Mutect2 error - Cannot construct fragment from more than two reads

Question

[Rozita]

Hi,

I'm trying to analyze WGS data and I'm currently running Mutect2, however, I've been receiving the following error message stating "Cannot construct fragment from more than two reads" and I'm not sure where things have gone wrong.

So far here's what I did:

I converted the FASTQ files to unaligned BAM format using FastqToSam, then sorted the BAM files by query using SortSam. I then used MarkIlluminaAdapters to read and re-write the BAM files with the new adapter-trimming tags. I also aligned the FASTQ reads to the genome using bwa-mem2. I then merged the 2 sets of files using the MergeBamAlignment tool to merge the read metadata, then marked the PCR and optical duplicates using GATK's MarkDuplicates with the flag REMOVE_DUPLICATES=TRUE (so I've removed the duplicates). I then used BaseRecalibrator to generate a recalibration table followed by ApplyBQSR. The error below is what I get when I run Mutect2

gatk Mutect2 -R Danio_rerio.GRCz11.dna.primary_assembly.fasta -I input_asoqueryname_bqsr_paired_reads.bam -O output_mutect2.vcf.gz

[29 June 2023 11:20:35 BST] org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2 done. Elapsed time: 1.00 minutes.
Runtime.totalMemory()=1884291072
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:1374)
        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)