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MuTect2: ERROR MESSAGE: Somehow the requested coordinate is not covered by the read. Too many deleti

Hi guys,

I use mutct2 to do somatic variant calls, and got this ERROR MESSAGE: Somehow the requested coordinate is not covered by the read. Too many deletions? I searched the forum and found people got similar errors before when doing UnifiedGenotyper or HaplotypeCaller. Now I got this from running mutetc2. What's strange is that some of my samples went through mutect2 successfully. I am doing a analysis of 14 samples and so far 1 succeeded and 1 got the error, the rest are still running or in queue

Any suggestion?

Thanks a lot for the help!

Ying

sean@honey03 mutectOut]$ more 12-BI_S12.mutect_log
INFO 10:13:01,304 HelpFormatter - --------------------------------------------------------------------------------
INFO 10:13:01,312 HelpFormatter - The Genome Analysis Toolkit (GATK) v3.5-0-g36282e4, Compiled 2015/11/25 04:03:56
INFO 10:13:01,313 HelpFormatter - Copyright (c) 2010 The Broad Institute
INFO 10:13:01,313 HelpFormatter - For support and documentation go to http://www.broadinstitute.org/gatk
INFO 10:13:01,318 HelpFormatter - Program Args: -nct 8 -T MuTect2 -R /data02/sean/refGenome/mgp/REL-1505-SNPs_Indels/GRCm38_68.fa -I:tumor /data02/sean/BI_Mouse_Exo
me/12-BI_S12_fixmate_sorted_Dedup_merged_realign_recal_merged.bam -I:normal /data02/sean/BI_Mouse_Exome/BI_control_S15_S16_merged.bam --dbsnp /data02/sean/
refGenome/mgp/REL-1505-SNPs_Indels/mgp.v5.merged.indels.snps_all.dbSNP142.rsIDonly.sorted.C57BL_6NJ.vcf -o /data02/sean/BI_Mouse_Exome/mutectOut/12-BI_S12_somati
c.vcf
INFO 10:13:01,326 HelpFormatter - Executing as sean@honey on Linux 2.6.32-431.el6.x86_64 amd64; OpenJDK 64-Bit Server VM 1.7.0_45-mockbuild_2013_12_10_15_14-b00.
INFO 10:13:01,326 HelpFormatter - Date/Time: 2016/02/03 10:13:01
INFO 10:13:01,326 HelpFormatter - --------------------------------------------------------------------------------
INFO 10:13:01,327 HelpFormatter - --------------------------------------------------------------------------------
INFO 10:13:01,588 GenomeAnalysisEngine - Strictness is SILENT
INFO 10:13:01,804 GenomeAnalysisEngine - Downsampling Settings: Method: BY_SAMPLE, Target Coverage: 1000
INFO 10:13:01,815 SAMDataSource$SAMReaders - Initializing SAMRecords in serial
INFO 10:13:01,937 SAMDataSource$SAMReaders - Done initializing BAM readers: total time 0.11
INFO 10:13:02,419 MicroScheduler - Running the GATK in parallel mode with 8 total threads, 8 CPU thread(s) for each of 1 data thread(s), of 48 processors available on this
machine
INFO 10:13:02,636 GenomeAnalysisEngine - Preparing for traversal over 2 BAM files
INFO 10:13:03,402 GenomeAnalysisEngine - Done preparing for traversal
INFO 10:13:03,403 ProgressMeter - [INITIALIZATION COMPLETE; STARTING PROCESSING]
INFO 10:13:03,403 ProgressMeter - | processed | time | per 1M | | total | remaining
INFO 10:13:03,404 ProgressMeter - Location | active regions | elapsed | active regions | completed | runtime | runtime
INFO 10:13:03,568 MuTect2 - Using global mismapping rate of 45 => -4.5 in log10 likelihood units
INFO 10:13:03,570 PairHMM - Performance profiling for PairHMM is disabled because HaplotypeCaller is being run with multiple threads (-nct>1) option
Profiling is enabled only when running in single thread mode

Using AVX accelerated implementation of PairHMM
INFO 10:13:12,307 VectorLoglessPairHMM - libVectorLoglessPairHMM unpacked successfully from GATK jar file
INFO 10:13:12,308 VectorLoglessPairHMM - Using vectorized implementation of PairHMM
INFO 10:13:33,410 ProgressMeter - 1:4347420 0.0 30.0 s 49.6 w 0.2% 5.2 h 5.2 h
INFO 10:14:03,413 ProgressMeter - 1:4889805 0.0 60.0 s 99.2 w 0.2% 9.3 h 9.3 h
INFO 10:14:33,415 ProgressMeter - 1:6248751 0.0 90.0 s 148.8 w 0.2% 10.9 h 10.9 h
......
......
......
INFO 15:12:07,667 ProgressMeter - 2:35036630 1.263075341E9 29.0 h 82.0 s 47.5% 61.0 h 32.0 h
INFO 15:13:07,669 ProgressMeter - 2:35293870 1.263075341E9 29.0 h 82.0 s 47.5% 61.0 h 32.0 h
INFO 15:13:11,896 GATKRunReport - Uploaded run statistics report to AWS S3

