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，用于染色质相互作用、可及性、基因的多组学分析 表达和细胞表面蛋白揭示细胞多样性的机制 ARIMA基因组学 项目摘要/摘要 人体内的所有细胞都携带相同的DNA序列，但单个细胞在身份上却高度不同， 形态、增殖和功能，导致组织、器官和 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，独特的 调控元件(RES)精确地指示每个细胞表达哪些基因以及何时表达。对这种基因进行定位 调控程序是分子生物学和基因组学的核心，因为错误的调控是疾病的主要原因 -映射不仅有助于诊断，而且能够进行干预和纠正错误调节的治疗。 单细胞ATAC测序(ScATAC)已经成为一种流行的图谱分析方法，用于描绘独有的Res 每一个细胞。当scATAC与单细胞RNA测序(ScRNA)同时进行时，研究人员 同时获得RES和基因表达，使他们能够对基因调控获得前所未有的洞察力 活细胞的程序。只有一个问题--在一个基因附近通常有多个RE 如果没有能力将特定的RES与其目标基因联系起来，就缺乏对基因调控的机械论观点， 限制了我们实现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)呈现三维图像 基因组的一部分，通常告知RE和它们的目标基因之间缺失的联系。事实上，有几个KOL--例如， Tomi Pastinen博士将scATAC、scRNA和scHiC称为能够真正描绘基因的“三种模式” 单个细胞的调控计划(见帕斯蒂宁博士的信和30多封其他支持信)。 认识到这一价值，几个学术实验室已经开发出scHiC协议，该协议已经被解开 对包括乳房在内的复杂微环境的基因调控的令人难以置信的详细机制洞察 癌症、前列腺癌、海马体-这些研究中的几个在本应用中进行了讨论。尽管 对scHiC数据的热情，由于(1)严重的实验，采用仅限于少数实验室 低效率导致过高的成本(每个样本超过20,000美元)，以及(2)因为目前的scHiC 协议涉及复杂的板式或组合索引工作流，这些工作流的设置和执行具有挑战性。 通过一个自筹资金的第一阶段计划，我们解决了问题(1)，以显著提高SCHiC的效率和 因此，将成本从早期的每电池10美元降低到每电池2美元，详细信息在中讨论 研究和商业计划。然后，我们使用我们严谨的产品开发专业知识将 将所得到的ScHiC化学制成试剂盒，该试剂盒被多个KOL广泛验证(参见Joe Ecker的信件， 谭龙志等人)。经过验证，这些KOL已成为使用ARIMA的scHiC的客户，推荐 TO作为A-scHiC工具包，在其单细胞工作流中取代了以前效率低下的学术协议。网络研讨会 这些早期采用者的会议演讲在社区中产生了涟漪，在短短几年内 几个月来，我们已经向数十个学术实验室销售了1,000个反应A-scHiC试剂盒(尽管没有任何营销活动 来自ARIMA)，他们已经将我们的工具包嵌入到平板和组合索引单细胞工作流中。 第二阶段计划的范围是解决问题(2)，以使人们能够广泛采用。特别是，我们建议 为了在A-scHiC化学的基础上建立我们所说的上游执行的sc3DGR化学 10倍基因组学(10XG)铬-即sc3DGR试剂盒的输出将成为10倍ATAC(Favor1)的输入 或10倍Multiome(Favor2)试剂盒，以同时捕获scHiC和scATAC(Favor1)，或scHiC、scATAC和 ScRNA(Favor2)。这种化学反应不仅会解决易用性问题(2)，因为它的 与市场领先者10XG的可匹配性，但重要的是，它将使来自 同时使用相同的单个单元，从而实现单元扰动、表征和筛选用例 用于精确测绘、诊断和治疗。一旦sc3DGR化学最终确定，我们将其转换为Robust 试剂盒，将由学术界和制药行业的14家KOL(见支持函)验证(AbbVie、阿斯利康和 基因泰克)。对于ARIMA来说，化学、信息学、简单的端到端工作流、总体工作流成本、 基于KOL的入市战略-所有这些都在其无缝商业化过程中发挥着主要作用 勾画单个细胞基因调控程序的越级技术。

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.