Question: CpG Methylation data from PacBio sequencing
0
gravatar for nchuang
5 months ago by
nchuang190
United States
nchuang190 wrote:

Sorry if this is a dumb question, but I am told by our sequencing core that you cannot detect methylation status of PCR amplicons with PacBio? Is that merely because the amplicons won't retain the methylated nucleotides? I was looking at the Nature Methods paper that is cited for this method of detection and I think they do use PCR amplification of custom fosmids prior to PacBio sequencing though.

ADD COMMENTlink modified 5 months ago by Antonio R. Franco4.0k • written 5 months ago by nchuang190

I was looking at the Nature Methods paper that is cited for this method of detection and I think they do use PCR amplification of custom fosmids prior to PacBio sequencing though.

Which paper? Which method?

ADD REPLYlink written 5 months ago by h.mon24k

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2879396/

For sequencing of the subsection of the fosmid (Fig. 5 and Supplementary Figs. 3 and 4), an ∼3.7 kb segment (corresponding to positions 12797-16484 within the fosmid) containing 13 instances of the GATC sequence context was PCR amplified from the fosmid using Phusion High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA)

ADD REPLYlink modified 5 months ago • written 5 months ago by nchuang190
1

You forgot to copy the relevant part

propagated in the dam+ E. coli strain TOP10

dam is a methylase. This will methylate your DNA.

ADD REPLYlink written 5 months ago by WouterDeCoster37k

Thank you this is what I was looking for. I don't know how to accept this as the answer, but perhaps it's not the direct answer to my posted question.

ADD REPLYlink written 5 months ago by nchuang190
1

If your question was "is it true that I cannot detect methylation from amplicons" then my answer below is probably what you should accept.

The comments here just explain why you were confused based on the paper.

ADD REPLYlink written 5 months ago by WouterDeCoster37k
3
gravatar for WouterDeCoster
5 months ago by
Belgium
WouterDeCoster37k wrote:

Is that merely because the amplicons won't retain the methylated nucleotides?

Yes. PCR will erase all modifications.

ADD COMMENTlink written 5 months ago by WouterDeCoster37k
0
gravatar for Antonio R. Franco
5 months ago by
Spain. Universidad de Córdoba
Antonio R. Franco4.0k wrote:

Neither you add methylated precursors in the PCR to maintain that status. That will be worthless in any case

ADD COMMENTlink modified 5 months ago • written 5 months ago by Antonio R. Franco4.0k

Do you mean a polymerase would incorporate a methylated C when the template strand is methylated? I don't think so...

ADD REPLYlink written 5 months ago by WouterDeCoster37k

Of course no. I was simply trying to indicate that if you don't add methylated precursors to your PCR it is impossible to include methylated bases to your DNA. And if you add such as methylated precursors they will not follow the original pattern in any case

ADD REPLYlink modified 5 months ago • written 5 months ago by Antonio R. Franco4.0k

Oh okay now I get it :)

ADD REPLYlink written 5 months ago by WouterDeCoster37k
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