# Running SCORPIUS on an AnnData object

#### Robrecht Cannoodt

#### 2019-01-12

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

    library(SCORPIUS)
    library(anndata)

    data(ginhoux)

    adata <- AnnData(
      X = ginhoux$expression,
      obs = ginhoux$sample_info
    )

    # short hand notation
    group_name <- adata$obs[["group_name"]]

    adata

    ## AnnData object with n_obs × n_vars = 245 × 2000
    ##     obs: '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(adata$X, 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
    )

    ## Warning: `aes_string()` was deprecated in ggplot2 3.0.0.
    ## ℹ Please use tidy evaluation idioms with `aes()`.
    ## ℹ See also `vignette("ggplot2-in-packages")` for more information.
    ## ℹ The deprecated feature was likely used in the SCORPIUS package.
    ##   Please report the issue at <https://github.com/rcannood/SCORPIUS/issues>.
    ## This warning is displayed once every 8 hours.
    ## Call `lifecycle::last_lifecycle_warnings()` to see where this warning was
    ## generated.

## 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
    )

    ## Warning: Using `size` aesthetic for lines was deprecated in ggplot2 3.4.0.
    ## ℹ Please use `linewidth` instead.
    ## ℹ The deprecated feature was likely used in the SCORPIUS package.
    ##   Please report the issue at <https://github.com/rcannood/SCORPIUS/issues>.
    ## This warning is displayed once every 8 hours.
    ## Call `lifecycle::last_lifecycle_warnings()` to see where this warning was
    ## generated.

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

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

    draw_trajectory_heatmap(expr_sel, traj$time, group_name)

Finally, these genes can also be grouped into modules as follows:

    modules <- extract_modules(scale_quantile(expr_sel), traj$time, verbose = FALSE)
    draw_trajectory_heatmap(expr_sel, traj$time, group_name, modules)

## Store outputs in AnnData

    adata$obsm[["X_mds"]] <- space
    adata$uns[["trajectory_path"]] <- traj$path
    adata$obs[["trajectory_pseudotime"]] <- traj$time
    adata$var[["trajectory_importance"]] <- gimp[match(adata$var_names, gimp$gene), ]$importance

    adata

    ## AnnData object with n_obs × n_vars = 245 × 2000
    ##     obs: 'group_name', 'trajectory_pseudotime'
    ##     var: 'trajectory_importance'
    ##     uns: 'trajectory_path'
    ##     obsm: 'X_mds'