Next-generation sequencing methods

ANRV353-GG09-20ARI25July200814:57Next-GenerationDNASequencingMethodsElaineR.MardisDepartmentsofGeneticsandMolecularMicrobiologyandGenomeSequencingCenter,WashingtonUniversitySchoolofMedicine,St.LouisMO63108;email:[e-mail].2008.9:387–402FirstpublishedonlineasaReviewinAdvanceonJune24,2008TheAnnualReviewofGenomicsandHumanGeneticsisonlineatgenom.annualreviews.orgThisarticle’sdoi:10.1146/annurev.genom.9.081307.164359Copyrightc2008byAnnualReviews.Allrightsreserved1527-8204/08/0922-0387$20.00KeyWordsmassivelyparallelsequencing,sequencing-by-synthesis,resequencingAbstractRecentscientificdiscoveriesthatresultedfromtheapplicationofnext-generationDNAsequencingtechnologieshighlightthestrikingimpactofthesemassivelyparallelplatformsongenetics.Thesenewmeth-odshaveexpandedpreviouslyfocusedreadoutsfromavarietyofDNApreparationprotocolstoagenome-widescaleandhavefine-tunedtheirresolutiontosinglebaseprecision.ThesequencingofRNAalsohastransitionedandnowincludesfull-lengthcDNAanalyses,serialanalysisofgeneexpression(SAGE)-basedmethods,andnoncodingRNAdis-covery.Next-generationsequencinghasalsoenablednovelapplicationssuchasthesequencingofancientDNAsamples,andhassubstantiallywidenedthescopeofmetagenomicanalysisofenvironmentallyderivedsamples.Takentogether,anastoundingpotentialexistsforthesetech-nologiestobringenormouschangeingeneticandbiologicalresearchandtoenhanceourfundamentalbiologicalknowledge.387ClickhereforquicklinkstoAnnualReviewscontentonline,including:•Otherarticlesinthisvolume•Topcitedarticles•Topdownloadedarticles•OurcomprehensivesearchFurtherANNUALREVIEWSAnnu.Rev.Genom.HumanGenet.2008.9:387-402.Downloadedfromarjournals.annualreviews.orgbyZhejiangUniversityon11/24/09.Forpersonaluseonly.ANRV353-GG09-20ARI25July200814:57INTRODUCTIONThesequencingofthereferencehumangenomewasthecapstoneformanyyearsofhardworkspentdevelopinghigh-throughput,high-capacityproductionDNAsequencingandas-sociatedsequencefinishingpipelines.Theap-proachused20,000largebacterialartificialchromosome(BAC)clonesthateachcontainedanapproximately100-kbfragmentofthehu-mangenome,whichtogetherprovidedanover-lappingsetortilingpaththrougheachhumanchromosomeasdeterminedbyphysicalmap-ping(31).InBAC-basedsequencing,eachBACcloneisamplifiedinbacterialculture,isolatedinlargequantities,andshearedtoproducesize-selectedpiecesofapproximately2−3kb.Thesepiecesaresubclonedintoplasmidvectors,am-plifiedinbacterialculture,andtheDNAisselectivelyisolatedpriortosequencing.Bygeneratingapproximatelyeightfoldoversam-pling(coverage)ofeachBACcloneinplasmidsubcloneequivalents,computer-aidedassemblycanlargelyrecreatetheBACinsertsequenceincontigs(contiguousstretchesofassembledsequencereads).Subsequentrefinement,in-cludinggapclosureandsequencequalityimprovement(finishing),producesasinglecon-tiguousstretchofhigh-qualitysequence(typi-callywithlessthan1errorper40,000bases).Sincethecompletionofthehumangenomeproject(HGP)(26,51),substantivechangeshaveoccurredintheapproachtogenomese-quencingthathavemovedawayfromBAC-basedapproachesandtowardwhole-genomesequencing(WGS),withchangesintheac-companyingassemblyalgorithms.IntheWGSapproach,thegenomicDNAissheareddi-rectlyintoseveraldistinctsizeclassesandplacedintoplasmidandfosmidsubclones.Oversam-plingtheendsofthesesubclonestogener-atepaired-endsequencingreadsprovidesthenecessarylinkinginformationtofuelwhole-genomeassemblyalgorithms.Thenetresultisthatgenomescanbesequencedmorerapidlyandmorereadily,buthighlypolymorphicorhighlyrepetitivegenomesremainquitefrag-mentedafterassembly.Despitethesedramaticchangesinsequenc-ingandassemblyapproaches,theprimarydataproductionformostgenomesequencingsincetheHGPhasreliedonthesametypeofcapillarysequencinginstrumentsasfortheHGP.How-ever,thatscenarioisrapidlychangingowingtotheinventionandcommercialintroductionofseveralrevolutionaryapproachestoDNAsequencing,theso-callednext-generationse-quencingtechnologies.Althoughtheseinstru-mentsonlybegantobecomecommerciallyavailablein2004,theyalreadyarehavingama-jorimpactonourabilitytoexploreandan-swergenome-widebiologicalquestions;morethan100next-generationsequencing–relatedmanuscriptshaveappearedtodateinthepeer-reviewedliterature.Thesetechnologiesarenotonlychangingourgenomesequencingap-proachesandtheassociatedtimelinesandcosts,butalsoacceleratingandalteringawideva-rietyoftypesofbiologicalinquirythathavehistoricallyusedasequencing-basedreadout,oreffectingatransitiontothistypeofread-out,asdetailedinthisreview.Furthermore,next-generationplatformsarehelpingtoopenentirelynewareasofbiologicalinquiry,includ-ingtheinvestigationofancientgenomes,thecharacterizationofecologicaldiversity,andtheidentificationofunknownetiologicagents.NEXT-GENERATIONDNASEQUENCINGThreeplatformsformassivelyparallelDNAsequencingreadproductionareinreasonablywidespreaduseatpresent:theRoche/454FLX(30)(),theIllumina/SolexaGenomeAnalyzer(7)(=203),andtheAppliedBiosystemsSOLiDTMSystem(