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Mutect2 Calls for non-tumor pooled samples - no variants pass filters

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13 comments

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    Gökalp Çelik

    Hi Ren Hamm

    Since you are using Mutect2 to call variants with allelic fractions in your pooled samples there is no need for adjusting ploidy for your samples. You might want to enable high sensitivity settings for Mutect2 to call very low fraction calls. We enable these settings under --mitochondria-mode so you can enable that mode to call and filter variant calls for your case. 

    Hard filtering with Mutect2 calls is not a recommended step as Mutect2's dynamics are different from germline. 

    If you can try with the mitochondria mode you may be able to get a better result for your variants. 

    I hope this helps. 

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    Ren Hamm

    I was able to successfully run everything in -mitochondia-mode, but FilterMutectCalls is still failing with the same error:

    java.lang.IllegalArgumentException: alpha must be greater than 0 but got NaN

     

    How should I approach solving this?

     

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    Gökalp Çelik

    Hi again.

    Can you try without additional mapping and base quality filters in FilterMutectCalls step?

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    Ren Hamm

    I ran everything again after removing all the additional filter parameters, and I am still recieving the same error.

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    Gökalp Çelik

    Hi Ren Hamm

    Can you tell us about how these samples were processed before Mutect2? Are these of DNA or RNA origin? What aligner did you use to map reads?

    The answer could be lying somewere along those lines.

     

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    Ren Hamm

    Of course! This DNA data is from WGS 150bp paired-end Illumina reads, which were aligned using BWA-mem. 

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    Gökalp Çelik

    Hi again.

    Did you happen to build GATK from source? It may be possible that you might have picked a source that is not fully tested. Can you try downloading the latest build and see if the problem still exitsts?

    Regards. 

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    Ren Hamm

    Redownloading the newest version seemed to fix the original issue, and I can successfully make Mutect2 mitocondrial mode vcfs that then can be filtered. However, I need to get allele frequency data for all the biallelic sites, but tools like vcftools code these files as polyploid and winnowing then down to only biallelic sites results in every variant being a "<NON_REF>" variant. How do I obtain accurate estimates of allele frequency from this data for biallelic sites?

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    Gökalp Çelik

    Hi Ren Hamm

    I think you are using the GVCF mode for Mutect2 which is still in beta stage and probably not even useful for you for this purpose. The GVCF output needs to be genotyped before you can actually pass it to any variant filtration workflow. 

    For that you can use GenotypeGVCFs tool with the beta option named

    --input-is-somatic true

    to make sure that tool considers this file as a Mutect2 output. Then you can use the genotyped file and check for INFO/AF or FORMAT/AF fields to gather allele fractions for your variants of interest. For the sake of simplicity you may also need to use 

    bcftools norm

    to split multiallelics to biallelics.

    Regards. 

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    Ren Hamm

    Thank you! I'm getting a strange error now, though.

    I am running this command:

    /global/scratch/users/laurenhamm/thesis/aim2/vcfs/gatk_reinstall/gatk-4.6.1.0/gatk --java-options "-Xmx64G" GenotypeGVCFs \
        -R /global/scratch/users/laurenhamm/thesis/ref-genomes/v3_mt-cs/Mgut_v3_mt-cs.fa \
        -V /global/scratch/users/laurenhamm/thesis/aim2/vcfs/GATK/pooled_vcfs/Mutect2/mitoMode_reinstall/gvcfs/BEL_2014_pool_reinstall.M2.g.vcf.gz \
        -O /global/scratch/users/laurenhamm/thesis/aim2/vcfs/GATK/pooled_vcfs/Mutect2/mitoMode_reinstall/vcfs/BEL_2014_pool_reinstall.vcf.gz\
        --input-is-somatic true \

     

    And this is the error file I receive:

