A scalable kit-based assay for multi-omic analyses of transcriptional protein binding and chromatin interactions from ultra-low input frozen and FFPE samples at single-cell resolution

基于试剂盒的可扩展测定，用于以单细胞分辨率对超低输入冷冻和 FFPE 样品中的转录蛋白结合和染色质相互作用进行多组学分析

• 批准号: 10487566
• 负责人: Anthony Schmitt
• 金额: $ 101.51万
• 依托单位: ARIMA GENOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-10 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10487566
• 关键词: 3-Dimensional; Address; Adoption; Algorithms; Area; Basic Science; Benchmarking; Binding; Binding Proteins; Bioinformatics; Biological; Biological Assay; Biopsy; Cell physiology; Cells; ChIP-seq; Characteristics; Chromatin; Chromatin Interaction Analysis by Paired-End Tag Sequencing; Clinical; Collaborations; Companions; DNA Sequence; Data Analyses; Development; Diagnosis; Disease; Ecosystem; Ensure; Evaluation; Freezing; Funding; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic Code; Genetic Transcription; Genome; Genome Mappings; Genomics; Human; Human Genome; Human body; Industry; Maps; Marketing; Measures; Mediating; Methods; Molecular; Performance; Phase; Play; Program Development; Proteins; Publishing; Reagent; Regulation; Regulator Genes; Regulatory Element; Research; Research Personnel; Resolution; Role; Sales; Sampling; Science; Shipping; Specific qualifier value; Technology; Testing; Time; Tissues; Traction; assay development; base; bioinformatics tool; cell type; chromatin immunoprecipitation; clinically relevant; commercialization; coronavirus disease; design; falls; human disease; improved; innovation; lens; meetings; multiple omics; next generation sequencing; novel strategies; preservation; product development; programs; prototype; research and development; single cell sequencing; success; tool

A scalable kit-based assay for multi-omic analyses of transcriptional protein binding and chromatin interactions from ultra-low input frozen and FFPE samples at single-cell resolution Arima Genomics Project Summary/Abstract Precise regulation of gene expression is paramount to establishing cellular identities, and mis-regulation of genes causes human disease. Cells regulate gene expression using regulatory elements (REs), short DNA sequences embedded throughout the genome, who are bound by transcriptional proteins (TBPs) to facilitate their regulatory function. Molecular mapping tools, such as Chromatin immunoprecipitation with sequencing (ChIP-seq), produce “maps” of REs along the genome and have been a ubiquitous approach towards understanding gene regulation. However, REs mapped using ChIP-seq are only understood in context of a linear genome. In reality, REs execute gene control within a three dimensional (3D) genome. Therefore to truly understand gene regulation – gene regulation must be mapped in 3D. Indeed, high throughput chromatin interaction capture (HiC) was developed to produce 3D interaction maps of all 3 billion bases in the human genome, however, HiC does measure transcriptional protein binding, nor whether an interaction is regulatory, thus having limited utility in advancing our understanding of 3D gene regulation. To truly obtain 3D gene regulation maps, a multi-omic assay that concurrently captures the binding of transcriptional proteins and their mediated interactions is necessary. Recently, novel approaches attempt to combine the molecular steps of ChIP-seq and chromatin interaction capture to measure transcriptional protein binding and mediated chromatin interactions in a single, multi-omic assay. However these approaches, termed ChIA-PET and HiChIP, do not efficiently capture chromatin interactions or transcriptional protein binding, respectively. Consequently, there is need for improved assays that produce true multi-omic maps of 3D gene regulation. To satisfy this unmet need, we have developed and commercialized our optimized minimal viable product (MVP) Arima-HiChIP (A-HiChIP) solution. This phase-1 product incorporates innovations designed to meet the needs of early adopter customers, achieving efficient multi-omic mapping of TBP and chromatin interactions in higher input frozen cells and tissues, and a defined subset of transcriptional proteins. We have also developed our phase-1 product for workflow integration, leveraging industry and academic partnerships to reduce barriers in ChIP and bioinformatics components of the workflow, respectively. Our team has deep expertise in the science of chromatin interaction capture, gene regulation, and its commercialization. In 2018, we commercialized Arima- HiC kits for studying general principles of chromatin interactions and within 2 years have accumulated 500+ customers, providing tools to enable published discoveries across a host of basic science and disease research. Based on VOC analytics, we shifted our focus to develop the A-HiChIP kit - a more relatable product to the gene regulation market that customers wanted and that represented a larger market opportunity. Indeed, after our self-funded phase-1 R&D and product developments, we launched our MVP A-HiChIP solution into the market and have seen remarkable success – measured by an increase in our revenue contributions, increased quality of revenue, and traction with key opinion leaders (KOLs), large consortia, and COVID research. However, our phase-1 A-HiChIP has known limitations. In particular, the product falls short of meeting the needs of researchers utilizing common clinical samples types or quantities in their research, or seeking single-cell resolution in their analyses of heterogeneous tissues. As part of this direct-2-phase II program, we propose to further develop our technology to overcome these limitations, meet customer need, and enable broader adoption and application of this powerful multi-omic assay in the form of our second-generation A-HiChIP solution. Specifically, we propose assay developments to enable compatibility with pervasive clinical sample characteristics - ultra-low cell inputs (<100K cells) and FFPE tissues. Further, we propose development of a first-of-its-kind single-cell HiChIP assay and companion bioinformatics tools. We also propose essential product developments, to ensure commercialization of a robust, premium- performance kit-based product that is optimally integrated into the bulk and single-cell sequencing ecosystems. Upon successful completion of these technical and product-oriented aims, we propose to benchmark and validate our phase-2 A-HiChIP solution through collaboration and prototype (beta) kit and bioinformatics evaluations with KOLs across customer segments.

