Multiomic genomic mapping with long read sequencing

项目总结 组蛋白翻译后修饰(PTM)、染色质相关蛋白的基因组图谱 DNA甲基化(DNAME)是生物医学研究和药物开发的一种强有力的方法。 当前的基因组分析(例如，芯片序列、切割和运行)依赖于第二代短读测序(SRS)， 其中短读数(&lt；500bp)限制了a)分析单个DNA上表观基因组特征的一致性的能力 分子和b)映射到基因组的重复区域。第三代长读测序(LRS)平台 能够对单个分子的长读数(10kb，甚至100kb)进行测序，并有望 通过克服SRS的显著限制，使基因组学发生革命性变化。通过保存较长的DNA片段， LRS允许使用单个分子上的特征之间的关系来解决混合分子中的异质性 人口。这与临床应用高度相关，因为它能够分析特定细胞的特征 不需要单细胞分析(这会产生非常稀疏的数据)。此外，排序 长阅读允许映射到基因组的具有挑战性和重复的区域，这些区域以前是 “不可映射”与SRS。使用LRS的表观遗传图谱分析的发展提供了前所未有的 破译混合种群内细胞染色质景观的机会，包括以前 无法映射的基因组区域。然而，使用LRS来测量表观遗传因素的分析是缺乏的。 在这里，EpiCypher正在与约翰·霍普金斯大学的LRS专家Winston Timp博士合作 开发Cutana-LRS，这是一个一流的多组学分析平台，利用LRS同时分析 组蛋白PTMS或CAPS和DNAME一次检测。Cutana-LRS的创新是一种 利用一种新的DNA甲基转移酶进行表观基因组图谱的专利、非破坏性方法 融合蛋白标记感兴趣的染色质特征。这种方法的灵感来自于相关的免疫拴系- EpiCypher正在开发和商业化的基于基因组图的方法(例如Cut&Run)。 在Cutana-LRS中，DNA分子被标记并完整地保存用于LRS，这将允许解析 数据类型内部/之间的异质性，并将提供对以前无法映射的基因组区域的访问。 总之，这些进展将为更好地理解基因调控机制和 转录反应，包括在人类疾病的背景下。在目标1中，我们将优化Cutana-LRS 并映射多个目标，包括在具有挑战性的地区内，同时还分析本地DNAME。在目标2中，我们 将通过优化跨不同目标、输入、测序的可靠协议来严格开发Cutana-LRS 平台，并采用有针对性的浓缩方法。在AIM 3中，我们将为商业推出做准备 Cutana-LRS，开发自动化方案，执行外部验证，并展示临床应用。 这项工作将使Cutana-LRS成为绘制和破译关系的革命性平台 在多种类型的染色质特征之间，可以访问以前无法映射的区域。

PROJECT SUMMARY Genomic mapping of histone post-translational modifications (PTMs), chromatin-associated proteins (CAPs), and DNA methylation (DNAme) is a powerful approach for biomedical research and drug development. Current genomics assays (e.g. ChIP-seq, CUT&RUN) rely on second generation short-read sequencing (SRS), wherein short reads (<500bp) limit the ability to a) analyze concordance of epigenomic features on a single DNA molecule and b) map to repetitive regions of the genome. Third generation long-read sequencing (LRS) platforms are capable of sequencing long reads (>10kb, even >100kb) from a single molecule, and are poised to revolutionize genomics by overcoming the significant limitations of SRS. By preserving long stretches of DNA, LRS allows relationships between features on a single molecule to be used to resolve heterogeneity within mixed populations. This is highly relevant for clinical applications, as it enables analysis of signatures of specific cells within a sample without the need for single cell assays (which generate very sparse data). Further, sequencing of long reads allows mapping to challenging and repetitive regions of the genome, which were previously “unmappable” with SRS. Development of epigenetic mapping assays that use LRS provides an unprecedented opportunity to decipher the chromatin landscape of cells within mixed populations, including within previously unmappable genomic regions. However, assays to measure epigenetic elements using LRS are lacking. Here, EpiCypher is collaborating with LRS expert Dr. Winston Timp at Johns Hopkins University to develop CUTANA-LRS, a first-in-class multiomics assay platform that leverages LRS to simultaneously profile histone PTMs or CAPs and DNAme in a single assay. The innovation of CUTANA-LRS is the development of a proprietary, nondestructive approach for epigenomic mapping that leverages a novel DNA methyltransferase fusion protein to label chromatin features of interest. This approach was inspired by related immunotethering- based approaches for genomic mapping that EpiCypher is developing and commercializing (e.g. CUT&RUN). In CUTANA-LRS, DNA molecules are labeled and preserved intact for LRS, which will allow resolution of heterogeneity within / between data types, and will provide access to previously unmappable genomic regions. Together, these advances will provide a pathway to better understand mechanisms of gene regulation and transcriptional response, including in the context of human disease. In Aim 1, we will optimize CUTANA-LRS and map multiple targets, including within challenging regions, while also profiling native DNAme. In Aim 2, we will rigorously develop CUTANA-LRS by optimizing robust protocols across diverse targets, inputs, sequencing platforms, and incorporate a targeted enrichment approach. In Aim 3, we will prepare for commercial launch of CUTANA-LRS, develop automated protocols, perform external validation, and demonstrate a clinical application. This work will establish CUTANA-LRS as a revolutionary platform for mapping and deciphering the relationships between multiple types of chromatin features with access to previously “unmappable” regions.