Quantitative and highly versatile chromatin accessibility platform

定量且高度通用的染色质可及性平台

• 批准号: 10065510
• 负责人: Michael-Christopher Keogh
• 金额: $ 87.64万
• 依托单位: EPICYPHER, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-05 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10065510
• 关键词: ATAC-seq; Aging; Basic Science; Biological Assay; Biotin; Cell Line; Cell Nucleus; Cells; Chromatin; Chromatin Structure; Clinical; Cultured Cells; DNA; DNA purification; DNase I hypersensitive sites sequencing; Data; Data Set; Development; Disease; Dissection; Dose; Enzymes; Epigenetic Process; Future; Genetic Transcription; Genomics; Goals; HCT116 Cells; Hepatocyte; Histones; Human Pathology; Industry; K-562; Label; Laboratories; Letters; Libraries; MCF7 cell; Malignant Neoplasms; Maps; Methodology; Methods; Monitor; New England; Nucleosomes; Nucleotides; Pharmaceutical Preparations; Pharmacotherapy; Phase; Polymerase; Positioning Attribute; Protocols documentation; Reagent; Recombinant DNA; Reproducibility; Research; Research Contracts; Saccharomyces cerevisiae; Sampling; Series; Services; Signal Transduction; Standardization; Sum; T-Lymphocyte; Technology; Testing; Time; Tissues; Transcriptional Regulation; Tube; Validation; Work; base; bioinformatics pipeline; bioinformatics tool; biomarker development; biomarker discovery; density; drug development; drug discovery; genomic locus; inhibitor/antagonist; innovation; insight; kidney cell; nervous system disorder; response; user-friendly

PROJECT SUMMARY Chromatin structure can directly regulate gene transcription by local nucleosome positioning / accessibility. Chromatin accessibility assays thus provide a powerful insight to transcriptional activity. The ability to compare chromatin accessibility in healthy and diseased tissue is a major drive: changes in this landscape are associated with a range of human pathologies including cancer, neurological disorders, and aging. However, current chromatin accessibility assays (e.g. ATAC-seq) lack compatibility with both fixed and native (i.e. unfixed) samples. Moreover, it remains challenging to normalize samples for cross-study comparisons, which significantly limits the development of epigenetics-focused drugs and undermines their future clinical dissection (e.g. biomarker development). Here, EpiCypher is partnering with New England Biolabs (NEB) to commercialize UniNicE-seqTM (Universal Nicking Enzyme Assisted Sequencing), a breakthrough chromatin accessibility platform. In contrast to current technologies, UniNicE-seq is fully compatible with native and fixed sample workflows, as well as being highly sensitive and requiring significantly less sequencing depth (>10-fold vs. ATAC-seq). The innovation of this technology is the application of DNA nicking and polymerase enzymes to incorporate biotin-labeled nucleotides into accessible chromatin regions for subsequent DNA purification, library sequencing, and genomic mapping. We have successfully used this approach to reliably generate high quality chromatin accessibility maps in both fixed and native cells. Importantly, these datasets corroborate those generated by current approaches (ATAC-seq and DNase-seq), demonstrating strong proof of concept for UniNicE-seq. Here, our goal is to commercialize UniNicE-seq kits and assay services. EpiCypher is an industry leader in the development of spike-in controls for epigenetics-focused genomic analyses. In Aim 1, we will leverage this expertise to develop recombinant DNA-based spike-in controls for quantitative UniNicE-seq assay normalization. This approach is essential to standardize assay methodology and for reliable cross-sample comparisons. In Aim 2, we will develop and rigorously validate our fully quantitative UniNicE-seq assays in a range of cell and tissue types, using both native and fixed sample workflows. This Aim will also include the development of user-friendly bioinformatic tooling to perform “one-click” analyses of UniNicE-seq samples (including sample normalization and comparisons). In Aim 3, we will develop and validate UniNicE-seq beta kits for commercial launch and end-to-end assay services. Together, these Aims will provide key reagents, methods, and application data as we begin to market our UniNicE-seq kits and end-to-end contract research services for chromatin research and drug discovery.

