1、A Field Guide Genome resources Sequence similarity淀粉样变性病Genome Resources淀粉样变性病Genomic Biology淀粉样变性病淀粉样变性病淀粉样变性病淀粉样变性病Genome Projects:microb淀粉样变性病淀粉样变性病淀粉样变性病Genome Resources淀粉样变性病A single query interface to SequencesSequences-RefSeqs-RefSeqs-GenBank-GenBank-Homologene-HomologeneMaps MapViewerMaps Ma
2、pViewerEntrez linksEntrez linksLocusLinkLocusLink will be replaced by Entrez Gene on MARCH 1,2005.Check Gene FAQ for current information.淀粉样变性病Entrez GeneLocusLinkA single query interface to SequencesSequences -RefSeqs -RefSeqs -GenBank -GenBank -Homologene -HomologeneMaps MapViewerMaps MapViewerEnt
3、rez linksEntrez linksEntrez Gene More organisms-all RefSeq genomes Entrez integration淀粉样变性病Gsnsym淀粉样变性病淀粉样变性病淀粉样变性病Global Entrez:NADH2nadh247淀粉样变性病Entrez Gene:NADH226 records淀粉样变性病Gene Record for Pongo NADH2Homo sapiens淀粉样变性病Display Exons/Introns:Gene Table淀粉样变性病Gene Table淀粉样变性病A Record With More Da
4、ta:Human HFE血色沉着病淀粉样变性病Gene Graphic LinksNM_NM_NP_NP_淀粉样变性病Introns/Exons:Gene Tablelinks to sequence淀粉样变性病A Record With More Data:Human HFE淀粉样变性病Entrez SNPhfegene name AND humanorgn 52血色沉着病淀粉样变性病Linking to SNP染色体定位基因定位序列定位淀粉样变性病SNP in Structure淀粉样变性病Link to OMIM淀粉样变性病Variants in OMIM淀粉样变性病Genome Res
5、ources淀粉样变性病Gene-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 reagentsUniGene淀粉样变性病A Cluster of ESTsquery5 E
6、ST hits3 EST hits淀粉样变性病Unigene淀粉样变性病UniGene Collections淀粉样变性病Example UniGene Cluster淀粉样变性病Histogram of cluster sizes for UniGene Hs build 177淀粉样变性病UniGene Cluster Hs.95351淀粉样变性病UniGene Cluster Hs.95351淀粉样变性病UniGene Cluster Hs.95351:expression淀粉样变性病UniGene Cluster Hs.95351:seqs淀粉样变性病Download sequence
7、sweb page淀粉样变性病Genome Resources淀粉样变性病The New HomologeneAutomated detection of homologs among the annotated genes of completely sequenced eukaryotic genomes.No longer UniGene based Protein similarities first Guided by taxonomic tree Includes orthologs and paralogs淀粉样变性病 Orthologs 和 Paralogs 是同源序列的两种类
8、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。淀粉样变性病gene duplicationParalogs vs Orthologsearly globin geneA-chain gene B-chain genefrog A chick A mouse Amouse B chick B frog Bparalogsorthologs orthologs淀粉样变性病The
9、New Homologene Homologene Build 37.2Species Number of genes input grouped groups淀粉样变性病RAG1 Homologenerag112recombination activating gene 淀粉样变性病RAG1 HomolgeneRAG1Amniota 淀粉样变性病Homolgene:RAG1淀粉样变性病淀粉样变性病Homolgene:RAG1淀粉样变性病Genome Resources淀粉样变性病淀粉样变性病淀粉样变性病淀粉样变性病MapViewer淀粉样变性病List View淀粉样变性病Human Map
10、Vieweradar腺甙脱氨酶淀粉样变性病MapViewer:Human ADAR4淀粉样变性病MV Hs ADAR3 UTR5 UTR淀粉样变性病Maps&Options-Sequence mapsSequence maps-Ab initioAssemblyRepeatsBES_CloneCloneNCI_CloneContigComponentCpG islanddbSNP haplotypeFosmidGenBank_DNAGenePhenotypeSAGE_TagSTSTCAG_RNATranscript(RNA)Hs_UniGeneHs_EST-Cytogenetic mapsCy
11、togenetic maps-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&O
