To find Differential expressed exons using xps package
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Biologist ▴ 90
@biologist-9801
Last seen 18 months ago

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, 

0.546665,

0.61691
0.322197
0.448327

0.609342

0.643494
0.509832
0.587659
0.481503
0.462129
0.515147
0.469252
0.645854
0.361072

0.548637

@cstrato Please help me. 

 

 

xps exons • 787 views
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cstrato ★ 3.9k
@cstrato-908
Last seen 3.0 years ago
Austria

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

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Correction: I do not mean quantile normalisation but median polish.

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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?

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Thank you Very much Christian. So to get the vocanoplot for differential expressed exons

volcanoplot(rma.ufr, mask = TRUE, labels="fSymbol") is this right?

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Yes

 

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And how to get a heatmap of differential expressed exons and normalised values?

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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

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