Dear list,

I am looking at Illumina beadarrays of 18 patients sampled at baseline ("A")
and after intervention ("B"), i.e. a total of 36 arrays. I think in this
case, a paired assessment should be done. I am interested in diff. exp. at
timepoint B over A.

Thus far, I have read the data using the beadarray package, and tried to
follow the limma manual's instructions for paired tests. I am, however,
unsure about the contrasts, and my results look weird to me.

I have done:

# read and normalize data using beadarray
library(beadarray)
dataFile = "data.txt"
qcFile = "control.txt"
sampleSheet = "SampleSheet.csv"
BSData = readBeadSummaryData(dataFile = dataFile, dec=",", qcFile = NULL,
skip = 0, qc.skip = 0)
BSData.quantile = normaliseIllumina(BSData, method = "quantile", transform =
"log2")
BSData.genes = BSData.quantile[which(fData(BSData)$Status == "Gene"), ]
expressed = apply(Detection(BSData.genes) < 0.05, 1, any)
BSData.filt = BSData.genes[expressed,]

# set up paired tests using limma - what's wrong here?
library(limma)
patients = c(17, 2, 18, 5, 3, 8, 6, 14, 4, 13, 7, 15, 9, 10, 11, 12, 17, 18,
2, 5, 3, 8, 6, 14, 1, 12, 4, 13, 7, 15, 9, 16, 10, 1, 11, 16)
patients = factor(patients)
treatment = c(rep("B",7), "A", "B", "A", "B", "A", "B", rep("A",10), "B",
"A", "B", "A", "B", "A", "B", "A", "A", rep("B", 4) )
treatment = factor(treatment, levels = c("A", "B"))
design = model.matrix(~patients+treatment)
fit = lmFit(exprs(BSData.filt), design)
ebFit=eBayes(fit)

# left out some annotation steps

# not sure what contrast is for what - does #19 stand for B-A??
topTable(ebFit, coef=19)


The primary issue is what contrast to choose - for normal, unpaired tests I
would have set up a contrast matrix for "B-A". When I look at the different
contrasts, I am missing patient #1.

> ebFit$contrasts
            (Intercept) patients2 patients3 patients4 patients5 patients6
(Intercept)           1         0         0         0         0         0
patients2             0         1         0         0         0         0
patients3             0         0         1         0         0         0
patients4             0         0         0         1         0         0
patients5             0         0         0         0         1         0
patients6             0         0         0         0         0         1
patients7             0         0         0         0         0         0
patients8             0         0         0         0         0         0
patients9             0         0         0         0         0         0
patients10            0         0         0         0         0         0
patients11            0         0         0         0         0         0
patients12            0         0         0         0         0         0
patients13            0         0         0         0         0         0
patients14            0         0         0         0         0         0
patients15            0         0         0         0         0         0
patients16            0         0         0         0         0         0
patients17            0         0         0         0         0         0
patients18            0         0         0         0         0         0
treatmentB            0         0         0         0         0         0
            patients7 patients8 patients9 patients10 patients11 patients12
(Intercept)         0         0         0          0          0          0
patients2           0         0         0          0          0          0
patients3           0         0         0          0          0          0
patients4           0         0         0          0          0          0
patients5           0         0         0          0          0          0
patients6           0         0         0          0          0          0
patients7           1         0         0          0          0          0
patients8           0         1         0          0          0          0
patients9           0         0         1          0          0          0
patients10          0         0         0          1          0          0
patients11          0         0         0          0          1          0
patients12          0         0         0          0          0          1
patients13          0         0         0          0          0          0
patients14          0         0         0          0          0          0
patients15          0         0         0          0          0          0
patients16          0         0         0          0          0          0
patients17          0         0         0          0          0          0
patients18          0         0         0          0          0          0
treatmentB          0         0         0          0          0          0
            patients13 patients14 patients15 patients16 patients17
patients18
(Intercept)          0          0          0          0          0
 0
patients2            0          0          0          0          0
 0
patients3            0          0          0          0          0
 0
patients4            0          0          0          0          0
 0
patients5            0          0          0          0          0
 0
patients6            0          0          0          0          0
 0
patients7            0          0          0          0          0
 0
patients8            0          0          0          0          0
 0
patients9            0          0          0          0          0
 0
patients10           0          0          0          0          0
 0
patients11           0          0          0          0          0
 0
patients12           0          0          0          0          0
 0
patients13           1          0          0          0          0
 0
patients14           0          1          0          0          0
 0
patients15           0          0          1          0          0
 0
patients16           0          0          0          1          0
 0
patients17           0          0          0          0          1
 0
patients18           0          0          0          0          0
 1
treatmentB           0          0          0          0          0
 0
            treatmentB
(Intercept)          0
patients2            0
patients3            0
patients4            0
patients5            0
patients6            0
patients7            0
patients8            0
patients9            0
patients10           0
patients11           0
patients12           0
patients13           0
patients14           0
patients15           0
patients16           0
patients17           0
patients18           0
treatmentB           1

Is contrast #19 the one I am looking for? Where is patient #1? The thing is
that when looking at pt #2 - #18 seperately, I do find a couple of genes
regulated pretty consistently, and these ones do not show up at all in the
overall comparison (contrast 19). In fact, there's pretty much no difference
using that contrast, which I find weird...

Perhaps anyone has an idea what's wrong? Many thanks!

Moritz (Bonn, Germany)


sessionInfo()
R version 2.12.1 (2010-12-16)
Platform: i386-pc-mingw32/i386 (32-bit)

locale:
[1] LC_COLLATE=German_Germany.1252  LC_CTYPE=German_Germany.1252
[3] LC_MONETARY=German_Germany.1252 LC_NUMERIC=C
[5] LC_TIME=German_Germany.1252

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base

other attached packages:
[1] illuminaHumanv4.db_1.8.0 org.Hs.eg.db_2.4.6       RSQLite_0.9-4

[4] DBI_0.2-5                AnnotationDbi_1.12.0     limma_3.6.9

[7] beadarray_2.0.6          Biobase_2.10.0

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