<html>
<head>
<meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1">
<style type="text/css" style="display:none;"><!-- P {margin-top:0;margin-bottom:0;} --></style>
</head>
<body dir="ltr">
<div id="divtagdefaultwrapper" dir="ltr" style="font-size: 12pt; color: rgb(0, 0, 0); font-family: Calibri,Helvetica,sans-serif,"EmojiFont","Apple Color Emoji","Segoe UI Emoji",NotoColorEmoji,"Segoe UI Symbol","Android Emoji",EmojiSymbols;">
<p style="margin-top:0; margin-bottom:0"><span id="divtagdefaultwrapper" style="font-size:12pt"></span></p>
<div style="margin-top:0; margin-bottom:0">Hello,</div>
<div style="margin-top:0; margin-bottom:0"><br>
</div>
<div style="margin-top:0; margin-bottom:0">I work for Dr. Faramarz Valafar at San Diego State University. Previously we have used Ensembl's VEP program on our vcf files of Mycobacterium tuberculosis sequences, using annotation from a cache file downloaded from
your website. However, recently we have developed additional annotations (mostly from running I-TASSER on ambiguously annotated genes) that we would like to include. To that end I converted our custom annotation file to a GFF3 format, and followed your website's
instructions for running VEP with that as the annotation source. This ran, but unfortunately it identified every variant as intergenic, even when they were within one of our annotated CDS features. I assume this is due to a formatting error on my part with
our GFF file, though I've been following the specifications described here <a previewremoved="true" href="https://github.com/The-Sequence-Ontology/Specifications/blob/master/gff3.md" target="_blank" rel="noopener noreferrer" id="LPlnk690491"><span id="LPlnk690491">https://github.com/The-Sequence-Ontology/Specifications/blob/master/gff3.md</span></a></div>
<div style="margin-top:0; margin-bottom:0"><br>
</div>
<div style="margin-top:0; margin-bottom:0">I'm using ensembl-vep version 91.3<br>
Here's a bit of our gff:<br>
<div>NC_000962.3 Modlin et. al. 2018 gene 1 1524 . + . ID=gene1;locus_tag=Rv0001;alias=dnaA;experiment=DESCRIPTION:Mutation analysis, gene expression[PMID: 10375628];Dbxref=GeneID:885041<br>
NC_000962.3 Modlin et. al. 2018 CDS 1 1524 . + . ID=CDS1;locus_tag=Rv0001;product=Chromosomal replication initiator protein DnaA;note=FunctionalCategory: information pathways<br>
NC_000962.3 Modlin et. al. 2018 gene 2052 3260 . + . ID=gene2;locus_tag=Rv0002;alias=dnaN;Dbxref=GeneID:887092<br>
NC_000962.3 Modlin et. al. 2018 CDS 2052 3260 . + . ID=CDS2;locus_tag=Rv0002;product=DNA polymerase III (beta chain) DnaN (DNA nucleotidyltransferase);note=FunctionalCategory: information pathways</div>
<br>
Here's a bit of our test input vcf:<br>
<div>##fileformat=VCFv4.0<br>
##source=pbhooverV1.0.0a8<br>
##INFO=<ID=RSR,Number=1,Type=Integer,Description="Reference-supporting reads"><br>
##INFO=<ID=VSR,Number=1,Type=Integer,Description="Variant-supporting reads"><br>
##INFO=<ID=VF,Number=1,Type=Float,Description="Variant frequency"><br>
##INFO=<ID=DP,Number=1,Type=Integer,Description="Read Depth"><br>
