I have normal tissue (replicate A1 and replicate A2) and abnormal tissue (replicate B1 and replicate B2). I would like to do gene expression analysis. I don't have close reference genome. I would like to denovo assemble all samples (normal tissue and abnormal tissue transcripts) and do read mapping each one to this assembled transcriptome. I can get FPKM for each replicates (replicate A1, A2, B1 and B2). Then I think I can get significant of genes among all four samples using cufflinks?
My question is:
1.By cufflinks pipeline I can get only significant genes (up regulated and down regulated) present in normal tissue vs abnormal tissue irrespective of replicate?
2.Can we know which replicate best among normal tissue (which is best whether replicate A1 or A2) and abnormal tissue (which is best replicate B1 or B2).? i.e can we compare replicate A1 and A2 and get best replicate for normal tissue, similarily for replicate B1 and B2 and get best replicate in abnormal tissue?