Workflow advice for totalVI in CITE-seq Multiplexed data

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1

Hi all,

I’m working with CITE-seq data where 4 samples were multiplexed, stained with antibodies in a single tube, and then distributed across 4 sequencing lanes. I’d like advice on the optimal analysis workflow.

Specifically:

  1. At which step should I split by lanes and then merge adata objects from the same biological sample?
  2. Is the following processing order reasonable?
  • Ambient RNA removal (CellBender)
  • Doublet removal
  • QC
  • Protein normalization (DSB)
  • HVG selection
  • totalVI integration (raw count)
  1. Since CellBender, DSB, and totalVI all perform some form of denoising, are there any conflicts between them?
  2. Should I use DSB normalization before totalVI, or instead normalize denoised_protein values from totalVI afterwards?

Any recommendations, best practices, or reference papers would be greatly appreciated. Thanks!

2

Cellbender and totalVI are fully compatible. TotalVI expects unnormalized counts and does its own normalization. So you shouldn’t combine DSB and totalVI in the same pipeline.