@bgicli/bgicli 2.1.1 → 2.2.0

package/data/skills/tooluniverse-metabolomics-analysis/SKILL.md

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+---
+name: tooluniverse-metabolomics-analysis
+description: Analyze metabolomics data including metabolite identification, quantification, pathway analysis, and metabolic flux. Processes LC-MS, GC-MS, NMR data from targeted and untargeted experiments. Performs normalization, statistical analysis, pathway enrichment, metabolite-enzyme integration, and biomarker discovery. Use when analyzing metabolomics datasets, identifying differential metabolites, studying metabolic pathways, integrating with transcriptomics/proteomics, discovering metabolic biomarkers, performing flux balance analysis, or characterizing metabolic phenotypes in disease, drug response, or physiological conditions.
+---
+
+# Metabolomics Analysis
+
+Comprehensive analysis of metabolomics data from metabolite identification through quantification, statistical analysis, pathway interpretation, and integration with other omics layers.
+
+## When to Use This Skill
+
+**Triggers**:
+- User has metabolomics data (LC-MS, GC-MS, NMR)
+- Questions about metabolite abundance or concentrations
+- Differential metabolite analysis requests
+- Metabolic pathway analysis
+- Multi-omics integration with metabolomics
+- Metabolic biomarker discovery
+- Flux balance analysis or metabolic modeling
+- Metabolite-enzyme correlation
+
+**Example Questions This Skill Solves**:
+1. "Analyze this LC-MS metabolomics data for differential metabolites"
+2. "Which metabolic pathways are dysregulated between conditions?"
+3. "Identify metabolite biomarkers for disease classification"
+4. "Correlate metabolite levels with enzyme expression"
+5. "Perform pathway enrichment for differential metabolites"
+6. "Integrate metabolomics with transcriptomics data"
+7. "Characterize the metabolic phenotype of this cell line"
+8. "Identify metabolites associated with drug response"
+
+---
+
+## Core Capabilities
+
+| Capability | Description |
+|-----------|-------------|
+| **Data Import** | LC-MS, GC-MS, NMR, targeted/untargeted platforms |
+| **Metabolite Identification** | Match to HMDB, KEGG, PubChem, spectral libraries |
+| **Quality Control** | Peak quality, blank subtraction, internal standard normalization |
+| **Normalization** | Probabilistic quotient, total ion current, internal standards |
+| **Statistical Analysis** | Univariate and multivariate (PCA, PLS-DA, OPLS-DA) |
+| **Differential Analysis** | Identify significant metabolite changes |
+| **Pathway Enrichment** | KEGG, Reactome, BioCyc metabolic pathway analysis |
+| **Metabolite-Enzyme Integration** | Correlate with expression data |
+| **Flux Analysis** | Metabolic flux balance analysis (FBA) |
+| **Biomarker Discovery** | Multi-metabolite signatures |
+
+---
+
+## Workflow Overview
+
+```
+Input: Metabolomics Data (Peak Table or Spectra)
+    |
+    v
+Phase 1: Data Import & Metabolite Identification
+    |-- Load peak table or process raw spectra
+    |-- Match features to metabolite databases (HMDB, KEGG)
+    |-- Annotate with chemical IDs, formulas, pathways
+    |-- Confidence scoring for IDs
+    |
+    v
+Phase 2: Quality Control & Filtering
+    |-- Assess peak quality (CV, blank ratios)
+    |-- Remove background peaks
+    |-- Filter low-quality metabolites
+    |-- Internal standard check
+    |
+    v
+Phase 3: Normalization
+    |-- Sample-wise normalization (TIC, PQN, internal standards)
+    |-- Batch effect correction
+    |-- Log-transform or scaling
+    |
+    v
+Phase 4: Exploratory Analysis
+    |-- PCA for sample clustering
+    |-- Quality assessment plots
+    |-- Outlier detection
+    |-- Sample correlation
+    |
+    v
+Phase 5: Differential Analysis
+    |-- Statistical testing (t-test, ANOVA, Wilcoxon)
+    |-- Fold change calculation
+    |-- Multiple testing correction
+    |-- Volcano plots, heatmaps
+    |
+    v
+Phase 6: Pathway Analysis
+    |-- Metabolite set enrichment (MSEA)
+    |-- Pathway topology analysis
+    |-- KEGG/Reactome pathway mapping
+    |-- Identify dysregulated pathways
+    |
+    v
+Phase 7: Multi-Omics Integration
+    |-- Correlate with enzyme expression (RNA/protein)
+    |-- Metabolite-gene associations
+    |-- Pathway-level integration
+    |-- Metabolic flux inference
+    |
+    v
+Phase 8: Generate Report
+    |-- Summary statistics
+    |-- Differential metabolites
+    |-- Pathway diagrams
+    |-- Multi-omics integration plots
+    |-- Biomarker panel
+```
+
+---
+
+## Phase Details
+
+### Phase 1: Data Import & Metabolite Identification
+
+**Objective**: Load data and identify metabolites from features.
