dS, dN, s and n values were estimated by the modiﬁed Nei–Gojobori method (26,27). If dN/dS > 1, the score (= 1 P-value) was plotted above the zero line (neutral), and if dN/dS < 1, the score [= (1 P-value)] was plotted below the zero line. The regions plotted above the red line indicate that the sites might be under positive selection (dN/dS > 1 and P < 0.01). Conversely, the regions plotted below the blue line indicate that the sites might be under negative (purifying) selection (dN/dS < 1 and P < 0.01). Locus maps: Comparative maps of orthologous gene loci (Figure 2C) Orthologs were detected for representative transcripts (one transcript per gene locus) in Evola. However, there could be transcript variants in gene loci that have diﬀerent exon–intron structures leading to produce diﬀerent protein isoforms. Thus, information on other transcripts besides the representative transcript among orthologous gene loci are shown in Locus maps. In the ﬁgures, exon/intron structure, coding sequence (CDS) and untranslated regions (UTR) for each transcript are visualized. H-Inv cluster ID (HIX, an identiﬁer of gene locus), Gene symbol, genomic location and a link to ‘G-integra’, an integrated genome browser of H-InvDB, are available. The ﬂag icon denotes the representative transcript. The blue diamond icon denotes the Representative Alternative Splicing Variant (RASV) that is another representative per transcript group consisting of the same alternative splicing pattern (28). Repre- sentative transcripts are also RASVs, and blue diamonds do not appear if there is only one splicing isoform. In the tables, the H-Inv transcript ID (HIX) and original accession numbers (DDBJ/EMBL/GenBank, Ensembl and RefSeq) of the representative transcript and other transcripts are listed. FUTURE DIRECTIONS As our update policy, orthology information in Evola is updated when H-InvDB annotation is updated. One major update and three minor updates per year are scheduled. At the next major update on December 2007, a new duplicate gene family view is planned to be integrated within Evola. Human duplicate gene family data was originally constructed based on both amino acid sequence similarity (29) and orthology information. In the current Evola (release 4.1), parts of human duplicate gene annotation have been already implemen- ted. The human duplicate genes are included in the alignments and phylogenetic trees of orthologs and homologs. Finally, we expect Evola to serve as a new database for evolutionary annotation of human genes. We sincerely welcome any requests and feedback from users. ACKNOWLEDGEMENTS We thank the members of Integrated Database Group, Japan Biological Information Research Center for their Nucleic Acids Research, 2008, Vol. 36, Database issue D791 helpful suggestions. We are also grateful to Craig Gough for critical reading of the manuscript. This work was supported by the Ministry of Economy, Trade and Industry of Japan (METI), and the Japan Biological Informatics Consortium (JBIC). Funding to pay the Open Access publication charges for this article was provided by JBIC. Conﬂict of interest statement. None declared. REFERENCES 1. Tatusov,R.L., Koonin,E.V. and Lipman,D.J. (1997) A genomic perspective on protein families. Science, 278, 631. 2. Wall,D.P., Fraser,H.B. and Hirsh,A.E. (2003) Detecting putative orthologs. Bioinformatics, 19, 1710–1711. 3. Fortna,A., Kim,Y., MacLaren,E., Marshall,K., Hahn,G., Meltesen,L., Brenton,M., Hink,R., Burgers,S., Hernandez- Boussard,T. et al. (2004) Lineage-speciﬁc gene duplication and loss in human and great ape evolution. PLoS. Biol., 2, E207. 4. O’Brien,K.P., Remm,M. and Sonnhammer,E.L. (2005) Inparanoid: a comprehensive database of eukaryotic orthologs. Nucleic Acids Res., 33, D476–D480. 5. Birney,E., Andrews,D., Caccamo,M., Chen,Y., Clarke,L., Coates,G., Cox,T., Cunningham,F., Curwen,V., Cutts,T. et al. (2006) Ensembl 2006. Nucleic Acids Res., 34, D556–D561. 6. Wheeler,D.L., Barrett,T., Benson,D.A., Bryant,S.H., Canese,K., Chetvernin,V., Church,D.M., DiCuccio,M., Edgar,R., Federhen,S. et al. (2007) Database resources of the National Center for Biotechnology Information. Nucleic Acids Res., 35, D5–D12. 7. Dufayard,J.F., Duret,L., Penel,S., Gouy,M., Rechenmann,F. and Perriere,G. (2005) Tree pattern matching in phylogenetic trees: automatic search for orthologs or paralogs in homologous gene sequence databases. Bioinformatics, 21, 2596–2603. 8. Li,H., Coghlan,A., Ruan,J., Coin,L.J., Heriche,J.K., Osmotherly,L., Li,R., Liu,T., Zhang,Z., Bolund,L. et al. (2006) TreeFam: a curated database of phylogenetic trees of animal gene families. Nucleic Acids Res., 34, D572–D580. 9. Imanishi,T., Itoh,T., Suzuki,Y., O’Donovan,C., Fukuchi,S., Koyanagi,K.O., Barrero,R.A., Tamura,T., Yamaguchi-Kabata,Y., Tanino,M. et al. (2004) Integrative annotation of 21,037 human genes validated by full-length cDNA clones. PLoS. Biol., 2, e162. 10. Yamasaki,C., Koyanagi,K.O., Fujii,Y., Itoh,T., Barrero,R., Tamura,T., Yamaguchi-Kabata,Y., Tanino,M., Takeda,J., Fukuchi,S. et al. (2005) Investigation of protein functions through data-mining on integrated human transcriptome database, H- Invitational database (H-InvDB). Gene, 364, 99–107. 11. Yamasaki,C., Murakami,K., Fujii,Y., Sato,Y., Harada,E., Takeda,J., Taniya,T., Sakate,R., Kikugawa,S., Shimada,M. et al. (2008) The H-Invitational Database (H-InvDB), A comprehensive annotation resource for human genes and transcripts. Nucleic Acids Res., 36, in press. 12. Fujii,Y., Itoh,T., Sakate,R., Koyanagi,K.O., Matsuya,A., Habara,T., Yamaguchi,K., Kaneko,Y., Gojobori,T., Imanishi,T. et al. (2005) A web tool for comparative genomics: G-compass. Gene, 364, 45–52. 13. Schwartz,S., Kent,W.J., Smit,A., Zhang,Z., Baertsch,R., Hardison,R.C., Haussler,D. and Miller,W. (2003) Human-mouse alignments with BLASTZ. Genome Res., 13, 103–107. 14. Kent,W.J. (2002) BLAT—the BLAST-like alignment tool. Genome. Res., 12, 656–664. 15. Altschul,S.F., Gish,W., Miller,W., Myers,E.W. and Lipman,D.J. (1990) Basic local alignment search tool. J. Mol. Biol., 215, 403–410. 16. Rice,P., Longden,I. and Bleasby,A. (2000) EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet., 16, 276–277. 17. Pearson,W.R. (2000) Flexible sequence similarity searching with the FASTA3 program package. Methods Mol. Biol., 132, 185–219.