Schematic representation of RAG-mediated V(D)J recombination showing the relative mapping positions of DP and RF read-pairs from recombined genomic and excised circular DNA. (A) Hypothetical genomic locus containing variable (blue box) and joining (red box) elements flanked by recombination signal sequence (RSS) motifs (blue and red triangles) are bound and brought into close association by the recombination activating gene (RAG) complex (gray). RAG-mediated double-stranded cleavage at RSS sites precedes genomic deletion (coding) end processing by the nonhomologous end joining (NHEJ) complex. Coding ends are covalently hair-pinned (gray circle section) and reopened prior to ligation. Secondarily, the excision circle (EC; signal) junction is ligated from unprocessed DNA ends with little modification. (B) Sequencing library fragments from recombined EC and genomic (g) DNA are shown. Read-pair sequences from fragments are mapped back to the reference genome, allowing junction-spanning reads to be identified. Gray arrows represent “standard” read-pairs generated from EC DNA or gDNA and map to the reference genome in standard orientation separated by a mapping distance equal to the initial fragment length. Deletion read-pairs (DPs) spanning a coding junction map to the reference genome with standard relative orientation—the forward read (green) mapping 5′ of and facing the reverse read (red)—but are separated by mapping distances greater than the initial fragment length. Reverse-forward read-pairs (RFs) spanning the signal junction map to the reference genome in an inverted relative read orientation so that the reverse read (red) maps 5′ of and faces away from the forward read (green). RF read-pairs are separated by the full length of the circle from which they originate rather than the initial library fragment length.
