ebi web resources i: databases and toolsbcb.unl.edu/yyin/teach/pbb/ebi-go.pdf · cellular...

EBI web resources I: databases and tools

YanbinYin

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Outline

• IntrotoEBI

• Databasesandwebtools– UniProt– GeneOntology

• HandsonPractice

MOSTMATERIALSAREFROM:http://www.ebi.ac.uk/training/online/course-list

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Threeinternationalnucleotidesequencedatabases

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TheEuropeanBioinformaticsInstitute (EBI)

Createdin1992aspartof EuropeanMolecularBiologyLaboratory (EMBL)

EMBLwascreatedin1974andisa molecularbiology researchinstitutionsupportedby20EuropeancountriesandAustralia

Wellcome TrustGenomeCampus, Hinxton,Cambridge,UKNeighborofWellcome TrustSangerInstitute

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http://www.ebi.ac.uk/

ResearchgroupsinEBI

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InterPro

UniProt

miRBase

MajordatabasesinEBI

EMBL-Bank (DNAandRNAsequences)Ensembl (genomes)ArrayExpress(microarray-basedgene-expressiondata)UniProt (proteinsequences)InterPro(proteinfamilies,domainsandmotifs)PDBe (macromolecularstructures)

Others,suchasIntAct (protein–proteininteractions)Reactome (pathways)ChEBI (smallmolecules)IntEnz (enzymeclassification)GO (geneontology)

GenBankGenomeMapView

GEOnr(GenPept)

CDDMMDB

SwissInstituteofBioinformaticsSangerInstitute

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http://www.ebi.ac.uk/training/online/course/nucleotide-sequence-data-resources-ebi

chromatograms

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SequencemightfirstenterENAasSRA (SequenceReadArchive)fragmented sequencereads;itmightbere-submittedasassembledWGS(WholeGenomeShotgun)sequenceoverlapcontigs;itmightbere-submittedagainwithfurtherassemblyasCON(Constructed)sequenceentries,withtheolderWGSentriesbeingconsignedtotheSequenceVersionArchive

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Dataisfirstsplitintoclasses,thenitissplitintointersectingslicesbytaxonomy

UniProt

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http://www.uniprot.org/help/uniparc

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SourcesofannotationfortheUniProtKnowledgebase

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Lifeasa ScientificCuratorhttp://www.ebi.ac.uk/about/jobs/career-profiles/scientific-curator

ScientificDatabaseCuratorjob:Cambridge,UnitedKingdomhttp://www.nature.com/naturejobs/science/jobs/589083-hgnc-gene-nomenclature-advisor

Curation generationhttp://cys.bios.niu.edu/yyin/teach/PBB/Bioinformatics%20Curation%20generation.pdf

Handsonpractice1:UniProt

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www.uniprot.orghttp://www.uniprot.org/help/abouthttp://www.uniprot.org/docs/uniprot_flyer.pdf

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WearegoingtodoIDmapping

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http://cys.bios.niu.edu/yyin/teach/PBB/at-id.txt

ChooseAraport hereandUniProtKB here

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TheseareUniProt IDs

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SelectthePALproteinsandalignthem

Clustal omegaprogramwillbecalledtoaligntheselectedproteinseqsMaytake1mintofinish

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ThisistheMSAresultpageToggletheseoptionsonwilladdcolorsinthealignment

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GobacktotheproteinlistpageSelectingoneproteinwillenabletheBLASTbutton

ChooseadvancedwillallowtochangeBLASTparameters

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Hereyoucanmakechanges

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WearegoingtosearchUniProt proteomesforhumanproteinsetClickonAdvancedyouwillseeapop-outwindow

Hereyoucanspecifysearchterms

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Clickheretogethelp

Clickheretoopenanewpage

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TheGeneOntology(GO)projectisacollaborativeefforttoaddresstheneedforconsistentdescriptionsofgeneproductsindifferentdatabases

Theprojectbeganasacollaborationbetweenthreemodelorganismdatabases, FlyBase (Drosophila),the Saccharomyces GenomeDatabase (SGD)andthe MouseGenomeDatabase (MGD),in1998

Threestructuredcontrolledvocabularies(ontologies)thatdescribegeneproductsintermsoftheirassociatedbiologicalprocesses,cellularcomponentsandmolecularfunctionsinaspecies-independent manner.

Therearethreeseparateaspectstothiseffort:

1,thedevelopmentandmaintenanceoftheontologies themselves;2,theannotation ofgeneproducts,whichentailsmakingassociationsbetweentheontologiesandthegenesandgeneproductsinthecollaboratingdatabases;and3,developmentoftools thatfacilitatethecreation,maintenanceanduseofontologies.

http://geneontology.org/page/documentation

GeneOntology

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GOisnotadatabaseofgenesequences,noracatalogofgeneproducts.Rather,GOdescribeshowgeneproductsbehave inacellularcontext.

GOisnotadictatedstandard,mandatingnomenclatureacrossdatabases.Groupsparticipatebecauseofself-interest,andcooperatetoarriveataconsensus.

GOisnotawaytounifybiologicaldatabases(i.e.GOisnota'federatedsolution').Sharingvocabularyisasteptowardsunification,butisnot,initself,sufficient.

