Decoding the antibody repertoire : high throughput sequencing of multiple transcripts from single B cells

Next-generation (high throughput) DNA sequencing of immunoglobulin variable region and T-cell receptor gene repertoires is providing critical information for understanding adaptive immune responses and for diagnostic and therapeutic applications¹⁻⁴. However, existing immune repertoire sequencing technologies yield data on only one of the two chains of immune receptors and thus cannot provide information on the identity of immune receptor pairs encoded by individual B or T lymphocytes⁵⁻⁷. This work directly addressed these limitations by developing two new technologies for sequencing the complementary DNA (cDNA) of multiple mRNA transcripts from isolated single cells with very high throughput. In these methods, cells are sequestered into individual compartments and lysed in situ to capture single-cell mRNA onto magnetic beads, then the magnetic beads are used as template for RT-PCR reactions inside emulsion droplets that physically link cDNA of multiple transcripts for subsequent analysis by high-throughput DNA sequencing. Experimental throughput of over 2x10⁶ cells in a single day with antibody heavy and light chain pairing accuracy greater than 97% was demonstrated with in vitro expanded human B cells. These new single-cell sequencing technologies were also applied for rapid discovery of new human antibodies and for analysis of the human immune response to vaccination. Finally we applied the techniques developed here to gain new insights regarding development of the antibody repertoire through high-throughput and high-resolution examination of naïve and memory B-cell compartments in healthy human donors.