Infinium HumanMethylation450 BeadChip Analysis

Sample DNA (1 μg) was bisulphite-converted using the EZ DNA methylation kit (Zymo Research) and analysed using the Infinium HumanMethylation450 BeadChip (Illumina Inc.). The samples and 450K BeadChips were processed according to the manufacturer’s protocol at Barts and The London Genome Centre, UK. Pre-processing of the 450K dataset was performed using Genome studio software v.2011.1 (Illumina Inc.). Quality control of bisulphite conversion was performed by calculating the ratio of unmethylated probe to methylated probe. Samples that had incomplete conversion (a ratio >0.2) were removed. Methylation status for each probe is given as a Beta value (β-value). The β-value is the ratio of the methylated probe intensity and the overall intensity (sum of the methylated and unmethylated probe intensities). Pre-processing of the data was then performed using R (version 2.15.0). Peak correction was performed [39 (link)] and probes that contained a minor allele frequency of >5 % within 50 bp of the target site were removed [59 (link)]. The Illumina annotation [3 (link)] and an enhanced annotation ([42 (link)] were added to the peak-corrected datasets (Additional file 2: Table S2). We excluded probes located on the X- and Y- chromosomes from further analysis. The dataset generated in this study has been deposited in the Gene Expression Omnibus (GEO) under accession GSE77241.