friends,

i have a file containing raw counts produced by featurecounts. now i need RPKM and VST...i read DEseq2 vignette but really i could not get what to do...supposing my raw counts file named counts.txt and placed in  /usr/data/nfs6/izadi/microarray/subread-1.4.6-p5-source/ and i installed DEseq2 package...please help me to calculate the RPKM and VST.

for example in the below i don't know what i should replace in the code

library("pasilla")

library("Biobase")

data("pasillaGenes")

countData <- counts(pasillaGenes)

colData <- pData(pasillaGenes)[,c("condition","type")] 

i am not familiar enough with R but i need RPKM and VST files...

thank you

We don't offer any functionality for helping you to calculate the feature size (the gene length), if you are importing a matrix of counts. So you would have to calculate the exonic basepairs for each gene on your own, using import() from rtracklayer and functions from the GRanges package. Or you could use software like RSEM or Cufflinks to calculate the FPKM estimate outside of R.

(Although if you do the fragment counting with the summarizeOverlaps() function, this information is included as rowRanges and we have a function fpkm() which will do the calculation for you.)

You can calculate VST values by first creating a DESeqDataSet and then using the varianceStabilizingTransformation() function on that object.

So you should first follow the vignette or workflow[1] example on creating a DESeqDataSet from matrix input:

http://www.bioconductor.org/help/workflows/rnaseqGene/

hmmmm, different user, but very similar question.... 

I've provided an answer here