Run DEXSeq successfully but got a result that padj==1 in all genes
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Entering edit mode
2406691063 • 0
@7d4e3803
Last seen 3 months ago
China

I used the "dexseq_prepare_annotation.py" provided by DEXSeq to transform my "gtf" and "dexseq_count.py" to get exon count successfully. Then I have run DEXSeq. To my surprise, all padj in result is "1". Can you tell me the errors in my analysis?

My codes in DEXSeq are following:

countFile<-list.files("D:/Users/Administrator/Desktop/cold/output/DT",pattern="txt$",full.names=TRUE)
gffFile <- list.files(D:/Users/Administrator/Desktop/cold/output/DT, pattern="gff$", full.names=TRUE) 
sampleTable <- data.frame(row.names=c(paste("treated", 1:5, sep=""), paste("untreated", 1:5, sep="")),
                          condition=rep(c("knockdown", "control"), c(5, 5)))
dxd <- DEXSeqDataSetFromHTSeq(
  countFile,
  sampleData=sampleTable,
  design= ~sample + exon + condition:exon,
  flattenedfile=gffFile

I got:

converting counts to integer mode
Warning message:
In DESeqDataSet(rse, design, ignoreRank = TRUE) :
  some variables in design formula are characters, converting to factors

Then I ran the code:

dxr <- DEXSeq(dxd)

I got the output:

-- note: fitType='parametric', but the dispersion trend was not well captured by the
   function: y = a/x + b, and a local regression fit was automatically substituted.
   specify fitType='local' or 'mean' to avoid this message next time.
Warning messages:
1: In MulticoreParam(workers = 1) :
  MulticoreParam() not supported on Windows, use SnowParam()
2: In vst(exp(alleffects), object) :
  Dispersion function not parametric, applying log2(x+ 1) instead of vst...

Finally, I ran the code;

NY<-as.data.frame(dxr)
NY$pvaue
NY$padj

But the output showed:

>NY$padj
   [1] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
  [67] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
 [133] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
 [199] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
 [ reached getOption("max.print") -- omitted 2320 entries ]
> NY$pvalue
   [1] 4.483972e-02 3.221151e-02 3.590747e-02 2.318606e-02 1.137251e-02 2.145131e-03 4.802402e-02 3.626523e-02 2.234388e-02 3.669871e-02
  [11] 2.046010e-02

I have no ideas with my errors. Can anyone help me solve the problem?

DEXSeq • 294 views
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Entering edit mode
ATpoint ★ 4.5k
@atpoint-13662
Last seen 5 hours ago
Germany

It means there is no evidence for differential expression. Either because there is none, or it's underpowered and/or technical variation introduces noise. Use PCA to explore your data and check for batch effects. You find info in DEXSeq and DESeq2 vignettes to get started.

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