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GenomeLocPArser error in SplitNCigarReads

efinlayefinlay IrelandMember

I don't understand the error message I'm getting and googling it or trying different reference and input files haven't helped.

ERROR MESSAGE: Badly formed genome loc: Parameters to GenomeLocParser are incorrect:The stop position 0 is less than start 38566563 in contig 10

Is the error within the bam file I am trying to split reads within or the reference genome I am aligning to?

Is there any quality control or cleam up method to check if the problem lies within my bam infile? I couldn't find one but when I ran BamIndexStats on the index file of the bam I was trying to use as my infile the line about contig 10 was

10 length= 104305016 Aligned= 61 Unaligned= 3

When I look at the .fai index of my reference genome it has the line

10 104305016 4 60 61

The .dict file also has

@SQ SN:10 LN:104305016 UR:file:/home/bmoran/bin/ens70/Btau.UMD3.1.70.short.fa M5:4c1ab0477783816857a28762f7630deb

When both input and reference files are using the length 104305016 for chromosome 10 I don't understand where the start 38566563 comes from in the error.

Thank you for your help and please let me know if I should supply any more information

Emma

My command

java -Xmx3g -jar /home/bmoran/bin/gatk3.1/GenomeAnalysisTK.jar

-T SplitNCigarReads

-R /home/bmoran/bin/ens70/Btau.UMD3.1.70.short.fa

-I 526_S19_align/526_S19.sort.rmdup.rgps.bam

-o 526_S19_align/526_S19.sort.rmdup.rgps.splitN.bam

-rf ReassignMappingQuality

-DMQ 60

-U ALLOW_N_CIGAR_READS

The complete error message.

INFO 11:56:59,997 GenomeAnalysisEngine - Strictness is SILENT
INFO 11:57:00,075 GenomeAnalysisEngine - Downsampling Settings: No downsampling
INFO 11:57:00,087 SAMDataSource$SAMReaders - Initializing SAMRecords in serial
INFO 11:57:00,112 SAMDataSource$SAMReaders - Done initializing BAM readers: total time 0.02
INFO 11:57:00,192 GenomeAnalysisEngine - Preparing for traversal over 1 BAM files
INFO 11:57:00,198 GenomeAnalysisEngine - Done preparing for traversal
INFO 11:57:00,198 ProgressMeter - [INITIALIZATION COMPLETE; STARTING PROCESSING]
INFO 11:57:00,198 ProgressMeter - Location processed.reads runtime per.1M.reads completed total.runtime remaining
INFO 11:57:00,227 ReadShardBalancer$1 - Loading BAM index data
INFO 11:57:00,228 ReadShardBalancer$1 - Done loading BAM index data

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

##### ERROR A USER ERROR has occurred (version 3.1-1-g07a4bf8):

ERROR
ERROR This means that one or more arguments or inputs in your command are incorrect.
ERROR The error message below tells you what is the problem.

##### ERROR

ERROR If the problem is an invalid argument, please check the online documentation guide
ERROR (or rerun your command with --help) to view allowable command-line arguments for this tool.
ERROR

##### ERROR Visit our website and forum for extensive documentation and answers to

ERROR commonly asked questions http://www.broadinstitute.org/gatk
ERROR

##### ERROR Please do NOT post this error to the GATK forum unless you have really tried to fix it yourself.

ERROR
ERROR MESSAGE: Badly formed genome loc: Parameters to GenomeLocParser are incorrect:The stop position 0 is less than start 38566563 in contig 10

##### ERROR ------------------------------------------------------------------------------------------

Answers

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin

    Can you please try to run this with the latest version (3.2)? If the error persists it may be a bug, as someone else reported a similar issue recently. If so we'll need a snippet of data to reproduce the error (see http://www.broadinstitute.org/gatk/guide/?1894 for instructions).

