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Help with genotyping in my vcf file

zgtmanzgtman Posts: 6Member

Dear GATK team,

Could you please help me how to explain genotyping in my vcf file. I have Illumina data and vcf caller was GATK. My variant frequency (Alt variant freq) is 99.7%. DP = 4622 (AD = 16, 4606) - so I would expect that this sample is alternate homozygous. But when I check PL value, which is - PL = 1655,0,323 - after calculating my likelihood -

REF= G ALT= A

P(D|GG) = 10 ^ -165.5 = small
P(D|AG) = 10 ^ 0  = 1
P(D|AA) = 10 ^ 32.3 = small

we can see it is heterozygous. Can anybody help me how to interpret my result? How it is possible that likelihoods show me heterozygous and coverage and VF show me homozygous?

Here is part of my vcf file:

chr13 32899193 . G A 1625.01 PASS AC=1;AF=0.5;AN=2;DP=4622;QD=0.35;TI=NM_000059;GI=BRCA2;FC=Silent GT:AD:DP:GQ:PL:VF:GQX 0/1:16,4606:5000:99:1655,0,323:0.997:99

Thank you for any explanation.

Paul.

Best Answer

Answers

  • Geraldine_VdAuweraGeraldine_VdAuwera Posts: 6,019Administrator, GATK Developer admin

    Hi Paul,

    That call does seem surprising considering the allele ratio. Have you looked at the data in IGV?

    Also, can you tell me which GATK caller was used (UnifiedGenotyper or HaplotypeCaller) and which version?

    Geraldine Van der Auwera, PhD

  • zgtmanzgtman Posts: 6Member
    edited February 27

    Dear Geraldine,

    at first thank you for response. Yeah I checked my BAMs file in IGV and there is no Strand Bias. The caller was UnifiedGenotyper - this is set up primary in Illumina (MiSeq) pipeline - GATK 1.6. I share bellow my vcf file (there are two position with weird genotype marked in bold).

    Thank you for any help!

