Introduction and Background
overweight or obese
DISEASE, EARLY PREGNANCY
With the advent of high-throughput assays, epigenome-wide DNA methylation studies have become more popular, and researchers are now investigating the effects on DNA methylation (DNAm) of a wide range of environmental exposures and physiological conditions, with particular interest in the contribution of epigenetic mechanisms such as DNAm to the early life origins of health and disease. The ability to perform EWAS is particularly useful in relation to conditions where associated differences in DNAm are likely to be fairly modest (We recently investigated the effect of an antenatal diet and lifestyle intervention, and of maternal early pregnancy BMI, on neonatal cord blood DNA methylation in infants of mothers who were overweight or obese in early pregnancy (While previous studies comparing different methods have also produced different findings, these have tended to consider only one element of the processing and analysis pipeline (such as normalisation, or batch correction) in isolation. In addition, they have tended to concentrate on the tendency for some methods to produce results which are likely to be spurious (false positives), while often being unable to definitively confirm this due to the lack of known truth regarding the presence and magnitude of differential methylation effects.We therefore set out to investigate the impact of different data-processing choices in a more systematic way, looking at the effect of combinations of data processing choices on findings specifically regarding statistically significant differentially methylated probes (DMPs), and of the behaviour of these combinations in cases where effects are known to be either absent or present, as well as their behaviour in relation to our effects of interest (maternal BMI and lifestyle intervention). We were able to create a scenario in which effects were known to be absent by randomly assigning samples to groupings. We could not similarly ensure a scenario where effects were known to be present (as the truth regarding the existence, location and magnitude of any effects in our samples is not known); however we investigated effects of infant sex as a rough proxy, since infant sex is known to have substantial effects on DNAm which can be detected by the 450K array (