Multiomic genomic mapping with long read sequencing

项目摘要 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有力方法。 目前的基因组学测定（例如ChIP-seq、CUT&RUN）依赖于第二代短读测序（SRS）， 其中短读段（<500 bp）限制了以下能力：a）分析单个DNA上的表观基因组特征的一致性， 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 能够对来自单个分子的长读段（> 10 kb，甚至> 100 kb）进行测序，并且准备 通过克服SRS的重大局限性来彻底改变基因组学。通过保存DNA的长片段， LRS允许使用单个分子上的特征之间的关系来解析混合细胞内的异质性。 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 而不需要单细胞测定（其产生非常稀疏的数据）。此外，测序 长读段允许映射到基因组的具有挑战性和重复性的区域，这些区域以前是 使用SRS“无法映射”。使用LRS的表观遗传作图测定的发展提供了前所未有的 有机会破译混合群体中细胞的染色质景观，包括以前的 无法定位的基因组区域然而，缺乏使用LRS测量表观遗传元件的测定方法。 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，这是一流的多组学检测平台，利用LRS同时分析 组蛋白PTM或CAP和DNAme。CUTANA-LRS的创新是一种 利用新型DNA甲基转移酶进行表观基因组作图的专有非破坏性方法 融合蛋白来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作图的方法（例如CUT&RUN）。 在CUTANA-LRS中，DNA分子被标记并完整保存用于LRS，这将允许解析 数据类型内/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提供一条途径， 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 和映射多个目标，包括在挑战性区域内，同时也分析天然DNAme。在目标2中， 将通过优化不同靶点、输入、测序的稳健方案，严格开发CUTANA-LRS 平台，并纳入有针对性的富集方法。在目标3中，我们将为商业推出 CUTANA-LRS，开发自动化方案，执行外部确认，并演示临床应用。 这项工作将建立CUTANA-LRS作为一个革命性的平台，映射和破译的关系 在多种类型的染色质特征之间，可以访问以前“不可映射”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.