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

Brandon J. Dekosky, Gregory C. Ippolito, Ryan P. Deschner, Jason J. Lavinder, Yariv Wine, Brandon M. Rawlings, Navin Varadarajan, Claudia Giesecke, Thomas Dörner, Sarah F. Andrews, Patrick C. Wilson, Scott P. Hunicke-Smith, C. Grant Willson, Andrew D. Ellington, George Georgiou

Research output: Contribution to journalArticle

282 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)166-169
Number of pages4
JournalNature Biotechnology
Volume31
Issue number2
DOIs
StatePublished - Feb 2013

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology
  • Molecular Medicine
  • Biomedical Engineering

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