Hello, I am doing RNAseq analysis for the first time. I have two samples, control and treatment of a plant variety collected at 14 days interval. I obtained few data sets of differentially expressed genes which had similar gene ID and were also the same transcripts. They were same except that, they differed in FPKM values and had different regulation, like one is up-regulated (12-fold) and other is down-regulated (13-fold). I assume that minor variation errors could be possible but such fold variation along with up-regulation and down-regulation cannot be overlooked. I also don’t think that they could be different fragments of the same transcript as they show different regulation. Can anyone suggest the reason for such data? Or is this mere an error.
Large log fold-changes are often observed in RNA-seq data that has undergone normalisation to FPKM expression levels, even as high as +90, but this is more due to the inadequacies of this normalisation strategy than anything else. For one, this normalisation is not performed across samples and is therefore not adequately adjusting for different library sizes.
If you can obtain raw counts, my advice is to get those, and then work from those using a 'better' normalisation strategy.