[BioC] Limma: Ignoring sample in differential analysis

[Daniel Brewer]

Dear all,
I am using limma to perform differential analysis between two
categories.  Some samples in my set do not fall in either category.  I
can see that there is two ways to approach this:
1) Set the non-category samples to zero in the design matrix e.g.
> design
         Low High
GSM89690   0    1
GSM89724   0    1
GSM89728   0    0
GSM89737   0    1
GSM89692   1    0
GSM89693   0    0
GSM89716   0    1
GSM89718   1    0
GSM89726   1    0
GSM89730   1    0
GSM89746   0    0
GSM89747   1    0
GSM89751   0    0
GSM89695   0    0
GSM89739   0    1
GSM89687   1    0
GSM89699   0    0
GSM89701   0    0
GSM89703   0    0
GSM89706   0    0
GSM89708   0    1
GSM89709   0    0
GSM89712   0    0
2) Subset the expression matrix to remove the non-category samples,
which results in the following design matrix:
> design2
         Low High
GSM89690   0    1
GSM89724   0    1
GSM89737   0    1
GSM89692   1    0
GSM89716   0    1
GSM89718   1    0
GSM89726   1    0
GSM89730   1    0
GSM89747   1    0
GSM89739   0    1
GSM89687   1    0
GSM89708   0    1

After running limma on these sets,
fit <- lmFit(SeminomaOnly,design2)
cont.matrix <- makeContrasts(HvsL=High-Low, levels=design2)
fit2 <- contrasts.fit(fit, cont.matrix)
fit3 <- eBayes(fit2)
topTable(fit3,adjust="BH"),
 I have found that these two approaches produce different results with
approach 1 producing which seems to be faulty results.

Could anyone explain if and why approach 1 is wrong?  It is easy enough
to subset the data in this situation, but when there is multiple
contrasts this might prove trickier.

Many thanks

Daniel Brewer
-- 
**************************************************************
Daniel Brewer, Ph.D.
Institute of Cancer Research
Molecular Carcinogenesis
Email: daniel.brewer at icr.ac.uk
**************************************************************

The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP.

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