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. Overall design: This study includes 4 samples, NSC probed with 100U M.CviPI and 0U control; GBM probed with 100U M.CviPI and 0U control.

人类肿瘤由兼具多样分子与表型特征的异质性细胞群构成。为探究表观遗传差异对瘤内细胞异质性的贡献程度，本研究开发了一种名为MAPit-patch的高通量检测方法。该方法以亚微克级用量的基因组DNA为起始材料，先对靶向序列进行多重扩增，再结合下一代亚硫酸氢盐测序（next generation bisulfite sequencing）完成实验流程，可在单分子水平以高分辨率实现染色质可及性与DNA甲基化的高可扩展性同步绘制。靶向区域的长测序读段可保留表观遗传信息的结构完整性，并提供充足的测序覆盖深度，首次检测到了此前被现有全基因组及群体整体分析方法所掩盖的表观遗传构型少数亚群。针对71个携带有癌症常见突变外显子的基因启动子队列开展分析后，MAPit-patch技术在神经干细胞（neural stem cell, NSC）与胶质母细胞瘤（glioblastoma, GBM）细胞群之间，发现了多个与基因表达改变相关的差异开放启动子与差异甲基化启动子。此外，在单个启动子的测序分子中也观察到了显著的表观遗传异质性，表明存在表观遗传特征迥异的细胞亚群。实验整体设计：本研究共包含4个样本，分别为经100U M.CviPI处理的神经干细胞样本及0U对照样本，以及经100U M.CviPI处理的胶质母细胞瘤样本及0U对照样本。