[Bioc-devel] No Significant hits!

[Sean Davis]

Hi, Pabita.

This question is better asked on the bioconductor support site (
HTTPS://support.bioconductor.org). However, finding no evidence for
differential expression is not uncommon. Whether it is expected or not is
hard to comment on without data.

Sean

On Jul 30, 2016 10:43, "Pabita Basnet" <saudpabita at gmail.com> wrote:

> 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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