Scalable single-cell workflow for multiomic analyses of chromatin interactions, accessibility, gene expression and cell surface proteins to unravel mechanisms of cellular diversity

可扩展的单细胞工作流程，用于染色质相互作用、可及性、基因表达和细胞表面蛋白的多组学分析，以揭示细胞多样性的机制

• 批准号: 10604121
• 负责人: Anthony Schmitt
• 金额: $ 101.34万
• 依托单位: ARIMA GENOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-15 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10604121
• 关键词: 3-Dimensional; ATAC-seq; Academia; Address; Adoption; Area; Bar Codes; Basic Science; Benchmarking; Biological; Biological Assay; Biology; Brain; Breast; Cell Separation; Cell Surface Proteins; Cells; Chemistry; Chromatin; Chromium; Communities; Complex; DNA Sequence; Data; Data Analyses; Development; Diagnosis; Disease; Ensure; Environment; Feeds; Funding; Gene Expression; Gene Expression Regulation; Genes; Genome; Genomics; Health; Heterogeneity; Hippocampus; Human; Human body; Individual; Informatics; Learning; Letters; Libraries; Link; Malignant neoplasm of prostate; Maps; Marketing; Measurement; Modality; Molecular Biology; Morphology; Mus; Neighborhoods; Nuclear; Nucleic Acid Regulatory Sequences; Organ; Organism; Output; Phase; Phenotype; Play; Population; Process; Program Development; Proliferating; Protocols documentation; Publishing; Reaction; Reagent; Regulator Genes; Regulatory Element; Research; Research Personnel; Resolution; Role; Sales; Sampling; Shipping; Source; Standardization; Technology; Testing; Tissues; Translating; Translational Research; Transposase; Validation; XCL1 gene; body system; cell type; combinatorial; commercial prototype; commercialization; cost; drug discovery; genomic platform; human disease; improved; indexing; innovation; insight; malignant breast neoplasm; manufacture; multiple omics; product development; programs; prostration; protein expression; prototype; research and development; screening; single-cell RNA sequencing; success; symposium; tool; webinar

Scalable single-cell workflow for multiomic analyses of chromatin interactions, accessibility, gene expression and cell surface proteins to unravel mechanisms of cellular diversity Arima Genomics Project Summary/Abstract All cells in the human body carry the same DNA sequence and yet individual cells are highly diverse in identity, morphology, proliferation, and function, leading to enormous heterogeneity in the context of tissues, organs, and organisms. Individual cells achieve this diversity via unique gene regulatory programs – where in, unique sets of regulatory elements (REs) precisely instruct each cell which genes to express and when. Mapping such gene regulatory programs are central to molecular biology and genomics, as mis-regulation is a major cause of disease – mapping not only helps in diagnosis but also enables therapies that can intervene and correct mis-regulation. Single cell ATAC sequencing (scATAC) has emerged as the popular mapping assay to delineate REs unique to each cell. When scATAC is performed alongside single cell RNA sequencing (scRNA), the researcher has access to both REs and gene expression, allowing them to obtain unprecedented insight into the gene regulatory programs of living cells. There is only one problem – there are often multiple REs in the neighborhood of a gene and without the ability to link specific REs to its target genes, a mechanistic view of gene regulation is lacking, limiting our ability to enable precise diagnosis and drug discovery programs. High throughput chromatin interaction capture assay and sequencing (HiC) presents a three-dimensional view of the genome, often informing the missing link between RE and their target genes. Indeed, several KOLs – e.g., Dr. Tomi Pastinen calls scATAC, scRNA and scHiC as the “trifecta of modalities” that can truly delineate gene regulatory programs of individual cells (see Dr. Pastinen’s letter and 30+ additional letters of support). Recognizing the value, several academic labs have developed scHiC protocols, which has already unraveled incredibly detailed mechanistic insights of gene regulation of complex microenvironments including breast cancer, prostate cancer, hippocampus – several of these studies are discussed in this application. Despite the enthusiasm around scHiC data, adoption has been restricted to a few labs because of (1) severe experimental inefficiencies that result in exorbitant costs (upwards of $20,000 per sample), and (2) because current scHiC protocols involve complex plate- or combinatorial indexing workflows that are challenging to setup and execute. Via a self-funded phase-1 program, we tackled problem (1) to drastically improve efficiency of scHiC and consequently, drive costs down from earlier $10 per cell, to <$2 per cell, details of which are discussed both in research and commercial plans. We then used our rigorous product development expertise to translate the resultant scHiC chemistry into kits that were extensively validated by multiple KOLs (see letters from Joe Ecker, Longzhi Tan and others). Upon validation, these KOLs have become customers using Arima’s scHiC, referred to as A-scHiC kits, in their single cell workflows instead of the inefficient former academic protocols. Webinars and conference presentations from these early adopters created a ripple in the community and in a span of few months, we have sold >1,000 reactions of A-scHiC kits to tens of academic labs (despite no marketing activity from Arima), who have embedded our kits within both the plate- and combinatorial indexing single cell workflows. The scope of phase-2 program is to tackle problem (2) to enable widespread adoption. In particular, we propose to build off the A-scHiC chemistry toward what we refer to as the sc3DGR chemistry that is performed upstream of 10X genomics (10XG) Chromium – i.e., the output of sc3DGR kit will be an input into the 10X ATAC (flavor1) or 10X Multiome (flavor2) kits, to concurrently capture scHiC and scATAC (flavor1), or, scHiC, scATAC and scRNA (flavor2), respectively. Such a chemistry will not only solve the ease to use problem (2) given its combability with the market leader 10XG, but importantly, it will enable multiomic analyses of the “trifecta” from the same individual cell concurrently, thus enabling cell perturbation, characterization and screening use-cases for precision mapping, diagnosis, and therapy. Once the sc3DGR chemistry is finalized, we translate it into robust kits, to be validated by 14 KOLs (see letters of support) across academia and pharma (AbbVie, AstraZeneca & Genentech). For Arima, the chemistry, the informatics, the easy end-to-end workflows, the overall workflow cost, the KOL-based go-to-market strategy – all play major factors in a seamless commercialization process of this leapfrog technology for delineating gene regulation programs of individual cells.

