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Estimation of alternative splicing isoform frequencies from RNA-Seq data.doc

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Estimation of alternative splicing isoform frequencies from RNA-Seq data

Nicolaeetal.AlgorithmsforMolecularBiology2011,6:9 /content/6/1/9 RESEARCH OpenAccess Estimationofalternativesplicingisoform frequenciesfromRNA-Seqdata MariusNicolae1*,SergheiMangul2,IonIM?ndoiu1andAlexZelikovsky2 Abstract Background:Massivelyparallelwholetranscriptomesequencing,commonlyreferredasRNA-Seq,isquickly becomingthetechnologyofchoiceforgeneexpressionprofiling.However,duetotheshortreadlengthdelivered bycurrentsequencingtechnologies,estimationofexpressionlevelsforalternativesplicinggeneisoformsremains challenging. Results:Inthispaperwepresentanovelexpectation-maximizationalgorithmforinferenceofisoform-andgene- specificexpressionlevelsfromRNA-Seqdata.Ouralgorithm,referredtoasIsoEM,isbasedondisambiguating informationprovidedbythedistributionofinsertsizesgeneratedduringsequencinglibrarypreparation,andtakes advantageofbasequalityscores,strandandreadpairinginformationwhenavailable.TheopensourceJava implementationofIsoEMisfreelyavailableat/software/IsoEM/. Conclusions:EmpiricalexperimentsonbothsyntheticandrealRNA-SeqdatasetsshowthatIsoEMhasscalable runningtimeandoutperformsexistingmethodsofisoformandgeneexpressionlevelestimation.Simulation experimentsconfirmpreviousfindingsthat,forafixedsequencingcost,usingreadslongerthan25-36basesdoes notnecessarilyleadtobetteraccuracyforestimatingexpressionlevelsofannotatedisoformsandgenes. Background methodssuchasquantitative PCRhaslongbeenasta- Ubiquitous regulatory mechanisms such as the use of ple of genetic studies. The completion of the human alternativetranscriptionstartandpolyadenylationsites, genome has been a key enabler for genome-wide GE alternative splicing, andRNAediting result inmultiple performed using expression microarrays. Since expres- messengerRNA(mRNA)isoformsbeinggeneratedfrom sion microarrays have limited capability of detecting a single genomic locus. Most prevalently, alternative alternative splicing events, specialized splicing arrays splicing is estimated to take place for over 90% of th