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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.

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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.

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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.

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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.

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Correspondence to Stephen W Turner.

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All of the authors are employees of Pacific Biosciences.

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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)

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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).

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