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CpG Methylation data from PacBio sequencing
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2.3 years ago
nchuang • 200
@nchuang18948

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.

sequencing next-gen pacbio epigenetics • 312 views
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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?

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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)

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You forgot to copy the relevant part

propagated in the dam+ E. coli strain TOP10

dam is a methylase. This will methylate your DNA.

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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.

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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.

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2.3 years ago
@WouterDeCoster

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

Yes. PCR will erase all modifications.

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2.3 years ago
@Antonio R. Franco13770

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

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

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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

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Oh okay now I get it :)

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