##FILTER=<ID=LOW,Description="Position with too low of depth"><br>
##FILTER=<ID=NO,Description="Does not meet criteria to call variant"><br>
##FILTER=<ID=HETERO,Description="Enough support to call reference and variant (mixed population)"><br>
#CHROM POS ID REF ALT QUAL FILTER INFO<br>
1 8 . A AGT 7.22 NO RSR=4;VSR=1;VF=0.2;DP=5<br>
etc.<br>
<div>1 2050 . C CC 11.36 NO RSR=15;VSR=2;VF=0.117647058824;DP=21<br>
1 2051 . A AA 13.36 NO RSR=18;VSR=2;VF=0.1;DP=20<br>
1 2051 . AA A 15.75 NO RSR=20;VSR=1;VF=0.047619047619;DP=21<br>
1 2052 . AT A 15.22 NO RSR=19;VSR=1;VF=0.05;DP=21<br>
1 2053 . TG T 33.02 NO RSR=15;VSR=2;VF=0.117647058824;DP=18<br>
1 2054 . GG G 45.75 NO RSR=17;VSR=3;VF=0.15;DP=21<br>
1 2056 . A ACG 12.46 NO RSR=17;VSR=1;VF=0.0555555555556;DP=21<br>
1 2057 . C CAC 13.28 NO RSR=20;VSR=1;VF=0.047619047619;DP=21</div>
<br>
</div>
<br>
</div>
<div style="margin-top:0; margin-bottom:0">I used these commands on our gff and vcf files:<br>
</div>
<div style="margin-top:0; margin-bottom:0">grep -v "#" mannotation-with-computation-4vep.gff | sort -k1,1 -k4,4n -k5,5n -t$'\t' | tabix/bgzip -c > mannotation-with-computation-4vep.gff.gz</div>
<div style="margin-top:0; margin-bottom:0">tabix/tabix -p gff mannotation-with-computation-4vep.gff.gz</div>
<div style="margin-top:0; margin-bottom:0">ensembl-vep/vep --force_overwrite --synonyms synonyms-hyp.txt --format vcf --vcf --species mycobacterium_tuberculosis --symbol --variant_class --flag_pick --everything -i test1-0006.vcf -gff mannotation-with-computation-4vep.gff.gz
-fasta H37Rv.fasta.gz -o test1-0006-annotated2.vcf</div>
<div style="margin-top:0; margin-bottom:0"><br>
</div>
<div style="margin-top:0; margin-bottom:0">And the output vcf looks like this:<br>
<div>##fileformat=VCFv4.0<br>
##source=pbhooverV1.0.0a8<br>
##INFO=<ID=RSR,Number=1,Type=Integer,Description="Reference-supporting reads"><br>
##INFO=<ID=VSR,Number=1,Type=Integer,Description="Variant-supporting reads"><br>
##INFO=<ID=VF,Number=1,Type=Float,Description="Variant frequency"><br>
##INFO=<ID=DP,Number=1,Type=Integer,Description="Read Depth"><br>
##FILTER=<ID=LOW,Description="Position with too low of depth"><br>
##FILTER=<ID=NO,Description="Does not meet criteria to call variant"><br>
##FILTER=<ID=HETERO,Description="Enough support to call reference and variant (mixed population)"><br>
##VEP="v91" time="2018-02-12 10:57:23" ensembl-variation=91.c78d8b4 ensembl-funcgen=91.4681d69 ensembl=91.18ee742 ensembl-io=91.923d668<br>
##INFO=<ID=CSQ,Number=.,Type=String,Description="Consequence annotations from Ensembl VEP. Format: Allele|Consequence|IMPACT|SYMBOL|Gene|Feature_type|Feature|BIOTYPE|EXON|INTRON|HGVSc|HGVSp|cDNA_position|CDS_position|Protein_position|Amino_acids|Codons|Existing_variation|DISTANCE|STRAND|FLAGS|PICK|VARIANT_CLASS|SYMBOL_SOURCE|HGNC_ID|CANONICAL|TSL|APPRIS|CCDS|ENSP|SWISSPROT|TREMBL|UNIPARC|SOURCE|GENE_PHENO|SIFT|PolyPhen|DOMAINS|HGVS_OFFSET|AF|AFR_AF|AMR_AF|EAS_AF|EUR_AF|SAS_AF|AA_AF|EA_AF|gnomAD_AF|gnomAD_AFR_AF|gnomAD_AMR_AF|gnomAD_ASJ_AF|gnomAD_EAS_AF|gnomAD_FIN_AF|gnomAD_NFE_AF|gnomAD_OTH_AF|gnomAD_SAS_AF|MAX_AF|MAX_AF_POPS|CLIN_SIG|SOMATIC|PHENO|PUBMED|MOTIF_NAME|MOTIF_POS|HIGH_INF_POS|MOTIF_SCORE_CHANGE|mannotation-with-computation.gff.gz"><br>
