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Nucleic Acids Research Advance Access originally published online on April 22, 2009
Nucleic Acids Research 2009 37(12):3829-3839; doi:10.1093/nar/gkp260
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Nucleic Acids Research, 2009, Vol. 37, No. 12 3829-3839
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Gene Regulation, Chromatin and Epigenetics

High-resolution genome-wide cytosine methylation profiling with simultaneous copy number analysis and optimization for limited cell numbers

Mayumi Oda1, Jacob L. Glass2, Reid F. Thompson2, Yongkai Mo3, Emmanuel N. Olivier1,4, Maria E. Figueroa5, Rebecca R. Selzer6, Todd A. Richmond6, Xinmin Zhang6, Luke Dannenberg6, Roland D. Green6, Ari Melnick5, Eli Hatchwell7, Eric E. Bouhassira4, Amit Verma3, Masako Suzuki2 and John M. Greally1,2,*

1Department of Medicine (Hematology), 2Department of Genetics (Computational Genetics), 3Department of Developmental and Molecular Biologyn, 4Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, 5Division of Hematology and Medical Oncology, Weill Cornell Medical Center, 1300 York Avenue, New York, NY, 6Roche-NimbleGen Inc., 504 S. Rosa Road, Madison, WI and 7Department of Pathology, State University of New York at Stony Brook, Stony Brook, NY, USA

*To whom correspondence should be addressed. Tel: 718 678 1234; Fax: 718 678 1020; Email: jgreally{at}aecom.yu.edu

Received December 10, 2008. Revised April 6, 2009. Accepted April 7, 2009.

Many genome-wide assays involve the generation of a subset (or representation) of the genome following restriction enzyme digestion. The use of enzymes sensitive to cytosine methylation allows high-throughput analysis of this epigenetic regulatory process. We show that the use of a dual-adapter approach allows us to generate genomic representations that includes fragments of <200 bp in size, previously not possible when using the standard approach of using a single adapter. By expanding the representation to smaller fragments using HpaII or MspI, we increase the representation by these isoschizomers to more than 1.32 million loci in the human genome, representing 98.5% of CpG islands and 91.1% of refSeq promoters. This advance allows the development of a new, high-resolution version of our HpaII-tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay to study cytosine methylation. We also show that the MspI representation generates information about copy-number variation, that the assay can be used on as little as 10 ng of DNA and that massively parallel sequencing can be used as an alternative to microarrays to read the output of the assay, making this a powerful discovery platform for studies of genomic and epigenomic abnormalities.


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