  • efinlayefinlay IrelandMember

    I downloaded the latest version and get the same error, I'll submit data

    [email protected]:/data/efinlay/previous_captures_Paulscript/testingfolder> java -Xmx3g -jar /data/efinlay/GenomeAnalysisTK.jar -T SplitNCigarReads -R /home/bmoran/bin/ens70/Btau.UMD3.1.70.short.fa -I 526_S19_align/526_S19.sort.rmdup.rgps.bam -o 526_S19_align/526_S19.sort.rmdup.rgps.splitN.bam -rf ReassignMappingQuality -DMQ 60 -U ALLOW_N_CIGAR_READS

    ERROR MESSAGE: Badly formed genome loc: Parameters to GenomeLocParser are incorrect:The stop position 0 is less than start 38566563 in contig 10
  • efinlayefinlay IrelandMember

    The data in the folder SplitNCigar_EmmaFinlay.tar.gz on ftp.broadinstitute.org
    Thank you

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin

    @efinlay Thanks for the test files! We have a fix for this going through code review right now. We'll make an announcement when it's available in the nightly builds.

  • SheilaSheila Broad InstituteMember, Broadie admin

    @efinlay‌

    Hi Emma,

    This bug has been fixed in the latest nightly build. Please download it here: http://www.broadinstitute.org/gatk/nightly

    -Sheila

  • efinlayefinlay IrelandMember

    Thank you very much, I'm no longer getting the error either

  • LavanyaLavanya Member

    Hi, I get to see the same error with GenomeAnalysisTK-3.2-2 version as below.
    ERROR MESSAGE: Badly formed genome loc: Parameters to GenomeLocParser are incorrect:The stop position 0 is less than start 12042 in contig chr1

    When I tried using with GenomeAnalysisTK-nightly-2014-09-23-g971ff9b.tar.bz2 from the link Sheila has shared above, I don't get the aforesaid error. Since we are planning to use a stable version for our analysis, is it advisable to use GenomeAnalysisTK-nightly-2014-09-23-g971ff9b.tar.bz2 for our pipelines or should we wait for stable release in future. Kindly suggest. Thanks.
    Regards

  • SheilaSheila Broad InstituteMember, Broadie admin

    @Lavanya‌

    Hi,

    As the nightly builds are a beta release, it is probably best to wait for the next stable release which should be in the next three weeks.

    -Sheila

  • LavanyaLavanya Member

    Hi Shiela,
    Can you please confirm whether a stable release has been released with the aforesaid bug fix? Thanks.

  • SheilaSheila Broad InstituteMember, Broadie admin

    @Lavanya‌

    Hi,

    Yes, there is a new release (version 3.3) that contains the fix. Please download it here: https://www.broadinstitute.org/gatk/auth?package=GATK

    -Sheila

  • naresh4836naresh4836 ROchester MNMember
    edited March 2015

    Hi Sheila,
    I using the version 3.3 i'm still getting the same error (in the SplitNCigarReads step)
    ERROR MESSAGE: Badly formed genome loc: Parameters to GenomeLocParser are incorrect:

    Regards
    Naresh

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin

    @naresh4836 It may be a different underlying issue causing your problem. Can you please post your command line and the full console output please?

  • naresh4836naresh4836 ROchester MNMember

    Command:
    /usr/java/jdk1.7.0_03/bin/java -XX:CompileThreshold=1000 -XX:ReservedCodeCacheSize=128m -Djava.io.tmpdir=$k3 -Xmx20g -Xms5g -jar GenomeAnalysisTK.jar -et NO_ET -T SplitNCigarReads -R /reference/sequence/human/ncbi/37.1/allchr.fa -I mergedsort.bam -o Aligned_sort_rg_md_splitNC.bam -rf ReassignOneMappingQuality -RMQF 255 -RMQT 60 -U ALLOW_N_CIGAR_READS

    Error:
    INFO 16:48:51,068 ProgressMeter - chr17:48151313 1.07580413E8 83.6 m 46.0 s 82.3% 101.6 m 18.0 m

