Marie-Claude.Blatter@isb-sib.ch Swiss-Prot group, Geneva SIB Swiss Institute of Bioinformatics

1. The UniProt knowledgebasewww.uniprot.orga hub of integrated protein datahttp://education.expasy.org/cours/Turin/ Marie-Claude.Blatter@isb-sib.ch Swiss-Prot group, Geneva SIB Swiss Institute of Bioinformatics

2. Protein sequences • > 180 billions of ‘different’ proteins on earth (∑ N species x M genes) • > 23.0 millions of ‘known and public’ protein sequences in 2012 • More than 99 % of the protein sequences are derived from the translation of nucleotide sequences (mRNA or DNA) • About 1 % come from direct protein sequencing (Edman, MS/MS…)

3. Science cover, february 2011

4. data knowledge proteinsequencefunctional information

5. UniProt consortium EBI : European Bioinformatics Institute (UK) SIB : Swiss Institute of Bioinformatics (CH) PIR : Protein information resource (US)

6. www.uniprot.org

7. UniProt databases

8. UniProt databases UniParc: proteinsequence archive (EMBL-ENA equivalentat the proteinlevel) Each entry contains a proteinsequence, taxonomic information, cross-links to otherdatabaseswhereyoufind the sequence (active or not) No annotation All the public patentedsequences are stored in UniParc (EPO, USPO, JPO) You can: query, Blast, download ~31 mo entries

9. UniProt databases UniRef 3 clusters of proteinsequenceswith 100, 90 and 50 % identity; useful to speed up sequencesimilaritysearch (BLAST) You can: query, Blast, download UniRef100 17 mo entries; UniRef90 11 mo entries; UniRef50 5 mo entries

10. UniProt databases UniMES: proteinsequencesderivedfrommetagenomicprojects (mostlyGlobal OceanSampling (GOS)) You can : download 12 mo entries, included in UniParc

11. UniProt databases The centerpiece

12. UniProtKB an encyclopedia on proteins composed of 2 sectionsUniProtKB/TrEMBL and UniProtKB/Swiss-Prot unreviewed and reviewed automatically annotated and manually annotated released every 4 weeks

13. UniProtKB • Origin of proteinsequences • UniProtKBproteinsequences are mainlyderivedfrom • INSDC (translatedsubmittedcodingsequences- CDS) • Ensembl (geneprediction) and RefSeqsequences • Sequences of PDB structures • Direct submission or sequencesscannedfromliterature • (includes direct proteinsequencing) • Notes:- UniProtis not doinganygeneprediction • - Most non-germlineimmunoglobulins, T-cell receptors , most patent sequences, highly over-representeddata (e.g. viral antigens), pseudogenessequences are excludedfromUniProtKB, - but stored in UniParc • - Data from the PIR database have been integrated in UniProtKBsince 2003. 85 % 15 %

14. Manual annotation of the sequence and associated biological information Swiss-Prot EMBL TrEMBL Automated extraction of proteinsequence (translated CDS), genename and references. Automated annotation

15. UniProtKB/TrEMBL unreviewed Automatic annotation released every 4 weeks

16. Protein and genenames Taxonomic information Automated annotation Function, Subcellular location, Catalyticactivity, Sequencesimilarities… Automated annotation transmembranedomains, signal peptide… References One proteinsequence One species Automated annotation Keywords and Gene Ontology Cross-references to over 125 databases UniProtKB/TrEMBL www.uniprot.org

17. UniProtKB/TrEMBLAutomatic annotation • Proteinsequence • -The quality of the protein sequences is dependent on the information provided by the submitter of the original nucleotide entry (CDS) or of the gene prediction pipeline (i.e. Ensembl). • - 100% identical sequences (same length, same organism are merged automatically). • Biologicalinformation • Sources of annotation • Provided by the submitter (EMBL, PDB, TAIR…) • From automated annotation (automated generated annotation rules (i.e. SAAS) and/or manually generated annotation rules (i.e. UniRule))

20. UniProtKB/TrEMBL Example of fullyautomatic annotation: SAAS • Rules are derived from the UniProtKB/Swiss-Prot manual annotation. • Fully automated rule generation based on C4.5 decision tree algorithm. • One annotation, one rule. • High stringency – require 99% or greater estimated precision to generate annotation(test on UniProtKB/Swiss-Prot) • Rules are produced, updated and validated at each release.

21. UniProtKB/Swiss-Prot reviewed manually annotated released every 4 weeks Manualbiocurationis essential to knowledge maintenance

22. Manual annotation Function, Subcellular location, Catalyticactivity, Disease, Tissue specificty, Pathway… Protein and genenames Taxonomic information MSKEKFERTKPHVNVGTIGHVDHGKTTLTAAITTVLAKTYGGAARAFDQIDNAPEEKARGITINTSHVEYDTPTRHYAHVDCPGHADYVKNMITGAAQMDGAILVVAATDGPMPQTREHILLGRQVGVPYIIVFLNKCDMVDDEELLELVEMEVRELLSQYDFPGDDTPIVRGSALKALE GDAEWEAKILELAGFLDSYIPEPERAIDKPFLLPIEDVFSISGRGTVVTGRVERGIIKVGEEVEIVGIKETQKSTCTGVEMFRKLLDEGRAGENVGVLLRGIKREEIERGQVLAKPGTIKPHTKFESEVYILSKDEGGRHTPFFKGYRPQFYFRTTDVTGTIELPEGVEMVMPGDNIKMV VTLIHPIAMDDGLRFAIREGGRTVGAGVVAKVLG Manual annotation Post-translational modifications, variants, transmembranedomains, signal peptide… References One proteinsequence One gene One species Alternative products: proteinsequencesproduced by alternative splicing, alternative promoter usage, alternative initiation… Manual annotation Keywords and Gene Ontology Cross-references to over 125 databases UniProtKB/Swiss-Prot www.uniprot.org