    Using GATK jar /global/scratch/users/laurenhamm/thesis/aim2/vcfs/gatk_reinstall/gatk-4.6.1.0/gatk-package-4.6.1.0-local.jar
    Running:
        java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -Xmx64G -jar /global/scratch/users/laurenhamm/thesis/aim2/vcfs/gatk_reinstall/gatk-4.6.1.0/gatk-package-4.6.1.0-local.jar GenotypeGVCFs -R /global/scratch/users/laurenhamm/thesis/ref-genomes/v3_mt-cs/Mgut_v3_mt-cs.fa -V /global/scratch/users/laurenhamm/thesis/aim2/vcfs/GATK/pooled_vcfs/Mutect2/mitoMode_reinstall/gvcfs/BEL_2014_pool_reinstall.M2.g.vcf.gz -O /global/scratch/users/laurenhamm/thesis/aim2/vcfs/GATK/pooled_vcfs/Mutect2/mitoMode_reinstall/vcfs/BEL_2014_pool_reinstall.vcf.gz --input-is-somatic true
    10:56:30.831 INFO  NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/global/scratch/users/laurenhamm/thesis/aim2/vcfs/gatk_reinstall/gatk-4.6.1.0/gatk-package-4.6.1.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
    SLF4J(W): Class path contains multiple SLF4J providers.
    SLF4J(W): Found provider [org.apache.logging.slf4j.SLF4JServiceProvider@336f49a1]
    SLF4J(W): Found provider [ch.qos.logback.classic.spi.LogbackServiceProvider@2c8b8de0]
    SLF4J(W): See https://www.slf4j.org/codes.html#multiple_bindings for an explanation.
    SLF4J(I): Actual provider is of type [org.apache.logging.slf4j.SLF4JServiceProvider@336f49a1]
    10:56:30.932 INFO  GenotypeGVCFs - ------------------------------------------------------------
    10:56:30.935 INFO  GenotypeGVCFs - The Genome Analysis Toolkit (GATK) v4.6.1.0
    10:56:30.935 INFO  GenotypeGVCFs - For support and documentation go to https://software.broadinstitute.org/gatk/
    10:56:30.935 INFO  GenotypeGVCFs - Executing as laurenhamm@n0001.savio2 on Linux v4.18.0-553.5.1.el8_10.x86_64 amd64
    10:56:30.935 INFO  GenotypeGVCFs - Java runtime: Java HotSpot(TM) 64-Bit Server VM v22.0.1+8-16
    10:56:30.935 INFO  GenotypeGVCFs - Start Date/Time: February 7, 2025, 10:56:30 AM PST
    10:56:30.935 INFO  GenotypeGVCFs - ------------------------------------------------------------
    10:56:30.936 INFO  GenotypeGVCFs - ------------------------------------------------------------
    10:56:30.936 INFO  GenotypeGVCFs - HTSJDK Version: 4.1.3
    10:56:30.936 INFO  GenotypeGVCFs - Picard Version: 3.3.0
    10:56:30.936 INFO  GenotypeGVCFs - Built for Spark Version: 3.5.0
    10:56:30.939 INFO  GenotypeGVCFs - HTSJDK Defaults.COMPRESSION_LEVEL : 2
    10:56:30.939 INFO  GenotypeGVCFs - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
    10:56:30.939 INFO  GenotypeGVCFs - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
    10:56:30.939 INFO  GenotypeGVCFs - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
    10:56:30.939 INFO  GenotypeGVCFs - Deflater: IntelDeflater
    10:56:30.940 INFO  GenotypeGVCFs - Inflater: IntelInflater
    10:56:30.940 INFO  GenotypeGVCFs - GCS max retries/reopens: 20
    10:56:30.940 INFO  GenotypeGVCFs - Requester pays: disabled
    10:56:30.940 INFO  GenotypeGVCFs - Initializing engine
    10:56:31.115 INFO  FeatureManager - Using codec VCFCodec to read file file:///global/scratch/users/laurenhamm/thesis/aim2/vcfs/GATK/pooled_vcfs/Mutect2/mitoMode_reinstall/gvcfs/BEL_2014_pool_reinstall.M2.g.vcf.gz
    10:56:31.404 INFO  GenotypeGVCFs - Done initializing engine
    10:56:31.404 WARN  GenotypeGVCFs - Note that the Mutect2 reference confidence mode is in BETA -- the likelihoods model and output format are subject to change in subsequent versions.
    10:56:31.494 INFO  ProgressMeter - Starting traversal
    10:56:31.495 INFO  ProgressMeter -        Current Locus  Elapsed Minutes    Variants Processed  Variants/Minute

