[BioC] limma: cannot repoduce older analysis

Philipp Pagel p.pagel at wzw.tum.de
Thu Jul 24 12:23:51 CEST 2008

	Dear list,

About 3 months ago I analyzed a simple two-color array experiment and got
results that looked quite reasonable and biologically sound. For some reason I
wanted to repeat the analysis and add a few plots that I had not included

When I got VERY different results in my toptable, I assumed I must have
changed something in my approach so I simply ran my original analysis
script again and found I was unable to reproduce the original toptable.
I have spent quite some time trying to debug the problem and have to say
that I am stuck. I have the original data files and the original
R-script. The normalization is 100% reproducible - i.e. the normalized
MALists seem to be identical. Yet when searching for differential
expression I get totally different results.

The only difference between the two runs lies in updates to R and limma in
the meantime. Unfortunately, I did not record which version of R, limma etc. I
had used, originally. My current environment is this:

	> sessionInfo()
	R version 2.7.1 (2008-06-23)


	attached base packages:
	[1] splines   stats     graphics  utils     datasets  grDevices methods   base

	other attached packages:
	[1] statmod_1.3.6   MASS_7.2-42     xtable_1.5-2    limma_2.14.2    lattice_0.17-10
	[6] cairoDevice_2.8

	loaded via a namespace (and not attached):
	[1] grid_2.7.1  tools_2.7.1

My search for differential expression seems pretty standard to me:

	MA$design <- modelMatrix(targets, ref="control")
	# flag out controls etc.
	MA$weights[MA$genes$Status != 'miRNA', ] = 0.0
	# sort spots by ID to put replicates next to each other
	MA2 <- MA[order(MA$genes$ID), ]

	dupfit <- duplicateCorrelation(MA2, ndups=4)
	fit <- lmFit(MA2, ndups=4, correlation=dupfit$consensus)
	fit <- eBayes(fit)
	tt <- topTable(fit, number=100)

I have siftet through the changelog of limma hoping to find a hint about
some changed default or behaviour in lmFit or eBayes but saw nothing
that seemed to expain my problem.

Any hints apprechiated.


Dr. Philipp Pagel
Lehrstuhl für Genomorientierte Bioinformatik
Technische Universität München
Wissenschaftszentrum Weihenstephan
85350 Freising, Germany

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