    ##fileformat=VCFv4.1
    ##FORMAT=<ID=GQX,Number=1,Type=Integer,Description="Minimum of {Genotype quality assuming variant position,Genotype quality assuming non-variant position}">
    ##FORMAT=<ID=AD,Number=.,Type=Integer,Description="Allelic depths for the ref and alt alleles in the order listed">
    ##FORMAT=<ID=DP,Number=1,Type=Integer,Description="Approximate read depth (reads with MQ=255 or with bad mates are filtered)">
    ##FORMAT=<ID=GQ,Number=1,Type=Float,Description="Genotype Quality">
    ##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">
    ##FORMAT=<ID=PL,Number=G,Type=Integer,Description="Normalized, Phred-scaled likelihoods for genotypes as defined in the VCF specification">
    ##FORMAT=<ID=VF,Number=1,Type=Float,Description="Variant Frequency, the ratio of the sum of the called variant depth to the total depth">
    ##INFO=<ID=TI,Number=.,Type=String,Description="Transcript ID">
    ##INFO=<ID=GI,Number=.,Type=String,Description="Gene ID">
    ##INFO=<ID=EXON,Number=0,Type=Flag,Description="Exon Region">
    ##INFO=<ID=FC,Number=.,Type=String,Description="Functional Consequence">
    ##INFO=<ID=AC,Number=A,Type=Integer,Description="Allele count in genotypes, for each ALT allele, in the same order as listed">
    ##INFO=<ID=AF,Number=A,Type=Float,Description="Allele Frequency, for each ALT allele, in the same order as listed">
    ##INFO=<ID=AN,Number=1,Type=Integer,Description="Total number of alleles in called genotypes">
    ##INFO=<ID=DP,Number=1,Type=Integer,Description="Approximate read depth; some reads may have been filtered">
    ##INFO=<ID=QD,Number=1,Type=Float,Description="Variant Confidence/Quality by Depth">
    ##FILTER=<ID=LowVariantFreq,Description="Low variant frequency < 0.20">
    ##FILTER=<ID=LowGQ,Description="GQ below < 30.00">
    ##FILTER=<ID=R8,Description="IndelRepeatLength is greater than 8">
    ##UnifiedGenotyper="analysis_type=UnifiedGenotyper input_file=[D:\Illumina\MiSeqAnalysis\55b039ca343a4b41924ba2cb00a36fc6\Data\Intensities\BaseCalls\Alignment\84-13_S10.bam, D:\Illumina\MiSeqAnalysis\55b039ca343a4b41924ba2cb00a36fc6\Data\Intensities\BaseCalls\Alignment\xxxx-12_S2.bam] read_buffer_size=null phone_home=NO_ET gatk_key=null read_filter=[] intervals=[D:\Illumina\MiSeqAnalysis\55b039ca343a4b41924ba2cb00a36fc6\Data\Intensities\BaseCalls\Alignment\intervals_M1.picard] excludeIntervals=null interval_set_rule=UNION interval_merging=ALL reference_sequence=E:\Genomes\Homo_sapiens\UCSC\hg19\Sequence\WholeGenomeFASTA\genome.fa nonDeterministicRandomSeed=false downsampling_type=BY_SAMPLE downsample_to_fraction=null downsample_to_coverage=5000 baq=OFF baqGapOpenPenalty=40.0 performanceLog=null useOriginalQualities=false BQSR=null quantize_quals=-1 defaultBaseQualities=-1 validation_strictness=SILENT unsafe=null num_threads=2 num_cpu_threads=null num_io_threads=null num_bam_file_handles=null read_group_black_list=null pedigree=[] pedigreeString=[] pedigreeValidationType=STRICT allow_intervals_with_unindexed_bam=false logging_level=INFO log_to_file=null help=false genotype_likelihoods_model=BOTH p_nonref_model=EXACT heterozygosity=0.0010 pcr_error_rate=1.0E-4 genotyping_mode=DISCOVERY output_mode=EMIT_VARIANTS_ONLY standard_min_confidence_threshold_for_calling=10.0 standard_min_confidence_threshold_for_emitting=10.0 noSLOD=false annotateNDA=false alleles=(RodBinding