title: Assessment of B Cell Repertoire in Humans.
abstract: The B cell receptor (BCR) repertoire is highly diverse. Repertoire diversity is achieved centrally by somatic recombination of immunoglobulin (Ig) genes and peripherally by somatic hypermutation and Ig heavy chain class-switching. Throughout these processes, there is selection for functional gene rearrangements, selection against gene combinations resulting in self-reactive BCRs, and selection for BCRs with high affinity for exogenous antigens after challenge. Hence, investigation of BCR repertoires from different groups of B cells can provide information on stages of B cell development and shed light on the etiology of B cell pathologies. In most instances, the third complementarity determining region of the Ig heavy chain (CDR-H3) contributes the majority of amino acids to the antibody/antigen binding interface. Although CDR-H3 spectratype analysis provides information on the overall diversity of BCR repertoires, this fairly simple technique analyzes the relative quantities of CDR-H3 regions of each size, within a range of approximately 10-80 bp, without sequence detail and thus is limited in scope. High-throughput sequencing (HTS) techniques on the Roche 454 GS FLX Titanium system, however, can generate a wide coverage of Ig sequences to provide more qualitative data such as V, D, and J usage as well as detailed CDR3 sequence information. Here we present protocols in detail for CDR-H3 spectratype analysis and HTS of human BCR repertoires.
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London School of Biomedical Science, London, UK.
authors.forename: Yu-Chang
authors.initials: YC
authors.lastname: Wu
authors.affiliation: Department of Pathology, Cardiff University, Cardiff, UK.
authors.forename: David
authors.initials: D
authors.lastname: Kipling
authors.affiliation: Department of Immunobiology, King's College London School of Medicine, Strand, London, SE1 9RT, UK. walters@kcl.ac.uk.
authors.forename: Deborah
authors.initials: D
authors.lastname: Dunn-Walters
mesh_terms: D001402:B-Lymphocytes / Q000378:metabolism*; D022801:Complementarity Determining Regions / Q000235:genetics; D005803:Genes, Immunoglobulin; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans
chemical_list: D022801:Complementarity Determining Regions
keywords: Antibody; B cell; B cell receptor; High-throughput sequencing; Immunoglobulin; Next generation sequencing; Repertoire; Spectratyping