Notice:
If you happen to see a question you know the answer to, please do chime in and help your fellow community members. We encourage our fourm members to be more involved, jump in and help out your fellow researchers with their questions. GATK forum is a community forum and helping each other with using GATK tools and research is the cornerstone of our success as a genomics research community.We appreciate your help!

Test-drive the GATK tools and Best Practices pipelines on Terra


Check out this blog post to learn how you can get started with GATK and try out the pipelines in preconfigured workspaces (with a user-friendly interface!) without having to install anything.

HaplotypeCaller error: Only one of refStart or refStop must be < 0, not both

Hi,

I am trying to run Haplotype Caller on picard-validated bam file (no errors). Several scaffolds give me the error below. There doesn't seem to be a problem with UnifiedGenotyper on the same data.

Any solutions would be greatly appreciated!

Thank you,

Sasha

ERROR ------------------------------------------------------------------------------------------

ERROR stack trace

org.broadinstitute.sting.utils.exceptions.ReviewedStingException: Only one of refStart or refStop must be < 0, not both (-1, -65)
at org.broadinstitute.sting.utils.clipping.ReadClipper.hardClipByReferenceCoordinates(ReadClipper.java:497)
at org.broadinstitute.sting.utils.clipping.ReadClipper.hardClipByReferenceCoordinatesLeftTail(ReadClipper.java:154)
at org.broadinstitute.sting.utils.clipping.ReadClipper.hardClipToRegion(ReadClipper.java:332)
at org.broadinstitute.sting.utils.clipping.ReadClipper.hardClipToRegion(ReadClipper.java:343)
at org.broadinstitute.sting.utils.activeregion.ActiveRegion.hardClipToActiveRegion(ActiveRegion.java:50)
at org.broadinstitute.sting.gatk.walkers.haplotypecaller.HaplotypeCaller.map(HaplotypeCaller.java:407)
at org.broadinstitute.sting.gatk.walkers.haplotypecaller.HaplotypeCaller.map(HaplotypeCaller.java:104)
at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.processActiveRegion(TraverseActiveRegions.java:249)
at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.callWalkerMapOnActiveRegions(TraverseActiveRegions.java:204)
at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.processActiveRegions(TraverseActiveRegions.java:179)
at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.traverse(TraverseActiveRegions.java:136)
at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.traverse(TraverseActiveRegions.java:29)
at org.broadinstitute.sting.gatk.executive.LinearMicroScheduler.execute(LinearMicroScheduler.java:74)
at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.execute(GenomeAnalysisEngine.java:281)
at org.broadinstitute.sting.gatk.CommandLineExecutable.execute(CommandLineExecutable.java:113)
at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:237)
at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:147)
at org.broadinstitute.sting.gatk.CommandLineGATK.main(CommandLineGATK.java:94)

Answers

  • Geraldine_VdAuweraGeraldine_VdAuwera admin Cambridge, MAMember, Administrator, Broadie admin

    I think we've seen this before... What version are you using?

  • mikheyevmikheyev Member

    v2.2-16-g9f648cb, Compiled 2012/12/04 03:46:58

  • Geraldine_VdAuweraGeraldine_VdAuwera admin Cambridge, MAMember, Administrator, Broadie admin

    I see -- please try again using the latest version of 2.3, I believe this is fixed.

  • mikheyevmikheyev Member

    I am afraid not... Still get the same error.

  • mikheyevmikheyev Member

    The stack trace is the same:
    INFO 22:04:42,418 ProgressMeter - pbar_scf7180000349862:49920 5.03e+04 24.0 m 8.0 h 26.0% 92.2 m 68.2 m
    INFO 22:05:12,425 ProgressMeter - pbar_scf7180000347691:1098 5.10e+04 24.5 m 8.0 h 26.6% 92.2 m 67.7 m

