The next generation sequencing Métodos Atuais Sequenciamento de próxima geração 1 - melhor custo-benefício para projetos de alta demanda de dados; 2 custo por pb muito menor; 3 sequenciamento muito mais rápido e eficiente (>40 Gbase/corrida). 4 versatilidade Genomas, expressão gênica, diagnóstico, gen. população, epigenética, metagenômica, entre outros!&
The next generation technologies Roche 454 Solid Illumina/Solexa Sequenciamento de segunda geração Ion Torrent PacBio Nanopore Sequenciamento de terceira geração Sequenciamento de quarta geração ()($$+$$& ()'*'+,*& CUSTO DO SEQUENCIAMENTO POR BASE ")($$+$$&,)($$+$$& ')($$+$$&!)($$+$$& Roche 454 Illumina Solid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http://genome.gov/splash.htm '&
!$$)$$$)$$$& *$)$$$)$$$& *()'B,)$%'& http://genome.gov/splash.htm CUSTO DO SEQUENCIAMENTO POR GENOMA C$)$$$)$$$& %$)$$$)$$$& B$)$$$)$$$& ($)$$$)$$$& "$)$$$)$$$&,$)$$$)$$$& '$)$$$)$$$& Roche 454 Illumina Solid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enoma humano U$ 2 bi; ~3 bilhões bp; 11 anos; ~10X Genoma U$ 3.000,00; ~3 bilhões bp; 2 dias; ~100X A evolução do sequenciamento de DNA %)*($& Mardis. Science 2011 Mardis, ER. A decade s perspective on DNA sequencing technology. Nature, 2011. Illumina HiSeq2500: 1 Tb,&
A evolução do sequenciamento de DNA!"#$%#!"& Kahn SD. Science 2011 Kahn, SD. On the future of Genomic Data. Science, 2011 Hoje Objetivo rastrear as variações genéticas em populações humanas Objetivo gerar conhecimento sobre o genoma de câncer com o intuito de gerar tratamentos e diagnósticos "&
Hoje Objetivo analisar o genoma de 10 mil espécies de vertebrados Objetivo analisar o genoma de 5 mil espécies de insetos e outros artrópodos Estocagem Montagem Análise Maiores problemas dos sequenciamentos em larga escala!!! (&
Roche 454 Solid Illumina/Solexa Sequenciamento de segunda geração Ion Torrent Amplificação e síntese Roche 454 2 nd generation 2004-2005 Roche 454 Life science Utiliza tecnologia de pirosequenciamento - 1986 HI/J=/<7@<K&3>&I><18/I@IL& B&
Princípio COBIOT-1093; NO. OF PAGES 9 Análise genômica 2013 2 Analytical Biotechnology Figure 1 (a) pyrosequencing Mecanismo do Pirosequenciamento (b) 454 sequencing polymerase polymerase 3 5 3 5 3 5 5 3 primer dntp dndp dntp dnmp apyrase ADP AMP ATP PPi ATP sulfurylase luciferase light N sulfurylase luciferase APS PPi ATP luciferin oxyluciferin time (c) Solexa (d) SOLiD ATP-sulfurilase Conversão PPi! ATP Luciferase Usa ATP p/ converter luciferina! oxyluciferina = LUZ Apirase degrada os ATPs e nucleotideos livres emulsion PCR primer polymerization laser detector %& Current Opin Basic principles of NGS techniques. (a) pyrosequencing: the incorporation of a new nucleotide generates detectable light. (b) 45 nucleotide incorporation is associated with the release of pyrophosphate resulting in a light signal. (c) Solexa: DNA fragments bui bridges and after the addition of the labeled terminators the sequencing cycle starts. (d) SOLiD: if the adapters are bound, emulsion to generate so-called bead clones.
Higher Education Press and Springer-Verlag Berlin Heidelberg 2010 523 Biotin tag Streptavidin Zhou et al., 2010. Protein Cell Figure 1. The GS FLX system working principle. (A) Prepare adapter ligated ssdna library (A-[insert]-B). (B) Emulsion based clonal amplification. (C) Depositing DNA beads into the PicoTiter plate. (D) Sequencing and base calling. (http://www.454.com) http://www.youtube.com/watch?v=bfnjxkhp8jc http://www.youtube.com/watch?v=jnqxglkozku The next-generation sequencing technology and application Protein & Cell C&
Roche GS FLX System& *&
SOLiD Life Technologies SOLiD - Sequencing by Oligonucleotide Ligation and Detection Life Technologies HI/J=/<7@<K&3>&A@K6R:<L& 2 Overview of Sequencing Technology Platforms 13 *-).(/)*0)./.12'3%) 8M0N##K17)I:/)=7I7)/O=#7:<1/<1#I:A@O- 1/78<:A:K>-:P/4P@/Q&!"#$%&'()*+,)!$& F i g. 2. 3 Generation of sequencing features. High-throughput sequencing systems have taken different approaches in the generation of the detectable sequencing features. ( a ) Emulsion PCR is applied in the GS FLX and SOLiD systems. Single enrichment bead and sequencing library fragment are emulsified inside an aqueous reaction bubble. PCR is then applied to populate the surface of the bead by clonal copies of the template. Beads with immobilized clonal DNA collections are
!!&
!'&
Amplificação Química da reação!,&
Sequenciamento Comparando metodologias 454, Solid Illumina!"&