+
+**Supported data types**:
+- **Peak tables**: Pre-processed metabolite abundance
+- **Raw spectra**: LC-MS (.mzML, .mzXML), GC-MS
+- **NMR spectra**: 1D/2D NMR data
+
+**Peak table format** (typical):
+```
+Sample_ID | Glucose | Lactate | Glutamine | ... | Cholesterol
+----------|---------|---------|-----------|-----|------------
+Control_1 | 125000  | 45000   | 78000     | ... | 23000
+Control_2 | 130000  | 43000   | 82000     | ... | 25000
+Disease_1 | 85000   | 92000   | 45000     | ... | 45000
+```
+
+**Data loading**:
+```python
+def load_metabolomics_data(file_path, file_type='peak_table'):
+    """
+    Load metabolomics data.
+
+    file_type options:
+    - 'peak_table': CSV/TSV with metabolites as columns
+    - 'mzml': Raw LC-MS data (requires processing)
+    - 'nmr': NMR spectra
+    """
+    import pandas as pd
+
+    if file_type == 'peak_table':
+        # Load peak table
+        data = pd.read_csv(file_path, index_col=0)
+        # Rows = samples, Columns = metabolites
+        return data
+
+    elif file_type == 'mzml':
+        # Process raw MS data (requires pymzml or similar)
+        # Peak detection, alignment, quantification
+        pass
+```
+
+**Metabolite identification**:
+```python
+def identify_metabolites(feature_data, mass_list, rt_list=None):
+    """
+    Match features to metabolite databases.
+
+    Uses accurate mass and retention time (if available).
+    Queries: HMDB, KEGG Compound, PubChem
+    """
+    from tooluniverse import ToolUniverse
+    tu = ToolUniverse()
+
+    identified_metabolites = []
+
+    for i, mass in enumerate(mass_list):
+        # Query HMDB by mass (±5 ppm tolerance)
+        hmdb_result = tu.run_one_function({
+            "name": "hmdb_search_by_mass",
+            "arguments": {
+                "mass": mass,
+                "mass_tolerance": 0.005  # 5 ppm
+            }
+        })
+
+        if hmdb_result and 'data' in hmdb_result:
+            matches = hmdb_result['data']
+            # Rank by confidence
+            # (exact mass match, pathway context, etc.)
+            identified_metabolites.append({
+                'feature_id': i,
+                'metabolite_name': matches[0]['name'],
+                'hmdb_id': matches[0]['accession'],
+                'formula': matches[0]['chemical_formula'],
+                'confidence': calculate_confidence(matches[0])
+            })
+
+    return identified_metabolites
+```
+
+**Confidence scoring**:
+```
+Level 1: Confirmed with authentic standard (MS + RT match)
+Level 2: Probable structure (accurate mass + MS/MS)
+Level 3: Tentative match (accurate mass only)
+Level 4: Unknown metabolite
+```
+
+### Phase 2: Quality Control & Filtering
+
+**Objective**: Remove low-quality features and background noise.
+
+**Quality control metrics**:
+```python
+def metabolomics_qc(data, sample_metadata):
+    """
+    Quality control for metabolomics data.
