PositionalAssembler errors #478
Description
Hi,
I am trying to run GRIDSS (version 2.10.2) on a matched tumour-normal pair (2 bam files for the tumour, separated as per readgroups, and 1 bam file for the normal).
gridss -Xmx12g -jar $app_JAR \
REFERENCE_SEQUENCE=$REF \
OUTPUT=$tumour_od/$tumour_id.vcf \
ASSEMBLY=$tumour_od/${tumour_id}_assembly.bam \
WORKING_DIR=$tumour_wd \
BLACKLIST=$WD/ENCFF220FIN.bed \
INPUT=$ASCEX_N/$normal_id/$normal_id.secondary.removed.bam \
INPUT=$ASCEX_T/$tumour_id.secondary.removed_0.bam \
INPUT=$ASCEX_T/$tumour_id.secondary.removed_1.bam
The bam files are trimmed, deduplicated, coordinate sorted and I removed the secondary alignments (the aligment was done with bwa mem, using the -M argument).
samtools view -h ${ASCEX}/${tumour_id}/${tumour_id}.sorted.clean.deduplicated.bam | grep -v -e 'XA:Z:' | samtools view -b > ${tumour_od}/${tumour_id}.secondary.removed.bam
GRIDSS did run and produced a vcf file, but I have got the two following error messages multiple times (~ 2000).
ERROR 2021-03-25 00:53:11 PositionalAssembler Error assembling chr1. This should not happen. Please raise an issue at https://github.com/PapenfussLab/gridss/issues. Attempting recovery by resuming assembly at chr1:40563013
java.lang.NullPointerException
at au.edu.wehi.idsv.debruijn.positional.NonReferenceContigAssembler$SupportLookup.contigBaseOffsetBounds(NonReferenceContigAssembler.java:855)
at au.edu.wehi.idsv.debruijn.positional.NonReferenceContigAssembler$SupportLookup.readPairEvidence(NonReferenceContigAssembler.java:818)
at au.edu.wehi.idsv.debruijn.positional.NonReferenceContigAssembler$SupportLookup.supportInterval(NonReferenceContigAssembler.java:800)
at au.edu.wehi.idsv.debruijn.positional.NonReferenceContigAssembler.lambda$callContig$7(NonReferenceContigAssembler.java:462)
at java.base/java.util.stream.ReferencePipeline$3$1.accept(ReferencePipeline.java:195)
at java.base/java.util.IdentityHashMap$KeySpliterator.forEachRemaining(IdentityHashMap.java:1443)
at java.base/java.util.stream.AbstractPipeline.copyInto(AbstractPipeline.java:484)
at java.base/java.util.stream.AbstractPipeline.wrapAndCopyInto(AbstractPipeline.java:474)
at java.base/java.util.stream.ReduceOps$ReduceOp.evaluateSequential(ReduceOps.java:913)
at java.base/java.util.stream.AbstractPipeline.evaluate(AbstractPipeline.java:234)
at java.base/java.util.stream.ReferencePipeline.collect(ReferencePipeline.java:578)
at au.edu.wehi.idsv.debruijn.positional.NonReferenceContigAssembler.callContig(NonReferenceContigAssembler.java:463)
at au.edu.wehi.idsv.debruijn.positional.NonReferenceContigAssembler.flushCallsOutsideRetainWindow(NonReferenceContigAssembler.java:198)
at au.edu.wehi.idsv.debruijn.positional.NonReferenceContigAssembler.ensureCalledContig(NonReferenceContigAssembler.java:225)
at au.edu.wehi.idsv.debruijn.positional.NonReferenceContigAssembler.hasNext(NonReferenceContigAssembler.java:162)
at au.edu.wehi.idsv.debruijn.positional.PositionalAssembler.flushIfRequired(PositionalAssembler.java:70)
at au.edu.wehi.idsv.debruijn.positional.PositionalAssembler.ensureAssembler(PositionalAssembler.java:110)
at au.edu.wehi.idsv.debruijn.positional.PositionalAssembler.ensureAssembler(PositionalAssembler.java:86)
at au.edu.wehi.idsv.debruijn.positional.PositionalAssembler.hasNext(PositionalAssembler.java:56)
at au.edu.wehi.idsv.AssemblyEvidenceSource.assembleChunk(AssemblyEvidenceSource.java:232)
at au.edu.wehi.idsv.AssemblyEvidenceSource.assembleChunk(AssemblyEvidenceSource.java:192)
at au.edu.wehi.idsv.AssemblyEvidenceSource.lambda$assembleBreakends$1(AssemblyEvidenceSource.java:93)
at java.base/java.util.concurrent.FutureTask.run(FutureTask.java:264)
at java.base/java.util.concurrent.ThreadPoolExecutor.runWorker(ThreadPoolExecutor.java:1135)
at java.base/java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:635)
at java.base/java.lang.Thread.run(Thread.java:844)
ERROR 2021-03-25 02:17:37 PositionalAssembler Error assembling chr6. This should not happen. Please raise an issue at https://github.com/PapenfussLab/gridss/issues. Attempting recovery by resuming assembly at chr6:95219770
java.util.NoSuchElementException
