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Cannot construct fragment from more than two reads

micknudsenmicknudsen DenmarkMember ✭✭

Hi,

I am running a Best Practices Mutect2 workflow, and after having upgraded to GATK 4.1.4.0 from 4.1.3.0, I am starting to see this error:

(...)
17:35:39.809 INFO  PairHMM - Using the OpenMP multi-threaded AVX-accelerated native PairHMM implementation
17:35:39.942 INFO  ProgressMeter - Starting traversal
17:35:39.943 INFO  ProgressMeter -        Current Locus  Elapsed Minutes     Regions Processed   Regions/Minute
17:35:50.272 INFO  ProgressMeter -       chr22:10938027              0.2                 36510         212082.5
17:36:00.317 INFO  ProgressMeter -       chr22:12587748              0.3                 42160         124158.2
17:36:10.409 INFO  ProgressMeter -       chr22:16564638              0.5                 55550         109400.6
17:36:20.431 INFO  ProgressMeter -       chr22:18088679              0.7                 60790          90086.0
17:36:30.395 INFO  VectorLoglessPairHMM - Time spent in setup for JNI call : 0.022855297
17:36:30.395 INFO  PairHMM - Total compute time in PairHMM computeLogLikelihoods() : 7.454791689
17:36:30.395 INFO  SmithWatermanAligner - Total compute time in java Smith-Waterman : 11.04 sec
17:36:30.516 INFO  Mutect2 - Shutting down engine
[October 9, 2019 5:36:30 PM CEST] org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2 done. Elapsed time: 0.86 minutes.
Runtime.totalMemory()=1360003072
java.lang.IllegalArgumentException: Cannot construct fragment from more than two reads
        at org.broadinstitute.hellbender.utils.Utils.validateArg(Utils.java:725)
        at org.broadinstitute.hellbender.utils.read.Fragment.create(Fragment.java:36)
        at java.util.stream.ReferencePipeline$3$1.accept(ReferencePipeline.java:193)
        at java.util.ArrayList$ArrayListSpliterator.forEachRemaining(ArrayList.java:1382)
        at java.util.stream.AbstractPipeline.copyInto(AbstractPipeline.java:481)
        at java.util.stream.AbstractPipeline.wrapAndCopyInto(AbstractPipeline.java:471)
        at java.util.stream.ReduceOps$ReduceOp.evaluateSequential(ReduceOps.java:708)
        at java.util.stream.AbstractPipeline.evaluate(AbstractPipeline.java:234)
        at java.util.stream.ReferencePipeline.collect(ReferencePipeline.java:499)
        at org.broadinstitute.hellbender.utils.genotyper.AlleleLikelihoods.groupEvidence(AlleleLikelihoods.java:595)
        at org.broadinstitute.hellbender.tools.walkers.mutect.SomaticGenotypingEngine.callMutations(SomaticGenotypingEngine.java:93)
        at org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2Engine.callRegion(Mutect2Engine.java:251)
        at org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2.apply(Mutect2.java:320)
        at org.broadinstitute.hellbender.engine.AssemblyRegionWalker.processReadShard(AssemblyRegionWalker.java:308)
        at org.broadinstitute.hellbender.engine.AssemblyRegionWalker.traverse(AssemblyRegionWalker.java:281)
        at org.broadinstitute.hellbender.engine.GATKTool.doWork(GATKTool.java:1048)
        at org.broadinstitute.hellbender.cmdline.CommandLineProgram.runTool(CommandLineProgram.java:139)
        at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMainPostParseArgs(CommandLineProgram.java:191)
        at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:210)
        at org.broadinstitute.hellbender.Main.runCommandLineProgram(Main.java:163)
        at org.broadinstitute.hellbender.Main.mainEntry(Main.java:206)
        at org.broadinstitute.hellbender.Main.main(Main.java:292)
Using GATK jar /home/michaelk/miniconda3/envs/moma-somatic-pipeline-gatk-4.1.4.0/share/gatk4-4.1.4.0-0/gatk-package-4.1.4.0-local.jar
Running:
    java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -Xmx8g -Djava.io.tmpdir=/scratch/2114474/tmp.xejWjcYSOO -jar /home/michaelk/miniconda3/envs/moma-somatic-pipeline-gatk-4.1.4.0/share/gatk4-4.1.4.0-0/gatk-package-4.1.4.0-local.jar Mutect2 -R /faststorage/project/MomaRAWfiles/BACKUP/reference/hg38/reference_hg38/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna -I output/raw_output/alignments/TUMOR.recalibrated.markdup.sorted.bam -tumor TUMOR -I output/raw_output/alignments/NORMAL.recalibrated.markdup.sorted.bam -normal NORMAL --bam-output /scratch/2114474/TUMOR_chr22.mutect.somatic.assembled.haplotypes.bam --f1r2-tar-gz /scratch/2114474/TUMOR_chr22.mutect.somatic.f1r2.tar.gz -pon /faststorage/project/MomaRAWfiles/BACKUP/reference/hg38/broad_bundle_hg38/1000g_pon.hg38.vcf.gz -L chr22 -O /scratch/2114474/TUMOR_chr22.mutect.somatic.vcf.gz

Both VCF file and BAM-out are produced and seem to be OK. If I run the exact same pipeline with GATK 4.1.3.0, the error does not occur.

I have not seen others report this error neither here nor as a GitHub issue. Any clue what could be going on?

Thanks!

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