Genome-wide DNA Methylation Profiling of TET2 Mutations

DNA was extracted from HEL cell clones using a DNA Mini Kit (QIAGEN) and sent for processing and hybridization to Infinium HumanMethylation450K BeadChips (Illumina) by Eurofins Genomics. Data processing and analysis were performed according to an established workflow (59 (link)). Specifically, raw intensity data files containing methylated (M) and unmethylated (U) intensity measurements were imported into R, and the minfi Bioconductor package (60 (link)) was used to calculate detection P values (detP), normalize data (using the preprocessFunnorm function), and generate β (β = M/[M+U+100]) and M (M = log₂[M/U]) values for individual CpG probes. Poorly performing probes (detP < 0.01) and those interrogating SNPs were removed, leaving 410,811 probes in the final data set. The limma Bioconductor package (61 (link)) was used to identify significantly differentially methylated probes based on TET2 mutation status (monoallelic versus biallelic) using M values. Resulting P values were adjusted to control for FDR (5%) (58 ), and significantly differentially methylated CpGs were defined as those with FDR-adjusted P < 0.05 and |log₂FC| ≥ 2. Unsupervised hierarchical clustering based on M values was performed in R with scaling by SD.