Entering edit mode
Philipp Pagel
▴
190
@philipp-pagel-2810
Last seen 10.3 years ago
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
before.
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)
x86_64-pc-linux-gnu
locale:
LC_CTYPE=en_US.utf8;LC_NUMERIC=C;LC_TIME=en_US.utf8;LC_COLLATE
=en_US.utf8;LC_MONETARY=C;LC_MESSAGES=en_US.utf8;LC_PAPER=en_US.utf8;L
C_NAME=C;LC_ADDRESS=C;LC_TELEPHONE=C;LC_MEASUREMENT=en_US.utf8;LC_IDEN
TIFICATION=C
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.
cu
Philipp
--
Dr. Philipp Pagel
Lehrstuhl f?r Genomorientierte Bioinformatik
Technische Universit?t M?nchen
Wissenschaftszentrum Weihenstephan
85350 Freising, Germany
http://mips.gsf.de/staff/pagel