    10:56:31.664 WARN  ReferenceConfidenceVariantContextMerger - Detected invalid annotations: When trying to merge variant contexts at location Chr_01:16712 the annotation AS_SB_TABLE=[10, 9|2, 0|0, 0] was not a numerical value and was ignored
    10:56:31.664 WARN  ReferenceConfidenceVariantContextMerger - Reducible annotation 'AS_SB_TABLE' detected, add -G StandardAnnotation -G AS_StandardAnnotation to the command to annotate in the final VC with this annotation.
    10:56:31.680 INFO  GenotypeGVCFs - Shutting down engine
    [February 7, 2025, 10:56:31 AM PST] org.broadinstitute.hellbender.tools.walkers.GenotypeGVCFs done. Elapsed time: 0.02 minutes.
    Runtime.totalMemory()=335544320
    org.broadinstitute.hellbender.exceptions.GATKException: Exception thrown at Chr_01:16712 [VC Unknown @ Chr_01:16712-16751 Q. of type=MIXED alleles=[AATAAAATTTAAATATGTTAATGAAATCATGTATTGACCC*, <NON_REF>, A] attr={AS_SB_TABLE=[10, 9|2, 0|0, 0], DP=23, ECNT=10, ECNTH=3, MBQ=[40, 40, 0], MFRL=[258, 366, 0], MMQ=[60, 60, 60], MPOS=[33, 50], OCM=0, POPAF=[2.40, 2.40], TLOD=[5.70, -1.070e+00]} GT=[[BEL_2014_pool AATAAAATTTAAATATGTTAATGAAATCATGTATTGACCC*|A|<NON_REF> DP 21 AD 19,2,0 {AF=0.135,0.041, F1R2=7,2,0, F2R1=9,0,0, FAD=19,3,0, PGT=0|1, PID=16712_AATAAAATTTAAATATGTTAATGAAATCATGTATTGACCC_A, PS=16712, SB=10,9,2,0}]] filters=
            at org.broadinstitute.hellbender.engine.VariantLocusWalker.lambda$traverse$0(VariantLocusWalker.java:144)
            at java.base/java.util.stream.ForEachOps$ForEachOp$OfRef.accept(ForEachOps.java:184)
            at java.base/java.util.stream.ReferencePipeline$3$1.accept(ReferencePipeline.java:212)
            at java.base/java.util.stream.ReferencePipeline$2$1.accept(ReferencePipeline.java:194)
            at java.base/java.util.stream.ReferencePipeline$3$1.accept(ReferencePipeline.java:212)
            at java.base/java.util.Iterator.forEachRemaining(Iterator.java:133)
            at java.base/java.util.Spliterators$IteratorSpliterator.forEachRemaining(Spliterators.java:1939)
            at java.base/java.util.stream.AbstractPipeline.copyInto(AbstractPipeline.java:556)
            at java.base/java.util.stream.AbstractPipeline.wrapAndCopyInto(AbstractPipeline.java:546)
            at java.base/java.util.stream.ForEachOps$ForEachOp.evaluateSequential(ForEachOps.java:151)
            at java.base/java.util.stream.ForEachOps$ForEachOp$OfRef.evaluateSequential(ForEachOps.java:174)
            at java.base/java.util.stream.AbstractPipeline.evaluate(AbstractPipeline.java:265)
            at java.base/java.util.stream.ReferencePipeline.forEachOrdered(ReferencePipeline.java:616)
            at org.broadinstitute.hellbender.engine.VariantLocusWalker.traverse(VariantLocusWalker.java:132)
            at org.broadinstitute.hellbender.engine.GATKTool.doWork(GATKTool.java:1119)
            at org.broadinstitute.hellbender.cmdline.CommandLineProgram.runTool(CommandLineProgram.java:150)
            at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMainPostParseArgs(CommandLineProgram.java:203)
            at org.broadinstitute.hellbender.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:222)
            at org.broadinstitute.hellbender.Main.runCommandLineProgram(Main.java:166)
            at org.broadinstitute.hellbender.Main.mainEntry(Main.java:209)
            at org.broadinstitute.hellbender.Main.main(Main.java:306)
    Caused by: java.lang.IllegalStateException: Key MBQ found in VariantContext field FORMAT at Chr_01:16712 but this key isn't defined in the VCFHeader.  We require all VCFs to have complete VCF headers by default.
            at htsjdk.variant.vcf.VCFEncoder.fieldIsMissingFromHeaderError(VCFEncoder.java:215)
            at htsjdk.variant.vcf.VCFEncoder.write(VCFEncoder.java:168)
            at htsjdk.variant.variantcontext.writer.VCFWriter.add(VCFWriter.java:250)
            at org.broadinstitute.hellbender.tools.walkers.GenotypeGVCFs.apply(GenotypeGVCFs.java:328)
            at org.broadinstitute.hellbender.engine.VariantLocusWalker.lambda$traverse$0(VariantLocusWalker.java:137)
            ... 20 more

     

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    Gökalp Çelik

    Can you post the header section of your input GVCF?

