In-Depth Determination and Analysis of the Human Paired Heavy and Light Chain Antibody Repertoire

High-throughput immune repertoire sequencing has emerged as a critical step in the understanding of adaptive responses following infection, vaccination or in autoimmunity.  However determination of native antibody variable heavy:light  pairs (VH:VL) remains a major challenge and no technologies exist to adequately interrogate the >10⁶ B cells in typical specimens. We developed a low-cost single-cell emulsion-based technology for sequencing antibody VH:VL repertoires from >2x10⁶ B cells per experiment with demonstrated pairing precision >97%.  A simple flow-focusing apparatus was used to sequester single B cells into emulsion droplets containing lysis buffer and magnetic beads for mRNA capture; subsequent emulsion RT-PCR generated VH:VL amplicons for NextGen sequencing.  Massive VH:VL repertoire analyses of human donors enabled rapid antibody discovery of HIV broadly neutralizing antibodies and cross-reactive influenza antibodies while also providing novel immunological insights including the identity, frequency, and pairing propensity of shared, or “public” VL genes, and detection of allelic inclusion (an implicated autoimmune mechanism) in healthy individuals.  As DNA sequencing technologies continue to progress, low-cost high-throughput single-cell antibody sequencing such as reported here may enable rapid antibody discovery and provide new insights into humoral immune responses associated with vaccine development, autoimmunity, infectious diseases, and other human disease states.