项目概要 染色质结构可通过局部核小体定位直接调控基因转录 / 因此，染色质检测的可及性为转录活性提供了强有力的洞察。 比较健康组织和患病组织中染色质可及性的能力是一个主要驱动力：这方面的变化 景观与一系列人类疾病有关，包括癌症、神经系统疾病和衰老。 然而，当前的染色质可及性测定（例如 ATAC-seq）缺乏与固定和天然的兼容性 （即未固定的）样本，标准化样本以进行交叉研究比较仍然具有挑战性， 这极大地限制了表观遗传学药物的开发并损害了它们未来的临床 解剖（例如生物标志物开发）。 EpiCypher 正在与 New England Biolabs (NEB) 合作，将 UniNicE-seqTM 商业化 （通用切口酶辅助测序），突破性的染色质可及性平台。 与当前技术相比，UniNicE-seq 与本机和固定样本工作流程完全兼容 其灵敏度高，并且需要的测序深度显着降低（与 ATAC-seq 相比，>10 倍）。 该技术的创新之处在于应用 DNA 切口和聚合酶来整合 将生物素标记的核苷酸插入可接近的染色质区域，用于后续 DNA 纯化、文库 我们已经成功地使用这种方法来可靠地生成高质量的结果。 重要的是，这些数据集证实了固定细胞和天然细胞中的染色质可及性图。 由当前方法（ATAC-seq 和 DNase-seq）生成，展示了强有力的概念证明 UniNicE-seq 在这里，我们的目标是将 UniNicE-seq 试剂盒和 EpiCypher 检测服务商业化。 作为开发用于表观遗传学基因组分析的掺入对照的行业领导者，我们的目标 1。 将利用这一专业知识开发基于重组 DNA 的掺入对照，用于定量 UniNicE-seq 该方法对于标准化测定方法和可靠的交叉样本至关重要。 在目标 2 中，我们将开发并严格验证我们的完全定量 UniNicE-seq 检测。 范围的细胞和组织类型，使用天然和固定样本工作流程。 开发用户友好的生物信息学工具来对 UniNicE-seq 样本进行“一键式”分析 （包括样本标准化和比较）在目标 3 中，我们将开发和验证 UniNicE-seq beta 试剂盒。 这些目标将共同提供关键的试剂、方法、 和应用数据，因为我们开始销售我们的 UniNicE-seq 试剂盒和端到端合同研究服务 染色质研究和药物发现。

项目成果

One-pot universal NicE-seq: all enzymatic downstream processing of 4% formaldehyde crosslinked cells for chromatin accessibility genomics.

• DOI: 10.1186/s13072-021-00427-2
• 发表时间: 2021-12-11
• 期刊: Epigenetics & chromatin
• 影响因子: 3.9
• 作者: Vishnu US;Estève PO;Chin HG;Pradhan S
• 通讯作者: Pradhan S

NicE-viewSeq: An Integrative Visualization and Genomics Method to Detect Accessible Chromatin in Fixed Cells.

NicE-viewSeq：一种用于检测固定细胞中可接近染色质的综合可视化和基因组学方法。

• DOI: 10.1007/978-1-0716-2899-7_16
• 发表时间: 2023
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Estève,Pierre-Olivier;Vishnu,UdayakumarS;Chin,HangGyeong;Pradhan,Sriharsa
• 通讯作者: Pradhan,Sriharsa

Universal NicE-Seq: A Simple and Quick Method for Accessible Chromatin Detection in Fixed Cells.

通用 NicE-Seq：一种简单快速的固定细胞染色质检测方法。

• DOI: 10.1007/978-1-0716-2899-7_3
• 发表时间: 2023
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Chin,HangGyeong;Vishnu,UdayakumarS;Sun,Zhiyi;Ponnaluri,VKChaithanya;Zhang,Guoqiang;Xu,Shuang-Yong;Benoukraf,Touati;Cejas,Paloma;Spracklin,George;Estève,Pierre-Olivier;Long,HenryW;Pradhan,Sriharsa
• 通讯作者: Pradhan,Sriharsa