[BioC] normalization and analysis of connected designs

[Ramon Diaz-Uriarte]

Dear Wolfgang,

> the package "vsn" has implemented single-channel normalization for cDNA
> microarrays since the end of 2001 and was published at ISMB 2002. We have
> run considerable comparison studies on data to validate it, where it
> compared favorably with log-ratio based methods. One of these comparisons
> is reported in that paper.

I apologize for not being clear: my response to Xavi regarding single-channel 
normalization was referring to single-channel normalization using quantile 
normalization. On reading my email, I see this is not at all clear from it; 
Xavi had mentioned single-channel using quantile normalization in the thread 
I was responding to, and that is what I had in mind. I have read two of your 
vsn papers (Bioiformatics, 2002, 18, S96-S104; Statistical Applications in 
Genetics and Molecular Biology, 2003, 2, article 3), and I have used the vsn 
package, but I understand that vsn is not using quantile normalization and 
that is why I did not mention it.

> The importance of spot-to-spot variation (relative to other kinds of
> varation) needs to be carefully considered. As a first approximation, the
> following calculation was pointed out to me by Gordon Smyth:
>
> Assume you have n arrays, with red and green values R1, G1, R2, .., Rn, Gn
> on the logarithmic scale. Assume var(Rk) = var(Gk) = sigma^2,
> corr(Gk,Rk)=rho and corr(Gk, Rj)=0 if j!=k. Then compare
> (1) var(R1-Gn) = 2 * sigma^2
> (2) var{ (R1-G1) - (R2-G2) - ... (Rn-Gn) } = 2n * (1-rho) sigma^2
>
> Whether (1) is larger than (2) or the other way round depends on n and the
> size of rho.

Thanks for pointing this out; I think I see the point.

Best,

Ramón


> -------------------------------------
> Wolfgang Huber
> Division of Molecular Genome Analysis
> German Cancer Research Center
> Heidelberg, Germany
> Phone: +49 6221 424709
> Fax:   +49 6221 42524709
> Http:  www.dkfz.de/mga/whuber
> -------------------------------------
>
> On Fri, 25 Jul 2003, Ramon Diaz-Uriarte wrote:
> > Dear Xavi,
> >
> > Thanks for your last two emails. I see your point, but it is my
> > understanding (which has improved a lot thanks to comments from Gordon
> > Smith) that one of the important reasons for dealing with ratios in cDNA
> > arrays is controlling spot-to-spot variation. In fact, this is mentioned
> > explictly in several papers (e.g., Yang & Thorne, 2003, p. 405). So,
> > regardless of the importance of competitive hybridization, spot-to-spot
> > variation is always there. I am not that familiar with Affy, but I think
> > that, because their setup is very different (e.g., multiple probes per
> > clone), the direct analogy "if we do it with Affy we ought to be able to
> > do it with cDNA" does not really hold just like that.
> >
> > By the way, the paper of Yang & Thorne, which Gordon Smith mentioned in a
> > previous email, contains discussion of single-channel normalization for
> > cDNA, and Natalie Thorne presented a very interested talk at the last RSS
> > meetings dealing with single-channel normalization. However, if I
> > understand correctly, there are still some issues that need to be
> > investigated more fully for single channel normalization and they are
> > working on it.
> >
> > Yang, Y. H., and Thorne, N. P. (2003). Normalization for two-color cDNA
> > microarray data. In: D. R. Goldstein (ed.), Science and Statistics: A
> > Festschrift for Terry Speed, IMS Lecture Notes - Monograph Series, Volume
> > 40, pp. 403-418.
> >
> >
> > Best,
> >
> > Ramón

-- 
Ramón Díaz-Uriarte
Bioinformatics Unit
Centro Nacional de Investigaciones Oncológicas (CNIO)
(Spanish National Cancer Center)
Melchor Fernández Almagro, 3
28029 Madrid (Spain)
Fax: +-34-91-224-6972
Phone: +-34-91-224-6900

http://bioinfo.cnio.es/~rdiaz