Optimizing a Massive Parallel Sequencing Workflow for Quantitative miRNA Expression Analysis 英文参考文献.docVIP

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Optimizing a Massive Parallel Sequencing Workflow for Quantitative miRNA Expression Analysis 英文参考文献.doc

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Optimizing a Massive Parallel Sequencing Workflow for Quantitative miRNA Expression Analysis 英文参考文献

OptimizingaMassiveParallelSequencingWorkflowfor QuantitativemiRNAExpressionAnalysis FrancescaCordero1,2.,MarcoBeccuti2.,MaddalenaArigoni1,SusannaDonatelli2,RaffaeleA.Calogero1* 1DepartmentofComputerSciences,UniversitydiTorino,Torino,Italy,2MolecularBiotechnologyCenter,UniversityofTorino,Torino,Italy Abstract Background: Massive Parallel Sequencing methods (MPS) can extend and improve the knowledge obtained by conventionalmicroarraytechnology,bothformRNAsandshortnon-codingRNAs,e.g.miRNAs.Theprocessingmethods usedtoextractandinterprettheinformationareanimportantaspectofdealingwiththevastamountsofdatagenerated fromshortreadsequencing.AlthoughthenumberofcomputationaltoolsforMPSdataanalysisisconstantlygrowing,their strengthsandweaknessesaspartofacomplexanalyticalpipe-linehavenotyetbeenwellinvestigated. Primary findings: A benchmark MPS miRNA dataset, resembling a situation in which miRNAs are spiked in biological replication experiments was assembled by merging a publicly available MPS spike-in miRNAs data set with MPS data derivedfromhealthydonorperipheralbloodmononuclearcells.Usingthisdatasetweobservedthatshortreadscounts estimationisstronglyunderestimatedincaseofduplicatesmiRNAs,ifwholegenomeisusedasreference.Furthermore,the sensitivityofmiRNAsdetectionisstronglydependentbytheprimarytoolusedintheanalysis.Withinthesixalignerstested, specifically devoted to miRNA detection, SHRiMP and MicroRazerS show the highest sensitivity. Differential expression estimationisquiteefficient.Withinthefivetoolsinvestigated,twoofthem(DESseq,baySeq)showaverygoodspecificity andsensitivityinthedetectionofdifferentialexpression. Conclusions:Theresultsprovidedbyouranalysisallowthedefinitionofaclearandsimpleanalyticaloptimizedworkflow formiRNAsdigitalquantitativeanalysis. Citation:CorderoF,BeccutiM,ArigoniM,DonatelliS,CalogeroRA(2012)OptimizingaMassiveParallelSequencingWorkflowforQuantitativemiRNAExpression Analysis.PLoSONE7(2):e31630.doi:10.1371/journal.pone.0031630 Editor:AnnaTramontano,U