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


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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  1. Zilberman, D. & Henikoff, S. Genome-wide analysis of DNA methylation patterns. Development 134, 3959–3965 (2007).

    Article  CAS  Google Scholar 

  2. Bibikova, M. et al. High-throughput DNA methylation profiling using universal bead arrays. Genome Res. 16, 383–393 (2006).

    Article  CAS  Google Scholar 

  3. Bibikova, M. et al. Human embryonic stem cells have a unique epigenetic signature. Genome Res. 16, 1075–1083 (2006).

    Article  CAS  Google Scholar 

  4. Irizarry, R.A. et al. Comprehensive high-throughput arrays for relative methylation (CHARM). Genome Res. 18, 780–790 (2008).

    Article  CAS  Google Scholar 

  5. Zhang, X. et al. Genome-wide high-resolution mapping and functional analysis of DNA methylation in arabidopsis. Cell 126, 1189–1201 (2006).

    Article  CAS  Google Scholar 

  6. Khulan, B. et al. Comparative isoschizomer profiling of cytosine methylation: the HELP assay. Genome Res. 16, 1046–1055 (2006).

    Article  CAS  Google Scholar 

  7. Eckhardt, F. et al. DNA methylation profiling of human chromosomes 6, 20 and 22. Nat. Genet. 38, 1378–1385 (2006).

    Article  CAS  Google Scholar 

  8. Rakyan, V.K. et al. DNA methylation profiling of the human major histocompatibility complex: a pilot study for the human epigenome project. PLoS Biol. 2, e405 (2004).

    Article  Google Scholar 

  9. Taylor, K.H. et al. Ultradeep bisulfite sequencing analysis of DNA methylation patterns in multiple gene promoters by 454 sequencing. Cancer Res. 67, 8511–8518 (2007).

    Article  CAS  Google Scholar 

  10. Meissner, A. et al. Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis. Nucleic Acids Res. 33, 5868–5877 (2005).

    Article  CAS  Google Scholar 

  11. Meissner, A. et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 454, 766–770 (2008).

    Article  CAS  Google Scholar 

  12. Cokus, S.J. et al. Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452, 215–219 (2008).

    Article  CAS  Google Scholar 

  13. Lister, R. et al. Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133, 523–536 (2008).

    Article  CAS  Google Scholar 

  14. Suzuki, M.M. & Bird, A. DNA methylation landscapes: provocative insights from epigenomics. Nat. Rev. Genet. 9, 465–476 (2008).

    Article  CAS  Google Scholar 

  15. Porreca, G.J. et al. Multiplex amplification of large sets of human exons. Nat. Methods 4, 931–936 (2007).

    Article  CAS  Google Scholar 

  16. Quail, M.A. et al. A large genome center's improvements to the Illumina sequencing system. Nat. Methods 5, 1005–1010 (2008).

    Article  CAS  Google Scholar 

  17. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    Article  CAS  Google Scholar 

  18. Park, I.H. et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–146 (2008).

    Article  CAS  Google Scholar 

  19. Maherali, N. et al. A high-efficiency system for the generation and study of human induced pluripotent stem cells. Cell Stem Cell 3, 340–345 (2008).

    Article  CAS  Google Scholar 

  20. Cowan, C.A., Atienza, J., Melton, D.A. & Eggan, K. Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science 309, 1369–1373 (2005).

    Article  CAS  Google Scholar 

  21. Jones, P.A. The DNA methylation paradox. Trends Genet. 15, 34–37 (1999).

    Article  CAS  Google Scholar 

  22. Hellman, A. & Chess, A. Gene body-specific methylation on the active X chromosome. Science 315, 1141–1143 (2007).

    Article  CAS  Google Scholar 

  23. Ball, M.P. et al. Targeted and genome-scale strategies reveal gene-body methylation signatures in human cells. Nat. Biotechnol. advance online publication, doi:10.1038/nbt.1533 (29 March 2009).

  24. Dennis, G. Jr. et al. DAVID: database for annotation, visualization, and integrated discovery. Genome Biol. 4, 3 (2003).

    Article  Google Scholar 

  25. Müller, F.J. et al. Regulatory networks define phenotypic classes of human stem cell lines. Nature 455, 401–405 (2008).

    Article  Google Scholar 

  26. Hardenbol, P. et al. Multiplexed genotyping with sequence-tagged molecular inversion probes. Nat. Biotechnol. 21, 673–678 (2003).

    Article  CAS  Google Scholar 

  27. Irizarry, R.A. et al. The human colon cancer methylome shows similar hypo- and hypermethylation at conserved tissue-specific CpG island shores. Nat. Genet. 41, 178–186 (2009).

    Article  CAS  Google Scholar 

  28. Gnirke, A. et al. Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat. Biotechnol. 27, 182–189 (2009).

    Article  CAS  Google Scholar 

  29. Gardiner-Garden, M. & Frommer, M. CpG islands in vertebrate genomes. J. Mol. Biol. 196, 261–282 (1987).

    Article  CAS  Google Scholar 

  30. Mikkelsen, T.S. et al. Dissecting direct reprogramming through integrative genomic analysis. Nature 454, 49–55 (2008).

    Article  CAS  Google Scholar 

  31. Li, R., Li, Y., Kristiansen, K. & Wang, J. SOAP: short oligonucleotide alignment program. Bioinformatics 24, 713–714 (2008).

    Article  CAS  Google Scholar 

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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.

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Authors and Affiliations



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).

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