name= source=UNBOUND) min_base_quality_score=17 max_deletion_fraction=0.05 max_alternate_alleles=3 min_indel_count_for_genotyping=5 min_indel_fraction_per_sample=0.25 indel_heterozygosity=1.25E-4 indelGapContinuationPenalty=10 indelGapOpenPenalty=45 indelHaplotypeSize=80 noBandedIndel=false indelDebug=false ignoreSNPAlleles=false includeGVCFData=false dbsnp=(RodBinding name= source=UNBOUND) comp=[] out=org.broadinstitute.sting.gatk.io.stubs.VCFWriterStub NO_HEADER=org.broadinstitute.sting.gatk.io.stubs.VCFWriterStub sites_only=org.broadinstitute.sting.gatk.io.stubs.VCFWriterStub debug_file=null metrics_file=null annotation=[] excludeAnnotation=[] filter_mismatching_base_and_quals=false"
    ##contig=<ID=chr1,length=249250621>
    ##contig=<ID=chr10,length=135534747>
    ##contig=<ID=chr11,length=135006516>
    ##contig=<ID=chr12,length=133851895>
    ##contig=<ID=chr13,length=115169878>
    ##contig=<ID=chr14,length=107349540>
    ##contig=<ID=chr15,length=102531392>
    ##contig=<ID=chr16,length=90354753>
    ##contig=<ID=chr17,length=81195210>
    ##contig=<ID=chr18,length=78077248>
    ##contig=<ID=chr19,length=59128983>
    ##contig=<ID=chr2,length=243199373>
    ##contig=<ID=chr20,length=63025520>
    ##contig=<ID=chr21,length=48129895>
    ##contig=<ID=chr22,length=51304566>
    ##contig=<ID=chr3,length=198022430>
    ##contig=<ID=chr4,length=191154276>
    ##contig=<ID=chr5,length=180915260>
    ##contig=<ID=chr6,length=171115067>
    ##contig=<ID=chr7,length=159138663>
    ##contig=<ID=chr8,length=146364022>
    ##contig=<ID=chr9,length=141213431>
    ##contig=<ID=chrM,length=16571>
    ##contig=<ID=chrX,length=155270560>
    ##contig=<ID=chrY,length=59373566>
    ##reference=file://E:\Genomes\Homo_sapiens\UCSC\hg19\Sequence\WholeGenomeFASTA\genome.fa
    ##source=GATK 1.6
    #CHROM  POS ID  REF ALT QUAL    FILTER  INFO    FORMAT  10
    chr13   32893197    .   AT  A   16087.00    LowVariantFreq;R8   AC=1;AF=0.5;AN=2;DP=4312;QD=3.73;TI=NM_000059;GI=BRCA2;FC=Noncoding GT:AD:DP:GQ:PL:VF:GQX   0/1:3633,679:4995:99:16087,0,55863:0.157:99
    **chr13 32899193    .   G   A   1625.01 PASS    AC=1;AF=0.5;AN=2;DP=4622;QD=0.35;TI=NM_000059;GI=BRCA2;FC=Silent    GT:AD:DP:GQ:PL:VF:GQX   0/1:16,4606:5000:99:1655,0,323:0.997:99**
    chr13   32900176    .   G   A   41738.01    PASS    AC=1;AF=0.5;AN=2;DP=4993;QD=8.36;TI=NM_000059;GI=BRCA2;FC=Silent    GT:AD:DP:GQ:PL:VF:GQX   0/1:1871,3122:5000:99:41738,0,54591:0.625:99
    chr13   32900177    .   A   AC  22236.00    PASS    AC=1;AF=0.5;AN=2;DP=4999;QD=4.45;TI=NM_000059;GI=BRCA2;FC=Noncoding GT:AD:DP:GQ:PL:VF:GQX   0/1:1879,3120:3463:99:22236,0,95745:0.624:99
    chr13   32900179    .   A   T   42153.01    PASS    AC=1;AF=0.5;AN=2;DP=4990;QD=8.45;TI=NM_000059;GI=BRCA2;FC=Silent    GT:AD:DP:GQ:PL:VF:GQX   0/1:1860,3130:5000:99:42153,0,49574:0.627:99
    chr13   32900183    .   T   A   41191.01    PASS    AC=1;AF=0.5;AN=2;DP=4988;QD=8.26;TI=NM_000059;GI=BRCA2;FC=Silent    GT:AD:DP:GQ:PL:VF:GQX   0/1:1874,3114:4992:99:41191,0,61505:0.624:99
    chr13   32900184    .   AGT A   23225.00    PASS    AC=1;AF=0.5;AN=2;DP=4996;QD=4.65;TI=NM_000059;GI=BRCA2;FC=Noncoding GT:AD:DP:GQ:PL:VF:GQX   0/1:1883,3113:3463:99:23225,0,55800:0.623:99
    chr13   32900191    .   G   C   41932.01    PASS    AC=1;AF=0.5;AN=2;DP=4984;QD=8.41;TI=NM_000059;GI=BRCA2;FC=Silent    GT:AD:DP:GQ:PL:VF:GQX   0/1:1866,3118:4991:99:41932,0,59194:0.626:99
