Thanks Tobias for your response

I am processing data obtained with Affymetrix mouse chips (430_2, previous
version)
The first filterning was done based on presence/absence calls, so only genes
present in 2/17 samples were used. It is a 2 condition set up, with 8 and 9
replicates for each condition. My definition of FDR in my previous question
was strictly limited to validation in 8+ independent qPCRs of 40+ randomly
selected genes obtained using a SAM cutoff of 5% FDR. So I am talking about
independently re-testing the reproducibility of gene expression, which is
the only way to really know your FDR. Using the Mas5 presence absence calls
filter leads to about 50% of the tested genes not being reproducible.

If I remove the filtering and redo the analysis at 5% FDR, I get all the the
previous "false positives" to become true positives. Which was not a
surprise to me since about 1/3 of MM probes are known to hybridize better
than PM probes, so I don't know what Mas5 presence/absence really means, but
definitely cannot reflect accurately the presence of a transcript if the MM
probe hybridizes better.

The problem is that I have a great loss of sensitivity (I have a lot of
positive controls so I know that), and I would like to increase that using a
filter that can come closer to really defining "present", because MM/PM does
not.
any ideas?
thanks

Lucia


On Mon, Aug 16, 2010 at 8:34 AM, Tobias Straub
<tstraub@med.uni-muenchen.de>wrote:

> Hi Lucia
>
> I am not sure if I completely understand your problem, just want to mention
> that I routinely apply non-specific filtering based on MAS5 calls with a
> very good outcome (based on a prior-knowledge training set). I do not like
> so much the alternative approach - filtering based on variance or IQR -  as
> it jeopardizes my preferred way of defining responders by applying a
> threshold on the local false discovery rate.
>
> Could you extend a bit on how you exactly filter based on MAS5 calls, how
> you define responders and non-responders in qPCR, how your "FDR disaster"
> exactly looks like.
>
> What is your model system by the way, which arrays you use?
>
> best regards
> T.
>
>
> On Aug 13, 2010, at 7:11 PM, Lucia Peixoto wrote:
>
> > Dear All,
> > I want to set up a non-specific filter to eliminate genes that are juts
> not
> > expressed from further statistical analysis. I've previously tried a
> filter
> > based on Mas5 presence/absence calls which turned out to be a disaster
> for
> > the FDR (as measured by lots of qPCRs), probably because 1/3 of the MM
> > probes actually hybridize better than PM, who knows.
> >
> > In any case, my plan is to set up a filter based both on raw fluorescent
> > intensity and IQR. I am trying to get as much sensitivity as possible
> > without increasing my FDR too much.
> > I was thinking that using the intensity distributions and box plots of
> the
> > raw data may be useful to determine what the best cutoffs to obtain the
> best
> > sensitivity will be.
> > Any advise on how to select appropriate cutoffs?
> >
> > Thank you very much in advance
> > Lucia
> >
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> >
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> ----------------------------------------------------------------------
> Dr. Tobias Straub ++4989218075439 Adolf-Butenandt-Institute, München D
>
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