Simultaneous mapping of epigenetic inter-haplotype, inter-cell and inter-individual variation via the discovery of jointly regulated CpGs in pooled sequencing data

In the post-GWAS era, great interest has arisen in the mapping of epigenetic inter-individual variation towards investigating the emergence of phenotype in health and disease. Relevant DNA methylation methodologies - epigenome-wide association studies (EWAS), methylation quantitative trait loci (mQTL) mapping and allele-specific methylation (ASM) analysis - can each map certain sources of epigenetic variation and all depend on matching phenotypic/genotypic data. Here, to avoid these requirements, we developed Binokulars, a novel randomization test that identifies signatures of joint CpG regulation from reads spanning multiple CpGs. We tested and benchmarked our novel approach against EWAS and ASM on pooled whole-genome bisulfite sequencing (WGBS) data from whole blood, sperm and combined. As a result, Binokulars simultaneously discovered regions associated with imprinting, cell type- and tissue-specific regulation, mQTL, ageing and other (still unknown) epigenetic processes. To verify examples of mQTL and polymorphic imprinting, we developed JRC_sorter, another novel tool that classifies regions based on epigenotype models, which we deployed on non-pooled WGBS data from cord blood. In the future, this approach can be applied on larger pools to simultaneously map and characterise inter-haplotype, inter-cell and inter-individual variation in DNA methylation in a cost-effective fashion, a relevant pursuit towards phenome-mapping in the post-GWAS era. ### Competing Interest Statement The authors have declared no competing interest.