23. UniProtKB/Swiss-ProtManual annotation 1. Protein sequence(merge available CDS, annotate sequence discrepancies, report sequencing mistakes…)2. Biological information(sequence analysis,extract literature information, ortholog data propagation, …)

24. UniProtKB/Swiss-Prot 1- Protein sequence curation

25. The displayed protein sequence: …canonical, representative, consensus…+alternative sequences (described within the entry) UniProtKB/Swiss-Prot a gene-centric view of the protein space 1 entry <-> 1 gene (1 species)

26. What is the current status? • At least 20% of Swiss-Prot entries required a minimal amount of curation effort so as to obtain the “correct” sequence. • Typical problems • unsolved conflicts • uncorrected initiation sites • frameshifts • wrong gene prediction • other ‘problems’

27. UCSC genome browser examples of CDS annotation submitted to INSDC…

28. UniProtKB/Swiss-Prot 2- Biological data curation

29. Extract literature informationand protein sequence analysis maximum usage of controlled vocabulary UniProtKB/Swiss-Prot gathers data form multiple sources: - publications (literature/Pubmed) - prediction programs (Prosite, TMHMM, …) - contacts with experts - other databases - nomenclature committees An evidence attribution system allows to easily trace the source of each annotation

30. Protein and gene names Synonyms useful for literature searching

31. General annotation (Comments) …enable researchers to obtain a summary of what is known about a protein… An evidence attribution system allows to easily trace the source of each annotation (Reference number, By similarity, Probable, Potential) www.uniprot.org

32. Human protein manual annotation: some statistics (June 2012)

33. Sequence annotation (Features) …enable researchers to obtain a summary of what is known about a protein… An evidence attribution system allows to easily trace the source of each annotation (Reference number, By similarity, Probable, Potential) www.uniprot.org

34. Find all the proteins localized in the cytoplasm (experimentally proven) which are phosphorylated on a serine (experimentally proven)

35. Ontologies www.uniprot.org

36. ‘Protein existence’ tag • The ‘Protein existence’ tag indicates what is the evidence for the existence of a given protein; • Different qualifiers: 1. Evidence at protein level (~18%) (MS, western blot (tissue specificity), immuno (subcellular location),…) 2. Evidence at transcript level (~19%) 3. Inferred from homology (~58 %) 4. Predicted (~5%) 5. Uncertain (mainly in TrEMBL) http://www.uniprot.org/docs/pe_criteria

37. Not sequence validation !

38. UniProtKB Additional information can be found in the cross-references (to more than 140 databases)

39. Family and domain Gene3D HAMAP InterPro PANTHER Pfam PIRSF PRINTS ProDom PROSITE SMART SUPFAM TIGRFAMs Organism-specific AGD ArachnoServer CGD ConoServer CTD CYGD dictyBase EchoBASE EcoGene euHCVdb EuPathDB FlyBase GeneCards GeneDB_Spombe GeneFarm GenoList Gramene H-InvDB HGNC HPA LegioList Leproma MaizeGDB MGI MIM neXtProt Orphanet PharmGKB PseudoCAP RGD SGD TAIR TubercuList WormBase Xenbase ZFIN Sequence EMBL IPI PIR RefSeq UniGene Proteomic PeptideAtlas PRIDE ProMEX Polymorphism dbSNP Genome annotation Ensembl EnsemblBacteria EnsemblFungi EnsemblMetazoa EnsemblPlants EnsemblProtists GeneID GenomeReviews KEGG NMPDR TIGR UCSC VectorBase Gene expression ArrayExpress Bgee CleanEx Genevestigator GermOnline Protein family/group Allergome CAZy MEROPS PeroxiBase PptaseDB REBASE TCDB Ontologies GO UniProtKB/Swiss-Prot: 129 explicit links 2D gel 2DBase-Ecoli ANU-2DPAGE Aarhus/Ghent-2DPAGE (no server) COMPLUYEAST-2DPAGE Cornea-2DPAGE DOSAC-COBS-2DPAGE ECO2DBASE (no server) OGP PHCI-2DPAGE PMMA-2DPAGE Rat-heart-2DPAGE REPRODUCTION-2DPAGE Siena-2DPAGE SWISS-2DPAGE UCD-2DPAGE World-2DPAGE and 14 implicit links! Phylogenomic dbs eggNOG GeneTree HOGENOM HOVERGEN InParanoid OMA OrthoDB PhylomeDB ProtClustDB 3D structure DisProt HSSP PDB PDBsum ProteinModelPortal SMR Other BindingDB DrugBank NextBio PMAP-CutDB PPI DIP IntAct MINT STRING Enzyme and pathway BioCyc BRENDA Pathway_Interaction_DB Reactome PTM GlycoSuiteDB PhosphoSite PhosSite

40. The UniProt web site www.uniprot.org Powerful search engine, google-like and easy-to-use, but also supports very directed field searches Scoring mechanism presenting relevant matches first Entry views, search result views and downloads are customizable The URL of a result page reflects the query; all pages and queries are bookmarkable, supporting programmatic access Search, Blast, Align, Retrieve, ID mapping

41. Search A very powerful text search tool with autocompletion and refinement options allowing to look for UniProt entries and documentation by biological information

42. The search interface guides users with helpful suggestions and hints

44. Result pages: highly customizable

45. Result pages: downloadable

47. The URL can be bookmarked and manually modified.

48. Query: sequence:(database:epo OR database:JPO or database:USPTO)

49. Blast A tool associated with the standard options to search sequences in different UniProt databases and data sets

50. Blast: customize the result display