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.

基于可扩展套件的基于转录蛋白结合和染色质的多OMIC分析的测定法 单细胞分辨率的超低输入冷冻和FFPE样品的相互作用 Arima基因组学 项目摘要/摘要 基因表达的精确调节对于建立细胞身份和基因的不正当至关重要 引起人类疾病。细胞使用调节元件（RES），短DNA序列调节基因表达 嵌入整个基因组，它们受转录蛋白（TBP）的约束，以促进其调节 功能。分子映射工具，例如染色质免疫沉淀（用测序）（芯片seq）产生 RES沿基因组的“地图”，是一种无处不在理解基因调节的方法。 但是，只有在线性基因组的上下文中，使用芯片序列映射的RES映射才能理解。实际上，res执行 基因控制在三维（3D）基因组中。因此，真正了解基因调节 - 基因 法规必须以3D映射。实际上，开发了高通量染色质相互作用捕获（HIC） 但是，要在人类基因组中产生所有30亿个碱基的3D相互作用图，但是，HIC确实测量了 转录蛋白结合，或相互作用是否是调节性的，因此效用有限 我们对3D基因调节的理解。为了真正获得3D基因调节图，一种多摩管测定法 同时需要捕获转录蛋白的结合及其介导的相互作用是必要的。 最近，新颖的方法试图结合ChIP-Seq和染色质相互作用的分子步骤 捕获以测量单个多摩管中的转录蛋白结合和介导的染色质相互作用 测定。但是，这些方法称为chia-pet和hichip，不能​​有效捕获染色质 相互作用或转录蛋白结合。因此，需要改进测定法 产生3D基因调节的真实多摩尼图。 为了满足这种未满足的需求，我们已经开发并商业化了我们优化的最小可行产品（MVP） Arima-Hichip（A-Hichip）解决方案。该阶段1产品融合了旨在满足需求的创新 早期采用者的客户，在更高的较高 输入冷冻细胞和组织，以及转录蛋白的定义子集。我们也发展了我们的 1阶段产品集成，利用行业和学术合作伙伴关系，以减少障碍 工作流的芯片和生物信息学组成部分。我们的团队在科学方面具有深厚的专业知识 染色质相互作用捕获，基因调节及其商业化。在2018年，我们将Arima-商业化 用于研究染色质相互作用的一般原理和2年内的HIC套件已积累了500多个 客户提供工具，以在许多基础科学和疾病研究中实现已发表的发现。 根据VOC分析，我们转移了重点以开发A -Hichip套件 - 一种更相关的基因产品 客户想要的监管市场代表了更大的市场机会。确实，在我们之后 自资助的第1阶段研发和产品开发，我们将MVP A-Hichip解决方案推向了市场 并看到了杰出的成功 - 通过我们的收入贡献增加，质量提高来衡量 收入以及关键意见领导者（KOLS），大财团和Covid研究的牵引力。但是，我们的 第1阶段A-Hichip具有已知局限性。特别是，该产品无法满足研究人员的需求 在其研究中使用常见的临床样品类型或数量，或在其中寻求单细胞分辨率 分析异质组织。 作为该直接2相II计划的一部分，我们建议进一步开发我们的技术来克服这些 局限性，满足客户需求，并能够更广泛地采用和应用此强大的多摩变测定法 以我们的第二代A-Hichip解决方案的形式。具体来说，我们提出评估发展，以实现 与普遍的临床样品特征的兼容性 - 超低细胞输入（<100K细胞）和FFPE组织。 此外，我们建议开发首先的单细胞Hichip分析和同伴生物信息学 工具。我们还提出了必不可少的产品开发，以确保商业化强大的，优质 基于性能套件的产品，可最佳地集成到批量和单细胞测序生态系统中。 成功完成这些以技术和产品为导向的目标后，我们建议进行基准测试和 通过协作和原型（Beta）套件和生物信息学验证我们的2阶段A-HICHIP解决方案 在客户群中对KOL进行评估。