Deep sequencing and human antibody repertoire analysis

Abstract

In the past decade, high-throughput DNA sequencing (HTS) methods and improved approaches for isolating antigen-specific B cells and their antibody genes have been applied in many areas of human immunology. This work has greatly increased our understanding of human antibody repertoires and the specific clones responsible for protective immunity or immune-mediated pathogenesis. Although the principles underlying selection of individual B cell clones in the intact immune system are still under investigation, the combination of more powerful genetic tracking of antibody lineage development and functional testing of the encoded proteins promises to transform therapeutic antibody discovery and optimization. Here, we highlight recent advances in this fast-moving field.

Figure 1

Typical workflow for high throughput sequence analysis of antibody variable gene repertoires. Usually, cells are separated from the starting tissue (often peripheral blood, but alternatively another tissue source containing B cells). RNA is extracted and reverse transcribed (or alternatively, genomic DNA can be used). Multiplex PCR for all antibody variable genes is performed. The antibody gene amplicon is isolated, libraries prepared, then subjected to high throughput sequencing. The resulting sequence sets must be processed and then analyzed.