[BioC] affycoretools repost

[Louisa A Rispoli/AS/EXP/UTIA]

Hi all-

I am fairly new to bioconductor and am very excited about the example
described in the affycoretools vignette since 1) it would make my mentor
very happy (almost the exact situation that we have in the data set), 2)
seemed like a simple solution to a issue I was not sure how to approach and
3) package seemed straightforward and easy to use.  This is probably a
stupid question arising from the fact that I have yet to fully understand R
and bioconductor but I have tried to spend a better portion of the day
trying to figure where I am going wrong and having no luck. I am trying to
compare samples (two treatment groups)  amplified two different ways and
see which differentially expressed genes are shared between the two
different methodologies. According to the PCA plot I need to compute the
expression values for each methodology separately and then combine the
data. I can not seem to create the new expression set properly to perform
the limma analysis.   I have attached a text file with my R code.

 If anyone could point me in the right direction (either the mistake I am
makeing or even the vignette that I should read closer) I would appreciate
the help. Sorry if this seems very silly but we do not have anyone here
familiar with Bioconductor to go to for help.

Thanks

Louisa

Sorry required a repost, forgot that attachments were stripped.

R version 2.7.0 (2008-04-22)
Copyright (C) 2008 The R Foundation for Statistical Computing
ISBN 3-900051-07-0
> library(affycoretools)
Loading required package: 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)'.

Loading required package: affyio
Loading required package: preprocessCore
Loading required package: limma
Loading required package: GOstats
Loading required package: graph
Loading required package: GO.db
Loading required package: AnnotationDbi
Loading required package: DBI
Loading required package: RSQLite
Loading required package: annotate
Loading required package: xtable
Loading required package: RBGL
Loading required package: Category
Loading required package: genefilter
Loading required package: survival
Loading required package: splines
Loading required package: biomaRt
Loading required package: RCurl

Attaching package: 'biomaRt'


        The following object(s) are masked from package:annotate :

         getGO

Loading required package: gcrma
Loading required package: matchprobes
Loading required package: annaffy
Loading required package: KEGG.db

Attaching package: 'annaffy'


        The following object(s) are masked from package:RCurl :

         getURL

Warning messages:
1: package 'DBI' was built under R version 2.7.1
2: package 'RSQLite' was built under R version 2.7.1
3: package 'xtable' was built under R version 2.7.1

> pd <-read.AnnotatedDataFrame("pData.txt", sep="\t", header=TRUE,
row.names=1)
> dat <- ReadAffy(phenoData= pd)
> pData(dat)
               amp  trt
PolyC-1.CEL  PolyA Ctrl
PolyC-2.CEL  PolyA Ctrl
PolyC-3.CEL  PolyA Ctrl
PolyC-4.CEL  PolyA Ctrl
PolyC-5.CEL  PolyA Ctrl
PolyC-6.CEL  PolyA Ctrl
PolyC-7.CEL  PolyA Ctrl
PolyC-8.CEL  PolyA Ctrl
PolyHS-1.CEL PolyA   HS
PolyHS-2.CEL PolyA   HS
PolyHS-3.CEL PolyA   HS
PolyHS-4.CEL PolyA   HS
PolyHS-5.CEL PolyA   HS
PolyHS-6.CEL PolyA   HS
PolyHS-7.CEL PolyA   HS
PolyHS-8.CEL PolyA   HS
WTC-1.CEL       WT Ctrl
WTC-2.CEL       WT Ctrl
WTC-3.CEL       WT Ctrl
WTC-4.CEL       WT Ctrl
WTC-5.CEL       WT Ctrl
WTC-6.CEL       WT Ctrl
WTC-7.CEL       WT Ctrl
WTC-8.CEL       WT Ctrl
WTHS-1.CEL      WT   HS
WTHS-2.CEL      WT   HS
WTHS-3.CEL      WT   HS
WTHS-4.CEL      WT   HS
WTHS-5.CEL      WT   HS
WTHS-6.CEL      WT   HS
WTHS-7.CEL      WT   HS
WTHS-8.CEL      WT   HS

> eset <-affystart(groups=rep(1:4, each = 8), groupsnames =
unique(paste(pData(pd)[,1],pData(pd)[,2], sep="-")), phenoData = pd)
Background correcting
Normalizing
Calculating Expression

> eset1 <-affystart(filenames=list.celfiles()[1:16], plot = FALSE,
pca=FALSE)
Background correcting
Normalizing
Calculating Expression

> eset2 <-affystart(filenames=list.celfiles()[17:32], plot=FALSE,
pca=FALSE)
Background correcting
Normalizing
Calculating Expression

> eset <- new("ExpressionSet", exprs=cbind(exprs(eset1), exprs(eset2)),
phenoData = pd, annotation=annotation(eset1))
Error in validObject(.Object) :
  invalid class "ExpressionSet" object: sampleNames differ between
assayData and phenoData

> eset1
ExpressionSet (storageMode: lockedEnvironment)
assayData: 24128 features, 16 samples
  element names: exprs
phenoData
  sampleNames: PolyC-1.CEL, PolyC-2.CEL, ..., PolyHS-8.CEL  (16 total)
  varLabels and varMetadata description:
    sample: arbitrary numbering
featureData
  featureNames: AFFX-BioB-3_at, AFFX-BioB-5_at, ..., BtAffx.29968.1.S1_at
(24128 total)
  fvarLabels and fvarMetadata description: none
experimentData: use 'experimentData(object)'
Annotation: bovine

> eset2
ExpressionSet (storageMode: lockedEnvironment)
assayData: 24128 features, 16 samples
  element names: exprs
phenoData
  sampleNames: WTC-1.CEL, WTC-2.CEL, ..., WTHS-8.CEL  (16 total)
  varLabels and varMetadata description:
    sample: arbitrary numbering
featureData
  featureNames: AFFX-BioB-3_at, AFFX-BioB-5_at, ..., BtAffx.29968.1.S1_at
(24128 total)
  fvarLabels and fvarMetadata description: none
experimentData: use 'experimentData(object)'
Annotation: bovine

"If we knew what we were doing, it wouldn't be called Research." - Albert
Einstein

Louisa Rispoli, Ph.D. Reproductive Physiology
Department of Animal Science
University of Tennessee, Knoxville
A105 Johnson Animal Research and Teaching Unit
1750 Alcoa Highway
Knoxville, TN 37920
phone:(865) 946-1874
fax:(865) 946-1010
email: lrispoli at utk.edu