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satijalab / seurat

R toolkit for single cell genomics
http://www.satijalab.org/seurat
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Mixscape issues. #6626

Closed kasumaz closed 2 years ago

kasumaz commented 2 years ago

Dear Seurat team,

I am trying to troubleshoot mixscape. To run 'Calculate perturbation signature (PRTB)' function, I entered the following code:

crispr <- CalcPerturbSig( object = crispr, assay = "RNA", slot = "data", gd.class ="sgrna_symbol", nt.cell.class = "sgrna_id", reduction = "pca", ndims = 40, num.neighbors = 20, split.by = "channel", new.assay.name = "PRTB")

I got the following error: Processing 1 Error in nn2(data = numeric(0), query = c(13.8771946372216, -5.71075258395218, : Cannot find more nearest neighbours than there are points

I am able to run the test eccite data from seuratdisk. I try to look around myself first before asking here. The seurat object here is crispr. I am able to do much of the other seurat functions on this object. But since the data is from a crispr-seq screen, it would have been good to determine which cells escaped KO. Any help would be greatly appreciated. Thanks a lot.

nmalwinka commented 2 years ago

I had that error too. I fixed that by combining several negative controls together (non-targeting gRNA cells + cells transfected with negative control gRNA that targets a non-expressed gene) as it seems there were not enough control cells.

kasumaz commented 2 years ago

@nmalwinka Thanks a lot. I will check it out and hope it does the trick. I can mention something here tomorrow when I check it out again :)

mhkowalski commented 2 years ago

Yes, I would check that you are labeling the NT cells correctly. The argument nt.cell.class should specify the non-targeting gRNA cell classification identity. Are your NT cells labeled as "sgrna_id"? For instance if the value of "sgrna_symbol" that corresponds to the control cells is "non-targeting", you should specify nt.cell.class = "non-targeting".

You can check the function documentation here as well. Please write back to us if you have additional questions.