Direct RNA sequencing(RNA测序)

Nature文章 RNA测序

Vol461|8October2009|doi:10.1038/nature08390

LETTERS

DirectRNAsequencing

FatihOzsolak1,AdamR.Platt1,DanR.Jones1,JeffreyG.Reifenberger1,LaurynE.Sass1,PeterMcInerney1,JohnF.Thompson1,JaysonBowers1,MirnaJarosz1&PatriceM.Milos1

Ourunderstandingofhumanbiologyanddiseaseisultimatelydependentonacompleteunderstandingofthegenomeanditsfunctions.Therecentapplicationofmicroarrayandsequencingtechnologiestotranscriptomicshaschangedthesimplisticviewoftranscriptomestoamorecomplicatedviewofgenome-widetranscriptionwherealargefractionoftranscriptsemanatesfromunannotatedpartsofgenomes1–7,andunderlinedourlimitedknowledgeofthedynamicstateoftranscription.Mostofthisbroadbodyofknowledgewasobtainedindirectlybecausecurrenttran-scriptomeanalysismethodstypicallyrequireRNAtobeconvertedtocomplementaryDNA(cDNA)beforemeasurements,eventhoughthecDNAsynthesisstepintroducesmultiplebiasesandartefactsthatinterferewithboththepropercharacterizationandquantificationoftranscripts8–18.Furthermore,cDNAsynthesisisnotparticularlysuitablefortheanalysisofshort,degradedand/orsmallquantityRNAsamples.HerewereportdirectsinglemoleculeRNAsequencingwithoutpriorconversionofRNAtocDNA.Weappliedthistechnologytosequencefemtomolequantitiesofpoly(A)1SaccharomycescerevisiaeRNAusingasurfacecoatedwithpoly(dT)oligonucleotidestocapturetheRNAsattheirnaturalpoly(A)tailsandinitiatesequencingbysynthesis.Weobservedtranscript39endheterogeneityandpolyadenylatedsmallnucleolarRNAs.Thisstudyprovidesapathtohigh-throughputandlow-costdirectRNAsequencingandachievingtheultimategoalofacom-prehensiveandbias-freeunderstandingoftranscriptomes.

cDNA-basedtranscriptomeanalysisapproachesbeingusedtodayexhibitseveralshortcomingsthatpreventusfromunderstandingtherealnatureoftranscriptomesandultimatelygenomebiology.Someoftheselimitationsare:(1)thetendencyofvariousreversetranscriptases(RT)togeneratespurioussecond-strandcDNAduetotheirDNA-dependentDNApolymeraseactivities9,10,18;(2)thegenerationofartefactualcDNAsduetotemplateswitching8,13,16,17orcontaminatingDNAandprimer-independentcDNAsynthesis11,12;and(3)theerror-prone15,19andinefficientnatureofRTsyieldinglowquantitiesofcDNA.Furthermore,mostRNAanalysistechnologiesrequirethesynthesisofnotjustthefirststrandcDNAbutalsoasecondstrandcDNAthatarebothsubjectedtofurtherligation/amplificationsteps,introducingyetmorebiases.TheselimitationsposeproblemsforthedeterminationofRNAstrandedness14,20,theidentificationofchimae-rictranscripts,quantificationofRNAspecies,andtheanalysisoflowquantity(,1nanogram)orshortRNAspecies,suchasthoseobtainedfromformalin-fixed,paraffin-embeddedtissuesamples.Becausealmostalltranscriptanalysistechnologiesinusetodaysufferfromthelimitationsbrieflysummarizedabove,thereisanever-growing

C

a

Fraction incorporated (%) 1007550250

b

C

VT

AUCGUGCAAAAAA

UGCACGUAAAAAA

AUACUCGAAAAAA

CGACUAUAAAAAA

UGACACUAAAAAA

AUCGUGCAAAAAA

C

VTUGCACGUAAAAAA

C

VT

C

VTC

VT

C

VT

C

VTUGACACUAAAAAA

G

TVTTTTTTATTTTTT

VT

CTTTTTT

VT

ATTTTTT

VT

ATTTTTT

VT

CGTTTTTT

VT

C

AVTTTTTTT

AUACUCGAAAAAA

CTTTTTT

CGACUAUAAAAAA

ATTTTTTATTTTTT

100200300400500600700

Time (s)Virtual terminator nucleotide analogue-A incorporationVirtual terminator nucleotide analogue-G misincorporation

AUCGUGCAAAAAA

GTTTTTT

UGCACGUAAAAAA

ATTTTTT

AUACUCGAAAAAA

CTTTTTT

CGACUAUAAAAAA

ATTTTTT

UGACACUAAAAAA

ATTTTTT

AUCGUGCAAAAAA

CGTTTTTT

UGCACGUAAAAAA

CATTTTTT

AUACUCGAAAAAA

CTTTTTT

CGACUAUAAAAAA

ATTTTTT

UGACACUAAAAAA

ATTTTTT

Figure1|DRSchemistryandsequencingsteps.a,Underoptimized

conditions,polymeraseexhibitsfastcorrectnucleotideincorporation(VT-A)andslowmisincorporation(VT-G)kinetics.b,DRSprocedure.Topleft:polyadenylatedand39-blockedRNAiscapturedonsurfacescoatedwithdT(50)oligonucleotide.A‘fill’stepisperformedwithnaturaldTTP,anda‘lock’stepwithfluorescentlylabelledVT-A,-Cand-Gnucleotides.Thesestepscorrectforanymisalignmentsthatmaybepresentinpoly(A/T)

duplexes,andensurethatthesequencingstartsinthetemplateratherthan

1

thepoly(A)tail.Imagingisperformedtolocatethetemplatepositions.Bottomleft:chemicalcleavageofthedye-nucleotidelinkerisperformedtopreparethetemplatesfornucleotideincorporation.Topright:incubationwithoneVTnucleotideandpolymeraseisperformed,followedbyimagingtolocatethetemplatesthatincorporatedthenucleotide.Bottomright:chemicalcleavageofthedyeallowsthesurfaceandRNAtemplatestobereadyforthenextnucleotideadditioncycle.

HelicosBioSciencesCorporation,OneKendallSquare,Cambridge,Massachusetts02139,USA.

814

Word文档免费下载Word文档免费下载:Direct RNA sequencing(RNA测序) (共6页,当前第1页)

你可能喜欢

  • 研究方案
  • 转录组测序
  • 高通量测序应用
  • RNA-seq
  • 基因组分析
  • 柯萨奇病毒
  • 转录组学

Direct RNA sequencing(RNA测序)相关文档

最新文档

返回顶部