Rna Seq Differential Expression

Question

[Empyrean]

Hi..

I have 5 rna seq samples for one group and 10 samples in another group. For this organism there is no reference transcriptome. I have assembled them using trinity and did assembly again by combining all samples to get master transcriptome. I have around 200k unique transcripts. I used RSEM to calculate abundance estimation and edgeR to get differential expression and normalized transcripts. Now i have 15 columns with the normalized fpkm values. If i wanted to get the differential expression between two groups, how can i use this information ? what analysis i should perform to obtain this

[Steve Lianoglou]

I share @Rm's confusion. You said you already did differential expression with edgeR -- so what are you trying to do again? If you're interested in differential expression, keep your data in a "count matrix" and w/ a proper design matrix and forget the FPKM values for now.

Answer 1

[Rm]

If i understand you correctly; you have already done DE using edgeR then why you need to do it again?

If not: Use DEB service (edger; deseq bayseq,) for DE analysis: http://www.ijbcb.org/DEB/php/onlinetool.php

Define 5 samples as group1 and the rest as group2 : then you can also use DEB or other tools like MEV, NOISeq for differential expression analysis.

[Empyrean]

Sorry for the confusion..i performed differential expression by giving 15 samples as individual input to edgeR and it gave me a file name Filename.normalized.FPKM and it has values ranging from 0 to 800,000 . I think its not a fold change. so, how can i get some meaningful data out of two groups or is there a way where i can do differential expression diffently by combining in to two groups?

[Steve Lianoglou]

@Empyrean: Sorry, you've got your wires crossed somewhere. edgeR most certainly did not give you an FPKM file. Please read through the edgeR User's Guide and adapt your analysis to the examples you see there. Your experimental design is not complex (just two groups), so it should be relatively straightforward.

[Rm]

simply submit your data to DEB server : you can perform edgeR analysis there.