Direct high-throughput amplification and sequencing of immunoglobulin genes from single human B cells.

Abstract

Single cell Ig gene amplification and sequencing has been widely used for the molecular and functional assessment of human antibody repertoires and has led to the identification of recombinant monoclonal antibodies with therapeutic potential against diverse pathogens 1, 2, 3. Due to the high reagent and Sanger sequencing costs, these antibody‐cloning strategies are limited to the analysis of relatively small B‐cell numbers and do not allow in‐depth repertoire measurements to assess the clonality and clonal evolution of B‐cell responses. A major goal in the field has been the development of platforms that allow the high‐throughput analysis of antibody repertoires at low costs 4. Next‐generation sequencing (NGS) of Ig heavy and light chain genes facilitates the high‐throughput analysis of antibody repertoires. So far only one platform preserves natural Ig gene associations at single‐cell level through linkage of Ig heavy and light chain amplicons before sequencing 5. However, due to the short NGS read‐lengths full‐length Ig genes are not readily available for cloning and recombinant monoclonal antibody production. Here, we describe a platform for the high‐throughput analysis of human antibody repertoires at single cell level that is fully compatible with direct Ig gene cloning and expression.