Issue with VCF format while using Pharmcat
1
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Entering edit mode
16 months ago

Hello everybody, I am using pharmcat tool's prerprocessor feature to preprocessmy vcf file using the command

> python3 pharmcat_vcf_preprocessor.py -vcf sample.vcf

But I think there is some issue with my vcf file as this command outputs an error

> Reading samples from sample.vcf ... Saving output to .
> 
> Processing sample.vcf ... The CHROM column does not conform with
> either "chr##" or "##" format.

Here is my vcf file format

> ##fileformat=VCFv4.2   
> ##FILTER=<ID=LowQual,Description="Low quality">  
> ##FORMAT=<ID=AD,Number=R,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=Integer,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">
> ##GATKCommandLine=<ID=HaplotypeCaller,CommandLine="HaplotypeCaller --output-mode EMIT_ALL_ACTIVE_SITES --output NA12801.vcf --input NA12801_dedup.bam --reference GCF_000001405.40_GRCh38.p14_genomic.fna
> --use-posteriors-to-calculate-qual false --dont-use-dragstr-priors false --use-new-qual-calculator true
> --annotate-with-num-discovered-alleles false --heterozygosity 0.001 --indel-heterozygosity 1.25E-4 --heterozygosity-stdev 0.01 --standard-min-confidence-threshold-for-calling 30.0 --max-alternate-alleles 6 --max-genotype-count 1024 --sample-ploidy 2 --num-reference-samples-if-no-call 0 --genotype-assignment-method USE_PLS_TO_ASSIGN --contamination-fraction-to-filter 0.0
> --all-site-pls false --flow-likelihood-parallel-threads 0 --flow-likelihood-optimized-comp false --flow-use-t0-tag false --flow-probability-threshold 0.003 --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 --flow-number-of-uncertain-flows-to-clip 0 --flow-nucleotide-of-first-uncertain-flow T --keep-boundary-flows false --gvcf-gq-bands 1 --gvcf-gq-bands 2 --gvcf-gq-bands 3
> --gvcf-gq-bands 4 --gvcf-gq-bands 5 --gvcf-gq-bands 6 --gvcf-gq-bands 7 --gvcf-gq-bands 8 --gvcf-gq-bands 9 --gvcf-gq-bands 10
> --gvcf-gq-bands 11 --gvcf-gq-bands 12 --gvcf-gq-bands 13 --gvcf-gq-bands 14 --gvcf-gq-bands 15 --gvcf-gq-bands 16 --gvcf-gq-bands 17 --gvcf-gq-bands 18 --gvcf-gq-bands 19 --gvcf-gq-bands 20 --gvcf-gq-bands 21 --gvcf-gq-bands 22 --gvcf-gq-bands 23 --gvcf-gq-bands 24 --gvcf-gq-bands 25 --gvcf-gq-bands 26 --gvcf-gq-bands 27 --gvcf-gq-bands 28 --gvcf-gq-bands 29 --gvcf-gq-bands 30 --gvcf-gq-bands 31 --gvcf-gq-bands 32 --gvcf-gq-bands 33 --gvcf-gq-bands 34 --gvcf-gq-bands 35 --gvcf-gq-bands 36 --gvcf-gq-bands 37 --gvcf-gq-bands 38 --gvcf-gq-bands 39 --gvcf-gq-bands 40 --gvcf-gq-bands 41 --gvcf-gq-bands 42 --gvcf-gq-bands 43 --gvcf-gq-bands 44 --gvcf-gq-bands 45 --gvcf-gq-bands 46 --gvcf-gq-bands 47 --gvcf-gq-bands 48 --gvcf-gq-bands 49 --gvcf-gq-bands 50 --gvcf-gq-bands 51 --gvcf-gq-bands 52 --gvcf-gq-bands 53 --gvcf-gq-bands 54 --gvcf-gq-bands 55 --gvcf-gq-bands 56 --gvcf-gq-bands 57 --gvcf-gq-bands 58 --gvcf-gq-bands 59 --gvcf-gq-bands 60 --gvcf-gq-bands 70 --gvcf-gq-bands 80 --gvcf-gq-bands 90 --gvcf-gq-bands 99 --floor-blocks false --indel-size-to-eliminate-in-ref-model 10 --disable-optimizations false --dragen-mode false --flow-mode NONE --apply-bqd false --apply-frd false --disable-spanning-event-genotyping false --transform-dragen-mapping-quality false --mapping-quality-threshold-for-genotyping 20 --max-effective-depth-adjustment-for-frd 0 --just-determine-active-regions false --dont-genotype false --do-not-run-physical-phasing false --do-not-correct-overlapping-quality false --use-filtered-reads-for-annotations false --use-flow-aligner-for-stepwise-hc-filtering false --adaptive-pruning false --do-not-recover-dangling-branches false
> --recover-dangling-heads 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 --pileup-detection-enable-indel-pileup-calling false --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 0.5 --pileup-detection-absolute-alt-depth 0.0 --pileup-detection-snp-adjacent-to-assembled-indel-range 5 --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 --bam-writer-type CALLED_HAPLOTYPES --dont-use-soft-clipped-bases false --override-fragment-softclip-check false
> --min-base-quality-score 10 --smith-waterman JAVA --emit-ref-confidence NONE --max-mnp-distance 0 --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 --minimum-mapping-quality 20 --disable-tool-default-annotations false --enable-all-annotations false --allow-old-rms-mapping-quality-annotation-data
> false",Version="4.3.0.0",Date="January 25, 2023 at 12:09:56 PM PKT">
> ##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=BaseQRankSum,Number=1,Type=Float,Description="Z-score from Wilcoxon rank sum test of Alt Vs. Ref base qualities">
> ##INFO=<ID=DP,Number=1,Type=Integer,Description="Approximate read depth; some reads may have been filtered">
> ##INFO=<ID=ExcessHet,Number=1,Type=Float,Description="Phred-scaled p-value for exact test of excess heterozygosity">
> ##INFO=<ID=FS,Number=1,Type=Float,Description="Phred-scaled p-value using Fisher's exact test to detect strand bias">
> ##INFO=<ID=InbreedingCoeff,Number=1,Type=Float,Description="Inbreeding coefficient as estimated from the genotype likelihoods per-sample when
> compared against the Hardy-Weinberg expectation">
> ##INFO=<ID=MLEAC,Number=A,Type=Integer,Description="Maximum likelihood expectation (MLE) for the allele counts (not necessarily the same as
> the AC), for each ALT allele, in the same order as listed">
> ##INFO=<ID=MLEAF,Number=A,Type=Float,Description="Maximum likelihood expectation (MLE) for the allele frequency (not necessarily the same
> as the AF), for each ALT allele, in the same order as listed">
> ##INFO=<ID=MQ,Number=1,Type=Float,Description="RMS Mapping Quality">
> ##INFO=<ID=MQRankSum,Number=1,Type=Float,Description="Z-score From Wilcoxon rank sum test of Alt vs. Ref read mapping qualities">
> ##INFO=<ID=QD,Number=1,Type=Float,Description="Variant Confidence/Quality by Depth">
> ##INFO=<ID=ReadPosRankSum,Number=1,Type=Float,Description="Z-score from Wilcoxon rank sum test of Alt vs. Ref read position bias">
> ##INFO=<ID=SOR,Number=1,Type=Float,Description="Symmetric Odds Ratio of 2x2 contingency table to detect strand bias">
> ##contig=<ID=NC_000001.11,length=248956422>
> ##contig=<ID=NT_187361.1,length=175055>
> ##contig=<ID=NT_187362.1,length=32032>
> ##source=HaplotypeCaller
> #CHROM    POS ID  REF ALT QUAL    FILTER  INFO    FORMAT  FATHER NC_000001.11 4373006 .   C   A   74.32   .   AC=2;AF=1.00;AN=2;DP=2;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=60.00;QD=25.36;SOR=2.303    GT:AD:DP:GQ:PL  1/1:0,2:2:6:86,6,0
> NC_000001.11  69169233    .   G   T   78.32   .   AC=2;AF=1.00;AN=2;DP=2;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=52.01;QD=28.73;SOR=2.303    GT:AD:DP:GQ:PL  1/1:0,2:2:6:90,6,0
> NC_000001.11  69169241    .   T   G   78.32   .   AC=2;AF=1.00;AN=2;DP=2;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=52.01;QD=30.97;SOR=2.303    GT:AD:DP:GQ:PL  1/1:0,2:2:6:90,6,0

