Non-canonical D1-D2 recombination produces two types of rearrangements and represents a conservative hidden stage of T-cell receptor beta chain generation

T-cell receptor (TCR) diversity is generated by VDJ recombination. The classical course of TCR beta (TRB) chain production starts with D and J segment recombination and finishes with subsequent recombination between the resulting DJ junction and V segment. In this study, we performed deep sequencing of poorly explored incomplete TRBD1 to TRBD2 rearrangements in T-cell genomic DNA. We reconstructed full repertoires of human incomplete TRB DD rearrangements and validated its authenticity by detecting excision circles with RSS (recombination signal sequence) junctions for the first time. The identified rearrangements generated in compliance with the classical 12/23 rule are common for humans, rats, and mice and contain typical VDJ recombination footprints. Detected bimodal distribution of DD junctions indicates two active recombination sites producing long and short DD rearrangements. Unlike long DD rearrangements, the short ones have unusual origin resulting from non-canonical intrachromosomal RSSs’ junctions formation. Identified DD rearrangements lead to deleting J1 and C1 segments and creating diverse hybrid D segments, which recombine further with J2 and V segments. Resulting functional TRB VDDJ rearrangements are present in the memory T-cells subset proving its participation in antigen recognition.