, bam_ntxt, next_ntxt); print_in_box(80,0,0,""); nfiles=0; while(1){ next_fn = strtok_r(nfiles==0?sam_used:NULL, FC_FLIST_SPLITOR, &tmp_ptr1); if(next_fn == NULL || strlen(next_fn)<1) break; //int is_first_read_PE = 0 , file_probe = is_certainly_bam_file(next_fn, &is_first_read_PE, NULL); print_in_box(89,0,0," %c[36m%s%c[0m",CHAR_ESC, global_context -> use_stdin_file?"":get_short_fname(next_fn),CHAR_ESC); nfiles++; } (*n_input_files) = nfiles; print_in_box(80,0,0,""); if(global_context -> annotation_file_screen_output[0]==0){ print_in_box(80,0,0," Output file : %s", get_short_fname(out)); print_in_box(80,0,0," Summary : %s.summary", get_short_fname(out)); } char * PEassignStr = malloc(nfiles * 6); char * PEexpectStr = malloc(nfiles * 6); int exp_all_same = 1, ass_all_same = 1; sprintf(PEexpectStr,"%s, ", (PE_exp[0]=='1')?"yes":"no"); char * Ystr = nfiles>5?"Y":"yes"; char * Nstr = nfiles>5?"N":"no"; if(PE_exp[1]){ for(x1=1; PE_exp[x1]; x1++) if(PE_exp[x1]!=PE_exp[0]) exp_all_same=0; if(!exp_all_same){ PEexpectStr[0]=0; for(x1=0; PE_exp[x1]; x1++) sprintf(PEexpectStr+strlen(PEexpectStr), "%s, ", (PE_exp[x1]=='1')?Ystr:Nstr); } } PEexpectStr[strlen(PEexpectStr)-2]=0; sprintf(PEassignStr,"%s, ", (PE_ass[0]=='1')?"yes":"no"); if(PE_ass[1]){ for(x1=1; PE_ass[x1]; x1++) if(PE_ass[x1]!=PE_ass[0]) ass_all_same=0; if(!ass_all_same){ PEassignStr[0]=0; for(x1=0; PE_ass[x1]; x1++) sprintf(PEassignStr+strlen(PEassignStr), "%s, ", (PE_ass[x1]=='1')?Ystr:Nstr); } } PEassignStr[strlen(PEassignStr)-2]=0; print_in_box(80,0,0," Paired-end : %s",PEexpectStr); print_in_box(80,0,0," Count read pairs : %s",PEassignStr); free(PEassignStr); free(PEexpectStr); if(global_context -> annotation_file_screen_output[0]) print_in_box(80,0,0," Annotation : %s",global_context -> annotation_file_screen_output); else print_in_box(80,0,0," Annotation : %s (%s)", get_short_fname(annot), is_GTF?"GTF":"SAF"); print_in_box(80,0,0," Dir for temp files : %s", global_context->temp_file_dir); if(global_context -> do_scRNA_table){ print_in_box(80,0,0,""); print_in_box(80,0,0," scRNA count table : .scRNA.table"); print_in_box(80,0,0," scRNA sample sheet : %s", get_short_fname(global_context->scRNA_sample_sheet)); print_in_box(80,0,0," scRNA barcode list : %s", get_short_fname(global_context->scRNA_cell_barcode_list)); } if(isReadSummaryReport){ print_in_box(80,0,0," Assignment details : .featureCounts%s", isReadSummaryReport == FILE_TYPE_BAM?".bam":(isReadSummaryReport == FILE_TYPE_SAM?".sam":"")); if(global_context -> read_details_path[0]) print_in_box(80,0,0," Details output path : %s", global_context ->read_details_path); else print_in_box(80,0,0," (Note that files are saved to the output directory)"); print_in_box(80,0,0,""); } if(global_context -> do_junction_counting) print_in_box(80,0,0," Junction Counting : .jcounts"); #ifdef MAKE_STANDALONE #endif if(global_context -> alias_file_name[0]) print_in_box(80,0,0," Chromosome alias file : %s", get_short_fname(global_context -> alias_file_name)); #ifdef MAKE_STANDALONE print_in_box(80,0,0,""); #endif print_in_box(80,0,0," Threads : %d", global_context->thread_number); print_in_box(80,0,0," Level : %s level", global_context->is_gene_level?"meta-feature":"feature"); // print_in_box(80,0,0," Paired-end : %s", global_context->is_paired_end_mode_assign?"yes":"no"); if(global_context -> do_not_sort && global_context->is_paired_end_mode_assign) { print_in_box(80,0,0," Sorting PE Reads : never"); print_in_box(80,0,0,""); } char * multi_mapping_allow_mode = "not counted"; if(global_context->is_multi_mapping_allowed) multi_mapping_allow_mode = global_context -> use_fraction_multi_mapping?"counted (fractional)": "counted"; print_in_box(80,0,0," Multimapping reads : %s", multi_mapping_allow_mode); if(global_context-> is_primary_alignment_only) print_in_box(80,0,0," Multiple alignments : primary alignment only"); print_in_box(80,0,0,"Multi-overlapping reads : %s", global_context->is_multi_overlap_allowed?"counted":"not counted"); if(global_context -> is_split_or_exonic_only) print_in_box(80,0,0," Split alignments : %s", (1 == global_context -> is_split_or_exonic_only)?"only split alignments":"only exonic alignments"); print_in_box(80,0,0," Min overlapping bases : %d", global_context -> fragment_minimum_overlapping); if(global_context -> max_missing_bases_in_read >= 0) print_in_box(80,0,0," Max missing bases : %d in reads", global_context -> max_missing_bases_in_read); if(global_context -> max_missing_bases_in_feature >= 0) print_in_box(80,0,0," Max missing bases : %d in features", global_context -> max_missing_bases_in_feature); if(global_context -> fractional_minimum_overlapping > 0.000001) print_in_box(81,0,0," Min overlapping frac. : %0.1f%%%% to reads", global_context -> fractional_minimum_overlapping*100); if(global_context -> fractional_minimum_feature_overlapping > 0.000001) print_in_box(81,0,0," Min overlapping frac. : %0.1f%%%% to features", global_context -> fractional_minimum_feature_overlapping*100); if(global_context -> read_shift_size >0) print_in_box(80,0,0," Read shift : %d to %s", global_context -> read_shift_size, global_context -> read_shift_type==READ_SHIFT_UPSTREAM?"upstream":( global_context -> read_shift_type==READ_SHIFT_DOWNSTREAM?"downstream":( global_context -> read_shift_type==READ_SHIFT_LEFT?"left":"right"))); if(global_context -> five_end_extension || global_context -> three_end_extension) print_in_box(80,0,0," Read extension : %d on 5' and %d on 3' ends", global_context -> five_end_extension , global_context -> three_end_extension); if(global_context -> reduce_5_3_ends_to_one) print_in_box(80,0,0," Read reduction : to %d' end" , global_context -> reduce_5_3_ends_to_one == REDUCE_TO_5_PRIME_END ?5:3); if(global_context -> is_duplicate_ignored) print_in_box(80,0,0," Duplicated Reads : ignored"); if(global_context -> long_read_minimum_length < 5000) print_in_box(80,0,0," Long read mode : yes"); //print_in_box(80,0,0," Read orientations : %c%c", global_context->is_first_read_reversed?'r':'f', global_context->is_second_read_straight?'f':'r' ); if(global_context->is_paired_end_mode_assign) { print_in_box(80,0,0,""); print_in_box(80,0,0," Chimeric reads : %s", global_context->is_chimertc_disallowed?"not counted":"counted"); print_in_box(80,0,0," Both ends mapped : %s", global_context->is_both_end_required?"required":"not required"); if(global_context->is_PE_distance_checked) print_in_box(80,0,0," Fragment length : %d - %d", global_context -> min_paired_end_distance, global_context -> max_paired_end_distance); } print_in_box(80,0,0,""); print_in_box(80,2,1,""); SUBREADputs(""); print_in_box(80,1,1,"Running"); print_in_box(80,0,0,""); if( global_context -> max_BAM_header_size > 32 * 1024 * 1024 ){ } if(global_context->BAM_chros_to_anno_table) print_in_box(80,0,0,"%ld chromosome name aliases are loaded.", global_context -> BAM_chros_to_anno_table ->numOfElements); free(sam_used); return 0; } void print_FC_results(fc_thread_global_context_t * global_context, char * out) { //print_in_box(89,0,1,"%c[36mAlignment assignment finished.%c[0m", CHAR_ESC, CHAR_ESC); print_in_box(80,0,0,""); #ifdef MAKE_STANDALONE print_in_box(80,0,PRINT_BOX_WRAPPED,"Summary of counting results can be found in file \"%s.summary\"", out); print_in_box(80,0,0,""); #endif print_in_box(80,2,1,""); SUBREADputs(""); return; SUBREADputs(""); } int fc_strcmp(const void * s1, const void * s2) { return strcmp((char*)s1, (char*)s2); } void junc_gene_free(void *vv){ fc_junction_gene_t *v = vv; free(v -> gene_name); free(v); } void register_junc_feature(fc_thread_global_context_t *global_context, char * feature_name, char * chro, unsigned int start, unsigned int stop){ HashTable * gene_table = HashTableGet(global_context -> junction_features_table, chro); //SUBREADprintf("REG %s : %p\n", chro, gene_table); if(NULL == gene_table){ gene_table = HashTableCreate(48367); HashTableSetDeallocationFunctions(gene_table, NULL, junc_gene_free); HashTableSetKeyComparisonFunction(gene_table, fc_strcmp); HashTableSetHashFunction(gene_table, fc_chro_hash); char * new_name = malloc(strlen(chro)+1); strcpy(new_name, chro); HashTablePut(global_context -> junction_features_table, new_name, gene_table); } fc_junction_gene_t * gene_info = HashTableGet(gene_table, feature_name); if(NULL == gene_info){ gene_info = malloc(sizeof(fc_junction_gene_t)); gene_info -> gene_name = strdup(feature_name); gene_info -> pos_first_base = start; gene_info -> pos_last_base = stop; HashTablePut(gene_table, gene_info -> gene_name, gene_info); }else{ gene_info -> pos_first_base = min(start, gene_info -> pos_first_base); gene_info -> pos_last_base = max(stop, gene_info -> pos_last_base); } } void free_bucket_table_list(void * pv){ gene_info_list_t * list = (gene_info_list_t*) pv; free(list -> genes); free(list); } int match_feature_name_column(char * infile, char * needed){ char * ptt = NULL; char lneeded[strlen(needed)+1]; strcpy(lneeded, needed); char * t1 = strtok_r(lneeded, ",", &ptt); while(t1){ if(strcmp(t1, infile)==0) return 1; t1 = strtok_r(NULL,",", &ptt); } return 0; } #define JUNCTION_BUCKET_STEP (128*1024) int locate_junc_features(fc_thread_global_context_t *global_context, char * chro, unsigned int pos, fc_junction_gene_t ** ret_info, int max_ret_info_size){ gene_info_list_t * list = NULL; char bucket_key[CHROMOSOME_NAME_LENGTH + 20]; if(global_context -> BAM_chros_to_anno_table) { char * anno_chro_name = HashTableGet( global_context -> BAM_chros_to_anno_table , chro); if(anno_chro_name){ sprintf(bucket_key, "%s:%u", anno_chro_name, pos - pos % JUNCTION_BUCKET_STEP); list = HashTableGet(global_context -> junction_bucket_table, bucket_key); } } if(list == NULL){ sprintf(bucket_key, "%s:%u", chro, pos - pos % JUNCTION_BUCKET_STEP); list = HashTableGet(global_context -> junction_bucket_table, bucket_key); } if(list == NULL && strlen(chro)>3 && memcmp(chro, "chr", 3)==0){ sprintf(bucket_key, "%s:%u", chro+3, pos - pos % JUNCTION_BUCKET_STEP); list = HashTableGet(global_context -> junction_bucket_table, bucket_key); } if(list == NULL){ sprintf(bucket_key, "chr%s:%u", chro, pos - pos % JUNCTION_BUCKET_STEP); list = HashTableGet(global_context -> junction_bucket_table, bucket_key); } int ret = 0; if(list){ int x1; for(x1 = 0; x1 < list -> used; x1++){ fc_junction_gene_t * gene_info = list -> genes[x1]; if(gene_info -> pos_first_base <= pos && gene_info -> pos_last_base >= pos){ if(ret < max_ret_info_size) ret_info [ret ++] = gene_info; } } } return ret; } // This function loads annotations from the file. // It returns the number of featres loaded, or -1 if something is wrong. // Memory will be allowcated in this function. The pointer is saved in *loaded_features. // The invoker must release the memory itself. #define MAX_ANNOT_LINE_LENGTH 1500000 int load_feature_info(fc_thread_global_context_t *global_context, const char * annotation_file, int file_type, fc_feature_info_t ** loaded_features) { unsigned int features = 0, xk1 = 0, lineno=0; char * file_line = malloc(MAX_ANNOT_LINE_LENGTH+1); autozip_fp anno_fp; int apret = autozip_open(annotation_file, &anno_fp); int is_GFF_warned = 0; if(apret < 0) return -1; HashTable * chro_name_table = HashTableCreate(1603); HashTableSetHashFunction(chro_name_table, fc_chro_hash); HashTableSetKeyComparisonFunction(chro_name_table, fc_strcmp_chro); global_context -> longest_chro_name = 0; if(global_context -> do_junction_counting){ global_context -> junction_bucket_table = HashTableCreate(76037); HashTableSetDeallocationFunctions(global_context -> junction_bucket_table, free, free_bucket_table_list); HashTableSetKeyComparisonFunction(global_context -> junction_bucket_table, fc_strcmp); HashTableSetHashFunction(global_context -> junction_bucket_table, fc_chro_hash); global_context -> junction_features_table = HashTableCreate(1603); HashTableSetDeallocationFunctions(global_context -> junction_features_table, free, (void (*)(void *))HashTableDestroy); HashTableSetKeyComparisonFunction(global_context -> junction_features_table, fc_strcmp); HashTableSetHashFunction(global_context -> junction_features_table, fc_chro_hash); } // first scan: get the chromosome size (that have exons), total number of features // also create chro_name_table : chro_name => fc_chromosome_index_info while(0) { int rchars = autozip_gets(&anno_fp, file_line, MAX_ANNOT_LINE_LENGTH); char * token_temp = NULL, *chro_name; fc_chromosome_index_info * chro_stab; unsigned int feature_pos = 0; if(rchars < 1) break; lineno++; if(is_comment_line(file_line, file_type, lineno-1))continue; if(file_type == FILE_TYPE_GTF) { chro_name = strtok_r(file_line,"\t",&token_temp); strtok_r(NULL,"\t", &token_temp); // lib_name (not needed) char * feature_type = strtok_r(NULL,"\t", &token_temp); if(match_feature_name_column(feature_type, global_context -> feature_name_column)) { strtok_r(NULL,"\t", &token_temp); // feature_start feature_pos = atoi(strtok_r(NULL,"\t", &token_temp));// feature_end features++; } else chro_name = NULL; } else { strtok_r(file_line,"\t", &token_temp); chro_name = strtok_r(NULL,"\t",&token_temp); strtok_r(NULL,"\t",&token_temp); // feature_start feature_pos = atoi(strtok_r(NULL,"\t", &token_temp));// feature_end features++; } if(chro_name) { if(strlen(chro_name)>=CHROMOSOME_NAME_LENGTH) chro_name[CHROMOSOME_NAME_LENGTH-1]=0; chro_stab = HashTableGet(chro_name_table, chro_name); if(chro_stab) { chro_stab -> chro_possible_length = max(chro_stab -> chro_possible_length , feature_pos+1); }else { char * tmp_chro_name = malloc(CHROMOSOME_NAME_LENGTH); term_strncpy(tmp_chro_name, chro_name, CHROMOSOME_NAME_LENGTH); chro_stab = calloc(sizeof(fc_chromosome_index_info),1); chro_stab -> chro_number = chro_name_table->numOfElements; chro_stab -> chro_possible_length = feature_pos+1; chro_stab -> reverse_table_start_index_size = 5000000; chro_stab -> reverse_table_start_index = NULL; HashTablePut(chro_name_table, tmp_chro_name, chro_stab); } chro_stab -> chro_features ++; } } //autozip_rewind(&anno_fp); unsigned int ret_features_size = 400000; fc_feature_info_t * ret_features = malloc(sizeof(fc_feature_info_t) * ret_features_size); char * tmpnameex = malloc(50001); lineno = 0; while(1) { int is_gene_id_found = 0; int rchars = autozip_gets(&anno_fp, file_line, MAX_ANNOT_LINE_LENGTH); if(rchars < 1) break; if(rchars >= MAX_ANNOT_LINE_LENGTH - 1){ SUBREADprintf("\nERROR: the %u-th line in your GTF file is extremely long (longer than %d bytes).\nThe program cannot parse this line.\n", lineno+1, MAX_ANNOT_LINE_LENGTH-1); return -2; } lineno++; char * token_temp = NULL; if(is_comment_line(file_line, file_type, lineno-1))continue; if(file_type == FILE_TYPE_RSUBREAD){ if(xk1 >= ret_features_size) { ret_features_size *=2; ret_features = realloc(ret_features, sizeof(fc_feature_info_t) * ret_features_size); } char * feature_name = strtok_r(file_line,"\t",&token_temp); int feature_name_len = strlen(feature_name); if(feature_name_len > FEATURE_NAME_LENGTH-2){ SUBREADprintf("WARNING: feature name on the %d-th line is longer than %d bytes. The name is truncated\n", lineno, FEATURE_NAME_LENGTH -2); feature_name[FEATURE_NAME_LENGTH -2 ] = 0; } srInt_64 genename_pos = unistr_cpy(global_context, (char *)feature_name, feature_name_len); // SUBREADprintf("REALL: '%s'=%d [%d] %p POS=%d\t\tOLD_NAME_POS=%d\n" , feature_name, feature_name_len , xk1, ret_features+xk1, genename_pos, xk1>0?ret_features[xk1-1].feature_name_pos:-1); ret_features[xk1].feature_name_pos = genename_pos; char * seq_name = strtok_r(NULL,"\t", &token_temp); int chro_name_len = strlen(seq_name); if(chro_name_len > CHROMOSOME_NAME_LENGTH) seq_name[CHROMOSOME_NAME_LENGTH -1 ] = 0; srInt_64 chro_name_pos = unistr_cpy(global_context, (char *)seq_name, chro_name_len); global_context -> longest_chro_name = max(chro_name_len, global_context -> longest_chro_name); char * start_ptr = strtok_r(NULL,"\t", &token_temp); char * end_ptr = strtok_r(NULL,"\t", &token_temp); if(start_ptr == NULL || end_ptr == NULL){ SUBREADprintf("\nWarning: the format on the %d-th line is wrong.\n", lineno); } srInt_64 tv1 = atoll(start_ptr); srInt_64 tv2 = atoll(end_ptr); if( isdigit(start_ptr[0]) && isdigit(end_ptr[0]) ){ if(strlen(start_ptr) > 10 || strlen(end_ptr) > 10 || tv1 > 0x7fffffff || tv2> 0x7fffffff){ SUBREADprintf("\nError: Line %d contains a coordinate greater than 2^31.\n", lineno); return -2; } if(tv1 >tv2){ SUBREADprintf("\nError: Line %d contains a feature that do not have a positive length.\n", lineno); return -2; } }else{ SUBREADprintf("\nError: Line %d contains a format error. The expected annotation format is SAF.\n", lineno); return -2; } ret_features[xk1].chro_name_pos_delta = chro_name_pos - ret_features[xk1].feature_name_pos; ret_features[xk1].start = atoi( start_ptr );// start if(ret_features[xk1].start>0x7fffffff) { ret_features[xk1].start = 0; print_in_box(80,0,0,"WARNING the %d-th line has a negative chro coordinate.", lineno); } ret_features[xk1].end = atoi( end_ptr );//end if(ret_features[xk1].end>0x7fffffff) { ret_features[xk1].end = 0; print_in_box(80,0,0,"WARNING the %d-th line has a negative chro coordinate.", lineno); } char * strand_str = strtok_r(NULL,"\t", &token_temp); if(strand_str == NULL) ret_features[xk1].is_negative_strand = 0; else ret_features[xk1].is_negative_strand = ('+' ==strand_str[0])?0:(('-' ==strand_str[0])?1:-1); if(global_context -> do_detection_call){ char * GCcontent = strtok_r(NULL,"\t", &token_temp); if(GCcontent){ int gclen = strlen(GCcontent); if(gclen>0)GCcontent[gclen-1]=0; HashTablePut(global_context -> GCcontent_table, strdup(feature_name) , strdup(GCcontent)); } } ret_features[xk1].sorted_order = xk1; int bin_location = ret_features[xk1].start / REVERSE_TABLE_BUCKET_LENGTH; fc_chromosome_index_info * chro_stab = HashTableGet(chro_name_table, seq_name); int feature_pos = ret_features[xk1].end; if(NULL == chro_stab){ char * tmp_chro_name = malloc(CHROMOSOME_NAME_LENGTH); term_strncpy(tmp_chro_name, seq_name, CHROMOSOME_NAME_LENGTH); chro_stab = calloc(sizeof(fc_chromosome_index_info),1); chro_stab -> chro_number = chro_name_table->numOfElements; chro_stab -> chro_possible_length = feature_pos+1; chro_stab -> reverse_table_start_index_size = 5000000; chro_stab -> reverse_table_start_index = calloc( chro_stab -> reverse_table_start_index_size / REVERSE_TABLE_BUCKET_LENGTH +2, sizeof(int)); HashTablePut(chro_name_table, tmp_chro_name, chro_stab); }else chro_stab -> chro_possible_length = max(feature_pos+1, chro_stab -> chro_possible_length); chro_stab -> chro_features ++; if( chro_stab -> chro_possible_length >= chro_stab -> reverse_table_start_index_size ) { int old_end = sizeof(int) *( chro_stab -> reverse_table_start_index_size / REVERSE_TABLE_BUCKET_LENGTH +2); chro_stab -> reverse_table_start_index_size = max(chro_stab -> reverse_table_start_index_size * 2, (int)(chro_stab -> chro_possible_length * 1.3)); int new_size = sizeof(int) *( chro_stab -> reverse_table_start_index_size / REVERSE_TABLE_BUCKET_LENGTH +2); chro_stab -> reverse_table_start_index = realloc( chro_stab -> reverse_table_start_index , new_size); memset(chro_stab -> reverse_table_start_index + old_end / sizeof(int), 0, new_size - old_end); } chro_stab -> reverse_table_start_index[bin_location]++; is_gene_id_found = 1; assert(feature_name); if(global_context -> do_junction_counting) register_junc_feature(global_context , feature_name, seq_name, ret_features[xk1].start, ret_features[xk1].end); xk1++; } else if(file_type == FILE_TYPE_GTF) { char feature_name_tmp[FEATURE_NAME_LENGTH]; sprintf(feature_name_tmp, "LINE_%07u", xk1 + 1); char * seq_name = strtok_r(file_line,"\t",&token_temp); strtok_r(NULL,"\t", &token_temp);// source char * feature_type = strtok_r(NULL,"\t", &token_temp);// feature_type if(match_feature_name_column(feature_type, global_context -> feature_name_column)) { if(xk1 >= ret_features_size) { ret_features_size *=2; ret_features = realloc(ret_features, sizeof(fc_feature_info_t) * ret_features_size); } char * start_ptr = strtok_r(NULL,"\t", &token_temp); char * end_ptr = strtok_r(NULL,"\t", &token_temp); if(start_ptr == NULL || end_ptr == NULL){ SUBREADprintf("\nWarning: the format on the %d-th line is wrong.\n", lineno); } srInt_64 tv1 = atoll(start_ptr); srInt_64 tv2 = atoll(end_ptr); if( isdigit(start_ptr[0]) && isdigit(end_ptr[0]) ){ if(strlen(start_ptr) > 10 || strlen(end_ptr) > 10 || tv1 > 0x7fffffff || tv2> 0x7fffffff){ SUBREADprintf("\nError: Line %d contains a coordinate greater than 2^31.\n", lineno); return -2; } }else{ SUBREADprintf("\nError: Line %d contains a format error. The expected annotation format is GTF/GFF.\n", lineno); return -2; } ret_features[xk1].start = atoi(start_ptr);// start ret_features[xk1].end = atoi(end_ptr);//end if(ret_features[xk1].start < 1 || ret_features[xk1].end<1 || ret_features[xk1].start > 0x7fffffff || ret_features[xk1].end > 0x7fffffff || ret_features[xk1].start > ret_features[xk1].end){ SUBREADprintf("\Error: the feature on the %d-th line has zero coordinate or zero lengths\n\n", lineno); return -2; } strtok_r(NULL,"\t", &token_temp);// score char * strand_str = strtok_r(NULL,"\t", &token_temp); ret_features[xk1].is_negative_strand = ('-' == strand_str[0])?1:( ('+' == strand_str[0])?0:-1 );//strand ret_features[xk1].sorted_order = xk1; strtok_r(NULL,"\t",&token_temp); // "frame" char * extra_attrs = strtok_r(NULL,"\t",&token_temp); // name_1 "val1"; name_2 "val2"; ... ret_features[xk1].extra_columns = NULL; if(extra_attrs && (strlen(extra_attrs)>2)) { int attr_val_len = GTF_extra_column_value(extra_attrs , global_context -> gene_id_column , feature_name_tmp, FEATURE_NAME_LENGTH); if(attr_val_len>0) is_gene_id_found=1; // printf("V=%s\tR=%d\n", extra_attrs , attr_val_len); if(global_context -> reported_extra_columns){ char * extcols = malloc(30); int extcols_size = 30, extcols_len = 0; char * this_exname_ptr=global_context -> reported_extra_columns; while(1){ int padd0, is_last=1; for(padd0=0; this_exname_ptr[padd0]; padd0++) if(this_exname_ptr[padd0]=='\t'){ this_exname_ptr[padd0]=0; is_last=0; break; } attr_val_len = GTF_extra_column_value(extra_attrs , this_exname_ptr , tmpnameex, 50000); if(attr_val_len<0){ attr_val_len=2; strcpy(tmpnameex,"NA"); } if(attr_val_len + extcols_len + 2 > extcols_size){ extcols_size = max(extcols_size*2, attr_val_len + extcols_len+2); extcols = realloc(extcols, extcols_size); } memcpy(extcols+extcols_len, tmpnameex, attr_val_len); extcols_len += attr_val_len; extcols[extcols_len]='\t'; extcols_len += 1; if(is_last)break; this_exname_ptr[padd0]='\t'; this_exname_ptr += padd0+1; } extcols[extcols_len-1]=0; ret_features[xk1].extra_columns = extcols; } } if(!is_gene_id_found) { if(!is_GFF_warned) { int ext_att_len = strlen(extra_attrs); if(extra_attrs[ext_att_len-1] == '\n') extra_attrs[ext_att_len-1] =0; SUBREADprintf("\nERROR: failed to find the gene identifier attribute in the 9th column of the provided GTF file.\nThe specified gene identifier attribute is '%s' \nAn example of attributes included in your GTF annotation is '%s'.\n\n", global_context -> gene_id_column, extra_attrs); } is_GFF_warned++; } int feature_name_len = strlen(feature_name_tmp); if(feature_name_len > FEATURE_NAME_LENGTH-2){ SUBREADprintf("WARNING: feature name on the %d-th line is longer than %d bytes. The name is truncated\n", lineno, FEATURE_NAME_LENGTH-2); feature_name_tmp[FEATURE_NAME_LENGTH -2 ] = 0; } ret_features[xk1].feature_name_pos = unistr_cpy(global_context, (char *)feature_name_tmp, feature_name_len); int chro_name_len = strlen(seq_name); if(chro_name_len > CHROMOSOME_NAME_LENGTH) seq_name[CHROMOSOME_NAME_LENGTH -1 ] = 0; srInt_64 chro_name_pos = unistr_cpy(global_context, (char *)seq_name, chro_name_len); global_context -> longest_chro_name = max(chro_name_len, global_context -> longest_chro_name); ret_features[xk1].chro_name_pos_delta = chro_name_pos - ret_features[xk1].feature_name_pos; int bin_location = ret_features[xk1].start / REVERSE_TABLE_BUCKET_LENGTH; fc_chromosome_index_info * chro_stab = HashTableGet(chro_name_table, seq_name); int feature_pos = ret_features[xk1].end; if(NULL == chro_stab){ char * tmp_chro_name = malloc(CHROMOSOME_NAME_LENGTH); term_strncpy(tmp_chro_name, seq_name, CHROMOSOME_NAME_LENGTH); chro_stab = calloc(sizeof(fc_chromosome_index_info),1); chro_stab -> chro_number = chro_name_table->numOfElements; chro_stab -> chro_possible_length = feature_pos+1; chro_stab -> reverse_table_start_index_size = 5000000; chro_stab -> reverse_table_start_index = calloc( chro_stab -> reverse_table_start_index_size / REVERSE_TABLE_BUCKET_LENGTH +2 , sizeof(int)); HashTablePut(chro_name_table, tmp_chro_name, chro_stab); }else chro_stab -> chro_possible_length = max(feature_pos+1, chro_stab -> chro_possible_length); chro_stab -> chro_features ++; if( chro_stab -> chro_possible_length >= chro_stab -> reverse_table_start_index_size ) { int old_end = sizeof(int) *( chro_stab -> reverse_table_start_index_size / REVERSE_TABLE_BUCKET_LENGTH +2); chro_stab -> reverse_table_start_index_size = max(chro_stab -> reverse_table_start_index_size * 2, (int)(chro_stab -> chro_possible_length * 1.3)); int new_size = sizeof(int) *( chro_stab -> reverse_table_start_index_size / REVERSE_TABLE_BUCKET_LENGTH +2); chro_stab -> reverse_table_start_index = realloc(chro_stab -> reverse_table_start_index, new_size); memset(chro_stab -> reverse_table_start_index + old_end / sizeof(int), 0, new_size - old_end); } chro_stab -> reverse_table_start_index[bin_location]++; if(global_context -> do_junction_counting) register_junc_feature(global_context , feature_name_tmp, seq_name, ret_features[xk1].start, ret_features[xk1].end); xk1++; } } } features = xk1; autozip_close(&anno_fp); free(file_line); free(tmpnameex); (*loaded_features) = ret_features; global_context -> exontable_nchrs = (int)chro_name_table-> numOfElements; global_context -> exontable_chro_table = chro_name_table; if(is_GFF_warned) return -2; if(features < 1){ if(global_context -> annotation_file_screen_output[0]){ SUBREADprintf("ERROR: no features were loaded in format %s. The annotation format can be specified by the 'isGTFAnnotationFile' option%s.\n", file_type == FILE_TYPE_GTF?"GTF":"SAF", file_type == FILE_TYPE_GTF?", and the required feature type can be specified by the 'GTF.featureType' option":""); }else{ SUBREADprintf("ERROR: no features were loaded in format %s. The annotation format can be specified by the '-F' option%s.\n", file_type == FILE_TYPE_GTF?"GTF":"SAF", file_type == FILE_TYPE_GTF?", and the required feature type can be specified by the '-t' option.":""); } SUBREADprintf("\n\n"); return -2; } print_in_box(80,0,0," Features : %d\n", features); return features; } int find_or_insert_gene_name(fc_thread_global_context_t * global_context, unsigned char * feature_name) { HashTable * genetable = global_context -> gene_name_table; srInt_64 gene_number = HashTableGet(genetable, feature_name) - NULL; if(gene_number>0) return gene_number-1; else { gene_number = genetable -> numOfElements; HashTablePut(genetable, feature_name, NULL+gene_number+1); global_context -> gene_name_array[gene_number] = feature_name; // real memory space of feature_name is in the "loaded_features" data structure. // now we only save its pointer. return gene_number; } } void register_reverse_table(int block_no, srInt_64 this_block_min_start, srInt_64 this_block_max_end, fc_chromosome_index_info * chro_inf) { unsigned int reversed_bucket_start = this_block_min_start / REVERSE_TABLE_BUCKET_LENGTH; unsigned int reversed_bucket_end = this_block_max_end / REVERSE_TABLE_BUCKET_LENGTH; assert(this_block_min_start <= this_block_max_end); assert(reversed_bucket_end < chro_inf -> chro_possible_length); int x1; for(x1 = reversed_bucket_start; x1 <= reversed_bucket_end; x1++) { chro_inf->reverse_table_start_index[x1] = min(chro_inf->reverse_table_start_index[x1], block_no); //chro_inf->reverse_table_end_index[x1] = max(chro_inf->reverse_table_end_index[x1], block_no+1); } } void feature_merge(void * arrv, int start, int items, int items2) { void ** arr = (void **) arrv; srInt_64 * ret_start = (srInt_64 *) arr[0]; srInt_64 * ret_end = (srInt_64 *) arr[1]; unsigned char * ret_strand = (unsigned char *) arr[2]; int * ret_entyrez = (int *) arr[3]; fc_feature_info_t ** old_info_ptr = (fc_feature_info_t **) arr[4]; int total_items = items+items2; srInt_64 * tmp_start = malloc(sizeof(srInt_64) * total_items); srInt_64 * tmp_end = malloc(sizeof(srInt_64) * total_items); unsigned char * tmp_strand = malloc(sizeof(char) * total_items); int * tmp_entyrez = malloc(sizeof(int) * total_items); fc_feature_info_t ** tmp_info_ptr = malloc(sizeof(fc_feature_info_t*) * total_items); int read_1_ptr = start; int read_2_ptr = start+items; int write_ptr; for(write_ptr=0; write_ptr= start+items)||(read_2_ptr < start+total_items && ret_start[read_1_ptr] >= ret_start[read_2_ptr])) { tmp_start[write_ptr] = ret_start[read_2_ptr]; tmp_end[write_ptr] = ret_end[read_2_ptr]; tmp_strand[write_ptr] = ret_strand[read_2_ptr]; tmp_entyrez[write_ptr] = ret_entyrez[read_2_ptr]; tmp_info_ptr[write_ptr] = old_info_ptr[read_2_ptr]; read_2_ptr++; } else { tmp_start[write_ptr] = ret_start[read_1_ptr]; tmp_end[write_ptr] = ret_end[read_1_ptr]; tmp_strand[write_ptr] = ret_strand[read_1_ptr]; tmp_entyrez[write_ptr] = ret_entyrez[read_1_ptr]; tmp_info_ptr[write_ptr] = old_info_ptr[read_1_ptr]; read_1_ptr++; } } memcpy(ret_start+ start, tmp_start, sizeof(srInt_64) * total_items); memcpy(ret_end+ start, tmp_end, sizeof(srInt_64) * total_items); memcpy(ret_strand+ start, tmp_strand, sizeof(char) * total_items); memcpy(ret_entyrez+ start, tmp_entyrez, sizeof(int) * total_items); memcpy(old_info_ptr+ start, tmp_info_ptr, sizeof(fc_feature_info_t*) * total_items); free(tmp_start); free(tmp_end); free(tmp_strand); free(tmp_entyrez); free(tmp_info_ptr); } int feature_sort_compare(void * arrv, int l, int r) { void ** arr = (void **) arrv; srInt_64 * ret_start = (srInt_64 *)arr[0]; srInt_64 ll = ret_start[l]; srInt_64 rl = ret_start[r]; if(ll==rl) return 0; else if(ll>rl) return 1; else return -1; } void feature_sort_exchange(void * arrv, int l, int r) { void ** arr = (void **) arrv; srInt_64 tmp; fc_feature_info_t * tmpptr; srInt_64 * ret_start = (srInt_64 *) arr[0]; srInt_64 * ret_end = (srInt_64 *) arr[1]; unsigned char * ret_strand = (unsigned char *) arr[2]; int * ret_entyrez = (int *) arr[3]; fc_feature_info_t ** old_info_ptr = (fc_feature_info_t **) arr[4]; tmp = ret_start[r]; ret_start[r]=ret_start[l]; ret_start[l]=tmp; tmp = ret_end[r]; ret_end[r]=ret_end[l]; ret_end[l]=tmp; tmp = ret_strand[r]; ret_strand[r]=ret_strand[l]; ret_strand[l]=tmp; tmp = ret_entyrez[r]; ret_entyrez[r]=ret_entyrez[l]; ret_entyrez[l]=tmp; tmpptr = old_info_ptr[r]; old_info_ptr[r]=old_info_ptr[l]; old_info_ptr[l]=tmpptr; } void sort_feature_info(fc_thread_global_context_t * global_context, unsigned int features, fc_feature_info_t * loaded_features, char *** sorted_chr_names, int ** sorted_entrezid, srInt_64 ** sorted_start, srInt_64 ** sorted_end, unsigned char ** sorted_strand, char ** anno_chr_2ch, char *** anno_chrs, srInt_64 ** anno_chr_head, srInt_64 ** block_end_index, srInt_64 ** block_min_start_pos, srInt_64 ** block_max_end_pos) { unsigned int chro_pnt; unsigned int xk1,xk2; int * ret_entrez = malloc(sizeof(int) * features); srInt_64 * ret_start = malloc(sizeof(srInt_64) * features); srInt_64 * ret_end = malloc(sizeof(srInt_64) * features); int current_block_buffer_size = 2000; srInt_64 * ret_block_end_index = malloc(sizeof(srInt_64) * current_block_buffer_size); srInt_64 * ret_block_min_start = malloc(sizeof(srInt_64) * current_block_buffer_size); srInt_64 * ret_block_max_end = malloc(sizeof(srInt_64) * current_block_buffer_size); unsigned char * ret_strand = malloc(features); char ** ret_char_name = malloc(sizeof(void *) * features); fc_feature_info_t ** old_info_ptr = malloc(sizeof(void *) * features); (*anno_chrs) = malloc(sizeof(void *) * global_context -> exontable_nchrs); (*anno_chr_head) = malloc(sizeof(srInt_64) * global_context -> exontable_nchrs); (*anno_chr_2ch) = malloc(sizeof(char) * global_context -> exontable_nchrs*2); unsigned int * chro_feature_ptr = calloc(sizeof(int) * global_context -> exontable_nchrs,1); fc_chromosome_index_info ** tmp_chro_info_ptrs = malloc(global_context -> exontable_nchrs * sizeof(fc_chromosome_index_info *)); global_context -> gene_name_array = malloc(sizeof(char *) * features); // there should be much less identical names. global_context -> gene_name_table = HashTableCreate(5000); HashTableSetHashFunction(global_context -> gene_name_table, HashTableStringHashFunction); HashTableSetKeyComparisonFunction(global_context -> gene_name_table, fc_strcmp); // init start positions of each chromosome block. if(1) { KeyValuePair * cursor; int bucket; unsigned int sum_ptr = 0; for(bucket=0; bucket < global_context -> exontable_chro_table -> numOfBuckets; bucket++) { cursor = global_context -> exontable_chro_table -> bucketArray[bucket]; while (1) { if (!cursor) break; fc_chromosome_index_info * tmp_chro_inf = cursor -> value; cursor = cursor->next; //tmp_chro_inf -> reverse_table_end_index = calloc(sizeof(int), tmp_chro_inf->chro_possible_length / REVERSE_TABLE_BUCKET_LENGTH +2); chro_feature_ptr [tmp_chro_inf -> chro_number] = tmp_chro_inf -> chro_features; tmp_chro_info_ptrs[tmp_chro_inf -> chro_number] = tmp_chro_inf; } } for(xk1 = 0; xk1 < global_context -> exontable_nchrs; xk1++) { unsigned int tmpv = chro_feature_ptr[xk1]; chro_feature_ptr[xk1] = sum_ptr; tmp_chro_info_ptrs[xk1] -> chro_feature_table_start = sum_ptr; // printf("SII=%u + %u\n", sum_ptr, tmpv); sum_ptr += tmpv; } } int current_block_id = 0, sort_i = 0; (*sorted_chr_names) = ret_char_name; (*sorted_entrezid) = ret_entrez; (*sorted_start) = ret_start; (*sorted_end) = ret_end; (*sorted_strand) = ret_strand; int curr_chro_number = 0; for(chro_pnt=0; chro_pnt < features; chro_pnt++) { char * this_chro_name = global_context -> unistr_buffer_space + loaded_features[chro_pnt].feature_name_pos + loaded_features[chro_pnt].chro_name_pos_delta; fc_chromosome_index_info * this_chro_info = HashTableGet(global_context -> exontable_chro_table , this_chro_name); assert(this_chro_info); unsigned int this_chro_number = this_chro_info -> chro_number; unsigned int this_chro_table_ptr = chro_feature_ptr[this_chro_number]; ret_char_name[this_chro_table_ptr] = this_chro_name;// (char *)loaded_features[chro_pnt].chro; ret_entrez[this_chro_table_ptr] = find_or_insert_gene_name(global_context, (unsigned char *)(global_context -> unistr_buffer_space + loaded_features[chro_pnt].feature_name_pos)); ret_start[this_chro_table_ptr] = loaded_features[chro_pnt].start; ret_end[this_chro_table_ptr] = loaded_features[chro_pnt].end; ret_strand[this_chro_table_ptr] = loaded_features[chro_pnt].is_negative_strand; old_info_ptr[this_chro_table_ptr] = &loaded_features[chro_pnt]; chro_feature_ptr[this_chro_number]++; } for(xk1 = 0; xk1 < global_context -> exontable_nchrs; xk1++) { fc_chromosome_index_info * tmp_chro_inf = tmp_chro_info_ptrs[xk1]; int bins_in_chr = ( tmp_chro_inf->chro_possible_length / REVERSE_TABLE_BUCKET_LENGTH +2); short * features_per_block_bins = malloc(sizeof(short)*bins_in_chr); for(xk2=0; xk2 reverse_table_start_index[xk2])))); //printf("CHR%d : SQR[%d]=%d (%d)\n", tmp_chro_inf -> chro_number,xk2, features_per_block_bins[xk2], tmp_chro_inf -> reverse_table_start_index[xk2] ); } memset(tmp_chro_inf -> reverse_table_start_index, 0xff, sizeof(int) *bins_in_chr); tmp_chro_inf -> chro_block_table_start = current_block_id; unsigned int this_block_items = 0; srInt_64 this_block_min_start = 0x7fffffff, this_block_max_end = 0; unsigned int this_chro_tab_end = tmp_chro_inf -> chro_features + tmp_chro_inf -> chro_feature_table_start; void * in_array[5]; in_array[0] = ret_start + tmp_chro_inf -> chro_feature_table_start; in_array[1] = ret_end + tmp_chro_inf -> chro_feature_table_start; in_array[2] = ret_strand + tmp_chro_inf -> chro_feature_table_start; in_array[3] = ret_entrez + tmp_chro_inf -> chro_feature_table_start; in_array[4] = old_info_ptr + tmp_chro_inf -> chro_feature_table_start; merge_sort(in_array, this_chro_tab_end - tmp_chro_inf -> chro_feature_table_start, feature_sort_compare, feature_sort_exchange, feature_merge); for(sort_i = tmp_chro_inf -> chro_feature_table_start; sort_i< this_chro_tab_end ; sort_i++) { // NOW THE FEATURES (ret_start, ret_end, ret_strand, ret_entrez, old_info_ptr) ARE ALL SORTED! //printf("NT=%lu\tCHRO=%d\n", ret_start[sort_i], tmp_chro_inf->chro_number); old_info_ptr[sort_i]->sorted_order = sort_i; int feature_bin_location = ret_start[sort_i] / REVERSE_TABLE_BUCKET_LENGTH; int block_bin_location = this_block_min_start / REVERSE_TABLE_BUCKET_LENGTH; if(this_block_items && (this_block_items > features_per_block_bins[block_bin_location] || feature_bin_location != block_bin_location))//global_context -> feature_block_size) { if(current_block_id >= current_block_buffer_size - 1) { current_block_buffer_size *= 1.3; ret_block_min_start = realloc(ret_block_min_start, sizeof(srInt_64)*current_block_buffer_size); ret_block_max_end = realloc(ret_block_max_end, sizeof(srInt_64)*current_block_buffer_size); ret_block_end_index = realloc(ret_block_end_index, sizeof(srInt_64)*current_block_buffer_size); } ret_block_end_index[current_block_id] = sort_i; // FIRST UNWANTED ID ret_block_min_start[current_block_id] = this_block_min_start; ret_block_max_end[current_block_id] = this_block_max_end; register_reverse_table(current_block_id, this_block_min_start, this_block_max_end, tmp_chro_inf); //printf("B=%d; ST=%ld, END=%ld, ITM=%d\n", current_block_id, this_block_min_start, this_block_max_end, this_block_items); current_block_id++; this_block_max_end = 0; this_block_items = 0; this_block_min_start = 0x7fffffff; } this_block_max_end = max(this_block_max_end, ret_end[sort_i]); this_block_min_start = min(this_block_min_start, ret_start[sort_i]); this_block_items ++; } if(this_block_items) { if(current_block_id >= current_block_buffer_size) { current_block_buffer_size *= 1.3; ret_block_min_start = realloc(ret_block_min_start, sizeof(srInt_64)*current_block_buffer_size); ret_block_max_end = realloc(ret_block_max_end, sizeof(srInt_64)*current_block_buffer_size); ret_block_end_index = realloc(ret_block_end_index, sizeof(srInt_64)*current_block_buffer_size); } ret_block_end_index[current_block_id] = this_chro_tab_end; // FIRST UNWANTED ID ret_block_min_start[current_block_id] = this_block_min_start; ret_block_max_end[current_block_id] = this_block_max_end; register_reverse_table(current_block_id, this_block_min_start, this_block_max_end, tmp_chro_inf); current_block_id++; } (*anno_chr_head) [curr_chro_number] = current_block_id; tmp_chro_inf -> chro_block_table_end = current_block_id; free(features_per_block_bins); } (*block_end_index) = ret_block_end_index; (*block_min_start_pos) = ret_block_min_start; (*block_max_end_pos) = ret_block_max_end; //print_in_box(80, 0,0,"The %u features are sorted.\n", sort_i); free(old_info_ptr); free(tmp_chro_info_ptrs); free(chro_feature_ptr); } int strcmp_slash(char * s1, char * s2) { char nch; while(0!=(nch = *(s1++))){ if(nch == '/') break; if(nch != (*s2)) return 1; s2++; } return nch != *s2; } #define NH_FRACTION_INT 65536 unsigned int calculate_multi_overlap_fraction(fc_thread_global_context_t * global_context, unsigned int fixed_fractional_count, int maximum_total_count){ //SUBREADprintf("SSSSFRAC = %d ; FIXED / MAX = %u , %d\n", global_context -> use_fraction_multi_mapping, fixed_fractional_count, maximum_total_count); if(global_context -> use_fraction_multi_mapping) return fixed_fractional_count / maximum_total_count; else return fixed_fractional_count; } unsigned int calc_fixed_fraction(int nh){ if(nh==1) return NH_FRACTION_INT; else if(nh == 2) return NH_FRACTION_INT>>1; else return NH_FRACTION_INT / nh; } int calc_float_fraction(read_count_type_t score, read_count_type_t * integer_count, double * float_count){ if(score % NH_FRACTION_INT == 0){ (*integer_count) = score / NH_FRACTION_INT; return 0; }else{ (*float_count) = score * 1./NH_FRACTION_INT; return 1; } } void print_read_wrapping(char * rl, int is_second){ int refill_spaces = 3; int read_length = 0, x1 = 0, spaces=0; for(x1 = 0; x1 < 3100; x1++){ if(rl[x1]==0 && rl[x1+1]==0)break; if(rl[x1]=='0' || rl[x1]=='\t') spaces++; read_length ++; } char *out_buf1 = malloc(read_length + spaces * refill_spaces + 1), out_buf2[100]; int ox=0; for(x1 = 0; x1 < 3000; x1++){ if(rl[x1]=='\n' || (rl[x1]==0 && rl[x1+1]==0)){ out_buf1[ox]=0; break; } else if((rl[x1]==0 && rl[x1+1]!=0) || rl[x1] == '\t'){ int x2; for(x2 = 0; x2 < refill_spaces ; x2++){ out_buf1[ox]=' '; ox++; } } else { out_buf1[ox]=rl[x1]; ox++; } } out_buf1[ox] = 0; x1=0; while(1){ int x2; for(x2 = 0; x2 < 67 ; x2 ++){ char nch = out_buf1[x1]; if(nch == 0) break; out_buf2[x2] = nch; x1++; } out_buf2[x2] = 0; print_in_box(80,0,PRINT_BOX_NOCOLOR_FOR_COLON," %s", out_buf2); if(out_buf1[x1] == 0)break; } free(out_buf1); } void disallocate_RG_tables(void * pt){ void ** t4 = pt; free(t4[0]); free(t4[1]); if(t4[2]){ HashTableDestroy(t4[2]); HashTableDestroy(t4[3]); } free(pt); } void process_pairer_reset(void * pairer_vp){ // SUBREADprintf("RESET COUNTERS\n"); SAM_pairer_context_t * pairer = (SAM_pairer_context_t *) pairer_vp; fc_thread_global_context_t * global_context = (fc_thread_global_context_t * )pairer -> appendix1; if(global_context -> sambam_chro_table) free(global_context -> sambam_chro_table); global_context -> sambam_chro_table = NULL; global_context -> sambam_chro_table_items = 0; if(global_context -> assign_reads_to_RG) free(global_context -> RGnames_set); if(global_context -> do_scRNA_table){ SUBREADprintf("ERROR: the BAM input is incompatible with scRNA\n"); assert(0); } int xk1, xk2; for(xk1=0; xk1 thread_number; xk1++) { for(xk2=0; xk2 exontable_exons; xk2++) { global_context -> thread_contexts[xk1].count_table[xk2] = 0; } global_context -> thread_contexts[xk1].del4_added_reads = 0; global_context -> thread_contexts[xk1].all_reads = 0; global_context -> thread_contexts[xk1].nreads_mapped_to_exon = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_ambiguous = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_nofeatures = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_unmapped = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_singleton = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_read_type = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_mappingquality = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_fragmentlength = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_chimericreads = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_multimapping = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_secondary = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_junction_condition = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_duplicate = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_overlapping_length = 0; global_context -> thread_contexts[xk1].read_counters.assigned_reads = 0; global_context -> thread_contexts[xk1].read_details_buff_used = 0; if(global_context -> do_junction_counting) { HashTableDestroy(global_context -> thread_contexts[xk1].junction_counting_table); global_context -> thread_contexts[xk1].junction_counting_table = HashTableCreate(131317); HashTableSetHashFunction(global_context -> thread_contexts[xk1].junction_counting_table,HashTableStringHashFunction); HashTableSetDeallocationFunctions(global_context -> thread_contexts[xk1].junction_counting_table, free, NULL); HashTableSetKeyComparisonFunction(global_context -> thread_contexts[xk1].junction_counting_table, fc_strcmp_chro); HashTableDestroy(global_context -> thread_contexts[xk1].splicing_point_table); global_context -> thread_contexts[xk1].splicing_point_table = HashTableCreate(131317); HashTableSetHashFunction(global_context -> thread_contexts[xk1].splicing_point_table,HashTableStringHashFunction); HashTableSetDeallocationFunctions(global_context -> thread_contexts[xk1].splicing_point_table, free, NULL); HashTableSetKeyComparisonFunction(global_context -> thread_contexts[xk1].splicing_point_table, fc_strcmp_chro); } if(global_context -> assign_reads_to_RG){ HashTableDestroy(global_context -> thread_contexts[xk1].RG_table); global_context -> thread_contexts[xk1].RG_table = HashTableCreate(97); HashTableSetHashFunction(global_context -> thread_contexts[xk1].RG_table,HashTableStringHashFunction); HashTableSetDeallocationFunctions(global_context -> thread_contexts[xk1].RG_table, free, disallocate_RG_tables); HashTableSetKeyComparisonFunction(global_context -> thread_contexts[xk1].RG_table, fc_strcmp_chro); } } if(global_context -> read_details_out_FP){ int tranc_ret = ftruncate(fileno(global_context -> read_details_out_FP), 0); if(0 != tranc_ret) SUBREADprintf("ERROR: Unable to truncate assignment detail file\n"); fseek(global_context -> read_details_out_FP, 0 , SEEK_SET); } } int is_value_contig_name(char * n, int l){ int x; for(x=0; x'~' || n[x]<'!') return 0; } return 1; } #ifdef __MINGW32__ #define this_memmem windows_memmem #else #define this_memmem memmem #endif void ** get_RG_tables(fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, char * rg_name); int compress_read_detail_BAM(fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, int write_start, int write_end, char * bam_buf); void scRNA_sample_SamBam_writers_add_header(void * k, void * v, HashTable * tab){ char *bin = tab -> appendix1; int bin_len = tab -> counter1; int txt_ptr = 0, old_ptr=0, xk1, bam_is_from_cellCounts = 0; void ** vv = v; simple_bam_writer * writer = vv[0]; ArrayList * chroname_size_list = ArrayListCreate(2000); for(txt_ptr=0; txt_ptr <= bin_len; txt_ptr++){ if(bin[txt_ptr] == '\n' || txt_ptr == bin_len){ if(this_memmem(bin+old_ptr, txt_ptr - old_ptr,"Per-sample-BAM-output:cellCounts", 31))bam_is_from_cellCounts = 1; else if(memcmp("@SQ\t",bin+old_ptr, 4)==0){ unsigned int seqlen = 0; char seqname[MAX_CHROMOSOME_NAME_LEN]; int seqname_len = 0, state = 0; for(xk1=3; xk1 + old_ptr < txt_ptr; xk1++){ int nch = bin[xk1 + old_ptr]; switch(state){ case 4: if(nch == '\t' ) state = 1; else seqname[seqname_len++]=nch; break; case 14: if(isdigit(nch)) seqlen = seqlen*10+nch-'0'; else state = nch == '\t'?1:0; break; case 13: if(nch == ':') state = 14; else state = nch == '\t'?1:0; break; case 12: if(nch == 'N') state = 13; else state = nch == '\t'?1:0; break; case 3: if(nch == ':') state = 4; else state = nch == '\t'?1:0; break; case 2: if(nch == 'N') state = 3; else state = nch == '\t'?1:0; break; case 1: if(nch == 'S' && seqname_len<1) state = 2; else if(nch == 'L' && seqlen<1) state = 12; else state = nch == '\t'?1:0; break; default: if(nch == '\t') state = 1; } } seqname[seqname_len]=0; if(seqlen && seqname_len){ ArrayListPush(chroname_size_list,strdup(seqname)); ArrayListPush(chroname_size_list,NULL+seqlen); } } old_ptr = txt_ptr+1; } } char * ncoline = "@CO\tPer-sample-BAM-output:cellCounts\n"; int binlen_wtr = bin_len; if(bin[bin_len-1]!='\n') binlen_wtr++; if(!bam_is_from_cellCounts) binlen_wtr += strlen(ncoline); simple_bam_write(&binlen_wtr,4,writer, 0); simple_bam_write(bin,bin_len, writer, 0); if(bin[bin_len-1]!='\n') simple_bam_write("\n",1, writer, 0); if(!bam_is_from_cellCounts) simple_bam_write(ncoline,strlen(ncoline), writer, 0); int seq_count = chroname_size_list -> numOfElements/2; simple_bam_write(&seq_count, 4, writer, 0); for(xk1 =0; xk1 < chroname_size_list -> numOfElements; xk1+=2){ char * seqname = ArrayListGet(chroname_size_list, xk1); unsigned int seqlen = ArrayListGet(chroname_size_list, xk1+1)-NULL; int seqname_len = 1+strlen(seqname); simple_bam_write(&seqname_len,4,writer,0); simple_bam_write(seqname,seqname_len,writer,0); simple_bam_write(&seqlen,4,writer,0); free(seqname); } ArrayListDestroy(chroname_size_list); simple_bam_write("",0,writer,1); writer -> total_chromosomes = seq_count; } int process_pairer_header (void * pairer_vp, int thread_no, int is_text, unsigned int items, char * bin, unsigned int bin_len){ SAM_pairer_context_t * pairer = (SAM_pairer_context_t *) pairer_vp; fc_thread_global_context_t * global_context = (fc_thread_global_context_t * )pairer -> appendix1; fc_thread_thread_context_t * thread_context = global_context -> thread_contexts; //SUBREADprintf("ENTER PROCESS (THRD %d): IS_TXT=%d, ITEMS = %d, CURRENT_ITEMS=%d\n", thread_no, is_text, items, global_context -> sambam_chro_table_items); if(global_context -> is_scRNA_BAM_FQ_out_generated && global_context -> do_scRNA_table && is_text) { global_context -> scRNA_sample_BAM_writers -> appendix1 = bin; global_context -> scRNA_sample_BAM_writers -> counter1 = bin_len; global_context -> scRNA_sample_BAM_writers -> appendix2 = global_context; HashTableIteration( global_context -> scRNA_sample_BAM_writers, scRNA_sample_SamBam_writers_add_header); } if(global_context -> is_read_details_out == FILE_TYPE_BAM){ int write_cursor; int first_block = 1; for(write_cursor = 0; write_cursor < bin_len; write_cursor += 55000){ int wlen = min(55000, bin_len - write_cursor); if( first_block ){ if(is_text)memcpy(thread_context -> read_details_buff, "BAM\1", 4); memcpy(thread_context -> read_details_buff + (is_text?4:0), is_text?(&bin_len):(&items), 4); } memcpy(thread_context -> read_details_buff + (first_block?4*(1+is_text):0), bin + write_cursor, wlen); int blen = compress_read_detail_BAM(global_context, thread_context, 0, wlen + (first_block?4*(1+is_text):0), thread_context -> bam_compressed_buff); fwrite( thread_context -> bam_compressed_buff, 1, blen, global_context -> read_details_out_FP); first_block = 0; } }else if( global_context -> is_read_details_out == FILE_TYPE_SAM && is_text ){ fwrite( bin, 1, bin_len, global_context -> read_details_out_FP); } if(is_text ){ if( global_context -> assign_reads_to_RG ){ global_context->RGnames_capacity = 10000; global_context->RGnames_ptr = 0; global_context->RGnames_set = malloc( global_context->RGnames_capacity ); int rcursor=0; for(;rcursor 0){ int id_len = id_end - id_start; if(global_context->RGnames_capacity < global_context->RGnames_ptr + id_len + 3){ global_context->RGnames_capacity = global_context->RGnames_capacity * 17 / 10; global_context->RGnames_set = realloc( global_context->RGnames_set , global_context->RGnames_capacity ); } memcpy(global_context->RGnames_set + global_context->RGnames_ptr, bin + id_start, id_len); global_context->RGnames_set[global_context->RGnames_ptr+id_len]='\t'; global_context->RGnames_ptr += id_len+1; } } for( ;rcursorRGnames_ptr>0){ global_context->RGnames_set[global_context->RGnames_ptr-1]=0; global_context->RGnames_ptr--; } //SUBREADprintf("RGList: %s\n", global_context->RGnames_set); int thread_no; for(thread_no = 0; thread_no < global_context -> thread_number; thread_no ++){ fc_thread_thread_context_t * RGthread_context = global_context -> thread_contexts + thread_no; int RGcursor = 0; char *lastRGptr = global_context->RGnames_set; for(; RGcursor < global_context->RGnames_ptr+1; RGcursor++){ if(global_context->RGnames_set[ RGcursor ] == '\t' || global_context->RGnames_set[ RGcursor ] == 0){ global_context->RGnames_set[ RGcursor ] = 0; if(strlen(lastRGptr)>0){ // SUBREADprintf("PUT 4Tab:'%s'\n", lastRGptr); get_RG_tables(global_context, RGthread_context, lastRGptr); lastRGptr = global_context->RGnames_set + RGcursor +1; if(RGcursor < global_context->RGnames_ptr) global_context->RGnames_set[ RGcursor ] = '\t'; } } } } } }else{ if(global_context -> sambam_chro_table) global_context -> sambam_chro_table = delay_realloc(global_context -> sambam_chro_table, global_context -> sambam_chro_table_items * sizeof(SamBam_Reference_Info), (items + global_context -> sambam_chro_table_items) * sizeof(SamBam_Reference_Info)); else global_context -> sambam_chro_table = malloc(items * sizeof(SamBam_Reference_Info)); int x1, bin_ptr = 0; for(x1 = global_context -> sambam_chro_table_items; x1 < global_context -> sambam_chro_table_items+items; x1++){ int l_name; memcpy(&l_name, bin + bin_ptr, 4); bin_ptr += 4; if( !is_value_contig_name(bin + bin_ptr, l_name)){ SUBREADprintf("The chromosome name contains unexpected characters: \"%s\" (%d chars)\nfeatureCounts has to stop running\n", bin + bin_ptr, l_name); return -1; } if(l_name >= MAX_CHROMOSOME_NAME_LEN){ SUBREADprintf("The chromosome name of \"%s\" contains %d characters, longer than the upper limit of %d\nfeatureCounts has to stop running\n", bin + bin_ptr , l_name, MAX_CHROMOSOME_NAME_LEN - 1); return -1; } memcpy(global_context -> sambam_chro_table[x1].chro_name , bin + bin_ptr, l_name); //SUBREADprintf("The %d-th is '%s'\n", x1, global_context -> sambam_chro_table[x1].chro_name); bin_ptr += l_name; memcpy(&global_context -> sambam_chro_table[x1].chro_length , bin + bin_ptr, 4); bin_ptr += 4; } global_context -> sambam_chro_table_items += items; } return 0; } void process_line_buffer(fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, char * bin1, char * bin2); void make_dummy(char * rname, char * bin1, char * out_txt2, SamBam_Reference_Info * sambam_chro_table){ char * tmptr = NULL; //SUBREADprintf("S=%s ", rname); char * realname = strtok_r(rname, "\027", &tmptr); //int len_name = strlen(realname); int r1_chro = atoi(strtok_r(NULL, "\027", &tmptr)); int r1_pos = atoi(strtok_r(NULL, "\027", &tmptr)); int r2_chro = atoi(strtok_r(NULL, "\027", &tmptr)); int r2_pos = atoi(strtok_r(NULL, "\027", &tmptr)); int HItag = atoi(strtok_r(NULL, "\027", &tmptr)); int mate_FLAG = 0; memcpy(&mate_FLAG, bin1 + 16, 4); mate_FLAG = 0xffff&(mate_FLAG >>16); int mate_tlen = 0; memcpy(&mate_tlen, bin1 + 32, 4); if(r1_chro<0) r1_pos=-1; if(r2_chro<0) r2_pos=-1; int my_chro = (mate_FLAG&0x40)? r2_chro : r1_chro; int my_pos = (mate_FLAG&0x40)? r2_pos : r1_pos; int mate_chro = (mate_FLAG&0x40)? r1_chro : r2_chro; int mate_pos = (mate_FLAG&0x40)? r1_pos : r2_pos; //int bin_mq_nl = (len_name+1); int my_flag = (mate_FLAG&0x40)? 0x80:0x40; my_flag |= 1; if(mate_FLAG & 8)my_flag |=4; if(mate_FLAG & 4)my_flag |=8; if(mate_FLAG & 0x10) my_flag |= 0x20; if(mate_FLAG & 0x20) my_flag |= 0x10; char HItagStr[20]; if(HItag>=0){ sprintf(HItagStr, "\tHI:i:%d", HItag); }else{ HItagStr[0]=0; } char * my_chro_str = "*"; if(my_chro >= 0) my_chro_str = sambam_chro_table[my_chro].chro_name; char * mate_chro_str = "*"; if(mate_chro >= 0) mate_chro_str = sambam_chro_table[mate_chro].chro_name; sprintf(out_txt2, "%s\t%d\t%s\t%d\t0\t*\t%s\t%d\t0\tN\tI\t%s", realname, my_flag, my_chro_str, max(0, my_pos), mate_chro_str, max(0,mate_pos), HItagStr); } int reverse_flag(int mf){ int ret = mf & 3; if(mf & 4) ret |= 8; if(mf & 8) ret |= 4; if((mf & 1)==0) ret |= 4; if(mf & 0x10) ret |= 0x20; if(mf & 0x20) ret |= 0x10; if(mf & 0x40) ret |= 0x80; if(mf & 0x80) ret |= 0x40; return ret; } int calc_total_frag_one_len(CIGAR_interval_t * intvs, int intvn, char * read_name){ int ret = 0, x1; for(x1 = 0; x1 < intvn; x1++){ int x2; //#warning "=========== DEBUG OUT ==============" if(0 && FIXLENstrcmp("V0112_0155:7:1101:20072:12961#ATCAC", read_name)==0){ SUBREADprintf("READ %s SINGLE: chro_len = %d, secs = %d\n" , read_name, intvs[x1].chromosomal_length, intvs[x1].insertions); } for(x2 = 0; x2 < intvs[x1].insertions; x2++) ret += intvs[x1].insertion_lengths[x2]; ret += intvs[x1].chromosomal_length; } return ret; } int calc_total_has_overlap(unsigned int r1_start, unsigned int r1_end, unsigned int r2_start, unsigned int r2_end, unsigned int * overlap_start, unsigned int * overlap_end){ if((r1_start <= r2_start && r1_end > r2_start) || (r2_start <= r1_start && r2_end > r1_start) ){ (*overlap_start) = max( r1_start, r2_start ); (*overlap_end) = min( r1_end, r2_end ); return 1; } return 0; } int calc_total_frag_len( fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, CIGAR_interval_t * CIGAR_intervals_R1, int CIGAR_intervals_R1_sections, CIGAR_interval_t * CIGAR_intervals_R2, int CIGAR_intervals_R2_sections, char * read_name){ if ( CIGAR_intervals_R1_sections == 0 && CIGAR_intervals_R2_sections > 0) return calc_total_frag_one_len( CIGAR_intervals_R2,CIGAR_intervals_R2_sections , read_name); else if( CIGAR_intervals_R1_sections > 0 && CIGAR_intervals_R2_sections== 0) return calc_total_frag_one_len( CIGAR_intervals_R1,CIGAR_intervals_R1_sections , read_name); else if( CIGAR_intervals_R1_sections == 0 && CIGAR_intervals_R2_sections== 0) return 0; if(CIGAR_intervals_R1_sections > 0 && CIGAR_intervals_R2_sections > 0 && strcmp(CIGAR_intervals_R1[0].chro, CIGAR_intervals_R2[0].chro )!=0 ) // two reads are from different chromosomes return calc_total_frag_one_len( CIGAR_intervals_R2,CIGAR_intervals_R2_sections , read_name) + calc_total_frag_one_len( CIGAR_intervals_R1,CIGAR_intervals_R1_sections , read_name); if(0 && FIXLENstrcmp("V0112_0155:7:1101:20072:12961#ATCAC", read_name)==0){ int xx; for(xx = 0; xx < CIGAR_intervals_R1_sections; xx++) SUBREADprintf("R1 SEC %d: %u + %d\n", xx, CIGAR_intervals_R1[xx].start_pos, CIGAR_intervals_R1[xx].chromosomal_length ); for(xx = 0; xx < CIGAR_intervals_R2_sections; xx++) SUBREADprintf("R2 SEC %d: %u + %d\n", xx, CIGAR_intervals_R2[xx].start_pos, CIGAR_intervals_R2[xx].chromosomal_length ); } unsigned int merged_section_count = 0; unsigned short merged_section_lengths[ MAXIMUM_INSERTION_IN_SECTION * 3 ]; unsigned int merged_section_indel_counts[ MAXIMUM_INSERTION_IN_SECTION * 3 ]; unsigned short merged_section_indel_lengths[ MAXIMUM_INSERTION_IN_SECTION * 3 ][ MAXIMUM_INSERTION_IN_SECTION ]; int R1_i = 0 , R2_i = 0; while (1){ //SUBREADprintf("FRAGDEBUG %s : %d < %d & %d < %d; MC=%d; INS1=%d INS2=%d\n", read_name, R1_i,CIGAR_intervals_R1_sections,R2_i,CIGAR_intervals_R2_sections, merged_section_count, CIGAR_intervals_R1[R1_i].insertions, CIGAR_intervals_R2[R2_i].insertions); if( R1_i >= CIGAR_intervals_R1_sections && R2_i >= CIGAR_intervals_R2_sections ) break; if( R1_i < CIGAR_intervals_R1_sections && R2_i < CIGAR_intervals_R2_sections){ // see if R1 and R2 overlap // if not: add R2 to specific sction; R2_i ++ // elif overlap: add the R1 first_half and/or R2 first_half or zero to specific section, and add overlapping part to overlapping section; DO NOT add the second specific half! // if R1_end > R2_end: R1_section_start = overlapping_end; R2_i ++ // elif R2_end > R1_end: R2_section_start = overlapping_end; R1_i ++ // elif R2_end == R1_end: R1_i++; R2_i++ unsigned int overlapping_start= 0 , overlapping_end = 0; int is_r1r2_overlap = 0; is_r1r2_overlap = calc_total_has_overlap( CIGAR_intervals_R1[R1_i].start_pos, CIGAR_intervals_R1[R1_i].start_pos + CIGAR_intervals_R1[R1_i].chromosomal_length , CIGAR_intervals_R2[R2_i].start_pos, CIGAR_intervals_R2[R2_i].start_pos + CIGAR_intervals_R2[R2_i].chromosomal_length , & overlapping_start , & overlapping_end); if( is_r1r2_overlap ){ if (CIGAR_intervals_R1[R1_i].start_pos > CIGAR_intervals_R2[R2_i].start_pos ){ //first half_R2 add special merged_section_lengths[merged_section_count] = overlapping_start - CIGAR_intervals_R2[R2_i].start_pos; int indel_i; for(indel_i = 0; indel_i < min(MAXIMUM_INSERTION_IN_SECTION,CIGAR_intervals_R2[R2_i].insertions); indel_i++){ if( CIGAR_intervals_R2[R2_i].insertion_start_pos[indel_i] >= overlapping_start ){ if(indel_i>0){ int insmov_i, ins_dist_i = 0; for(insmov_i = indel_i ; insmov_i < CIGAR_intervals_R2[R2_i].insertions; insmov_i++){ assert(MAXIMUM_INSERTION_IN_SECTION > ins_dist_i); assert(MAXIMUM_INSERTION_IN_SECTION > insmov_i); CIGAR_intervals_R2[R2_i].insertion_start_pos[ins_dist_i] = CIGAR_intervals_R2[R2_i].insertion_start_pos[insmov_i]; CIGAR_intervals_R2[R2_i].insertion_lengths[ins_dist_i] = CIGAR_intervals_R2[R2_i].insertion_lengths[insmov_i]; ins_dist_i++; } CIGAR_intervals_R2[R2_i].insertions = ins_dist_i; } break; } merged_section_indel_lengths[merged_section_count][indel_i] = CIGAR_intervals_R2[R2_i].insertion_lengths[indel_i]; } merged_section_indel_counts[merged_section_count] = indel_i; merged_section_count ++; }else if( CIGAR_intervals_R1[R1_i].start_pos < CIGAR_intervals_R2[R2_i].start_pos ){ //first half_R1 add special merged_section_lengths[merged_section_count] = overlapping_start - CIGAR_intervals_R1[R1_i].start_pos; int indel_i; for(indel_i = 0; indel_i < min(MAXIMUM_INSERTION_IN_SECTION,CIGAR_intervals_R1[R1_i].insertions); indel_i++){ if( CIGAR_intervals_R1[R1_i].insertion_start_pos[indel_i] >= overlapping_start ){ if(indel_i>0){ int insmov_i, ins_dist_i = 0; for(insmov_i = indel_i ; insmov_i < CIGAR_intervals_R1[R1_i].insertions; insmov_i++){ assert(MAXIMUM_INSERTION_IN_SECTION > insmov_i); CIGAR_intervals_R1[R1_i].insertion_start_pos[ins_dist_i] = CIGAR_intervals_R1[R1_i].insertion_start_pos[insmov_i]; CIGAR_intervals_R1[R1_i].insertion_lengths[ins_dist_i] = CIGAR_intervals_R1[R1_i].insertion_lengths[insmov_i]; ins_dist_i++; } CIGAR_intervals_R1[R1_i].insertions = ins_dist_i; } break; } merged_section_indel_lengths[merged_section_count][indel_i] = CIGAR_intervals_R1[R1_i].insertion_lengths[indel_i]; } merged_section_indel_counts[merged_section_count] = indel_i; merged_section_count ++; } merged_section_lengths[merged_section_count] = overlapping_end - overlapping_start; merged_section_indel_counts[merged_section_count] = 0; int indel_i_R1 = 0, indel_i_R2 = 0; while(1){ //SUBREADprintf("FRAGDEBUG: CC[%d] = %d ; II1=%d < %d; II2=%d < %d\n", merged_section_count, merged_section_indel_counts[merged_section_count], indel_i_R1, CIGAR_intervals_R1[R1_i].insertions , indel_i_R2, CIGAR_intervals_R2[R2_i].insertions); if( indel_i_R1 >= CIGAR_intervals_R1[R1_i].insertions || indel_i_R2 >= CIGAR_intervals_R2[R2_i].insertions ){ if(indel_i_R1 > 0){ int insmov_i, ins_dist_i = 0; for(insmov_i = indel_i_R1 ; insmov_i < min(MAXIMUM_INSERTION_IN_SECTION,CIGAR_intervals_R1[R1_i].insertions); insmov_i++){ assert(MAXIMUM_INSERTION_IN_SECTION > insmov_i); CIGAR_intervals_R1[R1_i].insertion_start_pos[ins_dist_i] = CIGAR_intervals_R1[R1_i].insertion_start_pos[insmov_i]; CIGAR_intervals_R1[R1_i].insertion_lengths[ins_dist_i] = CIGAR_intervals_R1[R1_i].insertion_lengths[insmov_i]; ins_dist_i++; } CIGAR_intervals_R1[R1_i].insertions = ins_dist_i; } if(indel_i_R2 > 0){ int insmov_i, ins_dist_i = 0; for(insmov_i = indel_i_R2 ; insmov_i < min(CIGAR_intervals_R2[R2_i].insertions,MAXIMUM_INSERTION_IN_SECTION); insmov_i++){ assert(MAXIMUM_INSERTION_IN_SECTION > insmov_i); CIGAR_intervals_R2[R2_i].insertion_start_pos[ins_dist_i] = CIGAR_intervals_R2[R2_i].insertion_start_pos[insmov_i]; CIGAR_intervals_R2[R2_i].insertion_lengths[ins_dist_i] = CIGAR_intervals_R2[R2_i].insertion_lengths[insmov_i]; ins_dist_i++; } CIGAR_intervals_R2[R2_i].insertions = ins_dist_i; } break; } if( CIGAR_intervals_R1[R1_i].insertion_start_pos[indel_i_R1] > CIGAR_intervals_R2[R2_i].insertion_start_pos[indel_i_R2] ) indel_i_R2 ++; else if( CIGAR_intervals_R1[R1_i].insertion_start_pos[indel_i_R1] < CIGAR_intervals_R2[R2_i].insertion_start_pos[indel_i_R2] ) indel_i_R1 ++; else{ if( CIGAR_intervals_R1[R1_i].insertion_lengths[ indel_i_R1 ] == CIGAR_intervals_R2[R2_i].insertion_lengths[ indel_i_R2 ] ){ merged_section_indel_lengths[merged_section_count][ merged_section_indel_counts[merged_section_count] ] = CIGAR_intervals_R1[R1_i].insertion_lengths[indel_i_R1]; merged_section_indel_counts[merged_section_count] ++; } indel_i_R2++; indel_i_R1++; } } merged_section_count ++; // add common if(CIGAR_intervals_R1[R1_i].start_pos + CIGAR_intervals_R1[R1_i].chromosomal_length > CIGAR_intervals_R2[R2_i].start_pos + CIGAR_intervals_R2[R2_i].chromosomal_length){ CIGAR_intervals_R1[R1_i].chromosomal_length -= ( overlapping_end - CIGAR_intervals_R1[R1_i].start_pos ); CIGAR_intervals_R1[R1_i].start_pos = overlapping_end; R2_i ++; }else if(CIGAR_intervals_R1[R1_i].start_pos + CIGAR_intervals_R1[R1_i].chromosomal_length < CIGAR_intervals_R2[R2_i].start_pos + CIGAR_intervals_R2[R2_i].chromosomal_length){ CIGAR_intervals_R2[R2_i].chromosomal_length -= ( overlapping_end - CIGAR_intervals_R2[R2_i].start_pos ); CIGAR_intervals_R2[R2_i].start_pos = overlapping_end; R1_i ++; }else{ R1_i ++; R2_i ++; } }else if(CIGAR_intervals_R1[R1_i].start_pos > CIGAR_intervals_R2[R2_i].start_pos){ merged_section_lengths[merged_section_count] = CIGAR_intervals_R2[R2_i].chromosomal_length; int indel_i; for(indel_i = 0; indel_i < CIGAR_intervals_R2[R2_i].insertions; indel_i++){ merged_section_indel_lengths[merged_section_count][indel_i] = CIGAR_intervals_R2[R2_i].insertion_lengths[indel_i]; } merged_section_indel_counts[merged_section_count] = CIGAR_intervals_R2[R2_i].insertions; merged_section_count ++; R2_i ++; }else{ merged_section_lengths[merged_section_count] = CIGAR_intervals_R1[R1_i].chromosomal_length; int indel_i; for(indel_i = 0; indel_i < CIGAR_intervals_R1[R1_i].insertions; indel_i++){ merged_section_indel_lengths[merged_section_count][indel_i] = CIGAR_intervals_R1[R1_i].insertion_lengths[indel_i]; } merged_section_indel_counts[merged_section_count] = CIGAR_intervals_R1[R1_i].insertions; merged_section_count ++; R1_i ++; } }else if(R1_i < CIGAR_intervals_R1_sections){ // add R1 section to specific section // R1_i ++ merged_section_lengths[merged_section_count] = CIGAR_intervals_R1[R1_i].chromosomal_length; int indel_i; for(indel_i = 0; indel_i < CIGAR_intervals_R1[R1_i].insertions; indel_i++){ merged_section_indel_lengths[merged_section_count][indel_i] = CIGAR_intervals_R1[R1_i].insertion_lengths[indel_i]; } merged_section_indel_counts[merged_section_count] = CIGAR_intervals_R1[R1_i].insertions; merged_section_count ++; R1_i ++; }else if(R2_i < CIGAR_intervals_R2_sections){ merged_section_lengths[merged_section_count] = CIGAR_intervals_R2[R2_i].chromosomal_length; int indel_i; for(indel_i = 0; indel_i < CIGAR_intervals_R2[R2_i].insertions; indel_i++){ merged_section_indel_lengths[merged_section_count][indel_i] = CIGAR_intervals_R2[R2_i].insertion_lengths[indel_i]; } merged_section_indel_counts[merged_section_count] = CIGAR_intervals_R2[R2_i].insertions; merged_section_count ++; R2_i ++; } } int ret = 0, x1, x2; for(x1 = 0; x1 < merged_section_count ; x1++){ ret += merged_section_lengths[x1]; for(x2 = 0; x2 < merged_section_indel_counts[x1]; x2++) ret += merged_section_indel_lengths[x1][x2]; // SUBREADprintf("FRAGDEBUG %s [%d] : len = %d , indels = %d\n" , read_name, x1, merged_section_lengths[x1] , merged_section_indel_counts[x1]); } return ret; } void get_readname_from_bin(char * bin, char ** read_name){ (*read_name) = bin + 36; } void parse_bin(SamBam_Reference_Info * sambam_chro_table, char * bin, char * bin2, char ** read_name, int * flag, char ** chro, srInt_64 * pos, int * mapq, char ** mate_chro, srInt_64 * mate_pos, srInt_64 * tlen, int * is_junction_read, int * cigar_sect, unsigned int * Starting_Chro_Points_1BASE, unsigned short * Starting_Read_Points, unsigned short * Section_Read_Lengths, char ** ChroNames, char * Event_After_Section, int * NH_value, int max_M, CIGAR_interval_t * intervals_buffer, int * intervals_i, int assign_reads_to_RG, char ** RG_ptr, int * ret_me_refID, int * ret_mate_refID){ int x1, len_of_S1 = 0; *cigar_sect = 0; *NH_value = 1; *flag = 0; *is_junction_read = 0; assert(bin||bin2); if(bin){ (*read_name) = bin + 36; memcpy(flag, bin + 16, 4); int cigar_opts = (*flag) & 0xffff; (*flag) = (*flag) >> 16; int refID, mate_refID; memcpy(&refID, bin + 4, 4); if(refID >= 0) (*chro) = sambam_chro_table[refID].chro_name; else (*chro) = NULL; (*pos) = 0; memcpy(pos, bin+8, 4); (*pos) ++; memcpy(mapq, bin+12, 4); int l_read_name = (*mapq)& 0xff; (*mapq) = ((*mapq)>>8)&0xff; int seq_len; memcpy(&seq_len, bin + 20,4); memcpy(&mate_refID, bin+24, 4); if(mate_refID>=0) (*mate_chro) = sambam_chro_table[mate_refID].chro_name; else (*mate_chro) = NULL; *ret_mate_refID = mate_refID; *ret_me_refID = refID; (*mate_pos)=0; memcpy(mate_pos, bin+28, 4); (*mate_pos)++; int tlen_int; memcpy(&tlen_int, bin+32, 4); (*tlen) = tlen_int; int * cigar_opt_ints = (int *)(bin + 36 + l_read_name); unsigned int chro_cursor = (*pos), section_start_chro = (*pos); unsigned short read_cursor = 0, this_section_length = 0, section_start_read = 0; if(intervals_buffer){ intervals_buffer[ *intervals_i ].start_pos = chro_cursor; intervals_buffer[ *intervals_i ].chro = *chro; } for(x1 = 0 ; x1 < cigar_opts; x1++){ int optype = cigar_opt_ints[x1]&0xf; int optval = (cigar_opt_ints[x1]>>4)& 0xfffffff; if(optype == 0 || optype == 7 || optype == 8){ // 'M' , '=', 'X' chro_cursor += optval; read_cursor += optval; this_section_length += optval; /* }else if(optype == 1){ // 'I' read_cursor += optval; }else if(optype == 2){ // 'D' chro_cursor += optval; */ }else if(optype == 1 || optype == 2 || optype == 3){ // 'I', 'D' or 'N' if(3 == optype) (*is_junction_read) = 1; char event_char=0; if(optype == 3) event_char = 'N'; if(optype == 2) event_char = 'D'; else if(optype == 1){ if(intervals_buffer && intervals_buffer[ *intervals_i ].insertions < MAXIMUM_INSERTION_IN_SECTION){ intervals_buffer[ *intervals_i ].insertion_start_pos[ intervals_buffer[ *intervals_i ].insertions ] = chro_cursor; intervals_buffer[ *intervals_i ].insertion_lengths[ intervals_buffer[ *intervals_i ].insertions ] = optval; intervals_buffer[ *intervals_i ].insertions ++; } event_char = 'I'; } if( (*cigar_sect) < max_M){ Event_After_Section[*cigar_sect] = event_char; Starting_Chro_Points_1BASE[*cigar_sect] = section_start_chro; Starting_Read_Points[*cigar_sect] = section_start_read; Section_Read_Lengths[*cigar_sect] = this_section_length; ChroNames[*cigar_sect] = (*chro); (*cigar_sect)++; if(intervals_buffer){ intervals_buffer[ *intervals_i ].chromosomal_length = chro_cursor - intervals_buffer[ *intervals_i ].start_pos; (*intervals_i) ++; } } if(optype == 2 || optype == 3)// N or D chro_cursor += optval; else read_cursor += optval; if(intervals_buffer && (*cigar_sect) < max_M){ intervals_buffer[ *intervals_i ].start_pos = chro_cursor; intervals_buffer[ *intervals_i ].chro = *chro; } section_start_chro = chro_cursor; section_start_read = read_cursor; this_section_length = 0; }else if(optype == 4){ // 'S' if(read_cursor==0) { read_cursor += optval; section_start_read = read_cursor; if(intervals_buffer){ if(intervals_buffer[ *intervals_i ].start_pos > optval) intervals_buffer[ *intervals_i ].start_pos -= optval; else intervals_buffer[ *intervals_i ].start_pos = 0; } }else len_of_S1 = optval; } // H and P do not have effect on cigar parsing. } if(this_section_length>0){ // add new section if( (*cigar_sect) < max_M){ if(intervals_buffer){ intervals_buffer[ *intervals_i ].chromosomal_length = chro_cursor - intervals_buffer[ *intervals_i ].start_pos + len_of_S1; (*intervals_i)++; } Starting_Chro_Points_1BASE[*cigar_sect] = section_start_chro; Starting_Read_Points[*cigar_sect] = section_start_read; Section_Read_Lengths[*cigar_sect] = this_section_length ; ChroNames[*cigar_sect] = (*chro); (*cigar_sect)++; } } int bin_ptr = 36 + l_read_name + seq_len + (seq_len+1)/2 + 4 * cigar_opts; int block_len; memcpy(&block_len, bin, 4); int found_NH = SAM_pairer_iterate_int_tags((unsigned char *)bin+bin_ptr, block_len + 4 - bin_ptr, "NH", NH_value); if(!found_NH) *(NH_value) = 1; if(assign_reads_to_RG){ char RG_type = 0; SAM_pairer_iterate_tags((unsigned char *)bin+bin_ptr, block_len + 4 - bin_ptr, "RG", &RG_type, RG_ptr); if(RG_type != 'Z') (*RG_ptr) = NULL; } //SUBREADprintf("FOUND=%d, NH=%d, TAG=%.