Sam Validation Error: Error: Record 8404072, Read Name Srx020270.6546275, Mapq Should Be 0 For Unmapped Read

Question

[Lds]

Hi, I had a error message when I use picard to replace read groups in a BAM file, like this:

$ java -jar AddOrReplaceReadGroups.jar I=SRX020270.sorted.bam O=SRX020270.resorted.bam SORTORDER=coordinate RGID=SRX020270 RGLB=bar RGPL=illumina RGSM=SRX020270 RGPU='run barcode' CREATEINDEX=True

[Sat Nov 19 13:17:11 CST 2011] net.sf.picard.sam.AddOrReplaceReadGroups INPUT=SRX020270.sorted.bam OUTPUT=SRX020270.resorted.bam SORTORDER=coordinate RGID=SRX020270 RGLB=bar RGPL=illumina RGPU=run barcode RGSM=SRX020270 CREATEINDEX=true TMPDIR=/tmp/ludongsheng VERBOSITY=INFO QUIET=false VALIDATIONSTRINGENCY=STRICT COMPRESSIONLEVEL=5 MAXRECORDSINRAM=500000 CREATEMD5FILE=false

INFO 2011-11-19 13:17:11 AddOrReplaceReadGroups Created read group ID=SRX020270 PL=illumina LB=bar SM=SRX020270

[Sat Nov 19 13:18:34 CST 2011] net.sf.picard.sam.AddOrReplaceReadGroups done. Elapsed time: 1.39 minutes. Runtime.totalMemory()=5798559744 Exception in thread "main" java.lang.RuntimeException: SAM validation error: ERROR: Record 8404072, Read name SRX020270.6546275, MAPQ should be 0 for unmapped read. at net.sf.samtools.SAMUtils.processValidationErrors(SAMUtils.java:334) at net.sf.samtools.BAMFileReader$BAMFileIterator.advance(BAMFileReader.java:469) at net.sf.samtools.BAMFileReader$BAMFileIterator.next(BAMFileReader.java:450) at net.sf.samtools.BAMFileReader$BAMFileIterator.next(BAMFileReader.java:417) at net.sf.samtools.SAMFileReader$AssertableIterator.next(SAMFileReader.java:629) at net.sf.samtools.SAMFileReader$AssertableIterator.next(SAMFileReader.java:607) at net.sf.picard.sam.AddOrReplaceReadGroups.doWork(AddOrReplaceReadGroups.java:91) at net.sf.picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:158) at net.sf.picard.cmdline.CommandLineProgram.instanceMainWithExit(CommandLineProgram.java:118) at net.sf.picard.sam.AddOrReplaceReadGroups.main(AddOrReplaceReadGroups.java:61)

I was told that read name SRX020270.6546275 was an unmapped read, so I checked out the entry for this read, and also the head line in SRX020270.sorted.bam.

$ samtools view SRX020270.sorted.bam | grep "SRX020270.6546275"

SRX020270.6546275 4 GL000198.1 90085 37 43M1D37M * 0 0 AGAATTCTTCAAAGAGTTCCAGATATCCACAGGCAGATTCTACAAATAAGTGTTTCAATACTGCTCTATCAAAAGACGTA BABA@?B?@BBBA@A@;?BBB@A<A?A@B@A>?AAA?>?@>@@?>A>@@@7@;;@?>@>@?:>A>@=<=@@=?@??>?;@ XT:A:U NM:i:4 X0:i:1 X1:i:0 XM:i:3 XO:i:1 XG:i:1 MD:Z:0C42^A3G29T3

$ samtools view -H SRX020270.sorted.bam

@SQ SN:1 LN:249250621

@SQ SN:2 LN:243199373

............

@SQ SN:GL000241.1 LN:42152

@SQ SN:GL000243.1 LN:43341

@SQ SN:GL000242.1 LN:43523

@SQ SN:GL000230.1 LN:43691

@SQ SN:GL000237.1 LN:45867

@SQ SN:GL000233.1 LN:45941

@SQ SN:GL000204.1 LN:81310

@SQ SN:GL000198.1 LN:90085

@SQ SN:GL000208.1 LN:92689

@SQ SN:GL000191.1 LN:106433

@SQ SN:GL000227.1 LN:128374

@SQ SN:GL000228.1 LN:129120

@SQ SN:GL000214.1 LN:137718

.....

@PG ID:bwa PN:bwa VN:0.5.9-r16

So, my question is, is there a way to fix this problem?

Answer 1

[toni]

Yes, there is and actually this is not a problem.

This is a BWA "feature". What you must keep in mind is that the only way to know if a read is unmapped is to check the flag. You may have a seq name or/and a mapQ>0, if the flag says it is unmapped, it is.

A simple solution to your problem is to set VALIDATION_STRINGENCY to LENIENT (you will be just warn) or SILENT (totally ignore) in your Picard commands.

[Lds]

Thank you so much, finally I've done with it.