I have received some RNA-seq data and asked to perform differential expression analysis, but I am not sure how to proceed due to the design of the experiment.
The authors have 18 tissue samples from 18 patients, 6 are healthy, 6 have a (non-cancer) disease, and another 6 are healthy but under a condition that is supposed to cause the disease. They ask for DE genes between the three conditions.
I can understand how to use DE software if I had 1 patient with 6 replicates per each condition. I also understand how to do it if I had 6 patients with paired normal and disease information per patient (even without replicates). But I am not sure how to proceed when I have 18 different patients, no paired, no replicates, just three groups of patients. Can I just assume that each group of 6 patients are analogous to 6 replicates for each condition and just use DESeq2 or edgeR? If not, what are the alternatives?
I would thank you for any guidance.
PS: Short story about this dataset: Both the sequencing and original DE analysis were originally performed by a hired professional company. The company reported three comparisons: conditions 1vs2, 1vs3, 2vs3. For each case, they reported hundreds of DE genes that they found by pooling the results from DESeq2, edgeR and DEGSeq. The authors tried to validate the top ranked genes sent by the company and nothing could be validated. Then they came to me. I read bad comments about DEGSeq here in this website so I tried to use DESeq2 and edgeR only, assuming three groups of 6 patients, and comparing conditions 1vs2, 1vs3, 2vs3, just as the company did. My result was only 1 DE gene under 1vs2 scenario, and nothing else (which seems to be less attractive to the authors than a long list of candidates). I also built a PCA plot trying to see if the 6 patients for each condition do actually cluster, but the result is a scattered plot that looks like all 18 patients are in the same cloud without any visible structure. Now I wonder: Is this experimental design good for DE analysis at all? If so, what is the right way to proceed?