DNA Methylation Profiling with Illumina 450k

In brief, bisulfite conversion using the Zymo EZ DNA Methylation Kit (ZymoResearch, Orange, CA, USA) as well as subsequent hybridization of the HumanMethylation450k Bead Chip (Illumina, San Diego, CA, USA), and scanning (iScan, Illumina) were performed according to the manufacturers’ protocols by applying standard settings. DNA methylation levels (β values) were determined by calculating the ratio of intensities between methylated (signal A) and unmethylated (signal B) sites. Specifically, the β value was calculated from the intensity of the methylated (M corresponding to signal A) and unmethylated (U corresponding to signal B) sites, as the ratio of fluorescent signals β = Max(M,0)/[Max(M,0) + Max(U,0) + 100]. Thus, β values range from 0 (completely unmethylated) to 1 (completely methylated) [82 (link)]. The epigenetic clock software implements a data normalization step that repurposes the BMIQ normalization method from Teschendorff [83 ] so that it automatically references each sample to a gold standard based on type II probes as detailed in [20 (link)].