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1、淀粉样变性病淀粉样变性病NCBI FieldGuideGenome ResourcesLocusLinkLocusLinkLocusLinkLocusLinkGene databaseGene databaseGene databaseGene databaseUniGeneUniGeneUniGeneUniGeneTrace ArchiveTrace ArchiveTrace ArchiveTrace ArchiveMap ViewerMap ViewerMap ViewerMap ViewerHomologeneHomologeneHomologeneHomologeneNCBI Fiel
2、dGuideGenomic BiologyNCBI FieldGuideNCBI FieldGuideNCBI FieldGuideNCBI FieldGuideGenome Projects:microbNCBI FieldGuideNCBI FieldGuideNCBI FieldGuideGenome ResourcesLocusLinkLocusLinkLocusLinkLocusLinkGene databaseGene databaseGene databaseGene databaseUniGeneUniGeneUniGeneUniGeneTrace ArchiveTrace A
3、rchiveTrace ArchiveTrace ArchiveMap ViewerMap ViewerMap ViewerMap ViewerHomologeneHomologeneHomologeneHomologeneNCBI FieldGuideA single query interface to SequencesSequences-RefSeqs-RefSeqs-GenBank-GenBank-HomologeneHomologeneMaps Maps MapViewerMapViewerEntrez linksEntrez linksLocusLinkLocusLink wil
4、l be replaced by Entrez Gene on MARCH 1,2005.Check Gene FAQ for current information.NCBI FieldGuideEntrez GeneLocusLinkA single query interface to SequencesSequences -RefSeqs -RefSeqs -GenBank -GenBank -HomologeneHomologeneMaps Maps MapViewerMapViewerEntrez linksEntrez linksEntrez Gene More organism
5、s-all RefSeq genomes Entrez integrationNCBI FieldGuideGsnsym淀粉样变性病NCBI FieldGuideNCBI FieldGuideGlobal Entrez:NADH2nadh247NCBI FieldGuideEntrez Gene:NADH226 recordsNCBI FieldGuideGene Record for Pongo NADH2Homo sapiensNCBI FieldGuideDisplay Exons/Introns:Gene TableNCBI FieldGuideGene TableNCBI Field
6、GuideA Record With More Data:Human HFE血色沉着病NCBI FieldGuideGene Graphic LinksNM_NM_NP_NP_NCBI FieldGuideIntrons/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 Fi
7、eldGuideSNP in StructureNCBI FieldGuideLink to OMIMNCBI FieldGuideVariants in OMIMNCBI FieldGuideGenome ResourcesLocusLinkLocusLinkLocusLinkLocusLinkGene databaseGene databaseGene databaseGene databaseTrace ArchiveTrace ArchiveTrace ArchiveTrace ArchiveMap ViewerMap ViewerMap ViewerMap ViewerHomolog
8、eneHomologeneHomologeneHomologeneUniGeneUniGeneNCBI 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 mapp
9、ing reagentsUniGeneNCBI FieldGuideA Cluster 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 Cl
10、uster Hs.95351NCBI FieldGuideUniGene Cluster Hs.95351:expressionNCBI FieldGuideUniGene Cluster Hs.95351:seqsNCBI FieldGuideDownload sequencesweb pageftp siteNCBI FieldGuideGenome ResourcesLocusLinkLocusLinkLocusLinkLocusLinkGene databaseGene databaseGene databaseGene databaseUniGeneUniGeneUniGeneUni
11、GeneTrace ArchiveTrace ArchiveTrace ArchiveTrace ArchiveMap ViewerMap ViewerMap ViewerMap ViewerHomologeneHomologeneHomologeneHomologeneNCBI FieldGuideThe New HomologeneAutomated detection of homologs among the annotated genes of completely sequenced eukaryotic genomes.No longer UniGene basedProtein
12、 similarities firstGuided by taxonomic treeIncludes 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 duplicationParalogs vs Ort
13、hologsearly 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 NCBI FieldGuideR
14、AG1 HomolgeneRAG1Amniota NCBI FieldGuideHomolgene:RAG1NCBI FieldGuideNCBI FieldGuideHomolgene:RAG1NCBI FieldGuideGenome ResourcesLocusLinkLocusLinkLocusLinkLocusLinkGene databaseGene databaseGene databaseGene databaseUniGeneUniGeneUniGeneUniGeneTrace ArchiveTrace ArchiveTrace ArchiveTrace ArchiveMap
15、 ViewerMap ViewerMap ViewerMap ViewerHomologeneHomologeneHomologeneHomologeneNCBI FieldGuideNCBI FieldGuideNCBI FieldGuideNCBI FieldGuideMapViewerNCBI FieldGuideList ViewNCBI FieldGuideHuman MapVieweradar腺甙脱氨酶NCBI FieldGuideMapViewer:Human ADAR4NCBI FieldGuideMV Hs ADAR3 UTR5 UTRNCBI FieldGuideMaps&
16、Options-Sequence mapsSequence maps-Ab initioAssemblyRepeatsBES_CloneCloneNCI_CloneContigComponentCpG islanddbSNP haplotypeFosmidGenBank_DNAGenePhenotypeSAGE_TagSTSTCAG_RNATranscript(RNA)Hs_UniGeneHs_EST-Cytogenetic mapsCytogenetic maps-IdeogramFISH CloneGene_CytogeneticMitelman BreakpointMorbid/Dise
17、ase-Genetic Maps-deCODEGenethonMarshfield-RH maps-GeneMap99-G3GeneMap99-GB4NCBI RHStandford-G3TNGWhitehead-RHWhitehead-YACMm_UniGeneMm_ESTRn_UniGeneRn_ESTSsc_UniGeneSsc_ESTBt_UniGeneBt_ESTGga_UniGeneGga_ESTVariationMaps&Options=SNPNCBI FieldGuideMapViewerUniGeneComponentRepeatsGeneNCBI FieldGuideMas
