Hi Sticklenator, It sounds like some of your alignments are hitting a limit in the BAM index format. Can you send a few of the offending SAM records (e.g. the ones named in your original error message)?
Thanks, Alec On 7/21/14, 7:36 AM, sticklena...@gmail.com wrote: > Hi all, > when using Picard 1.113 to verify my BAM files, I end up getting the error > "bin field of BAM record does not equal value computed based on alignment > start and end, and length of sequence to which read is aligned": > > java -jar ValidateSamFile.jar I= 70130.satrFamilyX.GQI35_unique.bam > net.sf.picard.sam.ValidateSamFile INPUT=70130.satrFamilyX.GQI35_unique.bam > MODE=VERBOSE MAX_OUTPUT=100 IGNORE_WARNINGS=false VALIDATE_INDEX=true > IS_BISULFITE_SEQUENCED=false MAX_OPEN_TEMP_FILES=8000 VERBOSITY=INFO > QUIET=false VALIDATION_STRINGENCY=STRICT COMPRESSION_LEVEL=5 > MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false > Executing on Linux 2.6.32-431.5.1.el6.x86_64 amd64; OpenJDK 64-Bit Server VM > 1.7.0_51-mockbuild_2014_01_10_10_19-b00; Picard version: 1.113(1962) > IntelDeflater > > ERROR: Record 1564981, Read name HWI-ST1206:32:C3VGTACXX:5:2305:12309:67018, > bin field of BAM record does not equal value computed based on alignment > start and end, and length of sequence to which read is aligned > ERROR: Record 1564982, Read name HWI-ST1206:32:C3VGTACXX:5:2305:13183:74032, > bin field of BAM record does not equal value computed based on alignment > start and end, and length of sequence to which read is aligned > ERROR: Record 1564983, Read name HWI-ST1206:32:C3VGTACXX:5:2306:8170:17042, > bin field of BAM record does not equal value computed based on alignment > start and end, and length of sequence to which read is aligned > > Specifically Picard finds 84554 reads with the same problem. These reads > occur almost in a consecutive genomic region (chrUn - which is composed of > sequences for which no chromosome anchoring information exist) close to the > end of the file. > > > Following the suggestions on a similar issue here in the forum > (http://sourceforge.net/p/samtools/mailman/message/31853465/), I first tried > to convert the SAM file into BAM using SamFormatConverter, which yielded the > following error: > > > java -jar SamFormatConverter.jar INPUT=out.sam OUTPUT=out.bam > > net.sf.picard.sam.SamFormatConverter INPUT=out.sam OUTPUT=out.bam > VERBOSITY=INFO QUIET=false VALIDATION_STRINGENCY=STRICT COMPRESSION_LEVEL=5 > MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false > Executing on Linux 2.6.32-431.5.1.el6.x86_64 amd64; OpenJDK 64-Bit Server VM > 1.7.0_51-mockbuild_2014_01_10_10_19-b00; Picard version: 1.113(1962) > IntelDeflater > INFO 2014-07-21 13:14:44 SamFormatConverter Processed > 1,000,000 records. Elapsed time: 00:00:24s. Time for last 1,000,000: 24s. > Last read position: chrUn:175,188,973 > net.sf.picard.sam.SamFormatConverter done. Elapsed time: 0.61 minutes. > Runtime.totalMemory()=3500146688 > To get help, see http://picard.sourceforge.net/index.shtml#GettingHelp > Exception in thread "main" java.lang.IllegalArgumentException: Value (65544) > to large to be written as ushort. > at net.sf.samtools.util.BinaryCodec.writeUShort(BinaryCodec.java:324) > at net.sf.samtools.BAMRecordCodec.encode(BAMRecordCodec.java:128) > at > net.sf.samtools.BAMFileWriter.writeAlignment(BAMFileWriter.java:133) > at > net.sf.samtools.SAMFileWriterImpl.addAlignment(SAMFileWriterImpl.java:177) > at > net.sf.picard.sam.SamFormatConverter.doWork(SamFormatConverter.java:73) > at > net.sf.picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:179) > at > net.sf.picard.cmdline.CommandLineProgram.instanceMainWithExit(CommandLineProgram.java:120) > at > net.sf.picard.sam.SamFormatConverter.main(SamFormatConverter.java:57) > > Although I have to say, that I used a SAM file generated by samtools from the > original BAM file. Using SamFormatConverter did not work here either. > > The second suggestion was to use FixBamFile, which similarly crashed: > > java -classpath sam-1.113.jar net.sf.samtools.FixBAMFile > 70130.satrFamilyX.GQI35_unique.bam fixed.bam > Exception in thread "main" java.lang.IllegalArgumentException: Value (65544) > to large to be written as ushort. > at net.sf.samtools.util.BinaryCodec.writeUShort(BinaryCodec.java:324) > at net.sf.samtools.BAMRecordCodec.encode(BAMRecordCodec.java:128) > at > net.sf.samtools.BAMFileWriter.writeAlignment(BAMFileWriter.java:133) > at > net.sf.samtools.SAMFileWriterImpl.addAlignment(SAMFileWriterImpl.java:177) > at net.sf.samtools.FixBAMFile.main(FixBAMFile.java:41) > > > Thus I was wondering 1) what the actual problem might be and 2) how to fix it? > Thanks a lot! > > > > > ------------------------------------------------------------------------------ > Want fast and easy access to all the code in your enterprise? Index and > search up to 200,000 lines of code with a free copy of Black Duck > Code Sight - the same software that powers the world's largest code > search on Ohloh, the Black Duck Open Hub! Try it now. > http://p.sf.net/sfu/bds > _______________________________________________ > Samtools-help mailing list > Samtools-help@lists.sourceforge.net > https://lists.sourceforge.net/lists/listinfo/samtools-help ------------------------------------------------------------------------------ Want fast and easy access to all the code in your enterprise? Index and search up to 200,000 lines of code with a free copy of Black Duck Code Sight - the same software that powers the world's largest code search on Ohloh, the Black Duck Open Hub! Try it now. http://p.sf.net/sfu/bds _______________________________________________ Samtools-help mailing list Samtools-help@lists.sourceforge.net https://lists.sourceforge.net/lists/listinfo/samtools-help
