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satijalab / seurat

R toolkit for single cell genomics
http://www.satijalab.org/seurat
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Should we merge loom objects or loom-originated Seurat objects before doing scVelo in RNA Velocity analysis? #6863

Closed denvercal1234GitHub closed 1 year ago

denvercal1234GitHub commented 1 year ago

Hi there,

I previously obtained clusters from the cellranger count outputs. Now, I want to perform RNA Velocity to see the differentiation relationship among these clusters using the loom files generated from the same cellranger count outputs.

If my original Seurat clusters were obtained by merging different cellranger count outputs (i.e., across donors etc), should I merge my loom files first (e.g., using loompy) or should I process each loom file separately then merge the loom-converted Seurat objects for performing RNA velocity?

Thank you very much for your help!

Related #5318, #3423

ollieeknight commented 1 year ago

RNA velocity analysis is quite tenuous, and current models might be incorrect.

Objects should be integrated by the best possible method - this is something you will have to play around with your data to find out - whether it should be from batch effects due to sequencing or donors. Then you will be in the best position to start RNA velocity analysis.

denvercal1234GitHub commented 1 year ago

Thank you @ollieeknight for your response.

I had determined that integration for our data was not needed to perform clustering.

I now would like to know if I merged the samples to obtain Seurat clustering, then now when I want to perform the RNA Velocity, should I (1) merge my loom files first (e.g., using loompy) or (2) should I process each loom file separately then merge the loom-converted Seurat objects for performing RNA velocity.

Thank you for your help.

ollieeknight commented 1 year ago

well I guess the best way might be to just try either one and use whichever one looks the best? I generally think that processing GEM wells/libraries separately is always the way to go (all QC, normalisation, etc)¬

rsatija commented 1 year ago

We have not tested this both ways, but I would hope that you would see similar if not identical results - though it would depend on which processing pipeline you want to use. If you want to use loom's, you should merge after processing. If you want to use Seurat's, you should merge before.

Note that in Seurat v5 onwards we are no longer actively supporting loom files.