18、ter map:repeatsNCBI FieldGuideGenePhenotypeVariationNCBI FieldGuideMaps&OptionsMaps&OptionsNCBI FieldGuideGenome ResourcesLocusLinkLocusLinkLocusLinkLocusLinkGene databaseGene databaseGene databaseGene databaseUniGeneUniGeneUniGeneUniGeneTrace ArchiveTrace ArchiveTrace ArchiveTrace ArchiveMap Viewer
19、Map ViewerMap ViewerMap ViewerHomologeneHomologeneHomologeneHomologeneNCBI FieldGuideNCBI FieldGuideNCBI FieldGuideStrongylocentrotus purpuratus TracesNCBI FieldGuideBLASTBasic Local Alignment Search ToolNCBI FieldGuideWeb AccessBLASTVASTEntrezTextSequenceStructureNCBI FieldGuideNCBI FieldGuideBasic
20、 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 annotate sequencesTo evaluate evolutionary relationshipsOther:model genomic structure(e.g.,Spidey)check primer sp
21、ecificity in silico:NCBIs toolNCBI FieldGuideBLAST Website StatsNCBI FieldGuideGlobal vs Local AlignmentSeq 1Seq 2Seq 1Seq 2Global alignmentLocal alignmentNCBI FieldGuideGlobal vs Local AlignmentSeq1:WHEREISWALTERNOW (16aa)Seq2:HEWASHEREBUTNOWISHERE(21aa)GlobalSeq1:1 W-HEREISWALTERNOW 16 W HERE Seq2
22、: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 BLASTStandard BLASTtraditional“contiguous”word hitposition independent scoring nucleotide,protein and translations(blastn,blastp,blastx,tblastn,tblastx)Megabl
23、astoptimized for large batch searchescan use discontiguous wordsPSI-BLASTconstructs PSSMs automatically;uses as queryvery sensitive protein searchRPS BLASTsearches a database of PSSMstool for conserved domain searchesNCBI FieldGuideWidely used similarity search toolHeuristic approach based on Smith
24、Waterman algorithmFinds best local alignmentsProvides statistical significanceAll combinations(DNA/Protein)query and database.DNA vs DNA blastnDNA translation vs Protein blastxProtein vs Protein blastpProtein vs DNA translation tblastnDNA translation vs DNA translation tblastx www,standalone,and net
25、work clientsBasic Local Alignment Search ToolNCBI FieldGuideTranslated BLASTQueryQueryDatabaseDatabaseProgramProgramNPucleotideroteinNNNNPPblastxtblastntblastxPPPPPPPPPPPPPPPPPPPPPPPPParticularly useful for nucleotide sequences withoutprotein annotations,such as ESTs or genomic DNANCBI FieldGuideHow
26、 BLAST WorksMake lookup table of“words”for queryScan database for hitsUngapped extensions of hits(initial HSPs)Gapped extensions(no traceback)Gapped extensions(traceback;alignment details)NCBI FieldGuideNucleotide WordsGTACTGGACATGGACCCTACAGGAAQuery:GTACTGGACAT TACTGGACATG ACTGGACATGG CTGGACATGGA TG
27、GACATGGAC GGACATGGACC GACATGGACCC ACATGGACCCTMake a lookuptable of words11-mer.828megablast711blastnminimumdefaultWORD SIZENCBI FieldGuideProtein WordsGTQITVEDLFYNIATRRKALKNQuery:Neighborhood WordsLTV,MTV,ISV,LSV,etc.GTQ TQI QIT ITV TVE VED EDL DLF .Make a lookuptable of wordsWord size=3(default)Wor
28、d 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 neighboring matches within 40 aaGTQITVEDLFYNI SEI YYNATCGCCATGCTTAATTGGGCTT CATGCTTAATT neighborhood wordsone exact matchtwo matches-A 40=b
29、lastp default NCBI FieldGuideBLASTP Summary YLS HFLSbjct 287 LEETYAKYLHKGASYFVYLSLNMSPEQLDVNVHPSKRIVHFLYDQEI 333 Query 1 IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESI 47Gapped extension with trace backGapped extension with trace backQuery 1 IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESI-LEV 50 +E
30、 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-scoring pair(HSP)High-scoring pair(HSP)HFL 18HFV 15 HFS 14HWL 13NFL 13DFL 12HWV 10etc YLS 15YLT 12 YVS 12YIT 10etc Neighborhood Neighborhood wo
31、rdswordsNeighborhood Neighborhood score thresholdscore thresholdT(-f)=11T(-f)=11Query:IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILEVexample query wordsexample 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|ra
32、w score=19-9=10CACGTAGCAAGCTTG-GTGTCA-r 1-q-3 NCBI FieldGuideScoring Systems-ProteinsPosition Independent MatricesPAM Matrices(Percent Accepted Mutation)Derived from observation;small dataset of alignments Implicit model of evolution All calculated from PAM1 PAM250 widely usedBLOSUM Matrices(BLOck S
