Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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


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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Figure 1: Principle and corresponding example of detecting DNA methylation during SMRT sequencing.
Figure 2: SMRT sequencing–mediated detection of methylated DNA bases.
Figure 3: Principal component analysis of cytosine, mC and hmC IPD and pulse width signatures.
Figure 4: IPD distributions for adenine and mA in synthetic DNA templates.
Figure 5: Comparison of SMRT sequencing kinetics for DNA samples propagated in dam+ E. coli and for the same samples after whole-genome amplification.


  1. Marinus, M.G. & Casadesus, J. Roles of DNA adenine methylation in host-pathogen interactions: mismatch repair, transcriptional regulation, and more. FEMS Microbiol. Rev. 33, 488–503 (2009).

    Article  CAS  Google Scholar 

  2. Cokus, S.J. et al. Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452, 215–219 (2008).

    Article  CAS  Google Scholar 

  3. Lister, R. et al. Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133, 523–536 (2008).

    Article  CAS  Google Scholar 

  4. Gardiner-Garden, M. & Frommer, M. CpG islands in vertebrate genomes. J. Mol. Biol. 196, 261–282 (1987).

    Article  CAS  Google Scholar 

  5. Saxonov, S., Berg, P. & Brutlag, D.L. A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters. Proc. Natl. Acad. Sci. USA 103, 1412–1417 (2006).

    Article  CAS  Google Scholar 

  6. Pomraning, K.R., Smith, K.M. & Freitag, M. Genome-wide high throughput analysis of DNA methylation in eukaryotes. Methods 47, 142–150 (2009).

    Article  CAS  Google Scholar 

  7. Jaenisch, R. & Bird, A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat. Genet. 33 (Suppl.), 245–254 (2003).

    Article  CAS  Google Scholar 

  8. Holliday, R. & Pugh, J.E. DNA modification mechanisms and gene activity during development. Science 187, 226–232 (1975).

    Article  CAS  Google Scholar 

  9. Riggs, A.D. X inactivation, differentiation, and DNA methylation. Cytogenet. Cell Genet. 14, 9–25 (1975).

    Article  CAS  Google Scholar 

  10. Li, E., Bestor, T.H. & Jaenisch, R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915–926 (1992).

    Article  CAS  Google Scholar 

  11. Razin, A. & Shemer, R. DNA methylation in early development. Hum. Mol. Genet. 4 Spec No, 1751–1755 (1995).

    Article  CAS  Google Scholar 

  12. Jones, P.A. & Baylin, S.B. The fundamental role of epigenetic events in cancer. Nat. Rev. Genet. 3, 415–428 (2002).

    Article  CAS  Google Scholar 

  13. Jones, P.A. & Laird, P.W. Cancer epigenetics comes of age. Nat. Genet. 21, 163–167 (1999).

    Article  CAS  Google Scholar 

  14. Robertson, K.D. DNA methylation and human disease. Nat. Rev. Genet. 6, 597–610 (2005).

    Article  CAS  Google Scholar 

  15. Lister, R. et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462, 315–322 (2009).

    Article  CAS  Google Scholar 

  16. Kriaucionis, S. & Heintz, N. The nuclear DNA base 5-hydroxymethylcytosine is present in purkinje neurons and the brain. Science 324, 929–930 (2009).

    Article  CAS  Google Scholar 

  17. Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930–935 (2009).

    Article  CAS  Google Scholar 

  18. Lister, R. & Ecker, J.R. Finding the fifth base: genome-wide sequencing of cytosine methylation. Genome Res. 19, 959–966 (2009).

    Article  CAS  Google Scholar 

  19. Meissner, A. et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 454, 766–770 (2008).

    Article  CAS  Google Scholar 

  20. Clark, S.J., Statham, A., Stirzaker, C., Molloy, P.L. & Frommer, M. DNA methylation: bisulphite modification and analysis. Nat. Protocols 1, 2353–2364 (2006).

