I am performing RNA Seq data analysis in order to identify differential gene expression analysis on a large number of samples (~200). Initially, I ran STAR with 40 samples (20 affected and 20 unaffected). I got a featureCount matrix. Now, I am performing DESeq2, but once I do the DEseq2 analysis with any of 6 samples (3 affected and 3 unaffected), it shows the some genes up and down regulation but once I increased the number of samples like 8 or 10 for DESeq2, it is not able to show the differential expression.
Please see the PCAplots. PCA plot2 which shows only 6 datasets in total (3 affected, 3 unaffected), datasets are separated better (still not the best) where I find Up and Down genes with small numbers of samples but there is no expression with 40 samples. Please see below statistical analysis and PCAs.
How we can do DGE if we have more samples.
Thanks in advance
DESeq2 analysis with 6 samples:
> summary (res) out of 20707 with nonzero total read count adjusted p-value < 0.1 LFC > 0 (up) : 27, 0.13% LFC < 0 (down) : 59, 0.28% outliers  : 0, 0% low counts  : 17677, 85%
DESeq2 analysis with 40 samples:
> summary (res) out of 42196 with nonzero total read count adjusted p-value < 0.1 LFC > 0 (up) : 0, 0% LFC < 0 (down) : 0, 0% outliers  : 0, 0% low counts  : 0, 0%