Hello all, I am working on a single nucleus RNA sequencing data of ALS cases and normal controls. I am comparing different regions of brain and spinal cord within ALS patient and between ALS and controls. First, I started to do the integration of the data using SCT+Seurat integration method which result in about 22 clusters. I did evaluate the stability of the clusters using Scclusteval package and hopefully most of the clusters were stable (resolution 0.65, k-param 100, PC 50). But I realized using Seurat integration is overcorrecting my data (I integrated pbmc sample with my dataset and they mixed together after integration). So, I decided to revise integration method and I tried Harmony integration. With harmony when I use scclusteval I'm getting very weird result. I cannot find reasonable number of stable clusters and the number of clusters are significantly lower compared to when I used Seurat integration. For one of my dataset I'm getting 8 clusters with these specification: pc 80, resolution 0.01, param 100 (all 8 are stable) however I expect more sub-clusters and these number could be the major number of clusters. For another dataset I have 5 clusters from which 2 are unstable. I am not sure why I'm getting this different result using these two integration method. I don't know which one I should trust. I really appreciate any comments. Paria

The integration method does not define the number of clusters. If you don't think 22 clusters makes sense, change the clustering resolution (in

`FindClusters()`

) to change the number of clusters. For any method, you have to try multiple resolution to fully understand your data.Thanks and sorry for the late response. I meant that when I use cluster evaluation tools it doesn't show that the clusters are stable. Do you think. I should necessarily used these kinds of tools? and what do you recommend? Thanks, Paria

Stable clusters are not necessarily biologically meaningful and vice versa.

I see. That's a good point. But if I do increase the resolution to get the expected number of clusters how to I make sure that they are stable? do I need to do so? Thanks