NCBI网站BLAST使用方法介绍

BLASTBasicLocalAlignmentSearchToolLushanWang2010.11.24生物信息的获取方式•1、以生物学信息为主检索数据——Entrez•2、以序列为主检索相关信息——BLAST•生物信息学时代BLAST相当于分子生物学进代的“PCR”技术DNAPolymeraseReplicationNNNNNH2OHHHHHHOPOPOPONNNNNH2OHOHHHHHOPOPOPO传统分子技术必然会让位于BLAST为主的生物信息技术Sanger’sddNTPSequencingWhatdoesthissequencemean?限制酶目标基因重组基因细胞转化宿主菌蛋白质分离纯化及性质测定传统分子生物学方法现代生物信息学方法BLASTGenefamilyOrProteinFamilyFunctionannotation几周的时间几分钟的时间BLAST计算机怎么会读我们读不懂的数据？BasicLocalAlignmentSearchTool•Whyusesequencesimilarity?•BLASTalgorithm•BLASTstatistics•BLASToutput•ExamplesWhyDoWeNeedSequenceSimilaritySearching?•Toidentifyandannotatesequences•Toevaluateevolutionaryrelationships•Other:–modelgenomicstructure(e.g.,Spidey)–checkprimerspecificityinsilico:NCBI’stool科学的方法：可以认我们研究我们不懂的数据！——比较的方法3000Myr1000Myr540MyrAlzheimer’sDiseaseAtaxiatelangiectasiaColoncancerPancreaticcarcinomaYeastBacteriaWormFlyHumanBLASTandMolecularEvolutionMLH1MutLBLASTScreening先找到相似的序列再找出相似序列间的关系GlobalvsLocalAlignmentSeq1Seq2Seq1Seq2GlobalalignmentLocalalignment如何找出序列间的相似性？GlobalvsLocalAlignmentSeq1:WHEREISWALTERNOW(16aa)Seq2:HEWASHEREBUTNOWISHERE(21aa)GlobalSeq1:1W--HEREISWALTERNOW16WHERESeq2:1HEWASHEREBUTNOWISHERE21LocalSeq1:1W--HERE5Seq1:1W--HERE5WHEREWHERESeq2:3WASHERE9Seq2:15WISHERE21TheFlavorsofBLAST•StandardBLAST–traditional“contiguous”wordhit–positionindependentscoring–nucleotide,proteinandtranslations(blastn,blastp,blastx,tblastn,tblastx)•Megablast–optimizedforlargebatchsearches–canusediscontiguouswords•PSI-BLAST–constructsPSSMsautomatically;usesasquery–verysensitiveproteinsearch•RPSBLAST–searchesadatabaseofPSSMs–toolforconserveddomainsearches•Widelyusedsimilaritysearchtool•HeuristicapproachbasedonSmithWatermanalgorithm•Findsbestlocalalignments•Providesstatisticalsignificance•Allcombinations(DNA/Protein)queryanddatabase.–DNAvsDNAblastn–DNAtranslationvsProteinblastx–ProteinvsProteinblastp–ProteinvsDNAtranslationtblastn–DNAtranslationvsDNAtranslationtblastx••Makelookuptableof“words”forquery•Scandatabaseforhits•Ungappedextensionsofhits(initialHSPs)•Gappedextensions(notraceback)•Gappedextensions(traceback;alignmentdetails)NucleotideWordsGTACTGGACATGGACCCTACAGGAAQuery:GTACTGGACATTACTGGACATGACTGGACATGGCTGGACATGGATGGACATGGACGGACATGGACCGACATGGACCCACATGGACCCTMakealookuptableofwords11-mer...828megablast711blastnminimumdefaultWORDSIZEProteinWordsGTQITVEDLFYNIATRRKALKNQuery:NeighborhoodWordsLTV,MTV,ISV,LSV,etc.GTQTQIQITITVTVEVEDEDLDLF...MakealookuptableofwordsWordsize=3(default)Wordsizecanonlybe2or3[-f11=blastpdefault]MinimumRequirementsforaHit•NucleotideBLASTrequiresoneexactmatch•ProteinBLASTrequirestwoneighboringmatcheswithin40aaGTQITVEDLFYNISEIYYNATCGCCATGCTTAATTGGGCTTCATGCTTAATTneighborhoodwordsoneexactmatchtwomatches[-A40=blastpdefault]BLASTPSummaryYLSHFLSbjct287LEETYAKYLHKGASYFVYLSLNMSPEQLDVNVHPSKRIVHFLYDQEI333Query1IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHP