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SUCEST SUCEST: Felipe Rodrigues da Silva Embrapa Recursos Genéticos e Biotecnologia o projeto genoma da cana-de-açúcar.

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Apresentação em tema: "SUCEST SUCEST: Felipe Rodrigues da Silva Embrapa Recursos Genéticos e Biotecnologia o projeto genoma da cana-de-açúcar."— Transcrição da apresentação:

1 SUCEST SUCEST: Felipe Rodrigues da Silva Embrapa Recursos Genéticos e Biotecnologia o projeto genoma da cana-de-açúcar.

2 SUCEST Volume de dados disponíveis publicamente

3 SUCEST Volume de dados disponíveis publicamente

4 SUCEST Genomas completos de organismos

5 SUCEST

6

7 SOPÃO de letrinhas... A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C A A A A A A A A A A T T T T T T T T T T G G G G G G C C C C C C C C C C C A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C A A A A A A A A A T T T T T T T T T G G G G G G G G C C C C C C C C C A A A A A A A A A A T T T T T T T T T T T G G G G G G G G G C C C C C C C C C C C

8 SUCEST Cana-de-açúcar Cultivada em mais de 90 países Ocupando cerca de 20 milhões de hectares Família das Gramíneas (Poace)

9 SUCEST A cana-de-açúcar no Brasil 25% da produção mundial 300 milhões de tons. 5 milhões de hectares plantados 14.5 milhões de tons. de açúcar 15.3 bilhões de litros de álcool 350 industrias 50 mil produtores 1.4 milhões de empregos direto 3.6 milhões de empregos indiretos

10 SUCEST Origem e tamanho Saccharum officinarum 2n = 80 Saccharum spontaneum 2n = 64 ou 2n = – 25% X S. berberi, S. sinence, S. robustum conjunto não-reduntante = 930 Mbp Sorgo = 760 Mbp Arroz = 430 Mbp 2C = Mbp2n = D'Hont, A. and Glaszmann, J. C Proc Int Soc Sugarcane Technol 24:

11 SUCEST Projeto Genoma Seqüenciamento Completo do Genoma –Região Gênica e Região Intergênica Estrutural Funcional EST – Expressed Sequence Tag –Regiões que codificam proteínas (Genes)

12 SUCEST Seqüenciamento Completo...ATGTTGGGCCACAGTTGACCATTGAAACTG GTTGACCATTGAAACTGACCTTGACGTAACGTGGTA.... Genomic DNA Biblioteca de BACs Mapa físico BAC a ser seqüenciado Clones Shotgun Seqüência...ATGTTGGGCCACAGTTGACCATTGAAACTGACCTTGACGTAACGTGGTA... Montagem

13 SUCEST EST – Expressed Sequence Tag Clonagem em E. coliSeqüenciamento ACCTGATGGCATTTCCATCAAGCTGACCTGGAAATCGTTGGCC DNA gene Bgene A Proteína NH 2 COOH NH 2 COOH 3´5´ mRNA Dogma Central da Biologia inserção em vetor cDNA

14 SUCEST

15 Total de Entradas 1,528,715 Homo sapiens 967,015 (63,4%) Plantas (total) 73,087 (4.8%) Mus musculus + domesticus (camundongo)306,544 Caenorhabditis elegans 72,521 Arabidopsis thaliana 36,173 Drosophila melanogaster 27,625 Oryza sativa (arroz) 25,844 Rattus sp. (rato) 20,311 Brugia malayi (nematoide parasita) 13,641 Toxoplasma gondii 10,671 Emericella nidulans 5,787 Schistosoma mansoni 3,659 Trypanosoma brucei rhodesiense 3,519 Danio rerio (zebrafish) 3,373 Saccharomyces cerevisiae 3,042 Zea mays (milho) 1,783 Leishmania major 1,692 Saccharum sp. 495 Outros ~ 20,000 GenBank - dbEST Março de 1998

16 SUCEST Os Objetivos do projeto SUCEST Identificar genes únicos (ou seqüenciar ESTs) Desenvolver um Banco de Dados para a cana-de-açúcar Disponibiilizar este Banco de Dados para grupos de Data Mining Análise funcional dos ESTs

17 SUCEST O Cronograma Data Meta Jul/1999 Distribuição dos Primeiros Clones Dec/ ,000 ESTs Jul/ ,000 ESTs Dec/ ,000 ESTs Jul/ ,000 ESTs Dec/ ,000 ESTs Jul/ ,000 ESTs Dec/ ,000 ESTs Jul/ ,000 ESTs

