| SoupX {SoupX} | R Documentation |
SoupX: Profile, quantify and remove ambient RNA expression from droplet based RNA-seq
Description
This package implements the method described in REF. First a few notes about nomenclature: soup - Used a shorthand to refer to the ambient RNA which is contained in the input solution to droplet based RNA-seq experiments and ends up being sequenced along with the cell endogenous RNAs that the experiment is aiming to quantify. channel - This refers to a single run input into a droplet based sequencing platform. For Chromium 10X 3' sequencing there are currently 8 "channels" per run of the instrument. Because the profile of the soup depends on the input solution, this is the minimal unit on which the soup should be estimated and subtracted.
Details
The essential step in performing background correction is deciding which genes are not expressed in a reasonable fraction of cells. This is because SoupX estimates the contamination fraction by comparing the expression of these non-expressed genes in droplets containing cells to the soup defined from empty droplets. For solid tissue, the set of Haemoglobin genes usually works well. The key properties a gene should have are: - it should be easy to identify when it is truly expressed (i.e., when it's expressed, it should be highly expressed) - it should be highly specific to a certain cell type or group of cell types so that when the expression level is low, you can be confident that the expression is coming from the soup and not a very low level of expression from the cell
Spike-in RNAs are the best case scenario. In the case where you do not have spike-ins and haemoglobin genes are not viable estimators, the user should begin by using the plotMarkerDistribution function to plot those genes with bi-modal distributions that have a pattern of expression across cells that is consistent with high cell-type specificity. The user should then select a set of genes that can be used for estimation from this list. One or two high quality genes is usually sufficient to obtain a good estimate for the average contamination level of a channel.