BMC Genomics 2009, 10:490 http://www.biomedcentral.com/1471-2164/10/490 closer match to the query and performs the appropriate ortholog prediction route (figure 1). In addition, for any that fail, a rescue was be performed by searching through the other route. CSRBH outperformed all other methods and produced 3% and 11% more successful ortholog assignments than the SRBH and RBH, respectively. In total 8,726 contigs were assigned an ortholog, totalling 3,762 non-redundant genes (see additional file 10). See table 2 and figure 3 for the number of successful ortholog assignments for all the different analysis methods used in this work. We propose that CSRBH should be the method of choice for research- ers working with non-model species which have no sequenced genome and partially sequenced genes. A weakness with this approach is that it does not deter- mine an orthologs absolute gene history and it will not report to a user whether the ortholog found was derived from a gene duplication event or from a speciation. That said, these methods will enable a user to quickly find the most likely functional homolog of a gene. Prediction of angiogenic genes Established angiogenic genes Previous groups have developed complex data mining methods using decision trees to identify genes of interest [50]. In this study, 4 simple strategies were combined to generate as a complete a set of angiogenic genes as possi- ble. First the cardiovascular and angiogenesis groups at Birmingham University used their knowledge to produce a list of angiogenic genes that included those from the recent literature [51] and the SABiosciences GEArray® Human Angiogenesis Microarray [52]. A total of 260 ang- iogenesis related genes were predicted this way and a list of 73 genes were contained in the successful CSRBH results (additional file 11, column D, labelled in house). However, it is possible many other genes have an undis- covered role in angiogenesis and, therefore, three further methods were employed to predict additional angiogenic genes. Table 2: A summary of the results for the bioinformatic analyses Number of contiguous sequences 16650 RBH successful orthologs 6779 SRBH successful orthologs 8145 CSRBH successful orthologs 8726 Putative angiogenic genes The second and third approaches used Perl programs to scan article abstracts from PubMed [53] and Gene Ontol- ogy processes from AMIGO [54] using the following ang- iogenic related keywords as baits: 'angiogenic, angiogenesis, neovascularization, neovascularisation, vas- culogenesis, hypoxia, endoth VEGF'. This predicted a fur- ther 737 angiogenic genes (additional file 11, columns E and F respectively). A fourth method used gene expression of ECs. During the activation of angiogenesis, ECs become activated and switch on several signalling pathways that cause prolifera- tion, migration and invasion [55,56]. This angiogenic phenotype leads to an up-regulation of EC genes. There- fore, genes up-regulated in the endothelial transcriptome could be angiogenic. The endothelium transcriptome has been extensively defined [15,16] and genes up-regulated in ECs were compared with the human/carp orthologs to identify additional putative angiogenic genes; 98 were found (additional file 11, columns G and H). Total angiogenic genes Combining the results from the four data mining meth- ods gave a total of 908 non-redundant predicted ang- iogenic genes (additional file 11). We postulate this gave us a comprehensive set and the best chance of discovering cold-induced angiogenic genes from the carp data. How- ever, it should be noted, and the authors fully acknowl- edge, that not every gene predicted here is a genuine angiogenesis gene and there will be some false positives. Very few bioinformatic predictions are 100% foolproof and this study is aimed at guiding the bench scientist into making more informed decisions before going into the laboratory. The number of different methods that pre- dicted a gene angiogenic now ranks the genes. For exam- ple, the gene HIF1A is a well-known angiogenic gene and was predicted as such by all four methods. Column I enti- tled "Number of methods" in additional file 11 gives the number of methods predicting a gene angiogenic and is ranked in a descending order. Conversely, genes that are not so well known to be angiogenic genes are only found by one or two methods. If this approach is employed and genes selected that were predicted by more than one Number of genes Number angiogenic genes Number of up-regulated genes 3762 908 567 Number of up-regulated angiogenic genes 135 This table displays the number of human protein orthologs found using CSRBH for the 16,650 cDNA carp contiguous sequences. 3,762 total none redundant genes were found and 908 of them were predicted as angiogenesis genes. From the re-analysis, 567 genes were up-regulated and 24% of them were predicted as angiogenesis genes. Page 7 of 17 (page number not for citation purposes)