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?