Questions about the pipeline

[vwtlin]

Thanks very much for developing such a comprehensive tool for QC. I was using some of these tools individually before and really excited to see your paper. It is really handy to use! I was not clear about the best practices in using some of them. For example, should I remove the ambient RNA first before identifying doublets? In your pipeline, it looks like analysis using SoupX and DoubletFinder is done in parallel.

I will be grateful if you could share your experience.

Cheers,
Weitao

[joshua-d-campbell]

Hi @vwtlin, thanks for trying out our tools! We generally recommend to run all cell QC tools on the original filtering matrix (i.e. the matrix with empty drops excluded) as this is what they were designed to do. That being said, sometimes QC'ing and filtering may need to be done in an iterative or step-wise fashion. For example, there have been a few single nucleus RNA-seq datasets where we first applied a filter for total counts/UMIs and features (e.g. total > 750 and detected > 500) before running the other ambient and doublet detection tools. We did this because we thought that CellRanger did not do a great job of distinguishing empty drops from true cells. I hope this helps but let me know if you have any specific questions on your data. I am going to move this to a Discussion since many people may have the same question.