title: Highly sensitive and unbiased approach for elucidating antibody repertoires.
abstract: Developing B lymphocytes undergo V(D)J recombination to assemble germ-line V, D, and J gene segments into exons that encode the antigen-binding variable region of Ig heavy (H) and light (L) chains. IgH and IgL chains associate to form the B-cell receptor (BCR), which, upon antigen binding, activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. The ability of V(D)J recombination to generate vast primary B-cell repertoires results from a combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B-cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or nonproductive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions, and also for following antibody affinity maturation processes.
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115; Department of Genetics, Harvard Medical School, Boston, MA 02115;
authors.forename: Sherry G
authors.initials: SG
authors.lastname: Lin
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115; Department of Genetics, Harvard Medical School, Boston, MA 02115;
authors.forename: Zhaoqing
authors.initials: Z
authors.lastname: Ba
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115; Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115.
authors.forename: Zhou
authors.initials: Z
authors.lastname: Du
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115; Department of Genetics, Harvard Medical School, Boston, MA 02115;
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115; Department of Genetics, Harvard Medical School, Boston, MA 02115; alt@enders.tch.harvard.edu jiazhi.hu@childrens.harvard.edu.
authors.forename: Jiazhi
authors.initials: J
authors.lastname: Hu
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115; Department of Genetics, Harvard Medical School, Boston, MA 02115; Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115 alt@enders.tch.harvard.edu jiazhi.hu@childrens.harvard.edu.
authors.forename: Frederick W
authors.initials: FW
authors.lastname: Alt
mesh_terms: D000818:Animals; D000906:Antibodies / Q000032:analysis*; D005821:Genetic Techniques; D051379:Mice; D060152:V(D)J Recombination
chemical_list: D000906:Antibodies
keywords: HTGTS-Rep-seq; V(D)J recombination; antibody repertoires