The Drosophila melanogaster transcriptome by paired-end RNA sequencing

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The Drosophila melanogaster transcriptome by paired-end
RNA sequencing
Bryce Daines,1,2,6 Hui Wang,2,6 Liguo Wang,3,6 Yumei Li,1,2 Yi Han,2 David Emmert,4
William Gelbart,4 Xia Wang,1,2 Wei Li,3,5 Richard Gibbs,1,2,7 and Rui Chen1,2,7
1DepartmentofMolecularandHumanGenetics,BaylorCollegeofMedicine,Houston,Texas77030,USA;2HumanGenome
SequencingCenter,BaylorCollegeofMedicine,Houston,Texas77030,USA;3DanL.DuncanCancerCenter,BaylorCollege
ofMedicine,Houston,Texas77030,USA;4DepartmentofMolecularandCellularBiology,HarvardUniversity,Cambridge,
Massachusetts02138,USA;5DepartmentofMolecularandCellularBiology,BaylorCollegeofMedicine,Houston,
Texas77030,USA
RNA-seq was used to generate an extensive map of the Drosophila melanogaster transcriptome by broad sampling of 10
developmentalstages.Intotal,142.2millionuniquelymapped64–100-bppaired-endreadsweregeneratedontheIllumina
GAIIyielding3563sequencingcoverage.Morethan95%ofFlyBasegenesand90%ofsplicingjunctionswereobserved.
Modificationsto30%ofFlyBasegenemodelsweremadebyextensionofuntranslatedregions,inclusionofnovelexons,
andidentificationofnovelsplicingevents.Atotalof319noveltranscriptswereidentified,representinga2%increaseover
the current annotation. Alternate splicing was observed in 31% of D. melanogaster genes, a 38% increase over previous
estimations,butsignificantlylessthanthatobservedinhigherorganisms.Muchofthissplicingissubtlesuchastandem
alternatesplicesites.
[Supplementalmaterialisavailableforthisarticle.ThesequencingdatafromthisstudyhavebeensubmittedtotheNCBI
Sequence Read Archive (http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi) under accession no. SRA012173 and to the
NCBIGeneExpressionOmnibus(http://www.ncbi.nlm.nih.gov/geo/)underaccessionno.GSE24324.]
High-throughputsequencingofcDNAlibraries(RNA-seq)isanef- splicingisoformsisenabledwithoutdependenceonpriorknowl-
fectiveandaccuratemethodfortranscriptomeprofiling.RNA-seq edge. While splicing variants can also be detected with custom
hasbeenappliedinmanyorganismsincludinghumans(Campbell microarrays, probes must be specifically designed to interrogate
etal.2008;CloonanandGrimmond2008;Cloonanetal.2008; exon–exonjunctionsandarethuslimitedinscopetoknownor
Marioni et al. 2008; Mortazavi et al. 2008; Mudge et al. 2008; predictedjunctions.Finally,directannotationofgenemodelsand
Nagalakshmietal.2008;Panetal.2008;Shendure2008;Sultan noveltranscriptdiscoveryarepossiblewithRNA-seq(Yassouretal.
etal.2008;Wangetal.2008;Filichkinetal.2009;Levinetal.2009; 2009).Thiscanbeaccomplishedbyalignmentofreadstoarefer-
Perkinsetal.2009;Wurtzeletal.2009).RNA-seqisnotdependent ence genome and inference of transcript models from coverage
onpriorknowledgeandrequiresnodesignwork,thusreducing andsplicingdata.Whilegenome-tilingarrayscanalsobeusedto
laborandenablingdenovodiscovery.Severaladditionalfeatures identifynoveltranscriptionalunits,RNA-seqisnotsubjecttothe
ofRNA-seqmakeitlikelytosupplantmicroarraysasthekeytech- inherentlimitationsofhybridizationplatforms.Duetotheseveral
nologyfortranscriptomeprofiling,includingaccurateandsensi- advantagesoftheRNA-seqapproachwe have appliedthis tech-
tivemeasuringofexpressionlevel,identificationofalternatesplic- nologytocharacterizethetranscriptomeofD.melanogaster.
ingisoforms,anddirectdiscoveryofnoveltranscripts(Graveley D. melanogaster is an important model organism and has
2008;Shendure2008). contributedsignificantlytoourunderstandingofgeneexpression
Highlyaccuratedigitalmeasurementofgeneexpressionby and development. The transcriptome of D. melanogaster is well
RNA-seqcanbeobtainedbycountingthenumberofsequencing annotated and functionally characterized. Analysis of transcript
reads which map to annotated transcripts from appropriately expression using spotted arrays has yielded insight into the de-
preparedlibraries(Mortazavietal.2008;Wangetal.2009).Inthis velopmental expression patterns of annotated D. melanogaster
way,RNA-seqoffersincreasedsensitivityandlargerdynamicrange transcripts;however,thisplatformislimitedtoasubsetofanno-
overmicroarray,effectivelydetectingraretranscriptswithgreater tated genes (Arbeitman et al. 2002). Additionally, tilling arrays
sensitivityandabundanttranscriptswithsuperiordiscrimination havebeenusedtodemonstratethat>29%ofunnanotatedintrons
(Marionietal.2008).Furthermore,RNA-seqgeneratesreadswhich andintergenicregionsaretranscribedwithinthefirst24hofde-
straddle exon–exon junctions (junction reads). When junction velopmentatspecifictimepointsindevelopment(Manaketal.
reads are properly aligned to a reference genome the associated 2006).Tillingarrays,however,arelimitedintheirresolutionbythe
splice event can be inferred. Thus, direct discovery of alternate size of probes used. Expressed sequence tags (ESTs) provide the
primary evidence for annotation efforts. Although more than
800,000 D. melanogaster ESTs have been sequenced, not all pre-
6Theseauthorscontributedequallytothiswork. dictedorannotatedgenesaresupportedbyESTs(Stapletonetal.