*s\n", found_NH, *(NH_value), 3 , bin+bin_ptr); }else{ (*read_name) = bin2 + 36; int mate_flag; memcpy(&mate_flag, bin2 + 16, 4); mate_flag = mate_flag >> 16; (*flag) = reverse_flag(mate_flag); int refID, mate_refID; memcpy(&refID, bin2 + 24, 4); memcpy(&mate_refID, bin2 + 4, 4); if(refID < 0) *chro = NULL; else (*chro) = sambam_chro_table[refID].chro_name; if(mate_refID < 0) *mate_chro = NULL; else (*mate_chro) = sambam_chro_table[mate_refID].chro_name; *ret_mate_refID = mate_refID; *ret_me_refID = refID; *pos=0; memcpy(pos, bin2+28, 4); (*pos)++; *mate_pos=0; memcpy(mate_pos, bin2+8, 4); (*mate_pos)++; (*tlen) = 0; memcpy(tlen, bin2+32, 4); (*tlen) = -(*tlen); if(assign_reads_to_RG){ char RG_type = 0; int block2_len = 0; memcpy(&block2_len, bin2, 4); int rname2len = 0, cigar2len = 0, seq2len = 0; memcpy(&rname2len, bin2+12, 1); memcpy(&cigar2len, bin2+16, 2); memcpy(&seq2len, bin2+20, 4); int bin2_ptr = 36 + rname2len + 4 * cigar2len + seq2len + (seq2len+1)/2; SAM_pairer_iterate_tags((unsigned char *)bin2+bin2_ptr, block2_len + 4 - bin2_ptr, "RG", &RG_type, RG_ptr); if(RG_type != 'Z') (*RG_ptr) = NULL; } } } /* typedef struct { char chromosome_name_left[CHROMOSOME_NAME_LENGTH + 1]; char chromosome_name_right[CHROMOSOME_NAME_LENGTH + 1]; unsigned int last_exon_base_left; unsigned int first_exon_base_right; } fc_junction_info_t; */ int calc_junctions_from_cigarInts(fc_thread_global_context_t * global_context, int alignment_masks , int cigar_sections, unsigned int * Starting_Chro_Points_1BASE, unsigned short * Starting_Read_Points, unsigned short * Section_Lengths, char ** ChroNames, char * Event_After_Section, fc_junction_info_t * junctions_current){ int x1, ret = 0; unsigned int last_base_pos = Starting_Chro_Points_1BASE[0] + Section_Lengths[0] - 1; for(x1 = 1; x1 < cigar_sections; x1++){ if(!ChroNames[x1]) continue; // NULL chro name for https://groups.google.com/forum/#!topic/subread/QDT6npjAZuE if(Event_After_Section[x1-1] == 'N'){ unsigned int first_base_pos = Starting_Chro_Points_1BASE[x1]; junctions_current[ret].last_exon_base_left = last_base_pos; junctions_current[ret].first_exon_base_right = first_base_pos; strcpy(junctions_current[ret].chromosome_name_left, ChroNames[x1]); strcpy(junctions_current[ret].chromosome_name_right, ChroNames[x1]); ret ++; } last_base_pos = Starting_Chro_Points_1BASE[x1] + Section_Lengths[x1] - 1; } return ret; } void add_fragment_supported_junction( fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, fc_junction_info_t * supported_junctions1, int njunc1, fc_junction_info_t * supported_junctions2, int njunc2, char * RG_name); void process_line_junctions(fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, char * bin1, char * bin2) { fc_junction_info_t supported_junctions1[global_context -> max_M], supported_junctions2[global_context -> max_M]; int is_second_read, njunc1=0, njunc2=0, is_junction_read, cigar_sections; int alignment_masks, mapping_qual, NH_value; char *RG_ptr=NULL; for(is_second_read = 0 ; is_second_read < 2; is_second_read++){ char * read_chr, *read_name, *mate_chr; srInt_64 read_pos, fragment_length = 0, mate_pos; unsigned int Starting_Chro_Points_1BASE[global_context -> max_M]; unsigned short Starting_Read_Points[global_context -> max_M]; unsigned short Section_Read_Lengths[global_context -> max_M]; char * ChroNames[global_context -> max_M]; char Event_After_Section[global_context -> max_M]; if(is_second_read && !global_context -> is_paired_end_mode_assign) break; char * RG_ptr_one = NULL; int me_refID, mate_refID; parse_bin(global_context -> sambam_chro_table, is_second_read?bin2:bin1, is_second_read?bin1:bin2 , &read_name, &alignment_masks , &read_chr, &read_pos, &mapping_qual, &mate_chr, &mate_pos, &fragment_length, &is_junction_read, &cigar_sections, Starting_Chro_Points_1BASE, Starting_Read_Points, Section_Read_Lengths, ChroNames, Event_After_Section, &NH_value, global_context -> max_M, NULL, NULL, global_context -> assign_reads_to_RG, &RG_ptr_one, &me_refID, &mate_refID); assert(cigar_sections <= global_context -> max_M); if(RG_ptr_one) RG_ptr = RG_ptr_one; int * njunc_current = is_second_read?&njunc2:&njunc1; fc_junction_info_t * junctions_current = is_second_read?supported_junctions2:supported_junctions1; (*njunc_current) = calc_junctions_from_cigarInts(global_context, alignment_masks , cigar_sections, Starting_Chro_Points_1BASE, Starting_Read_Points, Section_Read_Lengths, ChroNames, Event_After_Section, junctions_current); //if(0 && FIXLENstrcmp("HWI-ST212:219:C0C1TACXX:1:1101:13391:171460", read_name)==0){ // SUBREADprintf("JUNC_FOUND_IN_READ OF %s : %d\n", read_name , *njunc_current); //} } if(njunc1 >0 || njunc2>0) add_fragment_supported_junction(global_context, thread_context, supported_junctions1, njunc1, supported_junctions2, njunc2, RG_ptr); } void ** get_RG_tables(fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, char * rg_name){ void ** ret = HashTableGet(thread_context->RG_table, rg_name); if(ret) return ret; ret = malloc(sizeof(void *)*4); ret[0] = malloc(thread_context -> count_table_size * sizeof(read_count_type_t)); ret[1] = malloc(sizeof(fc_read_counters)); memset(ret[0], 0, thread_context -> count_table_size * sizeof(read_count_type_t)); memset(ret[1], 0, sizeof(fc_read_counters)); if(global_context -> do_junction_counting){ HashTable * junction_counting_table = HashTableCreate(131317); HashTableSetHashFunction(junction_counting_table,HashTableStringHashFunction); HashTableSetDeallocationFunctions(junction_counting_table, free, NULL); HashTableSetKeyComparisonFunction(junction_counting_table, fc_strcmp_chro); HashTable * splicing_point_table = HashTableCreate(131317); HashTableSetHashFunction(splicing_point_table,HashTableStringHashFunction); HashTableSetDeallocationFunctions(splicing_point_table, free, NULL); HashTableSetKeyComparisonFunction(splicing_point_table, fc_strcmp_chro); ret [2] = junction_counting_table; ret [3] = splicing_point_table; }else ret[2] = NULL; char * rg_name_mem = malloc(strlen(rg_name)+1); strcpy(rg_name_mem, rg_name); HashTablePut(thread_context->RG_table, rg_name_mem, ret); return ret; } void add_scRNA_read_tota1_no( fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, char * read_name, char * bin1, int step); void process_scRNAr2_line_buffer(fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, char * bin1, char * bin2){ char * read_name = bin1+36; add_scRNA_read_tota1_no(global_context, thread_context, read_name, bin1, 2); } int process_pairer_scRNAr2_output(void * pairer_vp, int thread_no, char * bin1, char * bin2){ SAM_pairer_context_t * pairer = (SAM_pairer_context_t *) pairer_vp; fc_thread_global_context_t * global_context = (fc_thread_global_context_t * )pairer -> appendix1; fc_thread_thread_context_t * thread_context = global_context -> thread_contexts + thread_no; process_scRNAr2_line_buffer(global_context, thread_context, bin1, bin2); return 0; } int process_pairer_output(void * pairer_vp, int thread_no, char * bin1, char * bin2){ SAM_pairer_context_t * pairer = (SAM_pairer_context_t *) pairer_vp; fc_thread_global_context_t * global_context = (fc_thread_global_context_t * )pairer -> appendix1; fc_thread_thread_context_t * thread_context = global_context -> thread_contexts + thread_no; if(pairer -> long_cigar_mode){ if(global_context -> max_M < 65536){ //SUBREADprintf("SWITCHED INTO LONG-READ MODE\n"); global_context -> max_M = 65536; } if(!global_context->is_read_too_long_to_SAM_BAM_shown &&(global_context -> is_read_details_out == FILE_TYPE_SAM || global_context -> is_read_details_out == FILE_TYPE_BAM)){ global_context -> is_read_details_out = 0; SUBREADprintf("ERROR: The read is too long to the SAM or BAM output.\nPlease use the 'CORE' mode for the assignment detail output.\n"); global_context->is_read_too_long_to_SAM_BAM_shown = 1; } } process_line_buffer(global_context, thread_context, bin1, bin2); if(0 && global_context -> do_junction_counting){ process_line_junctions(global_context, thread_context, bin1, bin2); } return 0; } void sort_bucket_table(fc_thread_global_context_t * global_context); void vote_and_add_count(fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, srInt_64 * hits_indices1, int nhits1, srInt_64 * hits_indices2, int nhits2, unsigned int total_frag_len, char ** hits_chro1, char ** hits_chro2, unsigned int * hits_start_pos1, unsigned int * hits_start_pos2, unsigned short * hits_length1, unsigned short * hits_length2, int fixed_fractional_count, char * read_name, char * RG_name, char * bin1, char * bin2); void add_bin_new_tags(char * oldbin, char **newbin, char ** tags, char * types, void ** vals){ int new_tags_length = 0; int tagi; for(tagi = 0; tags[tagi]; tagi++){ char type = types[tagi]; if(type == 'i') new_tags_length += 7; else new_tags_length += 4 + strlen((char *)vals[tagi]); } int oldbin_len; memcpy(&oldbin_len, oldbin, 4); oldbin_len += 4; int newbin_len = oldbin_len + new_tags_length; (*newbin) = malloc(newbin_len); memcpy(*newbin, oldbin, oldbin_len); newbin_len -= 4; memcpy(*newbin, &newbin_len, 4); newbin_len += 4; for(tagi = 0; tags[tagi]; tagi++){ memcpy( (*newbin) + oldbin_len, tags[tagi] ,2); (*newbin)[oldbin_len+2] = types[tagi]; if(types[tagi] == 'i'){ int intv = vals[tagi] - NULL; memcpy((*newbin) + oldbin_len + 3, &intv, 4); oldbin_len += 7; }else{ int vlen = strlen((char *)(vals[tagi]))+1; memcpy((*newbin) + oldbin_len + 3, vals[tagi], vlen); oldbin_len += 3 + vlen; } } } int compress_read_detail_BAM(fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, int write_start, int write_end, char * bam_buf){ if(global_context -> is_read_details_out == FILE_TYPE_SAM){ // there MUST be only one read in the buffer. int write_ptr = write_start; int tmplen = 0 ; int sam_ptr = 0; while(1){ if(write_ptr >= write_end) break; memcpy(&tmplen, thread_context -> read_details_buff + write_ptr, 4); tmplen +=4; int txtlen = convert_BAM_binary_to_SAM(global_context -> sambam_chro_table, thread_context -> read_details_buff + write_ptr, bam_buf + sam_ptr); bam_buf[sam_ptr + txtlen] = '\n'; bam_buf[sam_ptr + txtlen + 1] = 0; sam_ptr += txtlen + 1; write_ptr += tmplen; } return sam_ptr; }else{ // there may be multiple reads in the buffer. int bin_len = write_end - write_start; char * compressed_buff = bam_buf + 18; int compressed_size ; unsigned int CRC32; thread_context -> bam_file_output_stream.avail_out = 66600; thread_context -> bam_file_output_stream.avail_in = bin_len; //SUBREADprintf("COMPRESS PTR=%p , LEN=%d\n", thread_context -> read_details_buff + write_start , bin_len); CRC32 = FC_CRC32(thread_context -> read_details_buff + write_start , bin_len); int Z_DEFAULT_MEM_LEVEL = 8; thread_context -> bam_file_output_stream.zalloc = Z_NULL; thread_context -> bam_file_output_stream.zfree = Z_NULL; thread_context -> bam_file_output_stream.opaque = Z_NULL; deflateInit2(&thread_context -> bam_file_output_stream, bin_len?Z_BEST_SPEED:Z_DEFAULT_COMPRESSION, Z_DEFLATED, -15, Z_DEFAULT_MEM_LEVEL, Z_DEFAULT_STRATEGY); thread_context -> bam_file_output_stream.next_in = (unsigned char*) thread_context -> read_details_buff + write_start; thread_context -> bam_file_output_stream.next_out = (unsigned char*) compressed_buff; deflate(&thread_context -> bam_file_output_stream, Z_FINISH); deflateEnd(&thread_context -> bam_file_output_stream); compressed_size = 66600 -thread_context -> bam_file_output_stream.avail_out; bam_buf[0]=31; bam_buf[1]=-117; bam_buf[2]=8; bam_buf[3]=4; memset(bam_buf+4, 0, 5); bam_buf[9] = 0xff; // OS int tmpi = 6; memcpy(bam_buf+10, &tmpi, 2); //XLSN bam_buf[12]=66; // SI1 bam_buf[13]=67; // SI2 tmpi = 2; memcpy(bam_buf+14, &tmpi, 2); //BSIZE tmpi = compressed_size + 19 + 6; memcpy(bam_buf+16, &tmpi, 2); //BSIZE memcpy(bam_buf+18+compressed_size, &CRC32, 4); memcpy(bam_buf+18+compressed_size+4, &bin_len, 4); return 18+compressed_size+8; } } void write_read_detailed_remainder(fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context){ int write_bin_ptr = 0; int last_written_ptr = 0; int bam_compressed_buff_ptr = 0; if(thread_context -> read_details_buff_used <1)return; if(global_context -> is_read_details_out == FILE_TYPE_BAM && thread_context -> read_details_buff_used < 64000){ bam_compressed_buff_ptr = compress_read_detail_BAM(global_context, thread_context, 0, thread_context -> read_details_buff_used, thread_context -> bam_compressed_buff); }else while(1){ if(write_bin_ptr >= thread_context -> read_details_buff_used ) break; int tmplen = 0; memcpy(&tmplen, thread_context -> read_details_buff + write_bin_ptr, 4); if(tmplen < 9 || tmplen > 3*MAX_FC_READ_LENGTH){ SUBREADprintf("ERROR: Format error : len = %d\n", tmplen); //oexit(-1); return ; } tmplen +=4; write_bin_ptr += tmplen; if(write_bin_ptr - last_written_ptr > 64000 || write_bin_ptr >= thread_context -> read_details_buff_used || global_context -> is_read_details_out == FILE_TYPE_SAM){ bam_compressed_buff_ptr += compress_read_detail_BAM(global_context, thread_context, last_written_ptr, write_bin_ptr, thread_context -> bam_compressed_buff + bam_compressed_buff_ptr); last_written_ptr = write_bin_ptr; } } pthread_spin_lock(&global_context -> read_details_out_lock); fwrite(thread_context -> bam_compressed_buff, 1, bam_compressed_buff_ptr , global_context -> read_details_out_FP); pthread_spin_unlock(&global_context -> read_details_out_lock); thread_context -> read_details_buff_used =0; } int add_read_detail_bin_buff(fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, char * bin, int do_write){ int binlen=0; memcpy(&binlen, bin, 4); binlen += 4; if(binlen > MAX_FC_READ_LENGTH * 3){ if(!global_context->is_read_too_long_to_SAM_BAM_shown){ SUBREADprintf("ERROR: The read is too long to the SAM or BAM output.\nPlease use the 'CORE' mode for the assignment detail output.\n"); global_context->is_read_too_long_to_SAM_BAM_shown = 1; } return -1; } memcpy(thread_context -> read_details_buff + thread_context -> read_details_buff_used, bin, binlen); thread_context -> read_details_buff_used += binlen; if(do_write){ if(global_context -> is_read_details_out == FILE_TYPE_SAM || thread_context -> read_details_buff_used >= 55000) write_read_detailed_remainder(global_context, thread_context); } return 0; } int write_read_details_FP(fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, char * status, int feature_count, char * features, char * bin1, char * bin2){ int ret = 1; char * read_name; if(global_context -> is_read_details_out == FILE_TYPE_RSUBREAD){ get_readname_from_bin(bin1?bin1:bin2, &read_name); fprintf(global_context -> read_details_out_FP, "%s\t%s\t%d\t%s\n", read_name, status, feature_count, features?features:"NA"); }else{ char * out_bin1 = NULL, *out_bin2 = NULL; char * tags[4]; char types[4]; void * vals[4]; tags[0]="XS"; tags[1]=feature_count >0?"XN":NULL; tags[2]=feature_count >0?"XT":NULL; tags[3]=NULL; types[0]='Z'; types[1]='i'; types[2]='Z'; vals[0]=status; vals[1]=NULL+feature_count; vals[2]=features; if(bin1){ add_bin_new_tags(bin1, &out_bin1, tags, types, vals); add_read_detail_bin_buff(global_context, thread_context, out_bin1, bin2 == NULL); free(out_bin1); } if(bin2){ add_bin_new_tags(bin2, &out_bin2, tags, types, vals); add_read_detail_bin_buff(global_context, thread_context, out_bin2, 1); free(out_bin2); } } if(ret < 1) global_context -> disk_is_full = 1; return ret; } void warning_anno_BAM_chromosomes(fc_thread_global_context_t * global_context){ int x1; HashTable * BAM_chro_tab = HashTableCreate(1117); HashTableSetHashFunction(BAM_chro_tab,HashTableStringHashFunction); HashTableSetKeyComparisonFunction(BAM_chro_tab,fc_strcmp_chro ); for(x1 = 0; x1 < global_context -> sambam_chro_table_items; x1++){ char * BAM_chro = global_context -> sambam_chro_table[x1].chro_name; if( global_context -> BAM_chros_to_anno_table){ char * tmp_chro = HashTableGet(global_context -> BAM_chros_to_anno_table, global_context -> sambam_chro_table[x1].chro_name); if(tmp_chro) BAM_chro = tmp_chro; } HashTablePut(BAM_chro_tab, BAM_chro, NULL+1); } HashTable * ANNO_chro_tab = HashTableCreate(1117); HashTableSetHashFunction(ANNO_chro_tab,HashTableStringHashFunction); HashTableSetKeyComparisonFunction(ANNO_chro_tab,fc_strcmp_chro ); for(x1 = 0 ; x1 < global_context -> exontable_exons ; x1++) HashTablePut(ANNO_chro_tab, global_context -> exontable_chr[x1], NULL+1); if(global_context -> is_verbose){ warning_hash_hash(ANNO_chro_tab, BAM_chro_tab, "Chromosomes/contigs in annotation but not in input file"); warning_hash_hash(BAM_chro_tab, ANNO_chro_tab, "Chromosomes/contigs in input file but not in annotation"); } HashTableDestroy(BAM_chro_tab); HashTableDestroy(ANNO_chro_tab); } void add_scRNA_read_to_pool( fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, srInt_64 assign_target_number, char * read_name, char * read_bin, ArrayList * target_list ); void process_line_buffer(fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, char * bin1, char * bin2) { if(global_context -> is_input_bad_format) return; char * read_chr, *read_name, *mate_chr; srInt_64 read_pos, fragment_length = 0, mate_pos; unsigned int search_start = 0, search_end; int nhits1 = 0, nhits2 = 0, alignment_masks, search_block_id, search_item_id, mapping_qual; //long * hits_indices1 = thread_context -> hits_indices1, * hits_indices2 = thread_context -> hits_indices2; //unsigned int * hits_start_pos1 = thread_context -> hits_start_pos1 , * hits_start_pos2 = thread_context -> hits_start_pos2; //unsigned short * hits_length1 = thread_context -> hits_length1 , * hits_length2 = thread_context -> hits_length2; //char ** hits_chro1 = thread_context -> hits_chro1 , **hits_chro2 = thread_context -> hits_chro2; unsigned int total_frag_len =0; int cigar_sections, is_junction_read; unsigned int * Starting_Chro_Points_1BASE = thread_context -> proc_Starting_Chro_Points_1BASE; unsigned short * Starting_Read_Points = thread_context -> proc_Starting_Read_Points; unsigned short * Section_Read_Lengths = thread_context -> proc_Section_Read_Lengths; char ** ChroNames = thread_context -> proc_ChroNames; char * Event_After_Section = thread_context -> proc_Event_After_Section; CIGAR_interval_t * CIGAR_intervals_R1 = thread_context -> proc_CIGAR_intervals_R1; CIGAR_interval_t * CIGAR_intervals_R2 = thread_context -> proc_CIGAR_intervals_R2; int is_second_read; int maximum_NH_value = 1, NH_value; int skipped_for_exonic = 0; int first_read_quality_score = 0, CIGAR_intervals_R1_sections = 0, CIGAR_intervals_R2_sections = 0; if(thread_context -> thread_id == 0 && thread_context -> all_reads < 1){ warning_anno_BAM_chromosomes(global_context); } if(global_context -> need_calculate_overlap_len ){ memset( CIGAR_intervals_R1, 0, sizeof(CIGAR_interval_t) * global_context -> max_M ); memset( CIGAR_intervals_R2, 0, sizeof(CIGAR_interval_t) * global_context -> max_M ); } thread_context->all_reads++; //if(thread_context->all_reads>1000000) printf("TA=%llu\n%s\n",thread_context->all_reads, thread_context -> line_buffer1); char * RG_ptr; int me_refID =-1, mate_refID =-1, this_is_inconsistent_read_type = 0; for(is_second_read = 0 ; is_second_read < 2; is_second_read++) { if(is_second_read && !global_context -> is_paired_end_mode_assign) break; RG_ptr = NULL; parse_bin(global_context -> sambam_chro_table, is_second_read?bin2:bin1, is_second_read?bin1:bin2 , &read_name, &alignment_masks , &read_chr, &read_pos, &mapping_qual, &mate_chr, &mate_pos, &fragment_length, &is_junction_read, &cigar_sections, Starting_Chro_Points_1BASE, Starting_Read_Points, Section_Read_Lengths, ChroNames, Event_After_Section, &NH_value, global_context -> max_M , global_context -> need_calculate_overlap_len?(is_second_read?CIGAR_intervals_R2:CIGAR_intervals_R1):NULL, is_second_read?&CIGAR_intervals_R2_sections:&CIGAR_intervals_R1_sections, global_context -> assign_reads_to_RG, &RG_ptr, &me_refID, &mate_refID); // this will be done in the other function. if(global_context -> is_paired_end_mode_assign && (alignment_masks&1)==0) alignment_masks|=8; //#warning "========= DEBUG OUTPUT ==============" if(0 && FIXLENstrcmp("SEV0112_0155:7:1303:14436:74270", read_name)==0){ SUBREADprintf("RTEST:%s R_%d %p, %p FLAGS %d\n", read_name, 1+is_second_read, bin1, bin2, alignment_masks); } if(global_context -> assign_reads_to_RG && NULL == RG_ptr)return; if( ( alignment_masks & SAM_FLAG_PAIRED_TASK ) && !global_context -> any_reads_are_PE ) global_context -> any_reads_are_PE=1; if(((!global_context -> is_paired_end_reads_expected) && ( alignment_masks & SAM_FLAG_PAIRED_TASK )) || ((global_context -> is_paired_end_reads_expected) && 0 == ( alignment_masks & SAM_FLAG_PAIRED_TASK ))){ if(global_context -> is_mixed_PE_SE == 0) global_context -> is_mixed_PE_SE =1; if(!global_context -> is_paired_end_reads_expected){ SUBREADprintf("ERROR: Paired-end reads were detected in single-end read library : %s\n", global_context -> input_file_name); global_context -> is_input_bad_format = 1; return; } this_is_inconsistent_read_type = 1; } if(global_context -> do_scRNA_table)add_scRNA_read_tota1_no(global_context, thread_context, read_name, bin1, 0); if(is_second_read == 0) { //skip the read if unmapped (its mate will be skipped as well if paired-end) if( ((!global_context -> is_paired_end_mode_assign) && (alignment_masks & SAM_FLAG_UNMAPPED) ) || ((alignment_masks & SAM_FLAG_UNMAPPED) && (alignment_masks & SAM_FLAG_MATE_UNMATCHED) && global_context -> is_paired_end_mode_assign)) { if(RG_ptr){ void ** tab4s = get_RG_tables(global_context, thread_context, RG_ptr); fc_read_counters * sumtab = tab4s[1]; sumtab -> unassigned_unmapped++; }else thread_context->read_counters.unassigned_unmapped ++; if(global_context -> read_details_out_FP) write_read_details_FP(global_context , thread_context ,"Unassigned_Unmapped",0, NULL, bin1, bin2); if(global_context -> do_scRNA_table) add_scRNA_read_to_pool(global_context, thread_context, -1, read_name, bin1, NULL); return; // do nothing if a read is unmapped, or the first read in a pair of reads is unmapped. } } if(global_context -> do_scRNA_table)add_scRNA_read_tota1_no(global_context, thread_context, read_name, bin1, 1); if(((alignment_masks & SAM_FLAG_UNMAPPED) || (alignment_masks & SAM_FLAG_MATE_UNMATCHED)) && global_context -> is_paired_end_mode_assign && global_context -> is_both_end_required){ if(RG_ptr){ void ** tab4s = get_RG_tables(global_context, thread_context, RG_ptr); fc_read_counters * sumtab = tab4s[1]; sumtab -> unassigned_singleton++; }else thread_context->read_counters.unassigned_singleton ++; if(global_context -> read_details_out_FP) write_read_details_FP(global_context , thread_context ,"Unassigned_Singleton",0, NULL, bin1, bin2); if(global_context -> do_scRNA_table) add_scRNA_read_to_pool(global_context, thread_context, -1, read_name, bin1, NULL); return; } if(this_is_inconsistent_read_type){ if(global_context -> is_both_end_required && 0 == ( alignment_masks & SAM_FLAG_PAIRED_TASK )){ if(global_context -> read_details_out_FP) write_read_details_FP(global_context , thread_context ,"Unassigned_Singleton",0, NULL, bin1, bin2); if(RG_ptr){ void ** tab4s = get_RG_tables(global_context, thread_context, RG_ptr); fc_read_counters * sumtab = tab4s[1]; sumtab -> unassigned_singleton++; }else thread_context->read_counters.unassigned_singleton ++; if(global_context -> do_scRNA_table) add_scRNA_read_to_pool(global_context, thread_context, -1, read_name, bin1, NULL); return; // when running on PE mode, SE reads are seen as "only one end mapped" } } if(global_context -> min_mapping_quality_score>0) { //printf("SECOND=%d; FIRST=%d; THIS=%d; Q=%d\n", is_second_read, first_read_quality_score, mapping_qual, ); if(( mapping_qual < global_context -> min_mapping_quality_score && ! global_context -> is_paired_end_mode_assign)||( is_second_read && max( first_read_quality_score, mapping_qual ) < global_context -> min_mapping_quality_score)) { thread_context->read_counters.unassigned_mappingquality ++; if(global_context -> read_details_out_FP) { write_read_details_FP(global_context, thread_context, "Unassigned_MappingQuality", 0, NULL, bin1, bin2); } if(global_context -> do_scRNA_table) add_scRNA_read_to_pool(global_context, thread_context, -1, read_name, bin1, NULL); return; } if(is_second_read==0 && global_context -> is_paired_end_mode_assign) { first_read_quality_score = mapping_qual; } } if(is_second_read == 0 && global_context -> is_paired_end_mode_assign && (global_context -> is_PE_distance_checked || global_context -> is_chimertc_disallowed) ) { int is_half_mapped = (alignment_masks & SAM_FLAG_UNMAPPED) || (alignment_masks & SAM_FLAG_MATE_UNMATCHED); if(!is_half_mapped) { fragment_length = abs( fragment_length ); //get the fragment length int is_first_read_negative_strand = (alignment_masks & SAM_FLAG_REVERSE_STRAND_MATCHED)?1:0; int is_second_read_negative_strand = (alignment_masks & SAM_FLAG_MATE_REVERSE_STRAND_MATCHED)?1:0; if(mate_chr == read_chr && is_first_read_negative_strand!=is_second_read_negative_strand) { //^^^^^^^^^^^^^^^^^^^^ They are directly compared because they are both pointers in the same contig name table. // if(global_context -> is_PE_distance_checked && ((fragment_length > global_context -> max_paired_end_distance) || (fragment_length < global_context -> min_paired_end_distance))) { if(RG_ptr){ void ** tab4s = get_RG_tables(global_context, thread_context, RG_ptr); fc_read_counters * sumtab = tab4s[1]; sumtab -> unassigned_fragmentlength++; }else thread_context->read_counters.unassigned_fragmentlength ++; if(global_context -> read_details_out_FP) write_read_details_FP(global_context, thread_context, "Unassigned_FragmentLength", -1, NULL, bin1, bin2); if(global_context -> do_scRNA_table) add_scRNA_read_to_pool(global_context, thread_context, -1, read_name, bin1, NULL); return; } } else { if(global_context -> is_chimertc_disallowed) { if(RG_ptr){ void ** tab4s = get_RG_tables(global_context, thread_context, RG_ptr); fc_read_counters * sumtab = tab4s[1]; sumtab -> unassigned_chimericreads++; }else thread_context->read_counters.unassigned_chimericreads ++; if(global_context -> read_details_out_FP) write_read_details_FP(global_context, thread_context, "Unassigned_Chimera", -1, NULL, bin1, bin2); if(global_context -> do_scRNA_table) add_scRNA_read_to_pool(global_context, thread_context, -1, read_name, bin1, NULL); return; } } } } // This filter has to be put here because the 0x400 FLAG is not about mapping but about sequencing. // A unmapped read with 0x400 FLAG should be able to kill the mapped mate which may have no 0x400 FLAG. if(global_context -> is_duplicate_ignored) { if(alignment_masks & SAM_FLAG_DUPLICATE) { if(RG_ptr){ void ** tab4s = get_RG_tables(global_context, thread_context, RG_ptr); fc_read_counters * sumtab = tab4s[1]; sumtab -> unassigned_duplicate++; }else thread_context->read_counters.unassigned_duplicate ++; if(global_context -> read_details_out_FP) write_read_details_FP(global_context, thread_context, "Unassigned_Duplicate", -1, NULL, bin1, bin2); if(global_context -> do_scRNA_table) add_scRNA_read_to_pool(global_context, thread_context, -1, read_name, bin1, NULL); return; } } if(SAM_FLAG_UNMAPPED & alignment_masks) continue; if( NH_value > 1 ) { if(global_context -> is_multi_mapping_allowed == 0) { // now it is a NH>1 read! // not allow multimapping -> discard! if(RG_ptr){ void ** tab4s = get_RG_tables(global_context, thread_context, RG_ptr); fc_read_counters * sumtab = tab4s[1]; sumtab -> unassigned_multimapping++; }else thread_context->read_counters.unassigned_multimapping ++; if(global_context -> read_details_out_FP) write_read_details_FP(global_context, thread_context, "Unassigned_MultiMapping", -1, NULL, bin1, bin2); if(global_context -> do_scRNA_table) add_scRNA_read_to_pool(global_context, thread_context, -1, read_name, bin1, NULL); return; } } maximum_NH_value = max(maximum_NH_value, NH_value); // if a pair of reads have one secondary, the entire fragment is seen as secondary. if((alignment_masks & SAM_FLAG_SECONDARY_MAPPING) && (global_context -> is_primary_alignment_only)) { if(RG_ptr){ void ** tab4s = get_RG_tables(global_context, thread_context, RG_ptr); fc_read_counters * sumtab = tab4s[1]; sumtab -> unassigned_secondary++; }else thread_context->read_counters.unassigned_secondary ++; if(global_context -> read_details_out_FP) write_read_details_FP(global_context, thread_context, "Unassigned_Secondary", -1, NULL, bin1, bin2); if(global_context -> do_scRNA_table) add_scRNA_read_to_pool(global_context, thread_context, -1, read_name, bin1, NULL); return; } int is_this_negative_strand = (alignment_masks & SAM_FLAG_REVERSE_STRAND_MATCHED)?1:0; int is_fragment_negative_strand = is_this_negative_strand; if(1 || global_context -> is_paired_end_mode_assign){ // On 20 JULY 2020: If strand-specific counting is on, isPairedEnd = TRUE, countReadPairs = FALSE and the BAM file contains mixed reads, then the single-end reads and the R1 reads in read-pairs will be directly compared with the strand of the gene, but the R2 reads in read-pairs will be compared with the opposite strand of the gene. A read-pair will be counted twice no matter if the strand-specific mode is on or off. If the argument to the strand-specific option is "1", then R1 must have the same strand of the gene and R2 must have the opposite strand of the gene to be counted. int is_second_read_in_pair = alignment_masks & SAM_FLAG_SECOND_READ_IN_PAIR; //is_fragment_negative_strand = is_second_read_in_pair?(!is_this_negative_strand):is_this_negative_strand; if(is_second_read_in_pair) is_fragment_negative_strand = global_context -> is_second_read_straight?is_this_negative_strand:(!is_this_negative_strand); else is_fragment_negative_strand = global_context -> is_first_read_reversed?(!is_this_negative_strand):is_this_negative_strand; } int nhits = 0; int cigar_section_id; srInt_64 * hits_indices = is_second_read?thread_context -> hits_indices2:thread_context -> hits_indices1; unsigned int * hits_start_pos = is_second_read?thread_context -> hits_start_pos2:thread_context -> hits_start_pos1; unsigned short * hits_length = is_second_read?thread_context -> hits_length2:thread_context -> hits_length1; char ** hits_chro = is_second_read?thread_context -> hits_chro2:thread_context -> hits_chro1; if(global_context->is_split_or_exonic_only == 1 && !is_junction_read) { skipped_for_exonic ++; if(skipped_for_exonic == 1 + global_context -> is_paired_end_mode_assign){ if(global_context -> read_details_out_FP) write_read_details_FP(global_context, thread_context, (global_context->is_split_or_exonic_only == 2)?"Unassigned_Split":"Unassigned_NonSplit", -1, NULL, bin1, bin2); if(RG_ptr){ void ** tab4s = get_RG_tables(global_context, thread_context, RG_ptr); fc_read_counters * sumtab = tab4s[1]; sumtab -> unassigned_junction_condition++; }else thread_context->read_counters.unassigned_junction_condition ++; if(global_context -> do_scRNA_table) add_scRNA_read_to_pool(global_context, thread_context, -1, read_name, bin1, NULL); return; } } if(global_context->is_split_or_exonic_only == 2 && is_junction_read) { if(global_context -> read_details_out_FP) write_read_details_FP(global_context, thread_context,(global_context->is_split_or_exonic_only == 2)?"Unassigned_Split":"Unassigned_NonSplit", -1, NULL, bin1, bin2); if(RG_ptr){ void ** tab4s = get_RG_tables(global_context, thread_context, RG_ptr); fc_read_counters * sumtab = tab4s[1]; sumtab -> unassigned_junction_condition++; }else thread_context->read_counters.unassigned_junction_condition ++; if(global_context -> do_scRNA_table) add_scRNA_read_to_pool(global_context, thread_context, -1, read_name, bin1, NULL); return; } if(1) { if(0)SUBREADprintf("MAPPED R_%d to %s : CHR_POS=%u + %u, CHR_LEN=%u\n", is_second_read+1, global_context -> sambam_chro_table[me_refID]. chro_name, Starting_Chro_Points_1BASE[0], Section_Read_Lengths[0], global_context -> sambam_chro_table[me_refID] .chro_length ); if(global_context -> read_shift_size>0){ int shifting_applied_length = 0; int shifting_i; if((global_context -> read_shift_type == READ_SHIFT_UPSTREAM && (!is_this_negative_strand))|| (global_context -> read_shift_type == READ_SHIFT_DOWNSTREAM && is_this_negative_strand )) shifting_applied_length = -global_context -> read_shift_size; if((global_context -> read_shift_type == READ_SHIFT_UPSTREAM && is_this_negative_strand)|| (global_context -> read_shift_type == READ_SHIFT_DOWNSTREAM && (!is_this_negative_strand))) shifting_applied_length = global_context -> read_shift_size; if(global_context -> read_shift_type == READ_SHIFT_LEFT) shifting_applied_length = -global_context -> read_shift_size; if(global_context -> read_shift_type == READ_SHIFT_RIGHT) shifting_applied_length = global_context -> read_shift_size; if(shifting_applied_length < 0 && Starting_Chro_Points_1BASE[0] <=-shifting_applied_length) shifting_applied_length = - Starting_Chro_Points_1BASE[0]+1; if(shifting_applied_length > 0 && Starting_Chro_Points_1BASE[cigar_sections-1] + Section_Read_Lengths[cigar_sections-1] + shifting_applied_length > global_context -> sambam_chro_table[me_refID].chro_length +1 ) shifting_applied_length = global_context -> sambam_chro_table[me_refID].chro_length +1 - (Starting_Chro_Points_1BASE[cigar_sections-1] + Section_Read_Lengths[cigar_sections-1]); for(shifting_i = 0; shifting_i < cigar_sections ; shifting_i++) Starting_Chro_Points_1BASE[shifting_i] += shifting_applied_length; } if(global_context -> five_end_extension) { if(is_this_negative_strand){ int applied_ext = global_context -> five_end_extension; if( Starting_Chro_Points_1BASE[cigar_sections-1] + Section_Read_Lengths[cigar_sections-1] + applied_ext > global_context -> sambam_chro_table[me_refID].chro_length +1 ) applied_ext = global_context -> sambam_chro_table[me_refID].chro_length +1 - (Starting_Chro_Points_1BASE[cigar_sections-1] + Section_Read_Lengths[cigar_sections-1]); Section_Read_Lengths [cigar_sections - 1] += applied_ext; }else{ //SUBREADprintf("5-end extension: %d [%d]\n", Starting_Chro_Points_1BASE[0], Section_Lengths[0]); if( read_pos > global_context -> five_end_extension) { Section_Read_Lengths [0] += global_context -> five_end_extension; Starting_Chro_Points_1BASE [0] -= global_context -> five_end_extension; } else { Section_Read_Lengths [0] += read_pos-1; Starting_Chro_Points_1BASE [0] -= read_pos-1; } } } if(global_context -> three_end_extension) { if(is_this_negative_strand){ if( read_pos > global_context -> three_end_extension) { Section_Read_Lengths [0] += global_context -> three_end_extension; Starting_Chro_Points_1BASE [0] -= global_context -> three_end_extension; } else { Section_Read_Lengths [0] += read_pos - 1; Starting_Chro_Points_1BASE [0] -= read_pos - 1; } } else{ int applied_ext = global_context -> three_end_extension; if( Starting_Chro_Points_1BASE[cigar_sections-1] + Section_Read_Lengths[cigar_sections-1] + applied_ext > global_context -> sambam_chro_table[me_refID].chro_length +1 ) applied_ext = global_context -> sambam_chro_table[me_refID].chro_length +1 - (Starting_Chro_Points_1BASE[cigar_sections-1] + Section_Read_Lengths[cigar_sections-1]); Section_Read_Lengths [cigar_sections - 1] += applied_ext; } } if(global_context -> reduce_5_3_ends_to_one) { if((REDUCE_TO_5_PRIME_END == global_context -> reduce_5_3_ends_to_one) + is_this_negative_strand == 1) // reduce to 5' end (small coordinate if positive strand / large coordinate if negative strand) { Section_Read_Lengths[0]=1; } else { Starting_Chro_Points_1BASE[0] = Starting_Chro_Points_1BASE[cigar_sections-1] + Section_Read_Lengths[cigar_sections-1] - 1; Section_Read_Lengths[0]=1; } cigar_sections = 1; } for(cigar_section_id = 0; cigar_section_id exontable_chro_table, ChroNames[cigar_section_id]); if(this_chro_info == NULL) { if(global_context -> BAM_chros_to_anno_table) { char * anno_chro_name = HashTableGet( global_context -> BAM_chros_to_anno_table , ChroNames[cigar_section_id]); if(anno_chro_name) this_chro_info = HashTableGet(global_context -> exontable_chro_table, anno_chro_name); } if(this_chro_info == NULL && memcmp(ChroNames[cigar_section_id], "chr", 3)==0) { this_chro_info = HashTableGet(global_context -> exontable_chro_table, ChroNames[cigar_section_id]+3); // SUBREADprintf("INQ: %p : '%s'\n", this_chro_info , ChroNames[cigar_section_id]+3); } if(this_chro_info == NULL && strlen(ChroNames[cigar_section_id])<=2) { strcpy(thread_context -> chro_name_buff, "chr"); strcpy(thread_context -> chro_name_buff+3, ChroNames[cigar_section_id]); this_chro_info = HashTableGet(global_context -> exontable_chro_table, thread_context -> chro_name_buff); } } //SUBREADprintf("INF: %p : %s\n", this_chro_info , ChroNames[cigar_section_id]); if(this_chro_info) { start_reverse_table_index = min(start_reverse_table_index, this_chro_info-> chro_possible_length / REVERSE_TABLE_BUCKET_LENGTH); end_reverse_table_index = min(end_reverse_table_index, this_chro_info-> chro_possible_length / REVERSE_TABLE_BUCKET_LENGTH+ 1); while(start_reverse_table_index<=end_reverse_table_index) { search_start = this_chro_info -> reverse_table_start_index [start_reverse_table_index]; if(search_start<0xffffff00)break; start_reverse_table_index++; } if(search_start>0xffffff00) continue; //search_start = this_chro_info -> chro_block_table_start; search_end = this_chro_info -> chro_block_table_end;//reverse_table_end_index [end_reverse_table_index]; for(search_block_id=search_start;search_block_id exontable_block_min_start[search_block_id] > section_end_pos) break; if (global_context -> exontable_block_max_end[search_block_id] < section_begin_pos) continue; int search_item_start = 0, search_item_end = global_context -> exontable_block_end_index[search_block_id]; if(search_block_id>0)search_item_start = global_context -> exontable_block_end_index[search_block_id-1]; // search_item_id is the inner number of the exons. // the exontables in global_index has search_item_id as the index. for(search_item_id = search_item_start ; search_item_id < search_item_end; search_item_id++) { if (global_context -> exontable_stop[search_item_id] >= section_begin_pos) { if (global_context -> exontable_start[search_item_id] > section_end_pos) break; // there is an overlap >=1 between read and feature. // the overlap length is min(end_r, end_F) - max(start_r, start_F) + 1 int is_strand_ok =1; if(global_context->is_strand_checked){ if(global_context->is_strand_checked == 1) is_strand_ok = (is_fragment_negative_strand == global_context -> exontable_strand[search_item_id]); else// if(global_context->is_strand_checked == 2) is_strand_ok = (is_fragment_negative_strand != global_context -> exontable_strand[search_item_id]); //SUBREADprintf("%d = %d == %d\n", is_strand_ok, is_fragment_negative_strand, global_context -> exontable_strand[search_item_id]); } if(is_strand_ok){ if(nhits >= thread_context -> hits_number_capacity - 1){ //SUBREADprintf("RESIZE hits: %d\n", thread_context -> hits_number_capacity); thread_context -> hits_number_capacity = thread_context -> hits_number_capacity/2 * 3; thread_context -> hits_number_capacity = max(10, thread_context -> hits_number_capacity); thread_context -> hits_start_pos1 = realloc(thread_context -> hits_start_pos1 , sizeof(int) * thread_context -> hits_number_capacity); thread_context -> hits_start_pos2 = realloc(thread_context -> hits_start_pos2 , sizeof(int) * thread_context -> hits_number_capacity); thread_context -> hits_length1 = realloc(thread_context -> hits_length1, sizeof(short) * thread_context -> hits_number_capacity); thread_context -> hits_length2 = realloc(thread_context -> hits_length2, sizeof(short) * thread_context -> hits_number_capacity); thread_context -> hits_chro1 = realloc(thread_context -> hits_chro1, sizeof(char *) * thread_context -> hits_number_capacity); thread_context -> hits_chro2 = realloc(thread_context -> hits_chro2, sizeof(char *) * thread_context -> hits_number_capacity); thread_context -> hits_indices1 = realloc(thread_context -> hits_indices1, sizeof(srInt_64) * thread_context -> hits_number_capacity); thread_context -> hits_indices2 = realloc(thread_context -> hits_indices2, sizeof(srInt_64) * thread_context -> hits_number_capacity); thread_context -> scoring_buff_numbers = realloc(thread_context -> scoring_buff_numbers, sizeof(int)*2*thread_context -> hits_number_capacity); thread_context -> scoring_buff_flags = realloc(thread_context -> scoring_buff_flags, sizeof(int)*2*thread_context -> hits_number_capacity); thread_context -> scoring_buff_overlappings = realloc(thread_context -> scoring_buff_overlappings, sizeof(int)*2*thread_context -> hits_number_capacity); thread_context -> scoring_buff_exon_ids = realloc(thread_context -> scoring_buff_exon_ids, sizeof(srInt_64)*2*thread_context -> hits_number_capacity); if(global_context -> need_calculate_overlap_len){ thread_context -> scoring_buff_gap_chros = realloc(thread_context -> scoring_buff_gap_chros, sizeof(char *) * 2 * global_context -> max_M *2 * thread_context -> hits_number_capacity); thread_context -> scoring_buff_gap_starts = realloc(thread_context -> scoring_buff_gap_starts, sizeof(int) * 2 * global_context -> max_M *2 * thread_context -> hits_number_capacity); thread_context -> scoring_buff_gap_lengths = realloc(thread_context -> scoring_buff_gap_lengths, sizeof(short) * 2 * global_context -> max_M *2 * thread_context -> hits_number_capacity); } hits_indices = is_second_read?thread_context -> hits_indices2:thread_context -> hits_indices1; hits_start_pos = is_second_read?thread_context -> hits_start_pos2:thread_context -> hits_start_pos1; hits_length = is_second_read?thread_context -> hits_length2:thread_context -> hits_length1; hits_chro = is_second_read?thread_context -> hits_chro2:thread_context -> hits_chro1; //SUBREADprintf("RESIZE hits2: %d\n", thread_context -> hits_number_capacity); } if(nhits <= MAX_HIT_NUMBER - 1) { hits_indices[nhits] = search_item_id; if(global_context -> need_calculate_overlap_len) { hits_start_pos[nhits] = max(Starting_Chro_Points_1BASE[cigar_section_id], global_context -> exontable_start[search_item_id]); hits_length[nhits] = min(global_context -> exontable_stop[search_item_id] , section_end_pos)+1 - hits_start_pos[nhits] ; hits_chro[nhits] = ChroNames[cigar_section_id]; if(0 && FIXLENstrcmp("V0112_0155:7:1101:10214:3701", read_name)==0) SUBREADprintf("QNAME: [%d] %s %d ~ %d\n", nhits, hits_chro[nhits], hits_start_pos[nhits], hits_start_pos[nhits]+hits_length[nhits]); } nhits++; } else { SUBREADprintf("ERROR: the read overlapped with more than %d features.\n", nhits); global_context -> is_input_bad_format = 1; return ; } } } } } } } } if(is_second_read) nhits2 = nhits; else nhits1 = nhits; } // loop for is_second_read if(global_context -> do_junction_counting)// junction reads that passed the basic filters will be considered with the junction counting. Filters: Unmapped, Singleton, MAPQ, TemplateLength, Chimeric, Duplicate, Multimapping, Secondary alignment, Junction-containing status, process_line_junctions(global_context, thread_context, bin1, bin2); if(global_context -> need_calculate_fragment_len ) total_frag_len = calc_total_frag_len( global_context, thread_context, CIGAR_intervals_R1, CIGAR_intervals_R1_sections, CIGAR_intervals_R2, CIGAR_intervals_R2_sections , read_name); //SUBREADprintf("FRAGLEN: %s %d; CIGARS=%d,%d\n", read_name, total_frag_len, CIGAR_intervals_R1_sections,CIGAR_intervals_R2_sections); int fixed_fractional_count = ( global_context -> use_fraction_multi_mapping && ! global_context -> is_primary_alignment_only )?calc_fixed_fraction(maximum_NH_value): NH_FRACTION_INT; // we have hits_indices1 and hits_indices2 and nhits1 and nhits2 here // we also have fixed_fractional_count which is the value to add vote_and_add_count(global_context, thread_context, thread_context -> hits_indices1, nhits1, thread_context -> hits_indices2, nhits2, total_frag_len, thread_context -> hits_chro1, thread_context -> hits_chro2, thread_context -> hits_start_pos1, thread_context -> hits_start_pos2, thread_context -> hits_length1, thread_context ->hits_length2, fixed_fractional_count, read_name, RG_ptr, bin1, bin2); return; } void add_bitmap_overlapping(char * x1_bitmap, short start_base, short len){ int x1; int rl16 = start_base+len-16; for(x1 = start_base; x1 < start_base+len; x1++){ int bit = x1 % 8; int byte = x1 / 8; if(bit == 0 && x1 < rl16){ x1_bitmap[byte]=-1; x1_bitmap[byte+1]=-1; x1+=15; }else{ x1_bitmap[byte] |= (1<= 0 && njunc1 <= global_context -> max_M -1 ); assert(njunc2 >= 0 && njunc2 <= global_context -> max_M -1 ); int x1,x2, in_total_junctions = njunc2 + njunc1; HashTable * junction_counting_table, *splicing_point_table; if(RG_name){ void ** tab4s = get_RG_tables(global_context, thread_context, RG_name); junction_counting_table = tab4s[2]; splicing_point_table = tab4s[3]; }else{ junction_counting_table = thread_context -> junction_counting_table; splicing_point_table = thread_context -> splicing_point_table; } for(x1 = 0; x1 < in_total_junctions; x1 ++){ fc_junction_info_t * j_one = (x1 >= njunc1)?supported_junctions2+(x1-njunc1):(supported_junctions1+x1); if(j_one->chromosome_name_left[0]==0) continue; for(x2 = x1+1; x2 < in_total_junctions ; x2 ++){ fc_junction_info_t * j_two = (x2 >= njunc1)?supported_junctions2+(x2-njunc1):(supported_junctions1+x2); if(j_two->chromosome_name_left[0]==0) continue; if( j_one -> last_exon_base_left == j_two -> last_exon_base_left && j_one -> first_exon_base_right == j_two -> first_exon_base_right && strcmp(j_one -> chromosome_name_left, j_two -> chromosome_name_left) == 0 && strcmp(j_one -> chromosome_name_right, j_two -> chromosome_name_right) == 0 ) j_two -> chromosome_name_left[0]=0; } char * this_key = malloc(strlen(j_one->chromosome_name_left) + strlen(j_one->chromosome_name_right) + 36); sprintf(this_key, "%s\t%u\t%s\t%u", j_one->chromosome_name_left, j_one -> last_exon_base_left, j_one->chromosome_name_right, j_one -> first_exon_base_right); void * count_ptr = HashTableGet(junction_counting_table, this_key); srInt_64 count_junc = count_ptr - NULL; HashTablePut(junction_counting_table, this_key, NULL+count_junc + 1); // #warning "CONTINUE SHOULD BE REMOVED!!!." // continue; char * left_key = malloc(strlen(j_one->chromosome_name_left) + 16); char * right_key = malloc(strlen(j_one->chromosome_name_right) + 16); sprintf(left_key, "%s\t%u", j_one->chromosome_name_left, j_one -> last_exon_base_left); sprintf(right_key, "%s\t%u", j_one->chromosome_name_right, j_one -> first_exon_base_right); for( x2 = 0 ; x2 < 2 ; x2++ ){ char * lr_key = x2?right_key:left_key; count_ptr = HashTableGet(splicing_point_table, lr_key); count_junc = count_ptr - NULL; HashTablePut(splicing_point_table, lr_key, NULL + count_junc + 1); } } } int overlap_compare(void * arr, int L, int R){ unsigned int * pos = (unsigned int *)arr; return pos[ L*2 ] - pos[R*2]; } void overlap_exchange(void * arr, int L, int R){ unsigned int * pos = (unsigned int *)arr, tt; tt=pos[L*2]; pos[L*2] = pos[R*2]; pos[R*2] = tt; tt=pos[L*2+1]; pos[L*2+1] = pos[R*2+1]; pos[R*2+1] = tt; } int scRNA_get_sample_id(fc_thread_global_context_t *global_context, char * sbc, int read_laneno){ int x1; //SUBREADprintf("TOTAL_SBC=%ld\n", global_context -> scRNA_sample_barcode_list -> numOfElements); for(x1=0; x1 < global_context -> scRNA_sample_barcode_list -> numOfElements ; x1++ ){ char ** lane_and_barcode = ArrayListGet(global_context -> scRNA_sample_barcode_list, x1); int lane_no = lane_and_barcode[0]-(char*)NULL; // SUBREADprintf("KNOWN_LANE=%d, IN_LANE=%d, to\n", lane_no, read_laneno); if(read_laneno == lane_no){ int sample_no = lane_and_barcode[1]-(char*)NULL; char * knownbar = lane_and_barcode[2]; if(lane_and_barcode[3]){ int hd = hamming_dist_ATGC_max1_2p( sbc, knownbar ); if(hd<=2) return sample_no; }else{ int hd = hamming_dist_ATGC_max1( sbc, knownbar ); if(hd<=1) return sample_no; } } } return -1; } int scRNA_register_umi_id(fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, char * ubc){ int xk1=0,nch; for(xk1=0; 0!=(nch = ubc [xk1]); xk1++){ if(!isalpha(nch))break; } ubc[xk1]=0; int uno = HashTableGet(thread_context -> scRNA_registered_UMI_table, ubc ) -NULL -1; if(uno<0) { uno = thread_context -> scRNA_registered_UMI_table -> numOfElements; assert(strlen(ubc) <=MAX_UMI_LEN); HashTablePut( thread_context -> scRNA_registered_UMI_table, strdup(ubc) , NULL+ uno +1); } ubc[xk1]=nch; return uno; } #define IMPOSSIBLE_MEMORY_SPACE 0x5CAFEBABE0000000llu int scRNA_get_cell_id(fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, char * cbc){ //return -1; char tmpc [MAX_READ_NAME_LEN]; int xx1; ArrayList * ret=NULL; for(xx1=0;xx1<3;xx1++){ int xx2; if(xx1==1) ret = ArrayListCreate(100); if(xx1>0){ tmpc[0] = (xx1==2)?'S':'F'; for(xx2=0; xx2 known_cell_barcode_length/2 ; xx2++) tmpc[1+xx2] = cbc[2*xx2+xx1-1]; tmpc[1+global_context -> known_cell_barcode_length/2]=0; }else{ memcpy(tmpc, cbc, global_context -> known_cell_barcode_length); tmpc[global_context -> known_cell_barcode_length]=0; } void *xrawarr = HashTableGet(global_context -> scRNA_cell_barcode_head_tail_table, tmpc); if(xx1 == 0){ //if(xrawarr) SUBREADprintf("CAFE ? %p\n", xrawarr); srInt_64 xint = xrawarr - NULL; if(( xint & 0xFFFFFFFFF0000000llu)== IMPOSSIBLE_MEMORY_SPACE){ int only_cell_id = xint - IMPOSSIBLE_MEMORY_SPACE; // no memory was allocated. return only_cell_id; } }else{ ArrayList * rawarr = xrawarr; if(rawarr){ int xx3,xx2, found; for(xx2=0; xx2numOfElements; xx2++){ int bcno = ArrayListGet(rawarr, xx2)-NULL; found=0; for(xx3=0;xx3 numOfElements;xx3++){ if(ArrayListGet(ret, xx3)==NULL+bcno){ found=1; break; } } if(!found)ArrayListPush(ret, NULL+bcno); } } } } int tb1=-1; for(xx1=0; xx1 numOfElements; xx1++){ int tbcn = ArrayListGet(ret,xx1)-NULL; char * known_cbc = ArrayListGet(global_context -> scRNA_cell_barcodes_array, tbcn); int hc = hamming_dist_ATGC_max2( known_cbc, cbc ); // cbc[16]=0; if(hc <=3)SUBREADprintf("TEST_CBC %s ~ %s = %d\n", known_cbc, cbc, hc); if(hc==1){ tb1 = tbcn; break; } } //SUBREADprintf("CANDIDATE CELL BARCODES=%ld ; hit = %d\n", ret->numOfElements, tb1); ArrayListDestroy(ret); return tb1; } #define SCRNA_READ_NAME_SPLIT_CHAR '|' void scRNA_move_barcodes_to_tags(fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, char * inbin, char ** outbin_pr, char * fixed_cell_barcode, char * fixed_UMI){ int inbin_len=0; memcpy(&inbin_len, inbin, 4); char* outbin = malloc(inbin_len + 400); *outbin_pr = outbin; int l_read_name=0, new_l_read_name=0, x1; memcpy(&l_read_name, inbin+12, 1); char * BC_seq=NULL, * BC_qual=NULL, *UMI_seq=NULL, *UMI_qual=NULL, *RG=NULL; int BC_len=global_context -> known_cell_barcode_length, UMI_len=0, RG_len=0, field_i = 0; for(x1=1; x1 scRNA_input_mode == GENE_INPUT_BCL)){ field_i ++; if(field_i == 1){ new_l_read_name = x1+1; BC_seq = inbin+36 + x1+1; UMI_seq = BC_seq + BC_len; }else if(field_i == 2){ BC_qual = inbin+36 + x1+1; UMI_len = BC_qual - BC_seq - 1 - global_context -> known_cell_barcode_length; UMI_qual = BC_qual + BC_len; }else if(field_i == 5){ RG = inbin+36 + x1+1; RG_len = l_read_name - x1 - 2; } } } memcpy(outbin, inbin, 36+new_l_read_name); outbin[36+new_l_read_name-1]=0; memcpy(outbin + 12,&new_l_read_name, 1); memcpy(outbin + 36 + new_l_read_name, inbin + 36 + l_read_name, inbin_len +4 - 36 - l_read_name); int ext_ptr = inbin_len +4 - (l_read_name - new_l_read_name); for(x1 = 0;x1<7;x1++){ int this_len = BC_len; char * this_tag = "CR", * this_val = BC_seq; if(x1 == 1){this_tag = "CY"; this_val = BC_qual;} if(x1 == 2){this_tag = "CB"; this_val = fixed_cell_barcode;} if(x1 == 3){this_tag = "UR"; this_val = UMI_seq;} if(x1 == 4){this_tag = "UY"; this_val = UMI_qual;} if(x1 == 5){this_tag = "UB"; this_val = fixed_UMI;} if(x1 == 6){this_tag = "RG"; this_val = RG;} if(x1 == 3 || x1 == 4 || x1 == 5) this_len = UMI_len; if(x1 == 6) this_len = RG_len; outbin[ext_ptr]= this_tag[0]; outbin[ext_ptr+1]= this_tag[1]; outbin[ext_ptr+2]= 'Z'; memcpy(outbin+ext_ptr+3, this_val, this_len); outbin[ext_ptr+3+this_len]= 0; ext_ptr += 3+1+this_len; } ext_ptr -=4; // block_size excl itself memcpy(outbin, &ext_ptr, 4); } int scRNA_scan_read_name_str(fc_thread_global_context_t * global_context, char * read_name, char * read_bin, char ** sample_seq, char ** sample_qual, char ** BC_seq, char ** BC_qual, char ** UMI_seq, char ** UMI_qual, char ** lane_str, char ** RG, int * rname_trimmed_len){ char * testi; int field_i=0; if(NULL == read_name && read_bin) read_name = read_bin + 36; for(testi = read_name +1; * testi; testi ++){ if((*testi)== SCRNA_READ_NAME_SPLIT_CHAR || ((*testi)== ':' && global_context -> scRNA_input_mode == GENE_INPUT_BCL )){ field_i++; if(field_i == 1) { if(rname_trimmed_len) (*rname_trimmed_len)=testi-read_name; if(BC_seq)(*BC_seq) = testi+1; if(UMI_seq)(*UMI_seq) = testi+1+global_context -> known_cell_barcode_length; }else if(field_i == 2){ if(BC_qual)(*BC_qual) = testi+1; if(UMI_qual)(*UMI_qual) = testi+1+global_context -> known_cell_barcode_length; }else if(field_i == 3){ *sample_seq = testi + 1; if(RG)(*RG) = *sample_seq; }else if(field_i == 4){ if(sample_qual)(*sample_qual) = testi + 1; }else if(field_i == 5){ (*lane_str) = testi + 1; if(memcmp(*lane_str, "@RgLater@", 9)==0) (*lane_str) += 9; break; } } } if(field_i < 3 && read_bin){ int bin_len = 0; char tag_type = 0; int bintag_start = SAM_pairer_get_tag_bin_start(read_bin); memcpy(&bin_len, read_bin, 4); bin_len = bin_len +4 -bintag_start; if(BC_seq) SAM_pairer_iterate_tags((unsigned char*)read_bin+bintag_start, bin_len , "CR", &tag_type, BC_seq); if(UMI_seq) SAM_pairer_iterate_tags((unsigned char*)read_bin+bintag_start, bin_len , "UR", &tag_type, UMI_seq); if(BC_qual) SAM_pairer_iterate_tags((unsigned char*)read_bin+bintag_start, bin_len , "CY", &tag_type, BC_qual); if(UMI_qual) SAM_pairer_iterate_tags((unsigned char*)read_bin+bintag_start, bin_len , "UY", &tag_type, UMI_qual); if(RG) SAM_pairer_iterate_tags((unsigned char*)read_bin+bintag_start, bin_len , "RG", &tag_type, RG); } return field_i; } void scRNA_find_sample_cell_umi_from_readname(fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, char * read_name, char * read_bin, int * sample_id, char ** BC_seq, char ** UMI_seq, char ** RG){ int field_i = 0, laneno = 0; char * testi, * lane_str = NULL, *sample_barcode = NULL; if(sample_id)*sample_id = -1; field_i = scRNA_scan_read_name_str(global_context, read_name, read_bin, &sample_barcode, NULL, BC_seq, NULL, UMI_seq, NULL, &lane_str, RG, NULL); if(!sample_id){ if(!(UMI_seq && BC_seq))SUBREADprintf("ERROR: Cannot get UMI or BC: %s, %s\n", *UMI_seq, *BC_seq); return; } if(global_context -> scRNA_input_mode == GENE_INPUT_SCRNA_BAM){ *sample_id = 1; // on the BAM mode, every featureCounts run only has one sample }else if(global_context -> scRNA_input_mode == GENE_INPUT_SCRNA_FASTQ){ if(sample_barcode == NULL || memcmp(sample_barcode, "input#", 6) || !isdigit(sample_barcode[6])) SUBREADprintf("SPBCFMT_ERR %d // %s in %s // %s\n", field_i, sample_barcode, read_name, read_name +13 +global_context -> known_cell_barcode_length); else{ int lineno = atoi(sample_barcode +6) +1; *sample_id = (HashTableGet(global_context -> scRNA_lineno1B_to_sampleno1B_tab, NULL+lineno)-NULL); } }else{ if(field_i !=5 || (*lane_str)!='L') SUBREADprintf("LANESTR_ERR %d , %s\n", field_i, lane_str); for(testi = lane_str+1; *testi; testi++){ if(!isdigit(*testi))break; laneno = laneno*10 + (*testi)-'0'; } *sample_id = scRNA_get_sample_id(global_context, sample_barcode, laneno); //Rprintf("LOOKUP SAMPLE %d by %s-%s-%d\n", *sample_id, sample_barcode, lane_str, laneno); } } void add_scRNA_read_tota1_no( fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, char * read_name, char * bambin, int step){ int sample_id= -1; char * cell_bc = NULL, * umi = NULL; int known_sample_id = 0; if(global_context -> scRNA_rerun_on_persample_BAM) known_sample_id = global_context -> this_input_number+1; scRNA_find_sample_cell_umi_from_readname(global_context, thread_context, read_name, bambin, (known_sample_id>0)?NULL:&sample_id, &cell_bc, &umi, NULL); if(known_sample_id>0) sample_id = known_sample_id; if(global_context -> scRNA_UMI_length <1){ int umi_end_pos=0,nch; for(umi_end_pos=0; 0!=(nch = umi [umi_end_pos]); umi_end_pos++) if(!isalpha(nch))break; global_context -> scRNA_UMI_length = umi_end_pos; } //SUBREADprintf("QQVT 01 %s THR %d SPID=%d\n", read_name, thread_context -> thread_id, sample_id); if(sample_id>0){ if(step==0){ thread_context -> scRNA_reads_per_sample[sample_id-1] ++; }else if(step==1) thread_context -> scRNA_mapped_reads_per_sample[sample_id-1] ++; } if(step == 0){ if(global_context -> is_scRNA_BAM_FQ_out_generated){ void * pps[6]; void ** sample_bam_2fps = (void**)pps; if(sample_id<=0){ pps[0]=NULL; pps[1]=global_context -> scRNA_fastq_unassigned_writer; pps[2]=global_context -> scRNA_fastq_unassigned_writer+1; if(global_context -> scRNA_dual_index)pps[3]=global_context -> scRNA_fastq_unassigned_writer+2; else pps[3]=NULL; pps[4]=global_context -> scRNA_fastq_unassigned_writer+3; pps[5]=&global_context -> scRNA_fastq_unassigned_lock; }else sample_bam_2fps = HashTableGet(global_context -> scRNA_sample_BAM_writers, NULL+(sample_id-1) + 1); // sample_id-1: 0,1,2,... if(GENE_INPUT_SCRNA_FASTQ != global_context -> scRNA_input_mode){ parallel_gzip_writer_t **gz3fps = (parallel_gzip_writer_t **)sample_bam_2fps+1; parallel_gzip_writer_add_read_fqs_scRNA(gz3fps, bambin, thread_context -> thread_id); // I2 always has the same length as I1, hence no test is required. if( gz3fps[0]-> thread_objs[thread_context -> thread_id].in_buffer_used >= PARALLEL_GZIP_TXT_BUFFER_SIZE - PARALLEL_GZIP_TXT_BUFFER_MARGIN || gz3fps[1]-> thread_objs[thread_context -> thread_id].in_buffer_used >= PARALLEL_GZIP_TXT_BUFFER_SIZE - PARALLEL_GZIP_TXT_BUFFER_MARGIN || gz3fps[3]-> thread_objs[thread_context -> thread_id].in_buffer_used >= PARALLEL_GZIP_TXT_BUFFER_SIZE - PARALLEL_GZIP_TXT_BUFFER_MARGIN ){ parallel_gzip_zip_texts(gz3fps[0], thread_context -> thread_id, 0); parallel_gzip_zip_texts(gz3fps[1], thread_context -> thread_id, 0); if(gz3fps[2]) parallel_gzip_zip_texts(gz3fps[2], thread_context -> thread_id, 0); parallel_gzip_zip_texts(gz3fps[3], thread_context -> thread_id, 0); pthread_spin_lock(sample_bam_2fps[5]); parallel_gzip_writer_flush(gz3fps[0], thread_context -> thread_id); parallel_gzip_writer_flush(gz3fps[1], thread_context -> thread_id); if(gz3fps[2]) parallel_gzip_writer_flush(gz3fps[2], thread_context -> thread_id); parallel_gzip_writer_flush(gz3fps[3], thread_context -> thread_id); pthread_spin_unlock(sample_bam_2fps[5]); } } } } } void scRNA_do_one_batch_write_extend_rbin(fc_thread_global_context_t * global_context, char * rbin, int binlen, FILE * fp, char * fixedbc_seq, char * fixedumi_seq, srInt_64 gene_no, srInt_64 * genes){ char * cellbc_seq=NULL,*umi_seq=NULL, * cellbc_qual=NULL,*umi_qual=NULL, *sample_seq=NULL, *sample_qual=NULL, *lane_str=NULL; int rname_trimmed_len=0; scRNA_scan_read_name_str(global_context, NULL, rbin, & sample_seq, & sample_qual, & cellbc_seq, & cellbc_qual, & umi_seq, & umi_qual, &lane_str, NULL, &rname_trimmed_len); char new_rbin_stake[ binlen + 150 ]; // removed barcodes/qual from read names, add them to extra fields if they weren't there. Gene names are not put here. char * new_rbin = new_rbin_stake; int new_rbin_len = 0, n_cigar_op =0, l_read_name=0, l_seq=0; memcpy(new_rbin, rbin, 36); new_rbin_len += 36; memcpy(&n_cigar_op, rbin+16,2); memcpy(&l_seq, rbin+20,4); l_read_name=((unsigned char*)rbin)[12]; new_rbin[12] = rname_trimmed_len+1; memcpy(new_rbin+new_rbin_len, rbin+36, rname_trimmed_len); new_rbin[36+rname_trimmed_len]=0; new_rbin_len+= rname_trimmed_len+1; memcpy(new_rbin+new_rbin_len, rbin +36 + l_read_name, 4*n_cigar_op + l_seq + (l_seq+1)/2); new_rbin_len += 4*n_cigar_op + l_seq + (l_seq+1)/2; char * ext_bin_ptr = rbin + 36 + l_read_name +4*n_cigar_op + l_seq + (l_seq+1)/2; int CR_found=0, CB_found=0, CY_found=0, UR_found=0, UY_found=0, UB_found=0; while(ext_bin_ptr < rbin+binlen+4){ char * tagstr = NULL; int taglen = 0; if(ext_bin_ptr[0]=='C' && ext_bin_ptr[1]=='R' && ext_bin_ptr[2]=='Z'){ CR_found = 1; tagstr = cellbc_seq; taglen = global_context -> known_cell_barcode_length; }else if(ext_bin_ptr[0]=='C' && ext_bin_ptr[1]=='B' && ext_bin_ptr[2]=='Z'){ CB_found = 1; tagstr = fixedbc_seq; taglen = global_context -> known_cell_barcode_length; }else if(ext_bin_ptr[0]=='C' && ext_bin_ptr[1]=='Y' && ext_bin_ptr[2]=='Z'){ CY_found = 1; tagstr = cellbc_qual; taglen = global_context -> known_cell_barcode_length; }else if(ext_bin_ptr[0]=='U' && ext_bin_ptr[1]=='R' && ext_bin_ptr[2]=='Z'){ UR_found = 1; tagstr = umi_seq; taglen = global_context -> scRNA_UMI_length; }else if(ext_bin_ptr[0]=='U' && ext_bin_ptr[1]=='B' && ext_bin_ptr[2]=='Z'){ UB_found = 1; tagstr = fixedumi_seq; taglen = global_context -> scRNA_UMI_length; }else if(ext_bin_ptr[0]=='U' && ext_bin_ptr[1]=='Y' && ext_bin_ptr[2]=='Z'){ UY_found = 1; tagstr = umi_qual; taglen = global_context -> scRNA_UMI_length; } if(tagstr){ new_rbin[new_rbin_len++]=*(ext_bin_ptr++); new_rbin[new_rbin_len++]=*(ext_bin_ptr++); new_rbin[new_rbin_len++]=*(ext_bin_ptr++); int taglenold = strlen(ext_bin_ptr); memcpy(new_rbin+new_rbin_len,tagstr, taglen); *(new_rbin+new_rbin_len+taglen)=0; ext_bin_ptr += taglenold+1; new_rbin_len += taglen+1; }else{ int content_len = SAP_pairer_skip_tag_body_len(ext_bin_ptr); memcpy(new_rbin + new_rbin_len, ext_bin_ptr, content_len ); new_rbin_len += content_len; ext_bin_ptr += content_len; } } if(!CR_found){ new_rbin[new_rbin_len++]='C';new_rbin[new_rbin_len++]='R';new_rbin[new_rbin_len++]='Z'; memcpy(new_rbin+new_rbin_len, cellbc_seq, global_context -> known_cell_barcode_length); *(new_rbin+new_rbin_len+global_context -> known_cell_barcode_length)=0; new_rbin_len += global_context -> known_cell_barcode_length+1; } if(fixedbc_seq && !CB_found){ new_rbin[new_rbin_len++]='C';new_rbin[new_rbin_len++]='B';new_rbin[new_rbin_len++]='Z'; memcpy(new_rbin+new_rbin_len, fixedbc_seq, global_context -> known_cell_barcode_length); *(new_rbin+new_rbin_len+global_context -> known_cell_barcode_length)=0; new_rbin_len += global_context -> known_cell_barcode_length+1; } if(!CY_found){ new_rbin[new_rbin_len++]='C';new_rbin[new_rbin_len++]='Y';new_rbin[new_rbin_len++]='Z'; memcpy(new_rbin+new_rbin_len, cellbc_qual, global_context -> known_cell_barcode_length); *(new_rbin+new_rbin_len+global_context -> known_cell_barcode_length)=0; new_rbin_len += global_context -> known_cell_barcode_length+1; } if(!UR_found){ new_rbin[new_rbin_len++]='U';new_rbin[new_rbin_len++]='R';new_rbin[new_rbin_len++]='Z'; memcpy(new_rbin+new_rbin_len, umi_seq, global_context -> scRNA_UMI_length); *(new_rbin+new_rbin_len+global_context -> scRNA_UMI_length)=0; new_rbin_len += global_context -> scRNA_UMI_length+1; } if(fixedumi_seq && !UB_found){ new_rbin[new_rbin_len++]='U';new_rbin[new_rbin_len++]='B';new_rbin[new_rbin_len++]='Z'; memcpy(new_rbin+new_rbin_len, fixedumi_seq, global_context -> scRNA_UMI_length); *(new_rbin+new_rbin_len+global_context -> scRNA_UMI_length)=0; new_rbin_len += global_context -> scRNA_UMI_length+1; } if(!UY_found){ new_rbin[new_rbin_len++]='U';new_rbin[new_rbin_len++]='Y';new_rbin[new_rbin_len++]='Z'; memcpy(new_rbin+new_rbin_len, umi_qual, global_context -> scRNA_UMI_length); *(new_rbin+new_rbin_len+global_context -> scRNA_UMI_length)=0; new_rbin_len += global_context -> scRNA_UMI_length+1; } new_rbin_len-=4; memcpy(new_rbin, &new_rbin_len,4); fwrite(new_rbin, 1, new_rbin_len+4, fp); if(new_rbin!=new_rbin_stake)free(new_rbin); } //int cttt = 0; void add_scRNA_read_to_pool( fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, srInt_64 assign_target_number, char * read_name, char * read_bin, ArrayList * target_list ){ // the index of gene or the index of exon char * cell_barcode = NULL, * umi_barcode = NULL; int sample_id = -1, known_sample_id = 0; if(global_context -> scRNA_rerun_on_persample_BAM) known_sample_id = global_context -> this_input_number+1; scRNA_find_sample_cell_umi_from_readname(global_context, thread_context, read_name, read_bin, (known_sample_id >0)?NULL:&sample_id, &cell_barcode, &umi_barcode, NULL); if(known_sample_id >0) sample_id = known_sample_id; int cell_id = scRNA_get_cell_id(global_context, thread_context, cell_barcode); // int umi_id = scRNA_register_umi_id( global_context, thread_context, umi_barcode); //SUBREADprintf("P0\n"); thread_context -> scRNA_pooled_reads ++; if(sample_id >0)thread_context -> scRNA_has_valid_sample_index ++; if(cell_id >=0)thread_context -> scRNA_has_valid_cell_barcode ++; if(thread_context -> thread_id == 0 && thread_context -> scRNA_pooled_reads == 20000){ print_in_box(80,0,0," scRNA quality control in first 20,000 reads:"); print_in_box(80,0,0," %.1f pct reads have valid sample indices.", thread_context->scRNA_has_valid_sample_index*100./thread_context -> scRNA_pooled_reads); print_in_box(80,0,0," %.1f pct reads have valid cell barcodes.", thread_context->scRNA_has_valid_cell_barcode*100./thread_context -> scRNA_pooled_reads); print_in_box(80,0,0,""); } //if(sample_id >0) SUBREADprintf("P1 Cell=%s, Umi=%s, Lane=%d ==> sample %d\n", cell_barcode, umi_barcode, laneno, sample_id); if(sample_id >0){ int barcode_hashed_key; if(assign_target_number >=0 || target_list) thread_context -> scRNA_assigned_reads_per_sample[sample_id-1] ++; // has cell-bc, assigned : to hashed bin // has cell-bc, not assigned, has mapping location: to hashed bin, with 0-len gene list // no cell-bc, has mapping location: to the MAX - 1 bin, with 0-len gene list // no mapping location: to the MAX bin; only has rbins but no headers. if(cell_id >=0 && (assign_target_number>=0 || target_list))barcode_hashed_key = cell_id % global_context -> scRNA_barcode_batched_bin_no; else{ int chro_no = -1, chro_pos = -1; memcpy(&chro_no, read_bin + 4, 4); memcpy(&chro_pos, read_bin + 8, 4); if(cell_id >= 0 && chro_no>=0) barcode_hashed_key = cell_id % global_context -> scRNA_barcode_batched_bin_no; else if(cell_id <0 && chro_no>=0) barcode_hashed_key = global_context -> scRNA_barcode_batched_bin_no; else barcode_hashed_key = global_context -> scRNA_barcode_batched_bin_no+1; } pthread_spin_lock(global_context -> scRNA_barcode_batched_locks+barcode_hashed_key); FILE * myfp = global_context -> scRNA_barcode_batched_bins[barcode_hashed_key]; fwrite(&sample_id,1,4,myfp); srInt_64 itemno = 1; if(barcode_hashed_key<=global_context -> scRNA_barcode_batched_bin_no){ fwrite(&cell_id,1,4,myfp); if(assign_target_number<0){ itemno = target_list?target_list -> numOfElements:0; itemno = itemno | (1llu << 63); fwrite(&itemno,1,8,myfp); int x1; if(target_list)for(x1=0;x1 numOfElements;x1++){ srInt_64 geneno_0B = ArrayListGet(target_list, x1) -NULL; fwrite(&geneno_0B,1,8,myfp); } }else fwrite(&assign_target_number,1,8,myfp); fwrite(umi_barcode,1,global_context -> scRNA_UMI_length,myfp); } int read_bin_len=0; memcpy(&read_bin_len , read_bin, 4); if(barcode_hashed_key==global_context -> scRNA_barcode_batched_bin_no+1){ // the read is unmapped. It can still have a fixed cell barcode char * new_cellbc = NULL; if(cell_id>=0)new_cellbc = ArrayListGet(global_context -> scRNA_cell_barcodes_array, cell_id); scRNA_do_one_batch_write_extend_rbin(global_context, read_bin, read_bin_len, myfp, new_cellbc, NULL, -1, NULL); }else fwrite(read_bin, 1, read_bin_len+4, myfp); pthread_spin_unlock(global_context -> scRNA_barcode_batched_locks+barcode_hashed_key); } } int scRNA_do_one_batch_sort_compare(void * ar, int l, int r){ void ** arr = ar; void ** bin_ptrs = arr[0]; fc_thread_global_context_t * global_context = arr[1]; char * Lptr = bin_ptrs[l]; char * Rptr = bin_ptrs[r]; srInt_64 Lgenes=0, Rgenes=0; memcpy(&Lgenes, Lptr+8, 8); memcpy(&Rgenes, Rptr+8, 8); if(Lgenes & (1LLU<<63))Lgenes=Lgenes & 0x7fffffffllu; else Lgenes=0; if(Rgenes & (1LLU<<63))Rgenes=Rgenes & 0x7fffffffllu; else Rgenes=0; srInt_64 Lpos= ((0LLU+*(int*)(Lptr+16+Lgenes*8+global_context->scRNA_UMI_length+4))<<32) | *(unsigned int*)(Lptr+16+Lgenes*8+global_context->scRNA_UMI_length+4+4); srInt_64 Rpos= ((0LLU+*(int*)(Rptr+16+Rgenes*8+global_context->scRNA_UMI_length+4))<<32) | *(unsigned int*)(Rptr+16+Rgenes*8+global_context->scRNA_UMI_length+4+4); if(Lpos>Rpos)return 1; if(Lpos1)return ret; } return ret; } #define ADD_count_hash(bc,gn,no) HashTablePut(cellBCp0_genep0_P1_to_UMIs, NULL +1+(((1LLU*(bc))<<32)| (gn) ), HashTableGet( cellBCp0_genep0_P1_to_UMIs, NULL +1+(((1LLU*(bc))<<32)| (gn))) +(no) ) void scRNA_do_one_batch_UMI_merge_one_cell(ArrayList* structs, int sec_start, int sec_end, int is_UMI_step2, HashTable * filtered_CGU_table){ int x1; void ** app1 = structs -> appendix1; fc_thread_global_context_t * global_context = app1[0]; HashTable * cellBCp0_genep0_P1_to_UMIs = app1[2]; int sample_id = app1[3]-NULL; if(is_UMI_step2){ // NB: when this function is called, sec_end - sec_start MUST be >=2. for(x1 = sec_start; x1 supp_reads > str2 -> supp_reads){ ADD_count_hash(str1->cellbc, str1->gene_no,1); continue; } } str1 -> cellbc = -1; char replaced_key[40+MAX_UMI_LEN]; #ifdef __MINGW32__ int keyptr = sprintf(replaced_key,"%d-%I64d-", str1 -> cellbc, str1 -> gene_no); #else int keyptr = sprintf(replaced_key,"%d-%lld-", str1 -> cellbc, str1 -> gene_no); #endif memcpy(replaced_key+keyptr, str1 -> umi, global_context -> scRNA_UMI_length); replaced_key[keyptr+global_context -> scRNA_UMI_length]=0; HashTablePut(filtered_CGU_table, strdup(replaced_key), NULL-1); } }else{ ArrayList * accepted_list =NULL; HashTable * looktable = NULL; if(sec_end - sec_start >30){ looktable = StringTableCreate((sec_end - sec_start)/5); HashTableSetDeallocationFunctions(looktable, free, (void (*)(void *value))ArrayListDestroy); }else accepted_list = ArrayListCreate(sec_end - sec_start); for(x1=sec_start; x1 umi + hx * global_context -> scRNA_UMI_length/2 , global_context -> scRNA_UMI_length/2); test_ky[1+global_context -> scRNA_UMI_length/2]=0; test_accs = HashTableGet(looktable, test_ky); if(!test_accs)continue; for(x2=0; x2numOfElements; x2++){ struct cell_gene_umi_supp * acc_str = ArrayListGet(test_accs, x2); if(scRNA_hamming_max2_fixlen(acc_str -> umi, try_str -> umi, global_context -> scRNA_UMI_length)<2){ found=1; acc_str -> supp_reads += try_str -> supp_reads; try_str -> cellbc = -1; char replaced_key[55+MAX_UMI_LEN]; #ifdef __MINGW32__ int keyptr = sprintf(replaced_key,"%d-%d-%I64d-", sample_id, try_str -> cellbc, try_str -> gene_no); #else int keyptr = sprintf(replaced_key,"%d-%d-%lld-", sample_id, try_str -> cellbc, try_str -> gene_no); #endif memcpy(replaced_key+keyptr, try_str -> umi, global_context -> scRNA_UMI_length); replaced_key[keyptr+global_context -> scRNA_UMI_length]=0; HashTablePut(filtered_CGU_table, strdup(replaced_key), acc_str -> umi); break; } } if(found)break; } }else{ test_accs = accepted_list; for(x2=0; x2numOfElements; x2++){ struct cell_gene_umi_supp * acc_str = ArrayListGet(test_accs, x2); int hamming = scRNA_hamming_max2_fixlen(acc_str -> umi, try_str -> umi, global_context -> scRNA_UMI_length); if(hamming<2){ found=1; acc_str -> supp_reads += try_str -> supp_reads; try_str -> cellbc = -1; char replaced_key[55+MAX_UMI_LEN]; #ifdef __MINGW32__ int keyptr = sprintf(replaced_key,"%d-%d-%I64d-", sample_id, try_str -> cellbc, try_str -> gene_no); #else int keyptr = sprintf(replaced_key,"%d-%d-%lld-", sample_id, try_str -> cellbc, try_str -> gene_no); #endif memcpy(replaced_key+keyptr, try_str -> umi, global_context -> scRNA_UMI_length); replaced_key[keyptr+global_context -> scRNA_UMI_length]=0; HashTablePut(filtered_CGU_table, strdup(replaced_key), acc_str -> umi); break; } } } if(!found){ if(looktable){ for(hx = 0; hx<2; hx++){ char test_ky[MAX_UMI_LEN]; test_ky[0] = hx?'S':'F'; memcpy(test_ky +1, try_str -> umi + hx * global_context -> scRNA_UMI_length/2 , global_context -> scRNA_UMI_length/2); test_ky[1+global_context -> scRNA_UMI_length/2]=0; test_accs = HashTableGet(looktable, test_ky); if(!test_accs){ test_accs = ArrayListCreate(10); HashTablePut(looktable, strdup(test_ky), test_accs); } ArrayListPush(test_accs, try_str); } }else ArrayListPush(accepted_list, try_str); } } if(looktable)HashTableDestroy(looktable); else ArrayListDestroy(accepted_list); } } void scRNA_do_one_batch_UMI_merge_one_step(ArrayList* structs, int is_UMI_step2, HashTable * filtered_CGU_table){ void ** app1 = structs -> appendix1; fc_thread_global_context_t * global_context = app1[0]; HashTable * cellBCp0_genep0_P1_to_UMIs = app1[2]; srInt_64 x1, sec_start = 0; srInt_64 old_sec_key = -1; for(x1=0; x1<=structs -> numOfElements; x1++){ srInt_64 sec_key = -1; int is_umi_changed = 0; struct cell_gene_umi_supp * str1 =NULL; if(x1 numOfElements){ str1 = ArrayListGet(structs, x1); if(str1 -> cellbc <0) continue; sec_key = str1 -> cellbc; sec_key = sec_key << 32; if(is_UMI_step2 && sec_key == old_sec_key){ struct cell_gene_umi_supp * strold = ArrayListGet(structs, sec_start); is_umi_changed = memcmp(strold -> umi, str1-> umi, global_context-> scRNA_UMI_length); }else if(!is_UMI_step2) sec_key = sec_key | str1 -> gene_no; // gene_no itself is 64-bit, but it is nearly impossible to have two neighbouring // structures that have the same last 32-bit of gene_no. } if( (x1>sec_start && sec_key!=old_sec_key) || is_umi_changed){ // when x1 == numOfElements, sec_key is -1. If old_sec_key is also -1, no item is included in the list. If old_sec_key is >=0, the last sec is processed. struct cell_gene_umi_supp * str0 = ArrayListGet(structs, sec_start); if(x1 - sec_start>1 && str0->cellbc>=0) scRNA_do_one_batch_UMI_merge_one_cell(structs, sec_start, x1, is_UMI_step2, filtered_CGU_table); else if(is_UMI_step2 && str0->cellbc>=0) ADD_count_hash(str0->cellbc,str0->gene_no,1); sec_start = x1; } old_sec_key = sec_key; } } int scRNA_do_one_batch_tab_to_struct_list_compare(void * L_elem, void * R_elem, ArrayList * me){ struct cell_gene_umi_supp *L = L_elem, *R = R_elem; void ** app1 = me -> appendix1; fc_thread_global_context_t * global_context = app1[0]; int sort_by_geneid_then_umi = app1[1] - NULL; if(L->cellbc > R->cellbc) return 1; if(L->cellbc < R->cellbc) return -1; if(sort_by_geneid_then_umi){ if(L->gene_no>R->gene_no) return 1; if(L->gene_nogene_no) return -1; }else{ int umicmps = memcmp(L->umi, R->umi, global_context -> scRNA_UMI_length); if(umicmps) return umicmps; } if(L->supp_reads < R->supp_reads) return 1; if(L->supp_reads > R->supp_reads) return -1; // reversed by # supp reads if(sort_by_geneid_then_umi){ int umicmps = memcmp(L->umi, R->umi, global_context -> scRNA_UMI_length); if(umicmps) return umicmps; }else{ if(L->gene_no>R->gene_no) return 1; if(L->gene_nogene_no) return -1; } return 0; } void scRNA_do_one_batch_tab_to_struct_list(void *ky, void *val, HashTable * tab){ int supp_reads = val-NULL; ArrayList ** cell_gene_umi_list = tab -> appendix1; int UMI_length = tab -> counter1; struct cell_gene_umi_supp * new_item = malloc(sizeof(struct cell_gene_umi_supp)); char * kyptr = ky; int sample_id = atoi(kyptr); // one-based sample id for(; '-' != *kyptr; kyptr++); kyptr++; new_item -> cellbc = atoi(kyptr); for(; '-' != *kyptr; kyptr++); kyptr++; new_item -> gene_no = atoll(kyptr); for(; '-' != *kyptr; kyptr++); memcpy(new_item->umi, kyptr+1, UMI_length); new_item -> supp_reads = supp_reads; if(sample_id<1)SUBREADprintf("WRONG SAMPLE ID: %d from '%s'\n", sample_id, (char*)ky); ArrayListPush(cell_gene_umi_list[sample_id-1], new_item); } void scRNA_do_one_batch_write_UMIs(void * vcell_gene, void * vumis, HashTable * me){ FILE * fp = me->appendix1; vcell_gene --; fwrite(&vcell_gene,1,8,fp); fwrite(&vumis,1,8,fp); } srInt_64 two_long_hash(void * ky){ srInt_64 * ky2 = ky; return ky2[0]^ky2[1]; } int two_long_compare(void * k1, void * k2){ srInt_64 * k13 = k1, *k23 = k2; if(k13[0]!=k23[0])return 1; if(k13[1]!=k23[1])return 1; return 0; } #ifdef __MINGW32__ #define ADD_key_FMT1 "%d-%d-%I64d-%s" #else #define ADD_key_FMT1 "%d-%d-%lld-%s" #endif #define ADD_key_struct { char my_key [50+MAX_UMI_LEN]; \ sprintf(my_key,ADD_key_FMT1, sample_id, cell_no, gene_no, UMI_str); \ srInt_64 supp_reads = HashTableGet(supp_reads_SCGU, my_key)-NULL; \ if(1>supp_reads) HashTablePut(supp_reads_SCGU, strdup(my_key), NULL+1); \ else HashTablePutReplaceEx(supp_reads_SCGU, my_key, NULL+supp_reads+1, 0,0,0); } void * scRNA_do_one_batch(void * paramsp1){ srInt_64 x1; void ** params = paramsp1; fc_thread_global_context_t * global_context = params[0]; ArrayList * file_size_list = params[2]; char *temp_dir = global_context -> temp_file_dir; free(paramsp1); int me_max_Rbin_len = 0; int me_max_genes = 0; char ** bin_ptrs = malloc(sizeof(char*) * 1500000), * batch_content=NULL; int bin_ptr_size = 1500000; while(1){ int this_batch_no = -1; pthread_spin_lock(&global_context -> scRNA_do_one_batch_runner_lock); if(global_context -> scRNA_do_one_batch_runner_current < global_context -> scRNA_barcode_batched_bin_no +1){ int this_batch_sorted_idx = (global_context -> scRNA_do_one_batch_runner_current ++); srInt_64 this_batch_size_and_no = ArrayListGet(file_size_list, file_size_list->numOfElements-1 -this_batch_sorted_idx)-NULL; this_batch_no = (int)(this_batch_size_and_no&0xfffffllu); } if(me_max_genes > global_context -> scRNA_barcode_batched_max_genes) global_context -> scRNA_barcode_batched_max_genes = me_max_genes; if(me_max_Rbin_len > global_context->scRNA_barcode_batched_max_Rbin_len) global_context->scRNA_barcode_batched_max_Rbin_len = me_max_Rbin_len; pthread_spin_unlock(&global_context -> scRNA_do_one_batch_runner_lock); if(0>this_batch_no)break; char tmp_fname[MAX_FILE_NAME_LENGTH+80]; sprintf(tmp_fname, "%s/cellCounts-Splitted-Reads-%05d-%05d.bin", temp_dir, getpid(), this_batch_no); FILE * fp = fopen(tmp_fname, "rb"); fseek(fp, 0, SEEK_END); srInt_64 batch_fsize = ftello(fp); fseek(fp, 0, SEEK_SET); if(batch_content==NULL) batch_content = malloc(batch_fsize); srInt_64 batch_content_len = fread(batch_content, 1, batch_fsize, fp); fclose(fp); if(batch_content_len!=batch_fsize){ SUBREADprintf("ERROR: Cannot load file at once: %d!\n", this_batch_no); return NULL; } HashTable * supp_reads_SCGU = StringTableCreate(500000); HashTableSetDeallocationFunctions(supp_reads_SCGU, free, NULL); srInt_64 scanptr = 0; int rbin_no = 0; char UMI_str[MAX_UMI_LEN+1]; while(scanptr < batch_content_len-1){ int cell_no=0, sample_id=0; srInt_64 gene_no=0; if(bin_ptr_size<=rbin_no){ bin_ptr_size = bin_ptr_size*2; bin_ptrs = realloc(bin_ptrs, sizeof(char*)*bin_ptr_size); } bin_ptrs[rbin_no] = batch_content+scanptr; memcpy(&sample_id, batch_content+scanptr, 4); scanptr += 4; // sample_ID memcpy(&cell_no, batch_content+scanptr, 4); scanptr += 4; // cellbarcode_NO memcpy(&gene_no, batch_content+scanptr, 8); scanptr += 8; // gene_id if(gene_no & (1LLU<<63)){ int genes = (int)(gene_no & 0x7fffffffllu); if(genes > me_max_genes)me_max_genes=genes; memcpy(UMI_str, batch_content+scanptr+8*genes, global_context -> scRNA_UMI_length); UMI_str[global_context -> scRNA_UMI_length]=0; for(x1=0; x1 scRNA_UMI_length]=0; memcpy(UMI_str, batch_content+scanptr, global_context -> scRNA_UMI_length); ADD_key_struct; } scanptr += global_context -> scRNA_UMI_length ; // UMI str int rbinlen = 0; memcpy(&rbinlen, batch_content+scanptr, 4); if(me_max_Rbin_len < rbinlen) me_max_Rbin_len = rbinlen; scanptr += rbinlen +4; // read_bin // if(sample_id <0 || sample_id > 1000) SUBREADprintf("Wrong Sample: RNO=%d; ptr=%lld\n", rbin_no, scanptr); rbin_no++; } ArrayList ** cell_gene_umi_list = malloc(sizeof(void*)*global_context -> scRNA_sample_sheet_table -> numOfElements); for(x1 =0; x1< global_context -> scRNA_sample_sheet_table -> numOfElements; x1++){ cell_gene_umi_list[x1]=ArrayListCreate(2000000); ArrayListSetDeallocationFunction(cell_gene_umi_list[x1], free); } supp_reads_SCGU -> appendix1 = cell_gene_umi_list; supp_reads_SCGU -> appendix2 = global_context; supp_reads_SCGU -> counter1 = global_context -> scRNA_UMI_length; HashTableIteration(supp_reads_SCGU, scRNA_do_one_batch_tab_to_struct_list); HashTable * filtered_SCGU_table = StringTableCreate(max(10000,cell_gene_umi_list[0] -> numOfElements / 10)); HashTableSetDeallocationFunctions(filtered_SCGU_table, free, NULL); fp = fopen(tmp_fname, "wb"); for(x1 = 0; x1 < global_context -> scRNA_sample_sheet_table -> numOfElements; x1++){ HashTable * cellbcP0_to_geneno0B_P1_to_UMIs = HashTableCreate(500000); void * app1[3]; cell_gene_umi_list[x1] -> appendix1 = app1; app1[0] = global_context; app1[1] = NULL+1; // 0 : sorted by cell_bc, then UMIstr, then supported_reads, then gene // 1 : sorted by cell_bc, then gene, then supported_reads, then UMIstr // supported_reads : large -> small; the other: small -> large ArrayListSort(cell_gene_umi_list[x1], scRNA_do_one_batch_tab_to_struct_list_compare); scRNA_do_one_batch_UMI_merge_one_step(cell_gene_umi_list[x1], 0, filtered_SCGU_table); app1[1] = NULL+0; app1[2] = cellbcP0_to_geneno0B_P1_to_UMIs; ArrayListSort(cell_gene_umi_list[x1], scRNA_do_one_batch_tab_to_struct_list_compare); scRNA_do_one_batch_UMI_merge_one_step(cell_gene_umi_list[x1], 1, filtered_SCGU_table); cellbcP0_to_geneno0B_P1_to_UMIs -> appendix1 = fp; fwrite(&cellbcP0_to_geneno0B_P1_to_UMIs -> numOfElements,1,8,fp); HashTableIteration(cellbcP0_to_geneno0B_P1_to_UMIs, scRNA_do_one_batch_write_UMIs); HashTableDestroy(cellbcP0_to_geneno0B_P1_to_UMIs); } void * sort_base[2]; sort_base[0] = bin_ptrs; sort_base[1] = global_context; merge_sort(sort_base, rbin_no, scRNA_do_one_batch_sort_compare, scRNA_do_one_batch_sort_exchange, scRNA_do_one_batch_sort_merge); for(x1 = 0; x1 < rbin_no; x1++){ char * binptr = bin_ptrs[x1]; int cellid =0, sampleid = 0; srInt_64 gene_no =0, genes = 0, geneno_0 = 0; char * umi, * glist_ptr =NULL; memcpy(&sampleid, binptr, 4); memcpy(&cellid, binptr+4, 4); memcpy(&gene_no, binptr+8, 8); if(gene_no & (1LLU<<63)){ glist_ptr =binptr + 16; genes = (int)(gene_no & 0x7fffffff); memcpy(&geneno_0, binptr+16, 8); } umi = binptr + 16 + 8*genes; char SCGU_key [40+MAX_UMI_LEN]; #ifdef __MINGW32__ int keyptr = sprintf(SCGU_key,"%d-%d-%I64d-", sampleid, cellid, (gene_no & (1LLU<<63))? geneno_0: gene_no); #else int keyptr = sprintf(SCGU_key,"%d-%d-%lld-", sampleid, cellid, (gene_no & (1LLU<<63))? geneno_0: gene_no); #endif memcpy(SCGU_key+keyptr, umi, global_context -> scRNA_UMI_length); SCGU_key[keyptr+global_context -> scRNA_UMI_length] = 0; char * new_UMI = HashTableGet(filtered_SCGU_table, SCGU_key); if(new_UMI) umi = new_UMI; if(umi == NULL-1) umi="-----------------------------------------"; fwrite(&sampleid, 1, 4, fp); fwrite(&cellid, 1, 4, fp); fwrite(&gene_no, 1, 8, fp); if(gene_no & (1LLU<<63)) fwrite( glist_ptr, 1, 8*genes, fp ); fwrite(umi,1, global_context -> scRNA_UMI_length, fp); int binlen; memcpy(&binlen, binptr+16+8*genes+global_context -> scRNA_UMI_length,4 ); char * new_cellbc = NULL; if(cellid>=0)new_cellbc = ArrayListGet(global_context -> scRNA_cell_barcodes_array, cellid); scRNA_do_one_batch_write_extend_rbin(global_context, binptr+16+8*genes+global_context -> scRNA_UMI_length, binlen, fp, new_cellbc, umi[0]=='-'?NULL:umi, gene_no, (srInt_64*)glist_ptr); } fclose(fp); HashTableDestroy(supp_reads_SCGU); HashTableDestroy(filtered_SCGU_table); for(x1 =0; x1< global_context -> scRNA_sample_sheet_table -> numOfElements; x1++)ArrayListDestroy(cell_gene_umi_list[x1]); free(cell_gene_umi_list); } free(batch_content); free(bin_ptrs); return NULL; } unsigned int calc_score_overlaps(fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, char ** chros, unsigned int * start_poses, unsigned short * lens, int sections, char * read_name){ unsigned int in_intervals[ 2*sections ]; unsigned int out_intervals[ 2*sections ], x1; char used_interval[ sections ]; memset(used_interval, 0 , sections); unsigned int ret = 0; for(x1 = 0 ; x1 < sections ; x1++){ if( used_interval [x1] )continue; in_intervals[0] = start_poses[x1]; in_intervals[1] = start_poses[x1] + lens[x1]; used_interval[x1]=1; int x2, this_sections = 1; for(x2 = x1 + 1; x2 < sections; x2++){ if(strcmp( chros[x2], chros[x1] ) == 0){ in_intervals[this_sections*2] = start_poses[x2]; in_intervals[this_sections*2 + 1] = start_poses[x2] + lens[x2]; used_interval[x2]=1; this_sections++; } } basic_sort( in_intervals, this_sections, overlap_compare, overlap_exchange ); int merged_secs = mergeIntervals( in_intervals, out_intervals, this_sections ); for(x2 = 0; x2 < merged_secs; x2++) ret += ( out_intervals[x2*2+1] - out_intervals[x2*2] ); } return ret; } void vote_and_add_count(fc_thread_global_context_t * global_context, fc_thread_thread_context_t * thread_context, srInt_64 * hits_indices1, int nhits1, srInt_64 * hits_indices2, int nhits2, unsigned int total_frag_len, char ** hits_chro1, char ** hits_chro2, unsigned int * hits_start_pos1, unsigned int * hits_start_pos2, unsigned short * hits_length1, unsigned short * hits_length2, int fixed_fractional_count, char * read_name, char * RG_name, char * bin1, char * bin2){ if(global_context -> need_calculate_overlap_len == 0 && nhits2+nhits1==1) { srInt_64 hit_exon_id = nhits2?hits_indices2[0]:hits_indices1[0]; //SUBREADprintf("V_AND_A: '%p'\n", RG_name); if(RG_name){ void ** tab4s = get_RG_tables(global_context, thread_context, RG_name); fc_read_counters * sumtab = tab4s[1]; sumtab -> assigned_reads++; read_count_type_t * count_table = tab4s[0]; count_table[hit_exon_id] += fixed_fractional_count; }else{ thread_context->count_table[hit_exon_id] += fixed_fractional_count; thread_context->read_counters.assigned_reads ++; } thread_context->nreads_mapped_to_exon++; if(global_context -> read_details_out_FP){ int final_gene_number = global_context -> exontable_geneid[hit_exon_id]; char * final_feture_name = (char *)global_context -> gene_name_array[final_gene_number]; write_read_details_FP(global_context, thread_context, "Assigned", 1, final_feture_name, bin1, bin2); } if(global_context -> do_scRNA_table){ srInt_64 assignment_target_number = hit_exon_id; if(global_context->is_gene_level) assignment_target_number = global_context -> exontable_geneid[hit_exon_id]; add_scRNA_read_to_pool(global_context, thread_context, assignment_target_number, read_name, bin1, NULL); } } else if(global_context -> need_calculate_overlap_len == 0 && nhits2 == 1 && nhits1 == 1 && hits_indices2[0]==hits_indices1[0]) { srInt_64 hit_exon_id = hits_indices1[0]; if(RG_name){ void ** tab4s = get_RG_tables(global_context, thread_context, RG_name); fc_read_counters * sumtab = tab4s[1]; sumtab -> assigned_reads++; read_count_type_t * count_table = tab4s[0]; count_table[hit_exon_id] += fixed_fractional_count; }else{ thread_context->count_table[hit_exon_id] += fixed_fractional_count; thread_context->read_counters.assigned_reads ++; } thread_context->nreads_mapped_to_exon++; if(global_context -> read_details_out_FP) { int final_gene_number = global_context -> exontable_geneid[hit_exon_id]; char * final_feture_name = (char *)global_context -> gene_name_array[final_gene_number]; write_read_details_FP(global_context, thread_context, "Assigned", 1, final_feture_name, bin1, bin2); } if(global_context -> do_scRNA_table){ srInt_64 assignment_target_number = hit_exon_id; if(global_context->is_gene_level) assignment_target_number = global_context -> exontable_geneid[hit_exon_id]; add_scRNA_read_to_pool(global_context, thread_context, assignment_target_number, read_name, bin1, NULL); } } else { // Build a voting table. // The voting table should be: // total_length [nhit_final] = total_length_overlapping // final_id [nhit_final] = final_exon_id // if is_gene_leven, then decision_table_exon_ids[nhit_final] is the exon id where the count is added. // After all, the count is added to all hits where total_length has the maximum value. // If there are more than one locations having the same total_length, then the fragment is ambiguous. // Count is added when "-O" is specified. // merge feature : if a read overlaps with an EXON twice or more times (by >=2 segments in cigar), // then the total length of the overlapped bases is calculated. // // two ends in a fragment is considered individually; the overlapping bases are not added up. // unsigned int * scoring_numbers = thread_context -> scoring_buff_numbers; // size is : MAX_HIT_NUMBER *2 unsigned int * scoring_flags = thread_context -> scoring_buff_flags; // size is : MAX_HIT_NUMBER *2 unsigned int * scoring_overlappings = thread_context -> scoring_buff_overlappings; // size is : MAX_HIT_NUMBER *2 srInt_64 * scoring_exon_ids = thread_context -> scoring_buff_exon_ids; // size is : MAX_HIT_NUMBER *2 int scoring_count = 0, score_x1; if( global_context -> need_calculate_overlap_len ){ int end1, end2, hit_x1, hit_x2; char ** scoring_gap_chros = thread_context -> scoring_buff_gap_chros; unsigned int * scoring_gap_starts = thread_context -> scoring_buff_gap_starts; // size is : MAX_HIT_NUMBER *2; unsigned short * scoring_gap_lengths = thread_context -> scoring_buff_gap_lengths; // size is : MAX_HIT_NUMBER *2* global_context -> max_M*2 char used_hit1 [nhits1]; char used_hit2 [nhits2]; if( global_context -> fractional_minimum_feature_overlapping > 1E-10 || global_context -> max_missing_bases_in_feature >= 0){ memset(used_hit1 , 0 , nhits1); memset(used_hit2 , 0 , nhits2); for(end1 = 0; end1 < global_context -> is_paired_end_mode_assign + 1 ; end1++){ int allhits = end1?nhits2:nhits1; srInt_64 * hits_indices_X1 = end1?hits_indices2:hits_indices1; char * used_hit_X1 = end1?used_hit2:used_hit1; for(hit_x1 = 0; hit_x1 < allhits; hit_x1++){ if(used_hit_X1[hit_x1])continue; srInt_64 tested_exon_id = hits_indices_X1[hit_x1]; srInt_64 exon_span = global_context -> exontable_stop[tested_exon_id] +1; exon_span -= global_context -> exontable_start[tested_exon_id]; srInt_64 applied_overlapping_threshold_frac = 0, applied_overlapping_threshold_missing = 0; if(global_context -> max_missing_bases_in_feature >= 0){ if(exon_span <= global_context -> max_missing_bases_in_feature) applied_overlapping_threshold_missing = 0; else applied_overlapping_threshold_missing = 10000L * (exon_span - global_context -> max_missing_bases_in_feature); } applied_overlapping_threshold_frac = (srInt_64)(exon_span *10000.* global_context -> fractional_minimum_feature_overlapping + 0.9999); srInt_64 applied_overlapping_threshold = max(applied_overlapping_threshold_frac , applied_overlapping_threshold_missing); scoring_gap_chros[0 ] = (end1?hits_chro2:hits_chro1)[hit_x1]; scoring_gap_starts[0 ] = (end1?hits_start_pos2:hits_start_pos1)[hit_x1]; scoring_gap_lengths[0 ] = (end1?hits_length2:hits_length1)[hit_x1]; int gaps=1; for(end2 = 0; end2 < global_context -> is_paired_end_mode_assign + 1 ; end2++){ int allhits2 = end2?nhits2:nhits1; char * used_hit_X2 = end2?used_hit2:used_hit1; srInt_64 * hits_indices_X2 = end2?hits_indices2:hits_indices1; for(hit_x2 = 0; hit_x2 < allhits2; hit_x2++){ if(used_hit_X2[hit_x2]) continue; srInt_64 other_exon_id = hits_indices_X2[hit_x2]; if(other_exon_id == tested_exon_id){ used_hit_X2[ hit_x2 ]=1; scoring_gap_chros[ gaps ] = (end2?hits_chro2:hits_chro1)[hit_x2]; scoring_gap_starts[ gaps ] = (end2?hits_start_pos2:hits_start_pos1)[hit_x2]; scoring_gap_lengths[ gaps ] = (end2?hits_length2:hits_length1)[hit_x2]; gaps ++; } } } srInt_64 tested_exon_overlap_any_read = 10000L*calc_score_overlaps(global_context, thread_context, scoring_gap_chros, scoring_gap_starts, scoring_gap_lengths, gaps, read_name); if(applied_overlapping_threshold > tested_exon_overlap_any_read){ // remove this exon from lists for(end2 = 0; end2 < global_context -> is_paired_end_mode_assign + 1 ; end2++){ int allhits2 = end2?nhits2:nhits1; srInt_64 * hits_indices_X2 = end2?hits_indices2:hits_indices1; for(hit_x2 = 0; hit_x2 < allhits2; hit_x2++){ srInt_64 other_exon_id = hits_indices_X2[hit_x2]; if(other_exon_id == tested_exon_id){ hits_indices_X2[hit_x2] = -1; } } } } } } } memset(used_hit1 , 0 , nhits1); memset(used_hit2 , 0 , nhits2); for(end1 = 0; end1 < global_context -> is_paired_end_mode_assign + 1 ; end1++){ srInt_64 * hits_indices_X1 = end1?hits_indices2:hits_indices1; char * used_hit_X1 = end1?used_hit2:used_hit1; int nhit_X1 = end1?nhits2:nhits1; for( hit_x1 = 0 ; hit_x1 < nhit_X1; hit_x1 ++ ){ if(used_hit_X1[hit_x1])continue; int gaps = 0; srInt_64 tmp_exon_id = hits_indices_X1[hit_x1]; if(tmp_exon_id < 0) continue; srInt_64 score_merge_key; if (global_context -> is_gene_level ) score_merge_key = global_context -> exontable_geneid[tmp_exon_id]; else score_merge_key = tmp_exon_id; scoring_gap_chros[0 ] = (end1?hits_chro2:hits_chro1)[hit_x1]; scoring_gap_starts[0 ] = (end1?hits_start_pos2:hits_start_pos1)[hit_x1]; scoring_gap_lengths[0 ] = (end1?hits_length2:hits_length1)[hit_x1]; gaps=1; scoring_flags[scoring_count] = end1?2:1; scoring_numbers[scoring_count] =1; scoring_exon_ids[scoring_count] = tmp_exon_id; used_hit_X1[ hit_x1 ]=1; for(end2 = 0; end2 < global_context -> is_paired_end_mode_assign + 1 ; end2++){ srInt_64 * hits_indices_X2 = end2?hits_indices2:hits_indices1; char * used_hit_X2 = end2?used_hit2:used_hit1; int nhit_X2 = end2?nhits2:nhits1; for( hit_x2 = 0 ; hit_x2 < nhit_X2; hit_x2 ++ ){ if(used_hit_X2[hit_x2])continue; if(hits_indices_X2[hit_x2] < 0) continue; srInt_64 X2_merge_key; if (global_context -> is_gene_level ) X2_merge_key = global_context -> exontable_geneid[ hits_indices_X2[hit_x2] ]; else X2_merge_key = hits_indices_X2[hit_x2]; if( X2_merge_key == score_merge_key ){ used_hit_X2[ hit_x2 ]=1; scoring_gap_chros[ gaps ] = (end2?hits_chro2:hits_chro1)[hit_x2]; scoring_gap_starts[ gaps ] = (end2?hits_start_pos2:hits_start_pos1)[hit_x2]; scoring_gap_lengths[ gaps ] = (end2?hits_length2:hits_length1)[hit_x2]; if((scoring_flags[scoring_count] & (end2?2:1))== 0 ){ scoring_flags[scoring_count] |= end2?2:1; scoring_numbers[scoring_count] ++; } gaps ++; } } } scoring_overlappings [scoring_count] = calc_score_overlaps(global_context, thread_context, scoring_gap_chros, scoring_gap_starts, scoring_gap_lengths, gaps, read_name); if( global_context -> use_overlapping_break_tie ) scoring_numbers[scoring_count] = scoring_overlappings [scoring_count]; scoring_count++; } } }else{ int ends; for(ends =0 ; ends < global_context -> is_paired_end_mode_assign + 1 ; ends++){ int nhits = ends?nhits2:nhits1; srInt_64 * hits_indices = ends?hits_indices2:hits_indices1; int hit_x1; for(hit_x1 = 0; hit_x1 < nhits; hit_x1++){ srInt_64 tmp_exon_id = hits_indices[hit_x1], score_merge_key; int found = 0; if (global_context -> is_gene_level ) score_merge_key = global_context -> exontable_geneid[tmp_exon_id]; else score_merge_key = tmp_exon_id; for(score_x1 = 0; score_x1 < scoring_count; score_x1 ++){ srInt_64 score_x1_key ; if (global_context -> is_gene_level ) score_x1_key = global_context -> exontable_geneid[ scoring_exon_ids[score_x1] ]; else score_x1_key = scoring_exon_ids[score_x1] ; if( score_x1_key == score_merge_key ){ if((scoring_flags[score_x1] & ( ends?2:1 )) == 0) { scoring_flags[score_x1] |= (ends?2:1); scoring_numbers[score_x1] ++; } found = 1; break; } } if(0 == found){ scoring_exon_ids[scoring_count] = tmp_exon_id; scoring_flags[scoring_count] = ends?2:1; scoring_numbers[scoring_count] = 1; scoring_count++; } } } } int maximum_score = 0; int maximum_total_count = 0; int maximum_score_x1 = 0; srInt_64 applied_fragment_minimum_overlapping_overlap = 1, applied_fragment_minimum_overlapping_missing = 1; srInt_64 applied_fragment_minimum_overlapping = 1; int overlapping_total_count = 0; if( global_context -> fragment_minimum_overlapping > 1 || global_context -> need_calculate_fragment_len || global_context -> max_missing_bases_in_read >= 0){ if(global_context -> max_missing_bases_in_read >=0){ if(total_frag_len <= global_context -> max_missing_bases_in_read) applied_fragment_minimum_overlapping_missing = 0; else applied_fragment_minimum_overlapping_missing = 10000L * (total_frag_len - global_context -> max_missing_bases_in_read); } applied_fragment_minimum_overlapping_overlap = max( 10000L * global_context -> fragment_minimum_overlapping, 10000. * global_context -> fractional_minimum_overlapping * total_frag_len + 0.5); applied_fragment_minimum_overlapping = max(applied_fragment_minimum_overlapping_overlap , applied_fragment_minimum_overlapping_missing); } if(scoring_count == 0){ if(global_context -> read_details_out_FP) write_read_details_FP(global_context, thread_context,"Unassigned_NoFeatures",-1, NULL, bin1, bin2); if(RG_name){ void ** tab4s = get_RG_tables(global_context, thread_context, RG_name); fc_read_counters * sumtab = tab4s[1]; sumtab -> unassigned_nofeatures++; }else thread_context->read_counters.unassigned_nofeatures ++; if(global_context -> do_scRNA_table) add_scRNA_read_to_pool(global_context, thread_context, -1, read_name, bin1, NULL); }else{ for(score_x1 = 0; score_x1 < scoring_count ; score_x1++){ // #warning "======= DEBUG OUT ================" if(0 && FIXLENstrcmp("V0112_0155:7:1101:20072:12961", read_name)==0) SUBREADprintf("READ: %s FRAG_LEN=%d, THIS_OVERLAP=%d\n", read_name, total_frag_len, scoring_overlappings[score_x1]); if( applied_fragment_minimum_overlapping > 1 ) if( applied_fragment_minimum_overlapping > 10000L*scoring_overlappings[score_x1] ){ scoring_numbers[score_x1] = 0; continue; } if( maximum_score < scoring_numbers[score_x1] ){ maximum_total_count = 1; maximum_score = scoring_numbers[score_x1]; maximum_score_x1 = score_x1; }else if( maximum_score == scoring_numbers[score_x1] ) maximum_total_count++; overlapping_total_count ++; } if(maximum_total_count == 0){ if(global_context -> read_details_out_FP) write_read_details_FP(global_context, thread_context,"Unassigned_Overlapping_Length", -1, NULL, bin1, bin2); if(RG_name){ void ** tab4s = get_RG_tables(global_context, thread_context, RG_name); fc_read_counters * sumtab = tab4s[1]; sumtab -> unassigned_overlapping_length++; }else thread_context->read_counters.unassigned_overlapping_length ++; if(global_context -> do_scRNA_table) add_scRNA_read_to_pool(global_context, thread_context, -1, read_name, bin1, NULL); }else{ // final adding votes. if(1 == maximum_total_count && !global_context -> is_multi_overlap_allowed) { // simple add to the exon ( EXON_ID = decision_table_exon_ids[maximum_decision_no]) srInt_64 max_exon_id = scoring_exon_ids[maximum_score_x1]; if(RG_name){ void ** tab4s = get_RG_tables(global_context, thread_context, RG_name); fc_read_counters * sumtab = tab4s[1]; sumtab -> assigned_reads++; read_count_type_t * count_table = tab4s[0]; count_table[max_exon_id] += fixed_fractional_count; }else{ thread_context->count_table[max_exon_id] += fixed_fractional_count; thread_context->read_counters.assigned_reads ++; } thread_context->nreads_mapped_to_exon++; if(global_context -> read_details_out_FP) { int final_gene_number = global_context -> exontable_geneid[max_exon_id]; char * final_feture_name = (char *)global_context -> gene_name_array[final_gene_number]; write_read_details_FP(global_context, thread_context,"Assigned", 1, final_feture_name, bin1, bin2); } if(global_context -> do_scRNA_table){ srInt_64 assignment_target_number = max_exon_id; if(global_context->is_gene_level) assignment_target_number = global_context -> exontable_geneid[max_exon_id]; add_scRNA_read_to_pool(global_context, thread_context, assignment_target_number, read_name, bin1, NULL); } }else if(global_context -> is_multi_overlap_allowed) { #define GENE_NAME_LIST_BUFFER_SIZE (FEATURE_NAME_LENGTH * 50) char final_feture_names[GENE_NAME_LIST_BUFFER_SIZE]; int assigned_no = 0, xk1; final_feture_names[0]=0; int is_etc = 0; ArrayList * assigned_list = NULL; if(global_context -> do_scRNA_table)assigned_list = ArrayListCreate(20); for(xk1 = 0; xk1 < scoring_count; xk1++) { // This change was made on 31/MAR/2016 if( scoring_numbers[xk1] < 1 ) continue ; if( scoring_numbers[xk1] < maximum_score && global_context -> use_overlapping_break_tie ) continue ; srInt_64 tmp_voter_id = scoring_exon_ids[xk1]; srInt_64 assignment_target_number = tmp_voter_id; if(global_context->is_gene_level) assignment_target_number = global_context -> exontable_geneid[tmp_voter_id]; if(global_context -> do_scRNA_table)ArrayListPush(assigned_list, NULL+assignment_target_number); //if(1 && FIXLENstrcmp( read_name , "V0112_0155:7:1101:5467:23779#ATCACG" )==0) // SUBREADprintf("CountsFrac = %d ; add=%d\n", overlapping_total_count, calculate_multi_overlap_fraction(global_context, fixed_fractional_count, overlapping_total_count) ); if(RG_name){ void ** tab4s = get_RG_tables(global_context, thread_context, RG_name); read_count_type_t * count_table = tab4s[0]; count_table[tmp_voter_id] += calculate_multi_overlap_fraction(global_context, fixed_fractional_count, overlapping_total_count); }else thread_context->count_table[tmp_voter_id] += calculate_multi_overlap_fraction(global_context, fixed_fractional_count, overlapping_total_count); if(global_context -> read_details_out_FP) { if(strlen(final_feture_names)< (GENE_NAME_LIST_BUFFER_SIZE - 40 - FEATURE_NAME_LENGTH)) { int final_gene_number = global_context -> exontable_geneid[tmp_voter_id]; unsigned char * final_feture_name = global_context -> gene_name_array[final_gene_number]; strncat(final_feture_names, (char *)final_feture_name, GENE_NAME_LIST_BUFFER_SIZE-1); strncat(final_feture_names, ",", GENE_NAME_LIST_BUFFER_SIZE-1); }else{ is_etc ++; } assigned_no++; } } if(global_context -> do_scRNA_table && assigned_list->numOfElements>0) add_scRNA_read_to_pool(global_context, thread_context, -1, read_name, bin1, assigned_list); if(assigned_list)ArrayListDestroy(assigned_list); if(is_etc) sprintf(final_feture_names + strlen(final_feture_names), "... (%d names ommited),", is_etc); final_feture_names[GENE_NAME_LIST_BUFFER_SIZE-1]=0; if(RG_name){ void ** tab4s = get_RG_tables(global_context, thread_context, RG_name); fc_read_counters * sumtab = tab4s[1]; sumtab -> assigned_reads++; }else{ thread_context->read_counters.assigned_reads ++; } thread_context->nreads_mapped_to_exon++; if(global_context -> read_details_out_FP) { int ffnn = strlen(final_feture_names); if(ffnn>0) final_feture_names[ffnn-1]=0; // overlapped but still assigned write_read_details_FP(global_context, thread_context, "Assigned", assigned_no, final_feture_names, bin1, bin2); } } else { if(global_context -> read_details_out_FP) write_read_details_FP(global_context, thread_context,"Unassigned_Ambiguity", -1, NULL, bin1, bin2); if(RG_name){ fc_read_counters * sumtab = get_RG_tables(global_context, thread_context, RG_name)[1]; sumtab -> unassigned_ambiguous++; }else{ thread_context->read_counters.unassigned_ambiguous ++; } if(global_context -> do_scRNA_table) add_scRNA_read_to_pool(global_context, thread_context, -1, read_name, bin1, NULL); } } } } } void scRNA_merge_thread_reads_in(void *ky, void *val, HashTable * tab){ int * thread_umi_no_to_global_umi_no = tab->appendix1; int supp_reads_to_umi_in_cell = val-NULL; HashTable * merged_genep1_to_bcumip1_lists_table = tab->appendix2; HashTable * merged_genep1_to_bcumip1_reads_tab_table = tab->appendix3; srInt_64 gene_no = tab->counter1; srInt_64 cellno_locel_umino = (ky - NULL-1); srInt_64 cellno_global_umino = (cellno_locel_umino & 0xffffffff00000000llu) + thread_umi_no_to_global_umi_no[ cellno_locel_umino & 0xffffffff ]; ArrayList * merged_reads_gene_p1_list = HashTableGet(merged_genep1_to_bcumip1_lists_table , NULL+1+gene_no); if(NULL == merged_reads_gene_p1_list){ merged_reads_gene_p1_list = ArrayListCreate(10); HashTablePut(merged_genep1_to_bcumip1_lists_table, NULL+1+gene_no, merged_reads_gene_p1_list); } ArrayListPush( merged_reads_gene_p1_list, NULL+cellno_global_umino +1 ); // Rprintf("PUSH_GLB : %016llx\n", cellno_global_umino); HashTable * bc_umip1_to_reads_tab = HashTableGet(merged_genep1_to_bcumip1_reads_tab_table, NULL+1+gene_no); if(NULL == bc_umip1_to_reads_tab){ bc_umip1_to_reads_tab = HashTableCreate(10000); HashTablePut(merged_genep1_to_bcumip1_reads_tab_table, NULL+1+gene_no, bc_umip1_to_reads_tab); } int sup_reads = HashTableGet(bc_umip1_to_reads_tab, NULL+cellno_global_umino+1) - NULL; sup_reads += supp_reads_to_umi_in_cell; HashTablePut(bc_umip1_to_reads_tab, NULL+cellno_global_umino+1, NULL+sup_reads); } void scRNA_merge_thread_reads(void *ky, void *val, HashTable * tab){ int * thread_umi_no_to_global_umi_no = tab->appendix1; srInt_64 gene_no = ky-NULL -1; HashTable * merged_genep1_to_bcumip1_lists_table = tab->appendix2; HashTable * merged_genep1_to_bcumip1_reads_tab_table = tab->appendix3; HashTable * in_gene_cell_umi_table = val; //SUBREADprintf("scRNA_merge_thread_reads : %llu has %ld\n", gene_no, in_gene_cell_umi_table -> numOfElements); in_gene_cell_umi_table -> appendix3 = merged_genep1_to_bcumip1_reads_tab_table; in_gene_cell_umi_table -> appendix2 = merged_genep1_to_bcumip1_lists_table; in_gene_cell_umi_table -> appendix1 = thread_umi_no_to_global_umi_no; in_gene_cell_umi_table -> counter1 = gene_no; HashTableIteration(in_gene_cell_umi_table, scRNA_merge_thread_reads_in); } void scRNA_merge_thread_umitables(void *ky, void *val, HashTable * tab){ int * thread_umi_no_to_global_umi_no = tab->appendix1; HashTable * merged_umi_table = tab->appendix2; ArrayList * merged_umi_list = tab->appendix3; char * umicode = ky; int local_no = val-NULL-1; assert(local_no >= 0); int global_no = HashTableGet(merged_umi_table , umicode)-NULL-1; if(global_no<0){ char * newkey = strdup(umicode); global_no = merged_umi_table -> numOfElements; HashTablePut(merged_umi_table, newkey, NULL+global_no+1); assert(merged_umi_list -> numOfElements == global_no); ArrayListPush(merged_umi_list, newkey); } thread_umi_no_to_global_umi_no[ local_no ] = global_no; } #define MIN_EXPRESSED_UMIS_PER_CELL 100 #define MIN_EXPRESSED_UMIS_PER_GENE (3-2) void scRNA_merge_write_copy_gene_nos(void * ky, void * va , HashTable *tab){ HashTable * used_gene_table = tab -> appendix2; ArrayList * one_sampl_gene_to_cell_umis = va; srInt_64 UMIs = HashTableGet(used_gene_table, ky)-NULL; HashTablePut(used_gene_table, ky, NULL + UMIs + one_sampl_gene_to_cell_umis -> numOfElements); } int scRNA_merge_write_zero_gene(fc_thread_global_context_t * global_context, char * linebuf, ArrayList * high_confid_barcode_index_list){ int ret=0; srInt_64 x1; for(x1=0;x1numOfElements;x1++)ret += sprintf(linebuf + ret,"\t0"); return ret; } //#warning "======== SCRNA_ALLOWED_MAX_HAMMING_DIFF IS ZERO !! ========" #define SCRNA_ALLOWED_MAX_HAMMING_DIFF 1 int scRNA_reduce_cellno_compare(void * arr, int l, int r){ void **sd = arr; ArrayList * cellno_umino_p1_list = sd[0]; HashTable * cellno_umino_p1_to_reads_tab = sd[4]; srInt_64 off = sd[1]-NULL; srInt_64 bc_umi_p1_L = ArrayListGet(cellno_umino_p1_list, off+l) - NULL; srInt_64 bc_umi_p1_R = ArrayListGet(cellno_umino_p1_list, off+r) - NULL; int nreads_L = HashTableGet(cellno_umino_p1_to_reads_tab, NULL+bc_umi_p1_L) - NULL; int nreads_R = HashTableGet(cellno_umino_p1_to_reads_tab, NULL+bc_umi_p1_R) - NULL; if(nreads_L<1 || nreads_R<1) SUBREADprintf("ERROR: No known read counts: %d, %d\n", nreads_L, nreads_R); if(nreads_L>nreads_R) return -1; if(nreads_LelementList[off+l]; cellno_umino_p1_list->elementList[off+l] = cellno_umino_p1_list->elementList[off+r]; cellno_umino_p1_list->elementList[off+r] = ti; } void scRNA_reduce_cellno_merge(void * arr, int start, int items, int items2){ void **sd = arr; ArrayList * cellno_umino_p1_list = sd[0]; srInt_64 off = sd[1]-NULL; void ** tmpelem=malloc(sizeof(void*)*(items+items2)); int i1_cursor = start, i2_cursor = items + start, tmp_cursor=0; while(1){ if(i1_cursor == items + start && i2_cursor == items + items2 + start )break; int select_items_1 = (i2_cursor == start + items + items2) || (i1_cursor < items + start && scRNA_reduce_cellno_compare(arr, i1_cursor, i2_cursor) <= 0); if(select_items_1) tmpelem[tmp_cursor++] = cellno_umino_p1_list->elementList[off+(i1_cursor++)]; else tmpelem[tmp_cursor++] = cellno_umino_p1_list->elementList[off+(i2_cursor++)]; } memcpy(cellno_umino_p1_list -> elementList+off+start, tmpelem, sizeof(void*)*(items+items2)); free(tmpelem); } // #define DEBUG_FOR_EXACT #define MIN_UMIS_FOR_CANDIDATE_RESCUE 500 #define SCRNA_AMBIENT_RESCURE_MEDIAN_FRACTION 0.01 void scRNA_merged_ambient_rescure(fc_thread_global_context_t * global_context, HashTable * cellP1_to_geneP1_to_umis_tab, HashTable * cellnoP1_to_umis_tab, ArrayList * this_sample_45k_90k_barcode_no_P0, ArrayList * this_sample_ambient_rescure_candi, ArrayList * highconf_cellbc_list){ ArrayList * sorted_bcno_p1 = HashTableSortedIndexes( cellnoP1_to_umis_tab, 1); HashTable * highconf_cellbc_list_tab = ArrayListToLookupTable_Int(highconf_cellbc_list); srInt_64 x1, high_conf_cells = 0; for(x1=0; x1 < sorted_bcno_p1 -> numOfElements; x1++){ void * this_bc_pnt = ArrayListGet(sorted_bcno_p1 , x1); if(HashTableGet(highconf_cellbc_list_tab, this_bc_pnt)) high_conf_cells = x1+1; else break; // assuming that all high-umi barcodes are high-confident, this makes x1 being the # of total high-confidence barcodes. } #ifdef DEBUG_FOR_EXACT #warning "============= EXT 1 ===========" FILE * tfp = fopen("/tmp/del4-YangLiao-rescue-cand.txt","w"); #endif if(high_conf_cells >0){ srInt_64 median_umis = HashTableGet(cellnoP1_to_umis_tab, ArrayListGet(sorted_bcno_p1 , (high_conf_cells-1)/2))-NULL; srInt_64 median_umis_001_cut = (srInt_64)(median_umis *1. *SCRNA_AMBIENT_RESCURE_MEDIAN_FRACTION +0.50000001); for(x1=0; x1 < sorted_bcno_p1 -> numOfElements; x1++){ void * this_bc_pnt_p1 = ArrayListGet(sorted_bcno_p1 , x1); if(HashTableGet(highconf_cellbc_list_tab, this_bc_pnt_p1)){ continue; // it is in high-conf list } srInt_64 this_bc_umis = HashTableGet(cellnoP1_to_umis_tab, this_bc_pnt_p1) - NULL; if(this_bc_umis < median_umis_001_cut) break; if(this_bc_umis < MIN_UMIS_FOR_CANDIDATE_RESCUE) break; if(x1 >= 45000) break; ArrayListPush(this_sample_ambient_rescure_candi, this_bc_pnt_p1-1); } #ifdef DEBUG_FOR_EXACT #warning "============= EXT 2 ===========" for(x1=0; x1numOfElements; x1++){ int this_bc_no_p0 = ArrayListGet(this_sample_ambient_rescure_candi, x1)-NULL; srInt_64 this_bc_umis = HashTableGet(used_cell_barcode_tab, NULL+this_bc_no_p0+1) - NULL; fprintf(tfp,"CAND %d %d\n", this_bc_no_p0+1, this_bc_umis); } #endif } for(x1=45000; x1 < sorted_bcno_p1 -> numOfElements; x1++){ if(x1 >= 90000) break; ArrayListPush(this_sample_45k_90k_barcode_no_P0, ArrayListGet(sorted_bcno_p1 , x1)-1 ); #ifdef DEBUG_FOR_EXACT #warning "============= EXT 3 ===========" int this_bc_no_p1 = ArrayListGet(sorted_bcno_p1, x1)-NULL; int this_bc_umis = HashTableGet(used_cell_barcode_tab, NULL+this_bc_no_p1) - NULL; fprintf(tfp,"45K90K %d %d\n", this_bc_no_p1, this_bc_umis); #endif } ArrayListDestroy(sorted_bcno_p1); HashTableDestroy(highconf_cellbc_list_tab); #ifdef DEBUG_FOR_EXACT #warning "============= EXT 4 ===========" fclose(tfp); FILE * fp = fopen("/tmp/del4-YangLiao-from-python-rescue.txt","r"); x1=0; while(1){ char * tpm=NULL; char fl[100]; char * fr = fgets(fl, 99, fp); if(!fr) break; if(fl[0]!='4') continue; int bc_no = atoi(fl+7) -1; this_sample_45k_90k_barcode_no_P0 -> elementList[x1++] = NULL+bc_no; if(x1 >= 45000)break; } fclose(fp); fp = fopen("/tmp/del4-YangLiao-from-python-rescue.txt","r"); x1=0; this_sample_ambient_rescure_candi -> numOfElements = 0; while(1){ char * tpm=NULL; char fl[100]; char * fr = fgets(fl, 99, fp); if(!fr) break; if(fl[0]!='C') continue; int bc_no = atoi(fl+5) -1; ArrayListPush(this_sample_ambient_rescure_candi, NULL+bc_no); } fclose(fp); #endif } #define SCRNA_BOOTSTRAP_HIGH_INDEX 30 #define SCRNA_BOOTSTRAP_SAMPLING_TIMES 100 int scRNA_merged_bootstrap_a_sample(fc_thread_global_context_t * global_context, HashTable * cellP1_to_geneP1_to_umis_tab, HashTable * cellnoP1_to_umis_tab, ArrayList * highconf_cellbc_list){ ArrayList * sorted_idx = HashTableSortedIndexes( cellnoP1_to_umis_tab, 1); srInt_64 x2, x1; float scRNA_umi_cutoff = global_context -> scRNA_umi_cutoff; #define SCRNA_IDX_PRIME_NUMBER_BIG 11218439llu; srInt_64 this_total = 0, seed_rand = sorted_idx -> numOfElements/2; #ifdef DEBUG_FOR_EXACT #warning "============== THIS BUILD IS ONLY FOR DEBUGGING EXACT RESULTS !!!! =================" ArrayListSort(sorted_idx, NULL); FILE * dfp = fopen("/tmp/del4-YangLiao-for-resample.txt","w"); for(x2 = 0; x2 < sorted_idx -> numOfElements ; x2++){ int bc_no_p1 = ArrayListGet(sorted_idx, x2)-NULL; int bc_umis = HashTableGet(used_cell_barcode_tab, NULL+bc_no_p1) - NULL; fprintf(dfp,"%d\t%d\t%s\n", bc_no_p1, bc_umis, ArrayListGet(global_context -> scRNA_cell_barcodes_array, bc_no_p1-1)); } fclose(dfp); system("python /usr/local/work/liao/subread/scripts/Cellranger-replicate/CrepPY-resample.py"); FILE * rfp = fopen("/tmp/del4-YangLiao-from-resample.txt","r"); #endif int last_umi_no= -1; if(scRNA_umi_cutoff >= 0.0){ for(x1 = 0; x1 < sorted_idx -> numOfElements ; x1++){ void * cellbc_p1_ptr = ArrayListGet(sorted_idx,x1); srInt_64 this_umis = HashTableGet(cellnoP1_to_umis_tab, cellbc_p1_ptr )-NULL; if(this_umis >= scRNA_umi_cutoff-0.1){ ArrayListPush(highconf_cellbc_list, ArrayListGet( sorted_idx, x1 ) - 1 ); last_umi_no = this_umis; }else break; // #UMI-sorted so no need to scan more } }else{ for(x1 = 0; x1 < SCRNA_BOOTSTRAP_SAMPLING_TIMES; x1++){ ArrayList * resampled_list_of_umis = ArrayListCreate( sorted_idx->numOfElements ); #ifdef DEBUG_FOR_EXACT #warning "============== THIS BUILD IS ONLY FOR DEBUGGING EXACT RESULTS !!!! =================" for(x2 = 0; x2 < sorted_idx -> numOfElements ; x2++){ char fl [100]; fgets(fl, 99, rfp); int bc_no_p1 = atoi(fl); int this_umis = HashTableGet(cellnoP1_to_umis_tab, NULL+bc_no_p1); ArrayListPush(resampled_list_of_umis, NULL+this_umis); } ArrayListSort(resampled_list_of_umis, NULL); #else for(x2 = 0; x2 < sorted_idx -> numOfElements ; x2++){ seed_rand %= sorted_idx -> numOfElements; void * cellbc_p1_ptr = ArrayListGet(sorted_idx, seed_rand); seed_rand += SCRNA_IDX_PRIME_NUMBER_BIG; srInt_64 this_umis = HashTableGet( cellnoP1_to_umis_tab, cellbc_p1_ptr )-NULL; ArrayListPush(resampled_list_of_umis,NULL+this_umis); } #endif ArrayListSort( resampled_list_of_umis, NULL ); srInt_64 UMIs_30th_div10 = ArrayListGet(resampled_list_of_umis, resampled_list_of_umis -> numOfElements - SCRNA_BOOTSTRAP_HIGH_INDEX) -NULL; UMIs_30th_div10 = (srInt_64)(UMIs_30th_div10*1./10 + 0.500000001); for(x2 =0; x2< resampled_list_of_umis -> numOfElements; x2++){ srInt_64 lli = resampled_list_of_umis -> numOfElements -1 -x2; srInt_64 this_umis = ArrayListGet(resampled_list_of_umis, lli)-NULL; if(this_umis >= UMIs_30th_div10) this_total ++; else break; } ArrayListDestroy(resampled_list_of_umis); } double total_f = this_total*1. / SCRNA_BOOTSTRAP_SAMPLING_TIMES; if(0) SUBREADprintf("FINAL_5CODE SELECTION_IDX = %.5f\n",total_f); this_total = (int)(total_f + 0.500000001); #ifdef DEBUG_FOR_EXACT #warning "============== THIS BUILD IS ONLY FOR DEBUGGING EXACT RESULTS !!!! =================" sorted_idx = HashTableSortedIndexes( used_cell_barcode_tab, 1); #endif void * last_ptr =NULL; for(x1 = 0; x1 < min(sorted_idx -> numOfElements, this_total) ; x1++){ last_ptr = ArrayListGet( sorted_idx, x1 ); ArrayListPush(highconf_cellbc_list, last_ptr - 1 ); } last_umi_no = HashTableGet(cellnoP1_to_umis_tab ,last_ptr)-NULL; } ArrayListDestroy(sorted_idx); return last_umi_no; } void build_exon_name(fc_thread_global_context_t * global_context, fc_feature_info_t * loaded_features, int sorted_order, char * exon_name, HashTable * sorted_order_p1_to_i_p1_tab){ srInt_64 i = HashTableGet( sorted_order_p1_to_i_p1_tab , NULL+1+sorted_order )-NULL-1; sprintf(exon_name, "%s:fc@R@Spl:%s:fc@R@Spl:%u:fc@R@Spl:%u:fc@R@Spl:%c", global_context -> unistr_buffer_space + loaded_features[i].feature_name_pos, global_context-> unistr_buffer_space + loaded_features[i].feature_name_pos + loaded_features[i].chro_name_pos_delta, loaded_features[i].start, loaded_features[i].end, loaded_features[i].is_negative_strand == 1?'N':( loaded_features[i].is_negative_strand == 0? 'P':'X')); } void scRNA_merged_write_sparse_unique_genes(void * ky, void * va, HashTable * tab){ HashTable * unique_geneno1B_tab = tab -> appendix1; HashTable * used_cellnoP1_tab = tab -> appendix2; int cellbcP1 = ky-NULL; if(used_cellnoP1_tab && !HashTableGet(used_cellnoP1_tab, NULL+cellbcP1))return; HashTable * g2u = va; ArrayList * g2ul = HashTableKeys(g2u); int x1; for(x1=0; x1numOfElements; x1++){ void *geneno1B_ptr = ArrayListGet(g2ul,x1); if(!HashTableGet(unique_geneno1B_tab, ArrayListGet(g2ul,x1))) HashTablePut(unique_geneno1B_tab, geneno1B_ptr, NULL+1); tab -> counter1 += HashTableGet(g2u, geneno1B_ptr)-NULL; } ArrayListDestroy(g2ul); } int scRNA_merged_write_sparse_matrix(fc_thread_global_context_t * global_context, HashTable * cellP1_to_geneP1_to_umis_tab, HashTable * cellnoP1_to_umis_tab, ArrayList * used_cell_barcodes, int sample_index, char * tabtype, fc_feature_info_t* loaded_features, HashTable * sorted_order_p1_to_i_p1_tab){ int x1,x2; char ofname[MAX_FILE_NAME_LENGTH + 100]; sprintf(ofname,"%s.scRNA.%03d.%s.summary",global_context->input_file_name, sample_index+1,tabtype); sprintf(ofname,"%s.scRNA.%03d.%s.BCtab",global_context->input_file_name, sample_index+1,tabtype); FILE * ofp_bcs = fopen( ofname , "w" ); sprintf(ofname,"%s.scRNA.%03d.%s.GENEtab",global_context->input_file_name, sample_index+1,tabtype); FILE * ofp_genes = fopen( ofname , "w" ); sprintf(ofname,"%s.scRNA.%03d.%s.spmtx",global_context->input_file_name, sample_index+1,tabtype); FILE * ofp_mtx = fopen( ofname , "w" ); fprintf(ofp_mtx,"%%%%MatrixMarket matrix coordinate integer general\n"); HashTable * used_cellnoP1_tab = ArrayListToLookupTable_Int(used_cell_barcodes); HashTable * unique_NZ_geneno1B_table = HashTableCreate(10000); cellP1_to_geneP1_to_umis_tab -> counter1 = 0; cellP1_to_geneP1_to_umis_tab -> appendix1 = unique_NZ_geneno1B_table; cellP1_to_geneP1_to_umis_tab -> appendix2 = used_cellnoP1_tab; HashTableIteration(cellP1_to_geneP1_to_umis_tab, scRNA_merged_write_sparse_unique_genes); srInt_64 total_UMIs = cellP1_to_geneP1_to_umis_tab -> counter1; ArrayList * unique_NZ_genenosP1_list = HashTableKeys(unique_NZ_geneno1B_table); HashTableDestroy(unique_NZ_geneno1B_table); HashTableDestroy(used_cellnoP1_tab); ArrayListSort(unique_NZ_genenosP1_list, NULL); #ifdef __MINGW32__ fprintf(ofp_mtx, "%I64d %I64d %I64d\n", unique_NZ_genenosP1_list -> numOfElements , used_cell_barcodes -> numOfElements, total_UMIs ); #else fprintf(ofp_mtx, "%lld %lld %lld\n", unique_NZ_genenosP1_list -> numOfElements , used_cell_barcodes -> numOfElements, total_UMIs ); #endif for(x2=0; x2 < unique_NZ_genenosP1_list -> numOfElements; x2++){ int gene_index_0B = ArrayListGet(unique_NZ_genenosP1_list, x2) - NULL-1; if(global_context->is_gene_level){ char* gene_name = (char*)global_context -> gene_name_array [gene_index_0B]; fprintf(ofp_genes,"%s\n", gene_name); }else{ char exon_name[FEATURE_NAME_LENGTH+60]; build_exon_name(global_context, loaded_features, gene_index_0B, exon_name, sorted_order_p1_to_i_p1_tab); fprintf(ofp_genes,"%s\n", exon_name); } } for(x1 = 0; x1 < used_cell_barcodes -> numOfElements; x1++){ srInt_64 cellno = ArrayListGet(used_cell_barcodes, x1)-NULL; char * cellbc_seq = ArrayListGet(global_context -> scRNA_cell_barcodes_array, cellno); fprintf(ofp_bcs,"%s\n", cellbc_seq); } for(x1 = 0; x1 < used_cell_barcodes -> numOfElements; x1++){ srInt_64 cellno = ArrayListGet(used_cell_barcodes, x1)-NULL; HashTable * geneno1B_to_UMIs = HashTableGet(cellP1_to_geneP1_to_umis_tab, NULL+1+cellno); for(x2=0; x2 < unique_NZ_genenosP1_list -> numOfElements; x2++){ int geneno1B = ArrayListGet(unique_NZ_genenosP1_list, x2)-NULL; int this_umis = HashTableGet(geneno1B_to_UMIs, NULL+geneno1B) -NULL; if(this_umis>0)fprintf(ofp_mtx,"%d %d %d\n", x2+1, x1+1, this_umis); } } ArrayListDestroy(unique_NZ_genenosP1_list); fclose(ofp_bcs); fclose(ofp_genes); fclose(ofp_mtx); return 0; } void scRNA_merged_45K_to_90K_sum_SUM_Level2(void * GeneNo1B, void * vUMIs, HashTable * m2){ HashTable * summed_gene_to_umis = m2 -> appendix1; HashTablePut(summed_gene_to_umis, GeneNo1B, vUMIs + (HashTableGet(summed_gene_to_umis, GeneNo1B)-NULL)); } void scRNA_merged_45K_to_90K_sum_SUM(void * keyCellNoP1, void * Vgno_umi_tab, HashTable * me){ HashTable * summed_gene_to_umis = me -> appendix1; HashTable * bcid_look_tab = me -> appendix2; //fc_thread_global_context_t * global_context = me -> appendix3; HashTable * geneno1B_to_UMIs_tab = Vgno_umi_tab; if(!HashTableGet(bcid_look_tab, keyCellNoP1))return; geneno1B_to_UMIs_tab -> appendix1 = summed_gene_to_umis; HashTableIteration(geneno1B_to_UMIs_tab ,scRNA_merged_45K_to_90K_sum_SUM_Level2 ); } void scRNA_merged_45K_to_90K_sum_WRT(void * kyGeneID, void * valUMIs, HashTable * me){ fc_thread_global_context_t * global_context = me -> appendix1; FILE * ofp = me -> appendix2; void ** vp2 = me->appendix3; fc_feature_info_t * loaded_features = vp2[0]; HashTable * sorted_order_p1_to_i_p1_tab = vp2[1]; if(global_context -> is_gene_level){ unsigned char * gene_name = global_context -> gene_name_array[ kyGeneID - NULL-1 ]; fprintf(ofp, "%s\t%u\n", gene_name, (unsigned int) (valUMIs-NULL)); }else{ char exon_name[FEATURE_NAME_LENGTH+60]; build_exon_name(global_context, loaded_features, kyGeneID-NULL-1, exon_name, sorted_order_p1_to_i_p1_tab); fprintf(ofp,"%s\t%u\n", exon_name, (unsigned int) (valUMIs-NULL)); } } void scRNA_merged_45K_to_90K_sum(fc_thread_global_context_t * global_context, HashTable * cellP1_geneP1_UMIs_tab, ArrayList * bcid_P0_arr, int sample_no, fc_feature_info_t * loaded_features, HashTable * sorted_index_p1_to_i_p1_tab){ HashTable * summed_gene_to_umis = HashTableCreate( 3+cellP1_geneP1_UMIs_tab->numOfElements/6 ); HashTable * bcid_look_tab = ArrayListToLookupTable_Int(bcid_P0_arr); cellP1_geneP1_UMIs_tab -> appendix1 = summed_gene_to_umis; cellP1_geneP1_UMIs_tab -> appendix2 = bcid_look_tab; cellP1_geneP1_UMIs_tab -> appendix3 = global_context; HashTableIteration( cellP1_geneP1_UMIs_tab, scRNA_merged_45K_to_90K_sum_SUM ); char ofname[MAX_FILE_NAME_LENGTH + 100]; sprintf(ofname,"%s.scRNA.%03d.AmbSum",global_context->input_file_name, sample_no+1); FILE * write_fp = fopen(ofname,"w"); fprintf(write_fp,"GeneID\tUMIs\n"); summed_gene_to_umis -> appendix1 = global_context; summed_gene_to_umis -> appendix2 = write_fp; void * vp2[2]; vp2[0]=loaded_features; vp2[1]=sorted_index_p1_to_i_p1_tab; summed_gene_to_umis -> appendix3 = vp2; summed_gene_to_umis -> counter1 = sample_no; HashTableIteration( summed_gene_to_umis, scRNA_merged_45K_to_90K_sum_WRT ); HashTableDestroy(bcid_look_tab); HashTableDestroy(summed_gene_to_umis); fclose(write_fp); } void scRNA_merged_write_nozero_geneids_WRT(void *k, void *v, HashTable* me){ FILE * fp = me->appendix1; fc_thread_global_context_t * global_context = me->appendix2; void ** tv2 = me->appendix3; fc_feature_info_t * loaded_features = tv2[0]; HashTable * sorted_order_p1_to_i_p1_tab = tv2[1]; if(global_context -> is_gene_level){ unsigned char* gene_symbol = global_context -> gene_name_array [k-NULL-1]; fprintf(fp, "%s\n", gene_symbol); }else{ char exon_name[FEATURE_NAME_LENGTH+60]; build_exon_name(global_context, loaded_features, k-NULL-1, exon_name, sorted_order_p1_to_i_p1_tab); fprintf(fp,"%s\n", exon_name); } } void scRNA_merged_write_nozero_geneids(fc_thread_global_context_t * global_context, HashTable * no0genes, int samplenno, fc_feature_info_t * loaded_features, HashTable * sorted_order_p1_to_i_p1_tab){ char ofname[MAX_FILE_NAME_LENGTH + 100]; sprintf(ofname,"%s.scRNA.%03d.no0Genes",global_context->input_file_name, samplenno+1); FILE * fp = fopen( ofname , "w" ); no0genes -> appendix1 =fp; void * tv2[2]; no0genes -> appendix2 =global_context; tv2[0]=loaded_features; tv2[1]=sorted_order_p1_to_i_p1_tab; no0genes -> appendix3 =tv2; HashTableIteration(no0genes, scRNA_merged_write_nozero_geneids_WRT); fclose(fp); } void scRNA_merged_to_tables_write_build_UMIcount_in(void * ky, void * val, HashTable * tab){ tab -> counter1 += (val-NULL); } void scRNA_merged_to_tables_write_build_UMIcounts(void * ky, void * val, HashTable * tab){ HashTable * cellbcP1_to_umis_tab = tab -> appendix1; int cell_no = ky-NULL-1; HashTable * geneP1_to_counts_tab = val; geneP1_to_counts_tab -> counter1 = 0; HashTableIteration(geneP1_to_counts_tab, scRNA_merged_to_tables_write_build_UMIcount_in); HashTablePut(cellbcP1_to_umis_tab, NULL+1+cell_no, NULL+geneP1_to_counts_tab -> counter1); } // this function writes a single count table. // Rows: genes // Cols: Cell_Barcode +"."+ SampleName void scRNA_merged_to_tables_write( fc_thread_global_context_t * global_context, HashTable ** cellP1_to_geneP1_to_umis, fc_feature_info_t * loaded_features, srInt_64 nexons){ char ofname[MAX_FILE_NAME_LENGTH + 20]; sprintf(ofname,"%s.scRNA.SampleTable",global_context->input_file_name); FILE * sample_tab_fp = fopen( ofname , "w" ); int x1; fprintf(sample_tab_fp,"SampleName\tUMICutoff\tTotalReads\tMappedReads\tAssignedReads\tIndex\n"); for(x1 = 0; x1 < global_context -> scRNA_sample_sheet_table -> numOfElements ; x1++){ srInt_64 mapped_reads = 0, all_reads = 0, assigned_reads = 0; int thrid; for(thrid=0; thrid thread_number; thrid++){ mapped_reads += global_context -> thread_contexts[thrid].scRNA_mapped_reads_per_sample[x1]; assigned_reads += global_context -> thread_contexts[thrid].scRNA_assigned_reads_per_sample[x1]; all_reads += global_context -> thread_contexts[thrid].scRNA_reads_per_sample[x1]; } ArrayList * high_confid_barcode_index_list = ArrayListCreate(20000); ArrayList * this_sample_ambient_rescure_candi = ArrayListCreate(10000); ArrayList * this_sample_45k_90k_barcode_no_P0 = ArrayListCreate(90000 - 45000 + 100); HashTable * cellbcP1_to_umis_tab = HashTableCreate(cellP1_to_geneP1_to_umis[x1] -> numOfElements); cellP1_to_geneP1_to_umis[x1] -> appendix1 = cellbcP1_to_umis_tab; HashTableIteration(cellP1_to_geneP1_to_umis[x1], scRNA_merged_to_tables_write_build_UMIcounts); int applied_umi_cut = scRNA_merged_bootstrap_a_sample(global_context, cellP1_to_geneP1_to_umis[x1], cellbcP1_to_umis_tab, high_confid_barcode_index_list); global_context -> scRNA_applied_umi_cut[x1] = applied_umi_cut; scRNA_merged_ambient_rescure(global_context, cellP1_to_geneP1_to_umis[x1], cellbcP1_to_umis_tab, this_sample_45k_90k_barcode_no_P0, this_sample_ambient_rescure_candi, high_confid_barcode_index_list); int umi_cutoff = global_context -> scRNA_applied_umi_cut[x1]; char * this_sample_name = ArrayListGet(global_context -> scRNA_sample_id_to_name, x1); #ifdef __MINGW32__ fprintf(sample_tab_fp,"%s\t%d\t%I64d\t%I64d\t%I64d\t%d\n", this_sample_name, umi_cutoff, all_reads, mapped_reads, assigned_reads,x1+1); #else fprintf(sample_tab_fp,"%s\t%d\t%lld\t%lld\t%lld\t%d\n", this_sample_name, umi_cutoff, all_reads, mapped_reads, assigned_reads, x1+1); #endif srInt_64 xk1; HashTable * sorted_order_p1_to_i_p1_tab = HashTableCreate(nexons/4); for(xk1 = 0; xk1 < nexons ; xk1++){ HashTablePut(sorted_order_p1_to_i_p1_tab, NULL+loaded_features[xk1].sorted_order+1 , NULL+xk1+1 ); } #ifdef DEBUG_FOR_EXACT #warning " ======= Another debug ======" scRNA_merged_write_sparse_matrix(global_context, merged_tables_gene_to_cell_umis[x1], used_cell_barcode_tabs[x1], NULL, x1, "RawMatrix", loaded_features, sorted_order_p1_to_i_p1_tab); //scRNA_merged_write_sparse_matrix(global_context, merged_tables_gene_to_cell_umis[x1], used_cell_barcode_tabs[x1] this_sample_45k_90k_barcode_no_P0, x1, "AmbProfCells", loaded_features, sorted_order_p1_to_i_p1_tab); #endif scRNA_merged_write_sparse_matrix(global_context, cellP1_to_geneP1_to_umis[x1], cellbcP1_to_umis_tab, high_confid_barcode_index_list, x1, "HighConf", loaded_features, sorted_order_p1_to_i_p1_tab); scRNA_merged_write_sparse_matrix(global_context, cellP1_to_geneP1_to_umis[x1], cellbcP1_to_umis_tab, this_sample_ambient_rescure_candi, x1, "RescCand", loaded_features, sorted_order_p1_to_i_p1_tab); scRNA_merged_45K_to_90K_sum( global_context, cellP1_to_geneP1_to_umis[x1], this_sample_45k_90k_barcode_no_P0, x1 , loaded_features, sorted_order_p1_to_i_p1_tab); HashTable * no0genes = HashTableCreate(10000); cellP1_to_geneP1_to_umis[x1] -> appendix1 = no0genes; cellP1_to_geneP1_to_umis[x1] -> appendix2 = NULL; HashTableIteration(cellP1_to_geneP1_to_umis[x1], scRNA_merged_write_sparse_unique_genes); scRNA_merged_write_nozero_geneids(global_context, no0genes, x1, loaded_features, sorted_order_p1_to_i_p1_tab); HashTableDestroy(no0genes); ArrayListDestroy(this_sample_ambient_rescure_candi); ArrayListDestroy(this_sample_45k_90k_barcode_no_P0); ArrayListDestroy(high_confid_barcode_index_list); HashTableDestroy(cellbcP1_to_umis_tab); HashTableDestroy(sorted_order_p1_to_i_p1_tab); } fclose(sample_tab_fp); } void scRNA_find_gene_to_umi_do_merger(void * ky_genep1, void * val_arr_bc_umip1, HashTable * tab){ HashTable * bc_umi_to_genes_tab = tab -> appendix1; ArrayList * arr_bc_umip1 = val_arr_bc_umip1; int gene_no = ky_genep1-NULL-1; int x1; for(x1=0; x1 numOfElements; x1++){ void * bc_umip1 = ArrayListGet(arr_bc_umip1,x1); ArrayList * gene_list = HashTableGet(bc_umi_to_genes_tab, bc_umip1); if(!gene_list){ gene_list = ArrayListCreate(1); HashTablePut(bc_umi_to_genes_tab, bc_umip1, gene_list); } ArrayListPush(gene_list, NULL+gene_no); } } int scRNA_find_gene_to_umi_sortCompare(void * L_elem, void * R_elem, ArrayList * me){ void ** pnts = me -> appendix1; void * key_bc_umi_p1 = pnts[0]; HashTable * geneno_umi_bc_counts = pnts[1]; int geneno_L = L_elem-NULL; int geneno_R = R_elem-NULL; int nsupp_L = HashTableGet(HashTableGet(geneno_umi_bc_counts, NULL+1+ geneno_L), key_bc_umi_p1) - NULL; int nsupp_R = HashTableGet(HashTableGet(geneno_umi_bc_counts, NULL+1+ geneno_R), key_bc_umi_p1) - NULL; if(nsupp_L > nsupp_R) return -1; if(nsupp_L < nsupp_R) return 1; return 0; } void scRNA_find_gene_to_umi_sortByReads(void * key_bc_umi_p1, void * val_arr_genes, HashTable * tab){ ArrayList * arr_genes = val_arr_genes; if(arr_genes -> numOfElements<2) return; HashTable * geneno_umi_bc_counts = tab -> appendix1; void * pnts[2]; pnts[0]=key_bc_umi_p1; pnts[1]=geneno_umi_bc_counts; arr_genes -> appendix1 = pnts; ArrayListSort(arr_genes, scRNA_find_gene_to_umi_sortCompare); } void scRNA_find_gene_to_umi_mark_deletee(void * key_bc_umi_p1, void * val_arr_genes_sorted, HashTable * tab){ ArrayList * arr_genes = val_arr_genes_sorted; if(arr_genes -> numOfElements<2) return; HashTable * gene_bc_umi_to_deleted_genes_tab = tab->appendix1; HashTable * gene_to_bc_umi_p1_to_reads_tab = tab->appendix2; int gene1_no = ArrayListGet(arr_genes, 0)-NULL; int gene2_no = ArrayListGet(arr_genes, 1)-NULL; if(0){ int x1; for(x1=0; x1 numOfElements; x1++){ int gene_no = ArrayListGet(arr_genes, x1)-NULL; int nsupp = HashTableGet(HashTableGet(gene_to_bc_umi_p1_to_reads_tab , NULL+gene_no+1), key_bc_umi_p1) - NULL; int cellno = ( key_bc_umi_p1-NULL-1 ) >> 32; SUBREADprintf("TESTING_SORT : %d of %d have %d reads\n", gene_no, cellno, nsupp); } } int supp1 = HashTableGet(HashTableGet(gene_to_bc_umi_p1_to_reads_tab, NULL+gene1_no+1), key_bc_umi_p1)-NULL; int supp2 = HashTableGet(HashTableGet(gene_to_bc_umi_p1_to_reads_tab, NULL+gene2_no+1), key_bc_umi_p1)-NULL; ArrayList * to_del_genes = HashTableGet(gene_bc_umi_to_deleted_genes_tab, key_bc_umi_p1); if(NULL==to_del_genes){ to_del_genes = ArrayListCreate(3); HashTablePut(gene_bc_umi_to_deleted_genes_tab, key_bc_umi_p1, to_del_genes); } if(supp1 == supp2) ArrayListPush(to_del_genes, NULL+gene1_no); int x1; for(x1=1; x1< arr_genes->numOfElements; x1++) ArrayListPush(to_del_genes, ArrayListGet(arr_genes, x1)); } HashTable * scRNA_find_gene_to_umi_merger(fc_thread_global_context_t * global_context, HashTable * gene_to_bc_umi_p1_tab, HashTable * gene_to_bc_umi_p1_to_reads_tab){ HashTable * bc_umi_to_genes_tab = HashTableCreate( 1000000); HashTableSetDeallocationFunctions(bc_umi_to_genes_tab, NULL, (void (*) (void *))ArrayListDestroy); gene_to_bc_umi_p1_tab -> appendix1 = bc_umi_to_genes_tab; gene_to_bc_umi_p1_tab -> appendix2 = global_context; HashTableIteration(gene_to_bc_umi_p1_tab, scRNA_find_gene_to_umi_do_merger); bc_umi_to_genes_tab -> appendix1 = gene_to_bc_umi_p1_to_reads_tab; HashTableIteration(bc_umi_to_genes_tab, scRNA_find_gene_to_umi_sortByReads); HashTable * gene_bc_umi_to_deleted_genes_tab = HashTableCreate( 1000000); // bc_um1_p1 => ArrayList (deleted_gene_1, deleted_gene_2, ...) bc_umi_to_genes_tab -> appendix1 = gene_bc_umi_to_deleted_genes_tab; bc_umi_to_genes_tab -> appendix2 = gene_to_bc_umi_p1_to_reads_tab; HashTableIteration(bc_umi_to_genes_tab, scRNA_find_gene_to_umi_mark_deletee); HashTableDestroy(bc_umi_to_genes_tab); return gene_bc_umi_to_deleted_genes_tab ; } struct scRNA_merge_batches_worker_task{ int sample_id; int inbin_len; srInt_64 block_number; char inbin[MERGER_WORKER_BINSIZE]; }; struct scRNA_merge_batches_worker_current{ struct scRNA_merge_batches_worker_task * task; char outbin[MERGER_WORKER_BINSIZE]; int outbin_len; unsigned int crc32; z_stream strm; }; void * scRNA_merge_batches_worker(void * vp){ void **vpp = vp; fc_thread_global_context_t * global_context = vpp[0]; worker_master_mutex_t * worker_mut = vpp[1]; int my_worker_id = vpp[2] - NULL; struct scRNA_merge_batches_worker_current * my_current_job = vpp[3]; free(vp); int Z_DEFAULT_MEM_LEVEL = 8; worker_thread_start(worker_mut, my_worker_id); while(1){ if(worker_wait_for_job(worker_mut, my_worker_id)) break; if(!global_context -> is_scRNA_BAM_FQ_out_generated) continue; deflateInit2(&my_current_job -> strm , SAMBAM_COMPRESS_LEVEL_NORMAL, Z_DEFLATED, SAMBAM_GZIP_WINDOW_BITS, Z_DEFAULT_MEM_LEVEL, Z_DEFAULT_STRATEGY); struct scRNA_merge_batches_worker_task * current_input = my_current_job -> task; my_current_job -> strm.avail_in = current_input -> inbin_len; my_current_job -> strm.next_in = (unsigned char*)current_input -> inbin; my_current_job -> strm.avail_out = MERGER_WORKER_BINSIZE; my_current_job -> strm.next_out = (unsigned char*)my_current_job -> outbin; deflate(&my_current_job -> strm, Z_FINISH); my_current_job -> outbin_len = MERGER_WORKER_BINSIZE-my_current_job -> strm.avail_out; my_current_job -> crc32 = FC_CRC32(current_input -> inbin, current_input -> inbin_len); deflateEnd(&my_current_job -> strm); } return NULL; } void scRNA_save_BAM_result(fc_thread_global_context_t * global_context, struct scRNA_merge_batches_worker_current * finished_job){ if(!finished_job -> task)return; if(global_context -> is_scRNA_BAM_FQ_out_generated){ int sample_id = finished_job -> task -> sample_id; void ** fps = HashTableGet(global_context -> scRNA_sample_BAM_writers, NULL+sample_id); simple_bam_writer * wtr = fps[0]; int inbin_pos = 0; while(inbin_pos < finished_job -> task -> inbin_len){ int binlen = 0; memcpy(&binlen, finished_job -> task -> inbin+inbin_pos, 4); simple_bam_writer_update_index(wtr, finished_job -> task -> inbin+inbin_pos, binlen, finished_job -> task -> block_number, inbin_pos); inbin_pos += 4+binlen; } simple_bam_write_compressed_block(wtr, finished_job -> outbin, finished_job -> outbin_len, finished_job -> task -> inbin_len, finished_job -> crc32, finished_job -> task -> block_number); } finished_job -> task = NULL; } // return the number of RG result sets int fc_thread_merge_results(fc_thread_global_context_t * global_context, read_count_type_t * nreads , srInt_64 *nreads_mapped_to_exon, fc_read_counters * my_read_counter, HashTable * junction_global_table, HashTable * splicing_global_table, HashTable * RGmerged_table, fc_feature_info_t * loaded_features, srInt_64 nexons) { int xk1, xk2, ret = 0, sample_i; srInt_64 total_input_reads = 0 ; (*nreads_mapped_to_exon)=0; SAM_pairer_destroy(&global_context -> read_pairer); if(global_context -> do_scRNA_table){ int compress_workers = max(1,global_context-> thread_number-1); HashTable * cellnoP1_to_genenoP1_to_UMIs[global_context -> scRNA_sample_sheet_table -> numOfElements]; struct scRNA_merge_batches_worker_task * task_buffers = malloc(sizeof(struct scRNA_merge_batches_worker_task) * (1+compress_workers)* global_context->scRNA_sample_sheet_table -> numOfElements); int current_filling_worker_per_sample [global_context->scRNA_sample_sheet_table -> numOfElements]; struct scRNA_merge_batches_worker_current * worker_current_jobs = calloc(sizeof(struct scRNA_merge_batches_worker_current), compress_workers); ArrayList * file_size_list = ArrayListCreate(global_context-> scRNA_barcode_batched_bin_no +1); for(xk1=0; xk1 scRNA_barcode_batched_bin_no +2; xk1++){ if(xk1 scRNA_barcode_batched_bin_no +1){ srInt_64 batchsize = ftello(global_context -> scRNA_barcode_batched_bins[xk1]); ArrayListPush(file_size_list, NULL+( batchsize<<20 | xk1)); } fclose(global_context -> scRNA_barcode_batched_bins[xk1]); } ArrayListSort(file_size_list, NULL); srInt_64 block_numbers_current [global_context->scRNA_sample_sheet_table -> numOfElements]; for(xk1=0; xk1scRNA_sample_sheet_table -> numOfElements; xk1++){ cellnoP1_to_genenoP1_to_UMIs[xk1] = HashTableCreate(10000); HashTableSetDeallocationFunctions(cellnoP1_to_genenoP1_to_UMIs[xk1], NULL,(void (*) (void*))HashTableDestroy); current_filling_worker_per_sample[xk1] = 0; task_buffers[xk1].inbin_len = 0; block_numbers_current[xk1] = 0; } pthread_t *threads = malloc(sizeof(pthread_t)*global_context-> thread_number); for(xk1=0; xk1scRNA_sample_sheet_table -> numOfElements; xk2++) task_buffers[xk1*global_context->scRNA_sample_sheet_table -> numOfElements + xk2].sample_id = xk2+1; for(xk1=0; xk1 thread_number; xk1++){ void ** vpp = malloc(sizeof(void*)*3); vpp[0] = global_context; vpp[1] = global_context -> thread_contexts+xk1; vpp[2] = file_size_list; pthread_create(threads + xk1, NULL, scRNA_do_one_batch, vpp); } for(xk1=0; xk1 thread_number; xk1++) pthread_join(threads[xk1],NULL); ArrayListDestroy(file_size_list); worker_master_mutex_t worker_mut; worker_master_mutex_init(&worker_mut, max(1,global_context-> thread_number-1)); for(xk1=0; xk1 thread_number-1); xk1++){ void ** vpp = malloc(sizeof(void*)*4); vpp[0] = global_context; vpp[1] = &worker_mut; vpp[2] = NULL + xk1; vpp[3] = worker_current_jobs + xk1; pthread_create(threads + xk1, NULL, scRNA_merge_batches_worker, vpp); } FILE * input_fps[global_context -> scRNA_barcode_batched_bin_no+2]; char * last_rbin_buffer[global_context -> scRNA_barcode_batched_bin_no+1]; srInt_64 current_sorting_key[global_context -> scRNA_barcode_batched_bin_no+1]; for(xk1=0; xk1 scRNA_barcode_batched_bin_no+2; xk1++){ char tmp_fname[MAX_FILE_NAME_LENGTH+80]; sprintf(tmp_fname, "%s/cellCounts-Splitted-Reads-%05d-%05d.bin", global_context -> temp_file_dir, getpid(), xk1); input_fps[xk1] = fopen(tmp_fname,"rb"); if(xk1 == global_context -> scRNA_barcode_batched_bin_no+1)break; srInt_64 section1_items=0; for(sample_i = 0; sample_i < global_context -> scRNA_sample_sheet_table -> numOfElements; sample_i++){ int rrv = fread(§ion1_items,1, 8, input_fps[xk1]); for(xk2 = 0; xk2 < section1_items; xk2++){ srInt_64 cellbcP0_geneno0B=0, umis=0; rrv = rrv && fread(&cellbcP0_geneno0B,1,8,input_fps[xk1]); rrv = rrv && fread(&umis,1,8,input_fps[xk1]); int cellbc_no = cellbcP0_geneno0B>>32; int gene_no0B = (int)(cellbcP0_geneno0B&0xffffffffu); HashTable *gene_tab = HashTableGet(cellnoP1_to_genenoP1_to_UMIs[sample_i], NULL+cellbc_no+1); if(gene_tab==NULL){ gene_tab = HashTableCreate(300); HashTablePut(cellnoP1_to_genenoP1_to_UMIs[sample_i], NULL+cellbc_no+1, gene_tab); } HashTablePut(gene_tab, NULL+gene_no0B+1 , NULL+umis); } } last_rbin_buffer[xk1] = malloc( global_context -> scRNA_barcode_batched_max_genes *8 + global_context -> scRNA_barcode_batched_max_Rbin_len + 4 + MAX_UMI_LEN + 16 + 10000); int rlen = fread(last_rbin_buffer[xk1], 1, 16, input_fps[xk1]); if(rlen >0){ int binlen = 0; srInt_64 genes = 0; memcpy(&genes, last_rbin_buffer[xk1]+8, 8); if(genes & (1LLU<<63))genes = genes & 0x7fffffff; else genes= 0; int rrv = fread(last_rbin_buffer[xk1]+16, 1, 8*genes+ global_context -> scRNA_UMI_length + 4, input_fps[xk1]); memcpy(&binlen, last_rbin_buffer[xk1] +16 +8*genes+ global_context -> scRNA_UMI_length , 4); rrv = rrv && fread(last_rbin_buffer[xk1] + 16+ 8*genes+ global_context -> scRNA_UMI_length + 4, 1, binlen, input_fps[xk1]); srInt_64 sorting_key = *(int*)(last_rbin_buffer[xk1] + 16 +8*genes+global_context -> scRNA_UMI_length +4); sorting_key = sorting_key << 32; sorting_key |= *(int*)(last_rbin_buffer[xk1] + 16+ 8*genes+global_context -> scRNA_UMI_length +8); current_sorting_key[xk1] = sorting_key; }else current_sorting_key[xk1] = 0x7fffffffffffffffLLU; } int current_worker = 0; while(1){ int selected_fp_no = 0; srInt_64 selected_fp_key = current_sorting_key[0]; for(xk1=1; xk1 scRNA_barcode_batched_bin_no+1; xk1++){ if(current_sorting_key[xk1] < selected_fp_key){ selected_fp_key = current_sorting_key[xk1] ; selected_fp_no = xk1; } } if(selected_fp_key == 0x7fffffffffffffffLLU) break; int sample_id = 0, binlen = 0; srInt_64 genes = 0; memcpy(&sample_id, last_rbin_buffer[selected_fp_no], 4); memcpy(&genes, last_rbin_buffer[selected_fp_no]+8, 8); if(genes & (1LLU<<63)) genes = genes & 0x7fffffff; else genes = 0; memcpy(&binlen,last_rbin_buffer[selected_fp_no]+16+8*genes+global_context -> scRNA_UMI_length,4); struct scRNA_merge_batches_worker_task * tofill = task_buffers+(current_filling_worker_per_sample[sample_id-1] * global_context->scRNA_sample_sheet_table -> numOfElements +sample_id-1); memcpy(tofill->inbin + tofill-> inbin_len, last_rbin_buffer[selected_fp_no]+16+8*genes+global_context -> scRNA_UMI_length, binlen + 4); tofill -> inbin_len += (binlen + 4); //SUBREADprintf("ADDING BLOCKKK = %d WKR = %d IT THINK IT'S %d ; GENES=%d\n", tofill -> inbin_len, current_worker, tofill -> sample_id, genes); if(tofill-> inbin_len > 60000){ master_wait_for_job_done(&worker_mut, current_worker); struct scRNA_merge_batches_worker_current * my_finished_job = worker_current_jobs+current_worker; scRNA_save_BAM_result(global_context, my_finished_job); my_finished_job -> task = tofill; my_finished_job -> task -> block_number = (block_numbers_current[sample_id-1]++); my_finished_job -> outbin_len = 0; master_notify_worker(&worker_mut, current_worker); current_filling_worker_per_sample[sample_id-1] ++; if(current_filling_worker_per_sample[sample_id-1] == compress_workers +1) current_filling_worker_per_sample[sample_id-1] = 0; tofill = task_buffers+(current_filling_worker_per_sample[sample_id-1] * global_context->scRNA_sample_sheet_table -> numOfElements +sample_id-1); tofill -> inbin_len = 0; current_worker ++; if(current_worker == compress_workers) current_worker=0; } int rlen = fread(last_rbin_buffer[selected_fp_no], 1, 16, input_fps[selected_fp_no]); if(rlen >0){ int binlen = 0; srInt_64 genes = 0; memcpy(&genes, last_rbin_buffer[selected_fp_no]+8, 8); if(genes & (1LLU<<63))genes = genes & 0x7fffffff; else genes= 0; int rrv = fread(last_rbin_buffer[selected_fp_no]+16, 1, 8*genes+ global_context -> scRNA_UMI_length + 4, input_fps[selected_fp_no]); memcpy(&binlen, last_rbin_buffer[selected_fp_no] +16 +8*genes+ global_context -> scRNA_UMI_length , 4); rrv = rrv && fread(last_rbin_buffer[selected_fp_no] + 16+ 8*genes+ global_context -> scRNA_UMI_length + 4, 1, binlen, input_fps[selected_fp_no]); srInt_64 sorting_key = *(int*)(last_rbin_buffer[selected_fp_no] + 16+8*genes +global_context -> scRNA_UMI_length +4); sorting_key = sorting_key << 32; sorting_key |= *(int*)(last_rbin_buffer[selected_fp_no] + 16 +8*genes+global_context -> scRNA_UMI_length +8); current_sorting_key[selected_fp_no] = sorting_key; } else current_sorting_key[selected_fp_no] = 0x7fffffffffffffffLLU; } for(xk1=0; xk1 scRNA_barcode_batched_bin_no+1; xk1++){ fclose(input_fps[xk1]); free(last_rbin_buffer[xk1]); } for(xk1=0; xk1 scRNA_sample_sheet_table -> numOfElements; xk1++){ struct scRNA_merge_batches_worker_task * tofill = task_buffers+(current_filling_worker_per_sample[xk1] * global_context->scRNA_sample_sheet_table -> numOfElements +xk1); if(tofill->inbin_len<1) continue; master_wait_for_job_done(&worker_mut, current_worker); struct scRNA_merge_batches_worker_current * my_finished_job = worker_current_jobs+current_worker; scRNA_save_BAM_result(global_context, my_finished_job); my_finished_job -> task = tofill; my_finished_job -> task -> block_number = (block_numbers_current[xk1]++); my_finished_job -> outbin_len = 0; master_notify_worker(&worker_mut, current_worker); current_worker ++; if(current_worker == compress_workers) current_worker=0; } for(xk1=0; xk1 task)master_wait_for_job_done(&worker_mut, current_worker); scRNA_save_BAM_result(global_context, my_finished_job); current_worker ++; if(current_worker == compress_workers) current_worker=0; } //TODO: add "scRNA_barcode_batched_bin_no+1" bin into "all unmapped" for(xk1 = 0; xk1 < 1+compress_workers; xk1++) for(xk2 = 0; xk2 < global_context->scRNA_sample_sheet_table -> numOfElements;xk2++) task_buffers[ xk1 * global_context->scRNA_sample_sheet_table -> numOfElements + xk2 ].inbin_len = 0; current_worker = 0; FILE * notmapped_fp = input_fps[global_context -> scRNA_barcode_batched_bin_no+1]; while(1){ int sample_id = 0, binlen = 0; int rlen = fread(&sample_id, 1, 4, notmapped_fp); if(rlen < 4) break; struct scRNA_merge_batches_worker_task * tofill = task_buffers+(current_filling_worker_per_sample[sample_id -1] * global_context->scRNA_sample_sheet_table -> numOfElements +sample_id-1); int rrv = fread(&binlen, 1, 4, notmapped_fp); memcpy(tofill -> inbin + tofill -> inbin_len, &binlen, 4); tofill -> inbin_len += 4; rrv = rrv && fread(tofill -> inbin + tofill -> inbin_len, 1, binlen, notmapped_fp); tofill -> inbin_len += binlen; if(tofill-> inbin_len > 60000){ struct scRNA_merge_batches_worker_current * my_finished_job = worker_current_jobs+current_worker; if(my_finished_job -> task)master_wait_for_job_done(&worker_mut, current_worker); scRNA_save_BAM_result(global_context, my_finished_job); my_finished_job -> task = tofill; my_finished_job -> task -> block_number = (block_numbers_current[sample_id-1]++); my_finished_job -> outbin_len = 0; master_notify_worker(&worker_mut, current_worker); current_filling_worker_per_sample[sample_id-1] ++; if(current_filling_worker_per_sample[sample_id-1] == compress_workers +1) current_filling_worker_per_sample[sample_id-1] = 0; tofill = task_buffers+(current_filling_worker_per_sample[sample_id-1] * global_context->scRNA_sample_sheet_table -> numOfElements +sample_id-1); tofill -> inbin_len = 0; current_worker ++; if(current_worker == compress_workers) current_worker=0; } } for(xk1=0; xk1 scRNA_sample_sheet_table -> numOfElements; xk1++){ struct scRNA_merge_batches_worker_task * tofill = task_buffers+(current_filling_worker_per_sample[xk1] * global_context->scRNA_sample_sheet_table -> numOfElements +xk1); if(tofill->inbin_len<1) continue; master_wait_for_job_done(&worker_mut, current_worker); struct scRNA_merge_batches_worker_current * my_finished_job = worker_current_jobs+current_worker; scRNA_save_BAM_result(global_context, my_finished_job); my_finished_job -> task = tofill; my_finished_job -> task -> block_number = (block_numbers_current[xk1]++); my_finished_job -> outbin_len = 0; master_notify_worker(&worker_mut, current_worker); current_worker ++; if(current_worker == compress_workers) current_worker=0; } for(xk1=0; xk1 scRNA_applied_umi_cut = calloc(sizeof(int), global_context -> scRNA_sample_sheet_table -> numOfElements); scRNA_merged_to_tables_write(global_context , cellnoP1_to_genenoP1_to_UMIs , loaded_features, nexons); for(xk1=0; xk1 scRNA_sample_sheet_table -> numOfElements; xk1++) HashTableDestroy(cellnoP1_to_genenoP1_to_UMIs[xk1]); HashTable ** used_cell_no_tables = malloc(sizeof(HashTable*) * global_context -> scRNA_sample_sheet_table -> numOfElements); for(xk1=0; xk1 scRNA_sample_sheet_table -> numOfElements; xk1++){ used_cell_no_tables[xk1] = HashTableCreate(30000); used_cell_no_tables[xk1] -> appendix1 = malloc(sizeof(pthread_spinlock_t)); pthread_spin_init((pthread_spinlock_t*)used_cell_no_tables[xk1] -> appendix1,1); } for(xk1=0; xk1 scRNA_sample_sheet_table -> numOfElements; xk1++){ pthread_spin_destroy((pthread_spinlock_t*)used_cell_no_tables[xk1] -> appendix1); HashTableDestroy(used_cell_no_tables[xk1]); } free(used_cell_no_tables); } for(xk1=0; xk1 thread_number; xk1++) { if(global_context -> assign_reads_to_RG){ HashTable * thread_rg_tab = global_context -> thread_contexts[xk1].RG_table; int buck_i; for(buck_i = 0; buck_i < thread_rg_tab -> numOfBuckets; buck_i++){ KeyValuePair *cursor = thread_rg_tab -> bucketArray[buck_i]; while(cursor){ char * rg_name = (char *)cursor -> key; void ** rg_thread_tabs = cursor -> value; void ** rg_old_tabs = HashTableGet(RGmerged_table, rg_name); if(!rg_old_tabs){ rg_old_tabs = malloc(sizeof(char *)*4); // all_counts, sum_counts , junc_table, split_table rg_old_tabs[0] = calloc(global_context -> thread_contexts[xk1].count_table_size, sizeof(srInt_64)); rg_old_tabs[1] = calloc(1, sizeof(fc_read_counters)); if(global_context -> do_junction_counting){ HashTable * junction_counting_table = HashTableCreate(131317); HashTableSetHashFunction(junction_counting_table,HashTableStringHashFunction); HashTableSetDeallocationFunctions(junction_counting_table, free, NULL); HashTableSetKeyComparisonFunction(junction_counting_table, fc_strcmp_chro); HashTable * splicing_point_table = HashTableCreate(131317); HashTableSetHashFunction(splicing_point_table,HashTableStringHashFunction); HashTableSetDeallocationFunctions(splicing_point_table, free, NULL); HashTableSetKeyComparisonFunction(splicing_point_table, fc_strcmp_chro); rg_old_tabs[2] = junction_counting_table; rg_old_tabs[3] = splicing_point_table; }else rg_old_tabs[2] = NULL; HashTablePut(RGmerged_table, memstrcpy(rg_name), rg_old_tabs); } srInt_64 * rg_counts = rg_old_tabs[0]; fc_read_counters * rg_sum_reads = rg_old_tabs[1]; HashTable * rg_junc_tab = rg_old_tabs[2]; HashTable * rg_split_tab = rg_old_tabs[3]; srInt_64 * rg_thread_counts = rg_thread_tabs[0]; fc_read_counters * rg_thread_sum_reads = rg_thread_tabs[1]; HashTable * rg_thread_junc_table = rg_thread_tabs[2]; HashTable * rg_thread_split_table = rg_thread_tabs[3]; for(xk2=0; xk2 exontable_exons; xk2++) rg_counts[xk2] += rg_thread_counts[xk2]; rg_sum_reads->unassigned_ambiguous += rg_thread_sum_reads->unassigned_ambiguous; rg_sum_reads->unassigned_nofeatures += rg_thread_sum_reads->unassigned_nofeatures; rg_sum_reads->unassigned_overlapping_length += rg_thread_sum_reads->unassigned_overlapping_length; rg_sum_reads->unassigned_unmapped += rg_thread_sum_reads->unassigned_unmapped; rg_sum_reads->unassigned_singleton += rg_thread_sum_reads->unassigned_singleton; rg_sum_reads->unassigned_read_type += rg_thread_sum_reads->unassigned_read_type; rg_sum_reads->unassigned_mappingquality += rg_thread_sum_reads->unassigned_mappingquality; rg_sum_reads->unassigned_fragmentlength += rg_thread_sum_reads->unassigned_fragmentlength; rg_sum_reads->unassigned_chimericreads += rg_thread_sum_reads->unassigned_chimericreads; rg_sum_reads->unassigned_multimapping += rg_thread_sum_reads->unassigned_multimapping; rg_sum_reads->unassigned_secondary += rg_thread_sum_reads->unassigned_secondary; rg_sum_reads->unassigned_junction_condition += rg_thread_sum_reads->unassigned_junction_condition; rg_sum_reads->unassigned_duplicate += rg_thread_sum_reads->unassigned_duplicate; rg_sum_reads->assigned_reads += rg_thread_sum_reads->assigned_reads; if(global_context -> do_junction_counting){ int bucket_i; for(bucket_i = 0 ; bucket_i < rg_thread_junc_table -> numOfBuckets; bucket_i++){ KeyValuePair * cursor; cursor = rg_thread_junc_table -> bucketArray[bucket_i]; while(cursor){ char * junckey = (char *) cursor -> key; void * globval = HashTableGet(rg_junc_tab, junckey); char * new_key = memstrcpy(junckey); globval += (cursor -> value - NULL); HashTablePut(rg_junc_tab, new_key, globval); // new_key will be freed when it is replaced next time or when the global table is destroyed. cursor = cursor->next; } } for(bucket_i = 0 ; bucket_i < rg_thread_split_table -> numOfBuckets; bucket_i++){ KeyValuePair * cursor; cursor = rg_thread_split_table -> bucketArray[bucket_i]; while(cursor){ char * junckey = (char *) cursor -> key; void * globval = HashTableGet(rg_split_tab, junckey); char * new_key = memstrcpy(junckey); //if(xk1>0) //SUBREADprintf("MERGE THREAD-%d : %s VAL=%u, ADD=%u\n", xk1, junckey, globval - NULL, cursor -> value - NULL); globval += (cursor -> value - NULL); HashTablePut(rg_split_tab, new_key, globval); cursor = cursor->next; } } } // end : merge junc tables ret++; cursor = cursor -> next; } } } for(xk2=0; xk2 exontable_exons; xk2++) nreads[xk2]+=global_context -> thread_contexts[xk1].count_table[xk2]; total_input_reads += global_context -> thread_contexts[xk1].all_reads; (*nreads_mapped_to_exon) += global_context -> thread_contexts[xk1].nreads_mapped_to_exon; global_context -> read_counters.unassigned_ambiguous += global_context -> thread_contexts[xk1].read_counters.unassigned_ambiguous; global_context -> read_counters.unassigned_nofeatures += global_context -> thread_contexts[xk1].read_counters.unassigned_nofeatures; global_context -> read_counters.unassigned_overlapping_length += global_context -> thread_contexts[xk1].read_counters.unassigned_overlapping_length; global_context -> read_counters.unassigned_unmapped += global_context -> thread_contexts[xk1].read_counters.unassigned_unmapped; global_context -> read_counters.unassigned_singleton += global_context -> thread_contexts[xk1].read_counters.unassigned_singleton; global_context -> read_counters.unassigned_read_type += global_context -> thread_contexts[xk1].read_counters.unassigned_read_type; global_context -> read_counters.unassigned_mappingquality += global_context -> thread_contexts[xk1].read_counters.unassigned_mappingquality; global_context -> read_counters.unassigned_fragmentlength += global_context -> thread_contexts[xk1].read_counters.unassigned_fragmentlength; global_context -> read_counters.unassigned_chimericreads += global_context -> thread_contexts[xk1].read_counters.unassigned_chimericreads; global_context -> read_counters.unassigned_multimapping += global_context -> thread_contexts[xk1].read_counters.unassigned_multimapping; global_context -> read_counters.unassigned_secondary += global_context -> thread_contexts[xk1].read_counters.unassigned_secondary; global_context -> read_counters.unassigned_junction_condition += global_context -> thread_contexts[xk1].read_counters.unassigned_junction_condition; global_context -> read_counters.unassigned_duplicate += global_context -> thread_contexts[xk1].read_counters.unassigned_duplicate; global_context -> read_counters.assigned_reads += global_context -> thread_contexts[xk1].read_counters.assigned_reads; my_read_counter->unassigned_ambiguous += global_context -> thread_contexts[xk1].read_counters.unassigned_ambiguous; my_read_counter->unassigned_nofeatures += global_context -> thread_contexts[xk1].read_counters.unassigned_nofeatures; my_read_counter->unassigned_overlapping_length += global_context -> thread_contexts[xk1].read_counters.unassigned_overlapping_length; my_read_counter->unassigned_unmapped += global_context -> thread_contexts[xk1].read_counters.unassigned_unmapped; my_read_counter->unassigned_singleton += global_context -> thread_contexts[xk1].read_counters.unassigned_singleton; my_read_counter->unassigned_read_type += global_context -> thread_contexts[xk1].read_counters.unassigned_read_type; my_read_counter->unassigned_mappingquality += global_context -> thread_contexts[xk1].read_counters.unassigned_mappingquality; my_read_counter->unassigned_fragmentlength += global_context -> thread_contexts[xk1].read_counters.unassigned_fragmentlength; my_read_counter->unassigned_chimericreads += global_context -> thread_contexts[xk1].read_counters.unassigned_chimericreads; my_read_counter->unassigned_multimapping += global_context -> thread_contexts[xk1].read_counters.unassigned_multimapping; my_read_counter->unassigned_secondary += global_context -> thread_contexts[xk1].read_counters.unassigned_secondary; my_read_counter->unassigned_junction_condition += global_context -> thread_contexts[xk1].read_counters.unassigned_junction_condition; my_read_counter->unassigned_duplicate += global_context -> thread_contexts[xk1].read_counters.unassigned_duplicate; my_read_counter->assigned_reads += global_context -> thread_contexts[xk1].read_counters.assigned_reads; if(global_context -> do_junction_counting){ int bucket_i; for(bucket_i = 0 ; bucket_i < global_context -> thread_contexts[xk1].junction_counting_table -> numOfBuckets; bucket_i++){ KeyValuePair * cursor; cursor = global_context -> thread_contexts[xk1].junction_counting_table -> bucketArray[bucket_i]; while(cursor){ char * junckey = (char *) cursor -> key; void * globval = HashTableGet(junction_global_table, junckey); char * new_key = malloc(strlen(junckey)+1); strcpy(new_key, junckey); globval += (cursor -> value - NULL); HashTablePut(junction_global_table, new_key, globval); // new_key will be freed when it is replaced next time or when the global table is destroyed. cursor = cursor->next; } } for(bucket_i = 0 ; bucket_i < global_context -> thread_contexts[xk1].splicing_point_table -> numOfBuckets; bucket_i++){ KeyValuePair * cursor; cursor = global_context -> thread_contexts[xk1].splicing_point_table -> bucketArray[bucket_i]; while(cursor){ char * junckey = (char *) cursor -> key; void * globval = HashTableGet(splicing_global_table, junckey); char * new_key = malloc(strlen(junckey)+1); strcpy(new_key, junckey); //if(xk1>0) //SUBREADprintf("MERGE THREAD-%d : %s VAL=%u, ADD=%u\n", xk1, junckey, globval - NULL, cursor -> value - NULL); globval += (cursor -> value - NULL); HashTablePut(splicing_global_table, new_key, globval); cursor = cursor->next; } } } } if(0 == global_context -> is_input_bad_format){ if(global_context -> is_paired_end_reads_expected){ if(global_context -> is_mixed_PE_SE) print_in_box(80,0,0," WARNING: Single-end reads were found%s.", global_context -> is_strand_checked?" and excluded":""); else print_in_box(80,0,0," Paired-end reads are included."); if(!global_context -> is_paired_end_mode_assign) print_in_box(80,0,0, " The reads are assigned on the single-end mode."); }else{ // paired-end reads in a single-end lib will result in error. print_in_box(80,0,0," Single-end reads are included."); } char pct_str[10]; if(total_input_reads>0) sprintf(pct_str,"(%.1f%%%%)", (*nreads_mapped_to_exon)*100./total_input_reads); else pct_str[0]=0; int show_summary = 1; if(global_context -> assign_reads_to_RG){ if(RGmerged_table -> numOfElements) print_in_box(80,0,0," Total read groups : %ld", RGmerged_table -> numOfElements); else{ print_in_box(80,0,0," No read groups are found; no output is generated."); show_summary = 0; } } if(show_summary){ print_in_box(80,0,0," Total alignments : %llu", total_input_reads); print_in_box(pct_str[0]?81:80,0,0," Successfully assigned alignments : %llu %s", *nreads_mapped_to_exon,pct_str); } print_in_box(80,0,0," Running time : %.2f minutes", (miltime() - global_context -> start_time)/60); print_in_box(80,0,0,""); } return ret; } void get_temp_dir_from_out(char * tmp, char * out){ char * slash = strrchr(out,'/'); if(NULL == slash){ strcpy(tmp, "./"); }else{ memcpy(tmp, out, slash - out); tmp[slash - out]=0; } } void fc_thread_init_input_files(fc_thread_global_context_t * global_context, char * in_fnames, char ** out_ptr ){ if(global_context -> use_stdin_file){ #ifdef MAKE_STANDALONE char MAC_or_random[13]; (*out_ptr) = malloc(MAX_FILE_NAME_LENGTH); mac_or_rand_str(MAC_or_random); sprintf(*out_ptr, "%s/temp-core-%06u-%s.sam", global_context -> temp_file_dir, getpid(), MAC_or_random); SUBREADprintf("\nReading data from for featureCounts ...\n\n"); FILE * ifp = fopen(*out_ptr,"w"); while(1){ char nchar[100]; int rlen = fread(nchar, 1, 100, stdin); if(rlen > 0) fwrite(nchar, 1, rlen, ifp); else break; //if(rlen < 100)break; } fclose(ifp); #endif }else{ (*out_ptr) = malloc(strlen(in_fnames)+1); strcpy((*out_ptr), in_fnames); } } void fc_NCfree(void * vv){ char ** cc = vv; int i; for(i=0; cc[i]; i++) free(cc[i]); free(vv); } void scRNA_convert_ss_to_arr( void * key, void * hashed_obj, HashTable * tab ){ ArrayList * hashed_arr = hashed_obj ; fc_thread_global_context_t * global_context = tab->appendix1; ArrayListPush(global_context -> scRNA_sample_id_to_name, key); hashed_arr -> appendix1 = NULL+global_context -> scRNA_sample_id_to_name -> numOfElements; // One-based srInt_64 xx1; for(xx1 =0; xx1< hashed_arr -> numOfElements; xx1++){ char ** push_arr = malloc(sizeof(char*)*4); char ** sbc_lane_sample = ArrayListGet(hashed_arr, xx1); srInt_64 lane_sample_int = sbc_lane_sample[0]-(char*)NULL; ArrayListPush(global_context -> scRNA_sample_barcode_list, push_arr); push_arr[0] = NULL + lane_sample_int; push_arr[1] = NULL + global_context -> scRNA_sample_id_to_name -> numOfElements; push_arr[2] = sbc_lane_sample[1]; // Sample Barcode push_arr[3] = NULL + (sbc_lane_sample[1]!=NULL && strlen(sbc_lane_sample[1])>12); int line_no_in_sheet = sbc_lane_sample[2] - (char*)NULL; HashTablePut(global_context -> scRNA_lineno1B_to_sampleno1B_tab , NULL+line_no_in_sheet, NULL + global_context -> scRNA_sample_id_to_name -> numOfElements); //SUBREADprintf("Push_LineNo : %d -> %d\n", line_no_in_sheet, global_context -> scRNA_sample_id_to_name -> numOfElements); //SUBREADprintf("Push Barcode %s in Lane %ld for %s [%d]\n", push_arr[2], (sbc_lane_sample[0]-(char*)NULL), key, global_context -> scRNA_sample_id_to_name -> numOfElements); } } void scRNA_cell_barcode_tabel_destroy(void *a){ if(((a-NULL) & 0xfffffffff0000000llu ) ==IMPOSSIBLE_MEMORY_SPACE )return; ArrayListDestroy((ArrayList*)a); } void scRNA_make_barcode_HT_table( fc_thread_global_context_t * global_context ){ int xx1,xx2; global_context -> scRNA_cell_barcode_head_tail_table = StringTableCreate(600000); HashTableSetDeallocationFunctions( global_context -> scRNA_cell_barcode_head_tail_table, free, scRNA_cell_barcode_tabel_destroy); for(xx1=0;xx1 < global_context-> scRNA_cell_barcodes_array -> numOfElements; xx1++){ char * bc = ArrayListGet(global_context-> scRNA_cell_barcodes_array, xx1); int bcl =strlen(bc); if(global_context -> known_cell_barcode_length==0) global_context -> known_cell_barcode_length=bcl; if(bcl!=global_context -> known_cell_barcode_length){ // SUBREADprintf("The cell barcodes have variable lengths. This may be a new protocol and we don't support it yet.\n"); assert(bcl==global_context -> known_cell_barcode_length); } char bctmp[20]; HashTablePut(global_context -> scRNA_cell_barcode_head_tail_table, strdup(bc), NULL+xx1+IMPOSSIBLE_MEMORY_SPACE); for(xx2=0; xx2<2; xx2++){ bctmp[0] = xx2?'S':'F'; int xx3; for(xx3 = 0; xx3< global_context -> known_cell_barcode_length/2; xx3++) bctmp[xx3+1] = bc[ xx3*2+xx2 ]; bctmp[bcl/2+1]=0; ArrayList * array_of_codes = HashTableGet(global_context -> scRNA_cell_barcode_head_tail_table, bctmp); if(!array_of_codes){ array_of_codes = ArrayListCreate(4); HashTablePut(global_context -> scRNA_cell_barcode_head_tail_table, strdup(bctmp), array_of_codes); } ArrayListPush(array_of_codes, NULL+xx1); } } } void scRNA_close_sample_SamBam_writers(void *v){ void ** vv = v; simple_bam_writer * wtr = vv[0]; simple_bam_close(wtr); if(vv[1]){ parallel_gzip_writer_t* gzfp = vv[1]; parallel_gzip_writer_close(gzfp); free(gzfp); gzfp = vv[2]; parallel_gzip_writer_close(gzfp); free(gzfp); gzfp = vv[3]; // I2 if(gzfp){ parallel_gzip_writer_close(gzfp); free(gzfp); } gzfp = vv[4]; parallel_gzip_writer_close(gzfp); free(gzfp); } pthread_spinlock_t * gz_lock = vv[5]; pthread_spin_destroy(gz_lock); free(gz_lock); free(vv); } #define SORT_BAM_FROM_SCRNA 1 void scRNA_sample_SamBam_writers_new_files(void *k, void *v, HashTable * tab){ HashTable * fp_tab = tab -> appendix1; fc_thread_global_context_t * global_context = tab -> appendix2; ArrayList * scRNA_sample_id_to_name = tab -> appendix3; char * samplename = k; char fname [MAX_FILE_NAME_LENGTH+20], fnamet[MAX_FILE_NAME_LENGTH+20]; sprintf(fname, "%s.bam", samplename); sprintf(fnamet, "del4-cC-tmp0-%s.del", samplename); simple_bam_writer * wtr = simple_bam_create(fname); parallel_gzip_writer_t * gzipR1fq=NULL, * gzipI1fq=NULL, * gzipR2fq=NULL, *gzipI2fq =NULL; if(global_context -> scRNA_input_mode == GENE_INPUT_BCL || global_context -> scRNA_input_mode == GENE_INPUT_SCRNA_BAM){ gzipR1fq = calloc(sizeof(parallel_gzip_writer_t),1); gzipI1fq = calloc(sizeof(parallel_gzip_writer_t),1); if(global_context -> scRNA_dual_index)gzipI2fq = calloc(sizeof(parallel_gzip_writer_t),1); gzipR2fq = calloc(sizeof(parallel_gzip_writer_t),1); sprintf(fname, "%s_R1.fastq.gz", samplename); parallel_gzip_writer_init(gzipR1fq, fname, global_context -> thread_number); sprintf(fname, "%s_I1.fastq.gz", samplename); parallel_gzip_writer_init(gzipI1fq, fname, global_context -> thread_number); if(gzipI2fq){ sprintf(fname, "%s_I2.fastq.gz", samplename); parallel_gzip_writer_init(gzipI2fq, fname, global_context -> thread_number); } sprintf(fname, "%s_R2.fastq.gz", samplename); parallel_gzip_writer_init(gzipR2fq, fname, global_context -> thread_number); } pthread_spinlock_t * gzfp_lock = malloc(sizeof(pthread_spinlock_t)); pthread_spin_init(gzfp_lock, PTHREAD_PROCESS_PRIVATE); int x1; for(x1=0; x1 numOfElements; x1++){ char * sample_name = ArrayListGet( scRNA_sample_id_to_name, x1 ); if(strcmp(sample_name, samplename)==0){ void ** wtrptr = malloc(sizeof(void*)*6); wtrptr[0]=wtr; wtrptr[1]=gzipR1fq; wtrptr[2]=gzipI1fq; wtrptr[3]=gzipI2fq; wtrptr[4]=gzipR2fq; wtrptr[5]=gzfp_lock; HashTablePut(fp_tab, NULL+x1+1 , wtrptr); break; } } } void fc_thread_init_global_context(fc_thread_global_context_t * global_context, unsigned int buffer_size, unsigned short threads, int line_length, int min_pe_dist, int max_pe_dist, int is_gene_level, int is_overlap_allowed, char * strand_check_mode, char * output_fname, int is_sam_out, int is_both_end_required, int is_chimertc_disallowed, int is_PE_distance_checked, char *feature_name_column, char * gene_id_column, int min_map_qual_score, int is_multi_mapping_allowed, int is_SAM, char * alias_file_name, char * cmd_rebuilt, int is_input_file_resort_needed, int feature_block_size, int isCVersion, int fiveEndExtension, int threeEndExtension, int minFragmentOverlap, int is_split_or_exonic_only, int reduce_5_3_ends_to_one, char * debug_command, int is_duplicate_ignored, int is_not_sort, int use_fraction_multimapping, int useOverlappingBreakTie, char * pair_orientations, int do_junction_cnt, int max_M, int isRestrictlyNoOvelrapping, float fracOverlap, char * temp_dir, int use_stdin_file, int assign_reads_to_RG, int long_read_minimum_length, int is_verbose, float frac_feature_overlap, int do_detection_call, int max_missing_bases_in_read, int max_missing_bases_in_feature, int is_primary_alignment_only, char * Rpath, char * extra_column_names , char * annotation_file_screen_output, int read_shift_type, int read_shift_size, char * scRNA_sample_sheet, char * scRNA_cell_barcode_list, int is_scRNA_BAM_FQ_out_generated, int scRNA_input_mode, int scRNA_rerun_on_persample_BAM, float scRNA_umi_cutoff, int is_dual_index ) { int x1; myrand_srand(time(NULL)); memset(global_context, 0, sizeof(fc_thread_global_context_t)); global_context -> max_BAM_header_size = buffer_size; global_context -> all_reads = 0; global_context -> redo = 0; global_context -> read_details_out_FP = NULL; global_context -> reported_extra_columns = extra_column_names; global_context -> isCVersion = isCVersion; global_context -> is_read_details_out = is_sam_out; global_context -> is_multi_overlap_allowed = is_overlap_allowed; global_context -> restricted_no_multi_overlap = isRestrictlyNoOvelrapping; global_context -> is_gene_level = is_gene_level; global_context -> strand_check_mode = strand_check_mode; global_context -> is_both_end_required = is_both_end_required; global_context -> is_chimertc_disallowed = is_chimertc_disallowed; global_context -> is_PE_distance_checked = is_PE_distance_checked; global_context -> is_multi_mapping_allowed = is_multi_mapping_allowed; global_context -> is_primary_alignment_only = is_primary_alignment_only; global_context -> is_split_or_exonic_only = is_split_or_exonic_only; global_context -> is_duplicate_ignored = is_duplicate_ignored; global_context -> use_stdin_file = use_stdin_file; global_context -> assign_reads_to_RG = assign_reads_to_RG; global_context -> long_read_minimum_length = long_read_minimum_length; global_context -> is_verbose = is_verbose; global_context -> do_detection_call = do_detection_call; //global_context -> is_first_read_reversed = (pair_orientations[0]=='r'); //global_context -> is_second_read_straight = (pair_orientations[1]=='f'); global_context -> reduce_5_3_ends_to_one = reduce_5_3_ends_to_one; global_context -> do_not_sort = is_not_sort; global_context -> is_SAM_file = is_SAM; global_context -> use_fraction_multi_mapping = use_fraction_multimapping; global_context -> do_junction_counting = do_junction_cnt; global_context -> thread_number = threads; global_context -> min_mapping_quality_score = min_map_qual_score; global_context -> unistr_buffer_size = 1024*1024*2; global_context -> unistr_buffer_used = 0; global_context -> unistr_buffer_space = malloc(global_context -> unistr_buffer_size); global_context -> BAM_chros_to_anno_table = NULL; global_context -> cmd_rebuilt = cmd_rebuilt; global_context -> feature_block_size = feature_block_size; global_context -> five_end_extension = fiveEndExtension; global_context -> three_end_extension = threeEndExtension; global_context -> read_shift_type = read_shift_type; global_context -> read_shift_size = read_shift_size; global_context -> fragment_minimum_overlapping = minFragmentOverlap; global_context -> fractional_minimum_overlapping = fracOverlap; global_context -> fractional_minimum_feature_overlapping = frac_feature_overlap; global_context -> max_missing_bases_in_read = max_missing_bases_in_read; global_context -> max_missing_bases_in_feature = max_missing_bases_in_feature; global_context -> use_overlapping_break_tie = useOverlappingBreakTie; global_context -> need_calculate_fragment_len = ( global_context -> fractional_minimum_overlapping > 1E-10 ) || (global_context -> fractional_minimum_feature_overlapping > 1E-10) || ( global_context -> max_missing_bases_in_read >= 0 ) || ( global_context -> max_missing_bases_in_feature >= 0 ); global_context -> need_calculate_overlap_len = (global_context -> fractional_minimum_overlapping > 1E-10) || (global_context -> fragment_minimum_overlapping > 1) || global_context -> use_overlapping_break_tie || (global_context -> fractional_minimum_feature_overlapping > 1E-10) || ( global_context -> max_missing_bases_in_read >= 0 ) || ( global_context -> max_missing_bases_in_feature >= 0 ); global_context -> debug_command = debug_command; global_context -> max_M = max_M; global_context -> max_BAM_header_size = buffer_size; if(scRNA_sample_sheet){ global_context -> scRNA_umi_cutoff = scRNA_umi_cutoff; global_context -> do_scRNA_table = 1; global_context -> scRNA_sample_id_to_name = ArrayListCreate(64); global_context -> scRNA_lineno1B_to_sampleno1B_tab = HashTableCreate(10); strcpy(global_context->scRNA_sample_sheet,scRNA_sample_sheet); global_context-> scRNA_sample_sheet_table = input_BLC_parse_SampleSheet( global_context->scRNA_sample_sheet ); global_context-> scRNA_sample_sheet_table -> appendix1 = global_context; global_context -> scRNA_sample_barcode_list = ArrayListCreate(64); ArrayListSetDeallocationFunction(global_context -> scRNA_sample_barcode_list, free); HashTableIteration(global_context-> scRNA_sample_sheet_table, scRNA_convert_ss_to_arr); if(scRNA_cell_barcode_list){ strcpy(global_context->scRNA_cell_barcode_list,scRNA_cell_barcode_list); global_context-> scRNA_cell_barcodes_array = input_BLC_parse_CellBarcodes( global_context->scRNA_cell_barcode_list ); scRNA_make_barcode_HT_table( global_context ); //print_in_box(80,0,0,"Loaded %ld cell barcodes from the list.", global_context-> scRNA_cell_barcodes_array -> numOfElements); } global_context -> is_scRNA_BAM_FQ_out_generated = is_scRNA_BAM_FQ_out_generated; global_context -> scRNA_dual_index = is_dual_index ; global_context -> scRNA_input_mode = scRNA_input_mode; global_context -> scRNA_rerun_on_persample_BAM = scRNA_rerun_on_persample_BAM; global_context -> scRNA_barcode_batched_bin_no = 149; pthread_spin_init(&global_context -> scRNA_do_one_batch_runner_lock, PTHREAD_PROCESS_PRIVATE); global_context -> scRNA_barcode_batched_locks = malloc(sizeof(pthread_spinlock_t)*(global_context -> scRNA_barcode_batched_bin_no+2)); global_context -> scRNA_barcode_batched_bins = malloc(sizeof(FILE*)*(global_context -> scRNA_barcode_batched_bin_no+2)); for(x1=0; x1 scRNA_barcode_batched_bin_no+2; x1++){ char tmp_fname[MAX_FILE_NAME_LENGTH+20]; sprintf(tmp_fname, "%s/cellCounts-Splitted-Reads-%05d-%05d.bin", temp_dir, getpid(), x1); //SUBREADprintf("CREATE TEMP FILE %s\n" , tmp_fname); global_context -> scRNA_barcode_batched_bins[x1]=fopen(tmp_fname, "wb"); pthread_spin_init(global_context -> scRNA_barcode_batched_locks+x1, PTHREAD_PROCESS_PRIVATE); } }else{ global_context -> do_scRNA_table = 0; global_context-> scRNA_cell_barcodes_array = NULL; global_context-> scRNA_sample_sheet_table = NULL; } global_context -> read_counters.unassigned_ambiguous=0; global_context -> read_counters.unassigned_nofeatures=0; global_context -> read_counters.unassigned_overlapping_length=0; global_context -> read_counters.unassigned_unmapped=0; global_context -> read_counters.unassigned_read_type=0; global_context -> read_counters.unassigned_singleton=0; global_context -> read_counters.unassigned_mappingquality=0; global_context -> read_counters.unassigned_fragmentlength=0; global_context -> read_counters.unassigned_chimericreads=0; global_context -> read_counters.unassigned_multimapping=0; global_context -> read_counters.unassigned_secondary=0; global_context -> read_counters.unassigned_junction_condition=0; global_context -> read_counters.unassigned_duplicate=0; global_context -> read_counters.assigned_reads=0; global_context -> GCcontent_table = HashTableCreate(20000); HashTableSetHashFunction(global_context -> GCcontent_table, HashTableStringHashFunction); HashTableSetDeallocationFunctions(global_context -> GCcontent_table, free, free); HashTableSetKeyComparisonFunction(global_context -> GCcontent_table, fc_strcmp_chro); if(annotation_file_screen_output) strcpy(global_context -> annotation_file_screen_output, annotation_file_screen_output); else global_context ->annotation_file_screen_output[0]=0; if(alias_file_name && alias_file_name[0]) { strcpy(global_context -> alias_file_name,alias_file_name); global_context -> BAM_chros_to_anno_table = load_alias_table(alias_file_name); } else global_context -> alias_file_name[0]=0; global_context -> read_details_path[0]=0; if(Rpath)strcpy(global_context -> read_details_path, Rpath); strcpy(global_context -> feature_name_column,feature_name_column); strcpy(global_context -> gene_id_column,gene_id_column); strcpy(global_context -> output_file_name, output_fname); global_context -> output_file_path[0]=0; for( x1 = strlen(output_fname)-1; x1 >= 0; x1 --){ if(output_fname[x1]=='/'){ memcpy(global_context -> output_file_path, output_fname, x1); global_context -> output_file_path[x1]=0; break; } } if(0 == global_context -> output_file_path[0]){ strcpy(global_context -> output_file_path, "."); } if(temp_dir == NULL)get_temp_dir_from_out(global_context -> temp_file_dir, output_fname); else strcpy(global_context -> temp_file_dir, temp_dir); //SUBREADprintf("OFPP:%s, OFNN:%s\n", global_context -> output_file_path, global_context -> output_file_name); global_context -> min_paired_end_distance = min_pe_dist; global_context -> max_paired_end_distance = max_pe_dist; global_context -> thread_number = threads; global_context -> line_length = line_length; } int fc_thread_start_threads(fc_thread_global_context_t * global_context, int et_exons, int * et_geneid, char ** et_chr, srInt_64 * et_start, srInt_64 * et_stop, unsigned char * et_strand, char * et_anno_chr_2ch, char ** et_anno_chrs, srInt_64 * et_anno_chr_heads, srInt_64 * et_bk_end_index, srInt_64 * et_bk_min_start, srInt_64 * et_bk_max_end, int read_length) { int xk1; global_context -> read_length = read_length; global_context -> is_unpaired_warning_shown = 0; global_context -> is_stake_warning_shown = 0; global_context -> is_read_too_long_to_SAM_BAM_shown = 0; if(global_context -> is_read_details_out) { char tmp_fname[MAX_FILE_NAME_LENGTH+20], *modified_fname; int i=0; char * applied_detail_path = global_context -> output_file_path; if(global_context -> read_details_path[0]) applied_detail_path = global_context -> read_details_path; if( global_context -> input_file_unique ){ sprintf(tmp_fname, "%s/%s.featureCounts%s", applied_detail_path, global_context -> input_file_short_name, global_context -> is_read_details_out == FILE_TYPE_BAM?".bam":(global_context -> is_read_details_out == FILE_TYPE_SAM?".sam":"")); global_context -> read_details_out_FP = f_subr_open(tmp_fname, "w"); //SUBREADprintf("FCSSF=%s\n", tmp_fname); } else { sprintf(tmp_fname, "%s.featureCounts%s", global_context -> raw_input_file_name, global_context -> is_read_details_out == FILE_TYPE_BAM?".bam":(global_context -> is_read_details_out == FILE_TYPE_SAM?".sam":"")); modified_fname = tmp_fname; while(modified_fname[0]=='/' || modified_fname[0]=='.' || modified_fname[0]=='\\'){ modified_fname ++; } while(modified_fname[i]){ if(modified_fname[i]=='\\' || modified_fname[i]=='/'||modified_fname[i]==' ')modified_fname[i]='.'; i++; } char tmp_fname2[MAX_FILE_NAME_LENGTH*2+100]; sprintf(tmp_fname2, "%s/%s", applied_detail_path, modified_fname); global_context -> read_details_out_FP = f_subr_open(tmp_fname2, "w"); //SUBREADprintf("FCSSF=%s\n", tmp_fname2); } if(global_context -> read_details_out_FP){ pthread_spin_init(&global_context -> read_details_out_lock, 1); }else{ SUBREADprintf("Unable to create file '%s'; the read assignment details are not written.\n", tmp_fname); } } else global_context -> read_details_out_FP = NULL; global_context -> redo = 0; global_context -> exontable_geneid = et_geneid; global_context -> exontable_chr = et_chr; global_context -> exontable_start = et_start; global_context -> exontable_stop = et_stop; global_context -> exontable_strand = (char *)et_strand; global_context -> exontable_anno_chr_2ch = et_anno_chr_2ch; global_context -> exontable_anno_chrs = et_anno_chrs; global_context -> exontable_anno_chr_heads = et_anno_chr_heads; global_context -> exontable_block_end_index = et_bk_end_index; global_context -> exontable_block_max_end = et_bk_max_end; global_context -> exontable_block_min_start = et_bk_min_start; global_context -> sambam_chro_table_items = 0; global_context -> sambam_chro_table = NULL; global_context -> thread_contexts = malloc(sizeof(fc_thread_thread_context_t) * global_context -> thread_number); for(xk1=0; xk1 thread_number; xk1++) { // printf("CHRR_MALLOC\n"); global_context -> thread_contexts[xk1].thread_id = xk1; global_context -> thread_contexts[xk1].chunk_read_ptr = 0; global_context -> thread_contexts[xk1].count_table = calloc(sizeof(read_count_type_t), et_exons); global_context -> thread_contexts[xk1].count_table_size = et_exons; global_context -> thread_contexts[xk1].nreads_mapped_to_exon = 0; global_context -> thread_contexts[xk1].all_reads = 0; global_context -> thread_contexts[xk1].chro_name_buff = malloc(CHROMOSOME_NAME_LENGTH); global_context -> thread_contexts[xk1].read_counters.assigned_reads = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_ambiguous = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_nofeatures = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_unmapped = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_singleton = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_read_type = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_mappingquality = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_fragmentlength = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_chimericreads = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_multimapping = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_secondary = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_junction_condition = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_overlapping_length = 0; global_context -> thread_contexts[xk1].read_counters.unassigned_duplicate = 0; global_context -> thread_contexts[xk1].read_details_buff_used = 0; global_context -> thread_contexts[xk1].hits_number_capacity = 300 ; global_context -> thread_contexts[xk1].hits_start_pos1 = malloc(sizeof(int)* global_context -> thread_contexts[xk1].hits_number_capacity); global_context -> thread_contexts[xk1].hits_start_pos2 = malloc(sizeof(int)* global_context -> thread_contexts[xk1].hits_number_capacity); global_context -> thread_contexts[xk1].hits_length1 = malloc(sizeof(short)* global_context -> thread_contexts[xk1].hits_number_capacity); global_context -> thread_contexts[xk1].hits_length2 = malloc(sizeof(short)* global_context -> thread_contexts[xk1].hits_number_capacity); global_context -> thread_contexts[xk1].hits_chro1 = malloc(sizeof(char*)* global_context -> thread_contexts[xk1].hits_number_capacity); global_context -> thread_contexts[xk1].hits_chro2 = malloc(sizeof(char*)* global_context -> thread_contexts[xk1].hits_number_capacity); global_context -> thread_contexts[xk1].hits_indices1 = malloc(sizeof(srInt_64)* global_context -> thread_contexts[xk1].hits_number_capacity); global_context -> thread_contexts[xk1].hits_indices2 = malloc(sizeof(srInt_64)* global_context -> thread_contexts[xk1].hits_number_capacity); global_context -> thread_contexts[xk1].scoring_buff_numbers = malloc(sizeof(int)* global_context -> thread_contexts[xk1].hits_number_capacity * 2); global_context -> thread_contexts[xk1].scoring_buff_flags = malloc(sizeof(int)* global_context -> thread_contexts[xk1].hits_number_capacity * 2); global_context -> thread_contexts[xk1].scoring_buff_overlappings = malloc(sizeof(int)* global_context -> thread_contexts[xk1].hits_number_capacity * 2); global_context -> thread_contexts[xk1].scoring_buff_exon_ids =malloc(sizeof(srInt_64)* global_context -> thread_contexts[xk1].hits_number_capacity * 2); if(global_context -> read_details_out_FP){ global_context -> thread_contexts[xk1].read_details_buff = malloc(70000 + 2 * MAX_FC_READ_LENGTH * 3); global_context -> thread_contexts[xk1].bam_compressed_buff = malloc(70000 + 2 * MAX_FC_READ_LENGTH * 3); } if(global_context -> need_calculate_overlap_len){ global_context -> thread_contexts[xk1].scoring_buff_gap_chros = malloc( sizeof(char *) * global_context -> thread_contexts[xk1].hits_number_capacity * 2 * global_context -> max_M *2); global_context -> thread_contexts[xk1].scoring_buff_gap_starts = malloc( sizeof(unsigned int ) * global_context -> thread_contexts[xk1].hits_number_capacity * 2 * global_context -> max_M *2); global_context -> thread_contexts[xk1].scoring_buff_gap_lengths = malloc( sizeof(unsigned short) * global_context -> thread_contexts[xk1].hits_number_capacity * 2 * global_context -> max_M *2); } else global_context -> thread_contexts[xk1].scoring_buff_gap_chros = NULL; if(global_context -> do_junction_counting) { global_context -> thread_contexts[xk1].junction_counting_table = HashTableCreate(131317); HashTableSetHashFunction(global_context -> thread_contexts[xk1].junction_counting_table,HashTableStringHashFunction); HashTableSetDeallocationFunctions(global_context -> thread_contexts[xk1].junction_counting_table, free, NULL); HashTableSetKeyComparisonFunction(global_context -> thread_contexts[xk1].junction_counting_table, fc_strcmp_chro); global_context -> thread_contexts[xk1].splicing_point_table = HashTableCreate(131317); HashTableSetHashFunction(global_context -> thread_contexts[xk1].splicing_point_table,HashTableStringHashFunction); HashTableSetDeallocationFunctions(global_context -> thread_contexts[xk1].splicing_point_table, free, NULL); HashTableSetKeyComparisonFunction(global_context -> thread_contexts[xk1].splicing_point_table, fc_strcmp_chro); } if(global_context -> assign_reads_to_RG){ global_context -> thread_contexts[xk1].RG_table = HashTableCreate(97); HashTableSetHashFunction(global_context -> thread_contexts[xk1].RG_table,HashTableStringHashFunction); HashTableSetDeallocationFunctions(global_context -> thread_contexts[xk1].RG_table, free, disallocate_RG_tables); HashTableSetKeyComparisonFunction(global_context -> thread_contexts[xk1].RG_table, fc_strcmp_chro); } if(global_context -> do_scRNA_table){ global_context -> thread_contexts[xk1].scRNA_reads_per_sample = calloc(sizeof(srInt_64),global_context-> scRNA_sample_sheet_table ->numOfElements); global_context -> thread_contexts[xk1].scRNA_mapped_reads_per_sample = calloc(sizeof(srInt_64),global_context-> scRNA_sample_sheet_table ->numOfElements); global_context -> thread_contexts[xk1].scRNA_assigned_reads_per_sample = calloc(sizeof(srInt_64),global_context-> scRNA_sample_sheet_table ->numOfElements); global_context -> thread_contexts[xk1].scRNA_sample_bc_tables = malloc(sizeof(HashTable*) * global_context -> scRNA_sample_id_to_name -> numOfElements); global_context -> thread_contexts[xk1].scRNA_registered_UMI_table = StringTableCreate(100000); HashTableSetDeallocationFunctions(global_context -> thread_contexts[xk1].scRNA_registered_UMI_table, free, NULL); int xk2; for(xk2 = 0; xk2 < global_context -> scRNA_sample_id_to_name -> numOfElements; xk2++){ HashTable *al = HashTableCreate(2000); HashTableSetDeallocationFunctions(al, NULL, (void (*)(void*))HashTableDestroy); //SUBREADprintf("PUSH ARR for THR %d XK2 %d\n", xk1, xk2); global_context -> thread_contexts[xk1].scRNA_sample_bc_tables[xk2] = al; } global_context -> thread_contexts[xk1].scRNA_pooled_reads=0; global_context -> thread_contexts[xk1].scRNA_has_valid_sample_index =0; global_context -> thread_contexts[xk1].scRNA_has_valid_cell_barcode =0; } if(!global_context -> thread_contexts[xk1].count_table) return 1; } char new_fn[MAX_FILE_NAME_LENGTH+10]; char MAC_or_random[13]; mac_or_rand_str(MAC_or_random); char rand_prefix[MAX_FILE_NAME_LENGTH+100]; sprintf(rand_prefix, "%s/temp-core-%06u-%s.sam", global_context -> temp_file_dir, getpid(), MAC_or_random); if(global_context -> use_stdin_file) sprintf(new_fn, "<%s", global_context -> input_file_name ); else sprintf(new_fn, "%s", global_context -> input_file_name ); int pairer_error = SAM_pairer_create(&global_context -> read_pairer, global_context -> thread_number , global_context -> max_BAM_header_size/1024/1024+2, !global_context-> is_SAM_file, !( global_context -> is_read_details_out == FILE_TYPE_BAM ||global_context -> is_read_details_out == FILE_TYPE_SAM ) , !global_context -> is_paired_end_mode_assign, global_context ->is_paired_end_mode_assign && global_context -> do_not_sort, global_context -> assign_reads_to_RG ,0, new_fn, process_pairer_reset, process_pairer_header, process_pairer_output, rand_prefix, global_context, global_context -> long_read_minimum_length); return pairer_error; } void fc_thread_destroy_thread_context(fc_thread_global_context_t * global_context) { int xk1; if(global_context -> is_read_details_out)for(xk1=0; xk1 thread_number; xk1++) write_read_detailed_remainder(global_context, global_context -> thread_contexts+xk1); if(global_context -> is_read_details_out) { if( global_context -> is_read_details_out == FILE_TYPE_BAM ){ char bam_tail_block[1000]; int tail_size = compress_read_detail_BAM( global_context, global_context -> thread_contexts, 0,0,bam_tail_block); assert(tail_size > 0); //SUBREADprintf("TAIL SIZE=%d\n", tail_size); fwrite(bam_tail_block, 1, tail_size, global_context -> read_details_out_FP); } fclose(global_context -> read_details_out_FP); global_context -> read_details_out_FP = NULL; pthread_spin_destroy(&global_context -> read_details_out_lock); } for(xk1=0; xk1 thread_number; xk1++) { //printf("CHRR_FREE\n"); free(global_context -> thread_contexts[xk1].count_table); free(global_context -> thread_contexts[xk1].chro_name_buff); free(global_context -> thread_contexts[xk1].hits_start_pos1); free(global_context -> thread_contexts[xk1].hits_start_pos2); free(global_context -> thread_contexts[xk1].hits_length1); free(global_context -> thread_contexts[xk1].hits_length2); free(global_context -> thread_contexts[xk1].hits_chro1); free(global_context -> thread_contexts[xk1].hits_chro2); free(global_context -> thread_contexts[xk1].hits_indices1); free(global_context -> thread_contexts[xk1].hits_indices2); free(global_context -> thread_contexts[xk1].scoring_buff_numbers); free(global_context -> thread_contexts[xk1].scoring_buff_flags); free(global_context -> thread_contexts[xk1].scoring_buff_overlappings); free(global_context -> thread_contexts[xk1].scoring_buff_exon_ids); if(global_context -> thread_contexts[xk1].scoring_buff_gap_chros){ free(global_context -> thread_contexts[xk1].scoring_buff_gap_chros); free(global_context -> thread_contexts[xk1].scoring_buff_gap_starts); free(global_context -> thread_contexts[xk1].scoring_buff_gap_lengths); } if(global_context -> do_junction_counting){ HashTableDestroy(global_context -> thread_contexts[xk1].junction_counting_table); HashTableDestroy(global_context -> thread_contexts[xk1].splicing_point_table); } if(global_context -> assign_reads_to_RG) HashTableDestroy(global_context -> thread_contexts[xk1].RG_table); if(global_context -> is_read_details_out ){ free(global_context -> thread_contexts[xk1].read_details_buff); free(global_context -> thread_contexts[xk1].bam_compressed_buff); } if(global_context -> do_scRNA_table){ int xk2; for(xk2=0;xk2< global_context -> scRNA_sample_id_to_name -> numOfElements;xk2++) { HashTableDestroy(global_context -> thread_contexts[xk1].scRNA_sample_bc_tables[xk2]); } //HashTableDestroy(global_context -> scRNA_sample_BAM_writers); free(global_context -> thread_contexts[xk1].scRNA_reads_per_sample); free(global_context -> thread_contexts[xk1].scRNA_mapped_reads_per_sample); free(global_context -> thread_contexts[xk1].scRNA_assigned_reads_per_sample); free(global_context -> thread_contexts[xk1].scRNA_sample_bc_tables); HashTableDestroy(global_context -> thread_contexts[xk1].scRNA_registered_UMI_table); } } free(global_context -> thread_contexts); } void fc_thread_wait_threads(fc_thread_global_context_t * global_context) { int assign_ret = SAM_pairer_run(&global_context -> read_pairer); if(0 && assign_ret){ print_in_box(80,0,0,""); print_in_box(80,0,0," format error found in this file."); } global_context -> is_input_bad_format |= assign_ret; } void merge_repeated_extra_columns(char * cols){ if(cols[0]!=';')return; int is_diff = 0; int seglen = -1, laststart = 0; int xx; for(xx=0; ; xx++){ if(cols[xx]==';' || cols[xx]==0){ if(seglen <0)seglen = xx -1; else{ is_diff = (xx-laststart != seglen )|| memcmp(cols+laststart, cols+1, seglen); if(is_diff)break; } laststart = xx+1; } if(cols[xx]==0)break; } if(seglen>0 && !is_diff) cols[seglen+1]=0; } void BUFstrcat(char * targ, char * src, char ** buf){ int srclen = strlen(src); if( (*buf) == NULL){ (*buf) = targ; } memcpy((*buf), src, srclen); (*buf) += srclen; (**buf) = 0; } void fc_write_final_gene_results(fc_thread_global_context_t * global_context, int * et_geneid, char ** et_chr, srInt_64 * et_start, srInt_64 * et_stop, unsigned char * et_strand, char ** et_extra_columns, const char * out_file, int features, ArrayList * column_numbers, ArrayList * column_names, fc_feature_info_t * loaded_features, int header_out) { int xk1,xk4; int genes = global_context -> gene_name_table -> numOfElements; read_count_type_t *gene_columns; FILE * fp_out = f_subr_open(out_file,"w"); if(!fp_out){ SUBREADprintf("Failed to create file %s\n", out_file); return; } if(header_out) { fprintf(fp_out, "# Program:featureCounts v%s", SUBREAD_VERSION); if(global_context->cmd_rebuilt) fprintf(fp_out, "; Command:%s", global_context->cmd_rebuilt); fprintf(fp_out, "\n"); } int i_files; fprintf(fp_out,"Geneid\t%sChr\tStart\tEnd\tStrand\tLength%s%s", global_context->do_detection_call?"GCfraction\t":"", global_context -> reported_extra_columns?"\t":"", global_context -> reported_extra_columns?global_context -> reported_extra_columns:""); for(i_files=0; i_filesnumOfElements; i_files++) { char * next_fn = ArrayListGet(column_names, i_files); fprintf(fp_out,"\t%s", global_context -> use_stdin_file?"STDIN":next_fn); } fprintf(fp_out,"\n"); gene_columns = calloc(sizeof(read_count_type_t) , genes * column_names->numOfElements); unsigned int * gene_exons_number = calloc(sizeof(unsigned int) , genes); unsigned int * gene_exons_pointer = calloc(sizeof(unsigned int) , genes); unsigned int * gene_exons_start = malloc(sizeof(unsigned int) * features); unsigned int * gene_exons_end = malloc(sizeof(unsigned int) * features); char ** gene_exons_chr = malloc(sizeof(char *) * features); char ** gene_exons_extra_columns = malloc(sizeof(char *) * features); char * gene_exons_strand = malloc(features); for(xk1 = 0; xk1 < features; xk1++) { int gene_id = et_geneid[xk1]; gene_exons_number[gene_id]++; } unsigned int accumulative_no = 0; unsigned longest_gene_exons = 0; for(xk1 = 0 ; xk1 < genes; xk1++) { unsigned int this_gene_exons = gene_exons_number[xk1]; longest_gene_exons = max(longest_gene_exons, this_gene_exons); gene_exons_number[xk1] = accumulative_no; accumulative_no += this_gene_exons; } for(xk1 = 0; xk1 < features; xk1++) { int gene_id = et_geneid[xk1]; int gene_write_ptr = gene_exons_number[gene_id] + gene_exons_pointer[gene_id]; gene_exons_chr[gene_write_ptr] = et_chr[xk1]; gene_exons_start[gene_write_ptr] = et_start[xk1]; gene_exons_end[gene_write_ptr] = et_stop[xk1]; gene_exons_strand[gene_write_ptr] = et_strand[xk1]; if(global_context -> reported_extra_columns!=NULL)gene_exons_extra_columns[gene_write_ptr] = et_extra_columns[xk1]; gene_exons_pointer[gene_id]++; } for(xk1 = 0; xk1 < features; xk1++) { int gene_id = et_geneid[xk1], k_noempty = 0; for(i_files=0;i_files < column_names->numOfElements; i_files++) { srInt_64 * this_col = ArrayListGet(column_numbers, i_files); gene_columns[gene_id * column_names->numOfElements + k_noempty ] += this_col[xk1]; k_noempty++; } } char *is_occupied = malloc(longest_gene_exons); unsigned int * input_start_stop_list = malloc(longest_gene_exons * sizeof(int) * 2); unsigned int * output_start_stop_list = malloc(longest_gene_exons * sizeof(int) * 2); int disk_is_full = 0; char * out_chr_list = malloc(longest_gene_exons * (1+global_context -> longest_chro_name) + 1), * tmp_chr_list = NULL; char * out_start_list = malloc(11 * longest_gene_exons + 1), * tmp_start_list = NULL; char * out_end_list = malloc(11 * longest_gene_exons + 1), * tmp_end_list = NULL; char * out_strand_list = malloc(2 * longest_gene_exons + 1), * tmp_strand_list = NULL; char * out_extra_columns[MAX_EXTRA_COLS]; int out_extra_column_size[MAX_EXTRA_COLS]; int total_extra_cols = 0; if(global_context -> reported_extra_columns){ char * tnamep = global_context -> reported_extra_columns; total_extra_cols =1; while(*(tnamep++)) total_extra_cols += '\t' ==(*tnamep); for(xk1=0; xk1 gene_name_array [xk1]; for(xk2=0; xk2 reported_extra_columns && (xk3==xk2 || (0 == is_occupied[xk3] && strcmp(matched_chr, gene_exons_chr[xk3+gene_exons_number[xk1]])==0 && matched_strand == gene_exons_strand[xk3 + gene_exons_number[xk1]] ))){ char * this_col_ptr = NULL; char * this_col = strtok_r(gene_exons_extra_columns[xk3+gene_exons_number[xk1]], "\t", &this_col_ptr); for(xk4 = 0; xk4 < total_extra_cols; xk4++){ int exlen = strlen( this_col), ollen = strlen(out_extra_columns[xk4]); if(ollen + exlen +2 > out_extra_column_size[xk4]){ out_extra_column_size[xk4] = max(ollen + exlen +2, out_extra_column_size[xk4]); out_extra_columns[xk4] = realloc(out_extra_columns[xk4], out_extra_column_size[xk4]); } sprintf(out_extra_columns[xk4]+ollen,";%s", this_col); this_col = strtok_r(NULL, "\t", &this_col_ptr); } } if(xk3==xk2)continue; if((!is_occupied[xk3]) && strcmp(matched_chr, gene_exons_chr[xk3+gene_exons_number[xk1]])==0 && matched_strand == gene_exons_strand[xk3 + gene_exons_number[xk1]]) { is_occupied[xk3]=1; input_start_stop_list[gap_merge_ptr*2] = gene_exons_start[xk3+gene_exons_number[xk1]]; input_start_stop_list[gap_merge_ptr*2+1] = gene_exons_end[xk3+gene_exons_number[xk1]]+1; gap_merge_ptr++; } } { int merged_gaps = mergeIntervals(input_start_stop_list, output_start_stop_list, gap_merge_ptr); for(xk3=0; xk3 do_detection_call){ QCcontent = HashTableGet(global_context -> GCcontent_table, gene_symbol); QCtab = "\t"; if(!QCcontent)QCcontent="nan"; } int wlen = fprintf(fp_out, "%s\t%s%s%s\t%s\t%s\t%s\t%d", gene_symbol, QCcontent, QCtab, out_chr_list, out_start_list, out_end_list, out_strand_list, gene_nonoverlap_len); for(xk4 = 0; xk4numOfElements; i_files++) { read_count_type_t longlong_res = 0; double double_res = 0; int is_double_number = calc_float_fraction(gene_columns[i_files + column_names->numOfElements*xk1], &longlong_res, &double_res); if(is_double_number){ fprintf(fp_out,"\t%.2f", double_res); }else{ #ifdef __MINGW32__ fprintf(fp_out,"\t%I64u", (srInt_64)longlong_res); #else fprintf(fp_out,"\t%lld", (srInt_64)longlong_res); #endif } } fprintf(fp_out,"\n"); if(wlen < 6)disk_is_full = 1; } for(xk1=0; xk1numOfElements; i_files++) { char * next_fn = ArrayListGet(column_names, i_files); fprintf(fp_out,"\t%s", global_context -> use_stdin_file?"STDIN":next_fn); } fprintf(fp_out,"\n"); char * keys [] ={ "Assigned" , "Unassigned_Unmapped", "Unassigned_Read_Type", "Unassigned_Singleton", "Unassigned_MappingQuality", "Unassigned_Chimera", "Unassigned_FragmentLength", "Unassigned_Duplicate", "Unassigned_MultiMapping" , "Unassigned_Secondary", (global_context->is_split_or_exonic_only == 2)?"Unassigned_Split":"Unassigned_NonSplit", "Unassigned_NoFeatures", "Unassigned_Overlapping_Length", "Unassigned_Ambiguity"}; for(xk1=0; xk1<14; xk1++) { fprintf(fp_out,"%s", keys[xk1]); for(i_files = 0; i_files < column_names->numOfElements; i_files ++) { srInt_64 * array_0 = ArrayListGet(read_counters,i_files); srInt_64 * cntr = array_0 + xk1; #ifdef __MINGW32__ fprintf(fp_out,"\t%I64u", (srInt_64)*cntr); #else fprintf(fp_out,"\t%lld", (srInt_64)*cntr); #endif } int wlen = fprintf(fp_out,"\n"); if(wlen < 1)disk_is_full = 1; } fclose(fp_out); if(disk_is_full){ SUBREADprintf("ERROR: disk is full; the count file cannot be generated.\n"); unlink(out_file); } } void fc_write_final_results(fc_thread_global_context_t * global_context, const char * out_file, int features, ArrayList* column_numbers, ArrayList * column_names,fc_feature_info_t * loaded_features, int header_out) { /* save the results */ FILE * fp_out; int i, i_files = 0, disk_is_full =0; fp_out = f_subr_open(out_file,"w"); if(!fp_out){ SUBREADprintf("Failed to create file %s\n", out_file); return; } if(header_out) { fprintf(fp_out, "# Program:featureCounts v%s", SUBREAD_VERSION); if(global_context->cmd_rebuilt) fprintf(fp_out, "; Command:%s", global_context->cmd_rebuilt); fprintf(fp_out, "\n"); } char * next_fn; fprintf(fp_out,"Geneid\tChr\tStart\tEnd\tStrand\tLength"); if(global_context -> reported_extra_columns)fprintf(fp_out,"\t%s", global_context -> reported_extra_columns); for(i_files = 0; i_files < column_names -> numOfElements; i_files++){ next_fn = ArrayListGet(column_names, i_files); fprintf(fp_out,"\t%s", global_context -> use_stdin_file?"STDIN":next_fn); } fprintf(fp_out,"\n"); for(i=0;i unistr_buffer_space + loaded_features[i].feature_name_pos, global_context -> unistr_buffer_space + loaded_features[i].feature_name_pos + loaded_features[i].chro_name_pos_delta, loaded_features[i].start, loaded_features[i].end, loaded_features[i].is_negative_strand == 1?'-':( loaded_features[i].is_negative_strand == 0? '+':'.'), loaded_features[i].end-loaded_features[i].start+1, global_context -> reported_extra_columns ?"\t":"", global_context -> reported_extra_columns ?loaded_features[i].extra_columns:""); for(i_files=0; i_files < column_names -> numOfElements; i_files++) { srInt_64 * this_list = ArrayListGet(column_numbers, i_files); int sorted_exon_no = loaded_features[i].sorted_order; srInt_64 count_frac_raw = this_list[sorted_exon_no], longlong_res = 0; double double_res = 0; int is_double_number = calc_float_fraction(count_frac_raw, &longlong_res, &double_res); if(is_double_number){ fprintf(fp_out,"\t%.2f", double_res); }else{ #ifdef __MINGW32__ fprintf(fp_out,"\t%I64d", (srInt_64)longlong_res); #else fprintf(fp_out,"\t%lld", (srInt_64)longlong_res); #endif } } int wlen = fprintf(fp_out,"\n"); if(wlen < 1)disk_is_full = 1; } fclose(fp_out); if(disk_is_full){ SUBREADprintf("ERROR: disk is full; unable to write into the output file.\n"); unlink(out_file); } } static struct option long_options[] = { {"primary",no_argument, 0, 0}, {"readShiftSize", required_argument, 0, 0}, {"readShiftType", required_argument, 0, 0}, {"readExtension5", required_argument, 0, 0}, {"readExtension5", required_argument, 0, 0}, {"readExtension3", required_argument, 0, 0}, {"read2pos", required_argument, 0, 0}, {"minOverlap", required_argument, 0, 0}, {"fracOverlap", required_argument, 0, 0}, {"nonOverlap", required_argument, 0, 0}, {"nonOverlapFeature", required_argument, 0, 0}, {"fracOverlapFeature", required_argument, 0, 0}, {"splitOnly", no_argument, 0, 0}, {"nonSplitOnly", no_argument, 0, 0}, {"debugCommand", required_argument, 0, 0}, {"ignoreDup", no_argument, 0, 0}, {"donotsort", no_argument, 0, 0}, {"restrictedlyNoOverlap", no_argument, 0, 0}, {"fraction", no_argument, 0, 0}, {"order", required_argument, 0, 'S'}, {"genome", required_argument, 0, 'G'}, {"maxMOp", required_argument, 0, 0}, {"tmpDir", required_argument, 0, 0}, {"extraAttributes", required_argument, 0, 0}, {"largestOverlap", no_argument, 0,0}, {"countReadPairs", no_argument, 0, 0}, {"byReadGroup", no_argument, 0,0}, {"verbose", no_argument, 0,0}, {"detectionCall", no_argument, 0,0}, {"Rpath", required_argument, 0, 0}, {"scSampleSheet", required_argument, 0, 0}, {"scInputMode", required_argument, 0, 0}, {"scCellBarcodeFile", required_argument, 0, 0}, {0, 0, 0, 0} }; void print_usage() { SUBREADprintf("\nVersion %s\n\n", SUBREAD_VERSION); SUBREADputs("Usage: featureCounts [options] -a -o input_file1 [input_file2] ... \n"); SUBREADputs("## Mandatory arguments:"); SUBREADputs(""); SUBREADputs(" -a Name of an annotation file. GTF/GFF format by default. See"); SUBREADputs(" -F option for more format information. Inbuilt annotations"); SUBREADputs(" (SAF format) is available in 'annotation' directory of the"); SUBREADputs(" package. Gzipped file is also accepted."); SUBREADputs(""); SUBREADputs(" -o Name of output file including read counts. A separate file"); SUBREADputs(" including summary statistics of counting results is also"); SUBREADputs(" included in the output ('.summary'). Both files"); SUBREADputs(" are in tab delimited format."); SUBREADputs(""); SUBREADputs(" input_file1 [input_file2] ... A list of SAM or BAM format files. They can be"); SUBREADputs(" either name or location sorted. If no files provided,"); SUBREADputs(" input is expected. Location-sorted paired-end reads"); SUBREADputs(" are automatically sorted by read names."); SUBREADputs(""); SUBREADputs("## Optional arguments:"); SUBREADputs("# Annotation"); SUBREADputs(""); SUBREADputs(" -F Specify format of the provided annotation file. Acceptable"); SUBREADputs(" formats include 'GTF' (or compatible GFF format) and"); SUBREADputs(" 'SAF'. 'GTF' by default. For SAF format, please refer to"); SUBREADputs(" Users Guide."); SUBREADputs(""); SUBREADputs(" -t Specify feature type(s) in a GTF annotation. If multiple"); SUBREADputs(" types are provided, they should be separated by ',' with"); SUBREADputs(" no space in between. 'exon' by default. Rows in the"); SUBREADputs(" annotation with a matched feature will be extracted and"); SUBREADputs(" used for read mapping. "); SUBREADputs(""); SUBREADputs(" -g Specify attribute type in GTF annotation. 'gene_id' by "); SUBREADputs(" default. Meta-features used for read counting will be "); SUBREADputs(" extracted from annotation using the provided value."); SUBREADputs(""); SUBREADputs(" --extraAttributes Extract extra attribute types from the provided GTF"); SUBREADputs(" annotation and include them in the counting output. These"); SUBREADputs(" attribute types will not be used to group features. If"); SUBREADputs(" more than one attribute type is provided they should be"); SUBREADputs(" separated by comma."); SUBREADputs(""); SUBREADputs(" -A Provide a chromosome name alias file to match chr names in"); SUBREADputs(" annotation with those in the reads. This should be a two-"); SUBREADputs(" column comma-delimited text file. Its first column should"); SUBREADputs(" include chr names in the annotation and its second column"); SUBREADputs(" should include chr names in the reads. Chr names are case"); SUBREADputs(" sensitive. No column header should be included in the"); SUBREADputs(" file."); SUBREADputs(""); SUBREADputs("# Level of summarization"); SUBREADputs(""); SUBREADputs(" -f Perform read counting at feature level (eg. counting "); SUBREADputs(" reads for exons rather than genes)."); SUBREADputs(""); SUBREADputs("# Overlap between reads and features"); SUBREADputs(""); SUBREADputs(" -O Assign reads to all their overlapping meta-features (or "); SUBREADputs(" features if -f is specified)."); SUBREADputs(""); SUBREADputs(" --minOverlap Minimum number of overlapping bases in a read that is"); SUBREADputs(" required for read assignment. 1 by default. Number of"); SUBREADputs(" overlapping bases is counted from both reads if paired"); SUBREADputs(" end. If a negative value is provided, then a gap of up"); SUBREADputs(" to specified size will be allowed between read and the"); SUBREADputs(" feature that the read is assigned to."); SUBREADputs(""); SUBREADputs(" --fracOverlap Minimum fraction of overlapping bases in a read that is"); SUBREADputs(" required for read assignment. Value should be within range"); SUBREADputs(" [0,1]. 0 by default. Number of overlapping bases is"); SUBREADputs(" counted from both reads if paired end. Both this option"); SUBREADputs(" and '--minOverlap' option need to be satisfied for read"); SUBREADputs(" assignment."); SUBREADputs(""); SUBREADputs(" --fracOverlapFeature Minimum fraction of overlapping bases in a"); SUBREADputs(" feature that is required for read assignment. Value"); SUBREADputs(" should be within range [0,1]. 0 by default."); SUBREADputs(""); SUBREADputs(" --largestOverlap Assign reads to a meta-feature/feature that has the "); SUBREADputs(" largest number of overlapping bases."); SUBREADputs(""); SUBREADputs(" --nonOverlap Maximum number of non-overlapping bases in a read (or a"); SUBREADputs(" read pair) that is allowed when being assigned to a"); SUBREADputs(" feature. No limit is set by default."); SUBREADputs(""); SUBREADputs(" --nonOverlapFeature Maximum number of non-overlapping bases in a feature"); SUBREADputs(" that is allowed in read assignment. No limit is set by"); SUBREADputs(" default."); SUBREADputs(""); SUBREADputs(" --readExtension5 Reads are extended upstream by bases from their"); SUBREADputs(" 5' end."); SUBREADputs(""); SUBREADputs(" --readExtension3 Reads are extended upstream by bases from their"); SUBREADputs(" 3' end."); SUBREADputs(""); SUBREADputs(" --read2pos <5:3> Reduce reads to their 5' most base or 3' most base. Read"); SUBREADputs(" counting is then performed based on the single base the "); SUBREADputs(" read is reduced to."); SUBREADputs(""); SUBREADputs("# Multi-mapping reads"); SUBREADputs(""); SUBREADputs(" -M Multi-mapping reads will also be counted. For a multi-"); SUBREADputs(" mapping read, all its reported alignments will be "); SUBREADputs(" counted. The 'NH' tag in BAM/SAM input is used to detect "); SUBREADputs(" multi-mapping reads."); SUBREADputs(""); SUBREADputs("# Fractional counting"); SUBREADputs(""); SUBREADputs(" --fraction Assign fractional counts to features. This option must"); SUBREADputs(" be used together with '-M' or '-O' or both. When '-M' is"); SUBREADputs(" specified, each reported alignment from a multi-mapping"); SUBREADputs(" read (identified via 'NH' tag) will carry a fractional"); SUBREADputs(" count of 1/x, instead of 1 (one), where x is the total"); SUBREADputs(" number of alignments reported for the same read. When '-O'"); SUBREADputs(" is specified, each overlapping feature will receive a"); SUBREADputs(" fractional count of 1/y, where y is the total number of"); SUBREADputs(" features overlapping with the read. When both '-M' and"); SUBREADputs(" '-O' are specified, each alignment will carry a fractional"); SUBREADputs(" count of 1/(x*y)."); SUBREADputs(""); SUBREADputs("# Read filtering"); SUBREADputs(""); SUBREADputs(" -Q The minimum mapping quality score a read must satisfy in"); SUBREADputs(" order to be counted. For paired-end reads, at least one"); SUBREADputs(" end should satisfy this criteria. 0 by default."); SUBREADputs(""); SUBREADputs(" --splitOnly Count split alignments only (ie. alignments with CIGAR"); SUBREADputs(" string containing 'N'). An example of split alignments is"); SUBREADputs(" exon-spanning reads in RNA-seq data."); SUBREADputs(""); SUBREADputs(" --nonSplitOnly If specified, only non-split alignments (CIGAR strings do"); SUBREADputs(" not contain letter 'N') will be counted. All the other"); SUBREADputs(" alignments will be ignored."); SUBREADputs(""); SUBREADputs(" --primary Count primary alignments only. Primary alignments are "); SUBREADputs(" identified using bit 0x100 in SAM/BAM FLAG field."); SUBREADputs(""); SUBREADputs(" --ignoreDup Ignore duplicate reads in read counting. Duplicate reads "); SUBREADputs(" are identified using bit Ox400 in BAM/SAM FLAG field. The "); SUBREADputs(" whole read pair is ignored if one of the reads is a "); SUBREADputs(" duplicate read for paired end data."); SUBREADputs(""); SUBREADputs("# Strandness"); SUBREADputs(""); SUBREADputs(" -s Perform strand-specific read counting. A single integer"); SUBREADputs(" value (applied to all input files) or a string of comma-"); SUBREADputs(" separated values (applied to each corresponding input"); SUBREADputs(" file) should be provided. Possible values include:"); SUBREADputs(" 0 (unstranded), 1 (stranded) and 2 (reversely stranded)."); SUBREADputs(" Default value is 0 (ie. unstranded read counting carried"); SUBREADputs(" out for all input files)."); SUBREADputs(""); SUBREADputs("# Exon-exon junctions"); SUBREADputs(""); SUBREADputs(" -J Count number of reads supporting each exon-exon junction."); SUBREADputs(" Junctions were identified from those exon-spanning reads"); SUBREADputs(" in the input (containing 'N' in CIGAR string). Counting"); SUBREADputs(" results are saved to a file named '.jcounts'"); SUBREADputs(""); SUBREADputs(" -G Provide the name of a FASTA-format file that contains the"); SUBREADputs(" reference sequences used in read mapping that produced the"); SUBREADputs(" provided SAM/BAM files. This optional argument can be used"); SUBREADputs(" with '-J' option to improve read counting for junctions."); SUBREADputs(""); SUBREADputs("# Parameters specific to paired end reads"); SUBREADputs(""); SUBREADputs(" -p If specified, libraries are assumed to contain paired-end"); SUBREADputs(" reads. For any library that contains paired-end reads, the"); SUBREADputs(" 'countReadPairs' parameter controls if read pairs or reads"); SUBREADputs(" should be counted."); SUBREADputs(""); SUBREADputs(" --countReadPairs If specified, fragments (or templates) will be counted"); SUBREADputs(" instead of reads. This option is only applicable for"); SUBREADputs(" paired-end reads. For single-end data, it is ignored."); SUBREADputs(""); SUBREADputs(" -B Only count read pairs that have both ends aligned."); SUBREADputs(""); SUBREADputs(" -P Check validity of paired-end distance when counting read "); SUBREADputs(" pairs. Use -d and -D to set thresholds."); SUBREADputs(""); SUBREADputs(" -d Minimum fragment/template length, 50 by default."); SUBREADputs(""); SUBREADputs(" -D Maximum fragment/template length, 600 by default."); SUBREADputs(""); SUBREADputs(" -C Do not count read pairs that have their two ends mapping "); SUBREADputs(" to different chromosomes or mapping to same chromosome "); SUBREADputs(" but on different strands."); SUBREADputs(""); SUBREADputs(" --donotsort Do not sort reads in BAM/SAM input. Note that reads from "); SUBREADputs(" the same pair are required to be located next to each "); SUBREADputs(" other in the input."); SUBREADputs(""); SUBREADputs("# Number of CPU threads"); SUBREADputs(""); SUBREADputs(" -T Number of the threads. 1 by default."); SUBREADputs(""); SUBREADputs("# Read groups"); SUBREADputs(""); SUBREADputs(" --byReadGroup Assign reads by read group. \"RG\" tag is required to be"); SUBREADputs(" present in the input BAM/SAM files."); SUBREADputs(" "); SUBREADputs(""); SUBREADputs("# Long reads"); SUBREADputs(""); SUBREADputs(" -L Count long reads such as Nanopore and PacBio reads. Long"); SUBREADputs(" read counting can only run in one thread and only reads"); SUBREADputs(" (not read-pairs) can be counted. There is no limitation on"); SUBREADputs(" the number of 'M' operations allowed in a CIGAR string in"); SUBREADputs(" long read counting."); SUBREADputs(""); SUBREADputs("# Assignment results for each read"); SUBREADputs(""); SUBREADputs(" -R Output detailed assignment results for each read or read-"); SUBREADputs(" pair. Results are saved to a file that is in one of the"); SUBREADputs(" following formats: CORE, SAM and BAM. See Users Guide for"); SUBREADputs(" more info about these formats."); SUBREADputs(""); SUBREADputs(" --Rpath Specify a directory to save the detailed assignment"); SUBREADputs(" results. If unspecified, the directory where counting"); SUBREADputs(" results are saved is used."); SUBREADputs(""); SUBREADputs("# Miscellaneous"); SUBREADputs(""); SUBREADputs(" --tmpDir Directory under which intermediate files are saved (later"); SUBREADputs(" removed). By default, intermediate files will be saved to"); SUBREADputs(" the directory specified in '-o' argument."); SUBREADputs(""); SUBREADputs(" --maxMOp Maximum number of 'M' operations allowed in a CIGAR"); SUBREADputs(" string. 