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