I have the following RNA-seq set-up:
- 5 samples for comparison
- matrix with TMM-normalized FPKM values
Now, TMM-normalization allows comparison across samples, so I can compare gene X between samples 1-5. Can I also compare gene X with gene Y within, say, sample 2 or does the TMM-normalization make this inappropriate (e. g. using different scaling factors for each gene or anything like that)?
I have tried to read the TMM-normalization paper (cited below) and it seems that when there are more than two samples, it picks a reference sample and calculates a single scaling factor for each pairwise comparison between the reference and each non-reference sample. That would seem to suggest that the within-sample comparison like the one outlined above is possible. Is it?
Robinson, M. and A. Oshlack (2010). "A scaling normalization method for differential expression analysis of RNA-seq data." Genome Biology 11(3): R25.