Double coverage peak

[tramelliwe]

Hey! 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 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!

[Ben3]

Hello tramelliwe I can't really speak on whether the two peaks are common, but I can say that after doing a lot of amplicon sequencing I've had a lot of off-target amplification. Depending on what you're sequencing it can be expected and it happens even more with degenerate primers. Have you looked to see if any of your primers match up with that region? I can also check in with some friends that have more experience with this type of work.

[tramelliwe]

Thank you for your answer Ben3. Maybe I should clarify that my amplicon covers both peaks. I did check the specificity, but even with multiple mismatches, my primers do not bind other places on that locus. I did not do the library prep myself, maybe at that stage the fragments covering that zone were lost somehow?