0
$\begingroup$

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?

$\endgroup$
1
  • $\begingroup$ If the two odd samples each belong to the other's cluster, swapped labels seems like a reasonable hypothesis. You could look for tissue-specific genes (using something like GTex) to see if the expression patterns match the purported tissue as well. If you find high expression of liver-specific genes in a sample labelled as brain tissue and high expression of brain-specific genes in the liver sample, for example, you have good evidence to support the label swap hypothesis. $\endgroup$ – Nuclear Hoagie Feb 17 at 18:14
0
$\begingroup$

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.

$\endgroup$

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.