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.

Fig. 1.

Heavy and light family frequencies and pairing observed in 18,158 RCA reads. (A) Heavy (Left) and light chain (Right) family frequencies observed in the library. Only families at 1% chain frequency are shown. (B) Heavy and light chain family pairings occur in proportion to the abundance of their partner, indistinguishable from a null model of random assortment by χ² (P value: 0.9512). (C) Heavy and light chain families pairings in second and third round Panning show pairing preferences that cannot be explained by random assortment (P value: 0.00107).

Fig. 2.

CDR-H3 length and amino acid composition for the most common length bin. (A) Observed CDR-H3 length diversity from 65,240 amplicon and 22,769 RCA reads. (B) Position specific amino acid frequencies for 10,281 length 11 (determined by positions 95–102) CDR-H3s. (C) Number of nongermline encoded amino acids found in CDRs 1 and 2, by domain type.

Fig. 3.

Diversity estimates for heavy and light chain CDRs in the antibody library. (A and B) 10 capture-recapture rarefaction results for CDR1, 2, 3 and concatenated CDRs for heavy and light chain, respectively. (C and D) Percent recapture during rarefaction analysis.