10 by default. Both 'X' and '=' are treated as 'M'"); SUBREADputs(" and adjacent 'M' operations are merged in the CIGAR"); SUBREADputs(" string."); SUBREADputs(""); SUBREADputs(" --verbose Output verbose information for debugging, such as un-"); SUBREADputs(" matched chromosome/contig names."); SUBREADputs(""); SUBREADputs(" -v Output version of the program."); SUBREADputs(""); } int junckey_sort_compare(void * inptr, int i, int j){ char ** inp = (char **) inptr; int x1; int chrI=-1, chrJ=-1; if(atoi(inp[i])>0) chrI = atoi(inp[i]); if(atoi(inp[j])>0) chrJ = atoi(inp[j]); if(inp[i][0]=='X' && !isdigit(inp[i][1])&& !isalpha(inp[i][1])) chrI = 90; if(inp[i][0]=='Y' && !isdigit(inp[i][1])&& !isalpha(inp[i][1])) chrI = 91; if(inp[i][0]=='M' && !isdigit(inp[i][1])&& !isalpha(inp[i][1])) chrI = 99; if(inp[j][0]=='X' && !isdigit(inp[j][1])&& !isalpha(inp[j][1])) chrJ = 90; if(inp[j][0]=='Y' && !isdigit(inp[j][1])&& !isalpha(inp[j][1])) chrJ = 91; if(inp[j][0]=='M' && !isdigit(inp[j][1])&& !isalpha(inp[j][1])) chrJ = 99; if(memcmp(inp[i], "chr", 3)==0){ chrI=atoi(inp[i]+3); if(0 == chrI && inp[i][3] == 'X') chrI = 90; if(0 == chrI && inp[i][3] == 'Y') chrI = 91; if(0 == chrI && inp[i][3] == 'M') chrI = 99; } if(memcmp(inp[j], "chr", 3)==0){ chrJ=atoi(inp[j]+3); if(0 == chrJ && inp[j][3] == 'X') chrJ = 90; if(0 == chrJ && inp[j][3] == 'Y') chrJ = 91; if(0 == chrJ && inp[j][3] == 'M') chrJ = 99; } int len_I_long = 9; for(x1 = 0 ; x1 < FEATURE_NAME_LENGTH + 15 ; x1++){ int c1 = inp[i][x1]; int c2 = inp[j][x1]; if(c1 == '\t' && c2 != '\t') len_I_long = -1; else if(c1 != '\t' && c2 == '\t') len_I_long = 1; else if(c1 == '\t' && c2 == '\t') len_I_long = 0; if(len_I_long != 9) break; } if(chrI != chrJ || len_I_long != 0){ return (chrI * 100 + len_I_long) - (chrJ * 100); } for(x1 = 0 ; x1 < FEATURE_NAME_LENGTH + 15 ; x1++){ int c1 = inp[i][x1]; int c2 = inp[j][x1]; if(c1 != c2){ return c1 - c2; }else if(c1 == '\t' && c1 == c2){ int pos1 = atoi(inp[i]+x1+1); int pos2 = atoi(inp[j]+x1+1); if( pos1 == pos2) return strcmp(inp[i], inp[j]); else return pos1 - pos2; } if(c1 == 0 || c2 == 0)return c1 - c2; } return 0; } void junckey_sort_exchange(void * inptr, int i, int j){ char ** inp = (char **) inptr; char * tmpp = inp[j]; inp[j]=inp[i]; inp[i]=tmpp; } void junckey_sort_merge(void * inptr, int start, int items1, int items2){ char ** inp = (char **) inptr; char ** tmpp = malloc(sizeof(char *) * (items1+items2)); int read_1_ptr = start, read_2_ptr = start+items1, outptr = 0; while(1){ if(read_1_ptr == start+items1 && read_2_ptr == start+items1+items2) break; if((read_1_ptr == start+items1)||(read_2_ptr < start+items1+items2 && junckey_sort_compare(inptr, read_1_ptr, read_2_ptr) > 0 )) { // select 2 tmpp[outptr++]=inp[read_2_ptr++]; } else { // select 1 tmpp[outptr++]=inp[read_1_ptr++]; } } memcpy(inp + start, tmpp, sizeof(char *)*(items1+items2)); free(tmpp); } int junccmp(fc_junction_gene_t * j1, fc_junction_gene_t * j2){ if(strcmp( j1 -> gene_name, j2 -> gene_name ) == 0) return 0; return 1; } void fc_write_final_junctions(fc_thread_global_context_t * global_context, char * output_file_name, ArrayList * column_names, ArrayList * junction_global_table_list, ArrayList * splicing_global_table_list){ int infile_i, disk_is_full = 0; HashTable * merged_junction_table = HashTableCreate(156679); HashTableSetHashFunction(merged_junction_table,HashTableStringHashFunction); HashTableSetDeallocationFunctions(merged_junction_table, NULL, NULL); HashTableSetKeyComparisonFunction(merged_junction_table, fc_strcmp_chro); HashTable * merged_splicing_table = HashTableCreate(156679); HashTableSetHashFunction(merged_splicing_table,HashTableStringHashFunction); HashTableSetDeallocationFunctions(merged_splicing_table, NULL, NULL); HashTableSetKeyComparisonFunction(merged_splicing_table, fc_strcmp_chro); for(infile_i = 0 ; infile_i < column_names -> numOfElements ; infile_i ++){ KeyValuePair * cursor; int bucket; HashTable * spl_table = ArrayListGet(splicing_global_table_list, infile_i); for(bucket=0; bucket < spl_table -> numOfBuckets; bucket++) { cursor = spl_table -> bucketArray[bucket]; while (cursor) { char * ky = (char *)cursor -> key; unsigned int old_supp = HashTableGet(merged_splicing_table, ky) - NULL; old_supp += (cursor -> value - NULL); HashTablePut(merged_splicing_table, ky, NULL+old_supp); cursor = cursor -> next; } } } for(infile_i = 0 ; infile_i < column_names -> numOfElements ; infile_i ++){ KeyValuePair * cursor; int bucket; HashTable * junc_table = ArrayListGet(junction_global_table_list, infile_i); for(bucket=0; bucket < junc_table -> numOfBuckets; bucket++) { cursor = junc_table -> bucketArray[bucket]; while (cursor) { char * ky = (char *)cursor -> key; if(HashTableGet(merged_junction_table, ky)==NULL) HashTablePut(merged_junction_table, ky, NULL+1); cursor = cursor -> next; } } } char ** key_list; key_list = malloc(sizeof(char *) * merged_junction_table -> numOfElements); KeyValuePair * cursor; int bucket, ky_i = 0; for(bucket=0; bucket < merged_junction_table -> numOfBuckets; bucket++){ cursor = merged_junction_table -> bucketArray[bucket]; while (cursor){ char * ky = (char *)cursor -> key; key_list[ky_i ++] = ky; cursor = cursor -> next; } } merge_sort(key_list, merged_junction_table -> numOfElements , junckey_sort_compare, junckey_sort_exchange, junckey_sort_merge); char outfname[MAX_FILE_NAME_LENGTH]; sprintf(outfname, "%s.jcounts", output_file_name); int max_junction_genes = 3000; char * gene_names = malloc(max_junction_genes * FEATURE_NAME_LENGTH), * gene_name_tail; fc_junction_gene_t ** ret_juncs_small = malloc(sizeof(fc_junction_gene_t *) * max_junction_genes); fc_junction_gene_t ** ret_juncs_large = malloc(sizeof(fc_junction_gene_t *) * max_junction_genes); fc_junction_gene_t ** junction_key_list = malloc(sizeof(fc_junction_gene_t *)* max_junction_genes * 2); unsigned int * junction_support_list = malloc(sizeof(int)* max_junction_genes * 2); unsigned char * junction_source_list = malloc(sizeof(char)* max_junction_genes * 2 ); int ky_i1, ky_i2; FILE * ofp = fopen(outfname, "w"); char * tmpp = NULL; fprintf(ofp, "PrimaryGene\tSecondaryGenes\tSite1_chr\tSite1_location\tSite1_strand\tSite2_chr\tSite2_location\tSite2_strand"); for(infile_i=0; infile_i < column_names -> numOfElements; infile_i++) { char * next_fn = ArrayListGet(column_names, infile_i); fprintf(ofp,"\t%s", global_context -> use_stdin_file?"STDIN":next_fn); } fprintf(ofp, "\n"); for(ky_i = 0; ky_i < merged_junction_table -> numOfElements ; ky_i ++){ //SUBREADprintf("KY=%s\n", key_list[ky_i]); int unique_junctions = 0; char * chro_small = strtok_r( key_list[ky_i] , "\t", &tmpp); char * pos_small_str = strtok_r( NULL, "\t", &tmpp); char * chro_large = strtok_r( NULL, "\t", &tmpp); char * pos_large_str = strtok_r( NULL, "\t", &tmpp); unsigned int pos_small = atoi(pos_small_str); unsigned int pos_large = atoi(pos_large_str); int found_features_small = locate_junc_features(global_context, chro_small, pos_small, ret_juncs_small , max_junction_genes); int found_features_large = locate_junc_features(global_context, chro_large, pos_large, ret_juncs_large , max_junction_genes); char * strand = "NA"; if(global_context -> fasta_contigs){ char donor[3], receptor[3]; donor[2]=receptor[2]=0; int has = !get_contig_fasta(global_context -> fasta_contigs, chro_small, pos_small, 2, donor); has = has && !get_contig_fasta(global_context -> fasta_contigs, chro_large, pos_large-3, 2, receptor); if(has){ if(donor[0]=='G' && donor[1]=='T' && receptor[0]=='A' && receptor[1]=='G') strand = "+"; else if(donor[0]=='C' && donor[1]=='T' && receptor[0]=='A' && receptor[1]=='C') strand = "-"; }else if(!global_context ->is_junction_no_chro_shown){ global_context ->is_junction_no_chro_shown = 1; print_in_box(80,0,0, " WARNING contig '%s' is not found in the", chro_small); print_in_box(80,0,0, " provided genome file."); print_in_box(80,0,0,""); } } //SUBREADprintf("FOUND=%d, %d\n", found_features_small, found_features_large); gene_name_tail = gene_names; gene_names[0]=0; // rules to choose the primary gene: // (1) if some genes have one support but the other have multiple supporting reads: remove the lowly supported genes // (2) if all genes have only one support but from different ends of the fragment, then remove the genes that are assigned to the end having lower supporting fragments // (3) choose the gene that have the smallest coordinate. int max_supp = 0; for(ky_i1 = 0; ky_i1 < found_features_small + found_features_large; ky_i1++){ int is_duplicate = 0; fc_junction_gene_t * tested_key = (ky_i1 < found_features_small)?ret_juncs_small[ky_i1] :ret_juncs_large[ky_i1 - found_features_small]; for(ky_i2 = 0; ky_i2 < unique_junctions; ky_i2 ++){ if(junccmp( tested_key, junction_key_list[ky_i2] )==0){ junction_support_list[ ky_i2 ] ++; junction_source_list[ky_i2] |= ( (ky_i1 < found_features_small)? 1 : 2 ); is_duplicate = 1; max_supp = max(junction_support_list[ky_i2], max_supp); break; } } if(!is_duplicate){ junction_key_list[unique_junctions] = tested_key; junction_support_list[unique_junctions] = 1; junction_source_list[unique_junctions] = ( (ky_i1 < found_features_small)? 1 : 2 ); max_supp = max(junction_support_list[unique_junctions], max_supp); unique_junctions++; } } if(1 == max_supp){ if(found_features_small > 0 && found_features_large > 0){ char junc_key [FEATURE_NAME_LENGTH + 15]; sprintf(junc_key, "%s\t%u", chro_small, pos_small); unsigned int supp_small = HashTableGet(merged_splicing_table, junc_key) - NULL; sprintf(junc_key, "%s\t%u", chro_large, pos_large); unsigned int supp_large = HashTableGet(merged_splicing_table, junc_key) - NULL; if(supp_small !=supp_large){ for(ky_i2 = 0; ky_i2 < unique_junctions; ky_i2 ++){ if(supp_small > supp_large && junction_source_list[ky_i2] == 1) junction_key_list[ky_i2] = NULL; else if(supp_small < supp_large && junction_source_list[ky_i2] == 2) junction_key_list[ky_i2] = NULL; } } } } int smallest_coordinate_gene = 0x7fffffff; fc_junction_gene_t * primary_gene = NULL; for(ky_i2 = 0; ky_i2 < unique_junctions; ky_i2 ++){ fc_junction_gene_t * tested_key = junction_key_list[ky_i2]; if(tested_key != NULL && junction_support_list[ky_i2] == max_supp && tested_key -> pos_first_base < smallest_coordinate_gene){ primary_gene = tested_key; smallest_coordinate_gene = tested_key -> pos_first_base; } } if(primary_gene == NULL){ strcpy(gene_names, "NA"); }else{ strcpy(gene_names, primary_gene -> gene_name); } *(pos_small_str-1)='\t'; *(pos_large_str-1)='\t'; fprintf(ofp, "%s", gene_names); gene_name_tail = gene_names; gene_names[0]=0; for(ky_i2 = 0; ky_i2 < unique_junctions; ky_i2 ++){ fc_junction_gene_t * tested_key = junction_key_list[ky_i2]; if(tested_key && tested_key != primary_gene) gene_name_tail += sprintf(gene_name_tail, "%s,", tested_key -> gene_name); } if( gene_names[0] ) gene_name_tail[-1]=0; else strcpy(gene_names, "NA"); fprintf(ofp, "\t%s", gene_names); fprintf(ofp, "\t%s\t%s\t%s\t%s", chro_small, strand, chro_large, strand); chro_large[-1]='\t'; for(infile_i = 0 ; infile_i < column_names -> numOfElements ; infile_i ++){ HashTable * junc_table = ArrayListGet(junction_global_table_list, infile_i); srInt_64 count = HashTableGet(junc_table, key_list[ky_i]) - NULL; #ifdef __MINGW32__ fprintf(ofp,"\t%I64d", count); #else fprintf(ofp,"\t%lld", count); #endif } int wlen = fprintf(ofp, "\n"); if(wlen < 1) disk_is_full = 1; } fclose(ofp); free(junction_key_list); free(gene_names); free(ret_juncs_small); free(ret_juncs_large); free(junction_support_list); free(key_list); free(junction_source_list); //print_in_box(80,0,PRINT_BOX_CENTER,"Found %llu junctions in all the input files.", merged_junction_table -> numOfElements); //print_in_box(80,0,0,""); HashTableDestroy(merged_junction_table); HashTableDestroy(merged_splicing_table); if(disk_is_full){ unlink(outfname); SUBREADprintf("ERROR: disk is full; no junction counting table is generated.\n"); } } HashTable * scRNA_copy_loaded_features(srInt_64 nexons, fc_feature_info_t* loaded_features){ HashTable * ret = HashTableCreate(50000); srInt_64 x1; for(x1 =0; x1'2'; }else{ while('\0'!=(ch=*(fnames++)))if(ch == FC_FLIST_SPLITOR[0])ret++; while('\0'!=(ch=*(smodes++))){ if(ch == '.'){ if(numbs != 1) bad_fmt = 1; numbs = 0; ret--; }else if(ch >= '0' && ch <= '2') numbs++; } if(numbs != 1) bad_fmt = 1; } if(bad_fmt) SUBREADputs("Error: The strand mode list has a wrong format."); if(ret) SUBREADputs("Error: The length of strand mode list differs from the length of input file list"); ret |= bad_fmt; return ret; } int readSummary(int argc,char *argv[]){ /* This function counts the number of reads falling into each exon region. The order of exons in the output is the same as that of exons included in the annotation. The annotation, if provided as a file, should be sorted by chromosome name. Parameters passed from the featureCounts R function: 0: "readSummary" 1: ann 2: files[i] 3: fout 4: as.numeric(isPairedEnd) 5: min.distance 6: max.distance 7: as.numeric(tolower(file.type)=="sam") 8: as.numeric(allowMultiOverlap) 9: as.numeric(isGeneLevel) 10: as.numeric(nthreads) 11: as.numeric(isGTFannotation) 12: isStrandChecked 13: as.numeric(isReadSummaryReported) 14: as.numeric(isBothEndMapped) 15: as.numeric(isChimericDisallowed) 16: as.numeric(isPEDistChecked) 17: nameFeatureTypeColumn 18: nameGeneIDColumn 19: min.MappingQualityScore 20: as.numeric(isMultiMappingAllowed) # "1" : NH tag > 1 is allowed ; "0" : not allowd (by default) 21: Annotation Chromosome Alias Name File. If the file is not specified, set this value to NULL or a zero-length string. 22: Command line for CfeatureCounts header output; RfeatureCounts should set this value to NULL or a zero-length string or a space (' '). 23: as.numeric(isInputFileResortNeeded) 24: NOT IN USE: as.numeric(feature_block_size) # This parameter is no longer used. Give "14" for safe. 25: as.numeric(Five_End_Extension_Length) # 5' end extension 26: as.numeric(Three_End_Extension_Length) # 3' end extension 27: as.numeric(Minimum_Overlap_Between_Read_And_Feature) # 1 by default 28: as.numeric(is_Split_or_Exonic_Only) # 0 by default; 0: all reads are counted ; 1: only split (Cigar has "N") reads are counted ; 2: only exonic (no "N" in Cigar) are counted. 29: as.numeric(reduce_5_3_ends_to_one) # 0= no reduction; 1= reduce to 5' end; 2= reduce to 3' end 30: debug_command # This is for debug only; RfeatureCounts should pass a space (" ") to this parameter, disabling the debug command. 31: as.numeric(is_duplicate_ignored) # 0 = INCLUDE DUPLICATE READS; 1 = IGNORE DUPLICATE READS (0x400 FLAG IS SET) ; "0" by default. 32: as.numeric(do_not_sort) # 1 = NEVER SORT THE PE BAM/SAM FILES; 0 = SORT THE BAM/SAM FILE IF IT IS FOUND NOT SORTED. 33: as.numeric(fractionMultiMapping) # 1 = calculate fraction numbers if a read overlaps with multiple features or meta-features. "-M" must be specified when fractions are caculated. 34: as.numeric(useOverlappingBreakTie) # 1 = Select features or meta-features with a longer overlapping length; 0 = just use read-voting strategy: one overlapping read = 1 vote 35: Pair_Orientations # FF, FR, RF or RR. This parameter matters only if "-s" option is 1 or 2. 36: as.numeric(doJunctionCounting) # 1 = count the number of junction reads spaining each exon-exon pairs; 0 = do not. 37: file name of genome fasta (for determine the strandness of junctions by looking for GT/AG or CT/AC). 38: as.numeric(max_M_Ops) # maximum "M" sections allowed in the CIGAR string. This parameter is needed in parse_BIN() 39: as.numeric(is_Restrictly_No_Overlapping) # when "1", disable the voting-based tie breaking (e.g., when the reads are paired-end and one gene receives two votes but the other gene only has one.). "0" by default. 40: as.numeric(min_Fractional_Overlap) # A fractioal number. 0.00 : at least 1 bp overlapping 41: temp_directory # the directory to put temp files. "" by default, namely find it from the output file dir. 42: as.numeric(use_stdin_stdout) # only for CfeatureCounts. When use_stdin_stdout & 0x01 > 0, the input file is from stdin (stored in a temporary file); when use_stdin_stdout & 0x02 > 0, the output should be written to STDOUT instead of a file. 43: as.numeric(assign_reads_to_RG) # 1: reads with "RG" tags will be assigned to read groups' 0: default setting 44: as.numeric(long_read_minimum_length) # "1": treat the input BAM or SAM files as containing long reads. No multi-threading. "0": classic behaviour. 45: as.numeric(is_verbose) # 1: show the mismatched chromosome names on screet; 0: don't do so 46: as.numeric(frac_feature_overlap) # fraction of the feature to be overlapped with a read 47: as.numeric(do_detection_call) # do detectionCalls : put the GC fraction into the 2nd column. 48: as.numeric(max_missing_bases_in_read) # maximum # of bases in a read or fragment not overlapping with an exon ; efault value: "-1" means no limit 49: as.numeric(max_missing_bases_in_feature) # maximum # of bases in an exon not overlapping with a read or fragment ; default value: "-1" means no limit 50: as.numeric(is_Primary_Alignment_only) # "1" : only count the primary alignment (FLAG doesn't have 0x100 bit); "0" : count alignments no metter the 0x100 bit (by default) 51: Rpath : the path where the assignment details per read are stored. 52: AdditionalColumnList: the names of additional column names written after "Length". Comma deliminated. 53: annotation_file_screen_output : just for displaying the annotation file name or inbuilt (mm10/hg39/...) or R data.frame. 54: read_shift_type : how to shift reads? "upstream" : to the 5' end; "downstream" : to the 3' end; "left" : to the smaller coordinates in chromosome ; "right" : to the larger coordinates in chromosome. 55: as.numeric(read_shift_size) : how many bases to shift. Mush be a positive number or zero. */ int isCVersion, isChimericDisallowed, isPEDistChecked, minMappingQualityScore=0, isInputFileResortNeeded, feature_block_size = 20, reduce_5_3_ends_to_one, useStdinFile, assignReadsToRG, long_read_minimum_length, is_verbose, do_detectionCall, max_missing_bases_in_feature, max_missing_bases_in_read, is_Primary_Alignment_only, read_shift_size, read_shift_type, scRNA_input_mode; float fracOverlap, fracOverlapFeature, umi_cutoff; char **chr; srInt_64 *start, *stop; int *geneid; char *nameFeatureTypeColumn, *nameGeneIDColumn,*debug_command, *pair_orientations="fr", *temp_dir, *file_name_ptr =NULL, *strand_check_mode = NULL, *extra_column_names = NULL, *scRNA_sample_sheet = NULL, *scRNA_cell_barcode_list = NULL ; srInt_64 nexons; srInt_64 * anno_chr_head, * block_min_start, *block_max_end, *block_end_index; char ** anno_chrs, * anno_chr_2ch; char * fasta_contigs_fname, *annotation_file_screen_output; unsigned char * sorted_strand; int minPEDistance, maxPEDistance, isReadSummaryReport, isBothEndRequired, isMultiMappingAllowed, fiveEndExtension, threeEndExtension, minFragmentOverlap, isSplitOrExonicOnly, is_duplicate_ignored, doNotSort, fractionMultiMapping, useOverlappingBreakTie, doJuncCounting, max_M, isRestrictlyNoOvelrapping ,is_scRNA_BAM_FQ_out_generated, scRNA_rerun_on_persample_BAM; char * isPEassign, *is_paired_end_reads_expected; int isGTF, n_input_files=0,is_dual_index, scrna_total_BAM_no; char * alias_file_name = NULL, * cmd_rebuilt = NULL, * Rpath = NULL; int isMultiOverlapAllowed, isGeneLevel; isCVersion = ((argv[0][0]=='C')?1:0); isPEassign = argv[4]; minPEDistance = atoi(argv[5]); maxPEDistance = atoi(argv[6]); // isSAM = atoi(argv[7]); isMultiOverlapAllowed = atoi(argv[8]); isGeneLevel = atoi(argv[9]); unsigned short thread_number; if(argc > 10) thread_number = atoi(argv[10]); else thread_number = 4; if(argc > 11) isGTF = atoi(argv[11]); else isGTF = 0; if(argc > 12) strand_check_mode = argv[12]; else strand_check_mode = NULL; if(argc > 13) isReadSummaryReport = atoi(argv[13]); else isReadSummaryReport = 0; if(argc > 14) isBothEndRequired = atoi(argv[14]); else isBothEndRequired = 0; if(argc > 15) isChimericDisallowed = atoi(argv[15]); else isChimericDisallowed = 0; if(argc > 16) isPEDistChecked = atoi(argv[16]); else isPEDistChecked = 0; if(isPEDistChecked && 0==isBothEndRequired){ #ifdef MAKE_STANDALONE SUBREADprintf("ERROR: when the '-P' option is specified for checking fragment lengths, the '-B' option must also be specified to require both ends mapped.\n"); #else SUBREADprintf("ERROR: when parameter checkFragLength is set to TRUE, parameter requireBothEndMapped also needs to be set to TRUE.\n"); #endif return -1; } if(argc > 17) nameFeatureTypeColumn = argv[17]; else nameFeatureTypeColumn = "exon"; if(argc > 18) nameGeneIDColumn = argv[18]; else nameGeneIDColumn = "gene_id"; if(argc > 19) minMappingQualityScore = atoi(argv[19]); else minMappingQualityScore = 0; if(argc > 20) isMultiMappingAllowed = atoi(argv[20]); else isMultiMappingAllowed = 0; if(argc > 21) { alias_file_name = argv[21]; if(alias_file_name == NULL || alias_file_name[0]==' ' || alias_file_name[0]==0) alias_file_name = NULL; } else alias_file_name = NULL; if(argc > 22) { cmd_rebuilt = argv[22]; if(cmd_rebuilt == NULL || cmd_rebuilt[0]==' '||cmd_rebuilt[0]==0) cmd_rebuilt=NULL; } else cmd_rebuilt = NULL; if(argc>23) isInputFileResortNeeded = atoi(argv[23]); else isInputFileResortNeeded = 0; if(thread_number<1) thread_number=1; if(thread_number>FC_MAX_THREADS)thread_number=FC_MAX_THREADS; int Param_fiveEndExtension, Param_threeEndExtension; if(argc>25) Param_fiveEndExtension = atoi(argv[25]); else Param_fiveEndExtension = 0; if(argc>26) Param_threeEndExtension = atoi(argv[26]); else Param_threeEndExtension = 0; if(argc>27) minFragmentOverlap = atoi(argv[27]); else minFragmentOverlap = 1; if(minFragmentOverlap <1){ fiveEndExtension = 1 - minFragmentOverlap; threeEndExtension = 1 - minFragmentOverlap; minFragmentOverlap = 1; }else{ fiveEndExtension = Param_fiveEndExtension; threeEndExtension = Param_threeEndExtension; } if(argc>28) isSplitOrExonicOnly = atoi(argv[28]); else isSplitOrExonicOnly = 0; if(argc>29) reduce_5_3_ends_to_one = atoi(argv[29]); // 0 : no reduce; 1: reduce to 5' end; 2: reduce to 3' end. else reduce_5_3_ends_to_one = 0; if(argc>30 && strlen(argv[30])>0 && argv[30][0]!=' ') debug_command = argv[30]; else debug_command = " "; if(argc>31) is_duplicate_ignored = atoi(argv[31]); else is_duplicate_ignored = 0; if(argc>32) doNotSort = atoi(argv[32]); else doNotSort = 0; if(argc>33) fractionMultiMapping = atoi(argv[33]); else fractionMultiMapping = 0; if(argc>34) useOverlappingBreakTie = atoi(argv[34]); else useOverlappingBreakTie = 0; /*if(argc>35) "-S" is depreciated. pair_orientations = argv[35]; else pair_orientations = "FR"; */ if(argc>36) doJuncCounting = atoi(argv[36]); else doJuncCounting = 0; fasta_contigs_fname = NULL; if(argc>37) if(argv[37][0] != 0 && argv[37][0]!=' ') fasta_contigs_fname = argv[37]; if(argc>38) max_M = atoi(argv[38]); else max_M = 10; if(argc>39) isRestrictlyNoOvelrapping = atoi(argv[39]); else isRestrictlyNoOvelrapping = 0; if(argc>40) fracOverlap = atof(argv[40]); else fracOverlap= 0.0; if(argc>41){ if(strcmp("", argv[41])!=0)temp_dir = argv[41]; else temp_dir = NULL; } else temp_dir = NULL;// get_temp_dir_from_out(temp_dir, (char *)argv[3]); if(argc>42){ useStdinFile = (atoi(argv[42]) & 1)!=0; }else useStdinFile = 0; if(argc>43) assignReadsToRG = (argv[43][0]=='1'); else assignReadsToRG = 0; if(argc>44) long_read_minimum_length = atoi(argv[44])?1:1999999999; else long_read_minimum_length = 1999999999; if(long_read_minimum_length < 2 && isPEassign[0]=='1'){ SUBREADputs("ERROR: long read assignment can only be done on single-end mode"); return -1; } if(argc>45) is_verbose = (argv[45][0]=='1'); else is_verbose = 0; if(argc>46) fracOverlapFeature = atof(argv[46]); else fracOverlapFeature = 0.0; if(argc>47) do_detectionCall = (argv[47][0]=='1'); else do_detectionCall = 0; if(argc>48) max_missing_bases_in_read = atoi(argv[48]); else max_missing_bases_in_read = -1; if(argc>49) max_missing_bases_in_feature = atoi(argv[49]); else max_missing_bases_in_feature = -1; if(argc>50) is_Primary_Alignment_only = atoi(argv[50]); else is_Primary_Alignment_only = 0; if(argc>51 && argv[51]!=NULL && argv[51][0]!=0 && argv[51][0]!=' ') Rpath = argv[51]; else Rpath = NULL; if(argc>52 && argv[52]!=NULL && argv[52][0]!=0 && argv[52][0]!=' ') extra_column_names = argv[52]; else extra_column_names = NULL; annotation_file_screen_output = NULL; #ifndef MAKE_STANDALONE if(argc>53) annotation_file_screen_output = argv[53]; #endif if(argc>54){ read_shift_type = -1; if(strcmp(argv[54], "upstream")==0)read_shift_type = READ_SHIFT_UPSTREAM; if(strcmp(argv[54], "downstream")==0) read_shift_type = READ_SHIFT_DOWNSTREAM; if(strcmp(argv[54], "left")==0) read_shift_type = READ_SHIFT_LEFT; if(strcmp(argv[54], "right")==0) read_shift_type = READ_SHIFT_RIGHT; } else read_shift_type = READ_SHIFT_UPSTREAM; if(argc>55) read_shift_size = atoi(argv[55]); else read_shift_size = 0; if(argc>56 && strlen(argv[56])>0 && argv[56][0]!=' ') scRNA_sample_sheet = argv[56]; else scRNA_sample_sheet = NULL; if(argc>57 && strlen(argv[57])>0 && argv[57][0]!=' ') scRNA_cell_barcode_list = argv[57]; else scRNA_cell_barcode_list = NULL; if(argc>58 && strlen(argv[58])>0 && argv[58][0]!=' ') is_paired_end_reads_expected = argv[58]; else is_paired_end_reads_expected = "0"; if(argc>59 && strlen(argv[59])>0 && argv[59][0]!=' ') is_scRNA_BAM_FQ_out_generated = atoi(argv[59]); else is_scRNA_BAM_FQ_out_generated = 1; if(argc>60) scRNA_input_mode = (argv[60][0]-'0'); else scRNA_input_mode = GENE_INPUT_BCL; if(argc>61) scRNA_rerun_on_persample_BAM = (argv[61][0]-'0'); else scRNA_rerun_on_persample_BAM = 0; if(argc>62) umi_cutoff = atof(argv[62]); else umi_cutoff = -1; if(argc>63) is_dual_index = (strcmp(argv[63],"Dual-Index")==0); else is_dual_index =0; if(argc>64) scrna_total_BAM_no = atoi(argv[64]); else scrna_total_BAM_no =0; if(read_shift_size<0){ SUBREADprintf("ERROR: why the value for read_shift_size is negative?\n"); return -1; } if(read_shift_type<0){ SUBREADprintf("ERROR: why the value for read_shift_type is %s?\n", argv[54]); return -1; } if(SAM_pairer_warning_file_open_limit()) return -1; if(strand_check_mode != NULL && Input_Files_And_Strand_Mode_Pair(argv[2],strand_check_mode)) return -1; if(extra_column_names){ if(!isGTF){ SUBREADputs("ERROR: only GTF files contain additional attributes"); return -1; } int xk1, total_cols =1; for(xk1=0; extra_column_names[xk1]; xk1++) if(extra_column_names[xk1] == ';' || extra_column_names[xk1]==',' || extra_column_names[xk1]=='\t'){ extra_column_names[xk1]='\t'; total_cols ++; } if(total_cols>MAX_EXTRA_COLS){ SUBREADprintf("ERROR: there are more than %d additional attributes required\n", MAX_EXTRA_COLS); return -1; } } fc_thread_global_context_t global_context; fc_thread_init_global_context(& global_context, FEATURECOUNTS_BUFFER_SIZE, thread_number, MAX_LINE_LENGTH, minPEDistance, maxPEDistance,isGeneLevel, isMultiOverlapAllowed, strand_check_mode, (char *)argv[3] , isReadSummaryReport, isBothEndRequired, isChimericDisallowed, isPEDistChecked, nameFeatureTypeColumn, nameGeneIDColumn, minMappingQualityScore,isMultiMappingAllowed, 0, alias_file_name, cmd_rebuilt, isInputFileResortNeeded, feature_block_size, isCVersion, fiveEndExtension, threeEndExtension , minFragmentOverlap, isSplitOrExonicOnly, reduce_5_3_ends_to_one, debug_command, is_duplicate_ignored, doNotSort, fractionMultiMapping, useOverlappingBreakTie, pair_orientations, doJuncCounting, max_M, isRestrictlyNoOvelrapping, fracOverlap, temp_dir, useStdinFile, assignReadsToRG, long_read_minimum_length, is_verbose, fracOverlapFeature, do_detectionCall, max_missing_bases_in_read, max_missing_bases_in_feature, is_Primary_Alignment_only, Rpath, extra_column_names, annotation_file_screen_output, read_shift_type, read_shift_size, scRNA_sample_sheet, scRNA_cell_barcode_list, is_scRNA_BAM_FQ_out_generated, scRNA_input_mode, scRNA_rerun_on_persample_BAM, umi_cutoff,is_dual_index ); fc_thread_init_input_files( & global_context, argv[2], &file_name_ptr ); if( print_FC_configuration(&global_context, argv[1], file_name_ptr, argv[3], global_context.is_SAM_file, isGTF, & n_input_files, isReadSummaryReport, is_paired_end_reads_expected, isPEassign) ) return -1; // Loading the annotations. // Nothing is done if the annotation does not exist. fc_feature_info_t * loaded_features; print_in_box(84,0,0,"Load annotation file %s %c[0m...", get_short_fname(argv[1]), CHAR_ESC); nexons = load_feature_info(&global_context,argv[1], isGTF?FILE_TYPE_GTF:FILE_TYPE_RSUBREAD, &loaded_features); if(nexons<1){ if(nexons >= -1) SUBREADprintf("Failed to open the annotation file %s, or its format is incorrect, or it contains no '%s' features.\n",argv[1], nameFeatureTypeColumn); return -1; } sort_feature_info(&global_context, nexons, loaded_features, &chr, &geneid, &start, &stop, &sorted_strand, &anno_chr_2ch, &anno_chrs, &anno_chr_head, & block_end_index, & block_min_start, & block_max_end); if((!global_context.do_scRNA_table) || global_context.is_gene_level) global_context.lineno_2_sortedno_tab = NULL; else global_context.lineno_2_sortedno_tab = scRNA_copy_loaded_features(nexons, loaded_features); if(global_context.do_junction_counting){ sort_bucket_table(&global_context); } print_in_box(80,0,0," Meta-features : %d", global_context . gene_name_table -> numOfElements); print_in_box(80,0,0," Chromosomes/contigs : %d", global_context . exontable_nchrs); print_in_box(80,0,0,""); if(global_context.do_scRNA_table){ print_in_box(80,0,0,"Load scRNA-related files..."); print_in_box(80,0,0," scRNA samples : %d", global_context.scRNA_sample_sheet_table->numOfElements); print_in_box(80,0,0," scRNA cell barcodes : %d", global_context.scRNA_cell_barcodes_array -> numOfElements); print_in_box(80,0,0,""); } if(fasta_contigs_fname){ print_in_box(80,0,0,"Load FASTA contigs from %s...", get_short_fname(fasta_contigs_fname)); global_context.fasta_contigs = malloc(sizeof(fasta_contigs_t)); int ret_fq = read_contig_fasta(global_context.fasta_contigs, fasta_contigs_fname); if(ret_fq){ print_in_box(80,0,0," WARNING unable to open the FASTA file."); print_in_box(80,0,0,""); free(global_context.fasta_contigs); global_context.fasta_contigs = NULL; }else{ print_in_box(80,0,0," %lu contigs were loaded", global_context.fasta_contigs -> contig_table -> numOfElements); print_in_box(80,0,0,""); } }else global_context.fasta_contigs = NULL; global_context.exontable_exons = nexons; unsigned int x1, total_written_coulmns=0; char * tmp_pntr = NULL, *tmp_smode_ptr = NULL; char * strand_mode_list = strdup(global_context.strand_check_mode); char * file_list_used = malloc(strlen(file_name_ptr)+1); char * file_list_used2 = malloc(strlen(file_name_ptr)+1); char * is_unique = malloc(strlen(file_name_ptr)+1); strcpy(file_list_used, file_name_ptr); for(x1 = 0;;x1++){ char * test_fn = strtok_r(x1?NULL:file_list_used, FC_FLIST_SPLITOR, &tmp_pntr); if(NULL == test_fn) break; char * short_fname = get_short_fname(test_fn); strcpy(file_list_used2, file_name_ptr); is_unique[x1]=1; char * loop_ptr = NULL; int x2; for(x2 = 0;;x2++){ char * test_loopfn = strtok_r(x2?NULL:file_list_used2, FC_FLIST_SPLITOR, &loop_ptr); if(NULL == test_loopfn) break; if(x1==x2)continue; char * short_loop_fname = get_short_fname(test_loopfn); if(strcmp(short_loop_fname, short_fname)==0) { is_unique[x1] = 0; break; } } } free(file_list_used2); tmp_pntr = NULL; strcpy(file_list_used, file_name_ptr); char * next_fn = strtok_r(file_list_used, FC_FLIST_SPLITOR, &tmp_pntr); char * next_strand_mode = strtok_r(strand_mode_list, ".", &tmp_smode_ptr); int one_single_strand_mode = -1; if(NULL == strstr( global_context.strand_check_mode, "." )){ one_single_strand_mode = next_strand_mode[0] - '0'; assert(one_single_strand_mode >= 0 && one_single_strand_mode < 3); } ArrayList * table_columns = ArrayListCreate(n_input_files+1); ArrayList * table_column_names = ArrayListCreate(n_input_files+1); ArrayList * read_counters = ArrayListCreate(n_input_files+1); ArrayListSetDeallocationFunction(table_columns, free); ArrayListSetDeallocationFunction(table_column_names, free); ArrayListSetDeallocationFunction(read_counters, free); ArrayList * junction_global_table_list = NULL; ArrayList * splicing_global_table_list = NULL; if(global_context.do_junction_counting){ junction_global_table_list = ArrayListCreate(n_input_files+1); splicing_global_table_list = ArrayListCreate(n_input_files+1); ArrayListSetDeallocationFunction(junction_global_table_list, (void (*)(void *))HashTableDestroy); ArrayListSetDeallocationFunction(splicing_global_table_list, (void (*)(void *))HashTableDestroy); } int ret_int = 0; #ifdef MAKE_STANDALONE #define NO_SORT_OPTION_NAME "donotsort" #else #define NO_SORT_OPTION_NAME "autosort" #endif for(x1 = 0;;x1++){ int orininal_isPE = global_context.is_paired_end_mode_assign; if(next_fn==NULL || strlen(next_fn)<1 || global_context.disk_is_full || ret_int) break; int this_file_isPEassign = isPEassign[1]?isPEassign[x1] == '1' :(isPEassign[0]=='1'); int this_file_isPEexpected = is_paired_end_reads_expected[1]?is_paired_end_reads_expected[x1]=='1' :(is_paired_end_reads_expected[0]=='1'); global_context.is_paired_end_reads_expected = this_file_isPEexpected; global_context.is_paired_end_mode_assign = this_file_isPEassign; if(global_context.do_not_sort && 0==this_file_isPEassign){ print_in_box(80,0,0," WARNING the %s option is ignored when single-end reads", NO_SORT_OPTION_NAME); print_in_box(80,0,0," are being counted."); } read_count_type_t * column_numbers = calloc(nexons, sizeof(read_count_type_t)); HashTable * junction_global_table = NULL; HashTable * splicing_global_table = NULL; strcpy(global_context.input_file_name, next_fn); strcpy(global_context.raw_input_file_name, next_fn); global_context.this_input_number = x1; global_context.input_file_unique = is_unique[x1]; global_context.input_file_short_name = get_short_fname(next_fn); if(strstr( global_context.strand_check_mode, "." )){ global_context.is_strand_checked = next_strand_mode[0]-'0'; assert(global_context.is_strand_checked >=0 && global_context.is_strand_checked <=2); }else global_context.is_strand_checked = one_single_strand_mode; global_context.redo=0; if(global_context.is_scRNA_BAM_FQ_out_generated && global_context.scRNA_sample_sheet_table){ global_context.scRNA_sample_BAM_writers = HashTableCreate(global_context.scRNA_sample_sheet_table -> numOfElements); HashTableSetDeallocationFunctions(global_context.scRNA_sample_BAM_writers, NULL, scRNA_close_sample_SamBam_writers); global_context.scRNA_sample_sheet_table ->appendix1 = global_context.scRNA_sample_BAM_writers; global_context.scRNA_sample_sheet_table ->appendix2 = &global_context; global_context.scRNA_sample_sheet_table ->appendix3 = global_context.scRNA_sample_id_to_name; HashTableIteration( global_context.scRNA_sample_sheet_table, scRNA_sample_SamBam_writers_new_files); if(global_context.scRNA_input_mode == GENE_INPUT_BCL || global_context.scRNA_input_mode == GENE_INPUT_SCRNA_BAM){ int umfpi; for(umfpi=1; umfpi<=4; umfpi++){ char fname [MAX_FILE_NAME_LENGTH+20]; char * ftype = "R1"; if(3==umfpi && ! global_context.scRNA_dual_index) continue; if(2==umfpi) ftype = "I1"; if(3==umfpi) ftype = "I2"; if(4==umfpi) ftype = "R2"; sprintf(fname, "UnassignedReads%03d_%s.fastq.gz", scrna_total_BAM_no, ftype); parallel_gzip_writer_init(global_context.scRNA_fastq_unassigned_writer+(umfpi-1), fname, global_context.thread_number); } pthread_spin_init(&global_context.scRNA_fastq_unassigned_lock, PTHREAD_PROCESS_PRIVATE); } } if(global_context.do_junction_counting){ junction_global_table = HashTableCreate(156679); splicing_global_table = HashTableCreate(156679); HashTableSetHashFunction(junction_global_table,HashTableStringHashFunction); HashTableSetDeallocationFunctions(junction_global_table, free, NULL); HashTableSetKeyComparisonFunction(junction_global_table, fc_strcmp_chro); HashTableSetHashFunction(splicing_global_table,HashTableStringHashFunction); HashTableSetDeallocationFunctions(splicing_global_table, free, NULL); HashTableSetKeyComparisonFunction(splicing_global_table, fc_strcmp_chro); } HashTable * merged_RG_table = NULL; if(global_context.assign_reads_to_RG){ merged_RG_table = HashTableCreate(97); HashTableSetHashFunction(merged_RG_table,HashTableStringHashFunction); HashTableSetDeallocationFunctions(merged_RG_table, NULL, free); // the names are put into the column_names table, but the 4-pointer arrays are not used anymore. HashTableSetKeyComparisonFunction(merged_RG_table, fc_strcmp_chro); } fc_read_counters * my_read_counter = calloc(1, sizeof(fc_read_counters)); global_context.is_read_details_out = isReadSummaryReport; global_context.max_M = max_M; ret_int = ret_int || readSummary_single_file(& global_context, column_numbers, nexons, geneid, chr, start, stop, sorted_strand, anno_chr_2ch, anno_chrs, anno_chr_head, block_end_index, block_min_start, block_max_end, my_read_counter, junction_global_table, splicing_global_table, merged_RG_table, loaded_features); if(global_context.disk_is_full){ SUBREADprintf("ERROR: disk is full. Please check the free space in the output directory.\n"); } if(ret_int!=0){ // give up this file. if(global_context.do_junction_counting){ HashTableDestroy(junction_global_table); HashTableDestroy(splicing_global_table); } free(column_numbers); } else { // finished char * mem_file_name = memstrcpy(next_fn); if(!global_context.assign_reads_to_RG){ ArrayListPush(table_columns, column_numbers); ArrayListPush(table_column_names, mem_file_name); ArrayListPush(read_counters, my_read_counter); if(global_context.do_junction_counting){ ArrayListPush(junction_global_table_list,junction_global_table); ArrayListPush(splicing_global_table_list,splicing_global_table); } } if(global_context.assign_reads_to_RG){ int rgcur; char * rg_name = global_context.RGnames_set; for(rgcur = 0; rgcur < global_context.RGnames_ptr+1; rgcur ++){ if(global_context.RGnames_set[rgcur] == '\t'||global_context.RGnames_set[rgcur] == '\0'){ global_context.RGnames_set[rgcur] = 0; int rg_name_len = strlen(rg_name); if(rg_name_len > 0){ // SUBREADprintf("GET 4Tab:'%s'\n", rg_name); void ** tab4 = HashTableGet(merged_RG_table, rg_name); int file_len = strlen(mem_file_name); char * rg_file_name = malloc(rg_name_len + 3 + file_len); sprintf(rg_file_name, "%s:%s", mem_file_name, rg_name); ArrayListPush(table_column_names, rg_file_name); ArrayListPush(table_columns, tab4[0]); ArrayListPush(read_counters, tab4[1]); if(global_context.do_junction_counting){ ArrayListPush(junction_global_table_list,tab4[2]); ArrayListPush(splicing_global_table_list,tab4[3]); } rg_name = global_context.RGnames_set + rgcur + 1; } } } free(mem_file_name); } total_written_coulmns ++; } global_context.is_paired_end_mode_assign = orininal_isPE; next_fn = strtok_r(NULL, FC_FLIST_SPLITOR, &tmp_pntr); if(strstr( global_context.strand_check_mode, "." )) next_strand_mode = strtok_r(NULL, ".", &tmp_smode_ptr); if(global_context.assign_reads_to_RG) free(global_context.RGnames_set); if(merged_RG_table) HashTableDestroy(merged_RG_table); } free(file_list_used); free(is_unique); if(global_context.is_input_bad_format){ // SUBREADprintf("\nEEROR: The program has to terminate and no counting file is generated.\n\n"); }else if(!(global_context.disk_is_full||ret_int)){ print_in_box(80,0,0,"Write the final count table."); if(isGeneLevel){ char ** sorted_extra_columns = NULL; if(global_context.reported_extra_columns != NULL){ sorted_extra_columns = malloc(sizeof(char**) * nexons); int ii; for(ii = 0; ii < nexons; ii++){ sorted_extra_columns[loaded_features[ii].sorted_order] = loaded_features[ii].extra_columns; //SUBREADprintf("SSMQ: %d = %s\n", loaded_features[ii].sorted_order, loaded_features[ii].extra_columns); } } fc_write_final_gene_results(&global_context, geneid, chr, start, stop, sorted_strand, sorted_extra_columns, argv[3], nexons, table_columns, table_column_names, loaded_features, isCVersion); if(sorted_extra_columns) free(sorted_extra_columns); } else fc_write_final_results(&global_context, argv[3], nexons, table_columns, table_column_names, loaded_features, isCVersion); } if(global_context.do_junction_counting && global_context.is_input_bad_format == 0 && !(ret_int||global_context.disk_is_full)){ print_in_box(80,0,0,"Write the junction count table."); fc_write_final_junctions(&global_context, argv[3], table_column_names, junction_global_table_list, splicing_global_table_list); } if(global_context.is_input_bad_format == 0 && !(ret_int||global_context.disk_is_full)){ print_in_box(80,0,0,"Write the read assignment summary."); fc_write_final_counts(&global_context, argv[3], table_column_names, read_counters, isCVersion); } ArrayListDestroy(table_columns); ArrayListDestroy(table_column_names); ArrayListDestroy(read_counters); if(global_context.do_junction_counting){ ArrayListDestroy(junction_global_table_list); ArrayListDestroy(splicing_global_table_list); } free(file_name_ptr); if(global_context.is_input_bad_format == 0) print_FC_results(&global_context, (char *)argv[3]/*out file name*/); KeyValuePair * cursor; int bucket; for(bucket=0; bucket < global_context.exontable_chro_table -> numOfBuckets; bucket++) { cursor = global_context.exontable_chro_table -> bucketArray[bucket]; while (1) { if (!cursor) break; fc_chromosome_index_info * del_chro_info = cursor->value; free(del_chro_info->reverse_table_start_index); //free(del_chro_info->reverse_table_end_index); free((void *)cursor -> key); free(del_chro_info); cursor = cursor->next; } } if(global_context.read_details_out_FP) fclose(global_context. read_details_out_FP); HashTableDestroy(global_context.gene_name_table); HashTableDestroy(global_context.GCcontent_table); if(global_context.scRNA_sample_sheet_table){ HashTableDestroy(global_context.scRNA_sample_sheet_table); ArrayListDestroy(global_context.scRNA_sample_barcode_list); ArrayListDestroy(global_context.scRNA_sample_id_to_name); HashTableDestroy(global_context.scRNA_lineno1B_to_sampleno1B_tab); for(x1=0; x1 numOfBuckets; bucket++){ cursor = gene_feature_table -> bucketArray[bucket]; while(1){ if (!cursor) break; fc_junction_gene_t * gene = (fc_junction_gene_t *) cursor -> value; unsigned int x1; for(x1 = gene -> pos_first_base - gene -> pos_first_base % JUNCTION_BUCKET_STEP; x1 <= gene -> pos_last_base ; x1 += JUNCTION_BUCKET_STEP){ char bucket_key[CHROMOSOME_NAME_LENGTH + 20]; sprintf(bucket_key, "%s:%u", chro_name, x1); gene_info_list_t * list = HashTableGet(global_context -> junction_bucket_table, bucket_key); if(list == NULL){ list = malloc(sizeof(gene_info_list_t)); list -> space = 3; list -> used = 0; list -> genes = malloc(sizeof(void *) * list -> space); char * mem_bucket_key = malloc(strlen(bucket_key) + 1); strcpy(mem_bucket_key , bucket_key); HashTablePut(global_context -> junction_bucket_table, mem_bucket_key , list); } if(list -> used == list -> space){ list -> space = max(list -> space + 3, list -> space * 1.3); list -> genes = realloc(list -> genes , list -> space * sizeof(void *)); } list -> genes[list -> used++] = gene; } cursor = cursor -> next; } } } void sort_bucket_table(fc_thread_global_context_t * global_context){ KeyValuePair * cursor; int bucket; for(bucket=0; bucket < global_context -> junction_features_table -> numOfBuckets; bucket++){ cursor = global_context -> junction_features_table -> bucketArray[bucket]; while(1){ if (!cursor) break; HashTable * gene_feature_table = cursor -> value; char * chro_name = (char *)cursor -> key; register_buckets(global_context , gene_feature_table, chro_name); cursor = cursor -> next; } } } void scRNA_generate_BAM_FASTQ(fc_thread_global_context_t * global_context){ char MAC_or_random[13]; mac_or_rand_str(MAC_or_random); char rand_prefix[MAX_FILE_NAME_LENGTH+100]; sprintf(rand_prefix, "%s/temp-core-%06u-%s.sam", global_context -> temp_file_dir, getpid(), MAC_or_random); SAM_pairer_create(&global_context -> scRNA_read_pairer, global_context -> thread_number , global_context -> max_BAM_header_size/1024/1024+2, 1 /* is bam */, 0 /* do not drop seq/qual */ , 1 /*single end*/, 0 /*do not sort*/,0 /* no RG*/ ,0, global_context -> input_file_name, NULL, NULL, process_pairer_scRNAr2_output, rand_prefix, global_context, 9999); SAM_pairer_run(&global_context -> scRNA_read_pairer); SAM_pairer_destroy(&global_context -> scRNA_read_pairer); } int readSummary_single_file(fc_thread_global_context_t * global_context, read_count_type_t * column_numbers, srInt_64 nexons, int * geneid, char ** chr, srInt_64 * start, srInt_64 * stop, unsigned char * sorted_strand, char * anno_chr_2ch, char ** anno_chrs, srInt_64 * anno_chr_head, srInt_64 * block_end_index, srInt_64 * block_min_start , srInt_64 * block_max_end, fc_read_counters * my_read_counter, HashTable * junction_global_table, HashTable * splicing_global_table, HashTable * merged_RG_table, fc_feature_info_t * loaded_features) { int read_length = 0; int is_first_read_PE=0; char * line = (char*)calloc(MAX_LINE_LENGTH, 1); char * file_str = ""; int file_probe = is_certainly_bam_file(global_context->input_file_name, &is_first_read_PE, NULL); // a Singel-end SAM/BAM file cannot be assigned as a PE SAM/BAM file; // but a PE SAM/BAM file may be assigned as a SE file if the user wishes to do so. global_context->is_SAM_file = 1; if(file_probe == 1) global_context->is_SAM_file = 0; global_context->is_mixed_PE_SE = 0; global_context->any_reads_are_PE = 0; global_context -> start_time = miltime(); file_str = "SAM"; if(file_probe == 1) file_str = "BAM" ; if(file_probe == -1) file_str = "Unknown"; if(!global_context->redo) { print_in_box(80,0,0,"Process %s file %s...", file_str, global_context -> use_stdin_file? "":get_short_fname(global_context->input_file_name)); if(global_context->is_strand_checked) print_in_box(80,0,0," Strand specific : %s", global_context->is_strand_checked==1?"stranded":"reversely stranded"); } // Open the SAM/BAM file // Nothing is done if the file does not exist. int thread_error = fc_thread_start_threads(global_context, nexons, geneid, chr, start, stop, sorted_strand, anno_chr_2ch, anno_chrs, anno_chr_head, block_end_index, block_min_start , block_max_end, read_length); if(thread_error) return -1; fc_thread_wait_threads(global_context); if(global_context -> is_paired_end_reads_expected && !global_context -> any_reads_are_PE){ SUBREADprintf("ERROR: No paired-end reads were detected in paired-end read library : %s\n", global_context -> input_file_name); global_context -> is_input_bad_format=1; return -1; } srInt_64 nreads_mapped_to_exon = 0; fc_thread_merge_results(global_context, column_numbers , &nreads_mapped_to_exon, my_read_counter, junction_global_table, splicing_global_table, merged_RG_table, loaded_features, nexons); if(global_context -> do_scRNA_table){ scRNA_generate_BAM_FASTQ(global_context); free(global_context -> scRNA_applied_umi_cut); } fc_thread_destroy_thread_context(global_context); if(global_context -> sambam_chro_table) free(global_context -> sambam_chro_table); global_context -> sambam_chro_table = NULL; free(line); if(global_context -> is_input_bad_format) return -1; return 0; } #ifdef MAKE_STANDALONE int main(int argc, char ** argv) #else int feature_count_main(int argc, char ** argv) #endif { char * Rargv[64]; char annot_name[MAX_FILE_NAME_LENGTH]; char temp_dir[MAX_FILE_NAME_LENGTH]; char * out_name = malloc(MAX_FILE_NAME_LENGTH); char * fasta_contigs_name = malloc(MAX_FILE_NAME_LENGTH); char * alias_file_name = malloc(MAX_FILE_NAME_LENGTH); char * Rpath = malloc(MAX_FILE_NAME_LENGTH); char * scRNA_sample_sheet = malloc(MAX_FILE_NAME_LENGTH); char * scRNA_cell_barcode_list = malloc(MAX_FILE_NAME_LENGTH); int cmd_rebuilt_size = 2000; char * cmd_rebuilt = malloc(cmd_rebuilt_size); char max_M_str[8]; char nameFeatureTypeColumn[2000]; char nameGeneIDColumn[66]; int min_qual_score = 0; int min_dist = 50; int max_dist = 600; int read_shift_size = 0; char debug_command[15]; char max_missing_bases_in_read_str[15]; char max_missing_bases_in_feature_str[15]; char min_dist_str[15]; char max_dist_str[15]; char read_shift_size_str[15]; char read_shift_type[15]; char min_qual_score_str[15]; char feature_block_size_str[15]; char * Strand_Sensitive_Str = "0"; char * old_zero_smode = Strand_Sensitive_Str; char strFeatureFracOverlap[15]; char Pair_Orientations[3]; char * extra_column_names = NULL; char * very_long_file_names; char is_paired_end_reads_expected[2]; int is_Input_Need_Reorder = 0; int is_PE = 0; int is_SAM = 1; int is_primary_alignment_only = 0; int is_GeneLevel = 1; int is_Overlap = 0; int is_Both_End_Mapped = 0; int is_Restrictedly_No_Overlap = 0; int feature_block_size = 14; int is_ReadSummary_Report = 0; int is_Chimeric_Disallowed = 0; int is_PE_Dist_Checked = 0; int is_Multi_Mapping_Allowed = 0; int is_Split_or_Exonic_Only = 0; int is_duplicate_ignored = 0; int assign_reads_to_RG = 0; int do_not_sort = 0; int do_junction_cnt = 0; int do_detection_call = 0; int reduce_5_3_ends_to_one = 0; int use_fraction_multimapping = 0; int threads = 1; int isGTF = 1; int use_overlapping_length_break_tie = 0; char nthread_str[4]; int option_index = 0; int max_missing_bases_in_feature = -1; int max_missing_bases_in_read = -1; int scRNA_input_mode = GENE_INPUT_BCL; int c; int very_long_file_names_size = 200; int fiveEndExtension = 0, threeEndExtension = 0, minFragmentOverlap = 1; float fracOverlap = 0.0, fracOverlapFeature = 0.0; int std_input_output_mode = 0, long_read_mode = 0, is_verbose = 0; int is_scRNA_BAM_FQ_out_generated = 1; char strFiveEndExtension[11], strThreeEndExtension[11], strMinFragmentOverlap[11], fracOverlapStr[20], std_input_output_mode_str[16], long_read_mode_str[16]; very_long_file_names = malloc(very_long_file_names_size); very_long_file_names [0] = 0; fasta_contigs_name[0]=0; scRNA_cell_barcode_list[0]=0; scRNA_sample_sheet[0]=0; is_paired_end_reads_expected[0]='0'; is_paired_end_reads_expected[1]='\0'; alias_file_name[0]=0; debug_command[0] = 0; strcpy(read_shift_type,"upstream"); strcpy(nameFeatureTypeColumn,"exon"); strcpy(nameGeneIDColumn,"gene_id"); strcpy(temp_dir, ""); annot_name[0]=0;out_name[0]=0;Rpath[0]=0; cmd_rebuilt[0]=0; for(c = 0; c cmd_rebuilt_size) { cmd_rebuilt_size*=2; cmd_rebuilt = realloc(cmd_rebuilt, cmd_rebuilt_size); } sprintf(cmd_rebuilt+strlen(cmd_rebuilt), "\"%s\" ", argv[c]); } optind=0; opterr=1; optopt=63; strcpy(max_M_str, "10"); strcpy(Pair_Orientations,"fr"); while ((c = getopt_long (argc, argv, "G:A:g:t:T:o:a:d:D:LQ:pbF:fs:S:CBJPMOR:v?", long_options, &option_index)) != -1) switch(c) { case 'S': /* if(strlen(optarg)!=2 || (strcmp(optarg, "ff")!=0 && strcmp(optarg, "rf")!=0 && strcmp(optarg, "fr")!=0)){ SUBREADprintf("The order parameter can only be ff, fr or rf.\n"); print_usage(); return -1; } Pair_Orientations[0]=(optarg[0]=='r'?'r':'f'); Pair_Orientations[1]=(optarg[1]=='f'?'f':'r'); Pair_Orientations[2]=0; */ SUBREADprintf("The \"-S\" option has been depreciated.\n"); break; case 'G': strcpy(fasta_contigs_name , optarg); break; case 'J': do_junction_cnt = 1; break; case 'A': strcpy(alias_file_name, optarg); break; case 'M': is_Multi_Mapping_Allowed = 1; break; case 'v': core_version_number("featureCounts"); return 0; case 'Q': if(!is_valid_digit_range(optarg, "Q", 0 , 255)) STANDALONE_exit(-1); min_qual_score = atoi(optarg); break; case 't': strcpy(nameFeatureTypeColumn, optarg); break; case 'g': while((*optarg) == ' ') optarg++; strcpy(nameGeneIDColumn, optarg); break; case 'T': if(!is_valid_digit_range(optarg, "T", 1, FC_MAX_THREADS)) STANDALONE_exit(-1); threads = atoi(optarg); break; case 'd': if(!is_valid_digit(optarg, "d")) STANDALONE_exit(-1); min_dist = atoi(optarg); break; case 'D': if(!is_valid_digit(optarg, "D")) STANDALONE_exit(-1); max_dist = atoi(optarg); break; case 'p': is_paired_end_reads_expected[0]='1'; break; case 'C': is_Chimeric_Disallowed = 1; break; case 'P': is_PE_Dist_Checked = 1; break; case 'B': is_Both_End_Mapped = 1; break; case 'f': is_GeneLevel = 0; break; case 'F': isGTF = 1; if(strcmp("SAF", optarg)==0) isGTF=0; else if(strcmp("GTF", optarg)==0) isGTF=1; else SUBREADprintf("\nWarning: Unknown annotation format: %s. GTF format is used.\n\n", optarg); break; case 'O': is_Overlap = 1; break; case 'R': if(strcmp(optarg, "SAM")==0) is_ReadSummary_Report = FILE_TYPE_SAM; else if(strcmp(optarg, "BAM")==0) is_ReadSummary_Report = FILE_TYPE_BAM; else if(strcmp(optarg, "CORE")==0) is_ReadSummary_Report = FILE_TYPE_RSUBREAD; else{ SUBREADprintf("\nERROR: unknown output format: '%s'\n\n", optarg); STANDALONE_exit(-1); } break; case 's': Strand_Sensitive_Str = strdup(optarg); int xx; for(xx =0; Strand_Sensitive_Str[xx]!='\0'; xx++) if(Strand_Sensitive_Str[xx]==',') Strand_Sensitive_Str[xx]='.'; break; // case 'i': // term_strncpy(sam_name, optarg,299); // break; case 'o': term_strncpy(out_name, optarg,MAX_FILE_NAME_LENGTH-1); break; case 'a': term_strncpy(annot_name, optarg,MAX_FILE_NAME_LENGTH-1); break; case 'L': long_read_mode = 1; break; case 0 : // long options if(strcmp("countReadPairs", long_options[option_index].name)==0){ is_PE=1; } if(strcmp("primary", long_options[option_index].name)==0) { is_primary_alignment_only = 1; } if(strcmp("readExtension5", long_options[option_index].name)==0) { if(!is_valid_digit_range(optarg, "readExtension5", 0, 0x7fffffff)) STANDALONE_exit(-1); fiveEndExtension = atoi(optarg); fiveEndExtension = max(0, fiveEndExtension); } if(strcmp("readExtension3", long_options[option_index].name)==0) { if(!is_valid_digit_range(optarg, "readExtension3", 0, 0x7fffffff)) STANDALONE_exit(-1); threeEndExtension = atoi(optarg); threeEndExtension = max(0, threeEndExtension); } if(strcmp("fracOverlap", long_options[option_index].name)==0) { if(!is_valid_float(optarg, "fracOverlap")) STANDALONE_exit(-1); fracOverlap = atof(optarg); } if(strcmp("fracOverlapFeature", long_options[option_index].name)==0) { if(!is_valid_float(optarg, "fracOverlapFeature")) STANDALONE_exit(-1); fracOverlapFeature = atof(optarg); } if(strcmp("nonOverlapFeature", long_options[option_index].name)==0){ if(!is_valid_digit_range(optarg, "nonOverlapFeature", 0, 0x7fffffff)) STANDALONE_exit(-1); max_missing_bases_in_feature = atoi(optarg); } if(strcmp("nonOverlap", long_options[option_index].name)==0){ if(!is_valid_digit_range(optarg, "nonOverlap", 0, 0x7fffffff)) STANDALONE_exit(-1); max_missing_bases_in_read = atoi(optarg); } if(strcmp("scCellBarcodeFile", long_options[option_index].name)==0) { strcpy(scRNA_cell_barcode_list,optarg); } if(strcmp("scSampleSheet", long_options[option_index].name)==0) { strcpy(scRNA_sample_sheet,optarg); } if(strcmp("scInputMode", long_options[option_index].name)==0) { if(strcmp("FASTQ", optarg)==0) scRNA_input_mode=GENE_INPUT_SCRNA_FASTQ; if(strcmp("BAM", optarg)==0) scRNA_input_mode=GENE_INPUT_SCRNA_BAM; } if(strcmp("extraAttributes", long_options[option_index].name)==0) { extra_column_names = strdup(optarg); } if(strcmp("Rpath", long_options[option_index].name)==0) { strcpy(Rpath, optarg); } if(strcmp("minOverlap", long_options[option_index].name)==0) { if(!is_valid_digit(optarg, "minOverlap")) STANDALONE_exit(-1); minFragmentOverlap = atoi(optarg); } if(strcmp("debugCommand", long_options[option_index].name)==0) { strcpy(debug_command, optarg); } if(strcmp("ignoreDup", long_options[option_index].name)==0) { is_duplicate_ignored = 1 ; } if(strcmp("fraction", long_options[option_index].name)==0) { use_fraction_multimapping = 1; } if(strcmp("tmpDir", long_options[option_index].name)==0){ strcpy(temp_dir, optarg); } if(strcmp("maxMOp", long_options[option_index].name)==0){ if(!is_valid_digit_range(optarg, "maxMOp", 1 , 65555)) STANDALONE_exit(-1); strcpy(max_M_str, optarg); } if(strcmp("read2pos", long_options[option_index].name)==0) { if(optarg[0]=='3') reduce_5_3_ends_to_one = REDUCE_TO_3_PRIME_END; else if(optarg[0]=='5') reduce_5_3_ends_to_one = REDUCE_TO_5_PRIME_END; else{ SUBREADprintf("Invalide parameter to the --read2pos option: %s\n", optarg); STANDALONE_exit(-1); } } if(strcmp("largestOverlap", long_options[option_index].name)==0) { use_overlapping_length_break_tie = 1; } if(strcmp("detectionCall", long_options[option_index].name)==0) { do_detection_call = 1; } if(strcmp("donotsort", long_options[option_index].name)==0) { do_not_sort = 1; } if(strcmp("readShiftSize", long_options[option_index].name)==0) { if(!is_valid_digit_range(optarg, "readShiftSize", 1 , 0x7fffffff)) STANDALONE_exit(-1); read_shift_size = atoi(optarg); } if(strcmp("readShiftType", long_options[option_index].name)==0) { if(strcmp(optarg,"upstream")!=0 && strcmp(optarg,"downstream")!=0 && strcmp(optarg,"left")!=0 && strcmp(optarg,"right")!=0){ SUBREADprintf("Error: the readShiftType parameter can only be 'upstream', 'downstream', 'left' or 'right'\n"); STANDALONE_exit(-1); } strcpy(read_shift_type, optarg); } if(strcmp("splitOnly", long_options[option_index].name)==0) { if(is_Split_or_Exonic_Only == 2) { SUBREADprintf("Error: You can not specify both splitOnly and nonSplitOnly\n"); return -1; } is_Split_or_Exonic_Only = 1; } if(strcmp("restrictedlyNoOverlap", long_options[option_index].name)==0) { is_Restrictedly_No_Overlap = 1; } if(strcmp("nonSplitOnly", long_options[option_index].name)==0) { if(is_Split_or_Exonic_Only == 1) { SUBREADprintf("Error: You can not specify both splitOnly and nonSplitOnly\n"); return -1; } is_Split_or_Exonic_Only = 2; } if(strcmp("verbose", long_options[option_index].name)==0){ is_verbose = 1; } if(strcmp("byReadGroup", long_options[option_index].name)==0){ assign_reads_to_RG = 1; } break; case '?': default : print_usage(); return -1; break; } if(minFragmentOverlap<1) { fiveEndExtension = - minFragmentOverlap + 1; threeEndExtension = - minFragmentOverlap + 1; minFragmentOverlap = 1; } if(out_name[0]==0 || annot_name[0]==0) { print_usage(); return -1; } for(; optind < argc; optind++) { int curr_strlen = strlen(very_long_file_names); if( very_long_file_names_size - curr_strlen < MAX_FILE_NAME_LENGTH+1) { very_long_file_names_size *=2; //printf("CL=%d ; NS=%d\n", curr_strlen , very_long_file_names_size); very_long_file_names=realloc(very_long_file_names , very_long_file_names_size); } strcat(very_long_file_names, argv[optind]); strcat(very_long_file_names, FC_FLIST_SPLITOR); } very_long_file_names[strlen(very_long_file_names)-1]=0; std_input_output_mode = (strcmp(very_long_file_names, "") == 0?1:0); sprintf(strFiveEndExtension, "%d", fiveEndExtension); sprintf(strThreeEndExtension, "%d", threeEndExtension); sprintf(strMinFragmentOverlap, "%d", minFragmentOverlap); sprintf(nthread_str,"%d", threads); sprintf(min_dist_str,"%d",min_dist); sprintf(max_dist_str,"%d",max_dist); sprintf(min_qual_score_str,"%d", min_qual_score); sprintf(feature_block_size_str,"%d", feature_block_size); sprintf(fracOverlapStr, "%g", fracOverlap); sprintf(std_input_output_mode_str,"%d",std_input_output_mode); sprintf(long_read_mode_str, "%d", long_read_mode); sprintf(strFeatureFracOverlap, "%g", fracOverlapFeature); sprintf(max_missing_bases_in_feature_str, "%d", max_missing_bases_in_feature); sprintf(max_missing_bases_in_read_str, "%d", max_missing_bases_in_read); sprintf(read_shift_size_str, "%d", read_shift_size); Rargv[0] = "CreadSummary"; Rargv[1] = annot_name; Rargv[2] = very_long_file_names; Rargv[3] = out_name; Rargv[4] = is_PE?"1":"0"; Rargv[5] = min_dist_str; Rargv[6] = max_dist_str; Rargv[7] = is_SAM?"1":"0"; Rargv[8] = is_Overlap?"1":"0"; Rargv[9] = is_GeneLevel?"1":"0"; Rargv[10] = nthread_str; Rargv[11] = isGTF?"1":"0"; Rargv[12] = Strand_Sensitive_Str; Rargv[13] = is_ReadSummary_Report == 0 ? "0":(is_ReadSummary_Report == FILE_TYPE_RSUBREAD?"10":(is_ReadSummary_Report == FILE_TYPE_BAM?"500":"50")); Rargv[14] = is_Both_End_Mapped?"1":"0"; Rargv[15] = is_Chimeric_Disallowed?"1":"0"; Rargv[16] = is_PE_Dist_Checked?"1":"0"; Rargv[17] = nameFeatureTypeColumn; Rargv[18] = nameGeneIDColumn; Rargv[19] = min_qual_score_str; Rargv[20] = is_Multi_Mapping_Allowed?"1":"0"; Rargv[21] = alias_file_name; Rargv[22] = cmd_rebuilt; Rargv[23] = is_Input_Need_Reorder?"1":"0"; Rargv[24] = feature_block_size_str; Rargv[25] = strFiveEndExtension; Rargv[26] = strThreeEndExtension; Rargv[27] = strMinFragmentOverlap; Rargv[28] = is_Split_or_Exonic_Only == 1?"1":(is_Split_or_Exonic_Only == 2 ? "2":"0"); Rargv[29] = (reduce_5_3_ends_to_one == 0?"0":(reduce_5_3_ends_to_one==REDUCE_TO_3_PRIME_END?"3":"5")); Rargv[30] = debug_command; Rargv[31] = is_duplicate_ignored?"1":"0"; Rargv[32] = do_not_sort?"1":"0"; Rargv[33] = use_fraction_multimapping?"1":"0"; Rargv[34] = use_overlapping_length_break_tie?"1":"0"; Rargv[35] = Pair_Orientations; Rargv[36] = do_junction_cnt?"1":"0"; Rargv[37] = fasta_contigs_name; Rargv[38] = max_M_str; Rargv[39] = is_Restrictedly_No_Overlap?"1":"0"; Rargv[40] = fracOverlapStr; Rargv[41] = temp_dir; Rargv[42] = std_input_output_mode_str; Rargv[43] = assign_reads_to_RG?"1":"0"; Rargv[44] = long_read_mode_str; Rargv[45] = is_verbose?"1":"0"; Rargv[46] = strFeatureFracOverlap; Rargv[47] = do_detection_call?"1":"0"; Rargv[48] = max_missing_bases_in_read_str; Rargv[49] = max_missing_bases_in_feature_str; Rargv[50] = is_primary_alignment_only?"1":"0"; Rargv[51] = Rpath; Rargv[52] = extra_column_names; Rargv[54] = "NA"; // C featureCounts dosn't need the display_annotation_name. Rargv[54] = read_shift_type; Rargv[55] = read_shift_size_str; Rargv[56] = scRNA_sample_sheet; Rargv[57] = scRNA_cell_barcode_list; Rargv[58] = is_paired_end_reads_expected; Rargv[59] = is_scRNA_BAM_FQ_out_generated?"1":"0"; Rargv[60] = "3"; if(scRNA_input_mode == GENE_INPUT_SCRNA_FASTQ) Rargv[60] = "4"; if(scRNA_input_mode == GENE_INPUT_SCRNA_BAM) Rargv[60] = "5"; Rargv[61] = "0"; Rargv[62] = "-1"; Rargv[63] = "Single-Index"; int retvalue = -1; if(is_ReadSummary_Report && (std_input_output_mode & 1)==1) SUBREADprintf("ERROR: no detailed assignment results can be written when the input is from STDIN. Please remove the '-R' option.\n"); else retvalue = readSummary(64, Rargv); free(very_long_file_names); free(out_name); free(alias_file_name); free(fasta_contigs_name); if(old_zero_smode != Strand_Sensitive_Str)free(Strand_Sensitive_Str); free(cmd_rebuilt); free(Rpath); free(scRNA_sample_sheet); free(scRNA_cell_barcode_list); if(extra_column_names)free(extra_column_names); return retvalue; } Rsubread/src/removeDupReads.c0000644000175200017520000003246014136047615017237 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #include #include #include #include #include #include #include #include "subread.h" #include "sambam-file.h" #include "HelperFunctions.h" #include "removeDupReads.h" #include "input-files.h" extern unsigned int BASE_BLOCK_LENGTH; int generate_SAM_output = 0; int is_debug_mode = 0; unsigned long long read_status_space = 0; //inited later char * read_selection_list = NULL; int is_read_selected(unsigned int read_number) { if(read_number > read_status_space) return 1; unsigned int byte_offset = read_number/8; int bit_offset = read_number & 7; char old_8values = read_selection_list[byte_offset]; return ((old_8values >> bit_offset)&1); } void unselect_read_in_list(unsigned int read_number) { if(read_number >= read_status_space){ size_t old_size = read_status_space/8+1; read_status_space = max(read_status_space*14/10, read_number+1); size_t new_size = read_status_space/8+1; read_selection_list = realloc(read_selection_list, read_status_space); memset(read_selection_list+old_size,0xff, new_size-old_size); } unsigned int byte_offset = read_number/8; int bit_offset = read_number & 7; char old_8values = read_selection_list[byte_offset]; old_8values &= ~(1 << bit_offset); read_selection_list[byte_offset] = old_8values; } srInt_64 total_mapped_reads = 0; unsigned long long reads_passed_filter = 0; int parse_base_blocks(char * temp_prefix, chromosome_t * chromosomes, int threshold, int all_threads, int thread_no) { int chromosome_block_no = 0; int chromosome_no; unsigned long long parsed_reads = 0; unsigned int * read_voting_table; read_voting_table = malloc(sizeof(int) * BASE_BLOCK_LENGTH); if(!read_voting_table) { fatal_memory_size(); return 1; } for(chromosome_no=0 ; chromosomes[chromosome_no].chromosome_name[0]; chromosomes++ ) { int block_in_chro = chromosomes[chromosome_no].known_length / BASE_BLOCK_LENGTH+1; int i; for(i=0;iblock_start_base && read_record.pos <=block_start_base+BASE_BLOCK_LENGTH )){ // break_SAM subtracted 1 from pos SUBREADprintf("FATAL ERROR: unexpected temp file format : %d not between %d and %d.\n", read_record.pos, block_start_base, block_start_base+BASE_BLOCK_LENGTH); return -1; } if(read_record.mapped_segment_in_read==0)read_voting_table[read_record.pos-block_start_base] ++; } fseek(block_fp,0,SEEK_SET); while(!feof(block_fp)) { base_block_temp_read_t read_record; rrtv = fread(&read_record, sizeof(base_block_temp_read_t), 1, block_fp); if(rrtv < 1) break; if(read_record.mapped_segment_in_read==0 ){ parsed_reads ++; if(read_voting_table[read_record.pos-block_start_base]>= threshold) unselect_read_in_list(read_record.read_number); else reads_passed_filter++; } } fclose(block_fp); unlink(temp_file); } } if(total_mapped_reads!= parsed_reads){ SUBREADprintf("FATAL ERROR: parsed %llu reads, but found %llu reads in the temp files\n", total_mapped_reads, parsed_reads); return -1; } free(read_voting_table); return 0; } int parse_base_blocks_maybe_thread(char * temp_location, chromosome_t * chromosomes, int threshold, int threads) { return parse_base_blocks(temp_location, chromosomes,threshold, threads, 0); } unsigned long long written_reads=0; int input_file_type =0; int report_remainder(char *in_SAM_file, char *out_SAM_file) { int read_no = 0; SamBam_FILE * fp = SamBam_fopen(in_SAM_file, input_file_type==FILE_TYPE_BAM?SAMBAM_FILE_BAM:SAMBAM_FILE_SAM); FILE * out_fp =NULL; SamBam_Writer writer; memset(&writer,0, sizeof(writer)); int bam_retv = 1; if(generate_SAM_output) out_fp = f_subr_open(out_SAM_file,"w"); else bam_retv = SamBam_writer_create(&writer, out_SAM_file, -1, 0, 0, "."); if(!(out_fp || bam_retv == 0)) { SUBREADprintf("Unable to open the output file, '%s'.\n",out_SAM_file); return 1; } while(1) { char line_buffer [3000]; char * opret = SamBam_fgets(fp, line_buffer, 2999, 1);//(2999, fp, line_buffer, 0); if(!opret) break; int linelen = strlen(line_buffer) -1; line_buffer[linelen]=0; if(line_buffer[0]=='@') { if(out_fp==NULL){ SamBam_writer_add_header(&writer, line_buffer, 1); }else{ fwrite(line_buffer, linelen, 1, out_fp); fputc('\n', out_fp); } } else if((line_buffer[0] >='A' && line_buffer[0]<='Z') || (line_buffer[0] >='a' && line_buffer[0] <='z') || (line_buffer[0] >='0' && line_buffer[0] <='9') || line_buffer[0] =='_' || line_buffer[0] =='.') { /* if(is_read_selected(read_selection_list, read_no)) fputs("SEL ", out_fp); else fputs(" ", out_fp); */ if(is_read_selected(read_no)){ if(is_debug_mode) SUBREADprintf("DBG_KEPT\t%s\n", line_buffer); if(out_fp==NULL){ SamBam_writer_finish_header(&writer); SamBam_writer_add_read_line(&writer, -1, line_buffer, 1); }else{ fwrite(line_buffer, linelen, 1, out_fp); fputc('\n', out_fp); } char * flgstr = duplicate_TAB_record_field(line_buffer, 1, 0); if(( atoi(flgstr) & 4 )== 0) written_reads++; free(flgstr); }else if(is_debug_mode) SUBREADprintf("DBG_IGNORED\t%s\n", line_buffer); read_no ++; } } SamBam_fclose(fp); if(written_reads!=reads_passed_filter){ SUBREADprintf("FATAL ERROR: no expected reads written : %llu != %llu.\n", written_reads, reads_passed_filter); return -1; } if(out_fp==NULL) SamBam_writer_close(&writer); else fclose(out_fp); return 0; } // Giving temp_location 'NULL' makes the function to use the current directory to store temporary files. // Giving known_read_count '0' makas the function use its default value: known_read_count = 400,000,000, namely ~50MB memory is allocated to store the selection table (other parts of the program may use more memory) // This function returns 0 if everything is OK and -1 if memory is overused. int repeated_read_removal(char * in_SAM_file, int threshold, char * out_SAM_file, char * temp_location, int threads) { char temp_file_prefix[MAX_FILE_NAME_LENGTH]; unsigned int real_read_count = 0; chromosome_t * known_chromosomes; // Checking parameters unsigned int read_selection_list_size = read_status_space / 8 +1; // One bit for a read, one more byte for remainder. input_file_type = probe_file_type_fast(in_SAM_file); if(input_file_type!=FILE_TYPE_SAM && input_file_type!=FILE_TYPE_BAM){ SUBREADprintf("ERROR: The input file is neither a BAM file nor a SAM file.\n"); return -1; } SUBREADprintf("Repeated Read Removal\nInput=%s (%s)\nOutput=%s (%s)\nRemoval Threshold=%d\n\n", get_short_fname(in_SAM_file), input_file_type == FILE_TYPE_SAM? "SAM":"BAM", get_short_fname(out_SAM_file), generate_SAM_output?"SAM":"BAM" , threshold); // All parameters are validated. // Step 0: the read selection list and the chromosome list are initialized to be empty. read_selection_list = (char *) SUBREAD_malloc(sizeof(char) * read_selection_list_size); if(!read_selection_list) { fatal_memory_size(); return -1; } memset(read_selection_list, 0xff, sizeof(char) * read_selection_list_size); known_chromosomes = (chromosome_t *) SUBREAD_malloc(sizeof(chromosome_t) * XOFFSET_TABLE_SIZE); if(!known_chromosomes) { fatal_memory_size(); return -1; } // A chromosome_t is used if the first char of its name is not 0. known_chromosomes[0].chromosome_name[0]=0; // Step 1: scanning the SAM file, classify each read into a block. If a chro_name is unseen before, a new block is created for it and it is put into the chromosome list. Each block has a temporary file "temp-delrep-00000-XXXXXX-chrX-21" where chrX is the chromosome name, '21' is the block number, 00000 is the process id and XXXXXX is a randomly string. char mac_rand[13]; mac_or_rand_str(mac_rand); sprintf(temp_file_prefix, "%s/temp-delrep-%06u-%s-", temp_location==NULL?".":temp_location, getpid(), mac_rand); if(break_SAM_file(in_SAM_file, input_file_type == FILE_TYPE_BAM, temp_file_prefix, &real_read_count, NULL, known_chromosomes, 0 /* This 0 means that the sequence/quality/cigar fields are not needed in the temp files*/, 0, NULL, NULL, NULL, NULL, NULL, &total_mapped_reads, 0,1, 0)){ SUBREADprintf("ERROR: cannot parse the input file.\n"); return -1; }else SUBREADprintf("The input file contains %llu mapped reads.\n", total_mapped_reads); // Step 2: initialize the read voting table, scanning each temporary file. Each read in the temporary file is a vote to a location in the voting table. Then, each read in the temporary file is scanned again against the voting table. If the mapping location of this read receives >=threshold votes in the table, the read is removed from the read selection list. if(parse_base_blocks_maybe_thread(temp_file_prefix, known_chromosomes, threshold, threads)){ SUBREADprintf("ERROR: cannot process temperary reads.\n"); return -1; } // Step 3: rescanning the SAM file, copy a line to output file if its line number is marked 'selected' in the read selection list. if(report_remainder(in_SAM_file, out_SAM_file)){ SUBREADprintf("ERROR: cannot generate output files.\n"); return -1; } free(read_selection_list); free(known_chromosomes); SUBREADprintf("Finished. Processed %llu mapped reads; %llu (%.1f%%) reads were removed due to duplication.\n", total_mapped_reads, total_mapped_reads-written_reads, 100.*(total_mapped_reads-written_reads)/total_mapped_reads); return 0; } void print_usage_rrr(char * myname) { SUBREADprintf("\nremoveDup Version %s\n\n", SUBREAD_VERSION); SUBREADputs(" Remove duplicated reads."); SUBREADputs(""); SUBREADputs("Usage:"); SUBREADputs(" ./removeDup [options] -i -o \n"); SUBREADputs("Required arguments:"); SUBREADputs(""); SUBREADputs(" -i Name of input file in SAM/BAM format."); SUBREADputs(""); SUBREADputs(" -o Name of output SAM file including filtered reads. The format is"); SUBREADputs(" BAM unless '-S' is specified."); SUBREADputs(""); SUBREADputs("Optional arguments:"); SUBREADputs(""); SUBREADputs(" -S Generate the SAM format output."); SUBREADputs(""); SUBREADputs(" -r Specify the duplication cutoff. All the reads mapped to a location"); SUBREADputs(" are removed from the output if the number of reads mapped to this"); SUBREADputs(" location is equal or higher than the cutoff. 