Experimental and bioinformatics platform for epigenome analysis using nanopore sequencing

项目总结/摘要 基因组的表观遗传状态，包括核小体占有率、染色质可及性和DNA甲基化， 与从转录到DNA复制的DNA模板生物过程的调节高度相关， 修复.用于表征表观基因组的大多数现有技术利用下一代测序（NGS） 在捕获信号的生化反应之后（例如，亚硫酸氢盐转化、交联和 染色质免疫沉淀/ChIP）。这些技术的发展大大加速了 研究不同的表观遗传学事件，并导致了许多重要的生物学发现。为新任 纳米孔测序技术已被优化以传递单个DNA的稳健测序数据 具有长阅读长度的分子，它带来了新的可辨别的信息，这对于解决某些 具有挑战性但重要的表观遗传学问题。我们开发了一个实验方案MeSMLR-seq和一个 一系列的生物信息学方法来定义多种类型的表观遗传事件，包括核小体 占据，染色质可及性和DNA甲基化，在很少探索的基因组区域和生物 通过利用纳米孔测序的独特信息，利用这些实验和 生物信息学方法，我们的目标是询问以下问题，在表观遗传重编程 早期胚胎发育和原始生殖细胞发育：在目标1中，我们将研究表观遗传 转座因子表达和转座的调控。目标2将构建等位基因特异的 表观基因组，并鉴定等位基因之间具有显著表观基因组差异的基因组位点。目标3将 确定DNA链之间表观基因组的显著差异，并研究其调控作用 和动力学之间的联系这些研究预计将提供第一个实验性的 和生物信息学平台，以提高我们对具有复杂生物医学背景的表观基因组的理解， 全面的方式。

PROJECT SUMMARY / ABSTRACT The epigenetic status of genomes including nucleosome occupancy, chromatin accessibility and DNA methylation, is highly relevant to the regulation of DNA-template biological processes from transcription to DNA replication and repair. Most existing techniques for characterizing epigenome utilize Next Generation Sequencing (NGS) following the biochemical reactions that capture the signals (e.g., bisulfite conversion, cross-linking and chromatin immunoprecipitation/ChIP). The development of these techniques has dramatically accelerated the research of different epigenetics events and has led to many important biological findings. As the new technique nanopore sequencing has been optimized to convey robust sequencing data of single DNA molecules with long read length, it brings in new discernible information that is useful for addressing certain challenging but important epigenetics problems. We develop an experimental protocol MeSMLR-seq and a series of bioinformatics methods to define the multiple types of epigenetic events, including nucleosome occupancy, chromatin accessibility and DNA methylation, at the rarely explored genome regions and biological context by leveraging the unique information of nanopore sequencing. Utilizing these experimental and bioinformatics methods, we aim to interrogate the following problems during the epigenetic reprogramming of early embryonic development and primordial germ cell development: in Aim 1, we will study the epigenetic regulation of transposable element expression and transposition. Aim 2 will construct the allele-specific epigenome and identify the genome loci with significant epigenome difference between alleles. Aim 3 will identify significant difference of the epigenomes between DNA strands and investigate their regulatory roles and dynamics during epigenetic reprogramming. These studies are anticipated to provide the first experimental and bioinformatics platform for improve our understanding of epigenome with complex biomedical context in a comprehensive manner.