Hello fellow molecular biologists! I'm currently starting out doing a lot of Bisulfite Sequencing and as COBRA is not sufficiently working on my sequence, I do Sanger Sequencing instead, as suggested by my PI. The problem is I was told a single peak of "T" means the Cytosine is methylated, in a CpG context. However, thinking about it, I have trouble explaining how you would get to this conclusion.

My though was: Imagine you have:

5' CpG 3'

3' GpC 5'

Now if both are unmethylated you get:

5' TpG 3'

3' GpT 5'

If you now do a PCR, they give rise to two types of DNA molecules (TG/AC) and (CA/GT). If you perform Sanger Sequencing just with the forward primer, this would mean you get 50% "T" and 50% "C" at the original position. In other words, the reverse strand (3'-GC-5') should always restore the "C" at the original position, when I perfom a PCR.

So who is wrong? Should I expect 100% "T" or 50% "T" and "C" for a completely unmethylated CpG?

By the way, I asked ChatGPT. Initially he told me 100% "T" would be correct. As I explained my thought process, he agreed with me, but then cited imaginary papers with links that do not exist. :D

Thanks in advance! Best regards, Felix

  • $\begingroup$ I would not go to ChatGPT for trusted scientific advice, it has a tendency to make stuff up, as you have discovered. I would suggest going back and interrogating further what "single peak of T" means. It is not easy to tell what it is supposed to mean from your question. Notably: does 50% T count as a peak? I would say so, if I was expecting C. If it's not the CpG context that's methylated but rather just that Cs are methylated, you would instead expect 2 peaks of 50%, right? Possibly misunderstanding. $\endgroup$ Commented Apr 6, 2023 at 16:24
  • $\begingroup$ "single peak of T" should mean that the sequencing result really just shows a clean "T", without any major peak for "C", "G" or "A" (hence the C being unmethylated). To maybe elucidate that a little bit more: I'm really just aiming at analysing CpGs (not just Cs) and I try to conceptually get an understanding for what my result should be for a fully methylated CpG dyad, hemimethylated dyad (on one of the two strands) and fully unmethylated. So the question is very straightforward. What would the Sanger Sequencing result look like, if the CpG dyad is fully methylated? And why? $\endgroup$
    – Felix H.
    Commented Apr 6, 2023 at 20:35
  • $\begingroup$ Well, have you looked at the literature or wikipedia? $\endgroup$ Commented Apr 6, 2023 at 22:44
  • $\begingroup$ With all due respect, if your contribution is "read more" and have you looked on Wikipedia, then I seriously question the motif behind you answering this question. It's really not that trivial if you think about it - and yes, I have read some papers about data interpretation of BS-Seq data and of course Wikipedia as well. So far I do not have found a comprehensive answer to what the expectation would look like (some just take the relative difference between C and T at the "C" position of a CpG) and to me the answer seems to be more complicated. $\endgroup$
    – Felix H.
    Commented Apr 7, 2023 at 10:37
  • $\begingroup$ I am not trying to answer your question, or I would not have written in the comment field. I am trying to help you improve your question such that someone else can answer it (there are many people who are experts in this field but I'm not one of them). I'm sorry if this seems disrespectful. When someone feels prepared to answer your question, they will answer it in the "answer" field below. It is standard for people asking questions to directly cite the sources that they have already consulted and explain why they haven't answered the question; I am trying to help you to do that. $\endgroup$ Commented Apr 7, 2023 at 16:41


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