Sequencing - Index of

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Sequencing - Index of

High throughput screeningand –omics I


Different ways to address biological questions:Bottom up (classical):- Detailed analysis of single genes/proteins. Step by stepassembly of results to get an overview about processeswithin cells/organisms.Top down (modern):- Analysis of complete organisms.


- Omics ?Tyers & Mann, Nature 422, (13), 2003, 193-197


Steps that are analyzed by -omics:Graves, P.R. and Haystead, T.A.J., Micro Mol Biol Rev. Mar 2002 pp 39-63


Increasing complexityGenome:30.000 genesTranscriptome: 40-100.000 mRNAsProteome:100-400.000 proteins>1.000.000 interactionsHuman ProteomeProtein Interaction10 6Transcripts10 5Human Genomehttp://www.uta.edu/biology/michalak/classnotes/genomics/lect35.ppt


Todays program:High troughput sequencing:-454 Sequencing-Solexa SequencingMicroarrays and barcodingHigh troughput two hybrid experiments


DNA Sequences:August 2005 100 Gigabases were sequenced !http://www.ncbi.nlm.nih.gov/Genbank/index.html


Costs of DNA SequencingShendure, J., Mitra, R., Varma, C., and Church, G.M. (2004) Nature Genetics 5:335


DNA sequencingDNAShakeDNA fragments+ =Knownlocation(restrictionsite)source: robotics.stanford.edu/~serafim/cs262/Spring2003/Slides/Lecture9.ppt


Sanger Method for DNA Sequencing1. Start at primer2. Grow DNA chain3. Include dideoxynucleoside(modified a, c, g, t)4. Stops reaction at all possiblepoints5. Separate products with length,using gel electrophoresissource: robotics.stanford.edu/~serafim/cs262/Spring2003/Slides/Lecture9.ppt


Sequencing-strategies1. Hierarchical – Clone-by-clonei. Break genome into many long piecesii. Map each long piece onto the genomeiii. Sequence each piece with shotgunExample: Yeast, Worm, Human, Rat2. Online version of (1) – Walkingi. Break genome into many long piecesii. Start sequencing each piece with shotguniii. Construct map as you goExample: Rice genome3. Whole genome shotgunOne large shotgun pass on the whole genomeExample: Drosophila, Human (Celera), Neurospora, Mouse, Rat, Fugusource: robotics.stanford.edu/~serafim/cs262/Spring2003/Slides/Lecture9.ppt


Automatic Sequencing


Costs and time for sequencing ahuman genome (3.2 billion bp)2001 First human genomesequence draft: ~ 13 years and 300 million US$Technology ReviewMay 2005:~ 6 month and 20 to 30 million US$The Scientist(Vol. 20,2 p.67) 454: ~ 1 month and 900 000 US$ (1x coverage)The Scientist(Vol. 20,2 p.67) Solexa: ~ 6 month and 50 000 US$ (15x coverage)The Scientist(Vol. 20,2 p.67) Helicos (not released): ~25 hours ? US$


Shendure, J., Mitra, R., Varma, C., and Church, G.M. (2004)”Advanced sequencing technologies: Methods and Goals” Nature Genetics 5:335


Novel Sequencing-Technologies454 Biosciences (Roche)FastSequence more than 20 million bases per 5.5-hour instrument run.Cost-EffectiveBenefit from reduced cost per base compared to conventionalSanger technology.SimplePerform sequencing runs with an easy-to-use instrument requiringminimal steps.EfficientSequence a typical bacterial genome in days with one person —without cloning and colony picking.ConvenientUse the complete system solution — from sample preparation todata mapping or assembly.Solexa1 billion bases per 2 days runannounced for second half of 2006


