500ng of DNA from each sample was treated with sodium bisulfite in duplicate, using the EZ-96 DNA Methylation kit (Zymo Research, CA, USA). DNA methylation was quantified using the Illumina Infinium HumanMethylation450 BeadChip (Illumina Inc, CA, USA) run on an Illumina iScan System (Illumina, CA, USA) using the manufacturers’ standard protocol. Signal intensities for each probe were extracted using Illumina GenomeStudio software (Illumina, CA, USA) and imported into the R statistical programme using the methylumi and minfi packages41,42. Multidimensional scaling (MDS) plots of variable probes on the sex chromosomes were used to check that the predicted gender corresponded with the reported gender for each individual. Further data quality control and processing steps were conducted using the wateRmelon package43 in R. The pfilter function was used to filter firstly samples with >1% probes with a detection P value > 0.05 were removed and probes with a detection P value > 0.05 in at least 1% samples or/and a beadcount < 3 in 5% of samples were removed across all samples to control for poor quality probes. The dasen function was used
