How to call variant minimum 3 read coverage to make consensus?

Question

[Info.shi]

I have a query regarding consensus sequence assembly where reference bases are replaced with variants with a minimum of 3 read depths, using bcftools using the below command.

bcftools  mpileup -f  ref.fasta  mapped.bam | bcftools call -c | vcfutils.pl vcf2fq  -d 3 > cons.fq

Still, if I check the consensus sequence in IGV the variant is not in the consensus sequence at low coverage site.

Even I mapped reads in CDS sequence using "end to end " alignment so I already prohibit soft clipping.

I would like to compare pairwise sequence distance further so every variant is important even if ts low coverage side with a minimum of 3 reads depth.

Is there any parameter I am missing in my above command?

Kindly guide me! Thank you

Answer 1

[Istvan Albert]

To produce consensus use a different methodology as described here:

https://samtools.github.io/bcftools/bcftools.html#consensus

and also:

https://samtools.github.io/bcftools/howtos/consensus-sequence.html