淀粉样变性病精品课件

NCBI FieldGuide A Field Guide part 2 Genome resources Sequence similarity Apr, 2007Shandong University NCBI FieldGuide Genome Resources LocusLinkLocusLink Gene databaseGene database UniGeneUniGene Trace ArchiveTrace Archive Map ViewerMap Viewer HomologeneHomologene NCBI FieldGuide Genomic Biology NCBI FieldGuide NCBI FieldGuide NCBI FieldGuide NCBI FieldGuide Genome Projects: microb NCBI FieldGuide NCBI FieldGuide NCBI FieldGuide Genome Resources LocusLinkLocusLink Gene databaseGene database UniGeneUniGene Trace ArchiveTrace Archive Map ViewerMap Viewer HomologeneHomologene NCBI FieldGuide A single query interface to Sequences - RefSeqs - GenBank - Homologene Maps MapViewer Entrez links LocusLink LocusLink will be replaced by Entrez Gene on MARCH 1, 2005. Check Gene FAQ for current information. NCBI FieldGuide Entrez Gene LocusLink A single query interface to Sequences - RefSeqs - GenBank - Homologene Maps MapViewer Entrez links Entrez Gene More organisms - all RefSeq genomes Entrez integration NCBI FieldGuide Gsnsym 淀粉样变性病 NCBI FieldGuide NCBI FieldGuide Global Entrez: NADH2 nadh2 47 NCBI FieldGuide Entrez Gene: NADH2 26 records NCBI FieldGuide Gene Record for Pongo NADH2 Homo sapiens NCBI FieldGuide Display Exons/Introns: Gene Table NCBI FieldGuide Gene Table NCBI FieldGuide A Record With More Data: Human HFE 血色沉着病 NCBI FieldGuide Gene Graphic Links NM_ NP_ NCBI FieldGuide Introns/Exons: Gene Table links to sequence NCBI FieldGuide A Record With More Data: Human HFE NCBI FieldGuide Entrez SNP hfegene name AND humanorgn 52 血色沉着病 NCBI FieldGuide Linking to SNP 染色体定位 基因定位 序列定位 NCBI FieldGuide SNP in Structure NCBI FieldGuide Link to OMIM NCBI FieldGuide Variants in OMIM NCBI FieldGuide Genome Resources LocusLinkLocusLink Gene databaseGene database Trace ArchiveTrace Archive Map ViewerMap Viewer HomologeneHomologene UniGeneUniGene NCBI FieldGuide 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 reagents UniGene NCBI FieldGuide A Cluster of ESTs query 5 EST hits 3 EST hits NCBI FieldGuide Unigene NCBI FieldGuide UniGene Collections NCBI FieldGuide Example UniGene Cluster NCBI FieldGuide Histogram of cluster sizes for UniGene Hs build 177 NCBI FieldGuide UniGene Cluster Hs.95351 NCBI FieldGuide UniGene Cluster Hs.95351 NCBI FieldGuide UniGene Cluster Hs.95351: expression NCBI FieldGuide UniGene Cluster Hs.95351: seqs NCBI FieldGuide Download sequences web page ftp site NCBI FieldGuide Genome Resources LocusLinkLocusLink Gene databaseGene database UniGeneUniGene Trace ArchiveTrace Archive Map ViewerMap Viewer HomologeneHomologene NCBI FieldGuide The New Homologene Automated 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 NCBI 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 FieldGuide gene duplication Paralogs vs Orthologs early globin gene A-chain gene B-chain gene frog A chick A mouse Amouse B chick B frog B paralogs orthologs orthologs NCBI FieldGuide The New Homologene Homologene Build 37.2 Species Number of genes input grouped groups NCBI FieldGuide RAG1 Homologene rag1 12 recombination activating gene NCBI FieldGuide RAG1 Homolgene RAG1 Amniota NCBI FieldGuide Homolgene: RAG1 NCBI FieldGuide NCBI FieldGuide Homolgene: RAG1 NCBI FieldGuide Genome Resources LocusLinkLocusLink Gene databaseGene database UniGeneUniGene Trace ArchiveTrace Archive Map ViewerMap Viewer HomologeneHomologene NCBI FieldGuide NCBI FieldGuide NCBI FieldGuide NCBI FieldGuide MapViewer NCBI FieldGuide List View NCBI FieldGuide Human MapViewer adar 腺甙脱氨酶 NCBI FieldGuide MapViewer: Human ADAR 4 NCBI FieldGuide MV Hs ADAR 3 UTR 5 UTR NCBI FieldGuide Maps uses as query very sensitive protein search RPS BLAST searches a database of PSSMs tool for conserved domain searches NCBI 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 Protein blastx Protein vs Protein blastp Protein vs DNA translation tblastn DNA translation vs DNA translation tblastx www, standalone, and network clients Basic Local Alignment Search Tool NCBI FieldGuide Translated BLAST QueryDatabaseProgram NPucleotiderotein N N N N P P blastx tblastn tblastx PPPP PP PPPP PP PPPP PP PPPP PP Particularly useful for nucleotide sequences without protein annotations, such as ESTs or genomic DNA NCBI FieldGuide How 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 (traceback; alignment details) NCBI FieldGuide Nucleotide Words GTACTGGACATGGACCCTACAGGAAQuery: GTACTGGACAT TACTGGACATG ACTGGACATGG CTGGACATGGA TGGACATGGAC GGACATGGACC GACATGGACCC ACATGGACCCT Make a lookup table of words 11-mer . . . 828megablast 711blastn minimumdefaultWORD SIZE NCBI FieldGuide Protein Words GTQITVEDLFYNIATRRKALKNQuery: Neighborhood Words LTV, MTV, ISV, LSV, etc. GTQ TQI QIT ITV TVE VED EDL DLF . Make a lookup table of words Word size = 3 (default) Word size can only be 2 or 3 -f 11 = blastp default NCBI FieldGuide Minimum Requirements for a Hit Nucleotide BLAST requires one exact match Protein BLAST requires two neighboring matches within 40 aa GTQITVEDLFYNI SEI YYN ATCGCCATGCTTAATTGGGCTT CATGCTTAATT neighborhood words one exact match two matches -A 40 = blastp default NCBI FieldGuide BLASTP Summary YLS HFL Sbjct 287 LEETYAKYLHKGASYFVYLSLNMSPEQLDVNVHPSKRIVHFLYDQEI 333 Query 1 IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESI 47 Gapped extension with trace back Query 1 IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESI-LEV 50 +E YA YL K F+YLSL +SP+ +DVNVHP+K VHFL+ I + + Sbjct 287 LEETYAKYLHKGASYFVYLSLNMSPEQLDVNVHPSKRIVHFLYDQEIATSI 337 Final HSP +E YA YL K F+ L +SP+ +DVNVHP+K V + I High-scoring pair (HSP) HFL 18 HFV 15 HFS 14 HWL 13 NFL 13 DFL 12 HWV 10 etc YLS 15 YLT 12 YVS 12 YIT 10 etc Neighborhood words Neighborhood score threshold T (-f) =11 Query: IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILEV example query words NCBI FieldGuide Scoring Systems - Nucleotides A G C T A +1 3 3 -3 G 3 +1 3 -3 C 3 3 +1 -3 T 3 3 3 +1 Identity matrix CAGGTAGCAAGCTTGCATGTCA | | | raw score = 19-9 = 10 CACGTAGCAAGCTTG-GTGTCA -r 1 -q -3 NCBI FieldGuide Scoring Systems - Proteins Position Independent Matrices PAM Matrices (Percent Accepted Mutation) Derived from observation; small dataset of alignments Implicit model of evolution All calculated from PAM1 PAM250 widely used BLOSUM Matrices (BLOck SUbstitution 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 BLAST Position Specific Score Matrices (PSSMs) PSI- and RPS-BLAST NCBI FieldGuide A 4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4 X 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 X BLOSUM62 D F Negative for less likely substitutions D Y F Positive for more likely substitutions NCBI FieldGuide Position-Specific Score Matrix DAF-1 Serine/Threonine protein kinases catalytic loop 174PSSM scores54 NCBI 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 -1 438 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 -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 -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 -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 -3 Position-Specific Score Matrix catalytic loop ./blastpgp -i NP_499868.2 -d nr -j 3 -Q NP_499868.pssm NCBI FieldGuide Local Alignment Statistics High scores of local alignments between two random sequences follow the Extreme Value Distribution Score (S) Alignments (applies to ungapped alignments) E = Kmne-S or E = mn2-S K = scale for search space = scale for scoring system S = bitscore = (S - lnK)/ln2 Expect Value E = number of database hits you expect to find by chance, S your score expected number of random hits More info: /BLAST/tutorial/Altschul-1.html NCBI FieldGuide Advanced BLAST Options: Nucleotide Example Entrez Queries nucleotide allFilter NOT mammaliaOrganism green plantsOrganism biomol mrnaProperties gbdiv estProperties AND ratorganism Other Advanced e 10000 expect value -v 2000 descriptions -b 2000 alignments NCBI FieldGuide Advanced BLAST Options: Protein Matrix Selection PAM30 - most stringent BLOSUM45 - least stringent Example Entrez Queries proteins allFilter NOT mammaliaOrganism green plantsOrganism srcdb refseqProperties Other Advanced e 10000 expect value -v 2000 descriptions -b 2000 alignments Limit by taxon Mus musculusOrganism MammaliaOrganism ViridiplantaeOrganism NCBI 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-SQPQAIVTEDKTD 418 S+S SSS+S SS + + +S + + S S S+ + E K Sbjct: 29 SSSSSESSSSSSSSSESESESESESESSSSSSSSDSESSSSSSSDSESEAETKKEESKDS 88 Filtered Unfiltered Low Complexity Filtering NCBI FieldGuide Other BLAST Algorithms Megablast Discontiguous Megablast PSI-BLAST PHI-BLAST NCBI FieldGuide Megablast: NCBIs Genome