High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire.


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 (VH and VL) 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 × 104 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 VH:VL linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of VH:VL 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



IMMUNOGLOBULINS, B cell receptors, SEASONAL influenza, VIRUS disease vaccination, MESSENGER RNA, NUCLEOTIDE sequence, GENETIC transcription


Copyright 2013 Nature Biotechnology. This is a post-print version of a published paper that is available at: http://ev7su4gn4p.scholar.serialssolutions.com/?sid=google&auinit=BJ&aulast=DeKosky&atitle=High-throughput+sequencing+of+the+paired+human+immunoglobulin+heavy+and+light+chain+repertoire&id=doi:10.1038/nbt.2492&title=Nature+%26+technology&volume=31&issue=2&date=2013&spage=166&issn=1087-0156. Recommended citation: DeKosky, Brandon J., Gregory C. Ippolito, Ryan P. Deschner, Jason J. Lavinder, Yariv Wine, Brandon M. Rawlings, Navin Varadarajan et al. "High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire." Nature biotechnology 31, no. 2 (2013): 166. This item has been deposited in accordance with the publisher copyright and licensing terms and with the author's permission.