ERROR ------------------------------------------------------------------------------------------
ERROR stack trace

org.broadinstitute.gatk.utils.exceptions.ReviewedGATKException: Somehow the requested coordinate is not covered by the read. Too many deletions?
at org.broadinstitute.gatk.utils.sam.ReadUtils.getReadCoordinateForReferenceCoordinate(ReadUtils.java:474)
at org.broadinstitute.gatk.utils.sam.ReadUtils.getReadCoordinateForReferenceCoordinate(ReadUtils.java:420)
at org.broadinstitute.gatk.utils.sam.ReadUtils.getReadCoordinateForReferenceCoordinate(ReadUtils.java:411)
at org.broadinstitute.gatk.utils.clipping.ReadClipper.hardClipByReferenceCoordinates(ReadClipper.java:543)
at org.broadinstitute.gatk.utils.clipping.ReadClipper.hardClipByReferenceCoordinatesLeftTail(ReadClipper.java:177)
at org.broadinstitute.gatk.utils.clipping.ReadClipper.hardClipAdaptorSequence(ReadClipper.java:408)
at org.broadinstitute.gatk.utils.clipping.ReadClipper.hardClipAdaptorSequence(ReadClipper.java:411)
at org.broadinstitute.gatk.tools.walkers.cancer.m2.MuTect2.finalizeActiveRegion(MuTect2.java:1179)
at org.broadinstitute.gatk.tools.walkers.cancer.m2.MuTect2.assembleReads(MuTect2.java:1123)
at org.broadinstitute.gatk.tools.walkers.cancer.m2.MuTect2.map(MuTect2.java:535)
at org.broadinstitute.gatk.tools.walkers.cancer.m2.MuTect2.map(MuTect2.java:175)
at org.broadinstitute.gatk.engine.traversals.TraverseActiveRegions$TraverseActiveRegionMap.apply(TraverseActiveRegions.java:709)
at org.broadinstitute.gatk.engine.traversals.TraverseActiveRegions$TraverseActiveRegionMap.apply(TraverseActiveRegions.java:705)
at org.broadinstitute.gatk.utils.nanoScheduler.NanoScheduler$ReadMapReduceJob.run(NanoScheduler.java:471)
at java.util.concurrent.Executors$RunnableAdapter.call(Executors.java:471)
at java.util.concurrent.FutureTask.run(FutureTask.java:262)
at java.util.concurrent.ThreadPoolExecutor.runWorker(ThreadPoolExecutor.java:1145)
at java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:615)
at java.lang.Thread.run(Thread.java:744)

ERROR ------------------------------------------------------------------------------------------
ERROR A GATK RUNTIME ERROR has occurred (version 3.5-0-g36282e4):
ERROR
ERROR This might be a bug. Please check the documentation guide to see if this is a known problem.
ERROR If not, please post the error message, with stack trace, to the GATK forum.
ERROR Visit our website and forum for extensive documentation and answers to
ERROR commonly asked questions http://www.broadinstitute.org/gatk
ERROR
ERROR MESSAGE: Somehow the requested coordinate is not covered by the read. Too many deletions?
ERROR ------------------------------------------------------------------------------------------

[sean@honey03 mutectOut]$

Tagged:

Answers

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie

    Hi there,

    Can you please validate your bam files with Picard ValidateSamFile?

  • yingchen69yingchen69 nanjingMember

    @Geraldine_VdAuwera said:
    Hi there,

    Can you please validate your bam files with Picard ValidateSamFile?

    Sure I will try Picard ValidateSamFile once current run is finished.

    Thanks a lot,

    Ying

  • yingchen69yingchen69 nanjingMember

    Hi,

    I validated my 1st bam files with Picard ValidateSamFile. The log says It validated 50m records and no errors found.

    Thanks,

    Ying

  • SheilaSheila Broad InstituteMember, Broadie, Moderator

    @yingchen69
    Hi Ying,

    I wonder if this is related to multithreading. Can you please try running without -nct 8?

