Advanced DNA Methylation Analysis for Verification of Infinium HumanMethylation450 BeadChip Data

Genomewide methylation data are of increasing interest for identifying genomic regions important in cancers and other diseases. The HumanMethylation450 BeadChip from Illumina, Inc. is a widely used array for analyzing over 480,000 different CpG sites in parallel. After genomewide screening with such an array, it is important to analyze additional CpG sites within the identified target sequences in greater detail. This is particularly important when researching diseases involving heterogeneous CpG methylation within a CpG island. Therefore, a comprehensive method for analyzing methylation around an identified target with higher resolution and sensitivity is essential.

Pyrosequencing is well-established for quantifying DNA methylation at single base resolution and has been used in various studies to validate methylation data from other methods, including both HumanMethylation 27 and 450 BeadChip data. In these analyses, individual Pyrosequencing assays were designed for each identified target, a process that is time consuming when performed manually. We have developed a new assay design algorithm that is optimized for designing Pyrosequencing assays for specific methylation array targets.

We will discuss the following topics:

• Latest improvements to Pyrosequencing technology
• New predesigned assays for validating HumanMethylation450 BeadChip targets
• Results from starting materials including cell lines and fresh/frozen and FFPE tissues