10 by default."); SUBREADputs(""); SUBREADputs(" -t A directory storing temporary files generated by the program."); SUBREADputs(""); } struct option rem_long_options[]={{0,0,0,0}}; #ifdef MAKE_STANDALONE int main(int argc,char ** argv) #else // This function is actually useless because R uses repeated_read_removal() as the entry. However no main() function should be remained while building .so for R. int main_repeated_test(int argc,char ** argv) #endif { char c; char input_SAM_file[MAX_FILE_NAME_LENGTH]; char output_SAM_file[MAX_FILE_NAME_LENGTH]; char temp_path[MAX_FILE_NAME_LENGTH]; int threshold, optindex = 0; int threads; generate_SAM_output=0; read_status_space=40000000; read_selection_list=NULL; is_debug_mode=0; total_mapped_reads=0; reads_passed_filter=0; written_reads=0; input_file_type=0; input_SAM_file [0] = 0; output_SAM_file[0] = 0; temp_path[0]=0; threshold = 10; threads = 0; optind=0; opterr=1; optopt=63; if(argc<2) { print_usage_rrr(argv[0]); return 0; } while ((c = getopt_long (argc, argv, "Di:o:r:t:S?", rem_long_options, &optindex)) != -1) { switch (c) { case 'D': is_debug_mode=1; break; case 'i': strncpy(input_SAM_file, optarg,MAX_FILE_NAME_LENGTH-1); break; case 'o': strncpy(output_SAM_file, optarg,MAX_FILE_NAME_LENGTH-1); break; case 't': strncpy(temp_path, optarg,MAX_FILE_NAME_LENGTH-1); break; case 'T': threads = atoi(optarg); break; case 'S': generate_SAM_output = 1; break; case 'r': threshold = atoi(optarg); break; case '?': default: print_usage_rrr(argv[0]); } } return repeated_read_removal(input_SAM_file, threshold, output_SAM_file, temp_path[0]?temp_path:NULL, threads); } Rsubread/src/removeDupReads.h0000644000175200017520000000264014136047615017241 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #ifndef __REP_READ_REMO_H__ #define __REP_READ_REMO_H__ #include "subread.h" typedef struct{ unsigned short * repeating_times; } read_voting_table_t; // Giving temp_location 'NULL' makes the function to use the current directory to store temporary files. // Giving read_count '0' makas the function use its default value: read_count = 400,000,000, namely ~50MB memory is used to store the selection table (other parts of the program may use more memory) // Note that this function generates ( 4 * all_reads ) bytes of temporary files in the current director or the directory specified in temp_location. int repeated_read_removal(char * in_SAM_file, int threshold, char * out_SAM_file, char * temp_location, int threads); #endif Rsubread/src/sam2bed.c0000644000175200017520000000350214136047615015622 0ustar00biocbuildbiocbuild/*************************************************************** The Subread and Rsubread software packages are free software packages: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #define _GNU_SOURCE #include #include #include int sam2bed(int argc,char *argv[]){ /* if(argc == 1){ printf("Usage: sam2bed -n read_length sam_filename bed_filename\n"); exit(0); } */ FILE *fp, *fp_out; fp=fopen(argv[3],"r"); fp_out=fopen(argv[4],"w"); char * line = NULL; char strand; char * chr, *tok; int readlen, chr_start, chr_end, flag, mqs; int MAX_LINE_LENGTH = 100000; readlen = atoi(argv[2]); line = (char*)calloc(MAX_LINE_LENGTH, 1); while (fgets(line, MAX_LINE_LENGTH, fp)) { if(line[0] == '@') continue; tok = strtok(line,"\t"); if(tok == NULL) continue; flag = atoi(strtok(NULL,"\t")); chr = strtok(NULL,"\t"); if(chr[0] != '*'){ chr_start = atoi(strtok(NULL,"\t")) - 1; chr_end = chr_start + readlen; mqs = atoi(strtok(NULL,"\t")); if ((flag & 0x10) == 0){ strand = '+'; } else{ strand = '-'; } fprintf(fp_out,"%s\t%d\t%d\t%s\t%d\t%c\n", chr, chr_start, chr_end, ".",mqs,strand); } } if (line) free(line); fclose(fp); fclose(fp_out); return 0; } Rsubread/src/sambam-file.c0000644000175200017520000025554714136047615016504 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ /*************************************************************** The SamBam_reg2bin function was derived from the BAM specification. (The SAM Format Specication Working Group, September 7, 2011) ***************************************************************/ #include #include #include #include #include #include "subread.h" #include "core.h" #include "HelperFunctions.h" #include "gene-algorithms.h" #include "sambam-file.h" #include "input-files.h" #define Z_DEFAULT_MEM_LEVEL 8 int SamBam_fetch_next_chunk(SamBam_FILE *fp) { int x1, room = SAMBAM_INPUT_STREAM_SIZE - ( fp -> input_binary_stream_write_ptr - fp -> input_binary_stream_read_ptr); if(room < 65536) return -1; for(x1=0; x1 < fp->input_binary_stream_write_ptr - fp -> input_binary_stream_read_ptr; x1 ++) { fp -> input_binary_stream_buffer [x1] = fp -> input_binary_stream_buffer [x1 + fp->input_binary_stream_read_ptr - fp -> input_binary_stream_buffer_start_ptr]; } fp -> input_binary_stream_buffer_start_ptr = fp->input_binary_stream_read_ptr; char * in_buff = malloc(65537); unsigned int real_len = 0; int ret, have = 0; while (1){ int nchunk = 0; ret = PBam_get_next_zchunk(fp -> os_file, in_buff, 65536, & real_len); if(ret > 0) nchunk = SamBam_unzip(fp -> input_binary_stream_buffer + fp->input_binary_stream_write_ptr - fp -> input_binary_stream_read_ptr + have, 65536 , in_buff , ret, 0); else if(ret == -2){ SUBREADputs("ERROR: BAM format is broken."); return -2; } //printf("RET=%d; CHK=%d\n", ret, nchunk); if(nchunk>0) have += nchunk; if(have > 3000) break; if(feof(fp->os_file)){ fp->is_eof=1; break; } } free(in_buff); fp -> input_binary_stream_write_ptr += have; return have; } int is_paired_end_BAM(char * fn){ FILE * fp = fopen(fn, "rb"); if(!fp) return 0; unsigned char h2[2]; int retvv = fread(h2, 1,2,fp); if(retvv <2) return 0; int is_bam = (h2[0]==31) && (h2[1]==139); fclose(fp); SamBam_FILE * sambam_reader; char fline[3000]; sambam_reader = SamBam_fopen(fn, is_bam?SAMBAM_FILE_BAM:SAMBAM_FILE_SAM); while(1){ char * is_ret = SamBam_fgets(sambam_reader, fline, 2999, 1); if(!is_ret) return 0; if(fline[0]!='@') break; } char * tok = NULL; strtok_r(fline, "\t", &tok); int flags = atoi(strtok_r(NULL, "\t", &tok)); SamBam_fclose(sambam_reader); return flags & 1; } SamBam_FILE * SamBam_fopen(char * fname , int file_type) { SamBam_FILE * ret = (SamBam_FILE *)malloc(sizeof(SamBam_FILE)); memset(ret, 0, sizeof(SamBam_FILE)); ret -> file_type = file_type; if(file_type ==SAMBAM_FILE_SAM) { ret -> os_file = f_subr_open(fname, "rb"); if(!ret -> os_file) { free(ret); return NULL; } ret -> header_length = 0; char nch0=-1, nchold=-1; while(!feof(ret -> os_file)){ nchold = nch0; nch0=fgetc(ret -> os_file); if(nchold=='\n' && nch0!='@') break; if(nch0!='@' && ret -> header_length == 0){ ret -> header_length = 0; break; } ret -> header_length++; } //SUBREADprintf("The SAM header has %d bytes\n", ret -> header_length); fseek(ret -> os_file,0,SEEK_SET); } else { ret -> os_file = f_subr_open(fname, "rb"); if(ret -> os_file == NULL) { free(ret); return NULL; } unsigned char first_ch = fgetc(ret->os_file); unsigned char second_ch = fgetc(ret->os_file); if(first_ch!=31 || second_ch!=139) { free(ret); SUBREADprintf("Not a BAM file! %d %d\n", first_ch, second_ch); return NULL; } fseek(ret->os_file, 0, SEEK_SET); ret -> input_binary_stream_buffer = (char *)malloc(SAMBAM_INPUT_STREAM_SIZE); ret -> input_binary_stream_read_ptr = 0; ret -> input_binary_stream_write_ptr = 0; ret -> input_binary_stream_buffer_start_ptr = 0; ret -> bam_file_stage = BAM_FILE_STAGE_HEADER; ret -> is_eof = 0; SB_FETCH(ret); if(SB_EOF(ret)) { free(ret->input_binary_stream_buffer); free(ret); SUBREADprintf("FEOF 0.\n"); return NULL; } int magic_4 = 0; memcpy(&magic_4 , SB_READ(ret), 4); SB_RINC(ret, 4); if(magic_4 != 21840194) // this number is the four bytes of "BAM1" { free(ret->input_binary_stream_buffer); free(ret); SUBREADprintf("FEOF 4 == %d.\n", magic_4); return NULL; } int l_text = 0; memcpy(&l_text, SB_READ(ret), 4); SB_RINC(ret, 4); ret -> bam_file_next_section_start = ret -> input_binary_stream_read_ptr + l_text; ret -> header_length = ret -> bam_file_next_section_start; } return ret; } char cigar_op_char(int ch) { if(ch<9) return "MIDNSHP=X"[ch]; else { SUBREADprintf("Unknwon opt was found in the CIGAR string: '%c'.\n", ch); return 'M'; } } char read_int_char(int ch) { assert(ch<16); return "=ACMGRSVTWYHKDBN"[ch]; } void SamBam_fclose(SamBam_FILE * fp) { if(fp->file_type==SAMBAM_FILE_SAM) { fclose(fp->os_file); free(fp); } else { fclose(fp->os_file); free(fp -> input_binary_stream_buffer); free(fp -> bam_chro_table); free(fp); } } int SamBam_feof(SamBam_FILE * fp) { if(fp->file_type ==SAMBAM_FILE_SAM) return feof(fp->os_file); else return SB_EOF(fp); } void SamBam_read_ref_info(SamBam_FILE * ret) { unsigned int ref_info_size = 0; ret ->bam_chro_table_size = 0; //printf("CKK0\n"); SB_FETCH(ret); if(SB_EOF(ret)) return; //printf("CKK1\n"); memcpy(&ref_info_size, SB_READ(ret),4); SB_RINC(ret, 4); int xk1; ret -> bam_chro_table = malloc(sizeof(SamBam_Reference_Info) * ref_info_size); for(xk1=0;xk1 bam_chro_table[xk1].chro_name, SB_READ(ret), ref_readin_len); ret -> bam_chro_table[xk1].chro_name[ref_readin_len] = 0; SB_RINC(ret, ref_readin_len + ref_skip_len); memcpy(&(ret -> bam_chro_table[xk1].chro_length), SB_READ(ret),4); SB_RINC(ret, 4); //SUBREADprintf("CHRO[%d] : %s [%d]\n", xk1+1, ret -> bam_chro_table[xk1].chro_name , ret -> bam_chro_table[xk1].chro_length); } ret ->bam_chro_table_size = ref_info_size; } char * SamBam_fgets(SamBam_FILE * fp, char * buff , int buff_len, int seq_needed) { if(fp->file_type==SAMBAM_FILE_SAM){ char * ret = fgets(buff, buff_len, fp->os_file); int strlenbuff = 0; if(ret){ strlenbuff = strlen(buff); ///if(buff[0]!='@') // if(strlenbuff < 100)SUBREADprintf("WRONG LOAD: '%s'\n", buff); } if(strlenbuff < 1 || ret == NULL) return NULL; else{ if(ret[strlenbuff-1]!='\n') { while(1) { int ch = getc(fp->os_file); if(ch == EOF || ch == '\n')break; } ret[strlenbuff-1] = '\n'; } if(fp -> is_paired_end < 10){ if(buff[0]!='@'){ int tabs = 0,x1=0, tmpi=0; for(x1 = 0; x1 < strlenbuff; x1++){ if(buff[x1] == '\t'){ if(tabs == 1){ // SUBREADprintf("TMPI_SAM = %d\n", tmpi); fp -> is_paired_end = 10 + (tmpi & 1); break; } else tabs ++; }else{ if(tabs == 1)tmpi = tmpi * 10 + buff[x1]-'0'; } } } } if(buff[0] == '@'){ fp -> header_length = ftello(fp->os_file) + strlenbuff+1; } return ret; } } else { int xk1; // decrypt the BAM mess. if(fp-> bam_file_stage == BAM_FILE_STAGE_HEADER) { char nch; xk1=0; SB_FETCH(fp); if(SB_EOF(fp)) return NULL; while(1) { if(fp -> input_binary_stream_read_ptr >= fp -> bam_file_next_section_start) break; SB_FETCH(fp); nch = *(SB_READ(fp)); SB_RINC(fp,1); //printf("%c", nch); if(nch == '\r')continue; if(SB_EOF(fp)||nch == '\n' || nch <0) break; if(xk1 < buff_len-2) { buff[xk1]=nch; xk1++; } } buff[xk1]='\n'; buff[xk1+1]=0; //SUBREADprintf("BUFF=%s\n========================================================================\n\n", buff); //printf("RL=%d , PTR %d >? RECORD_START %d\n\n\n", xk1, fp -> input_binary_stream_read_ptr , fp -> bam_file_next_section_start); if(fp -> input_binary_stream_read_ptr >= fp -> bam_file_next_section_start) { SamBam_read_ref_info(fp); fp -> bam_file_stage = BAM_FILE_STAGE_ALIGNMENT; fp -> header_length = fp-> input_binary_stream_read_ptr; } return buff; } else { SamBam_Alignment *aln = &fp->aln_buff; int chunk_ptr = 0; SB_FETCH(fp); if(SB_EOF(fp)) return NULL; fp -> is_paired_end = 10 + ((*(fp -> input_binary_stream_buffer + fp -> input_binary_stream_read_ptr - fp -> input_binary_stream_buffer_start_ptr + 18)) & 1); //SUBREADprintf("FLAG=%d\n", *(fp -> input_binary_stream_buffer + fp -> input_binary_stream_read_ptr - fp -> input_binary_stream_buffer_start_ptr + 18) ); int text_len = PBam_chunk_gets(SB_READ(fp) , &chunk_ptr, fp -> input_binary_stream_write_ptr - fp -> input_binary_stream_read_ptr , fp -> bam_chro_table, buff , buff_len, aln, seq_needed); SB_RINC(fp, chunk_ptr); if(text_len>0) return buff; return NULL; } } } int PBam_get_next_zchunk(FILE * bam_fp, char * buffer, int buffer_length, unsigned int * real_len) { unsigned char ID1, ID2, CM, FLG; unsigned short XLEN; int BSIZE=-1, rlen, is_file_broken = 0, rrtv; if(feof(bam_fp)) return -1; rrtv = fread(&ID1, 1, 1, bam_fp); if(rrtv <1) return -1; rrtv = fread(&ID2, 1, 1, bam_fp); if(rrtv <1) return -1; rrtv = fread(&CM, 1, 1, bam_fp); if(rrtv <1) return -1; rrtv = fread(&FLG, 1, 1, bam_fp); if(rrtv <1) return -1; if(feof(bam_fp)) return -1; if(ID1!=31 || ID2!=139 || CM!=8 || FLG!=4) { //SUBREADprintf("4CHR = %d, %d, %d, %d\n", ID1, ID2, CM, FLG); return -1; } fseeko(bam_fp, 6, SEEK_CUR); rrtv = fread(&XLEN,1, 2, bam_fp ); if(rrtv < 2) return -1; int XLEN_READ = 0; while(1) { unsigned char SI1, SI2; unsigned short SLEN, BSIZE_MID; rrtv = fread(&SI1, 1, 1, bam_fp); if(rrtv <1) return -1; rrtv = fread(&SI2, 1, 1, bam_fp); if(rrtv <1) return -1; rlen = fread(&SLEN, 2, 1, bam_fp); if(rlen < 1) is_file_broken = 1; if(SI1==66 && SI2== 67 && SLEN == 2) { rrtv = fread(&BSIZE_MID, 1,2 , bam_fp); if(rrtv <2) return -1; BSIZE = BSIZE_MID; } else fseeko(bam_fp, SLEN, SEEK_CUR); XLEN_READ += SLEN + 4; if(XLEN_READ>=XLEN) break; } if(BSIZE>19) { int CDATA_LEN = BSIZE - XLEN - 19; int CDATA_READING = min(CDATA_LEN, buffer_length); rrtv = fread(buffer, 1, CDATA_READING, bam_fp); if(rrtv < CDATA_READING) return -1; if(CDATA_READING0) { int chro_name_len; unsigned int chro_len; (*reminder_byte_len) = chunk_len - (*chunk_ptr); if( (*chunk_ptr) < chunk_len-4) { memcpy(&chro_name_len, chunk + (*chunk_ptr),4); (*chunk_ptr)+=4; if( (*chunk_ptr) <= chunk_len-chro_name_len-4) { char * chro_name = chunk + (*chunk_ptr); (*chunk_ptr)+=chro_name_len; memcpy(&chro_len, chunk + (*chunk_ptr),4); (*chunk_ptr)+=4; (*reminder_byte_len) =0; //todo: insert item if(0==(* table_items)) { (*table_size) = 50; (*bam_chro_table) = malloc(sizeof(SamBam_Reference_Info)*50); } else if((*table_size) <= (* table_items)) { (*table_size) *= 2; (*bam_chro_table) = realloc((*bam_chro_table),sizeof(SamBam_Reference_Info)*(*table_size)); } SamBam_Reference_Info * new_event = (*bam_chro_table) + (* table_items); if(strlen(chro_name)>=BAM_MAX_CHROMOSOME_NAME_LEN) chro_name[BAM_MAX_CHROMOSOME_NAME_LEN-1]=0; strcpy(new_event->chro_name, chro_name); new_event -> chro_length = chro_len; (* table_items)++; //SUBREADprintf("CHRO %d/%d added\n", (* table_items),(remainder_chrs)); remainder_chrs --; } else break; } else break; } if(remainder_chrs) { (*state) = 3; (*header_txt_remainder) = remainder_chrs; return 1; } else{ (*state) = 4; return 0; } } return -1; } int convert_BAM_binary_to_SAM( SamBam_Reference_Info * chro_table, char * bam_bin, char * sam_txt ){ int bin_len = 0; memcpy(&bin_len, bam_bin, 4); bin_len += 4; int sam_ptr = 0, tmpint = 0; sam_ptr += sprintf(sam_txt + sam_ptr, "%s\t", bam_bin+36); memcpy(&tmpint, bam_bin + 16 ,4); sam_ptr += sprintf(sam_txt + sam_ptr, "%d\t", (tmpint >> 16) & 0xffff); int cigar_opts = tmpint & 0xffff; memcpy(&tmpint, bam_bin + 4 ,4); int r1chro = tmpint; sam_ptr += sprintf(sam_txt + sam_ptr, "%s\t", tmpint<0?"*":chro_table[tmpint].chro_name); memcpy(&tmpint, bam_bin + 8 ,4); sam_ptr += sprintf(sam_txt + sam_ptr, "%d\t", tmpint+1); memcpy(&tmpint, bam_bin + 12 ,4); sam_ptr += sprintf(sam_txt + sam_ptr, "%d\t", (tmpint >> 8) & 0xff); int name_len = tmpint & 0xff; int cigar_i; for(cigar_i = 0; cigar_i < cigar_opts; cigar_i ++){ unsigned int cigarint=0; memcpy(&cigarint, bam_bin + name_len + 36 + cigar_i * 4,4); sam_ptr += sprintf(sam_txt + sam_ptr, "%u%c", cigarint >> 4, "MIDNSHP=X"[cigarint&0xf]); } sam_ptr += sprintf(sam_txt + sam_ptr, "%s\t", cigar_i<1?"*":""); memcpy(&tmpint, bam_bin + 24, 4); //SUBREADprintf("CHRO_IDX=%d\n", tmpint); sam_ptr += sprintf(sam_txt + sam_ptr, "%s\t", tmpint<0?"*":((tmpint == r1chro)?"=":chro_table[tmpint].chro_name)); memcpy(&tmpint, bam_bin + 28, 4); sam_ptr += sprintf(sam_txt + sam_ptr, "%d\t", tmpint+1); memcpy(&tmpint, bam_bin + 32, 4); sam_ptr += sprintf(sam_txt + sam_ptr, "%d\t", tmpint); int seq_len; memcpy(&seq_len, bam_bin + 20,4); int seqi, flex_ptr=name_len + 36 + cigar_opts * 4; for(seqi=0; seqi> ( 4*!(seqi %2) )) & 15 ]; if(seqi %2) flex_ptr++; } sam_txt[sam_ptr++]='\t'; if(seqi %2) flex_ptr++; for(seqi=0; seqi chunk_limit) return -1; memcpy(&block_size, chunk+(*chunk_ptr), 4); //SUBREADprintf("PBSIZE=%u\n", block_size); (*chunk_ptr)+=4; unsigned int next_start = block_size+(*chunk_ptr); int ref_id; memcpy(&ref_id, chunk+(*chunk_ptr), 4); (*chunk_ptr)+=4; if(ref_id == -1) aln -> chro_name = NULL; else aln -> chro_name = bam_chro_table[ref_id].chro_name; memcpy(&(aln -> chro_offset), chunk+(*chunk_ptr), 4); (*chunk_ptr)+=4; unsigned int comb1; memcpy(&comb1, chunk+(*chunk_ptr), 4); (*chunk_ptr)+=4; aln -> mapping_quality = 0xff & (comb1 >> 8); unsigned int comb2; memcpy(&comb2, chunk+(*chunk_ptr), 4); (*chunk_ptr)+=4; aln -> flags = 0xffff&(comb2 >> 16); unsigned int read_len; memcpy(&read_len, chunk+(*chunk_ptr), 4); (*chunk_ptr)+=4; unsigned int mate_ref_id; memcpy(&mate_ref_id, chunk+(*chunk_ptr), 4); (*chunk_ptr)+=4; if(mate_ref_id == -1) aln -> mate_chro_name = NULL; else aln -> mate_chro_name = bam_chro_table[mate_ref_id].chro_name; memcpy(&(aln -> mate_chro_offset), chunk+(*chunk_ptr), 4); (*chunk_ptr)+=4; memcpy(&(aln -> templete_length), chunk+(*chunk_ptr), 4); (*chunk_ptr)+=4; int read_name_len = comb1 & 0xff; assert(read_name_len < BAM_MAX_READ_NAME_LEN); memcpy(aln -> read_name, chunk+(*chunk_ptr), read_name_len); aln -> read_name[read_name_len] = 0; (*chunk_ptr)+=read_name_len; int cigar_ops = comb2 & 0xffff; aln -> cigar[0]=0; for(xk1=0; xk1 chunk_limit) return -1; memcpy(&cigar_piece, chunk+(*chunk_ptr),4); (*chunk_ptr)+=4; sprintf(cigar_piece_buf, "%u%c", cigar_piece>>4, cigar_op_char(cigar_piece&0xf)); if(strlen(cigar_piece_buf)+strlen(aln->cigar)cigar, cigar_piece_buf); else { SUBREADprintf("WARNING: cigar string is too long to the buffer.\n"); SUBREADprintf("Please only use the compressed BAM format.\n"); assert(0); return -1; } } char read_2_seq = 0; int seq_qual_bytes = read_len + (read_len /2)+(read_len%2); if(seq_needed) memcpy( aln-> buff_for_seq, chunk+(*chunk_ptr), seq_qual_bytes); (*chunk_ptr) += seq_qual_bytes; char extra_tags [CORE_ADDITIONAL_INFO_LENGTH]; extra_tags[0]=0; int extra_len = 0; while( (*chunk_ptr) < next_start) { char extag[2]; char extype; int delta, need_tag = 1; memcpy(extag, chunk+(*chunk_ptr), 2); extype = chunk[2+(*chunk_ptr)]; (*chunk_ptr)+=3; //fprintf(stderr, "COL_EXTYPE: %c\n", extype); if(extype == 'Z' || extype == 'H') { delta = 0; // 'Z' columns are NULL-terminated. while(chunk[delta + (*chunk_ptr)]) delta++; delta += 1; } else if(extype == 'A' || extype == 'c' || extype=='C') delta=1; else if(extype == 'i' || extype=='I' || extype == 'f') delta=4; else if(extype == 's' || extype=='S') delta=2; else if(extype == 'B') { extype = chunk[(*chunk_ptr)]; // fprintf(stderr, "B_EXTYPE: %c\n", extype); (*chunk_ptr)++; if(extype == 'A' || extype=='Z') delta=1; else if(extype == 'c' || extype=='C') delta=1; else if(extype == 'i' || extype=='I' || extype == 'f') delta=4; else if(extype == 's' || extype=='S') delta=2; else break; int array_len; need_tag = 0; memcpy(&array_len, chunk+(*chunk_ptr), 4); (*chunk_ptr)+=4; delta *= array_len; } else{ // fprintf(stderr, "NO_EXTYPE: %c\n", extype); break; } if(need_tag){ if(extype == 'c' || extype=='C' || extype == 'i' || extype=='I' || extype == 's' || extype=='S'){ int tmpi = 0; memcpy(&tmpi, chunk+(*chunk_ptr),delta); if(tmpi >= 0 && extra_len < CORE_ADDITIONAL_INFO_LENGTH - 18){ int sret = sprintf(extra_tags + strlen(extra_tags), "\t%c%c:i:%d", extag[0], extag[1], tmpi); extra_len += sret; } }else if(extype == 'Z'){ if(extra_len < CORE_ADDITIONAL_INFO_LENGTH - 7 - delta){ sprintf(extra_tags + strlen(extra_tags), "\t%c%c:Z:", extag[0], extag[1]); extra_len += 6; *(extra_tags + strlen(extra_tags)+delta-1) = 0; memcpy(extra_tags + strlen(extra_tags), chunk + (*chunk_ptr), delta - 1); extra_len += delta - 1; } }else if(extype == 'A'){ if(extra_len < CORE_ADDITIONAL_INFO_LENGTH - 8){ int sret = sprintf(extra_tags + strlen(extra_tags), "\t%c%c:A:%c", extag[0], extag[1], *(chunk + *chunk_ptr) ); extra_len += sret; } } } if((*chunk_ptr) + delta > chunk_limit) return -1; (*chunk_ptr)+=delta; } if(next_start > chunk_limit) return -1; (*chunk_ptr) = next_start; if(seq_needed) { for(xk1=0;xk1 buff_for_seq[xk1/2]; } if(xk1 < BAM_MAX_READ_LEN) aln -> sequence[xk1] = read_int_char(0xf&(read_2_seq >> (xk1%2?0:4))); } aln -> sequence[min(BAM_MAX_READ_LEN-1,read_len)] = 0; if(read_len >= BAM_MAX_READ_LEN-1) SUBREADprintf("WARNING: read is too long to the buffer\n"); for(xk1=0;xk1 buff_for_seq[(read_len /2)+(read_len%2) + xk1] ; if(xk1 < BAM_MAX_READ_LEN) aln -> seq_quality[xk1] = 33+read_2_seq; } aln -> seq_quality[min(BAM_MAX_READ_LEN-1,read_len)] = 0; if(aln -> seq_quality[0]==' ') strcpy(aln -> seq_quality, "*"); } else { aln -> sequence[0]='N'; aln -> sequence[1]=0; aln -> seq_quality[0]='#'; aln -> seq_quality[1]=0; } char * chro_name = "*"; char * cigar = "*"; unsigned int chro_offset = 0; if(aln -> chro_name){ chro_name = aln -> chro_name; chro_offset = aln -> chro_offset+1; if(aln -> cigar[0]) cigar = aln -> cigar; } char * mate_chro_name = "*"; unsigned int mate_chro_offset = 0; if(aln -> mate_chro_name) { if(aln -> mate_chro_name == chro_name) mate_chro_name = "="; else mate_chro_name = aln -> mate_chro_name; mate_chro_offset = aln -> mate_chro_offset+1; } long long int templete_length = aln -> templete_length; //fprintf(stderr, "HN_TAG=%d\n", nh_val ); #ifdef __MINGW32__ int plen = snprintf(buff, buff_len-1, "%s\t%u\t%s\t%u\t%d\t%s\t%s\t%u\t%I64d\t%s\t%s%s\n%c", aln -> read_name, aln -> flags , chro_name, chro_offset, aln -> mapping_quality, cigar, mate_chro_name, mate_chro_offset, templete_length, aln -> sequence , aln -> seq_quality, extra_tags, 0); #else int plen = snprintf(buff, buff_len-1, "%s\t%u\t%s\t%u\t%d\t%s\t%s\t%u\t%lld\t%s\t%s%s\n%c", aln -> read_name, aln -> flags , chro_name, chro_offset, aln -> mapping_quality, cigar, mate_chro_name, mate_chro_offset, templete_length, aln -> sequence , aln -> seq_quality, extra_tags, 0); #endif //fprintf(stderr,"%s", buff); return plen; } int PBum_load_header(FILE * bam_fp, SamBam_Reference_Info** chro_tab, char * remainder_reads_data , int * remainder_reads_data_len) { char * CDATA = malloc(80010); char * PDATA = malloc(1000000); int chro_tab_size = 0, chro_tab_items = 0, chro_tab_state = 0, header_remainder = 0, remainder_byte_len = 0, bam_is_broken = 0; z_stream strm; while(1) { unsigned int real_len = 0; int rlen = PBam_get_next_zchunk(bam_fp,CDATA,80000, & real_len); if(rlen<0){ bam_is_broken = (rlen == -2); if(bam_is_broken){ SUBREADprintf("BAM file format error.\n"); free(CDATA); free(PDATA); return -1; } break; } strm.zalloc = Z_NULL; strm.zfree = Z_NULL; strm.opaque = Z_NULL; strm.avail_in = 0; strm.next_in = Z_NULL; int ret = inflateInit2(&strm, SAMBAM_GZIP_WINDOW_BITS); if (ret != Z_OK) { free(CDATA); free(PDATA); return -1; } strm.avail_in = (unsigned int)rlen; strm.next_in = (unsigned char *)CDATA; strm.avail_out = 1000000 - remainder_byte_len; strm.next_out = (unsigned char *)(PDATA + remainder_byte_len); ret = inflate(&strm, Z_FINISH); int have = 1000000 - strm.avail_out; int PDATA_ptr=0; inflateEnd(&strm); ret = PBam_chunk_headers(PDATA, &PDATA_ptr, have, chro_tab, &chro_tab_size, &chro_tab_items, &chro_tab_state, &header_remainder,&remainder_byte_len); memcpy(PDATA , PDATA + have - remainder_byte_len, remainder_byte_len); if(ret<0) { SUBREADprintf("Header error.\n"); free(CDATA); free(PDATA); return -1; } else if(ret == 0) { //SUBREADprintf("Header loaded = %d\n", (chro_tab_items)); remainder_byte_len=0; } if(chro_tab_state>3){ if(remainder_reads_data && PDATA_ptr < have) { memcpy(remainder_reads_data , PDATA + PDATA_ptr, have - PDATA_ptr); (*remainder_reads_data_len) = have - PDATA_ptr ; } break; } } free(CDATA); free(PDATA); return 0; } int test_bamview(int argc, char ** argv) { if(argc>1) { SamBam_FILE * fp = SamBam_fopen(argv[1], SAMBAM_FILE_BAM); assert(fp); /* while(1) { char buf[3000]; char * buf2 = SamBam_fgets(fp,buf, 3000); //SUBREADprintf(">>%s<<\n",buf); //if(buf2) // fwrite(buf,strlen(buf), 1, stdout); //else break; } */ SamBam_fclose(fp); } return 0; } int SamBam_writer_create(SamBam_Writer * writer, char * BAM_fname, int threads, int sort_reads_by_coord, int is_temp_BAM_file, char * tmpfname) { memset(writer, 0, sizeof(SamBam_Writer)); if(BAM_fname) { writer -> bam_fp = f_subr_open(BAM_fname, "wb"); if(sort_reads_by_coord){ char tname[MAX_FILE_NAME_LENGTH]; sprintf(tname, "%s.bai", BAM_fname); writer -> BAI_fp = f_subr_open(tname, "wb"); worker_master_mutex_init(&writer->sorted_notifier, threads); } if(!writer -> bam_fp) return -1; } #ifdef MAKE_STANDALONE else writer -> bam_fp = stdout; #endif writer -> threads = threads; writer -> sort_reads_by_coord = sort_reads_by_coord; writer -> fastest_compression = is_temp_BAM_file; writer -> compressed_chunk_buffer = malloc(70000); writer -> chunk_buffer = malloc(70000); writer -> chunk_buffer_max_size = 70000; strcpy(writer -> tmpf_prefix, tmpfname); if(threads>= 2){ int x1; writer -> threads_chunk_buffer = malloc(sizeof(char *) * threads) ; writer -> threads_chunk_buffer_compressed = malloc(sizeof(char *) * threads) ; writer -> threads_chunk_buffer_used = malloc(sizeof(long long) * threads); writer -> threads_output_stream = malloc(sizeof(z_stream) * threads); writer -> threads_chunk_buffer_max_size = malloc(sizeof(long long) * threads); memset(writer -> threads_chunk_buffer_used, 0, sizeof(long long) * threads); for(x1 = 0; x1 < threads ; x1++){ writer -> threads_chunk_buffer [x1] = malloc(70000); writer -> threads_chunk_buffer_compressed [x1] = malloc(70000); writer -> threads_chunk_buffer_max_size[x1] = 70000; } subread_init_lock(&writer -> thread_bam_lock); } writer -> chromosome_name_table = HashTableCreate(1603); writer -> chromosome_id_table = HashTableCreate(1603); writer -> chromosome_len_table = HashTableCreate(1603); writer -> header_plain_text_buffer = malloc(100000000); writer -> header_plain_text_buffer_max = 100000000; writer -> header_plain_text_buffer_used = 0; //memset(writer -> header_plain_text_buffer , 0 , 100000000); HashTableSetHashFunction(writer -> chromosome_name_table , fc_chro_hash); HashTableSetKeyComparisonFunction(writer -> chromosome_name_table , fc_strcmp_chro); HashTableSetDeallocationFunctions(writer -> chromosome_name_table , free, NULL); return 0; } void SamBam_writer_chunk_header(SamBam_Writer * writer, int compressed_size) { // the four magic characters fputc(31, writer -> bam_fp); fputc(139, writer -> bam_fp); fputc(8, writer -> bam_fp); fputc(4, writer -> bam_fp); time_t time_now = 0; fwrite(&time_now,4,1, writer -> bam_fp); int tmp_i; // Extra flags and OS fputc(0, writer -> bam_fp); fputc(0xff, writer -> bam_fp); // Extra length tmp_i = 6; fwrite(&tmp_i,2,1, writer -> bam_fp); // SI1 and SI2 magic numbers, and SLEN fputc(66, writer -> bam_fp); fputc(67, writer -> bam_fp); tmp_i = 2; fwrite(&tmp_i,2,1, writer -> bam_fp); tmp_i = compressed_size + 19 + 6; fwrite(&tmp_i,2,1, writer -> bam_fp); } unsigned int SamBam_CRC32(char * dat, int len) { unsigned int crc0 = crc32(0, NULL, 0); unsigned int ret = crc32(crc0, (unsigned char *)dat, len); return ret; } void SamBam_writer_add_chunk(SamBam_Writer * writer, int thread_no) { int compressed_size ; unsigned int CRC32; z_stream * this_stream = thread_no < 0 ? &writer ->output_stream:writer -> threads_output_stream + thread_no; long long * this_buffer_used = thread_no < 0 ? &writer ->chunk_buffer_used : writer -> threads_chunk_buffer_used + thread_no; char * this_buffer = thread_no < 0 ? writer ->chunk_buffer: writer -> threads_chunk_buffer[thread_no]; char * this_compressed_chunk_buffer = thread_no < 0 ? writer ->compressed_chunk_buffer : writer -> threads_chunk_buffer_compressed[thread_no]; if(*this_buffer_used < 1){ // magic block with 0 byte data(EOF) subread_lock_occupy(&writer -> thread_bam_lock); fwrite( "\x1f\x8b\x08\x04\x00\x00\x00\x00\x00\xff\x06\x00\x42\x43\x02\x00\x1b\x00\x03\x00\x00\x00\x00\x00\x00\x00\x00\x00",1,28,writer -> bam_fp); writer -> current_BAM_pos = ftello(writer -> bam_fp); subread_lock_release(&writer -> thread_bam_lock); return; } this_stream -> avail_out = 70000; this_stream -> avail_in = (* this_buffer_used); CRC32 = SamBam_CRC32(this_buffer , * this_buffer_used); this_stream -> zalloc = Z_NULL; this_stream -> zfree = Z_NULL; this_stream -> opaque = Z_NULL; deflateInit2(this_stream, writer -> fastest_compression? SAMBAM_COMPRESS_LEVEL_FASTEST : SAMBAM_COMPRESS_LEVEL_NORMAL, Z_DEFLATED, SAMBAM_GZIP_WINDOW_BITS, Z_DEFAULT_MEM_LEVEL, Z_DEFAULT_STRATEGY); this_stream -> next_in = (unsigned char *)this_buffer; this_stream -> next_out = (unsigned char *)this_compressed_chunk_buffer; deflate(this_stream, Z_FINISH); deflateEnd(this_stream); compressed_size = 70000 - this_stream -> avail_out; subread_lock_occupy(&writer -> thread_bam_lock); SamBam_writer_chunk_header(writer, compressed_size); int chunk_write_size = fwrite(this_compressed_chunk_buffer, 1, compressed_size, writer -> bam_fp); fwrite(&CRC32 , 4, 1, writer -> bam_fp); fwrite(this_buffer_used , 4, 1, writer -> bam_fp); writer -> current_BAM_pos = ftello(writer -> bam_fp); subread_lock_release(&writer -> thread_bam_lock); if(chunk_write_size < compressed_size){ if(!writer -> is_internal_error)SUBREADputs("ERROR: no space left in the output directory."); writer -> is_internal_error = 1; } (* this_buffer_used) = 0; } double sambam_t1 = 0; void SamBam_writer_write_header(SamBam_Writer * writer) { int header_ptr=0, header_block_start = 0; while(header_ptr < writer->header_plain_text_buffer_used) { if(( header_ptr - header_block_start > 55000 || header_ptr >= writer->header_plain_text_buffer_used-1) && writer -> header_plain_text_buffer[header_ptr] == '\n') { writer -> chunk_buffer_used = 0; if(header_block_start == 0) // the very first block { memcpy(writer -> chunk_buffer, "BAM\1",4); writer -> chunk_buffer_used = 4; memcpy(writer -> chunk_buffer + writer -> chunk_buffer_used, &writer -> header_plain_text_buffer_used, 4); writer -> chunk_buffer_used += 4; } memcpy(writer -> chunk_buffer + writer -> chunk_buffer_used , writer -> header_plain_text_buffer + header_block_start, header_ptr - header_block_start+1); writer -> chunk_buffer_used += header_ptr - header_block_start + 1; SamBam_writer_add_chunk(writer, -1); header_block_start = header_ptr + 1; } header_ptr ++; } free(writer -> header_plain_text_buffer); writer -> header_plain_text_buffer = NULL; // reference sequences writer -> chunk_buffer_used = 0; memcpy(writer -> chunk_buffer, & writer -> chromosome_name_table -> numOfElements, 4); writer -> chunk_buffer_used = 4; for( header_ptr=0 ; header_ptr < writer -> chromosome_name_table -> numOfElements ; header_ptr ++) { //printf("D=%d\n", writer -> chromosome_id_table -> numOfElements); char * chro_name = HashTableGet(writer -> chromosome_id_table, NULL + 1 + header_ptr); unsigned int chro_len = HashTableGet(writer -> chromosome_len_table, NULL + 1 + header_ptr) - NULL - 1; assert(chro_name); int chro_name_len = strlen(chro_name)+1; memcpy(writer -> chunk_buffer + writer -> chunk_buffer_used , &chro_name_len, 4); writer -> chunk_buffer_used += 4; strcpy(writer -> chunk_buffer + writer -> chunk_buffer_used , chro_name); writer -> chunk_buffer_used += chro_name_len; memcpy(writer -> chunk_buffer + writer -> chunk_buffer_used , &chro_len, 4); writer -> chunk_buffer_used += 4; if(header_ptr == writer -> chromosome_name_table -> numOfElements - 1 || writer -> chunk_buffer_used > 55000) { SamBam_writer_add_chunk(writer, -1); writer -> chunk_buffer_used = 0; } } } void SamBam_writer_finalise_one_thread(SamBam_Writer * writer); void SamBam_writer_sort_bins_to_BAM(SamBam_Writer * writer); int SamBam_writer_close(SamBam_Writer * writer) { if(writer -> writer_state == 0) // no reads were added { if(writer -> header_plain_text_buffer) SamBam_writer_write_header(writer); } else SamBam_writer_finalise_one_thread(writer); if(writer -> sort_reads_by_coord){ SamBam_writer_sort_bins_to_BAM(writer); worker_master_mutex_destroy(&writer->sorted_notifier); } writer -> chunk_buffer_used = 0; SamBam_writer_add_chunk(writer, -1); // fputc(0, writer -> bam_fp); writer -> output_stream.next_in= NULL; writer -> output_stream.avail_in= 0; writer -> output_stream.next_out= NULL; writer -> output_stream.avail_out= 0; free(writer -> chunk_buffer); free(writer -> compressed_chunk_buffer); if(writer -> threads >=2){ int x1; for(x1 = 0 ; x1 < writer -> threads; x1 ++){ free(writer -> threads_chunk_buffer[x1]); free(writer -> threads_chunk_buffer_compressed[x1]); } free(writer -> threads_output_stream); free(writer -> threads_chunk_buffer); free(writer -> threads_chunk_buffer_compressed); free(writer -> threads_chunk_buffer_used); } HashTableDestroy(writer -> chromosome_name_table); HashTableDestroy(writer -> chromosome_id_table); HashTableDestroy(writer -> chromosome_len_table); #ifdef MAKE_STANDALONE if(stdout != writer -> bam_fp) #endif fclose(writer -> bam_fp); if(writer -> BAI_fp!=NULL) fclose(writer -> BAI_fp); return 0; } int SamBam_writer_add_header(SamBam_Writer * writer, char * header_text, int add_chro) { int new_text_len = strlen(header_text); if(writer -> header_plain_text_buffer_max <= writer -> header_plain_text_buffer_used + new_text_len + 1) { //return 0; writer -> header_plain_text_buffer_max *=2; writer -> header_plain_text_buffer = realloc(writer -> header_plain_text_buffer , writer -> header_plain_text_buffer_max); //printf("REAL: %d : %llX\n",writer -> header_plain_text_buffer_max, (long long ) writer -> header_plain_text_buffer); } strcpy(writer -> header_plain_text_buffer + writer -> header_plain_text_buffer_used, header_text); writer -> header_plain_text_buffer_used += new_text_len; strcpy(writer -> header_plain_text_buffer + writer -> header_plain_text_buffer_used, "\n"); writer -> header_plain_text_buffer_used ++; // SUBREADprintf("ADJHEADERXCD BIN=%s\n", header_text); if(add_chro && memcmp(header_text, "@SQ",3)==0) { char * chro = NULL; int chro_len = -1; char * toktmp = NULL; char * ret_tmp = strtok_r(header_text, "\t", &toktmp); while(1){ if(!ret_tmp) break; if(memcmp(ret_tmp,"SN:", 3)==0) chro = ret_tmp + 3; else if(memcmp(ret_tmp,"LN:", 3)==0) chro_len = atoi(ret_tmp + 3); ret_tmp = strtok_r(NULL, "\t", &toktmp); } if(chro && (chro_len>0)) SamBam_writer_add_chromosome(writer, chro, chro_len, 0); } //if(writer -> header_plain_text_buffer_used %97==0) printf("MV=%d\n",writer -> header_plain_text_buffer_used); return 0; } int SamBam_writer_add_chromosome(SamBam_Writer * writer, char * chro_name, unsigned int chro_length, int add_header) { unsigned int chro_id = writer -> chromosome_name_table -> numOfElements; //SUBREADprintf("ADJHEADER_CHRO %s of %u\n", chro_name, chro_length); //assert(strlen(chro_name) < 30); char * chro_name_space = malloc(strlen(chro_name)+1); strcpy(chro_name_space , chro_name); HashTablePut(writer -> chromosome_name_table, chro_name_space, NULL+1+chro_id); HashTablePut(writer -> chromosome_id_table, NULL+1+chro_id, chro_name_space); HashTablePut(writer -> chromosome_len_table, NULL+1+chro_id, NULL + 1 + chro_length); if(add_header) { char * line_buf = malloc(1000); snprintf(line_buf,999, "@SQ\tSN:%s\tLN:%u", chro_name , chro_length); SamBam_writer_add_header(writer, line_buf, 0); free(line_buf); } return 0; } int SamBam_compress_cigar(char * cigar, int * cigar_int, int * ret_coverage, int max_secs) { int tmp_int=0; int cigar_cursor = 0, num_opt = 0; int coverage_len = 0; (* ret_coverage) = 0; if(cigar[0]=='*') return 0; while(1) { char nch = cigar[cigar_cursor++]; if(!nch)break; if(isdigit(nch)) { tmp_int = tmp_int*10+(nch-'0'); } else { int int_opt=0; if(nch == 'M' || nch == 'N' || nch == 'D') coverage_len += tmp_int; //if(nch == 'M' ||nch == 'D' || nch == '=' || nch == 'X') coverage_len += tmp_int; for(; int_opt<8; int_opt++) if("MIDNSHP=X"[int_opt] == nch)break; cigar_int[num_opt ++] = (tmp_int << 4) | int_opt; tmp_int = 0; //SUBREADprintf("CIGARCOM: %d-th is %c\n", num_opt, nch); if(num_opt>=max_secs)break; } } (*ret_coverage) = coverage_len; return num_opt; } void SamBam_read2bin(char * read_txt, char * read_bin) { int bin_cursor = 0, txt_cursor = 0; while(1) { char nch = read_txt[txt_cursor++]; if(!nch)break; int fourbit; for(fourbit=0;fourbit<15;fourbit++) if("=ACMGRSVTWYHKDBN"[fourbit] == nch)break; if(bin_cursor %2 == 0) read_bin[bin_cursor/2] = fourbit<<4; else read_bin[bin_cursor/2] |= fourbit; bin_cursor++; } } int SamBam_compress_additional(char * additional_columns, char * bin) { int col_cursor = 0 , col_len = strlen(additional_columns); int bin_cursor = 0; while(col_cursor 780) break; } bin[bin_cursor + str_len] =0; bin_cursor += str_len + 1; col_cursor += str_len; } else if(datatype=='A') { bin[bin_cursor+2]='A'; bin[bin_cursor+3]=additional_columns[col_cursor+5]; col_cursor += 6; bin_cursor += 4; } else if(datatype=='B') //array { char celltype = additional_columns[col_cursor+5]; int * items = (int *)(&bin[bin_cursor+4]); bin[bin_cursor+2]='B'; bin[bin_cursor+3]=celltype; bin_cursor += 4 + 4; col_cursor += 7; (*items) = 0; int last_cursor = col_cursor; while(1){ if(additional_columns[col_cursor] == ',' || additional_columns[col_cursor] == '\t' || additional_columns[col_cursor] == 0) { // add new item char cell_buff [30]; if((col_cursor - last_cursor) < 29) { memcpy(cell_buff, additional_columns + last_cursor, (col_cursor - last_cursor)); cell_buff[(col_cursor - last_cursor)] = 0; int intv = 0; float fltv = 0; if(celltype == 'i')intv = atoi(cell_buff); else fltv = atof(cell_buff); if(bin_cursor < 780){ memcpy(bin + bin_cursor, (celltype == 'i')?(void *)&intv:(void *)&fltv, 4); bin_cursor += 4; (*items) ++; } } last_cursor = col_cursor+1; } if(additional_columns[col_cursor] == '\t' || additional_columns[col_cursor] == 0) break; col_cursor++; } } if(bin_cursor>750) break; continue; } col_cursor++; } return bin_cursor; } int SamBam_reg2bin(int beg, int end) { --end; if (beg>>14 == end>>14) return ((1<<15)-1)/7 + (beg>>14); if (beg>>17 == end>>17) return ((1<<12)-1)/7 + (beg>>17); if (beg>>20 == end>>20) return ((1<<9)-1)/7 + (beg>>20); if (beg>>23 == end>>23) return ((1<<6)-1)/7 + (beg>>23); if (beg>>26 == end>>26) return ((1<<3)-1)/7 + (beg>>26); return 0; } void SamBam_writer_finish_header( SamBam_Writer * writer ){ if(writer -> writer_state == 0){ if(writer -> header_plain_text_buffer) SamBam_writer_write_header(writer); writer -> writer_state = 10; } } #define FC_MAX_CIGAR_SECTIONS 96 // The caller has to free the returned pointer. // It returns NULL if no such field. char * duplicate_TAB_record_field(char * rline, int fno, int to_end){ int i, fldi = 0, start_pos = -1; if(fno >=1) for(i=0;rline[i] && rline[i] != '\n'; i++){ int nch = rline[i]; if(nch == '\t'){ fldi++; if(fldi == fno) start_pos = i+1; if(fldi == fno+1) break; } } else{ start_pos = rline[0]>0?0:-1; if(start_pos == 0) for(i=0;rline[i] && rline[i] != '\n' && rline[i] != '\t'; i++); else i=-1; } int end_pos =i; if(to_end){ end_pos = strlen(rline); if(end_pos<1) return NULL; if(rline[end_pos-1]=='\n') end_pos--; } if(start_pos<0 || end_pos <= start_pos) return NULL; char * ret = malloc(end_pos - start_pos +1); memcpy(ret, rline+start_pos, end_pos -start_pos); ret[end_pos -start_pos] = 0; return ret; } int SamBam_writer_add_read_bin(SamBam_Writer * writer, int thread_no, char * rbin, int committable){ char * this_chunk_buffer=NULL; srInt_64 *this_chunk_buffer_used=NULL; if(thread_no >= 0){ if(writer -> sort_reads_by_coord && writer -> threads_chunk_buffer_max_size[thread_no] < writer -> threads_chunk_buffer_used[thread_no] + 12000){ writer -> threads_chunk_buffer_max_size[thread_no] = writer -> threads_chunk_buffer_max_size[thread_no] * 7/4; writer -> threads_chunk_buffer[ thread_no ] = realloc(writer -> threads_chunk_buffer[ thread_no ], writer -> threads_chunk_buffer_max_size[thread_no]); } this_chunk_buffer = writer -> threads_chunk_buffer[ thread_no ]; this_chunk_buffer_used = writer -> threads_chunk_buffer_used + thread_no; }else{ if(writer -> sort_reads_by_coord && writer -> chunk_buffer_max_size < writer -> chunk_buffer_used + 12000){ //SUBREADprintf("0THR_REALLOCATE MEM : %d -> %d\n", writer -> chunk_buffer_max_size, writer -> chunk_buffer_max_size * 7/4); writer -> chunk_buffer_max_size = writer -> chunk_buffer_max_size * 7/4; writer -> chunk_buffer = realloc(writer -> chunk_buffer, writer -> chunk_buffer_max_size); } this_chunk_buffer = writer -> chunk_buffer; this_chunk_buffer_used = &writer -> chunk_buffer_used; } //SUBREADprintf("WRTXBIN: PTR %p of USED %lld at read %p\n", this_chunk_buffer, *this_chunk_buffer_used, rbin); int reclen=0; memcpy(&reclen, rbin,4); memcpy(this_chunk_buffer+(*this_chunk_buffer_used), rbin, reclen+4); (*this_chunk_buffer_used)+= reclen+4; if((*this_chunk_buffer_used)>55000 && committable && !writer -> sort_reads_by_coord) SamBam_writer_add_chunk(writer, thread_no); //SUBREADprintf("WRTXBIN: FIN WTR %p of USED %lld\n", this_chunk_buffer, *this_chunk_buffer_used); return 0; } int SamBam_writer_add_read_line(SamBam_Writer * writer, int thread_no, char * rline, int committable){ char * read_name, * flag_str, *chro_name, *chro_position_str, * mapping_quality_str, * cigar, * next_chro_name, *next_chro_position_str, *temp_len_str, *read_text, *qual_text, *additional_columns; read_name = duplicate_TAB_record_field(rline, 0,0); flag_str = duplicate_TAB_record_field(rline, 1,0); chro_name = duplicate_TAB_record_field(rline, 2,0); chro_position_str = duplicate_TAB_record_field(rline, 3,0); mapping_quality_str = duplicate_TAB_record_field(rline, 4,0); cigar = duplicate_TAB_record_field(rline, 5,0); next_chro_name = duplicate_TAB_record_field(rline, 6,0); next_chro_position_str = duplicate_TAB_record_field(rline, 7,0); temp_len_str = duplicate_TAB_record_field(rline, 8,0); read_text = duplicate_TAB_record_field(rline, 9,0); qual_text = duplicate_TAB_record_field(rline, 10,0); additional_columns = duplicate_TAB_record_field(rline, 11,1); if(qual_text==NULL){ SUBREADprintf("FATAL ERROR : bad read format: %s, %s, %s, %s\n", read_name, flag_str, chro_name, rline); return -1; } SamBam_writer_add_read(writer, thread_no, read_name, atoi(flag_str), chro_name, atoi(chro_position_str), atoi(mapping_quality_str), cigar, next_chro_name, atoi(next_chro_position_str), atoi(temp_len_str), strlen(read_text), read_text, qual_text, additional_columns, committable); if(additional_columns) free(additional_columns); free(qual_text); free(read_text); free(temp_len_str); free(next_chro_position_str); free(next_chro_name); free(cigar); free(mapping_quality_str); free(chro_position_str); free(chro_name); free(flag_str); free(read_name); return 0; } int SamBam_writer_add_read(SamBam_Writer * writer, int thread_no, char * read_name, unsigned int flags, char * chro_name, unsigned int chro_position, int mapping_quality, char * cigar, char * next_chro_name, unsigned int next_chro_position, int temp_len, int read_len, char * read_text, char * qual_text, char * additional_columns, int committable) { assert(writer -> writer_state!=0); if(!qual_text || !read_text) { SUBREADprintf("ERROR: sam file is incomplete.\n"); return 1; } char additional_bin[1000]; int cigar_opts[FC_MAX_CIGAR_SECTIONS], xk1, cover_length = 0; int cigar_opt_len = SamBam_compress_cigar(cigar, cigar_opts, & cover_length, FC_MAX_CIGAR_SECTIONS); int read_name_len = 1+strlen(read_name) ; int additional_bin_len = 0; if(additional_columns) additional_bin_len = SamBam_compress_additional(additional_columns, additional_bin); int record_length = 4 + 4 + 4 + 4 + /* l_seq: */ 4 + 4 + 4 + 4 + /* read_name:*/ read_name_len + cigar_opt_len * 4 + (read_len + 1) /2 + read_len + additional_bin_len; char * this_chunk_buffer; srInt_64 * this_chunk_buffer_used; if(thread_no >= 0){ if(writer -> sort_reads_by_coord && writer -> threads_chunk_buffer_max_size[thread_no] < writer -> threads_chunk_buffer_used[thread_no] + 12000){ writer -> threads_chunk_buffer_max_size[thread_no] = writer -> threads_chunk_buffer_max_size[thread_no] * 7/4; writer -> threads_chunk_buffer[ thread_no ] = realloc(writer -> threads_chunk_buffer[ thread_no ], writer -> threads_chunk_buffer_max_size[thread_no]); } this_chunk_buffer = writer -> threads_chunk_buffer[ thread_no ]; this_chunk_buffer_used = writer -> threads_chunk_buffer_used + thread_no; }else{ if(writer -> sort_reads_by_coord && writer -> chunk_buffer_max_size < writer -> chunk_buffer_used + 12000){ //SUBREADprintf("REALLOCATE MEM : %d -> %d\n", writer -> chunk_buffer_max_size, writer -> chunk_buffer_max_size * 7/4); writer -> chunk_buffer_max_size = writer -> chunk_buffer_max_size * 7/4; writer -> chunk_buffer = realloc(writer -> chunk_buffer, writer -> chunk_buffer_max_size); } this_chunk_buffer = writer -> chunk_buffer; this_chunk_buffer_used = &writer -> chunk_buffer_used; } memcpy(this_chunk_buffer + (*this_chunk_buffer_used) , & record_length , 4); (*this_chunk_buffer_used) += 4; int bin = SamBam_reg2bin(chro_position -1, chro_position-1+cover_length); //if(chro_position>0)SUBREADprintf("CREATE_BIN=%d\n", bin); int refID = HashTableGet(writer -> chromosome_name_table, chro_name) - NULL - 1; int bin_mq_nl = (bin<<16) | (mapping_quality << 8) | read_name_len ; int fag_nc = (flags<<16) | cigar_opt_len; int nextRefID = -1; if(next_chro_name[0] != '*' && next_chro_name[0]!='=') nextRefID = HashTableGet(writer -> chromosome_name_table, next_chro_name) - NULL - 1; else if(next_chro_name[0] == '=') nextRefID = refID; chro_position--; next_chro_position--; memcpy(this_chunk_buffer + (*this_chunk_buffer_used) , & refID , 4); (*this_chunk_buffer_used) += 4; memcpy(this_chunk_buffer + (*this_chunk_buffer_used) , & chro_position , 4); (*this_chunk_buffer_used) += 4; memcpy(this_chunk_buffer + (*this_chunk_buffer_used) , & bin_mq_nl , 4); (*this_chunk_buffer_used) += 4; memcpy(this_chunk_buffer + (*this_chunk_buffer_used) , & fag_nc , 4); (*this_chunk_buffer_used) += 4; memcpy(this_chunk_buffer + (*this_chunk_buffer_used) , & read_len , 4); (*this_chunk_buffer_used) += 4; memcpy(this_chunk_buffer + (*this_chunk_buffer_used) , & nextRefID , 4); (*this_chunk_buffer_used) += 4; memcpy(this_chunk_buffer + (*this_chunk_buffer_used) , & next_chro_position , 4); (*this_chunk_buffer_used) += 4; memcpy(this_chunk_buffer + (*this_chunk_buffer_used) , & temp_len , 4); (*this_chunk_buffer_used) += 4; strcpy(this_chunk_buffer + (*this_chunk_buffer_used) , read_name); (*this_chunk_buffer_used) += read_name_len; memcpy(this_chunk_buffer + (*this_chunk_buffer_used) , cigar_opts, 4*cigar_opt_len); (*this_chunk_buffer_used) += 4*cigar_opt_len; SamBam_read2bin(read_text , this_chunk_buffer + (*this_chunk_buffer_used)); (*this_chunk_buffer_used) += (read_len + 1) /2; memcpy(this_chunk_buffer + (*this_chunk_buffer_used), qual_text, read_len); for(xk1=0; xk155000 && committable && !writer -> sort_reads_by_coord) SamBam_writer_add_chunk(writer, thread_no); return 0; } int SamBam_unzip(char * out, int outlen , char * in , int inlen, int sync_only) { z_stream strm; strm.zalloc = Z_NULL; strm.zfree = Z_NULL; strm.opaque = Z_NULL; strm.avail_in = 0; strm.next_in = Z_NULL; int ret = inflateInit2(&strm, SAMBAM_GZIP_WINDOW_BITS); if (ret != Z_OK) return -1; strm.avail_in = (unsigned int)inlen; strm.next_in = (unsigned char *)in; strm.avail_out = outlen; strm.next_out = (unsigned char *)out; ret = inflate(&strm, sync_only?Z_SYNC_FLUSH:Z_FINISH); if(ret != Z_STREAM_END && ret!=0) { inflateEnd(&strm); SUBREADprintf("DATA ERROR! code=%d\n", ret); assert(0); return -1; } int have = outlen - strm.avail_out; inflateEnd(&strm); //SUBREADprintf("DECOMPRESS GENERATED=%d\n", have); return have; } int SamBam_writer_sort_buff_one_compare(void * Lbin, void * Rbin, ArrayList * me){ unsigned long long * Lv = Lbin, *Rv = Rbin; if(*Lv > *Rv) return 1; else if(*Lv < *Rv) return -1; return 0; } // return total reads in bin. int SamBam_writer_sort_buff_one_write(SamBam_Writer * writer, char * bin, int binlen, int thread_id){ int bin_cursor = 0; ArrayList* sort_linear_pos = ArrayListCreate(1000000); ArrayListSetDeallocationFunction(sort_linear_pos, free); int ii_reads = 0; while(bin_cursor < binlen){ int this_block_len = 0; memcpy(&this_block_len, bin + bin_cursor, 4); char * key_binpos = malloc(12); memcpy(key_binpos, bin + bin_cursor+8, 4); memcpy(key_binpos+4, bin + bin_cursor+4, 4); memcpy(key_binpos+8, &bin_cursor, 4); ArrayListPush(sort_linear_pos, key_binpos); bin_cursor +=4 + this_block_len; ii_reads ++; } ArrayListSort(sort_linear_pos, SamBam_writer_sort_buff_one_compare); char * nbin = NULL; if(binlen >0 && binlen < 0x7fffffff)nbin=malloc(binlen); int nb_cursor = 0, xx, wlen=0; for(xx=0; xx threads>1) subread_lock_occupy(&writer -> thread_bam_lock); sprintf(tmpfname, "%s-%06d.sortedbin", writer -> tmpf_prefix, writer -> sorted_batch_id++); if(writer -> threads>1) subread_lock_release(&writer -> thread_bam_lock); FILE * tofp = fopen(tmpfname, "wb"); if(tofp){ if(binlen>0)wlen = fwrite(nbin, binlen,1, tofp); fclose(tofp); } free(nbin); if(wlen < 1 && binlen>0) { SUBREADprintf("ERROR: no space (%d bytes) in the temp directory (%s).\nThe program cannot run properly.\n", binlen, tmpfname); writer ->is_internal_error = 1; return -1; } return ii_reads; } unsigned long long SamBam_writer_sort_bins_to_BAM_FP_pos(FILE * fp){ int intvals[3]; unsigned long long ret = 0; int rlen = fread(intvals, sizeof(int),3,fp); if(rlen>0 && intvals[0]<10000){ int chro_no, poss; chro_no=intvals[1]; poss=intvals[2]; ret =((1LLU*chro_no) << 32)| poss; if(ret == SUBREAD_MAX_ULONGLONG) ret = ret- 10 ; fseek(fp, -12, SEEK_CUR); return ret; } return SUBREAD_MAX_ULONGLONG; } int SamBam_writer_calc_cigar_span(char * bin){ int cops, rname_len, ret = 0; cops = *(short *)(bin+12); rname_len = bin[8]; int ii; char * binbase = bin+32+rname_len; for(ii = 0; ii < cops ; ii++){ unsigned int copt = 0; copt = *(int*)(binbase+4*ii); int copt_char = copt & 0xf; unsigned int copt_len = copt >> 4; if(copt_char == 0 || copt_char == 2 || copt_char == 3 || copt_char == 7 || copt_char == 8) ret += copt_len; } return ret; } #define MAX_ALLOWED_GAP_IN_BAI_CHUNK 5 // the number of blocks, not the file positions. void SamBam_writer_sort_bins_to_BAM_test_bins(SamBam_Writer * writer, HashTable * bin_tab, ArrayList * bin_list, ArrayList * win16k_list, int block_len, void ***last_chunk_ptr, int chro_no){ int inbin_pos = writer -> chunk_buffer_used - block_len ; // point to the byte AFTER "block_len" int. int pos=0, bin_mq_nl = 0, binno=0; memcpy(&pos, writer -> chunk_buffer + inbin_pos + 4, 4); memcpy(&bin_mq_nl, writer -> chunk_buffer + inbin_pos + 8,4); binno = bin_mq_nl>>16; int cigar_span = SamBam_writer_calc_cigar_span(writer -> chunk_buffer + inbin_pos); int this_w16_no = (pos + cigar_span) >>14; // WIN is calculated on 0-based pos. unsigned long long this_Vpos = writer -> this_bam_block_no<<16 | (inbin_pos-4); // if this read is after the maximum coordinate in the win16k list: all elements before last one and this one starts at this read. if(this_w16_no >=win16k_list->numOfElements){ int bbi; for(bbi = win16k_list->numOfElements; bbi <=this_w16_no; bbi++) ArrayListPush(win16k_list, NULL+ this_Vpos); } // a read only belongs to ONE bin which is the smallest bin that can FULLY cover the full length of this read. ArrayList * this_bin_chunks = HashTableGet(bin_tab, NULL+binno+1); if(NULL == this_bin_chunks){ this_bin_chunks = ArrayListCreate(5); HashTablePut(bin_tab, NULL+binno+1, this_bin_chunks); ArrayListPush(bin_list, NULL+binno); } int found = 0; // a bin is not necessarily continuous. Say, a top-level bin only contains a few reads (most reads a in low-level bins), but their locations are everywhere. if(this_bin_chunks -> numOfElements > 0){ //SUBREADprintf("RESLOCS : %ld == %ld\n", this_Vpos, this_bin_chunks -> elementList [ this_bin_chunks -> numOfElements - 1 ] - NULL); long long diff = this_Vpos >>16; diff -=(this_bin_chunks -> elementList [ this_bin_chunks -> numOfElements - 1] - NULL)>>16; if(diff < MAX_ALLOWED_GAP_IN_BAI_CHUNK){ this_bin_chunks -> elementList [ this_bin_chunks -> numOfElements -1] = NULL+this_Vpos + block_len+4; *last_chunk_ptr = this_bin_chunks -> elementList + this_bin_chunks -> numOfElements -1; found = 1; } } // if the last chunk in this bin isn't good to be extended (too far from the file location of the new read), a new chunk is created. if(!found){ ArrayListPush(this_bin_chunks, NULL + this_Vpos); ArrayListPush(this_bin_chunks, NULL + this_Vpos + block_len+4); *last_chunk_ptr = this_bin_chunks -> elementList + this_bin_chunks -> numOfElements -1; } } void * SamBam_writer_sorted_compress(void * vptr0){ void ** vptr = vptr0; SamBam_Writer * writer = vptr[0]; int this_thread_no = vptr[1]-NULL; subread_lock_t * initlock = vptr[2]; worker_thread_start(&writer->sorted_notifier, this_thread_no); subread_lock_release(initlock); free(vptr0); struct SamBam_sorted_compressor_st * me = writer -> writer_threads + this_thread_no; while(1){ int termed = worker_wait_for_job(&writer->sorted_notifier, this_thread_no); if(termed)break; me->CRC32_plain = SamBam_CRC32(me->plain_text, me->text_size); me->strm.next_in = (unsigned char*)me->plain_text; me->strm.avail_in = me->text_size; me->strm.next_out = (unsigned char*)me->zipped_bin; me->strm.avail_out = 70000; int deret = deflate(&me->strm,Z_FINISH); if(deret >=0){ deflateReset(&me->strm); me->bin_size = 70000 - me->strm.avail_out; me->last_job_done = 1; }else{ SUBREADprintf("Error: cannot compress BAM block #%d , which is %llu, had %d => 70000 [ %d ] bytes , return = %d\n", this_thread_no, me -> bam_block_no, me->text_size, me->bin_size, deret); } } return NULL; } void SamBam_thread_wait_merge_write(SamBam_Writer * writer, int thread_no){ master_wait_for_job_done(&writer -> sorted_notifier, thread_no); if(writer -> writer_threads[thread_no].last_job_done){ srInt_64 fpos = ftello(writer -> bam_fp); HashTablePut(writer -> block_no_p1_to_vpos_tab, NULL+1+writer -> writer_threads[thread_no].bam_block_no, NULL+fpos); SamBam_writer_chunk_header(writer, writer -> writer_threads[thread_no].bin_size); int rlen = fwrite( writer -> writer_threads[thread_no].zipped_bin ,1, writer -> writer_threads[thread_no].bin_size, writer -> bam_fp); if(rlen != writer -> writer_threads[thread_no].bin_size){ SUBREADprintf("ERROR: cannot write output files.\n"); assert(0); } rlen = fwrite(&writer -> writer_threads[thread_no].CRC32_plain , 4, 1, writer -> bam_fp); rlen = fwrite(&writer -> writer_threads[thread_no].text_size , 4, 1, writer -> bam_fp); writer -> writer_threads[thread_no].text_size = 0; writer -> writer_threads[thread_no].bin_size = 0; writer -> writer_threads[thread_no].bam_block_no = -1; writer -> writer_threads[thread_no].last_job_done = 0; } } void SamBam_thread_set_new_job(SamBam_Writer * writer, int thread_no){ memcpy(writer -> writer_threads[thread_no].plain_text, writer ->chunk_buffer, writer->chunk_buffer_used); writer -> writer_threads[thread_no].text_size = writer->chunk_buffer_used; writer -> writer_threads[thread_no].bam_block_no = writer -> this_bam_block_no; writer -> chunk_buffer_used = 0; master_notify_worker(&writer -> sorted_notifier, thread_no); } void SamBam_writer_submit_sorted_compressing_task(SamBam_Writer * writer){ SamBam_thread_wait_merge_write(writer, writer -> sorted_compress_this_thread_no); SamBam_thread_set_new_job(writer, writer -> sorted_compress_this_thread_no); writer -> sorted_compress_this_thread_no++; if(writer -> sorted_compress_this_thread_no == writer -> threads) writer -> sorted_compress_this_thread_no=0; writer -> this_bam_block_no ++; } void SamBam_writer_sort_bins_to_BAM_write_1R(SamBam_Writer * writer, FILE * fp, HashTable * bin_tab, ArrayList * bin_list, ArrayList * win16k_list, int chro_no){ int block_len=0; int rlen = fread(&block_len, 4,1,fp); //SUBREADprintf("WBIN=%d\n", block_len); if(rlen<1 || block_len >= 10000){ SUBREADprintf("ERROR: sorted bin files are broken. RLEN=%d , BLKLEN=%d\n", rlen, block_len); assert(rlen >=1); } memcpy(writer -> chunk_buffer + writer -> chunk_buffer_used, &block_len, 4); writer ->chunk_buffer_used += 4; rlen = fread(writer -> chunk_buffer + writer -> chunk_buffer_used, 1, block_len ,fp); if(rlen < block_len){ SUBREADprintf("ERROR: sorted bin files are broken.\n"); assert(rlen >=block_len); } writer -> chunk_buffer_used+= rlen; void ** last_chunk_ptr = NULL; SamBam_writer_sort_bins_to_BAM_test_bins(writer, bin_tab, bin_list, win16k_list, block_len, &last_chunk_ptr, chro_no); if(writer -> chunk_buffer_used>55000){ assert(writer -> chunk_buffer_used< 65000); SamBam_writer_submit_sorted_compressing_task(writer); } } #define SAMBAM_MERGE_MAX_FPS (350) void SamBam_writer_one_thread_merge_sortedbins(SamBam_Writer * writer){ int new_bins = 0; if(writer -> sorted_batch_id > SAMBAM_MERGE_MAX_FPS){ int merge_i; for(merge_i = 0; merge_i < writer -> sorted_batch_id; merge_i+= SAMBAM_MERGE_MAX_FPS){ char tfp[MAX_FILE_NAME_LENGTH+50]; int this_size = min(SAMBAM_MERGE_MAX_FPS, writer -> sorted_batch_id - merge_i); if(this_size < 2) break; //SUBREADprintf("CREATE_MERGE %d\n", new_bins + writer -> sorted_batch_id); sprintf(tfp , "%s-%06d.sortedbin", writer -> tmpf_prefix, writer -> sorted_batch_id + (new_bins++) ); FILE * outbinfp = fopen(tfp,"wb"); FILE ** sb_fps = malloc(sizeof(FILE *) * this_size); unsigned long long * current_min_fps = malloc(sizeof(unsigned long long) * this_size); unsigned long long current_min = SUBREAD_MAX_ULONGLONG; int current_min_batch = -1, bii; for(bii = 0; bii < this_size; bii++){ char tfpx[MAX_FILE_NAME_LENGTH+50]; current_min_fps[bii] = SUBREAD_MAX_ULONGLONG; sprintf(tfpx , "%s-%06d.sortedbin", writer -> tmpf_prefix, bii + merge_i); sb_fps[bii] = fopen(tfpx,"rb"); current_min_fps[bii] = SamBam_writer_sort_bins_to_BAM_FP_pos(sb_fps[bii]); if(current_min_fps[bii] < SUBREAD_MAX_ULONGLONG && current_min_fps[bii] < current_min){ current_min = current_min_fps[bii]; current_min_batch = bii; } } char * mtmp = malloc(10000); while(1){ if(current_min_batch>-1){ int blk_len = 0; int rrlen = fread(&blk_len, 4, 1, sb_fps[current_min_batch]); if(rrlen <1) assert(rrlen >0); assert(blk_len < 10000); rrlen = fread(mtmp, blk_len, 1, sb_fps[current_min_batch]); if(rrlen <1) assert(rrlen >0); fwrite(&blk_len,4,1, outbinfp); fwrite(mtmp, blk_len,1, outbinfp); current_min_fps[current_min_batch] = SamBam_writer_sort_bins_to_BAM_FP_pos(sb_fps[current_min_batch]); current_min = SUBREAD_MAX_ULONGLONG; current_min_batch = -1; }else break; for(bii = 0; bii < this_size; bii++){ if(current_min_fps[bii] < SUBREAD_MAX_ULONGLONG && current_min_fps[bii] < current_min){ current_min = current_min_fps[bii]; current_min_batch = bii; } } } free(mtmp); for(bii = 0; bii < this_size; bii++){ fclose(sb_fps[bii]); sprintf(tfp , "%s-%06d.sortedbin", writer -> tmpf_prefix, merge_i + bii); //SUBREADprintf(" DEL_MERGE %d\n", bii); unlink(tfp); } fclose(outbinfp); } writer -> sorted_batch_id += new_bins; } } #define SAMBAM_renew_BAItabs { if(bin_chunks_table != NULL) { HashTableDestroy(bin_chunks_table); ArrayListDestroy(bins_list); ArrayListDestroy(win16k_list) ; } \ bin_chunks_table = HashTableCreate(10000); bins_list = ArrayListCreate(10000); win16k_list = ArrayListCreate(10000);\ HashTableSetDeallocationFunctions(bin_chunks_table, NULL, (void(*)(void *))ArrayListDestroy); } #define SAMBAM_write_empty_chros(nSTART, nEND) {int iikk; for(iikk=(nSTART); iikk<(nEND);iikk++){ int wwwlen = fwrite("\0\0\0\0\0\0\0\0", 1,8,writer -> BAI_fp); if(wwwlen < 8){ assert(wwwlen==8);}} } int level_min_binno[] = {0, 1, 9, 73, 585, 4681}; int SamBam_writer_merge_chunks_compare(void * vL, void * vR, ArrayList * me){ long long * lL = vL; long long * lR = vR; if( (*lL) > (*lR)) return 1; if( (*lL) < (*lR)) return -1; return 0; } void SamBam_writer_merge_chunks(ArrayList * chs){ ArrayList * intvs = ArrayListCreate(chs -> numOfElements/2); ArrayListSetDeallocationFunction(intvs, free); int i; for(i=0; i numOfElements; i+=2){ long long * ll = malloc(sizeof(long long)*2); ll[0] = ArrayListGet(chs, i)-NULL; ll[1] = ArrayListGet(chs, i+1)-NULL; ArrayListPush(intvs, ll); } chs -> numOfElements = 0; ArrayListSort(intvs, SamBam_writer_merge_chunks_compare); long long * ll0 = ArrayListGet(intvs, 0); ArrayListPush(chs, NULL+ll0[0]); ArrayListPush(chs, NULL+ll0[1]); for(i=1; i numOfElements; i++){ long long last_end = ArrayListGet(chs, chs -> numOfElements-1) -NULL; long long * ll_this = ArrayListGet(intvs, i); long long dist = ll_this[0]>>16; dist -= last_end>>16; if(dist < MAX_ALLOWED_GAP_IN_BAI_CHUNK) chs-> elementList[chs -> numOfElements-1] = NULL+max(ll_this[1],last_end); else{ ArrayListPush(chs, NULL+ll_this[0]); ArrayListPush(chs, NULL+ll_this[1]); } } //SUBREADprintf("MERGE CHUNKS : %ld -> %ld\n", intvs-> numOfElements, chs -> numOfElements/2); ArrayListDestroy(intvs); } void SamBam_writer_optimize_bins_level(HashTable *bin_tab, ArrayList *bin_arr, HashTable * new_tab, ArrayList * new_arrs, int this_level){ int i,j, my_min_binno = level_min_binno[this_level], my_parent_min_binno = -1, my_child_min_binno = 999999; if(this_level>0) my_parent_min_binno = level_min_binno[this_level-1]; if(this_level<5) my_child_min_binno = level_min_binno[this_level+1]; // copy all parents and ancestors and children and descendant bins into new table & list for(i=0; i< bin_arr -> numOfElements; i++){ int binno = ArrayListGet(bin_arr, i)-NULL; if(binno < my_min_binno || binno >= my_child_min_binno){ ArrayList * old_bin_arr = HashTableGet(bin_tab, NULL+1+binno); if(old_bin_arr -> numOfElements > 1){ HashTablePut(new_tab, NULL+1+binno, ArrayListDuplicate(old_bin_arr)); ArrayListPush(new_arrs, NULL+binno); } } } // scan this_level bins. If small enough -> put to parent bin and delete it. // Otherwise put it into new tab&list. for(i=0; i< bin_arr -> numOfElements; i++){ int binno = ArrayListGet(bin_arr, i)-NULL; if(binno < my_min_binno || binno >= my_child_min_binno )continue; ArrayList * small_bin_arr = HashTableGet(bin_tab, NULL+1+binno); if(small_bin_arr -> numOfElements < 2) continue; long long min_start_Voff = SUBREAD_MAX_LONGLONG; long long max_end_Voff = -1; for(j =0 ; j< small_bin_arr -> numOfElements; j+=2){ long long this_start = ArrayListGet(small_bin_arr, j)-NULL; long long this_end = ArrayListGet(small_bin_arr, j+1)-NULL; min_start_Voff = min(min_start_Voff, this_start); max_end_Voff = max(max_end_Voff, this_end); } long long dist = max_end_Voff >> 16; dist -= min_start_Voff >>16; if(dist < MAX_ALLOWED_GAP_IN_BAI_CHUNK) { int parent_binno = my_parent_min_binno +((binno - my_min_binno)>>3); ArrayList * parent_bin_arr = HashTableGet(new_tab, NULL+1+parent_binno); if(NULL == parent_bin_arr){ parent_bin_arr = ArrayListCreate(10); HashTablePut(new_tab, NULL+1+parent_binno, parent_bin_arr); ArrayListPush(new_arrs, NULL+parent_binno); } for(j =0 ; j< small_bin_arr -> numOfElements; j++) ArrayListPush(parent_bin_arr, ArrayListGet(small_bin_arr, j)); } else { HashTablePut(new_tab, NULL+1+binno, ArrayListDuplicate(small_bin_arr)); ArrayListPush(new_arrs, NULL + binno); } } // look into all parent_level bins, reduce its size for(i=0; i< new_arrs -> numOfElements; i++){ int binno = ArrayListGet(new_arrs, i)-NULL; if(binno >=my_min_binno || binno < my_parent_min_binno) continue; ArrayList * large_bin_arr = HashTableGet(new_tab, NULL+1+binno); SamBam_writer_merge_chunks(large_bin_arr); } //SUBREADprintf("Tab size : %ld => %ld\n",bin_tab -> numOfElements, new_tab -> numOfElements); HashTableDestroy(bin_tab); ArrayListDestroy(bin_arr); } void SamBam_writer_optimize_bins(HashTable *bin_tab, ArrayList *bin_arr, HashTable ** new_tab, ArrayList ** new_arrs){ int this_level; for(this_level = 5; this_level > 2; this_level --){ // SUBREADprintf("OPTIMIZING AT LEVEL %d\n", this_level); HashTable * new_bin_tab = HashTableCreate(2000); HashTableSetDeallocationFunctions(new_bin_tab, NULL, (void(*)(void *))ArrayListDestroy); ArrayList * new_bin_list = ArrayListCreate(2000); SamBam_writer_optimize_bins_level(bin_tab, bin_arr, new_bin_tab, new_bin_list, this_level); bin_tab = new_bin_tab; bin_arr = new_bin_list; (*new_tab) = new_bin_tab; (*new_arrs) = new_bin_list; } } void SamBam_write_sorted_thread_collect(SamBam_Writer * writer){ int thread_i; if(writer -> chunk_buffer_used>0) SamBam_writer_submit_sorted_compressing_task(writer); for(thread_i = 0; thread_i < writer -> threads; thread_i++){ //SUBREADprintf("FINISHED %d-th CHRO, MERGING THR %d\n", old_chro_no, writer->sorted_compress_this_thread_no ); SamBam_thread_wait_merge_write(writer, writer->sorted_compress_this_thread_no); writer->sorted_compress_this_thread_no ++; if(writer->sorted_compress_this_thread_no == writer -> threads) writer->sorted_compress_this_thread_no = 0; } } #define SAMBAM_Block2Vpos(v) (v)=((HashTableGet(writer -> block_no_p1_to_vpos_tab, NULL+(v>>16)+1)-NULL )<<16 )|(v & 0xffff); void SamBam_write_BAI_for_1chr(SamBam_Writer * writer, HashTable ** bin_chunks_table, ArrayList ** bins_list, ArrayList ** win16k_list){ SamBam_write_sorted_thread_collect(writer); if(1){ HashTable * new_bin_tab = NULL; ArrayList * new_bin_list = NULL; SamBam_writer_optimize_bins(*bin_chunks_table ,*bins_list,&new_bin_tab,&new_bin_list); *bin_chunks_table = new_bin_tab; *bins_list = new_bin_list; } int n_bins = (*bins_list)->numOfElements; int win16_nos = (*win16k_list)->numOfElements; //SUBREADprintf("WIN_ITEMS=%d\t\t\tOLD_CHRO=%d NEW_CHRO=%d CURR_BATCH=%d\n", win16_nos, old_chro_no, chro_no , current_min_batch); fwrite(&n_bins, 4, 1, writer -> BAI_fp); int bini; for(bini = 0; bini < n_bins; bini ++){ int bin_no = ArrayListGet(*bins_list, bini)-NULL; ArrayList * chunks_list = HashTableGet(*bin_chunks_table, NULL+ bin_no+1); int n_chunks = chunks_list -> numOfElements/2; fwrite(&bin_no, 4, 1, writer -> BAI_fp); fwrite(&n_chunks, 4, 1, writer -> BAI_fp); int chunk_i; for(chunk_i = 0; chunk_i < 2* n_chunks; chunk_i +=2){ long long int Voff_st = ArrayListGet(chunks_list, chunk_i) - NULL; long long int Voff_en = ArrayListGet(chunks_list, chunk_i+1) - NULL; SAMBAM_Block2Vpos(Voff_st); SAMBAM_Block2Vpos(Voff_en); fwrite(&Voff_st, 8, 1, writer -> BAI_fp); fwrite(&Voff_en, 8, 1, writer -> BAI_fp); } } fwrite(&win16_nos, 4, 1, writer -> BAI_fp); for(bini = 0; bini < win16_nos; bini++){ long long int Voff = ArrayListGet(*win16k_list , bini) - NULL; SAMBAM_Block2Vpos(Voff); fwrite(&Voff, 8, 1, writer -> BAI_fp); } } #define SAMBAM_reset_sorting_writer { HashTableRemoveAll( writer -> block_no_p1_to_vpos_tab ); } // MUST be called by THREAD 0! void SamBam_writer_sort_bins_to_BAM(SamBam_Writer * writer){ SamBam_writer_one_thread_merge_sortedbins(writer); FILE ** sb_fps = malloc(sizeof(FILE *) * writer -> sorted_batch_id); unsigned long long * current_min_fps = malloc(sizeof(unsigned long long) * writer -> sorted_batch_id); int bii; unsigned long long current_min = SUBREAD_MAX_ULONGLONG; int current_min_batch = -1; writer -> chunk_buffer_used = 0; for(bii = 0; bii < writer -> sorted_batch_id; bii++){ char tfp[MAX_FILE_NAME_LENGTH+40]; current_min_fps[bii] = SUBREAD_MAX_ULONGLONG; sprintf(tfp , "%s-%06d.sortedbin", writer -> tmpf_prefix, bii); sb_fps[bii] = fopen(tfp,"rb"); if(sb_fps[bii]!