Next-Generation-Sequencing(NGS)-高通量测序

NextGenerationSequencing(NGS,2nd~Generationsequencing)SangerSequencing(1stgenerationsequencing)1432Gelelectrophoresis5Chainterminationordideoxymethod(F.Sanger)5Steps:•Denaturation→Primerattachment→extensionofbases→Termination→GelelectrophoresisddNTP:•2’,3’-dideoxynucleotide•No!3’hydroxylresidueRunfourseparatereactionseachwithdifferentddNTPsRunonagelinfourseparatelanesReadthegelfromthebottomupSangerSequencing(1stgenerationsequencing)SoWhat’sWrongWithIt?Thedideoxy(Sanger)methodisgoodonlyfor500-750bpreactionsExpensiveTakesawhileThehumangenomeisabout3billionbpHumanGenomeProjectBeganin1990Why?•Humanevolution•Natureversusnurture•CausesofdiseaseShotgunSequencingUsedtosequencewholegenomesSteps:•DNAisbrokenuprandomlyintosmallerfragments•Dideoxymethodproducesreads•LookforoverlapofreadsStrandSequenceFirstShotgunSequenceAGCATGCTGCAGTCATGCT--------------------------TAGGCTASecondShotgunSequenceAGCATG--------------------------CTGCAGTCATGCTTAGGCTAReconstructionAGCATGCTGCAGTCATGCTTAGGCTANextGenerationSequencing:WhyNow?NGSisageneraltermreferingtoallpost-Sangersequencingtechnologiesthatenablemassivesequencingatlowcost.NGSmaybefurtherdividedintopolony-sequencingbasedtechnologieswhichrequiretheamplificationofDNApriortosequencing,andsinglemoleculesequencingwhichdonot.Motivationfornewtechnologiesdrivesitsrootsnotonlyfrompotentiallycommercialusagesuchasinpersonalisedmedicine,butalsofromgovernmentsupportedprojectssuchastheHGPorthe1000genomesprojectsaimingtosequencethegenomesof1000individualsaroundtheworldwithpricetagforgenomesequencingsinglegenomessetto50,000$.•Throughput,scalability,speed,andresolutionShortreadsapplications:Otherthande-novosequencing.Potentialapplicationsincludere-sequencing,andalsogeneexpressionanalysis,bothcanmakeuseofshortreadswhichareofferedbyallcurrenttechnologies.Sodespitetheread-lengthbarrierofthenewtechnologies,sequencersstillbecamecommercial.Advancementsinchemistry,microscopyandotherrelatedtechnologiesenabledthenewsequencingtechnologies.WhatistheNGS?Next-generationsequencingreferstonon-Sanger-basedhigh-throughputDNAsequencingtechnologies.MillionsorbillionsofDNAstrandscanbesequencedinparallel,yieldingsubstantiallymorethroughputandminimizingtheneedforthefragment-cloningmethodsthatareoftenusedinSangersequencingofgenomes.•Rapid(sequenceanentiregenomeinlessthanoneday)•Lowcostincomparisontotraditionaltechniques(Sangersequencing)TheserecenttechnologiesallowustosequenceDNAandRNAmuchmorequicklyandcheaplythanthepreviouslyusedSangersequencing.NextGenerationSequencingApplicationsdeNovosequencing(genomes,transcriptomes)Resequencing(genomes,exomes,customsequencecapture)RNA-seq(mRNA,miRNA,degradome)Chip-Seq(Chromatinimmuno-precipitationfollowedbysequencing)Methyl-seqGenomeDNARNAProteinDNAassemblyGenepredictionSequencealignmentSequencealignmentExpressionprofilingClassificationSNPdiscoveryExpressionquantificationProteinprofilingGenomecomparisonGeneclassificationAvailableNGSplatformsRoche454(2-generation,454FLX)Illumina/Solexa(2-generation,MiSeqorHiSeq)ABISOLiD(2-generation)IonTorrentPGM™(2.5-generation,LifeTechnologies)PacBio(3-generation)HeliScope(3-generation)OxfordNanopore(3-generation)…NGSPlatformsOverviewDifferindesignandchemistriesFundamentallyrelated-sequencingofthousandstomillionsofclonallyamplifiedmoleculesinamassivelyparallelmannerOrdersofmagnitudemoreinformation-willcontinuetoevolveAttractiveforclinicalapplications–individualsequencingassayscostlyandlaborious-serial“genebygene”analysisOligoH+PPiPacBioFluorescenceReal-timesequencingExperimentalSpace:Next-GenPlatform•Roche454FLX+:0.5-1x106reads/sample,800-1000nt–Small–MediumGenomedenovosequencing–LongAmplicon/Transcriptome•MiSeq:1-2x106readspersample,50–250nt–SmallgenomedeNovo/SmallAmplicon•HiSeq:10-100x106readspersample,50–150nt–CountingApplications:RNA-seq,ChIP-seq,RIP-seq,Methyl-seq–Largegenomedenovoandresequencing•PacBio:0.075x106reads/sample,1000–3000nt–WholetranscriptWhat,WhenandWhy???Sanger:Smallprojects(lessthan1Mbp)454-pyrosequencing:De-novosequencing,metagenomicsIllumina(Solexa),SOLiD,Heliscope:Geneexpression,protein-DNAinteractionsRe-sequencingTemplatePreparationTheDNA(orcDNA)youwishtosequenceisfragmentedAdapterfragmentsareligatedontotheendsoftheDNAfragmentsThepreparedlibraryisclonallyamplifiedRoche454,IonTorrent:EmulsionPCRIllumina/Solexa,SOLiD:ClustergenerationConceptualoverviewofsamplemultiplexing“Barcode”sequencing:smallbacterial/viralgenome,exomesMultiplexingenableslargesamplenumberstobesimultaneouslysequencingduringasingleexperiment.AdapterBarcodeDNABarcodeAdapter454GS_FLX(Pyrosequencing,Roche)DiphosphateMiSeqorHiSeq(Illumina/Solexa)①PreparegenomicDNA②AttachDNAtosurface③Bridgeamplification④Fragmentbecomedoublestranded⑤Denaturethedoublestrandedmolecules⑥Completeamplification4typeofOligoMiSeqorHiSeq(Illumina/Solexa)⑦Determinefirstbase⑧Imagefirstbase⑨Determinesecondbase⑩Imagesecondbase11Sequencereadsovermultiplecycles12Aligndata.50milliionclusters/flowcelleach1000copiesofthesametemplate1bill