orthologs are defined as genes in different species deriving from a single gene in the last common ancestor ( 1 ) , and are therefore likely to have the same function .
if an ortholog undergoes duplication in one species , the copies are referred to as inparalogs ( 2 ) .
inparalogs are by definition co - orthologs to one or more orthologs in another species . in contrast , two genes deriving from a duplication that predated the speciation event between the species are referred to as outparalogs .
the inparanoid program was developed to identify clusters of inparalogs while avoiding inclusion of outparalogs .
inparanoid is one of the first comprehensive ortholog databases ( 3,4 ) , but nowadays more than 15 different ortholog databases exist ( 5 ) .
a reason for the multitude of ortholog databases is that different research questions have different needs .
for instance , the cogs database ( 6 ) contains very large clusters of orthologs that often contain outparalogs ( 7 ) . at the other extreme , the homologene database ( 8) often places inparalogs in different clusters .
however , the average user is normally interested in simply finding all orthologs in species y to a gene in species x , including all inparalogs but excluding outparalogs .
two benchmarks have recently been published that try to objectively assess the quality of different ortholog databases ( 9,10 ) . in both these tests , which look either at accuracy of functional annotation or at inferred accuracy ,
this suggests that inparanoid is successful at balancing the false - negative and false - positive rate , and is appropriate as a general - purpose orthology tool .
this release contains a number of fixes to minor bugs that could lead to incorrect cluster merging or bootstrap values .
the website has been completely reconstructed and has new front- and back - ends , yet looks very similar to the old site .
the new design makes it much faster for the user , and allows easier updating of the system . with the new back - end
the data was gathered from three different sources : ensembl , ncbi and model organism databases ( mods ) .
we only considered eukaryotic genomes sequenced to a coverage greater than 6x , with < 1% unknown amino acids ( x in the protein sequences ) .
most mod data was packaged and uploaded by the staffs at tair , wormbase , flybase , zfin , dictybase , sgd and mgi to us directly , but three mods were downloaded from their repositories . before running inparanoid
, each proteome was made non - redundant by keeping only the longest transcript from each gene .
if this is not done first , different transcripts from the same gene can end up in different clusters if they exist in more than one species .
together , we have formed an informal consortium of mods that want to cross - reference each other using orthologs from inparanoid .
we particularly welcome this system as it allows us to use the most complete and recent set of proteins for each organism , and ensures that we use identifiers that work in the mods so that web links to proteins are valid .
we hope that more mods will join in and provide their proteomes in a new and robust xml format that will be introduced for the next release . from ensembl ,
data was obtained for aedes aegypti ( transcripts for 15 419 genes ) , anopheles gambiae ( 13 277 ) , apis mellifera ( 13 448 ) , bos taurus ( 22 280 ) , canis familiaris ( 19 314 ) , ciona intestinalis ( 14 278 ) , gallus gallus ( 16 715 ) , gasterosteus aculeatus ( 20 879 ) , homo sapiens ( 22 983 ) , macaca mulatta ( 22 045 ) , monodelphis domestica ( 19 597 ) , pan troglodytes ( 20 982 ) , rattus norvegicus ( 23 299 ) , takifugu rubripes ( 22 008 ) , tetraodon nigroviridis ( 28 005 ) and xenopus tropicalis ( 18 473 ) .
apis mellifera was taken from ensembl release 38 and all other proteomes from release 43 . from ncbi , we obtained candida glabrata ( 5192 ) , cryptococcus neoformans ( 6487 ) , debaromyces hansenii ( 6318 ) , entamoeba histolytica ( 9772 ) , escherichia coli k12 ( 4243 ) , entamoeba histolytica ( 9772 ) , kluyveromyces lactis ( 5336 ) , yarrowia lipolytica ( 6544 ) .
the mods uploaded proteomes for arabidopsis thaliana ( 26 819 ) , caenorhabditis briggsae ( 19 334 ) , caenorhabditis elegans ( 20 084 ) , caenorhabditis remanei ( 25 595 ) , danio rerio , ( 12 303 ) , dictyostelium discoideum ( 13 523 ) , drosophila melanogaster ( 13 854 ) , mus musculus ( 23 132 ) , saccharomyces cerevisiae ( 5792 )
. we obtained from other mods oryza sativa ( 77 853 ) ( from http://www.gramene.org ) , drosophila pseudoobscura ( 9871 ) ( from http://www.flybase.org ) , and schizosaccharomyces pombe ( 5003 ) ( http://www.sanger.ac.uk ) .
blast comparisons using these datasets were performed between each pair of species , involving four whole proteome runs per species pair ( normal runs both ways plus two self - self runs ) .
for the 35 proteomes this amounts to 595 species pairs , requiring 1225 whole - proteome blast searches .
the pairwise blast results were used as the input for the inparanoid ortholog clustering procedure ( 3 ) .
the output from inparanoid 6 is available as xml , sql , html and native format for downloading at the inparanoid homepage , and is searchable via the web interface .
ncbi  blast comparisons using these datasets were performed between each pair of species , involving four whole proteome runs per species pair ( normal runs both ways plus two self - self runs ) .
for the 35 proteomes this amounts to 595 species pairs , requiring 1225 whole - proteome blast searches .
the pairwise blast results were used as the input for the inparanoid ortholog clustering procedure ( 3 ) .
the output from inparanoid 6 is available as xml , sql , html and native format for downloading at the inparanoid homepage , and is searchable via the web interface .
the 35 species present in the inparanoid database result in 595 pairwise ortholog lists . the information in these lists was used to generate a phylogenetic tree that reflects the level of orthology between the different species .
we calculated the orthology distance from species a to b , dab , by 