    Article  CAS  Google Scholar 

  21. Hayatsu, H. & Shiragami, M. Reaction of bisulfite with the 5-hydroxymethyl group in pyrimidines and in phage DNAs. Biochemistry 18, 632–637 (1979).

    Article  CAS  Google Scholar 

  22. Huang, Y. et al. The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS One 5, e8888 (2010).

    Article  Google Scholar 

  23. Tardy-Planechaud, S., Fujimoto, J., Lin, S.S. & Sowers, L.C. Solid phase synthesis and restriction endonuclease cleavage of oligodeoxynucleotides containing 5-(hydroxymethyl)-cytosine. Nucleic Acids Res. 25, 553–559 (1997).

    Article  CAS  Google Scholar 

  24. Clarke, J. et al. Continuous base identification for single-molecule nanopore DNA sequencing. Nat. Nanotechnol. 4, 265–270 (2009).

    Article  CAS  Google Scholar 

  25. Eid, J. et al. Real-time DNA sequencing from single polymerase molecules. Science 323, 133–138 (2009).

    Article  CAS  Google Scholar 

  26. Levene, M.J. et al. Zero-mode waveguides for single-molecule analysis at high concentrations. Science 299, 682–686 (2003).

    Article  CAS  Google Scholar 

  27. Wong, I., Patel, S.S. & Johnson, K.A. An induced-fit kinetic mechanism for DNA replication fidelity: direct measurement by single-turnover kinetics. Biochemistry 30, 526–537 (1991).

    Article  CAS  Google Scholar 

  28. Hsu, G.W., Ober, M., Carell, T. & Beese, L.S. Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase. Nature 431, 217–221 (2004).

    Article  CAS  Google Scholar 

  29. Berman, A.J. et al. Structures of phi29 DNA polymerase complexed with substrate: the mechanism of translocation in B-family polymerases. EMBO J. 26, 3494–3505 (2007).

    Article  CAS  Google Scholar 

  30. Lundquist, P.M. et al. Parallel confocal detection of single molecules in real time. Opt. Lett. 33, 1026–1028 (2008).

    Article  Google Scholar 

  31. Foquet, M. et al. Improved fabrication of zero-mode waveguides for single-molecule detection. J. Appl. Phys. 103, 034301 (2008).

    Article  Google Scholar 

  32. Korlach, J. et al. Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures. Proc. Natl. Acad. Sci. USA 105, 1176–1181 (2008).

    Article  CAS  Google Scholar 

  33. Korlach, J. et al. Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides. Nucleosides Nucleotides Nucleic Acids 27, 1072–1082 (2008).

    Article  CAS  Google Scholar 

  34. Jolliffe, I.T. Principal Component Analysis 2nd edn. (Springer-Verlag, New York, 2002).

Download references


We thank the entire staff at Pacific Biosciences, in particular J. Londry and D. Kolesnikov for sample preparation; E. Mollova, M. Berhe and J. Yen for running sequencing experiments; J. Sorenson, J. Chin, A. Kislyuk and D. Holden for help with data analysis; and E. Schadt and J. Eid for helpful discussions. This work was supported by US National Human Genome Research Institute grant 1RC2HG005618-01.

Author information

Authors and Affiliations



B.A.F., K.J.T., J.K., J.H.L. and S.W.T. designed the experiments. E.C.O. and T.A.C. prepared fosmid library constructs. B.A.F. conducted the sequencing experiments. D.R.W. and B.A.F. analyzed data. B.A.F., J.K., S.W.T., E.C.O., D.R.W. and T.A.C. wrote the manuscript.

Corresponding author

Correspondence to Stephen W Turner.

Ethics declarations

Competing interests

All of the authors are employees of Pacific Biosciences.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Note 1 (PDF 511 kb)

Supplementary Data

IPD values at fosmid GATC positions (XLS 58 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Flusberg, B., Webster, D., Lee, J. et al. Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat Methods 7, 461–465 (2010).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research