title: Application of circular consensus sequencing and network analysis to characterize the bovine IgG repertoire.
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. In particular, 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 in CDR3s increased with CDR3 length and we observed that cysteine residues were centrally located in 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 resulting in base insertions and deletions in 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 in bovine CDR3s provide further evidence that disulfide bond formation is likely 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.
authors.affiliation: Genetics and Breeding Unit, United States Meat Animal Research Center, ARS, USDA, Clay Center, NE, USA.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Larsen
authors.forename: Timothy P L
authors.initials: TP
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000940:Antigenic Variation / Q000276:immunology; D002417:Cattle / Q000235:genetics* / Q000276:immunology*; D016000:Cluster Analysis; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics; D016384:Consensus Sequence / Q000235:genetics*; D019277:Entropy; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D000596:Amino Acids; D022801:Complementarity Determining Regions; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains