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Sequenciamento de ácidos nucleicos em larga escala como novo paradigma para
geração de conhecimento
Prof. Dr. Rinaldo Wellerson PereiraPrograma de Pós Graduação em Ciências
Genômicas e Biotecnologia
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Apresentação
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Grupo de Pesquisa
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Contatos
https://www.facebook.com/pages/Genética-Prof-Rinaldo-Pereira/173846522641806
http://rinaldogenetica.com
rinaldo.ucb rinaldo_pereira
https://www.facebook.com/pages/Ci%C3%AAncias-Gen%C3%B4micas-UCB-Bras%C3%ADlia/199850806721852
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Planejamento
• Dia 31/08/11 – Métodos e Genomas– Metodologia NGS (Next Generation Sequencing)
• Dia 01/08/11 - Sequenciamento de Genomas – Sequenciamento de Genomas Completos– Sequenciamento de regiões de interesse (targeted resequencing)
• Dia 02/08/11 – RNAseq – Epigenética– Sequenciamento Global de RNAS– Sequenciamento de DNA tratado com bissulfito– Sequencimento de DNA proveniente de Chip (ChipSeq)
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Sequenciamento de DNA
Método de Sanger
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Sequenciamento com Dye Terminators
(Fluorescente)
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Sequenciamento de DNAMétodo de Sanger - Automatização
96 poços 384 poços
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Sequenciamento de DNADetecção Simultânea de Quatro Cores
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Sequenciamento de DNA
• 1977 -2004 – Evolução e consolidação do método de Sanger
• 2004 - ……. – Plataformas de NGS – Next Generation Sequencing (Na verdade pode ser: “Now Generation Sequencing”)
• Segunda Geração • Terceira Geração
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• Sanger • NGS
Shendure, 2008 – Nature Biotechnology
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Human genome at ten: the sequence explosion. Nature, 464: 670-671. 2010.
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Venter, 2010 - NatureHuman genome at ten: the sequence explosion. Nature, 464: 670-671. 2010.
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Wetterstrand KA. DNA Sequencing Costs: Data from the NHGRI Large-Scale Genome Sequencing Program Available at: www.genome.gov/sequencingcosts. Accessed [08/28/11]
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Wetterstrand KA. DNA Sequencing Costs: Data from the NHGRI Large-Scale Genome Sequencing Program Available at: www.genome.gov/sequencingcosts. Accessed [08/28/11]
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Do que falamos quando falamos sobre NGS?
Zhang et al., The impact of next-generation sequencing on genomics. J Genet Genomics. 2011, 38(3):95-109.
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Mais e novas possibilidades
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http://454.com/products/gs-flx-system/index.asp - Acesso em 28/08/11
http://www.454.com/flash/roche-gene.asp
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Genome Analyser IIx
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HiSeq 1000
http://www.illumina.com/systems/hiseq_1000.ilmn, acesso em 28/08/11
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HiSeq 2000
http://www.illumina.com/systems/hiseq_2000.ilmn, acesso em 28/08/11
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MiSeq Personal Sequencing System
http://www.illumina.com/systems/miseq.ilmn , acesso em 28/08/11
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5500 SOLiDTM Sequencer
5500xl SOLiDTM Sequencer
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50Mb 100x
>100 M reads/amostra
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Ion Personal Genome Machine-PGM
JM Rothberg et al. Nature 475, 348-352 (2011) doi:10.1038/nature10242
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Read length – 1000 bases35-45 Mb/Run
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Sequenciamento de GenomasDe Mendel a Sanger
Gregor Mendel Frederick GriffithMorgan, Sturtevant, Bridges Avery, Mcarty, McLeod
Watson, Crick, Franklin, Wilkins Zamenick, Hoagland, Brenner
Nirenberg, Khorana, Holley Sanger
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A ideia geral em se sequenciar genoma
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They made the cover!
Sacharomyces cerevisae25/10/1996
Caenorhabditis elegans
11/12/1998
Drosophila melanogaster24/03/2000
Arabidopsis thaliana
14/12/2000
Homo sapiens sapiens15/02/2001 e 16/02/2001
Haemophilusinfluenza
28/07/1995
Pan troglodytes01/09/2005
Mus muscullus
05/12/2002
Rattus novergicus
01/04/2005
Gallus gallus
09/12/2004
Cannis familiaris
05/12/2005
Apis mellifera
26/10/2006
Monodelphisdomestica10/05/2007
Tetraedon Nigrovirids21/10/2004
Finalização doGenoma Humano
Macaca mulatta
13/04/2007
ZeaMays
20/11/2009
Bostaurus
24/04/2009
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O que se extrai de um genoma?
A ponta do Iceberg! O início de uma longa jornada!
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Sequenciamentos de Genomas na era NGS
Até amanhã!Divirtam-se!