Empty VCF file with bcftools call

Question

[AP]

Hello,

I am trying to produce a vcf file using bcftools call but it produces an empty vcf file containing only the header. In short, here is what I do:

1. Alignment with BWA
2. With samtools, make sorted.bam files
3. With samtools, index the sorted.bam files
4. Run samtools mpileup in the following way: samtools mpileup -C 50 -E -t SP -t DP -u -I –f /genome/refgenome.fa -b bam_list.txt > output.bcf
5. Run bcftools call: bcftools call -v -c output.bcf > output.vcf

I am using versions 1.3.1 of samtools, bcftools and htslib. I tried reinstalling these programs but it did not change the issue. I also tried with versions 1.2. Same problem. As far as I know, the bcf file seems fine, it contains lots of data and is 20GB.

I tried producing a basic vcf file using bcftools view: bcftools view output.cf > output.vcf and it works. The vcf file seems completely normal.

Could anyone help me with this? Why would bcftools call produce an empty output?

Thanks

[reza.jabal]

How big is your vcf file? whats is the last line of your vcf file? Have you tried to run bcftools on Galaxy with your vcf file?

[AP]

Thanks for your answer. My BAM files varie in size from 150MB to 2GB. My bcf file is ~ 22GB. I am running my analysis on a HPC and have plenty of power. The last line of my "empty" vcf file is just the end of the header with CHROM POS ID ...etc. I did not try to run bcftools on Galaxy but I am not sure that it will help. I suspect an issue with mpileup or bcftools call.

Please let me know if you have other suggestions.

[reza.jabal]

Ok, I guess the bam_list.txt might be the issue. If you're running your analysis on cluster make a flat file containing your bam locations. Then include the following to your pipeline:

cat BAM_Path | while read path sample; do
samtools mpileup -C 50 -E -t SP -t DP -u -I –f /genome/refgenome.fa -b "$location" > output.bcf...

Hope this helps!

Answer 1

[AP]

For some reasons, an empty line was added at the bottom of the index genome file. Removing it solved the problem…

[reza.jabal]

well-done on sharing this.