Annotator Long alignments of similar DNA sequences Greedy algorithm Concatenation of query sequences Faster than blastn; less sensitive NCBI FieldGuide MegaBLAST 18(3):440-5 W = word size; # matches in template t = template length NCBI FieldGuide Discontiguous (Cross-species) MegaBLAST NCBI FieldGuide Discontiguous Word Options NCBI FieldGuide MegaBLAST vs Discontiguous MegaBLAST NM_017460 Homo sapiens cytochrome P450, family 3, subfamily A, polypeptide 4 (CYP3A4), transcript variant 1, mRNA (2768 letters) vs Drosophila NCBI FieldGuide MegaBLAST vs Discontiguous MegaBLAST MegaBLAST = “No significant similarity found.” Discontiguous megaBLAST = NCBI FieldGuide Another 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), Mammaliaorgn NCBI FieldGuide Ex: Discontiguous MegaBLAST NCBI FieldGuide Ex: BLASTN NCBI FieldGuide PSI-BLAST Example: Confirming relationships of purine nucleotide metabolism proteins Position-specific Iterated BLAST NCBI FieldGuide gi|113340|sp|P03958|ADA_MOUSE ADENOSINE DEAMINASE (ADENOSINE MAQTPAFNKPKVELHVHLDGAIKPETILYFGKKRGIALPADTVEELRNIIGMDKPLSLPGF VIAGCREAIKRIAYEFVEMKAKEGVVYVEVRYSPHLLANSKVDPMPWNQTEGDVTPDDVVD EQAFGIKVRSILCCMRHQPSWSLEVLELCKKYNQKTVVAMDLAGDETIEGSSLFPGHVEAY RTVHAGEVGSPEVVREAVDILKTERVGHGYHTIEDEALYNRLLKENMHFEVCPWSSYLTGA VRFKNDKANYSLNTDDPLIFKSTLDTDYQMTKKDMGFTEEEFKRLNINAAKSSFLPEEEKK PSI-BLAST 0.005 E value cutoff for PSSM NCBI FieldGuide RESULTS: Initial BLASTP Same results as protein-protein BLAST; different format NCBI FieldGuide Results of First PSSM Search Other purine nucleotide metabolizing enzymes not found by ordinary BLAST NCBI FieldGuide Tenth PSSM Search: Convergence Just below threshold, another nucleotide metabolism enzyme Check to add to PSSM NCBI FieldGuide Reverse PSI-BLAST (RPS)-BLAST NCBI FieldGuide Adenosine/AMP Deaminase Domain AMP Deaminases . . . NCBI FieldGuide PHI-BLAST gi|231729|sp|P30429|CED4_CAEEL CELL DEATH PROTEIN 4 MLCEIECRALSTAHTRLIHDFEPRDALTYLEGKNIFTEDHSELISKMSTRLERIANFLRIYRRQASE LIDFFNYNNQSHLADFLEDYIDFAINEPDLLRPVVIAPQFSRQMLDRKLLLGNVPKQMTCYIREYHV IKKLDEMCDLDSFFLFLHGRAGSGKSVIASQALSKSDQLIGINYDSIVWLKDSGTAPKSTFDLFTDI LKSEDDLLNFPSVEHVTSVVLKRMICNALIDRPNTLFVFDDVVQEETIRWAQELRLRCLVTTRDVEI ASQTCEFIEVTSLEIDECYDFLEAYGMPMPVGEKEEDVLNKTIELSSGNPATLMMFFKSCEPKTFEK GAxxxxGKST NCBI FieldGuide Whats New? NCBI FieldGuide BLAST Databases Nucleotide refseq_rna = NM_*, XM_* refseq_genomic = NC_*, NG_* env_nt environmental samplefilter, e.g., 16S rRNA Protein refseq = NP_*, XP_* env_nr NCBI FieldGuide New Formatter Select lower caseSelect red NCBI FieldGuide New Formatter gray line = same database hit hsps color-coded independently NCBI FieldGuide BLAST Output: Alignments & Filter low complexity sequence filtered NCBI FieldGuide Advanced Options Limit to Organism protein allfilter N Example Entrez Queries proteins allFilter NOT mammaliaOrganism ray finned fishesOrganism srcdb