Accessible high-throughput single-cell genome sequencing

项目摘要 大多数实体瘤的发生和发展除了点突变外，还涉及复杂的基因组重排。 突变-所有这些都可能导致癌症进展和对治疗药物的耐药性的演变。 单细胞全基因组测序（scWGS）已被证明是非常宝贵的识别肿瘤 亚群和推进我们的克隆动力学和肿瘤进化的理解。尽管价值 由于scWGS的存在，缺乏提供足够的小区吞吐量（即功率）的商业可用选项 和/或基因组覆盖率来完全分类和表征肿瘤样品内的克隆群体。这 当罕见的、可能具有治疗抗性的亚群存在于 肿瘤可能逃避检测使用现有的方法。 在这里，我们将通过开发利用广泛可用的 仪器和现成的试剂。我们之前描述了原理证明技术， 包含在每个细胞的细胞核（原位）内的scWGS文库，然后通过细胞条形码化进行 使用我们的自定义工作流程。我们将扩展、开发和调整这些技术，以提供三个工作流程 这将通过提供易于获得的数量级来满足癌症研究社区的需求 与当前选项相比，scWGS分析的小区吞吐量、小区覆盖范围和成本节约得到改进。的 第一个是集中在高细胞吞吐量使用广泛可用的仪器;第二个利用我们的高， 内容化学，以产生高覆盖率的单细胞基因组谱，减少试剂和改善 最后，第三种方法实施靶捕获以靶向外显子序列或其他靶向序列。 以降低测序成本。其中每一个都是针对满足特定领域的需求， 癌症研究，从基础科学到临床，并分享可及性的主题。

PROJECT SUMMARY Initiation and evolution of most solid tumors involve complex genomic rearrangements in addition to point mutations – all of which can contribute to cancer progression and to the evolution of resistance to therapeutics. Single-cell whole genome sequencing (scWGS) has proven invaluable for the identification of tumor subpopulations and in advancing our understanding of clonal dynamics and tumor evolution. Despite the value of scWGS, there is a dearth of commercially-available options that provide enough cell throughput (i.e. power) and/or genomic coverage to fully catalogue and characterize clonal populations within a tumor sample. This can be particularly problematic when rare, possible therapy-resistant, subpopulations are present within a tumor that may elude detection using existing methodologies. Here we will solve the accessibility problem by developing technologies that leverage widely-available instrumentation and off-the-shelf reagents. We previously described proof-of-principle technologies to construct scWGS libraries contained within the nucleus (in situ) of each cell that are then carried through cell barcoding using our custom workflow. We will extend, develop, and adapt these technologies to provide three workflows that will meet the needs of the cancer research community by providing readily-accessible order-of-magnitude improvements to cell throughput, cell coverage and cost savings for scWGS analysis over current options. The first is focused on high cell throughput using widely available instrumentation; the second leverages our high- content chemistry to produce high-coverage single cell genomic profiles with reduced reagents and improved throughput; and finally, the third approach implements target capture to target exonic sequence or other regions of interest to reduce sequencing costs. Each of these is targeted to meet the needs of specific areas of cancer research, from basic science to clinical, and share the theme of accessibility.