18 SUCEST As Bibliotecas de cDNA Tecidos / Órgãos –Raiz –Meristema –Caule –Sementes –Flores –Cartucho da Folha –Zona de Transição Folha-Raiz –Gema Lateral –Calli –Plântulas imaturas –Plântulas infectadas com Herbaspirillum rubrisubalbicans –Plântulas infectadas com Gluconacetobacter diazotroficans Variedades –SP –SP –SP –RB –RB –PB5211 X P

19 SUCEST Os Laboratórios de Seqüenciamento USP (SP) (3) BIOINFORMATICA UNICAMP (CA) UNESP (BT) (2) ESALQ (PI) (3) USP (RP) (1) UNAERP (RP) (1) UMC (MC) (1) UNIVAP (SJ) (1) UNESP (JB) (2) UFSCAR (AR) (1) UFSCAR (SC) (1) USP (SC) (1) UNESP (RC) (1) IAC (CA) (1) IAC (CO) (1) UNICAMP (CA) (1) RIO DE JANEIROPERNAMBUCOALAGOAS ABI

20 SUCEST EST – Expressed Sequence Tag Clonagem em E. coliSeqüenciamento ACCTGATGGCATTTCCATCAAGCTGACCTGGAAATCGTTGGCC DNA gene Bgene A Proteína NH 2 COOH NH 2 COOH 3´5´ mRNA Dogma Central da Biologia inserção em vetor cDNA reads clones clones

21 SUCEST Limpeza das seqüências remoção de seqüências ribossômicas remoção de seqüências de vetor remoção da região de poliA corte por qualidade eliminação das derrapagens

22 SUCEST poliA AGGGGAGAATTTATGATCCCCTAGTACACCCGGCAGGACCGGTCCGGAATTCCCCGGTCGACCCAC GCGTCCGCTACAACAACAGCAGCAGCTTCCATTTACCTTGTCGGCTGTTGCAACCGCTGCTGCCTA CCACCAGCAACTACAGCTGCTACCAGTTAACCCATTGGCACTGGCTAACCCATTGGCTGCTGCCTT CCTGCAGCAGCAACAATTGCTGCCATTCAACCAGATGTCTTTGATGAACCCTGCCTTGTCGTGGTA GCAACCCATCGTTGGAGGTGCCATCTTCTAGAATACAAATGAGTTGTACTTGATAACAATGTTCTT GTGTCGGCGTGTGCAACTTCCCAGAAATAATCAATACATTGATTGAGATTTANAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAATATAATTAAAATAAAAAAATTTATAAAAAAAAAAAAATAATT TTTTTTTATAAAAAATAAATATAAAATAAAAAGGGGGGGCCGTTTTAAAGGAACAAAGTTTAAGAC CGGGGGTATGAAAGGGAAAATTTTTTTATATAGGGCCCCAAAATTAAATACATGGGCCGGTGTTAA CAACGGCGGGAGGGAAAAAACCTGGGGGTTACCAATTTAAAGCCGTGGAAAAAATCCCTTTTTTCA AGTGGGGTAAAAAGAAAAGGCCCCACCCATCGCCCTTCCAAAAATTGCCCCCCTTAAAGGAAAAAG GACACCCCCTTTTGGGCGCATATAACCGGGGGGGTGGGGGTACCCCCAAGGGAACTTATATTTTTC AGGCCTCATAGCCCTTTTTTTTTTTTTTTTTTTTTTTTTCAAGGTAGCGGGTTTCCCAGGAAAATT AAAAGGGGGGTCCTTTTGGGTAATAATGTTTTN

