I am using Seurat package for the analysis of my single-cell RNA-seq. I read their basic PBMCs tutorial https://satijalab.org/seurat/v3.1/pbmc3k_tutorial.html. However, I get confused about some steps. I would highly appreciate having your answers on my following questions:
1) Is FindVariableFeatures() working on Normalized-Data or on Scaled_Data?
As in the seurat tutorial it is after NormalizeData() and before ScaleData(), but in kallisto | bustools tutorial (file://filestore.soton.ac.uk/users/fsgh1d18/mydesktop/Kallisto_bustools/BUS_notebooks_R-master/docs/10xv2.html) it is after both NormalizeData() and ScaleData().
2) Is FindMarkers() working on Normalized_Data or Scaled_Data or clusters obtained by RunTSNE()/RunUMAP()?
As FindMarkers() finds marker genes of the clusters, so I assume that the input data for FindMarkers() should be clusters obtained by RunTSNE()/RunUMAP() which these functions are also using scaled_data or selected_PCAs (obtained from RunPCA function), see below. Therefore, in practice, FindMarkers() is also using Scaled_Data, while it should work on Normalized_Data.
RunPCA() on Scaled_Data --> Selection of PCAs --> RunTSNE()/RunUMAP() --> FindMarkers()
3) If FindMarkers() works on Normalized_Data (rather than Scaled_Data), how it can be explained that FindMarkers() has inputs of clusters obtained by RunTSNE()/RunUMAP() which these functions are also using scaled_data (not Normalized_Data)?
Many thanks for any help and clarifications.