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

Nature Biotechnology volume 31, pages 166–169 (2013)Cite this article

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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 (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.

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Figure 1: Overview of the high-throughput methodology for paired VH:VL antibody repertoire analysis.
Figure 2: VH:VL gene family usage of unique CDR-H3:CDR-L3 pairs identified by high-throughput sequencing of cell populations from three different individuals in separate experiments using the workflow in Figure 1.

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Acknowledgements

We thank B. Iverson and M. Pogson for insightful discussions and thoughtful advice, C. Das for assistance with antibody expression, and S. Reddy for help with initial studies. This work was funded by fellowships to B.J.D. from the Hertz Foundation, the University of Texas Donald D. Harrington Foundation and the National Science Foundation, and also by the US National Institutes of Health U19AI057234-09 (P.C.W.), U19AI057234-09 (G.G.), and U54AI057156 (G.G. and A.E.D.).

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Authors and Affiliations

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

    Brandon J DeKosky, Ryan P Deschner, Yariv Wine, Brandon M Rawlings, C Grant Willson & George Georgiou

  2. Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas, USA

    Gregory C Ippolito

  3. Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA

    Jason J Lavinder, Scott P Hunicke-Smith & Andrew D Ellington

  4. Department of Chemical & Biomolecular Engineering, University of Houston, Houston, Texas, USA

    Navin Varadarajan

  5. Department of Medicine, Rheumatology, and Clinical Immunology, Charité University Medicine, Berlin, Germany

    Claudia Giesecke & Thomas Dörner

  6. German Rheumatism Research Center (DRFZ), Berlin, Germany

    Claudia Giesecke & Thomas Dörner

  7. Department of Medicine, Section of Rheumatology, University of Chicago, Chicago, Illinois, USA

    Sarah F Andrews & Patrick C Wilson

  8. Department of Chemistry & Biochemistry, University of Texas at Austin, Austin, Texas, USA

    C Grant Willson & Andrew D Ellington

  9. Department of Biomedical Engineering, University of Texas at Austin, Austin, Texas, USA

    George Georgiou

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  1. Brandon J DeKosky
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Contributions

B.J.D. and G.G. developed the methodology and designed the experiments. B.J.D., G.C.I. and G.G. wrote the manuscript; B.J.D., G.C.I., R.P.D., J.J.L., Y.W., B.M.R., C.G. and S.F.A. performed the experiments; B.J.D. carried out the bioinformatic analysis; S.P.H.-S. performed Illumina sequencing; G.C.I., N.V., T.D., P.C.W., C.G.W. and A.D.E. helped design experiments; B.J.D., G.C.I., J.J.L., Y.W., S.P.H.-S., A.D.E. and G.G. analyzed the data.

Corresponding author

Correspondence to George Georgiou.

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Competing interests

G.G., B.J.D., A.D.E. and S.P.H.-S. declare competing financial interests in the form of a provisional patent application filed by the University of Texas, Austin.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–7 and Supplementary Figs. 1–3 (PDF 252 kb)

Supplementary Data

Supplementary Data sets (zip file) (ZIP 1607 kb)

Supplementary Video 1

Real-time video was used to observe MOPC-315 plasmacytoma cell lysis inside sealed microwells (MOV 9212 kb)

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DeKosky, B., Ippolito, G., Deschner, R. et al. High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire. Nat Biotechnol 31, 166–169 (2013). https://doi.org/10.1038/nbt.2492

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