Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Ultra-high-throughput sequencing of the immune receptor repertoire from millions of lymphocytes

Nature Protocols volume 11, pages 429–442 (2016)Cite this article

Subjects

Abstract

High-throughput sequencing of the variable domains of immune receptors (antibodies and T cell receptors (TCRs)) is of key importance in the understanding of adaptive immune responses in health and disease. However, the sequencing of both immune receptor chains (VH+VL or TCRβ/δ+TCRα/γ) at the single-cell level for typical samples containing >104 lymphocytes is problematic, because immune receptors comprise two polypeptide chains that are encoded by separate mRNAs. Here we present a technology that allows rapid and low-cost determination of a paired immune receptor repertoire from millions of cells with high precision (>97%). Flow focusing is used to encapsulate single cells in emulsions containing magnetic beads for mRNA capture. The mRNA transcripts are then reverse-transcribed, physically linked to their partners by overlap extension PCR, and interrogated by high-throughput paired-end Illumina sequencing. This protocol describes the construction and operation of the flow-focusing device in detail, as well as the bioinformatics pipeline used to interpret the data. The entire procedure can be performed by a single researcher in under 12 h of effort per sample.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Overview of the workflow.
Figure 2: Diagram of the flow-focusing device.
Figure 3
Figure 4: Nozzle geometry.
Figure 5: Optimization of nested PCR conditions.

Similar content being viewed by others

References

  1. Finco, O. & Rappuoli, R. Designing vaccines for the twenty-first century society. Front. Immunol. 5, 12 (2014).

    Article  Google Scholar 

  2. Georgiou, G. et al. The promise and challenge of high-throughput sequencing of the antibody repertoire. Nat. Biotechnol. 32, 158–168 (2014).

    Article  CAS  Google Scholar 

  3. Logan, A.C. et al. High-throughput VDJ sequencing for quantification of minimal residual disease in chronic lymphocytic leukemia and immune reconstitution assessment. Proc. Natl. Acad. Sci. USA 108, 21194–21199 (2011).

    Article  CAS  Google Scholar 

  4. Mascola, J.R. & Haynes, B.F. Hiv-1 neutralizing antibodies: understanding nature′s pathways. Immunol. Rev. 254, 225–244 (2013).

    Article  Google Scholar 

  5. Warren, E.H., Matsen, F.A.t. & Chou, J. High-throughput sequencing of B- and T-lymphocyte antigen receptors in hematology. Blood 122, 19–22 (2013).

    Article  CAS  Google Scholar 

  6. Brezinschek, H.P., Foster, S.J., Dorner, T., Brezinschek, R.I. & Lipsky, P.E. Pairing of variable heavy and variable kappa chains in individual naive and memory B cells. J. Immunol. 160, 4762–4767 (1998).

    CAS  PubMed  Google Scholar 

  7. Meijer, P.J . et al. Isolation of human antibody repertoires with preservation of the natural heavy and light chain pairing. J. Mol. Biol. 358, 764–772 (2006).

    Article  CAS  Google Scholar 

  8. Poulsen, T.R., Meijer, P.J., Jensen, A., Nielsen, L.S. & Andersen, P.S. Kinetic, affinity, and diversity limits of human polyclonal antibody responses against tetanus toxoid. J. Immunol. 179, 3841–3850 (2007).

    Article  CAS  Google Scholar 

  9. Smith, K. et al. Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat. Protoc. 4, 372–384 (2009).

    Article  CAS  Google Scholar 

  10. Howie, B. et al. High-throughput pairing of T cell receptor alpha and beta sequences. Sci. Transl. Med. 7, 301ra131 (2015).

    Article  Google Scholar 

  11. DeKosky, B.J. et al. High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire. Nat. Biotechnol. 31, 166–169 (2013).

