DNA Methylation Analysis of Twin Samples

Genomic DNA from peripheral blood was bisulfite converted using the EZ DNA methylation Kit (ZYMO research), and DNAm status was assessed using the Infinium 450 K HumanMethylation BeadChip (Illumina) according to the manufacturer’s instructions at the Norwegian Microarray Consortium in Oslo. In order to minimize the batch effect on intra-pair DNAm differences, co-twins were processed together on the same chip. Data normalization was done using the free R package minfi, which employs subset quantile within-array normalization (24 (link)). The level of DNAm was summarized by calculating the “beta” value defined by the Illumina’s formula as β = M/(M + U + 100). We also performed QC using minfi to calculate the detection p-value defined as the proportion of control probes, which have intensities greater than that probe on the same array. A β value with its assigned detection p-value >0.01 was treated as missing. CpGs with more than 5% missing data were dropped from the subsequent analysis.
To adjust for differences in cell type composition between co-twins, we applied a statistical algorithm integrated in minfi (25 (link), 26 (link)). All downstream analyses were based on this cell type adjusted dataset.