Hi,
for my PhD I have several patients with multiple biopsies being available, so the multi sample variant calling is really very interesting for me.
I did follow the latest tutorial on running Mutect2 v4.1.2.0 with first creating a panel of normals with 40 healthy samples sequenced on the same machine with the same library prep,
Then performed joint variant calling according to the tutorial with a later step of filtering.
From orthagonal validation of those exact samples with ddPCR I know that these variants are actually present in the samples, but in they get filtered out with two filters: multiallelic, which I kind of understand, as there are a lot of different variants at this position (as this position allows resistance to treatment), but it also says normal_artifact, which I do not understand at all.
First of all, I would love to know if there is a way for me to not filter out these variants, especially as they are pretty "common" in some samples (vaf around 0.4)
And secondly I would like to understand why the calculated AF for most these alleles is actually not 0 in the tumour, when the AD field says, that no supporting read was found
In general, this is unfortunate, because I was really hoping Mutect2 would be an easy solution.
I would love to understand why this happened and if I can change the behaviour
##fileformat=VCFv4.2
##FILTER=<ID=PASS,Description="All filters passed">
##FILTER=<ID=base_qual,Description="alt median base quality">
##FILTER=<ID=clustered_events,Description="Clustered events observed in the tumor">
##FILTER=<ID=contamination,Description="contamination">
##FILTER=<ID=duplicate,Description="evidence for alt allele is overrepresented by apparent duplicates">
##FILTER=<ID=fragment,Description="abs(ref - alt) median fragment length">
##FILTER=<ID=germline,Description="Evidence indicates this site is germline, not somatic">
##FILTER=<ID=haplotype,Description="Variant near filtered variant on same haplotype.">
##FILTER=<ID=low_allele_frac,Description="Allele fraction is below specified threshold">
##FILTER=<ID=map_qual,Description="ref - alt median mapping quality">
##FILTER=<ID=multiallelic,Description="Site filtered because too many alt alleles pass tumor LOD">
##FILTER=<ID=n_ratio,Description="Ratio of N to alt exceeds specified ratio">
##FILTER=<ID=normal_artifact,Description="artifact_in_normal">
##FILTER=<ID=numt_chimera,Description="NuMT variant with too many ALT reads originally from autosome">
##FILTER=<ID=numt_novel,Description="Alt depth is below expected coverage of NuMT in autosome">
##FILTER=<ID=orientation,Description="orientation bias detected by the orientation bias mixture model">
##FILTER=<ID=panel_of_normals,Description="Blacklisted site in panel of normals">
##FILTER=<ID=position,Description="median distance of alt variants from end of reads">
##FILTER=<ID=slippage,Description="Site filtered due to contraction of short tandem repeat region">
##FILTER=<ID=strand_bias,Description="Evidence for alt allele comes from one read direction only">
##FILTER=<ID=strict_strand,Description="Evidence for alt allele is not represented in both directions">
##FILTER=<ID=weak_evidence,Description="Mutation does not meet likelihood threshold">
##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=GQ,Number=1,Type=Integer,Description="Genotype Quality">
##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">
##FORMAT=<ID=PGT,Number=1,Type=String,Description="Physical phasing haplotype information, describing how the alternate alleles are phased in relation to one another">
