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<p>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.</p>

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TIRTL-seq: deep, quantitative and affordable paired TCR repertoire sequencing

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年11月24日

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  • Mikhail V. Pogorelyy,
  • Allison M. Kirk,
  • Samir Adhikari,
  • Anastasia A. Minervina,
  • Balaji Sundararaman,
  • Kasi Vegesana,
  • David C. Brice,
  • Zachary B. Scott,
  • Joshua Wolf,
  • Aditya Gaur,
  • James M. Hoffman,
  • Tomi Mori,
  • Li Tang,
  • Elaine I. Tuomanen,
  • Hana Hakim,
  • Randall T. Hayden,
  • Diego R. Hijano,
  • Kim J. Allison,
  • E. Kaitlynn Allen,
  • Walid Awad,
  • Resha Bajracharya,
  • Brandi L. Clark,
  • Lee-Ann Van de Velde,
  • Taylor L. Wilson,
  • Ronald H. Dallas,
  • Ashleigh Gowen,
  • Amanda Cole,
  • Jamie Russell-Bell,
  • Ashley Castellaw,
  • Chun-Yang Lin,
  • Maureen A. McGargill,
  • Richard J. Webby,
  • Gang Wu,
  • Paul G. Thomas

摘要

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.