title: Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire.
abstract: Antibody repertoire diversity, potentially as high as 10(11) 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 10(6) reads, including more than 1.9 x 10(5) 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 10(4) 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 10(10). The results provide insights into the role of IgM diversification, display library construction, and productive germline usages in antibody libraries and the humoral repertoire.
authors.affiliation: Research Informatics, Rinat-Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA 94080, USA.
authors.forename: Jacob
authors.initials: J
authors.lastname: Glanville
authors.forename: Wenwu
authors.initials: W
authors.lastname: Zhai
authors.forename: Jan
authors.initials: J
authors.lastname: Berka
authors.forename: Dilduz
authors.initials: D
authors.lastname: Telman
authors.forename: Gabriella
authors.initials: G
authors.lastname: Huerta
authors.forename: Gautam R
authors.initials: GR
authors.lastname: Mehta
authors.forename: Irene
authors.initials: I
authors.lastname: Ni
authors.forename: Li
authors.initials: L
authors.lastname: Mei
authors.forename: Purnima D
authors.initials: PD
authors.lastname: Sundar
authors.forename: Giles M R
authors.initials: GM
authors.lastname: Day
authors.forename: David
authors.initials: D
authors.lastname: Cox
authors.forename: Arvind
authors.initials: A
authors.lastname: Rajpal
authors.forename: Jaume
authors.initials: J
authors.lastname: Pons
mesh_terms: D000916:Antibody Diversity / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D007075:Immunoglobulin M / Q000145:classification / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D007075:Immunoglobulin M