1、NCBI FieldGuideA Field Guide part 2 Genome resources Sequence similarityApr,2007Shandong UniversityNCBI FieldGuideGenome ResourcesNCBI FieldGuideGenomic BiologyNCBI FieldGuideNCBI FieldGuideNCBI FieldGuideNCBI FieldGuideGenome Projects:microbNCBI FieldGuideNCBI FieldGuideNCBI FieldGuideGenome Resour
2、cesNCBI FieldGuideA single query interface to SequencesSequences-RefSeqs-RefSeqs-GenBank-GenBank-Homologene-HomologeneMaps MapViewerMaps MapViewerEntrez linksEntrez linksLocusLinkLocusLink will be replaced by Entrez Gene on MARCH 1,2005.Check Gene FAQ for current information.NCBI FieldGuideEntrez Ge
3、neLocusLinkA single query interface to SequencesSequences -RefSeqs -RefSeqs -GenBank -GenBank -Homologene -HomologeneMaps MapViewerMaps MapViewerEntrez linksEntrez linksEntrez Gene More organisms-all RefSeq genomes Entrez integrationNCBI FieldGuideGsnsym淀粉样变性病NCBI FieldGuideNCBI FieldGuideGlobal Ent
4、rez:NADH2nadh247NCBI FieldGuideEntrez Gene:NADH226 recordsNCBI FieldGuideGene Record for Pongo NADH2Homo sapiensNCBI FieldGuideDisplay Exons/Introns:Gene TableNCBI FieldGuideGene TableNCBI FieldGuideA Record With More Data:Human HFE血色沉着病NCBI FieldGuideGene Graphic LinksNM_NM_NP_NP_NCBI FieldGuideInt
5、rons/Exons:Gene Tablelinks to sequenceNCBI FieldGuideA Record With More Data:Human HFENCBI FieldGuideEntrez SNPhfegene name AND humanorgn 52血色沉着病NCBI FieldGuideLinking to SNP染色体定位基因定位序列定位NCBI FieldGuideSNP in StructureNCBI FieldGuideLink to OMIMNCBI FieldGuideVariants in OMIMNCBI FieldGuideGenome Re
6、sourcesNCBI FieldGuideGene-oriented clusters of expressed sequences Automatic clustering using MegaBlast Each cluster represents a unique gene Informed by genome hits Information on tissue types and map locations Useful for gene discovery and selection of mapping reagentsUniGeneNCBI FieldGuideA Clus
7、ter of ESTsquery5 EST hits3 EST hitsNCBI FieldGuideUnigeneNCBI FieldGuideUniGene CollectionsNCBI FieldGuideExample UniGene ClusterNCBI FieldGuideHistogram of cluster sizes for UniGene Hs build 177NCBI FieldGuideUniGene Cluster Hs.95351NCBI FieldGuideUniGene Cluster Hs.95351NCBI FieldGuideUniGene Clu
8、ster Hs.95351:expressionNCBI FieldGuideUniGene Cluster Hs.95351:seqsNCBI FieldGuideDownload sequencesweb pageftp siteNCBI FieldGuideGenome ResourcesNCBI FieldGuideThe New HomologeneAutomated detection of homologs among the annotated genes of completely sequenced eukaryotic genomes.No longer UniGene
9、based Protein similarities first Guided by taxonomic tree Includes orthologs and paralogsNCBI FieldGuide Orthologs 和 Paralogs 是同源序列的两种类型。Orthologs（垂直同源基因）是指来自于不同物种的由垂直家系（物种形成）进化而来的蛋白，并且典型的保留与原始蛋白有相同的功能。Paralogs（平行同源基因）是那些在一定物种中的来源于基因复制的蛋白，可能会进化出新的与原来有关的功能。请参考文献获得更多的信息。NCBI FieldGuidegene duplication
10、Paralogs vs Orthologsearly globin geneA-chain gene B-chain genefrog A chick A mouse Amouse B chick B frog Bparalogsorthologs orthologsNCBI FieldGuideThe New Homologene Homologene Build 37.2Species Number of genes input grouped groupsNCBI FieldGuideRAG1 Homologenerag112recombination activating gene N
