[BioC] Problem with RMA using pdInfoBuilder, oligo and limma

[Anne-Marie Madore]

Hi,

 
I am a Ph.D. student from Quebec, Canada. I'm a beginner with R and
Bioconductor. Until now the only experience I have is in analyzing
microarray data using affy and limma packages. Now I'm trying to analyze
Rat Gene 10 st arrays and I would like to run RMA analysis and Smyth
moderated t test on those arrays. Since no cdf official package is
available for those arrays, after reading many of the questions and
responses on this mailing list, I decided to use pdInfoBuilder, oligo
and limma packages to run analysis. 

I built my package according to info available at this web site:
http://article.gmane.org/gmane.science.biology.informatics.conductor/189
63/match=oligo+s
Below is the output of the installation.

The problem is, after running RMA and moderated T-test, I get expression
and differential expression measured for all probe separately but not
the calculated expression for all probes representing a gene. When I run
RMA, I got only two steps, Background correcting and Normalizing but not
Calculating expression. Do you know how I can get differential
expression calculated for each gene? I don't know if the problem is in
the package I built or if I can use some code to answer this question.
Below I list warning messages I have when I check my package after
installation with R CMD check and codes used to analyze expression
arrays.

Many thanks for your help,

Anne-Marie Madore
Universite Laval/UQAC


## installing the package in cmd command shell

c:\Program Files\R\R-2.8.1\bin>R CMD INSTALL pd.ragene.1.0.st.v1
installing to 'c:/PROGRA~1/R/R-28~1.1/library'


---------- Making package pd.ragene.1.0.st.v1 ------------
  adding build stamp to DESCRIPTION
  installing NAMESPACE file and metadata
  installing R files
  installing inst files
  preparing package pd.ragene.1.0.st.v1 for lazy loading
Loading required package: RSQLite
Loading required package: DBI
Loading required package: oligoClasses
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)'.

  no man files in this package
  installing indices
  installing help
  adding MD5 sums

* DONE (pd.ragene.1.0.st.v1)



## If I run a check (R CMD check pd.ragene.st.v1) I get three warning
messages and one note: 

1.       * checking R files for non-ASCII characters ... WARNING 
Found the following files with non-ASCII characters: all.R Portable
packages must use only ASCII characters in their R code, except perhaps
in comments.

2.       * checking whether the name space can be loaded with stated
dependencies ... WARNING
Error in initDbConnection() : could not find function "dbConnect" Error:
.onLoad failed in 'loadNamespace' for 'pd.ragene.1.0.st.v1' Execution
halted A namespace must be able to be loaded with just the base
namespace loaded:
otherwise if the namespace gets loaded by a saved object, the session
will be unable to start. 
Probably some imports need to be declared in the NAMESPACE file.

3.       * checking R code for possible problems ... NOTE
closeDb: no visible binding for global variable 'dbCon' 

4.       * checking for missing documentation entries ... WARNING
Undocumented code objects: 
pd.ragene.1.0.st.v1
All user-level objects in a package should have documentation entries.
See the chapter 'Writing R documentation files' in manual 'Writing R
Extensions'.


## analyzing the package

> library(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(pdInfoBuilder)
Loading required package: RSQLite
Loading required package: DBI
Loading required package: affxparser
Loading required package: oligo
Loading required package: splines
Loading required package: preprocessCore
Loading required package: AnnotationDbi
Loading required package: oligoClasses
oligo Package - Series 1.5.x
> setwd("D:/Anne-Marie/Doctorat/puces ADN macrophages/puces rat/Annie
Dube/Analyse")
> library("pd.ragene.1.0.st.v1")
> library(oligo)
> library(limma)
> library(genefilter)
Loading required package: survival
> library(geneplotter)
Loading required package: lattice
Loading required package: annotate
Loading required package: xtable
KernSmooth 2.22 installed
Copyright M. P. Wand 1997
> cel.files <- list.celfiles(".", full.names = TRUE)
> basename(cel.files)
 [1] "AD_Ctrl_1.CEL"    "AD_Ctrl_2.CEL"    "AD_Ctrl_3.CEL"
"AD_Ctrl_5.CEL"   
 [5] "AD_Ctrl_6.CEL"    "AD_Traite_10.CEL" "AD_Traite_11.CEL"
"AD_Traite_7.CEL" 
 [9] "AD_Traite_8.CEL"  "AD_Traite_9.CEL" 
> test <- read.celfiles(cel.files)
Platform design info loaded.
> phenoData(test) <- read.AnnotatedDataFrame("phenoData.txt", header =
TRUE, row.name=1)
> class(test)
[1] "GeneFeatureSet"
attr(,"package")
[1] "oligoClasses"
> class(phenoData)
[1] "standardGeneric"
attr(,"package")
[1] "methods"
> eset <- rma(test)
Background correcting
Normalizing
> e <- exprs(eset)
> design <- model.matrix(~factor(eset$Key))
> fit <- lmFit(eset, design)
> ebayes <- eBayes(fit)
> 
> 
## also a try with slightly the same method as used to make the link
between phenol data and .CEL with affy package

> pd <- read.AnnotatedDataFrame("pheno.txt", header = TRUE, row.names =
1)
> pData(pd)
                 Phenotype
AD_Ctrl_1.CEL      Control
AD_Ctrl_2.CEL      Control
AD_Ctrl_3.CEL      Control
AD_Ctrl_5.CEL      Control
AD_Ctrl_6.CEL      Control
AD_Traite_7.CEL    Traited
AD_Traite_8.CEL    Traited
AD_Traite_9.CEL    Traited
AD_Traite_10.CEL   Traited
AD_Traite_11.CEL   Traited
> a <- read.celfiles(filenames = rownames(pData(pd)), phenoData = pd,
verbose = TRUE)
Platform design info loaded.
> eset <- rma(a)
Background correcting
Normalizing
> exprs.eset <- exprs(eset)
> population.groups <- factor (c(rep("Control",5), rep ("Traited",5)))
> design <- model.matrix (~population.groups)
> fit <- lmFit (eset, design)
> fit.eBayes <- eBayes (fit)
>
> sessionInfo()
R version 2.8.1 (2008-12-22) 
i386-pc-mingw32 

locale:
LC_COLLATE=English_United States.1252;LC_CTYPE=English_United
States.1252;LC_MONETARY=English_United
States.1252;LC_NUMERIC=C;LC_TIME=English_United States.1252

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

other attached packages:
 [1] geneplotter_1.20.0        annotate_1.20.1           xtable_1.5-4

 [4] lattice_0.17-17           genefilter_1.22.0         survival_2.34-1

 [7] limma_2.16.3              pd.ragene.1.0.st.v1_0.0.1
pdInfoBuilder_1.6.0      
[10] oligo_1.6.0               oligoClasses_1.4.0
AnnotationDbi_1.4.2      
[13] preprocessCore_1.4.0      affxparser_1.14.2         RSQLite_0.7-1

[16] DBI_0.2-4                 Biobase_2.2.1            

loaded via a namespace (and not attached):
[1] grid_2.8.1         KernSmooth_2.22-22 RColorBrewer_1.0-2