I have RNAseq data for time-course experiment with 5 time points with three replicate in each.
Some replicates have twice lower sequencing depth than others. I want to check if these low sequencing depth replicates will affect somehow DE analysis. For this, I have normalized raw counts in replicates by (library size*10e6) and did plotPCA. On PCA I see good clustering of samples by time points, and replicates with lowers seq. depth don't appear to be outliers. The same trend is for RLE plots.
Is this enough to assume that differences is sequencing depth will not affect DE analysis? I will use Deseq2 for DE.