7Correspondingauthors. 2002a,b). Furthermore, these ESTefforts have focused primarily
[email protected]. onembryoandadultlibraries,specificallyadultheadandgonad
[email protected].
tissues, leaving intermediate stages of development underrep-
Articlepublishedonlinebeforeprint.Article,Supplementalmaterial,andpub-
licationdateareathttp://www.genome.org/cgi/doi/10.1101/gr.107854.110. resented.ContinuationofSanger-basedESTsequencingeffortsis
21:000–000 (cid:2) 2011 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/11; www.genome.org GenomeResearch 1
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Daines et al.
notanidealsolutionforimprovementofgenemodelsbecauseof setofcuratedmodels.Otherannotationsbasedonexperimental
its higher cost in comparison to next-generation sequencing and computational analysis are also available; a prominent ex-
platforms.Morerecently,highthroughputsequencinghasbeen ampleisthegenemodels(MB7)producedbythemodENCODE
recognized as an ideal solution for transcriptome profiling with consortium (http://www.modENCODE.org/). For robustness we
basepairresolution.InD.melanogasterthe59-endSAGEmethod used both of these annotations as references throughout our
has been combined with Illumina sequencing to generate a de- analysisofRNA-seqdata.
tailedprofileoftranscriptionstartsitesthroughoutdevelopment Read alignment with BLATenabled the direct detection of
(Ahsanetal.2009).RNA-seqwillenableinterrogationofalltran- junction reads. These reads are essential to the identification of
scripts,includingpotentiallyunannotatedtranscriptswithoutthe exon–exon junctions and alternate splicing. To maximize sensi-
need for further EST characterization (Torres et al. 2008). We tivity for detecting junction reads we used two independent
therefore propose that characterization of the D. melanogaster computationalapproaches(seeSupplementalmaterials).Approx-
transcriptome by RNA-seq technology will greatly facilitate on- imately6%ofalluniquelyalignedreadswerejunctionreads,of
goingannotationefforts. which, 94% are consistent with annotated FlyBase events, sug-
gesting high concordance between experimental evidence and
existing annotations. In total, 90.6% of annotated junctions
Results
(46,820)wereobservedwith93.6%consistencybetweenthetwo
approaches(seeSupplementalTable1;SupplementalFig.1).Ad-
ExtensivetranscriptomeprofilingofD.melanogasterbyRNA-seq
ditionally, junction reads supported ;120,000 candidate novel
In order to gain a broad sampling of the D. melanogaster tran- junctions.Analyticalmethodswereusedtoreducetheseto7776
scriptome, RNA-seq experiments were performed at all stages of well-supportedcandidates(seeSupplementalmaterials),ofwhich
theD.melanogasterlifecycle.Poly(A)+transcriptsfrom10distinct 2012(25.9%)arereportedinthemodENCODEMB7genemodels.
stages during the live cycle of D. melanogaster were isolated to To determine the technical robustness of RNA-seq we con-
generatedcDNAlibrarieswhichweresequencedontheIllumina sidered the positive predictive value (PPV) and sensitivity of the
GAIIinstrument.AsshowninTable1,12independentcDNAli- platform.ThePPVofRNA-seqforexpressedsequencesisdefinedas
braries were generated, including embryonic, larval, pupal, and thepercent ofuniquely alignedreads whichoverlapwithanno-
adult.Somelibrarieswerestagedasspecificwindows:2–4-hem- tated transcripts. Across all samples, 97.6% of uniquely aligned
bryo,14–16-hembryo,thirdinstarlarva,3-dpupa,and17-dadult. readsoverlapwithannotatedsequences,suggestingthatRNA-seqis
Additionallibrarieswerederivedfrombroadlystagedmixedsam- highlyspecifictoexpressedsequences(Table1).Thesensitivityof
ples: embryo, larvae, and pupa. Three-day-old male and female RNA-seqiscalculatedasthepercentageofannotatedbasescovered
adultsweresequencedseparatelyfordiscoveryofsex-specificvar- bysequencedreadsfortheentiretranscriptomeandforeachgene
iation.Finally,onelibraryofmixed-agepupalRNAwassequenced individually.Withallpooledreadsconsidered,93.4%and91.9%
inreplicateasavalidationofthetechnology.Theselibrarieswere of FlyBase and modENCODE annotated bases were observed re-
selectedtorepresentabroaddiversityofRNAmolecules(Arbeitman spectively,suggestingahighdegreeofsensitivity(Fig.1A).Random
etal.2002).Atotalof272millionpaired-endreadsof64–100bpin sampling of pooled reads from all samples was used to simulate
lengthwereobtained. transcriptomecoverageatvariousreaddepths.Thecurveofthese
SequencedreadswerealignedtotheD.melanogastergenome plotsisasymptotic,suggestingthatdeepersequencingwouldoffer
(UCSCdm3/FlyBaser5.23)usingBLAT(Kent2002).Intotal,180.5 lowreturnsatthebase-pairlevel.Themajorityofannotatedgenes
million reads were aligned to the genome with 142.2 million arewellcoveredbyRNA-seqreads.Forexample,>70%ofannotated
mapped touniquelocations,representing >3563sequencecov- genesarecoveredfor>95%oftheirlength(Fig.1B).Weconclude
erage of the 30Mb D. melanogaster transcriptome. The current that RNA-seq is a robust technology for transcriptome profiling
FlyBaseannotation(v5.23)wasusedtocalculatedigitalcountsof includingthecharacterizationofgenemodels.
