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ANOVA-Like Differential Expression (ALDEx) Analysis for Mixed Population RNA-Seq.doc

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ANOVA-Like Differential Expression (ALDEx) Analysis for Mixed Population RNA-Seq

ANOVA-LikeDifferentialExpression(ALDEx)Analysisfor MixedPopulationRNA-Seq AndrewD.Fernandes1,JeanM.Macklaim2,5,ThomasG.Linn3,GregorReid3,4,5,GregoryB.Gloor2,5 * 1YouKaryote Genomics, London, Ontario, Canada, 2Department of Biochemistry, The University of Western Ontario, London, Ontario, Canada, 3Department of MicrobiologyImmunology,TheUniversityofWesternOntario,London,Ontario,Canada,4DepartmentofSurgery,TheUniversityofWesternOntario,London,Ontario, Canada,5CanadianResearchDevelopmentCentreforProbiotics,LawsonHealthResearchInstitute,London,Ontario,Canada Abstract Experimentalvarianceisamajorchallengewhendealingwithhigh-throughputsequencingdata.Thisvariancehasseveral sources:samplingreplication,technicalreplication,variabilitywithinbiologicalconditions,andvariabilitybetweenbiological conditions. The high per-sample cost of RNA-Seq often precludes the large number of experiments needed to partition observedvarianceintothesecategoriesasperstandardANOVAmodels.Weshowthatthepartitioningofwithin-condition to between-condition variation cannot reasonably be ignored, whether in single-organism RNA-Seq or in Meta-RNA-Seq experiments,andfurtherfindthatcommonly-usedRNA-Seqanalysistools,asdescribedintheliterature,donotenforcethe constraintthatthesumofrelativeexpressionlevelsmustbeone,andthusreportexpressionlevelsthataresystematically distorted.Thesetwofactorsleadtomisleadinginferencesifnotproperlyaccommodated.Asitisusuallyonlythebiological between-conditionandwithin-conditiondifferencesthatareofinterest,wedevelopedALDEx,anANOVA-likedifferential expressionprocedure,toidentifygeneswithgreaterbetween-towithin-conditiondifferences.Weshowthatthepresence ofdifferentialexpressionandthemagnitudeofthesecomparativedifferencescanbereasonablyestimatedwithevenvery smallsamplesizes. Citation:FernandesAD,MacklaimJM,LinnTG,ReidG,GloorGB(2013)ANOVA-LikeDifferentialExpression(ALDEx)AnalysisforMixedPopulationRNA-Seq.PLoS ONE8(6):e67019.doi:10.1371/journal.pone.0067019 Editor:JohnParkinson,Hospit