Autopure system

Whole blood samples were collected for all individuals during the follow-up assessment. Genomic DNA was extracted from buffy-coats with the use of the semi-automated salt precipitation protocol with Autopure System (Qiagen, Valencia, CA, USA). Genomic DNA (500 ng/sample) was bisulfite converted with EZ Methylation Gold Kit (Zymo Research, Irvine, CA, USA) and analyzed using the Infinium HumanMethylation450 BeadChips (Illumina, San Diego, CA, USA) array according to manufacturer's protocol. Obtained DNA methylation data was subjected to quality control with two different pipelines, combination of MethylAid (van Iterson et al., 2014 (link)) and minfi tools (Aryee et al., 2014 (link)). Probes with low bead count (<3 beads), high detection p-value (>0.01), zero signal, missing in >5% of samples and cross-reactive ones as reported before (Chen et al., 2013 (link)) were removed from the dataset, leaving 453,014 probes for further analysis. Data was normalized with the use of functional normalization (Fortin et al., 2014 (link)) and obtained beta values were further logit transformed.

DNA was isolated from buffy coats using the salt precipitation method applying either a manual protocol (Miller et al., 1988) or a semi‐automated protocol based on the Autopure System (Qiagen, Hilden, Germany). Bisulfite treatment of 500 ng genomic DNA was carried out with the EZ‐96 DNA methylation kit (Zymo Research, Orange County, CA, USA) following the manufacturer's protocol. Genomewide DNA methylation was measured using the Infinium HumanMethylation450K BeadChip (Illumina, San Diego, CA, USA) at the Leiden University Medical Center and in accordance with the manufacturer's instructions. Data preprocessing was performed using the free r package minfi (Maksimovic et al., 2012), and a DNA methylation level was summarized for each CpG site by calculating a ‘beta’ value ranging from 0 to 100%. CpG probes were treated as missing if the detection P‐value > 0.01, and CpG sites with more than 5% missing data were excluded from the analysis.
DNA methylation data have been deposited in the NCBI GEO database (http://www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE61496 and GSE73115.

The semi-automated salt precipitation protocol with Autopure System (Qiagen) was used to extract genomic DNA from leukocytes in the buffy coat. Genomic DNA (500 ng/sample) was bisulfite-converted with EZ Methylation Gold Kit (Zymo Research) and analysed using the Infinium HumanMethylation450 BeadChips (Illumina) array according to the manufacturer’s protocol. Quality control for obtained DNA methylation data was performed with two different pipelines, a combination of MethylAid [69 (link)] and minfi tools [70 (link)]. Probes with low bead count (< 3 beads), high detection p value (> 0.01), and zero signal and missing in > 5% of samples were removed from further analysis. Additionally, cross-reactive probes identified previously by Chen et al. [71 (link)] were removed from the dataset. Four hundred fifty-three thousand fourteen good quality probes remained for further EWAS analyses. Normalisation of DNA methylation data, in order to control for technical variation, was done with the use of functional normalization (FunNorm) [72 (link)], and obtained β values (the proportion of DNA methylation) were further logit-transformed giving M values  for each probe.