Relative Abundance Preprocessing

relative-abundance-preprocessing

Summary

Normalize microbiome and metabolome count tables to relative abundance (RA) or centered log-ratio (CLR) transformed compositions before predictive modeling. This preprocessing step standardizes feature scales and handles zero-inflation in compositional microbiome data, enabling fair comparison of transformation effects on downstream prediction performance.

When to use

When you have raw count tables from 16S rRNA sequencing (microbiome) or LC-MS/MS metabolomics (metabolome) and need to train neural network or regression models for microbe-metabolite relationship prediction. Use this skill when comparing whether CLR or RA normalization produces better-predicted metabolite counts or prediction correlations in a specific disease cohort (e.g., IBD vs. controls).

When NOT to use

• Input data is already normalized or transformed (e.g., already CLR, relative abundance, or log-scaled). Check data descriptors and apply 'None' normalization flag in tools like MiMeNet.
• Metabolite data lacks sufficient compositional structure (e.g., proteomics absolute abundance, single-metabolite standards rather than untargeted profiles). CLR is designed for compositional data where features sum to a constant per sample.
• Microbiome data consists of functional profiles or pathways that are not compositional in nature. RA/CLR transformations assume zero-sum constraints; independent abundance measurements should not be transformed.

Inputs

• Raw microbiome count table (16S rRNA, taxonomic or functional features; samples × microbes)
• Raw metabolome count table (LC-MS/MS; samples × metabolites)
• Sample metadata (optional: disease status, cohort labels for downstream enrichment)

Outputs

• CLR-transformed microbiome matrix (samples × microbes, log-centered compositions)
• CLR-transformed metabolome matrix (samples × metabolites, log-centered compositions)
• Relative-abundance microbiome matrix (samples × microbes, proportions ≤1)
• Relative-abundance metabolome matrix (samples × metabolites, proportions ≤1)
• Feature-retention report (count of features removed at 10% prevalence threshold)

How to apply

Load paired microbiome and metabolome count matrices (samples × features). Apply one or both transformations in parallel: (1) relative abundance (RA): divide each feature by sample total and scale to proportions; (2) centered log-ratio (CLR): add pseudocount +1, log-transform, and center by subtracting the geometric mean of log-transformed features. Remove features present in <10% of samples before transformation to reduce sparsity. Train identical downstream models (e.g., MiMeNet neural networks with fixed hyperparameters: ReLU, L2=0.001, dropout=0.5, layer size=512, 1 hidden layer) on each transformed dataset using identical cross-validation folds (10 iterations of 10-fold CV). Compare prediction performance using mean Spearman correlation coefficients (SCCs) and counts of well-predicted metabolites at the 95th percentile of background SCC distribution. Document whether CLR or RA produces higher correlations and more well-predicted metabolites in your specific cohort (e.g., IBD PRISM showed comparable CLR/RA correlations, but CLR improved cystic fibrosis and soil).

Related tools

• MiMeNet (accepts normalized microbiome and metabolome inputs (RA, CLR, or None) via -micro_norm and -metab_norm flags; outputs prediction correlations and module assignments stratified by transformation type) — https://github.com/YDaiLab/MiMeNet
• MelonnPan (linear regression-based metabolite prediction tool that can be trained on transformed microbial features; uses Elastic Net with relative-abundance or other normalized inputs) — https://github.com/biobakery/melonnpan
• scikit-learn (preprocessing utilities for log-transformation, scaling, and feature filtering in Python pipelines)
• pandas (data frame manipulation for feature filtering, composition normalization, and pseudocount addition in Python)

Examples

python MiMeNet_train.py -micro data/IBD/microbiome_PRISM.csv -metab data/IBD/metabolome_PRISM.csv -micro_norm CLR -metab_norm CLR -num_run_cv 10 -num_cv 10 -output IBD_CLR_normalized

Evaluation signals

• Feature prevalence: verify that all retained features are present in ≥10% of samples; count removed features and confirm <10% prevalence cutoff was applied
• Composition constraint: for CLR, verify that log-transformed, centered feature values sum to ~0 per sample (within floating-point tolerance); for RA, verify all feature rows sum to ~1.0 per sample
• Pseudocount addition: confirm pseudocount +1 was added to all counts before log-transformation to handle zeros; raw count tables must not contain exact zeros after transformation
• Spearman correlation improvement: compare mean SCC between CLR and RA across identical CV folds; CLR should show equal or improved correlations vs. RA in most datasets; document dataset-specific differences (e.g., IBD comparable, CF improved, soil improved)
• Well-predicted metabolite count consistency: verify that well-predicted metabolite counts are reproducible across repeated CV iterations; higher counts for CLR would indicate stronger metabolite predictability

Limitations

• CLR transformation is undefined for samples with all-zero features; pseudocount +1 is a heuristic choice and may not be optimal for all microbiome compositionality profiles.
• Relative abundance normalization discards absolute abundance information; rare but biologically important features may be downweighted relative to highly abundant taxa even after RA scaling.
• No universally optimal transformation: IBD (PRISM) showed comparable CLR and RA correlations, while cystic fibrosis and soil showed CLR advantage; transformation choice may be dataset- or disease-context dependent. Prior benchmarking on similar cohorts is recommended.
• The 10% prevalence filter is arbitrary and may remove biologically relevant rare features; tune this threshold based on study design and statistical power.
• External validation datasets (e.g., LifeLines-DEEP for IBD) may have different baseline feature prevalence, requiring re-filtering or tolerance adjustment to maintain consistency.

Evidence

• [methods] Remove features present in <10% of samples: "Any input or output feature that is present in less than 10% of samples was removed"
• [methods] CLR transformation with pseudocount +1: "centered log-ratio (CLR) transformation with pseudocount +1, and relative abundance (RA) normalization"
• [methods] Parallel transformation comparison approach: "Prepare two parallel input transformations: centered log-ratio (CLR) transformation with pseudocount +1, and relative abundance (RA) normalization"
• [readme] MiMeNet tool configuration for RA vs CLR: "Transform the microbial features into relative abundance (RA) or center log-ratio (CLR). If the data is already transformed, apply 'None' to skip transformation."
• [other] Dataset-specific transformation performance findings: "In the IBD (PRISM) dataset, prediction correlations were comparable between CLR and relative abundance transformations, while CLR showed increased correlations in cystic fibrosis and soil datasets"