Accessible high-throughput single-cell genome sequencing

项目总结 大多数实体肿瘤的发生和发展除了点外，还涉及复杂的基因组重排 突变--所有这些都可能导致癌症的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)已被证明在肿瘤鉴定中具有非常重要的价值。 这有助于我们进一步了解克隆动力学和肿瘤进化。尽管价值不菲 在scwg中，缺乏商业上可用的选项来提供足够的小区吞吐量(即功率)。 和/或基因组覆盖，以完全编目和表征肿瘤样本中的克隆群体。这 当罕见的、可能具有治疗抵抗力的亚群出现在 使用现有方法可能无法检测到的肿瘤。 在这里，我们将通过开发利用广泛可用的技术来解决可访问性问题 仪器和现成的试剂。我们之前描述了构建以下组件的原则证明技术 每个细胞的细胞核内(原位)包含的scWGS文库，然后通过细胞条形码进行携带 使用我们的定制工作流程。我们将扩展、开发和调整这些技术以提供三个工作流 它将通过提供易于访问的数量级来满足癌症研究社区的需求 在小区吞吐量、小区覆盖率和scWGS分析的成本节约方面比目前的方案有所改进。这个 第一个是使用广泛可用的仪器来关注高小区吞吐量；第二个是利用我们的高 内容化学用更少的试剂和改进的方法生产高覆盖率的单细胞基因组图谱 最后，第三种方法实现对目标外显子序列或其他序列的目标捕获 以降低测序成本。其中每一项都是为了满足特定领域的需求 癌症研究，从基础科学到临床，并分享可访问性的主题。

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.