Nucleic Acids Research
Evola: Ortholog database of all human genes in H-InvDB with manual curation of phylogenetic trees
Akihiro Matsuya, Ryuichi Sakate, Yoshihiro Kawahara, Kanako O. Koyanagi, Yoshiharu Sato, Yasuyuki Fujii, Chisato Yamasaki,
Takuya Habara, Hajime Nakaoka, Fusano Todokoro, Kaori Yamaguchi, Toshinori Endo, Satoshi OOta, Wojciech Makalowski,
Kazuho Ikeo, Yoshiyuki Suzuki, Kousuke Hanada, Katsuyuki Hashimoto, Momoki Hirai, Hisakazu Iwama, Naruya Saitou, AikoT. Hiraki, Lihua Jin, Yayoi Kaneko, Masako Kanno, Katsuhiko Murakami, Akiko Ogura Noda, Naomi Saichi, Ryoko
Sanbonmatsu, Mami Suzuki, Jun-ichi Takeda, Masayuki Tanaka, Takashi Gojobori, Tadashi Imanishi and Takeshi Itoh
Nucleic Acids Res. 36:787-792, 2008. First published 3 Nov 2007;
doi:10.1093/nar/gkm878

The full text of this article, along with updated information and services is available online at
http://nar.oxfordjournals.org/cgi/content/full/36/suppl_1/D787
References

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Published online 3 November 2007

Nucleic Acids Research, 2008, Vol. 36, Database issue D787–D792
doi:10.1093/nar/gkm878

Evola: Ortholog database of all human genes in
H-InvDB with manual curation of phylogenetic trees
Akihiro Matsuya1,2, Ryuichi Sakate1,3,*, Yoshihiro Kawahara1,3, Kanako O.
Koyanagi4, Yoshiharu Sato1,3, Yasuyuki Fujii1,3, Chisato Yamasaki1,3,
Takuya Habara1,3, Hajime Nakaoka5, Fusano Todokoro1,6, Kaori Yamaguchi1,3,
Toshinori Endo4, Satoshi OOta7, Wojciech Makalowski8, Kazuho Ikeo9,
Yoshiyuki Suzuki9, Kousuke Hanada9, Katsuyuki Hashimoto10, Momoki Hirai11,
Hisakazu Iwama12, Naruya Saitou13, Aiko T. Hiraki1,3, Lihua Jin9, Yayoi Kaneko1,3,
Masako Kanno1,3, Katsuhiko Murakami1,3, Akiko Ogura Noda1,3, Naomi Saichi1,3,
Ryoko Sanbonmatsu1,3, Mami Suzuki1,3, Jun-ichi Takeda1,3, Masayuki Tanaka1,3,
Takashi Gojobori3,9, Tadashi Imanishi3 and Takeshi Itoh3,141Integrated Database Group, Japan Biological Information Research Center, Japan Biological Informatics
Consortium, 2Government & Public Corporation Information Systems, Hitachi, Co., Ltd., 3Integrated Database
Group, Biological Information Research Center, National Institute of Advanced Industrial Science and Technology,
Tokyo, 4Graduate School of Information Science and Technology, Hokkaido University, Hokkaido, 5C’s Lab Co.,
Ltd., Hokkaido, 6DYNACOM Co., Ltd, Chiba, 7BioResource Center, RIKEN, Ibaraki, Japan, 8Institute of
Bioinformatics, University of Muenster, Muenster, Germany, 9Center for Information Biology and DNA Data Bankof Japan, National Institute of Genetics, Shizuoka, 10Department of Biomedical Resources, National Institute of
Biomedical Innovation, Osaka, 11International Research and Educational Institute for Integrated Medical Sciences,
Tokyo Women’s Medical University, Tokyo, 12Kagawa University, Kagawa, 13Department of Population Genetics,
National Institute of Genetics, Shizuoka and 14Division of Genome and Biodiversity Research, National Institute ofAgrobiological Sciences, Ibaraki, Japan
Received August 15, 2007; Revised September 27, 2007; Accepted October 1, 2007

