Permutation-based significance analysis reduces the type 1 error rate in bisulfite sequencing data analysis of human umbilical cord blood samples

Background: The aim of this study was to detect differential methylation in umbilical cord blood that is associated with maternal and pregnancy-related variables, such as maternal age and gestational weight gain. These have been studied earlier with 450K microarrays but not with bisulfite sequencing. Methods: Reduced representation bisulfite sequencing (RRBS) analysis was performed on 200 umbilical cord blood samples. Altogether 24 clinical and technical covariates were included in a binomial mixed effects model, which was fit separately for each high-coverage CpG site, followed by spatial and multiple testing adjustment of P values. Inflation of spatially adjusted P values was discovered in a permutation analysis, which was then applied for empirical type 1 error control. Results: Empirical type 1 error control decreased the number of findings associated with each covariate to zero or a small fraction of the number that would have been discovered with standard cutoffs. In this collection of samples, some differential methylation was associated with sex, the usage of epidural anesthetic during delivery, 1 minute Apgar points, maternal age and height, gestational weight gain, maternal smoking, and maternal insulin-treated diabetes, but not with the birth weight of the newborn infant, maternal pre-pregnancy BMI, the number of earlier miscarriages, the mode of delivery, labor induction, or the cosine transformed month of birth. Conclusions: The autocorrelation-adjusted Z-test is a convenient tool for detecting differentially methylated regions, but the significance should either be determined empirically or before the spatial adjustment. With appropriate significance thresholds, the detected differentially methylated regions were reproducible across studies, technologies, and statistical models. Our RRBS data analysis workflow is available in https://github.com/EssiLaajala/RRBS_workflow. Keywords: DNA methylation, bisulfite sequencing, RRBS, umbilical cord blood, pregnancy, sex, spatial correlation, type 1 error, differential methylation, analysis workflow