Hi, We have 2 groups of control samples that belong to 2 different batches, so I used DESeq2 with the desgin ~Batch+Condition for the test. We are pretty happy with the results. Then we want to visualize the counts with heatmap. I used the function limma::removeBatchEffect to get the counts to plot but we still see very clear different pattern between the 2 batches. Is it expected and should we be worry about this? Here's the code I used the get the couns

vsd <- vst(dds)
vsd$Batch <- as.factor(vsd$Batch)
assay(vsd) <- limma::removeBatchEffect(assay(vsd), vsd$Batch)
top <- assay(vsd)[top_index,] %>% as.data.frame()
pheatmap(top,annotation_col = annot_col, scale = "row", cluster_cols = F,  show_rownames = F

Thanks a lot.

You have only one condition in the second batch, so it's not possible to remove the batch effect. You can probably adjust for it somewhat, but you are likely also removing some biological signal, as batch and treatment are correlated. In the future you want to make sure you have both conditions in both batches, which will make batch and condition orthogonal.