11、CBI FieldGuideRAG1 HomolgeneRAG1Amniota NCBI FieldGuideHomolgene:RAG1NCBI FieldGuideNCBI FieldGuideHomolgene:RAG1NCBI FieldGuideGenome ResourcesNCBI FieldGuideNCBI FieldGuideNCBI FieldGuideNCBI FieldGuideMapViewerNCBI FieldGuideList ViewNCBI FieldGuideHuman MapVieweradar腺甙脱氨酶NCBI FieldGuideMapViewer
12、:Human ADAR4NCBI FieldGuideMV Hs ADAR3 UTR5 UTRNCBI FieldGuideMaps&Options-Sequence mapsSequence maps-Ab initioAssemblyRepeatsBES_CloneCloneNCI_CloneContigComponentCpG islanddbSNP haplotypeFosmidGenBank_DNAGenePhenotypeSAGE_TagSTSTCAG_RNATranscript(RNA)Hs_UniGeneHs_EST-Cytogenetic mapsCytogenetic ma
13、ps-IdeogramFISH CloneGene_CytogeneticMitelman BreakpointMorbid/Disease-Genetic Maps-deCODEGenethonMarshfield-RH maps-GeneMap99-G3GeneMap99-GB4NCBI RHStandford-G3TNGWhitehead-RHWhitehead-YACMm_UniGeneMm_ESTRn_UniGeneRn_ESTSsc_UniGeneSsc_ESTBt_UniGeneBt_ESTGga_UniGeneGga_ESTVariationMaps&Options=SNPNC
14、BI FieldGuideMapViewerUniGeneComponentRepeatsGeneNCBI FieldGuideMaster map:repeatsNCBI FieldGuideGenePhenotypeVariationNCBI FieldGuideMaps&OptionsMaps&OptionsNCBI FieldGuideGenome ResourcesNCBI FieldGuideNCBI FieldGuideNCBI FieldGuideStrongylocentrotus purpuratus TracesNCBI FieldGuideBasic Local Ali
15、gnment Search ToolNCBI FieldGuideWeb AccessBLASTVASTEntrezTextSequenceStructureNCBI FieldGuideNCBI FieldGuideBasic Local Alignment Search Tool Why use sequence similarity?BLAST algorithm BLAST statistics BLAST output ExamplesNCBI FieldGuideWhy Do We Need Sequence Similarity Searching?To identify and
16、 annotate sequences To evaluate evolutionary relationships Other:model genomic structure(e.g.,Spidey)check primer specificity in silico:NCBIs toolNCBI FieldGuideBLAST Website StatsNCBI FieldGuideGlobal vs Local AlignmentSeq 1Seq 2Seq 1Seq 2Global alignmentLocal alignmentNCBI FieldGuideGlobal vs Loca
17、l AlignmentSeq1:WHEREISWALTERNOW (16aa)Seq2:HEWASHEREBUTNOWISHERE(21aa)GlobalSeq1:1 W-HEREISWALTERNOW 16 W HERE Seq2:1 HEWASHEREBUTNOWISHERE 21LocalSeq1:1 W-HERE 5 Seq1:1 W-HERE 5 W HERE W HERESeq2:3 WASHERE 9 Seq2:15 WISHERE 21NCBI FieldGuideThe Flavors of BLAST Standard BLAST traditional“contiguou
18、s”word hit position independent scoring nucleotide,protein and translations(blastn,blastp,blastx,tblastn,tblastx)Megablast optimized for large batch searches can use discontiguous words PSI-BLAST constructs PSSMs automatically;uses as query very sensitive protein search RPS BLAST searches a database
19、 of PSSMs tool for conserved domain searchesNCBI FieldGuide Widely used similarity search tool Heuristic approach based on Smith Waterman algorithm Finds best local alignments Provides statistical significance All combinations(DNA/Protein)query and database.DNA vs DNA blastn DNA translation vs Prote
20、in blastx Protein vs Protein blastp Protein vs DNA translation tblastn DNA translation vs DNA translation tblastx www,standalone,and network clientsBasic Local Alignment Search ToolNCBI FieldGuideTranslated BLASTQueryQueryDatabaseDatabaseProgramProgramNPucleotideroteinNNNNPPblastxtblastntblastxPPPPP
