| Autor: |
GIanviIIe, 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 |
| Předmět: |
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| Zdroj: |
Proceedings of the National Academy of Sciences of the United States of America; 12/1/2009, Vol. 106 Issue 48, p20216-20221, 6p |
| Abstrakt: |
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. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
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