ABSTRACT
Orthologs are genes in different species thatevolved from a common ancestral gene by speciation. Currently, with the rapid growth of transcriptome data of various species, more reliableorthology information is prerequisite for further
studies. However, detection of orthologs could beerroneous if pairwise distance-based methods,
such as reciprocal BLAST searches, are utilized.
Thus, as a sub-database of H-InvDB, an integrated
database of annotated human genes (http://h-invi
tational.jp/), we constructed a fully curated database of evolutionary features of human genes,
called ‘Evola’. In the process of the ortholog
detection, computational analysis based on conserved genome synteny and transcript sequencesimilarity was followed by manual curation byresearchers examining phylogenetic trees. In total,
18 968 human genes have orthologs among 11

vertebrates (chimpanzee, mouse, cow, chicken,
zebrafish, etc.), either computationally detected or
manually curated orthologs. Evola provides aminoacid sequence alignments and phylogenetic trees oforthologs and homologs. In ‘dN/dS view’, natural
selection on genes can be analyzed between humanand other species. In ‘Locus maps’, all transcript
variants and their exon/intron structures can becompared among orthologous gene loci. We expect
the Evola to serve as a comprehensive and reliabledatabase to be utilized in comparative analyses
for obtaining new knowledge about human genes.
Evola is available at http://www.h-invitational.jp/
evola/.

INTRODUCTION
A large number of genome and transcript sequences
accumulated in the last decade give us an opportunity*To whom correspondence should be addressed. Tel: +81 3 3599 8800; Fax: +81 3 3599 8801; Email: rsakate@ni.aist.go.jp
ß 2007 The Author(s)This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 functionaland evolutionary studies. Despite the past eﬀorts todevelop bioinformatics methods for analyzing a large
number of sequences, it is still a challenge to comprehensively identify orthologs between species. A numberof automated pairwise distance-based methods forortholog 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 mightlead 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 complementary to the H-Invitational database (H-InvDB), wasdeveloped to provide orthology information for theoriginally annotated human genes in H-InvDB. Evolafeatures its ortholog detection in which genome syntenybased computational analysis was followed by manual
curation of molecular phylogenetic trees. Evola diﬀers inthis 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 andsynteny (Ensembl-Compara and Homologene) and phylogenetic trees (HOGENOM and TreeFam). The concept
of Evola is that genomic region (gene locus) is a unit ofgenes 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 annotatedhuman 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 thehuman genes. With several updates afterwards and asubsequent All Human Genes Evolutionary Annotation(AHG-EV) meeting in 2006, the current strategy ofevolutionary annotation (computational analysis andmanual curation) in Evola has been established.
Orthology information for human and other 11 vertebrates is currently included in the Evola: human,
chimpanzee, macaque, mouse, rat, dog, cow, opossum,
chicken, zebraﬁsh, Tetraodon and Fugu. Several visualization 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 oneof the databases listed in the Comparisonof Orthology Predictions project of the HUGO
Gene Nomenclature Committee (HGNC, http://www.
genenames.org/).

ORTHOLOG DETECTION
Computational analysis: Ortholog detection basedon conserved genomic synteny and pairwise distanceSpecies for ortholog detection were selected with consideration 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), chimpanzee (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 otherprimates (between more similar genome sequences), andC = 2 between human and non-primate vertebrates(between less similar genome sequences).
For human transcripts, H-InvDB representative transcripts (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 andcoverage to the genome, number of exons, etc. of alltranscripts 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 genomealignment. A gene pair with the maximum length wasselected 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 acidsequences of the corresponding human genes. Finally, if
the length of the alignable region between human andother species ortholog candidates was 80 a.a., they weredeﬁned as computationally detected orthologs.
Manual curation: Examination of phylogenetic
trees by experts
Homologs of human genes (amino acid sequences) wereobtained from UniProt (http://www.uniprot.org/) andhuman RefSeq (NP) by FASTY similarity searches withthe option of E-value of <1e 5. For each human gene, aNucleic Acids Research, 2008, Vol. 36, Database issue D789

sequence set consisting of both the computationallydetected orthologs and the homologs was prepared. For
these sequence sets, phylogenetic trees were constructed by
the neighbor-joining (NJ) method (18). In detail, multipleamino acid sequence alignments and phylogenetic trees
were constructed by ClustalW (19) with the options ofbootstrap = 1000,
seed = 1,
kimura,tossgaps,
bootlabels = node.
Phylogenetic trees were examined by experts in theﬁeld of molecular evolution, who attended the evolutionary annotation meetings described in the introduction.
The trees were drawn by NJplot (http://pbil.univ-lyon1.fr/
software/njplot.html) and the default rooting was used.
Discarding or re-rooting the tree was judged by theexperts if necessary. All the ortholog pairs of human andother species detected by the computational analysis were
examined (Figure 1). The primary principles of manual
curation in Evola to be checked were as follows. [1]Phylogenetic topology between gene tree and species tree
is consistent. As a gene tree, the minimum sub-clade
including the pair (a part of the tree) was examined. As a
species tree of reference, a phylogenetic tree indicating thetrifurcation among primates, rodents and Laurasiatherian(dog, cow, etc.) species (20) was used, because thephylogenetic relationship has been controversial among
them (21). In fact, we found that ((human–mouse)–dog)clades for some genes and ((human–dog)–mouse) clades
for other genes. [2] Outgroup includes either two or
more species that are phylogenetically distant from all
the species in the sub-clade, or human and otherspecies. In the latter case, human duplicate genes might
exist. [3] Available bootstrap values of the corresponding
three branches (one between the sub-clade and outgroup,
and its two descendants) are all 900. The gene pairsconsistent with all the principles were deﬁned as ‘manuallycurated orthologs’, otherwise their annotation status
remained to be ‘computationally detected orthologs’.