Custos S6A/&T/<1/4&U:4&V/<:2/&?<6A>I@I&DSTV?G 1 Tb de dados!(&
ION TORRENT Não utiliza scanner e câmeras 4'()*56 76 )89:#9(;93) 4'()*3'2'( 76 )89:#9(;93)./J=/<7@6O:4&4W0@O:+&7:20671:&/&/7:<X2@7:&!B&
DETECÇÃO DO SINAL T8@0N&&?446>&:U&2@74:Q/AAI& T8@0N&&?446>&:U&2@74:Q/AAI& 1 dntp de cada vez ñ utiliza nucleotídeos modificados e cascatas enzimáticas ñ utiliza detecção óptica (fluorescência e quimioluminescência)!%&
DETECÇÃO DO SINAL Y.ZEF&[&4:<&8/<I@RP/&<@/AO-!\/71&746<I@I1:4& DETECÇÃO DO SINAL!C&
DETECÇÃO DO SINAL 8M0N##QQQ)>:=1=3/)7:2#=I/4#@:<1:44/<1& PacBio RS sequencer Sequenciamento de terceira geração X Amplificação e síntese!*&
'$&
'!&
Detecção de modificações epigenéticas Custos S6A/&T/<1/4&U:4&V/<:2/&?<6A>I@I&DSTV?G 1Gb of data ''&
Nanopore Sequenciamento de quarta geração X Amplificação e síntese Constituintes Principais Camada lipídica ',&
Mecanismo Constituintes Principais Exonuclease '"&
Nanopore genome sequencer makes its debut : Nature News & Comment Métodos do futuro próximo Commendations for Nature News & Comment in the 2012 Online Media Find Awards out more 14/08/12 11:33 NATURE NEWS Nanopore genome sequencer makes its debut Technique promises it will produce a human genome in 15 minutes. Erika Check Hayden 17 February 2012 Technology that its parent company says will sequence a human genome in just 15 minutes opened its first data run to scrutiny today. 9]U:4O&^6<:0:4/&F/78<:A:K@/I+& 36I/O&@<&9]U:4O+&_`&/]0/71I&1:&I1641& Oxford Nanopore Technologies, based in Oxford, UK, revealed the initial results from its GridION system I/AA@<K&@1I&</Q&2678@</&@<&18/&I/7:<O& at the Advances in Genome Biology and Technology meeting in Marco Island, Florida. The firm expects 86AU&:U&18@I&>/64&6<O&6AI:&0A6<I&1:& A6=<78&18/&Q:4AOaI&b4I1&2@<@61=4@c/O+& to start selling its new machine in the second half of this year and also plans to launch the world s first O@I0:I63A/&I/J=/<7/4&d&18/&5@<Y9^& miniaturized, disposable sequencer the MinION which will retail for less than US$900. d&q8@78&q@aa&4/16@a&u:4&a/ii&186<&_. e*$$)& Given its flexibility, scalability and low entry price, this technology could have a seriously disruptive effect on the sequencing industry, says Daniel MacArthur, a geneticist who blogs about the genomics industry. That industry is already seeing significant jockeying for position with Swiss drug giant Roche last month launching a takeover bid for the manufacturer of the sector s dominant technology: Illumina of San Diego, California (see Roche takeover bid poses challenge to Fast track: nanopore sequencing identifies Illumina). In the same month, up-and-coming company individual bases as a strand of DNA is passed Ion Torrent Systems of Guilford, Connecticut, vowed to through a pore. begin selling a machine by the end of the year that can IEMEDIA SOLUTIONS sequence an entire human genome in a day for less than $1,000 per sequence.and last April, Pacific Biosciences of Menlo Park, California, launched its own sequencing technology. Oxford Nanopore s system uses nanopore sequencing to rapidly read DNA sequences. A strand of DNA is fed through a biological pore and the various bases are identfied by measuring the difference in their electrical conductivity as they pass through the pore (see Personal genomes: Standard and pores). The launch of the nanopore machines marks the end of a decadeslong wait. Nanopore technology was first mooted in the early 1990s, Related stories http://www.nature.com/news/nanopore-genome-sequencer-makes-its-debut-1.10051 Página 1 de 4 '(&
F8/&3/I1&4/I=A1I&7:P/4/O&63:=1&,!(!&36I/I&:U&18/&4/6O&I164R<K&U4:2&63:=1&'!C$18&36I/&1:& "**$18&36I/)&Y<&18/&6A@K</O&0641&:U&18/&4/6O&2=939)>.%)?@A)&/9(B2C)D'!'*#,!(!G&Q@18&K60I& 7:P/4@<K&63:=1&!(f&D"*B#,!(!G&:U&36I/I)&gh?.F&@I&04:363A>&<:1&18/&4@K81&6004:678&6<O&@1I& K60&0/<6A1>&@I&<:1&:0R2@c/O&U:4&^6<:0:4/&4/6OI)&& 'B&
Comparação diferenttes plataformas Plataf. Roche/454 s GS FLX Comp (bp) Tempo de corrida (dias) Gb por corrida Prós Contras 500-1kb 0,35 0,45 Reads longos; agilidade *Illumina 100-150 9 40 Plataforma mais utilizada Life/APG s SOLiD 3 50 14 50 Sistema de correção de erros Alto custo dos reagentes; alto erro PacificBioscience 3-20kb? 1 10Kb Alto erro Nanopore 100kb 15 min? 8Kb Alto erro Baixa capacidade multiplexar amostras Demora na corrida * HiSeq2000 gera um output com 600 Gb por corrida. Maior output atual. '%&
Curso de Férias Análise genômica 2014 15-18 de Julho de 2014 Instituto de Biociências, UNESP, Botucatu, SP 'C&