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    Ren Hamm

    ##fileformat=VCFv4.2
    ##ALT=<ID=NON_REF,Description="Represents any possible alternative allele not already represented at this location by REF and ALT">
    ##FORMAT=<ID=AD,Number=R,Type=Integer,Description="Allelic depths for the ref and alt alleles in the order listed">
    ##FORMAT=<ID=AF,Number=A,Type=Float,Description="Allele fractions of alternate alleles in the tumor">
    ##FORMAT=<ID=DP,Number=1,Type=Integer,Description="Approximate read depth (reads with MQ=255 or with bad mates are filtered)">
    ##FORMAT=<ID=F1R2,Number=R,Type=Integer,Description="Count of reads in F1R2 pair orientation supporting each allele">
    ##FORMAT=<ID=F2R1,Number=R,Type=Integer,Description="Count of reads in F2R1 pair orientation supporting each allele">
    ##FORMAT=<ID=FAD,Number=R,Type=Integer,Description="Count of fragments supporting each allele.">
    ##FORMAT=<ID=GQ,Number=1,Type=Integer,Description="Genotype Quality">
    ##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">
    ##FORMAT=<ID=MIN_DP,Number=1,Type=Integer,Description="Minimum DP observed within the GVCF block">
    ##FORMAT=<ID=PGT,Number=1,Type=String,Description="Physical phasing haplotype information, describing how the alternate alleles are phased in relation to one another; will always be heterozygous and is not intended to describe called alleles">
    ##FORMAT=<ID=PID,Number=1,Type=String,Description="Physical phasing ID information, where each unique ID within a given sample (but not across samples) connects records within a phasing group">
    ##FORMAT=<ID=PL,Number=G,Type=Integer,Description="Normalized, Phred-scaled likelihoods for genotypes as defined in the VCF specification">
    ##FORMAT=<ID=PS,Number=1,Type=Integer,Description="Phasing set (typically the position of the first variant in the set)">
    ##FORMAT=<ID=SB,Number=4,Type=Integer,Description="Per-sample component statistics which comprise the Fisher's Exact Test to detect strand bias.">
    ##FORMAT=<ID=TLOD,Number=A,Type=Float,Description="Log 10 likelihood ratio score of variant existing versus not existing">
    ##GATKCommandLine=<ID=Mutect2,CommandLine="Mutect2 --mitochondria-mode true --emit-ref-confidence GVCF --output /global/scratch/users/laurenhamm/thesis/aim2/vcfs/GATK/pooled_vcfs/Mutect2/mitoMode_reinstall/BEL_2014_pool_reinstall.M2.g.vcf.gz --input /global/scratch/users/laurenhamm/thesis/aim2/paleomix/bamOuts/BEL_2014_pool.GEAsamples.bam --reference /global/scratch/users/laurenhamm/thesis/ref-genomes/v3_mt-cs/Mgut_v3_mt-cs.fa --f1r2-median-mq 50 --f1r2-min-bq 20 --f1r2-max-depth 200 --flow-likelihood-parallel-threads 0 --flow-likelihood-optimized-comp false --trim-to-haplotype true --exact-matching false --flow-use-t0-tag false --flow-remove-non-single-base-pair-indels false --flow-remove-one-zero-probs false --flow-quantization-bins 121 --flow-fill-empty-bins-value 0.001 --flow-symmetric-indel-probs false --flow-report-insertion-or-deletion false --flow-disallow-probs-larger-than-call false --flow-lump-probs false --flow-retain-max-n-probs-base-format false --flow-probability-scaling-factor 10 --flow-order-cycle-length 4 --keep-boundary-flows false --genotype-pon-sites false --genotype-germline-sites false --af-of-alleles-not-in-resource -1.0 --mutect3-training-mode false --mutect3-ref-downsample 10 --mutect3-alt-downsample 20 --mutect3-non-artifact-ratio 1 --mutect3-dataset-mode ILLUMINA --tumor-lod-to-emit 3.0 --initial-tumor-lod 2.0 --pcr-snv-qual 40 --pcr-indel-qual 40 --base-qual-correction-factor 5 --max-population-af 0.01 --downsampling-stride 1 --callable-depth 10 --max-suspicious-reads-per-alignment-start 0 --normal-lod 2.2 --ignore-itr-artifacts false --gvcf-lod-band -2.5 --gvcf-lod-band -2.0 --gvcf-lod-band -1.5 --gvcf-lod-band -1.0 --gvcf-lod-band -0.5 --gvcf-lod-band 0.0 --gvcf-lod-band 0.5 --gvcf-lod-band 1.0 --minimum-allele-fraction 0.0 --independent-mates false --flow-mode NONE --disable-adaptive-pruning false --kmer-size 10 --kmer-size 25 --dont-increase-kmer-sizes-for-cycles false --allow-non-unique-kmers-in-ref false --num-pruning-samples 1 --min-dangling-branch-length 4 --recover-all-dangling-branches false --max-num-haplotypes-in-population 128 --min-pruning 2 --adaptive-pruning-initial-error-rate 0.001 --pruning-lod-threshold 2.302585092994046 --pruning-seeding-lod-threshold 9.210340371976184 --max-unpruned-variants 100 --linked-de-bruijn-graph false --disable-artificial-haplotype-recovery false --enable-legacy-graph-cycle-detection false --debug-assembly false --debug-graph-transformations false --capture-assembly-failure-bam false --num-matching-bases-in-dangling-end-to-recover -1 --error-correction-log-odds -Infinity --error-correct-reads false --kmer-length-for-read-error-correction 25 --min-observations-for-kmer-to-be-solid 20 --likelihood-calculation-engine PairHMM --base-quality-score-threshold 18 --dragstr-het-hom-ratio 2 --dont-use-dragstr-pair-hmm-scores false --pair-hmm-gap-continuation-penalty 10 --expected-mismatch-rate-for-read-disqualification 0.02 --pair-hmm-implementation FASTEST_AVAILABLE --pcr-indel-model CONSERVATIVE --phred-scaled-global-read-mismapping-rate 45 --disable-symmetric-hmm-normalizing false --disable-cap-base-qualities-to-map-quality false --enable-dynamic-read-disqualification-for-genotyping false --dynamic-read-disqualification-threshold 1.0 --native-pair-hmm-threads 4 --native-pair-hmm-use-double-precision false --flow-hmm-engine-min-indel-adjust 6 --flow-hmm-engine-flat-insertion-penatly 45 --flow-hmm-engine-flat-deletion-penatly 45 --pileup-detection false --use-pdhmm false --use-pdhmm-overlap-optimization false --make-determined-haps-from-pd-code false --print-pileupcalling-status false --fallback-gga-if-pdhmm-fails true --pileup-detection-enable-indel-pileup-calling false --pileup-detection-active-region-phred-threshold 0.0 --num-artificial-haplotypes-to-add-per-allele 5 --artifical-haplotype-filtering-kmer-size 10 --pileup-detection-snp-alt-threshold 0.1 --pileup-detection-indel-alt-threshold 0.1 --pileup-detection-absolute-alt-depth 0.0 --pileup-detection-snp-adjacent-to-assembled-indel-range 5 --pileup-detection-snp-basequality-filter 12 --pileup-detection-bad-read-tolerance 0.0 --pileup-detection-proper-pair-read-badness true --pileup-detection-edit-distance-read-badness-threshold 0.08 --pileup-detection-chimeric-read-badness true --pileup-detection-template-mean-badness-threshold 0.0 --pileup-detection-template-std-badness-threshold 0.0 --pileup-detection-filter-assembly-alt-bad-read-tolerance 0.0 --pileup-detection-edit-distance-read-badness-for-assembly-filtering-threshold 0.12 --bam-writer-type CALLED_HAPLOTYPES --dont-use-soft-clipped-bases false --override-fragment-softclip-check false --min-base-quality-score 10 --smith-waterman FASTEST_AVAILABLE --max-mnp-distance 1 --force-call-filtered-alleles false --reference-model-deletion-quality 30 --soft-clip-low-quality-ends false --allele-informative-reads-overlap-margin 2 --smith-waterman-dangling-end-match-value 25 --smith-waterman-dangling-end-mismatch-penalty -50 --smith-waterman-dangling-end-gap-open-penalty -110 --smith-waterman-dangling-end-gap-extend-penalty -6 --smith-waterman-haplotype-to-reference-match-value 200 --smith-waterman-haplotype-to-reference-mismatch-penalty -150 --smith-waterman-haplotype-to-reference-gap-open-penalty -260 --smith-waterman-haplotype-to-reference-gap-extend-penalty -11 --smith-waterman-read-to-haplotype-match-value 10 --smith-waterman-read-to-haplotype-mismatch-penalty -15 --smith-waterman-read-to-haplotype-gap-open-penalty -30 --smith-waterman-read-to-haplotype-gap-extend-penalty -5 --flow-assembly-collapse-hmer-size 0 --flow-assembly-collapse-partial-mode false --flow-filter-alleles false --flow-filter-alleles-qual-threshold 30.0 --flow-filter-alleles-sor-threshold 3.0 --flow-filter-lone-alleles false --flow-filter-alleles-debug-graphs false --min-assembly-region-size 50 --max-assembly-region-size 300 --active-probability-threshold 0.002 --max-prob-propagation-distance 50 --force-active false --assembly-region-padding 100 --padding-around-indels 75 --padding-around-snps 20 --padding-around-strs 75 --max-extension-into-assembly-region-padding-legacy 25 --max-reads-per-alignment-start 50 --enable-legacy-assembly-region-trimming false --interval-set-rule UNION --interval-padding 0 --interval-exclusion-padding 0 --interval-merging-rule ALL --read-validation-stringency SILENT --seconds-between-progress-updates 10.0 --disable-sequence-dictionary-validation false --create-output-bam-index true --create-output-bam-md5 false --create-output-variant-index true --create-output-variant-md5 false --max-variants-per-shard 0 --lenient false --add-output-sam-program-record true --add-output-vcf-command-line true --cloud-prefetch-buffer 40 --cloud-index-prefetch-buffer -1 --disable-bam-index-caching false --sites-only-vcf-output false --help false --version false --showHidden false --verbosity INFO --QUIET false --use-jdk-deflater false --use-jdk-inflater false --gcs-max-retries 20 --gcs-project-for-requester-pays  --disable-tool-default-read-filters false --max-read-length 2147483647 --min-read-length 30 --minimum-mapping-quality 20 --disable-tool-default-annotations false --enable-all-annotations false",Version="4.6.1.0",Date="January 18, 2025, 1:23:52?PM PST">
    ##GVCFBlock-10--5=minGQ=-10(inclusive),maxGQ=-5(exclusive)
    ##GVCFBlock-15--10=minGQ=-15(inclusive),maxGQ=-10(exclusive)
    ##GVCFBlock-20--15=minGQ=-20(inclusive),maxGQ=-15(exclusive)
    ##GVCFBlock-2147483648--25=minGQ=-2147483648(inclusive),maxGQ=-25(exclusive)
    ##GVCFBlock-25--20=minGQ=-25(inclusive),maxGQ=-20(exclusive)
    ##GVCFBlock-5-0=minGQ=-5(inclusive),maxGQ=0(exclusive)
    ##GVCFBlock0-5=minGQ=0(inclusive),maxGQ=5(exclusive)
    ##GVCFBlock10-2147483647=minGQ=10(inclusive),maxGQ=2147483647(exclusive)
    ##GVCFBlock5-10=minGQ=5(inclusive),maxGQ=10(exclusive)
    ##INFO=<ID=AS_SB_TABLE,Number=1,Type=String,Description="Allele-specific forward/reverse read counts for strand bias tests. Includes the reference and alleles separated by |.">
    ##INFO=<ID=AS_UNIQ_ALT_READ_COUNT,Number=A,Type=Integer,Description="Number of reads with unique start and mate end positions for each alt at a variant site"  ##INFO=<ID=CONTQ,Number=1,Type=Float,Description="Phred-scaled qualities that alt allele are not due to contamination">
    ##INFO=<ID=DP,Number=1,Type=Integer,Description="Approximate read depth; some reads may have been filtered">
    ##INFO=<ID=ECNT,Number=1,Type=Integer,Description="Number of potential somatic events in the assembly region">
    ##INFO=<ID=ECNTH,Number=A,Type=Integer,Description="Number of somatic events in best supporting haplotype for each alt allele">
    ##INFO=<ID=END,Number=1,Type=Integer,Description="Stop position of the interval">
    ##INFO=<ID=GERMQ,Number=1,Type=Integer,Description="Phred-scaled quality that alt alleles are not germline variants">
