Dear all,

Happy Holidays!

I would appreciate any help in designing my 'cl' file for use with SAM, EBAM or RankProd. I can't seem to figure out from the Vignettes exactly how I should set mine up. I think I'm close but I would appreciate any insights.

Here is how my data is organized:
I have isolated white blood cells from human volunteers and infected them (or not) with my infectious parasite. I am interested in figuring out genes that are differentially expressed. In a given experiment, I have four conditions:
(a) Macrophages alone (not infected).
(b) Macrophages infected.
(c) Macrophages and Tcells together, not infected.
(d) Macrophages and Tcells together, infected.
I have repeated this setup with four separate individuals (same procedure, same kind of parasite, different days). Therefore I have 16 Affy arrays (4 sets of 4).

How should I design my 'cl' file?

Hopefully this helps:
>TotalData <- ReadAffy()
>show(TotalData)

AffyBatch object
size of arrays=1164x1164 features (169370 kb)
cdf=HG-U133_Plus_2 (54675 affyids)
number of samples=16
number of genes=54675
annotation=hgu133plus2

> eset_rma <- rma(TotalData)
> pData(eset_rma)

NE_MDM-INF1.CEL	1
NE_MDM-INF2.CEL	2
NE_MDM-INF3.CEL	3
NE_MDM-INF4.CEL	4
NE_MDM-T-1.CEL		5
NE_MDM-T-2.CEL		6
NE_MDM-T-3.CEL		7
NE_MDM-T-4.CEL		8
NE_MDM-T-INF-1.CEL	9
NE_MDM-T-INF-2.CEL	10
NE_MDM-T-INF-3.CEL	11
NE_MDM-T-INF-4.CEL	12
NE_MDM1.CEL		13
NE_MDM2.CEL		14
NE_MDM3.CEL		15
NE_MDM4.CEL		16
Note: MDM=macrophage, INF=infected, 1-4 = different donors

Given the above, this is my stab at a 'cl' file:
\> chipnumber <- length(sampleNames(TotalData))
> K <- chipnumber/2
> eset.cl <- c((1:K), (((-K/2)-1):-K), (-1:(-K/2)))
> eset.cl
 [1] 1  2  3  4  5  6  7  8 -5 -6 -7 -8 -1 -2 -3 -4

The comparisons that I am most interested in are infected macrophages vs. non-infected and infected MDM+T vs. non-infected MDM+T. I am not so interested in comparisons between donors. I can't figure out whether I should break my data up into two sets and run SAM, etc. separately or whether to run everything at once.

Related question: is there a way to take the 4 arrays for each condition and to generate an average for each condition? In effect, is there a way to reduce my 16 pieces of expression data down to 4 that each is the averages across the 4 individuals for each condition?

All help is much, much appreciated!! Thank you!
---Nick

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