33、Ubstitution Matrices)Derived from observation;large dataset of highly conserved blocks Each matrix derived separately from blocks with a defined percent identity 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
34、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 0-2-3-2 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-
35、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 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 Field
36、GuidePosition-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 435 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 43
37、8 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 1-2-2-2 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
38、 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 1-5-5-3-4-3 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
39、-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 -3-3-5-6-3-4-5-6-5 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
40、-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_499868.pssm NCBI FieldGuideLocal Alignment StatisticsHigh scores of local alignments between two random sequencesfollow the Extreme Value
41、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 ValueExpect ValueE=number of database hits you expect to find by chance,Syour scoreexpected number of random hitsMore info:www.ncbi.nlm.nih
42、.gov/BLAST/tutorial/Altschul-1.html NCBI FieldGuideAdvanced BLAST Options:NucleotideExample Entrez Queriesnucleotide allFilter NOT mammaliaOrganismgreen plantsOrganismbiomol mrnaPropertiesgbdiv estProperties AND ratorganismOther Advancede 10000 expect value-v 2000 descriptions-b 2000 alignmentsNCBI
43、FieldGuideAdvanced BLAST Options:ProteinMatrix SelectionPAM30-most stringentBLOSUM45-least stringentExample Entrez Queriesproteins allFilter NOT mammaliaOrganismgreen plantsOrganismsrcdb refseqPropertiesOther Advancede 10000 expect value-v 2000 descriptions-b 2000 alignmentsLimit by taxonMus musculu
44、sOrganismMammaliaOrganismViridiplantaeOrganismNCBI FieldGuide sp|P27476|NSR1_YEAST NUCLEAR LOCALIZATION SEQUENCE BINDING PROTEIN(P67)Length=414 Score=40.2 bits(92),Expect=0.013 Identities=35/131(26%),Positives=56/131(42%),Gaps=4/131(3%)Query:362 STTSLTSSSTSGSSDKVYAHQMVRTDSREQKLDAFLQPLSKPLS-SQPQAIVTE
45、DKTD 418 S+S SSS+S SS+S +S S S+E K Sbjct:29 SSSSSESSSSSSSSSESESESESESESSSSSSSSDSESSSSSSSDSESEAETKKEESKDS 88FilteredUnfilteredLow Complexity FilteringNCBI FieldGuideOther BLAST Algorithms MegablastDiscontiguous MegablastPSI-BLASTPHI-BLASTNCBI FieldGuideMegablast:NCBIs Genome Annotator Long alignments
46、 of similar DNA sequencesGreedy algorithmConcatenation of query sequencesFaster than blastn;less sensitiveNCBI FieldGuideMegaBLAST&Word SizeTrade-off:sensitivity vs speed23blastp828megablast711blastnminimumdefaultWORD SIZENCBI FieldGuideDiscontiguous MegablastUses discontiguous word matchesBetter fo
47、r cross-species comparisonsNCBI FieldGuideTemplates for Discontiguous WordsW=11,t=16,coding:1101101101101101W=11,t=16,non-coding:1110010110110111W=12,t=16,coding:1111101101101101W=12,t=16,non-coding:1110110110110111W=11,t=18,coding:101101100101101101W=11,t=18,non-coding:111010010110010111W=12,t=18,c
48、oding:101101101101101101W=12,t=18,non-coding:111010110010110111W=11,t=21,coding:100101100101100101101W=11,t=21,non-coding:111010010100010010111W=12,t=21,coding:100101101101100101101W=12,t=21,non-coding:111010010110010010111 Reference:Ma,B,Tromp,J,Li,M.PatternHunter:faster and more sensitive homology
49、 search.Bioinformatics March,2002;18(3):440-5 W=word size;#matches in templatet=template lengthNCBI FieldGuideDiscontiguous(Cross-species)MegaBLASTNCBI FieldGuideDiscontiguous Word OptionsNCBI FieldGuideMegaBLAST vs Discontiguous MegaBLASTNM_017460Homo sapiens cytochrome P450,family 3,subfamily A,po
50、lypeptide 4(CYP3A4),transcript variant 1,mRNA(2768 letters)vs DrosophilaNCBI FieldGuideMegaBLAST vs Discontiguous MegaBLAST MegaBLAST=“No significant similarity found.”Discontiguous megaBLAST=NCBI FieldGuideAnother Example.Discontiguous megaBLAST=numerous hits.Query:NM_078651 Drosophila melanogaster