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New DNA Sequencing Technologies Viikki Science Park 1999 Lars Paulin New DNA sequencing technologies DNA Sequencing and Genomics Institute of Biotechnology University ofof HelsinkiHelsinki http://www.biocenter.helsinki.fi/bi/DNA/ Lars Paulin Institute of Biotechnology University of Helsinki DNADNA SequencingSequencing LaboratoryLaboratory Cultivator 2, Viikinkaari 4 Started in 1990 with DNA Synthesis 1991 DNA Sequencing 1994 EU Yeast Genome Project 1999 - 2000 High-throughput pipeline 1999 – 2002 Five EST Sequencing Projects 2003 First Microbe Genome Project – Move together with Microarray Laboratory to Cultivator 2 2006 Genome Sequencer 20 Core Facility – Service DNA sequencing and whole projects – Collaborative projects ”Research hotel” – Develope high-throughput methods – Consulting Lars Paulin Institute of Biotechnology University of Helsinki DNA Sequencing and Microarray Pipe-Line Bacterial growth Sequencers – QPix Colony Picker – ABI 3130 16-Capillary Sequencer – QFill 2 Microplate filler – ABI 3730 48-Capillary Sequencer – 4 Multidrop 96 and 384 well – Genome Sequencer 20 – 4 Titramax shakers Centrifuges PCR – 2 Eppendorf 5810 – 1 Eppendorf 5804 – 1 Tetrad MjResearch [1 x 96, 3 x (2x48)] – 1 Beckman – 2 Tetrad MjResearch ( 4 x 96 ) Other – 1 Eppendorf Mastercycler (384) – Tecan SpectraFluor Microplate Reader – 1 ABI 9700 ( 2 x 384 ) – ImageMaster VDS, GE Healthcare – 4 ABI 9700 ( 96 ) – 8 Servers for analysis Robotics Phred, Staden Program, – Qiagen Biorobot 9600 AceDB, ARB etc. – Qiagen Biorobot 8000 – SUN cluster – ABI Prism 7000 – Qiagen Biorobot 3000 ( 2m ) – 2 Tecan Genesis RSP100 – LightCycler 480, Roche low volume pipetting – Coulter Counter Z2 – Tecan Freedom Evo – Bioanalyzer low volume pipetting – LIMS, Sapphire – Biomek NXp , 96-pipettor Lars Paulin Institute of Biotechnology University of Helsinki WhatWhat cancan wewe do?do? All kinds of service High-throughput – Sequencing projects Picking 3 500 colonies / h Fragment analysis – petri discs, 22 x 22 cm plate – T-RLFP Growth ~50 x 96 / day SNP detection – 70 ml - 1 ml Colony picking PCR ~5 000 / day Plasmid, cosmid, fosmid, BAC – 50 - 100 ml purification PCR purification 12 x 96 / day PCR, PCR purification – 2 x 100 ml rDNA sequencing Plasmid, Cosmid, Fosmid, BAC purification EST, SAGE sequencing – 96 / 384 well Whole genome sequencing Sequencing 0,5 - 1 Mb / 24h Metagenomic sequencing Gridding 12 membranes / day MLST-Sequencing – 1536 spots in duplicate, 384 etc. controls Genome Sequencer FLX Lars Paulin Institute of Biotechnology University of Helsinki ESTEST ProjectsProjects 19991999 –– 20022002 Barley Birch 44 829 ESTs Wood, Cold, Oxidative 13 448 clusters stress, Pathogen – 7 062 cluster 1 73 881 ESTs 20 103 clusters – 10 554 cluster 1 Gerbera Spruce Genome Res 2005, 15,475-86 Plant Mol Biol 2007, 65, 311-328. 16 994 ESTs 8 432 ESTs 11 266 clusters 5 817 clusters – 8 817 cluster 1 – 4 343 cluster 1 Total Poplar Genome Biol, 2005, 6, r101 13 845 ESTs 157 981 ESTs in Databases 8 043 clusters ~260 000 sequencing reactions – 5 860 cluster 1 Lars Paulin Institute of Biotechnology University of Helsinki Leuconostoc gasicomitatum genome project Paulin, BjörkrothBjörkroth andand Auvinen First described by Johanna Björkroth – Appl Environ Microbiol. 