HolobiomicsLab Reconstruct the metabolic correlation network and module identification with WGCNA + Dynamic Tree Cut
License: restricted — no clear open-source license detected for the underlying tool; verify licensing before commercial use or redistribution.
Summary
Build a weighted metabolic correlation network from normalized metabolite abundance data, then partition it into co-expression modules using hierarchical clustering and dynamic branch cutting. This skill enables discovery of functionally coherent metabolite groups and identification of sample trait associations within metabolic modules.
When to use
Apply this skill after variance-stabilizing normalization and imputation of metabolomic data when you have a MultiAssayExperiment object containing normalized metabolite abundances and wish to identify groups of co-abundant metabolites that may share biochemical pathways or regulatory mechanisms. Use it as a precursor to trait association analysis or when exploring the modular structure of the metabolome.
When NOT to use
- Input metabolite data has not been normalized (e.g., raw intensity values still contain batch effects or variance heterogeneity) — apply variance-stabilizing normalization first.
- Missing data prevalence exceeds the imputation tolerance — validate that k-nearest neighbor imputation with appropriate cutoff has already been performed.
- The research question requires single-metabolite differential abundance testing rather than co-expression discovery — use univariate statistical tests instead.
Inputs
- MultiAssayExperiment object with normalized and imputed metabolite measurements (norm_imputed assay)
- Metabolite rowData with SUB_PATHWAY annotations
Outputs
- Module color vector (dendrogram branch assignments)
- Metabolite-to-module membership table
- Named module assignments mapping metabolites to pathway-derived module labels
How to apply
Load the normalized and imputed MultiAssayExperiment object (norm_imputed assay) containing metabolite abundance data. Compute a biweight midcorrelation similarity matrix across all metabolites, chosen for its robustness to outliers compared to absolute correlation. Apply soft thresholding with power β=3 to convert the similarity matrix to an adjacency matrix, then calculate dissimilarity as 1 − adjacency. Perform hierarchical clustering on the dissimilarity matrix to construct a dendrogram. Apply dynamic branch cutting (rather than fixed-height cutoffs, which perform suboptimally on complex dendrograms) with a minimum module size threshold of 5 metabolites to identify stable clusters. Extract the module color vector and metabolite-to-module assignments from the dendrogram. For interpretability, identify the most abundant SUB_PATHWAY annotation within each module and assign module names accordingly. Output the result as a table mapping metabolite IDs to module names or colors.
Related tools
- MetaboDiff (Provides high-level wrapper functions and data structures (MultiAssayExperiment) for correlation network construction and module detection in metabolomic workflows) — https://github.com/andreasmock/MetaboDiff
- WGCNA (Supplies the underlying hierarchical clustering, soft thresholding, and adjacency computation algorithms for weighted correlation network analysis)
- R (Execution environment for all computational steps; requires version 4.0.2 or higher for MetaboDiff compatibility)
- MultiAssayExperiment (Unified data container that integrates assay matrices, row annotations (metabolite metadata), and column annotations (sample traits) for modular analysis)
Examples
# After loading norm_imputed MAE object:
bicor_matrix <- bicor(t(assay(mae, "norm_imputed")));
adj_matrix <- (0.5 + 0.5 * bicor_matrix)^3;
diss_matrix <- 1 - adj_matrix;
hc <- hclust(as.dist(diss_matrix), method="average");
module_colors <- cutreeDynamic(dendro=hc, distM=diss_matrix, minClusterSize=5, method="hybrid");
Evaluation signals
- Dendrogram structure is tree-like and readable; no single massive module dominates (absence of this suggests poor parameter choice or extreme data skew).
- Each identified module contains at least 5 metabolites (the hard minimum threshold) and is labeled with a consistent SUB_PATHWAY annotation, indicating biochemical coherence.
- Module membership is reproducible: re-running with the same parameters produces identical module colors and assignments.
- Modules show expected size distribution (neither fragmented into singletons nor merged into one giant cluster); visual inspection of the dendrogram confirms clean branch separation.
- Module-level trait associations (e.g., correlation of module eigenvector with sample phenotype) are stronger than random expectation, validating functional relevance.
Limitations
- Soft thresholding power (β=3) is fixed in the described workflow; sensitivity to this parameter is not explored — practitioners may need to test alternative powers for their data.
- Minimum module size of 5 metabolites is a default choice; very small sample sizes or sparse networks may require adjustment, and very large networks may merge biologically distinct clusters.
- Dynamic branch cutting depends on dendrogram topology and can be sensitive to tied distances or numerical precision; results should be validated against prior knowledge or alternative clustering methods.
- Module naming by 'most abundant SUB_PATHWAY' is heuristic and may not capture all metabolites in a module or reflect their primary function — manual curation is recommended.
- No explicit handling of missing data within the correlation step; relies entirely on upstream k-nearest neighbor imputation, which assumes data are missing at random.
Evidence
- [methods] biweight midcorrelation as a similarity measure robust to outliers: "Biweight midcorrelation was used as a similiarity measure as it is more robust to outliers than the absolute correlation coefficient"
- [methods] soft thresholding with power β=3 to convert similarity to adjacency: "Apply soft thresholding with power β=3 to convert similarity to adjacency, then compute dissimilarity as 1 − adjacency."
- [methods] dynamic branch cutting outperforms fixed-height cutoffs on complex dendrograms: "We employed the dynamic branch cut method developed by Langfelder and colleagues, as constant height cutoffs exhibit suboptimal performance on complicated dendrograms."
- [methods] minimum module size threshold of 5 metabolites: "modules are detected by applying a branch cutting method with a minimal module size of 5 metabolites"
- [readme] MetaboDiff offers exploration of sample traits in metabolic correlation networks: "As a key functionality, MetaboDiffs offers the exploration of sample traits in a data-derived metabolic correlation network."