Deep sequencingbased expression analysis shows major advances in robustness resolution and interlWord下载.docx
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*Towhomcorrespondenceshouldbeaddressed.Tel:
+31715269421;
Fax:
+31715268285;
Email:
p.a.c.hoen@lumc.nl
ReceivedAugust12,2008.RevisedSeptember16,2008.AcceptedSeptember29,2008.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALSANDMETHODS
RESULTS
DISCUSSION
SUPPLEMENTARYDATA
FUNDING
REFERENCES
Thehippocampalexpressionprofilesofwild-typemiceandmicetransgenicfor
C-doublecortin-likekinasewerecomparedwithSolexa/Illuminadeepsequencingtechnologyandfivedifferentmicroarrayplatforms.WithIllumina'
sdigitalgeneexpressionassay,weobtained
2.4millionsequencetagspersample,theirabundancespanningfourordersofmagnitude.Resultswerehighlyreproducible,evenacrosslaboratories.WithadedicatedBayesianmodel,wefounddifferentialexpressionof3179transcriptswithanestimatedfalse-discoveryrateof8.5%.Thisisamuchhigherfigurethanfoundformicroarrays.TheoverlapindifferentiallyexpressedtranscriptsfoundwithdeepsequencingandmicroarrayswasmostsignificantforAffymetrix.ThechangesinexpressionobservedbydeepsequencingwerelargerthanobservedbymicroarraysorquantitativePCR.Relevantprocessessuchascalmodulin-dependentproteinkinaseactivityandvesicletransportalongmicrotubuleswerefoundaffectedbydeepsequencingbutnotbymicroarrays.Whileundetectablebymicroarrays,antisensetranscriptionwasfoundfor51%ofallgenesandalternativepolyadenylationfor47%.Weconcludethatdeepsequencingprovidesamajoradvanceinrobustness,comparabilityandrichnessofexpressionprofilingdataandisexpectedtoboostcollaborative,comparativeandintegrativegenomicsstudies.
INTRODUCTION
Geneexpressionmicroarraysareatpresentthedefaulttechnologyfortranscriptomeanalysis.Sincetheyrelyonsequence-specificprobehybridization,theysufferfrombackgroundandcross-hybridizationproblemsandmeasureonlytherelativeabundancesoftranscripts
(1).Moreover,onlypredefinedsequencesaredetected.Incontrast,tag-basedsequencingmethodslikeSAGE(SerialAnalysisofGeneExpression)measureabsoluteabundanceandarenotlimitedbyarraycontent
(2).However,laboriousandcostlycloningandsequencingstepshavethusfargreatlylimitedtheuseofSAGE.Thishasradicallychangedwiththeintroductionofdeepsequencingtechnology,enablingthesimultaneoussequencingofuptomillionsofdifferentDNAmolecules.ThesharedideabehindthedifferentdeepsequencingapproachesistheclonaldetectionofsingleDNAmoleculesatphysicallyisolatedlocations(3–5).WeusedtheSolexa/Illumina1GGenomeAnalyzer,inwhichadaptersequences,ligatedtobothendsoftheDNAmolecule,areboundtoaglasssurfacecoatedwithcomplementaryoligonucleotides.Thisisfollowedbysolid-phaseDNAamplificationandsequencing-by-synthesis(6).Thesystemyieldsmillionsofshortreads(currentlyupto36bp),andisthereforeverysuitablefortag-basedtranscriptomesequencing.ThetechnologyisalsoreferredtoasDigitalGeneExpressiontagprofiling(DGE),andisessentiallyanimprovedversionoftheearlierMassivelyParallelSignatureSequencing(MPSS)technology(3,7).
ThefirststepsoftheprocedurearesimilartoclassicalLONG-SAGE.Tworestrictionenzymesareusedtogeneratetags,cuttingatthemost3'
CATGand17bpdownstreamofthefirstenzymesite.UnlikeinclassicalSAGE,tagsareneitherconcatenatednorcloned,butsequencedimmediately.Theunprecedentedsequencingdepthnowenablestheanalysisofindividualbiologicalsamples,whilepoolingofsampleswaspreviouslytheonlyaffordableoptioninSAGE.Ourresultsincludeastrikingexampleoftheintrinsichazardsofpoolinginexpressionprofiling.
Thebiologicalquestionaddressedinthecurrentstudywastheidentificationoftranscriptsdifferentiallyexpressedinthehippocampusbetweenwild-typeandtransgenicmiceoverexpressingasplicevariantofthedoublecortin-likekinase-1(Dclk1)gene.Thissplicevariant,
C-doublecortin-likekinase(DCLK)-short,makesthekinaseconstitutivelyactive(8),andcausessubtlebehavioralphenotypes(Schenketal.,inpreparation).TheexactsameRNAsampleshavebeenanalyzedbeforeonfivedifferentgenome-widemicroarrayexpressionprofilingplatforms(9),whichdetectedfewdifferencesinexpressionbetweenthetwogroups.WereportherethatDGEdetectsalotmoresmall,yetsignificantdifferencesbetweenthetwogroupsofmice,includingthoseinantisensetranscriptsandtranscriptswithdifferent3'
-untranslatedregions(UTRs).Furthermore,wediscusstheadvantagesofdeepsequencingovermicroarrayexpressionprofiling.
