I am trying to analyze Affymetrix data (treated (group1) vs Non-Treated
(group2))
I would like to know up-regulated probesets in group1 (treated)?

1. I would like to know that I set up the design matrix correctly.
2. I did group1 (treated)  - group1 (non-treated). So positive M values
means up-regulated in group1 (treated), is that right? Or I switched
treated to Non-treated?
3. Output numbers look like to have opposite signs.
4. If I go down to positive M values, then all P-values are 1.
5  Does 4 mean there's no up-regulated probe sets at significant level?

Data and R codes are available http://binf.gmu.edu/wmoon/diff.

6. How can I remove 'Absent' flagged probesets? Or is it OK to leave
them?
Thank you.

Wonjong


Target file: 141PD.txt

Name	FileName	Target
CSA1	1A-1_SA1_141PD.CEL	CSA
CSA2	2A-1_SA2_141PD.CEL	CSA
CSA3	3A-1_SA4_141PD.CEL	CSA
CSA4	4A-1_SA5_141PD.CEL	CSA
Non5	5A-1_Non1_141PD.CEL	Non
Non6	6Ar-1_Non2_141PD.CEL	Non
Non7	7A-1_Non4_141PD.CEL	Non
Non8	8A-1_Non5_141PD.CEL	Non


library(limma) # Loads limma library.
targets <- readTargets("141PD.txt") 
library(affy); data <- ReadAffy(filenames=targets$FileName) # Reads CEL
files 
eset <- rma(data) # Normalizes data with 'rma' 
esign <- model.matrix(~ -1+factor(c(1,1,1,1,2,2,2,2))) 
colnames(design) <- c("group1", "group2") 
fit <- lmFit(eset, design)
contrast.matrix <- makeContrasts(group1-group2,levels=design) 
fit2 <- contrasts.fit(fit, contrast.matrix)
fit2 <- eBayes(fit2) # Computes moderated t-statistics and log-odds 
topTable(fit2, coef=1, adjust="fdr", sort.by="M", number=10)


output
                         ID     M    A     t  P.Value adj.P.Val     B
21231   Pf.4.224.0_CDS_x_at -4.49 3.99 -37.7 2.59e-10  5.90e-06 10.27
21230   Pf.4.223.0_CDS_x_at -4.32 4.10 -30.8 1.32e-09  1.50e-05  9.77
22728           X03144.1_at -3.57 4.39 -23.4 1.17e-08  5.74e-05  8.86
22101    Pf.7.64.0_CDS_a_at -5.03 4.64 -23.2 1.22e-08  5.74e-05  8.84
612        AF306408.1_RC_at -3.51 3.52 -22.6 1.50e-08  5.74e-05  8.73
20063 Pf.13_1.84.0_CDS_a_at -5.03 4.54 -22.6 1.51e-08  5.74e-05  8.73
20855      Pf.2.36.0_CDS_at -2.58 4.65 -20.2 3.73e-08  1.21e-04  8.24
22524     Pf.9.267.0_CDS_at -4.16 4.05 -18.5 7.27e-08  2.04e-04  7.84
21350   Pf.5.119.0_CDS_x_at -4.55 5.32 -18.3 8.07e-08  2.04e-04  7.78
20078 Pf.13_1.99.0_CDS_x_at -2.36 6.50 -17.9 9.52e-08  2.17e-04  7.67

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