High definition profiling of mammalian DNA methylation by array capture and single molecule bisulfite sequencing
- Emily Hodges1,2,6,
- Andrew D. Smith1,3,6,
- Jude Kendall1,
- Zhenyu Xuan1,
- Kandasamy Ravi1,
- Michelle Rooks1,2,
- Michael Q. Zhang1,
- Kenny Ye4,
- Arindam Bhattacharjee5,
- Leonardo Brizuela5,
- W. Richard McCombie1,
- Michael Wigler1,
- Gregory J. Hannon1,2,7 and
- James B. Hicks1,7
- 1 Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA;
- 2 Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA;
- 3 Molecular and Computational Biology, University of Southern California, Los Angeles, California 90089, USA;
- 4 Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, New York 10461, USA;
- 5 Agilent Technologies, Inc., Santa Clara, California 95051, USA
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↵6 These authors contributed equally to this work.
Abstract
DNA methylation stabilizes developmentally programmed gene expression states. Aberrant methylation is associated with disease progression and is a common feature of cancer genomes. Presently, few methods enable quantitative, large-scale, single-base resolution mapping of DNA methylation states in desired regions of a complex mammalian genome. Here, we present an approach that combines array-based hybrid selection and massively parallel bisulfite sequencing to profile DNA methylation in genomic regions spanning hundreds of thousands of bases. This single molecule strategy enables methylation variable positions to be quantitatively examined with high sampling precision. Using bisulfite capture, we assessed methylation patterns across 324 randomly selected CpG islands (CGI) representing more than 25,000 CpG sites. A single lane of Illumina sequencing permitted methylation states to be definitively called for >90% of target sties. The accuracy of the hybrid-selection approach was verified using conventional bisulfite capillary sequencing of cloned PCR products amplified from a subset of the selected regions. This confirmed that even partially methylated states could be successfully called. A comparison of human primary and cancer cells revealed multiple differentially methylated regions. More than 25% of islands showed complex methylation patterns either with partial methylation states defining the entire CGI or with contrasting methylation states appearing in specific regional blocks within the island. We observed that transitions in methylation state often correlate with genomic landmarks, including transcriptional start sites and intron-exon junctions. Methylation, along with specific histone marks, was enriched in exonic regions, suggesting that chromatin states can foreshadow the content of mature mRNAs.
Footnotes
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↵7 Corresponding authors.
E-mail hicks{at}cshl.edu; fax (516) 367-8381.
E-mail hannon{at}cshl.edu; fax (516) 367-8874.
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[Supplemental material is available online at http://www.genome.org. The ChIP-seq and bisulfite sequence data from this study have been submitted to NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under series accession no. GSE17001.]
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Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.095190.109.
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- Received April 20, 2009.
- Accepted July 2, 2009.
- Copyright © 2009 by Cold Spring Harbor Laboratory Press
