Dear Hungjen,

I perform direct Sanger's sequencing (i.e. without TA cloning) on a regular basis and analyse peak height ratios to obtain % methylation levels at CpG sites. You can refer to the supplemental file of my recent manuscript for further details. Attaching the paper for your reference. Hope it helps. 

Sincerely,

Pooja

If I understand correctly, you have generated a PCR product from your two lines and have sequenced directly from the PCR product? If so, it is very tricky to quantitate the ratio of C (me) to T (unme) in these output chromatograms. I'm not sure how quantitative this approach is. As Pooja says above, it is more useful to clone the PCR products (into pGemT-Easy or equivalent T-cloning), transform e.coli, pick many individual clones and sequence from the plasmids.  In this way you are sequencing individual molecules, instead of a mixture. Then all you have to do is calculate the ratio of C:T to get a methylation % in each CpG position within your PCR product. hope this help, Donncha.

Dear Dr. Dunican

I would like to share my little experience based on TA cloning vs. direct Sanger's sequencing approach. Like you said, TA cloning is the most preferred way to quantify methylation because you either get a C or a T at a given CpG site. So the information we get is totally different i.e out of 20 strands represented by 20 different colonies, how many have C and how many T. But we select colonies randomly, and are therefore introducing selection bias at this step. Also, cloning can be extremely time consuming and more expensive as one has to screen at least 20 clones per sample. I have tried doing both cloning and direct sequencing and haven't seen much difference in CpG methylation levels using either methods. I would say direct sequencing works like pyrosequencing, the latter being the most preferred and highly accurate way to understand methylation signatures currently. So yes, one may choose whether to opt for direct sequencing or cloning. 

Best regards.

Dear Hungjen,

imho the best approach is to use the Sanger seq you already did only to test the quality of bisulfite conversion, i.e. to assess how many non-CpG C were converted into T. If quality is ok you should definitely send the amplicon for NGS sequencing. You don't even need to modify your current amplification protocol. Amplicon NGS sequencing (ultradeep sequencing) is cheap and can give you QUANTITATIVE data, which is very hard to get with TA cloning, as TA requires a large number of colonies to be Sanger sequenced to avoid the bias mentioned by Pooja.

Best,

Rocco