2000, 66, 3764-72. Food spoiling bacteria Leuconostoc gasicomitatum – can grow at + 6 oC – marinaded poultry products 1 953 872 bp – fish and meat 1 923 genes Björkroth , J. Lars Paulin Institute of Biotechnology University of Helsinki LactobacillusLactobacillus rhamnosusrhamnosus GGGG Widely used probiote Biggest Lactobacillus genome so far – 3 009 877 bp – 3 120 ORFs 2 129 ORFS with assigned functions 991 ORFs without assigned functions – 5 rRNA operons – G+C % 46,69 – Annotation done at ERGO http://ergo.integratedgenomics.com/ERGO/ – Agilent microarrays done – The sequence announced at the conference: SSA GutImpact 3rd Platform meeting on Foods for Intestinal Health 29.- 31.8.2007, Haikko Manor & Spa, Finland Lars Paulin Institute of Biotechnology University of Helsinki GenomeGenome ProjectsProjects L. gasicomitatum,1,9 Mb H. bizzozeronii, 1,7 Mb - Shotgun, fosmids - 454, plasmids - Status: finished, annotated - Status: closing phase C. pneumoniae, 1,3 Mb H. salomonis, 1,71,7 MbMb - PCR products - 454, plasmids - Status: almost finished - Status: closing phase L. rhamnosus , 3 Mb E. carotovora, 5 Mb - Shotgun, fosmids, 454 - 454, fosmids - 2 Strains, GG and Lc - Status: closing phase - Status: 2 finished, 1 annotated S. islandicus, 2,7 Mb L. oligofermentans, 1,8 Mb S. islandicus, 2,7 Mb - shotgun, 454 - 454 , fosmids - Status: closing phase - Status: closing phase A. brierleyi, 2,7 Mb C. botulinum, 3,8 Mb A. brierleyi, 2,7 Mb - Shotgun, 454 - 454, fosmids - Status: closing phase - Status: closing phase Lars Paulin Institute of Biotechnology University of Helsinki ShortShort HistoryHistory ofof DNADNA SequencingSequencing 1977 1998 – Maxam-Gilbert – First 96 Capillary – Sanger instruments MegaBace, ABI 3700 1986 2000 – First Automated DNA 2000 Sequencer ABI 370 (373) – ABI 3100, 16 Capillary 1988 2002 – Pharmacia ALF – ABI 3730, 48 or 96 Capillary 1995 2005 – ABI 377 2005 Up to 96 lanes – Genome Sequencer GS20 1996 2006 – First Capillary DNA – Solexa (Illumina) Sequencer ABI 310 2007 – SOLiD Lars Paulin Institute of Biotechnology University of Helsinki ssDNA tai denaturoitu plasmidi AACGGTACACG SangerSanger DNADNA SequencingSequencing 3' 5' Alukkeen hybridisointi 5' 3' AACGGTACACG 1. Template 3' 5' – ssDNA or dsDNA Sekvensointireaktiot A C G T 2. Primer annealing dATP+ddATP dATP dATP dATP dCTP dCTP+ddCTP dCTP dCTP dGTP dGTP dGTP+ddGTP dGTP – Sequencing primer dTTP dTTP dTTP dTTP+ddTTP TTGCCddA TTGCCATGTGddC TTGCCATGTddG TTGCCATGddT 3. Elongation TTGCddC TTGCCATddG TTGCCAddT TTGddC TTddG TddT – DNA polymerase ddT Steps 2 and 3 can be done Geelielektroforeesi ja autoradiografia repeatedly => cycle sequencing A C G T 3' C 4. Electrophoresis G T G T A C C G T T 5' deoksi TTP dideoksi TTP Lars Paulin Institute of Biotechnology University of Helsinki Incorporating Labels Labelled primers •1 or 4 labels Labelled deoxynucleotides •1 label Labelled dideoxynucleotides •1 or 4 labels •BigDye, ET DEOKSINUKLEOTIDI ALUKE terminators TEMPLAATTI DIDEOKSINUKLEOTIDI SYNTETISOITU JUOSTE Lars Paulin Institute of Biotechnology University of Helsinki AutomatedAutomated DNADNA SequencingSequencing Single-dye systems 4-dye systems slab-gel systems capillary systems 1. 