Help!
I have no replicate data. I spent a lot of time doing downregulated/upregulated and then do Pathway Analysis.

Method 1: Rsubread and the edgeR quasi-likelihood pipeline:
it does not give me a Dispersion estimation. As a result, I can not continue to calculate
res <- glmQLFTest(fit, contrast=B.LvsP) , neither pathway analysis

Method 2: DESEQ2 R does not calculate dds
Link: bioconductor.org/packages/release/workflows/vignettes/RnaSeqGeneEdgeRQL/inst/doc/edgeRQL.html

Method 3:can not draw voom plot and gives error fit.cont <- eBayes(fit.cont). " no residual"

Link: combine-australia.github.io/RNAseq-R/06-rnaseq-day1.html#Testing_relative_to_a_threshold_(TREAT)

Method 4" Page 24 gives a solution for no replication data. I do not know what is housekeeping index is and what I should add to my R pipeline

y0 <- estimateDisp(y1[housekeeping,], trend="none", tagwise=FALSE)
Error in h(simpleError(msg, call)) :
error in evaluating the argument 'i' in selecting a method for function '[': object 'housekeeping' not found

Link: www.bioconductor.org/packages/devel/bioc/vignettes/edgeR/inst/doc/edgeRUsersGuide.pdf

Method 5: it is not easy to follow
hLink:www.rstudio.com/wp-content/uploads/2015/02/data-wrangling-cheatsheet.pdf

I appreciate it if you help me by example or by way to follow comments

Thanks in advance



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