I am learning myselves to run a paired t-test in limma. Everything goes fine, but i do encounter a warning, which for me is very vague... > fit2 <- contrasts.fit(fit, cont.matrix) Warning message: In any(contrasts[-est, ]) : coercing argument of type 'double' to logical (find the complete code is used below; warning happens at almost the end) I do know that warnings are not equal to errors, but still i would like to know what this warning means, and if possible how to avoid it. Thanks, Guido > library(affy) Loading required package: Biobase Welcome to Bioconductor Vignettes contain introductory material. To view, type 'openVignette()'. To cite Bioconductor, see 'citation("Biobase")' and for packages 'citation(pkgname)'. > library(limma) > targets <- readTargets("targets_A23_paired.txt") > targets FileName SibShip Strain Treatment 1 KOcontrol2.CEL 1 KO Con 2 KOcontrol3.CEL 2 KO Con 3 KOcontrol5.CEL 3 KO Con 4 KOWY7.CEL 1 KO WY 5 KOWY8.CEL 2 KO WY 6 KOWY9.CEL 3 KO WY 7 WTcontrol20.CEL 4 WT Con 8 WTcontrol21.CEL 5 WT Con 9 WTcontrol22.CEL 6 WT Con 10 WTWY25.CEL 4 WT WY 11 WTWY26.CEL 5 WT WY 12 WTWY27.CEL 6 WT WY > data <- ReadAffy(filenames=targets$FileName) > eset <- rma(data) Background correcting Normalizing Calculating Expression > eset ExpressionSet (storageMode: lockedEnvironment) assayData: 22690 features, 12 samples element names: exprs phenoData sampleNames: KOcontrol2.CEL, KOcontrol3.CEL, ..., WTWY27.CEL (12 total) varLabels and varMetadata description: sample: arbitrary numbering featureData featureNames: 1415670_at, 1415671_at, ..., AFFX-TrpnX-M_at (22690 total) fvarLabels and fvarMetadata description: none experimentData: use 'experimentData(object)' Annotation: moe430a > TS <- paste(targets$Strain, targets$Treatment, sep=".") > TS [1] "KO.Con" "KO.Con" "KO.Con" "KO.WY" "KO.WY" "KO.WY" "WT.Con" "WT.Con" "WT.Con" "WT.WY" "WT.WY" "WT.WY" > TS <- factor(TS, levels=c("WT.Con","WT.WY","KO.Con","KO.WY")) > SibShip <- factor(targets$SibShip) > TS [1] KO.Con KO.Con KO.Con KO.WY KO.WY KO.WY WT.Con WT.Con WT.Con WT.WY WT.WY WT.WY Levels: WT.Con WT.WY KO.Con KO.WY > SibShip [1] 1 2 3 1 2 3 4 5 6 4 5 6 Levels: 1 2 3 4 5 6 > design <- model.matrix(~0+TS+SibShip) > design TSWT.Con TSWT.WY TSKO.Con TSKO.WY SibShip2 SibShip3 SibShip4 SibShip5 SibShip6 1 0 0 1 0 0 0 0 0 0 2 0 0 1 0 1 0 0 0 0 3 0 0 1 0 0 1 0 0 0 4 0 0 0 1 0 0 0 0 0 5 0 0 0 1 1 0 0 0 0 6 0 0 0 1 0 1 0 0 0 7 1 0 0 0 0 0 1 0 0 8 1 0 0 0 0 0 0 1 0 9 1 0 0 0 0 0 0 0 1 10 0 1 0 0 0 0 1 0 0 11 0 1 0 0 0 0 0 1 0 12 0 1 0 0 0 0 0 0 1 attr(,"assign") [1] 1 1 1 1 2 2 2 2 2 attr(,"contrasts") attr(,"contrasts")$TS [1] "contr.treatment" attr(,"contrasts")$SibShip [1] "contr.treatment" > fit <- lmFit(eset, design) Coefficients not estimable: SibShip6 Warning message: Partial NA coefficients for 22690 probe(s) > cont.matrix <- makeContrasts(WTwyvWTc=TSWT.WY-TSWT.Con, KOwyvKOc=TSKO.WY-TSKO.Con, Diff=(TSWT.WY-TSWT.Con)-(TSKO.WY-TSKO.Con), levels=design) > fit2 <- contrasts.fit(fit, cont.matrix) Warning message: In any(contrasts[-est, ]) : coercing argument of type 'double' to logical > fit2 <- eBayes(fit2) > topTable(fit2, coef=1, adjust="BH") ID logFC AveExpr t P.Value adj.P.Val B 17177 1449065_at 4.528581 6.713129 66.51179 3.781733e-16 8.580752e-12 24.96342 7192 1422997_s_at 4.298410 8.311977 56.45944 2.444477e-15 2.773259e-11 23.90403 16494 1448382_at 3.386068 9.658716 50.20860 9.290666e-15 6.124439e-11 23.04600 13289 1431833_a_at 3.538370 7.856618 49.48486 1.095883e-14 6.124439e-11 22.93425 7120 1422925_s_at 3.149781 8.072523 48.58693 1.349590e-14 6.124439e-11 22.79159 18706 1450643_s_at 2.847376 7.907519 44.87659 3.329207e-14 1.258995e-10 22.15116 296 1415965_at 3.749143 5.293939 43.70413 4.497336e-14 1.457779e-10 21.93016 12200 1428005_at 3.232843 6.882881 42.99986 5.408650e-14 1.534028e-10 21.79272 9048 1424853_s_at 4.716858 8.082727 40.20307 1.160545e-13 2.845272e-10 21.20948 6721 1422526_at 2.772829 8.060643 39.92966 1.253976e-13 2.845272e-10 21.14905 > > sessionInfo() R version 2.9.0 (2009-04-17) i386-pc-mingw32 locale: LC_COLLATE=English_United Kingdom.1252;LC_CTYPE=English_United Kingdom.1252;LC_MONETARY=English_United Kingdom.1252;LC_NUMERIC=C;LC_TIME=English_United Kingdom.1252 attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] moe430acdf_2.4.0 limma_2.18.0 affy_1.22.0 Biobase_2.4.1 loaded via a namespace (and not attached): [1] affyio_1.12.0 preprocessCore_1.6.0 tools_2.9.0 ------------------------------------------------ Guido Hooiveld, PhD Nutrition, Metabolism & Genomics Group Division of Human Nutrition Wageningen University Biotechnion, Bomenweg 2 NL-6703 HD Wageningen the Netherlands tel: (+)31 317 485788 fax: (+)31 317 483342 internet: http://nutrigene.4t.com email: guido.hooiveld@wur.nl [[alternative HTML version deleted]]