 and used the average orthology distances ( dab + dba)/2 to construct a upgma tree , shown in figure 1 .
, it agrees with the standard taxonomic species tree , but we noted a few exceptions .
opossum ( m. domesticus ) , a marsupial mammal , is clustered together with placental mammals , and the zebrafish d. rerio clustered as an outgroup to the land animals rather than together with other fish .
the latter anomaly is very minor as all fish are still neighbors in the tree , but the placement of opossum is surprising .
if this placement is correct , then marsupials could have evolved from a particular lineage of placental mammals .
k. lactis , s. cerevisiae and d. hansenii are clustered together , while c. glabrata is placed outside this group .
these are arranged differently in the orthophylogram in that c. glabrata has traded place with d. hansenii , which now is placed as an outgroup to k. lactis , s. cerevisiae and c. glabrata .
surprisingly , the green plants are placed as a subgroup among single - cell organisms , next to the fungal group . 

for instance , on average 91.2% of the proteins in h. sapiens and p. troglodytes are orthologous .
the tree topology generally corresponds to the standard taxonomy , but a few exceptions were noted ( see text ) .
for instance , on average 91.2% of the proteins in h. sapiens and p. troglodytes are orthologous .
the tree topology generally corresponds to the standard taxonomy , but a few exceptions were noted ( see text ) .
it is worth noting that on average only 91.2% of the proteins in h. sapiens and chimpanzee p. troglodytes are orthologous .
this is surprisingly low since the genome - wide nucleotide divergence between human and chimpanzee is estimated to only 1.23% ( 12 ) .
the much higher difference observed for orthologs is not due to unique proteins in either proteome , as the fraction of homologs reported by inparanoid is 96.7% for human and 99.3% for chimpanzee .
this is , however , often caused by incomplete sequencing or errors in gene annotation .
the average number of inparalogs per cluster ranges from 1.001 ( in drosophila pseudoobscura when compared to d. melanogaster ) to 7.160 ( in o. sativa when compared to d. rerio ) .
the overall mean number of inparalogs per species was 1.54 , and the median was 1.25 .
the highly duplicated genome of o. sativa is responsible for all average cluster sizes of four , and generates a separate peak in the distribution around five .
in fact , o. sativa had on average more than four inparalogs per ortholog group when compared to every other non - plant species .
it is surprising that the average number of inparalogs in o. sativa was so high when compared with d. rerio ; when compared with d. rerio 's phylogenetic neighbors the number was only around five .
although the rice proteome clearly contains the largest number of genes , our figures are probably somewhat overestimated .
evidence for this is that we were not able to find shared gene identifiers between any rice proteins in the mod .
this problem will be resolved in the future by collaborating directly with the rice mod staff to get a better - annotated rice proteome . 

figure 2.histogram of average number of inparalogs / cluster per species for all species  species comparisons in inparanoid 6 .
the peak around five inparalogs / cluster is entirely caused by o. sativa , rice .
histogram of average number of inparalogs / cluster per species for all species  species comparisons in inparanoid 6 .
the peak around five inparalogs / cluster is entirely caused by o. sativa , rice .
the inparanoid database is freely available at http://inparanoid.sbc.su.se . in addition to the data which is available to search /
browse using the web interface , fasta files containing all proteins , protein description files , ortholog tables in raw , sql and xml format are available for each pairwise inparanoid analysis .
 the inparanoid eukaryotic ortholog database ( http://inparanoid.sbc.su.se/ ) has been updated to version 6 and is now based on 35 species . 
 we collected all available  
 complete eukaryotic proteomes and escherichia coli , and calculated ortholog groups for all 595 species pairs using the inparanoid program . 
 this resulted in 2 642 187 pairwise ortholog groups in total . 
 the orthology - based species relations are presented in an orthophylogram . 
 inparanoid clusters contain one or more orthologs from each of the two species . 
 multiple orthologs in the same species , i.e. inparalogs , result from gene duplications after the species divergence . 
 a new inparanoid website has been developed which is optimized for speed both for users and for updating the system . 
 the xml output format has been improved for efficient processing of the inparanoid ortholog clusters . 
INTRODUCTION
DATA AND IMPLEMENTATION
InParanoid clustering
INPARANOID CONTENT
DATA AVAILABILITY