Multiomic genomic mapping with long read sequencing

使用长读长测序进行多组基因组作图

• 批准号: 10685064
• 负责人: JONATHAN MICHAEL BURG
• 金额: $ 127.29万
• 依托单位: EPICYPHER, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10685064
• 关键词: Multiomic; genomic; mapping; long; read

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.

项目概要 组蛋白翻译后修饰 (PTM)、染色质相关蛋白的基因组作图 (CAP) 和 DNA 甲基化 (DNAme) 是生物医学研究和药物开发的强大方法。 当前的基因组学检测（例如 ChIP-seq、CUT&RUN）依赖于第二代短读长测序 (SRS)， 短读长 (<500bp) 限制了 a) 分析单个 DNA 表观基因组特征一致性的能力 分子区域和 b) 映射到基因组的重复序列。 能够对单个分子的长读长（>10kb，甚至>100kb）进行测序，并准备好 通过克服 SRS 的重大限制，彻底改变基因组学。 LRS 允许使用单个分子上的特征之间的关系来解决混合内的异质性 这与临床应用高度相关，因为它可以分析特定细胞的特征。 无需进行单细胞分析（产生非常稀疏的数据）。 长区域的分析允许映射到基因组的挑战性和重复性，这在以前是 使用 SRS 进行“不可映射” 使用 LRS 开发的表观遗传图谱提供了前所未有的检测方法。 有机会破译混合群体中细胞的染色质景观，包括以前的细胞群体 然而，缺乏使用 LRS 测量表观遗传元件的分析方法。 EpiCypher 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检测平台，利用 LRS 同时分析 CUTANA-LRS 的创新在于开发了一种单次检测中的组蛋白 PTM 或 CAP 和 DNAme。 利用新型 DNA 甲基转移酶进行表观基因组图谱绘制的专有非破坏性方法 这种方法的灵感来自于相关的免疫束缚。 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以用于 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和 转录反应，包括在人类疾病的背景下，我们将优化 CUTANA-LRS。 并绘制区域多个目标，包括具有挑战性的区域，同时还对目标 2 中的原生 DNAme 进行分析。 将通过优化跨不同目标、输入、测序的稳健协议来严格开发 CUTANA-LRS 平台，并采用有针对性的丰富方法，在目标 3 中，我们将准备商业发布。 CUTANA-LRS，开发自动化协议，执行外部验证并演示临床应用。 这项工作将把 CUTANA-LRS 打造成一个革命性的平台，用于绘制和破译关系 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。