Contrasts stage (`contrasts`)

A stage for calculation, visualization and reporting of differentially expressed markers (“contrasts”). This stage is basically the same as the cluster_markers stage, but all output is related to individual comparisons of levels of cell groupings. Hence “contrasts”, a term known from bulk RNA-seq where sample groups are compared -> they are put in contrast.

⚙️ Config files: config/single_sample/contrasts.yaml, config/integration/contrasts.yaml

📋 HTML report target (in config/pipeline.yaml): DRAKE_TARGETS: ["report_contrasts"]

📜 Example report for PBMC 1k data (used config)

📜 Example report for integrated data (used config)

Cluster markers config is stored in the config/single_sample/contrasts.yaml and config/integration/contrasts.yaml files (the location of this file is different for the single-sample and integration pipelines). As for the pipeline config, directory with this file is read from environment variables:

SCDRAKE_SINGLE_SAMPLE_CONFIG_DIR for the single-sample pipeline.
SCDRAKE_INTEGRATION_CONFIG_DIR for the integration pipeline.

Options named in lowercase are set upon scdrake load or attach. Then the actual directory used depends on whether you run run_single_sample_r() or run_integration_r().

Parameters for this stage are almost identical to those for cluster_markers stage (see vignette("stage_cluster_markers")). You just need to replace CLUSTER_MARKERS with CONTRASTS in parameter names 🙂

The different parameters are below.

CONTRASTS_SOURCES:
  - dea_cluster_int_graph_louvain_r0.4:
      source_column: "cluster_int_graph_louvain_r0.4"
      description: "DEA of all groups in graph-based clustering (Louvain alg. with r = 0.4) with blocking on batch"
      contrasts: "all"
    dea_cluster_int_graph_louvain_r0.8:
     source_column: "cluster_int_graph_louvain_r0.8"
     description: "DEA of some groups in graph-based clustering (Louvain alg. with r = 0.8)"
     contrasts:
       - target: "2"
         reference: "1"
       - target: "3"
         reference: "2"
         name: "cl3_vs_cl4"

Type: list of named lists

This parameter is similar to CLUSTER_MARKERS_SOURCES, but an additional parameter contrasts is used. It is used to specify which levels in source_column will be compared (differential expression).

If "all", all combinations of levels in source_column will be compared. For example, if source_column is "cluster_kmeans_k3" (k-means with k = 3), it contains three levels, and the following comparisons (contrasts) will be made: 1_vs_2, 1_vs_3, and 2_vs_3. Note that it could lead to an excessive amount of comparisons for graph-based clustering.
If list of lists, the following character scalar items can/must be set:
- target (required): target level of source_column.
- reference (required): reference level of source_column.
- name (optional): unique contrast name. If not specified, will be created as {target}_vs_{reference}.

CONTRASTS_SOURCES_DEFAULTS

Same as CLUSTER_MARKERS_SOURCES_DEFAULTS, but the following parameters for statistical tests cannot be used, and hardcoded values are used for them internally:

LFC_DIRECTION ("any")
PVAL_TYPE ("any")
MIN_PROP (null)

Here you can find description of the most important targets for this stage. However, for a full overview, you have to inspect the source code of the get_contrasts_subplan() function.

As in the config for this stage, all target names are similar to those in the stage cluster_markers after replacing cluster_markers with contrasts (with few exceptions, see below). Please, refer to vignette("stage_cluster_markers").

`SingleCellExperiment` objects (SCE)

sce_dimred_contrasts, sce_final_contrasts, sce_contrasts

Tibbles with parameters

contrasts_params, contrasts_test_params, contrasts_heatmap_params, contrasts_plot_params, contrasts_dimred_plot_params

Tibbles with cluster markers (contrasts) test results

contrasts_raw: a tibble, same as cluster_markers_raw. Used to extract results for contrasts of interest.

contrasts: a tibble with extracted results (in markers column) for contrasts of interest. Contrasts are identified by columns target, reference, or contrast_name, and their cell grouping of origin by source_column. The name column comes from list names in CONTRASTS_SOURCES.

contrasts_out: same as contrasts, but markers are coerced to dataframes and column names are normalized to snake_case.

contrasts_for_tables: same as contrasts_out, but markers dataframes are prepared for HTML output. See cluster_markers_for_tables in vignette("stage_cluster_markers").

Heatmaps

seu_for_contrasts_heatmaps, contrasts_heatmaps_df

Marker plots

contrasts_plots_top

Dimensionality reduction plots

contrasts_dimred_plots

Other targets

config_contrasts: a list holding parameters for this stage.

Document generated: 2024-09-27 15:33:06 UTC+0000

SingleCellExperiment objects (SCE)