    Thanks,
    Sheila

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie

    Also, try to narrow down the error to a subset of reads so that you don't have to re-run on the full bam every time.

  • yingchen69yingchen69 nanjingMember

    Hi,

    Thanks a lot for the suggestions. I tried to run on the individual chromosomes where the errors happened with no multithreading, and it succeeded!

    Is this a bug? This only happened to my 2 samples, and my other 12 samples had no issue at all.

    Thanks,

    Ying

  • SheilaSheila Broad InstituteMember, Broadie, Moderator

    @yingchen69
    Hi Ying,

    I am happy to hear things are working now! :smile: We have had users report issues with multi-threading, but because it is so finnicky, we prefer users to use Queue for parallelism.

    -Sheila

  • wungjaeleewungjaelee Northwestern UniversityMember

    Hi,

    I had the same problem as Ying while running mutect2 with -nct 8.
    Is it best to avoid parallelization? Or is there a safe threshold for -nct? Would lowering the number of cpu threads generally mean more error-prone result?

    Thanks so much,
    Wung Jae Lee

  • wungjaeleewungjaelee Northwestern UniversityMember

    The error messages I get are

    1291 org.broadinstitute.gatk.utils.exceptions.ReviewedGATKException: Somehow the requested coordinate is not covered by the read. Alignment 11113116 | 58S92M
    1292 at org.broadinstitute.gatk.utils.sam.ReadUtils.getReadCoordinateForReferenceCoordinate(ReadUtils.java:580)
    1293 at org.broadinstitute.gatk.utils.sam.ReadUtils.getReadCoordinateForReferenceCoordinate(ReadUtils.java:436)
    1294 at org.broadinstitute.gatk.utils.sam.ReadUtils.getReadCoordinateForReferenceCoordinate(ReadUtils.java:427)
    1295 at org.broadinstitute.gatk.utils.clipping.ReadClipper.hardClipByReferenceCoordinates(ReadClipper.java:543)
    1296 at org.broadinstitute.gatk.utils.clipping.ReadClipper.hardClipByReferenceCoordinatesLeftTail(ReadClipper.java:180)
    1297 at org.broadinstitute.gatk.utils.clipping.ReadClipper.hardClipToRegion(ReadClipper.java:375)
    1298 at org.broadinstitute.gatk.utils.clipping.ReadClipper.hardClipToRegion(ReadClipper.java:352)
    1299 at org.broadinstitute.gatk.utils.activeregion.ActiveRegion.trim(ActiveRegion.java:482)
    1300 at org.broadinstitute.gatk.utils.activeregion.ActiveRegion.trim(ActiveRegion.java:437)
    1301 at org.broadinstitute.gatk.tools.walkers.haplotypecaller.ActiveRegionTrimmer$Result.getCallableRegion(ActiveRegionTrimmer.java:383)
    1302 at org.broadinstitute.gatk.tools.walkers.cancer.m2.MuTect2.map(MuTect2.java:551)
    1303 at org.broadinstitute.gatk.tools.walkers.cancer.m2.MuTect2.map(MuTect2.java:176)
    1304 at org.broadinstitute.gatk.engine.traversals.TraverseActiveRegions$TraverseActiveRegionMap.apply(TraverseActiveRegions.java:709)
    1305 at org.broadinstitute.gatk.engine.traversals.TraverseActiveRegions$TraverseActiveRegionMap.apply(TraverseActiveRegions.java:705)
    1306 at org.broadinstitute.gatk.utils.nanoScheduler.NanoScheduler$ReadMapReduceJob.run(NanoScheduler.java:471)
    1307 at java.util.concurrent.Executors$RunnableAdapter.call(Executors.java:511)
    1308 at java.util.concurrent.FutureTask.run(FutureTask.java:266)
    1309 at java.util.concurrent.ThreadPoolExecutor.runWorker(ThreadPoolExecutor.java:1142)
    1310 at java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:617)
    1311 at java.lang.Thread.run(Thread.java:745)
    1312 ##### ERROR ------------------------------------------------------------------------------------------
    1313 ##### ERROR A GATK RUNTIME ERROR has occurred (version 3.6-0-g89b7209):
    1314 ##### ERROR
    1315 ##### ERROR This might be a bug. Please check the documentation guide to see if this is a known problem.
    1316 ##### ERROR If not, please post the error message, with stack trace, to the GATK forum.
    1317 ##### ERROR Visit our website and forum for extensive documentation and answers to
    1318 ##### ERROR commonly asked questions https://www.broadinstitute.org/gatk
    1319 ##### ERROR
    1320 ##### ERROR MESSAGE: Somehow the requested coordinate is not covered by the read. Alignment 11113116 | 58S92M
    1321 ##### ERROR ------------------------------------------------------------------------------------------

    If there's a way to just bypass those coordinates, please let me know!

