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MultiParanoid Automatic Clustering of Orthologs and Inparalogs Shared by Multiple Proteomes Andrey Alexeyenko Ivica Tamas Gang Liu Erik L.L. Sonnhammer Stockholm Homologs: orthologs and paralogs MultiParanoid Homologs: genes that have descended from a common ancestral gene. Manifested by a sequence similarity. We do not believe in sequence similarity without a shared ancestry. Ancestral gene Gene 1 BLAST hit. Low e-value Gene2 Orthologs are related via a speciation S Paralogs are related via a gene duplication. May or may not be in the same species D Homologs: orthologs and paralogs MultiParanoid Inparalogs ~ co-orthologs paralogs that were duplicated after the speciation and hence are orthologs to the other species’ genes Outparalogs = not co-orthologs paralogs that were duplicated before the speciation Orthology, paralogy and proposed classification for paralog subtypes Sonnhammer ELL and Koonin EV Trends in Genetics Volume 18, Issue 12 , 1 December 2002, Pages 619-620 Orthologs for functional genomics MultiParanoid Orthologs are more likely than outparalogs to have identical/similar biochemical functions and biological roles Orthologs are optimal to discover gene function via model organism counterparts Benchmarking ortholog identification methods using functional genomics data. Hulsen T, Huynen MA, de Vlieg J, Groenen PM. Genome Biol. 2006;7(4):R31. Epub 2006 Apr 13. “…the InParanoid program is the best ortholog identification method in terms of identifying functionally equivalent proteins.” Outline 1.InParanoid 2. The world of ortholog resources MultiParanoid 3. Why MultiParanoid 4. Limitations 5. Future development S Inparalogs Orthologs Outparalogs MultiParanoid Homologs: orthologs and paralogs D S D MultiParanoid InParanoid P r o t e o m e A P r o t e o m e B Reciprocally best hits ~ seed orthologs Inparalogs Automatic clustering of orthologs and in-paralogs from pairwise species comparisons Maido Remm, Christian E. V. Storm and Erik L. L. Sonnhammer Journal of Molecular Biology 314, 5 , 14 December 2001, Pages 1041-1052 Resources using InParanoid Eukaryotic Ortholog Groups MultiParanoid 3409 diseases Multi-species ortholog resources “Massive download” friendly: Clusters of Orthologous Groups Tree-based, best for detailed analysis MultiParanoid HOVERGEN release 47 Any cluster of more than 2 species’ genes is controversial in terms of orthology as the speciation gives rise to a pair of species. MultiParanoid S D D D S S MultiParanoid algorithm 1. Take >2 species with maximally close speciation points MultiParanoid 2. Generate 2-species InParanoid clusters 3. Find protein counterparts across the clusters A-B B-C A-C InParanoid cluster A-C ? InParanoid cluster A-B InParanoid cluster B-C MultiParanoid validation The MultiParanoid output was benchmarked on a manually curated set of 221 human-fly-worm clusters: - 214 MultiParanoid clusters found - 177 (almost) identical -The rest controversial mainly due to: MultiParanoid - differences between pairwise and multiple alignments - the curator’s perception and InParanoid settings However: tree conflicts InParanoid cluster membership Genes: Fly Worm Human MultiParanoid vs. Clusters of Orthologous Groups and Tree conflict Other Short match Outparalog MultiParanoid Weak homolog 14000 12000 10000 8000 6000 4000 2000 0 0 2000 4000 MP not KOG 6000 8000 10000 12000 14000 KOG not MP Tree conflict Other Short match Outparalog Weak homolog 14000 12000 10000 8000 6000 4000 MP not OrthoMCL 2000 0 0 2000 4000 6000 8000 10000 12000 14000 OrthoMCL not MP Current MultiParanoid release MultiParanoid ??? C.elegans D.melanogaster C.intestinalis H.sapiens 40451 protein sequences classified into 7695 clusters http://multiparanoid.cgb.ki.se/ A solution: expansion of MultiParanoid clusters MultiParanoid 1. Process all the possible 3-species combinations: 2. Merge respective cluster members across the clades: But still, orthology is a pairwise concept! MultiParanoid The speciation gives rise to a pair of species. Post-processing (bootstrap, synteny, tree manual curation etc.) MultiParanoid How the ortholog resources cope with it? Clusters of Orthologous Groups HOVERGEN release 47 Cluster size ~ outparalogs/orthologs ratio Overview and comparison of ortholog databases Alexeyenko A, Lindberg J, Pérez-Bercoff Å, Sonnhammer ELL MultiParanoid Drug Discovery Today:Technologies (2006) v. 3; 2, 137-143 •EGO •COG/KOG •HomoloGene •InParanoid/MultiParanoid •HOPS •KEGG •OrthoMCL •ENSEMBL Compara •PhiGs •MGD •HOGENOM •HOVERGEN •INVHOGEN •TreeFam •OrthologID How to reconcile… …the demand for multi-species clusters and pair-wise gene relations? The common feature is a single ancestor gene at the root point: MultiParanoid S D D D S D S 2 new terms: Pseudo-proteome: a union of MultiParanoid proteomes of the same clade Cluster of pseudo-inparalogs: a within-clade gene family MultiParanoid Pseudo–proteome A (reptiles) Pseudo–proteome B (mammals) Another view: “gene-family”-wise: LCA MultiParanoid D S D S S D … and all the members of the same cluster ascend to a single gene in the last common ancestor (LCA) of the two major clades The clustering can be done at different levels MultiParanoid S • • • D S D Orthologs For example: Fungi vs. animals Insects vs. mammals Rodents vs. primates S S D Having more than one species in a pseudo-proteome reduces misassignments in case of gene loss. Closer pseudo-proteomes increase resolution. Lineage(~pseudo-proteome)-specific expansions should be also available Conclusions • Most of the ortholog resources may build clusters in form of gene trees, but only InParanoid seems to correctly delineate ortholog/inparalog groups • MultiParanoid algorithm has relieved the problem of “hidden outparalogs”, but the number/content of species remains limited MultiParanoid • The “LCA-Paranoid” concept: the long waited solution? – Each of the two clade-specific cluster parts may be regarded as a multispecies cluster – When (in future) all possible “clade<->clade” clustering solutions will be found, each gene would receive a complete set of orthologs at a desirable level of LCA – With sufficient number of complete proteomes, it would be possible to date each gene pair’s point of divergence