CellCycleScoring error

Question

[marco.barr]

Hi, everyone,

I'm trying to assess the effect of the cell cycle on my normalized scRNA data using Seurat but I'm encountering this error.

seurat_phase <- CellCycleScoring(seurat_phase,
                             g2m.features = cc.genes$g2m_genes,
                             s.features = cc.genes$s_genes)

  Error in `GetAssayData()`:
  ! GetAssayData doesn't work for multiple layers in v5 assay.
  Run `rlang::last_trace()` to see where the error occurred.

Should I perhaps split by sample or use JoinLayers() or what else could I do? Thank you very much for your help

[Nitin Narwade]

Yes, I think you need to join the layers. I was trying to perform DGE on the split layers and it was throwing the same error. So, I guess before calculating the score, which I am sure uses the GetAssayData() internally and needs the collapsed layers.

Description form Seurat_v5 documentation:

Once integrative analysis is complete, you can rejoin the layers - which collapses the individual datasets together and recreates the original counts and data layers. You will need to do this before performing any differential expression analysis.......

Regards,

Nitin N,

[marco.barr]

Thank you very much for your help. However, I have a question. In my dataset, I have a control sample and a patient sample (which I noticed differ significantly in the number of cells). Should I, for example, 'play around' by using JoinLayers() and SplitObject() depending on whether I want to facilitate certain filters, and then proceed with doublet identification and removal? For cluster assignment and annotation, would it be preferable to split again?