23 SUCEST poliA AGGGGAGAATTTATGATCCCCTAGTACACCCGGCAGGACCGGTCCGGAATTCCCCGGTCGACCCAC GCGTCCGCTACAACAACAGCAGCAGCTTCCATTTACCTTGTCGGCTGTTGCAACCGCTGCTGCCTA CCACCAGCAACTACAGCTGCTACCAGTTAACCCATTGGCACTGGCTAACCCATTGGCTGCTGCCTT CCTGCAGCAGCAACAATTGCTGCCATTCAACCAGATGTCTTTGATGAACCCTGCCTTGTCGTGGTA GCAACCCATCGTTGGAGGTGCCATCTTCTAGAATACAAATGAGTTGTACTTGATAACAATGTTCTT GTGTCGGCGTGTGCAACTTCCCAGAAATAATCAATACATTGATTGAGATTTANAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAATATAATTAAAATAAAAAAATTTATAAAAAAAAAAAAATAATT TTTTTTTATAAAAAATAAATATAAAATAAAAAGGGGGGGCCGTTTTAAAGGAACAAAGTTTAAGAC CGGGGGTATGAAAGGGAAAATTTTTTTATATAGGGCCCCAAAATTAAATACATGGGCCGGTGTTAA CAACGGCGGGAGGGAAAAAACCTGGGGGTTACCAATTTAAAGCCGTGGAAAAAATCCCTTTTTTCA AGTGGGGTAAAAAGAAAAGGCCCCACCCATCGCCCTTCCAAAAATTGCCCCCCTTAAAGGAAAAAG GACACCCCCTTTTGGGCGCATATAACCGGGGGGGTGGGGGTACCCCCAAGGGAACTTATATTTTTC AGGCCTCATAGCCCTTTTTTTTTTTTTTTTTTTTTTTTTCAAGGTAGCGGGTTTCCCAGGAAAATT AAAAGGGGGGTCCTTTTGGGTAATAATGTTTTN

24 SUCEST Quality trimming CGGAAGACTGGAGTCGTCGCTGCGGCACCGGTCCGGAATTCCCGGGTCGACCCACGCGTCCGGCCG CCGCCACCGCATCCCTTGCAGCCCCAATCCCCCACGGCGACCATGGCCGGCGCGCAGGAGTCCCTG TCCCTGGTGGGCACGATGCGTGGCCACAACGGCGAGGTGACGGCGATCGCCACCCCGATCGACAAC TCGCCGTTCATCGTCTCCTCCTCCCGCGACAAGTCCGTGCTGGTGTGGGACCTGCAAAACCCGGTC CACTCCACCCCGGAATCCGGCGCCACCGCCGACTACGGCGTCCCCTTCCGCCGCCTCACCGGCCAC TCCCACTTCGTCCAGGACGTCGTCCTCAGCTCCGACGGCCAGTTCGCCCTCTCCGGCTCCTGGGAC GGCGAGCTCCGCCTCTGGGACCTCTCCACCGGCGTCACCACCCGCCGCTTCGTCGGCCACGAGAAG GACGTCCTCTCCGTCGCCTTCTCCGTCGACAACCGCCAGATCGTCTCCGCGTCCCGCGACAAGACC ATCAAGCTCTGGAACACCCTCGGTGAGTGCAAGTACACCATTGGTGGCGACCTCGGCGGCGGGGAG GGCCACAACGGGTGGGTCTCCTGCGTCAGGTTCTTCCCCAACACCTTTCAGGCCACCATTGTCTCC GGATTCTGGGACCGCACCGTCAGGTCTGGAACCTTACCAACTGCAAGCTGCGATGCACTCTCGATG CCCACGCGGCTATGTTAACGCCGTCGCC ACGTX: <10 ACGTX: >=10 and <15 ACGTX: >=15 and <20 ACGTX: >=20 and <25 ACGTX: >=25 and <30 ACGTX: >= bases

25 SUCEST Quality trimming CGGAAGACTGGAGTCGTCGCTGCGGCACCGGTCCGGAATTCCCGGGTCGACCCACGCGTCCGGCCG CCGCCACCGCATCCCTTGCAGCCCCAATCCCCCACGGCGACCATGGCCGGCGCGCAGGAGTCCCTG TCCCTGGTGGGCACGATGCGTGGCCACAACGGCGAGGTGACGGCGATCGCCACCCCGATCGACAAC TCGCCGTTCATCGTCTCCTCCTCCCGCGACAAGTCCGTGCTGGTGTGGGACCTGCAAAACCCGGTC CACTCCACCCCGGAATCCGGCGCCACCGCCGACTACGGCGTCCCCTTCCGCCGCCTCACCGGCCAC TCCCACTTCGTCCAGGACGTCGTCCTCAGCTCCGACGGCCAGTTCGCCCTCTCCGGCTCCTGGGAC GGCGAGCTCCGCCTCTGGGACCTCTCCACCGGCGTCACCACCCGCCGCTTCGTCGGCCACGAGAAG GACGTCCTCTCCGTCGCCTTCTCCGTCGACAACCGCCAGATCGTCTCCGCGTCCCGCGACAAGACC ATCAAGCTCTGGAACACCCTCGGTGAGTGCAAGTACACCATTGGTGGCGACCTCGGCGGCGGGGAG GGCCACAACGGGTGGGTCTCCTGCGTCAGGTTCTTCCCCAACACCTTTCAGGCCACCATTGTCTCC GGATTCTGGGACCGCACCGTCAGGTCTGGAACCTTACCAACTGCAAGCTGCGATGCACTCTCGATG CCCACGCGGCTATGTTAACGCCGTCGC ACGTX: <10 ACGTX: >=10 and <15 ACGTX: >=15 and <20 ACGTX: >=20 and <25 ACGTX: >=25 and <30 ACGTX: >= bases