GeneOntologycoversthreedomains:

cellularcomponent,thepartsofacelloritsextracellularenvironment;

molecularfunction,theelementalactivitiesofageneproductatthemolecularlevel,suchasbindingorcatalysis;

biologicalprocess,operationsorsetsofmoleculareventswithadefinedbeginningandend,pertinenttothefunctioningofintegratedlivingunits:cells,tissues,organs,andorganisms

ThescopeofGO

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ThestructureofGOcanbedescribedintermsofagraph,whereeachGOtermisanode,andtherelationshipsbetweenthetermsareedgesbetweenthenodes.GOislooselyhierarchical,with'child'termsbeingmorespecializedthantheir'parent'terms,butunlikeastricthierarchy,atermmayhavemorethanoneparentterm

http://geneontology.org/page/ontology-structure

28http://www.ebi.ac.uk/training/online/course/go-quick-tour/what-can-i-do-go

id: GO:0000016 name: lactase activity namespace: molecular_function def: "Catalysis of the reaction: lactose + H2O = D-glucose + D-galactose." [EC:3.2.1.108] synonym: "lactase-phlorizin hydrolase activity" BROAD [EC:3.2.1.108] synonym: "lactose galactohydrolase activity" EXACT [EC:3.2.1.108] xref: EC:3.2.1.108 xref: MetaCyc:LACTASE-RXN xref: Reactome:20536 is_a: GO:0004553 ! hydrolase activity, hydrolyzing O-glycosyl compounds

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Enrichmentanalysis:usestatisticalteste.g.FisherexacttestExample:inhumangenomebackground(20,000genetotal),40genesareinvolvedinp53signalingpathway.Agivengenelisthasfoundthat3outof300belongtop53signalingpathway.Then weaskthequestionif3/300ismorethanrandomchancecomparingtothehumanbackgroundof40/20000

http://david.abcc.ncifcrf.gov/helps/functional_annotation.html#E4

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UniProt-GOannotation(GOA)

http://www.ebi.ac.uk/training/online/course/uniprot-goa-quick-tour/what-uniprot-goa

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The reference usedtomaketheannotation(e.g. ajournalarticle)An evidencecode denotingthetypeofevidenceuponwhichtheannotationisbasedThedateandthecreatoroftheannotation

Gene product: Actin, alpha cardiac muscle 1, UniProtKB:P68032GO term: heart contraction ; GO:0060047 (biological process) Evidence code: Inferred from Mutant Phenotype (IMP) Reference: PMID 17611253Assigned by: UniProtKB, June 6, 2008

UniProt-GOAformat

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Ifyouhaveanewgenome/transcriptome sequenced,howdoyouperformaGOannotationforit?

1. FindaclosetmodelorganismwhichhasbeenannotatedbyGO2. BLASTyourdataagainstthisclosestorganism3. TransfertheGOannotationofthebestmatchtoyourquerysequences

Forinstance,ifwewanttoannotateferntranscriptome withGOfunctiondescriptions….

1. FindArabidopsisUniProt proteindataset2. FindtheArabidopsisGOAassociationfile3. BLASTx fernreads(orassembledUniGenes)againsttheUniProt set4. AnalyzeBLASTresulttolinkfernreadsGOterms

TheideaofGOannotationfornewsequences

Handsonpractice2:GOannotation

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http://geneontology.org/

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http://amigo1.geneontology.org/cgi-bin/amigo/blast.cgi

Getanexampleproteinsequencefilefromhttp://cys.bios.niu.edu/yyin/teach/PBB/csl-pr.fa

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Thisiseasy.Nowlet’strytogetalistofdifferentiallyexpressedgenesandthenfindwhat’scommoninthislistofgenesintermsoffunctions.

We’regonna useNCBIGEOwebsitetogetthegenelistandthenfeedthegenelisttoGOenrichmentanalysistools

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GotoNCBIhomepage,searchGEODataSets withkeyword“GDS4831”,andhitsearch

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Choose“Compare2setsofsamples”

Choose“Valuemeansdifference”Choose“8+fold”Choose“higher”

ThengotoStep2

SelecttochoosegroupA:threesamplesforCOP1depletionandHuh7cellline

GroupB:threesamplesfornegativecontrolandHuh7cellline

Hitok,andgotoStep3

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Total256geneprofilesarefoundwith8+foldhigherexpressioninCOP1depletionthaninnegativecontrolinHuh7cellline

Togetthelistofgenes,chooseGenedatabaseandhitFinditems

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Total225genescorrespondto256geneprofilesTodownloadthelistofGeneIDs,hitSendto,chooseUIlistasformatandhitCreatefile

Afilenamed“gene_result.txt”willbeautomaticallydownloadedtoyourlocalcomputerFindoutwhereitisdownloadedto,openitusingnotepad++

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Viewthefileusingnotepad++

NextwewilluseDAVIDtoperformfunctionenrichmentanalysis

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The Databasefor Annotation, Visualizationand IntegratedDiscovery (DAVID )

Hitstartanalysis

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UploadthelistofGeneIDs

SelectENTREZ_GENE_ID

ClickonGenelist

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ThisallowsyoutoviewfunctionalannotationfromvariousresourcesincludingGO

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IfyouhaveclickedonFunctionalAnnotationtool,youareatthispage

Allthesecanbechangedbyusers(toshowornottoshowandshowwhat)

Uncheckthis

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SelectjustGO

Clickherewillopenanewwindowtoshowthe225differentiallyexpressedgenesareenrichedinwhatGO

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GenesareenrichedinwhatGOcategories(comparedtothegenomebackground)?

Nextlecture: EBI web resources II (ENSEMBL

and InterPro)

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