serial analysis of gene expression - sage. aaaaaa tttttt aaaaaa tttttt serial analysis of gene...
TRANSCRIPT
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Serial Analysis of Gene Expression - SAGE
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AAAAAA
TTTTTT
AAAAAA
TTTTTT
Serial Analysis of Gene Expression - SAGE
AAAAAA
TTTTTTAAAAAA
TTTTTT
GTAC
GTAC
AAAAAA
TTTTTTAAAAAA
TTTTTT
GTAC
GTAC
CATG
CATG
AAAAAA
TTTTTTAAAAAA
TTTTTT
GTAC
GTAC
CATG
CATG
AGGYTGTACXXXXXXXXXXXXXXXTCCRACATGXXXXXXXXXXXXXXXXX
TE AE Tag
TCCRACATGXXXXXXXXXXXXXXXXXAGGYTGTACXXXXXXXXXXXXXXX
TE AE Tag
Clivagem com a enzima de ancoragem (AE) – Nla IIILigação às esferas de estreptoavidina
Dividir e ligar os“linkers” (A+B)
Clivar com a enzima para a produção das Tags (TE) Mme I - Gerar extremidades coesivas
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AGGYTGTACXXXXXXXXXXXXXXXTCCRACATGXXXXXXXXXXXXXXXXX
TE AE Tag
TCCRACATGXXXXXXXXXXXXXXXXXAGGYTGTACXXXXXXXXXXXXXXX
TE AE Tag
AGGYTGTACXXXXXXXXXXXXXXXTCCRACATGXXXXXXXXXXXXXXXXX XXXXXXXXXXXXXXXCATGTYGGA
XXXXXXXXXXXXXXXXXGTACARCCT
Ditag
Ligar e amplificar com os iniciadores A e B
----CATGXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXCATGXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXCATG--------GTACXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXGTACXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXGTAC----
AE Tag1 Tag2 AE Tag1 Tag2 AE
Ditag Ditag
Clivar com a enzima de ancoragemIsolar os “ditags”Concatenar e clonar
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TÉCNICA1) 2)
3)
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4)
5)
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6)
7) 8)
9)
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10)
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Contagem das Tags
Anotação das Tags
Análisedos dados
Experimento de SAGE
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A Bioinformática têm papel essencial para o SAGE em três funções básicas:
•Extração e gerenciamento dos dados•Anotação das tags•Análise estatística (distribuição e comparações)
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Extração das Tags
Dados estão no formato de cromatogramas
Software de Base Calling (Ex.: Phred) para gerar a sequência do concatâmero no formato texto com seu valor de qualidade
Extração e contagem das tags
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Contagem das Tags
Localizar CATG
Extração dos Ditags (20 – 34 pb)
Descartar ditags duplicadas
Obter as 10-17 bases extremas, sendo o complemento reverso a sequencia da direita
Descartar adaptadores (linkers) de sequências
Contagem das tags
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Exemplo
>SAGE-WT1-A0001-A01.abd 1047 0 1047 ABI
GGCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGCCCTCTAATGCATGTTGACGTGCACTTCCGTAGCCTCATGTTTTATGGAATCACCTATTATGCCATGACTTTTTCAAAACTAGGCTGTGCCATGTTTACACAGTATGCACACATCTTCCATGGATGTGGACAGAAAATCCTCCAACATGATGGCAA
A tag em azul deverá ser a o complemento reverso da sequência.
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Contagem das Tags
Softwares que fazem a extração e contagem das tag:
SAGE 300 – Zhang et al. (1997)SAGE 2000 – Invitrogen, Inc. (I-SAGE)eSAGE – Margulies & Innis (2000)USAGE (On-line) – van Kampen el at. (2000)SAGEnhaft (On-line) – Beissbarth et al. (2004)
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Anotação de SAGE Tags
Tag, etiqueta, marcador, assinatura...
É uma sequência de 10-17 nt.
Uma Tag possui informação suficiente para a identificação de um transcrito único.
Mais de 1 gene pode estar relacionado a mesma tag.
1 gene pode estar relacionado a 1 ou mais tags diferentes.
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Erros de amostragem;
Erros de sequenciamento;
Possibilidade de tags não unívocas;
Transcritos que não geram tags utilizando uma dada enzima;
Sequências repetitivas;
Principais Problemas
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Aumento do número de tags coletadas.
Uso de tags mais longas.
Uso de diferentes enzimas de restrição.
Como Resolver?
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Biblioteca de SAGE
Short SAGE:
Tag Freq.
GCAGACCATA 1451AACAGTTCCA 931GCCAACTCGG 2CGTGCGGATT 1
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Gerar a lista das CSTs (Confident SAGE Tag):
Remover tags com frequência igual a 1;Remover linkers de sequências
Biblioteca de SAGE
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Extração das Tags virtuais:
Tag Virtual: é uma “tag” extraída computacionalmente.
Encontrada nos 10 nt posterior ao sítio da enzima NlaIII na região 3' UTR em mRNA.
Cauda de poli-A: quantidade de “A” nas ultimas bases.
Sinal de poli-A: AATAAA e ATAAAA nas últimas 50 bases.
Anotação de SAGE Tags
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Relacionar Tag ao Gene
Pode-se encontrar mais de uma tag relacionada a um mesmo gene.
Qual a melhor Tag para o Gene?
RankingTag com alta expressão (Maior pontuação).Tag virtual interna (Menor pontuação).
SAGE Tags x Tag Virtual
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Onde encontrar os transcritos?
Banco de dados públicos: UniGene, RefSeq.Caso não tenha, pode-se usar organismo filogeneticamente próximos para obter uma anotação prévia.
Extração das Tags Virtuais