RNAseq biological replicates not clustering in PCA plots

Question

I have RNAseq data from 4 samples with 3 biological replicates per sample. I am currently trying to do the differential expression analysis with DESeq2 but the biological replicates will not cluster together when I make the PCA plot or correlation heatmap. This is my first time with RNASeq analysis and so am not sure what the best route forward is? I would like to avoid repeating the experiment with new samples if possible!
My pipeline prior to DESeq2 was the following:
FastQC quality check -> Trimmomatic -> Kallisto
I used tximport to convert kallisto files into suitable format for DESeq2

swbarnes2 · Answer

PC1 is 81% of the variance?
This PCA plot confirms that different tissues are different.  You probably already knew that.  I'd make more PCA plots that are tissue specific.  That will be more informative than these.
Personally, I'd also not do DESeq on all of these samples together, unless the goal of your experiment really is to learn the differences between these two tissues.

neonglow · Answer

Clustering of replicates looks decent enough to me, so you should be abl to push ahead, but I agree the tissues are grouping, which could mask any differences based on sex or genotype.
You might consider the EdgeR package for DE analysis here. It allows for flexibility when making complex comparisons while accounting for tissue/batch effects. I've had good luck with using it to compare across batch effects from complex experiments like this.

RNAseq biological replicates not clustering in PCA plots

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