Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine

Journal name:
Nature Methods
Volume:
9,
Pages:
75–77
Year published:
DOI:
doi:10.1038/nmeth.1779
Received
Accepted
Published online

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.

At a glance

Figures

  1. Principle of selective chemical labeling of 5-hmC followed by SMRT DNA sequencing.
    Figure 1: Principle of selective chemical labeling of 5-hmC followed by SMRT DNA sequencing.
  2. Effects of 5-hmC modifications on polymerase kinetics in SMRT DNA sequencing.
    Figure 2: Effects of 5-hmC modifications on polymerase kinetics in SMRT DNA sequencing.

    (ac) Using synthetic DNA templates with known positions of 5-hmC (triangles), the graphs show IPD ratios, at each template position, of the modified DNA template over a control DNA template of identical sequence but lacking 5-hmC. Conditions were 5-hmC untreated (a), upon coupling with glucose azide (b) and upon additional coupling with the disulfide-containing biotin linker, followed by cleavage of the disulfide bond (c).

  3. Example of 5-hmC detection by SMRT sequencing from mESC genomic DNA.
    Figure 3: Example of 5-hmC detection by SMRT sequencing from mESC genomic DNA.

    (a) The raw SMRT sequencing read. A.u., arbitrary units. (b) Sequencing subreads from the SMRTbell template, mapped onto mouse chromosome 1 over the sequencing time course. Pauses appear as discontinuities as the polymerase temporarily stops progressing along the DNA template. F, forward strand reads; R, reverse strand reads. (c) Subreads are grouped and annotated by IPD values in a heat-map scale, identifying a hemi-hydroxymethylated CG position in this DNA molecule. Arrows indicate consistent pausing of the polymerase (that is, large IPD value) at the same genomic position across multiple intramolecular subreads, indicating the presence of a 5-hmC adduct.

References

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Author information

  1. These authors contributed equally to this work.

    • Chun-Xiao Song &
    • Tyson A Clark

Affiliations

  1. Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois, USA.

    • Chun-Xiao Song,
    • Xing-Yu Lu,
    • Qing Dai &
    • Chuan He
  2. Pacific Biosciences, Menlo Park, California, USA.

    • Tyson A Clark,
    • Andrey Kislyuk,
    • Stephen W Turner &
    • Jonas Korlach

Contributions

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.

Competing financial 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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Supplementary information

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    Supplementary Figures 1–3 and Supplementary Tables 1–3

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