Ultrahigh-throughput single-cell genetic analysis and sorting with PACS

描述（由申请人提供）：Torrent Bio, Inc.的目标是将一种能够使用PCR对单细胞进行超高通量筛选的技术商业化。该系统在许多方面类似于荧光活化细胞分选（FACS），不同之处在于，它不是分选完整的细胞，而是将单个细胞分离到悬浮在油中的单个皮升体积的水滴中，并对其裂解物进行PCR反应。使用多路TaqMan PCR检测，该系统能够询问每个细胞特定基因、突变或非编码rna组合的表达。此外，与FACS类似，该系统可以将细胞裂解液分选到不同的容器中，允许使用微阵列，下一代测序或其他方法恢复具有独特转录指纹的细胞裂解物进行额外分析。该系统的主要优点是将FACS的高通量单细胞筛选和分选能力与PCR的灵敏度相结合。在本提案中，我们的目标是进一步提高工作实验室原型的单细胞通量，并演示使用多路TaqMan检测从混合细胞群中靶向和分选乳腺癌细胞的完整PACS工作流程。PACS方法的智力优势源于它能够提供快速和低成本的解决方案，用于大规模平行单细胞基因和转录分析大量异质细胞群体。细胞异质性及其对生物功能和疾病的影响对人类免疫学、干细胞生物学和癌症研究至关重要。通过分析群体中的单个细胞，可以识别对人类健康和发育至关重要的罕见细胞群体或短暂细胞状态，否则无法通过集合测量观察到。PACS系统的商业化将使对这些罕见细胞的基因组和转录组的分析比以往任何时候都更加详细和全面。

DESCRIPTION (provided by applicant): The goal of Torrent Bio, Inc. is to commercialize a technology capable of ultrahigh-throughput screening of single-cells using PCR. The system is in many ways analogous to Fluorescent Activated Cell Sorting (FACS), except that rather than sorting intact cells, it isolates individual cells into individual aqueous picoliter-volume drops suspended in oil and performs PCR reactions on their lysates. Using multiplexed TaqMan PCR assays, the system is able to interrogate each cell for the expression of specific combinations of genes, mutations or non-coding RNAs. Moreover, similar to FACS, this system can sort the cell lysate into different containers, allowing the lysates of the cells with a unique transcriptional fingerprint to be recovered for additional analysis using microarrays, Next Gen Sequencing, or other methods. The principal advantage of this system is that it combines the high-throughput single-cell screening and sorting capacity of FACS with the sensitivity of PCR. In this proposal, we aim to further improve the single-cell throughput of a working lab prototype and demonstrate the complete PACS workflow using a multiplexed TaqMan assay to target and sort breast cancer cells from a mixed cell population. The intellectual merits of the PACS approach stem from its ability to deliver a rapid and low-cost solution for massively parallel single-cell geneti and transcriptional analysis on large heterogeneous populations of cells. Cellular heterogeneity and its impact on biological function and disease is crucially important to questions in human immunology, stem cell biology and cancer research. By analyzing individual cells within a population, it is possible to identify rare cell populations or transient cell states, critical to uman health and development, that are otherwise unobservable by ensemble measurements. Commercialization of the PACS system will enable a more detailed and comprehensive analysis of the genomes and transcriptomes of these rare cells than ever before.