12、ptions=SNP淀粉样变性病MapViewerUniGeneComponentRepeatsGene淀粉样变性病Master map:repeats淀粉样变性病GenePhenotypeVariation淀粉样变性病Maps&OptionsMaps&Options淀粉样变性病Genome Resources淀粉样变性病淀粉样变性病淀粉样变性病Strongylocentrotus purpuratus Traces淀粉样变性病Basic Local Alignment Search Tool淀粉样变性病Web AccessBLASTVASTEntrezTextSequenceStructur
13、e淀粉样变性病淀粉样变性病Basic Local Alignment Search Tool Why use sequence similarity?BLAST algorithm BLAST statistics BLAST output Examples淀粉样变性病Why Do We Need Sequence Similarity Searching?To identify and annotate sequences To evaluate evolutionary relationships Other:model genomic structure(e.g.,Spidey)chec
14、k primer specificity in silico:NCBIs tool淀粉样变性病BLAST Website Stats淀粉样变性病Global vs Local AlignmentSeq 1Seq 2Seq 1Seq 2Global alignmentLocal alignment淀粉样变性病Global vs Local AlignmentSeq1:WHEREISWALTERNOW (16aa)Seq2:HEWASHEREBUTNOWISHERE(21aa)GlobalSeq1:1 W-HEREISWALTERNOW 16 W HERE Seq2:1 HEWASHEREBUTN
15、OWISHERE 21LocalSeq1:1 W-HERE 5 Seq1:1 W-HERE 5 W HERE W HERESeq2:3 WASHERE 9 Seq2:15 WISHERE 21淀粉样变性病The Flavors of BLAST Standard BLAST traditional“contiguous”word hit position independent scoring nucleotide,protein and translations(blastn,blastp,blastx,tblastn,tblastx)Megablast optimized for larg
16、e batch searches can use discontiguous words PSI-BLAST constructs PSSMs automatically;uses as query very sensitive protein search RPS BLAST searches a database of PSSMs tool for conserved domain searches淀粉样变性病 Widely used similarity search tool Heuristic approach based on Smith Waterman algorithm Fi
17、nds best local alignments Provides statistical significance All combinations(DNA/Protein)query and database.DNA vs DNA blastn DNA translation vs Protein blastx Protein vs Protein blastp Protein vs DNA translation tblastn DNA translation vs DNA translation tblastx www,standalone,and network clientsBa
18、sic Local Alignment Search Tool淀粉样变性病Translated BLASTQueryQueryDatabaseDatabaseProgramProgramNPucleotideroteinNNNNPPblastxtblastntblastxPPPPPPPPPPPPPPPPPPPPPPPPParticularly useful for nucleotide sequences withoutprotein annotations,such as ESTs or genomic DNA淀粉样变性病How BLAST Works Make lookup table o
19、f“words”for query Scan database for hits Ungapped extensions of hits(initial HSPs)Gapped extensions(no traceback)Gapped extensions(traceback;alignment details)淀粉样变性病Nucleotide WordsGTACTGGACATGGACCCTACAGGAAQuery:GTACTGGACAT TACTGGACATG ACTGGACATGG CTGGACATGGA TGGACATGGAC GGACATGGACC GACATGGACCC ACAT
20、GGACCCTMake a lookuptable of words11-mer.828megablast711blastnminimumdefaultWORD SIZE淀粉样变性病Protein WordsGTQITVEDLFYNIATRRKALKNQuery: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 淀
21、粉样变性病Minimum 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=blastp default 淀粉样变性病BLASTP Summary YLS HFLSbjct 287 LEET
22、YAKYLHKGASYFVYLSLNMSPEQLDVNVHPSKRIVHFLYDQEI 333 Query 1 IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESI 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 LEETYAKYLHKGASYFVYLS
23、LNMSPEQLDVNVHPSKRIVHFLYDQEIATSI 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 wordswordsNeighborhood Neighborhood score thresholdscore thresholdT
