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: 10546355
• 负责人: JONATHAN MICHAEL BURG
• 金额: $ 40.64万
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10546355
• 关键词: Antibodies; Automation; Benchmarking; Biological Assay; Biomedical Research; Blood specimen; Cell Differentiation process; Cell Line; Cells; Cellular Assay; Centromere; ChIP-seq; Chimeric Proteins; Chromatin; DNA; DNA Methylation; DNA Modification Methylases; DNA sequencing; Data; Data Set; Development; Elements; Enzymes; Epigenetic Process; GTP-Binding Protein alpha Subunits, Gs; Gene Expression Regulation; Generations; Genetic Transcription; Genome; Genomic Segment; Genomic approach; Genomics; Heterogeneity; Histones; Label; Maps; Measures; Methylation; Pathway interactions; Phase; Population; Post-Translational Protein Processing; Production; Protein Methylation; Proteins; Protocols documentation; Repetitive Sequence; Resolution; Sampling; Signal Transduction; Stretching; Universities; Validation; Work; base; biomarker discovery; bisulfite sequencing; cancer cell; cell type; clinical application; clinically relevant; data analysis pipeline; drug development; epigenomics; human disease; innovation; interest; multiple omics; novel; paralogous gene; peripheral blood; preservation; research and development; response; sequencing platform; single molecule; targeted sequencing; telomere

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)， 其中短读长 (<500bp) 限制了 a) 分析单个 DNA 表观基因组特征一致性的能力 分子和 b) 映射到基因组的重复区域。第三代长读长测序（LRS）平台 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 的重大局限性，彻底改变基因组学。通过保存长段DNA， 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 人口。这与临床应用高度相关，因为它可以分析特定细胞的特征 无需进行单细胞测定（产生非常稀疏的数据）。进一步地，测序 长读长可以映射到基因组中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 的表观遗传作图分析的开发提供了前所未有的 有机会破译混合群体中细胞的染色质景观，包括以前的细胞群体 无法映射的基因组区域。然而，缺乏使用 LRS 测量表观遗传元件的测定方法。 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 同时进行分析 一次测定中的组蛋白 PTM 或 CAP 和 DNAme。 CUTANA-LRS 的创新在于开发了 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 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。在目标 1 中，我们将优化 CUTANA-LRS 绘制多个目标图谱，包括具有挑战性的区域内的目标，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。