DATABASE CONTENTS
Evola contains two ortholog datasets: (1) more comprehensive set of orthologs (computational analysis); and(2) more reliable orthologs (computational analysis
supported by manual curation). In the current Evola(release 4.1), orthology information for 18 968 humangenes is available among 11 vertebrates: chimpanzee,
macaque, mouse, rat, dog, cow, opossum, chicken,
zebraﬁsh, Tetraodon and Fugu (Table 1). Manuallycurated orthologs occupied 25.4% of all computationallydetected ortholog pairs (24 122/94 935) (release 4.1, 2007).
Evola is a sub-database of H-InvDB (9,10,11), andorthology information in Evola is, as ‘Evolutionary
annotation’, a part of the comprehensive human geneannotations in H-InvDB. Thus, orthology information
can be utilized with close reference to other annotation in
H-InvDB. For example, 2090 human genes with orthology
information belonged to H-Inv protein similarity
categories of ‘hypothetical proteins’ (similarity category
IV–VI). Molecular functions of these hypotheticalFigure 1. An example of manually curated gene pair from H.sapiens(red underline) and Macaca.sp (blue underline). In this case, conditionsof phylogenetic topologies, outgroup species (light gray background)and bootstrap values (two circles) are suﬃcient (refer to the text). Thus,
the pair was deﬁned as a manually curated ortholog.

Table 1. Number of orthologs provided in Evola (release 4.1, June
2007)
Species

Genes

Human genes

Homo sapiens (Human)
Pan troglodytes (Chimpanzee)
Macaca sp. (Macaque)a
Mus musculus (Mouse)
Rattus norvegicus (Rat)
Canis familiaris (Dog)Bos taurus (Cow)
Monodelphis domestica (Opossum)
Gallus gallus (Chicken)
Danio rerio (Zebraﬁsh)
Tetraodon nigroviridis (Tetraodon)
Takifugu rubripes (Fugu)

18 96816 36812 037
15 57015 63214 7309375
13 2019266
12 33411 5059738–
15 615
12 35214 57414 30213 91610 181
13 58810 73810 4689820
9459

Numbers of genes of both human and other species are listed. Owing tolineage-speciﬁc duplication or loss, the numbers are usually diﬀerent(for example, 15 570 mouse genes are orthologous to 14 574 humangenes). 18 968 human genes have at least one ortholog among other 11
species.
a
Macaca mulatta, Macaca fascicularis, Macaca fuscata, etc. are
included.

proteins can be analyzed using model species. Moreover,cross references between Evola and other annotationsin H-InvDB (protein–protein interaction (PPI), expression, polymorphism, disease, etc.) can produce valuable
information contributing to the comprehensive understanding of the human genes.
We aimed to develop user-friendly interfaces thatprovide easy access to a variety of orthology information
in Evola. Users can search orthologs in the top pageof Evola as well as in the search systems of H-InvDB[simple search, advanced search and navigation system(Navi)]. Users can download data for each human gene on
the main page as well as all the data of Evola in thedownload page. On the main page of Evola (Figure 2),

D790 Nucleic Acids Research, 2008, Vol. 36, Database issue

Figure 2. Evola main page. This page is divided into left and right frames. In the left frame, tables of orthologs, download data, Gene ontology, andInterPro are listed. Three green buttons are links to show ‘Alignment’ (A), ‘dN/dS view’ (B) and ‘Locus maps’ (C) in the right frame.

