I have around 100 chromatograms (
.ab1 files) from Sanger sequencing a genome at loci believed to have an indel.
I'm new to interpreting this kind of data in general, but I've read a bit on the general idea—mostly in guides like this. What I'm not able to figure out from the resources I've checked is how to identify insertions and deletions in a chromatogram.
I'm most concerned with heterozygous indels, and these seem to be simpler. (And since my samples are (supposed to be) larger indels that are more rare and more likely to be het, I think.) A heterozygous indel, according to my understanding, would like something like consecutive heterozygous SNPs—overlapping peaks, maybe not with the same height, but with the same horizontal placement—until the end of the sample.
In other words, something like the top track in the open window in this image.
(I'm trying to do this manually, and not with packages like this for several reasons, including: I want to see how this is actually done, I want to do it as thoroughly as possible—I will probably check my results with an automated package—and I was asked to do it like this.)
How can evidence of (het) indels like this be distinguished from noise in the chromatogram? My guess is it includes things like
- the overlapping peaks last until the end of the sample (and don't start at the beginning, but somewhere in the middle); and
- the peaks are still at the same horizontal positions; i.e., one is not shifted right or left relative to the other?
Is this correct?
With my own chromatograms, I have a lot of doubt about distinguishing noise from indel. Here's an example, visualized with 4Peaks. (Full chromatogram here.)
On the left, the chromatogram appears high quality. There are sharp, regularly spaced, single peaks. On the right, there appear to be two sets of peaks lined up with each other. Does this constitute evidence for an indel? Does the homopolymer region—I've noticed in many of these samples, the putative indel seems to start in a homopolymer region—cast doubt on this?
I've also noticed that in some situations like this, there appear to be not two but three peaks.
Is one of these just noise and can be disregarded? Or does this cast doubt on the validity of the data—which is supposed to be coming from the diploid human genome?