Sam to Bam using Picard - help!

[Kath]

Hi,

I am trying to convert from sam to bam using Picard but am getting an error message (see below). Can anyone interpret this for me?

Thanks!

Kath

[skgtkaf@login01 13655]$ java -jar /home/skgtkaf/ngs/picard-tools-1.35/SamFormatConverter.jar I=13655_q20.sam O=13655_q20.bam
[Wed Nov 24 10:54:33 CET 2010] net.sf.picard.sam.SamFormatConverter INPUT=13655_q20.sam OUTPUT=13655_q20.bam TMP_DIR=/tmp/skgtkaf VERBOSITY=INFO QUIET=false VALIDATION_STRINGENCY=STRICT COMPRESSION_LEVEL=5 MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false
[Wed Nov 24 10:54:33 CET 2010] net.sf.picard.sam.SamFormatConverter done.
Runtime.totalMemory()=128843776
Exception in thread "main" net.sf.samtools.SAMFormatException: Error parsing text SAM file. CIGAR should have zero elements for unmapped read.; Line 165
Line: GA04_0001:4:1:1082:7316#0 133 chr12 27877005 0 35M43S =27877005 0 AAANNATNTNNTNNAATGANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCTTANNANNACNNNNNNNNNN %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% XC:i:35
at net.sf.samtools.SAMTextReader.reportErrorParsingLine(SAMTextReader.java:220)
at net.sf.samtools.SAMTextReader.access$500(SAMTextReader.java:40)
at net.sf.samtools.SAMTextReader$RecordIterator.parseLine(SAMTextReader.java:424)
at net.sf.samtools.SAMTextReader$RecordIterator.next(SAMTextReader.java:268)
at net.sf.samtools.SAMTextReader$RecordIterator.next(SAMTextReader.java:240)
at net.sf.samtools.SAMFileReader$AssertableIterator.next(SAMFileReader.java:604)
at net.sf.samtools.SAMFileReader$AssertableIterator.next(SAMFileReader.java:582)
at net.sf.picard.sam.SamFormatConverter.doWork(SamFormatConverter.java:65)
at net.sf.picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:156)
at net.sf.picard.sam.SamFormatConverter.main(SamFormatConverter.java:73)

[frozenlyse]

What aligner are you using? From the SAM flag of that read (133) that read is unaligned and so picard wants it to have no defined CIGAR (which makes sense!) Have you tried samtools? May its less strict

[furious]

I think you should change the VALIDATION_STRINGENCY option, read more about it here: http://seqanswers.com/forums/showthread.php?t=4246

[Kath]

Thanks for the suggestions. I used BWA to align the reads. I didn't realise you could use SAMtools to convert from SAM to BAM.

[Kath]

Ok, setting VALIDATION_STRINGENCY=LENIENT allows the .bam file to be produced. Thanks very much for your help!

[muzz56]

How did you set the validation=lenient?

[jstjohn]

How did you set the validation=lenient?

just include that string "VALIDATION_STRINGENCY=LENIENT" somewhere in your command.