[BioC] Exclude probes that show sd above 0.1between replicatevalues

[Naomi Altman]

Personally, I use single channel analysis because I feel that spots 
that are "off" under some conditions and "on" under others are most 
interesting.  Using log2 is still a problem, however.

--Naomi

At 09:19 AM 3/1/2007, J.delasHeras at ed.ac.uk wrote:

>Hi Jan,
>
>not sure if I am understanding.
>I am with you about the variability of replicate spots... as long as
>they can be measured reliably, as you say. The question I guess is
>where do you take these measurements: at the intensity or at the ratio
>level? If you're looking at the variability based on ratios (M
>values), replicate spots with no signal in one channel tend to have
>wildly varying M values (all quite high, in absolute value). Wouldn't
>a filtering based solely on variation at M value level discard those
>spots? For these kind of spots the M value is irrelevant (I mean, how
>much is something divided by *almost* nothing?), we don't really have
>a use for the actual number, except for the fact that it should be
>large.
>
>As you say, I guess that any analysis depends on what you're after,
>but most "general" approaches I see mentioned don't seem to care about
>this particular case when signal is missing only in one channel. In
>fact, some people just remove any spot where the signal is not
>detectable in both channels, which for my purposes would be a disaster
>[1]. I have my own approach to deal with this, and I am reasonably
>happy, but I am very curious to see how other people approach this
>issue.
>
>[1] We had a while ago a demo of teh software Acuity at our centre.
>The guy contacted us before asking if we'd have some real data we'd
>like to use in teh demo. He chose some of my data, which I thought was
>great, as I had already analysed it using my usual tools. His demo
>picked up genes I knew to be upregulated... but my "top genes" that
>I've continued to use in my experiments were all missing, as they had
>been left behind in one of the filtering steps, either the low
>intensity filter (applied on *either* channel, or the standard
>deviation filter on log2 ratios,, not sure which ones, probably
>both)... it took me a while to convince him that I really really
>didn't want those spots removed, which surprised me. Is most people
>really throwing away these kind of spots?
>
>Jose
>
>
>Quoting J.Oosting at lumc.nl:
>
> > Hi Jose,
> >
> > IMHO you should use the variability of replicate spots whenever
> > possible. Limma can handle this nicely and for the analysis of
> > differential expression I always leave in the replicate spots, and I let
> > limma handle them.
> >
> > For presentation purposes (i.e. heatmaps) it is usually handy to have
> > averaged values per gene, and I think that removing genes that cannot be
> > measured reliably is a way of improving the visualizations.
> >
> > Any data-manipulation is context dependent, and especially the effects
> > of removing data points should be considered case by case. If you're
> > interested in on/off phenomena you should not remove 'empty' spots.
> >
> > Regards,
> >
> > Jan
> >
> >>
> >> If you look at the variation on M values alone (it's a
> >> MAList), and throw away those with high variation... that
> >> sounds like a reasonable thing to do, except that when you
> >> have spots with no signal in only one of the channels, the
> >> variation is probably quite high too, and you'd remove them.
> >> However, they are probably quite an interesting class of
> >> spots to keep (genes that become silenced, or activated,
> >> after treatment, not merely down/upregulated).
> >>
> >> I'm mostly studying experiments when I am interested mostly
> >> in these cases of activation/silencing, and not so much in
> >> up/downregulation alone. I wonder how people account for
> >> these situations...
> >>
> >> Jose
> >>
> >
> >
>
>
>
>--
>Dr. Jose I. de las Heras                      Email: J.delasHeras at ed.ac.uk
>The Wellcome Trust Centre for Cell Biology    Phone: +44 (0)131 6513374
>Institute for Cell & Molecular Biology        Fax:   +44 (0)131 6507360
>Swann Building, Mayfield Road
>University of Edinburgh
>Edinburgh EH9 3JR
>UK
>
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Naomi S. Altman                                814-865-3791 (voice)
Associate Professor
Dept. of Statistics                              814-863-7114 (fax)
Penn State University                         814-865-1348 (Statistics)
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