This vignette assumes that you have a Seurat object at the ready. As an example, we create one from the ginhoux dataset containing 248 dendritic cell progenitors.

library(SCORPIUS)
library(Seurat)

data(ginhoux)

counts <- t(round(2^ginhoux$expression))
srt <- CreateSeuratObject(counts = counts, meta.data = ginhoux$sample_info)

Normalise with Seurat

Seurat is used to normalise the dataset. Since the dimensionality reduction method is scale-invariant, the scaling step in Seurat is not required.

srt <- NormalizeData(srt)

expression <- t(as.matrix(srt@assays$RNA@data))

Also fetch some metadata from the Seurat object. Change group_name to whatever column in srt@meta.data you are interested in.

group_name <- srt@meta.data$group_name

Reduce dimensionality of the dataset

SCORPIUS uses Torgerson multi-dimensional scaling to reduce the dataset to three dimensions. This technique attempts to place the cells in a space such that the distance between any two points in that space approximates the original distance between the two cells as well as possible.

The distance between any two samples is defined as their correlation distance, namely 1 - (cor(x, y)+1)/2. The reduced space is constructed as follows:

space <- reduce_dimensionality(expression, dist = "spearman", ndim = 3)

The new space is a matrix that can be visualised with or without colouring of the different cell types.

draw_trajectory_plot(space, progression_group = group_name, contour = TRUE)

Inferring a trajectory through the cells

The main goal of SCORPIUS is to infer a trajectory through the cells, and orden the cells according to the inferred timeline.

SCORPIUS infers a trajectory through several intermediate steps, which are all executed as follows:

traj <- infer_trajectory(space)

The result is a list containing the final trajectory path and the inferred timeline for each sample time.

The trajectory can be visualised with respect to the samples by passing it to draw_trajectory_plot:

draw_trajectory_plot(
  space, 
  progression_group = group_name,
  path = traj$path,
  contour = TRUE
)

Finding candidate marker genes

We search for genes whose expression is seems to be a function of the trajectory timeline that was inferred, as such genes might be good candidate marker genes for dendritic cell maturation.

gimp <- gene_importances(expression, traj$time, num_permutations = 0, num_threads = 8)
gene_sel <- gimp[1:50,]
expr_sel <- expression[,gene_sel$gene]

To visualise the expression of the selected genes, use the draw_trajectory_heatmap function.

Running SCOPIUS on a Seurat object

Normalise with Seurat

Reduce dimensionality of the dataset

Inferring a trajectory through the cells

Finding candidate marker genes