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Nat Methods. 2010 Jun;7(6):461-5. doi: 10.1038/nmeth.1459. Epub 2010 May 9.

Direct detection of DNA methylation during single-molecule, real-time sequencing.

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1
Pacific Biosciences, Menlo Park, California, USA.

Abstract

We describe the direct detection of DNA methylation, without bisulfite conversion, through single-molecule, real-time (SMRT) sequencing. In SMRT sequencing, DNA polymerases catalyze the incorporation of fluorescently labeled nucleotides into complementary nucleic acid strands. The arrival times and durations of the resulting fluorescence pulses yield information about polymerase kinetics and allow direct detection of modified nucleotides in the DNA template, including N6-methyladenine, 5-methylcytosine and 5-hydroxymethylcytosine. Measurement of polymerase kinetics is an intrinsic part of SMRT sequencing and does not adversely affect determination of primary DNA sequence. The various modifications affect polymerase kinetics differently, allowing discrimination between them. We used these kinetic signatures to identify adenine methylation in genomic samples and found that, in combination with circular consensus sequencing, they can enable single-molecule identification of epigenetic modifications with base-pair resolution. This method is amenable to long read lengths and will likely enable mapping of methylation patterns in even highly repetitive genomic regions.

PMID:
20453866
PMCID:
PMC2879396
DOI:
10.1038/nmeth.1459
[Indexed for MEDLINE]
Free PMC Article
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