    Thanks,
    Wung Jae

  • SheilaSheila Broad InstituteMember, Broadie, Moderator

    @wungjaelee
    Hi Wung Jae,

    We have had reports of odd errors from users who used -nct or -nt. However, we have a replacement for Queue that should be easier to use :smile:
    Have a look at WDL which can be used in place of parallelism.

    -Sheila

  • cordiercordier portland, orMember

    Is this issue still being looked into? I haven't seen it under current issues or past issues on the issue tracker.

    I get this error, along with another, using GATK 3.6 / MuTect 2 with -nct on a Linux cluster running CentOS 6.5.

    The error that appears regularly with MuTect 2 (also 3.6) is the documented "We encountered a non-standard non-IUPAC base in the provided reference '10'" issue. The issue arises using the 'Homo_sapiens_assembly19.fasta' reference, available on the NCBI FTP (ftp://ftp.ncbi.nlm.nih.gov/sra/reports/Assembly/GRCh37-HG19_Broad_variant/).

    Note that I haven't used another reference, but this reference does run correctly most of the time – I can run the MuTect 2 under the exact same settings with the same data and whether I get this error appears to be random with a frequency of maybe 30%.

    Any update or advice on how to navigate it (without installing having to manage more software) would be great.

    Thanks

  • cordiercordier portland, orMember

    @cordier said:
    Is this issue still being looked into? I haven't seen it under current issues or past issues on the issue tracker.

    I get this error, along with another, using GATK 3.6 / MuTect 2 with -nct on a Linux cluster running CentOS 6.5.

    The error that appears regularly with MuTect 2 (also 3.6) is the documented "We encountered a non-standard non-IUPAC base in the provided reference '10'" issue. The issue arises using the 'Homo_sapiens_assembly19.fasta' reference, available on the NCBI FTP (ftp://ftp.ncbi.nlm.nih.gov/sra/reports/Assembly/GRCh37-HG19_Broad_variant/).

    Note that I haven't used another reference, but this reference does run correctly most of the time – I can run the MuTect 2 under the exact same settings with the same data and whether I get this error appears to be random with a frequency of maybe 30%.

    Any update or advice on how to navigate it (without installing having to manage more software) would be great.

    Thanks

    Also, wondering if this issue is more prevalent when the coverage of your BAMs is relatively low – say 30x?

  • SheilaSheila Broad InstituteMember, Broadie, Moderator

    @cordier
    Hi,

    Does this issue only occur when you use -nct? Can you please post the exact command you ran? As for issues involving multi-threading, we are moving to WDL which is much more stable.

    -Sheila

  • cordiercordier portland, orMember

    Hi Sheila,

    No, the issue does occur when run with a single thread. The boilerplate for each command I'm running is:

        java -Xmx6G \
        -Djava.io.tmpdir=$tmpDir \
        -jar $GATK -T MuTect2 \
        -R $PIPELINE_REF/Homo_sapiens_assembly19.fasta \
        -I:tumor $experiment.tumor.data.bam \
        -I:normal $experiment.normal.data.bam \
        --dbsnp $PIPELINE_REF/dbsnp_138.hg19_modified.vcf \
        --cosmic $PIPELINE_REF/b37_cosmic_v54_120711_modified.vcf \
        --tumor_lod 10.0 \
        --contamination_fraction_to_filter $contamination \ # Contamination parse from ContEst output
        -o $experiment..raw.snps.indels.vcf \
        --log_to_file $PIPELINE_LOG/log_mutect2_$experiment.txt \
        --graphOutput $PIPELINE_LOG/assembly_graph_info.txt \
        -nct 20
    

    I've also attempted to run it with no NCT using the interval flag:

        java -Xmx6G \
        -Djava.io.tmpdir=$tmpDir \
        -jar $GATK -T MuTect2 \
        -R $PIPELINE_REF/Homo_sapiens_assembly19.fasta \
        -I:tumor $experiment.tumor.data.bam \
        -I:normal $experiment.normal.data.bam \
        --dbsnp $PIPELINE_REF/dbsnp_138.hg19_modified.vcf \
        --cosmic $PIPELINE_REF/b37_cosmic_v54_120711_modified.vcf \
        --tumor_lod 10.0 \
       -L $interval
        --contamination_fraction_to_filter $contamination \ # Contamination parse from ContEst output
        -o $experiment..raw.snps.indels.vcf \
        --log_to_file $PIPELINE_LOG/log_mutect2_$experiment.txt \
        --graphOutput $PIPELINE_LOG/assembly_graph_info.txt
    

    Because the issue persists in a single thread, running MuTect per chromosome effectively increases the likelihood that an error will occur.

