Dear All,

I would be grateful for your advice on bcftools calling issue I am having. I have 3 bam files and following changes I see in the genome browser at a particular location.

• REF -> ALT
• G -> GT (in first two bams)
• G -> GA (in third bam)

Visually they look clean and no issues. When I call individually all changes are detected properly. But when calling jointly by bcftools mpileup the G->GT is detected but G->GA is not and more disturbingly the genotype for the 3rd bam is marked as 0/0 . There seem to be no heterozygous sites here. bcftools norm with reference did not fix it.

The commands used are

bcftools mpileup -a AD -f ref.fa    -P Illumina bam1.bam bam2.bam bam3.bam| bcftools call  --ploidy-file Ploidy.txt  --multiallelic-caller --variants-only

Thanks so much!