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

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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.