at com.google.common.collect.TreeRangeSet.span(TreeRangeSet.java:173)
at au.edu.wehi.idsv.debruijn.positional.NonReferenceContigAssembler$SupportLookup.singleReadEvidence(NonReferenceContigAssembler.java:811)
at au.edu.wehi.idsv.debruijn.positional.NonReferenceContigAssembler$SupportLookup.supportInterval(NonReferenceContigAssembler.java:797)
at au.edu.wehi.idsv.debruijn.positional.NonReferenceContigAssembler.lambda$callContig$7(NonReferenceContigAssembler.java:462)
at java.base/java.util.stream.ReferencePipeline$3$1.accept(ReferencePipeline.java:195)
at java.base/java.util.IdentityHashMap$KeySpliterator.forEachRemaining(IdentityHashMap.java:1443)
at java.base/java.util.stream.AbstractPipeline.copyInto(AbstractPipeline.java:484)
at java.base/java.util.stream.AbstractPipeline.wrapAndCopyInto(AbstractPipeline.java:474)
at java.base/java.util.stream.ReduceOps$ReduceOp.evaluateSequential(ReduceOps.java:913)
at java.base/java.util.stream.AbstractPipeline.evaluate(AbstractPipeline.java:234)
at java.base/java.util.stream.ReferencePipeline.collect(ReferencePipeline.java:578)
at au.edu.wehi.idsv.debruijn.positional.NonReferenceContigAssembler.callContig(NonReferenceContigAssembler.java:463)
at au.edu.wehi.idsv.debruijn.positional.NonReferenceContigAssembler.ensureCalledContig(NonReferenceContigAssembler.java:232)
at au.edu.wehi.idsv.debruijn.positional.NonReferenceContigAssembler.hasNext(NonReferenceContigAssembler.java:162)
at au.edu.wehi.idsv.debruijn.positional.PositionalAssembler.flushIfRequired(PositionalAssembler.java:70)
at au.edu.wehi.idsv.debruijn.positional.PositionalAssembler.ensureAssembler(PositionalAssembler.java:110)
at au.edu.wehi.idsv.debruijn.positional.PositionalAssembler.ensureAssembler(PositionalAssembler.java:86)
at au.edu.wehi.idsv.debruijn.positional.PositionalAssembler.hasNext(PositionalAssembler.java:56)
at au.edu.wehi.idsv.AssemblyEvidenceSource.assembleChunk(AssemblyEvidenceSource.java:232)
at au.edu.wehi.idsv.AssemblyEvidenceSource.assembleChunk(AssemblyEvidenceSource.java:192)
at au.edu.wehi.idsv.AssemblyEvidenceSource.lambda$assembleBreakends$1(AssemblyEvidenceSource.java:93)
at java.base/java.util.concurrent.FutureTask.run(FutureTask.java:264)
at java.base/java.util.concurrent.ThreadPoolExecutor.runWorker(ThreadPoolExecutor.java:1135)
at java.base/java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:635)
at java.base/java.lang.Thread.run(Thread.java:844)
I did try to do queryname sorting, as it was recommended on the manual with samtools:
samtools sort -l 9 -m 4G -@ 8 -n ${ASCEX_T}/${tumour_id}.secondary.removed_0.bam -o ${tumour_od}/${tumour_id}.samtools.queryname.sorted_0.bam
As GRIDSS failed, I also tried the queryname soring with picard-tools:
java -Xmx12G -jar $picard_jar SortSam \
I=${ASCEX_T}/${tumour_id}.secondary.removed_0.bam \
O=${tumour_od}/${tumour_id}.queryname.sorted_0.bam \
SORT_ORDER=queryname
But in both cases GRIDSS failed with the same error message:
ERROR 2021-03-24 06:36:09 CallVariants Fatal exception thrown by worker thread.
htsjdk.samtools.SAMException: Requesting earlier reference sequence: 0 < 7
at htsjdk.samtools.reference.ReferenceSequenceFileWalker.get(ReferenceSequenceFileWalker.java:87)
at picard.analysis.SinglePassSamProgram.makeItSo(SinglePassSamProgram.java:141)
at picard.analysis.CollectMultipleMetrics.doWork(CollectMultipleMetrics.java:563)
at gridss.analysis.CollectGridssMetrics.doWork(CollectGridssMetrics.java:68)
at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:295)
at au.edu.wehi.idsv.SAMEvidenceSource.execute(SAMEvidenceSource.java:156)
at au.edu.wehi.idsv.SAMEvidenceSource.ensureMetrics(SAMEvidenceSource.java:140)
at gridss.CallVariants$1$1.call(CallVariants.java:55)
at gridss.CallVariants$1$1.call(CallVariants.java:51)
at java.base/java.util.concurrent.FutureTask.run(FutureTask.java:264)
at java.base/java.util.concurrent.ThreadPoolExecutor.runWorker(ThreadPoolExecutor.java:1135)
at java.base/java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:635)
at java.base/java.lang.Thread.run(Thread.java:844)
Any help would be much appreciated on the bam file preprocessing. Thank you!