Herzig et al. BMC Genomics 2010, 11:100 http://www.biomedcentral.com/1471-2164/11/100 Page 12 of 19 Figure 6 TRDJ gene sequence alignments and phylogenetic tree. Alignments of TRDJ gene (A) nucleotide and (B) deduced amino acid sequences, using bovine (bo) genomic TRDJ sequences identified here and previously classified ovine (ov), swine (sw), human (hu) and murine (mo) TRDJ sequences (described in Methods) are shown with identities indicated by a dot (.) and gaps indicated by a dash (-). The Neighbor- Joining method [47] was used to infer evolutionary history of the above mentioned sequences. The optimal tree (C) with the sum of branch length = 2.43145392 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [48]. The Maximum Composite Likelihood method [49] was used to compute evolutionary distances. Complete deletion to eliminate gaps was performed and the final dataset included a total of 42 positions. Last, for the sake of completeness we note that cDNA evidence for bovine TRDV2, TRDV3 and TRDV4 genes reported here has already been demonstrated [17] and has been confirmed [20]. Identification of the TRDD genes within the bovine genome allowed us to evaluate TRDD gene usage in cDNA sequences that had been previously sequenced in our laboratory. CDR3 of 73 TRDV1, 17 TRDV2, 17 TRDV3 and 36 TRDV4 containing sequences were ana- lyzed (Table 6). All D genes were represented in tran- scripts containing genes of the four TRDV subgroups. P nucleotides were observed flanking untrimmed TRDD regions and extensive N-regions were frequently found between TRDD regions. Some cases were ambiguous because nucleotides could be attributed to either P- or N-region but also to the presence of more than one TRDD gene. Therefore, we applied the criteria that at least six nucleotides of a particular TRDD gene must be present to claim usage of that gene with reasonable con- fidence. Examples of TRDD gene usage with nucleotide and deduced amino acid sequences are shown (Figure 7). These demonstrated the incorporation of one to five TRDD genes within a single CDR3. Only cases in which the order of TRDD genes in the CDR3 corresponded to that found in the genome were retained. Discussion In order to evaluate and characterize the bovine T cell receptor delta gene repertoire and their genomic orga- nization we annotated the TRD genes in the Btau_3.1 assembly. Here we describe the existence of two TRDV2 genes, as well as a single gene each for