Issue Running GATK Mitochondrial Pipeline

[jingxiant]

Hi GATK team,

I encountered an issue while running the GATK mitochondrial pipeline using Dockstore. The error occured during the step where the pipeline merges merges the aligned BAM file with the unmapped BAM file. During this step, I received the following error message:

Exception in thread "main" htsjdk.samtools.SAMFormatException: SAM validation error: WARNING: Record 1, Read name E00577:220:HVMHYCCXY:5:1101:10277:44327, Paired read should be m
arked as first of pair or second of pair.
at htsjdk.samtools.SAMUtils.processValidationErrors(SAMUtils.java:455)
at htsjdk.samtools.BAMFileReader$BAMFileIterator.advance(BAMFileReader.java:821)
at htsjdk.samtools.BAMFileReader$BAMFileIterator.(BAMFileReader.java:792)
at htsjdk.samtools.BAMFileReader$BAMFileIterator.(BAMFileReader.java:780)
at htsjdk.samtools.BAMFileReader.getIterator(BAMFileReader.java:483)
at htsjdk.samtools.SamReader$PrimitiveSamReaderToSamReaderAdapter.iterator(SamReader.java:478)
at picard.sam.SamToFastq.doWork(SamToFastq.java:191)
at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:295)
at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:103)
at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:113)
[main] Version: 0.7.15-r1140
[main] CMD: /usr/gitc/bwa mem -K 100000000 -p -v 3 -t 2 -Y /cromwell-executions/MitochondriaPipeline/05543c3b-ff39-4764-bd26-1172b8eb4165/call-AlignAndCall/AlignAndCall/d3f232c0-
c13b-4df8-9343-6d740c7562dd/call-AlignToMt/AlignmentPipeline/c8aab115-e001-40f1-9dc5-083d1f22304d/call-AlignAndMarkDuplicates/inputs/-595724031/Homo_sapiens_assembly38.chrM.fasta
/dev/stdin -
[main] Real time: 0.931 sec; CPU: 0.004 sec
[Mon Nov 11 06:13:47 UTC 2024] picard.sam.MergeBamAlignment done. Elapsed time: 0.00 minutes.
Runtime.totalMemory()=3014656000
To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp
Exception in thread "main" picard.PicardException: First record in unmapped bam is not first of pair: E00577:220:HVMHYCCXY:5:1101:10277:44327
at picard.sam.AbstractAlignmentMerger.mergeAlignment(AbstractAlignmentMerger.java:414)
at picard.sam.SamAlignmentMerger.mergeAlignment(SamAlignmentMerger.java:181)
at picard.sam.MergeBamAlignment.doWork(MergeBamAlignment.java:356)
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 have already tried sorting the BAM file by query name and also running the FixMateInformation tool, but the error persists.

Has anyone else encountered this error or have suggestions for how to resolve it?