Ambient RNA expression correction

Question

[Pac314]

Should ambient RNA expression correction (e.g. with SoupX) occur before or after initial QC for low-quality cells (e.g. filtering using RNA counts, mitochondrial fraction etc. when performing single-cell RNAseq analysis?

[swbarnes2]

Isn't ambient RNA filtered out by keeping only reads with cell barcodes that have a relatively high abundance?

[rpolicastro]

Ambient RNA would have the same cell barcode as a valid cell-containing droplet since the RNA gets partitioned into the droplet with the cell.

Answer 1

[pankaj.agarwal.duke]

Generally I correct for ambient RNA before the initial QC. It is the first step after getting the expression matrix from CellRanger for 10X assay. You would want to do the initial QC on cells that have been "decontaminated"