geneexpressionforeachoftheannotated14,797FlyBasegenes. Weobserved,however,thatnotallannotatedgenesarewell
TheFlyBaseannotationisoftenconsideredthe‘‘goldstandard’’of represented by RNA-seq reads. In total, 1133 genes (7.7%) are
D.melanogastergenemodelsbecauseitisthemostcomprehensive coveredat<25%byuniquereads.Todeterminethelimitationsof
Table1. RNA-collectionandsequencingyields6903coverageoftheD.melanogastertranscriptome
Readlength Readsproduced Sequencedbases Alignedreads Readsaligned Uniquereadsoverlapping
(bp) (millions) (Mb) (millions) uniquely(millions) exons(%)
Earlyembryo(E2–4hr) 62 16.0 989.9 10.5 8.9 98.7
Lateembryo(E14–16hr) 75 22.9 1715.7 4.8 3.3 97.7
Embryo(E2–16hr) 75 20.3 1520.6 13.0 10.0 98.0
Embryo(E2–16hr100) 100 7.3 730.8 6.9 6.0 98.7
3rdinstarlarva(L3i) 75 27.3 2044.8 21.1 12.5 96.4
3rdinstarlarva(L3i100) 100 14.6 1455.8 13.1 7.7 99.0
Larva(Larva) 75 33.2 2489.7 26.3 20.7 98.6
3-dpupa(P3d) 75 25.0 1872.5 18.3 14.4 95.9
Pupa(Pupa) 75 30.5 2286.3 25.8 22.5 96.5
Maleadult(MA3d) 75 19.8 1481.9 6.4 5.3 96.6
Femaleadult(FA3d) 75 24.6 1847.9 11.0 9.6 98.8
17-dadult(A17d) 75 30.7 2301.4 23.4 21.2 97.9
Total 272.0 20,737.2 180.5 142.2 97.6
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The D. melanogaster transcriptome by RNA-seq
Figure1. DeepRNA-seqcovers93%oftheannotatedD.melanogastertranscriptome.(A)93.4%and91.9%oftheFlyBaseandmodENCODEan-
notatedtranscriptomeisobservedbypooledRNA-seqreads.Simulatedreaddensitiesindicatethatthecurrentdepthofsequencingapproachessatu-
ration.(B)AnnotatedtranscriptsarewellcoveredbyRNA-seqreads.Morethan70%ofannotatedgenesarecoveredfor>95%oftheirlength.(C)Most
unobservedgenescanbecategorizedintoclasseswhicharenotexpectedtobeobservedduetosize,genomicduplication,orlackofpolyadenylation.(D)
AnovelexonisidentifiedinwupA,whichissupportedbytwonoveljunctionswith487and428reads,respectively.(E)RT-PCRdesignedtovalidatethe
noveljunctionsuccessfullyamplifiesanappropriatelysizedproductfromcDNAbutnotfromgenomicDNA.
RNA-seqitisimportanttounderstandthenatureoftheseunder- cantmodificationwastheadditionofexonsequences,whichaf-
representedgenes.Thelackofgenecoveragemaybeduetotwo fected 25% of gene models (3692). For example, a novel exon
primary causes: either reads were generated but could not be identifiedinwupAisdepicted(Fig.1D).RT-PCRexperimentswere
mappeduniquelytothereferencesequence,ornoreadsoriginated designedtoconfirmtheexpressionofthisnovelexon.Subsequent
fromthegeneinquestion.Themajorityofunderrepresentedgenes isolationand sequencingofan appropriatelysized PCRproduct
fallintoclasseswhicharenotexpectedtobeobservedduetose- confirmedtheprediction.Additionally,theuntranslatedregions
quencefeaturesandlimitationsofthechosenmethodology(Fig. (UTRs)of>8%ofgenes(1274)wereextendedbyRNA-seqcoverage
1C). For example, tRNAs, histones, 5SrRNAs, and Ste/Su(Ste) are ingeneswithpreviouslyunannotatedUTRs.TheabilityofRNA-
highlysimilarandoccuringeneclusters.Readsfromthesegenes seqtoaccuratelydefine59and39UTRsislimitedduetobiasesin
arenotlikelytomapuniquelytothereferencegenome.Further- sequencing;therefore,werestrictedtheseanalysistogeneswhose
more,unpolyadenylatedtranscriptssuchasmiRNAandsnoRNA UTRswerepreviouslyunannotated.
arenotlikelytobeobservedbecauseofthepoly(A)selectionused
intheRNApurificationstrategy(seeMethods).Finally,128gus-
RNA-seqdetects319noveltranscripts
tatory,odorant,andionotropicreceptors(smallmoleculeswhose
expressionistightlyrestrictedtospecificneuronalcells)werenot Inadditiontomodifyingcurrentgenemodels,RNA-seqenables
observedinourdatapotentiallyduetotheirrestrictedexpression the identification of novel transcripts. Evidence from junction
patternalthoughsizeselectionmayalsoplayarole(seeMethods). reads and read coverage was combined to identify 319 novel
Weconcludethatunderrepresentedgenesarepredominantlydue transcriptsmappingcompletelyoutsideofFlyBasegeneannota-
to explainable limitations of the library preparation and read tions (see Methods). These novel genes are distributed across
mappingmethodologiesemployed. chromosomes roughly as expectedby chromosome size and be-
tween strands in nearly equal proportions (Fig. 2A). Several fea-
turesstandoutforthesenovelgenescomparedtoannotatedgenes.
