Infinium methylationepic v1.0 b4 manifest file

All microarrays were scanned with an Illumina HiScan system. For the EPIC array data, we used the most current manifest file, “Infinium MethylationEPIC v1.0 B4 Manifest File,” released by Illumina on May 26, 2017 and consisting of 865,918 probes, whereas for the 450K we used the “HumanMethylation450 v1.2 Manifest File” with 485,577 probes. Both manifest files are available at https://support.illumina.com/downloads.html. In addition to unprocessed (raw) data, we used data preprocessed in three different ways (1) color corrected/background subtracted in Genome Studio (GS), (2) quantile-normalized using “preprocessQuantile” [29 (link)], (3) normal-expontential out-of-band (noob)-normalized with “preprocessNoob” [30 (link)]. Raw data and data that were to be quantile or noob normalized were uploaded directly into R from IDAT files using the ‘minfi’ package function “read.metharray” [8 ]. For the color correction/background subtracted preprocessing, data were background subtracted/color corrected with GenomeStudio, and then uploaded into R with the package ‘methylumi,’ function ‘lumiMethyR’ [5 ].

Output data following the QC and RnBeads analyses pertaining to the individual CpG sites, CpG islands, genes, promoters and tiling were shared between both cohorts, and a sample-size weighted meta-analysis was performed using METAL^{13 (link)}. Specifically, we used the FDR-adjusted p-values, the difference in the methylation means between the case and control groups (to determine the direction of effect on methylation), and the number of individuals (weights) in the sample-size weighted meta-analysis. We considered the commonly used p-value threshold of p ≤ 9.9 × 10⁻⁸ for epigenome-wide significance to identify epigenome-wide significant associations^{25 ,59 (link)}. In addition, we required differentially methylated CpG sites, islands, genes, and promoters to affect methylation levels in the same direction in both cohorts and reach epigenome-wide significance in at least one of the three adjusted models. Differentially methylated CpGs identified with p ≤ 9.9 × 10⁻⁸ were reported with CpG locations mapped to Human Genome build 37 and annotated using the Illumina Infinium MethylationEPIC v1.0 B4 Manifest File. Manhattan and Quantile-quantile (QQ) plots were drawn following meta-analysis for each adjusted model, using the R package ‘qqman’ (v0.1.8). Volcano plots were drawn using the R package ‘ggplot2’ (v3.3.2).