##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.">
##GATKCommandLine=<ID=FilterMutectCalls,CommandLine="FilterMutectCalls --output /home/shollizeck/CASCADE/analysis/CA99/tumor/joined/mutect2/CA99_somatic.filtered.vcf.gz --contamination-table /home/shollizeck/CASCADE/analysis/CA99/tumor/joined/mutect2/contamination.table --tumor-segmentation /home/shollizeck/CASCADE/analysis/CA99/tumor/joined/mutect2/segments.table --orientation-bias-artifact-priors /home/shollizeck/CASCADE/analysis/CA99/tumor/joined/mutect2/readOrientationModel.tar.gz --variant /home/shollizeck/CASCADE/analysis/CA99/tumor/joined/mutect2/CA99_somatic.unfiltered.vcf.gz --reference /data/reference/indexes/human/hg19/fasta/Homo_sapiens.GRCh37.73.dna.toplevel.fa --threshold-strategy OPTIMAL_F_SCORE --f-score-beta 1.0 --false-discovery-rate 0.05 --initial-threshold 0.1 --mitochondria-mode false --max-events-in-region 2 --max-alt-allele-count 1 --unique-alt-read-count 0 --min-median-mapping-quality 30 --min-median-base-quality 20 --max-median-fragment-length-difference 10000 --min-median-read-position 1 --max-n-ratio Infinity --min-reads-per-strand 0 --autosomal-coverage 0.0 --max-numt-fraction 0.85 --min-allele-fraction 0.0 --contamination-estimate 0.0 --log-snv-prior -13.815510557964275 --log-indel-prior -16.11809565095832 --log-artifact-prior -2.302585092994046 --normal-p-value-threshold 0.001 --min-slippage-length 8 --pcr-slippage-rate 0.1 --distance-on-haplotype 100 --long-indel-length 5 --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 --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",Version="4.1.2.0",Date="August 12, 2019 10:14:09 AM UTC">
##GATKCommandLine=<ID=Mutect2,CommandLine="Mutect2 --f1r2-tar-gz /home/shollizeck/CASCADE/analysis/CA99/tumor/joined/mutect2/10-f1r2.tar.gz --normal-sample CA99 --panel-of-normals /data/reference/dawson_labs/Mutect2/PanelOfNormals/pon.vcf.gz --germline-resource /data/reference/dawson_labs/Mutect2/af-only-gnomad.raw.sites.GRCh37.73.vcf.gz --output /home/shollizeck/CASCADE/analysis/CA99/tumor/joined/mutect2/CA99_10.somatic.unfiltered.vcf.gz --intervals 10 --input /home/shollizeck/CASCADE/analysis/CA99/germline/Bam/CA99_merged.markdups.bam --input /home/shollizeck/CASCADE/analysis/CA99/tumor/CA99-26/Bam/CA99-26_merged.markdups.bam --input /home/shollizeck/CASCADE/analysis/CA99/tumor/CA99-31/Bam/CA99-31_merged.markdups.bam --input /home/shollizeck/CASCADE/analysis/CA99/tumor/CA99-41/Bam/CA99-41_merged.markdups.bam --input /home/shollizeck/CASCADE/analysis/CA99/tumor/CA99-57/Bam/CA99-57_merged.markdups.bam --input /home/shollizeck/CASCADE/analysis/CA99/tumor/CA99-11/Bam/CA99-11_merged.markdups.bam --input /home/shollizeck/CASCADE/analysis/CA99/tumor/CA99-59/Bam/CA99-59_merged.markdups.bam --input /home/shollizeck/CASCADE/analysis/CA99/tumor/CA99-47/Bam/CA99-47_merged.markdups.bam --input /home/shollizeck/CASCADE/analysis/CA99/tumor/CA99-55/Bam/CA99-55_merged.markdups.bam --reference /data/reference/indexes/human/hg19/fasta/Homo_sapiens.GRCh37.73.dna.toplevel.fa --f1r2-median-mq 50 --f1r2-min-bq 20 --f1r2-max-depth 200 --genotype-pon-sites false --genotype-germline-sites false --af-of-alleles-not-in-resource -1.0 --mitochondria-mode false --tumor-lod-to-emit 3.0 --initial-tumor-lod 2.0 --pcr-snv-qual 40 --pcr-indel-qual 40 --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 --genotype-filtered-alleles false --disable-adaptive-pruning false --dont-trim-active-regions false --max-disc-ar-extension 