heterogeneity in cluster when using Seurat Doheatmap

Question

[Jie]

Hi, I am trying to identify clusters from my data (scRNA-seq for ~10k heart immune cells ) using Seurat4 in R server.

When I attempted to find clusters, I firstly tested resolution=0.4 in FindClusters() and got . ( min.pct = 0.2, logfc.threshold = 0.25 for FindAllMarkers(), I keep these parameters unchanged)

Then I used Doheatmap() to show top20 markers, and I found obvious subclusters in several top clusters. So I increased my resolution to 0.8, 1.0, 1.5, but still it seem weird.

The figure.1~3 refer to res=0.4, 0.8, 1.5 , we can see subclusters.

SO I would like to have your advice on the causes of this situation and what to do next.

My exploration:

1. I have used clustree to find the proper resolution, we consider 1.5 too high and unnecessary, so increasing resolution might not be a good way to solve the problem.
2. I subset the cluster0 and re-run the pipeline (from FindVariableFeatures to Doheatmap), the heatmap looks better, however the marker genes are different from last step. And is this a good way?

Any help or discussion will be appreciated.

[fracarb8]

Having multiple bars doesn't mean you have sub-clusters. If you change the order of the genes you will obtain different bars. What is important in your heatmap, is that each clusters have a unique pattern which indicate different expression profiles. Furthermore, if your cluster contains similar cell types (e.g. fibroblast and myo-fibroblast or TCD8 and NK), you expect to see both shared (faith in colour) and not shared (intense colour) bars.

[Jie]

Thanks for your reply! :）