Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire

• Published in: Proceedings of the National Academy of Sciences - PNAS 2009-12, Vol.106 (48), p.20216-20221
• Main Authors: Glanville, Jacob, Zhai, Wenwu, Berka, Jan, Telman, Dilduz, Huerta, Gabriella, Mehta, Gautam R, Ni, Irene, Mei, Li, Sundar, Purnima D, Day, Giles M.R, Cox, David, Rajpal, Arvind, Pons, Jaume
• Format: Article
• Language: English
• Subjects: Amino acids; Antibodies; Antibody binding sites; Antibody diversity; Antibody Diversity - genetics; Antigens; Bacteriophages; Biological Sciences; Gene Library; Germ cells; Humans; Immune system; Immunoglobulin M - classification; Immunoglobulin M - genetics; Immunoglobulins; Immunology; Libraries; Methods; Molecules; Sequence Analysis, DNA - methods; Sequencing
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0909775106

Antibody repertoire diversity, potentially as high as 10¹¹ unique molecules in a single individual, confounds characterization by conventional sequence analyses. In this study, we present a general method for assessing human antibody sequence diversity displayed on phage using massively parallel pyrosequencing, a novel application of Kabat column-labeled profile Hidden Markov Models, and translated complementarity determining region (CDR) capture-recapture analysis. Pyrosequencing of domain amplicon and RCA PCR products generated 1.5 x 10⁶ reads, including more than 1.9 x 10⁵ high quality, full-length sequences of antibody variable fragment (Fv) variable domains. Novel methods for germline and CDR classification and fine characterization of sequence diversity in the 6 CDRs are presented. Diverse germline contributions to the repertoire with random heavy and light chain pairing are observed. All germline families were found to be represented in 1.7 x 10⁴ sequences obtained from repeated panning of the library. While the most variable CDR (CDR-H3) presents significant length and sequence variability, we find a substantial contribution to total diversity from somatically mutated germline encoded CDRs 1 and 2. Using a capture-recapture method, the total diversity of the antibody library obtained from a human donor Immunoglobulin M (IgM) pool was determined to be at least 3.5 x 10¹⁰. The results provide insights into the role of IgM diversification, display library construction, and productive germline usages in antibody libraries and the humoral repertoire.