用于转录蛋白结合和染色质的多组学分析的可扩展的基于试剂盒的测定 单细胞分辨率下超低输入冷冻和FFPE样本的相互作用 Arima Genomics 项目总结/摘要 基因表达的精确调节对于建立细胞特性至关重要，而基因的错误调节 导致人类疾病。细胞利用调控元件（RE）、短DNA序列来调节基因表达 嵌入在整个基因组中，它们被转录蛋白（TBP）结合，以促进它们的调控。 功能分子作图工具，如染色质免疫沉淀测序（ChIP-seq），产生 RE的“地图”沿基因组沿着，并已成为一个普遍存在的方法来了解基因调控。 然而，使用ChIP-seq映射的RE仅在线性基因组的背景下被理解。实际上，RE执行 三维（3D）基因组内的基因控制。因此，要真正了解基因调控-基因 必须在3D中绘制规则。事实上，开发了高通量染色质相互作用捕获（HiC）。 然而，为了制作人类基因组中所有30亿个碱基的3D相互作用图，HiC确实测量了 转录蛋白结合，也不知道相互作用是否是调节性的，因此在推进基因工程中具有有限的效用。 我们对3D基因调控的理解为了真正获得3D基因调控图谱， 同时捕获转录蛋白的结合及其介导的相互作用是必需的。 最近，新的方法试图将ChIP-seq和染色质相互作用的分子步骤联合收割机结合起来 捕获，以测量转录蛋白结合和介导的染色质相互作用，在一个单一的，多组 比色法然而，这些被称为ChIA-PET和HiChIP的方法不能有效地捕获染色质 相互作用或转录蛋白结合。因此，需要改进的测定， 产生真正的3D基因调控的多组图谱。 为了满足这一未满足的需求，我们开发了优化的最小可行产品（MVP）并将其商业化。 Arima-HiChIP（A-HiChIP）溶液。这一第一阶段的产品采用了创新设计，以满足需求 早期采用者的客户，实现了TBP和染色质相互作用的高效多组学映射， 输入冷冻细胞和组织，以及转录蛋白的定义子集。我们还开发了我们的 第一阶段的工作流程集成产品，利用行业和学术界的合作伙伴关系，减少 分别是工作流程的ChIP和生物信息学组件。我们的团队在科学上有很深的专业知识 染色质相互作用捕获，基因调控及其商业化。2018年，我们将Arima商业化- 用于研究染色质相互作用一般原理的HiC试剂盒，2年内已积累500+ 客户，提供工具，使发表的发现在一系列基础科学和疾病研究。 基于VOC分析，我们将重点转移到开发A-HiChIP试剂盒-一种与基因更相关的产品 监管市场，客户想要的，这代表了一个更大的市场机会。其实，在我们的 自资的第一阶段研发和产品开发，我们推出了我们的MVP A-HiChIP解决方案进入市场 并取得了显著的成功--从我们的收入贡献增加、质量提高 收入，以及与关键意见领袖（KOL），大型财团和COVID研究的牵引力。但我们的 阶段1A-HiChIP具有已知的局限性。特别是，该产品福尔斯不能满足研究人员的需要 在他们的研究中利用常见的临床样本类型或数量，或在他们的研究中寻求单细胞分辨率， 异质组织的分析。 作为这个直接-2-阶段II计划的一部分，我们建议进一步发展我们的技术，以克服这些问题。 限制，满足客户需求，并使这种功能强大的多组学检测更广泛地采用和应用 我们的第二代A-HiChIP解决方案。具体而言，我们提出了检测方法的发展， 与普遍的临床样品特征-超低细胞输入（<100 K细胞）和FFPE组织的相容性。 此外，我们建议开发一种首创的单细胞HiChIP检测方法和配套的生物信息学方法。 工具.我们还提出必要的产品开发，以确保一个强大的，优质的商业化- 这是一种基于高性能试剂盒的产品，可最佳地集成到批量和单细胞测序生态系统中。 在成功完成这些技术和产品导向的目标后，我们建议制定基准， 通过协作和原型（beta）试剂盒和生物信息学验证我们的第2阶段A-HiChIP解决方案 与跨客户群的KOL进行评估。