RNA-seqdataprovidesevidenceformodifying30%
First, these novel genes are on average smaller than annotated
ofgeneannotations
genes.TheFlyBasemediangenesizeis1560bp,whilethemedian
ThedepthofourRNA-seqdataenablesustoevaluatenotonlythe sizeofthesenoveltranscriptsis315bp(Fig.2B).Second,thema-
accuracyofcurrentgenemodelannotationsbutalsotheircom- jorityofnoveltranscriptscontainonlytwoexons(Fig.2C).Third,
pleteness. While RNA-seq and current gene models are largely thesenovelgenesareoftenexpressedinstageand/orsex-specific
consistent,asignificantportionofcurrentmodelsareincomplete. patterns (Fig. 2D). For example, many novel transcripts are
Evidence from junction reads and coverage was used to extend expressed in male but not female adults, although they are ob-
annotatedgenemodels(seeMethods).Evenunderstringentcri- servedinmixedlarvaandpupasamples(Fig.2D,‘‘Male’’).Other
teria,30%ofgenes(4418)underwentsomelevelofmodification clusters express most abundantly in a specific stage (Fig. 2D,
duringthisprocess(seeSupplementalTable2).Themostsignifi- ‘‘Larva’’).
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Daines et al.
Figure2. RNA-seqdetects319noveltranscripts.(A)NoveltranscriptsdetectedbyRNA-seqareidentifiedonallchromosomesdistributedasexpected
bychromosomesize.(B)Noveltranscripts(mediansize315bp)aremuchsmallerthanannotatedFlyBasetranscripts(mediansize1560bp).(C)The
majorityofnoveltranscriptsidentifiedinthisstudyhavetwoexons.(D)Clusteringidentifiesmanynoveltranscriptsexpressedatspecifictimepointsorin
sex-specificpatterns.Forexample,manynoveltranscriptsareexpressedinmalebutnotfemaleadults,althoughtheyareobservedinmixedlarvaandpupa
samples(Male).Otherclustersexpressmostabundantlyinaspecificstage(Larva)Anoveltranscriptisdepicted,andtheassociatedjunctionissupported
by50sequencedreads.ExperimentalvalidationbyRT-PCRwasobtainedinpupaecDNA(seeSupplementalFig.2).
Forvalidationoftheseresults,noveltranscriptsderivedfrom Supplemental Fig. 3). The largest differences between the two
RNA-seqwerecomparedtotworecentlydescribeddatasets.One platformsoccurforgeneswhichweredetectedatverylowlevelson
study using comparativegenomic techniques published a set of thearraybutwereidentifiedinarangeofexpressionbyRNA-seq.
146novelintronsconfirmedbyESTsorRT-PCR(Hilleretal.2009). RNA-seqrobustnesswasalsoobservedintechnicalreplicates.Total
RNA-seqidentified55%oftheseintrons(80of146).Additionally, RNAextractedfrompupa-stagedflieswassplittoderivetwosepa-
RNA-seqidentifies61%predictedcoding introns(57 of94) and ratesequencinglibraries.Theselibrariesweresequencedandana-
18% of predicted introns of messenger-like-noncoding-RNAs lyzedinparallel,andthecorrelationcoefficientofthesereplicates
(mlncRNAs) from the same study (23 of 129). These results are wascalculatedasR=0.99(Fig.3D).Finally,wefoundthatbyran-
consistentwiththeauthors’ownexperimentalvalidationrateat dom sub-sampling of RNA-seq reads at various depths, gene ex-
;60%,whichwaslowestforthemlncRNAsdueprimarilytolow pressionlevelsarehighlyrobustandR=0.99correlationcouldbe
andstage-specificexpression.Thusweconcludethatourcurrent obtainedwithasfewas100,000reads(seeSupplementalFig.3).
depthandbreadthofsequencingisadequateforidentificationof To determine which genes are expressed in each develop-
somenoveltranscripts.Second,significantoverlaphasbeenob- mental stage, the read counts and normalized expression levels
served between our data set and the novel genes identified by werecalculatedforeachannotatedfeature(seeMethods).Libraries
modENCODE.Ofthenoveltranscriptsidentified,45%(144outof weregroupedaccordingtothemajorstagesasappropriatetocon-
319) are supportedby themodENCODE annotation. Finally, by siderthetotalnumberofgenesobservedineach.Intotal,84.4%of
RT-PCRthejunctionssupportingnineofthesenoveltranscripts FlyBase genes (12,490) are expressedtwofold above background,
werevalidated.AnexamplenoveltranscriptisillustratedinFigure calculatedastheaveragecoverageofintergenicregions,inatleast
2E; this junction, which was supported by only 50 sequenced one sample. Furthermore, 48.8% of FlyBase genes (7214) were
reads,was validatedby RT-PCRin thepupaecDNA.Theexperi- expressedtwofoldabovebackgroundinallstages,suggestingthat
mentalevidenceforthesevalidationsisincludedintheSupple- thisgroupofgenesisbroadlyexpressedacrosstheD.melanogaster
mentalmaterial(seeSupplementalFig.2). lifecycle(Fig.3A).Onaverage,67.5%ofgenes(9995)areobserved
withineachstage.SincethemajorityofD.melanogastergenesare
developmentallyregulated(Arbeitmanetal.2002),wesoughtto
RNA-seqprovidesanextensivedigitalexpressionprofile
identify genes which are differentially regulated during devel-
ofD.melanogasterdevelopment
opment and those which are invariant in their expression. De-
RNA-seq provides ‘‘digital’’ reading of gene expression levels velopmentallyregulatedgeneswereidentifiedbyconsideringthe
(Mortazavietal.2008).To assesswhetherRNA-seqisconsistent difference between maximum and minimum expression levels
withprevioustechnologyinmeasuringgeneexpressionweana- across all samples. The majority of genes expressed above back-
lyzed RNA expression in parallel on the microarray platform. A ground(85.7%;10,702of12,490)exhibitfourfoldorgreaterdif-
singleRNAlibrarygeneratedpreviouslyandanalyzedontheAffy- ferencesinexpressionlevelbetweentheirhighestandloweststages
metrix microarray was reanalyzed by RNA-seq. Expression levels ofexpression,suggestingdevelopmentalregulation(Fig.3B).Ex-
wereevaluatedandnormalizedappropriatelyforeachplatformand pressedgeneswhichexhibitconsistentexpressionwereidentified
thecorrelation coefficient calculated as R = 0.76 (Spearman), in- bycalculatingthecoefficientofvariation(Fig.3C).GeneOntology
dicatinga strong positive correlation between theplatforms (see (GO) analysiswasperformed onthe3% of genes exhibiting the
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The D. melanogaster transcriptome by RNA-seq
Figure3. Sex-biasedexpressionoccursinone-thirdofgenes.(A)Onaverage,9995genesareobservedineachstage,7214genesaresharedbetween
allstages,and12,490genesareexpressedinoneormorestages.(B)85.7%ofgenesexhibitgreaterthanfourfolddifferenceinexpressionbetween
maximumandminimumexpressiontimepoints.(C)Thedistributionofcoefficientofvariationcalculatedforeachgeneidentifiesgeneswhoseexpression
ishighlyconsistentacrossdevelopment.(D)TechnicalreplicatesofpupaRNA-seqexhibitnearlyperfectcorrelation(R=0.99).(E)Femalesandearly
embryosexhibitahighdegreeofcorrelation(R=0.80).(F)5251exhibitfourfolddifferenceinexpressionlevelbetweenmalesandfemales:1088genesup-
regulatedinfemalesareconsistentwiththeirexpressioninearlyembryos(red),suggestingtheirimportanceinembryonicdevelopment,3486genesare
up-regulatedinmales(blue).(G)Geneswithoutaknownorthologareabundantlyexpressedinmales,manyofwhichhaveknownmale-specificfunctions
includingseminalfluidproteinsandmale-specifictranscripts.(H)Manygenesonunassembledheterochromatin(chrU)exhibitmale-specificexpression.