##INFO=<ID=mannotation-with-computation.gff.gz,Number=.,Type=String,Description="mannotation-with-computation.gff.gz (overlap)"><br>
#CHROM POS ID REF ALT QUAL FILTER INFO<br>
1 8 . A AGT 7.22 NO RSR=4;VSR=1;VF=0.2;DP=5;CSQ=GT|intergenic_variant|MODIFIER|||||||||||||||||||1|insertion||||||||||||||||||||||||||||||||||||||||||||</div>
etc.<br>
<div>1 2050 . C CC 11.36 NO RSR=15;VSR=2;VF=0.117647058824;DP=21;CSQ=C|intergenic_variant|MODIFIER|||||||||||||||||||1|insertion||||||||||||||||||||||||||||||||||||||||||||<br>
1 2051 . A AA 13.36 NO RSR=18;VSR=2;VF=0.1;DP=20;CSQ=A|intergenic_variant|MODIFIER|||||||||||||||||||1|insertion||||||||||||||||||||||||||||||||||||||||||||<br>
1 2051 . AA A 15.75 NO RSR=20;VSR=1;VF=0.047619047619;DP=21;CSQ=-|intergenic_variant|MODIFIER|||||||||||||||||||1|deletion||||||||||||||||||||||||||||||||||||||||||||<br>
1 2052 . AT A 15.22 NO RSR=19;VSR=1;VF=0.05;DP=21;CSQ=-|intergenic_variant|MODIFIER|||||||||||||||||||1|deletion||||||||||||||||||||||||||||||||||||||||||||<br>
1 2053 . TG T 33.02 NO RSR=15;VSR=2;VF=0.117647058824;DP=18;CSQ=-|intergenic_variant|MODIFIER|||||||||||||||||||1|deletion||||||||||||||||||||||||||||||||||||||||||||<br>
1 2054 . GG G 45.75 NO RSR=17;VSR=3;VF=0.15;DP=21;CSQ=-|intergenic_variant|MODIFIER|||||||||||||||||||1|deletion||||||||||||||||||||||||||||||||||||||||||||<br>
1 2056 . A ACG 12.46 NO RSR=17;VSR=1;VF=0.0555555555556;DP=21;CSQ=CG|intergenic_variant|MODIFIER|||||||||||||||||||1|insertion||||||||||||||||||||||||||||||||||||||||||||<br>
1 2057 . C CAC 13.28 NO RSR=20;VSR=1;VF=0.047619047619;DP=21;CSQ=AC|intergenic_variant|MODIFIER|||||||||||||||||||1|insertion||||||||||||||||||||||||||||||||||||||||||||</div>
<br>
I've also tried adding '<span>Is_circular=true</span>' to the attribute column of the first entry in the GFF, replacing 'locus_tag' with 'Name', and capitalizing 'alias', in case those deviations from the format described in the GFF documentation were the
problem. I also added 'biotype' attributes to the GFF, after seeing this discussion in the forums:
<a previewremoved="true" id="LPlnk609331" href="http://lists.ensembl.org/pipermail/dev/2018-January/012867.html" class="OWAAutoLink">
http://lists.ensembl.org/pipermail/dev/2018-January/012867.html</a>, though I was unsure if that advice was meant for the GFF or VCF, or whether it was applicable to whole genome reads vs transcripts. None of this changed the resulting output.
<br>
<br>
</div>
<div style="margin-top:0; margin-bottom:0">Do you have an example of a GFF annotation file that's worked with VEP, so I can compare it with ours to see what I've done wrong? Or is there a tool we can use to create our own cache files?<br>
<br>
Thank you for your assistance. <br>
</div>
<br>
<p></p>
</div>
</body>
</html>