[BioC] Vsn transformation repeatability

[Paolo Innocenti]

Hi all,

We are attempting to identify significantly differentially expressed 
genes between two treatments of female Drosophila melanogaster, using 
results from Affymetrix Drosophila2 microarray chip hybridization (8 
chips, 4 per treatment). The end result is that the number of 
significantly differentially expressed genes for a particular FDR 
changes with each run, despite the input data and the program code 
remaining unchanged.

We are using ”vsnrma” for pre-processing, which utilizes a search 
algorithm to find the maximum likelihood transformed values. The 
procedure produces slightly different calibrated GLOG2-transformed 
expression values on each run, despite the untransformed expression 
values and the R code being identical each time. Our first guess is that 
the search algorithm is unable to locate the likelihood peak and settles 
on a slightly different set of transformed values each time. Is this a 
possibility? If so, is there anything we can do to fix it? E.g. increase 
the number of iterations, or modify the search algorithm.

Many thanks in advance.
Paolo Innocenti


 > library(affy)
Loading required package: Biobase
Loading required package: tools

Welcome to Bioconductor

   Vignettes contain introductory material. To view, type
   'openVignette()'. To cite Bioconductor, see
   'citation("Biobase")' and for packages 'citation(pkgname)'.

 > library(vsn)
Loading required package: lattice
Loading required package: limma
 > data <- ReadAffy(celfile.path="./celfiles")
 > sampleNames(data) <- c("z1","z2","z3","z4","m1","m2","m3","m4")
 > eset1 <- vsnrma(data)
100% done.4 x 8 matrix (1 stratum).   0% done.
Please use 'meanSdPlot' to verify the fit.
Calculating Expression
 > eset2 <- vsnrma(data)
100% done.4 x 8 matrix (1 stratum).   0% done.
Please use 'meanSdPlot' to verify the fit.
Calculating Expression
 > exprs(eset1)[1,]
       z1       z2       z3       z4       m1       m2       m3       m4
12.52630 12.23441 12.54579 12.35582 12.45190 12.35984 12.33933 12.35031
 > exprs(eset2)[1,]
       z1       z2       z3       z4       m1       m2       m3       m4
12.52422 12.24059 12.53899 12.35949 12.44828 12.35460 12.34465 12.35277
 > head(exprs(eset1))==head(exprs(eset2))
                 z1    z2    z3    z4    m1    m2    m3    m4
1616608_a_at FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE
1622892_s_at FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE
1622893_at   FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE
1622894_at   FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE
1622895_at   FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE
1622896_at   FALSE FALSE FALSE FALSE FALSE FALSE FALSE FALSE
 > sessionInfo()
R version 2.8.0 (2008-10-20)
i686-pc-linux-gnu

locale:
LC_CTYPE=en_US.UTF-8;LC_NUMERIC=C;LC_TIME=en_US.UTF-8;LC_COLLATE=en_US.UTF-8;LC_MONETARY=C;LC_MESSAGES=en_US.UTF-8;LC_PAPER=en_US.UTF-8;LC_NAME=C;LC_ADDRESS=C;LC_TELEPHONE=C;LC_MEASUREMENT=en_US.UTF-8;LC_IDENTIFICATION=C

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

other attached packages:
[1] drosophila2cdf_2.3.0 vsn_3.8.0            limma_2.16.2
[4] lattice_0.17-15      affy_1.20.0          Biobase_2.2.0

loaded via a namespace (and not attached):
[1] affyio_1.10.0        grid_2.8.0           preprocessCore_1.4.0


-- 
Paolo Innocenti
Department of Animal Ecology, EBC
Uppsala University
Norbyvägen 18D
75236 Uppsala, Sweden