I'm aiming to correct a single-base substitution using prime editing. To assess the efficiency and deleterious effects, I am performing a targeted sequencing of a 686 bp-long amplicon.
It is the first time I'm analyzing the sequencing outcomes, and I have a question left unanswered. When I'm aligning my reads using BWA or Bowtie, there are two distinct coverage peaks, with a region of downsampling in between. This might indicate a deletion, but this strange profile is present in all my samples, including my controls. If I use a software specialized in Cas9 efficiency, it will filter out the reads that do not cover my cut site, and I'm thus left with a single, good-looking peak.
My question is the following. Is it common to have this downsampled region, those two peaks? Is it simply a PCR-related artifact? I've suspected a lack of specificity of my primers, but this does not seem to be the case. I attach two images: the overview of the coverage (with or without filtering) and how the individual reads look like for the unfiltered ones.
Thank you for your help!
