D790 Nucleic Acids Research, 2008, Vol. 36, Database issue Figure 2. Evola main page. This page is divided into left and right frames. In the left frame, tables of orthologs, download data, Gene ontology, and InterPro are listed. Three green buttons are links to show ‘Alignment’ (A), ‘dN/dS view’ (B) and ‘Locus maps’ (C) in the right frame. the following information for a human gene is available in the left frame: gene name, ortholog list with annotation status, download of sequences, alignments and phyloge- netic trees, Gene ontology (22) and InterPro (23). In addition to the set of original ClustalW alignments, another set of alignments, including properly aligned sequences only (24), was also constructed and provided. In the latter sets, sequences with distinctively low identity to other sequences in an alignment were excluded. Based on both alignment sets, phylogenetic trees were constructed by the neighbor-joining method (18) and the NJML+ method (25). In the right frame of the main page, Evola features the three views described below. Users can switch among the views. Alignment: Multiple alignments of orthologs and homologs (Figure 2A) Amino acid sequence alignments of orthologs and homologs are displayed. Users can switch from ‘Alignment of Orthologs’ (default) to ‘Alignment of Orthologs and Homologs’, or vice versa. Each amino acid residue is color coded as deﬁned in ClustalX (19). Accession numbers and species names of orthologs (human and other species) are colored in their species colors deﬁned in Evola (human in red, mouse in gray, etc.). Accession numbers of homologs are linked to the original data sources of UniProt or RefSeq. While species are labeled by their scientiﬁc names (Homo, Mus, etc.), users can activate a popup window giving a species common name by placing the mouse cursor over homolog accession numbers (for example, ‘Q5R508_Pongo’). InterPro data in the left frame include positional information on a human gene, and they can be utilized to detect conserved domains in the proteins. dN/dS view: Window analysis detecting regions under positive or negative selection (Figure 2B) Users can select one or more species for which to show the plots in the graph. In the lower frame under the graph, the pairwise nucleotide sequence alignment of CDSs is shown. The sequence positions (a.a. or codon) appearing in the graph and alignment are those of human genes. The nonsynonymous to synonymous substitution rate ratio (dN/dS) is a commonly used measure of natural selection. In order to visualize positively and negatively selected regions, sliding window analysis was conducted (a 20 codon window with 1 codon stepping; result for the ﬁrst window appears as a plot at 11th codon of the human gene). The statistical signiﬁcance (P-value) of the diﬀerence between the number of nonsynonymous substitution (n) per synonymous substitutions (s): n/s, and the number of nonsynonymous sites (N) per synonymous sites (S): N/S was calculated by Fisher’s exact test.