24、(-f)=11T(-f)=11Query:IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILEVexample query wordsexample query words淀粉样变性病Scoring 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 淀粉样变性病Scoring Systems-Protei
25、nsPosition 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 SUbstitution Matrices)Derived from observation;large dataset of highly conserved blo
26、cks Each matrix derived separately from blocks with a defined percent identity cutoff BLOSUM62-default matrix for BLASTPosition Specific Score Matrices(PSSMs)PSI-and RPS-BLAST淀粉样变性病A 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
27、 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-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
28、-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 substitutions淀粉样变性病Position-Specific Score MatrixDAF-1Serine/Threonine protein kinases catalytic loop174PSSM scores
29、54淀粉样变性病 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 0-1 438 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
30、-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 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-
31、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 1-1-5-4-4 451 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-
32、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 5-1-1 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 Matrixc
33、atalytic loop./blastpgp-i NP_499868.2-d nr-j 3-Q NP_499868.pssm 淀粉样变性病Local 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 sc
34、oring 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:淀粉样变性病Advanced BLAST Options:NucleotideExample Entrez Queriesnucleotide allFilter NOT mammaliaOrganismgreen plantsOrganismbiomol mrnaPr
35、opertiesgbdiv estProperties AND ratorganismOther Advancede 10000 expect value-v 2000 descriptions-b 2000 alignments淀粉样变性病Advanced BLAST Options:ProteinMatrix SelectionPAM30-most stringentBLOSUM45-least stringentExample Entrez Queriesproteins allFilter NOT mammaliaOrganismgreen plantsOrganismsrcdb re
36、fseqPropertiesOther Advancede 10000 expect value-v 2000 descriptions-b 2000 alignmentsLimit by taxonMus musculusOrganismMammaliaOrganismViridiplantaeOrganism淀粉样变性病 sp|P27476|NSR1_YEAST NUCLEAR LOCALIZATION SEQUENCE BINDING PROTEIN(P67)Length=414 Score=40.2 bits(92),Expect=0.013 Identities=35/131(26%
37、),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 Filtering淀粉样变性病Other BLAST Algorithms Megablast Discontigu
38、ous Megablast PSI-BLAST PHI-BLAST淀粉样变性病Megablast:NCBIs Genome Annotator Long alignments of similar DNA sequences Greedy algorithm Concatenation of query sequences Faster than blastn;less sensitive淀粉样变性病MegaBLAST&Word SizeTrade-off:sensitivity vs speed23blastp828megablast711blastnminimumdefaultWORD S
39、IZE淀粉样变性病Discontiguous Megablast Uses discontiguous word matches Better for cross-species comparisons淀粉样变性病Templates 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:101
40、101100101101101W=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,coding:100101101101100101101W=12,t=21,non-coding:111010010110010010111 Reference:M