26 SUCEST Quality trimming CGGAAGACTGGAGTCGTCGCTGCGGCACCGGTCCGGAATTCCCGGGTCGACCCACGCGTCCGGCCG CCGCCACCGCATCCCTTGCAGCCCCAATCCCCCACGGCGACCATGGCCGGCGCGCAGGAGTCCCTG TCCCTGGTGGGCACGATGCGTGGCCACAACGGCGAGGTGACGGCGATCGCCACCCCGATCGACAAC TCGCCGTTCATCGTCTCCTCCTCCCGCGACAAGTCCGTGCTGGTGTGGGACCTGCAAAACCCGGTC CACTCCACCCCGGAATCCGGCGCCACCGCCGACTACGGCGTCCCCTTCCGCCGCCTCACCGGCCAC TCCCACTTCGTCCAGGACGTCGTCCTCAGCTCCGACGGCCAGTTCGCCCTCTCCGGCTCCTGGGAC GGCGAGCTCCGCCTCTGGGACCTCTCCACCGGCGTCACCACCCGCCGCTTCGTCGGCCACGAGAAG GACGTCCTCTCCGTCGCCTTCTCCGTCGACAACCGCCAGATCGTCTCCGCGTCCCGCGACAAGACC ATCAAGCTCTGGAACACCCTCGGTGAGTGCAAGTACACCATTGGTGGCGACCTCGGCGGCGGGGAG GGCCACAACGGGTGGGTCTCCTGCGT ACGTX: <10 ACGTX: >=10 and <15 ACGTX: >=15 and <20 ACGTX: >=20 and <25 ACGTX: >=25 and <30 ACGTX: >= bases

27 SUCEST Resultado de blastX >gi| |sp|P49027|GBLP_ORYSA GUANINE NUCLEOTIDE-BINDING PROTEIN BETA SUBUNIT-LIKE PROTEIN (GPB-LR) (RWD) pir||T03764 protein RWD - rice dbj|BAA | (D38231) RWD [Oryza sativa] Length = 334 Score = 315 bits (798), Expect = 4e-85 Identities = 150/170 (88%), Positives = 156/170 (91%) Frame = +1 Query: 109 MAGAQESLSLVGTMRGHNGEVTAIATPIDNSPFIVSSSRDKSVLVWDLQNPVHSTPESGA 288 MAGAQESL L G M GHN VTAIATPIDNSPFIVSSSRDKS+LVWDL NPV + E Sbjct: 1 MAGAQESLVLAGVMHGHNDVVTAIATPIDNSPFIVSSSRDKSLLVWDLTNPVQNVGEGAG 60 Query: 289 TADYGVPFRRLTGHSHFVQDVVLSSDGQFALSGSWDGELRLWDLSTGVTTRRFVGHEKDV YGVPFRRLTGHSHFVQDVVLSSDGQFALSGSWDGELRLWDLSTGVTTRRFVGH+KDV Sbjct: 61 ASEYGVPFRRLTGHSHFVQDVVLSSDGQFALSGSWDGELRLWDLSTGVTTRRFVGHDKDV 120 Query: 469 LSVAFSVDNRQIVSASRDKTIKLWNTLGECKYTIGGDLGGGEGHNGWVSC 618 LSVAFSVDNRQIVSASRD+TIKLWNTLGECKYTIGGDLGGGEGHNGWVSC Sbjct: 121 LSVAFSVDNRQIVSASRDRTIKLWNTLGECKYTIGGDLGGGEGHNGWVSC 170 read trimmado

