Infinium methylation 450k

Peripheral whole blood DNA was extracted using standard techniques. Following bisulfite conversion, DNA methylation analysis of the samples was performed using the Illumina Infinium methylation 450k or EPIC bead chip arrays (San Diego, CA), according to the manufacturer’s protocol. The resulting methylated and unmethylated signal intensity data were imported into R 3.4.2 for analysis. Normalization was performed using Illumina normalization method with background correction using the minfi package^{40 (link)}. Probes with detection p value > 0.01, those located on chromosomes X and Y, those known to contain single-nucleotide polymorphisms at the CpG interrogation or single-nucleotide extension, and probes known to cross-react with chromosomal locations other than their target regions were removed. Arrays with more than 5% failed probe rate were excluded from the analysis. All of the samples were examined for genome-wide methylation density, and those deviating from a bimodal distribution were excluded. Factor analysis using a principal component analysis was performed to rule out batch effect or unexplained variations. Outliers were identified in this analysis and removed. No batch effect was observed by factor analysis. All of the patients and controls clustered together and did not separate based on the batch variable.

DNA methylation and copy number analyses were performed using the Infinium Methylation450k and EPIC BeadChip array platforms (Illumina, USA). All analyses were performed according to the manufacturer’s instructions. In brief, DNA was extracted from FFPE tumor samples using the Maxwell RSC FFPE Plus DNA Purification Kit (Promega, USA). After bisulfite conversion using the Zymo EZ Methylation Kit (Zymo Research Irvine, USA), the Infinium HD FFPE DNA Restore Kit was used for DNA restoration. The beadchips were scanned on the iScan system (Illumina, USA). The unprocessed output data (.idat files) from the iScan reader were checked for general quality measures as indicated by the manufacturer. DNA methylation based classification was performed for both 450 k and 850 k data using the publically available “brain tumor classifier”, version v11b4 (https://www.molecularneuropathology.org/mnp) [7 (link)].