We are investigating the effect of TF-mediated mutations on genome. In the validation part, we narrowed our regions to 1. We sequenced this region and upstream of it and obtained ~100 k coverage with paired end reads. This is our experimental setup. So we simple want to compare mutation frequency between TF-binding and upstream regions. (For now lets assume, I am interested in SNV/small indels).
I have two questions. One is about mapping and the other is about mutation calling.
1) Mapping (forgive my ignorance about it) So assume 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,