Hi folks,
I am working with 3 related plant species from the same genus, which I would like to investigate their differences in response to an insect. For each species, I have only one time-point and a total of 6 replicates (3 for treatment and 3 for control). I have a reference genome and annotation, but for the other two I have to perform de novo assembly with Trinity. I am done with mapping (Salmon) and read counts (htseq-count for species with genome ref. and Corset for species with de novo assembly). Now the question is how should I normalise reads in order to let comparison among the three species possible?
I am very confused with these two ideas:
Normalise each species individually and run DEA (individually) for each species. After DEA, use Orthofinder or reciprocal blast search to find otholog groups. For this idea, It seems very straightforward but are the DEA results comparable (e.g., is it possible to say that 4-fold up-regulation of gene A in species 1 is higher than 2-fold up-regulation of its orthologues in species 2 and 3 since they are separately normalised?).
First create a reference genome for the three species (orthofinder/reciprocal blast search), normalise reads from the three species together and run DEA for each species individually. I assume that if all samples are normalised together, they are comparable? And I can say if
I am sorry if the questions sound silly, but I am a wet-lab biologist and need to be sure that I do proper analysis.
Thank you very much in advance and all advice/suggestions are most welcome!
