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Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine


We describe strand-specific, base-resolution detection of 5-hydroxymethylcytosine (5-hmC) in genomic DNA with single-molecule sensitivity, combining a bioorthogonal, selective chemical labeling method of 5-hmC with single-molecule, real-time (SMRT) DNA sequencing. The chemical labeling not only allows affinity enrichment of 5-hmC–containing DNA fragments but also enhances the kinetic signal of 5-hmC during SMRT sequencing. We applied the approach to sequence 5-hmC in a genomic DNA sample with high confidence.

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Figure 1
Figure 2: Effects of 5-hmC modifications on polymerase kinetics in SMRT DNA sequencing.
Figure 3: Example of 5-hmC detection by SMRT sequencing from mESC genomic DNA.


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This study was supported partly by US National Institutes of Health grants GM071440 (to C.H.) and 1RC2HG005618-01 (National Human Genome Research Institute to S.T.) and The University of Chicago. We thank K. Spittle, M. Boitano, J. Eid, J. Wegener and K. Luong for assistance in data acquisition and figure generation.

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Authors and Affiliations



C.H., C.-X.S., J.K. and S.W.T. designed experiments. C.-X.S. performed the labeling and pulldown of synthetic template and mESC sample. T.A.C. prepared library constructs and conducted the sequencing experiments. A.K. analyzed data. Q.D., C.-X.S. and X.-Y.L. carried out the chemical synthesis. X.-Y.L. validated mESC hits. C.H., C.-X.S. and J.K. wrote the paper.

Corresponding authors

Correspondence to Chuan He or Jonas Korlach.

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Competing interests

T.A.C., S.W.T. and J.K. are employees of Pacific Biosciences, which commercializes single-molecule, real-time sequencing technologies.

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Song, CX., Clark, T., Lu, XY. et al. Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine. Nat Methods 9, 75–77 (2012).

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