(I)GenomicPCRresultssuggestCG40968,CG40583,CG40992,andCG41561arelinkedtochromosomeY.
most consistent expression (Carmona-Saez et al. 2007; Nogales- andfemaleadults(Fig.3E,F).Ofthese,1088genesareup-regulated
Cadenasetal.2009).Manyofthesegenesareinvolvedwithbasic infemaleovermalebutconsistentbetweenfemaleandembryonic
cellularprocesses(246genes,P=1.47310(cid:2)16)andfunctions,such expression(Fig.3E,F).GOanalysisofthesegenesidentifiedfunc-
astranslation(14genes,P=1.39310(cid:2)6). tions related to embryonic development, including 45 genes in-
Ithasbeenreportedthatasignificantnumberofgenesare volvedinoogenesis(P=5.99310(cid:2)13),25genesinvolvedwithDNA
differentially expressed between males and females (Arbeitman replication (P = 4.5 3 10(cid:2)16), and 17 genes involved with rRNA
et al. 2002). We observed 5251 genes (35.5%) that exhibited processing(P=1.08310(cid:2)9).Intotal,3486genesareup-regulatedin
fourfolddifferenceintheirexpressionlevelsbetween3-d-oldmale malesoverfemales(Fig.3F).GOanalysisidentifiedmanyexpected
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Daines et al.
functional groups, including 27 genes involved with sensory Table2. Alternatesplicingisobservedin36%ofD.melanogaster
perception of chemical stimulus (P = 2.297 3 10(cid:2)6), eight genes genes
involvedwithpost-matingbehavior(P=4.64310(cid:2)6),38genesin-
Event Annotated Observed Total Increase
volvedwithmicrotubule-basedmovement(P=8.06310(cid:2)6),and32
genesinvolvedwithoxidationreduction(P=1.16310(cid:2)5). Skippedexon(SE) 3763 90% 6464 72%
Anotherclassofgenesup-regulatedinmalesconsistsofgenes Retainedintron(RI) 1605 80% 2222 38%
withoutanidentifiedorthologamongthe12DrosophilaGenomes Alternatedonorsplice 2963 78% 4714 59%
site(ADSS)
Project(Fig.3G;Clarketal.2007).Of1043geneswithoutanan-
Alternateacceptor 1978 73% 3697 87%
notated Drosophila ortholog, nearly 200 exhibited male-specific splicesite(AASS)
expressionpatterns;incontrast,lessthan20genesexhibitedafe- Alternatefirstexon(AFE) 1082 70% 1270 17%
male-specificexpressionpattern.Itisconceivablethatthesenon- Alternatelastexon(ALE) 27 70% 83 207%
conserved genes are more rapidly evolving and that their male- Alternatesplicinggenes 3353 86% 4618 38%
specific expression can be related to the forces of male-driven
Manyspeciesofalternatesplicingeventswereidentifiedandcharacter-
evolution.Manyknownmale-specifictranscriptsandspermfac-
izedinthisstudy,includingskippedexons(SE),retainedintron(RI),al-
torsdonothaveanidentifiableorthologamongDrosophilaspecies, ternate donor splice site (ADSS), alternate acceptor splice site (AASS),
supporting this hypothesis; however, several genes do not have alternatefirstexon(AFE),andalternatelastexon(ALE).Ofallannotated
describedmolecularfunctionsanditisreasonabletopostulatethat alternatesplicingevents86%weredetectedwithRNA-seqdata.Addi-
tionally,a38%increasewasobservedinthenumberofgenesdetected
someofthesemayalsobelinkedtomalereproduction(seeSup-
withalternatespliceisoforms.
plementalTable3).
commonannotatedeventistheSE,ofwhichweobserved90%of
IdentificationofnovelD.melanogasterY–linkedgenes
events (3390). The SE was also the most common novel event
We observed that many genes with male-specific patterns of ex- observed,ofwhichweobserved3074newevents.Theotherclasses
pressionarelocatedonchrU(Fig.3H).TheD.melanogasterchrU werealsobroadlyobserved(Table2).Of3353geneswith anno-
consistsofunassembledcontigswhicharelargelyheterochromatic tatedsplicingeventsprofiledinthisstudy(23%ofallgenes)we
andhavenotbeenassignedtophysicalgenomiclocationsdueto detectedannotatedalternatesplicingeventsin86%(2899genes).
