Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire.
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.
Glanville
Jacob
J
Research Informatics, Rinat-Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA 94080, USA.
Zhai
Wenwu
W
Berka
Jan
J
Telman
Dilduz
D
Huerta
Gabriella
G
Mehta
Gautam R
GR
Ni
Irene
I
Mei
Li
L
Sundar
Purnima D
PD
Day
Giles M R
GM
Cox
David
D
Rajpal
Arvind
A
Pons
Jaume
J
D000916:Antibody Diversity / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D007075:Immunoglobulin M / Q000145:classification / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
D007075:Immunoglobulin M