Humanmethylationepic beadchip

Genome-wide DNAm data profiled from whole blood samples was available for 9873 individuals in GS:SFHS using the Illumina Human-MethylationEPIC BeadChip [16 ]. DNAm data for individuals was initially released in two waves (wave 1_N = 5101; wave 2_N = 4450). Quality control (QC) and normalisation were conducted using R packages ShinyMethyl [30 (link)] and watermelon [31 (link)]. Details of the protocol are described in Supplementary Materials.

The saliva was collected using the Real Saliva DNA Sample Collection Kit (Ref. RBMSAL01) for mothers and the Pediatric Genotek DNA Sample Collection Kit OC-175 for children. The DNA was extracted using the Maxwell extraction kit (Maxwell^® 16 Buccal Swab LEV DNA Purification Kit-Cat.#AS1295, Promega Corporation, Madison, WI, USA) at The University Hospital N.S. de Candelaria (Tenerife, Spain). The quality of DNA samples was assessed with the TapeStation instrument and their concentration and purity with spectrophotometry. Library preparation and methylation sequencing were conducted at the University of Michigan Epigenomics Core in Ann Arbor, United States. We performed an EWAS using the Illumina Human Methylation EPIC BeadChip. Given that DNA derived from saliva shows cellular heterogeneity, the value of the epithelial cells was calculated using the estimated LC function from ewastools R-package [39 (link), 40 (link)], with the Houseman algorithm. The process (bisulfite conversion, hybridization, methylation value correction, probes and samples out of range removed) left us with 115 mother and child pairs and 771,785 probes of CpGs. Complete details of the procedure can be found in a previous study [13 (link)].

Blood samples used to generate the DNAm CRP score were collected at the Generation Scotland baseline appointment (between 2006 and 2011) and DNA methylation was profiled using the Illumina Human-MethylationEPIC BeadChip in two different sets. Pre-processing and quality control steps for both sets of methylation data have previously been fully reported (Madden et al., 2020 , Stevenson et al., 2020 (link)).
Full details of the calculation of the DNAm CRP score in GS have been reported previously (Stevenson et al., 2020 (link)). Briefly, methylation beta values were extracted for 6 CpG sites shown to have the strongest evidence of a functional association with serum CRP levels as shown by Lighthart and colleagues (n = 8863 and 4111 of European and African ancestries, respectively) (Ligthart et al., 2016 (link)). One of the 7 CpG sites (cg06126421) in the original study was unavailable in the GS dataset and was therefore not included in the current analysis resulting in 6 CpG sites. The beta values for the six CpG sites associated with serum CRP were then multiplied by their respective regression weights and summed to generate a single score for each STRADL participant (Stevenson et al., 2020 (link)). As all the EWAS regression weights were negative, a higher DNAm CRP score corresponds to a score closer to zero.

DNA was extracted from bulk brain tissue using the DNeasy extraction kit from Qiagen (Qiagen, Hilden, Germany). The genomic DNA samples were stored at −20 °C. For the microarray analysis, the samples were randomized based on AUD case/control status and sex, and pipetted on processing plates. Due to the sample and different group sizes, samples from each brain region were processed on separate plates. Epigenome-wide methylation levels were determined using the Illumina HumanMethylationEPIC Beadchip and Illumina HiScan array scanning systems (Illumina, San Diego, CA).

The DNA methylation samples were measured using the Illumina HumanMethylation EPIC beadchip, which includes more than 850,000 CpGs. We pre-processed the samples using the SeSAMe 2 pipeline described in Welsh et al. (2023), which was found to perform the best and produced the largest percentage of reliable CpG probes in a recent comparison of various pre-processing and normalization pipelines [17 ]. Supplementary Table 2 shows the number of CpGs at each pre-processing step.
First, we removed CpGs that overlap with single nucleotide polymorphism (SNP), non-CpG probes, cross-reactive probes [18 (link)], and probes located on X or Y chromosomes. Samples and probes were further filtered using the iterative Greedy-cut algorithm (with a p-value threshold of 0.01) in RnBeads R package, which iteratively removes the probe or sample with the highest fraction of unreliable measurements one at a time [19 (link)]. Next, we removed additional probes that had missing values in more than 5% of samples or were masked by the pOOBAH (P-value with out-of-band array hybridization) algorithm in SeSAMe R package in more than 20% of samples. Finally, we performed noob (normal-exponential using out-of-band probes) background correction and a non-linear dye-bias correction [20 (link)]. These analyses were performed using the RnBeads and SeSAMe R packages.

Peripheral whole blood was collected by the lancet and capillary method into lysis buffer and DNA extract, and 500 ng of DNA of bisulfite converted using the EZ DNA Methylation kit (Zymo Research) according to the manufacturer’s instructions. Bisulfite-converted DNA samples were randomly assigned to a chip well on the Infinium HumanMethylationEPIC BeadChip, amplified, hybridized onto the array, stained, washed, and imaged with the Illumina iScan SQ instrument to obtain raw image intensities. DNA methylation data for longitudinal sampling of participant’s pre- and post-COVID-19 and pre- and post-vaccination time points were assayed for each participant at separate times.

DNA was isolated from 1 to 2 million cells using DNAQuik DNA Extraction protocol and the Qiagen DNeasy Kit. 300 ng of DNA was treated with sodium bisulfite using Zymo EZ-96 DNA Methylation Kit. The methylation of ~850,000 CpG sites was determined using Illumina Human MethylationEPIC BeadChip, and data preanalyzed by GenomeStudio 2011.1.