difficultyinassembly.ChromosomeYiswhollyheterochromatic; Additionally, we observed many novel alternatesplicing events.
therefore,muchofitssequenceiscurrentlyunassembledandas- ThelargestincreaseoverannotationistheALEeventofwhichwe
signedaschromosomeU.Onlyeightgenesareannotatedonchro- detected64novelevents,atwofoldincrease.
mosomeYaccording tothecurrentFlyBase annotation. We hy- Alternate splicing is an essential feature of sexual determi-
pothesizedthatgeneswithmale-specificexpressionannotatedon nationanddifferentiationinD.melanogaster,andithasbeenesti-
chrU might potentially be linked to chromosome Y. To test for matedthatasmanyas22%ofmulti-transcriptgenesexhibitsex-
Y-linkage,PCRprimersweredesignedforeightgenesexhibiting biasedsplicingpatterns(Telonis-Scottetal.2009).Inourdataset
male-specificexpression.AmplificationofgenomicDNAinmales 3-d-old males and females were sequenced separately to enable
butnotinfemalesisindicativeofY-linkage.PCRproductswere assessmentofsex-specificalternatesplicing.Ofalternatelyspliced
obtainedonlyinmalesforfouroftheeighttestedgenes:CG41561, genes,2308wereexpressedinbothmalesandfemalesatasufficient
CG40968,CG40583,CG40992(Fig.3I).Evidencelinkingthreeof level to enable examination of alternate splicing. Of genes with
these genes to the Y chromosome has been reported previously annotated alternate splice isoforms, differential splicing was ob-
(Carvalhoetal.2000,2001,2003;Vibranovskietal.2008;Krsticevic servedin23.5%.Inarecentreport,interrogationof417geneswith
etal.2009).AssignmentofCG41561tochromosomeYisbelievedto exon-microarraysledtoidentificationof33genesshowntohave
beanoveldiscoveryasnoFlyBaseorGenBankrecordlinksthisgene sex-biasedsplicing.Amongthesecandidategenes,weconfirmed14
tochromosomeY. (Telonis-Scottetal.2009),mostunconfirmedgeneswereexpressed
belowourthresholdinoneortheothersex(seeSupplementalTable
Alternatesplicingisobservedin31%ofD.melanogastergenes 4).Therefore,ouranalysisisquitesensitive,identifiedhundredsof
specifictranscripts,andconfirmsthatwidespreadsex-specificreg-
Akeyfeatureofeukaryoticgeneexpressionisalternatesplicing.In
ulationofalternatesplicingoccursinD.melanogaster.
humans, as many as 95%of transcriptsare believedtobe alter-
natelyspliced;incontrast,inthecurrentFlyBaseannotationonly
TandemalternatesplicingintheD.melanogastertranscriptome
26%ofgeneshavemultiplespliceisoforms.Ouranalysisestimates
thatalternatesplicingoccursinatleast4618genes(31%)ofthe Astrikingobservationregardingalternatesplicingisthatmanyof
D.melanogastergenome,a38%increaseoverFlyBaseestimates.The the120,000candidatenoveljunctionsoccurnearannotatedjunc-
detectionofjunctionreadsinRNA-seqdataenablesthediscovery tions,exhibitinga3-bpperiodicityintheirdistancefromannotated
ofthealternatesplicingeventssummarizedinTable2.Weprofiled junctions(Fig.4A).Infact,whenthesearefurtherpartitionedinto
theextentofdefinedalternatesplicingevents:skippedexons(SE), coding and noncoding junctions, this periodicity is strongly ob-
retainedintrons(RI),alternatedonorsplicesite(ADSS),alternate servedonlyincodingregions(Fig.4B).Theseobservationsledusto
acceptorsplicesite(AASS),andalternatelastexon(ALE)(seeSup- considermechanismswhichmightresultintheobservedspacing.
plemental materials). Alternate first exons (AFE), a transcrip- One explanation for this observation is the existence of tandem
tionallyregulatedeventdetectablebyRNA-seq,werealsoprofiled. alternatesplicesites(TASSs)intheD.melanogastergenome.Subtly
Eachofthesesplicingeventshasthecapacitytogeneratetranscript differentalternatespliceisoformscanbeobservedwhenmultiple
diversitybymodifyingthecodingandregulatorysequencesofa potentialsplicingsitesarelocatedinclosephysicalproximity.We
transcript(Wangetal.2008). profiled two well-described classes of TASS: the GYNGYN donor
We observed 80% of annotated alternate splicing events site and the NAGNAG alternate acceptor site (Hiller et al. 2004,
(9181)acrossallcategoriesofsplicingeventsexamined.Themost 2006,2007).ThoughmanythousandTASSsitesexistwithinthe
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The D. melanogaster transcriptome by RNA-seq
transcriptomeofD.melanogasteronlyasmallfractionareknownto
splicealternately. Furthermore,splicingatNAGNAGsisfar more
commonthanatGYNGYNs,with135andsevenannotatedsites,
respectively.Fromthe7776noveljunctionsidentified,tandemal-
ternatesplicingwasobservedat90%ofannotatedNAGNAGsites
and29%ofGYNGYNsites.Inadditiontoannotatedsites,weob-
servealternatesplicingat242novelNAGNAGsplicingsitesand22
novelGYNGYNsites(seeSupplementalTable5).
