BMC Genomics 2009, 10:490 http://www.biomedcentral.com/1471-2164/10/490 From the 8,145 successful RBHs, only 7,020 could be compared as only these had both human and zebrafish genes present in the Homologene data. This fact alone gives support to using this approach as a user supplies all genes, and as such, all are present. From these ortholog assignments, 85% agreed with Homologene orthologous groups. A manual investigation of some of those that dis- agreed suggests Homologene does not always perform well for a proportion of duplicated genes. For instance, one example, the zebrafish actc1l gene best matched the human ACTC1 gene with 99% identity over the full align- ment length of the gene but Homologene put actc1l in the ACTB orthologous group (id: 110648) where alignment percent identity was less at 94% (additional file 5). The full results of the comparison can be viewed in additional file 6. In addition, ten alignments were chosen randomly and manually investigated (additional file 7) to see which source of data was most realistic. The results of this sug- gested 8 out of 10 ortholog assignments were correct with RBH whilst Homologene put the genes into different orthologous groups. A full comparison of RBH with Homologene A full analysis was also performed that took all human proteins in the Refseq database of proteins and RBH searched against the full database of zebrafish Refseq pro- teins. On July 22nd 2009, there were 29,428 proteins in the human database. 19,648 of these proteins resulted in a successful RBH analysis against zebrafish. Build 64 of Homologene (16th July 2009) contained 19,571 human proteins. Therefore 33% of the human proteome was not represented in Homologene at this time and only 12,035 of the 19,571 human proteins had a zebrafish homolog protein in the same orthologous group. There were 653 human/zebrafish orthologs identified by Homologene only and not with the RBH procedure. In contrast, there were 8,266 human/zebrafish orthologs found using the RBH but not with in Homologene. 11,382 human proteins were found in both Homologene and in the RBH analysis. 10,099 of these agreed, an 89% agreement. This is very similar to the result above with only the carp data. These findings, particularly the fact that Homologene only had 12,035 human proteins with a zebrafish ortholog and the findings of Altenhoff [34], give a user some further support and confidence to using a RBH ortholog assignment approach rather than phylogenetic tree methods for analysing large amounts of data from a non-model species in a quick and fairly accurate manner. The SRBH approach is endorsed by the fact other research- ers have used closely related species to predict genes and sequence motifs [39,40]. However, it should be noted that the comparisons done here with Homologene only vali- dates the RBH method but not the CSRBH as, to defini- tively validate the CSRBH approach, the carp genome and annotation need to be more complete. In addition, the CSRBH approach does not attempt to bet- ter or replace the efforts of databases such as Homolo- gene. One reason is that it does not attempt to deal with paralogs and other sources of gene histories but only with the most functional homologs (orthologs), though some attention was given to the teleost genome duplication event. So there are examples where Homologene better assigns orthologs than the methods used here. Having said that, with a non-model species not present in Homol- ogene, this approach is excellent at giving a good approx- imation of the human functional homologs of researchers' genes. CSRBH, combining RBH and SRBH to find further orthologs Although adding the zebrafish intermediary step (SRBH) improved ortholog assignment by 8%, 581 assignments that failed using this procedure were successfully found using a carp vs. human RBH alone (figure 1, route 1). Inspection of a random sample of these found two exam- ples of why this occurred. BLAST of carp EST EC394432 vs. zebrafish best hit the gene smyhc3 and the reciprocal BLAST vs. carp found EST AB231800 as the best hit. How- ever, smyhc3 was the fifth best hit to AB231800, thus fail- ing the SRBH analysis (additional file 8). However, this gene is successfully assigned a human ortholog from an RBH analysis (figure 1, route 1). Even more compelling was the situation with EST AU052068 (additional file 9). It aligns with the human protein NP_003745 as the best hit and the alignment was highly similar with a percent identity of 91% over 200 amino acids. The same EST best aligned to the zebrafish protein NP_956083 with 37% identity over 200 amino acids. Hence, the EST is significantly more similar to the human protein rather than the zebrafish. The human pro- tein NP_003745 was BLAST searched against zebrafish to see if the full-length protein found a likely ortholog in zebrafish. The best hit to the human protein was NP_956083, the same as found by searching EST AU052068. Therefore, there is evidence that either some genes do not exist in zebrafish and do so in carp or are not currently found in zebrafish as the genome is not yet com- pletely sequenced or annotated. Another piece of evidence is, if a simple BLAST search is performed against the mul- tiple species NR database of proteins at the NCBI [35], 24% of carp sequences preferentially match a human pro- tein, whilst 30% match a zebrafish protein. This supports the use of a Conditional Stepped Reciprocal Best Hit (CSRBH) approach, which uses whichever species is a Page 6 of 17 (page number not for citation purposes)