Errors with Picard CollectRnaSeqMetrics

Question

[bertb]

Hello,

I'm trying to run a QC on my bam files for an RNAseq, but when I run the command:

find *U*.bam -exec echo java -jar ~/path/picard.jar CollectRnaSeqMetrics I={} O=picard/{}.RNA_Metrics REF_FLAT=$RNA_HOME/refs/sacCer3.ncbiRefSeq.ref_flat.txt STRAND=SECOND_READ_TRANSCRIPTION_STRAND RIBOSOMAL_INTERVALS=$RNA_HOME/refs/ref_ribosome.interval_list \; | sh

I receive an error about missing an SM tag:

Exception in thread "main" htsjdk.samtools.SAMFormatException: Error parsing SAM header. @RG line missing SM tag. Line:
@RG ID:UMH; File /home/path/to/file/UMH.bam; Line number 19
    at htsjdk.samtools.SAMTextHeaderCodec.reportErrorParsingLine(SAMTextHeaderCodec.java:265)
    at htsjdk.samtools.SAMTextHeaderCodec.access$200(SAMTextHeaderCodec.java:43)
    at htsjdk.samtools.SAMTextHeaderCodec$ParsedHeaderLine.requireTag(SAMTextHeaderCodec.java:346)
    at htsjdk.samtools.SAMTextHeaderCodec.parseRGLine(SAMTextHeaderCodec.java:175)
    at htsjdk.samtools.SAMTextHeaderCodec.decode(SAMTextHeaderCodec.java:108)
    at htsjdk.samtools.BAMFileReader.readHeader(BAMFileReader.java:667)
    at htsjdk.samtools.BAMFileReader.<init>(BAMFileReader.java:298)
    at htsjdk.samtools.BAMFileReader.<init>(BAMFileReader.java:176)
    at htsjdk.samtools.SamReaderFactory$SamReaderFactoryImpl.open(SamReaderFactory.java:396)
    at htsjdk.samtools.SamReaderFactory$SamReaderFactoryImpl.open(SamReaderFactory.java:208)
    at picard.analysis.SinglePassSamProgram.makeItSo(SinglePassSamProgram.java:96)
    at picard.analysis.SinglePassSamProgram.doWork(SinglePassSamProgram.java:84)
    at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:295)
    at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:103)
    at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:113)

I found some posts that talked about removing the SM tag, and whether or not that was possible/required, but didn't know how I might apply that here. I'm also not sure how to view the BAM file to inspect the @RG line.

Any help would be appreciated!

Thanks,

Answer 1

[Pierre Lindenbaum]

Your SAM header has a @RG line without the SM: (sample) attribute.

See :

• https://gatk.broadinstitute.org/hc/en-us/articles/360035890671-Read-groups
• http://broadinstitute.github.io/picard/command-line-overview.html#AddOrReplaceReadGroups
• Adding Read Groups To Bam Files

. I'm also not sure how to view the BAM file to inspect the @RG line.

samtools view -H UMH.bam | grep '^@RG'

[bertb]

Ok, I'm going to try that next. Thanks!