Consistentwithpreviousreports,weobservedthatthemost
importantcharacteristicofdeterminingthedominantsplicesiteat
aNAGNAGarethebasesN andN (Fig.4E;Sinhaetal.2009).For
1 2
example,CAGisastrongacceptorsitewhileGAGisaweakac-
ceptorsite.Whenweaksitesorstrongsitesarepaired,alternate
splicingfrequentlyoccurs.Conversely,whenaweakandastrong
sitearepaired,constitutivesplicingisthenorm.Themostabun-
dantalternatesplicepairCAGCAGdemonstratedalternatesplic-
ing at 53.2% of observed sites, suggesting that while N N are
1 2
highlyinformativeforsplicesitestrength,otherfactors(cisor
trans) likelycontributetodeterminealternatesplicing.Theseob-
servationssuggestthatTASSsareacommonmechanismforalter-
natesplicinginD.melanogaster.
Discussion
WehavegeneratedthefirstmapofDrosophilatranscriptionbased
onpaired-endRNA-seq.Theextensivenatureofthesedataisil-
lustrated by the broad range of 10 distinct developmental time
points sampled. Additionally, from the 142.2 million uniquely
mapped sequencing reads, 95% of annotatedgenes and 90% of
annotatedjunctionswereobservedinthesedata.
RNA-seqdataprovidesdirectexperimentalevidenceforvali-
datingandmodifyingcurrentgenemodelsandidentifyingnovel
genes.Inourdatasets,atotalof97.5%ofuniquelymappedreads
overlappedwithannotatedmodelssuggestingahighlevelofcon-
cordancebetweenexperimentalevidenceandcurrentannotations.
Even using stringent criteria, ;30% of FlyBase genes underwent
somelevelofmodificationwhenthecoverageandjunctionreads
datawerecombinedwithannotatedgenemodels.Thesechanges
includeextensivemodificationtocodingandnoncodingexonsin
25%ofgenemodelsandtheextensionofpreviouslyunannotated
UTRsfor8%ofgenemodels.Furthermore,weidentified319novel
transcriptsacrossthegenome.Itremainstobedeterminedwhether
thesetranscriptshavephenotypicconsequencesorrepresentnoisy
transcription.Itseemslikelythatnarrowerdevelopmentaltime
pointsandtissue-specificsamplesmaybenecessarytoidentifythe
fullrangeoftranscriptsintheD.melanogastertranscriptome.This
Figure4. RNA-seqdetectsabundantsubtlealternatespliceisoforms.
isbasedonourobservationthatnoveltranscriptsareusuallysmall,
(A) Distribution of novel splicing junctions near annotated exon–exon
junctionsapproximatesthefrequencyofreadindelevents.Thedistance oflowabundance,andexhibitstageandsex-specificexpression
betweennovelsplicesitesandannotatedsplicesitesisplottedseparately patterns.Fromtheseobservationsweconcludethat,whileexisting
for canonical and noncanonical novel junctions. (B) Canonical novel models are largely complete, RNA-seq data sets are valuable re-
junctionsincodingregionsconserveframe.Theportionofnoveljunctions
sourcesforthecontinuedrefinementofgenemodelsandidenti-
whichconserveframeiscalculatedforallcandidatejunctionsacrosstwo
ficationofnoveltranscripts.
partions:canonical/noncanonicalandcoding/noncoding.Onlycanonical
noveljunctionswithincodingregionsconserveframemorethanexpected Ourdataprovideinterestinginsightsintotheextentofalter-
bychance(Binomialtestn=34,060,P-value=2.2310(cid:2)16).(C)Con- natesplicinginD.melanogasterandhighlightmechanismswhich
stitutivelysplicingNAGNAGscanbeexonic(E)andintronic(I),namedfor contributetothesubtlevarietyofspliceisoforms.Weidentified
wherethesecondNAGisincorporated.AlternatesplicingNANAGsresultin
7776potentialnovelspliceeventswithstrongexperimentalsup-
bothE andIstates.(D) ConstitutivelysplicingGYNGYNs canbeoftwo
forms: exonic (e) or intronic (i), named for where the first GYN is in- port.ManyoftheseeventsarewellsupportedbyRNA-seqreadsbut
corporated.AlternatesplicingGYNGYNsresultinbotheandistates.Dia- havenotbeenvalidatedbyadditionalexperimentalmethodolo-
gramadaptedfromHilleretal.(2006).(E)ForeachpossibleNAGNAGsplice gies.Onagene-by-genebasis,interestedresearchersmaywantto
siteN (A,T,G,orC)andN (A,T,G,orC),thegenomiccount(circular
1 2 validatealternatesplicingeventswhichoccurintheirparticular
diameter)andtheproportionofexonic(E),intronic(I),andalternative(EI)
splicing (circle color) was calculated. The most abundant splicing site, geneofinterest.Weestimatethatalternatesplicingoccursin5014
CAGGAG,exhibitedalmostexclusivelyconstitutiveexonicsplicing. genes(36%)oftheD.melanogastergenome,a61%increaseover
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Daines et al.
FlyBase estimates. In comparison to mammals, where alternate cDNA Synthesis kit (Invitrogen) and random hexamer primers
splicing is estimated to occur in 95% of genes, this number (Invitrogen,3mg/mL).Ona2%agrosegel,200–400-bpcDNAswere
isrelativelylow.Interestingly,however,weobservedextensive selected and used as the DNA template for the Illumina library
amounts of subtle alternate splicing, including 264 novel TASS construction. The quality and quantity of the resulting double-
sitesidentified. strandedcDNAswasassessedusingtheNanodrop7500spectro-
We observed that sex-specific differences are extensive in photometer(Nanodrop).
