This Small Business Innovation Research (SBIR) Phase I project will enable a completely new approach to biology. The fundamental unit of biology is the single cell, and each cell contains complex genetic machinery. Many fundamental questions could be answered if biologists could perform genetic analysis on large numbers of single cells derived from tissues. The vast majority of useful applications would require detection of multiple genetic loci in single cells. For example, immunologists would like to measure co-expression of multiple T cell receptors and inflammatory molecules in hundreds of thousands of single T cells circulating in human blood. Currently, biology lacks elegant tools to perform this type of analysis. The current project aims to solve this problem with an innovative approach for analysis multiple genetic loci in hundreds of thousands of single cells analyzed in parallel. The technology uses a device to isolate single cells into aqueous-in-oil picoliter microdroplets, amplifies and links two or more genetic loci by intermolecular hybridization, and then sequences linked loci in reversed emulsions by next-generation sequencing. This enables far more complicated biological analysis than is possible if analyzing only a single locus in a single cell, or a single locus across many single cells. The broader impact/commercial potential of this project includes commercial applications in genetics and immunology research as well as molecular diagnostics and pharmaceutical development. Immunology researchers worldwide are eager to understand T cell and B cell immune repertoires. Immune repertoires respond to factors such as infectious disease, age, and obesity, so immune repertoire profiling is of great interest worldwide. The innovation of this proposed research is to link subunits of antibody genes to reveal a more complete immune repertoire profile. A similar method could be used to link subunits of T cell receptor genes. The platform will also enable unique and innovative approaches to a number of currently intractable problems in molecular diagnostics, including noninvasive prenatal diagnosis, noninvasive molecular typing of solid tumors, and inflammatory response to allograft procedures. Finally, T cells and antibodies are increasingly used as therapy for disease, and immune repertoire profiling technology will be critical to the development of such therapies. In summary, the technology developed in this project will be marketed to research immunologists, leading to fundamental improvements in our understanding of immunology. Eventually, the technology could be extended to the fields of molecular diagnostics and immune therapy, which could help cure intractable diseases
