High-throughput DNA methylation profiling using universal bead arrays

Abstract

We have developed a high-throughput method for analyzing the methylation status of hundreds of preselected genes simultaneously and have applied it to the discovery of methylation signatures that distinguish normal from cancer tissue samples. Through an adaptation of the GoldenGate genotyping assay implemented on a BeadArray platform, the methylation state of 1536 specific CpG sites in 371 genes (one to nine CpG sites per gene) was measured in a single reaction by multiplexed genotyping of 200 ng of bisulfite-treated genomic DNA. The assay was used to obtain a quantitative measure of the methylation level at each CpG site. After validating the assay in cell lines and normal tissues, we analyzed a panel of lung cancer biopsy samples (N = 22) and identified a panel of methylation markers that distinguished lung adenocarcinomas from normal lung tissues with high specificity. These markers were validated in a second sample set (N = 24). These results demonstrate the effectiveness of the method for reliably profiling many CpG sites in parallel for the discovery of informative methylation markers. The technology should prove useful for DNA methylation analyses in large populations, with potential application to the classification and diagnosis of a broad range of cancers and other diseases.

High-definition CpG methylation of novel genes in gastric carcinogenesis identified by next-generation sequencing

“…24 This panel includes 1505 CpG sites from 807 cancer-related genes. We used 500 ng of genomic DNA from formalin-fixed, paraffin-embedded for each bisulfite conversion.…”

“…Methylation ratios were calculated using the Methylation Module in BeadStudio (Illumina) after normalization to a background derived by averaging the signals of an internal negative control. Data from DNA methylation assays performed with Illumina GoldenGate BeadArrays 24 were obtained as a β-value of 0.0-1.0 indicating the methylation level at each CpG site. For statistical analyses, β-values were converted to M-values using the formula M = log2 (beta/(1-beta)) to improve linearity and normality.…”

Distinct DNA methylation patterns in cirrhotic liver and hepatocellular carcinoma

“…40 The complete dataset is provided in Supporting Information S1. To assess the technical robustness, one cell line (Huh7) …”

High-content analysis of cancer genome DNA alterations

“…Several groups have been developing array-based methods for genome-wide detection of methylation or other epigenetic alterations such as histone modifications [5,41,42]. These emerging methods have not yet yielded the depth of biological insight that methods that are lower in gene content, but technologically more robust, have yielded.…”

“…to study DNA alterations on a genome-wide scale, including array CGH with very high genome resolution [4], new methylation detection systems [5], and last but not least, single-molecule sequencing technologies [6] (Box 1). In this review we focus on the recent biological discoveries that have been made with these new tools along with the large-scale efforts using older tools.…”