Amplicon based sequencing Mutation Calling

[iscancilga]

Hi!

We are investigating the effect of TF-mediated mutations on genome. In the validation part, we narrowed our regions to 1. We sequenced a region and upstream of it and obtained ~100 k coverage with paired end reads. We want to compare mutation frequency between TF-binding and upstream regions. (interested in SNV/small indels).

I have two questions. One is about mapping and the other is about mutation calling.

1) Mapping: I use bowtie2 with default conditions. What if my reads are not mapped due to those mutations, so that those reads are discarded ? Shouldn't I decrease the stringency of mapping in mutation calling cases ?

I used bowtie2 with default settings and obtained %97 mapping.

2) Mutation calling: I used samtools mpileup —> bcftools call but did not get any mutations in the ranges of our region of interest. I found UNDR ROVER caller that might work for me but any suggestion would be really appreciated.

TL;DR I am looking for a mutation caller that is focused on targeted region mutations.

Thank you very much