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

    org.broadinstitute.sting.utils.exceptions.ReviewedStingException: Only one of refStart or refStop must be < 0, not both (-1, -65)
    at org.broadinstitute.sting.utils.clipping.ReadClipper.hardClipByReferenceCoordinates(ReadClipper.java:497)
    at org.broadinstitute.sting.utils.clipping.ReadClipper.hardClipByReferenceCoordinatesLeftTail(ReadClipper.java:154)
    at org.broadinstitute.sting.utils.clipping.ReadClipper.hardClipToRegion(ReadClipper.java:332)
    at org.broadinstitute.sting.utils.clipping.ReadClipper.hardClipToRegion(ReadClipper.java:343)
    at org.broadinstitute.sting.utils.activeregion.ActiveRegion.hardClipToActiveRegion(ActiveRegion.java:50)
    at org.broadinstitute.sting.gatk.walkers.haplotypecaller.HaplotypeCaller.map(HaplotypeCaller.java:397)
    at org.broadinstitute.sting.gatk.walkers.haplotypecaller.HaplotypeCaller.map(HaplotypeCaller.java:107)
    at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.processActiveRegion(TraverseActiveRegions.java:285)
    at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.callWalkerMapOnActiveRegions(TraverseActiveRegions.java:230)
    at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.processActiveRegions(TraverseActiveRegions.java:205)
    at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.traverse(TraverseActiveRegions.java:131)
    at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.traverse(TraverseActiveRegions.java:28)
    at org.broadinstitute.sting.gatk.executive.LinearMicroScheduler.execute(LinearMicroScheduler.java:74)
    at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.execute(GenomeAnalysisEngine.java:281)
    at org.broadinstitute.sting.gatk.CommandLineExecutable.execute(CommandLineExecutable.java:113)
    at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:237)
    at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:147)
    at org.broadinstitute.sting.gatk.CommandLineGATK.main(CommandLineGATK.java:94)

    ERROR ------------------------------------------------------------------------------------------
    ERROR A GATK RUNTIME ERROR has occurred (version 2.3-4-g57ea19f):
    ERROR
    ERROR Please visit the wiki to see if this is a known problem
    ERROR If not, please post the error, 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: Only one of refStart or refStop must be < 0, not both (-1, -65)
    ERROR ------------------------------------------------------------------------------------------
  • Geraldine_VdAuweraGeraldine_VdAuwera admin Cambridge, MAMember, Administrator, Broadie admin

    Ok, then we'll need you to please isolate and upload a bam snippet containing the region where the issue occurs. There is an FAQ article with instructions on how to do this if you need them (How to submit a detailed bug report).

  • mikheyevmikheyev Member

    OK, I submitted a bug report with the relevant data in a file called bugreport.tar.gz

    Although the entire merged bam has no errors according to ValidateSamFile, the subset does give a lot of "Mate not found for paired read errors"

  • Geraldine_VdAuweraGeraldine_VdAuwera admin Cambridge, MAMember, Administrator, Broadie admin

    Thanks Sasha, we'll take a look at it. Note that it might take a little longer than usual because of the holiday, but hopefully we should have an answer for you within a few days.

  • ebanksebanks ✭✭✭✭ Broad InstituteMember, Broadie, Dev ✭✭✭✭

    Hi Sasha,

    Because you are using a non-standard reference, we need your various reference files too (.fasta, .fai, and .dict).

  • mikheyevmikheyev Member

    Hi Geraldine,
    I uploaded the reference file to bugreport_reference.tar.gz. I wasn't sure is you needed the entire reference, so I uploaded it all of it. The actual contig that has the problem is called pbar_scf7180000347691.

    Happy New Year!

    Sasha

  • Geraldine_VdAuweraGeraldine_VdAuwera admin Cambridge, MAMember, Administrator, Broadie admin

    Hi Sasha,

    FYI, we've found the source of your problem and the developer is working on a fix.

    Happy New Year to you too!

  • thibaultthibault admin Broad InstituteMember, Broadie, Moderator, Dev admin

    This bug has been fixed for the next release (2.4).

    Thanks for reporting this.

  • mikheyevmikheyev Member

    Thank you very much! Do you have any ideas when the next release is coming out?

    Sasha

  • Geraldine_VdAuweraGeraldine_VdAuwera admin Cambridge, MAMember, Administrator, Broadie admin

    No guarantees, but the next release is tentatively scheduled for early February...

  • andrewseverinandrewseverin Member

    Any Idea what was causing the bug? And any update when 2.4 will be released?

  • Geraldine_VdAuweraGeraldine_VdAuwera admin Cambridge, MAMember, Administrator, Broadie admin

    Version 2.4 is in the release pipeline, should pop out later tonight or tomorrow morning.

  • johnomicsjohnomics Member

    Unfortunately, I'm still getting this error with 2.5-2:

    java -Xmx20g -jar /biosoft/src/GenomeAnalysisTK-2.5-2-gf57256b/GenomeAnalysisTK.jar -T HaplotypeCaller -R ref.fa -o out.vcf -L 1.intervals -bamout out.bam -hets 0.01 -maxNumHaplotypesInPopulation 4 -I 1.bam -I 2.bam -I 3.bam