refseqProperties Nucleotide only: biomol mrnaProperties biomol genomicProperties OtherAdvanced e 10000expect value -v 2000descriptions -b 2000alignments -e 10000 -v 2000 NCBI FieldGuide Genome BLAST Examples NCBI FieldGuide Example Search Pathways: Hemochromatosis Gene OMIMOMIMGeneGene “hemochromatosis” nucleotide sequence Genome BLAST Map Viewer SNP Protein Domains text search sequence search NCBI FieldGuide Sample NCBI FieldGuide Example: Human Genome BLAST TGCCTCCTTTGGTGAAGGTGACACATCATGTGACCTCTTCAGTGA CCACTCTACGGTGTCGGGCCTTGAACTACTACCCCCAGAAC ATCACCATGAAGTGGCTGAAGGATAAGCAGCCAATGGATGCCAAG GAGTTCGAACCTAAAGACGTATTGCCCAATGGGGATGGGAC CTACCAGGGCTGGATAACCTTGGCTGTACCCCCTGGGGAAGAGC Human EST NCBI FieldGuide Human Genome BLAST: Results NCBI FieldGuide Human Genome BLAST: MapViewer Entrez GeneEntrez Gene NCBI FieldGuide Map Oligos Onto Genome CCATGGCGACCCTGGAAAAGCNNNNNNNNNNCAGCAGCGGCTGTGCCTGCGG -W 7 e 1000 forward primerreverse primer NCBI FieldGuide Genome BLAST Results NCBI FieldGuide Primer Alignments forward primer reverse primer NCBI FieldGuide MapViewer NCBI FieldGuide MapViewer NCBI FieldGuide Sequence View (sv) forward reverse NCBI FieldGuide /refseq/LocusLink/LL_tmpl.gz A Few Good Files /refseq/release/genomic.fna.gz /repository/UniGene/Hs.seq.uniq.gz /pub/mmdb/cdd/ /toolbox/ncbi_tools/ncbi.tar.gz /gene/DATA/gene_info.gz NCBI FieldGuide Service Addresses BLAST General H Wayne Matten NCBI FieldGuide yf*Lr7XDk0Qwc$Ip5VBh)Nua#Gm2Tzf*Lr7YEk0Qwc%Jp5VBi-Oua#Gn3Tzf*Ls8YEk0Qxd%Jp5WCi-Oua!Hn3Tzf(Ms8YEl1Rxd%Jq6WCi-Ovb!Hn3TAg(Ms8ZFl1Rxd&Kq6WCi+Pvb!Ho4UAg(Mt9ZFl1Rye&Kq6WDj+Pvb$Io4UAg)Nt9ZFl2Sye&Kr7XDj+Pwc$Io4UBh)Nt9#Gm2Sye*Lr7XDj0Qwc$Io5VBh)Nua#Gm2Szf*Lr7XEk0Qwc%Jp5VBh-Oua#Gm3Tzf*Lr8YEk0Qxd%Jp5VCi-Oua#Hn3Tzf(Ms8YEk1Rxd%Jp6WCi- Oub!Hn3TAg(Ms8YFl1Rxd%Kq6WCi+Pvb!Hn4UAg(Ms9ZFl1Rxe&Kq6WDj+Pvb!Io4UAg(Nt9ZFl2Sye&Kq7XDj+Pvc$Io4UBh)Nt9ZGm2Sye&Lr7XDj+Qwc$Io5VBh)Nta#Gm2Syf*Lr7XEk0Qwc$Jp5VBh)Oua#Gm2Tzf*Lr8YEk0Qwd%Jp5VBi-Oua#Hn3Tzf*Ms8YEk0Rxd%Jp5WCi-Oub!Hn3Tzg(Ms8YEl1Rxd%Kq6WCi-Pvb!Hn3UAg(Ms8ZFl1Rxe&Kq6WCj+Pvb!Ho4UAg(Nt9ZFl1Sye&Kq6XDj+Pvc$Io4UAh)Nt9ZFm2Sye&Kr7XDj+Qwc$Io4VBh)Nt9#Gm2Syf*Lr7XDk0Qwc$Ip5VBh)Nua#Gm2Tzf*Lr7YEk0Qwc%Jp5VBi-Oua#Gn3Tzf*Ls8YEk0Qxd%Jp5WCi- Oua!Hn3Tzf(Ms8YEl1Rxd%Jq6WCi-Ovb!Hn3UAg(Ms8ZFl1Rxd&Kq6WCi+Pvb!Ho4UAg(Mt9ZFl1Rye&Kq6XDj+Pvb$Io4UAg)Nt9ZFl2Sye&Kr7XDj+Pwc$Io4UBh)Nt9#Gm2Sye*Lr7XDj0Qwc$Io5VBh)Nua#Gm2Szf*Lr7XEk0Qwc%Jp5VBh-Oua#Gm3Tzf*Lr8YEk0Qxd%Jp5VCi-Oua#Hn3Tzf(Ms8YEk1Rxd%Jp6WCi-Ovb!Hn3TAg(Ms8YFl1Rxd%Kq6WCi+Pvb!Hn4UAg(Ms9ZFl1Rye&Kq6WDj+Pvb!Io4UAg(Nt9ZFl2Sye&Kq7XDj+Pvc$Io4UBh)Nt9ZGm2Sye&Lr7XDj+Qwc$Io5VBh)Nta#Gm2Syf*Lr7XEk0Qwc$Jp5VBh)Oua#Gm2Tzf*Lr8YEk0Qwd%Jp5VBi- Oua#Hn3Tzf*Ms8YEk0Rxd%Jp6WCi-Oub!Hn3Tzg(Ms8YEl1Rxd%Kq6WCi-Pvb!Hn3UAg(Ms9ZFl1Rxe&Kq6WCj+Pvb!Ho4UAg(Nt9ZFl1Sye&Kq6XDj+Pvc$Io4UAh)Nt9ZFm2Sye&Kr7XDj+Qwc$Io4VBh)Nt9#Gm2Syf*Lr7XDk0Qwc$Ip5VBh)Nua#Gm2Tzf*Lr7YEk0Qwc%Jp5VBi-Oua#Gn3Tzf*Ls8YEk0Rxd%Jp5WCi-Oua!Hn3Tzf(Ms8YEl1Rxd%