BMC Genomics 2009, 10:490 http://www.biomedcentral.com/1471-2164/10/490 puted databases including Homologene [32,33], it could only deal with one sequence at a time. Additionally, the BLASTO form did not permit the user to change BLAST fil- tering options which, as seen below, makes a difference to the quality and quantity of successful ortholog predic- tions. Ortholog assignments, RBH and filtering options A graph based, nearest neighbour [28] approach, RBH, was considered the best option since constructing multi- ple sequence alignments and phylogenetic trees from incomplete nucleotide sequences would be inaccurate and not amenable to high throughput. Strengthening this decision, a performance assessment of different ortholog prediction methods by Altenhoff and Dessimoz 2009 [34] found the RBH performed well in comparison with the other methods. Reciprocal Best Hit (RBH) [28,29] is a technique using BLAST to first search a query sequence against a genome. If the resulting best hit matches the original query sequence from a reciprocal BLAST then it is an ortholog termed RBH. A diagrammatical representation of the RBH process of carp vs. human is shown in figure 1, labelled route 1. An article by Moreno-Hagelsieb and Latimer 2008 [29] tested ortholog RBH searching using different BLAST options. The tests utilised bacterial genomes, neighbour- ing orthologs and paralogs to estimate error rates of ortholog assignments by RBH. They found the soft filter- ing option -F "m S" with the addition of Smith-Waterman (SW) alignments (-s T) were optimal for RBH success. These options were tested for eukaryotes using carp vs. human RBH searches. Four different BLAST options were tested: soft filtering (-F "m S") with and without SW (-s T) and hard filtering with and without SW. The results revealed very little difference in the number of successful RBH with or without the SW algorithm against their find- ings of 10%. In contrast, as with prokaryotes, soft filtering increased the number of RBHs by 2% (209 RBHs). Table 1 shows the number of successful orthologs found using carp vs. human RBH for the different filtering options. Fig- TFhigisufriegu1re shows the routes to ortholog assignment This figure shows the routes to ortholog assignment. Reciprocal Best Hit (RBH), labelled route 1, conveys if two genes in carp and human find each other as the best hit in the other species. Stepped Reciprocal Best Hit, SRBH (route 2), extends RBH by using a closely related species, zebrafish, to enhance ortholog assignments. This is possible as the zebrafish has many more sequences in the databases, its genome is significantly more annotated and it has a genome sequence available. The Con- ditional Stepped Reciprocal Best Hit combines both RBH and SRBH as some sequences are still missing from zebrafish. Page 3 of 17 (page number not for citation purposes)