21、PPPPPPPPPPPPPPPPPPPParticularly useful for nucleotide sequences withoutprotein annotations,such as ESTs or genomic DNANCBI FieldGuideHow BLAST Works Make lookup table of“words”for query Scan database for hits Ungapped extensions of hits(initial HSPs)Gapped extensions(no traceback)Gapped extensions(t
22、raceback;alignment details)NCBI FieldGuideNucleotide WordsGTACTGGACATGGACCCTACAGGAAQuery:GTACTGGACAT TACTGGACATG ACTGGACATGG CTGGACATGGA TGGACATGGAC GGACATGGACC GACATGGACCC ACATGGACCCTMake a lookuptable of words11-mer.828megablast711blastnminimumdefaultWORD SIZENCBI FieldGuideProtein WordsGTQITVEDLF
23、YNIATRRKALKNQuery:Neighborhood WordsLTV,MTV,ISV,LSV,etc.GTQ TQI QIT ITV TVE VED EDL DLF .Make a lookuptable of wordsWord size=3(default)Word size can only be 2 or 3-f 11=blastp default NCBI FieldGuideMinimum Requirements for a Hit Nucleotide BLAST requires one exact match Protein BLAST requires two
24、neighboring matches within 40 aaGTQITVEDLFYNI SEI YYNATCGCCATGCTTAATTGGGCTT CATGCTTAATT neighborhood wordsone exact matchtwo matches-A 40=blastp default NCBI FieldGuideBLASTP Summary YLS HFLSbjct 287 LEETYAKYLHKGASYFVYLSLNMSPEQLDVNVHPSKRIVHFLYDQEI 333 Query 1 IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVH
25、FLHEESI 47Gapped extension with trace backGapped extension with trace backQuery 1 IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESI-LEV 50 +E YA YL K F+YLSL+SP+DVNVHP+K VHFL+I+Sbjct 287 LEETYAKYLHKGASYFVYLSLNMSPEQLDVNVHPSKRIVHFLYDQEIATSI 337 Final HSPFinal HSP +E YA YL K F+L+SP+DVNVHP+K V +I High-scor
26、ing pair(HSP)High-scoring pair(HSP)HFL 18HFV 15 HFS 14HWL 13NFL 13DFL 12HWV 10etc YLS 15YLT 12 YVS 12YIT 10etc Neighborhood Neighborhood wordswordsNeighborhood Neighborhood score thresholdscore thresholdT(-f)=11T(-f)=11Query:IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILEVexample query wordsexampl
27、e query wordsNCBI FieldGuideScoring Systems-Nucleotides A G C TA+1 3 3-3G 3+1 3-3C 3 3+1-3T 3 3 3+1Identity matrixCAGGTAGCAAGCTTGCATGTCA|raw score=19-9=10CACGTAGCAAGCTTG-GTGTCA-r 1-q-3 NCBI FieldGuideScoring Systems-ProteinsPosition Independent MatricesPAM Matrices(Percent Accepted Mutation)Derived
28、from observation;small dataset of alignments Implicit model of evolution All calculated from PAM1 PAM250 widely usedBLOSUM Matrices(BLOck SUbstitution Matrices)Derived from observation;large dataset of highly conserved blocks Each matrix derived separately from blocks with a defined percent identity
29、 cutoff BLOSUM62-default matrix for BLASTPosition Specific Score Matrices(PSSMs)PSI-and RPS-BLASTNCBI FieldGuideA 4R-1 5 N-2 0 6D-2-2 1 6C 0-3-3-3 9Q-1 1 0 0-3 5E-1 0 0 2-4 2 5G 0-2 0-1-3-2-2 6H-2 0 1-1-3 0 0-2 8I-1-3-3-3-1-3-3-4-3 4 L-1-2-3-4-1-2-3-4-3 2 4K-1 2 0-1-3 1 1-2-1-3-2 5M-1-1-2-3-1 0-2-3-
30、2 1 2-1 5F-2-3-3-3-2-3-3-3-1 0 0-3 0 6P-1-2-2-1-3-1-1-2-2-3-3-1-2-4 7S 1-1 1 0-1 0 0 0-1-2-2 0-1-2-1 4T 0-1 0-1-1-1-1-2-2-1-1-1-1-2-1 1 5W-3-3-4-4-2-2-3-2-2-3-2-3-1 1-4-3-2 11Y-2-2-2-3-2-1-2-3 2-1-1-2-1 3-3-2-2 2 7V 0-3-3-3-1-2-2-3-3 3 1-2 1-1-2-2 0-3-1 4X 0-1-1-1-2-1-1-1-1-1-1-1-1-1-2 0 0-2-1-1-1 A
