I have recently explored the use of alternative CDFs from affyprobeminer
(APM) or a 36 array dataset derived using the Affy rat2302 chipset. I used
both the Affy cdf and the transcript-level affyprobeminer cdf. I
preprocessed using RMA, filtered using an A/P filter, and statistically
analyzed using an appropriate lme model followed by qvalue FDR correction. I
set my FDR threshold at 5%. I eliminated duplicate genes by picking the one
with the lowest p-value.

Using the Affy cdf, I got ~2000 sig. genes, which APM ~1000. If I choose
only those EntrezGene identifiers present on both cdfs, my number sig. with
the APM cdf was ~1000 and there was a 90% overlap with the Affy sig. list.
My conclusion from the latter observation is that I am measuring largely the
same transcripts/genes with both CDFs.

I was interested in the ~1000 genes which are annotated with the Affy CDF
but not the APM cdf. Following the logic behind APM, I would assume that
these would be largely incorrectly annotated probesets or probesets that are
not really measuring any "real" transcript. This list should, then, consist
largely of random genes. To test this hypothesis, I used the Category
package to test for over-representation of GO and KEGG categories in my
various lists. What I found was a huge degree of overlap between: 1. the
affy genes also annotated with APM, 2. the affy genes not annotated with
APM, 3. the genes derived solely from APM.

My conclusion from this latest observation is that APM is not annotating a
large number of genes/transcripts that are in fact real. Assuming that APM
is correctly throwing out some "junk" probesets, is it throwing out the baby
with the bathwater?

I'd be interested to hear the thoughts and experiences of others. I've
certainly run into occasions where Affy annotated probesets turn out to
represent introns or something other than they purport to be, and I was
hoping that APM would solve this problem, but I don't want to use it if it
means a massive loss of truly significant data.

Mark



-- 
Mark W. Kimpel MD ** Neuroinformatics ** Dept. of Psychiatry
Indiana University School of Medicine

15032 Hunter Court, Westfield, IN 46074

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