    Article  CAS  Google Scholar 

  12. Turchaninova, M.A. et al. Pairing of T-cell receptor chains via emulsion PCR. Eur. J. Immunol. 43, 2507–2515 (2013).

    Article  CAS  Google Scholar 

  13. Busse, C.E., Czogiel, I., Braun, P., Arndt, P.F. & Wardemann, H. Single-cell based high-throughput sequencing of full-length immunoglobulin heavy and light chain genes. Eur. J. Immunol. 44, 597–603 (2014).

    Article  CAS  Google Scholar 

  14. DeKosky, B.J. et al. In-depth determination and analysis of the human paired heavy- and light-chain antibody repertoire. Nat. Med. 21, 86–91 (2015).

    Article  CAS  Google Scholar 

  15. Doria-Rose, N.A. et al. Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies. Nature 509, 55–62 (2014).

    Article  CAS  Google Scholar 

  16. Berkland, C., Kim, K. & Pack, D.W. Fabrication of PLG microspheres with precisely controlled and monodisperse size distributions. J. Control. Release 73, 59–74 (2001).

    Article  CAS  Google Scholar 

  17. Berkland, C., Pollauf, E., Pack, D.W. & Kim, K. Uniform double-walled polymer microspheres of controllable shell thickness. J. Control. Release 96, 101–111 (2004).

    Article  CAS  Google Scholar 

  18. Lavinder, J.J. et al. Identification and characterization of the constituent human serum antibodies elicited by vaccination. Proc. Natl. Acad. Sci. USA 111, 2259–2264 (2014).

    Article  CAS  Google Scholar 

  19. Beltramello, M. et al. The human immune response to dengue virus is dominated by highly cross-reactive antibodies endowed with neutralizing and enhancing activity. Cell Host Microbe 8, 271–283 (2010).

    Article  CAS  Google Scholar 

  20. Walker, L.M. et al. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science 326, 285–289 (2009).

    Article  CAS  Google Scholar 

  21. Liao, H.X. et al. High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies. J. Virol. Methods 158, 171–179 (2009).

    Article  CAS  Google Scholar 

  22. Wrammert, J. et al. Rapid cloning of high-affinity human monoclonal antibodies against influenza virus. Nature 453, 667–671 (2008).

    Article  CAS  Google Scholar 

  23. Wardemann, H. et al. Predominant autoantibody production by early human B cell precursors. Science 301, 1374–1377 (2003).

    Article  CAS  Google Scholar 

  24. Carlson, C.S. et al. Using synthetic templates to design an unbiased multiplex PCR assay. Nat. Commun. 4, 2680 (2013).

    Article  Google Scholar 

  25. Ippolito, G.C. et al. Antibody repertoires in humanized NOD-scid-il2rγnull mice and human B cells reveals human-like diversification and tolerance checkpoints in the mouse. PLoS One 7, e35497 (2012).

    Article  CAS  Google Scholar 

  26. Lim, T.S. et al. V-gene amplification revisited: an optimised procedure for amplification of rearranged human antibody genes of different isotypes. N. Biotechnol. 27, 108–117 (2010).

    Article  CAS  Google Scholar 

  27. Brochet, X., Lefranc, M.P. & Giudicelli, V. IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis. Nucleic Acids Res. 36, W503–W508 (2008).

    Article  CAS  Google Scholar 

  28. Edgar, R.C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460–2461 (2010).

    Article  CAS  Google Scholar 

  29. Greiff, V., Miho, E., Menzel, U. & Reddy, S.T. Bioinformatic and statistical analysis of adaptive immune repertoires. Trends Immunol 36, 738–749 (2014).

    Article  Google Scholar 

  30. Saha, S. & Raghava, G.P. Algpred: prediction of allergenic proteins and mapping of IGE epitopes. Nucleic Acids Res. 34, W202–W209 (2006).

