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.

项目摘要 染色质结构可以通过局部核小体定位直接调节基因转录 / 可访问性。因此，染色质可及性测定为转录活动提供了有力的见解。 比较健康和解剖组织中染色质可及性的能力是一个主要动力：改变 景观与包括癌症，神经系统疾病和衰老在内的一系列人类病理有关。 但是，当前的染色质可及性评估（例如ATAC-SEQ）与固定和天然 （即未固定的）样品。此外，将样本归一化以进行跨研究的比较仍然是挑战 这显着限制了以表观遗传学为中心的药物的发展，并破坏了他们未来的临床 解剖（例如生物标志物开发）。 在这里，Epicypher与新英格兰Biolabs（NEB）合作，以商业化UnInice-Seqtm （通用划痕酶辅助测序），一个突破性的染色质可及性平台。在 与当前技术形成鲜明对比的是，UnInice-Seq与本地和固定样本工作流程完全兼容 是高度敏感的，需要明显较少的测序深度（> 10倍与ATAC-SEQ）。这 这项技术的创新是DNA入口和聚合酶酶合并的应用 生物素标记的核苷酸进入可访问的染色质区域，用于随后的DNA纯化，文库 测序和基因组映射。我们已经成功地使用了这种方法来可靠地产生高质量 固定细胞和天然细胞中的染色质可及性图。重要的是，这些数据集证实了这些数据集 由当前方法（ATAC-SEQ和DNASE-SEQ）产生，证明了有力的概念证明 uninice-seq。在这里，我们的目标是将UnInice-Seq套件和测定服务商业化。 Epicypher是一个 开发以表观遗传学为重点基因组分析的尖峰控制控制的行业领导者。在AIM 1中，我们 将利用这种专业知识来开发基于重组DNA的SPIKE-IN控制，以进行定量的UnInice-Seq 测定归一化。这种方法对于标准化测定方法和可靠的跨样本至关重要 比较。在AIM 2中，我们将开发和严格验证我们在 使用天然和固定样品工作流程的细胞和组织类型范围。这个目标还将包括 开发用户友好的生物信息学工具，以执行对UnInice-Seq样品的“一键”分析 （包括样本归一化和比较）。在AIM 3中，我们将开发和验证UnInice-Seq Beta套件 用于商业发布和端到端测定服务。这些目标在一起将提供关键的试剂，方法， 和应用数据开始销售我们的UnInice-Seq套件和端到端合同研究服务 Chormetin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