Direct detection of DNA methylation during single-molecule, real-time sequencing
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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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Advanced next generation sequencing platforms such as Single Molecule Real-Time sequencing can be employed to reliably detect certain methylation signatures, as done for C. autoethanogenum.
“Advanced next generation sequencing platforms such as Single Molecule Real-Time (SMRT) sequencing can be employed to reliably detect certain methylation signatures (Flusberg et al., 2010; Fang et al., 2012) as it has been done for C. autoethanogenum (Utturkar et al., 2015).”
From Gas Fermentation—A Flexible Platform for Commercial Scale Production of Low-Carbon-Fuels and Chemicals from Waste and Renewable Feedstocks (2016) · cites this paper as Flusberg et al., 2010
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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
- PMC
- PMC2879396
- 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
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- 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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