I have questions regarding merging of files in cuffmerge and usage of groups in cuffdiff. I have 5 samples in condition1 and 4 samples in condition2 and 3 samples are normal samples. I would like carry out differential expression analysis using cuffdiff. So I have following questions:
i) Can cuffdiff carry out differential analysis for three conditions- Condition1, Condition2 and Normal, together? I am asking this to know what is best method to input these conditions in cuffdiff- pairwise or all conditions together?
ii) If I choose to go with pairwise comparisons - Condition1 vs Condtion2 or Normal vs Condition1 or Normal vs Condition2, is it correct to use transcript.gtf files from all 12 samples to create a merged gtf file which can be used further in either of the mentioned comparisons? Thanks for your help.