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ARS Home » Plains Area » Clay Center, Nebraska » U.S. Meat Animal Research Center » Genetics and Animal Breeding » Research » Publications at this Location » Publication #283318
Title: Application of circular consensus sequencing and network analysis to characterize the bovine IgG repertoire

Author
item Larsen, Peter
item Smith, Timothy - Tim

Submitted to: BMC Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/4/2012
Publication Date: 9/14/2012
Citation: Larsen, P.A., Smith, T.P. 2012. Application of circular consensus sequencing and network analysis to characterize the bovine IgG repertoire. BioMed Central (BMC) Immunology. 13:52 doi:10.1186/1471-2172-13-52.

Interpretive Summary: Cattle immune systems produce a large number of antibodies that respond to a variety of antigens. New DNA sequencing technologies (i.e., high-throughput sequencing) provide unique approaches to antibody-based research areas including antibody discovery and engineering, disease surveillance, and host immune response to vaccines. One of these technologies, single-molecule circular consensus sequencing, permits the sequencing of antibodies at previously unattainable depths of coverage and accuracy. We approached the bovine antibody repertoire with the goal of describing the diversity of expressed antibodies. In this paper we present the results of the first high-throughput analysis of expressed antibodies from cattle. We describe the diversity observed within the portion of the antibody that physically interacts with antigens and visualize this diversity using a network-based approach. Our results show that cattle immune systems use a number of interesting methods to increase antibody diversity. We hypothesize that additional study of bovine antibody sequences from controlled challenge experiments will help with the development of new vaccines and can be used to screen for the presence or absence of diseases.

Technical Abstract: Background: Vertebrate immune systems generate diverse repertoires of antibodies capable of mediating response to a variety of antigens. Next generation sequencing methods provide unique approaches to a number of immuno-based research areas including antibody discovery and engineering, disease surveillance, and host immune response to vaccines. Single-molecule circular consensus sequencing permits the sequencing of antibody repertoires at previously unattainable depths of coverage and accuracy. We approached the bovine immunoglobulin G (IgG) repertoire with the objective of characterizing diversity of expressed IgG transcripts. Here we present single-molecule real-time sequencing data of expressed IgG heavy-chain repertoires of four individual cattle. We describe the diversity observed within antigen binding regions and visualize this diversity using a network-based approach. Results: We generated 49,945 high quality cDNA sequences, each spanning the entire IgG variable region from four Bos taurus calves. From these sequences we identified 49,521 antigen binding regions using the automated Paratome web server. Approximately 9% of all unique complementarity-determining 2 (CDR2) sequences were of variable lengths. A bimodal distribution of unique CDR3 sequence lengths was observed, with common lengths of 5–6 and 21–25 amino acids. The average number of cysteine residues within CDR3s increased with CDR3 length and we observed that cysteine residues were centrally located within CDR3s. We identified 19 extremely long CDR3 sequences (up to 62 amino acids in length) within IgG transcripts. Network analyses revealed distinct patterns among the expressed IgG antigen binding repertoires of the examined individuals. Conclusions: We utilized circular consensus sequencing technology to provide baseline data of the expressed bovine IgG repertoire that can be used for future studies important to livestock research. Somatic mutation within CDR2 further diversifies the bovine antibody repertoire. In contrast to previous studies, our data indicate that unusually long CDR3 sequences are not unique to IgM antibodies in cattle. Centrally located cysteine residues within bovine CDR3s provide further evidence that disulfide bond formation is of structural importance. We hypothesize that network or cluster-based analyses of expressed antibody repertoires from controlled challenge experiments will help identify novel natural antigen binding solutions to specific pathogens of interest.