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In-depth determination and analysis of the human paired heavy- and light-chain antibody repertoire

Nature Medicine volume 21, pages 86–91 (2015)Cite this article

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Abstract

High-throughput immune repertoire sequencing has emerged as a critical step in the understanding of adaptive responses following infection or vaccination or in autoimmunity. However, determination of native antibody variable heavy-light pairs (VH-VL pairs) remains a major challenge, and no technologies exist to adequately interrogate the >1 × 106 B cells in typical specimens. We developed a low-cost, single-cell, emulsion-based technology for sequencing antibody VH-VL repertoires from >2 × 106 B cells per experiment with demonstrated pairing precision >97%. A simple flow-focusing apparatus was used to sequester single B cells into emulsion droplets containing lysis buffer and magnetic beads for mRNA capture; subsequent emulsion RT-PCR generated VH-VL amplicons for next-generation sequencing. Massive VH-VL repertoire analyses of three human donors provided new immunological insights including (i) the identity, frequency and pairing propensity of shared, or 'public', VL genes, (ii) the detection of allelic inclusion (an implicated autoimmune mechanism) in healthy individuals and (iii) the occurrence of antibodies with features, in terms of gene usage and CDR3 length, associated with broadly neutralizing antibodies to rapidly evolving viruses such as HIV-1 and influenza.

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Figure 1: Technical workflow for ultra-high throughput VH-VL sequencing from single B cells.
Figure 2: Heavy-light V-gene pairing landscape of CD3−CD19+CD20+CD27+ peripheral memory B cells in two healthy human donors.
Figure 3: VH pairing statistics for representative promiscuous and public light chains.
Figure 4: Frequency of VL transcript allelic inclusion in two donors (n = 184 and n = 64 allelically included antibodies from n = 37,995 and n = 19,096 VH-VL clusters detected across replicates in donor 1 and donor 2, respectively).

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Acknowledgements

We thank C. Berkland, M. Singh and N. Dormer for critical help and advice. We thank B. Iverson and D. Derryberry for insightful feedback, M. Ronalter, C. Das, M. Wirth and Y. Wine for help with experiments, J. Lavinder for reviewing the manuscript, O. Lungu for help with data analysis, B. Briney for sharing an unpublished primer sequence, L. Morris (National Institute for Communicable Diseases of the National Health Laboratory Service, South Africa) and P. Kwong (Vaccine Research Center, NIAID, USA) for sharing CAP256 samples, C. McHenry for PacBio sequencing, and J. Wheeler and S. Hunicke-Smith for Illumina MiSeq sequencing. 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 by the US Defense Threat Reduction Agency (DTRA) HDTRA1-12-C-0105 (G.G.). A.D.E. would like to acknowledge funding from US National Security Science and Engineering Faculty Fellowship (FA9550-10-1-0169) and grants from the DTRA (HDTRA1-12-C-0007) and the Welch Foundation (F-1654). The content is solely the responsibility of the authors and do not necessarily represent the official views of the sponsors.

Author information

Authors and Affiliations

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

    Brandon J DeKosky, Takaaki Kojima, Alexa Rodin, Wissam Charab & George Georgiou

  2. Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan

    Takaaki Kojima

  3. Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA

    Gregory C Ippolito & George Georgiou

  4. Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas, USA

    Andrew D Ellington

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

    George Georgiou

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

    George Georgiou

Authors
  1. Brandon J DeKosky
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  2. Takaaki Kojima
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  4. Wissam Charab
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Contributions

B.J.D. and G.G. developed the methodology and wrote the manuscript; B.J.D., T.K., G.C.I., A.D.E. and G.G. designed the experiments; B.J.D., T.K., A.R. and W.C. performed the experiments; B.J.D. carried out the bioinformatic analysis; and B.J.D. and T.K. analyzed the data.

Corresponding author

Correspondence to George Georgiou.

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Competing interests

G.G., B.J.D. and A.D.E. declare competing financial interests in the form of a provisional patent for high-throughput sequencing of multiple transcripts from single cells, filed by the University of Texas, Austin, to the US Patent and Trademark Office and under the Patent Cooperation Treaty.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Tables 1–4 (PDF 2761 kb)

Supplementary Data Set 1

Sequenced VH:VL clusters for the all replicates of Donors 1, 2, and 3 (XLSX 18013 kb)

Supplementary Data Set 2

Quantitative list of CDR-L3 junctions observed in Donors 1, 2, and 3, sorted by prevalence in the three datasets (XLSX 8399 kb)

Supplementary Data Set 3

FASTA file containing allelically included pairs observed in all donors (TXT 215 kb)

Supplementary Data Set 4

FASTA file containing allelically included pairs with stop codons (TXT 4 kb)

Supplementary Data Set 5

FASTA file containing full-length VH:VL sequences from PacBio data that encoded VRC26-class antibodies (TXT 5 kb)

Supplementary Data Set 6

FASTA file containing Sanger sequences of VH and VL plasmids used in HEK293 transfections (TXT 26 kb)

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DeKosky, B., Kojima, T., Rodin, A. et al. In-depth determination and analysis of the human paired heavy- and light-chain antibody repertoire. Nat Med 21, 86–91 (2015). https://doi.org/10.1038/nm.3743

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