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;
0.482065,
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Correction: I do not mean quantile normalisation but median polish.
Thank you Very much Christian. So to get the vocanoplot for differential expressed exons
volcanoplot(rma.ufr, mask = TRUE, labels="fSymbol") is this right?
And is it possible to get a heatmap of differential expressed exons and normalised values?
Thank you Very much Christian. So to get the vocanoplot for differential expressed exons
volcanoplot(rma.ufr, mask = TRUE, labels="fSymbol") is this right?
Yes
And how to get a heatmap of differential expressed exons and normalised values?
Regarding heatmaps you have already got some answers, e.g. C: How to import normalized file (using xps) to limma for differential expressed ge
I am not the right person to ask regarding heatmaps since I have mostly used the commercial
program 'Spotfire'.
Regards,
Christian