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Nat Biotechnol. 2013 Feb;31(2):166-9. doi: 10.1038/nbt.2492. Epub 2013 Jan 20.

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

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1
Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, USA.

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.

PMID:
23334449
PMCID:
PMC3910347
DOI:
10.1038/nbt.2492
[Indexed for MEDLINE]
Free PMC Article
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