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: 9911357
• 负责人: Michael-Christopher Keogh
• 金额: $ 91.04万
• 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 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9911357
• 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 染色质结构可通过局部核小体定位直接调控基因转录/ 可访问性。因此，染色质可及性测定提供了对转录活性的有力洞察。 比较健康和患病组织中染色质可及性的能力是一个主要的驱动力： 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，目前的染色质可及性测定（例如ATAC-seq）缺乏与固定的和天然的染色质可及性测定的相容性。 (i.e.未固定的）样品。此外，将样本标准化以进行交叉研究比较仍然具有挑战性， 这大大限制了以表观遗传学为重点的药物的发展，并破坏了它们未来的临床应用。 解剖（例如生物标志物开发）。 在这里，EpiCypher与新英格兰Biolabs（NEB）合作，将UniNiceE-seqTM商业化 （通用切口酶辅助测序），一个突破性的染色质可及性平台。在 与现有技术相比，UniNiceE-seq还完全兼容原生和固定样品工作流程， 因为其是高度敏感的并且需要显著更少的测序深度（>10倍于ATAC-seq）。的 这项技术的创新是应用DNA切口和聚合酶来整合 生物素标记的核苷酸进入可接近的染色质区域，用于随后的DNA纯化、文库 测序和基因组作图。我们已经成功地使用这种方法来可靠地生成高质量的 固定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 通过当前方法（ATAC-seq和DNase-seq）生成，证明了 UniNiceE-seq.在这里，我们的目标是商业化UniNiceE-seq试剂盒和检测服务。EpiCypher是一个 在开发用于表观遗传学基因组分析的加标质控品方面处于行业领先地位。目标1： 将利用这一专业知识开发基于重组DNA的定量UniNiceE-seq加标质控品 测定标准化。这种方法对于标准化分析方法和可靠的交叉样品分析至关重要。 比较。在目标2中，我们将开发并严格验证我们的完全定量UniNiceE-seq检测方法， 一系列细胞和组织类型，使用天然和固定样品工作流程。这一目标还将包括 开发用户友好的生物信息学工具，对UniNiceE-seq样本进行“一键”分析 （包括样品标准化和比较）。在目标3中，我们将开发和验证UniNiceE-seq beta试剂盒 用于商业发射和端到端分析服务。总之，这些目标将提供关键的试剂，方法， 和应用数据，因为我们开始营销我们的UniNiceE-seq试剂盒和端到端的合同研究服务， 染色质研究和药物发现。