F19159E7d01 - Systems Biology Prof. Hilson Academic year...

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Unformatted text preview: Systems Biology Prof. Hilson Academic year 2009 - 2010 1 Systems Biology Systems Biology 3. Sequencing technologies 3. Sequencing technologies Prof. Prof. Pierre Hilson Pierre Hilson VIB Department of Plant Systems Biology VIB Department of Plant Systems Biology University of Ghent University of Ghent Academic year Academic year 2009 2009-2010 2010- Next generation DNA sequencing Outline- 454- ABI Solid- Solexa- Helicos cDNA analysis- cDNA analysis- RNA sequencing Systems Biology Prof. Hilson Academic year 2009 - 2010 2 Next generation sequencing technologies ¤ Massively parallel sequencing instruments – Eliminate the rate limiting step in sequencing DNA cloning • In situ amplification / no amplification In situ amplification / no amplification – 10 5 to 10 7 sequencing reactions in parallel – “shorter” sequence runs: 30 to 100s bases – Total capacity of 100 MB to several GB per run ! – Reduced cost of 5-10 K€ per (human) genome within a year ! ¤ Next generation sequencing instruments – 454 (Roche): pyrosequencing of DNA amplified on beads 454 (Roche): pyrosequencing of DNA amplified on beads – ABI: Sequencing-by- ligation of DNA amplified on beads – Solexa (Illumina): Sequencing-by-Synthesis of DNA on amplified slides – … and a panoply of upcoming, fiercely competing, technologies Genome sequencing in microfabricated high-density picolitre reactors ¤ Paper presents highly parallel sequencing system Margulies et. al., Nature 437: 376-380 (2005) – emulsion method for DNA amplification on beads – Base calling using pyrosequencing ¤ 454 Sequencing instrument – instrument for massively parallel sequencing by synthesis – throughput 50 to 100 times greater than capillary electrophoresis instruments – Performance • 1st gen. : 250.000 reads of ~100 bases or 25 Mi bases • 2nd gen. : 500.000 reads of ~200 bases or 100 Mi bases • 3rd gen. (2008): 2,5 million reads of ~400 bases or 1 billion bases Systems Biology Prof. Hilson Academic year 2009 - 2010 3 Speeding up sequencing Conventional Cloning and sequencing Massive parallel sequencing Reprinted from: Rogers and Venter, Nature 437: 326-327 (2005) Multiplex amplification: emulsion PCR ¤ Genomic DNA preparation – DNA is isolated, fragmented, ligated to adapters and separated into single strands ¤ PCR amplification – Fragments are bound to beads with one fragment per bead – beads are captured in the droplets of a PCR-reaction- mixture-in-oil emulsion Reprinted from: Shendure et. al., Science. 309: 1728-1732 (2005) – PCR amplification occurs within each droplet – resulting in beads each carrying 10 million copies of a unique DNA template Systems Biology Prof. Hilson Academic year 2009 - 2010 4 Fibre-optic slide preparation ¤ Beads carrying single- stranded DNA clones – are deposited into wells ¤ Smaller beads carrying immobilized enzymes Reprinted from: Shendure et. al., Science. 309: 1728-1732 (2005) required for pyrophosphate sequencing – are deposited into each well...
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This note was uploaded on 05/30/2010 for the course WE CMBIBI0200 taught by Professor Pierrehilson during the Spring '10 term at Ghent University.

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F19159E7d01 - Systems Biology Prof. Hilson Academic year...

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