微量DNA的鸟枪法测序

APPLIEDANDENVIRONMENTALMICROBIOLOGY,Nov.2011,p.8071–80790099-2240/11/$12.00doi:10.1128/AEM.05610-11

Copyright©2011,AmericanSocietyforMicrobiology.AllRightsReserved.

Vol.77,No.22

EvaluationofaTransposaseProtocolforRapidGenerationof

ShotgunHigh-ThroughputSequencingLibraries

fromNanogramQuantitiesofDNA

RachelMarine,1ShawnW.Polson,1JacquesRavel,2GrahamHatfull,3DanielRussell,3

MatthewSullivan,4FrazSyed,5MichaelDumas,1andK.EricWommack1*

UniversityofDelaware,DelawareBiotechnologyInstitute,Newark,Delaware197111;UniversityofMarylandSchoolof

Medicine,InstituteforGenomeSciences,Baltimore,Maryland212012;UniversityofPittsburg,Departmentof

BiologicalSciences,Pittsburgh,Pennsylvania152603;UniversityofArizona,EcologyandEvolutionaryBiologyDeptartment,Tucson,Arizona857214;

andEpicenterBiotechnologies,Madison,Wisconsin537135

Received26May2011/Accepted3September2011

ConstructionofDNAfragmentlibrariesfornext-generationsequencingcanprovechallenging,especiallyforsampleswithlowDNAyield.ProtocolsdevisedtocircumventtheproblemsassociatedwithlowstartingquantitiesofDNAcanresultinampli cationbiasesthatskewthedistributionofgenomesinmetagenomicdata.Moreover,samplethroughputcanbeslow,ascurrentlibraryconstructiontechniquesaretime-consum-ing.ThisstudyevaluatedNextera,anewtransposon-basedmethodthatisdesignedforquickproductionofDNAfragmentlibrariesfromasmallquantityofDNA.Thesequencereaddistributionacrossninephagegenomesinamockviralassemblagemetpredictionsforsixoftheleast-abundantphages;however,therankorderofthemostabundantphagesdifferedslightlyfrompredictions.DenovogenomeassembliesfromNexteralibrariesprovidedlongcontigsspanningoverhalfofthephagegenome;infourcaseswherefull-lengthgenomesequenceswereavailableforcomparison,consensussequenceswerefoundtomatchover99%ofthegenomewithnear-perfectidentity.AnalysisofareasoflowandhighsequencecoveragewithinphagegenomesindicatedthatGCcontentmayin http://parisonsofphagegenomespreparedusingbothNexteraandastandard454FLXTitaniumlibrarypreparationprotocolsuggestedthatthecoveragebiasesaccordingtoGCcontentobservedwithintheNexteralibrarieswerelargelyattributabletobiasintheNexteraprotocolratherthantothe454sequencingtechnology.Nevertheless,givensuitablesequencecoverage,theNexteraprotocolproducedhigh-qualitydataforgenomicstudies.Formetagenomicsanalyses,effectsofGCampli cationbiaswouldneedtobeconsidered;however,thelibrarypreparationstandardizationthatNexteraprovidesshouldbene tcomparativemetagenomicanalyses.

Theextensiveavailabilityandlowcostofhigh-throughputDNAsequencing(HTS)hasrevolutionizedthelifesciences,enablingthesequencingoflargegenomesandopeningentirelynewapproachestogeneexpressionanalysis,mutationmap-ping,andanalysisofnoncodingRNAs(15–17,21).Inmicro-biology,HTShasgivenrisetothenewsubdisciplineofmet-agenomics,whichutilizessequenceinformationtoexploretheecologyandfunctionalcapabilitiesofentiremicrobialcommu-nities.Byavoidingcultivationorenrichment,metagenomicsallowsrelativelyunbiasedassessmentofthetaxonomiccom-positionandgeneticcontentofanautochthonousmicrobialcommunity(27).

Methodologicalrequirementsforanymetagenomicanalysisinclude(i)con rmationthattheoriginalsampleisfreefromcontaminationwithallochthonousornontargetmicroorgan-isms;(ii)unbiasedisolationofnucleicacidsfromallpopula-tionsofmicroorganismswithinasample;and(iii)isolationofenoughnucleicacidtomeettherequirementsofagivenHTSplatform(e.g.,Illuminaor454).Inparticular,obtainingsuf -cientamountsofnucleicacidhasbeenachallengeforthoseenvironmentswheremicrobialdensityisloworformicroor-ganismswithsmallgenomesizessuchasviruses.While454sequencingofgenomesfromcultivatedvirusescanbeachievedusingaslittleas1ngofDNA(11),sequencingoflargergenomesorviralmetagenomesfromenvironmentalsamplesrequiresalargeramountofstartingtemplate.

Todate,issuesofyieldhavebeenaddressedbystrategiesdesignedtononselectivelyamplifyenvironmentalDNA.Forexample,thelinkeradaptershotgunlibrary(LASL)protocolinvolvesshearingofenvironmentalDNAfollowedbyligationofadaptersequencesandsubsequentPCRampli cationofDNAfragmentsbytheuseofprimerstargetedtotheadaptersequences(5,25)(Fig.1).Despitethesuccessofthisapproach,theprotocolistime-consumingandcontainsseveralindepen-dentsteps,someofwhich(e.g.,shearingandgelpuri cationoffragments)canposerisksforcross-samplecontamination(5).Analternativeandsimplerapproachtoampli cationofenvi-ronmentalDNAhasbeentousecommerciallyavailablewhole-genomeampli cationkits,e.g.,REPLI-g(Qiagen)orGenomi-phi(GEHealthcare)(9,23).Thesekitsutilizethehighly

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*Correspondingauthor.Mailingaddress:DelawareBiotechnologyInstitute,UniversityofDelaware,Newark,DE19711.Phoneandfax:(302)831-4362.E-mail:wommack@dbi.udel.edu.

Supplementalmaterialforthisarticlemaybefoundathttp:///.

Publishedaheadofprinton23September2011.

Theauthorshavepaidafeetoallowimmediatefreeaccesstothisarticle.

微量DNA的鸟枪法测序

8072MARINEETAL.FIG.1.OverviewofsequencingstrategiesusedtoprepareviralgenomicDNAsamplesfor454sequencing.

processive,strand-displacingDNApolymeraseofbacterio-phagephi29toproducemicrogramquantitiesofDNAfromaslittleas10ngofinitialDNAtemplate(8)(Fig.1).Despitetheef ciencyandeaseofuseofmultiple-displacementampli ca-tion(MDA)proceduresinmetagenomics,thisapproachcanintroducesigni cantbiasindownstreamsequencelibraries.Forexample,circulargenomesarepreferentiallyampli edoverlinearones(24);moreover,asaconsequenceofstranddisplacementandtheformationofmultiplereplicationforks,chimericsequencescanoccurwithinmetagenomelibrariescreatedfromMDA-treatedenvironmentalDNA,makingcom-parisonsbetweenmetagenomespreparedusingthisprotocolessentiallynonquantitative(14,20,28).Thus,aneasyandquickprotocolthatproducessuf cienthigh-qualityfragmentedDNAforHTSfromsmallstartingquantitiesofinitialsampleDNAishighlydesirableforthoseresearchersinterestedinapplyingmetagenomicapproachestothestudyofmicroorgan-ismswithi …… 此处隐藏：35067字，全部文档内容请下载后查看。喜欢就下载吧 ……