The identification of single copy (1-to-1) orthologs in any group of organisms is important for functional classification and phylogenetic studies. The Metazoa are no exception, but only recently has there been a wide-enough distribution of taxa with sufficiently high quality sequenced genomes to gain confidence in the wide-spread single copy status of a gene.Here, we present a phylogenetic approach for identifying overlooked single copy orthologs from multigene families and apply it to the Metazoa. Using 18 sequenced metazoan genomes of high quality we identified a robust set of 1,126 orthologous groups that have been retained in single copy since the last common ancestor of Metazoa. We found that the use of the phylogenetic procedure increased the number of single copy orthologs found by over a third more than standard taxon-count approaches. The orthologs represented a wide range of functional categories, expression profiles and levels of divergence.To demonstrate the value of our set of single copy orthologs, we used them to assess the completeness of 24 currently published metazoan genomes and 62 EST datasets. We found that the annotated genes in published genomes vary in coverage from 79% (Ciona intestinalis) to 99.8% (human) with an average of 92%, suggesting a value for the underlying error rate in genome annotation, and a strategy for identifying single copy orthologs in larger datasets. In contrast, the vast majority of EST datasets with no corresponding genome sequence available are largely under-sampled and probably do not accurately represent the actual genomic complement of the organisms from which they are derived.

UNLABELLED: Multiple sequence alignment (MSA) is a central tool in most modern biology studies. However, despite generations of valuable tools, human experts are still able to improve automatically generated MSAs. In an effort to automatically identify the most reliable MSA for a given protein family, we propose a very simple protocol, named AQUA for 'Automated quality improvement for multiple sequence alignments'. Our current implementation relies on two alignment programs (MUSCLE and MAFFT), one refinement program (RASCAL) and one assessment program (NORMD), but other programs could be incorporated at any of the three steps. AVAILABILITY: AQUA is implemented in Tcl/Tk and runs in command line on all platforms. The source code is available under the GNU GPL license. Source code, README and Supplementary data are available at http://www.bork.embl.de/Docu/AQUA.