TIRTL-seq: deep, quantitative and affordable paired TCR repertoire sequencing
The specificity of T cells is determined by T cell receptor (TCR) α and β chain sequences. While bulk TCR sequencing enables cost-effective repertoire profiling without chain pairing information, single-cell approaches provide paired data but are costly and limited in throughput. Here we present throughput-intensive rapid TCR library sequencing (TIRTL-seq), an experimental and computational methodology for paired TCR repertoire sequencing (TCR-seq). TIRTL-seq is based on the parallel generation of hundreds of TCR libraries in 384-well plates at less than US$200 per plate, allowing cohort-scale paired TCR-seq studies. We benchmarked TIRTL-seq against state-of-the-art bulk TCR-seq and 10x Genomics Chromium technologies on longitudinal samples and identified severe acute respiratory syndrome coronavirus 2- and Epstein-Barr virus-specific clonal expansions after infection with distinct dynamics. TIRTL-seq offers a universal protocol scalable from a single cell to millions of T cells per sample, simultaneously delivering both precise clonal frequency estimation and accurate TCR chain pairing, combining the strengths of bulk and single-cell TCR-seq.
Authors
- Paul Glyndwr Thomas (ORCID: https://orcid.org/0000-0001-7955-0256)
- Richard J. Webby (ORCID: https://orcid.org/0000-0002-4397-7132)
- Ashleigh Gowen (ORCID: https://orcid.org/0000-0003-3132-6364)
- Balaji Sundararaman (ORCID: https://orcid.org/0000-0001-8559-1660)
- E. Kaitlynn Allen (ORCID: https://orcid.org/0000-0002-3067-751X)
- Jamie Russell-Bell
- Randall T. Hayden (ORCID: https://orcid.org/0000-0002-3073-4115)
- Resha Bajracharya
- Anastasia A. Minervina (ORCID: https://orcid.org/0000-0001-9884-6351)
- Chun-Yang Lin (ORCID: https://orcid.org/0000-0002-3064-0178)
- Kim Allison (ORCID: https://orcid.org/0000-0003-1344-1465)
- Allison M. Kirk (ORCID: https://orcid.org/0000-0002-4286-3678)
- Gang Wu (ORCID: https://orcid.org/0000-0002-1678-5864)
- Hana Hakim (ORCID: https://orcid.org/0000-0001-7146-5452)
- Lee-Ann Van de Velde (ORCID: https://orcid.org/0009-0000-6205-1151)
- Elaine Tuomanen (ORCID: https://orcid.org/0000-0003-0349-8716)
- Amanda Cole (ORCID: https://orcid.org/0000-0001-6179-7752)
- Ronald H. Dallas (ORCID: https://orcid.org/0000-0002-3259-6801)
- Brandi L. Clark
- David C. Brice (ORCID: https://orcid.org/0000-0002-0294-9987)
- Aditya H. Gaur (ORCID: https://orcid.org/0000-0002-6639-5231)
- Diego R. Hijano (ORCID: https://orcid.org/0000-0002-2903-666X)
- Ashley Castellaw (ORCID: https://orcid.org/0000-0002-4149-4209)
- Taylor L. Wilson (ORCID: https://orcid.org/0000-0002-2040-7655)
- Kasi Vegesana (ORCID: https://orcid.org/0000-0001-8847-3774)
- James M. Hoffman (ORCID: https://orcid.org/0000-0002-2625-7766)
- Mikhail V. Pogorelyy (ORCID: https://orcid.org/0000-0003-0773-1204)
- Walid H. Awad
- Samir Adhikari (ORCID: https://orcid.org/0000-0003-0406-2498)
- Maureen A. McGargill (ORCID: https://orcid.org/0000-0002-0916-0372)
- Joshua Wolf (ORCID: https://orcid.org/0000-0002-9912-3646)
- Li Tang (ORCID: https://orcid.org/0009-0007-3391-6655)
- Tomi Mori
- Zachary B. Scott
Institutions
- St. Jude Children's Research Hospital (US)
Publication Details
- Journal
- Nature Methods
- Published
- 2025-11-24
- DOI
- https://doi.org/10.1038/s41592-025-02907-9
- Citations
- 5
- Primary Topic
- T-cell and B-cell Immunology
- Type
- article
- Field-Weighted Citation Impact
- 3.99
Funders
- American Association of Immunologists
- American Lebanese Syrian Associated Charities