Entering edit mode
> In limma manual it is written that correlation should be negative
for
> dye swaps- why is it positive?- is it a question of wrong model
matrix
> or is it something wrong with my samples?
I concur with Juan Pedro that the most likely cause of this problem is
mislabeling of the data; often they are already 'swapped back' for
convenience, which is of course wrong.
Regarding dye-bias, it would have be to really extreme to get the
thing
you're seeing.
But more generally, if you are worried about gene-specific dyebias
(and
basically everyone should be), you could take a look at the dyebias
package.
It implements a method published recently by us (Margaritis et al.,
Mol. Sys.
Biol. 2009, doi:10.1038/msb.2009.21).
You can use the dyebias package to (1) recognize if there is any gene-
specific
dyebias; (2) which genes are affected most badly; (3) which slides are
affected most badly; and (4) to correct it. Including a Dye effect in
Limma
is useful, but not enough: linear models cannot cope with the fact
that the
dye bias artefact depends not only on the gene, but also on the
hybridization
(for details, see the paper). The latter was not realized previously.
We frequently achieve variance reductions of M of over 50%. The only
prerequisite is a set of dye-swaps. If you run into difficulties using
the
package, please feel free to drop me a line.
Regards,
Philip
--
Philip Lijnzaad, PhD
Holstege Genomics Laboratory
Dept. of Biomedical Genetics
University Medical Center (UMC), Utrecht
Stratenum room 2.211 (on Tuesdays and Thursdays not in after 15.00)
MSN chat (*NOT* email): philip_lijnzaad at hotmail.com
P.O. Box 85060, 3508 AB Utrecht
(Universiteitsweg 100, 3584 CG Utrecht)
The Netherlands
tel: +31 (0)8875 68464
fax: +31 (0)8875 68479
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