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Old 08-06-2014, 12:31 PM   #1
spyf89
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Location: St. Louis

Join Date: Aug 2014
Posts: 2
Default Missing chromosomes in vcf output

**UPDATE**

Problem solved. The local version of the .fai index file I was using only contained chromosome 1. Version on the server that I accidentally checked multiple times was fine. Whoops.

Hello everyone!

I have bam files for several histone ChIP-seq and RNA-seq experiments and I am attempting to call SNPs. I used the following command successfully with the ChIP-seq data:

Code:
samtools mpileup -uD -f hg19.fa INPUT.bam | bcftools view -bcvg - > INPUT_raw.bcf; 
bcftools view DL237_INPUT_raw.bcf | vcfutils.pl varFilter -D100 > INPUT.vcf;
For the RNA-seq data, the sequencing center changed their pipeline and I needed a different ENSEMBL build for mpileup:

Code:
samtools mpileup -uD -f Homo_sapiens.GRCh37.72.dna_rm.toplevel.fa.gz RNA.bam | bcftools view -bcvg - > RNA_raw.bcf; 
bcftools view RNA_raw.bcf | vcfutils.pl varFilter -D100 > RNA.vcf;
Everything seemed to run smoothly until the final step. I get no errors and no indication of a problem.
The output VCF starts out just like all the others but doesn't have entries for any chromosomes except for chromosome 1.
If I take out the vcfutils.pl varFilter step and run:

Code:
samtools mpileup -uD -f Homo_sapiens.GRCh37.72.dna_rm.toplevel.fa.gz RNA.bam | bcftools view -bcvg - > RNA_raw.bcf; 
bcftools view RNA_raw.bcf > RNA.vcf;
I end up with a MASSIVE vcf file with SNPs called for all chromosomes and no errors. The problem seems to be in the vcfutils.pl step but I have used this script successfully with other files. I'm not sure what else to try/where the problem might be. Any advice? And please let me know if any additional information would be helpful! *Note* I am limitedly familiar with this pipeline and am doing this as a favor for a lab mate who is out of town.

Last edited by spyf89; 08-13-2014 at 08:01 AM.
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