28 SUCEST Resultado de blastX >gi| |sp|P49027|GBLP_ORYSA GUANINE NUCLEOTIDE-BINDING PROTEIN BETA SUBUNIT-LIKE PROTEIN (GPB-LR) (RWD) pir||T03764 protein RWD - rice dbj|BAA | (D38231) RWD [Oryza sativa] Length = 334 Score = 352 bits (893), Expect(2) = e-100 Identities = 168/192 (87%), Positives = 175/192 (90%) Frame = +1 Query: 109 MAGAQESLSLVGTMRGHNGEVTAIATPIDNSPFIVSSSRDKSVLVWDLQNPVHSTPESGA 288 MAGAQESL L G M GHN VTAIATPIDNSPFIVSSSRDKS+LVWDL NPV + E Sbjct: 1 MAGAQESLVLAGVMHGHNDVVTAIATPIDNSPFIVSSSRDKSLLVWDLTNPVQNVGEGAG 60 Query: 289 TADYGVPFRRLTGHSHFVQDVVLSSDGQFALSGSWDGELRLWDLSTGVTTRRFVGHEKDV YGVPFRRLTGHSHFVQDVVLSSDGQFALSGSWDGELRLWDLSTGVTTRRFVGH+KDV Sbjct: 61 ASEYGVPFRRLTGHSHFVQDVVLSSDGQFALSGSWDGELRLWDLSTGVTTRRFVGHDKDV 120 Query: 469 LSVAFSVDNRQIVSASRDKTIKLWNTLGECKYTIGGDLGGGEGHNGWVSCVRFFPNTFQA 648 LSVAFSVDNRQIVSASRD+TIKLWNTLGECKYTIGGDLGGGEGHNGWVSCVRF PNTFQ Sbjct: 121 LSVAFSVDNRQIVSASRDRTIKLWNTLGECKYTIGGDLGGGEGHNGWVSCVRFSPNTFQP 180 Query: 649 TIVSGFWDRTVR 684 TIVSG WDRTV+ Sbjct: 181 TIVSGSWDRTVK 192 read inteiro

29 SUCEST Determinação do limiar de qualidade

30 SUCEST Quality trimming CGGAAGACTGGAGTCGTCGCTGCGGCACCGGTCCGGAATTCCCGGGTCGACCCACGCGTCCGGCCG CCGCCACCGCATCCCTTGCAGCCCCAATCCCCCACGGCGACCATGGCCGGCGCGCAGGAGTCCCTG TCCCTGGTGGGCACGATGCGTGGCCACAACGGCGAGGTGACGGCGATCGCCACCCCGATCGACAAC TCGCCGTTCATCGTCTCCTCCTCCCGCGACAAGTCCGTGCTGGTGTGGGACCTGCAAAACCCGGTC CACTCCACCCCGGAATCCGGCGCCACCGCCGACTACGGCGTCCCCTTCCGCCGCCTCACCGGCCAC TCCCACTTCGTCCAGGACGTCGTCCTCAGCTCCGACGGCCAGTTCGCCCTCTCCGGCTCCTGGGAC GGCGAGCTCCGCCTCTGGGACCTCTCCACCGGCGTCACCACCCGCCGCTTCGTCGGCCACGAGAAG GACGTCCTCTCCGTCGCCTTCTCCGTCGACAACCGCCAGATCGTCTCCGCGTCCCGCGACAAGACC ATCAAGCTCTGGAACACCCTCGGTGAGTGCAAGTACACCATTGGTGGCGACCTCGGCGGCGGGGAG GGCCACAACGGGTGGGTCTCCTGCGTCAGGTTCTTCCCCAACACCTTTCAGGCCACCATTGTCTCC GGATTCTGGGACCGCACCGTCAGGTCTGGAACCTTACCAACTGCAAGCTGCGATGCACTCTCGATG CCCACGCGGCTATGTTAACGCCGTCGCC ACGTX: <10 ACGTX: >=10 and <15 ACGTX: >=15 and <20 ACGTX: >=20 and <25 ACGTX: >=25 and <30 ACGTX: >= bases

31 SUCEST Quality trimming CGGAAGACTGGAGTCGTCGCTGCGGCACCGGTCCGGAATTCCCGGGTCGACCCACGCGTCCGGCCG CCGCCACCGCATCCCTTGCAGCCCCAATCCCCCACGGCGACCATGGCCGGCGCGCAGGAGTCCCTG TCCCTGGTGGGCACGATGCGTGGCCACAACGGCGAGGTGACGGCGATCGCCACCCCGATCGACAAC TCGCCGTTCATCGTCTCCTCCTCCCGCGACAAGTCCGTGCTGGTGTGGGACCTGCAAAACCCGGTC CACTCCACCCCGGAATCCGGCGCCACCGCCGACTACGGCGTCCCCTTCCGCCGCCTCACCGGCCAC TCCCACTTCGTCCAGGACGTCGTCCTCAGCTCCGACGGCCAGTTCGCCCTCTCCGGCTCCTGGGAC GGCGAGCTCCGCCTCTGGGACCTCTCCACCGGCGTCACCACCCGCCGCTTCGTCGGCCACGAGAAG GACGTCCTCTCCGTCGCCTTCTCCGTCGACAACCGCCAGATCGTCTCCGCGTCCCGCGACAAGACC ATCAAGCTCTGGAACACCCTCGGTGAGTGCAAGTACACCATTGGTGGCGACCTCGGCGGCGGGGAG GGCCACAACGGGTGGGTCTCCTGCGT ACGTX: <10 ACGTX: >=10 and <15 ACGTX: >=15 and <20 ACGTX: >=20 and <25 ACGTX: >=25 and <30 ACGTX: >= bases

