bulk-wgcna-analysis-with-omicverse

Bulk WGCNA analysis with omicverse

Overview

Activate this skill for users who want to reproduce the WGCNA workflow from t_wgcna.ipynb. It guides you through loading expression data, configuring PyWGCNA, constructing weighted gene co-expression networks, and inspecting modules of interest.

Instructions

1. Prepare the environment
   • Import omicverse as ov, scanpy as sc, matplotlib.pyplot as plt, and pandas as pd.
   • Set plotting defaults via ov.plot_set().
2. Load and filter expression data
   • Read expression matrices (e.g., from expressionList.csv).
   • Calculate median absolute deviation with from statsmodels import robust and gene_mad = data.apply(robust.mad).
   • Keep the top variable genes (e.g., data = data.T.loc[gene_mad.sort_values(ascending=False).index[:2000]]).
3. Initialise PyWGCNA
   • Create pyWGCNA_5xFAD = ov.bulk.pyWGCNA(name=..., species='mus musculus', geneExp=data.T, outputPath='', save=True).
   • Confirm pyWGCNA_5xFAD.geneExpr looks correct before proceeding.
4. Preprocess the dataset
   • Run pyWGCNA_5xFAD.preprocess() to drop low-expression genes and problematic samples.
5. Construct the co-expression network
   • Evaluate soft-threshold power: pyWGCNA_5xFAD.calculate_soft_threshold().
   • Build adjacency and TOM matrices via calculating_adjacency_matrix() and calculating_TOM_similarity_matrix().
6. Detect gene modules
   • Generate dendrograms and modules: calculate_geneTree(), calculate_dynamicMods(kwargs_function={'cutreeHybrid': {...}}).
   • Derive module eigengenes with calculate_gene_module(kwargs_function={'moduleEigengenes': {'softPower': 8}}).
   • Visualise adjacency/TOM heatmaps using plot_matrix(save=False) if needed.
7. Inspect specific modules
   • Extract genes from modules with get_sub_module([...], mod_type='module_color').
   • Build sub-networks using get_sub_network(mod_list=[...], mod_type='module_color', correlation_threshold=0.2) and plot them via plot_sub_network(...).
8. Update sample metadata for downstream analyses
   • Load sample annotations updateSampleInfo(path='.../sampleInfo.csv', sep=',').
   • Assign colour maps for metadata categories with setMetadataColor(...).
9. Analyse module–trait relationships
   • Run analyseWGCNA() to compute module–trait statistics.
   • Plot module eigengene heatmaps and bar charts with plotModuleEigenGene(module, metadata, show=True) and barplotModuleEigenGene(...).
10. Find hub genes
    • Identify top hubs per module using top_n_hub_genes(moduleName='lightgreen', n=10).
11. Defensive validation

    # Before WGCNA: verify enough genes remain after MAD filtering
    assert data.shape[0] >= 1000, f"Only {data.shape[0]} genes after filtering — WGCNA needs >1000 for meaningful modules"
    # Verify expression values are numeric and non-negative
    assert data.dtypes.apply(lambda d: d.kind in 'iuf').all(), "Expression matrix contains non-numeric columns"
    # Verify enough samples for network construction
    assert data.shape[1] >= 6, f"Only {data.shape[1]} samples — WGCNA needs >=6 samples for reliable co-expression"
    

12. Troubleshooting tips
    • Large datasets may require increasing save=False to avoid writing many intermediate files.
    • If module detection fails, confirm enough genes remain after MAD filtering and adjust deepSplit or softPower.
    • Ensure metadata categories have assigned colours before plotting eigengene heatmaps.

Examples

• "Build a WGCNA network on the 5xFAD dataset, visualise modules, and extract hub genes from the lightgreen module."
• "Load sample metadata, update colours for sex and genotype, and plot module eigengene heatmaps."
• "Create a sub-network plot for the gold module using a correlation threshold of 0.2."

References

• Tutorial notebook: t_wgcna.ipynb
• Tutorial dataset: data/5xFAD_paper/
• Quick copy/paste commands: reference.md