First of all I would like to apologise if this is a straight forward question/ similar to another thread.
I have been given some RNA-seq data to do my dissertation (i say this as there is no possibility for more sequencing!), and have assembled a de novo transcriptome. Now I want to look at differentially expressed genes.
Unfortunately, there are no replicates, and nestedness within the design.
There are 6 individuals- normal male, infected male (intersex1), infected male (intersex2), normal female, normal female infected, infected female (intersex3). From this I want to look at the differences between males/females, infected/uninfected and normal males/females/intersex1/2/3. However, I also have 4 different tissue types, from each of these individuals which I also want to compare.
So my question is, what would be the best way to approach such analysis?
I can't really remove any explanatory factors, as they are all important- and even then i still probably do not have enough replicates. I am not entirely sure what a 'reasonable' dispersion value would be, as this is not a controlled experiment (environmental samples). So perhaps the method which appeals to me most would be to identify the non DE genes and calculate the dispersion value from that?
Any advice/ suggestions would be greatly appreciated!