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name: topexpressinggenes description: Identifies and visualizes the top expressing genes per cluster in T/B cells, followed by pathway enrichment analysis. Provides quick cluster characterization by highlighting the most highly expressed genes and their biological functions.

TopExpressingGenes Process Configuration

Purpose

Identifies and visualizes the top expressing genes per cluster in T/B cells, followed by pathway enrichment analysis. Provides quick cluster characterization by highlighting the most highly expressed genes and their biological functions.

When to Use

• After: SeuratClustering and TOrBCellSelection processes
• Use cases: Quick cluster characterization, identifying dominant gene programs, pathway enrichment
• Optional process: Enable only when cluster-level expression profiling is needed

Configuration Structure

Process Enablement

Copy[TopExpressingGenes]
cache = true

Input Specification

Copy[TopExpressingGenes.in]
srtobj = ["SeuratClustering"]

Note: srtobj accepts the output from SeuratClustering or SeuratSubClustering.

Environment Variables

Core Parameters

Copy[TopExpressingGenes.envs]
# Number of top expressing genes to identify per cluster
n = 250

# Enrichment style
enrich_style = "enrichr"  # Options: "enrichr", "clusterprofiler"

# Enrichment databases
dbs = ["KEGG_2021_Human", "MSigDB_Hallmark_2020"]

Enrichment Plot Settings

Copy[TopExpressingGenes.envs.enrich_plots_defaults]
# Plot type: "bar", "dot", "lollipop", "network", "enrichmap", "wordcloud"
plot_type = "bar"
devpars = {res = 100, width = 800, height = 600}
top_term = 10  # Top enriched pathways to show
ncol = 1

Configuration Examples

Minimal Configuration

Copy[TopExpressingGenes]

[TopExpressingGenes.in]
srtobj = ["SeuratClustering"]

Top 10 Genes with Custom Databases

Copy[TopExpressingGenes]

[TopExpressingGenes.in]
srtobj = ["SeuratClustering"]

[TopExpressingGenes.envs]
n = 10
dbs = ["GO_Biological_Process_2025", "Reactome_Pathways_2024"]

Network Visualization

Copy[TopExpressingGenes.envs.enrich_plots."Network"]
plot_type = "network"
top_term = 15

[TopExpressingGenes.envs.enrich_plots."Enrichmap"]
plot_type = "enrichmap"

Common Patterns

Pattern 1: Quick Cluster Overview

Copy[TopExpressingGenes]

[TopExpressingGenes.in]
srtobj = ["SeuratClustering"]

[TopExpressingGenes.envs]
n = 10
dbs = ["MSigDB_Hallmark_2020"]

Pattern 2: Detailed Profile

Copy[TopExpressingGenes.envs]
n = 250
enrich_style = "clusterprofiler"

[TopExpressingGenes.envs.enrich_plots]
"KEGG" = {plot_type = "bar", dbs = ["KEGG_2021_Human"]}
"Reactome" = {plot_type = "network"}

Pattern 3: Multiple Visualizations

Copy[TopExpressingGenes.envs]
n = 50

[TopExpressingGenes.envs.enrich_plots."Bar"]
plot_type = "bar"

[TopExpressingGenes.envs.enrich_plots."Word Cloud"]
plot_type = "wordcloud"

Difference from ClusterMarkers

Recommendation: Use both processes:

1. TopExpressingGenes: Quick overview of dominant programs
2. ClusterMarkers: Rigorous marker identification

Dependencies

• Upstream: SeuratClustering, TOrBCellSelection (for TCR route)
• Downstream: None (terminal analysis process)

Validation Rules

• n: Positive integer (typically 10-500)
• dbs: Valid enrichit/Enrichr database names or local GMT paths
• enrich_style: "enrichr" or "clusterprofiler"
• plot_type: Valid scplotter plot type

Troubleshooting

Ribosomal/Mitochondrial Gene Dominance

Issue: Housekeeping genes (RPS, RPL, MT-) dominate

Solutions: Increase n, use ClusterMarkers, filter genes in SeuratPreparing

Empty Enrichment Results

Issue: No pathways enriched

Solutions: Increase n to 100-500, verify species (UPPERCASE=human, TitleCase=mouse)

Plot Rendering Errors

Issue: Plots fail to render

Solutions: Reduce top_term (5-15), use simpler plots (bar, dot)

Performance Issues

Issue: Process too slow

Solutions: Reduce n, use fewer databases, disable enrichment: dbs = []

External References

Databases (enrichit)

• KEGG_2021_Human - KEGG pathways
• MSigDB_Hallmark_2020 - Hallmark gene sets
• GO_Biological_Process_2025 - GO Biological Process
• Reactome_Pathways_2024 - Reactome pathways
• See: https://pwwang.github.io/enrichit/reference/FetchGMT.html

Plot Types (scplotter)

• bar - Bar chart
• dot - Dot plot
• lollipop - Lollipop plot
• network - Network visualization
• enrichmap - Enrichment map
• wordcloud - Word cloud

Enrichment Styles

• enrichr - Fisher's exact test
• clusterprofiler - Hypergeometric test

See Also

• TopExpressingGenesOfAllCells - Top genes before T/B selection
• ClusterMarkers - Differential expression analysis