D.melanogasteratthegeneregulatorylevelandatthelevelofal- DNA sequencing libraries were generated using the size-
selectedcDNAsaccordingtothemanufacturer’sprotocol.Cluster
ternatesplicing.Ofgeneswithannotatedalternatespliceisoforms,
generation and sequencing was performed on the Illumina
differentialsplicingwasobservedin23.5%.Thissuggeststhatex-
cluster station and Illumina GA II following manufacturer’s
tensive sex-specific regulation occurs at the level of alternate
instructions.
splicing.Furthermore,whilethemajorityofgenes(85.7%)appear
to be developmentally regulated, there is a significantdegree of
sex-specificregulationingeneexpression;morethanone-thirdof Readalignment
genes are differentially expressed between males and females.
Severalalignmentprogramswrittenspecificallyformappingnext-
From this we conclude that both gene regulation and alternate
generation sequencing data were considered for this study, in-
splicingcontributesignificantlytothedifferencesbetweenmales
cludingSOAP,SOAP2,Bowtie/Tophat,andBWA.BLATwaschosen
andfemalesinD.melanogaster.
fortwoimportantreasons:First,itoutperformedtheotheralign-
Severaldirectionsremaintofurtherutilizethesedata.Forex-
ment algorithms in the number of reads aligned and in our as-
ample,itisreasonablethatevidenceforRNAeditingcanbeextracted
sessment of alignment quality. Short-read alignment programs,
fromourreads(Stapletonetal.2006).Additionally,recentstudies while efficient, offer their substantial increases in speed at the
havedemonstratedthatdenovoassemblyoftranscriptsfromRNA- expense of sensitivity and accuracy. Second, BLAT natively sup-
seqdataisapotentialavenueforgeneannotation(Yassouretal. portsalignmentsofintron-sizedgapsandthedirectdiscoveryof
2009).Finally,forsimplicitywehaveignoredallnovelnoncanonical junctionreads.Smallindelsoftwotothreebasepairswereper-
splicingeventsdetectedinourdatadespitesomeeventshavingvery mittedasthesemightcorrespondtopolymorphismorsequencing
highsupport,andexaminationofthesemayshedadditionallight error;largerindelswerefilteredout.Alignmentswithgaps>39bp
onthevariationintroducedbyalternatesplicingintheD.melanogaster were additionally processed to determine whether they corre-
genome. spondedtointrons.WhileBLATdoesnotmakeuseofpaired-end
information,customscriptsweredevelopedtodisambiguateread
pairs with multiple mapping locations if a unique concordant
Methods
alignment could be identified. Read pairs mapping to separate
chromosomeswerediscarded,asthesearelikelyartifactsoflibrary
Stocksandstaging
preparationoralignmenterrors(McManusetal.2010).
CSflieswererearedatroomtemperature(23°C–24°C)forcollec-
tion.Flieswerestagedtogenerate12separatecollections.Forearly
embryo(E2–4hr),adultsweresetfor2hofegglayingongrapejuice Modifyinggenemodels
plates.Embryoswerethencollectedandallowedtoageforanad-
Areference-guidedassemblyapproachwhichjoinedoverlapping
ditional2h,resultinginembryosfrom2to4hinage.Lateembryos
readstoformblocksofcoveragewasusedsimilartoapproaches
(E14–16hr)werecollectedasabove,butallowedtoagefor14h.The
previouslydescribed(Denoeudetal.2008).Noveljunctionswere
broadlystagedembryos(E2–16hrandE2–16hr100)weresetfor14h
incorporatedintoannotatedgenemodelsiftheysharedacommon
ofegglayingongrapejuiceplates,followedby2hofagingresulting
splicesitewiththemodel.Noveljunctionswerealsoincorporated
inembryosfrom2to16hinage.Forthirdinstarlarva(L3iand
iftheirassociatedblockofreadcoverageoverlappedwiththean-
L3i100),;30wanderinglarvaewerecollected.Forpupacollections,
notatedexonsofthatlocusandtheywerederivedfromtheap-
third instar wandering larvae were collected into new vials and propriatestrand.Additionally,59and39UTRswereextendedbased
approximately30pupaewerecollectedat24,48,72,96,and108h.
oncompositecoverageonlyiftheUTRwaspreviouslyannotated
Mixed pupa sample (P) consisted of equimolar amounts of total
as10bporsmaller.
RNAfromeachofthesecollections,whereas3-dpupa(P3d)con-
sistedofonlythe72-hcollection.Three-day-oldadults(MA3dand
FA3d)werecollectedwhenemergingandseparatedasmalesand Identifyingnoveltranscripts
virginfemalesintonewvials,followedby48hofaging.Seventeen-
Areference-guidedassemblyapproachwhichjoinedoverlapping
day-oldadultswerecollectedseparately,allowedtoagefor17d,and
readstoformblocksofcoveragewasused.Readsmappingoutside
totalRNAfromeachwascombinedinequimolaramounts.
ofannotatedFlyBasegeneswhichcouldnotbeconnectedtogene
models were considered for the detection of novel transcripts.
RNAisolation Noveltranscriptswererequiredtocontainatleastonecanonical
intronandbecoveredby20ormorereads.
Whole-bodysampleswerehomogenizedin1mLofTRIzol(Invitro-
gen)per50–100mgoftissueusingaglass-Teflonhomogenizer.RNA
waspurifiedaccordingtothesuggestedmanufacturer’sprotocol
Geneexpressioncounts
(Invitrogen).
Thenumberofreadsuniquelyaligningtotranscribedregionsof
each gene was calculated for all genes in the annotated tran-
Librarypreparation
scriptome.Thegenereadcountwascalculatedasthenumberof
Foreachlibrary,mRNAwaspurifiedfrom20mgoftotalRNAusing uniquereadswhich aligned to the genomewithin theexons of
an mRNA purification kit (Invitrogen). Double-stranded cDNAs eachgene.Theexpressionlevelofeachgenewascalculatedinreads
were made from mRNA using the Superscript Double-Stranded perkilobasepermillionreads(RPKM)accordingtotheformula:
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The D. melanogaster transcriptome by RNA-seq
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