    Thanks,
    Ben

  • cordiercordier portland, orMember

    Note, I wrote in the interval argument above - so there is an error there with the lack of a '\' for the multiline argument that does not exist in the actual code that is run.

  • SheilaSheila Broad InstituteMember, Broadie, Moderator

    @cordier
    Hi Ben,

    Thanks for the information. Can you submit a bug report so we can have a look? Instructions are here.

    Thanks,
    Sheila

  • hi, is there any solution for this problem? I use GATK 3.6 or 3.7 with -nct 30, the error still there

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie
    Try the latest nightly build, but if that doesn't work you will have to drop that option.

    Development effort on that tool has been moved to the GATK 4 project which is due to be released in a few months.
  • qunosenqunosen qunosen@gmail.comMember
    edited December 2017

    @Geraldine_VdAuwera & @Sheila,
    Hi Geraldine_VdAuwera & Sheila,

    I got the same problem as Ying and yingchen69 when running mutect2(GATK3.6) with "-nct" option, but it can work properly with "-nct" option while calling for another sample data ,would give me some suggestions?
    ERROR stack trace
    org.broadinstitute.gatk.utils.exceptions.ReviewedGATKException: Somehow the requested coordinate is not covered by the read. Too many deletions?
    at org.broadinstitute.gatk.utils.sam.ReadUtils.getReadCoordinateForReferenceCoordinate(ReadUtils.java:490)
    at org.broadinstitute.gatk.utils.sam.ReadUtils.getReadCoordinateForReferenceCoordinate(ReadUtils.java:436)
    at org.broadinstitute.gatk.utils.sam.ReadUtils.getReadCoordinateForReferenceCoordinate(ReadUtils.java:427)
    at org.broadinstitute.gatk.utils.clipping.ReadClipper.hardClipByReferenceCoordinates(ReadClipper.java:543)
    at org.broadinstitute.gatk.utils.clipping.ReadClipper.hardClipByReferenceCoordinatesLeftTail(ReadClipper.java:177)
    at org.broadinstitute.gatk.utils.clipping.ReadClipper.hardClipAdaptorSequence(ReadClipper.java:408)
    at org.broadinstitute.gatk.utils.clipping.ReadClipper.hardClipAdaptorSequence(ReadClipper.java:411)
    at org.broadinstitute.gatk.tools.walkers.cancer.m2.MuTect2.finalizeActiveRegion(MuTect2.java:1201)
    at org.broadinstitute.gatk.tools.walkers.cancer.m2.MuTect2.assembleReads(MuTect2.java:1145)
    at org.broadinstitute.gatk.tools.walkers.cancer.m2.MuTect2.map(MuTect2.java:536)
    at org.broadinstitute.gatk.tools.walkers.cancer.m2.MuTect2.map(MuTect2.java:176)
    at org.broadinstitute.gatk.engine.traversals.TraverseActiveRegions$TraverseActiveRegionMap.apply(TraverseActiveRegions.java:709)
    at org.broadinstitute.gatk.engine.traversals.TraverseActiveRegions$TraverseActiveRegionMap.apply(TraverseActiveRegions.java:705)
    at org.broadinstitute.gatk.utils.nanoScheduler.NanoScheduler$ReadMapReduceJob.run(NanoScheduler.java:471)
    at java.util.concurrent.Executors$RunnableAdapter.call(Executors.java:511)
    at java.util.concurrent.FutureTask.run(FutureTask.java:266)
    at java.util.concurrent.ThreadPoolExecutor.runWorker(ThreadPoolExecutor.java:1149)
    at java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:624)
    at java.lang.Thread.run(Thread.java:748)

    ERROR ------------------------------------------------------------------------------------------
    ERROR A GATK RUNTIME ERROR has occurred (version 3.6-0-g89b7209):
    ERROR
    ERROR This might be a bug. Please check the documentation guide to see if this is a known problem.
    ERROR If not, please post the error message, with stack trace, to the GATK forum.
    ERROR Visit our website and forum for extensive documentation and answers to
    ERROR commonly asked questions https://www.broadinstitute.org/gatk
    ERROR
    ERROR MESSAGE: Somehow the requested coordinate is not covered by the read. Too many deletions?
    ERROR ------------------------------------------------------------------------------------------

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie

    Please don’t post the same question in two places. We’ll answer you in the other thread.

  • qunosenqunosen qunosen@gmail.comMember
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