    ##### ERROR stack trace 
    org.broadinstitute.sting.utils.exceptions.ReviewedStingException: Only one of refStart or refStop must be < 0, not both (-9, -1)
            at org.broadinstitute.sting.utils.clipping.ReadClipper.hardClipByReferenceCoordinates(ReadClipper.java:522)
            at org.broadinstitute.sting.utils.clipping.ReadClipper.hardClipByReferenceCoordinatesRightTail(ReadClipper.java:193)
            at org.broadinstitute.sting.utils.clipping.ReadClipper.hardClipAdaptorSequence(ReadClipper.java:389)
            at org.broadinstitute.sting.utils.clipping.ReadClipper.hardClipAdaptorSequence(ReadClipper.java:392)
            at org.broadinstitute.sting.utils.fragments.FragmentUtils.mergeOverlappingPairedFragments(FragmentUtils.java:231)
            at org.broadinstitute.sting.utils.fragments.FragmentUtils.mergeOverlappingPairedFragments(FragmentUtils.java:202)
            at org.broadinstitute.sting.gatk.walkers.haplotypecaller.HaplotypeCaller.finalizeActiveRegion(HaplotypeCaller.java:814)
            at org.broadinstitute.sting.gatk.walkers.haplotypecaller.HaplotypeCaller.assembleReads(HaplotypeCaller.java:636)
            at org.broadinstitute.sting.gatk.walkers.haplotypecaller.HaplotypeCaller.map(HaplotypeCaller.java:564)
            at org.broadinstitute.sting.gatk.walkers.haplotypecaller.HaplotypeCaller.map(HaplotypeCaller.java:135)
            at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.processActiveRegion(TraverseActiveRegions.java:564)
            at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.processActiveRegions(TraverseActiveRegions.java:521)
            at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.traverse(TraverseActiveRegions.java:251)
            at org.broadinstitute.sting.gatk.traversals.TraverseActiveRegions.traverse(TraverseActiveRegions.java:70)
            at org.broadinstitute.sting.gatk.executive.LinearMicroScheduler.execute(LinearMicroScheduler.java:100)
            at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.execute(GenomeAnalysisEngine.java:286)
            at org.broadinstitute.sting.gatk.CommandLineExecutable.execute(CommandLineExecutable.java:113)
            at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:245)
            at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:152)
            at org.broadinstitute.sting.gatk.CommandLineGATK.main(CommandLineGATK.java:91)
    ##### ERROR ------------------------------------------------------------------------------------------
    ##### ERROR A GATK RUNTIME ERROR has occurred (version 2.5-2-gf57256b):
    ##### ERROR
    ##### ERROR Please check the documentation guide to see if this is a known problem
    ##### ERROR If not, please post the error, 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: Only one of refStart or refStop must be < 0, not both (-9, -1)
    ##### ERROR ------------------------------------------------------------------------------------------
    

    I'm running 45 jobs using different intervals files. So far three have completed successfully, eleven have failed with this error, and 31 are still running.

    I have used the UnifiedGenotyper to call SNPs on the same BAM files and reference with no problem.

  • rpoplinrpoplin ✭✭✭ Member ✭✭✭

    Hi there,

    Thanks for the info. Can you please upload a bam snippet which reproduces this problem so we can jump in and debug? Geraldine has instructions for uploading bams to us earlier in this thread.

    Thanks so much,

  • bredesonbredeson ✭✭ Member ✭✭

    I've run into this same error with GATKv2.5-2. I've uploaded a tar ball by the name of GATK-v2.5-2_refStart-Stop_lt0.tar.gz to the public ftp server. The tar ball contains BAM files for the target region, the scaffold, a sh script with the command line and the exact error output from the run.

    The history of the bam files:
    The data was aligned with smalt, sorted with samtools, read groups added and duplicates marked with picard, and ValidateSamFile run on all. Due to ValidateSameFile reports, I ran FixMateInformation and samtools calmd to fix mate-pair info and re-calculate NM tags. ValidateSamFile only now reports errors for missing mates (located on different scaffolds).

  • Geraldine_VdAuweraGeraldine_VdAuwera admin Cambridge, MAMember, Administrator, Broadie admin

    Thanks @bredeson, we'll have a look at this and let you know when we have a fix.

  • Geraldine_VdAuweraGeraldine_VdAuwera admin Cambridge, MAMember, Administrator, Broadie admin

    Hi @bredeson,

    Thanks for the great test files, we were able to identify the bug. In brief, this occurred because we were reverting reads with soft clips that would produce reads with negative (or 0) alignment starts.

    The fix is currently going through code review; once it's accepted it will be available in the next nightly build (if you need to use it urgently) and will be in the next release (2.6).

  • bredesonbredeson ✭✭ Member ✭✭

    Awesome, thank you GATK Team for your hard work!

Sign In or Register to comment.