Probing chromatin accessibility with small molecule DNA intercalation and nanopore sequencing

Genome-wide identification of chromatin organization and structure has been generally probed by measuring accessibility of the underlying DNA to nucleases or methyltransferases. These methods either only observe the positioning of a single nucleosome or rely on large enzymes to modify or cleave the DNA. We developed adduct sequencing (Add-seq), a method to probe chromatin accessibility by treating chromatin with the small molecule angelicin, which preferentially intercalates into DNA not bound to core nucleosomes. We show that Nanopore sequencing of the angelicin-modified DNA is possible and allows visualization and analysis of long single molecules with distinct chromatin structure. The angelicin modification can be detected from the Nanopore current signal data using a neural network model trained on unmodified and modified chromatin-free DNA. Applying Add-seq to Saccharomyces cerevisiae nuclei, we identified expected patterns of accessibility around annotated gene loci in yeast. We also identify individual clusters of single-molecule reads displaying different chromatin structure at specific yeast loci, which demonstrates heterogeneity in the chromatin structure of the yeast population. Thus, using Add-seq, we are able to profile DNA accessibility in the yeast genome across long molecules.全基因组范围内的染色质组织与结构鉴定，通常通过检测基因组DNA对核酸酶或甲基转移酶的可及性来实现。此类方法要么仅能观测单个核小体的定位，要么依赖大型酶类对DNA进行修饰或切割。我们开发了加合物测序（adduct sequencing, Add-seq），该方法通过小分子补骨脂素（angelicin）处理染色质来检测染色质可及性——补骨脂素可优先插入未与核心核小体结合的DNA区域。我们证实，对经补骨脂素修饰的DNA进行纳米孔（Nanopore）测序是可行的，该技术可实现具有不同染色质结构的长读长单分子的可视化与分析。借助基于未修饰及无染色质的修饰DNA训练得到的神经网络模型，可从纳米孔电流信号数据中识别补骨脂素修饰位点。我们将加合物测序应用于酿酒酵母（Saccharomyces cerevisiae）细胞核，成功在酵母已注释的基因座周围检测到预期的染色质可及性模式。此外，我们还在特定酵母基因座处鉴定到显示出不同染色质结构的单分子读段簇，这表明酵母群体的染色质结构存在异质性。综上，借助加合物测序技术，我们可基于长读长单分子对酵母基因组的DNA可及性进行全景分析。