Dear all,

I am using GATK (v4.1.9.0) AnnotateVcfWithBamDepth to get the DP for all variants in ClinVar VCF in a retina RNA-seq BAM file. However, the tool returns zero depth for all variants in the VCF, even though I checked multiple variants in IGV and I saw that they are covered pretty well.

This is the code:

java -Xms128g -Xmx128g -jar gatk.jar AnnotateVcfWithBamDepth -V clinvar_20230717_PLP_IRD_withchr_adj.vcf -I Retina1.bam -O clinvar_adj_Retina1.vcf

Below, you can see the headers of input BAM, input vcf and output vcf files: BAM header (I didn't put chromosomes on purpose to not give too long information):

@PG ID:TopHat   VN:2.0.10   CL:/opt/software/ngs/bin/tophat -p 24 -G /home/ngsworkspace//references/RNASeq/gencode_hg19_v19_final.gtf -r 170 --mate-std-dev 50 --segment-length 25 --transcriptome-index /home/ngsworkspace//references/RNASeq/bowtie2/transIndex/hg19_gencode_v19_final_tophat2 --library-type fr-unstranded -N 5 --read-gap-length 2 --read-edit-dist 5 --max-insertion-length 3 --max-deletion-length 3 -o /home/ngsworkspace//RNASeqAnalysis/Project_HumanRetina_Final/Tuxedo_analysis/tophat2_RNA1 /home/ngsworkspace//references/RNASeq/bowtie2/genomeIndex/hg19_gencode_v19_final /home/ngsworkspace//RNASeqAnalysis/Project_HumanRetina_Final/trim/RNA1_R1_val_1.fq.gz /home/ngsworkspace//RNASeqAnalysis/Project_HumanRetina_Final/trim/RNA1_R2_val_2.fq.gz
@PG ID:samtools PN:samtools PP:TopHat   VN:1.12-2-gd515e1b  CL:samtools view -H Retina1.bam

ClinVar input vcf file header and the first variant - Here, I added chr notations otherwise tool cannot find chromosomes:

##fileformat=VCFv4.1
##fileDate=2023-07-17
##source=ClinVar
##reference=GRCh37
##ID=<Description="ClinVar Variation ID">
##INFO=<ID=AF_ESP,Number=1,Type=Float,Description="allele frequencies from GO-ESP">
##INFO=<ID=AF_EXAC,Number=1,Type=Float,Description="allele frequencies from ExAC">
##INFO=<ID=AF_TGP,Number=1,Type=Float,Description="allele frequencies from TGP">
##INFO=<ID=ALLELEID,Number=1,Type=Integer,Description="the ClinVar Allele ID">
##INFO=<ID=CLNDN,Number=.,Type=String,Description="ClinVar's preferred disease name for the concept specified by disease identifiers in CLNDISDB">
##INFO=<ID=CLNDNINCL,Number=.,Type=String,Description="For included Variant : ClinVar's preferred disease name for the concept specified by disease identifiers in CLNDISDB">
##INFO=<ID=CLNDISDB,Number=.,Type=String,Description="Tag-value pairs of disease database name and identifier, e.g. OMIM:NNNNNN">
##INFO=<ID=CLNDISDBINCL,Number=.,Type=String,Description="For included Variant: Tag-value pairs of disease database name and identifier, e.g. OMIM:NNNNNN">
##INFO=<ID=CLNHGVS,Number=.,Type=String,Description="Top-level (primary assembly, alt, or patch) HGVS expression.">
##INFO=<ID=CLNREVSTAT,Number=.,Type=String,Description="ClinVar review status for the Variation ID">
##INFO=<ID=CLNSIG,Number=.,Type=String,Description="Clinical significance for this single variant; multiple values are separated by a vertical bar">
##INFO=<ID=CLNSIGCONF,Number=.,Type=String,Description="Conflicting clinical significance for this single variant; multiple values are separated by a vertical bar">
##INFO=<ID=CLNSIGINCL,Number=.,Type=String,Description="Clinical significance for a haplotype or genotype that includes this variant. Reported as pairs of VariationID:clinical significance; multiple values are separated by a vertical bar">
##INFO=<ID=CLNVC,Number=1,Type=String,Description="Variant type">
##INFO=<ID=CLNVCSO,Number=1,Type=String,Description="Sequence Ontology id for variant type">
##INFO=<ID=CLNVI,Number=.,Type=String,Description="the variant's clinical sources reported as tag-value pairs of database and variant identifier">
##INFO=<ID=DBVARID,Number=.,Type=String,Description="nsv accessions from dbVar for the variant">
##INFO=<ID=GENEINFO,Number=1,Type=String,Description="Gene(s) for the variant reported as gene symbol:gene id. The gene symbol and id are delimited by a colon (:) and each pair is delimited by a vertical bar (|)">
##INFO=<ID=MC,Number=.,Type=String,Description="comma separated list of molecular consequence in the form of Sequence Ontology ID|molecular_consequence">
##INFO=<ID=ORIGIN,Number=.,Type=String,Description="Allele origin. One or more of the following values may be added: 0 - unknown; 1 - germline; 2 - somatic; 4 - inherited; 8 - paternal; 16 - maternal; 32 - de-novo; 64 - biparental; 128 - uniparental; 256 - not-tested; 512 - tested-inconclusive; 1073741824 - other">
##INFO=<ID=RS,Number=.,Type=String,Description="dbSNP ID (i.e. rs number)">
#CHROM  POS ID  REF ALT QUAL    FILTER  INFO
chr1    879375  950448  C   T   .   .   ALLELEID=929884;CLNDISDB=MedGen:CN517202;CLNDN=not_provided;CLNHGVS=NC_000001.10:g.879375C>T;CLNREVSTAT=criteria_provided,_single_submitter;CLNSIG=Pathogenic;CLNVC=single_nucleotide_variant;CLNVCSO=SO:0001483;GENEINFO=SAMD11:148398;MC=SO:0001587|nonsense;ORIGIN=1;RS=761448939