+
+    QC metrics:
+    - Coefficient of variation (CV) in QC samples
+    - Blank ratios (signal in samples vs blanks)
+    - Missing values per metabolite
+    - Total ion current per sample
+    """
+    # 1. CV in QC samples (should be < 30%)
+    qc_samples = sample_metadata['sample_type'] == 'QC'
+    qc_data = data[qc_samples]
+
+    cv_per_metabolite = qc_data.std() / qc_data.mean()
+    high_cv = cv_per_metabolite > 0.3
+
+    print(f"Metabolites with CV > 30%: {high_cv.sum()}")
+
+    # 2. Blank subtraction
+    blank_samples = sample_metadata['sample_type'] == 'Blank'
+    blank_data = data[blank_samples]
+
+    # Calculate blank ratios
+    blank_means = blank_data.mean()
+    sample_means = data[~blank_samples & ~qc_samples].mean()
+    blank_ratio = sample_means / blank_means
+
+    # Filter: keep metabolites with sample/blank > 3
+    keep_metabolites = blank_ratio > 3
+
+    # 3. Missing values (remove if >50% missing)
+    missing_per_metabolite = (data == 0).sum() / data.shape[0]
+    keep_metabolites &= (missing_per_metabolite < 0.5)
+
+    # Filter data
+    filtered_data = data.loc[:, keep_metabolites]
+
+    return filtered_data
+```
+
+### Phase 3: Normalization
+
+**Objective**: Account for technical variation and enable fair comparison.
+
+**Normalization methods**:
+
+**1. Total Ion Current (TIC)**:
+```python
+def normalize_tic(data):
+    """
+    Normalize by total ion current.
+    Assumes total metabolite abundance is similar across samples.
+    """
+    tic = data.sum(axis=1)
+    median_tic = tic.median()
+    norm_factors = median_tic / tic
+    normalized = data.multiply(norm_factors, axis=0)
+    return normalized
+```
+
+**2. Probabilistic Quotient Normalization (PQN)**:
+```python
+def normalize_pqn(data, reference_sample=None):
+    """
+    Probabilistic quotient normalization.
+    More robust than TIC to large metabolite changes.
+    """
+    import numpy as np
+
+    # Use median sample as reference
+    if reference_sample is None:
+        reference = data.median(axis=0)
+    else:
+        reference = data.loc[reference_sample]
+
+    # Calculate quotients
+    quotients = data.div(reference, axis=1)
+
+    # Median quotient per sample
+    norm_factors = quotients.median(axis=1)
+
+    # Normalize
+    normalized = data.div(norm_factors, axis=0)
+
+    return normalized
+```
+
+**3. Internal Standard Normalization**:
+```python
+def normalize_internal_standard(data, is_metabolite):
+    """
+    Normalize by spiked-in internal standard.
+    Most accurate if added before sample processing.
+    """
+    is_abundance = data[is_metabolite]
+    norm_factors = is_abundance.median() / is_abundance
+    normalized = data.multiply(norm_factors, axis=0)
+
+    # Remove internal standard from data
+    normalized = normalized.drop(columns=[is_metabolite])
+
+    return normalized
+```
+
+**Transformation**:
+```python
+def transform_data(data, method='log'):
+    """
+    Transform metabolite abundances.
+
+    Methods:
+    - 'log': log2 transform (stabilize variance)
+    - 'pareto': Pareto scaling (mean-center, divide by sqrt(std))
+    - 'auto': Auto-scaling (mean-center, divide by std)
+    """
+    import numpy as np
+
+    if method == 'log':
+        # Add small constant to avoid log(0)
+        transformed = np.log2(data + 1)
+
+    elif method == 'pareto':
+        # Pareto scaling (common in metabolomics)
+        mean = data.mean(axis=0)
+        std = data.std(axis=0)
+        transformed = (data - mean) / np.sqrt(std)
+
+    elif method == 'auto':
+        # Auto-scaling (z-score)
+        mean = data.mean(axis=0)
+        std = data.std(axis=0)
+        transformed = (data - mean) / std
+
+    return transformed
+```
+
+### Phase 4: Exploratory Analysis
+
+**Objective**: Visualize data structure and detect outliers.
+
+**PCA**:
+```python
+def perform_pca_metabolomics(data, sample_groups):
+    """
+    Principal component analysis for sample clustering.
