Hello, I'm very new to rna-seq analysis and I would love to get some help regarding batch effect in my data. Data is comprised of 40 individuals (blood samples) 3 conditions (control, infection 1, infection 2. each individual has 3 replicates for each conditions- overall 120 *3 samples) the preparation was done in 4 - 96 wells plates. each plate comprised of a single condition (+ leftovers from the previous condition) so for example - the first plate is only control. the second is the leftovers of the control and infection 1. and so on...
after running PCA I observed batch effect:
as you can see there is a obvious plate-effect. I would like to correct between the same condition on different plates and so when i try using limma's removeBatcheffect I dont know how to define my batches nor my covariates.
Any help will be greatly appreciated. Thanks.