=NULL){ current_min_fps[bii] = SamBam_writer_sort_bins_to_BAM_FP_pos(sb_fps[bii]); if(current_min_fps[bii] < SUBREAD_MAX_ULONGLONG && current_min_fps[bii] < current_min){ current_min = current_min_fps[bii]; current_min_batch = bii; } } } ArrayList * bins_list = NULL; // [Bin_Number_0, Bin_Number_1, ...] ArrayList * win16k_list = NULL; // [ Voffset_0, Voffset_1, ... ] HashTable * bin_chunks_table=NULL; SAMBAM_renew_BAItabs; int chro_no = (int)(current_min >> 32); int old_chro_no = -1, last_written_BAI_chro = -1; int wlen = fwrite("BAI\1", 4, 1, writer -> BAI_fp); if(wlen < 1) assert(wlen >0); int thread_i, n_ref = writer -> chromosome_name_table -> numOfElements; subread_lock_t initlocks[writer -> threads]; wlen = fwrite(&n_ref, 4, 1, writer -> BAI_fp); SAMBAM_write_empty_chros(0, chro_no); writer -> block_no_p1_to_vpos_tab = HashTableCreate(100000); writer -> writer_threads = calloc(sizeof(struct SamBam_sorted_compressor_st), writer -> threads); for(thread_i = 0; thread_i < writer -> threads; thread_i++){ memset(&(writer -> writer_threads[thread_i].strm),0,sizeof(z_stream)); deflateInit2(&(writer -> writer_threads[thread_i].strm), writer -> fastest_compression? SAMBAM_COMPRESS_LEVEL_FASTEST : SAMBAM_COMPRESS_LEVEL_NORMAL , Z_DEFLATED, SAMBAM_GZIP_WINDOW_BITS, Z_DEFAULT_MEM_LEVEL, Z_DEFAULT_STRATEGY); subread_init_lock(initlocks+thread_i); subread_lock_occupy(initlocks+thread_i); void ** thparam = malloc(sizeof(void*)*3); thparam[0] = writer; thparam[1] = NULL + thread_i; thparam[2] = initlocks+thread_i; pthread_create(&writer -> writer_threads[thread_i].thread_stub, NULL,SamBam_writer_sorted_compress,thparam); } for(thread_i = 0; thread_i < writer -> threads; thread_i++){ subread_lock_occupy(initlocks+thread_i); subread_destroy_lock(initlocks+thread_i); } SAMBAM_reset_sorting_writer; while(1){ // GO THROUGH EACH READ and write into BAM //if(current_min_batch<0)SUBREADprintf("FINALTEST: OLD/NEW = %d , %d\n", old_chro_no ,chro_no); if(old_chro_no >=0 && chro_no != old_chro_no){ // if there is old_chro && if has to write. Note: old_chro_no<0 means we reached the "unmapped" part of the sorted temp files //SUBREADprintf("\n====== W1CHR %d =====\n", old_chro_no); SamBam_write_BAI_for_1chr(writer, &bin_chunks_table, &bins_list, &win16k_list); last_written_BAI_chro = old_chro_no; SAMBAM_write_empty_chros(old_chro_no +1, chro_no < 0?n_ref:chro_no); SAMBAM_renew_BAItabs; SAMBAM_reset_sorting_writer; } if(current_min_batch>-1){ SamBam_writer_sort_bins_to_BAM_write_1R(writer, sb_fps[current_min_batch], bin_chunks_table, bins_list, win16k_list, chro_no); current_min_fps[current_min_batch] = SamBam_writer_sort_bins_to_BAM_FP_pos(sb_fps[current_min_batch]); current_min = SUBREAD_MAX_ULONGLONG; current_min_batch = -1; old_chro_no = chro_no; }else{ SamBam_write_sorted_thread_collect(writer); break; // all finished. quit. } for(bii = 0; bii < writer -> sorted_batch_id; bii++){ if(current_min_fps[bii] < SUBREAD_MAX_ULONGLONG && current_min_fps[bii] < current_min){ current_min = current_min_fps[bii]; current_min_batch = bii; } } chro_no = (int)(current_min >> 32); } for(bii = 0; bii < writer -> sorted_batch_id; bii++){ if(!sb_fps[bii]) continue; char tfp[MAX_FILE_NAME_LENGTH+40]; sprintf(tfp , "%s-%06d.sortedbin", writer -> tmpf_prefix, bii); fclose(sb_fps[bii]); unlink(tfp); } if(bin_chunks_table != NULL) { HashTableDestroy(bin_chunks_table); ArrayListDestroy(bins_list); ArrayListDestroy(win16k_list); } terminate_workers(&writer->sorted_notifier); for(thread_i = 0; thread_i < writer -> threads; thread_i++){ pthread_join(writer -> writer_threads[thread_i].thread_stub, NULL); deflateEnd(&(writer -> writer_threads[thread_i].strm)); } HashTableDestroy(writer -> block_no_p1_to_vpos_tab); free(writer->writer_threads); free(current_min_fps); free(sb_fps); } void SamBam_writer_finalise_thread(SamBam_Writer * writer, int thread_id){ if(writer -> threads < 2){ if(writer -> sort_reads_by_coord){ SamBam_writer_sort_buff_one_write(writer, writer ->chunk_buffer, writer -> chunk_buffer_used, -1); writer -> chunk_buffer_used= 0; }else{ if(writer -> chunk_buffer_used) SamBam_writer_add_chunk(writer, thread_id); } }else{ if(writer -> sort_reads_by_coord){ SamBam_writer_sort_buff_one_write(writer, writer -> threads_chunk_buffer[thread_id], writer -> threads_chunk_buffer_used[thread_id], thread_id); writer -> threads_chunk_buffer_used [thread_id] = 0; }else{ if(writer -> threads_chunk_buffer_used[thread_id]) SamBam_writer_add_chunk(writer, thread_id); } } } void SamBam_writer_finalise_one_thread(SamBam_Writer * writer){ if(writer -> threads < 2){ if(writer -> sort_reads_by_coord){ if(writer -> chunk_buffer_used>0){ SamBam_writer_sort_buff_one_write(writer, writer -> chunk_buffer, writer -> chunk_buffer_used, -1); writer -> chunk_buffer_used = 0; } }else{ if(writer -> chunk_buffer_used) SamBam_writer_add_chunk(writer, -1); } } } unsigned int FC_CRC32(char * dat, int len){ unsigned int crc0 = crc32(0, NULL, 0); unsigned int ret = crc32(crc0, (unsigned char *)dat, len); return ret; } void simple_bam_writer_deallocate_index_per_chro(void * p){ struct simple_bam_writer_index_per_chro * ch = p; HashTableDestroy(ch->index_binP1_table); ArrayListDestroy(ch->index_binP0_list); ArrayListDestroy(ch->win16k_list); free(ch); } struct simple_bam_writer_index_per_chro * simple_bam_writer_new_index_per_chro(){ struct simple_bam_writer_index_per_chro * ret = malloc(sizeof(struct simple_bam_writer_index_per_chro )); ret -> index_binP1_table = HashTableCreate(4000); HashTableSetDeallocationFunctions(ret -> index_binP1_table, NULL, (void (*) (void*)) ArrayListDestroy); ret -> index_binP0_list = ArrayListCreate(20000); ret -> win16k_list = ArrayListCreate(20000); return ret; } void simple_bam_writer_update_index(simple_bam_writer * writer, char * rbin, int binlen, srInt_64 block_number, int inbin_pos){ int chro_no=0; memcpy(&chro_no, rbin + 4, 4); if(chro_no<0)return; unsigned int pos, bin_mq_nl=0; pos = *(unsigned int*)(rbin + 8); bin_mq_nl = *(unsigned int*)(rbin + 12); struct simple_bam_writer_index_per_chro * index_chro = HashTableGet(writer -> index_per_chro, NULL+chro_no+1); if(NULL==index_chro){ index_chro = simple_bam_writer_new_index_per_chro(); HashTablePut(writer -> index_per_chro, NULL+chro_no+1, index_chro); } unsigned int binno = bin_mq_nl>>16; int cigar_span = SamBam_writer_calc_cigar_span(rbin +4); int this_w16_no = (pos + cigar_span) >>14; // WIN is calculated on 0-based pos. unsigned long long this_Vpos = block_number<<16 | inbin_pos; ArrayList * win16k_list = index_chro -> win16k_list; // if this read is after the maximum coordinate in the win16k list: all elements before last one and this one starts at this read. if(this_w16_no > win16k_list->numOfElements){ int bbi; for(bbi = win16k_list->numOfElements; bbi <=this_w16_no; bbi++) ArrayListPush(win16k_list, NULL+ this_Vpos); } ArrayList * this_bin_chunks = HashTableGet(index_chro -> index_binP1_table, NULL+binno+1); if(NULL == this_bin_chunks){ this_bin_chunks = ArrayListCreate(4); HashTablePut(index_chro -> index_binP1_table, NULL+binno+1, this_bin_chunks); ArrayListPush(index_chro -> index_binP0_list, NULL+binno); } int found = 0; // a bin is not necessarily continuous. Say, a top-level bin only contains a few reads (most reads a in low-level bins), but their locations are everywhere if(this_bin_chunks -> numOfElements > 0){ long long diff = this_Vpos >>16; diff -=(this_bin_chunks -> elementList [ this_bin_chunks -> numOfElements - 1] - NULL)>>16; if(diff < MAX_ALLOWED_GAP_IN_BAI_CHUNK){ this_bin_chunks -> elementList [ this_bin_chunks -> numOfElements - 1] = NULL+this_Vpos + binlen + 4; found = 1; } } // if the last chunk in this bin isn't good to be extended (too far from the file location of the new read), a new chunk is created. if(!found){ ArrayListPush(this_bin_chunks, NULL + this_Vpos); ArrayListPush(this_bin_chunks, NULL + this_Vpos + binlen+4); } } void simple_bam_write_compressed_block(simple_bam_writer * writer,char *obuf, int olen, int ilen, unsigned int crcval, srInt_64 block_number){ if(block_number >= 0)HashTablePut(writer -> bam_blockP1_to_offset0B_table, NULL+1+block_number, NULL+ftello(writer -> bam_FP)); fwrite("\x1f\x8b\x8\x4\0\0\0\0\0\0\x6\0", 1, 12, writer -> bam_FP); fwrite("\x42\x43\x2\0", 1, 4, writer -> bam_FP); int BSIZE = olen+19+6; fwrite(&BSIZE, 1, 2, writer -> bam_FP); fwrite(obuf, 1, olen, writer -> bam_FP); fwrite(&crcval, 1, 4, writer -> bam_FP); fwrite(&ilen, 1, 4, writer -> bam_FP); } void simple_bam_write(void * bin, int binlen, simple_bam_writer * writer, int force_flush){ while(binlen > 0 || (force_flush && writer->inbin_len)){ int concatinate_binlen = min(binlen, 63000 - writer->inbin_len); memcpy(writer->inbin+writer->inbin_len, bin, concatinate_binlen); writer->inbin_len += concatinate_binlen; bin += concatinate_binlen; binlen -= concatinate_binlen; if(writer->inbin_len >=63000 || force_flush){ deflateInit2(&writer -> strm, Z_BEST_SPEED, Z_DEFLATED, -15, Z_DEFAULT_MEM_LEVEL, Z_DEFAULT_STRATEGY); char obuf[MERGER_WORKER_BINSIZE]; writer->strm.next_in = (unsigned char *)writer->inbin; writer->strm.avail_in = writer->inbin_len; writer->strm.next_out = (unsigned char *)obuf; writer->strm.avail_out = MERGER_WORKER_BINSIZE; deflate(&writer->strm, Z_FINISH); int have = MERGER_WORKER_BINSIZE-writer->strm.avail_out; simple_bam_write_compressed_block(writer, obuf, have, writer->inbin_len, FC_CRC32(writer->inbin, writer->inbin_len), -1); writer->inbin_len=0; deflateEnd(&writer -> strm); } } } simple_bam_writer * simple_bam_create(char * fname){ simple_bam_writer * ret = calloc(sizeof(simple_bam_writer), 1); ret -> bam_FP = fopen(fname, "wb"); ret -> bam_blockP1_to_offset0B_table = HashTableCreate(100000); simple_bam_write("BAM\1", 4, ret, 0); char bainame [strlen(fname)+10]; strcpy(bainame , fname); strcat(bainame, ".bai"); ret -> bai_FP = fopen(bainame, "wb"); fwrite("BAI\1", 1, 4, ret -> bai_FP); ret -> index_per_chro = HashTableCreate(1000); HashTableSetDeallocationFunctions(ret -> index_per_chro , NULL , simple_bam_writer_deallocate_index_per_chro); return ret; } #define vpos_to_rpos rposone = (vposone & 0xffff ) + ( (HashTableGet(writer -> bam_blockP1_to_offset0B_table, NULL+1+(vposone >>16)) - NULL) << 16 ) #define BAM_EOF_MARKER "\x1f\x8b\x08\x04\0\0\0\0\0\xff\x06\0\x42\x43\x02\0\x1b\0\x03\0\0\0\0\0\0\0\0\0" void simple_bam_close(simple_bam_writer * writer){ fwrite(BAM_EOF_MARKER, 1, 28, writer -> bam_FP); fclose(writer -> bam_FP); fwrite(&writer -> total_chromosomes, 1, 4, writer -> bai_FP); int chri; for(chri=0; chri total_chromosomes; chri++){ struct simple_bam_writer_index_per_chro *this_idx = HashTableGet(writer -> index_per_chro , NULL+1+chri); if(NULL == this_idx ){ fwrite("\0\0\0\0\0\0\0\0", 1, 8, writer -> bai_FP);//0 intervals and 0 bins }else{ HashTable * new_tab=NULL; ArrayList * new_arr=NULL; SamBam_writer_optimize_bins(this_idx -> index_binP1_table, this_idx -> index_binP0_list ,& new_tab, & new_arr); this_idx -> index_binP1_table = new_tab; this_idx -> index_binP0_list = new_arr; fwrite(&this_idx -> index_binP0_list->numOfElements ,1, 4, writer -> bai_FP); int bini; for(bini = 0; bini < this_idx -> index_binP0_list -> numOfElements; bini ++){ int binno = ArrayListGet(this_idx -> index_binP0_list, bini)-NULL; ArrayList * bingaps = HashTableGet(this_idx -> index_binP1_table, NULL+1+binno); srInt_64 gapi = bingaps -> numOfElements/2; fwrite(&binno, 1, 4, writer -> bai_FP); fwrite(&gapi ,1, 4, writer -> bai_FP); for(gapi = 0; gapi < bingaps -> numOfElements; gapi++){ srInt_64 rposone, vposone = ArrayListGet(bingaps , gapi)-NULL; vpos_to_rpos; fwrite(&rposone, 1, 8, writer -> bai_FP); } } fwrite(&this_idx -> win16k_list -> numOfElements ,1, 4, writer -> bai_FP); for(bini = 0; bini < this_idx -> win16k_list -> numOfElements; bini ++){ srInt_64 rposone, vposone = ArrayListGet(this_idx -> win16k_list , bini )-NULL; vpos_to_rpos; fwrite(&rposone, 1, 8, writer -> bai_FP); } } } HashTableDestroy(writer -> index_per_chro); fclose(writer -> bai_FP); free(writer); } Rsubread/src/sambam-file.h0000644000175200017520000002335214136047615016474 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #ifndef _SAMBAM_FILE_H_ #define _SAMBAM_FILE_H_ #include #include "HelperFunctions.h" typedef unsigned char BS_uint_8; typedef unsigned short BS_uint_16; typedef unsigned int BS_uint_32; #define BAM_MAX_CHROMOSOME_NAME_LEN 200 #define BAM_MAX_CIGAR_LEN (30000) #define BAM_MAX_READ_NAME_LEN 256 #define BAM_MAX_READ_LEN 3000 #define SAMBAM_FILE_SAM 10 #define SAMBAM_FILE_BAM 20 #define BAM_FILE_STAGE_HEADER 10 #define BAM_FILE_STAGE_ALIGNMENT 20 #define SAMBAM_COMPRESS_LEVEL_FASTEST Z_NO_COMPRESSION #define SAMBAM_COMPRESS_LEVEL_NORMAL Z_BEST_SPEED #define SAMBAM_GZIP_WINDOW_BITS -15 #define SAMBAM_INPUT_STREAM_SIZE 140000 #define TEST_BAD_BAM_CHUNKS 9999925 typedef struct { char read_name[BAM_MAX_READ_NAME_LEN]; char * chro_name; unsigned int chro_offset; unsigned short flags; char * mate_chro_name; unsigned int mate_chro_offset; int templete_length; unsigned char mapping_quality; int NH_number; char cigar[BAM_MAX_CIGAR_LEN]; char sequence[BAM_MAX_READ_LEN]; char seq_quality[BAM_MAX_READ_LEN]; char buff_for_seq[BAM_MAX_READ_LEN*2]; } SamBam_Alignment; #define SB_FETCH(a) if((a) -> input_binary_stream_write_ptr - (a) -> input_binary_stream_read_ptr < 3000){int test_rlen_2 = SamBam_fetch_next_chunk(a); if(test_rlen_2 == -2){(a)->is_bam_broken = 1;}} #define SB_EOF(a) ((a)-> is_eof && ( (a) -> input_binary_stream_write_ptr <= (a) -> input_binary_stream_read_ptr )) #define SB_READ(a) ((a) -> input_binary_stream_buffer + (a) -> input_binary_stream_read_ptr - (a) -> input_binary_stream_buffer_start_ptr) #define SB_RINC(a, len) ((a) -> input_binary_stream_read_ptr) += len typedef struct { FILE * os_file; int file_type; int bam_file_stage; unsigned long long bam_file_next_section_start; unsigned long long input_binary_stream_read_ptr; unsigned long long input_binary_stream_write_ptr; unsigned long long input_binary_stream_buffer_start_ptr; unsigned long long header_length; SamBam_Reference_Info * bam_chro_table; int bam_chro_table_size; SamBam_Alignment aln_buff; char * input_binary_stream_buffer; int is_eof; int is_paired_end; int is_bam_broken; } SamBam_FILE; struct SamBam_sorted_compressor_st{ char plain_text[66000]; char zipped_bin[70000]; int text_size, bin_size; unsigned int CRC32_plain; z_stream strm; pthread_t thread_stub; srInt_64 bam_block_no; int last_job_done; }; typedef struct { FILE * bam_fp; FILE * BAI_fp; long long current_BAM_pos; char tmpf_prefix[MAX_FILE_NAME_LENGTH]; z_stream output_stream; char * chunk_buffer; char * compressed_chunk_buffer; char * header_plain_text_buffer; int header_plain_text_buffer_used; int header_plain_text_buffer_max; long long chunk_buffer_used; long long chunk_buffer_max_size; int writer_state; int is_internal_error; int sort_reads_by_coord; int fastest_compression; int sorted_batch_id; unsigned int crc0; int threads; z_stream * threads_output_stream; char ** threads_chunk_buffer; char ** threads_chunk_buffer_compressed; long long * threads_chunk_buffer_used; long long * threads_chunk_buffer_max_size; HashTable * chromosome_name_table; HashTable * chromosome_id_table; HashTable * chromosome_len_table; subread_lock_t thread_bam_lock; worker_master_mutex_t sorted_notifier; HashTable * block_no_p1_to_vpos_tab; //int sorted_compress_plain_text_used; int sorted_compress_this_thread_no; srInt_64 this_bam_block_no; struct SamBam_sorted_compressor_st * writer_threads; } SamBam_Writer; // This function reads the next BAM section from the bam_fp. The buffer has a variable length but should be at least 64K bytes. // I recommend you to allocate 80KB of memory. // This function returns the size of the compressed data ( CDATA ). It returns < 0 if EOF. int PBam_get_next_zchunk(FILE * bam_fp, char * buffer, int buffer_length, unsigned int * real_len); // load the header of a BAM file (the header is important to load BAM reads) // this function puts the File Pointer to the first read chunk in the BAM. // It returns 0 if finished loading, or non-zero if wrong. // If the chunk contains read data after the chromosome table, the read data is copied into remainder_read_data, and its lengtb is returned in remainder_read_data_len. int PBum_load_header(FILE * bam_fp, SamBam_Reference_Info** chro_tab, char * remainder_read_data, int * remainder_read_data_len); // load a new line from the BAM buffer (chunk) at chunk_ptr. // if seq_needed==0, then no sequence nor quality str will be loaded. // it returns the length (without "\0" after the tail) of the SAM string. int PBam_chunk_gets(char * chunk, int *chunk_ptr, int chunk_limit, SamBam_Reference_Info * bam_chro_table, char * buff , int buff_len, SamBam_Alignment*aln, int seq_needed); // This function opens a file, either SAM or BAM, in read-only mode. // The "file_type" parameter specifies which type of file it is: SAMBAM_FILE_BAM or SAMBAM_FILE_SAM. SamBam_FILE * SamBam_fopen(char * fname , int file_type); // This function closes any opened file and releases memory footprint. It works just like "fclose()". void SamBam_fclose(SamBam_FILE * fp); // This function tells if a file is exhausted. // Note that a non-exhausted file can still contain no more alignment results. // Hence, it is recommended to check the return value of SamBam_fgets() to tell if the file has reached its end. int SamBam_feof(SamBam_FILE * fp); // This function works like fgets except it decode the BAM file. // If the buffer is not long enough to store the line, the remainder of this line is omitted and the next call will read the next alignment. // A very important difference between fgets and SamBam_fgets is that this function returns NULL when there are no more lines. // It is recommended to use the return value as the indicator of EOF like: /** * SamBam_FILE * fp = SamBam_fopen("my.bam", SAMBAM_FILE_BAM); * while(1) * { * char buf[3000]; * char * ret = SamBam_fgets(fp, buf, 3000); * if(ret) puts(buf); * else break; * } * SamBam_fclose(fp); */ char * SamBam_fgets(SamBam_FILE * fp , char * buff , int buff_len , int seq_needed); int SamBam_writer_create(SamBam_Writer * writer, char * BAM_fname, int threads, int sort_reads_by_coord, int is_tmp_BAM, char * tmpfname); int SamBam_writer_close(SamBam_Writer * writer); int SamBam_writer_add_header(SamBam_Writer * writer, char * header_text, int add_chro); int SamBam_writer_add_chromosome(SamBam_Writer * writer, char * chro_name, unsigned int chro_length, int add_header_too); int SamBam_writer_add_read_bin(SamBam_Writer * writer, int thread_no, char * rbin, int committable); int SamBam_writer_calc_cigar_span(char * bin); int SamBam_writer_add_read_fqs_scRNA(gzFile * outfp, char * bambin); int SamBam_writer_add_read(SamBam_Writer * writer, int threadno, char * read_name, unsigned int flags, char * chro_name, unsigned int chro_position, int mapping_quality, char * cigar, char * next_chro_name, unsigned int next_chro_pos, int temp_len, int read_len, char * read_text, char * qual_text, char * additional_columns, int can_submit); void SamBam_writer_optimize_bins(HashTable *bin_tab, ArrayList *bin_arr, HashTable ** new_tab, ArrayList ** new_arrs); int is_badBAM(char * fn); int SamBam_unzip(char * out, int out_max_len , char * in , int inlen, int sync_only); int SamBam_fetch_next_chunk(SamBam_FILE *fp); int SamBam_compress_cigar(char * cigar, int * cigar_int, int * ret_coverage, int max_secs); char cigar_op_char(int ch); void SamBam_read2bin(char * read_txt, char * read_bin); int convert_BAM_binary_to_SAM(SamBam_Reference_Info * chro_table, char * bam_bin, char * sam_txt); int is_paired_end_BAM(char * fn); void SamBam_writer_finalise_thread(SamBam_Writer * writer, int thread_id); void SamBam_writer_finish_header( SamBam_Writer * writer ); void SamBam_writer_finalise_one_thread(SamBam_Writer * writer); int SamBam_writer_add_read_line(SamBam_Writer * writer, int thread_no, char * rline, int commitable); char *duplicate_TAB_record_field(char * rline, int fld_no, int toend); int SamBam_reg2bin(int beg, int end); unsigned int SamBam_CRC32(char * dat, int len); struct simple_bam_writer_index_per_chro{ HashTable * index_binP1_table; ArrayList * index_binP0_list; ArrayList * win16k_list; }; #define MERGER_WORKER_BINSIZE 66000 typedef struct { FILE * bam_FP; FILE * bai_FP; z_stream strm; char inbin[MERGER_WORKER_BINSIZE]; int inbin_len; int total_chromosomes; HashTable * bam_blockP1_to_offset0B_table; HashTable * index_per_chro; } simple_bam_writer; //#define MAX_ALLOWED_GAP_IN_BAI_CHUNK 10 // 10 blocks unsigned int FC_CRC32(char * dat, int len); void simple_bam_writer_deallocate_index_per_chro(void * p); struct simple_bam_writer_index_per_chro * simple_bam_writer_new_index_per_chro(); void simple_bam_write_compressed_block(simple_bam_writer * writer,char *obuf, int olen, int ilen, unsigned int crcval, srInt_64 block_number); void simple_bam_write(void * bin, int binlen, simple_bam_writer * writer, int force_flush); simple_bam_writer * simple_bam_create(char * fname); void simple_bam_close(simple_bam_writer * writer); void simple_bam_writer_update_index(simple_bam_writer * writer, char * rbin, int binlen, srInt_64 block_number, int inbin_pos); #endif Rsubread/src/seek-zlib.c0000644000175200017520000007161014136047615016177 0ustar00biocbuildbiocbuild#include #include "core.h" #include "seek-zlib.h" #include "input-files.h" #include "gene-algorithms.h" #define SEEKGZ_INIT_TEXT_SIZE (1024*1024) #define SEEKGZ_BINBUFF_SIZE (1*1024*1024) unsigned long long seekgz_ftello(seekable_zfile_t * fp){ unsigned long long ret = ftello(fp -> gz_fp); ret -= fp -> stem.avail_in; return ret; } unsigned int crc_pos(char * bin, int len){ unsigned int crc0 = 0;//crc32(0, NULL, 0); unsigned int CRC32 = crc32(crc0, (unsigned char *) bin, len); return CRC32; } void seekgz_try_read_some_zipped_data(seekable_zfile_t * fp, int * is_eof){ if(feof(fp->gz_fp)){ if(is_eof) (*is_eof)=1; return; } assert(fp -> stem.avail_in >= 0); if(fp -> stem.avail_in < SEEKGZ_BINBUFF_SIZE / 2 ) { if(fp -> in_zipped_buff_read_ptr > 0 && fp -> stem.avail_in > 0){ int i; for(i = 0 ; i < fp -> stem.avail_in ; i ++){ fp -> in_zipped_buffer[i] = fp -> in_zipped_buffer[i + fp -> in_zipped_buff_read_ptr]; } } fp -> in_zipped_buff_read_ptr = 0; int readlen = fread(fp -> in_zipped_buffer + fp -> stem.avail_in, 1 , SEEKGZ_BINBUFF_SIZE - fp -> stem.avail_in , fp -> gz_fp); if(readlen>0) fp -> stem.avail_in += readlen; fp -> stem.next_in = (unsigned char *)fp -> in_zipped_buffer; } } int seekgz_get_next_zipped_char(seekable_zfile_t * fp){ seekgz_try_read_some_zipped_data(fp, NULL); int ret = -1; if(fp -> stem.avail_in > 0) { ret = fp -> in_zipped_buffer [ fp -> in_zipped_buff_read_ptr ++]; fp -> stem.next_in = (unsigned char *)(fp -> in_zipped_buffer + fp -> in_zipped_buff_read_ptr); fp -> stem.avail_in --; if(ret<0) ret=256+ret; } return ret; } int seekgz_skip_gzfile_header(seekable_zfile_t * fp, int tail_size){ int id1, id2; if(tail_size){ for(id1=0; id10; XLEN--) seekgz_get_next_zipped_char(fp); } for(id1 = 3; id1 <=4; id1++){ if(FLG & (1< next_block_file_offset = seekgz_ftello(fp); fp -> next_block_file_bits = 0; fp -> rolling_dict_window_used = 0; return 0; } int seekgz_load_more_blocks( seekable_zfile_t * fp , int bytes_ahead, subread_lock_t * read_lock ); int seekgz_open(const char * fname, seekable_zfile_t * fp, FILE * old_fp){ memset(fp, 0, sizeof(seekable_zfile_t)); if(old_fp) fp -> gz_fp = old_fp; else fp -> gz_fp = f_subr_open(fname, "rb"); if(NULL==fp -> gz_fp)return -1; fp -> in_zipped_buffer = malloc(SEEKGZ_BINBUFF_SIZE); subread_init_lock(&fp -> write_lock); //fp -> txt_buffer_size = SEEKGZ_INIT_TEXT_SIZE; fp -> blocks_in_chain = 0; fp -> block_chain_current_no = 0; // no blocks in the chain fp -> rolling_dict_window_used = 0; fp -> current_block_txt_read_ptr = 0; fp -> stem.zalloc = Z_NULL; fp -> stem.zfree = Z_NULL; fp -> stem.opaque = Z_NULL; fp -> stem.avail_in = 0; fp -> stem.next_in = Z_NULL; if( old_fp ){ fp -> stem.avail_in = 2; fp -> in_zipped_buffer[0] = 0x1f; fp -> in_zipped_buffer[1] = 0x8b; } int ret = seekgz_skip_gzfile_header(fp,0); if(ret) return 1; ret = inflateInit2(&(fp -> stem), -15); if(ret) return 1; fp -> next_block_file_offset = seekgz_ftello( fp ); fp -> next_block_file_bits = 0; ret = seekgz_load_more_blocks(fp, 300000, NULL); //SUBREADprintf("Gzip : opened %s of %p\n", fname, fp); return ret; } void seekgz_tell(seekable_zfile_t * fp, seekable_position_t * pos){ if(fp -> blocks_in_chain>0){ seekable_decompressed_block_t * curr_block = fp -> block_rolling_chain+fp -> block_chain_current_no; pos -> block_gzfile_offset = curr_block -> block_start_in_file_offset; pos -> block_gzfile_bits = curr_block -> block_start_in_file_bits; memcpy(pos -> dict_window, curr_block -> block_dict_window, curr_block -> block_dict_window_size); pos -> block_dict_window_size = curr_block -> block_dict_window_size; pos -> in_block_text_offset = fp -> current_block_txt_read_ptr; }else{ pos -> block_gzfile_offset = 0xffffffffffffffffllu; } } int seekgz_load_1_block( seekable_zfile_t * fp , int empty_block_no); void seekgz_seek(seekable_zfile_t * fp, seekable_position_t * pos){ //SUBREADprintf("SEEK:gzip -> fpos=%llu, block_offset=%d\n", pos -> block_gzfile_offset, pos -> in_block_text_offset); // if(pos -> block_gzfile_offset== 0xffffffffffffffffllu){ fp -> block_chain_current_no = 0; fp -> blocks_in_chain = 0; fp -> stem.avail_in = 0; fseeko(fp->gz_fp, 0, SEEK_END); fgetc(fp->gz_fp); return ; } fseeko(fp->gz_fp, pos -> block_gzfile_offset - (pos -> block_gzfile_bits?1:0), SEEK_SET); if(Z_OK!=inflateReset(&fp->stem)) SUBREADprintf("FATAL: UNABLE TO INIT STREAM.\n\n\n"); if(pos -> block_dict_window_size>0){ if(pos -> block_gzfile_bits){ char nch = fgetc(fp->gz_fp); //fprintf(stderr, "SEEK 2 FPPOS:%llu, NCH=%d\n", ftello(fp->gz_fp) , nch); inflatePrime(&fp->stem, pos -> block_gzfile_bits, nch>>(8-pos -> block_gzfile_bits)); } if(Z_OK != inflateSetDictionary(&fp->stem, (unsigned char *)pos -> dict_window, pos -> block_dict_window_size)) SUBREADprintf("FATAL: UNABLE TO RESET STREAM.\n\n\n"); } fp -> stem.avail_in = 0; int ii; for(ii = 0; ii < fp -> blocks_in_chain ; ii++){ int iv = ii + fp -> block_chain_current_no; if(iv >=SEEKGZ_CHAIN_BLOCKS_NO) iv-=SEEKGZ_CHAIN_BLOCKS_NO; free(fp -> block_rolling_chain[iv].block_txt); free(fp -> block_rolling_chain[iv].linebreak_positions); } fp -> block_chain_current_no = 0; fp -> blocks_in_chain = 0; fp -> in_zipped_buff_read_ptr = 0; fp -> current_block_txt_read_ptr = 0; memcpy(fp -> rolling_dict_window, pos -> dict_window, pos -> block_dict_window_size); fp -> rolling_dict_window_used= pos -> block_dict_window_size; fp -> next_block_file_offset = pos -> block_gzfile_offset; fp -> next_block_file_bits = pos -> block_gzfile_bits; seekgz_load_more_blocks(fp, 30000, NULL); fp -> current_block_txt_read_ptr = pos -> in_block_text_offset; //SUBREADprintf("SEK: %d WIN=%d BLK (%d / %d), %d AVI\n", fp -> blocks_in_chain, pos -> block_dict_window_size, fp -> current_block_txt_read_ptr , fp -> block_rolling_chain[0].block_txt_size, fp -> stem.avail_in); } void seekgz_find_linebreaks(seekable_zfile_t * fp, int empty_block_no){ int lbks= 0, arrsize = 5000; unsigned int * arr = malloc(sizeof(int) * arrsize); int ii; for(ii = 0; ii < fp -> block_rolling_chain[empty_block_no].block_txt_size ; ii++){ char nch = fp-> block_rolling_chain[empty_block_no].block_txt[ii]; if(nch == '\n'){ if(lbks >= arrsize){ arrsize*=2; arr = realloc(arr, sizeof(int) * arrsize); } arr[lbks++] = ii; } } fp -> block_rolling_chain[empty_block_no].linebreak_positions = arr; fp -> block_rolling_chain[empty_block_no].linebreaks = lbks; } void seekgz_update_current_window(seekable_zfile_t * fp , char * txt, int tlen){ int fp_start=0, new_start=0, cplen=0; int moveback = 0; if(tlen + fp -> rolling_dict_window_used > SEEKGZ_ZLIB_WINDOW_SIZE) moveback = tlen + fp -> rolling_dict_window_used - SEEKGZ_ZLIB_WINDOW_SIZE; if(moveback>0 && moveback < fp -> rolling_dict_window_used){ int ii; for(ii = 0; ii < fp -> rolling_dict_window_used - moveback; ii++){ fp -> rolling_dict_window[ii] = fp -> rolling_dict_window[ii + moveback]; } } fp -> rolling_dict_window_used = moveback < fp -> rolling_dict_window_used ? (fp -> rolling_dict_window_used - moveback):0; if(tlen > SEEKGZ_ZLIB_WINDOW_SIZE){ fp_start =0; new_start = tlen - SEEKGZ_ZLIB_WINDOW_SIZE; cplen = SEEKGZ_ZLIB_WINDOW_SIZE; }else{ new_start = 0; fp_start = fp -> rolling_dict_window_used; cplen = tlen; } memcpy(fp -> rolling_dict_window + fp_start, txt + new_start, cplen); fp -> rolling_dict_window_used += cplen; } int seekgz_eof(seekable_zfile_t * fp){ if(fp -> stem.avail_in < 1 && feof(fp->gz_fp))return 1; return 0; } #define SEEKGZ_ONE_BLOCK_TEXT_INIT_SIZE (256*1024) // 1block ret < 0 : bad error // 1block ret > 0 : data is loaded // 1block ret == 0 : no data is loaded // A block MUST have at least 10000 bytes otherwise multiple blocks are concatinated #define MINIMUM_GZIP_BLOCK_SIZE 10000 int seekgz_load_1_block( seekable_zfile_t * fp , int empty_block_no){ if(seekgz_eof(fp))return 0; char * out_txt = malloc(SEEKGZ_ONE_BLOCK_TEXT_INIT_SIZE); int out_txt_size = SEEKGZ_ONE_BLOCK_TEXT_INIT_SIZE; int out_txt_used = 0; memcpy(fp -> block_rolling_chain[empty_block_no].block_dict_window, fp -> rolling_dict_window, fp -> rolling_dict_window_used); fp -> block_rolling_chain[empty_block_no].block_dict_window_size = fp -> rolling_dict_window_used; //fp -> rolling_dict_window_used = 0; fp -> block_rolling_chain[empty_block_no].block_start_in_file_offset = fp -> next_block_file_offset; fp -> block_rolling_chain[empty_block_no].block_start_in_file_bits = fp -> next_block_file_bits; int is_data_error = 0; int is_eof = 0; while(1){ int is_block_end = 0; int is_gzip_unit_end = 0; while(1){ seekgz_try_read_some_zipped_data(fp, &is_eof); if(fp -> stem.avail_in < 1 && is_eof)break; if(out_txt_size < out_txt_used *4/3){ out_txt_size *= 2; out_txt = realloc(out_txt,out_txt_size); } int old_avail_in = fp -> stem.avail_in; fp -> stem.avail_out = out_txt_size - out_txt_used; fp -> stem.next_out = (unsigned char *)out_txt + out_txt_used; void * old_next_in = fp -> stem.next_in; //SUBREADprintf("i INFLATINHG: FILEPOS=%llu IN_AVAIL=%d OUT_AVAIL=%d\n", seekgz_ftello(fp), fp -> stem.avail_in, fp -> stem.avail_out); int ret_ifl = inflate(&(fp -> stem), Z_BLOCK); int have = (out_txt_size - out_txt_used) - fp -> stem.avail_out; if(ret_ifl != Z_OK && ret_ifl != Z_STREAM_END){ SUBREADprintf("ERR INFLATINHG: RET=%d BY IN %d zipped bytes ==> %d PLAIN TXT\n", ret_ifl, old_avail_in - fp -> stem.avail_in, have); SUBREADprintf("ERR INFLATINHG: BEND=%d\n", ( fp -> stem.data_type & 128 )&& !(fp -> stem.data_type & 64)); } //if(ret_ifl != Z_STREAM_END){ // SUBREADprintf("INF: STREAM_END\n"); //} if(ret_ifl != Z_OK && ret_ifl != Z_STREAM_END){ is_data_error = 1; break; } int zipped_data_used = (void *)fp -> stem.next_in - old_next_in; fp -> in_zipped_buff_read_ptr += zipped_data_used; if( ( fp -> stem.data_type & 128 )&& !(fp -> stem.data_type & 64)) is_block_end = 1; if(ret_ifl == Z_STREAM_END) is_gzip_unit_end = 1; //if(is_block_end) // fp -> rolling_dict_window_used = 0; //else seekgz_update_current_window(fp, out_txt + out_txt_used, have); out_txt_used += have; if(is_block_end){ fp -> next_block_file_offset = seekgz_ftello(fp); fp -> next_block_file_bits = fp->stem.data_type & 7; } if(is_gzip_unit_end){ seekgz_skip_gzfile_header(fp, 8); inflateReset(&fp->stem); } if(is_block_end || is_gzip_unit_end) break; } if(out_txt_used >= MINIMUM_GZIP_BLOCK_SIZE || is_data_error ) break; if(fp -> stem.avail_in < 1 && is_eof)break; } if(is_data_error) return -1; //SUBREADprintf("o END_1BLK: GOT %d PLAIN TXT i; first char: '%c' fp==%p\n\n", out_txt_used, out_txt[0], fp); if(out_txt_used>0){ fp -> block_rolling_chain[empty_block_no].block_txt = out_txt; fp -> block_rolling_chain[empty_block_no].block_txt_size = out_txt_used; seekgz_find_linebreaks(fp, empty_block_no); return out_txt_used; }else{ free(out_txt); return 0; } } int seekgz_load_more_blocks( seekable_zfile_t * fp , int bytes_ahead, subread_lock_t * read_lock ){ int loaded_bytes=0, ret=0; while(1){ int empty_block_no = -1; subread_lock_occupy(&fp -> write_lock ); if(read_lock) subread_lock_occupy(read_lock); if(fp -> blocks_in_chain < SEEKGZ_CHAIN_BLOCKS_NO){ empty_block_no = fp-> block_chain_current_no + fp -> blocks_in_chain; if(empty_block_no >= SEEKGZ_CHAIN_BLOCKS_NO) empty_block_no -= SEEKGZ_CHAIN_BLOCKS_NO; } if(read_lock) subread_lock_release(read_lock); if(empty_block_no < 0 || (bytes_ahead>=0 && loaded_bytes >= bytes_ahead)){ subread_lock_release(&fp -> write_lock ); break; } int oneblk_ret = seekgz_load_1_block(fp, empty_block_no); //SUBREADprintf("LOAD_1BLK: RET=%d EMPTY_NO=%d\n", oneblk_ret, empty_block_no); // 1block ret < 0 : bad error // 1block ret > 0 : no data is loaded // 1block ret == 0 : normal if(oneblk_ret < 0){ subread_lock_release(&fp -> write_lock ); ret = oneblk_ret; break; }else if(0 < oneblk_ret){ if(read_lock) subread_lock_occupy(read_lock); fp -> blocks_in_chain++; if(read_lock) subread_lock_release(read_lock); loaded_bytes+= oneblk_ret; }else if(seekgz_eof(fp)){ subread_lock_release(&fp -> write_lock ); break; } subread_lock_release(&fp -> write_lock ); } return ret; } int seqs = 0; int seekgz_preload_buffer( seekable_zfile_t * fp , subread_lock_t * read_lock){ int do_preload = 0; seqs+=1;// (unsigned int)(fp->rolling_dict_window[0]); if(( read_lock || !fp -> has_multi_thread_accessed) && fp -> blocks_in_chain < 3 && !seekgz_eof(fp) ) do_preload =1; else if(read_lock && fp -> blocks_in_chain < SEEKGZ_CHAIN_BLOCKS_NO ){ if(seqs >= 2000) { do_preload = 1; seqs = 0; } } if(!fp -> has_multi_thread_accessed && read_lock) fp -> has_multi_thread_accessed =1; if(do_preload){ int decompress_ret = seekgz_load_more_blocks(fp, -1, read_lock); return decompress_ret; } return 0; } // return : > 0 : N bytes loaded // == 0 : EOF // < 0 : data error int seekgz_gets(seekable_zfile_t * fp, char * buff, int buff_len){ //if(fp -> blocks_in_chain<3)SUBREADprintf("GTS: %d BLK, %d AVI\n", fp -> blocks_in_chain, fp -> stem.avail_in); int line_write_ptr = 0, is_end_line = 0; if(fp->blocks_in_chain < 1 && seekgz_eof(fp)){ return 0; } while(1){ int consumed_bytes; assert(fp->blocks_in_chain>0); seekable_decompressed_block_t *cblk = fp -> block_rolling_chain+fp -> block_chain_current_no; if( cblk -> linebreaks >0 && fp-> current_block_txt_read_ptr <= cblk->linebreak_positions[cblk -> linebreaks-1] ){ if(fp-> current_block_txt_read_ptr <=cblk->linebreak_positions[0]){ consumed_bytes = cblk->linebreak_positions[0] - fp-> current_block_txt_read_ptr +1; }else{ int high_idx = cblk -> linebreaks - 1 , low_idx = 0, idx = -1; while(1){ if(high_idx <= low_idx+1) { idx = min(low_idx, high_idx); break; } int mid_idx = (high_idx+low_idx)/2; if(cblk->linebreak_positions[mid_idx] > fp -> current_block_txt_read_ptr) high_idx = mid_idx; else if( cblk->linebreak_positions[mid_idx] < fp -> current_block_txt_read_ptr ) low_idx = mid_idx; else{ idx = high_idx; break; } } if(idx>0)idx--; while( cblk->linebreak_positions[idx+1] < fp -> current_block_txt_read_ptr) idx++; assert( idx < cblk -> linebreaks-1 ); consumed_bytes = cblk->linebreak_positions[idx+1] - fp-> current_block_txt_read_ptr +1; } is_end_line = 1; } else consumed_bytes = cblk -> block_txt_size - fp-> current_block_txt_read_ptr ; if(buff){ int cp_bytes = min(consumed_bytes, buff_len - line_write_ptr); memcpy( buff + line_write_ptr , cblk-> block_txt + fp-> current_block_txt_read_ptr, cp_bytes ); line_write_ptr += cp_bytes; if(buff)buff[line_write_ptr] = 0; } //SUBREADprintf("OUT_TXT in BLC %d / %d %p ENDL=%d CUNS_BYTES=%d / %d / %d : '''%s'''\n", fp -> block_chain_current_no, fp -> blocks_in_chain, fp, is_end_line, consumed_bytes, fp-> current_block_txt_read_ptr, cblk -> block_txt_size , buff); fp-> current_block_txt_read_ptr += consumed_bytes; if( fp-> current_block_txt_read_ptr >= cblk -> block_txt_size ){ //SUBREADprintf("CHAIN CONSUME_0 : %d ( - 1 ) %p\n", fp -> block_chain_current_no, cblk->block_txt); free(cblk->block_txt); //SUBREADprintf("CHAIN CONSUME_X : %d ( - 1 ) %p\n", fp -> block_chain_current_no, cblk->linebreak_positions); free(cblk->linebreak_positions); //SUBREADprintf("CHAIN CONSUME_Z : %d ( - 1 )\n", fp -> block_chain_current_no); fp-> current_block_txt_read_ptr = 0; fp -> block_chain_current_no++; if(fp -> block_chain_current_no>= SEEKGZ_CHAIN_BLOCKS_NO) fp -> block_chain_current_no=0; fp->blocks_in_chain --; } if(is_end_line) break; } return line_write_ptr; } int seekgz_next_int8(seekable_zfile_t * fp){ if(fp -> blocks_in_chain<1){ seekgz_load_more_blocks(fp, -1, NULL); if(fp -> blocks_in_chain<1) return -1; } seekable_decompressed_block_t *cblk = fp -> block_rolling_chain+fp -> block_chain_current_no; int ret = cblk->block_txt[fp-> current_block_txt_read_ptr]; fp-> current_block_txt_read_ptr++; if(fp-> current_block_txt_read_ptr == cblk -> block_txt_size){ free(cblk->block_txt); free(cblk->linebreak_positions); fp-> current_block_txt_read_ptr = 0; fp -> block_chain_current_no++; if(fp -> block_chain_current_no>= SEEKGZ_CHAIN_BLOCKS_NO) fp -> block_chain_current_no=0; fp->blocks_in_chain --; } return ret<0?256+ret:ret; } int seekgz_next_char(seekable_zfile_t * fp){ // MUST BE PROTECTED BY read_lock //SUBREADprintf("gen_next_char: block_in_chain=%d\n", fp -> blocks_in_chain); //if(fp -> blocks_in_chain<3)SUBREADprintf("1CH: %d BLK, %d AVI\n", fp -> blocks_in_chain, fp -> stem.avail_in); if(fp -> blocks_in_chain<1){ return EOF; } seekable_decompressed_block_t *cblk = fp -> block_rolling_chain+fp -> block_chain_current_no; int ret = cblk->block_txt[fp-> current_block_txt_read_ptr]; fp-> current_block_txt_read_ptr++; if(fp-> current_block_txt_read_ptr == cblk -> block_txt_size){ free(cblk->block_txt); free(cblk->linebreak_positions); fp-> current_block_txt_read_ptr = 0; fp -> block_chain_current_no++; if(fp -> block_chain_current_no>= SEEKGZ_CHAIN_BLOCKS_NO) fp -> block_chain_current_no=0; fp->blocks_in_chain --; } return ret; } void seekgz_close(seekable_zfile_t * fp){ int ii; fclose(fp -> gz_fp); free(fp -> in_zipped_buffer); for(ii = 0; ii < fp -> blocks_in_chain ; ii++){ int iv = ii + fp -> block_chain_current_no; if(iv >=SEEKGZ_CHAIN_BLOCKS_NO) iv-=SEEKGZ_CHAIN_BLOCKS_NO; free(fp -> block_rolling_chain[iv].block_txt); free(fp -> block_rolling_chain[iv].linebreak_positions); } inflateEnd(&fp -> stem); subread_destroy_lock(&fp -> write_lock); } int autozip_open(const char * fname, autozip_fp * fp){ int ret = -1; int need_seek=1; memset(fp, 0, sizeof(autozip_fp)); strcpy(fp -> filename, fname); FILE * tstfp = fopen(fname,"rb"); if(!tstfp) return ret; int cc1, cc2; cc1 = fgetc(tstfp); cc2 = fgetc(tstfp); if(cc2 == 0x8b && cc1 == 0x1f){ //fclose(tstfp); fp -> is_plain = 0; fp -> zlib_fp = NULL; int iret = 0; if(need_seek) iret = seekgz_open(fname, &fp -> gz_fp, tstfp); else{ fp -> zlib_fp = gzopen(fname, "rb"); if(fp -> zlib_fp == NULL) iret=-1; } if(iret >= 0) ret = 1; else ret = -1; }else{ if(cc1 != EOF && cc2 != EOF){ fp -> first_chars[0] = cc1; fp -> first_chars[1] = cc2; fp -> is_first_chars = 2; } fp -> plain_fp = tstfp; fp -> is_plain = 1; ret = 0; } return ret; } void autozip_close(autozip_fp * fp){ if(fp -> is_plain) fclose(fp -> plain_fp); else if(fp->zlib_fp) gzclose(fp -> zlib_fp); else seekgz_close(&fp -> gz_fp); } int autozip_gets(autozip_fp * fp, char * buf, int buf_size){ int ret = 0; if(fp -> is_plain) { int base0 = 0; if( fp -> is_first_chars ){ buf[0]=fp -> first_chars[0]; buf[1]=fp -> first_chars[1]; base0 = 2; fp -> is_first_chars = 0; } buf[2]=0; char * retc = fgets(buf + base0, buf_size, fp -> plain_fp); if(retc == NULL && base0 == 0) ret = 0; else ret = strlen(buf); }else{ if(fp -> zlib_fp){ char *rets = gzgets(fp -> zlib_fp, buf, buf_size); if(rets) ret = strlen(buf); else ret = 0; }else{ seekgz_preload_buffer(&fp->gz_fp, NULL); ret = seekgz_gets(&fp -> gz_fp, buf, buf_size); } } //SUBREADprintf("READBUF '%s'\n", buf); return ret; } int autozip_getch(autozip_fp * fp){ int ret = 0; if(fp -> is_plain) { if( fp -> is_first_chars ) return fp -> first_chars[2-( fp -> is_first_chars -- ) ]; ret = fgetc(fp -> plain_fp); if(ret==EOF)ret = -1; }else{ if(fp -> zlib_fp) ret = gzgetc (fp -> zlib_fp); else ret = seekgz_next_int8(&fp -> gz_fp); } return ret; } void autozip_rewind(autozip_fp * fp){ char fname [MAX_FILE_NAME_LENGTH+1]; if(fp->zlib_fp){ SUBREADprintf("File opened as non-seekable.\n"); return; } strcpy(fname, fp -> filename); autozip_close(fp); autozip_open(fname, fp); } void parallel_gzip_writer_init(parallel_gzip_writer_t * pzwtr, char * output_filename, int total_threads){ memset(pzwtr, 0, sizeof(parallel_gzip_writer_t)); pzwtr -> threads = total_threads; pzwtr -> thread_objs = calloc(sizeof(parallel_gzip_writer_thread_t), total_threads); pzwtr -> os_file = f_subr_open(output_filename, "wb"); fputc(31, pzwtr -> os_file); fputc(139, pzwtr -> os_file); fputc(8, pzwtr -> os_file); fputc(0, pzwtr -> os_file); fputc(0, pzwtr -> os_file); // MTIME x 4 fputc(0, pzwtr -> os_file); fputc(0, pzwtr -> os_file); fputc(0, pzwtr -> os_file); fputc(4, pzwtr -> os_file); // XFL : fastest fputc(255, pzwtr -> os_file); // OS=unknown int x1; for(x1=0; x1 thread_objs[x1].thread_no = x1; deflateInit2(&pzwtr -> thread_objs[x1].zipper, SAMBAM_COMPRESS_LEVEL_NORMAL, Z_DEFLATED, SAMBAM_GZIP_WINDOW_BITS, 8, Z_DEFAULT_STRATEGY); } pzwtr -> CRC32 = crc32(0, NULL, 0); } #define CHECK_SIZE parallel_gzip_writer_thread_t *tho = pzwtr -> thread_objs + thread_no;\ if(tlen + tho -> in_buffer_used >= PARALLEL_GZIP_TXT_BUFFER_SIZE){\ SUBREADprintf("Insufficient gzip buffer.\n"); return; \ } void parallel_gzip_writer_add_text_qual(parallel_gzip_writer_t * pzwtr, char * text, int tlen, int thread_no){ CHECK_SIZE ; int ii; for(ii=0; ii'/') nch--; tho -> in_buffer[tho -> in_buffer_used +ii] = nch; } tho -> in_buffer_used += tlen; } void parallel_gzip_writer_add_text(parallel_gzip_writer_t * pzwtr, char * text, int tlen, int thread_no){ CHECK_SIZE ; memcpy(tho -> in_buffer + tho -> in_buffer_used, text, tlen); tho -> in_buffer_used += tlen; } void parallel_gzip_zip_texts(parallel_gzip_writer_t * pzwtr, int thread_no, int eof_marker){ parallel_gzip_writer_thread_t *tho = pzwtr -> thread_objs + thread_no; int write_txt_ptr = 0; tho -> out_buffer_used = 0; tho -> CRC32 = crc_pos(tho -> in_buffer, tho -> in_buffer_used); while(tho -> in_buffer_used - write_txt_ptr > 0 || eof_marker){ tho -> zipper . next_in = (unsigned char*)tho -> in_buffer + write_txt_ptr; tho -> zipper . avail_in = tho -> in_buffer_used - write_txt_ptr; tho -> zipper . next_out = (unsigned char*)tho -> out_buffer + tho -> out_buffer_used; tho -> zipper . avail_out = PARALLEL_GZIP_ZIPPED_BUFFER_SIZE - tho -> out_buffer_used; int defret = deflate(&tho -> zipper, eof_marker?Z_FINISH:Z_FULL_FLUSH); int consumed_input = tho -> in_buffer_used - write_txt_ptr - tho -> zipper . avail_in; int generated_output = PARALLEL_GZIP_ZIPPED_BUFFER_SIZE - tho -> out_buffer_used - tho -> zipper.avail_out; if(defret == Z_OK || defret == Z_STREAM_END ){ write_txt_ptr += consumed_input; tho -> out_buffer_used += generated_output; }else{ SUBREADprintf("Cannot compress the zipped output: %d with in_len=%d, consumed=%d and out_aval=%d\n", defret, tho -> in_buffer_used, consumed_input, tho -> zipper.avail_out); break; } if(eof_marker)break; } tho -> plain_length = tho -> in_buffer_used; tho -> in_buffer_used =0; } // because we have to keep sync between three fastq files, the flush function has to be manually called three times at the same time point. // otherwise R1, I2 and R2 files will have inconsistent read orders. // the outer program has to check if any of the three in_buffers is full. void parallel_gzip_writer_flush(parallel_gzip_writer_t * pzwtr, int thread_no){ parallel_gzip_writer_thread_t *tho = pzwtr -> thread_objs + thread_no; if(tho -> out_buffer_used>0){ int fret = fwrite(tho -> out_buffer, 1, tho -> out_buffer_used, pzwtr -> os_file); if(fret != tho ->out_buffer_used) SUBREADprintf("Cannot write the zipped output: %d\n", fret); if(tho -> plain_length){ unsigned int CRCnew = crc32_combine( pzwtr -> CRC32, tho -> CRC32, tho -> plain_length ); pzwtr -> plain_length += tho -> plain_length; pzwtr -> CRC32 = CRCnew; } } tho -> out_buffer_used =0; tho -> plain_length = 0; } void plgz_finish_in_buffers(parallel_gzip_writer_t * pzwtr){ int x1; for(x1=0; x1 threads; x1++){ if(pzwtr -> thread_objs[x1].in_buffer_used<1) continue; parallel_gzip_zip_texts(pzwtr, x1, 0); parallel_gzip_writer_flush(pzwtr, x1); } } void parallel_gzip_writer_close(parallel_gzip_writer_t * pzwtr){ int x1; plgz_finish_in_buffers(pzwtr); // write the last "Z_FINISH" 0-len block to tell gzip that the zipped chunk end pzwtr -> thread_objs[0].in_buffer_used = 0; parallel_gzip_zip_texts(pzwtr, 0, 1); parallel_gzip_writer_flush(pzwtr, 0); for(x1=0; x1 threads; x1++) deflateEnd(&pzwtr -> thread_objs[x1].zipper); fwrite(&pzwtr -> CRC32, 4, 1, pzwtr -> os_file); fwrite(&pzwtr -> plain_length, 4, 1, pzwtr -> os_file); // write the first (lowest) 4 bytes in a 64-bit integer -- as gzip file format required original (uncompressed) input data modulo 2^32. fclose(pzwtr -> os_file); free(pzwtr -> thread_objs); } int parallel_gzip_writer_add_read_fqs_scRNA(parallel_gzip_writer_t**outfps, char * bambin, int thread_no){ int reclen=0; parallel_gzip_writer_t * outR1fp = outfps[0]; parallel_gzip_writer_t * outI1fp = outfps[1]; parallel_gzip_writer_t * outI2fp = outfps[2]; parallel_gzip_writer_t * outR2fp = outfps[3]; memcpy(&reclen, bambin,4); int flag = 0, l_seq = 0, l_read_name = 0, n_cigar_ops = 0; memcpy(&l_read_name, bambin+12,1); memcpy(&n_cigar_ops, bambin+16,2); memcpy(&flag, bambin+18,2); memcpy(&l_seq, bambin+20,4); int x1=0; parallel_gzip_writer_add_text(outR2fp,"@",1,thread_no); parallel_gzip_writer_add_text(outR1fp,"@",1,thread_no); parallel_gzip_writer_add_text(outI1fp,"@",1,thread_no); if(outI2fp) parallel_gzip_writer_add_text(outI2fp,"@",1,thread_no); char * readname = bambin+36; parallel_gzip_writer_add_text(outR1fp,readname, 12,thread_no); parallel_gzip_writer_add_text(outR2fp,readname, 12,thread_no); parallel_gzip_writer_add_text(outI1fp,readname, 12,thread_no); if(outI2fp) parallel_gzip_writer_add_text(outI2fp,readname, 12,thread_no); parallel_gzip_writer_add_text(outR1fp,"\n",1,thread_no); parallel_gzip_writer_add_text(outR2fp,"\n",1,thread_no); parallel_gzip_writer_add_text(outI1fp,"\n",1,thread_no); if(outI2fp) parallel_gzip_writer_add_text(outI2fp,"\n",1,thread_no); //SUBREADprintf("WRITEFQ RNAME '%s'\n", bambin+36); char * R1seq = bambin+36+13; int R1len = 0; for(R1len=0; R1seq[R1len] && R1seq[R1len]!='|' ;R1len++); char * R1qual = R1seq + R1len + 1; parallel_gzip_writer_add_text(outR1fp,R1seq, R1len,thread_no); parallel_gzip_writer_add_text(outR1fp,"\n+\n",3,thread_no); parallel_gzip_writer_add_text_qual(outR1fp,R1qual, R1len,thread_no); parallel_gzip_writer_add_text(outR1fp,"\n",1,thread_no); char * I1seq = R1qual + R1len + 1; int I1I2len = 0; for(I1I2len=0; I1seq[I1I2len] && I1seq[I1I2len]!='|' ;I1I2len++); int I1len = I1I2len; if(outI2fp) I1len /=2; char * I2seq = I1seq + I1len; char * I1qual = I1seq + I1I2len + 1; char * I2qual = I1seq + I1I2len + I1len + 1; parallel_gzip_writer_add_text(outI1fp,I1seq, I1len,thread_no); parallel_gzip_writer_add_text(outI1fp,"\n+\n",3,thread_no); parallel_gzip_writer_add_text_qual(outI1fp,I1qual, I1len,thread_no); parallel_gzip_writer_add_text(outI1fp,"\n",1,thread_no); if(outI2fp){ parallel_gzip_writer_add_text(outI2fp,I2seq, I1len,thread_no); parallel_gzip_writer_add_text(outI2fp,"\n+\n",3,thread_no); parallel_gzip_writer_add_text_qual(outI2fp,I2qual, I1len,thread_no); parallel_gzip_writer_add_text(outI2fp,"\n",1,thread_no); } char oseq[l_seq+1]; int seqbase = 36+l_read_name+n_cigar_ops*4; for(x1=0; x1> (((x1%2)?0:1) *4) )&0xf]; oseq[l_seq]=0; if(flag & 16) reverse_read(oseq,l_seq, GENE_SPACE_BASE); parallel_gzip_writer_add_text(outR2fp, oseq, l_seq,thread_no); parallel_gzip_writer_add_text(outR2fp,"\n+\n",3,thread_no); seqbase = 36+l_read_name+n_cigar_ops*4 + ( l_seq+1 )/2; for(x1=0; x1 #include #include #include #include "subread.h" #define PARALLEL_GZIP_TXT_BUFFER_MARGIN (2* MAX_FC_READ_LENGTH + 500) #define PARALLEL_GZIP_TXT_BUFFER_SIZE (1024*1024) #define PARALLEL_GZIP_ZIPPED_BUFFER_SIZE (PARALLEL_GZIP_TXT_BUFFER_SIZE *9/8 ) typedef struct{ int thread_no; int in_buffer_used; int out_buffer_used; unsigned int CRC32; unsigned int zipped_CRC32; int plain_length; char in_buffer[PARALLEL_GZIP_TXT_BUFFER_SIZE]; char out_buffer[PARALLEL_GZIP_ZIPPED_BUFFER_SIZE]; z_stream zipper; } parallel_gzip_writer_thread_t; typedef struct{ int threads; srInt_64 plain_length; unsigned int CRC32; FILE * os_file; parallel_gzip_writer_thread_t * thread_objs; } parallel_gzip_writer_t; void parallel_gzip_writer_init(parallel_gzip_writer_t * pzwtr, char * output_filename, int total_threads); void parallel_gzip_writer_add_text(parallel_gzip_writer_t * pzwtr, char * text, int tlen, int thread_no); // because we have to keep sync between three fastq files, the flush function has to be manually called three times at the same time point. // otherwise R1, I2 and R2 files will have inconsistent read orders. // the outer program has to check if any of the three in_buffers is full. void parallel_gzip_zip_texts(parallel_gzip_writer_t * pzwtr, int thread_no, int for_eof_marker); void parallel_gzip_writer_flush(parallel_gzip_writer_t * pzwtr, int thread_no); void parallel_gzip_writer_close(parallel_gzip_writer_t * pzwtr); int parallel_gzip_writer_add_read_fqs_scRNA(parallel_gzip_writer_t**outfps, char * bambin, int thread_no); void parallel_gzip_writer_add_text_qual(parallel_gzip_writer_t * pzwtr, char * text, int tlen, int thread_no); // returns 0 if OK; returns 1 if the file is not indexable; returns -1 if file doesn't exist. int seekgz_open(const char * fname, seekable_zfile_t * fp, FILE * old_fp); // returns length in bytes if OK (length includes the line break at the end); returns 0 if EOF int seekgz_gets(seekable_zfile_t * fp, char * buf, int buf_size); void seekgz_tell(seekable_zfile_t * fp, seekable_position_t * pos); void seekgz_seek(seekable_zfile_t * fp, seekable_position_t * pos); // Diff: seekgz_next_char returns EOF for EOF but seekgz_next_int8 returns -1 for EOF int seekgz_next_char(seekable_zfile_t * fp); int seekgz_next_int8(seekable_zfile_t * fp); void seekgz_close(seekable_zfile_t * fp); // returns length in bytes if OK (length includes the line break at the end); returns 0 if EOF int autozip_gets(autozip_fp * fp, char * buf, int buf_size); // return -1 for EOF int autozip_getch(autozip_fp * fp); void autozip_close(autozip_fp * fp); // return -1 if error, return 0 if plain text, return 1 if gzipped int autozip_open(const char * fname, autozip_fp * fp); void autozip_rewind(autozip_fp * fp); int seekgz_preload_buffer( seekable_zfile_t * fp , subread_lock_t * read_lock); // returns length in bytes if OK (length includes the line break at the end); returns 0 if EOF int seekgz_gets(seekable_zfile_t * fp, char * buff, int buff_len); #endif Rsubread/src/sorted-hashtable.c0000644000175200017520000016256614136047615017556 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #include "sorted-hashtable.h" #include "gene-algorithms.h" #include #include #include #ifndef MACOS #ifndef FREEBSD #include #endif #endif #include #include"core.h" #define _gehash_hash(k) ((unsigned int)(k)) int gehash_create(gehash_t * the_table, size_t expected_size, char is_small_table) { return gehash_create_ex(the_table, expected_size, is_small_table, SUBINDEX_VER0, 3, 0); } unsigned int calculate_buckets_by_size( size_t expected_size, int version_number, int is_small_table, int index_gap ){ if(expected_size ==0) expected_size = GEHASH_DEFAULT_SIZE; int expected_bucket_number = expected_size / GEHASH_BUCKET_LENGTH; if(index_gap >= 3) expected_bucket_number /=3; if(SUBINDEX_VER1 > version_number) { if(expected_bucket_number < 10111 && !is_small_table)expected_bucket_number = 10111; else if(is_small_table) expected_bucket_number = 4; } else if(expected_bucket_number <= 0x3ffff )expected_bucket_number = 0x3ffff+4; for (;;expected_bucket_number++) { int j, valid_v; valid_v = 1; int test_prime = 13; if(SUBINDEX_VER1 > version_number && is_small_table) test_prime = 3; for(j=2; j<=test_prime;j++) { if (expected_bucket_number % j == 0) valid_v = 0; } if (valid_v) break; } //SUBREADprintf("EXP_BUCKS=%u\n",expected_bucket_number); return expected_bucket_number; } int gehash_create_ex(gehash_t * the_table, size_t expected_size, char is_small_table, int version_number, int index_gap, int padding) { int expected_bucket_number; int i; memset(the_table, 0, sizeof(gehash_t)); if(expected_size ==0) expected_size = GEHASH_DEFAULT_SIZE; expected_bucket_number = calculate_buckets_by_size(expected_size, version_number, is_small_table, index_gap); // calculate the number of buckets for creating the data structure the_table -> version_number = version_number; the_table -> current_items = 0; the_table -> is_small_table = is_small_table; the_table -> buckets_number = expected_bucket_number; the_table -> buckets = (struct gehash_bucket *) malloc( expected_bucket_number * sizeof(struct gehash_bucket ) ); the_table -> padding = padding; if(!the_table -> buckets) { SUBREADputs(MESSAGE_OUT_OF_MEMORY); return 1; } for(i=0; i buckets [i].item_keys = NULL; the_table -> buckets [i].current_items = 0; the_table -> buckets [i].space_size = 0; } the_table -> index_gap = index_gap; return 0; } int _gehash_resize_bucket(gehash_t * the_table , int bucket_no, char is_small_table) { int new_bucket_length; struct gehash_bucket * current_bucket = &(the_table -> buckets [bucket_no]); gehash_key_t * new_item_keys = NULL; short * new_new_item_keys = NULL; gehash_data_t * new_item_values; if (current_bucket->space_size<1) { if(is_small_table) new_bucket_length = 5 ; else new_bucket_length = 2 ; if(the_table->version_number == SUBINDEX_VER0) new_item_keys = (gehash_key_t *) malloc(sizeof(gehash_key_t) * new_bucket_length); else new_new_item_keys = (short *) malloc(sizeof(short)*new_bucket_length); new_item_values = (gehash_data_t *) malloc(sizeof(gehash_data_t) * new_bucket_length); if(!((new_item_keys|| new_new_item_keys) && new_item_values)) { SUBREADputs(MESSAGE_OUT_OF_MEMORY); return 1; } /*if(the_table->version_number == SUBINDEX_VER0) bzero(new_item_keys, sizeof(gehash_key_t) * new_bucket_length); else bzero(new_new_item_keys, sizeof(short) * new_bucket_length); bzero(new_item_values, sizeof(gehash_data_t) * new_bucket_length); */ if(the_table->version_number == SUBINDEX_VER0) current_bucket->item_keys = new_item_keys; else current_bucket->new_item_keys = new_new_item_keys; current_bucket->item_values = new_item_values; current_bucket->space_size = new_bucket_length; } else { if(is_small_table) //new_bucket_length = (int)max(15,current_bucket->space_size*1.5+1); new_bucket_length = (int)(current_bucket->space_size*5); else new_bucket_length = max((int)(current_bucket->space_size*1.5), (int)(current_bucket->space_size+4)); if(the_table->version_number == SUBINDEX_VER0) current_bucket->item_keys = (gehash_key_t *) realloc(current_bucket->item_keys , sizeof(gehash_key_t) * new_bucket_length); else current_bucket->new_item_keys = (short *) realloc(current_bucket->new_item_keys, sizeof(short)*new_bucket_length); current_bucket->item_values = (gehash_data_t *) realloc(current_bucket->item_values, sizeof(gehash_data_t) * new_bucket_length); if(!((current_bucket->new_item_keys||current_bucket->new_item_keys) && current_bucket->item_values)) { SUBREADputs(MESSAGE_OUT_OF_MEMORY); return 1; } current_bucket->space_size = new_bucket_length; } return 0; } #define _gehash_get_bucket(tab, key) ( (tab) -> buckets + _gehash_hash( key ) % (tab) -> buckets_number ) #define _gehash_get_bucketNO(tab, key) ( _gehash_hash( key ) % (tab) -> buckets_number ) void gehash_try_insert_measure(unsigned int * bucket_sizes, int bucket_no, gehash_key_t key){ unsigned int buck_this = _gehash_hash(key)%bucket_no; //if(buck_this == 16305862)SUBREADprintf("Insert bucket : %u/%u -> %u at PTR=%p\n", buck_this, bucket_no, bucket_sizes[buck_this] , bucket_sizes); bucket_sizes[buck_this]++; } int gehash_insert(gehash_t * the_table, gehash_key_t key, gehash_data_t data, unsigned int * bucket_sizes) { struct gehash_bucket * current_bucket; int is_fault = 0; //SUBREADprintf("Insert subread : %u/%u\n", _gehash_hash(key)% the_table->buckets_number, the_table->buckets_number); current_bucket = _gehash_get_bucket (the_table, key); if(the_table->version_number == SUBINDEX_VER0) { if (current_bucket->current_items >= current_bucket->space_size) { int bucket_number; if(bucket_sizes){ SUBREADprintf("Bucket size was wrongly calculated.\n"); return 1; } bucket_number = _gehash_hash(key) % the_table -> buckets_number; is_fault = _gehash_resize_bucket(the_table, bucket_number, the_table->is_small_table); if(is_fault) return 1; } current_bucket->item_keys[current_bucket->current_items] = key; } else { short step_number = key/the_table -> buckets_number; if(current_bucket-> new_item_keys == NULL && bucket_sizes){ //this will go only once when all the bucktes are NULL. unsigned int * memory_sizes = malloc(sizeof(int) * the_table -> buckets_number); memset(memory_sizes, 0xff, sizeof(int) * the_table -> buckets_number); unsigned int grp_bytes = 0, grps = 0, this_grp_bucks = 0, xk1, bucks_per_grp = the_table -> buckets_number / GEHASH_MEM_PTR_NO +2; for(xk1=0; xk1< the_table -> buckets_number ; xk1++){ unsigned int buck_size =(sizeof(short) + sizeof(gehash_data_t)) * bucket_sizes[xk1]; grp_bytes += buck_size; this_grp_bucks ++; if( this_grp_bucks >= bucks_per_grp ){ memory_sizes[grps++] = grp_bytes; grp_bytes=0; this_grp_bucks=0; } } if(this_grp_bucks) memory_sizes[grps] = grp_bytes; for(xk1=0; xk1 < GEHASH_MEM_PTR_NO; xk1++){ unsigned int current_bytes = memory_sizes[xk1]; if(current_bytes<0xff000000u){ if(the_table -> malloc_ptr[xk1]!=NULL){ SUBREADprintf("ERROR : no-NULL ptr : %p\n",the_table -> malloc_ptr[xk1]); } the_table -> malloc_ptr[xk1] = malloc(current_bytes); if(!the_table -> malloc_ptr[xk1]){ SUBREADputs(MESSAGE_OUT_OF_MEMORY); return 1; } } } grp_bytes = 0; grps = 0; this_grp_bucks = 0; for(xk1=0; xk1< the_table -> buckets_number ; xk1++){ struct gehash_bucket * mem_bucket = the_table -> buckets+xk1; memset(mem_bucket, 0, sizeof(struct gehash_bucket)); mem_bucket -> new_item_keys = (short*)((char*)the_table -> malloc_ptr[grps] + grp_bytes); mem_bucket -> item_values = (gehash_data_t*)((char *)mem_bucket -> new_item_keys + sizeof(short) * bucket_sizes[xk1]); mem_bucket -> space_size = bucket_sizes[xk1]; unsigned int buck_size =(sizeof(short) + sizeof(gehash_data_t)) * bucket_sizes[xk1]; grp_bytes += buck_size; this_grp_bucks ++; if( this_grp_bucks >= bucks_per_grp ){ grps++; grp_bytes=0; this_grp_bucks=0; } } the_table -> free_item_only = 2; free(memory_sizes); } if (current_bucket->current_items >= current_bucket->space_size) { if(bucket_sizes){ SUBREADprintf("Bucket [%d] size was wrongly calculated : %d >= %u.\n", _gehash_get_bucketNO (the_table, key), current_bucket->current_items, current_bucket->space_size); return 1; } int bucket_number; bucket_number = _gehash_hash(key) % the_table -> buckets_number; is_fault = _gehash_resize_bucket(the_table, bucket_number, the_table->is_small_table); if(is_fault) return 1; } current_bucket->new_item_keys[current_bucket->current_items] = step_number; } current_bucket->item_values[current_bucket->current_items] = data; current_bucket->current_items ++; the_table ->current_items ++; return 0; } int gehash_insert_limited(gehash_t * the_table, gehash_key_t key, gehash_data_t data, int limit, int replace_prob) { struct gehash_bucket * current_bucket; int occurance = 0, xk1 ; assert(the_table->version_number == SUBINDEX_VER0); current_bucket = _gehash_get_bucket (the_table, key); for(xk1=0; xk1current_items ; xk1++) if(current_bucket->item_keys[xk1]==key) occurance++; if(occurance >= limit) { if(myrand_rand()%32768 < replace_prob) return 1; int replacement_id = myrand_rand()%occurance; occurance = 0; for(xk1=0; xk1current_items ; xk1++) if(current_bucket->item_keys[xk1]==key) { if(occurance == replacement_id) { current_bucket->item_values[xk1]=data; return 0; } occurance++; } } gehash_insert(the_table, key, data, NULL); return 0; } #define INDEL_SEGMENT_SIZE 5 #define _index_vote(key) (((unsigned int)(key))%GENE_VOTE_TABLE_SIZE) #define _index_vote_tol(key) (((unsigned int)(key)/INDEL_SEGMENT_SIZE)%GENE_VOTE_TABLE_SIZE) #define is_quality_subread(scr) ((scr)>15?1:0) size_t gehash_go_q_tolerable(gehash_t * the_table, gehash_key_t key, int offset, int read_len, int is_reversed, gene_vote_t * vote,gene_vote_number_t weight, gene_quality_score_t quality ,int max_match_number, int indel_tolerance, int subread_number, int max_error_bases, int subread_len, unsigned int low_border, unsigned int high_border) { if(max_error_bases >= 10) return 0; char error_pos_stack[10]; // max error bases = 10; gehash_key_t mutation_key; int ret = 0; ret+=gehash_go_q(the_table, key, offset, read_len, is_reversed, vote, indel_tolerance, subread_number, low_border, high_border); int error_bases ; for (error_bases=1; error_bases <= max_error_bases; error_bases++) { int i, j; for(i=0; i=0; i--) if (mutation_stack[i] <= 3) { int base_to_change=-1; mutation_key = key; for (j = 0; j < error_bases; j++) { base_to_change = error_pos_stack[j]; int new_value = mutation_stack[j]; mutation_key = mutation_key & ~(0x3 << (2*base_to_change)); mutation_key = mutation_key | (new_value << (2*base_to_change)); } for (j = i+1; j=0; i--) if (error_pos_stack[i] < subread_len - (error_bases - i)) { error_pos_stack[i] ++; for (j = i+1; j> ((15-i)*2) )&3) == 3) // if it is 'T' at i-th base { C_poses[availableC++] = i; } } for (error_bases= min(availableC,max_error_bases); error_bases <= min(availableC,max_error_bases); error_bases++) { assert(error_bases < 5); int j; for(i=0; i=0; i--) if (mutation_stack[i] <1) { int base_to_change=-1; mutation_key = key; for (j = 0; j < error_bases; j++) { base_to_change = C_poses[error_pos_stack[j]]; int new_value = mutation_stack[j]?2:3; //(C or T) mutation_key = mutation_key & ~(0x3 << (2*(15-base_to_change))); mutation_key = mutation_key | (new_value << (2*(15-base_to_change))); } for (j = i+1; j=0; i--) if (error_pos_stack[i] < availableC - (error_bases - i)) { error_pos_stack[i] ++; for (j = i+1; jmax_mask = vote->masks[i][j]; vote->max_vote = vote->votes[i][j]; vote->max_position =vote->pos[i][j]; vote->max_coverage_start = vote->coverage_start [i][j]; vote->max_coverage_end = vote->coverage_end [i][j]; memcpy(vote->max_indel_recorder, vote->indel_recorder[i][j], 3*MAX_INDEL_TOLERANCE * sizeof(*vote->max_indel_recorder)); } size_t gehash_go_q(gehash_t * the_table, gehash_key_t raw_key, int offset, int read_len, int is_reversed, gene_vote_t * vote, int indel_tolerance, int subread_number, unsigned int low_border, unsigned int high_border) { //SUBREADprintf("Q=%u, OFFSET=%d, B=%u ~ %u\n", raw_key, offset, low_border, high_border); if(the_table->version_number == SUBINDEX_VER0) { gehash_key_t key = raw_key; struct gehash_bucket * current_bucket; int i=0, items; gehash_key_t *current_keys;//, *endp12; current_bucket = _gehash_get_bucket (the_table, key); items = current_bucket -> current_items; current_keys = current_bucket -> item_keys; if(!items) return 0; #define SPEED_UP_DENOMINAOR 3 int jump_step = items / SPEED_UP_DENOMINAOR; int last_accepted_index = 0; if(jump_step<1) jump_step=1; if(key > current_keys[0]) { while(1) { while(1) { int next_p = last_accepted_index + jump_step; if(next_p >= items) break; if(current_keys[next_p]>=key) break; last_accepted_index = next_p; } if(jump_step>SPEED_UP_DENOMINAOR) jump_step /= SPEED_UP_DENOMINAOR; else if(jump_step>1) jump_step = 1; else break; } last_accepted_index++; } if(current_keys[last_accepted_index]!=key) return 0; short offset_from_5 = offset; //short offset_from_5 = is_reversed?(read_len - offset - 16):offset ; if(*(current_bucket -> item_values+last_accepted_index) > 0xffff0000) // no position should be greater than this. { // assumed to be non-informative subread. vote -> noninformative_subreads++; return 0; } if (indel_tolerance <1) for (; last_accepted_indexitem_values[last_accepted_index] - offset; int offsetX = _index_vote(kv); int datalen = vote -> items[offsetX]; unsigned int * dat = vote -> pos[offsetX]; if (kv > 0xffff0000) continue; for (i=0;i vote -> last_subread_cluster[offsetX][i])) { gene_vote_number_t test_max = (vote->votes[offsetX][i]); test_max += 1; vote->votes[offsetX][i] = test_max; if (offset_from_5 < vote->coverage_start [offsetX][i]) vote->coverage_start [offsetX][i] = offset_from_5; if (offset_from_5 +16 > vote->coverage_end [offsetX][i]) vote->coverage_end [offsetX][i] = offset_from_5+16; vote -> last_subread_cluster[offsetX][i] = subread_number + 1; vote->max_vote = max(vote->max_vote , test_max); i = 9999999; } } if (i < 9999999 && datalen high_border) continue; vote -> items[offsetX] ++; dat[i] = kv; vote->votes[offsetX][i]=1; vote->masks[offsetX][i]= (is_reversed?IS_NEGATIVE_STRAND:0); vote->coverage_start [offsetX][i] = offset_from_5; vote->coverage_end [offsetX][i] = offset_from_5+16; vote -> last_subread_cluster[offsetX][i] = subread_number + 1; if(vote->max_vote==0) vote->max_vote = 1; } } else { // We duplicated all codes for indel_tolerance >= 1 for the minimal impact to performance. //int ii_end = (indel_tolerance % INDEL_SEGMENT_SIZE)?(indel_tolerance - indel_tolerance%INDEL_SEGMENT_SIZE+INDEL_SEGMENT_SIZE):indel_tolerance; for (; last_accepted_indexitem_values[last_accepted_index] - offset; int ii_end = (indel_tolerance % INDEL_SEGMENT_SIZE)?(indel_tolerance - indel_tolerance%INDEL_SEGMENT_SIZE+INDEL_SEGMENT_SIZE):indel_tolerance; int iix; i=0; for(iix = 0; iix<=ii_end; iix = iix>0?-iix:(-iix+INDEL_SEGMENT_SIZE)) { int offsetX = _index_vote_tol(kv+iix); int datalen = vote -> items[offsetX]; if(!datalen)continue; unsigned int * dat = vote -> pos[offsetX]; for (i=0;i= -indel_tolerance && dist0 <= indel_tolerance && is_reversed == (0!=(vote -> masks[offsetX][i]&IS_NEGATIVE_STRAND))) { int toli = vote -> toli[offsetX][i]; if(di >= 46494104 && di <= 46496104 ){ SUBREADprintf("VOTES: at %u, subread_no = %d , last_cluster = %d , toli = %d\n", di, subread_number , vote -> last_subread_cluster[offsetX][i] , toli); } if( toli > 0 && subread_number + 1 == vote -> last_subread_cluster[offsetX][i] ){ int move_dist = 0; if( toli >=3 ) move_dist = vote -> indel_recorder[offsetX][i][toli-3+2]; int new_dist = move_dist; move_dist -= vote -> indel_recorder[offsetX][i][toli+2]; new_dist -= dist0; if(abs(move_dist) > abs(new_dist)){ toli -= 3; vote -> toli[offsetX][i] = toli; vote -> last_subread_cluster[offsetX][i]--; vote -> votes[offsetX][i] --; } } if(subread_number + 1 <= vote -> last_subread_cluster[offsetX][i]) continue; gene_vote_number_t test_max = (vote->votes[offsetX][i]); test_max += 1; vote -> votes[offsetX][i] = test_max; /* if (offset_from_5 < vote->coverage_start [offsetX][i]) { vote->coverage_start [offsetX][i] = offset_from_5; }*/ if (offset_from_5 +16 > vote->coverage_end [offsetX][i]) { vote->coverage_end [offsetX][i] = offset_from_5+16; } if (dist0 != vote->current_indel_cursor[offsetX][i]) { toli +=3; if (toli < indel_tolerance*3) { vote -> toli[offsetX][i] = toli; vote -> indel_recorder[offsetX][i][toli] = subread_number+1; vote -> indel_recorder[offsetX][i][toli+1] = subread_number+1; vote -> indel_recorder[offsetX][i][toli+2] = dist0; if(toli < indel_tolerance*3-3) vote -> indel_recorder[offsetX][i][toli+3]=0; } vote->current_indel_cursor [offsetX][i] = (char)dist0; } else vote -> indel_recorder[offsetX][i][toli+1] = subread_number+1; vote -> last_subread_cluster[offsetX][i] = subread_number + 1; vote->max_vote = max(vote->max_vote , test_max); i = 9999999; } } if (i==9999999){ break; } } if (i < 9999999) { if (kv < low_border || kv > high_border) continue; int offsetX2 = _index_vote_tol(kv); int datalen2 = vote -> items[offsetX2]; unsigned int * dat2 = vote -> pos[offsetX2]; if (datalen2 items[offsetX2] ++; dat2[datalen2] = kv; vote -> masks[offsetX2][datalen2]=(is_reversed?IS_NEGATIVE_STRAND:0); vote -> votes[offsetX2][datalen2]=1; vote -> toli[offsetX2][datalen2]=0; vote -> last_subread_cluster[offsetX2][datalen2] = subread_number + 1; // data structure of recorder: // {unsigned char subread_start; unsigned char subread_end, char indel_offset_from_start} // All subread numbers are added with 1 for not being 0. vote -> indel_recorder[offsetX2][datalen2][0] = vote -> indel_recorder[offsetX2][datalen2][1] = subread_number+1; vote -> indel_recorder[offsetX2][datalen2][2] = 0; vote -> indel_recorder[offsetX2][datalen2][3] = 0; vote->current_indel_cursor [offsetX2][datalen2] = 0; vote->coverage_start [offsetX2][datalen2] = offset_from_5; vote->coverage_end [offsetX2][datalen2] = offset_from_5+16; if (vote->max_vote==0) vote->max_vote = 1; } } } } return 1; //return match_end-match_start; } else { // VER_1 // VER_2 struct gehash_bucket * current_bucket; int i = 0, items; short *current_keys;//, *endp12; short key = raw_key / the_table->buckets_number; current_bucket = _gehash_get_bucket (the_table, raw_key); items = current_bucket -> current_items; current_keys = current_bucket -> new_item_keys; if(!items) return 0; //#warning "======== MAKE SURE THAT '-1' IS CORRECT ============" int imin=0, imax=items - 1; int last_accepted_index; while(1) { last_accepted_index=(imin+imax)/2; short current_key = current_keys[last_accepted_index]; if(current_key>key) { imax = last_accepted_index - 1; } else if(current_key item_values+last_accepted_index) > 0xffff0000) // no position should be greater than this. { // assumed to be non-informative subread. vote -> noninformative_subreads++; return 0; }*/ int subread_number_P1 = subread_number + 1; int of_p_16 = offset + 16; { int ii_end = INDEL_SEGMENT_SIZE; if(indel_tolerance>5) ii_end=(indel_tolerance % INDEL_SEGMENT_SIZE)?(indel_tolerance - indel_tolerance%INDEL_SEGMENT_SIZE+INDEL_SEGMENT_SIZE):indel_tolerance; for (; last_accepted_indexitem_values[last_accepted_index] - offset; offsetX2 = _index_vote_tol(kv); datalen = vote -> items[offsetX2]; datalen2 = datalen; offsetX = offsetX2; unsigned int * dat2, *dat; dat = dat2 = vote -> pos[offsetX2]; //SUBREADprintf("You can find KV at %u\n", kv); for(iix = 0; iix<=ii_end; iix = iix>0?-iix:(-iix+INDEL_SEGMENT_SIZE)) { if(iix) { offsetX = _index_vote_tol(kv+iix); datalen = vote -> items[offsetX]; if(!datalen)continue; dat = vote -> pos[offsetX]; }else if(!datalen)continue; for (i=0;i= -indel_tolerance && dist0 <= indel_tolerance && is_reversed == (0!= vote->masks[offsetX][i])) { // if(di >= 46494104 && di <= 46496104 ){ // SUBREADprintf("VOTES: at %u, subread_no = %d , last_cluster = %d , toli = %d\n", di, subread_number , vote -> last_subread_cluster[offsetX][i] , toli); // } int toli = vote -> toli[offsetX][i]; if( subread_number_P1 == vote -> last_subread_cluster[offsetX][i] && toli >0){ int move_dist = 0; if( toli >=3 ) move_dist = vote -> indel_recorder[offsetX][i][toli-3+2]; int new_dist = move_dist; move_dist -= vote -> indel_recorder[offsetX][i][toli+2]; new_dist -= dist0; if(abs(move_dist) > abs(new_dist)){ toli -= 3; vote -> toli[offsetX][i] = toli; vote -> last_subread_cluster[offsetX][i]--; vote -> votes[offsetX][i] --; } } if(subread_number_P1 <= vote -> last_subread_cluster[offsetX][i]) continue; gene_vote_number_t test_max = (vote->votes[offsetX][i]); test_max += 1; vote -> votes[offsetX][i] = test_max; if (offset +16 > vote->coverage_end [offsetX][i]) vote->coverage_end [offsetX][i] = of_p_16; if (dist0 == vote->current_indel_cursor[offsetX][i]) vote -> indel_recorder[offsetX][i][toli+1] = subread_number_P1; else { toli +=3; if (toli < MAX_INDEL_SECTIONS*3) { vote -> toli[offsetX][i] = toli; vote -> indel_recorder[offsetX][i][toli] = subread_number_P1; vote -> indel_recorder[offsetX][i][toli+1] = subread_number_P1; vote -> indel_recorder[offsetX][i][toli+2] = dist0; if(toli < MAX_INDEL_SECTIONS*3-3) vote -> indel_recorder[offsetX][i][toli+3]=0; } vote->current_indel_cursor [offsetX][i] = (char)dist0; } vote -> last_subread_cluster[offsetX][i] = subread_number_P1; if(vote->max_vote < test_max)vote->max_vote = test_max; i = 9999999; break; } } if (i==9999999)break; } if (i < 9999999) { if (kv < low_border || kv > high_border) continue; if (datalen2 items[offsetX2] ++; dat2[datalen2] = kv; vote -> masks[offsetX2][datalen2]=(is_reversed?IS_NEGATIVE_STRAND:0); vote -> votes[offsetX2][datalen2]=1; vote -> toli[offsetX2][datalen2]=0; // data structure of recorder: // {unsigned char subread_start; unsigned char subread_end, char indel_offset_from_start} // All subread numbers are added with 1 for not being 0. vote -> indel_recorder[offsetX2][datalen2][0] = vote -> indel_recorder[offsetX2][datalen2][1] = subread_number_P1; vote -> indel_recorder[offsetX2][datalen2][2] = 0; vote -> indel_recorder[offsetX2][datalen2][3] = 0; vote->current_indel_cursor [offsetX2][datalen2] = 0; vote->coverage_start [offsetX2][datalen2] = offset; vote->coverage_end [offsetX2][datalen2] = of_p_16; vote -> last_subread_cluster[offsetX2][datalen2] = subread_number_P1; if (vote->max_vote==0) vote->max_vote = 1; } } else i=0; } } return 1; } } size_t gehash_go_X(gehash_t * the_table, gehash_key_t raw_key, int offset, int read_len, int is_reversed, gene_vote_t * vote, int indel_tolerance, int subread_number, unsigned int low_border, unsigned int high_border, int run_round, unsigned int * shift_indel_locs, unsigned int * shift_indel_NO){ if(0)if(the_table->version_number == SUBINDEX_VER0){ SUBREADprintf("ERROR: the version of the index is too old.\n"); assert(the_table->version_number != SUBINDEX_VER0); return -1; } // VER_1 // VER_2 struct gehash_bucket * current_bucket; int i = 0, items; short *current_keys;//, *endp12; short key = raw_key / the_table->buckets_number; current_bucket = _gehash_get_bucket (the_table, raw_key); items = current_bucket -> current_items; current_keys = current_bucket -> new_item_keys; if(!items) return 0; //#warning "======== MAKE SURE THAT '-1' IS CORRECT ============" int imin=0, imax=items - 1; int last_accepted_index; while(1) { last_accepted_index=(imin+imax)/2; short current_key = current_keys[last_accepted_index]; if(current_key>key) { imax = last_accepted_index - 1; } else if(current_key5) ii_end=(indel_tolerance % INDEL_SEGMENT_SIZE)?(indel_tolerance - indel_tolerance%INDEL_SEGMENT_SIZE+INDEL_SEGMENT_SIZE):indel_tolerance; //for (; last_accepted_indexitem_values[last_accepted_index] - offset; offsetX = offsetX2 = _index_vote_tol(kv); datalen = datalen2 = vote -> items[offsetX]; unsigned int * dat2, *dat; dat = dat2 = vote -> pos[offsetX2]; int found_some = 0; //SUBREADprintf("You can find KV at %u\n", kv); for(iix = 0; iix<=ii_end; iix = iix>0?-iix:(-iix+INDEL_SEGMENT_SIZE)) { if(iix) { offsetX = _index_vote_tol(kv+iix); datalen = vote -> items[offsetX]; if(!datalen)continue; dat = vote -> pos[offsetX]; } else if(!datalen) continue; for (i=0;i0 && vote -> marked_shift_indel[offsetX][i])? 0: indel_tolerance ; if( dist0 >= -applied_indel_tol && dist0 <= applied_indel_tol && is_reversed == vote->masks[offsetX][i]){ int toli = vote -> toli[offsetX][i]; if(run_round == 0 && toli >0 && dist0 ==0 && ! vote -> marked_shift_indel[offsetX][i]){ vote -> marked_shift_indel[offsetX][i] = 1; shift_indel_locs[(* shift_indel_NO)++] = dat[i]; } if( subread_number_P1 == vote -> last_subread_cluster[offsetX][i] && toli >0){ int move_dist = 0; if( toli >=3 ) move_dist = vote -> indel_recorder[offsetX][i][toli-3+2]; int new_dist = move_dist; move_dist -= vote -> indel_recorder[offsetX][i][toli+2]; new_dist -= dist0; if(abs(move_dist) > abs(new_dist)){ toli -= 3; vote -> toli[offsetX][i] = toli; vote -> last_subread_cluster[offsetX][i]--; vote -> votes[offsetX][i] --; } } if(subread_number_P1 <= vote -> last_subread_cluster[offsetX][i]) continue; gene_vote_number_t test_max = (vote->votes[offsetX][i]); test_max += 1; vote -> votes[offsetX][i] = test_max; if (offset +16 > vote->coverage_end [offsetX][i]) vote->coverage_end [offsetX][i] = of_p_16; if (dist0 == vote->current_indel_cursor[offsetX][i]){ vote -> indel_recorder[offsetX][i][toli+1] = subread_number_P1; } else { toli +=3; if (toli < MAX_INDEL_SECTIONS*3) { vote -> toli[offsetX][i] = toli; vote -> indel_recorder[offsetX][i][toli] = subread_number_P1; vote -> indel_recorder[offsetX][i][toli+1] = subread_number_P1; vote -> indel_recorder[offsetX][i][toli+2] = dist0; if(toli < MAX_INDEL_SECTIONS*3-3) vote -> indel_recorder[offsetX][i][toli+3]=0; } vote->current_indel_cursor [offsetX][i] = (char)dist0; } vote -> last_subread_cluster[offsetX][i] = subread_number_P1; if(vote->max_vote < test_max)vote->max_vote = test_max; found_some = 1; break; } } if(found_some) break; } if (!found_some) { if (kv >=low_border && kv <= high_border && datalen2 items[offsetX2] ++; dat2[datalen2] = kv; vote -> masks[offsetX2][datalen2] = is_reversed; vote -> votes[offsetX2][datalen2] = 1; vote -> toli[offsetX2][datalen2] = 0; vote -> marked_shift_indel[offsetX2][datalen2] = 0; if(run_round>0){ int kk; for(kk = 0; kk < * shift_indel_NO ; kk++){ if( kv >= shift_indel_locs[kk] - indel_tolerance && kv <= shift_indel_locs[kk] + indel_tolerance ){ vote -> marked_shift_indel[offsetX2][datalen2] = 1; break; } } } // data structure of recorder: // {unsigned char subread_start; unsigned char subread_end, char indel_offset_from_start} // All subread numbers are added with 1 for not being 0. vote -> indel_recorder[offsetX2][datalen2][0] = vote -> indel_recorder[offsetX2][datalen2][1] = subread_number_P1; vote -> indel_recorder[offsetX2][datalen2][2] = 0; vote -> indel_recorder[offsetX2][datalen2][3] = 0; vote->current_indel_cursor [offsetX2][datalen2] = 0; vote->coverage_start [offsetX2][datalen2] = offset; vote->coverage_end [offsetX2][datalen2] = of_p_16; vote -> last_subread_cluster[offsetX2][datalen2] = subread_number_P1; if (vote->max_vote==0) vote->max_vote = 1; } } if(! scan_step){ last_accepted_index++; if(last_accepted_index == items || current_keys[last_accepted_index]!= key){ scan_step = 1; last_accepted_index = start_scan_idx; } } if(scan_step){ last_accepted_index --; if(last_accepted_index <0 || current_keys[last_accepted_index]!