    ERROR ------------------------------------------------------------------------------------------
    ERROR A USER ERROR has occurred (version nightly-2015-03-25-g84882ed):
    ERROR
    ERROR This means that one or more arguments or inputs in your command are incorrect.
    ERROR The error message below tells you what is the problem.
    ERROR
    ERROR If the problem is an invalid argument, please check the online documentation guide
    ERROR (or rerun your command with --help) to view allowable command-line arguments for this tool.
    ERROR
    ERROR Visit our website and forum for extensive documentation and answers to
    ERROR commonly asked questions http://www.broadinstitute.org/gatk
    ERROR
    ERROR Please do NOT post this error to the GATK forum unless you have really tried to fix it yourself.
    ERROR
    ERROR MESSAGE: Badly formed genome loc: Parameters to GenomeLocParser are incorrect:The stop position 48264257 is less than start 48264258 in contig chr17
    ERROR ------------------------------------------------------------------------------------------
  • naresh4836naresh4836 ROchester MNMember

    Hello Geraldine,

    Can you please look at the error log file i send you.

  • SheilaSheila Broad InstituteMember, Broadie admin
  • naresh4836naresh4836 ROchester MNMember

    Hi Sheila,

    I'm still getting the same error
    /usr/java/jdk1.7.0_03/bin/java -XX:CompileThreshold=1000 -XX:ReservedCodeCacheSize=128m -Djava.io.tmpdir=$k3 -Xmx20g -Xms5g -jar GenomeAnalysisTK.jar -et NO_ET -T SplitNCigarReads -R /reference/sequence/human/ncbi/37.1/allchr.fa -I mergedsort.bam -o Aligned_sort_rg_md_splitNC.bam -rf ReassignOneMappingQuality -RMQF 255 -RMQT 60 -U ALLOW_N_CIGAR_READS --validation_strictness SILENT -fixNDN

    ERROR ------------------------------------------------------------------------------------------
    ERROR A USER ERROR has occurred (version nightly-2015-03-25-g84882ed):
    ERROR
    ERROR This means that one or more arguments or inputs in your command are incorrect.
    ERROR The error message below tells you what is the problem.
    ERROR
    ERROR If the problem is an invalid argument, please check the online documentation guide
    ERROR (or rerun your command with --help) to view allowable command-line arguments for this tool.
    ERROR
    ERROR Visit our website and forum for extensive documentation and answers to
    ERROR commonly asked questions http://www.broadinstitute.org/gatk
    ERROR
    ERROR Please do NOT post this error to the GATK forum unless you have really tried to fix it yourself.
    ERROR
    ERROR MESSAGE: Badly formed genome loc: Parameters to GenomeLocParser are incorrect:The stop position 48264257 is less than start 48264258 in contig chr17
    ERROR ------------------------------------------------------------------------------------------

    --Naresh

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin

    @naresh4836 Can you please run Picard ValidateSAMFile on your data? I wonder if there's something wrong with some of the data in the file.

  • naresh4836naresh4836 ROchester MNMember

    Hello Geraldine,

    I got the following errors when i ran Picard ValidateSAMFile

    Mate alignment does not match alignment start of mate
    Mate negative strand flag does not match read negative strand flag of mate
    Mate reference index (MRNM) does not match reference index of mate

    Regards
    Naresh

  • SheilaSheila Broad InstituteMember, Broadie admin

    @naresh4836
    Hi Naresh,

    You can use Picard's FixMateInformation to fix this error.

    -Sheila

  • naresh4836naresh4836 ROchester MNMember

    Hello Sheila,

    I tried the GATK splitNcigar step on the different RNASEQ gsnap aligned bam file(Some sample ran fine with out any error).I got a similar error

    Command
    /usr/java/jdk1.7.0_03/bin/java -XX:CompileThreshold=1000 -XX:ReservedCodeCacheSize=128m -Djava.io.tmpdir=gsnap -Xmx20g -Xms5g -jar GenomeAnalysisTK.jar -et NO_ET -K /GenomeAnalysisTK/3.1-1/Hossain.Asif_mayo.edu.key -T SplitNCigarReads -R /human/ncbi/hg19/allchr.fa -I Aligned_sort_rg_md_nochrstar_bamtools_isPairedFiltered.bam -o Aligned_sort_rg_md_splitNC.bam -rf ReassignOneMappingQuality -RMQF 255 -RMQT 60 -U ALLOW_N_CIGAR_READS