the following information for a human gene is available in
the left frame: gene name, ortholog list with annotation
status, download of sequences, alignments and phylogenetic trees, Gene ontology (22) and InterPro (23).
In addition to the set of original ClustalW alignments,
another set of alignments, including properly aligned
sequences only (24), was also constructed and provided.
In the latter sets, sequences with distinctively low
identity to other sequences in an alignment were excluded.
Based on both alignment sets, phylogenetic trees wereconstructed by the neighbor-joining method (18) and the
NJML+ method (25).
In the right frame of the main page, Evola features
the three views described below. Users can switch among
the views.
Alignment: Multiple alignments of orthologs
and homologs (Figure 2A)
Amino acid sequence alignments of orthologs andhomologs are displayed. Users can switch from
‘Alignment of Orthologs’ (default) to ‘Alignment ofOrthologs and Homologs’, or vice versa. Each aminoacid residue is color coded as deﬁned in ClustalX (19).
Accession numbers and species names of orthologs(human and other species) are colored in their species
colors deﬁned in Evola (human in red, mouse in gray,etc.). Accession numbers of homologs are linked to theoriginal data sources of UniProt or RefSeq. While species
are labeled by their scientiﬁc names (Homo, Mus, etc.),users can activate a popup window giving a species
common name by placing the mouse cursor over homolog
accession numbers (for example, ‘Q5R508_Pongo’).
InterPro data in the left frame include positional
information on a human gene, and they can be utilized
to detect conserved domains in the proteins.
dN/dS view: Window analysis detecting regions
under positive or negative selection (Figure 2B)
Users can select one or more species for which to show
the plots in the graph. In the lower frame under the graph,the pairwise nucleotide sequence alignment of CDSs isshown. The sequence positions (a.a. or codon) appearing
in the graph and alignment are those of human genes.
The nonsynonymous to synonymous substitution rate
ratio (dN/dS) is a commonly used measure of natural
selection. In order to visualize positively and negatively
selected regions, sliding window analysis was conducted(a 20 codon window with 1 codon stepping; result forthe ﬁrst window appears as a plot at 11th codon of thehuman gene). The statistical signiﬁcance (P-value) of
the diﬀerence between the number of nonsynonymous
substitution (n) per synonymous substitutions (s): n/s, andthe number of nonsynonymous sites (N) per synonymoussites (S): N/S was calculated by Fisher’s exact test.

Nucleic Acids Research, 2008, Vol. 36, Database issue D791

dS, dN, s and n values were estimated by the modiﬁed
Nei–Gojobori method (26,27). If dN/dS > 1, the score(= 1 P-value) was plotted above the zero line (neutral),and if dN/dS < 1, the score [= (1 P-value)] was plotted
below the zero line. The regions plotted above the redline indicate that the sites might be under positive
selection (dN/dS > 1 and P < 0.01). Conversely, the regions
plotted below the blue line indicate that the sites might
be under negative (purifying) selection (dN/dS < 1 and
P < 0.01).

helpful suggestions. We are also grateful to Craig Goughfor critical reading of the manuscript. This work was
supported by the Ministry of Economy, Trade andIndustry of Japan (METI), and the Japan Biological
Informatics Consortium (JBIC). Funding to pay the Open
Access publication charges for this article was provided by
JBIC.

Locus maps: Comparative maps of orthologous gene loci(Figure 2C)

REFERENCES

Orthologs were detected for representative transcripts(one transcript per gene locus) in Evola. However,there could be transcript variants in gene loci that havediﬀerent exon–intron structures leading to produce
diﬀerent protein isoforms. Thus, information on othertranscripts besides the representative transcript among
orthologous gene loci are shown in Locus maps. In theﬁgures, exon/intron structure, coding sequence (CDS)
and untranslated regions (UTR) for each transcript are
visualized. H-Inv cluster ID (HIX, an identiﬁer ofgene locus), Gene symbol, genomic location and a linkto ‘G-integra’, an integrated genome browser of H-InvDB,
are available. The ﬂag icon denotes the representativetranscript. The blue diamond icon denotes theRepresentative Alternative Splicing Variant (RASV) thatis another representative per transcript group consistingof the same alternative splicing pattern (28). Representative transcripts are also RASVs, and blue diamonds
do not appear if there is only one splicing isoform. In thetables, the H-Inv transcript ID (HIX) and original
accession numbers (DDBJ/EMBL/GenBank, Ensembl
and RefSeq) of the representative transcript and othertranscripts are listed.
FUTURE DIRECTIONS
As our update policy, orthology information in Evolais updated when H-InvDB annotation is updated.
One major update and three minor updates per year
are scheduled. At the next major update on December
2007, a new duplicate gene family view is planned to
be integrated within Evola. Human duplicate gene family
data was originally constructed based on both aminoacid sequence similarity (29) and orthology information.
In the current Evola (release 4.1), parts of human
duplicate gene annotation have been already implemented. The human duplicate genes are included in thealignments and phylogenetic trees of orthologs andhomologs. Finally, we expect Evola to serve as a new
database for evolutionary annotation of human genes.
We sincerely welcome any requests and feedback
from users.
ACKNOWLEDGEMENTS
We thank the members of Integrated Database Group,Japan Biological Information Research Center for their

Conﬂict of interest statement. None declared.

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