Hi Christian,

scheme.exon <- import.exon.scheme("Scheme",filedir=scmdir,layoutfile=paste(libdir,"HuGene-2_0-st.clf",sep="/"),schemefile=paste(libdir,"HuGene-2_0-st.pgf",sep="/"),probeset=paste(anndir,"HuGene-2_0-st-v1.na35.hg19.probeset.csv",sep="/"),transcript=paste(anndir,"HuGene-2_0-st-v1.na35.hg19.transcript.csv",sep="/"),verbose=TRUE)

##Reading CEL Info
#Import CEL-files by giving the path where all .CEL files are present

celfiles <- dir(path = "D:/GeneST_Analyse/ProjectName/CELfiles", pattern = "*.CEL$", all.files = FALSE, full.names = FALSE, recursive = FALSE, ignore.case = FALSE)
data.exon <- import.data(scheme.exon,"tmp_data_exon",filedir=rootdir,celfiles=celfiles,celdir=celdir,verbose=FALSE)

unlist(treeNames(data.exon))

[1] "X_8__HuGene_2_0_st_.cel" "E__HuGene_2_0_st_.cel"  
[3] "GG7__HuGene_2_0_st_.cel" "J__HuGene_2_0_st_.cel"  
[5] "O__HuGene_2_0_st_.cel"   "T__HuGene_2_0_st_.cel"

data.rma <- rma(data.exon, "tmp_exonRMA", filedir=rootdir, verbose=FALSE, exonlevel="affx+core", option="transcript", background="antigenomic")

prefltr <- PreFilter(mad=c(0.5), lothreshold=c(7.0,0.02,"mean"), hithreshold=c(10.5,80.0,"percent"))

str(prefltr)

rma.pfr <- prefilter(data.rma, "tmp_exonPrefilter", filedir=rootdir , filter=prefltr, minfilters=2, verbose=FALSE)

tmp <- validData(rma.pfr)

head(tmp)

dim(tmp[tmp[,"FLAG"]==1,])

[1] 5295    2

The data show that 5295 exons of the 44797 exons on the Human Gene 2.0 ST Array are selected for further
analysis

unifltr <- UniFilter(foldchange=c(1.5,"both"), unifilter=c(0.05,"pval"))

Here I want the FoldChange to be >= 1.5 and P.Value <= 0.05

rma.ufr <- unifilter(data.rma, "tmp_exonUnifilter", filedir=rootdir , filter=unifltr, group=c("GrpA","GrpA","GrpA","GrpB","GrpB","GrpB"), xps.fltr=rma.pfr, verbose=FALSE)

tmp2 <- validData(rma.ufr)

Now when I see the values of "tmp2" I see there are some exons with values like the following. the down regulated values should be in negative(-). Could you please help me in this. And the Volcano plot gives only the differentially expressed exons? Looking forward to your response.

And a small doubt. As I want the differential expressed exons 

In the command RMA the option should be exon or probset and what should be the exonlevel? Imworking on Human Gene 2.0 ST array;

If you look at the example shown in Chapter 6.2 of vignette 'xps.pdf' you will see the columns Mean1, Mean2, FoldChange.
Then you will see that a FoldChange of 0.7028 means that the gene is downregulated, i.e. 1/0.7028 = -1.3-fold downregulation.

Regarding the Volcano plot: Once again please read the help file '?volcanoplot' first. For parameter 'mask'
it says: if TRUE draw only points for transcripts satisfying the univariate test.

Regarding function rma():
- for parameter exonlevel, exonlevel="affx+core" is ok, but you coluld also use 'core' only.
- for parameter option the default is "transcript". If you want to get exons, you should use "probeset"

However, please note the following important issue:

When using HuGene-2.0 arrays you should only compute the transcript levels since it does NOT make any 
sense to compute the exon levels for the following reason:
As an example I use the gene 'agrin (AGRN)' with the transcript_cluster_id '16657598':
The annotation 'HuGene-2_0-st-v1.na35.hg19.transcript.csv' shows that it has 34 'total_probes'.
However, the annotation 'HuGene-2_0-st-v1.na35.hg19.probeset.csv' shows that each probeset (exon)
has a 'probe_count' of 1 only!!! For this reason you cannot do quantile normalization. 

Please discuss this issue with your colleagues!

Regards,
Christian