    Error

    ERROR ------------------------------------------------------------------------------------------
    ERROR A USER ERROR has occurred (version nightly-2015-05-02-g6d40932):
    ERROR
    ERROR This means that one or more arguments or inputs in your command are incorrect.
    ERROR The error message below tells you what is the problem.
    ERROR
    ERROR If the problem is an invalid argument, please check the online documentation guide
    ERROR (or rerun your command with --help) to view allowable command-line arguments for this tool.
    ERROR
    ERROR Visit our website and forum for extensive documentation and answers to
    ERROR commonly asked questions http://www.broadinstitute.org/gatk
    ERROR
    ERROR Please do NOT post this error to the GATK forum unless you have really tried to fix it yourself.
    ERROR
    ERROR MESSAGE: Badly formed genome loc: Parameters to GenomeLocParser are incorrect:The stop position 42376807 is less than start 42376808 in contig chr10
    ERROR ------------------------------------------------------------------------------------------

    INFO 13:39:58,091 ProgressMeter - chr10:42382840 2.22022517E8 100.8 m 27.0 s 55.5% 3.0 h 80.7 m

    Can you please give your input how to slove this error

    Regards
    Naresh

  • SheilaSheila Broad InstituteMember, Broadie admin

    @naresh4836
    Hi Naresh,

    Are you running on the same bam file from the previous comments where you already validated and fixed the mate information? You can try adding -fixNDN to your command above.

    -Sheila

  • naresh4836naresh4836 ROchester MNMember

    Hello Sheila,

    This is a different sample. I tried with -fixNDN still the error persists

    Command:
    /usr/java/jdk1.7.0_03/bin/java -XX:CompileThreshold=1000 -XX:ReservedCodeCacheSize=128m -Djava.io.tmpdir=gsnap -Xmx20g -Xms5g -jar GenomeAnalysisTK.jar -et NO_ET -K /GenomeAnalysisTK/3.1-1/Hossain.Asif_mayo.edu.key -T SplitNCigarReads -R /human/ncbi/hg19/allchr.fa -I Aligned_sort_rg_md_nochrstar_bamtools_isPairedFiltered.bam -o Aligned_sort_rg_md_splitNC.bam -rf ReassignOneMappingQuality -RMQF 255 -RMQT 60 -U ALLOW_N_CIGAR_READS -fixNDN

    Error

    ERROR ------------------------------------------------------------------------------------------
    ERROR A USER ERROR has occurred (version nightly-2015-05-02-g6d40932):
    ERROR
    ERROR This means that one or more arguments or inputs in your command are incorrect.
    ERROR The error message below tells you what is the problem.
    ERROR
    ERROR If the problem is an invalid argument, please check the online documentation guide
    ERROR (or rerun your command with --help) to view allowable command-line arguments for this tool.
    ERROR
    ERROR Visit our website and forum for extensive documentation and answers to
    ERROR commonly asked questions http://www.broadinstitute.org/gatk
    ERROR
    ERROR Please do NOT post this error to the GATK forum unless you have really tried to fix it yourself.
    ERROR
    ERROR MESSAGE: Badly formed genome loc: Parameters to GenomeLocParser are incorrect:The stop position 42376807 is less than start 42376808 in contig chr10
    ERROR ------------------------------------------------------------------------------------------

    INFO 14:27:39,188 ProgressMeter - chr10:42382747 2.67018824E8 2.3 h 31.0 s 55.5% 4.2 h 111.6 m

    Regards
    Naresh

  • SheilaSheila Broad InstituteMember, Broadie admin

    @naresh4836
    Hi Naresh,

    And, Picard's Validate Sam File gives no errors?

