[BioC] Two channel data vs. one colour data for PCA, heatmaps and clustering

john herbert arraystruggles at gmail.com
Mon Nov 14 13:08:48 CET 2011

Hi Maciej  and Samuel,
I have tried your options and they seem to work pretty good. I
normalised global loess and converted back to log2(R and G), then did
eyayes diff-exp as a one colour array. The results are not exactly the
same as the straight two colour but similar enough for me to be
confident in such an approach and to use for PCA/heatmaps.

Thank you very much for your help.

Kind regards,


2011/11/14 Samuel Wuest <wuests at tcd.ie>:
> Hi John,
> I would also recommend using the limma package for your type of
> analysis. After appropriate normalizations/bg-corrections, you can
> extract log-intensities from an MAList object using the exprs.MA()
> function. This will return a matrix object that you can use for
> pca/heatmaps as you've used before. It s possible that you have to
> relabel the columns and rows of the matrix accordingly (you probably
> want to use the info in the $targets  and $genes slots of the MAList
> object, but there might be other ways to do that).
> The only drawback here is that correlations can occur between the two
> channels of an array, so for testing for differential gene expression
> in an unconnected design -after splitting the channels- I would refer
> to Chapter 9 of the limma user guide (see: limmaUsersGuide() ).
> Cheers, Sam
> -----------------------------------------------------
> Samuel Wuest
> Smurfit Institute of Genetics
> Trinity College Dublin
> Dublin 2, Ireland
> Phone: +353-1-896 2444
> Web: http://www.tcd.ie/Genetics/wellmer-2/index.html
> Email: wuests at tcd.ie
> ------------------------------------------------------
> On 14 November 2011 10:52, mjonczyk <mjonczyk at biol.uw.edu.pl> wrote:
>> Dear John,
>> I suppose that for the two-colour experiment you have also "A" (average
>> expression) values.
>> I don't know what package you have used but limma has RG.MA function
>> which transforms MA data to RG (i.e. unlogged intensities).
>> So you could construct MAlist object from your data, transform it to RGlist,
>> (maybe take a log2) and you will have data for both channels.
>> HTH,
>> Maciej Jończyk
>>> Dear Bioconductor.
>>> In the past I have produced some PCA plots and heatmaps using one
>>> colour data. On the PCA, it is useful to separate out the different
>>> sample groups using the normalised expression values (say normal
>>> coloured green and treatment coloured red).
>>> However, this sort of analyses does not seem possible with two colour
>>> as you have a sinlge log2 normalised ratio (M value) as input to PCA
>>> and heatmap functions.
>>> Does anyone have experience of doing PCA and/or heatmaps with 2 colour
>>> data? Any info/advice appreciated.
>>> John.
>> --
>> Maciej Jonczyk,
>> Department of Plant Molecular Ecophysiology
>> Faculty of Biology, University of Warsaw
>> 02-096 Warsaw, Miecznikowa 1
>> Poland
>> --
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