The 70 manually curated RefOGs as a quality assessment tool. Five databases were used to illustrate the validating power of the benchmark set. The performance of each database was evaluated at two levels: gene (focus on mispredicted genes; upper panel) and Group (focus on fusions/fissions; lower panel) level. A: Gene count – for each database we identified the OG with the largest overlap with each RefOG and calculated how many genes were not predicted in the OG (missing genes) and how many genes were over-predicted in the OG (erroneously assigned genes) and E: group count – for each method we counted the number of OGs that members of the same RefOG have been separated (RefOG fission) and how many of those OGs include more than three erroneously assigned genes (RefOG fusion). To increase the resolution of our comparison, three different measurements for each level were provided, resulting in six different scoring schemes. B: Percentage of accurately predicted RefOGs in gene level (RefOGs with no mispredicted genes); C: number of erroneously assigned and missing genes; D: percentage of affected RefOGs by erroneously assigned and missing genes; F: percentage of accurately predicted RefOGs in grouplevel (all RefOG members belong to one OG and are not fused with any proteins); G: number of fusions and fissions; and J: percentage of affected RefOGs by fusion and fission events. Databases are aligned from the more to the less accurate, taking into account the total number of errors (length of the bar in total). Black bars indicate identical scores.

The impact of species coverage and genome annotation. A: Comparison of the performance of 34-species and 12-species OGs using RefOGs. We measure the percentage of orthologs recovered (coverage), missing genes and erroneously assigned genes for each reference species for those datasets [yellow bar: publicly available OGs in eggNOG (same measurements as Fig. 2) and green bar: customized OGs of the 12 selected species using same genome annotations as the public eggNOG]. The reference species are highlighted by black letters, while the unconsidered species that complete the set of 34 eggNOG species are written in gray letters. Numbers in parentheses show the total number of orthologs per species in the benchmarking set. The gray boxes enclosing the colored bars correspond to 100% coverage. Notice that the coverage is always higher for the 34-species OGs compared to the 12-species OGs except in the cases of C. elegans and Ciona (marked by asterisk), which are separated by long branches in both datasets. B: Comparison of the public eggNOG (yellow bar), 12-species-old-annotation OGs (green bar) and 12-species-new-annotation OGs (purple bar) at the gene level. Hatched boxes label the fraction of mispredicted genes of 34-species- and 12-species-old-annotation datasets that do not exist in Ensembl v60 genome annotations, indicating the high number of errors due to old genome annotations. C: Comparison of public eggNOG (yellow bar), 12-species-old-annotation OGs (green bar) and 12-species-new-annotation OGs (purple bar) at the group level. Notice that the 12-species datasets (either with old or new annotation) always introduce a larger number of fission events than the 34-species OGs, highlighting again the importance of species coverage.

Mucins: a challenging family for orthology prediction. This figure shows the phylogenetic tree and domain architecture of aligned mucins. The identification of cnidarian (an outgroup for bilaterians) mucin2/5 orthologs separates the gel-forming mucins from other mucins, defining a bilaterian-specific OG (gray box). An alternative topology of Hydra in respect to the LCA of bilaterian species (shown schematically in the red box) would propose that those two different classes of mucins should be clustered together at the bilaterian level. The bilaterian OG can be further resolved at the vertebrate level into OG.A (blue) and OG.B (red), illustrating the hierarchical nature of OGs. This family, besides its large size due to vertebrate-specific duplications, exemplify five additional problems that often lead to orthology misassignment: (1) uneven evolutionary rate illustrated as branch lengths, lowering the sequence similarity among members of the family; (2) quality of genome annotation: the particular zebrafish protein can be either a derived member of the mucin family or a erroneous gene prediction; (3) repeated domains: the domain combination VWD-C8-VWC, which is the core of the family, is repeated multiple times within the protein; (4) complexity of domain architectures: there are multiple unique domain combinations (e.g. the VWD domain is combined with the F5-F8 type C domain only in the Drosophila ortholog); and (5) low complexity regions: internal repeats within the amino acid sequences and other low complexity features impede the correct sequence alignment of the mucins. *Possible orthologous sequence at the LCA of cnidarians bilaterians.

The impact of biological complexity in orthology assignment. To evaluate the impact of important caveats in orthology prediction, the RefOGs were classified based on their family size, rate of evolution, alignment quality and domain complexity. A: Family size (reveals the impact of paralogy): the RefOGs were separated into (i) small (less than 14 members), (ii) medium (more than 14 members, but less than 40), and (iii) large (more than 40 genes). B: Rate of evolution: the RefOGs were classified based on the MeanID score (described as the “FamID” in 33), an evolutionary rate score derived from the MSA of each family. There are: (i) slow-evolving (MeanID >0.7), (ii) medium-evolving (MeanID <0.7, but >0.5), and (iii) fast-evolving (MeanID <0.5) RefOGs. C: Quality of alignment: we classified the families based on their norMD score 34 into: (i) high-quality alignment (norMD >0.6), and (ii) low-quality alignment 44, 45. We can observe that high amino acid divergence correlates with an increasing number of mispredicted genes. D: Domain architecture complexity; each RefOG is associated with the average number of domains, which is equal to the sum of predicted domains of the members of one RefOG divided by the family size. There are three levels of complexity, starting from (i) none or one domain on average, to (ii) two to four, to (iii) more than four. We observe that the performance of the five databases correlates with the biological complexity of RefOGs; as families increasing their complexity (more members, fast-evolving or multiple domains), the accuracy of predictions drops. (+) and (−) symbolize erroneously assigned and missing genes, respectively. Significant correlations (Table S5 of Supporting Information) between the distribution of missing/erroneously assigned genes and the tested factor are indicated in bold [(+), (−)]. Figures S2 and S4 of Supporting Information show similar observations at the group level (fusions/fissions of RefOGs).