D284 Nucleic Acids Research, 2011, Vol. 39, Database issue evolutionary annotation of the derived orthologous groups, as well as improved text-based searches, querying on the phyletic proﬁle of orthologous gene copy numbers, and searches by sequence homology. OrthoDB is freely accessible from http://cegg.unige.ch/ orthodb, and now referenced with link-outs from a number of resources including UniProt (24) and FlyBase (25). METHODOLOGY Orthology is deﬁned relative to the last common ancestor of the species being considered, thereby determining the hierarchical nature of orthologous classiﬁcations (1–3) (Supplementary Figure S1). This is explicitly addressed in OrthoDB (10) by application of the orthology delinea- tion procedure at each radiation point of the considered phylogeny, empirically computed over the super-alignment of single-copy orthologs using a maximum-likelihood approach corroborated with known taxonomies. The OrthoDB implementation employs a BRH clustering algorithm based on all-against-all Smith–Waterman (26) protein sequence comparisons computed using PARALIGN (27). Gene set pre-processing selects the longest protein-coding transcript of alternatively spliced genes and of very similar gene copies (>97% identity). The newly optimized procedure triangulates BRHs with an e-value cutoff of 1e-3 to pro- gressively build the clusters, (non-triangulated BRHs are considered with an e-value cutoff of 1e-6), and requiring an overall minimum sequence alignment overlap of 30 amino acids to avoid domain walking. These core clusters are further expanded to include all more closely related within-species in-paralogs, and the previously identiﬁed very similar gene copies. Inspections of the OrthoDB orthologous classiﬁcations as part of several genome projects (28–31) and other comparative genomic studies (32–35) have conﬁrmed their biological relevance and acceptable accuracy. UPDATED DATABASE CONTENT Gene sets The complete predicted protein-coding gene sets were retrieved from publically available genomic resources including 44 vertebrates from Ensembl (36) (Release 58, May 2010), 25 arthropods from AphidBase (37), BeetleBase (38), FlyBase (25), Hymenoptera Genome Database, SilkDB (39), VectorBase (40) and wFleaBase (41) (current releases in July 2010), and 46 fungi from UniProt (24) (August 2010 release). Gene sets for an additional ﬁve animal species were retrieved for orthology delineation across metazoa: lancelet, polyp, sea anemone, sea urchin, and worm (current releases in July 2010). For full details of the genome assembly and gene set releases used for each species, please see Supplementary Table S1. Gene annotations Annotations describing putative functional attributes were sourced from UniProt (24), as well as from species-speciﬁc Table 1. OrthoDB species and gene content Lineage Species count Vertebrates 44 Arthropods 25 Fungi 46 Total 793 077 422 677 396 089 Input genes 18 024 16 907 8611 Average SD Count % 2884 5280 3632 748 195 94 18 474 325 685 77 20 428 331 623 84 14 088 Classiﬁed genesa Orthologous groupsa Statistics describing the input gene sets and OrthoDB classiﬁcations. aTotal counts of genes and orthologous groups at the root of each lineage. resources including Mouse Genome Informatics (MGI) (42), FlyBase (25) and Saccharomyces Genome Database (SGD) (43). UniProt identiﬁer cross-referencing allowed mapping of gene annotations to the gene sets retrieved from Ensembl and other sources. The UniProt data were also employed to comprehensively map gene names and synonyms, as well as secondary gene identiﬁers and cross-referenced database gene identiﬁers, e.g. RefSeq, Entrez GeneID, GenBank, Protein Data Bank and Mendelian Inheritance in Man, as well as assigned Gene Ontology (GO) (44) attributes. The species-speciﬁc model organism databases (MGI, FlyBase and SGD) provided mapping to additional gene synonyms and identiﬁers as well as selected controlled-vocabulary gene phenotypes from relevant experimental data (Supplementary Table S2). Protein domain signatures were retrieved from InterPro (45) matches to the UniProt Archive (UniParc) of non-redundant protein sequences. Orthologous groups Analysis of the selected eukaryotic species focused on resolving orthologous relations at each radiation of the three sampled lineages, as well as delineating metazoan orthologous groups by analyzing the vertebrates and arthropods with ﬁve additional animal species. For the complete sets of vertebrates, arthropods and fungi, 87% of a total of 1 611 843 genes were classiﬁed into 18 474, 20 428 and 14 088 orthologous groups, respectively (Table 1). The greater spans and faster evolutionary rates across the arthropod and fungal phylogenies (33,46,47) may limit the detection of very distant homology, leading to the observed lower proportions of classiﬁed genes compared to the vertebrates. Additional factors that may inﬂuence the proportions of classiﬁed genes include the completeness and coverage of genome sequencing as well as quality and consistency of gene repertoire predictions (e.g. variable strategies applied to arthropods). NEW OrthoDB FEATURES Functional annotations of orthologous groups Orthology delineation aims to identify groups of genes descended from a common ancestor, thereby enabling tentative functional attributes ascribed to one or more members to be generally extrapolated to describe the group as a whole. Protein-coding genes from model organisms are by far the best studied and therefore provide the most comprehensive annotations and insights