Problem with generating consensus sequence using samtool mpileup

[jimguo22]

Hi everyone,

I am trying to generate a consensus sequence from a bam file generate. I used the
samtools mpileup -uf ref.fa aln.bam | bcftools view -cg - | vcfutils.pl vcf2fq > cns.fq

and obtained the following error message:

Error: Could not parse --min-ac -g
[mpileup] 1 samples in 1 input files
Use of uninitialized value $l in numeric lt (<) at /usr/local/bin/vcfutils.pl line 566.
Use of uninitialized value $l in numeric lt (<) at /usr/local/bin/vcfutils.pl line 566.
<mpileup> Set max per-file depth to 8000

Does anyone know what might have gone wrong? Thanks,
Jim

[dpryan]

The parameters for bcftools have changed, use "bcftools consensus" instead.

[SNPsaurus]

I actually had this problem in a class I teach that had the old pipeline in the instructions (for creating your own mtDNA sequence). As dpryan says, there is a bcftools consensus that you should use. Just to add a minor point--the error can be resolved by using bcftools call instead of view and piping that to vcfutils, but better to use consensus.

[GenoMax]

A google search ("samtools consensus sequence") brings up the web page for the old samtools and biostars/SeqAnswers threads as top hits: http://samtools.sourceforge.net/mpileup.shtml. It would be best to have Heng Li (or someone who has access to this page) to indicate clearly that this commanline example is only applicable for samtools (prior to v.1.0 i.e. 0.1.x) at the top of that page.

For new versions of samtools/bcftools the relevant information can be found here: https://samtools.github.io/bcftools/...html#consensus