title: High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire.
abstract: Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy- and light-chain variable regions (V(H) and V(L)) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 10(4) capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed in situ. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion V(H):V(L) linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of V(H):V(L) pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets-peripheral blood IgG(+) B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccination.
authors: Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, USA.
authors: Brandon J
authors: BJ
authors: DeKosky
authors: Gregory C
authors: GC
authors: Ippolito
authors: Ryan P
authors: RP
authors: Deschner
authors: Jason J
authors: JJ
authors: Lavinder
authors: Yariv
authors: Y
authors: Wine
authors: Brandon M
authors: BM
authors: Rawlings
authors: Navin
authors: N
authors: Varadarajan
authors: Claudia
authors: C
authors: Giesecke
authors: Thomas
authors: T
authors: Dörner
authors: Sarah F
authors: SF
authors: Andrews
authors: Patrick C
authors: PC
authors: Wilson
authors: Scott P
authors: SP
authors: Hunicke-Smith
authors: C Grant
authors: CG
authors: Willson
authors: Andrew D
authors: AD
authors: Ellington
authors: George
authors: G
authors: Georgiou
mesh_terms: D016175:B-Lymphocyte Subsets / Q000166:cytology / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000097:blood / Q000235:genetics*; D007147:Immunoglobulin Light Chains / Q000097:blood / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007252:Influenza Vaccines / Q000008:administration & dosage / Q000276:immunology; D008297:Male; D011947:Receptors, Antigen, B-Cell / Q000235:genetics* / Q000276:immunology; D055815:Young Adult
chemical_list: D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D007252:Influenza Vaccines; D011947:Receptors, Antigen, B-Cell