0FB5649Ad01 - Systems Biology Prof Pierre Hilson Systems...

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Systems Biology Prof. Pierre Hilson Academic year 2009 - 2010 1 Systems Biology 2. Introduction to Genomics Prof. Pierre Hilson VIB Department of Plant Systems Biology University of Ghent Academic year 2009-2010 The human genome project 1990 1995 2005 2000 Human Genome Project Technological innovations High throughput automation Large scale genome sequencing <1Mb/year >1000 Mb/year 1000-fold 10.000 Mb/year Genomics revolution
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Systems Biology Prof. Pierre Hilson Academic year 2009 - 2010 2 DNA sequencing technology ¤ 70’s: two different DNA sequencing methods Enzymatic chain termination method (Sanger) • Method proved more robust and automatable – Chemical base specific cleavage method (Maxam and Gilbert) ¤ 80’s: the first automated sequencers – 4-color dye terminators: single readout for 4 different reactions – Automated data acquisition ¤ 90’s: various technological innovations for large-scale sequencing – Fully automated large-scale sequencing made possible by eliminating the human interface ¤ Excellent overview C. A. Hutchison III, Nucleic Acids Res. (2007) Automated DNA sequencers ¤ Four-color fluorescence-based sequence detection – Each base specific chain termination reaction uses a different fluorophore – simultaneous detection of the products of the 4 reactions in a single run ¤ Automated DNA sequence gel readers – First generation: slab gel-based DNA sequencers • Difficulties in lane tracking causing considerable losses in data • Manual loading – Second generation: capillary DNA sequencers • Automated loading, allowing unattended operation and perfect lane tracking • 15 * 96 samples/day = ~1 to 1,5 million bases of raw sequence/day • Instruments currently used for genome sequencing
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Systems Biology Prof. Pierre Hilson Academic year 2009 - 2010 3 Genome sizes and sequence reads Organism Genome size range Sequence reads (7x coverage - 700 bases/read) bacteria 500kb – 8Mb 5.000-80.000 fungi 10Mb – 40Mb 100.000-400.000 invertebrates 100Mb – 300Mb 1-3 million vertebrates 400Mb – 3.000Mb 4-30 million plants 100Mb – 50.000Mb 1-500 million `Courtesy of http://www.mun.ca/biology/scarr/377_gel_file.htm
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Systems Biology Prof. Pierre Hilson Academic year 2009 - 2010 4 The Broad Institute of MIT and Harvard Innovations for large-scale sequencing 1. Construction of physical maps High throughput fingerprinting of BAC clones 2. Improvements of the dideoxy sequencing technique Improved fluorescent labeling and sequencing enzymes 3. Software for automated interpretation of fluorograms Automated data analysis is a prerequisite 4. Improved sequencing strategies Shotgun sequencing, improved shotgun libraries 5. Automation of sample preparation and analysis Robots made the up scaling feasible
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Systems Biology Prof. Pierre Hilson Academic year 2009 - 2010 5 High throughput fingerprint analysis of large-insert clones Marra et al., Genome Res 7: 1072–1084 (1997) ¤ A method for constructing physical maps – Physical maps provide uninterrupted sources of material for sequencing • bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes
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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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0FB5649Ad01 - Systems Biology Prof Pierre Hilson Systems...

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