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1、Growth of GenBank(GeneBank-NCBI,2009)Data generated by 1000 Genome Project had surpassed total data deposited in NCBI For the sequencing cost dramatically decreased sequencing technology is widly used in reseach Current Challenge:Data minning and disclose biological issue1950196019701980199020002010

2、Brief History of SequencingDiscovery of DNA structure(1953)Development of Sanger Sequencing(1977)Invention of Automated FluorescentSequencer(1987)Invention of CapillarySequencer(1996)Invention of Applied BiosystemsSolid System(2007)Invention of Illumina Genome Analyzer System(2006)Invention of 454 G

3、S 20 Sequencer(2005)Invention of 3rd generation sequencing(2009)OUTLINESequencing principle of NGSSolexa data analysisSingle molecule sequencing technologyNext Generation Sequencing TechnologyApplied Biosystems/SOLIDSequencing-By-LigationRoche/454 Genome Sequencer FLXSequencing-By-SynthesisIllumina/

4、HiSeq 2000Sequencing-By-SynthesisIllumina/HiSeq 2000Sequencing-By-SynthesisHIGHEST OUTPUTInitially capable of up to 200 Gb per runFASTEST DATA RATE25 Gb/day 10 days for 2 x 100 bpHIGHEST NUMBER OF READSOne billion single-end reads*Two billion paired-end reads*Illumina Solexa FlowcellA flowcell conta

5、ins eight lanesLane 1Lane 8Each tile is imaged four times per cycle Image from 1 tileSimplest Sequencing WorkflowSIMPLIFIED SAMPLE PREPParallel sample processingAutomated sequencingHiSeq 2000 SEQUENCINGDATA PROCESSING&ANALYSISSimple,efficient data analysisAutomated clustergenerationcBot CLUSTER GENE

6、RATIONSequencing Overview/SolexaSequencing Overview/SolexaSequencing Overview/SolexaSequencing Overview/SolexaSequencing Overview/SolexaSBS（sequencing-by-synthesis）Sequencing Overview/SolexaSequencing Overview/SolexaCluster Generation:Initial ExtensionCluster Generation:AmplificationSequencing Overv

7、iew/SolexaOVERVIEW OF THE METHOD8oxoG-P7U-P5OHOH1.GraftingU8oxo-G2.Cluster ampnOHOHUU3.USER Linearisation4PO-OHOH=4.ddNTP()block&Hyb SBS 3SBS READ 15.De-phosphorylateP5-PO4-(PNK)OHOHUSERFpg6.Re-synthesis ofP5-strand(Isothermal amp)OH8.ddNTP block&Hyb SBS 8SBS READ 2PNK7.Linearise P7(Fpg)OHSOLiDSCALA

8、BLE OUTPUTProvides 100 Gb&2000M reads per runUNIFORM COVERAGEimprove coverage to discover rare variants in difficult(GC/AT-rich)regionsHIGH ACCURACYDetection of causative variation enabledat low coverage and cost per sampleAB SOLID 3 System Slide20emPCRemPCRSequencingSequencingDNA LibraryDNA Library

9、Preparation Preparation DNA Library PreparationGenome fragmented by nebulizationAdaptor ligationEmulsion PCR AmplificationMix PCR aqueous phase into a water-in-oil emulsion and carry out emulsion PCRSequencing by SynthesisBeads attached to glass surface in a random arraySequencing by synthesis Photo

10、ns Generated are Captured by CameraSequencing Image CreatedAB/SOLID Workflow DNA Fragment1.Create library of DNA fragments:隨機(jī)Fragment Library(directed resequencing)Mate Pair Library(whole genome sequencing)1mbeadForwardAdapterReverseAdapter1mbeadForwardAdapterReverseAdapterInternal AdapterSequencing

11、 OverviewSOLIDCreate a fragment libraryTemplate is generated through random shearingComplex samples(genomes,exons or amplicon pools)can be concatenated and sheared for directed resequencing of known populations of genomesFragmented templateComplex sampleLigate P1 and P2 primers to endSequencing Over

12、viewSOLIDSequencing OverviewSOLID2.Emulsion PCR+Templates Primers P11,000,000 individual readswith 400 bases read lengthHigh SpeedThe whole sequencing reaction finished in 10 hoursRoche/454 Titanium PicoTiterPlate2 Samples/Run4 Samples/Run16 Samples/Run41emPCRemPCRSequencingSequencingDNA LibraryDNA

13、LibraryPreparation Preparation DNA Library PreparationGenome fragmented by nebulizationAdaptor ligationsstDNA library created with adaptersA/B fragments selected using avidin-biotin purificationEmulsion PCR AmplificationAnneal sstDNA to an excess of DNA capture beadsEmulsify beads and PCR reagents i

14、n water-in-oil microreactorsClonal amplification occurs inside microreactorsSequencing By SynthesisLoad beads into PicoTiter PlateSequencing by synthesis Photons Generated are Captured by CameraSequencing Image CreatedRoche/454 GS FLX Workflow sstDNA sstDNA librarylibrarygDNAgDNAGenome fragmented by

15、 nebulizationNo cloning;no colony picking sstDNA library created with adaptersA/B fragments selected using avidin-biotin purification1.DNA library preparationSequencing OverviewGS Flx(454)2.Emulsion Based Clonal AmplificationClonallyClonally-amplified sstDNA attached to amplified sstDNA attached to

16、beadbeadsstDNA librarysstDNA libraryAnneal sstDNA to an excess of DNA capture beadsEmulsify beads and PCR reagents in water-in-oil microreactorsBreak microreactors,enrich for DNA-positive beadsClonal amplification occurs inside microreactorsSequencing OverviewGS Flx(454)3.Loading DNA Beads into the PicoTiterPlateLoad beads into Pico Titer PlateLoad Enzyme beadsWell diameter:average of 44 mSequencing OverviewGS Flx(454)The sequencing instrument consists of the following major subsystems:(a)a flui