Hi all
I am using DEXSeq to analyse genome wide exon usage in RNA-Seq sample from
maize. I have two factors data. One is condition (weedy/weed-free) and
thiamethoxam treatment (treated/untreated). Each has 3 replicates. I wanted
to see the differential exon usage for weedy versus weed-free, treated
versus untreated and for interaction between condition and treatment. Here
is my sample table :
condition treatment replicate
WC_RepI weedy untreated 1
WC_RepII weedy untreated 2
WC_RepIII weedy untreated 3
WCT_RepI weedy treated 1
WCT_RepII weedy treated 2
WCT_RepIII weedy treated 3
WFC_RepI weed-free untreated 1
WFC_RepII weed-free untreated 2
WFC_RepIII weed-free untreated 3
WFT_RepI weed-free treated 1
WFT_RepII weed-free treated 2
WFT_RepIII weed-free treated 3
The DEXSeq dataset is
dxd1= DEXSeqDataSetFromHTSeq(
countFiles,
sampleData=SampleTable,
design=~sample+exon+treatment:exon+condition:exon,
flattenedfile=annotfile)
The formula that I used for all 3 analysis;
For weedy vs weed-free,
formulaFullModel=~sample+exon+treatment:exon+condition:exon
formulaReducedModel=~sample+exon+treatment:exon
dxd_e1=estimateDispersions(dxd1,formula=formulaFullModel)
dxd_t1=testForDEU(dxd_e1,reducedModel=formulaReducedModel,fullModel=formulaFullModel)
dxd_fc1=estimateExonFoldChanges(dxd_t1,fitExpToVar = "condition")
dxr3=DEXSeqResults(dxd_fc1)
For treated vs untreated,
formulaFullModel_t=~sample+exon+condition:exon+treatment:exon
formulaReducedModel_t=~sample+exon+condition:exon
dxd_e2=estimateDispersions(dxd1,formula=formulaFullModel_t)
dxd_t2=testForDEU(dxd_e2,reducedModel=formulaReducedModel_t,fullModel=formulaFullModel_t)
dxd_fc2=estimateExonFoldChanges(dxd_t2,fitExpToVar = "treatment")
dxr2=DEXSeqResults(dxd_fc2)
For interaction,
colData(dxd1)$mix<-paste0(colData(dxd1)$condition,colData(dxd1)$treatment)
formulaFullModel_i=~sample+exon+condition:exon+treatment:exon+condition:treatment:exon
formulaReducedModel_i=~sample+exon+condition:exon+treatment:exon
dxd_e3=estimateDispersions(dxd1,formula=formulaFullModel_i)
dxd_t3=testForDEU(dxd_e3,reducedModel=formulaReducedModel_i,fullModel=formulaFullModel_i)
dxd_fc3=estimateExonFoldChanges(dxd_t3,fitExpToVar = "mix")
dxr3=DEXSeqResults(dxd_fc3)
For first two analysis I got same result with no significant hits for
padj<0.1
While, for interaction I got 1 significant hit at padj<0.1.
I am just wondering if such results can be expected. Did I do wrong
somewhere in the steps?
I will really appreciate your suggestions.
My session info :
R version 3.3.1 (2016-06-21)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows >= 8 x64 (build 9200)
locale:
[1] LC_COLLATE=English_Canada.1252 LC_CTYPE=English_Canada.1252
[3] LC_MONETARY=English_Canada.1252 LC_NUMERIC=C
[5] LC_TIME=English_Canada.1252
attached base packages:
[1] stats4 parallel stats graphics grDevices utils datasets
[8] methods base
other attached packages:
[1] genefilter_1.54.2 DEXSeq_1.18.4
[3] RColorBrewer_1.1-2 AnnotationDbi_1.34.4
[5] DESeq2_1.12.3 SummarizedExperiment_1.2.3
[7] GenomicRanges_1.24.2 BiocInstaller_1.22.3
[9] GenomeInfoDb_1.8.3 IRanges_2.6.1
[11] S4Vectors_0.10.2 Biobase_2.32.0
[13] BiocGenerics_0.18.0 BiocParallel_1.6.3
Thanks!
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