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.

项目摘要 组蛋白翻译后修饰（PTM）的基因组图，染色质相关蛋白 （CAPS）和DNA甲基化（DNAME）是生物医学研究和药物开发的强大方法。 当前的基因组学测定（例如ChIP-Seq，Cut＆Run）依靠第二代短阅读测序（SRS）， 其中简短读数（<500bp）限制了a）分析单个DNA上表观基因组特征的一致性 分子和b）向基因组的重复区域地图。第三代长阅读测序（LRS）平台 能够从单个分子中测序长读（> 10kb，甚至> 100kb），并被中毒 通过克服SR的重大局限性来彻底改变基因组学。通过保留长长的DNA， LRS允许单个分子上的特征之间的关系用于解决混合物中的异质性 人群。这与临床应用高度相关，因为它可以分析特定细胞的特定标志 在不需要单细胞测定的样本中（这会产生非常稀疏的数据）。此外，测序 长期读取使映射可以挑战和重复的基因组的重复区域，以前是 SRS“无法实现”。使用LRS的表观遗传学映射评估的开发提供了前所未有的 混合种群中细胞的染色质景观的机会，包括以前 无法欣赏的基因组区域。但是，缺乏使用LRS测量表观遗传元件的测定方法。 在这里，Epicypher与Johns Hopkins University的LRS专家Winston Timp博士合作 开发Cutana-LRS，这是一种一流的多组学测定平台，利用LRS简单介绍 Hisstone PTMS或CAPS和DNAME单个测定法。 Cutana-LRS的创新是 具有新型DNA甲基转移酶的表观基因组映射的专有，非破坏性方法 融合蛋白可以标记感兴趣的染色质特征。这种方法的灵感来自相关的免疫纤维 Epicypher正在开发和商业化（例如剪切和运行）的基于基因组映射的方法。 在Cutana-LRS中，DNA分子被标记并完整地保留了LR，这将允许解决 数据类型之间 /之间的异质性，并将提供对以前无法实现的基因组区域的访问。 这些进步将共同提供更好地理解基因调节机制的途径和 转录反应，包括在人类疾病的背景下。在AIM 1中，我们将优化Cutana-lrs 并绘制多个目标，包括挑战区域内，同时还对本机dname进行了分析。在AIM 2中，我们 将通过优化潜水员目标，输入，测序的强大协议来严格开发Cutana-LRS 平台，并结合了目标富集方法。在AIM 3中，我们将为商业发布做准备 Cutana-LRS，开发自动化协议，执行外部验证并演示临床应用。 这项工作将建立cutana-lrs作为绘制和破译关系的革命平台 在多种类型的染色质特征之间，可以访问以前的“无法实现”区域。