I have to do differential expression analsysis of RNA-seq data. I have 2 different conditions, and 5 animals in each condition, and I want to see the DEGs between both conditions.
Around 5 years ago, this analysis with my same data was done using the limma and puma libraries in R and conducting a two-way ANOVA2, obtaining more than 100 DEGS.
Now I have to repeat the analysis using the new genome reference. However, after my analysis with DESeq2 (and previously alignment using STAR and counting reads per feature using htseq-count), I only obtain 5 DEGs.
What's going on? Which method do you think is best?