= key) break; } } } return 1; } void select_best_vote(gene_vote_t * vote) { int i,j; for (i=0; iitems[i]; j++) { if(vote -> votes[i][j] == vote->max_vote) { vote -> max_position = vote -> pos[i][j]; vote -> max_mask = vote -> masks[i][j]; vote -> max_coverage_start = vote->coverage_start[i][j]; vote -> max_coverage_end = vote->coverage_end[i][j]; } } } short indel_recorder_copy(gene_vote_number_t *dst, gene_vote_number_t * src) { short all_indels = 0; // memcpy(dst, src, 3*MAX_INDEL_TOLERANCE); return; int i=0; while(src[i] && (i<3*MAX_INDEL_TOLERANCE-2)) { dst[i] = src[i]; i++; dst[i] = src[i]; i++; dst[i] = src[i]; all_indels = dst[i]; i++; } dst[i] = 0; return all_indels; } void finalise_vote(gene_vote_t * vote) { if(vote->max_tmp_indel_recorder) { indel_recorder_copy(vote->max_indel_recorder, vote->max_tmp_indel_recorder); } // memcpy(vote->max_indel_recorder, vote->max_tmp_indel_recorder, sizeof(char)*3 * MAX_INDEL_TOLERANCE); } int gehash_exist(gehash_t * the_table, gehash_key_t key) { struct gehash_bucket * current_bucket; int items; gehash_key_t * keyp, *endkp; assert(the_table->version_number == SUBINDEX_VER0); current_bucket = _gehash_get_bucket (the_table, key); items = current_bucket -> current_items; if(items <1)return 0; keyp = current_bucket -> item_keys; endkp = keyp + items; while(1) { if(*keyp == key) return 1; if(++keyp >= endkp) break; } return 0; } size_t gehash_update(gehash_t * the_table, gehash_key_t key, gehash_data_t data_new) { struct gehash_bucket * current_bucket; size_t matched; int items; gehash_key_t * keyp, *endkp; assert(the_table->version_number == SUBINDEX_VER0); current_bucket = _gehash_get_bucket (the_table, key); matched = 0; items = current_bucket -> current_items; keyp = current_bucket -> item_keys; endkp = keyp + items; while(1) { if(*keyp == key) { current_bucket -> item_values[keyp-current_bucket -> item_keys] = data_new; matched++; } keyp +=1; if(keyp >= endkp) break; } return matched; } size_t gehash_get(gehash_t * the_table, gehash_key_t key, gehash_data_t * data_result, size_t max_result_space) { struct gehash_bucket * current_bucket; size_t matched; int items; gehash_key_t * keyp, *endkp; if(max_result_space<1) return 0; assert(the_table->version_number == SUBINDEX_VER0); current_bucket = _gehash_get_bucket (the_table, key); matched = 0; items = current_bucket -> current_items; if(items<1) return 0; keyp = current_bucket -> item_keys; endkp = keyp + items; while(1) { if(*keyp == key) { data_result [matched] = current_bucket -> item_values[keyp-current_bucket -> item_keys]; matched +=1; if(matched >= max_result_space) break; } keyp +=1; if(keyp >= endkp) break; } return matched; } size_t gehash_remove(gehash_t * the_table, gehash_key_t key) { struct gehash_bucket * current_bucket; int i; size_t removed; assert(the_table->version_number == SUBINDEX_VER0); current_bucket = _gehash_get_bucket (the_table, key); if(current_bucket -> current_items < 1) return 0; removed = 0; for(i=0; ; i++) { while(current_bucket -> item_keys [i+removed] == key && i+removed < current_bucket -> current_items) removed += 1; if(i+removed >= current_bucket -> current_items) break; if(removed) { current_bucket -> item_keys [i] = current_bucket -> item_keys [i + removed]; current_bucket -> item_values [i] = current_bucket -> item_values [i + removed]; } } current_bucket -> current_items -= removed; the_table-> current_items -= removed; return removed; } size_t gehash_get_hpc(gehash_t * the_table, gehash_key_t key, gehash_data_t * data_result, size_t max_result_space) { return -1; } void gehash_insert_sorted(gehash_t * the_table, gehash_key_t key, gehash_data_t data) { SUBREADprintf("UNIMPLEMENTED! gehash_insert_sorted \n"); } // Data Struct of dumpping: // { // size_t current_items; // size_t buckets_number; // struct // { // size_t current_items; // size_t space_size; // gehash_key_t item_keys [current_items]; // gehash_data_t item_values [current_items] // } [buckets_number]; // } // unsigned int load_int32(FILE * fp) { int ret; int read_length; read_length = fread(&ret, sizeof(int), 1, fp); if(read_length<=0)assert(0); return ret; } srInt_64 load_int64(FILE * fp) { srInt_64 ret; int read_length; read_length = fread(&ret, sizeof(srInt_64), 1, fp); if(read_length<=0)assert(0); return ret; } int gehash_load_option(const char fname [], int option_no, int * result){ char tabname[MAX_FILE_NAME_LENGTH]; char magic_chars[8]; int found = 0, rrtv; sprintf(tabname, "%s.00.b.tab", fname); FILE * fp = f_subr_open(tabname, "rb"); if(fp == NULL){ sprintf(tabname, "%s.00.c.tab", fname); fp = f_subr_open(tabname, "rb"); } if(fp){ rrtv = fread(magic_chars,1,8,fp); if(rrtv < 8) return -1; if(memcmp(magic_chars, "2subindx",7)==0) { while(1) { short option_key, option_length; rrtv = fread(&option_key, 2, 1, fp); if(rrtv < 1) return -1; if(!option_key) break; rrtv = fread(&option_length, 2, 1, fp); if(rrtv < 1) return -1; if(option_key == option_no){ *result = 0; rrtv = fread(result ,option_length,1,fp); if(rrtv < 1) return -1; found = 1; } else fseek(fp, option_length, SEEK_CUR); } } fclose(fp); } return found; } int gehash_load(gehash_t * the_table, const char fname []) { int i, read_length, rrtv; char magic_chars[8]; magic_chars[7]=0; if(0)if(sizeof( size_t ) != 8|| sizeof(long int ) != 8|| sizeof(int ) != 4){ SUBREADprintf("LINT: %zd , INT: %zd , SIZD : %zd\n", sizeof(long int ), sizeof(int ), sizeof(size_t)); //return -1; } memset(the_table -> malloc_ptr ,0, sizeof(void*) * GEHASH_MEM_PTR_NO); the_table -> index_gap = 0; FILE * fp = f_subr_open(fname, "rb"); if (!fp) { SUBREADprintf ("Table file '%s' is not found.\n", fname); return 1; } rrtv = fread(magic_chars,1,8,fp); if(rrtv !=8){ SUBREADprintf("Error: the index magic string cannot be found. It may contain format errors or file '%s' may be truncated.\n", fname); assert(rrtv==8); } if(memcmp(magic_chars, "2subindx",8)!=0) { print_in_box(80,0,0,""); print_in_box(80,0,0,"WARNING your reference index was built under an old version of subread"); print_in_box(80,0,0," package. Please rebuild the index for the reference genome."); print_in_box(80,0,0,""); } if(memcmp(magic_chars+1, "subindx",7)==0) { if('1'==magic_chars[0]) the_table -> version_number = SUBINDEX_VER1; else if('2'==magic_chars[0]) the_table -> version_number = SUBINDEX_VER2; else assert(0); if(SUBINDEX_VER2 == the_table -> version_number) { while(1) { short option_key, option_length; rrtv = fread(&option_key, 2, 1, fp); if(rrtv <1){ SUBREADprintf("Error: the index header cannot be fully loaded. It may contain format errors or file '%s' may be truncated.\n", fname); return -1; } //SUBREADprintf("READOPT_TAB = %04X\n", option_key); if(!option_key) break; rrtv = fread(&option_length, 2, 1, fp); if(rrtv <1){ SUBREADprintf("Error: the index header cannot be fully loaded. It may contain format errors or file '%s' may be truncated.\n", fname); return -1; } rrtv = 999; if(option_key == SUBREAD_INDEX_OPTION_INDEX_GAP) rrtv = fread(&(the_table -> index_gap),2,1,fp); else if (option_key ==SUBREAD_INDEX_OPTION_INDEX_PADDING) rrtv = fread(&(the_table -> padding),2,1,fp); else fseek(fp, option_length, SEEK_CUR); if(rrtv <1){ SUBREADprintf("Error: the index header cannot be fully loaded. It may contain format errors or file '%s' may be truncated.\n", fname); return -1; } } assert(the_table -> index_gap); } else if(SUBINDEX_VER1 == the_table -> version_number) the_table -> index_gap = 3; the_table -> current_items = load_int64(fp); if(the_table -> current_items < 1 || the_table -> current_items > 0xffffffffllu){ SUBREADputs("ERROR: the index format is unrecognizable."); assert(the_table -> current_items >0 && the_table -> current_items <=0xffffffffllu); } unsigned int * bucket_bytes = malloc(sizeof(int) * GEHASH_MEM_PTR_NO); memset(bucket_bytes, 0xff, sizeof(int) * GEHASH_MEM_PTR_NO); the_table -> buckets_number = load_int32(fp); the_table -> buckets = (struct gehash_bucket * )malloc(sizeof(struct gehash_bucket) * the_table -> buckets_number); if(!the_table -> buckets) { SUBREADputs("Creating buckets."); SUBREADputs(MESSAGE_OUT_OF_MEMORY); return 1; } srInt_64 fp_curr = ftello(fp); unsigned int accued_bytes = 0, grp_i = 0, curr_bucks = 0, per_group_bucks = the_table -> buckets_number/ GEHASH_MEM_PTR_NO + 2; for(i=0; i buckets_number ; i++){ unsigned int current_items = load_int32(fp); load_int32(fp);//useless for loading : space size unsigned int current_bytes = current_items*( sizeof(short) + sizeof(gehash_data_t) ); accued_bytes += current_bytes; curr_bucks ++; if(curr_bucks >= per_group_bucks){ //SUBREADprintf("Allocating %d : %d buckets : %u\n", grp_i, i, accued_bytes); bucket_bytes[grp_i++] = accued_bytes; accued_bytes = 0; curr_bucks = 0; } #ifdef __MINGW32__ { //if(i%500000 == 0)SUBREADprintf("ESTMHUGE %d/%d\n", i, the_table -> buckets_number); char * buffkk = malloc(current_bytes ); fread(buffkk,current_bytes ,1, fp); free(buffkk); } #else fseeko(fp, current_bytes, SEEK_CUR); #endif } if(curr_bucks) bucket_bytes[grp_i++] = accued_bytes; fseeko(fp, (off_t)fp_curr, SEEK_SET); for(i=0; i buckets_number); unsigned int current_bytes = bucket_bytes[i]; if(current_bytes<0xff000000u){ the_table -> malloc_ptr[i] = malloc(current_bytes); //SUBREADprintf("Allocating %d buckets : %u\n", i, current_bytes); if(!the_table -> malloc_ptr[i]){ SUBREADputs(MESSAGE_OUT_OF_MEMORY); return 1; } } } grp_i = 0; curr_bucks = 0; accued_bytes = 0; for(i=0; i buckets_number ; i++){ //if(i%50000 == 0)SUBREADprintf("FILLHUGE %d/%d\n", i, the_table -> buckets_number); struct gehash_bucket * current_bucket = the_table -> buckets+i; current_bucket -> current_items = load_int32(fp); load_int32(fp);//useless for lo: space size current_bucket -> space_size = current_bucket -> current_items; unsigned int current_bytes = current_bucket -> current_items*( sizeof(short) + sizeof(gehash_data_t) ); current_bucket -> new_item_keys = (short*)(the_table -> malloc_ptr[grp_i] + accued_bytes); current_bucket -> item_values = (gehash_data_t*)(the_table -> malloc_ptr[grp_i] + accued_bytes + current_bucket -> current_items * sizeof(short)); read_length = fread(current_bucket -> new_item_keys, sizeof(short), current_bucket -> current_items, fp); read_length += fread(current_bucket -> item_values, sizeof(gehash_data_t), current_bucket -> current_items, fp); if(read_length < 2* current_bucket -> current_items){ SUBREADprintf("ERROR: the index is incomplete.\n"); return 1; } accued_bytes += current_bytes; curr_bucks ++; if(curr_bucks >= per_group_bucks){ curr_bucks=0; accued_bytes=0; grp_i++; } } int rval = fread(&(the_table -> is_small_table), sizeof(char), 1, fp); if (rval != 1)SUBREADprintf("ERROR: cannot find the table table.\n"); free(bucket_bytes); fclose(fp); return 0; } else { fclose(fp); the_table -> index_gap = 3; fp = f_subr_open(fname, "rb"); the_table -> version_number = SUBINDEX_VER0; the_table -> current_items = load_int64(fp); the_table -> buckets_number = load_int32(fp); the_table -> buckets = (struct gehash_bucket * )malloc(sizeof(struct gehash_bucket) * the_table -> buckets_number); if(!the_table -> buckets) { SUBREADputs(MESSAGE_OUT_OF_MEMORY); return 1; } for (i=0; i buckets_number; i++) { struct gehash_bucket * current_bucket = &(the_table -> buckets[i]); current_bucket -> current_items = load_int32(fp); current_bucket -> space_size = load_int32(fp); current_bucket -> space_size = current_bucket -> current_items; current_bucket -> item_keys = (gehash_key_t *) malloc ( sizeof(gehash_key_t) * current_bucket -> space_size); current_bucket -> item_values = (gehash_data_t *) malloc ( sizeof(gehash_data_t) * current_bucket -> space_size); if(!(current_bucket -> item_keys&¤t_bucket -> item_values)) { SUBREADputs(MESSAGE_OUT_OF_MEMORY); return 1; } if(current_bucket -> current_items > 0) { read_length = fread(current_bucket -> item_keys, sizeof(gehash_key_t), current_bucket -> current_items, fp); if(read_length < current_bucket -> current_items){ SUBREADprintf("ERROR: the index is incomplete.\n"); return 1; } read_length = fread(current_bucket -> item_values, sizeof(gehash_data_t), current_bucket -> current_items, fp); if(read_length < current_bucket -> current_items){ SUBREADprintf("ERROR: the index is incomplete.\n"); return 1; } } } read_length = fread(&(the_table -> is_small_table), sizeof(char), 1, fp); if(read_length!=1){ SUBREADprintf("ERROR: the index is incomplete.\n"); return 1; } fclose(fp); return 0; } } void gehash_sort(gehash_t * the_table) { int i; for (i=0; i buckets_number; i++) { struct gehash_bucket * current_bucket = &(the_table -> buckets[i]); int ii, jj; gehash_key_t tmp_key; gehash_data_t tmp_val; if(current_bucket -> current_items>=1) { for(ii=0;ii current_items -1; ii++) { for (jj = ii+1; jj < current_bucket -> current_items; jj++) { if (current_bucket -> item_keys[ii] > current_bucket -> item_keys[jj]) { tmp_key = current_bucket -> item_keys[ii]; current_bucket -> item_keys[ii] = current_bucket -> item_keys[jj]; current_bucket -> item_keys[jj] = tmp_key; tmp_val = current_bucket -> item_values[ii]; current_bucket -> item_values[ii] = current_bucket -> item_values[jj]; current_bucket -> item_values[jj] = tmp_val; } } } } } } void write_cell(short option_key, short option_length, char * option_value, FILE * fp) { fwrite(&option_key, 2, 1, fp); fwrite(&option_length, 2, 1, fp); fwrite(option_value, option_length, 1, fp); } void write_options(FILE * fp, gehash_t * the_table) { short option_key, option_length; short option_value; option_key = SUBREAD_INDEX_OPTION_INDEX_PADDING; option_length = 2; option_value = the_table -> padding; write_cell(option_key, option_length, (char *) &option_value,fp); option_key = SUBREAD_INDEX_OPTION_INDEX_GAP; option_length = 2; option_value = the_table -> index_gap; write_cell(option_key, option_length, (char *) &option_value,fp); option_key = 0; fwrite(&option_key, 2, 1, fp); } int is_1_greater_than_2(int bucket_i, int all_buckets , short k1, gehash_data_t v1, short k2, gehash_data_t v2){ if(k1 > k2) return 1; if(k1 == k2){ unsigned int real_key = 1u*k1*1u*all_buckets; real_key += bucket_i; if((real_key%791)%2==0 && v1>v2) return 1; if((real_key%791)%2==1 && v1version_number == SUBINDEX_VER2) { fwrite("2subindx",1,8,fp); write_options(fp, the_table); } assert(sizeof(srInt_64 ) == 8); assert(sizeof(int ) == 4); fwrite(& (the_table -> current_items ), sizeof(srInt_64), 1, fp); fwrite(& (the_table -> buckets_number), sizeof(int), 1, fp); print_in_box(80,0,0,"Save current index block... "); for (i=0; i buckets_number; i++) { struct gehash_bucket * current_bucket = &(the_table -> buckets[i]); if(current_bucket -> current_items > maximum_bucket_size) maximum_bucket_size = current_bucket -> current_items; } #define SORT_LANE_NUMBER 16 short * sort_space_new_key[SORT_LANE_NUMBER]; gehash_data_t * sort_space_data [SORT_LANE_NUMBER]; int items_in_sort[SORT_LANE_NUMBER] ; int items_in_merge[SORT_LANE_NUMBER] ; if(the_table->version_number > SUBINDEX_VER0) { for(xx=0;xx buckets_number; i++) { struct gehash_bucket * current_bucket = &(the_table -> buckets[i]); gehash_data_t tmp_val=0; if(i % (the_table -> buckets_number/10) == 0) print_window_scrolling_bar("", 1.0*i/the_table -> buckets_number, 74, &scroll_counter); if(current_bucket -> current_items>=1) { if(the_table->version_number == SUBINDEX_VER0) { for(ii=0;ii current_items -1; ii++) { gehash_key_t tmp_key; for (jj = ii+1; jj < current_bucket -> current_items; jj++) { if(is_1_greater_than_2(i, the_table -> buckets_number, current_bucket -> item_keys[ii], current_bucket -> item_values[ii], current_bucket -> item_keys[jj], current_bucket -> item_values[jj] )) { tmp_key = current_bucket -> item_keys[ii]; current_bucket -> item_keys[ii] = current_bucket -> item_keys[jj]; current_bucket -> item_keys[jj] = tmp_key; tmp_val = current_bucket -> item_values[ii]; current_bucket -> item_values[ii] = current_bucket -> item_values[jj]; current_bucket -> item_values[jj] = tmp_val; } } } } else { if(1){ memset(items_in_sort, 0, sizeof(int)*SORT_LANE_NUMBER); for(ii=0;ii current_items;ii++) { int sort_lane_no = ii%SORT_LANE_NUMBER; int xx_th_item = items_in_sort[sort_lane_no] ++; sort_space_new_key[sort_lane_no][xx_th_item] = current_bucket -> new_item_keys[ii]; sort_space_data[sort_lane_no][xx_th_item] = current_bucket -> item_values[ii]; } // sort the individual lanes. for(xx=0;xx buckets_number, sort_space_new_key[xx][ii], sort_space_data[xx][ii], sort_space_new_key[xx][jj], sort_space_data[xx][jj] )) { tmp_key = sort_space_new_key[xx][ii]; sort_space_new_key[xx][ii] = sort_space_new_key[xx][jj]; sort_space_new_key[xx][jj] = tmp_key; tmp_val = sort_space_data[xx][ii]; sort_space_data[xx][ii]=sort_space_data[xx][jj]; sort_space_data[xx][jj]=tmp_val; } } } } // items_in_sort : array, each element for a lane // items_in_merge : how many items in each lane that have been merged (ie, read_ptr for each lane) // sort_space_new_key : N lanes each has been sorted, each has many keys (ie subreads) // sort_space_data : N lanes, each has many values (ie positions) // current_bucket -> new_item_keys : One array containing sorted keys (write_ptr is ii) // current_bucket -> item_values: One array containing sorted values (write_ptr is ii)) memset(items_in_merge, 0, sizeof(int)*SORT_LANE_NUMBER); // tmp_key : the current key being examed. A larger key is put AFTER a smaller key. for(ii=0;ii current_items;ii++) { int tmp_key=0x7fffffff; int selected_lane = 0, trying_lane; for(trying_lane=0;trying_lane= items_in_sort[trying_lane]) continue; if(tmp_key >= 0x10000 ||is_1_greater_than_2( i, the_table -> buckets_number, sort_space_new_key[selected_lane][items_in_merge[selected_lane]], sort_space_data[selected_lane][items_in_merge[selected_lane]], sort_space_new_key[trying_lane][items_in_merge[trying_lane]], sort_space_data[trying_lane][items_in_merge[trying_lane]] )){ selected_lane=trying_lane; tmp_key = sort_space_new_key[trying_lane][ii_in_trying_lane]; } } current_bucket -> new_item_keys[ii] = sort_space_new_key[selected_lane][items_in_merge[selected_lane]]; current_bucket -> item_values[ii] = sort_space_data[selected_lane][items_in_merge[selected_lane]]; items_in_merge[selected_lane]++; //printf("V [%d] [%d] =%d\n",i , ii, tmp_key); } } } } if(0) // debug { int inidx=0; srInt_64 real_old = -1; for(xx = 0; xx < current_bucket -> current_items; xx++){ unsigned int real_key = 1u * current_bucket -> new_item_keys[xx] *1u * the_table -> buckets_number; real_key += i; if(real_old != real_key) inidx=0; if(1||real_key >= 0xffffff00 || real_key <5000) SUBREADprintf("SUBREAD_%010u POS_%010u INIDX_%04d BUCKET_%04d_ITEM_%08d\n",real_key ,current_bucket -> item_values[xx], inidx, i, xx); real_old = real_key; inidx++; } } int is_full = 0; int write_len = fwrite(& (current_bucket -> current_items), sizeof(int), 1, fp); if(write_len<1) is_full = 1; write_len = fwrite(& (current_bucket -> space_size), sizeof(int), 1, fp); if(write_len<1) is_full = 1; if(the_table->version_number == SUBINDEX_VER0) fwrite(current_bucket -> item_keys, sizeof(gehash_key_t), current_bucket -> current_items, fp); else{ write_len = fwrite(current_bucket -> new_item_keys, sizeof(short), current_bucket -> current_items, fp); if(write_len < current_bucket -> current_items) is_full = 1; } write_len = fwrite(current_bucket -> item_values, sizeof(gehash_data_t), current_bucket -> current_items, fp); if(write_len < current_bucket -> current_items) is_full = 1; if(is_full){ fclose(fp); SUBREADprintf("ERROR: Unable to write into the output file. Please check the disk space in the output directory.\n"); return 1; } } if(the_table->version_number > SUBINDEX_VER0) { for(xx=0;xx is_small_table), sizeof(char), 1, fp); fclose(fp); if(write_len < 1){ SUBREADprintf("ERROR: Unable to write into the output file. Please check the disk space in the output directory.\n"); return 1; } print_in_box(80,0,0,""); return 0; } void gehash_destory(gehash_t * the_table) { int i, is_ptr = 0; for(i = 0; i < GEHASH_MEM_PTR_NO; i++) if(the_table -> malloc_ptr[i]){ free(the_table -> malloc_ptr[i]); is_ptr=1; } if(!is_ptr) for (i=0; i buckets_number; i++) { struct gehash_bucket * current_bucket = &(the_table -> buckets[i]); if (current_bucket -> space_size > 0) { if(0==the_table->free_item_only)free (current_bucket -> item_keys); free (current_bucket -> item_values); } } free (the_table -> buckets); the_table -> current_items = 0; the_table -> buckets_number = 0; } Rsubread/src/sorted-hashtable.h0000644000175200017520000001255414136047615017552 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #ifndef _SORTED_HASHTABLE_H_ #define _SORTED_HASHTABLE_H_ #include #include #include "subread.h" #include "gene-algorithms.h" #include "gene-value-index.h" #define GEHASH_DEFAULT_SIZE 2000000000 #if __WORDSIZE == 64 || _WIN64 #define GEHASH_BUCKET_LENGTH (100 - 69) #else #define GEHASH_BUCKET_LENGTH 100 #endif #define gehash_fast_t gehash_t #define gehash_destory_fast gehash_destory // This function creates a new hash table. The invoter may provide the expected size of the table or -1 for a default size (2 billions) // This function returns 0 if success or an errno // The version of the hash table created by this function must be SUBINDEX_VER0. int gehash_create(gehash_t * the_table, size_t expected_size, char is_small_table); // The EX version creates a hashtable with the given version number int gehash_create_ex(gehash_t * the_table, size_t expected_size, char is_small_table, int version_number, int GENE_SLIDING_STEP, int padding); // This function puts a data item into the table. If there is duplication, it insert another copy into the table but do not overlap on the old one. int gehash_insert(gehash_t * the_table, gehash_key_t key, gehash_data_t data, unsigned int * bucket_sizes); void gehash_try_insert_measure(unsigned int * bucket_sizes, int bucket_no, gehash_key_t key); // This function does what gehash_insert does, but insert nothing if the key has occured max_key_occurance times. int gehash_insert_limited(gehash_t * the_table, gehash_key_t key, gehash_data_t data, int max_key_occurance, int prob_replace); // This function queries the table and put the matched data item into data_result. // This function returns 0 if not found, or the number of matched items. // The invoter is in charge of allocating memory for results. size_t gehash_get(gehash_t * the_table, gehash_key_t key, gehash_data_t * data_result, size_t max_result_space); // Test existance, disregarding numbers. // Return 1 if exist, 0 if not. int gehash_exist(gehash_t * the_table, gehash_key_t key); size_t gehash_go_q_CtoT(gehash_t * the_table, gehash_key_t key, int offset, int read_len, int is_reversed, gene_vote_t * vote,gene_vote_number_t weight, int max_match_number, int indel_tolerance, int subread_number,int max_error_bases, unsigned int low_border, unsigned int high_border); size_t gehash_go_q_tolerable(gehash_t * the_table, gehash_key_t key, int offset, int read_len, int is_reversed, gene_vote_t * vote, gene_vote_number_t weight, gene_quality_score_t quality, int max_match_number, int indel_tolerance, int subread_number,int max_error_bases, int subread_len, unsigned int low_border, unsigned int high_border); size_t gehash_go_q(gehash_t * the_table, gehash_key_t key, int offset, int read_len, int is_reversed, gene_vote_t * vote,int indel_tolerance, int subread_number, unsigned int low_border, unsigned int high_border); size_t gehash_go_X(gehash_t * the_table, gehash_key_t key, int offset, int read_len, int is_reversed, gene_vote_t * vote,int indel_tolerance, int subread_number, unsigned int low_border, unsigned int high_border, int run_round, unsigned int * shift_indel_locations, unsigned int * shift_indel_NO); // This function performs the same functionality, but runs only on AMD-64 cpus, and the length of each key must be 4 bytes. size_t gehash_get_hpc(gehash_t * the_table, gehash_key_t key, gehash_data_t * data_result, size_t max_result_space); // This function removes all items under the key. It returns the number of items that has been removed in this call. size_t gehash_remove(gehash_t * the_table, gehash_key_t key); // Free all memory that is allocated for the table. Only the table structure itself is not freed. void gehash_destory(gehash_t * the_table); // This function conpletely dumps a table into a disk file. // It returns 0 if success, otherwise -1. int gehash_dump(gehash_t * the_table, const char fname []); void finalise_vote(gene_vote_t * vote); // This function loads a dumpped hash table. // The invoker does not need to initialise the table; it will be initialised in the function. // It returns 0 if success, otherwise -1. int gehash_load(gehash_t * the_table, const char fname []); void gehash_prealloc(gehash_t * the_table); size_t gehash_update(gehash_t * the_table, gehash_key_t key, gehash_data_t data_new); short indel_recorder_copy(gene_vote_number_t *dst, gene_vote_number_t* src); void assign_best_vote(gene_vote_t * vote, int i, int j); void select_best_vote(gene_vote_t * vote); void gehash_sort(gehash_t * the_table); int gehash_load_option(const char fname [], int option_no, int * result); // calculate # of buckets for estimaing their sizes. unsigned int calculate_buckets_by_size(size_t exp_size, int version, int is_small_tab, int index_gap); #endif Rsubread/src/sublog.c0000644000175200017520000000572714136047615015613 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #include #include #include #include #include #include #include "subread.h" #include "sublog.h" #define MINIMUM_LOG_LEVEL SUBLOG_LEVEL_INFO void remove_ESC_effects(char * txt) { int x1; int ocur = 0; int state = 0; int trimmed = 0; // return 0; for(x1=0;x1<1199;x1++) { if(!txt[x1])break; if((state == 0) && (txt[x1]==CHAR_ESC)) { state = 1; trimmed = 1; continue; } if(state == 0){ if(x1>ocur) txt[ocur] = txt[x1]; ocur++; } if((state == 1) && (txt[x1]=='m')) state = 0; } if(trimmed) txt[ocur]=0; } int is_ESC_removed() { #if defined(MAKE_STANDALONE) || defined(RUNNING_ENV) return !isatty(fileno(stderr)); #else return 1; #endif } void sublog_printf(int stage, int level, const char * pattern, ...) { va_list args; va_start(args , pattern); if(level0) SUBREADprintf("%s",vsbuf); free(vsbuf); } else { #if defined(MAKE_STANDALONE) || defined(RUNNING_ENV) vfprintf(stderr, pattern , args); fflush(stderr); #endif } va_end(args); } int sambamout_fprintf(FILE * fp, const char * pattern, ...) { int ret; va_list args; va_start(args , pattern); //printf("FP=%llu\n", (long long)fp); #ifdef MAKE_STANDALONE if(fp == NULL) fp = stdout; #endif assert(fp); ret = vfprintf(fp, pattern , args); va_end(args); return ret; } Rsubread/src/sublog.h0000644000175200017520000000254614136047615015614 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #ifndef _SUBLOG_H_ #define _SUBLOG_H_ #define SUBLOG_STAGE_DEV1 16 #define SUBLOG_STAGE_DEV_ALPHA 256 #define SUBLOG_STAGE_DEV_BETA 4096 #define SUBLOG_STAGE_DEV_RC 65536 #define SUBLOG_STAGE_RELEASED 1048576 #define SUBLOG_LEVEL_NIL 10 #define SUBLOG_LEVEL_DEBUG 20 #define SUBLOG_LEVEL_DETAILS 110 #define SUBLOG_LEVEL_INFO 120 #define SUBLOG_LEVEL_ABNORMAL 210 #define SUBLOG_LEVEL_WARNING 220 #define SUBLOG_LEVEL_ERROR 310 #define SUBLOG_LEVEL_FATAL 900 void sublog_printf(int stage, int level, const char * pattern, ...); void sublog_fwrite(int stage, int level, const char * pattern, ...); int sambamout_fprintf(FILE * fp, const char * pattern, ...); #endif Rsubread/src/subread.h0000644000175200017520000004532714136047615015752 0ustar00biocbuildbiocbuild/*************************************************************** The Subread software package is free software package: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Subread is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. Authors: Drs Yang Liao and Wei Shi ***************************************************************/ #ifndef _SUBREAD_H_ #define _SUBREAD_H_ #include #include #include #include #ifndef MAKE_STANDALONE #ifndef RUNNING_ENV #include #endif #endif #include "hashtable.h" #define SCRNA_HIGHEST_REPORTED_ALIGNMENTS 30 #define MAX_SCRNA_FASTQ_FILES 256 #define SCRNA_FASTA_SPLIT1 "|Rsd:cCounts:mFQs|" #define SCRNA_FASTA_SPLIT2 "|Rsd:cCounts:1mFQ|" #define SAM_FLAG_PAIRED_TASK 0x01 #define SAM_FLAG_FIRST_READ_IN_PAIR 0x40 #define SAM_FLAG_SECOND_READ_IN_PAIR 0x80 #define SAM_FLAG_MATE_UNMATCHED 0x08 #define SAM_FLAG_MATCHED_IN_PAIR 0x02 #define SAM_FLAG_REVERSE_STRAND_MATCHED 0x10 #define SAM_FLAG_MATE_REVERSE_STRAND_MATCHED 0x20 #define SAM_FLAG_SECONDARY_MAPPING 0x100 #define SAM_FLAG_DUPLICATE 0x400 #define SAM_FLAG_UNMAPPED 0x04 #define SUBREAD_MAX_ULONGLONG 0xffffffffffffffffllu #define SUBREAD_MAX_LONGLONG 0x7fffffffffffffffll #define FUSION_BREAK_POINT 2 #define FUSION_JUNCTION 1 #define SPLICING_JUNCTION 0 #define RUN_ALIGN 0 #define RUN_FINAL 1 #define MAX_THREADS 40 #define FC_MAX_THREADS 64 #define MAX_EVENTS_IN_READ 8 //#warning "============== REMOVE '* 15' FROM THE NEXT LINE ================" #define MAX_READ_LENGTH ( 1210 ) #define MAX_READ_NAME_LEN 200 #define MAX_CHROMOSOME_NAME_LEN 200 #define MAX_FILE_NAME_LENGTH (1000) #define FEATURE_NAME_LENGTH 256 #define INPUT_BLC_MAX_READS 20 #define MAX_BARCODE_LEN 32 //#warning "============== REMOVE '*1.2' FROM THE NEXT LINE ================" #define MULTI_THREAD_OUTPUT_ITEMS (4096 * 3/5 *3) #define EXON_LONG_READ_LENGTH 160 #define EXON_MAX_CIGAR_LEN 256 #define FC_CIGAR_PARSER_ITEMS 11 #define FC_LONG_READ_RECORD_HARDLIMIT (8*1024*1024) #define MAX_INDEL_SECTIONS 7 //#define XBIG_MARGIN_RECORD_SIZE 24 #define MAX_INSERTION_LENGTH 200 #define MAX_DELETION_LENGTH 1000 //#define BASE_BLOCK_LENGTH 15000000 //#define NEED_SUBREAD_STATISTIC #define IS_MIN_POS_NEGATIVE_STRAND 4 #define IS_MAX_POS_NEGATIVE_STRAND 12 #define IS_PAIRED_HINTED 16 #define IS_R1_CLOSE_TO_5 1 #define IS_REVERSED_HALVES 2 #define IS_PROCESSED_READ 32 #define IS_PROCESSED_READ_R2 64 #define IS_PAIRED_MATCH 128 #define IS_NEGATIVE_STRAND_R1 256 #define IS_NEGATIVE_STRAND_R2 512 #define IS_FUSION 1024 #define IS_NEGATIVE_STRAND 2048 #define IS_RECOVERED_JUNCTION_READ 4096 #define IS_FINALISED_PROCESSING 8192 #define IS_RECOVERED_JUNCTION_READ_STEP4 (8192*2) #define IS_BREAKEVEN_READ (8192*4) #define IS_R1R2_EQUAL_LEN 1024 #ifndef MAKE_CELLCOUNTS #define USE_POSIX_MUTEX_LOCK #endif #if defined(MACOS) || defined(FREEBSD) || defined(USE_POSIX_MUTEX_LOCK) typedef pthread_mutex_t subread_lock_t; #define pthread_spinlock_t pthread_mutex_t #define pthread_spin_lock pthread_mutex_lock #define pthread_spin_unlock pthread_mutex_unlock #define pthread_spin_init(a, b) pthread_mutex_init(a, NULL) #define pthread_spin_destroy(a) pthread_mutex_destroy(a) #define strnlen(a,l) strlen(a) #else typedef pthread_spinlock_t subread_lock_t; #endif #ifndef SRINT_64_DEFINED #define SRINT_64_DEFINED typedef long long srInt_64; typedef unsigned long long srUInt_64; #endif #ifdef __MINGW32__ #define ftello ftello64 #define fseeko fseeko64 #endif #if defined(MAKE_STANDALONE) || defined(RUNNING_ENV) #define STANDALONE_exit(i) exit(i) #define SUBREADprintf(...) fprintf(stderr, __VA_ARGS__) #define SUBREADputs(x) fprintf(stderr, "%s\n", x) #define SUBREADputchar(x) fputc(x, stderr) #define SUBREADfflush(x) fflush(x) #define CORE_SOFT_BR_CHAR '\n' #else #define STANDALONE_exit(i) return i; int safeRprintf(char *fmt, ...); void msgqu_printf(const char * fmt, ...); #define SUBREADprintf msgqu_printf #define SUBREADputs(x) msgqu_printf("%s\n",(x)) #define SUBREADputchar(X) msgqu_printf("%c",(X)) #define SUBREADfflush(X) #define CORE_SOFT_BR_CHAR '\n' #endif #ifndef NONONO_DONOTDEF #define QUALITY_KILL 198 #define QUALITY_KILL_SUBREAD 160 #define MAX_QUALITY_TO_CALL_JUNCTION 2195 #define MAX_QUALITY_TO_EXPLORER_JUNCTION 209 #else #define TEST_TARGET "" #endif #define SNP_CALLING_ONLY_HIGHQUAL 1 #define MESSAGE_OUT_OF_MEMORY "Out of memory. If you are using Rsubread in R, please save your working environment and restart R. \n" #define fatal_memory_size(a) SUBREADputs(MESSAGE_OUT_OF_MEMORY); //#define QUALITY_KILL 175 //#define QUALITY_KILL_SUBREAD 150 typedef long long subread_read_number_t; typedef unsigned int gehash_key_t; typedef unsigned int gehash_data_t; //typedef float gene_quality_score_t; typedef int gene_quality_score_t; //typedef unsigned char gene_vote_number_t; typedef short gene_vote_number_t; #define XOFFSET_TABLE_SIZE 250000 #define ANCHORS_NUMBER 259 #define MAX_ALIGNMENT_PER_ANCHOR 2 #define BEXT_RESULT_LIMIT 16 #define SEARCH_BACK 0 #define SEARCH_FRONT 1 //#define LARGE_GENE_VOTE_TABLE #ifdef LARGE_GENE_VOTE_TABLE #warning "Using LARGE_GENE_VOTE_TABLE" #define GENE_VOTE_SPACE 173 #define GENE_VOTE_TABLE_SIZE 331 #else /* #warning "============ ADD '-21' from next line ================" #define GENE_VOTE_SPACE (24 - 21) #warning "============ ADD '-13' from next line ================" #define GENE_VOTE_TABLE_SIZE (30 - 13) */ #define GENE_VOTE_SPACE 24 #define GENE_VOTE_TABLE_SIZE 30 #endif #define MAX_ANNOTATION_EXONS 30000 #define MAX_EXONS_PER_GENE 400 #define MAX_EXON_CONNECTIONS 10 #define MAX_GENE_NAME_LEN 128 #define MAX_INDEL_TOLERANCE 7 #define SUBINDEX_VER0 100 #define SUBINDEX_VER1 200 #define SUBINDEX_VER2 201 #define SUBREAD_INDEX_OPTION_INDEX_GAP 0x0101 #define SUBREAD_INDEX_OPTION_INDEX_PADDING 0x0102 #define CHAR_ESC 27 //#define base2int(c) ((c)=='A'?0:((c)=='T'?3:((c)=='C'?2:1))) #define base2int(c) ((c)<'G'?((c)=='A'?0:2):((c)=='G'?1:3)) /* #define base2int(c) (("\x3\x3\x3\x3\x3\x3\x3\x3" "\x3\x3\x3\x3\x3\x3\x3\x3" "\x3\x3\x3\x3\x3\x3\x3\x3" "\x3\x3\x3\x3\x3\x3\x3\x3" "\x3\x3\x3\x3\x3\x3\x3\x3" "\x3\x3\x3\x3\x3\x3\x3\x3" "\x3\x3\x3\x3\x3\x3\x3\x3" "\x3\x3\x3\x3\x3\x3\x3\x3" "\x3"\ "\x0\x3\x2\x3\x3\x3\x1\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3\x3")[(int)(c)]) // A B C D E F G */ //#define int2base(c) ((c)==1?'G':((c)==0?'A':((c)==2?'C':'T'))) //#define int2base(c) ("AGCT"[(c)]) #define int2base(c) (1413695297 >> (8*(c))&0xff) #define color2int(c) ((c) - '0') #define int2color(c) ("0123"[(c)]) #define remove_backslash(str) { int xxxa=0; while(str[xxxa]){ if(str[xxxa]=='/'){str[xxxa]='\0'; break;} xxxa++;} /* SUBREADprintf("BSRRR %s\n", str);*/ } /* #define get_base_error_prob64(a) ((a) < '@'-1?1:pow(10., -0.1*((a)-'@'))) #define get_base_error_prob33(a) ((a) < '!'-1?1:pow(10., -0.1*((a)-'!'))) */ #define SUBREAD_malloc(a) malloc(a) #define FASTQ_PHRED33 1 #define FASTQ_PHRED64 0 #define IS_DEBUG 0 typedef struct { char gene_name [MAX_GENE_NAME_LEN]; // The chromosome name is not stored in this data structure // All coordinates are translated into the linear location in the entire referenced genome, usually 0 ~ 3.2G unsigned int start_offset; unsigned int end_offset; // All exons are marked with the linear location in the entire referenced genome, usually 0 ~ 3.2G // This marks the end of the list: exon_ends [total_number_of_exons] = 0 // It shouldn't be equal to 0, should it be? unsigned int exon_starts [MAX_EXONS_PER_GENE]; unsigned int exon_ends [MAX_EXONS_PER_GENE]; } gene_t; struct gehash_bucket { int current_items; int space_size; union { short * new_item_keys; gehash_key_t * item_keys; }; gehash_data_t * item_values; }; #ifdef __MINGW32__ #define GEHASH_MEM_PTR_NO (64) #else #define GEHASH_MEM_PTR_NO (64*1024) #endif typedef struct { int version_number; unsigned long long int current_items; int buckets_number; char is_small_table; struct gehash_bucket * buckets; int index_gap; int padding; char * malloc_ptr [GEHASH_MEM_PTR_NO]; int free_item_only; } gehash_t; typedef struct{ unsigned int memory_block_size; unsigned int start_base_offset; unsigned int start_point; unsigned int length; unsigned char * values; unsigned int values_bytes; void * appendix1; void * appendix2; } gene_value_index_t; typedef struct { gene_vote_number_t max_vote; int max_vote_IJ; gehash_data_t max_position; gene_quality_score_t max_quality; gene_vote_number_t max_indel_recorder[MAX_INDEL_TOLERANCE*3]; gene_vote_number_t * max_tmp_indel_recorder; int max_mask; gene_vote_number_t noninformative_subreads; unsigned short items[GENE_VOTE_TABLE_SIZE]; unsigned int pos [GENE_VOTE_TABLE_SIZE][GENE_VOTE_SPACE]; int masks [GENE_VOTE_TABLE_SIZE][GENE_VOTE_SPACE]; int marked_shift_indel[GENE_VOTE_TABLE_SIZE][GENE_VOTE_SPACE]; gene_vote_number_t votes [GENE_VOTE_TABLE_SIZE][GENE_VOTE_SPACE]; gene_quality_score_t quality [GENE_VOTE_TABLE_SIZE][GENE_VOTE_SPACE]; gene_vote_number_t last_subread_cluster [GENE_VOTE_TABLE_SIZE][GENE_VOTE_SPACE]; gene_vote_number_t indel_recorder [GENE_VOTE_TABLE_SIZE][GENE_VOTE_SPACE][MAX_INDEL_TOLERANCE*3]; char current_indel_cursor[GENE_VOTE_TABLE_SIZE][GENE_VOTE_SPACE]; char toli[GENE_VOTE_TABLE_SIZE][GENE_VOTE_SPACE]; int topK_votes[SCRNA_HIGHEST_REPORTED_ALIGNMENTS]; int topK_IJ[SCRNA_HIGHEST_REPORTED_ALIGNMENTS ]; #ifdef MAKE_FOR_EXON short coverage_start [GENE_VOTE_TABLE_SIZE][GENE_VOTE_SPACE]; short coverage_end [GENE_VOTE_TABLE_SIZE][GENE_VOTE_SPACE]; short max_coverage_start; short max_coverage_end; //#warning Switch "MAKE_FOR_EXON" is turned on. It may cost more time. Do not turn it on unless you want to detect junction reads. #endif } gene_vote_t ; typedef struct{ unsigned int pos; char len; } indel_record_t; typedef struct{ int count; int support; } indel_result_t; typedef struct{ unsigned char best_len; unsigned int offsets [BEXT_RESULT_LIMIT]; unsigned char is_reverse [BEXT_RESULT_LIMIT]; } gene_best_record_t; typedef struct{ int max_len; unsigned int * max_positions; unsigned char * is_counterpart; gene_vote_number_t * max_votes; gene_quality_score_t * max_quality; gene_quality_score_t * max_final_quality; short * masks; char * max_indel_recorder; char * span_coverage; #ifdef REPORT_ALL_THE_BEST gene_best_record_t * best_records; #endif char max_indel_tolerance; short indel_recorder_length; unsigned char *repeated_regions; } gene_allvote_t; typedef struct{ int total_offsets; char *read_names; unsigned int *read_offsets; HashTable * read_name_to_index; int padding; } gene_offset_t; #define EXON_BUFFER_SIZE 3000 struct thread_input_buffer { char read_names [EXON_BUFFER_SIZE][121]; char read [EXON_BUFFER_SIZE][1201]; char quality [EXON_BUFFER_SIZE][1201]; int rl[EXON_BUFFER_SIZE]; int write_pointer; int read_pointer; unsigned int read_id[EXON_BUFFER_SIZE]; }; #define SEEKGZ_CHAIN_BLOCKS_NO 15 #define SEEKGZ_ZLIB_WINDOW_SIZE (32*1024) #define PAIRER_GZIP_WINDOW_BITS -15 #define PAIRER_DEFAULT_MEM_LEVEL 8 typedef struct { unsigned long long block_start_in_file_offset; unsigned int block_start_in_file_bits; char block_dict_window[SEEKGZ_ZLIB_WINDOW_SIZE]; // copied from the rolling window before this block is decompressed. unsigned int block_dict_window_size; char * block_txt; unsigned int * linebreak_positions; int linebreaks; int block_txt_size; } seekable_decompressed_block_t; typedef struct { FILE * gz_fp; z_stream stem; char * in_zipped_buffer; unsigned int in_zipped_buff_read_ptr; unsigned int current_block_txt_read_ptr; int blocks_in_chain; int has_multi_thread_accessed; int block_chain_current_no; seekable_decompressed_block_t block_rolling_chain[SEEKGZ_CHAIN_BLOCKS_NO]; int internal_error; subread_lock_t write_lock; unsigned int rolling_dict_window_used; char rolling_dict_window[SEEKGZ_ZLIB_WINDOW_SIZE]; unsigned long long next_block_file_offset; // for the next block after ALL blocks in the chain unsigned int next_block_file_bits; // for the next block after ALL blocks in the chain } seekable_zfile_t; typedef struct{ char dict_window[SEEKGZ_ZLIB_WINDOW_SIZE]; unsigned long long block_gzfile_offset; unsigned int block_gzfile_bits; unsigned int block_dict_window_size; unsigned int in_block_text_offset; } seekable_position_t; typedef struct { unsigned long long read_number; int lane_id; union{ seekable_position_t ** pos_of_bclgzs; unsigned long long * pos_of_bcls; }; union{ seekable_position_t * pos_of_filtergz; unsigned long long pos_of_filter; }; int is_EOF; } input_BLC_pos_t; typedef struct { unsigned long long read_number; int total_bases_in_each_cluster; int single_read_lengths[INPUT_BLC_MAX_READS+1]; int single_read_is_index[INPUT_BLC_MAX_READS]; int current_lane, bcl_is_gzipped, filter_is_gzipped; char bcl_format_string[MAX_FILE_NAME_LENGTH]; char filter_format_string[MAX_FILE_NAME_LENGTH]; union{ seekable_zfile_t ** bcl_gzip_fps; FILE ** bcl_fps; }; union{ seekable_zfile_t * filter_gzip_fp; FILE * filter_fp; }; subread_lock_t read_lock; int is_EOF; } input_BLC_t; #define MAX_SCRNA_READ_LENGTH 160 typedef struct{ char read_text[MAX_SCRNA_READ_LENGTH]; char qual_text[MAX_SCRNA_READ_LENGTH]; int read_len; int is_last_read_in_dataset; int file_no; int guessed_lane_no; } input_mFQ_cached_read_t; typedef struct { char filename[MAX_FILE_NAME_LENGTH+1]; int is_plain; FILE * plain_fp; seekable_zfile_t gz_fp; gzFile zlib_fp; int is_first_chars; unsigned char first_chars[2]; } autozip_fp; /* typedef pthread_mutex_t cellCounts_lock_t; #define cellCounts_init_lock(pp) pthread_mutex_init(pp, NULL) #define cellCounts_destroy_lock pthread_mutex_destroy #define cellCounts_lock_occupy pthread_mutex_lock #define cellCounts_lock_release pthread_mutex_unlock */ typedef struct{ int is_spin_lock; pthread_spinlock_t spinlock; pthread_mutex_t mutexlock; } cellCounts_lock_t; void cellCounts_init_lock(cellCounts_lock_t * lock, int is_spin); void cellCounts_destroy_lock(cellCounts_lock_t * lock); int cellCounts_lock_occupy(cellCounts_lock_t * lock); int cellCounts_lock_release(cellCounts_lock_t * lock); /* #define cellCounts_init_lock(pp) pthread_spin_init(pp,PTHREAD_PROCESS_SHARED) #define cellCounts_destroy_lock pthread_spin_destroy #define cellCounts_lock_occupy pthread_spin_lock #define cellCounts_lock_release pthread_spin_unlock */ typedef struct { char ** files1; char ** files2; char ** files3; int total_files; int current_file_no; int current_guessed_lane_no; srInt_64 current_read_no; autozip_fp autofp1; autozip_fp autofp2; autozip_fp autofp3; } input_mFQ_t; typedef struct { char chro_name[MAX_CHROMOSOME_NAME_LEN]; unsigned int chro_length; } SamBam_Reference_Info; typedef struct{ int current_BAM_file_no; srInt_64 section_start_pos; int in_section_offset; srInt_64 current_read_no; } input_scBAM_pos_t; typedef struct { FILE * os_file; char * BAM_file_names[MAX_SCRNA_FASTQ_FILES]; char section_buff[66000]; char align_buff[FC_LONG_READ_RECORD_HARDLIMIT]; int current_BAM_file_no; int total_BAM_files; int in_section_offset; int section_bin_bytes; int chro_table_size; SamBam_Reference_Info * chro_table; srInt_64 section_start_pos; srInt_64 current_read_no; subread_lock_t read_lock; } input_scBAM_t; typedef struct { int read_no_in_chunk; int reads_available_in_chunk; // -1 : EOF of all input reads : no next chunk available. // This can be set to -1 only when calling cacheBCL_netx_chunk(). int chunk_no; int chunk_start_lane; int chunk_end_lane; int reads_per_chunk; int last_chunk_in_cache; int total_bases_in_each_cluster; int single_read_lengths[INPUT_BLC_MAX_READS+1]; int single_read_is_index[INPUT_BLC_MAX_READS]; int current_lane, bcl_is_gzipped, filter_is_gzipped; int all_threads; char bcl_format_string[MAX_FILE_NAME_LENGTH]; char filter_format_string[MAX_FILE_NAME_LENGTH]; int bcl_no_is_used[MAX_READ_LENGTH]; autozip_fp * bcl_gzip_fps; autozip_fp filter_fp; subread_lock_t read_lock; char ** bcl_bin_cache; int flt_bin_cache_size; char * flt_bin_cache; char * lane_no_in_chunk; int is_EOF; } cache_BCL_t; typedef struct{ union{ srInt_64 pos_file1, pos_file2, pos_file3; seekable_position_t zpos_file1; }; seekable_position_t zpos_file2; seekable_position_t zpos_file3; int current_file_no; srInt_64 current_read_no; } input_mFQ_pos_t; typedef struct{ union{ unsigned long long simple_file_position; seekable_position_t seekable_gzip_position; input_BLC_pos_t BCL_position; input_scBAM_pos_t scBAM_position; input_mFQ_pos_t mFQ_position; }; char gzfa_last_name[MAX_READ_NAME_LEN]; } gene_inputfile_position_t; typedef struct{ unsigned int small_key; unsigned int big_key; } paired_exon_key; typedef struct{ unsigned int supporting_reads; char is_fusion; char big_pos_neg; char small_pos_neg; } fusion_record; double miltime(); typedef struct{ char chromosome_name[MAX_CHROMOSOME_NAME_LEN]; unsigned long known_length; } chromosome_t; typedef struct{ unsigned char record_type; unsigned char mapping_quality; unsigned short read_pos; unsigned short read_len; unsigned short flags; unsigned int read_number; unsigned int pos; unsigned short mapped_segment_in_read; char strand; // 0 = positive, 1 = negative } base_block_temp_read_t; typedef struct{ unsigned char record_type; unsigned int pos; short type; } VCF_temp_read_t; typedef struct { char filename [MAX_FILE_NAME_LENGTH * MAX_SCRNA_FASTQ_FILES * 3]; int space_type ; int file_type ; void * input_fp; // can be system (FILE * sam or fastq or fasta), (seekable_zfile_t *) char gzfa_last_name[MAX_READ_NAME_LEN]; unsigned long long read_chunk_start; union{ cache_BCL_t bcl_input; input_mFQ_t scRNA_fq_input; input_scBAM_t scBAM_input; }; } gene_input_t; struct explorer_section_t { unsigned int start_pos; short read_pos_start; short read_pos_end; char is_neg_strand; short indels; short indel_pos; short all_indel_poses[10]; short all_indels[10]; }; struct explorer_record_t { struct explorer_section_t cigar_record[6]; short b_search_tail; }; FILE * f_subr_open(const char * fname, const char * mode); void myrand_srand(unsigned long long seed); int myrand_rand(); #define abs(a) ((a)>=0?(a):-(a)) #define max(a,b) ((a)<(b)?(b):(a)) #define min(a,b) ((a)>(b)?(b):(a)) #endif Rsubread/src/tx-unique.c0000644000175200017520000004023314136047615016246 0ustar00biocbuildbiocbuild#include #include #include #include #include #include "tx-unique.h" void txunique_gene_free(void * v_gene){ txunique_gene_t * gene = v_gene; ArrayListDestroy(gene -> transcript_list); free(gene); } int txunique_init_context(txunique_context_t * context){ memset(context, 0 , sizeof(txunique_context_t)); strcpy(context -> gene_name_column_name, "gene_id"); strcpy(context -> transcript_id_column_name, "transcript_id"); strcpy(context -> used_feature_type, "exon"); context -> gene_table = HashTableCreate(62333); HashTableSetKeyComparisonFunction(context -> gene_table, my_strcmp); HashTableSetHashFunction(context -> gene_table, HashTableStringHashFunction); HashTableSetDeallocationFunctions(context -> gene_table, NULL, txunique_gene_free); context -> result_table = HashTableCreate(62333); HashTableSetKeyComparisonFunction(context -> result_table, my_strcmp); HashTableSetHashFunction(context -> result_table, HashTableStringHashFunction); HashTableSetDeallocationFunctions(context -> result_table, free, NULL); return 0; } void txunique_free_trans(void * v_trans){ txunique_transcript_t * trans = v_trans; ArrayListDestroy(trans -> exon_list); free(trans); } int txunique_do_add_exon(char * gene_name, char * transcript_id, char * chrome_name, unsigned int start, unsigned int end, int is_negative_strand, void * v_context){ int tx_i; txunique_context_t * context = v_context; txunique_gene_t * tag_gene = HashTableGet(context -> gene_table, gene_name); if(!tag_gene){ tag_gene = malloc(sizeof(txunique_gene_t)); strncpy(tag_gene -> gene_name, gene_name, FEATURE_NAME_LENGTH-1); tag_gene -> transcript_list = ArrayListCreate(6); ArrayListSetDeallocationFunction(tag_gene -> transcript_list, txunique_free_trans); HashTablePut(context -> gene_table, tag_gene -> gene_name, tag_gene); } txunique_transcript_t * tag_tx = NULL; //SUBREADprintf("NEW EXON: %s of %s : %s %u ~ %u ; txs = %lu in %lu\n", gene_name, transcript_id, chrome_name, start, end, tag_gene -> transcript_list -> numOfElements, tag_gene -> transcript_list -> capacityOfElements); for(tx_i = 0; tx_i < tag_gene -> transcript_list -> numOfElements; tx_i++){ txunique_transcript_t * try_tx = ArrayListGet(tag_gene -> transcript_list , tx_i); if(strcmp(try_tx -> transcript_id, transcript_id) == 0){ tag_tx = try_tx; break; } } if(!tag_tx){ tag_tx = malloc(sizeof(txunique_transcript_t)); strncpy(tag_tx -> transcript_id, transcript_id, FEATURE_NAME_LENGTH-1); tag_tx -> exon_list = ArrayListCreate(6); ArrayListSetDeallocationFunction(tag_tx -> exon_list, free); ArrayListPush(tag_gene -> transcript_list, tag_tx); } txunique_exon_t * tag_exon = malloc(sizeof(txunique_exon_t)); strncpy(tag_exon -> chro_name, chrome_name, MAX_CHROMOSOME_NAME_LEN -1); tag_exon -> exon_start = start; tag_exon -> exon_stop = end; tag_exon -> is_negative_strand = is_negative_strand; ArrayListPush(tag_tx -> exon_list, tag_exon); return 0; } int txunique_load_annotation(txunique_context_t * context){ int loaded_features = load_features_annotation(context -> input_GTF_file_name, FILE_TYPE_GTF, context -> gene_name_column_name, context -> transcript_id_column_name, context -> used_feature_type, context, txunique_do_add_exon); if(loaded_features<1) return -1; return 0; } int txunique_process_flat_comp( void * ex1p, void * ex2p, ArrayList *me ){ txunique_exon_t * ex1 = ex1p; txunique_exon_t * ex2 = ex2p; if(ex1 -> exon_start < ex2 -> exon_start)return -1; if(ex1 -> exon_start > ex2 -> exon_start)return 1; return 0; } void debug_print_exs(ArrayList * exs){ int ex_i; for(ex_i=0; ex_i numOfElements; ex_i++){ txunique_exon_t *ex = ArrayListGet(exs,ex_i); SUBREADprintf(" %s (%s) : %u ~ %u\n", ex->chro_name, ex->is_negative_strand?"NEG":"POS", ex->exon_start, ex->exon_stop); } } ArrayList * txunique_process_flat_exons(ArrayList * exs){ ArrayList * ret = ArrayListCreate(5); ArrayListSetDeallocationFunction(ret, free); if(exs->numOfElements < 1) return ret; //SUBREADputs("Before Sorting"); //debug_print_exs(exs); int ex_i; ArrayListSort(exs , txunique_process_flat_comp); //SUBREADputs("Before Flatten"); //debug_print_exs(exs); txunique_exon_t * memex = malloc(sizeof(txunique_exon_t)); memcpy(memex, ArrayListGet(exs,0), sizeof(txunique_exon_t)); ArrayListPush(ret, memex); for(ex_i=1; ex_i numOfElements; ex_i++){ txunique_exon_t * lastex = ArrayListGet(ret, ret -> numOfElements - 1); txunique_exon_t * tryex = ArrayListGet(exs, ex_i); if(tryex -> exon_start > lastex -> exon_stop + 1){ txunique_exon_t * memex = malloc(sizeof(txunique_exon_t)); memcpy(memex, tryex, sizeof(txunique_exon_t)); ArrayListPush(ret, memex); } else lastex -> exon_stop = max(tryex -> exon_stop, lastex -> exon_stop); } //SUBREADputs("After Flatten"); //debug_print_exs(ret); return ret; } struct _txunique_tmp_edges{ int is_exon_start; int nsupp; unsigned int base_open_end; }; void debug_print_edges(ArrayList * exs){ int ex_i; for(ex_i=0; ex_i numOfElements; ex_i++){ struct _txunique_tmp_edges *ex = ArrayListGet(exs,ex_i); SUBREADprintf(" %u : %s - nsup=%d\n", ex->base_open_end, ex->is_exon_start?"START":"END ", ex->nsupp); } } int txunique_process_gene_edge_comp(void * e1p, void * e2p, ArrayList *me){ struct _txunique_tmp_edges * e1 = e1p; struct _txunique_tmp_edges * e2 = e2p; if(e1 -> base_open_end < e2 -> base_open_end) return -1; if(e1 -> base_open_end > e2 -> base_open_end) return 1; if(e1 -> is_exon_start && !e2 -> is_exon_start) return -1; if(e2 -> is_exon_start && !e1 -> is_exon_start) return 1; return 0; } void txunique_process_gene_chro(txunique_context_t * context, char * chro, int strand_mode, txunique_gene_t * gene){ int tx_i, ex_i; ArrayList **flatten_trans = malloc(gene -> transcript_list -> numOfElements*sizeof(void *)); assert(flatten_trans); ArrayList * edge_list = ArrayListCreate(6); ArrayListSetDeallocationFunction(edge_list, free); for(tx_i = 0; tx_i < gene -> transcript_list -> numOfElements; tx_i ++){ txunique_transcript_t * try_tx = ArrayListGet(gene -> transcript_list , tx_i); ArrayList * used_exons = ArrayListCreate(6); ArrayListSetDeallocationFunction(used_exons, free); for(ex_i = 0; ex_i < try_tx -> exon_list -> numOfElements; ex_i ++){ txunique_exon_t * try_ex = ArrayListGet(try_tx -> exon_list, ex_i); if(strand_mode != try_ex -> is_negative_strand|| strcmp(try_ex -> chro_name, chro)!=0)continue; txunique_exon_t * memex = malloc(sizeof(txunique_exon_t)); memcpy(memex, try_ex, sizeof(txunique_exon_t)); ArrayListPush(used_exons, memex); } //SUBREADprintf("===== Process : %s : %s : %s : %s\n", gene -> gene_name, try_tx -> transcript_id, chro, strand_mode?"NEG":"POS"); flatten_trans[tx_i] = txunique_process_flat_exons(used_exons); ArrayListDestroy(used_exons); for(ex_i = 0; ex_i < flatten_trans[tx_i] -> numOfElements; ex_i ++){ txunique_exon_t * try_ex = ArrayListGet(flatten_trans[tx_i], ex_i); struct _txunique_tmp_edges * edge_start = malloc(sizeof(struct _txunique_tmp_edges)), *edge_end = malloc(sizeof(struct _txunique_tmp_edges)); edge_start -> is_exon_start = 1; edge_start -> base_open_end = try_ex -> exon_start; edge_start -> nsupp = 0; edge_end -> is_exon_start = 0; edge_end -> base_open_end = try_ex -> exon_stop + 1; edge_end -> nsupp = 0; ArrayListPush(edge_list, edge_start); ArrayListPush(edge_list, edge_end); } } if(edge_list -> numOfElements >0){ ArrayListSort(edge_list, txunique_process_gene_edge_comp); ArrayList * combined_edge_list = ArrayListCreate(6); ArrayListSetDeallocationFunction(combined_edge_list, free); struct _txunique_tmp_edges * merged_edge = ArrayListGet(edge_list, 0); merged_edge -> nsupp = 1; for(ex_i = 1; ex_i <= edge_list -> numOfElements; ex_i ++){ struct _txunique_tmp_edges * tmpedge = NULL; if(ex_i < edge_list -> numOfElements) tmpedge = ArrayListGet(edge_list, ex_i); if(NULL == tmpedge || merged_edge -> is_exon_start != tmpedge -> is_exon_start || merged_edge -> base_open_end != tmpedge -> base_open_end){ struct _txunique_tmp_edges * memedge = malloc(sizeof(struct _txunique_tmp_edges)); memcpy(memedge, merged_edge, sizeof(struct _txunique_tmp_edges)); ArrayListPush(combined_edge_list, memedge); if(tmpedge){ merged_edge = tmpedge; merged_edge -> nsupp = 1; } }else merged_edge -> nsupp++; } //debug_print_edges(combined_edge_list); for(tx_i = 0; tx_i < gene -> transcript_list -> numOfElements; tx_i ++){ txunique_transcript_t * try_tx = ArrayListGet(gene -> transcript_list , tx_i); ArrayList * flatten_exons = flatten_trans[tx_i]; unsigned int total_bases = 0, unique_bases = 0, unique_start = 0, total_start = 0; int overlapping_count = 0, txex_i = 0, is_on = 0; for(ex_i = 0; ex_i < combined_edge_list -> numOfElements; ex_i ++){ txunique_exon_t * txex = NULL; if(txex_i < flatten_exons -> numOfElements) txex = ArrayListGet(flatten_exons, txex_i); struct _txunique_tmp_edges *edge = ArrayListGet(combined_edge_list, ex_i); //SUBREADprintf(" TXN %s, Edge: %u (%s), Exon [%d]: %u ~ %u, %s, depth=%d, uniq_base=%u, all_base=%u\n", try_tx->transcript_id, edge -> base_open_end, edge -> is_exon_start?"START":"END ", txex_i, txex?txex->exon_start:0, txex?txex->exon_stop:0 , is_on?"ON":"OFF", overlapping_count, unique_bases, total_bases); if(total_start<1) assert(!is_on); if(!is_on) assert(total_start < 1); if(edge -> is_exon_start){ overlapping_count += edge -> nsupp; if(txex && edge -> base_open_end == txex -> exon_start) is_on = 1; if(unique_start >0){ unique_bases += edge -> base_open_end - unique_start; unique_start = 0; assert(overlapping_count > 1); }else if(overlapping_count == 1&& is_on) unique_start = edge -> base_open_end; if(total_start<1 && is_on)total_start = edge -> base_open_end; } if(is_on) assert(overlapping_count>0); if(!edge -> is_exon_start){ if(txex && edge -> base_open_end == txex -> exon_stop+1) is_on = 0; overlapping_count -= edge -> nsupp; if(unique_start >0){ unique_bases += edge -> base_open_end - unique_start; unique_start =0; assert(overlapping_count ==0); } else if(overlapping_count == 1 && is_on) unique_start = edge -> base_open_end; if(total_start && !is_on){ total_bases += edge -> base_open_end - total_start; total_start = 0; } } if(overlapping_count<1) assert(!is_on); if(!is_on) assert(total_start < 1); if(total_start<1) assert(!is_on); if(txex && edge-> is_exon_start==0){ if(edge-> base_open_end > txex -> exon_stop) txex_i++; } //SUBREADprintf(" %s, Edge: %u (%s), Exon [%d]: %u ~ %u, %s, depth=%d, uniq_base=%u, all_base=%u\n\n", try_tx->transcript_id, edge -> base_open_end, edge -> is_exon_start?"START":"END ", txex_i, txex?txex->exon_start:0, txex?txex->exon_stop:0 , is_on?"ON":"OFF", overlapping_count, unique_bases, total_bases); } assert(overlapping_count == 0); assert(total_start == 0); assert(unique_start == 0); assert(is_on == 0); char * hash_key = malloc(strlen(try_tx->transcript_id) + strlen(gene -> gene_name)+20); sprintf(hash_key, "%s\t%s\nALL", gene -> gene_name, try_tx->transcript_id); int old_all_bases = HashTableGet(context -> result_table, hash_key) - NULL; if(old_all_bases < 1)old_all_bases = 1; HashTablePut(context -> result_table, hash_key, NULL + old_all_bases + total_bases); hash_key = malloc(strlen(try_tx->transcript_id) + strlen(gene -> gene_name)+20); sprintf(hash_key, "%s\t%s\nUNIQUE", gene -> gene_name, try_tx->transcript_id); old_all_bases = HashTableGet(context -> result_table, hash_key) - NULL; if(old_all_bases < 1)old_all_bases = 1; HashTablePut(context -> result_table, hash_key, NULL + old_all_bases + unique_bases); } ArrayListDestroy(combined_edge_list); } ArrayListDestroy(edge_list); free(flatten_trans); } void txunique_process_write_gene(void * key, void * hashed_obj, HashTable * tab){ txunique_context_t * context = tab -> appendix1; FILE * out_fp = tab -> appendix2; int tx_i; txunique_gene_t * gene = hashed_obj; for(tx_i = 0; tx_i < gene -> transcript_list -> numOfElements; tx_i ++){ txunique_transcript_t * try_tx = ArrayListGet(gene -> transcript_list , tx_i); char hash_key [ FEATURE_NAME_LENGTH * 2 + 20]; sprintf(hash_key, "%s\t%s\nALL", gene->gene_name, try_tx -> transcript_id); int all_bases = HashTableGet(context -> result_table, hash_key)-NULL-1; sprintf(hash_key, "%s\t%s\nUNIQUE", gene->gene_name, try_tx -> transcript_id); int unique_bases = HashTableGet(context -> result_table, hash_key)-NULL-1; fprintf(out_fp, "%s\t%s\t%d\t%d\n", gene->gene_name, try_tx -> transcript_id, unique_bases, all_bases); } } void txunique_process_gene(void * key, void * hashed_obj, HashTable * tab){ txunique_context_t * context = tab -> appendix1; txunique_gene_t * gene = hashed_obj; int tx_i, ex_i, ch_i; ArrayList * chro_list = ArrayListCreate(5); for(tx_i = 0; tx_i < gene -> transcript_list -> numOfElements; tx_i ++){ txunique_transcript_t * try_tx = ArrayListGet(gene -> transcript_list , tx_i); for(ex_i = 0; ex_i < try_tx -> exon_list -> numOfElements; ex_i ++){ txunique_exon_t * try_ex = ArrayListGet(try_tx -> exon_list, ex_i); int found_chro = 0; for(ch_i = 0; ch_i < chro_list->numOfElements; ch_i++){ char * t_chro = ArrayListGet(chro_list, ch_i); if(strcmp(t_chro, try_ex -> chro_name)==0){ found_chro = 1; break; } } if(!found_chro) ArrayListPush(chro_list, try_ex -> chro_name); } } for(ch_i = 0; ch_i < chro_list -> numOfElements; ch_i ++){ int is_negative_strand; char * chro = ArrayListGet(chro_list, ch_i); for(is_negative_strand = 0; is_negative_strand<2; is_negative_strand++) txunique_process_gene_chro(context, chro, is_negative_strand, gene); } } int txunique_find_unique_bases(txunique_context_t * context){ context -> gene_table -> appendix1 = context; HashTableIteration(context -> gene_table, txunique_process_gene); return 0; } int txunique_write_output_file(txunique_context_t * context){ FILE * out_fp = fopen(context -> output_file_name, "w" ); fprintf(out_fp, "Gene_ID\tTranscript_ID\tUnique_Bases\tAll_Bases\n"); if(!out_fp){ SUBREADprintf("ERROR: unable to write output file : '%s'\n", context -> output_file_name); return 1; } context -> gene_table -> appendix1 = context; context -> gene_table -> appendix2 = out_fp; HashTableIteration(context -> gene_table, txunique_process_write_gene); fclose(out_fp); return 0; } int txunique_destroy_context(txunique_context_t * context){ HashTableDestroy(context -> gene_table); HashTableDestroy(context -> result_table); return 0; } int txunique_parse_options(txunique_context_t * context, int argc, char ** argv){ int c; optind = 1; opterr = 1; optopt = 63; while ((c = getopt (argc, argv, "a:o:g:t:f:h"))!=-1){ switch(c){ case 'a': strcpy(context -> input_GTF_file_name, optarg); break; case 'o': strcpy(context -> output_file_name, optarg); break; case 'g': strcpy(context -> gene_name_column_name, optarg); break; case 't': strcpy(context -> transcript_id_column_name, optarg); break; case 'f': strcpy(context -> used_feature_type, optarg); break; default: SUBREADputs("./txUnique -a -o { -g -t -f }"); break; } } if( context -> input_GTF_file_name[0]==0 || context -> output_file_name[0] == 0 ){ SUBREADputs("The GTF file name and the output file name must be specified."); return 1; } return 0; } #ifdef MAKE_STANDALONE int main(int argc, char ** argv){ #else int TxUniqueMain(int argc, char ** argv){ #endif txunique_context_t context; int ret = 0; ret = ret || txunique_init_context(&context); ret = ret || txunique_parse_options(&context, argc, argv); ret = ret || txunique_load_annotation(&context); ret = ret || txunique_find_unique_bases(&context); ret = ret || txunique_write_output_file(&context); ret = ret || txunique_destroy_context(&context); if(!ret) SUBREADputs("All finished."); return ret; } Rsubread/src/tx-unique.h0000644000175200017520000000174014136047615016253 0ustar00biocbuildbiocbuild#ifndef __TX_UNIQUE_H_ #define __TX_UNIQUE_H_ #include "subread.h" #include "hashtable.h" #include "input-files.h" #include "HelperFunctions.h" typedef struct{ char chro_name[MAX_CHROMOSOME_NAME_LEN]; unsigned int exon_start; unsigned int exon_stop; int is_negative_strand; } txunique_exon_t; typedef struct{ char transcript_id[FEATURE_NAME_LENGTH]; ArrayList * exon_list; } txunique_transcript_t; typedef struct{ char gene_name[FEATURE_NAME_LENGTH]; ArrayList * transcript_list; } txunique_gene_t; typedef struct{ char input_GTF_file_name[MAX_FILE_NAME_LENGTH]; char output_file_name[MAX_FILE_NAME_LENGTH]; char gene_name_column_name[FEATURE_NAME_LENGTH]; char transcript_id_column_name[FEATURE_NAME_LENGTH]; char used_feature_type[FEATURE_NAME_LENGTH]; HashTable * gene_table; // gene_id => array of transcripts [ (transcript_id, list_of_exons) ] HashTable * result_table; // "$gene_id\t$transcript_id\nALL|UNIQUE" => NULL + number + 1 } txunique_context_t; #endif Rsubread/vignettes/0000755000175200017520000000000014142061021015342 5ustar00biocbuildbiocbuildRsubread/vignettes/Rsubread.Rnw0000644000175200017520000002277714136047615017640 0ustar00biocbuildbiocbuild%\VignetteIndexEntry{Rsubread Vignette} %\VignetteDepends{} %\VignetteKeywords{Read mapping} %\VignettePackage{Rsubread} \documentclass[12pt]{article} \usepackage{hyperref} \newcommand{\R}[1]{{\texttt{#1}}} \newcommand{\C}[1]{{\texttt{#1}}} \textwidth=6.2in \textheight=8.5in \oddsidemargin=0.2in \evensidemargin=0.2in \headheight=0in \headsep=0in \begin{document} \title{Rsubread package: high-performance read alignment, quantification and mutation discovery} \author{Wei Shi} \date{2 January 2019} \maketitle \section{Introduction} This vignette provides a brief description to the Rsubread package. For more details, please refer to the Users Guide which can brought up in your R session via the following commands: \\ \noindent\R{> library(Rsubread)}\\ \R{> RsubreadUsersGuide()}\\ The Rsubread package provides facilities for processing short and long reads generated from the sequencing technologies. These facilities include quality assessment, read alignment, read summarization, exon-exon junction detection, absolute expression calling and SNP discovery. The Subread aligner (\R{align} function) is a highly efficient and accurate aligner for mapping genomic DNA and RNA sequencing reads. It adopts a novel mapping paradigm called ``seed-and-vote". Under this paradigm, a number of 16mers (called seeds or subreads) are extracted from each read and they were mapped to the reference genome to vote for the mapping location of the read. Read mapping performed under this paradigm has been found to be more efficient and accurate than that carried out under the conventional ``seed-and-extend" paradigm (Liao et al. 2013). Another aligner included in Rsubread is the Subjunc aligner (\R{subjunc} function). It was also developed under the ``seed-and-vote" paradigm, but different from the Subread aligner it performs full alignment for exon spanning reads and reports exon-exon junctions in addition to the read mapping results. The Subread aligner is recommended for gene-level expression analysis. For other types of RNA-seq analyses such as alternative splicing analysis, the Subjunc aligner should be used. An important step in processing next-gen sequencing data is to assign mapped reads to genomic features such as genes, exons and genomic windows. This package includes a general-purpose read summarization function \R{featureCounts} that takes mapped reads as input and assigns them to genomic features. In-built annotations are provided for users convenience. Different from microarray technologies, the next-gen sequencing technologies do not provide Present/Absent calls for genomic features such as genes. We have developed an algorithm to use the background noise measured from the RNA-seq data to call absolutely expressed genes. The function \R{detectionCall} returns a detection p value for each gene from the read mapping results. We have also developed a new SNP calling algorithm which is being implemented in function \R{exactSNPs}. Our results showed that it compared favorably to competing methods, but was an order of magnitude faster. This package also includes some other useful functions such as quality assessment (\R{qualityScores}, \R{atgcContent}), duplicate read removal (\R{removeDupReads}) and mapping percentage calculation (\R{propmapped}). \section{Read alignment} An index needs to be built first and then alignments can be carried out. Building the index is an one-off operation. The generated index can be re-used in subsequent read alignments.\\ {\noindent\bf Step 1: Index building \\} The Rsubread package includes a dummy reference sequence that was generated by concatenating 900 100bp reads that were taken from a pilot dataset generated from the SEquencing Quality Control (SEQC) project. We further extracted 100 reads from the same dataset and combine them with the 900 reads to make a read dataset for mapping. Below is the command for building an index for the reference sequence: \begin{scriptsize} <<>>= library(Rsubread) ref <- system.file("extdata","reference.fa",package="Rsubread") buildindex(basename="reference_index",reference=ref) @ \end{scriptsize} The generated index files were saved to the current working directory. Rsubread creates a hash table for indexing the reference genome. Keys in the hash table are the 16bp sequences and hash values are their corresponding chromosomal locations. Color space index can be built by setting the \R{colorsapce} argument to \R{TRUE}.\\ A unique feature of Rsubread is that it allows users to control the computer memory usage in read mapping process. Users can do this by tuning the amount of memory (in MB) to be used in read mapping.\\ {\noindent\bf Step 2: read mapping\\} After the index was successfully built, we map the read dataset (including 1,000 reads) to the reference sequence: \begin{scriptsize} <<>>= reads <- system.file("extdata","reads.txt.gz",package="Rsubread") align.stat <- align(index="reference_index",readfile1=reads,output_file="alignResults.BAM",phredOffset=64) @ \end{scriptsize} Map paired-end reads: \begin{scriptsize} <<>>= reads1 <- system.file("extdata","reads1.txt.gz",package="Rsubread") reads2 <- system.file("extdata","reads2.txt.gz",package="Rsubread") align.stat2 <- align(index="reference_index",readfile1=reads1,readfile2=reads2, output_file="alignResultsPE.BAM",phredOffset=64) @ \end{scriptsize} \section{Counting mapped reads for genomic features} The \R{featureCounts} function is a general-purpose read summarization function that assigns mapped reads (RNA-seq or gDNA-seq reads) to genomic features such as genes, exons, promoters, gene bodies and genomic windows. This function takes as input a set of files that contain read mapping results and an annotation file that includes genomic features. It automatically detects the format of input read files (supported formats include SAM and BAM). Input reads can be name-sorted or location-sorted. Users do not need to resort the reads before feeding them to \R{featureCounts}. In-built NCBI RefSeq gene annotations for genomes mm9, mm10, hg19 and hg38 are provided for convenience. These annotations include chromosomal coordinates of exons of each gene. When these annotations are used for summarization, only reads overlapping with exons will be counted by \R{featureCounts}. Users can use {\texttt{getInBuiltAnnotation}} function to retrieve these annotations. Below gives the example code of assigning reads and fragments, generated in the last section, to two artificial genes. Assign single end reads to genes: \begin{scriptsize} <<>>= ann <- data.frame( GeneID=c("gene1","gene1","gene2","gene2"), Chr="chr_dummy", Start=c(100,1000,3000,5000), End=c(500,1800,4000,5500), Strand=c("+","+","-","-"), stringsAsFactors=FALSE) ann fc_SE <- featureCounts("alignResults.BAM",annot.ext=ann) fc_SE @ \end{scriptsize} Assign fragments (read pairs) to the two genes: \begin{scriptsize} <<>>= fc_PE <- featureCounts("alignResultsPE.BAM",annot.ext=ann,isPairedEnd=TRUE) fc_PE @ \end{scriptsize} \section{Finding exon junctions} The RNA-seq technology provides a unique opportunity to identify the alternative splicing events that occur during the gene transcription process. The \R{subjunc} function can be used to detect exon-exon junctions. It first extracts a number of subreads (16mers) from each read, maps them to the reference genome and identifies the two best mapping locations for each read (representing potential locations of exons spanned by the read). Then, it builds a junction table including all putative junctions. Finally, it carries out a verification step to remove false positives in junction detection by realigning all the reads. The donor (`GT') and receptor sites(`AG'), are required to be present when calling exon-exon junctions. Output of this function includes the discovered exon-exon junctions and also read mapping results. \section{Base quality scores} Quality scores give the probabilities of read bases being incorrectly called, which is useful for examining the quality of sequencing data. The \R{qualityScores} function can be used to quickly retrieve and display the quality score data extracted from a read file. \begin{scriptsize} <<>>= x <- qualityScores(filename=reads,offset=64,nreads=1000) x[1:10,1:10] @ \end{scriptsize} \section{GC content} The \R{atgcContent} function returns fractions of A, T, G and C bases at each base location of reads or in the entire dataset. %\begin{scriptsize} %<<>>= %## Fraction of A,T,G and C bases in all the reads %x <- atgcContent(filename=reads) %x %## Fraction of A,T,G and C bases at each read location %xb <- atgcContent(filename=reads,basewise=TRUE) %xb[,1:5] %@ %\end{scriptsize} \section{Mapping percentage} Function \R{propmapped} returns the proportion of mapped reads included in a SAM/BAM file. For paired end reads, it can return the proportion of mapped fragments (ie. read pairs). \begin{scriptsize} <<>>= propmapped("alignResults.BAM") @ \end{scriptsize} \section{Citation} Yang Liao, Gordon K Smyth and Wei Shi (2013). The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Research, 41(10):e108.\\ \noindent{Yang Liao, Gordon K Smyth and Wei Shi (2014). featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics, 30(7):923-30} \section{Authors} Wei Shi and Yang Liao \\ Bioinformatics Division \\ The Walter and Eliza Hall Institute of Medical Research \\ 1G Royal Parade, Parkville, Victoria 3052 \\ Australia \\ \section{Contact} Please post to the Bioconductor Support site (\url{https://support.bioconductor.org/}) if you have any questions or suggestions. \end{document}

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