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Hello

I am working with canine genomes and donwloaded the reference file (ftp://ftp.ensembl.org/pub/release-73/fasta/canis_familiaris/dna/Canis_familiaris.CanFam3.1.73.dna.toplevel.fa.gz) and variation VCF file (ftp://ftp.ensembl.org/pub/release-73/variation/vcf/canis_familiaris/Canis_familiaris.vcf.gz) from Ensembl. I was able to index the reference files and perform the alignment and Mark duplicate steps of the pipeline for the canine genomes.

I extracted SNPs that were marked either deleteions or duplications from the variation VCF file using grep -E "^#|deletion|insertion" Canis_familiaris.vcf > Canis_familiaris.indels.vcf to use in the Indel Realigner steps. The header of the file is as follows: ##fileformat=VCFv4.1 ##fileDate=20130826 ##source=ensembl;version=73;url=http://e73.ensembl.org/canis_familiaris ##reference=ftp://ftp.ensembl.org/pub/release-73/fasta/canis_familiaris/dna/ ##INFO=<ID=TSA,Number=0,Type=String,Description="Type of sequence alteration. Child of term sequence_alteration as defined by the sequence ontology project."> ##INFO=<ID=E_MO,Number=0,Type=Flag,Description="Multiple_observations.http://www.ensembl.org/info/docs/variation/data_description.html#evidence_status"> ##INFO=<ID=E_ESP,Number=0,Type=Flag,Description="Exome_Sequencing_Project.http://www.ensembl.org/info/docs/variation/data_description.html#evidence_status"> ##INFO=<ID=E_1000G,Number=0,Type=Flag,Description="1000Genomes.http://www.ensembl.org/info/docs/variation/data_description.html#evidence_status"> ##INFO=<ID=E_HM,Number=0,Type=Flag,Description="HapMap.http://www.ensembl.org/info/docs/variation/data_description.html#evidence_status"> ##INFO=<ID=E_Freq,Number=0,Type=Flag,Description="Frequency.http://www.ensembl.org/info/docs/variation/data_description.html#evidence_status"> ##INFO=<ID=E_C,Number=0,Type=Flag,Description="Cited.http://www.ensembl.org/info/docs/variation/data_description.html#evidence_status"> ##INFO=<ID=dbSNP_131,Number=0,Type=Flag,Description="Variants (including SNPs and indels) imported from dbSNP [remapped from build CanFam2.0]"> 1 8252 rs8962840 C CT . . dbSNP_131;TSA=insertion 1 9702 rs8457244 CA C . . dbSNP_131;TSA=deletion 1 18289 rs8444620 GT G . . dbSNP_131;TSA=deletion 1 36381 rs8471229 GT G . . dbSNP_131;TSA=deletion 1 52452 rs8469855 AT A . . dbSNP_131;TSA=deletion 1 55767 rs8285977 G GA . . dbSNP_131;TSA=insertion 1 60065 rs8723538 TC T . . dbSNP_131;TSA=deletion 1 62067 rs8395051 TA T . . dbSNP_131;TSA=deletion

However, when I run the filrst step of Indel Realignment -T RealignerTargetCreator -nt 16 -R canFam3.1_bwa075/Canis_familiaris.CanFam3.1.73.dna.toplevel.fa -I AF23.rg.prmdup.bam -l INFO -S SILENT -o AF23.indel.intervals --known canFam3.1_bwa075/Canis_familiaris.indels.vcf

I get the error message: ##### ERROR A USER ERROR has occurred (version 2.7-2-g6bda569): ##### ERROR ##### ERROR This means that one or more arguments or inputs in your command are incorrect. ##### ERROR The error message below tells you what is the problem. ##### ERROR ##### ERROR If the problem is an invalid argument, please check the online documentation guide ##### ERROR (or rerun your command with --help) to view allowable command-line arguments for this tool. ##### ERROR ##### ERROR Visit our website and forum for extensive documentation and answers to ##### ERROR commonly asked questions http://www.broadinstitute.org/gatk ##### ERROR ##### ERROR Please do NOT post this error to the GATK forum unless you have really tried to fix it yourself. ##### ERROR ##### ERROR MESSAGE: Your input file has a malformed header: We never saw the required CHROM header line (starting with one #) for the input VCF file

I guess it is because the VCF file is missing the standard header (#CHROM POS ID ...). I was wondering if there was any way I could add the header line to the VCF file to be able to use in GATK? Would it also be possible to use the original VCF with all variations in the Indel Realignment steps or would you recommend I subset it for insertions/deletions as I did in this case?

Thank you very much for your help and suggestions!!!

Yours sincerely Shanker Swaminathan

Hello,

I'm currently using DepthOfCoverage with -L option and a BED intervals file. I would to get gene report also. I'm not familiar with RefSeq format for the gene list.

I work with HGNC and Ensembl genes. I could get a gene list file with the following format:

HGNC chr start end

But this doesn't seem to work. Any suggestions on how I could achieve that or how I could generate a valid gene list file with my current intervals?

Thank you.