可扩展的单细胞工作流程，用于染色质相互作用、可及性、基因 表达和细胞表面蛋白，以阐明细胞多样性的机制 Arima Genomics 项目总结/摘要 人体内的所有细胞都携带相同的DNA序列，但单个细胞的身份却高度多样化， 形态，增殖和功能，导致组织，器官和组织中的巨大异质性。 有机体单个细胞通过独特的基因调控程序实现这种多样性-其中， 调控元件（RE）精确地指示每个细胞表达哪些基因以及何时表达。定位这样的基因 调控程序是分子生物学和基因组学的核心，因为错误调控是疾病的主要原因 - 映射不仅有助于诊断，而且还能够进行干预和纠正错误调节的治疗。 单细胞ATAC测序（scATAC）已经成为流行的作图测定，以描绘独特的RE， 每个细胞。当scATAC与单细胞RNA测序（scRNA）一起进行时，研究人员 获得RE和基因表达，使他们能够获得前所未有的基因调控的洞察力。 活细胞的程序只有一个问题--在一个基因附近经常有多个RE 并且没有将特定RE连接到其靶基因的能力，缺乏基因调控的机制观点， 限制了我们进行精确诊断和药物发现计划的能力。 高通量染色质相互作用捕获分析和测序（HiC）呈现三维视图 基因组，往往通知RE和它们的靶基因之间的缺失环节。事实上，几个KOL-例如， 博士Tomi Pastinen将scATAC、scRNA和scHiC称为“三种模式”，可以真正描述基因表达。 单个细胞的监管计划（参见Pastinen博士的信和30多封额外的支持信）。 认识到这一价值，几个学术实验室已经开发出scHiC协议，该协议已经解开 令人难以置信的详细机制的见解，基因调控的复杂微环境，包括乳腺癌 癌症、前列腺癌、海马-在本申请中讨论了这些研究中的几个。尽管 关于scHiC数据的热情，采用已被限制在少数实验室，因为（1）严重的实验 效率低下导致过高的成本（每个样品高达20，000美元），以及（2）由于当前的scHiC 方案涉及复杂的板或组合索引工作流程，其对设置和执行具有挑战性。 通过自筹资金的第一阶段计划，我们解决了问题（1），以大幅提高scHiC的效率， 因此，将成本从早期的每单元10美元降低到每单元<2美元， 研究和商业计划。然后，我们利用我们严格的产品开发专业知识， 将得到的scHiC化学转化为试剂盒，该试剂盒被多个KOL广泛验证（参见来自Joe Ecker的信件， Longzhi Tan等人）。经验证，这些KOL已成为使用Arima scHiC的客户， 作为A-scHiC试剂盒，在他们的单细胞工作流程中，而不是效率低下的前学术协议。网络研讨 这些早期采用者的会议演示在社区中产生了涟漪， 几个月来，我们已经向数十个学术实验室出售了> 1，000个A-scHiC试剂盒反应（尽管没有营销活动 来自Arima），他们将我们的试剂盒嵌入平板和组合索引单细胞工作流程中。 第二阶段计划的范围是解决问题（2），以实现广泛采用。特别是，我们建议 为了将A-scHiC化学构建为我们所称的在上游进行的sc 3DGR化学， 10 X基因组学（10 XG）铬-即，sc 3DGR套件的输出将输入到10 X ATAC（flavor 1） 或10 X Multiome（flavor 2）试剂盒，以同时捕获scHiC和scATAC（flavor 1），或scHiC、scATAC和 scRNA（flavor2）。这样的化学反应将不仅解决易于使用的问题（2）， 与市场领导者10 XG的兼容性，但重要的是，它将使多组学分析的“三连胜”，从 从而实现细胞扰动、表征和筛选用例 用于精确绘图诊断和治疗一旦sc 3DGR化学完成，我们将其转化为鲁棒的 试剂盒，将由学术界和制药公司（AbbVie，AstraZeneca & Genentech）。对于Arima来说，化学，信息学，简单的端到端工作流程，整体工作流程成本， 基于KOL进入市场战略--所有这些都在这一无缝商业化过程中发挥着重要作用 跨越式技术，用于描绘单个细胞的基因调控程序。