454 Sequencingsource: http://www.454.com/


454 Sequencingsource: http://www.454.com/


454 Sequencingsource: http://www.454.com/


454 Sequencingsource: http://www.454.com/


454 Sequencingsource: http://www.454.com/


Is 454 DNA sequencing technology mature?• The short answer is NO.• The “sweet spot” currently is in bacterial genomicsbecause of short sequences (~100 nt read length)• You will collect 30+ million bases in a run. Like it or not,what do you want to do with them?• Some applications are too small.• Some applications are too large


Novel Sequencing-Technologies454 Biosciences (Roche)FastSequence more than 20 million bases per 5.5-hour instrument run.Cost-EffectiveBenefit from reduced cost per base compared to conventionalSanger technology.SimplePerform sequencing runs with an easy-to-use instrument requiringminimal steps.EfficientSequence a typical bacterial genome in days with one person —without cloning and colony picking.ConvenientUse the complete system solution — from sample preparation todata mapping or assembly.Solexa1 billion bases per 2 days runannounced for second half of 2006


Solexa Sequencingsource: http://www.solexa.com/


Solexa Sequencing


Solexa Sequencing


Solexa Sequencing


Solexa Sequencing


Solexa Sequencing


Solexa Sequencing


Solexa Sequencing


Solexa Sequencing


DNA Microarrays• Microarrays are composed of short DNA oligomers attached to aninert substrate– glass slide, nylon membrane (historically)• Typically contain a grid of 10 5 -10 6 features (spots) each with adifferent DNA molecule• Fluorescently-labeled DNA or RNA hybridizes to complementaryprobes• Hybridized array is scanned with a laser to produce a signal for eachspot


Oligo-Array-ExperimentsExtraktion derpoly-A-RNAAmplifikationund Markierungder RNAFragmentierungHybridisierungFärbung


Different results using different machinesNucleic Acids Research, 2003, Vol. 31, No. 19, 5676-56845684


Barcoding: Gene knockout libraries for yeastReplacement ofyeast ORFs withkanMX geneflanked by uniqueoligo barcodes-“Yeast DeletionProject Consortium”similar RNAi libraries in other systemssource http://www.cbs.dtu.dk/~workman/IntroSysBio/01%20Introduction.ppt


Systematic phenotypingBarcode(UPTAG):DeletionStrain:CTAACTC TCGCGCA TCATAATyfg1Δ yfg2Δ yfg3Δ…Rich mediaGrowth 6hrsin minimal media(how many doublings?)Harvest and label genomic DNAsource http://www.cbs.dtu.dk/~workman/IntroSysBio/01%20Introduction.ppt


Systematic phenotyping with abarcode array(Ron Davis and others)These oligo barcodes are alsospotted on a DNA microarrayGrowth time in minimal media:– Red: 0 hours– Green: 6 hourssource http://www.cbs.dtu.dk/~workman/IntroSysBio/01%20Introduction.ppt


2-hybrid: Genetic approach to study protein interactionsFields S, Song O. Nature. 1989 Jul 20;340(6230):245-6comprehensive studies done by:Uetz P et al., Nature (2000) 403:623-627Ito T et al., PNAS (2001) 98:4569-4574


How does it work ?Think of the principle of a transcription factor


Large scale made easyLEU2TRP1HIS3 ADE2 LacZ HIS3 ADE2 LacZMATa; Δleu2; Δtrp1; Δade2; Δhis3MATα; Δleu2; Δtrp1; Δade2; Δhis3LEU2TRP1HIS3 ADE2 LacZHIS3 ADE2 LacZMATa/ MATα; Δleu2/Δleu2; Δtrp1/Δtrp1; Δade2/Δade2; Δhis3/Δhis3


(a) 6000 haploid yeast transformantsplated in array format. Eachexpresses one yeast ORF as a fusionto the Gal4 activation domain.(b) Positives obtained in a screenusing a Gal4 DNA binding domainfusion to the Pcf11 protein (part of thepre-mRNA cleavage andpolyadenylation factor 1A ) fivepolypeptides in all). Three othercomponents of factor 1A wereidentified as positives.


Tyers & Mann, Nature 422, (13), 2003, 193-197

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