I am trying to analyze human RNA-seq data with limma. I have two conditions: affected by disease and not affected. More concretely, I have mother (VAN012, not affected), father (VAN013, affected), paternal grandma (VAN0092, affected), a kid (VAN011, girl, affected) and two unrelated and not affected controls (Ctl1, Ctl2, both males); I also have 2 replicates for each sample. My goal is to find DE genes in these two conditions. I am using limma for this task and after building MDS plot I see this pic:
Two conditions are not clustered at all, for example I see that not affected mother (VAN012) is very close to affected daughter (VAN011) and affected father (VAN013) is close to not affected control (Ctl2).
I guess, I will not obtain any good results with this dataset (as I said I am using limma, in my linear model I also include sex and family relation). So my question is, what is the proper way of doing RNA-seq analysis with such data?