    ##INFO=<ID=MBQ,Number=R,Type=Integer,Description="median base quality by allele">
    ##INFO=<ID=MFRL,Number=R,Type=Integer,Description="median fragment length by allele">
    ##INFO=<ID=MMQ,Number=R,Type=Integer,Description="median mapping quality by allele">
    ##INFO=<ID=MPOS,Number=A,Type=Integer,Description="median distance from end of read">
    ##INFO=<ID=NALOD,Number=A,Type=Float,Description="Log 10 odds of artifact in normal with same allele fraction as tumor">
    ##INFO=<ID=NCount,Number=1,Type=Integer,Description="Count of N bases in the pileup">
    ##INFO=<ID=NLOD,Number=A,Type=Float,Description="Normal log 10 likelihood ratio of diploid het or hom alt genotypes">
    ##INFO=<ID=OCM,Number=1,Type=Integer,Description="Number of alt reads whose original alignment doesn't match the current contig.">
    ##INFO=<ID=PON,Number=0,Type=Flag,Description="site found in panel of normals">
    ##INFO=<ID=POPAF,Number=A,Type=Float,Description="negative log 10 population allele frequencies of alt alleles">
    ##INFO=<ID=ROQ,Number=1,Type=Float,Description="Phred-scaled qualities that alt allele are not due to read orientation artifact">
    ##INFO=<ID=RPA,Number=R,Type=Integer,Description="Number of times tandem repeat unit is repeated, for each allele (including reference)">
    ##INFO=<ID=RU,Number=1,Type=String,Description="Tandem repeat unit (bases)">
    ##INFO=<ID=SEQQ,Number=1,Type=Integer,Description="Phred-scaled quality that alt alleles are not sequencing errors">
    ##INFO=<ID=STR,Number=0,Type=Flag,Description="Variant is a short tandem repeat">
    ##INFO=<ID=STRANDQ,Number=1,Type=Integer,Description="Phred-scaled quality of strand bias artifact">
    ##INFO=<ID=STRQ,Number=1,Type=Integer,Description="Phred-scaled quality that alt alleles in STRs are not polymerase slippage errors">
    ##INFO=<ID=TLOD,Number=A,Type=Float,Description="Log 10 likelihood ratio score of variant existing versus not existing">
    ##MutectVersion=2.2
    ##contig=<ID=Chr_01,length=13883608>
    ##contig=<ID=Chr_02,length=20544351>
    ##contig=<ID=Chr_03,length=23796107>
    ##contig=<ID=Chr_04,length=22814073>
    ##contig=<ID=Chr_05,length=21770955>
    ##contig=<ID=Chr_06,length=20891190>
    ##contig=<ID=Chr_07,length=21879443>
    ##contig=<ID=Chr_08,length=27686485>
    ##contig=<ID=Chr_09,length=25153474>
    ##contig=<ID=Chr_10,length=22115060>
    ##contig=<ID=Chr_11,length=33601899>
    ##contig=<ID=Chr_12,length=25847345>
    ##contig=<ID=Chr_13,length=30765978>
    ##contig=<ID=Chr_14,length=28290211>
    ##contig=<ID=scaffold_133,length=127285>
    ##contig=<ID=scaffold_189,length=98462>
    ##contig=<ID=scaffold_197,length=95235> ##contig=<ID=scaffold_301,length=65824>
    ##contig=<ID=scaffold_321,length=62769>
    ##contig=<ID=scaffold_337,length=60860>
    ##contig=<ID=scaffold_347,length=59054>
    ##contig=<ID=scaffold_373,length=57196>
    ##contig=<ID=scaffold_407,length=53677>
    ##contig=<ID=IM767_cs_124,length=86899>
    ##contig=<ID=IM767_cs_298,length=24204>
    ##contig=<ID=IM767_cs_299,length=19788>
    ##contig=<ID=IM62v2_mt,length=525671>
    ##filtering_status=Warning: unfiltered Mutect 2 calls.  Please run FilterMutectCalls to remove false positives.
    ##source=Mutect2
    ##tumor_sample=BEL_2014_pool

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    Gökalp Çelik

    Hi Ren Hamm

    As I said this GVCF for somatic calls is quite beta and this is a bug that we can recreate as well. A fix will be in the works shortly but it may not become alive until the next point release. In the meantime we recommend running Mutect2 without the GVCF mode. 

    Regards. 

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