450k beadchip

In both PACE and CHARGE, DNA methylation was measured using the Illumina450K Beadchip (Illumina, Inc, CA, USA). In PACE, newborn DNA was extracted from umbilical cord blood in all cohorts except for NFCS, which used neonatal phlebotomy [3 (link)]. In CHARGE, DNA was extracted from whole blood in 14 cohorts, isolated CD4⁺ T cells in GOLDN, and monocytes (CD14⁺) in MESA [2 (link)]. In all PACE and CHARGE cohorts, DNA was subjected to bisulfite conversion using Zymo EZ DNA methylation (Zymo Research, CA, USA). Each cohort performed its own quality control removing low quality samples and low quality CpGs, and normalized their untransformed methylation β-values as previously described [2 (link),3 (link)].

De-identified saliva samples (n=45) from children were obtained from other studies. All samples were de-identified and numerically labeled. This study was approved by the UNM IRB. DNA was extracted using the Qiagen kit and submitted for testing on the Illumina 450k BeadChip. For 4 samples, DNA was analyzed by three different methods in parallel: MeDIP followed by bisulfite conversion and then sequencing by either the IonProton at the ATG facility at UNM, or the Illumina MiSeq in the lab of Darrell Dinwiddie at UNM. Parallel aliquots of the same DNA were submitted for Illumina BeadChip. Resulting sequences were aligned using Interactive Genome Viewer (GV; (http://software.broadinstitute.org/software/igv/). Results from the Illumina BeadChip were further analyzed using RNBeads. Methylation patterns of brain, keratinocytes and whole blood for cell type deconvolution were from the Genome Omnibus (GEO). Please see figure legends for more detail on methods.

Genome-wide DNA methylation analysis was performed on DNAs from 48 fresh-frozen samples. Microarray data were collected at Expression Analysis Inc. (Durham, NC, USA) using the IlluminaHumanMethylation450 Beadchip (Illumina) and preprocessed using the Bioconductor methylumi software package (version 2.14.0; PMID 18467348) as described previously.^{40 (link)} Genome-wide methylation data were deposited into the GEO database under the accession number GSE77954. Methylation values were reported as M-values (log 2 ratios of methylated to unmethylated probes). To determine probe-wise methylation scores, we associated each probe with the nearest annotated transcriptional start site, focusing on probes within the putative promoter regions of annotated genes (Supplementary Table S6).
To subgroup TCGA tumors into MLH1 methylation high/low categories, we obtained raw Illumina 450k Beadchip microarray data for 292 tumor samples from the TCGA and preprocessed it as described above. As described previously, we examined probe cg00893636 located in the CpG island of the bidirectional MLH1/EPM2AIP1 promoter. The normalized methylation scores (M-value) were transformed to z-scores by centering on the mean and scaling to unit variance. As expected, visual inspection revealed a strongly bimodal distribution and samples with positive scores were assigned the MLH1/CIMP+ subgroup label.