    chr13   32900194    .   A   C   41444.01    PASS    AC=1;AF=0.5;AN=2;DP=4976;QD=8.33;TI=NM_000059;GI=BRCA2;FC=Silent    GT:AD:DP:GQ:PL:VF:GQX   0/1:1862,3114:4996:99:41444,0,57067:0.626:99
    chr13   32900195    .   G   T   41497.01    PASS    AC=1;AF=0.5;AN=2;DP=4993;QD=8.31;TI=NM_000059;GI=BRCA2;FC=Silent    GT:AD:DP:GQ:PL:VF:GQX   0/1:1870,3123:4996:99:41497,0,62223:0.625:99
    chr13   32900253    rs80358676  A   T   86.01   PASS    AC=1;AF=0.5;AN=2;DP=29;QD=2.97;TI=NM_000059;GI=BRCA2;FC=Missense_Q147H;EXON GT:AD:DP:GQ:PL:VF:GQX   0/1:22,7:32:99:116,0,512:0.241:86
    chr13   32900256    .   T   A   27379.01    PASS    AC=1;AF=0.5;AN=2;DP=4966;QD=5.51;TI=NM_000059;GI=BRCA2;FC=Nonsense_C148X;EXON   GT:AD:DP:GQ:PL:VF:GQX   0/1:2780,2186:4999:99:27379,0,86322:0.440:99
    chr13   32900257    .   A   C   14784.01    PASS    AC=1;AF=0.5;AN=2;DP=4985;QD=2.97;TI=NM_000059;GI=BRCA2;FC=Missense_T149P;EXON   GT:AD:DP:GQ:PL:VF:GQX   0/1:2823,2162:4999:99:14784,0,89260:0.434:99
    chr13   32900261    .   A   T   27270.01    PASS    AC=1;AF=0.5;AN=2;DP=4948;QD=5.51;TI=NM_000059;GI=BRCA2;FC=Missense_H150L;EXON   GT:AD:DP:GQ:PL:VF:GQX   0/1:2789,2159:4975:99:27270,0,94949:0.436:99
    chr13   32900262    .   T   A   18838.01    PASS    AC=1;AF=0.5;AN=2;DP=4981;QD=3.78;TI=NM_000059;GI=BRCA2;FC=Missense_H150Q;EXON   GT:AD:DP:GQ:PL:VF:GQX   0/1:2848,2133:4996:99:18838,0,96431:0.428:99
    chr13   32900263    .   G   C   28584.01    PASS    AC=1;AF=0.5;AN=2;DP=4983;QD=5.74;TI=NM_000059;GI=BRCA2;FC=Missense_V151L;EXON   GT:AD:DP:GQ:PL:VF:GQX   0/1:2782,2201:4991:99:28584,0,94296:0.442:99
    chr13   32900267    .   CA  C   7539.00 PASS    AC=1;AF=0.5;AN=2;DP=4990;QD=1.51;TI=NM_000059;GI=BRCA2;FC=Frameshift;EXON   GT:AD:DP:GQ:PL:VF:GQX   0/1:2852,2138:4079:99:7539,0,75685:0.428:99
    chr13   32903685    rs2126042   C   T   83048.01    PASS    AC=1;AF=0.5;AN=2;DP=4988;QD=16.65;TI=NM_000059;GI=BRCA2;FC=Silent   GT:AD:DP:GQ:PL:VF:GQX   0/1:2517,2471:5000:99:83048,0,81547:0.495:99
    chr13   32906729    rs144848    A   C   82275.01    PASS    AC=1;AF=0.5;AN=2;DP=4955;QD=16.60;TI=NM_000059;GI=BRCA2;FC=Missense_N372H;EXON  GT:AD:DP:GQ:PL:VF:GQX   0/1:2414,2541:4962:99:82275,0,81204:0.513:99
    chr13   32907535    .   CT  C   52527.00    R8  AC=1;AF=0.5;AN=2;DP=3566;QD=14.73;TI=NM_000059;GI=BRCA2;FC=Noncoding    GT:AD:DP:GQ:PL:VF:GQX   0/1:1757,1809:4998:99:52527,0,30569:0.507:99
    chr13   32910880    .   C   A   45754.01    PASS    AC=1;AF=0.5;AN=2;DP=4978;QD=9.19;TI=NM_000059;GI=BRCA2;FC=Missense_D796E;EXON   GT:AD:DP:GQ:PL:VF:GQX   0/1:3148,1830:4993:99:45754,0,109737:0.368:99
    chr13   32910884    .   C   A   46134.01    PASS    AC=1;AF=0.5;AN=2;DP=4986;QD=9.25;TI=NM_000059;GI=BRCA2;FC=Missense_L798I;EXON   GT:AD:DP:GQ:PL:VF:GQX   0/1:3138,1848:4989:99:46134,0,108587:0.371:99
    chr13   32912202    .   C   G   4111.01 LowVariantFreq  AC=1;AF=0.5;AN=2;DP=4986;QD=0.82;TI=NM_000059;GI=BRCA2;FC=Missense_A1237G;EXON  GT:AD:DP:GQ:PL:VF:GQX   0/1:4463,523:5000:99:4111,0,149815:0.105:99
    chr13   32913055    rs206075    A   G   183782.01   PASS    AC=2;AF=1.0;AN=2;DP=4997;QD=36.78;TI=NM_000059;GI=BRCA2;FC=Synonymous_L1521L;EXON   GT:AD:DP:GQ:PL:VF:GQX   1/1:8,4989:5000:99:183782,14074,0:0.998:99
