HTGTS-TCR-seq for profiling of mouse and human T-cell receptor a and ß gene rearrangements and diversity [HTGTS-TCRseq]

T-cell receptor (TCR) diversity is generated through V(D)J recombination, which are essential for adaptive immunity. Although many methods have been applied, a simple method to detect TCR rearrangement at the DNA level remains lacking. Here we introduce HTGTS-TCR-seq, a novel approach derived from high-throughput genome-wide translocation sequencing (HTGTS), to comprehensively and unbiasedly analyze TCR recombination. Unlike multiplex PCR requiring extensive V-J segment primers, HTGTS-TCR-seq enables unbiased TCR profiling through targeted enrichment and deep sequencing by a single primer. This method identifies biased V(D)J segment usage in developing T cells through V/J primer-based amplification and provides unique complementarity determining region 3 (CDR3) profiles in mature T cells via J primers. HTGTS-TCR-seq detects rearrangement at both alleles, enabling quantification of DJß and VßDJß rearrangement products with productive and nonproductive profile. Applied to human peripheral blood mononuclear cells (PBMCs), HTGTS-TCR-seq reveals a shared CDR3 motif between humans and mice. HTGTS-TCR-seq is a robust tool for T cell immunity research, with potential application in immune responses analysis, cancer research, and autoimmune diseases investigation. Overall design: We employed HTGTS-TCR-seq to asess rearrangement events and TCR repertoire changes across developmental stages and T cell subsets at DNA level. DN3 thymocytes, preselection DP thymocytes, splenic CD4+ and CD8+ T cells from mice and CD4+ and CD8+ T cells from human PBMCs were FACS sorted. Total thymocytes from young and aged mice were collected. Genome DNA were extracted, sonicated and utilized to conduct HTGTS-TCR-seq.