A few precisions regarding my previous posting
1- our cDNA data correlates at 0.72 (comparing >6000 genes averaged
over patients) with data from another completely independent group
using Affy U133 chips and different lab technicians, pathologists and
2- running SAM @ q<0.05, more than 30% of the genes are called
significant in *both* data sets. Affy data were normalised with MAS5,
I don't have access to the CEL files.
3- 7/7 genes with average fold-change >2.0, and high SAM rank, were
confirmed by RT-PCR in our data set
4- RT-PCR gave mixed results for two genes ranking high with SAM but
with fold-change 1.6. By mixed result I mean that RT-PCR data are
clearly correlated with microarray but give lower fold-change
5- I searched for spatial biased with box-plots, and did find some,
but much below the magnitude that could explain the 30% result.
6- we are talking about paired sample SAM comparisons. I call SAM with
cl <- rep(1, N) #paired samples
sam1 <- sam(exprs, cl, B=1000, rand=123, q.version=1)
To summarize, the data seems correct. The questions are whether SAM is
appropriate on these data sets and others, whether q-values mean what
they are supposed to, what is the relevance of calling a gene
regulated on a purely statistical basis, etc.
>Since SAM computes a regularised t-statistic, I think, you should
>also check that the normal-distribution assumption does at least
I though SAM use a computed permutation-based null distribution of the
moderated t-statistics in order to avoid hypothesis about this
distribution? Am I missing somenthing here?
Thanks you all for your input!
On Tue, 10 May 2005, Sean Davis wrote:
> > settings or hybridisation protocols etc. I would check if after
> > normalisation such large differences between the groups are
> > using boxplots, Scatter-Plots etc. (many examples for such control
> > procedures can be found on the Bioconductor website , especially
> > the pages containing material for courses and workshops). If so,
> > might think about other methods for normalisation or combining the
> > groups data in another way, if they happen to be too different.
> > Another reason for large differences could be that there might
> > be huge biological differences between the two groups. For
> > when analyzing T- versus B-lymphocytes, one usually observes large
> > percentages > 20% of differentially expressed genes, since in that
> > case we were comparing very different cell types with each other.
> > However, I would not expect such striking differences between a
> > and the related physiological tissue.
> Actually, having a large proportion of differentially-expressed
> between tumor and normal is certainly possible. You got the same
> results with two different data sets if I read your original post
> correctly, so go back to check quality of data, statistical biases,
> etc., but it seems quite possible that your results are correct.
> will, of course, have to think about validation strategies, but....
Vincent Detours, Ph.D.
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