    -Sheila

  • naresh4836naresh4836 ROchester MNMember

    Hello Sheila,

    I got the following errors when i ran Picard ValidateSAMFile

    Mate alignment does not match alignment start of mate
    Mate negative strand flag does not match read negative strand flag of mate
    Mate reference index (MRNM) does not match reference index of mate

    So i ran Picard fixmate step then it gave an error

    To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp
    Exception in thread "main" htsjdk.samtools.SAMException: Found two records that are paired, not supplementary, and second of the pair
    at htsjdk.samtools.SamPairUtil$SetMateInfoIterator.advance(SamPairUtil.java:420)
    at htsjdk.samtools.SamPairUtil$SetMateInfoIterator.next(SamPairUtil.java:454)
    at htsjdk.samtools.SamPairUtil$SetMateInfoIterator.next(SamPairUtil.java:360)
    at picard.sam.FixMateInformation.doWork(FixMateInformation.java:195)
    at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:187)
    at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:95)
    at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:105)

    Regards
    Naresh

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin

    @naresh4836 This error suggests that there are problems in your data. You should consider using a different aligner because the one you are using is producing malformed records.

  • naresh4836naresh4836 ROchester MNMember

    I tried Picard ValidateSAMFile on an another sample and I got the same error.But i tried GATK SPLITNCIGAR on this sample and it just ran fine with out giving any error.

    Regards
    Naresh

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin

    @naresh4836 It's possible to run another tool on malformed data without getting an error if that particular tool doesn't access the part of the data that is malformed. But this could mask issues that will pop up later in your analysis. Generally speaking it's safer to deal with problems up front. If your aligner is causing malformed data, you should either eliminate the malformed data, fix it or switch to a different aligner.

  • humburghumburg SydneyMember

    I have the same problem:

    ##### ERROR ------------------------------------------------------------------------------------------
    ##### ERROR A USER ERROR has occurred (version 3.7-0-gcfedb67):
    ##### ERROR
    ##### ERROR This means that one or more arguments or inputs in your command are incorrect.
    ##### ERROR The error message below tells you what is the problem.
    ##### ERROR
    ##### ERROR If the problem is an invalid argument, please check the online documentation guide
    ##### ERROR (or rerun your command with --help) to view allowable command-line arguments for this tool.
    ##### ERROR
    ##### ERROR Visit our website and forum for extensive documentation and answers to
    ##### ERROR commonly asked questions https://software.broadinstitute.org/gatk
    ##### ERROR
    ##### ERROR Please do NOT post this error to the GATK forum unless you have really tried to fix it yourself.
    ##### ERROR
    ##### ERROR MESSAGE: Badly formed genome location: Parameters to GenomeLocParser are incorrect:The stop position 27867039 is less than start 27867040 in contig chr6
    ##### ERROR ------------------------------------------------------------------------------------------
    

    Running picard ValidateSamFile on the BAM in question reports no errors. I've tried to identify the offending read but have only been able to reproduce the problem with a BAM file that contains at least two reads. Each of these on its own is fine. BAM files containing one of these (but not both) reads as well as some other reads also appear to be fine but if both reads are present I get the above error. In case it helps, the CIGAR strings for these reads are 194M51N236M and 14S50M3I23M1D5M1D6M2I14M3D27M5N5I7M11N62I1M6D18M54N186M28S.

  • SheilaSheila Broad InstituteMember, Broadie admin

    @humburg
    Hi,

    Can you post the exact command you ran? Please also post the entire stack trace you get before the error message.

    Thanks,
    Sheila

  • humburghumburg SydneyMember

    @Sheila

    Here is the complete output:

    INFO  09:15:23,010 HelpFormatter - ----------------------------------------------------------------------------------
    INFO  09:15:23,015 HelpFormatter - The Genome Analysis Toolkit (GATK) v3.7-0-gcfedb67, Compiled 2016/12/12 11:21:18
    INFO  09:15:23,015 HelpFormatter - Copyright (c) 2010-2016 The Broad Institute
    INFO  09:15:23,015 HelpFormatter - For support and documentation go to https://software.broadinstitute.org/gatk
    INFO  09:15:23,015 HelpFormatter - [Thu May 18 09:15:22 AEST 2017] Executing on Linux 2.6.32-642.3.1.el6.x86_64 amd64
    INFO  09:15:23,015 HelpFormatter - Java HotSpot(TM) 64-Bit Server VM 1.8.0_121-b13
    INFO  09:15:23,020 HelpFormatter - Program Args: -T SplitNCigarReads -R <path to reference>/GRCh38_noALT_decoy.fa -I bam/test.bam -o bam/test.split.bam -U ALLOW_N_CIGAR_READS -fixNDN --allow_potentially_misencoded_quality_scores
    INFO  09:15:23,033 HelpFormatter - Executing as <user>@<server> on Linux 2.6.32-642.3.1.el6.x86_64 amd64; Java HotSpot(TM) 64-Bit Server VM 1.8.0_121-b13.
    INFO  09:15:23,033 HelpFormatter - Date/Time: 2017/05/18 09:15:23
    INFO  09:15:23,033 HelpFormatter - ----------------------------------------------------------------------------------
    INFO  09:15:23,033 HelpFormatter - ----------------------------------------------------------------------------------
    INFO  09:15:23,200 GenomeAnalysisEngine - Strictness is SILENT
    INFO  09:15:24,261 GenomeAnalysisEngine - Downsampling Settings: No downsampling
    INFO  09:15:24,268 SAMDataSource$SAMReaders - Initializing SAMRecords in serial
    INFO  09:15:24,389 SAMDataSource$SAMReaders - Done initializing BAM readers: total time 0.12
    INFO  09:15:25,550 GenomeAnalysisEngine - Preparing for traversal over 1 BAM files
    INFO  09:15:25,554 GenomeAnalysisEngine - Done preparing for traversal
    INFO  09:15:25,555 ProgressMeter - [INITIALIZATION COMPLETE; STARTING PROCESSING]
    INFO  09:15:25,555 ProgressMeter -                 | processed |    time |    per 1M |           |   total | remaining
    INFO  09:15:25,555 ProgressMeter -        Location |     reads | elapsed |     reads | completed | runtime |   runtime
    INFO  09:15:25,630 ReadShardBalancer$1 - Loading BAM index data
    INFO  09:15:25,632 ReadShardBalancer$1 - Done loading BAM index data
    ##### ERROR ------------------------------------------------------------------------------------------
    ##### ERROR A USER ERROR has occurred (version 3.7-0-gcfedb67):
    ##### ERROR
    ##### ERROR This means that one or more arguments or inputs in your command are incorrect.
    ##### ERROR The error message below tells you what is the problem.
    ##### ERROR
    ##### ERROR If the problem is an invalid argument, please check the online documentation guide
    ##### ERROR (or rerun your command with --help) to view allowable command-line arguments for this tool.
    ##### ERROR
    ##### ERROR Visit our website and forum for extensive documentation and answers to
    ##### ERROR commonly asked questions https://software.broadinstitute.org/gatk
    ##### ERROR
    ##### ERROR Please do NOT post this error to the GATK forum unless you have really tried to fix it yourself.
    ##### ERROR
    ##### ERROR MESSAGE: Badly formed genome location: Parameters to GenomeLocParser are incorrect:The stop position 27867039 is less than start 27867040 in contig chr6
    ##### ERROR ------------------------------------------------------------------------------------------
    
  • SheilaSheila Broad InstituteMember, Broadie admin

    @humburg
    Hi,

    Can you submit a bug report so I can take a look? Instructions are here.

    Thanks,
    Sheila

  • humburghumburg SydneyMember
    edited May 2017

    @Sheila
    I uploaded an archive with the relevant files (bugreport_humburg_20170522.tar.gz) to the ftp server.

    Another thing I noticed while looking at this is that for the reads that are split successfully, the MD tag is not adjusted accordingly. This leaves the BAM records in an inconsistent state.

    Issue · Github
    by Sheila

    Issue Number
    2097
    State
    open
    Last Updated
    Assignee
    Array
    Milestone
    Array
  • SheilaSheila Broad InstituteMember, Broadie admin

    @humburg
    Hi,

    Thanks. I will have a look soon.

    -Sheila

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin

    Hi @humburg, we are aware that the SplitNCigarReads tool as implemented in GATK 3.x is fairly buggy. We have reimplemented it in the soon-to-be released GATK4 and it's much better, so we won't make any improvements or fixes to the GATK 3.x version anymore. You're welcome to try out the GATK4 version; we'll have documentation coming out on it within the next couple of weeks. In the meantime, our apologies for the inconvenience.

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