32 SUCEST Quality trimming CGGAAGACTGGAGTCGTCGCTGCGGCACCGGTCCGGAATTCCCGGGTCGACCCACGCGTCCGGCCG CCGCCACCGCATCCCTTGCAGCCCCAATCCCCCACGGCGACCATGGCCGGCGCGCAGGAGTCCCTG TCCCTGGTGGGCACGATGCGTGGCCACAACGGCGAGGTGACGGCGATCGCCACCCCGATCGACAAC TCGCCGTTCATCGTCTCCTCCTCCCGCGACAAGTCCGTGCTGGTGTGGGACCTGCAAAACCCGGTC CACTCCACCCCGGAATCCGGCGCCACCGCCGACTACGGCGTCCCCTTCCGCCGCCTCACCGGCCAC TCCCACTTCGTCCAGGACGTCGTCCTCAGCTCCGACGGCCAGTTCGCCCTCTCCGGCTCCTGGGAC GGCGAGCTCCGCCTCTGGGACCTCTCCACCGGCGTCACCACCCGCCGCTTCGTCGGCCACGAGAAG GACGTCCTCTCCGTCGCCTTCTCCGTCGACAACCGCCAGATCGTCTCCGCGTCCCGCGACAAGACC ATCAAGCTCTGGAACACCCTCGGTGAGTGCAAGTACACCATTGGTGGCGACCTCGGCGGCGGGGAG GGCCACAACGGGTGGGTCTCCTGCGTCAGGTTCTTCCCCAACACCTTTCAGGCCACCATTGTCTCC GGATTCTGGGACCGCACCGTCAGGTCTGGAACCTTACCAACTGCAAGCTGCGATGCACTCTCGATG CCCACGCGGCTATGTTAACGCCGTCGCC ACGTX: <10 ACGTX: >=10 and <15 ACGTX: >=15 and <20 ACGTX: >=20 and <25 ACGTX: >=25 and <30 ACGTX: >=30 base 684

33 SUCEST Quality trimming CGGAAGACTGGAGTCGTCGCTGCGGCACCGGTCCGGAATTCCCGGGTCGACCCACGCGTCCGGCCG CCGCCACCGCATCCCTTGCAGCCCCAATCCCCCACGGCGACCATGGCCGGCGCGCAGGAGTCCCTG TCCCTGGTGGGCACGATGCGTGGCCACAACGGCGAGGTGACGGCGATCGCCACCCCGATCGACAAC TCGCCGTTCATCGTCTCCTCCTCCCGCGACAAGTCCGTGCTGGTGTGGGACCTGCAAAACCCGGTC CACTCCACCCCGGAATCCGGCGCCACCGCCGACTACGGCGTCCCCTTCCGCCGCCTCACCGGCCAC TCCCACTTCGTCCAGGACGTCGTCCTCAGCTCCGACGGCCAGTTCGCCCTCTCCGGCTCCTGGGAC GGCGAGCTCCGCCTCTGGGACCTCTCCACCGGCGTCACCACCCGCCGCTTCGTCGGCCACGAGAAG GACGTCCTCTCCGTCGCCTTCTCCGTCGACAACCGCCAGATCGTCTCCGCGTCCCGCGACAAGACC ATCAAGCTCTGGAACACCCTCGGTGAGTGCAAGTACACCATTGGTGGCGACCTCGGCGGCGGGGAG GGCCACAACGGGTGGGTCTCCTGCGTCAGGTTCTTCCCCAACACCTTTCAGGCCACCATTGTCTCC GGATTCTGGGACCGCACCGTCAGGTCTGGAACCTTACCAACTGCAAGCTGCGATGCACTCTCGATG CCCACGCGGCTATGTTAACGCCGTCGCC 719 bases antesdif.homol.dif.depois

