Function to identify perturbed and non-perturbed gRNA expressing cells that accounts for multiple treatments/conditions/chemical perturbations.
Usage
RunMixscape(
object,
assay = "PRTB",
slot = "scale.data",
labels = "gene",
nt.class.name = "NT",
new.class.name = "mixscape_class",
min.de.genes = 5,
min.cells = 5,
de.assay = "RNA",
logfc.threshold = 0.25,
iter.num = 10,
verbose = FALSE,
split.by = NULL,
fine.mode = FALSE,
fine.mode.labels = "guide_ID",
prtb.type = "KO"
)Arguments
- object
An object of class Seurat.
- assay
Assay to use for mixscape classification.
- slot
Assay data slot to use.
- labels
metadata column with target gene labels.
- nt.class.name
Classification name of non-targeting gRNA cells.
- new.class.name
Name of mixscape classification to be stored in metadata.
- min.de.genes
Required number of genes that are differentially expressed for method to separate perturbed and non-perturbed cells.
- min.cells
Minimum number of cells in target gene class. If fewer than this many cells are assigned to a target gene class during classification, all are assigned NP.
- de.assay
Assay to use when performing differential expression analysis. Usually RNA.
- logfc.threshold
Limit testing to genes which show, on average, at least X-fold difference (log-scale) between the two groups of cells. Default is 0.25 Increasing logfc.threshold speeds up the function, but can miss weaker signals.
- iter.num
Number of normalmixEM iterations to run if convergence does not occur.
- verbose
Display messages
- split.by
metadata column with experimental condition/cell type classification information. This is meant to be used to account for cases a perturbation is condition/cell type -specific.
- fine.mode
When this is equal to TRUE, DE genes for each target gene class will be calculated for each gRNA separately and pooled into one DE list for calculating the perturbation score of every cell and their subsequent classification.
- fine.mode.labels
metadata column with gRNA ID labels.
- prtb.type
specify type of CRISPR perturbation expected for labeling mixscape classifications. Default is KO.
Value
Returns Seurat object with with the following information in the meta data and tools slots:
- mixscape_class
Classification result with cells being either classified as perturbed (KO, by default) or non-perturbed (NP) based on their target gene class.
- mixscape_class.global
Global classification result (perturbed, NP or NT)
- p_ko
Posterior probabilities used to determine if a cell is KO (default). Name of this item will change to match prtb.type parameter setting. (>0.5) or NP
- perturbation score
Perturbation scores for every cell calculated in the first iteration of the function.