Item

Abstract

Single-cell PCR and sequencing of full-length Ig heavy (Igh) and Igk and Igl light chain genes is a powerful tool to measure the diversity of antibody repertoires and allows the functional assessment of B-cell responses through direct Ig gene cloning and the generation of recombinant mAbs. However, the current methodology is not high-throughput compatible. Here we developed a two-dimensional bar-coded primer matrix to combine Igh and Igk/Igl chain gene single-cell PCR with next-generation sequencing for the parallel analysis of the antibody repertoire of over 46 000 individual B cells. Our approach provides full-length Igh and corresponding Igk/Igl chain gene-sequence information and permits the accurate correction of sequencing errors by consensus building. The use of indexed cell sorting for the isolation of single B cells enables the integration of flow cytometry and Ig gene sequence information. The strategy is fully compatible with established protocols for direct antibody gene cloning and expression and therefore advances over previously described high-throughput approaches to assess antibody repertoires at the single-cell level.