31、 R N D C Q E G H I L K M F P S T W Y V XBLOSUM62DFNegative for less likely substitutionsDYFPositive for more likely substitutionsNCBI FieldGuidePosition-Specific Score MatrixDAF-1Serine/Threonine protein kinases catalytic loop174PSSM scores54NCBI FieldGuide A R N D C Q E G H I L K M F P S T W Y V 43
32、5 K -1 0 0-1-2 3 0 3 0-2-2 1-1-1-1-1-1-1-1-2 436 E 0 1 0 2-1 0 2-1 0-1-1 0 0 0-1 0 0-1-1-1 437 S 0 0-1 0 1 1 0 1 1 0-1 0 0 0 2 0-1-1 0-1 438 N -1 0-1-1 1 0-1 3 3-1-1 1-1 0 0-1-1 1 1-1 439 K -2 1 1-1-2 0-1-2-2-1-2 5 1-2-2-1-1-2-2-1 440 P -2-2-2-2-3-2-2-2-2-1-2-1 0-3 7-1-2-3-1-1 441 A 3-2 1-2 0-1 0 1-
33、2-2-2 0-1-2 3 1 0-3-3 0 442 M -3-4-4-4-3-4-4-5-4 7 0-4 1 0-4-4-2-4-1 2 443 A 4-4-4-4 0-4-4-3-4 4-1-4-2-3-4-1-2-4-3 4 444 H -4-2-1-3-5-2-2-4 10-6-5-3-4-3-2-3-4-5 0-5 445 R -4 8-3-4 0-1-2-3-2-5-4 0-3-2-4-3-3 0-4-5 446 D -4-4-1 8-6-2 0-3-3-5-6-3-5-6-4-2-3-7-5-5 447 I -4-5-6-6-3-4-5-6-5 3 5-5 1 1-5-5-3-
34、4-3 1 448 K 0 0 1-3-5-1-1-3-3-5-5 7-4-5-3-1-2-5-4-4 449 S 0-3-2-3 0-2-2-3-3-4-4-2-4-5 2 6 2-5-4-4 450 K 0 3 0 1-5 0 0-4-1-4-3 4-3-2 2 1-1-5-4-4 451 N -4-3 8-1-5-2-2-3-1-6-6-2-4-5-4-1-2-6-4-5 452 I -3-5-5-6 0-5-5-6-5 6 2-5 2-2-5-4-3-5-3 3 453 M -4-4-6-6-3-4-5-6-5 0 6-5 1 0-5-4-3-4-3 0 454 V -3-3-5-6-
35、3-4-5-6-5 3 3-4 2-2-5-4-3-5-3 5 455 K -2 1 1 4-5 0-1-2 1-4-2 4-3-2-3 0-1-5-2-3 456 N 1 1 3 0-4-1 1 0-3-4-4 3-2-5-2 2-2-5-4-4 457 D -3-2 5 5-1-1 1-1 0-5-4 0-2-5-1 0-2-6-4-5 458 L -3-1 0-3 0-3-2 3-4-2 3 0 1 1-2-2-3 5-1-3Position-Specific Score Matrixcatalytic loop./blastpgp-i NP_499868.2-d nr-j 3-Q NP
36、_499868.pssm NCBI FieldGuideLocal Alignment StatisticsHigh scores of local alignments between two random sequencesfollow the Extreme Value DistributionScore(S)Alignments(applies to ungapped alignments)E=Kmne-S or E=mn2-SK=scale for search space =scale for scoring system S=bitscore=(S-lnK)/ln2Expect
37、ValueExpect ValueE=number of database hits you expect to find by chance,Syour scoreexpected number of random hitsMore info:www.ncbi.nlm.nih.gov/BLAST/tutorial/Altschul-1.html NCBI FieldGuideAdvanced BLAST Options:NucleotideExample Entrez Queriesnucleotide allFilter NOT mammaliaOrganismgreen plantsOr
38、ganismbiomol mrnaPropertiesgbdiv estProperties AND ratorganismOther Advancede 10000 expect value-v 2000 descriptions-b 2000 alignmentsNCBI FieldGuideAdvanced BLAST Options:ProteinMatrix SelectionPAM30-most stringentBLOSUM45-least stringentExample Entrez Queriesproteins allFilter NOT mammaliaOrganism
39、green plantsOrganismsrcdb refseqPropertiesOther Advancede 10000 expect value-v 2000 descriptions-b 2000 alignmentsLimit by taxonMus musculusOrganismMammaliaOrganismViridiplantaeOrganismNCBI FieldGuide sp|P27476|NSR1_YEAST NUCLEAR LOCALIZATION SEQUENCE BINDING PROTEIN(P67)Length=414 Score=40.2 bits(9
40、2),Expect=0.013 Identities=35/131(26%),Positives=56/131(42%),Gaps=4/131(3%)Query:362 STTSLTSSSTSGSSDKVYAHQMVRTDSREQKLDAFLQPLSKPLS-SQPQAIVTEDKTD 418 S+S SSS+S SS+S +S S S+E K Sbjct:29 SSSSSESSSSSSSSSESESESESESESSSSSSSSDSESSSSSSSDSESEAETKKEESKDS 88FilteredUnfilteredLow Complexity FilteringNCBI FieldGu