34 SUCEST Determinação do limiar de qualidade

35 SUCEST Exemplo de derrapagem

36 SUCEST todos os reads 291,689 reads ±186.3 comprimento médio ±161.3 # médio bases >= 20/read 283,216 reads remoção de ribossômicos 283,216 reads busca de vetores 275,436 reads corte de vector + poliA 273,728 reads corte por qualidade 273,728 reads corte de vetores em extremidade 258,107 reads corte de derrapagens 256,101 reads corte de poliA em extremidade reads trimados 237,954 reads ±139.8 avg. read size ±120.1 avg bases >= 20/read remoção de seqs de baixa qualidade

37 SUCEST cluster size (reads)HSXphrapXCAP3X HS total common >= total

38 SUCEST Discrepância interna

39 SUCEST Discrepância interna

40 SUCEST Teste de consistência interna

41 SUCEST Teste de consistência interna

42 SUCEST Teste de consistência interna

43 SUCEST Teste de consistência externa

44 SUCEST Teste de consistência externa

45 SUCEST Teste de consistência externa

46 SUCEST Números totais Total sequences 291,689 cDNA clones sequenced (5or 3) 260,352 5 end sequences 259,325 3 end sequences 32,364 Total high-quality sequences 237,954 Success index (%) 81.6 Average insert size (bp) 1,250 Average sequence size (bp) 864 / 642 Bases with phred quality = 20/read 399

47 SUCEST Números totais Trotal sequences analyzed 237,954 Number of contigs 26,803 Number of singletons 16,338 Number of sugarcane assembled sequences (SAS) 43,141 Number of assembled sequences matching to known genes 27,833 (64.5%) Number of clones with full length inserts 14,409 (

48 SUCEST Contribuição específica por biblioteca Número de ESTsSAScontigssingletons% contribuição AD18,1371,4741, AM15, AM26, CL63, FL18,4121,7531, FL35, FL47,2891, FL55, FL83, HR15, LB13, LB25, LR16, LR22, LV13, RT14, RT25, RT34, RZ12, RZ23, RZ36, SB17,4071,3131, SD14, SD24, ST14, ST34, % dos SAS são formados por reads oriúndos de uma única biblioteca 38% dos SAS tecido-especícos são singletons

49 SUCEST Classificação funcional

50 SUCEST Porcentagem por órgão

51 SUCEST SAS tecido- específicas Número de ESTs Melhor hit biblioteca 360 (Y17556) alpha kafirin [Sorghum bicolor] SD 103 (A23207) zein zA1 [Zea mays] SD 42 (AF232008) beta-glucosidase aggregating factor precursor [Zea mays] RT 24 (AC007789) putative low molecular early light-inducible protein [Oryza sativa] SD 22 (AP002820) putative peroxidase [Oryza sativa] RT 19 (X56337) alpha-amylase [Oryza sativa] CL 18 (AP000374) cyclopropane fatty acid synthase [Arabidopsis thaliana] FL

52 SUCEST Total de Entradas 1,528,715 Homo sapiens 967,015 (63,4%) Plantas (total) 73,087 (4.8%) Mus musculus + domesticus (camundongo)306,544 Caenorhabditis elegans 72,521 Arabidopsis thaliana 36,173 Drosophila melanogaster 27,625 Oryza sativa (arroz) 25,844 Rattus sp. (rato) 20,311 Brugia malayi (nematoide parasita) 13,641 Toxoplasma gondii 10,671 Emericella nidulans 5,787 Schistosoma mansoni 3,659 Trypanosoma brucei rhodesiense 3,519 Danio rerio (zebrafish) 3,373 Saccharomyces cerevisiae 3,042 Zea mays (milho) 1,783 Leishmania major 1,692 Saccharum sp. 495 Outros ~ 20,000 GenBank - dbEST Março de 1998

53 SUCEST Total de Entradas 7,692,809 Homo sapiens 3,369,459 (43.8%) Plantas (total) 1,099,102 (14.3 %) Glycine max (soja)160,500 Arabidopsis thaliana113,000 Medicago truncatula (barrel medic)112,458 Lycopersicon esculentum (tomate) 107,226 Zea mays (milho) 86,999 Oryza sativa (arroz) 72,657 Hordeum vulgare (cevada) 68,480 Chlamydomonas reinhardtii 64,973 Sorghum bicolor 62,642 Triticum aestivum (trigo) 58,141 Pinus taeda (loblolly pine) 34,896 Lotus japonicus 27,078 Solanum tuberosum (batata) 26,177 Gossypium arboreum 20,978 Sorghum propinquum 17,974 Mesembryanthemum (ice plant) 14,033 Gossypium hirsutum (algodão) 9,438 Secale cereale 8,123 Saccharum sp. 495 Outras Plantas (67 spp.) GenBank - dbEST Março de 2001

