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.