ABSTRACT: 204 placentas from three cohorts were processed for DNAme arrays in Vancouver, Canada. The three cohorts consisted of: (i) V-NORM, a normative population of term pregnancies recruited at BC Women’s Hospital (BCWH) in Vancouver, Canada (n=35), as part of a study on Epigenetics in Pregnancy Complications (EPIC); ii) V-SSRI, a population of pregnant individuals recruited in Vancouver, Canada in the 20th week of gestation (n=64), with/without clinical depression, and with/without the use of selective serotonin reuptake inhibitors (SSRIs); and (iii) QF-2011, a population of term pregnancies that were exposed to sudden-onset stress during gestation due to catastrophic flooding in the Australian state of Queensland in early January 2011 (n=105). DNA samples from all three cohorts were run on Illumina Infinium MethylationEPIC arrays in Vancouver, BC, Canada. Processing included DNA purification after extraction using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA), bisulfite conversion using the EZ DNAme Kit (Zymo Research, Orange, CA, USA), and hybridization to and processing of the Illumina Infinium MethylationEPIC BeadChip arrays according to the manufacturer’s protocol (Illumina, San Diego, CA, USA). Samples from the three cohorts were randomized and run in 3 array batches across 44 eight-sample chips as illustrated in Supp Figure 5. In the case of the V-SSRI and QF-2011 cohorts, sample exposure groups (SSRI exposed/non-exposed and objective flood stress high/low) and infant sex were carefully distributed across array chips (1-44) and rows (1-8) to minimize potential batch effects. DNAme data from raw IDAT files were read into R v4.2.2(54) and annotated with the Illumina Infinium MethylationEPIC v1.0 B4 Manifest. Several data quality control checks were undertaken using the R packages minfi(55,56), wateRmelon(57,58), and ewastools(59). First, each sample was assessed at 17 Illumina control probes to evaluate bisulfite conversion efficiency and array run quality; all samples passed the manufacturer-recommended thresholds at the control probes. Next, average total (methylated + unmethylated) fluorescence intensity was assessed between samples, and between array batches. All samples had similar total fluorescence, though samples run on the EPIC array in Batch 3 had slightly higher average intensities than those in Batch 1 and 2. Sample sex was assessed with the ewastools package(59), using the mean total fluorescence intensity (methylated + unmethylated) of the X and Y chromosome probes, normalized to the per-sample mean autosomal total fluorescence intensity, and was confirmed to match the clinically-reported sex of the infant in all cases. Sample genetic identity was assessed using the 59 SNP (‘rs’) probes on the EPIC array with the call_genotypes() and enumerate_sample_donors() functions (ewastools)(59). Finally, DNAme beta value density plots of all samples were visually assessed to determine overall similarity of the beta value distributions between samples, with no outliers identified.