And this is the header and the first variant of output vcf:

##fileformat=VCFv4.2
##GATKCommandLine=<ID=AnnotateVcfWithBamDepth,CommandLine="AnnotateVcfWithBamDepth  --output /home/user/clinvar_adj_Retina1.vcf --variant /home/user/clinvar_20230717_PLP_IRD_withchr_adj.vcf --input /home/user/Retina1.bam  --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="4.1.9.0",Date="August 27, 2023 7:06:15 PM CEST">
##ID=<Description="ClinVar Variation ID">
##INFO=<ID=AF_ESP,Number=1,Type=Float,Description="allele frequencies from GO-ESP">
##INFO=<ID=AF_EXAC,Number=1,Type=Float,Description="allele frequencies from ExAC">
##INFO=<ID=AF_TGP,Number=1,Type=Float,Description="allele frequencies from TGP">
##INFO=<ID=ALLELEID,Number=1,Type=Integer,Description="the ClinVar Allele ID">
##INFO=<ID=BAM_DEPTH,Number=1,Type=Integer,Description="pooled bam depth">
##INFO=<ID=CLNDISDB,Number=.,Type=String,Description="Tag-value pairs of disease database name and identifier, e.g. OMIM:NNNNNN">
##INFO=<ID=CLNDISDBINCL,Number=.,Type=String,Description="For included Variant: Tag-value pairs of disease database name and identifier, e.g. OMIM:NNNNNN">
##INFO=<ID=CLNDN,Number=.,Type=String,Description="ClinVar's preferred disease name for the concept specified by disease identifiers in CLNDISDB">
##INFO=<ID=CLNDNINCL,Number=.,Type=String,Description="For included Variant : ClinVar's preferred disease name for the concept specified by disease identifiers in CLNDISDB">
##INFO=<ID=CLNHGVS,Number=.,Type=String,Description="Top-level (primary assembly, alt, or patch) HGVS expression.">
##INFO=<ID=CLNREVSTAT,Number=.,Type=String,Description="ClinVar review status for the Variation ID">
##INFO=<ID=CLNSIG,Number=.,Type=String,Description="Clinical significance for this single variant; multiple values are separated by a vertical bar">
##INFO=<ID=CLNSIGCONF,Number=.,Type=String,Description="Conflicting clinical significance for this single variant; multiple values are separated by a vertical bar">
##INFO=<ID=CLNSIGINCL,Number=.,Type=String,Description="Clinical significance for a haplotype or genotype that includes this variant. Reported as pairs of VariationID:clinical significance; multiple values are separated by a vertical bar">
##INFO=<ID=CLNVC,Number=1,Type=String,Description="Variant type">
##INFO=<ID=CLNVCSO,Number=1,Type=String,Description="Sequence Ontology id for variant type">
##INFO=<ID=CLNVI,Number=.,Type=String,Description="the variant's clinical sources reported as tag-value pairs of database and variant identifier">
##INFO=<ID=DBVARID,Number=.,Type=String,Description="nsv accessions from dbVar for the variant">
##INFO=<ID=GENEINFO,Number=1,Type=String,Description="Gene(s) for the variant reported as gene symbol:gene id. The gene symbol and id are delimited by a colon (:) and each pair is delimited by a vertical bar (|)">
##INFO=<ID=MC,Number=.,Type=String,Description="comma separated list of molecular consequence in the form of Sequence Ontology ID|molecular_consequence">
##INFO=<ID=ORIGIN,Number=.,Type=String,Description="Allele origin. One or more of the following values may be added: 0 - unknown; 1 - germline; 2 - somatic; 4 - inherited; 8 - paternal; 16 - maternal; 32 - de-novo; 64 - biparental; 128 - uniparental; 256 - not-tested; 512 - tested-inconclusive; 1073741824 - other">
##INFO=<ID=RS,Number=.,Type=String,Description="dbSNP ID (i.e. rs number)">
##fileDate=2023-07-17
##reference=GRCh37
##source=AnnotateVcfWithBamDepth
##source=ClinVar
#CHROM  POS ID  REF ALT QUAL    FILTER  INFO
chr1    879375  950448  C   T   .   .   ALLELEID=929884;BAM_DEPTH=0;CLNDISDB=MedGen:CN517202;CLNDN=not_provided;CLNHGVS=NC_000001.10:g.879375C>T;CLNREVSTAT=criteria_provided,_single_submitter;CLNSIG=Pathogenic;CLNVC=single_nucleotide_variant;CLNVCSO=SO:0001483;GENEINFO=SAMD11:148398;MC=SO:0001587|nonsense;ORIGIN=1;RS=761448939

Unfortunately, the BAM file was downloaded from a study and I can't have any further information about the actual data. It would be really great if there is something I can do on the code, VCF and/or BAM file to fix the issue.

Thank you very much in advance for your help.