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

Dear Guido,

The warning is not a problem, it does not affect your results. However I will fix the code so the warning does not occur in this situation.

Best wishes
Gordon