TY - JOUR
T1 - A facile technology for the high-throughput sequencing of the paired VH:VL and TCRβ:TCRα repertoires
AU - Tanno, Hidetaka
AU - McDaniel, Jonathan R.
AU - Stevens, Christopher A.
AU - Voss, William N.
AU - Li, Jie
AU - Durrett, Russell
AU - Lee, Jiwon
AU - Gollihar, Jimmy
AU - Tanno, Yuri
AU - Delidakis, George
AU - Pothukuchy, Arti
AU - Ellefson, Jared W.
AU - Goronzy, Jörg J.
AU - Maynard, Jennifer A.
AU - Ellington, Andrew D.
AU - Ippolito, Gregory C.
AU - Georgiou, George
N1 - Funding Information:
We thank R. Salinas for assistance with FACS; S.C. Harrison (Harvard Medical School) for HA proteins; and the Genome Sequencing and Analysis Facility at the University of Texas at Austin for performing Illumina sequencing. Funding for this work was provided by U.S. Defense Threat Reduction Agency (DTRA) HDTRA1-12-C-0105 (G.G.) and HDTRA1-18-1-0030 (A.D.E.); National Institutes of Health (NIH) grant 5R21CA191239-02 (A.D.E.) and grant U19 AI057266 (G.G. and J.J.G.); Texas Health Catalyst program no. 2056003826 (G.C.I.); and the Welch Foundation (F-1654 to A.D.E. and F-1767 to J.A.M.). H.T. was supported by UTHealth Innovation for Cancer Prevention Research Training Program Post-Doctoral Fellowship (Cancer Prevention and Research Institute of Texas grant no. RP160015), Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellowships for Research Abroad, and Uehara Memorial Foundation Research Fellowship. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Cancer Prevention and Research Institute of Texas.
Publisher Copyright:
© 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).
Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.
PY - 2020/4
Y1 - 2020/4
N2 - Natively paired sequencing (NPS) of B cell receptors [variable heavy (VH) and light (VL)] and T cell receptors (TCRβ and TCRα) is essential for the understanding of adaptive immunity in health and disease. Despite many recent technical advances, determining the VH:VL or TCRβ:α repertoire with high accuracy and throughput remains challenging. We discovered that the recently engineered xenopolymerase, RTX, is exceptionally resistant to cell lysate inhibition in single-cell emulsion droplets. We capitalized on the characteristics of this enzyme to develop a simple, rapid, and inexpensive in-droplet overlap extension reverse transcription polymerase chain reaction method for NPS not requiring microfluidics or other specialized equipment. Using this technique, we obtained high yields (5000 to >20,000 per sample) of paired VH:VL or TCRβ:α clonotypes at low cost. As a demonstration, we performed NPS on peripheral blood plasmablasts and T follicular helper cells following seasonal influenza vaccination and discovered high-affinity influenza-specific antibodies and TCRβ:α.
AB - Natively paired sequencing (NPS) of B cell receptors [variable heavy (VH) and light (VL)] and T cell receptors (TCRβ and TCRα) is essential for the understanding of adaptive immunity in health and disease. Despite many recent technical advances, determining the VH:VL or TCRβ:α repertoire with high accuracy and throughput remains challenging. We discovered that the recently engineered xenopolymerase, RTX, is exceptionally resistant to cell lysate inhibition in single-cell emulsion droplets. We capitalized on the characteristics of this enzyme to develop a simple, rapid, and inexpensive in-droplet overlap extension reverse transcription polymerase chain reaction method for NPS not requiring microfluidics or other specialized equipment. Using this technique, we obtained high yields (5000 to >20,000 per sample) of paired VH:VL or TCRβ:α clonotypes at low cost. As a demonstration, we performed NPS on peripheral blood plasmablasts and T follicular helper cells following seasonal influenza vaccination and discovered high-affinity influenza-specific antibodies and TCRβ:α.
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U2 - 10.1126/sciadv.aay9093
DO - 10.1126/sciadv.aay9093
M3 - Article
C2 - 32426460
AN - SCOPUS:85084663519
VL - 6
JO - Sci Adv
JF - Sci Adv
SN - 2375-2548
IS - 17
M1 - aay9093
ER -