41、a,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 length淀粉样变性病Discontiguous(Cross-species)MegaBLAST淀粉样变性病Discontiguous Word Options淀粉样变性病MegaBLAST vs Discontiguous MegaBLASTNM_017460Homo sapiens c
42、ytochrome P450,family 3,subfamily A,polypeptide 4(CYP3A4),transcript variant 1,mRNA(2768 letters)vs Drosophila淀粉样变性病MegaBLAST vs Discontiguous MegaBLAST MegaBLAST=“No significant similarity found.”Discontiguous megaBLAST=淀粉样变性病Another Example.Discontiguous megaBLAST=numerous hits.Query:NM_078651 Dro
43、sophila melanogaster CG18582-PA(mbt)mRNA,(3244 bp)/note=mushroom bodies tiny;synonyms:Pak2,STE20,dPAK2 MegaBLAST=“No significant similarity found.”Database:nr(nt),Mammaliaorgn淀粉样变性病Ex:Discontiguous MegaBLAST淀粉样变性病Ex:BLASTN淀粉样变性病PSI-BLASTExample:Confirming relationships of purinenucleotide metabolism
44、 proteinsPosition-specific Iterated BLAST淀粉样变性病gi|113340|sp|P03958|ADA_MOUSE ADENOSINE DEAMINASE(ADENOSINEMAQTPAFNKPKVELHVHLDGAIKPETILYFGKKRGIALPADTVEELRNIIGMDKPLSLPGFVIAGCREAIKRIAYEFVEMKAKEGVVYVEVRYSPHLLANSKVDPMPWNQTEGDVTPDDVVDEQAFGIKVRSILCCMRHQPSWSLEVLELCKKYNQKTVVAMDLAGDETIEGSSLFPGHVEAYRTVHAGEVGSP
45、EVVREAVDILKTERVGHGYHTIEDEALYNRLLKENMHFEVCPWSSYLTGAVRFKNDKANYSLNTDDPLIFKSTLDTDYQMTKKDMGFTEEEFKRLNINAAKSSFLPEEEKKPSI-BLAST0.005E value cutoff for PSSM淀粉样变性病RESULTS:Initial BLASTPSame results as protein-protein BLAST;different format淀粉样变性病Results of First PSSM SearchOther purine nucleotide metabolizing
46、 enzymes not found by ordinary BLAST淀粉样变性病Tenth PSSM Search:ConvergenceJust below threshold,another nucleotide metabolism enzymeCheck to add to PSSM淀粉样变性病Reverse PSI-BLAST(RPS)-BLAST淀粉样变性病Adenosine/AMP Deaminase DomainAMP Deaminases.淀粉样变性病PHI-BLASTgi|231729|sp|P30429|CED4_CAEEL CELL DEATH PROTEIN 4M
47、LCEIECRALSTAHTRLIHDFEPRDALTYLEGKNIFTEDHSELISKMSTRLERIANFLRIYRRQASELIDFFNYNNQSHLADFLEDYIDFAINEPDLLRPVVIAPQFSRQMLDRKLLLGNVPKQMTCYIREYHVIKKLDEMCDLDSFFLFLHGRAGSGKSVIASQALSKSDQLIGINYDSIVWLKDSGTAPKSTFDLFTDILKSEDDLLNFPSVEHVTSVVLKRMICNALIDRPNTLFVFDDVVQEETIRWAQELRLRCLVTTRDVEIASQTCEFIEVTSLEIDECYDFLEAYGMPMPVGE
48、KEEDVLNKTIELSSGNPATLMMFFKSCEPKTFEKGAxxxxGKST淀粉样变性病Whats New?Whats New?淀粉样变性病BLAST DatabasesNucleotide refseq_rna=NM_*,XM_*refseq_genomic=NC_*,NG_*env_nt environmental samplefilter,e.g.,16S rRNAProtein refseq=NP_*,XP_*env_nr淀粉样变性病New FormatterSelect lower caseSelect red淀粉样变性病New Formatter gray line=s
49、ame database hit hsps color-coded independently淀粉样变性病BLAST Output:Alignments&Filterlow complexity sequence filteredlow complexity sequence filtered淀粉样变性病Advanced OptionsLimit to Organismprotein allfilter Nprotein allfilter NExample Entrez Queriesproteins allFilter NOT mammaliaOrganismray finned fish
50、esOrganismsrcdb refseqProperties Nucleotide only:biomol mrnaPropertiesbiomol genomicPropertiesOtherAdvancede 10000expect value-v 2000descriptions-b 2000alignments-e 10000-v 2000-e 10000-v 2000淀粉样变性病Genome BLAST Examples淀粉样变性病Example Search Pathways:HemochromatosisGene“hemochromatosis”nucleotide sequ
