Cited by 1,460Epigenetics and DNA MethylationDNA and Nucleic Acid ChemistryGenomics and Phylogenetic Studies

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

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Flusberg, Benjamin A, Webster, Dale R, Lee, Jessica H, Travers, Kevin J, Olivares, Eric C, Clark, Tyson A, Korlach, Jonas, Turner, Stephen W·2010·Nature Methods

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.

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Authors
Flusberg, B. A., Webster, D. R., Lee, J. H., Travers, K. J., Olivares, E. C., Clark, T. A., Korlach, J., & Turner, S. W.
Affiliations
Pacific Biosciences (United States) (US)
Corresponding author
Turner, Stephen W
Venue
Nature Methods
Published
2010-05-09
Volume
7
Issue
6
Pages
461-465
ISSN
  • 1548-7091 (ISSN-L)
  • 1548-7105
OpenAlex
W1984783889
Cited by
1,460
References
37
Citations by year
2026: 55 · 2025: 73 · 2024: 81 · 2023: 84 · 2022: 111 · 2021: 119 · 2020: 120 · 2019: 109 · 2018: 93 · 2017: 83 · 2016: 83 · 2015: 92 · 2014: 74 · 2013: 72 · 2012: 77
FWCI
35.85
Type
journal article
Language
en
Open access
Locations
  • Nature Methods (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Institutes of Health (5rc2hg005618-02)
  • National Human Genome Research Institute (1RC2HG005618-01, RC2 HG005618)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
Keywords
DNA methylation, single-molecule real-time sequencing, polymerase kinetics, 5-methylcytosine, 5-hydroxymethylcytosine, N6-methyladenine, epigenetic modifications, circular consensus sequencing, Escherichia coli, modified nucleotides
MeSH terms
DNA-Directed DNA Polymerase, Escherichia coli, Kinetics, Sequence Analysis, DNA, DNA Methylation, Principal Component Analysis

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