Question: Differential Expression Analysis On Rpkms - Contrasts And Contrasts Of Contrasts
3
gravatar for Federico Giorgi
6.7 years ago by
Columbia University
Federico Giorgi420 wrote:

Dear all,

I'm looking for a tool (better would be a Bioconductor package) able to perform differential expression analysis on a table of RPKMs. I've seen so far only solutions implementing raw read counts, like DEGSeq, edgeR and BaySeq. And yeah, in this particular case I cannot transform RPKMs back to read counts.

Do you have any idea? Furthermore, a great advantage would be the capability of calculating significance of "contrasts of contrasts" analysis, i.e. of tetrafactorial designs.

E.g. having 4 conditions: WT control, WT treated, mutant control, mutant treated.

DE1 = WT treated vs. WT control
DE2 = mutant treated vs. mutant control

DE1DE2 = (mutant trated vs. mutant control) vs. (WT treated vs. WT control)

Something like the great limma does for microarrays. But on RNASeq RPKMs.

Thanks a lot for any hint! :-)

R rna • 2.2k views
ADD COMMENTlink modified 10 months ago by Biostar ♦♦ 20 • written 6.7 years ago by Federico Giorgi420
1

if you are more familiar with LIMMA, it now can handle RNAseq data. Check out the user guide: http://bioconductor.org/packages/release/bioc/vignettes/limma/inst/doc/usersguide.pdf

ADD REPLYlink written 6.7 years ago by Dave Bridges1.3k

Thanks Dave! Unfortunately, limma explicitly requires read counts, not RPKMs. I cite page 3 of the user guide: "The approach is to convert a table of sequence read counts into an expression object which can then be analysed as for microarray data."

ADD REPLYlink written 6.7 years ago by Federico Giorgi420

Do you want to go ahead with cufflinks output (which compute FPKM) data by some bioconductor package? .I am not sure, but i think all R packages are based on read count data and are suitable for differential expressions for genes only not for differential isoforms expression.

ADD REPLYlink written 6.7 years ago by Ngsnewbie360

I do use cummeRbund, but it's a mere wrapper of cuffdiff output (although very nice-looking), and it has no function of conversion to original raw read counts per isoform. AFAIK.

ADD REPLYlink written 6.7 years ago by Federico Giorgi420
0
gravatar for Federico Giorgi
6.7 years ago by
Columbia University
Federico Giorgi420 wrote:

Some useful thoughts on my question:

http://seqanswers.com/forums/showthread.php?t=18622

Conclusion: use edgeR and raw read counts. No complex designs for RPKMs

ADD COMMENTlink written 6.7 years ago by Federico Giorgi420
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