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UniProtKB

Sandra Orchard. UniProtKB. Importance of reference protein sequence databases. Completeness and minimal redundancy A non redundant protein sequence database, with maximal coverage including splice isoforms, disease variant and PTMs.

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UniProtKB

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  1. Sandra Orchard UniProtKB

  2. Importance of reference protein sequence databases • Completeness and minimal redundancy A non redundant protein sequence database, with maximal coverage including splice isoforms, disease variant and PTMs. Low degree of redundancy for facilitating peptide assignments • Stabilityand consistency Stable identifiers and consistent nomenclature Databases are in constant change due to a substantial amount of work to improve their completeness and the quality of sequence annotation • High quality protein annotation Detailed information on protein function, biological processes, molecular interactions and pathways cross-referenced to external source

  3. Summary of protein sequence databases Updated from Nesvizhskii, A. I., and Aebersold, R. (2005) Interpretation of shotgun proteomic data: the protein inference problem. Mol. Cell. Proteomics. 4,1419–1440l

  4. UniProtKB • UniProt Knowledgebase: • 2 sections • UniProtKB/Swiss-Prot Non-redundant, high-quality manual annotation - reviewed • UniProtKB/TrEMBL Redundant, automatically annotated - unreviewed www.uniprot.org Master headline

  5. Collaboration projects between database resources • CCDS project, a collaboration between Ensembl, NCBI, UCSC and UniProt, aims to provide a standard set of gene predictions for the human and mouse genomes • Considerable communication effort between curators from different groups is on-going • Ensembl and UniProt collaboration to cover the gaps in gene predictions in UniProtKB (one sequence for each protein coding transcript in Ensembl) • Ensembl high quality gene/transcript models (quality checks remove gene models with erroneous structures or supported by dubious evidence – e.g. cDNA fragments with short/wrongly annotated ORF) • UniProtKB high quality protein sequences

  6. Complete proteome data sets in UniProtKB • Ensembl sequences have now been incorporated for an increasing number of species: human, mouse, rat , zebrafish, chicken, dog pig and cow. • Tagged with Complete proteome keyword by release 2011_06 of 31st May • Now increasingly >1 proteome/species – most completely annotated tagged as ‘Reference Proteome’

  7. Manual annotation of the human proteome(UniProtKB/Swiss-Prot) A draft of the complete human proteome has been available in UniProtKB/Swiss-Prot since 2008 Manually annotated representation of 20,242 protein coding genes with ~ 36,000 protein sequences - an additional 38,484 UniProtKB/TrEMBL form the complete proteome set Approximately 63,000 single amino acid polymorphisms (SAPs), mostly disease-linked 80,000 post-translational modifications (PTMs) Close collaboration with NCBI, Ensembl, Sanger Institute and UCSC to provide the authoritative set to the user community

  8. Manual annotation of UniProtKB/Swiss-Prot Splice variants Sequence Sequence features UniProtKB Ontologies Annotations References Nomenclature

  9. Sequence curation, stable identifiers, versioning and archiving • For example – erroneous gene model predictions, frameshifts • …. ..premature stop codons, read-throughs, erroneous initiator methionines….. Master headline

  10. Splice variants Master headline

  11. Identification of amino acid variants ..and of PTMs … and also Master headline

  12. Domain annotation Binding sites Master headline

  13. Protein nomenclature Master headline

  14. Master headline

  15. Annotation - >30 defined fields Controlled vocabularies used whenever possible… Master headline

  16. ..and also imported from external resources Binary interactions taken from the IntAct database Interactors of human p53 Master headline

  17. Controlled vocabulary usage increasing – for example from the Gene Ontology Annotation for human Rhodopsin Master headline

  18. Evidence at protein level There is experimental evidence of the existence of a protein (e.g. Edman sequencing, MS, X-ray/NMR structure, good quality protein-protein interaction , detection by antibodies) Evidence at transcript level The existence of a protein has not been proven but there is expression data (e.g. existence of cDNAs, RT-PCR or Northern blots) that indicates the existence of a transcript. Inferred from homology The existence of a protein is likely because orthologs exist in closely related species 4 Predicted 5 Uncertain Sequence evidence Type of evidence that supports the existence of a protein

  19. UniProtKB/TrEMBL • Multiple entries for the same protein (redundancy) can arise in UniProtKB/TrEMBL due to: • Erroneous gene model predictions • Sequence errors (Frame shifts) • Polymorphisms • Alternative start sites • Isoforms • Apart from 100% identical sequences all merged sequences are analysed by a curator so they can be annotated accordingly.

  20. Automatic Annotation • Automated clean-up of annotation from original nucleotide sequence entry • Additional value added by using automatic annotation • Recognises common annotation belonging to a closely related family within UniProtKB/Swiss-Prot • Identifies all members of this family using pattern/motif/HMMs in InterPro • Transfers common annotation to related family members in TrEMBL Master headline

  21. ← Taxonomy ← Publication ← Name (non-standard) ← Sequence

  22. InterPro Master headline

  23. Master headline

  24. Text-based searching • Logical operators ‘&’ (and), ‘|’ Searching UniProt – Simple Search Master headline

  25. Searching UniProt – Advanced Search Master headline

  26. Each linked to the UniProt entry Searching UniProt – Search Results Master headline

  27. Searching UniProt – Search Results Master headline

  28. Searching UniProt – Search Results Master headline

  29. Searching UniProt – Blast Search Master headline

  30. Searching UniProt – Blast Search Master headline

  31. Alignment with query sequence Searching UniProt – Blast Results Master headline

  32. Searching UniProt – Blast Results Master headline

  33. Finding a complete proteome in UniProtKB

  34. Complete Proteomes

  35. MS Proteomics • Require each sequence (inc isoforms) to be present in the dataset as an separate entity for search engines to access • For higher organisms, with isoforms, expanded set made available on ftp site • Fastafiles by FTP • One file per species containing canonical + isoform sequences

  36. ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? Master headline

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