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

Abstract

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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Figure 1: Targeted bisulfite sequencing with padlock probes.
Figure 2: Normalization of padlock-capturing efficiency.
Figure 3: Patterns of CpG methylation in fibroblasts and pluripotent cells.

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Acknowledgements

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.

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Authors and Affiliations

Authors

Contributions

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.

Corresponding authors

Correspondence to Yuan Gao or Kun Zhang.

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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.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Tables 1–6,8. (PDF 993 kb)

Supplementary Table 7

Sequences and annotations of all padlock probes. (XLS 12767 kb)

Supplementary Data

Perl scripts and related data files for probe design and data analysis. (ZIP 19536 kb)

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Deng, J., Shoemaker, R., Xie, B. et al. Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nat Biotechnol 27, 353–360 (2009). https://doi.org/10.1038/nbt.1530

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  • DOI: https://doi.org/10.1038/nbt.1530

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