Non-canonical D1-D2 rearrangements are bimodal and represent a conservative hidden stage of VDJ recombination in TCR beta locus

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. Here we reconstructed full human TRB DD rearrangements repertoires for the first time. We validated its authenticity by detecting excision circles with RSS (recombination signal sequence) junctions. The found rearrangements generated in compliance with the classical 12/23 rule are common for humans, rats, and mice, and contain typical VDJ footprints: random nucleotide deletions and insertions. Detected bimodal distribution of DD junctions indicates two active recombination sites producing long and short rearrangements. Unlike long DD rearrangements, the short ones have unusual origin resulting from non-canonical intrachromosomal RSSs junctions formation. Identified DD rearrangement leads to the deletion of J1 and C1 segments and creates a diverse hybrid D segment that can further recombine with J2 and V segments. However, in most complete TRB genes, the traces of DD junctions are blurred by cutting in DDJ and VDDJ rearrangements which made this stage of VDJ recombination mostly hidden in the final completely rearranged TRB gene. ### Competing Interest Statement The authors have declared no competing interest.