41、ideOther BLAST Algorithms Megablast Discontiguous Megablast PSI-BLAST PHI-BLASTNCBI FieldGuideMegablast:NCBIs Genome Annotator Long alignments of similar DNA sequences Greedy algorithm Concatenation of query sequences Faster than blastn;less sensitiveNCBI FieldGuideMegaBLAST&Word SizeTrade-off:sensi
42、tivity vs speed23blastp828megablast711blastnminimumdefaultWORD SIZENCBI FieldGuideDiscontiguous Megablast Uses discontiguous word matches Better for cross-species comparisonsNCBI FieldGuideTemplates for Discontiguous WordsW=11,t=16,coding:1101101101101101W=11,t=16,non-coding:1110010110110111W=12,t=1
43、6,coding:1111101101101101W=12,t=16,non-coding:1110110110110111W=11,t=18,coding:101101100101101101W=11,t=18,non-coding:111010010110010111W=12,t=18,coding:101101101101101101W=12,t=18,non-coding:111010110010110111W=11,t=21,coding:100101100101100101101W=11,t=21,non-coding:111010010100010010111W=12,t=21,
44、coding:100101101101100101101W=12,t=21,non-coding:111010010110010010111 Reference:Ma,B,Tromp,J,Li,M.PatternHunter:faster and more sensitive homology search.Bioinformatics March,2002;18(3):440-5 W=word size;#matches in templatet=template lengthNCBI FieldGuideDiscontiguous(Cross-species)MegaBLASTNCBI F
45、ieldGuideDiscontiguous Word OptionsNCBI FieldGuideMegaBLAST vs Discontiguous MegaBLASTNM_017460Homo sapiens cytochrome P450,family 3,subfamily A,polypeptide 4(CYP3A4),transcript variant 1,mRNA(2768 letters)vs DrosophilaNCBI FieldGuideMegaBLAST vs Discontiguous MegaBLAST MegaBLAST=“No significant sim
46、ilarity found.”Discontiguous megaBLAST=NCBI FieldGuideAnother Example.Discontiguous megaBLAST=numerous hits.Query:NM_078651 Drosophila melanogaster CG18582-PA(mbt)mRNA,(3244 bp)/note=mushroom bodies tiny;synonyms:Pak2,STE20,dPAK2 MegaBLAST=“No significant similarity found.”Database:nr(nt),Mammaliaor
47、gnNCBI FieldGuideEx:Discontiguous MegaBLASTNCBI FieldGuideEx:BLASTNNCBI FieldGuidePSI-BLASTExample:Confirming relationships of purinenucleotide metabolism proteinsPosition-specific Iterated BLASTNCBI FieldGuidegi|113340|sp|P03958|ADA_MOUSE ADENOSINE DEAMINASE(ADENOSINEMAQTPAFNKPKVELHVHLDGAIKPETILYFG
48、KKRGIALPADTVEELRNIIGMDKPLSLPGFVIAGCREAIKRIAYEFVEMKAKEGVVYVEVRYSPHLLANSKVDPMPWNQTEGDVTPDDVVDEQAFGIKVRSILCCMRHQPSWSLEVLELCKKYNQKTVVAMDLAGDETIEGSSLFPGHVEAYRTVHAGEVGSPEVVREAVDILKTERVGHGYHTIEDEALYNRLLKENMHFEVCPWSSYLTGAVRFKNDKANYSLNTDDPLIFKSTLDTDYQMTKKDMGFTEEEFKRLNINAAKSSFLPEEEKKPSI-BLAST0.005E value cuto
49、ff for PSSMNCBI FieldGuideRESULTS:Initial BLASTPSame results as protein-protein BLAST;different formatNCBI FieldGuideResults of First PSSM SearchOther purine nucleotide metabolizing enzymes not found by ordinary BLASTNCBI FieldGuideTenth PSSM Search:ConvergenceJust below threshold,another nucleotide
50、 metabolism enzymeCheck to add to PSSMNCBI FieldGuideReverse PSI-BLAST(RPS)-BLASTNCBI FieldGuideAdenosine/AMP Deaminase DomainAMP Deaminases.NCBI FieldGuidePHI-BLASTgi|231729|sp|P30429|CED4_CAEEL CELL DEATH PROTEIN 4MLCEIECRALSTAHTRLIHDFEPRDALTYLEGKNIFTEDHSELISKMSTRLERIANFLRIYRRQASELIDFFNYNNQSHLADFL
