Transcript Uniprot - European Bioinformatics Institute

UniProtKB
Sandra Orchard
EBI is an Outstation of the European Molecular Biology Laboratory.
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
• Stability and 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
Summary of protein sequence databases
Database
Description
Species
UniProtKB
Expertly curated section (UniProtKB/Swiss-Prot) and computerannotated section (UniProtKB/TrEMBL); minimum level of redundancy;
high level of integration with other databases; stable identifiers;
diversity of sources including large scale genomics, small scale
cloning and sequencing, protein sequencing, PDB, predicted
sequences from Ensembl and RefSeq
Many
UniRef100
Assembled from UniProtKB, Ensembl and RefSeq; merges 100%
identical sequences; stable identifiers
Many
Ensembl
Predictions using automated genome annotation pipeline; explicitly
linked to nucleotide and protein sequences; stable reference; merge
their annotations with Vega annotations at transcript level; extensive
quality checks to remove erroneous gene models ; high level of
integration with other databases
Over 50 Eukaryotic genomes
Ensembl Genomes: Metazoa,
Plants and Fungi, Protists,
Bacteria and Archaea
RefSeq
NCBI creates from existing data; ongoing curation; non-redundant;
explicitly linked nucleotide and protein sequences; stable reference;
high level of integration with other databases
Limited to fully sequenced
organisms
Entrez protein (NCBInr)
Assembled from GenBank and RefSeq coding sequence translations
and UniProt KB ; annotations extracted from source curated
databases; high degree of sequence redundancy
Many
Updated from Nesvizhskii, A. I., and Aebersold, R. (2005) Interpretation of shotgun proteomic data: the protein inference problem. Mol. Cell. Proteomics. 4,1419–1440l
UniProtKB
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UniProt Knowledgebase:
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2 sections
UniProtKB/Swiss-Prot Non-redundant, highquality manual annotation - reviewed
UniProtKB/TrEMBL Redundant, automatically
annotated - unreviewed
www.uniprot.org
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
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’
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
Manual annotation of UniProtKB/Swiss-Prot
Splice variants
Sequence
Sequence
features
UniProtKB
Ontologies
Annotations
Nomenclature
References
Sequence curation, stable identifiers, versioning and archiving
For example – erroneous gene model predictions, frameshifts
….
..premature stop codons, read-throughs, erroneous initiator methionines…..
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Splice variants
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Identification of amino acid variants
..and of PTMs
… and also
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Domain annotation
Binding sites
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Protein nomenclature
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Annotation - >30 defined fields
Controlled vocabularies used whenever possible…
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..and also imported from external resources
Binary interactions taken from the IntAct database
Interactors of human p53
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Controlled vocabulary usage increasing – for example
from the Gene Ontology
Annotation for human Rhodopsin
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Sequence evidence
Type of evidence that supports the existence of a protein
1 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)
2 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.
3 Inferred from homology
The existence of a protein is likely because orthologs exist in closely
related species
4 Predicted
5 Uncertain
UniProtKB/TrEMBL
Multiple entries for the same protein (redundancy)
can arise in UniProtKB/TrEMBL due to:
o Erroneous gene model predictions
o Sequence errors (Frame shifts)
o Polymorphisms
o Alternative start sites
o Isoforms
Apart from 100% identical sequences all merged
sequences are analysed by a curator so they can be
annotated accordingly.
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
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← Taxonomy
← Publication
← Name (non-standard)
← Sequence
InterPro
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Searching UniProt – Simple Search
• Text-based searching
• Logical operators ‘&’ (and), ‘|’
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Searching UniProt – Advanced Search
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Searching UniProt – Search Results
Each linked to the UniProt entry
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Searching UniProt – Search Results
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Searching UniProt – Search Results
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Searching UniProt – Blast Search
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Searching UniProt – Blast Search
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Searching UniProt – Blast Results
Alignment with query sequence
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Searching UniProt – Blast Results
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Finding a complete proteome in UniProtKB
Complete
Proteomes
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
• Fasta files by FTP
• One file per species containing canonical + isoform sequences
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