    Article  CAS  Google Scholar 

  31. Magoc, T. & Salzberg, S.L. Flash: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27, 2957–2963 (2011).

    Article  CAS  Google Scholar 

  32. Zhang, J., Kobert, K., Flouri, T. & Stamatakis, A. Pear: a fast and accurate Illumina paired-end read merger. Bioinformatics 30, 614–620 (2014).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank A. Kennedy for his assistance in making the borosilicate glass nozzles. Funding for this work was provided by Defense Threat Reduction Agency HDTRA1-12-C-0105 (G.G.), National Institutes of Health (NIH) Grant R01AI096228 (G.G.), and fellowships to B.J.D. from the Hertz Foundation, the University of Texas Donald D. Harrington Foundation and the National Science Foundation. H.T. was supported by Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellowships for Research Abroad.

Author information

Author notes

  1. Brandon J DeKosky

    Present address: Present address: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.,

  2. Jonathan R McDaniel and Brandon J DeKosky: These authors contributed equally to this work.

Authors and Affiliations

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

    Jonathan R McDaniel, Brandon J DeKosky, Hidetaka Tanno & George Georgiou

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

    Jonathan R McDaniel, Hidetaka Tanno, Andrew D Ellington & George Georgiou

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

    Andrew D Ellington

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

    George Georgiou

Authors
  1. Jonathan R McDaniel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Brandon J DeKosky
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hidetaka Tanno
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andrew D Ellington
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. George Georgiou
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.R.M., B.J.D. and G.G. wrote the manuscript. J.R.M., B.J.D., H.T., A.D.E and G.G. designed the experiments. J.R.M., B.J.D. and H.T. performed the experiments and conducted bioinformatic analyses. All authors edited and approved the manuscript.

Corresponding author

Correspondence to George Georgiou.

Ethics declarations

Competing interests

G.G., B.J.D. and A.D.E. declare competing financial interests in the form of a patent filed by the University of Texas at Austin.

Integrated supplementary information

Supplementary Figure 1 Alignment of custom-designed glass nozzle.

A) PBS/PBS aqueous phases in oil; nozzle aperture is properly aligned. B) PBS/Lysis Buffer aqueous phases in oil; nozzle aperture is properly aligned. The presence of surfactants greatly reduces the width of the aqueous stream. C) PBS/Lysis Buffer aqueous phases in oil; aperture is off-center. D) PBS/Lysis Buffer aqueous phases in oil; aperture is so far off-center that the aqueous phase is coming into contact with the curvature of the glass nozzle. In all pictures, the stream is flowing right to left.

Supplementary Figure 2 Emulsions collected from flow-focusing device before and after centrifugation.

The top layer of mineral oil containing microfines (small emulsions) should be removed and discarded. The bottom layer of larger emulsions contains cells and beads, which should be retained. The color will change depending on the concentration of the poly(dT) beads.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2, Supplementary Table 1 (PDF 476 kb)

Formation of the custom made glass nozzle. (MOV 6903 kb)

Aqueous/oil interface for misaligned glass nozzle.

If the concentric metal tubes are not centered on the aperture of the nozzle, the aqueous stream will flicker or appear asymmetric. If the needle-to-aperture distance is too great (>2-3 mm), the aqueous stream will flicker and may form a second node. (MOV 2845 kb)

Aqueous/oil interface during the transition from the PBS/PBS aqueous phase to the PBS/Lysis Buffer aqueous phase.

The introduction of surfactant into the aqueous phase greatly reduces the diameter of the stream. The transition is also typically accompanied by air bubbles that are introduced while switching syringes. Sample collection should begin once the bubbles cease and the stream becomes stable. (MOV 1053 kb)

Supplementary Methods

Bioinformatics script written in Python to parse and pair the VH and VL sequences following IMGT gene annotation. (TXT 17 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

McDaniel, J., DeKosky, B., Tanno, H. et al. Ultra-high-throughput sequencing of the immune receptor repertoire from millions of lymphocytes. Nat Protoc 11, 429–442 (2016). https://doi.org/10.1038/nprot.2016.024

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2016.024

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research