I really need help for a problem I have come across since I received another batch of RNA-seq data which I have combined with the first batch. Within both batches I have the same organ but different biological replicates... for example, 2 replicates of the lungs in batch 1 and 2 and the third and fourth replicate of the lungs in batch 2.
With the metadata, replicate info and the column order of the counts table aligning with the row order of the metadata sheet, all samples model well with DESeq2 with regards to replicates etc... apart from one organ.. the small intestine. The small intestine is actually recognised as two separate samples and not replicates (or the same organ) across different ages. The small intestine samples are actually separated according to the batches so that in a PCA plot for example, there are separate coloured dots for one set of small intestine samples and another, as if they are two different organs.
This is not happening with the other organs and all organs are recognised correctly as one sample in terms of one organ within which there are different replicates for different ages.
Is this a known issue/bug? Could this result from mistakes in the metadata sheet? I have checked this over and unless I am missing something really obvious, I cannot see any inconsistencies in the metadata table... Any help would be greatly appreciated..
I am also happy to provide code although I don't know what to add as the steps are a standard DESeq2 pipeline!