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Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming

Nature Biotechnology volume 27, pages 353360 (2009) | Download Citation



Current DNA methylation assays are limited in the flexibility and efficiency of characterizing a large number of genomic targets. We report a method to specifically capture an arbitrary subset of genomic targets for single-molecule bisulfite sequencing for digital quantification of DNA methylation at single-nucleotide resolution. A set of ~30,000 padlock probes was designed to assess methylation of ~66,000 CpG sites within 2,020 CpG islands on human chromosome 12, chromosome 20, and 34 selected regions. To investigate epigenetic differences associated with dedifferentiation, we compared methylation in three human fibroblast lines and eight human pluripotent stem cell lines. Chromosome-wide methylation patterns were similar among all lines studied, but cytosine methylation was slightly more prevalent in the pluripotent cells than in the fibroblasts. Induced pluripotent stem (iPS) cells appeared to display more methylation than embryonic stem cells. We found 288 regions methylated differently in fibroblasts and pluripotent cells. This targeted approach should be particularly useful for analyzing DNA methylation in large genomes.

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We thank George Church, Billy Jin Li, Jay Shendure for inputs related to padlock probes; Huidong Shi, Billy Jin Li and Madeleine Ball for suggestions on methylation analysis; Ruiqiang Li for suggestions on read mapping; James Sprague for assistance on gene expression profiling, Colleen Ludka for assistance on Illumina sequencing. This work was supported by the UCSD new faculty startup fund, and partially by NIH/NIDA R01-DA025779 (to K.Z.). J.D. was sponsored by a CIRM post-doctoral fellowship.

Author information


  1. Department of Bioengineering, University of California at San Diego, La Jolla, California, USA.

    • Jie Deng
    • , Athurva Gore
    •  & Kun Zhang
  2. Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California, USA.

    • Robert Shoemaker
    •  & Wei Wang
  3. Center for the Study of Biological Complexity, Virginia Commonwealth University, Richmond, Virginia, USA.

    • Bin Xie
    •  & Yuan Gao
  4. Department of Computer Science, Virginia Commonwealth University, Richmond, Virginia, USA.

    • Yuan Gao
  5. Genomics Solution Unit, Agilent Technologies Inc., Santa Clara, California, USA.

    • Emily M LeProust
  6. Department of Anatomy, University of Wisconsin-Madison, Madison, Wisconsin, USA.

    • Jessica Antosiewicz-Bourget
    • , Junying Yu
    •  & James Thomson
  7. The Stowers Medical Institute, Harvard Stem Cell Institute and Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Dieter Egli
    •  & Kevin Eggan
  8. Massachusetts General Hospital Cancer Center and Center for Regenerative Medicine, Harvard Stem Cell Institute, Boston, Massachusetts, USA.

    • Nimet Maherali
    •  & Konrad Hochedlinger
  9. Division of Pediatric Hematology/Oncology, Children's Hospital Boston and Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

    • In-Hyun Park
    •  & George Q Daley


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K.Z. and Y.G. oversaw the project. J.D. and K.Z. designed and performed experiments related to padlock probe preparation, target capture, sequencing library construction and various validation assays. B.X. and Y.G. performed Illumina sequencing. E.M.L. provided oligonucleotide libraries. J.A.-B., D.E., N.M., I.-H.P., J.Y. G.Q.D., K.E. K.H. J.T. provided DNA/RNA from stem cells and fibroblasts. J.D., R.S., A.G. W.W., Y.G., and K.Z. performed data analysis. J.D. and K.Z wrote the manuscript.

Competing interests

K.Z. is a co-inventor in a patent application related to the method described in this publication. E.L. is an employee of Agilent Technology, which manufactures and sells oligonucleotide libraries.

Corresponding authors

Correspondence to Yuan Gao or Kun Zhang.

Supplementary information

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    Supplementary Text and Figures

    Supplementary Figures 1–8, Supplementary Tables 1–6,8.

Excel files

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    Supplementary Table 7

    Sequences and annotations of all padlock probes.

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    Supplementary Data

    Perl scripts and related data files for probe design and data analysis.

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