Question: Statistical comparison of low replicate affy data

0

Matthew Hannah •

**940**wrote:Hi,
I'm looking at how different analyses of affy data perform on a sample
data
set of 2 conditions (untr vs. trt) with 3 biological reps (Ua, Ub, Uc
vs.
Ta, Tb, Tc). I've computed RMA and GCRMA expression measures as
standard and
so have 2 exprSets containing these values.
I've looked at fold changes and the treatment leads to many (1000-RMA,
1600 -
GCRMA) pairwise 2x changes (Ua-Ta, Ub-Tb, Uc-Tc all >2). In order to
estimate
the false positive rate I made pairwise comparisons within groups (Ua-
Ub, Ua-Ub,
Ub-Uc and same for T) and was suprised to see that with only 3 reps
there were
very few genes that met the 2x criteria by chance (<5 - RMA, <10
GCRMA). What
are peoples views on estimating false positives in this way?
I now want to make some statistical comparisons of the data both
paired and un-
paired. I was thinking of making these comparisons first Ua-Ta..
(paired) and
then Uabc-Tabc (unpaired) and then permutate the data so to compare
Ua,Tb,Uc -
Ta,Ub,Tc....etc in various combinations paired and un-paired. Would
this provide
reliable false positive rates?
I have looked into the BioC packages and guess I'll use a t-test with
multest
correction, LPE (although this doesn't say it accepts RMA-type data?)
and SAM,
EBAM & EBAM.WILC from siggenes. Are there others I should also
consider?
My request for help is if people have experience of applying these
tests to
affy data, specifically in the form of the RMA style exprSets my data
is
currently in, could they possibly post or send the r-scripts they
used. I've
obviously searched BioC and help but my attempts so far have returned
errors
and I can't help but think I'm missing something obvious (need to get
rid of
the affy ID's?) and obviously help would speed things up a great deal.
For example
> cl <- c(0,0,0,1,1,1)
> rmasam <- sam(esetrma, cl)
returned - Error in var(v) : missing observations in cov/cor
> rmaebw <- ebam.wilc(esetrma, cl)
returned - Error in 2^data : non-numeric argument to binary operator
Obviously if anyone is interested in what results I (eventually)
obtain then
let me know.
Thanks,
Matt