Performs differential gene expression analysis using both single-cell and pseudo-bulk approaches across all annotated cell types.
The single-cell method uses Seurat's FindMarkers, while pseudo-bulk testing uses DESeq2 on aggregated expression profiles.
Outputs a merged data frame with DGE statistics from both methods per condition and cell type.
Usage
DO.MultiDGE(
sce_object,
assay = "RNA",
method_sc = "wilcox",
group_by = "condition",
annotation_col = "annotation",
sample_col = "orig.ident",
ident_ctrl = "ctrl",
min_pct = 0,
logfc_threshold = 0,
only_pos = FALSE,
min_cells_group = 3,
...
)Arguments
- sce_object
The seurat or SCE object
- assay
Specified assay in Seurat or SCE object, default "RNA"
- method_sc
method to use for single cell DEG analysis, see FindMarkers from Seurat for options, default "wilcox"
- group_by
Column in meta data containing groups used for testing, default "condition"
- annotation_col
Column in meta data containing information of cell type annotation
- sample_col
Column in meta data containing information of sample annotation, default "orig.ident"
- ident_ctrl
Name of the condition in group_by to test against as ctrl, default "ctrl"
- min_pct
only test genes that are detected in a minimum fraction of min.pct cells in either of the two populations, default is 0
- 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.
- only_pos
Only return positive markers, default FALSE
- min_cells_group
Minimum number of cells in one of the groups, default 3
- ...
Additional arguments passed to FindMarkers function
Value
Dataframe containing statistics for each gene from the single cell and the Pseudobulk DGE approach.
Examples
sce_data <- readRDS(system.file("extdata", "sce_data.rds", package = "DOtools"))
DGE_result <- DO.MultiDGE(sce_data,
sample_col = "orig.ident",
method_sc = "wilcox",
annotation_col = "annotation",
ident_ctrl = "healthy")
#> Names of identity class contain underscores ('_'), replacing with dashes ('-')
#> This message is displayed once every 8 hours.
#> Centering and scaling data matrix
#> 2025-07-26 09:21:15 - Corrected annotation names in pseudo-bulk object by replacing '-' with '_'.
#> 2025-07-26 09:21:15 - Starting DGE single cell method analysis
#> 2025-07-26 09:21:15 - Comparing disease with healthy in: Monocytes
#> 2025-07-26 09:21:15 - Skipping Monocytes since one comparison has fewer than 3 cells!
#> 2025-07-26 09:21:15 - Comparing disease with healthy in: CD4_T_cells
#> 2025-07-26 09:21:15 - Comparing disease with healthy in: NK
#> 2025-07-26 09:21:16 - Comparing disease with healthy in: CD8_T_cells
#> 2025-07-26 09:21:16 - Skipping CD8_T_cells since one comparison has fewer than 3 cells!
#> 2025-07-26 09:21:16 - Comparing disease with healthy in: B_cells
#> 2025-07-26 09:21:16 - Comparing disease with healthy in: pDC
#> 2025-07-26 09:21:16 - Finished DGE single cell method analysis
#> 2025-07-26 09:21:16 - Starting DGE pseudo bulk method analysis
#> 2025-07-26 09:21:16 - Comparing disease with healthy in: B_cells
#> 2025-07-26 09:21:16 - Skipping B_cells since one comparison has fewer than 3 cells!
#> 2025-07-26 09:21:16 - Comparing disease with healthy in: CD4_T_cells
#> 2025-07-26 09:21:16 - Skipping CD4_T_cells since one comparison has fewer than 3 cells!
#> 2025-07-26 09:21:16 - Comparing disease with healthy in: Monocytes
#> 2025-07-26 09:21:16 - Skipping Monocytes since one comparison has fewer than 3 cells!
#> 2025-07-26 09:21:16 - Comparing disease with healthy in: NK
#> 2025-07-26 09:21:16 - Skipping NK since one comparison has fewer than 3 cells!
#> 2025-07-26 09:21:16 - Comparing disease with healthy in: pDC
#> 2025-07-26 09:21:16 - Skipping pDC since one comparison has fewer than 3 cells!
#> 2025-07-26 09:21:16 - Comparing disease with healthy in: CD8_T_cells
#> 2025-07-26 09:21:16 - Skipping CD8_T_cells since one comparison has fewer than 3 cells!
#> 2025-07-26 09:21:16 - Finished DGE pseudo bulk method analysis
#> 2025-07-26 09:21:16 - DGE pseudo bulk result is empty...