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

The specificity of T cells is determined by T cell receptor (TCR) alpha and beta chain sequences. While bulk TCR sequencing enables cost-effective repertoire profiling without chain pairing information, single-cell approaches provide paired data but are limited in throughput and costly. Here, we present TIRTL-seq (Throughput-Intensive Rapid TCR Library sequencing), an experimental and computational methodology for paired TCR repertoire sequencing. TIRTL-seq is based on the parallel generation of hundreds of TCR libraries in 384-well plates at less than USD200 per plate allowing cohort-scale paired TCR-seq studies. We benchmarked TIRTL-seq against state-of-the-art 5RACE bulk TCR-seq and 10x Genomics Chromium technologies on longitudinal samples and identified SARS-CoV-2- and EBV-specific CD8+ T cell expansions post-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.