Comprehensive high-throughput arrays for relative methylation (CHARM)

TLDR

The study was designed to rigorously compare the specificity of three high‑throughput DNA methylation array methods (MeDIP, HELP, and McrBC) and to introduce the comprehensive high‑throughput arrays for relative methylation (CHARM) platform. The authors validated the methods using 1,466 Illumina probes on the GoldenGate assay and quantitative methylation pyrosequencing, and developed CHARM’s tiling array design and statistical algorithms to average neighboring genomic information. While MeDIP, HELP, and McrBC each have biases—CpG island enrichment, incomplete coverage, and location imprecision—the CHARM approach, validated with Illumina probes and pyrosequencing, achieves near‑perfect sensitivity (≈100%) at 90% specificity, is method‑independent, cost‑effective, and provides highly quantitative, locus‑level genome‑wide epigenetic discrimination.

Abstract

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.