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MuTect error

uthrabalajiuthrabalaji DALLASMember

Hello GATK team,

I get the following error when I run MuTect (version 1.1.7).

ERROR ------------------------------------------------------------------------------------------
ERROR A USER ERROR has occurred (version 3.1-0-g72492bb):
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: SAM/BAM file SAMFileReader{new_alignment/NB13_new.rgID.rmdup.sorted.bam} appears to be using the wrong encoding for quality scores: we encountered an extremely high quality score of 62; please see the GATK --help documentation for options related to this error
ERROR ------------------------------------------------------------------------------------------

I understand from the other posts that -allowPotentiallyMisencodedQuals can be used to overcome this problem. But I am not sure how to use it as an option with MuTect.

This is the GATK pipeline I have used.
1. BWA alignment
2. SAM to BAM (samtools)
3. Sort the BAM (samtools)
4. Remove duplicates (picard: MarkDuplicates)
5. Add read groups (picard: AddOrReplaceReadGroups)

Am I missing anything here?

I really appreciate any inputs.

Thank you,
Uthra

Answers

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin

    Hi Uthra,

    The right way to use -allowPotentiallyMisencodedQuals is to not use it at all ;)

    Seriously, it's almost always a bad idea. That error occurs for a good reason.

    So, can you post the MuTect command you ran? If it has -fixMisencodedQuals in it, try removing it and tell us what happens then.

    Note that we do recommend running Indel Realignment and BQSR from GATK on your data before running MuTect. You run them separately on each file (tumor and normal) then you run Indel Realignment again on the T/N pair together, with the -nWayOut argument.

  • uthrabalajiuthrabalaji DALLASMember

    Hi @Geraldine_VdAuwera ,

    This is the mutect command I ran:

    /usr/lib/jvm/jre-1.7.0-openjdk.x86_64/bin/java -Xmx8g -jar /home/uthrabalaji/software/mutect-1.1.7.jar --analysis_type MuTect --reference_sequence /media/sf_F_DRIVE/Homo_sapiens/ReferenceFiles/Homo_sapiens_assembly19.fasta --dbsnp /ReferenceFiles/dbsnp_132_b37.leftAligned.vcf --cosmic /ReferenceFiles/b37_cosmic_v54_120711.vcf --intervals /ReferenceFiles/exome_targeted_regions_b37.bed --enable_extended_output --input_file:normal Normal.bam --input_file:tumor Tumor.bam --out MuTect_call_stats.txt --coverage_file MuTect_coverage.wig > mutect.log 2>&1

    Thanks,
    Uthra

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin

    Ah ok, then try with the -fixMisencodedQuals argument added please.

  • uthrabalajiuthrabalaji DALLASMember

    Hi @Geraldine_VdAuwera ,

    I tried to run MuTect with the -fixMisencodedQuals option and this is what I got.

    ERROR ------------------------------------------------------------------------------------------
    ERROR A USER ERROR has occurred (version 3.1-0-g72492bb):
    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: Bad input: while fixing mis-encoded base qualities we encountered a read that was correctly encoded; we cannot handle such a mixture of reads so unfortunately the BAM must be fixed with some other tool
    ERROR ------------------------------------------------------------------------------------------

    I would really appreciate any inputs on how to proceed with fixing the BAM.

    Thank you,
    Uthra

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin

    Ah, that's unfortunate. Do you know if the files contain information from multiple sequencing runs?

  • uthrabalajiuthrabalaji DALLASMember

    Hi @Geraldine_VdAuwera ,

    No, this bam is from a single run.

    Thanks,
    Uthra

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