    chr13   32913196    .   G   A   1104.01 PASS    AC=1;AF=0.5;AN=2;DP=4197;QD=0.26;TI=NM_000059;GI=BRCA2;FC=Synonymous_K1568K;EXON    GT:AD:DP:GQ:PL:VF:GQX   0/1:1752,2445:4996:99:1134,0,64062:0.583:99
    chr13   32913198    .   A   T   10935.01    LowVariantFreq  AC=1;AF=0.5;AN=2;DP=4962;QD=2.20;TI=NM_000059;GI=BRCA2;FC=Missense_Y1569F;EXON  GT:AD:DP:GQ:PL:VF:GQX   0/1:4243,719:4998:99:10935,0,142672:0.145:99
    chr13   32913199    .   C   A   2129.01 LowVariantFreq  AC=1;AF=0.5;AN=2;DP=4988;QD=0.43;TI=NM_000059;GI=BRCA2;FC=Nonsense_Y1569X;EXON  GT:AD:DP:GQ:PL:VF:GQX   0/1:4557,431:5000:99:2159,0,155010:0.086:99
    chr13   32915005    rs206076    G   C   196037.01   PASS    AC=2;AF=1.0;AN=2;DP=4928;QD=39.78;TI=NM_000059;GI=BRCA2;FC=Synonymous_V2171V;EXON   GT:AD:DP:GQ:PL:VF:GQX   1/1:0,4928:4929:99:196037,14573,0:1.000:99
    **chr13 32929283    .   C   A   5164.01 PASS    AC=1;AF=0.5;AN=2;DP=4996;QD=1.03;TI=NM_000059;GI=BRCA2;FC=Missense_N2431K;EXON  GT:AD:DP:GQ:PL:VF:GQX   0/1:80,4916:5000:99:5164,0,1152:0.984:99**
    chr13   32929387    rs169547    T   C   192362.01   PASS    AC=2;AF=1.0;AN=2;DP=4995;QD=38.51;TI=NM_000059;GI=BRCA2;FC=Missense_V2466A;EXON GT:AD:DP:GQ:PL:VF:GQX   1/1:4,4991:4996:99:192362,14254,0:0.999:99
    chr13   32936646    rs9534262   T   C   199213.01   PASS    AC=2;AF=1.0;AN=2;DP=4991;QD=39.91;TI=NM_000059;GI=BRCA2;FC=Silent   GT:AD:DP:GQ:PL:VF:GQX   1/1:16,4975:4999:99:199213,14371,0:0.997:99
    chr13   32953388    rs4942486   T   C   202167.01   PASS    AC=2;AF=1.0;AN=2;DP=4996;QD=40.47;TI=NM_000059;GI=BRCA2;FC=Silent   GT:AD:DP:GQ:PL:VF:GQX   1/1:6,4990:5000:99:202167,14781,0:0.999:99
    chr13   32968844    rs80359195;rs80359196   A   G   163441.01   PASS    AC=2;AF=1.0;AN=2;DP=4999;QD=32.69;TI=NM_000059;GI=BRCA2;FC=Missense_Y3092C;EXON GT:AD:DP:GQ:PL:VF:GQX   1/1:31,4968:5000:99:163441,13800,0:0.994:99
    chr17   41223031    .   T   A   151274.01   PASS    AC=2;AF=1.0;AN=2;DP=4984;QD=30.35;TI=NM_007298,NR_027676,NM_007299,NM_007297,NM_007294,NM_007300;GI=BRCA1,BRCA1,BRCA1,BRCA1,BRCA1,BRCA1;FC=Missense_R530W,Silent,Missense_R530W,Missense_R1587W,Missense_R1634W,Missense_R1655W;EXON    GT:AD:DP:GQ:PL:VF:GQX   1/1:200,4784:5000:99:151274,7096,0:0.960:99
    chr17   41244403    .   A   G   144.01  LowVariantFreq  AC=1;AF=0.5;AN=2;DP=4991;QD=0.03;TI=NM_007298,NR_027676,NM_007299,NM_007297,NM_007294,NM_007300;GI=BRCA1,BRCA1,BRCA1,BRCA1,BRCA1,BRCA1;FC=Silent,Silent,Silent,Missense_S1002P,Missense_S1049P,Missense_S1049P;EXON GT:AD:DP:GQ:PL:VF:GQX   0/1:4580,411:5000:99:174,0,153873:0.082:99
    chr17   41251931    rs799923    G   A   76630.01    PASS    AC=1;AF=0.5;AN=2;DP=4934;QD=15.53;TI=NM_007298,NR_027676,NM_007299,NM_007297,NM_007294,NM_007300;GI=BRCA1,BRCA1,BRCA1,BRCA1,BRCA1,BRCA1;FC=Silent,Silent,Silent,Silent,Silent,Silent    GT:AD:DP:GQ:PL:VF:GQX   0/1:2372,2562:4997:99:76630,0,68452:0.519:99
    chr17   41276152    .   C   CAT 107888.00   PASS    AC=1;AF=0.5;AN=2;DP=4730;QD=22.81;TI=NR_027676,NM_007299,NM_007297,NM_007294,NM_007300;GI=BRCA1,BRCA1,BRCA1,BRCA1,BRCA1;FC=Noncoding,Noncoding,Noncoding,Noncoding,Noncoding    GT:AD:DP:GQ:PL:VF:GQX   0/1:2112,2618:5000:99:107888,0,76063:0.553:99
    
    Post edited by zgtman on
  • zgtmanzgtman Posts: 6Member

    yeah this is really ancient version... I can see the newest 2.8-1 version.. I try to update a new version and I let you know if it helps.

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