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Nucleic Acids Research 2005 33(18):5868-5877; doi:10.1093/nar/gki901
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Published online 13 October 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Article

Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis

Alexander Meissner1, Andreas Gnirke2, George W. Bell1, Bernard Ramsahoye3, Eric S. Lander1,2 and Rudolf Jaenisch1,*

1Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology, Nine Cambridge Center Cambridge, MA 02142, USA 2Broad Institute of MIT and Harvard Cambridge, MA, USA 3John Hughes Bennett Laboratory, Division of Oncology, School of Molecular and Clinical Medicine, University of Edinburgh, Western General Hospital Edinburgh, UK

*To whom correspondence should be addressed. Tel: +1 617 258 5186; Fax: +1 617 258 6505; Email: jaenisch{at}wi.mit.edu

Received August 23, 2005. Revised September 28, 2005. Accepted September 28, 2005.

We describe a large-scale random approach termed reduced representation bisulfite sequencing (RRBS) for analyzing and comparing genomic methylation patterns. BglII restriction fragments were size-selected to 500–600 bp, equipped with adapters, treated with bisulfite, PCR amplified, cloned and sequenced. We constructed RRBS libraries from murine ES cells and from ES cells lacking DNA methyltransferases Dnmt3a and 3b and with knocked-down (kd) levels of Dnmt1 (Dnmt[1kd,3a–/–,3b–/–]). Sequencing of 960 RRBS clones from Dnmt[1kd,3a–/–,3b–/–] cells generated 343 kb of non-redundant bisulfite sequence covering 66212 cytosines in the genome. All but 38 cytosines had been converted to uracil indicating a conversion rate of >99.9%. Of the remaining cytosines 35 were found in CpG and 3 in CpT dinucleotides. Non-CpG methylation was >250-fold reduced compared with wild-type ES cells, consistent with a role for Dnmt3a and/or Dnmt3b in CpA and CpT methylation. Closer inspection revealed neither a consensus sequence around the methylated sites nor evidence for clustering of residual methylation in the genome. Our findings indicate random loss rather than specific maintenance of methylation in Dnmt[1kd,3a–/–,3b–/–] cells. Near-complete bisulfite conversion and largely unbiased representation of RRBS libraries suggest that random shotgun bisulfite sequencing can be scaled to a genome-wide approach.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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