Explanation for RNA-seq samples not clustering in PCA as expected

Question

A colleague is analysing RNA-seq data - the study design is 2 treatments, 3 replicates, 3 tissues. In their PCA plot the samples clustered neatly by tissue. Except for two samples - two tissue samples originating from the same animal clustered with the wrong tissue.
I suggested that the two samples had likely been switched accidently somewhere along the way. They think its explained simply by biological variation which I'm not convinced of. Are there any technical explanations for why samples could cluster on a PCA in such a way?

Phoenix Mu · Answer

It's hard to say what is going on without looking at the plots. But it is a reasonable guess that samples are mislabelled and I think it is worth checking the experimental log carefully. It is also possible that the two samples are of low quality. People often use PCA to check and remove outliers from their RNA-seq libraries. So you may also check the RNA-seq library quality for those two samples.

Answered by Phoenix Mu on March 5, 2021

Explanation for RNA-seq samples not clustering in PCA as expected

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