NGS tips: FPKM for short genes in cufflinks
Don't trust Cufflinks FPKM for short genes Here is what Cole (author of Cufflinks) commented to the observation of " very high RPKM values from Cufflink ": This issue has been discussed elsewhere on this board. As Nicholas points out, RNA-Seq really isn't reliable for very short transcripts. The reason is that all the fragments that map to these transcripts come from the "tail" of the distribution of library fragment lengths. That is, fragments that map to microRNAs are much, much shorter than most fragments in the library - by design in the RNA-Seq protocol, which size selects away very short inserts. Thus, Cufflinks infers that even though relatively few fragments actually mapped to the microRNAs, there were probably TONS of individual microRNA molecules in the transcriptome before all of the various size selection parts of the protocol kicked in. Cufflinks accordingly increases the FPKM of these short transcripts to compensate for the bias agai...