Kindly help me in resolving his error. Any help would be highly appreciated.

Thanks in advance

Pharmcat VCF • 1.1k views
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Entering edit mode
16 months ago
> ##GATKCommandLine=<ID=HaplotypeCaller,CommandLine="HaplotypeCaller --output-mode EMIT_ALL_ACTIVE_SITES --output NA12801.vcf --input NA12801_dedup.bam --reference GCF_000001405.40_GRCh38.p14_genomic.fna
> --use-posteriors-to-calculate-qual false --dont-use-dragstr-priors false --use-new-qual-calculator true
> --annotate-with-num-discovered-alleles false --heterozygosity 0.001 --indel-heterozygosity 1.25E-4 --heterozygosity-stdev 0.01 --standard-min-confidence-threshold-for-calling 30.0 --max-alternate-alleles 6 --max-genotype-count 1024 --sample-ploidy 2 --num-reference-samples-if-no-call 0 --genotype-assignment-method USE_PLS_TO_ASSIGN --contamination-fraction-to-filter 0.0
> --all-site-pls false --flow-likelihood-para

does it mean that some lines really start with " --use-posteriors-to-calculate-qual ..." ? if so, this is wrong, the lines have been folded to fit on xxx columns, the file was most likely edited in a text editor.

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No, it's like this

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Metalines should start with ##. Try concatenating the split header lines back to a single line.

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Entering edit mode

taniamahmood38 : Do the lines in your file actually wrap around like that (and do not start with ## or is it a side effect of how you tried to apply code formatting in main post?

> ##FORMAT=<ID=PL,Number=G,Type=Integer,Description="Normalized, Phred-scaled likelihoods for genotypes as defined in the VCF
> specification">
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Lines in my actual file starts with ##. You are right its just a side effect in the post

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lines have been broken by something. try to fix those lines, you should only have lines starting with # in the header.

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Sorry it's the effect when I applied the code while writing the post. Actual file is totally fine no lines are broken in the main file.

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