Hi everyone,
I have a question regarding the use of RSEM for estimating isoform expression levels from RNA-Seq data. I have 5 different RNA-Seq assemblies (4 are SE and the remaining one is PE) from different species of the same tissue. The species are divided in two groups (A and B) and I'm interested in looking at the expression profiles of some genes between the two groups. When I use Trinity's Transcript Quantification protocol with RSEM, I get very different FPKM values for the SE assemblies compared to the PE assembly. For one gene I'm getting the following values (and it is very similar with the other genes):
SE assemblies: 424 (group A), 381.8 (group A), 471 (group A), 115.3 (group B)
PE assemblies: 1.74 (group B)
As you can see, there's a difference of two orders of magnitude between assemblies, and this difference occurs within the same species group (B). So, does the FPKM values from SE and PE libraries can be directly compared? Is there a way to do a correction for the different amount of reads between SE and PE libraries?
Any suggestion is very welcome, thanks!!
I didn't understand. Did you use trinity to separately assemble each of the 5 samples? I think assembly performance vary a lot between SE and PE. Maybe that is the problem.
A: Are Fpkm And Rpkm Values Equivalent?
Thanks @genomax, however I'm using fpkm values for both single-end and paired-end libraries, and I don't understand why there are such great differences in fpkm values.