MATERIALSANDMETHODS
Samples
Wild-typemaleC57/BL6jandtransgenicmalemiceoverexpressingDCLK-shortwithaC57/BL6jbackgroundwereindividuallyhoused7dayspriortothestartoftheexperiment.Animalswerehousedunderstandardconditions,12h/12hlight/darkcycleandhadaccesstofoodandwateradlibitum.Wild-type(N=4)andtransgenic(N=4)tissuesampleswerecollectedbytakingthebrainfromtheskullandquicklydissectingoutbothhippocampi.Dissectionwasperformedat0°
CtopreventdegradationofRNA.Hippocampiwereputdirectlyinpre-chilledtubescontainingTrizolreagent(InvitrogenLifeTechnologies,Carlsbad,CA,USA).AllanimaltreatmentswereapprovedbytheLeidenUniversityAnimalCareandUseCommittee(UDEC#01022).
RNAextraction
Aftertransfertoice-coldTrizol,hippocampiwerehomogenizedusingatissuehomogenizer(Salm&
Kipp,Breukelen,TheNetherlands)andtotalRNAwasisolatedaccordingtothemanufacturer'
sprotocol.Afterprecipitation,RNAwaspurifiedwithQiagen'
sRNeasykitwithon-columnDNasedigestion.ThequalityoftheRNAwasassessedwiththeRNA6000LabchipkitincombinationwiththeAgilent2100Bioanalyzer(AgilentTechnologies,PaloAlto,CA,USA),usingtheEukaryoteTotalRNANanoassayaccordingtothemanufacturer'
sinstructions.
Sequencetagpreparation
SequencetagpreparationwasdonewithIllumina'
sDigitalGeneExpressionTagProfilingKitaccordingtothemanufacturer'
sprotocol(version2.1B).AschematicoverviewoftheprocedureisgiveninSupplementaryFigure1.1.pdfOnemicrogramoftotalRNAwasincubatedwitholigo-dTbeadstocapturethepolyadenlyatedRNAfraction.First-andsecond-strandcDNAsynthesiswereperformedwhiletheRNAwasboundtothebeads.Whileonthebeads,samplesweredigestedwithNlaIIItoretainacDNAfragmentfromthemost3'
CATGtothepoly(A)-tail.Subsequently,theGEXadapter1wasligatedtothefree5'
endoftheRNA,andadigestionwithMmeIwasperformed,whichcuts17bpdownstreamoftheCATGsite.Atthispoint,thefragmentsdetachfromthebeads.Afterdephosphorylationandphenolextraction,theGEXadapter2wasligatedtothe3'
endofthetag.APCRamplifcationwith15cyclesusingPhusionpolymerase(Finnzymes)wasperformedwithprimerscomplementarytotheadaptersequencestoenrichthesamplesforthedesiredfragments.Theresultingfragmentsof85bpwerepurifiedbyexcisionfroma6%polyacrylamideTBEgel.TheDNAwaselutedfromthegeldebriswith1xNEBuffer2bygentlerotationfor2hatroomtemperature.GeldebriswereremovedusingSpin-XCelluloseAcetateFilter(2ml,0.45µ
m)andtheDNAwasprecipitatedbyadding10µ
lof3Msodiumacetate(pH5.2)and325µ
lofethanol(–20°
C),followedbycentrifugationat14000r.p.m.for20min.Afterwashingthepelletwith70%ethanol,theDNAwasresuspendedin10µ
lof10mMTris–HCl,pH8.5andquantifiedtheDNAwithaNanodrop1000spectrophotometer.
SequencingusingSolexa/IlluminaWholeGenomeSequencer
Clustergenerationwasperformedafterapplying4pMofeachsampletotheindividuallanesoftheIllumina1Gflowcell.Afterhybridizationofthesequencingprimertothesingle-strandedproducts,18cyclesofbaseincorporationwerecarriedoutonthe1Ganalyzeraccordingtothemanufacturer'
sinstructions.ImageanalysisandbasecallingwereperformedusingtheIlluminaPipeline,wheresequencetagswereobtainedafterpurityfiltering.Thiswasfollowedbysortingandcountingtheuniquetags.Therawdata(tagsequencesandcounts)havebeensubmittedtoGeneExpressionOmnibus(GEO)underseriesGSE10782[NCBIGEO].
IlluminaDGEtagannotation
AlltagswereannotatedusingadatabaseprovidedbyIllumina.Briefly,apreprocesseddatabaseofallpossibleCATG+17-nttagsequenceswascreated,usingmousegenome(mm8versionfromUCSCsite)andmousetranscriptome(allrefseq,mRNAandESTsfoundinGenBankasofNovember2006andUnigeneversionMm159).AlltagswereclassifiedbasedonthelocationandorientationintheoriginalsequenceasoutlinedinSupplementaryTable1.Thegenomewasusedasabackbonefortagcl
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