This Article OPEN ACCESS ARTICLE Full Text Full Text (PDF) Supplemental Research Data All Versions of this Article: gr.7301508v1 18/5/780    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Irizarry, R. A. Articles by Feinberg, A. P. PubMed PubMed Citation Articles by Irizarry, R. A. Articles by Feinberg, A. P. Social Bookmarking                   What's this?

Methods

Comprehensive high-throughput arrays for relative methylation (CHARM)

¹ Department of Biostatistics, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205, USA; ² Department of Medicine and Center for Epigenetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA; ³ Orion Genomics, LLC, St. Louis, Missouri 63108, USA

This study was originally conceived to test in a rigorous way the specificity of three major approaches to high-throughput array-based DNA methylation analysis: (1) MeDIP, or methylated DNA immunoprecipitation, an example of antibody-mediated methyl-specific fractionation; (2) HELP, or HpaII tiny fragment enrichment by ligation-mediated PCR, an example of differential amplification of methylated DNA; and (3) fractionation by McrBC, an enzyme that cuts most methylated DNA. These results were validated using 1466 Illumina methylation probes on the GoldenGate methylation assay and further resolved discrepancies among the methods through quantitative methylation pyrosequencing analysis. While all three methods provide useful information, there were significant limitations to each, specifically bias toward CpG islands in MeDIP, relatively incomplete coverage in HELP, and location imprecision in McrBC. However, we found that with an original array design strategy using tiling arrays and statistical procedures that average information from neighboring genomic locations, much improved specificity and sensitivity could be achieved, e.g., 100% sensitivity at 90% specificity with McrBC. We term this approach "comprehensive high-throughput arrays for relative methylation" (CHARM). While this approach was applied to McrBC analysis, the array design and computational algorithms are fractionation method-independent and make this a simple, general, relatively inexpensive tool suitable for genome-wide analysis, and in which individual samples can be assayed reliably at very high density, allowing locus-level genome-wide epigenetic discrimination of individuals, not just groups of samples. Furthermore, unlike the other approaches, CHARM is highly quantitative, a substantial advantage in application to the study of human disease.

⁵ Corresponding authors.

E-MAIL afeinberg{at}jhu.edu; (410) 614-9819.

E-mail rafa{at}jhu.edu; fax (410) 955-0958.

[Supplemental material is available online at www.genome.org.]

Article published online before print. Article publication date are at http://www.genome.org/cgi/doi/10.1101/gr.7301508.

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?