Samtools mpileup returns empty vcf file

Question

[shreyasibiswas88]

Hello everyone,

I am using samtools mpileup on multi bam files to genrate vcf file. mpileup seems to be working with only 6 bam files but not on more than 6 files. If I enter more than 6 files, samtools finishes without error in approx 2min and the vcf file generated is empty except for the header. I have used this command line in the past to call vcf from as many as 10 bam files. Now I need to increase it to 58 bam files. But it is failing if I include more than 6 bam files.

the command line I am using

/home/sb47/Programs/samtools-1.3.1/samtools mpileup -uv \
    -l /scratch/sb47cp/global_seq_Rnorvegicus/858_positions \
    -f /scratch/sb47cp/Ref_5_71/Rattus_norvegicus.Rnor_5.0.71.dna_sm.toplevel.fa \
    -b bams.list  |  /home/sb47/Programs/samtools-1.3.1/bcftools-1.3.1/bcftools call -c \
    > /scratch/sb47cp/FranceIce_858loci.vcf

bams.list is the list of bam files.

Can anyone please help me with this? I unable to figure out why it is not working.

Answer 1

[Ram]

From a first glance, I can see that the bcftools command is not proper. When input is stdin (being piped in from a previous command), you need the stdin indicator - as specified in the specs:

BCFtools is designed to work on a stream. It regards an input file "-" as the standard input (stdin) and outputs to the standard output (stdout). Several commands can thus be combined with Unix pipes.

Your bcftools command should be: bcftools call -c - >out_file.vcf

[shreyasibiswas88]

Thank you for your response. I followed your suggestion. Unfortunately it still doesn't work. i have successfully used the above command in the past without the - as suggested by you in generating vcf from 10 bam files.

[Ram]

That is strange. Maybe check out the bams.list to ensure line endings are fine and the BAM files exist at the right locations?

[shreyasibiswas88]

I have checked and re-checked without any success. It is bizarre to me as it has worked in the past. I have written to the authors too. But haven't heard anything back. Thank you for your suggestions.