2. 1. 1. 2. A C G T ELECTROPHORESIS DATA COLLECTION RAW DATA PROSESSING PROCESSED DATA LOW CAPACITY HIGH CAPACITY Lars Paulin Institute of Biotechnology University of Helsinki Lars Paulin Institute of Biotechnology University of Helsinki StrategiesStrategies forfor GenomeGenome SequencingSequencing Shotgun approach Libraries – random sequencing – usually made by of different sized random shearing of libraries genomic DNADNA – assembly using – 2 kb, 4-6 kb, 10 kb different software plasmid librarieslibraries – closing of gaps using – fosmid or cosmid different methods libraries with 30 - 50 kb inserts Lars Paulin Institute of Biotechnology University of Helsinki Whole Genome Shotgun Sequencing Sheared DNA: Whole Genome: ~ 2 kb ~ 3 Mb Random Sequencing Reads Templates Both ends Lars Paulin Institute of Biotechnology University of Helsinki Shotgun Sequencing :ASSEMBLY Contig 1 Contig 2 Single Low Base Consensus sequence Quality Stranded Sequence Quality Miss-Assembly Region Gap (Inverted) • 0.5 -1.0 X (2 reads/kb) - ‘Skimming’ • 6.5 - 8.0 X (~18 reads/kb) - ‘pre-finished’ • 3.5 - 4.0 X (~9 reads/kb) -’half-shotgun’ • 10 X (22-24 reads/kb) - ‘deep shotgun’ Lars Paulin Institute of Biotechnology University of Helsinki PhredPhred,, PhrapPhrap andand StadenStaden PackagePackage ProgramProgram Phred and Phrap Staden Program University of Washington Cambridge, Sanger Center Phil Green, http://www.phrap.org/ Roger Staden, Phred quality score: http://staden.sourceforge.net/ QV = - 10 * log10( Pe ) where Pe is the probability that Trace editing the base call is an error. Phred Pe Accuracy of Phrap assembly and Gap4 score the base call editing 10 1 in 10 90% – display of traces from sequencers 20 1 in 100 99% – display of traces from sequencers 30 1 in 1,000 99.9% – translations, orfs, RE etc. 40 1 in 10,000 99.99% – good capacity 50 1 in 100,000 99.999% Lars Paulin Institute of Biotechnology University of Helsinki NewNew DNADNA SequencingSequencing TechnologyTechnology Parallel Sequencing Technology Massive throughput Fast sequencing No cloning step PCR Currently three systems ready – Genome Sequencer (http://www.454.com/,http://www.roche.com) 454 Life Sciences, Roche Launched in October 2005 – Solexa (http://www.illumina.com) Illumina Launched 2006 – SOLiD (http://www.appliedbiosystems.com) Applied Biosystems Launched in October 2007 Lars Paulin Institute of Biotechnology University of Helsinki Lars Paulin Institute of Biotechnology University of Helsinki GenomeGenome SequencerSequencer (http://www.454.com/,http://www.roche.com) Genome Sequencer GS20;FLX – Manufacturer 454 Life Science – Marketing Roche Parallel Sequencing – Shotgun sequencing No plasmid libraries Linkers ligated to fragments Emulsion PCR Picotiter plate, 1 600 000 wells – Pyrosequencing (Nyren, P. et al Anal Biochem. 1993, 208,171-5) Detection with sensitive CCD camera Run time ca. 4,5 h; 7,5 h Read lenght 100 -120 bp; 250 – 300 bp Raw sequence ca. 25 – 35 Mb/run; 80 – 100 Mb/run Lars Paulin Institute of Biotechnology University of Helsinki GenomeGenome SequencerSequencer GSGS 20/FLX20/FLX Lars Paulin Institute of Biotechnology University of
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