25 --max-gga-ar-extension 300 --padding-around-indels 150 --padding-around-snps 20 --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 --max-unpruned-variants 100 --debug-assembly false --debug-graph-transformations false --capture-assembly-failure-bam false --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 --pair-hmm-gap-continuation-penalty 10 --pair-hmm-implementation FASTEST_AVAILABLE --pcr-indel-model CONSERVATIVE --phred-scaled-global-read-mismapping-rate 45 --native-pair-hmm-threads 4 --native-pair-hmm-use-double-precision false --bam-writer-type CALLED_HAPLOTYPES --dont-use-soft-clipped-bases false --min-base-quality-score 10 --smith-waterman JAVA --emit-ref-confidence NONE --max-mnp-distance 1 --min-assembly-region-size 50 --max-assembly-region-size 300 --assembly-region-padding 100 --max-reads-per-alignment-start 50 --active-probability-threshold 0.002 --max-prob-propagation-distance 50 --force-active 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 --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="4.1.2.0",Date="July 29, 2019 12:11:09 AM UTC">
##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 events in this haplotype">
##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">
##INFO=<ID=MFRL,Number=R,Type=Integer,Description="median fragment length">
##INFO=<ID=MMQ,Number=R,Type=Integer,Description="median mapping quality">
##INFO=<ID=MPOS,Number=A,Type=Integer,Description="median distance from end of read">
##INFO=<ID=NALOD,Number=A,Type=Float,Description="Negative 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=.,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">
##INFO=<ID=UNIQ_ALT_READ_COUNT,Number=1,Type=Integer,Description="Number of ALT reads with unique start and mate end positions at a variant site">
##MutectVersion=2.2
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##filtering_status=These calls have been filtered by FilterMutectCalls to label false positives with a list of failed filters and true positives with PASS.
##normal_sample=CA99
##source=FilterMutectCalls
##source=Mutect2
##tumor_sample=CA99-11
##tumor_sample=CA99-26
##tumor_sample=CA99-31
##tumor_sample=CA99-41
##tumor_sample=CA99-47
##tumor_sample=CA99-55
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##bcftools_viewCommand=view -h CASCADE/analysis/CA99/tumor/joined/mutect2/CA99_somatic.filtered.vcf.gz; Date=Wed Sep 4 13:01:27 2019
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT CA99 CA99-11 CA99-26 CA99-31 CA99-41 CA99-47 CA99-55 CA99-57 CA99-59
10 43615014 . G A,C,T . multiallelic;normal_artifact CONTQ=93;DP=1438;ECNT=1;GERMQ=93;MBQ=37,37,37,37;MFRL=360,407,325,369;MMQ=60,60,60,60;MPOS=35,41,29;NALOD=0.957,1.52,1.52;NLOD=26.39,28.55,28.56;POPAF=6.00,6.00,6.00;ROQ=21;SEQQ=93;STRANDQ=93;TLOD=318.68,206.55,109.89 GT:AD:AF:DP:F1R2:F2R1:SB 0/0:95,1,0,0:0.018,9.934e-03,9.933e-03:96:39,0,0,0:56,1,0,0:49,46,1,0 0/1/2/3:104,69,1,0:0.394,5.737e-03,5.589e-03:174:59,30,0,0:43,36,0,0:49,55,34,36 0/1/2/3:86,0,32,4:7.869e-03,0.263,0.037:122:47,0,16,3:36,0,16,1:50,36,19,17 0/1/2/3:115,2,2,0:0.024,0.022,8.101e-03:119:57,1,2,0:56,1,0,0:63,52,2,2 0/1/2/3:189,0,3,1:5.039e-03,0.020,0.010:193:97,0,3,1:91,0,0,0:96,93,4,0 0/1/2/3:166,0,3,30:4.824e-03,0.020,0.151:199:89,0,2,18:75,0,1,11:82,84,15,18 0/1/2/3:184,2,19,17:0.013,0.089,0.080:222:97,0,13,7:84,2,6,10:80,104,18,20 0/1/2/3:101,0,35,0:7.072e-03,0.257,7.072e-03:136:49,0,21,0:50,0,14,0:53,48,17,18 0/1/2/3:85,56,0,0:0.390,6.877e-03,6.877e-03:141:47,28,0,0:38,25,0,0:42,43,27,29