54 SUCEST Total de Entradas 12,845,578 Homo sapiens 4,691,979 (36.5%) Plantas (total) 2,279,170 (17.4 %) Glycine max (soja)284,714 Triticum aestivum (trigo) 256,593 Hordeum vulgare (cevada) 240,882 Zea mays (milho)180,587 Arabidopsis thaliana 174,624 Medicago truncatula (barrel medic) 170,500 Lycopersicon esculentum (tomate) 148,346 Chlamydomonas reinhardtii 130,324 Oryza sativa (arroz) 108,429 Solanum tuberosum (batata) 94,420 Sorghum bicolor 84,712 Lactuca sativa (alface) 68,188 Pinus taeda (loblolly pine) 60,226 Physcomitrella patens 50,250 Helianthus annuus (girassol) 44,961 Gossypium arboreum (algodão) 38,894 Lotus japonicus 32,096 Sorghum propinquum 21,387 Saccharum sp. 495 Outras Plantas (78 spp.) GenBank - dbEST Setembro de 2002

55 SUCEST Genetics and Molecular Biology 1.The libraries that made SUCEST 2.Bioinformatics of the sugarcane EST project 3.Trimming and clustering sugarcane ESTs 4.The sugarcane signal transduction (SUCAST) catalogue: prospecting signal transduction in sugarcane 5.In silico characterization and expression analyses of sugarcane putative sucrose non-fermenting-1 (SNF1) related kinases 6.Identification of like protein in sugarcane (Saccharum officinarum) 7.A search for homologues of plant photoreceptor genes and their signaling partners in the sugarcane expressed sequence tag (Sucest) database 8.Phylogenetic relationships between Arabidopsis and sugarcane bZIP transcriptional regulatory factors 9.Identification of sugarcane cDNAs encoding components of the cell cycle machinery 10.Dissecting the sugarcane expressed sequence tag (SUCEST) database: unraveling flower-specific genes 11.Molecular chaperone genes in the sugarcane expressed sequence database (SUCEST) 12.Oxidative stress response in sugarcane 13.In silico differential display of defense-related expressed sequence tags from sugarcane tissues infected with diazotrophic endophytes 14.Mechanisms of sugarcane response to herbivory 15.Base excision repair in sugarcane 19.Preliminary analysis of microsatellite markers derived from sugarcane expressed sequence tags (ESTs) 20.Sequence polymorphism from EST data in sugarcane: a fine analysis of 6-phosphogluconate dehydrogenase genes 21.A search for markers of sugarcane evolution 22.Sugarcane genes related to mitochondrial function 23.Mitochondrial and chloroplast localization of FtsH-like proteins in sugarcane based on their phylogenetic profile 24.Patterns of expression of cell wall related genes in sugacane 25.Expression of sugarcane genes induced by inoculation with Gluconacetobacter diazotrophicus and Herbaspirillum rubrisubalbicans 26.Identifying sugarcane expressed sequences associated with nutrient transporters and peptide metal chelators 27.Prospecting sugarcane genes involved in aluminum tolerance 28. N-glycosylation in sugarcane 29.Sugarcane expressed sequences tags (ESTs) encoding enzymes involved in lignin biosynthesis pathways 30.Biosynthesis of secondary metabolites in sugarcane 31.Identification of sugarcane genes involved in the purine synthesis pathway 32.A new member of the chalcone synthase (CHS) family in sugarcane 33.Classification. expression pattern and comparative analysis of sugarcane expressed analysis of sugarcane expressed sequences tags (ESTs) encoding glycine-rich proteins (GRPs) 34.Identification. classification and expression pattern analysis of sugarcane cysteine proteinases 35.Identification of metalloprotease gene families in sugarcane 36.Sugarcane phytocystatins: Identification. classification and expression pattern analysis 16.DNA repair-related genes in sugarcane expressed sequence tags (ESTs) 17.Distribution of DNA repair-related ESTs in sugarcane 18.Survey of transposable elements in sugarcane expressed sequence tags (ESTs)

56 SUCEST Genetics and Molecular Biology

57 SUCEST Grupo do SUCEST

58 SUCEST Uma parte do LBI

59 SUCEST Uma parte do LBI

60 SUCEST Os trimmadores

61 Grupo Genoma - CBMEG

62

63 SUCEST

64

65


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