Multiplex mapping of chromatin accessibility and DNA methylation within targeted single molecules identifies epigenetic heterogeneity in neural stem cells and glioblastoma

Human tumors are comprised of heterogeneous cell populations that display diverse molecular and phenotypic features. To examine the extent to which epigenetic differences contribute to intratumoral cellular heterogeneity, we have developed a high-throughput method, termed MAPit-patch. The method uses multiplexed amplification of targeted sequences from sub-microgram input quantities of genomic DNA followed by next generation bisulfite sequencing. This provides highly scalable and simultaneous mapping of chromatin accessibility and DNA methylation on single molecules at high resolution. Long sequencing reads from targeted regions maintains the structural integrity of epigenetic information and provides substantial depth of coverage, detecting for the first time minority subpopulations of epigenetic configurations formerly obscured by existing genome-wide and population-ensemble methodologies. Analyzing a cohort of 71 promoters of genes with exons commonly mutated in cancer, MAPit-patch uncovered several differentially accessible and methylated promoters that are associated with altered gene expression between neural stem cell (NSC) and glioblastoma (GBM) cell populations. Additionally, substantial epigenetic heterogeneity was observed across the sequenced molecules at individual promoters, indicating the presence of epigenetically distinct cellular subpopulations.