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I've sequenced two genes of 15 dogs for SNP analysis. the chromatograms of one gene looked perfect but the ones of the second gene didn't look as good. To be precise, the chromatograms of one exon looked relatively good with only a few spots where the bases seemed unreadable. But the chromatograms of the second exon were full of that. So there must have been an error somewhere and my question is where this could have happened. My hypothesis is that I simply made a mistake during the mixing of the chemicals at some point.

Further useful information: The error must have happened befor the first pcr since i checked the pcr product with a fragment analyzer and the bands were only barely or not visible. Additionally, I noticed that many imprecisions seen in the chromatograms were only found at either the forward or backward strand.

Here is an image of several of the chromatograms of the exon that didn't work:

enter image description here

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Looks like a dye blob. A better job of clean up might help.

https://www.nucleics.com/DNA_sequencing_support/DNA-sequencing-dye-blobs.html

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You almost give the answer yourself: the PCR doesn't look good. This might lead to insufficient PCR product to base your sequencing on. If you might have done something wrong: repeat. Otherwise try a few different conditions to get a decent amount of DNA.

Certain pieces of DNA will just sequence better than others for the same reasons PCRs sometimes work better or worse, that's why the reverse sequences might look better.

I don't know what caused the strange effect in the middle of your chromatogram. It might be a starting artifact, but also the rest of the chromatogram looks quite bad indeed.

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  • $\begingroup$ Since i only had very limited time, I couldn't retry. I was just interested in whether one could pinpoint the problem exactly but it doesn't seem so. $\endgroup$ – Lukeception Sep 6 '16 at 7:32
  • $\begingroup$ Just for fun you could look at the absolute signal strength (or the scale) of your sequencing results. Probably your sequencing results are a lot closer to the noise than for a correct sequencing run. $\endgroup$ – VonBeche Sep 6 '16 at 9:12

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