Humanmethylation27 beadchip

For all subjects, peripheral blood samples were collected at the visit time. The buffy coat and plasma samples were separated within two hours and stored at -80°C immediately. DNA was extracted from the buffy coat using Qiagen^® QiAamp^® Blood Kit. The HumanMethylation27 BeadChip and the HumanMethylation450 BeadChip from Illumina (San Diego, CA, USA) were used for the genome wide DNA methylation profiling in the initial and the second EWAS panels respectively, as described previously (Wang et al., 2010 (link); Xu et al, 2013 (link)). The methylation levels of the selected CpG sites for replications were determined by pyrosequencing technology (Wang et al, 2010 (link)), which is based on the principle of sequencing by synthesis. For methylation analysis, pyrosequencing not only offers individual methylation high-resolution results for all CpG sites, but also has built-in control for bisulfite treatment to improve accuracy and reproducibility. For each CpG site, a 100–350-bp region was amplified by polymerase chain reaction (PCR) using a pair of primers complementary to the bisulfite-treated DNA sequence. Therefore, in addition to the targeted CpG site, the pyrosequencing assay may involve several surrounding CpG sites.

Illumina Human Methylation27 Beadchip data on HGSOC from The Cancer Genome Atlas data portal (http://cancergenome.nih.gov/dataportal) (“TCGA Cohort”) was used for independent validation of correlations observed in the Hammersmith cohort. Level 2 expression data on Affymetrix HGU133A microarrays, level 3 methylation data and annotated clinical data were obtained. The expression microarray data was pre-processed and normalised across samples.