How to find DEG within a cluster between two groups without any replication information in scRNA-Seq analysis?

Question

[Zahra]

Hi all

I have a Seurat object containing the information about the cluster of cells and their condition but not the replication information, now I want to find the DEG within a cluster between two conditions. but without the replication information, I can not perform the Pseudobulk method. What alternative method do you suggest?

Thanks for any help

[piyushjo]

What do you mean by "replication" information? Do you mean "replicates"? Or do you mean "DNA replication"?

[Zahra]

Thanks for your reply. I mean the biological replicates information. Indeed the dataset is for the 3 conditions (ctrl, treat1, treat2) and each condition has 3 biological replicates. The number of clusters for each condition is 8.

What I exactly want: finding DEG between two conditions within one cluster.

As I know the Pseudobulk method needs information about biological replicates. In this situation, I can't use this method for my dataset. Now I want to know what can I do alternatively?

[piyushjo]

Ok.

I don't think you need to care about replicates here. If you are just merging the data and your replicates form separate cluster, then it will suggest batch effect. However, if you used seurat integration you can find that even different conditions get clustered together because it can "correct" biological effects too.

If you just merged the data and are getting cells from different condition clustering together, there is low chance that you will find any meaningful DEG. However, if you used integration and then got different conditions clustering, than you can create a new variable that has information about cluster (seurat_cluster) and sample (orig.ident) like this:

seurat.obj$New_var <- paste0(seurat.obj$orig.ident,"_",seurat,obj$seurat_cluster)

And then use this variable for calling DEG. if You just want to focus on one specific cluster, you can subset the object to that cluster and than simply use seurat.obj$orig.ident variable to call DEG.

I hope this helps.