D788 Nucleic Acids Research, 2008, Vol. 36, Database issue for large-scale comparative analyses. In particular, detection of orthologs, groups of genes in diﬀerent species that evolved by speciation, accelerates functional and evolutionary studies. Despite the past eﬀorts to develop bioinformatics methods for analyzing a large number of sequences, it is still a challenge to compre- hensively identify orthologs between species. A number of automated pairwise distance-based methods for ortholog detection have been proposed, as represented by the reciprocal best BLAST hits (RBH) method (1) and the reciprocal smallest distance (RSD) method (2). However, as genes might have frequently undergone duplications and losses in evolutionary lineages leading to human (3), pairwise distance-based methods might lead to erroneous inferences of phylogenetic relationships and thus of orthologs. Thus, phylogenetic tree-based detection can be the most plausible solution to provide more reliable orthologs. Here this database ‘Evola’, a sub-database comple- mentary to the H-Invitational database (H-InvDB), was developed to provide orthology information for the originally annotated human genes in H-InvDB. Evola features its ortholog detection in which genome synteny- based computational analysis was followed by manual curation of molecular phylogenetic trees. Evola diﬀers in this way from other ortholog databases such as Inparanoid (4), Ensembl-Compara (5), Homologene (6), HOGENOM (7) and TreeFam (8). These databases are based on BLAST hits (Inparanoid), BLAST hits and synteny (Ensembl-Compara and Homologene) and phy- logenetic trees (HOGENOM and TreeFam). The concept of Evola is that genomic region (gene locus) is a unit of genes that are duplicated or lost. In collaboration with H-InvDB, Evola enables users to compare gene structure, transcript variants, upstream/downstream region of the genome among species. H-InvDB is an integrated database of annotated human genes providing annotation of human full-length enriched cDNAs (9,10,11). At the meetings of the Human Full-Length cDNA Annotation Invitational held in Japan (2002 and 2003), Evola started with H-InvDB to annotate evolutionary features of the human genes. With several updates afterwards and a subsequent All Human Genes Evolutionary Annotation (AHG-EV) meeting in 2006, the current strategy of evolutionary annotation (computational analysis and manual curation) in Evola has been established. Orthology information for human and other 11 verte- brates is currently included in the Evola: human, chimpanzee, macaque, mouse, rat, dog, cow, opossum, chicken, zebraﬁsh, Tetraodon and Fugu. Several visua- lization tools are incorporated into the database, including sequence alignment viewer, natural selection plot and graphical representation of orthologous gene loci among diﬀerent species. Evola is now one of the databases listed in the Comparison of Orthology Predictions project of the HUGO Gene Nomenclature Committee (HGNC, http://www. genenames.org/). ORTHOLOG DETECTION Computational analysis: Ortholog detection based on conserved genomic synteny and pairwise distance Species for ortholog detection were selected with con- sideration of completeness of their genome assemblies (chromosome level), abundance of transcript sequences ( 20 000) and importance in biology (intensively studied or a representative of a phylogenetic clade). Whole genome sequence assemblies of human (hg18), chimpan- zee (panTro2), macaque (rheMac2), mouse (mm8), rat (rn4), dog (canFam2), cow (rn4), opossum (monDom4), chicken (galGal3), zebraﬁsh (danRer4), Tetraodon (tetNig1) and Fugu (fr1) were downloaded from UCSC (http://genome.ucsc.edu/). Conserved syntenic regions were detected by a modiﬁed pairwise genome alignment method (12) using BLASTZ (13) with the options of C = 2, T = 4, Y = 3400 between human and other primates (between more similar genome sequences), and C = 2 between human and non-primate vertebrates (between less similar genome sequences). For human transcripts, H-InvDB representative tran- scripts (HITs) were used. Other vertebrates’ transcripts (mRNAs) were downloaded from DDBJ (http://www. ddbj.nig.ac.jp/) release 66, Ensembl (http://www.ensembl. org/) release 38 and RefSeq (http://www.ncbi.nlm. nih.gov/RefSeq/) release 17, and their genomic locations (one location per transcript) were detected on cognate genomes by a hybrid method using BLAT (14), BLAST (15) and est2genome (16) as they were used to detect genomic locations of human transcripts in H-InvDB. Representative transcripts (one transcript per gene locus) were determined in consideration of percent identity and coverage to the genome, number of exons, etc. of all transcripts in each locus (9,10,11). Thus, in Evola, representative transcripts were deﬁned as genes. Lengths of overlapping exons of each gene pair between human and other species were calculated in the genome alignment. A gene pair with the maximum length was selected as the best assignment (not a minimum length was deﬁned). Every gene in a species was assigned to a gene in the other species. If two human genes were assigned to one mouse gene, this was deﬁned as a two-to-one ortholog. As a result, Evola contains not only one-to-one orthologs but also many-to-many orthologs. For all the assignment pairs, coding sequences (CDSs) and amino acid (a.a.) sequences of other species were predicted by FASTY (17). They were predicted by comparing with the amino acid sequences of the corresponding human genes. Finally, if the length of the alignable region between human and other species ortholog candidates was 80 a.a., they were deﬁned as computationally detected orthologs. Manual curation: Examination of phylogenetic trees by experts Homologs of human genes (amino acid sequences) were obtained from UniProt (http://www.uniprot.org/) and human RefSeq (NP) by FASTY similarity searches with the option of E-value of <1e 5. For each human gene, a