Nucleic Acids Research, 2011, Vol. 39, Database issue D571 Table 1. Global species and sequence content in PhEVER All Viruses Bacteria Archaea Eukarya Eukarya (excl. Anopheles)d No. of species 3476 2426 937 70 43 – No. of proteinsa 4 515 271 82 929 (2718 m.p.c) 3 207 914 158 919 1 065 509 580 567 No. of proteins in fam.b Data source 333 618 (1%) 82 784 (100%) 232 066 (7%) 6702 (4%) 12 066 (1%) 11 201 (2%) – RefSeq Genome Reviews Genome Reviews Ensembl+Genome Reviews – aProteins correspond to translated annotated CDS. The number of proteins in the protein database is therefore equal to the number of CDS in the nucleic database. bNumber of proteins associated to a family, followed by the proportion of proteins associated to a family in the taxonomic group. c2718 mature peptides are added to the 80 210 proteins translated from all CDS. dThe genome of A. gambiae contains data from different haplotypes and presents therefore a high level of redundancy. appropriate genetic codes. For viral genomes presenting polypeptides which further maturate in vivo into mature peptides, the mature peptides were added to the set of translated proteins according to the annotations speciﬁed in the given genome (Figure 1). Table 1 lists the global content of the databases as well as their original sources. Annotations were extracted from UniProtKB via the cross-references found in the CDS (21). Subsequently, se- quences in the nucleic and protein PhEVER database were clustered into families and were assigned a family acces- sion number. Alignments and phylogenies were built for each of these families according to state-of-the-art proced- ures (Figure 1). The classiﬁcation of all organisms present in the database was retrieved from the taxonomy database at National Centre for Biotechnology Information (11) and is available on the PhEVER web interface. CLUSTERING INTO FAMILIES The clustering of proteins into homologous families was constructed using an automated procedure similar to the one described in (14) and implemented within a parallel framework in a software package called SiLiX. Brieﬂy, sequences were assigned to protein families by simple tran- sitive link using the following criteria. A similarity search of all translated proteins and mature peptides against all was performed using BLASTP2 similarity search with the BLOSUM62 substitution matrix, a 10 4 e-value threshold and the ‘m S’ ﬁlter option (22,23). For each pair of se- quences, HSPs which were not compatible with a global alignment were removed. Two sequences were included in the same family if the sum of the remaining HSPs covered >80% of the proteins length (and at least 100 amino acids) and if their identity was 35%. These two criteria were previously shown to provide a good trade-off between the ability of clustering sequences from divergent organisms and the quality of resulting alignments for sub- sequent phylogenetic analyses (14). Finally, only families containing at least one viral sequence were integrated in the database. For each of the families, a small description built from the gene annotations ordered by frequency is available on the web interface. Figure 2 presents the dis- tribution of viral species, proteins and families according to each viral group (A) as well as a Venn diagram representing the families content (B). Figure 2A presents PhEVER’s broad taxonomical distribution covering all Baltimore groups (24). This distribution is naturally biased towards dsDNA, ssDNA and positive-sense ssRNA viruses reﬂecting the bias in genomic sequencing efforts. Indeed these groups contain long-studied viral families—either for their medical interest or for their economical impacts—such as Caudovirales, Poxviridae, Herpesvirales, Flaviviridae, Picornavirales or Parvoviridae. Figure 2 (B) shows a large number of orphan families indicating that a signiﬁcant proportion of viral proteins (32%) do not contain any homologs with proteins from known genomes. These proteins, among which some might possibly be caused by annota- tion errors, are unﬁt for comparative functional analysis and should be the focus of future experimental studies to validate them and to provide with crucial information on viral mechanisms. Figure 2B also shows the small number of families sharing sequences from both viruses and eukaryotes compared to the relatively high number of families sharing sequences from both viruses and bacteria. This observation may be due to different underlying biological mechanisms but might also be an indicator of a still low coverage of the Eukarya domain. DETECTION OF HORIZONTAL GENE TRANSFER One of the applications of PhEVER is the detection of horizontal gene transfer events by comparing a gene family tree with the expected species tree. The discrepancies between the gene family history and the species phylogeny can then be an indication of possible events including a gene duplication, a gene loss or a hori- zontal gene transfer. The quality of the gene phylogenies is therefore essential and in this perspective, we implemented a procedure based on a rigorous methodology. First, the clustering into families was built with criteria leading to conservative families. Second, maximum likelihood phylo- genetic trees were inferred for all families based on conserved aligned blocks. In our databases, for all families containing at least three sequences, pre-computed alignments and phylogenies are therefore already avail- able. This allows for a simple and accurate overview of any family without the need of heavy computations and