Tab ROI Analysis
The ROI DEG tab analyses gene expression within hand-drawn ROI polygons on the napari canvas, computes per-region summary statistics, and runs differential expression analysis between regions with volcano plot export.
| Control | Description |
|---|---|
| Calculate Expression | Computes per-cell expression statistics for each ROI polygon on the Shapes layer; outputs per-region mean, median, std, min, and max, plus all pairwise Welch's t-tests with Benjamini-Hochberg FDR correction |
| Results area | Read-only text display of per-region statistics |
| Export CSV | Saves per-cell data (region ID, cell ID, centroid coordinates, expression value) to a CSV file; enabled after calculation |
| Control | Description |
|---|---|
| Method | Statistical test for differential expression: wilcoxon or t-test
|
| Filter by cluster | When checked, restricts DEG to cells in the currently active cluster filter |
| Run ROI DEG | Runs differential expression between all pairs of ROI regions |
| DEG results area | Read-only display of the top 50 differentially expressed genes |
| Export DEG CSV... | Saves the full DEG results table to a CSV file; enabled after running DEG |
| Save Volcano Plot(s)... | Generates one PNG volcano plot per region pair (LFC threshold 1.0, p-value threshold 0.01, 300 dpi), named roi_volcano_<a>_vs_<b>.png, and saves them to a chosen directory; enabled after running DEG |
- In the napari canvas, select the "ROI polygons" Shapes layer and use the polygon drawing tool to draw two or more ROI regions.
- Select the gene of interest in the Coloring tab's Gene dropdown — that is the gene this tab analyses. You do not need to click Apply Cell Coloring; only the dropdown value is read.
- Click Calculate Expression to compute per-region statistics.
- Review the results in the text area, then click Export CSV to save per-cell data if needed.
- For differential expression between regions, click Run ROI DEG.
- Export the full DEG table with Export DEG CSV... or save per-pair volcano plots with Save Volcano Plot(s)....
- At least two ROI polygons are required to run DEG analysis.
- ROI polygons are automatically saved when the viewer closes and restored on the next load.
- A cell belongs to an ROI when its centroid falls inside the drawn polygon (
spatialdata.polygon_query). A centroid landing exactly on the boundary counts as inside. Where two ROIs overlap, a cell is assigned to the last one drawn — each cell carries one region label. - A polygon drawn across its own edge (a figure-eight) is repaired before the test, and every lobe is kept.
- When Filter by cluster is checked, only cells belonging to the active cluster selection contribute to the DEG test. Note that this checkbox affects the DEG analysis only — Calculate Expression always follows the cluster filter set in the Coloring tab, whether or not this box is ticked.
- DEG results are cached to
<dataset>/viewer_cache/roi_deg_cache.parquetand restored on the next launch, so reopening the dataset does not mean re-running the test. - A progress bar under Run ROI DEG tracks the pairwise comparisons.