+    """
+    from sklearn.decomposition import PCA
+    import matplotlib.pyplot as plt
+
+    # PCA
+    pca = PCA(n_components=2)
+    pca_result = pca.fit_transform(data)
+
+    # Plot
+    plt.figure(figsize=(8, 6))
+    for group in sample_groups.unique():
+        mask = sample_groups == group
+        plt.scatter(pca_result[mask, 0], pca_result[mask, 1], label=group)
+
+    plt.xlabel(f'PC1 ({pca.explained_variance_ratio_[0]:.1%})')
+    plt.ylabel(f'PC2 ({pca.explained_variance_ratio_[1]:.1%})')
+    plt.legend()
+    plt.title('PCA - Metabolomics Data')
+```
+
+**PLS-DA** (Partial Least Squares Discriminant Analysis):
+```python
+def plsda_analysis(X, y, n_components=2):
+    """
+    PLS-DA for supervised dimensionality reduction.
+    Better separation than PCA for classification tasks.
+    """
+    from sklearn.cross_decomposition import PLSRegression
+    from sklearn.preprocessing import LabelEncoder
+
+    # Encode labels
+    le = LabelEncoder()
+    y_encoded = le.fit_transform(y)
+
+    # PLS
+    pls = PLSRegression(n_components=n_components)
+    X_pls = pls.fit_transform(X, y_encoded)[0]
+
+    # Plot
+    plt.scatter(X_pls[:, 0], X_pls[:, 1], c=y_encoded)
+    plt.xlabel('PLS Component 1')
+    plt.ylabel('PLS Component 2')
+    plt.title('PLS-DA')
+
+    return X_pls
+```
+
+### Phase 5: Differential Metabolite Analysis
+
+**Objective**: Identify metabolites with significant abundance changes.
+
+**Statistical testing**:
+```python
+def differential_metabolites(data, group1_samples, group2_samples):
+    """
+    Identify differential metabolites between two groups.
+    """
+    from scipy import stats
+    import numpy as np
+
+    results = []
+
+    for metabolite in data.columns:
+        # Extract abundances
+        group1 = data.loc[group1_samples, metabolite]
+        group2 = data.loc[group2_samples, metabolite]
+
+        # Statistics
+        mean1 = group1.mean()
+        mean2 = group2.mean()
+        fold_change = mean2 / mean1
+        log2fc = np.log2(fold_change)
+
+        # t-test
+        t_stat, p_value = stats.ttest_ind(group1, group2, equal_var=False)
+
+        results.append({
+            'metabolite': metabolite,
+            'fold_change': fold_change,
+            'log2FC': log2fc,
+            'mean_group1': mean1,
+            'mean_group2': mean2,
+            'p_value': p_value,
+            't_statistic': t_stat
+        })
+
+    results_df = pd.DataFrame(results)
+
+    # Multiple testing correction
+    from statsmodels.stats.multitest import multipletests
+    results_df['adj_p_value'] = multipletests(results_df['p_value'], method='fdr_bh')[1]
+
+    # Significance
+    results_df['significant'] = (
+        (results_df['adj_p_value'] < 0.05) &
+        (np.abs(results_df['log2FC']) > 1.0)
+    )
+
+    return results_df
+```
+
+**Volcano plot**:
+```python
+def plot_metabolite_volcano(de_results):
+    """Visualize differential metabolite results."""
+    plt.figure(figsize=(8, 6))
+
+    # Non-significant
+    non_sig = de_results[~de_results['significant']]
+    plt.scatter(non_sig['log2FC'], -np.log10(non_sig['p_value']),
+                c='gray', alpha=0.5, s=20)
+
+    # Significant
+    sig = de_results[de_results['significant']]
+    plt.scatter(sig['log2FC'], -np.log10(sig['p_value']),
+                c='red', alpha=0.7, s=30)
+
+    plt.axhline(-np.log10(0.05), color='blue', linestyle='--')
+    plt.axvline(-1, color='blue', linestyle='--')
+    plt.axvline(1, color='blue', linestyle='--')
+
+    plt.xlabel('log2 Fold Change')
+    plt.ylabel('-log10(p-value)')
+    plt.title('Differential Metabolites')
+```
+
+### Phase 6: Metabolic Pathway Analysis
+
+**Objective**: Interpret metabolite changes at pathway level.
+
+**Metabolite Set Enrichment Analysis (MSEA)**:
+```python
+def pathway_enrichment_metabolites(metabolite_list, organism='human'):
+    """
+    Perform pathway enrichment for differential metabolites.
+
+    Uses KEGG metabolic pathways.
+    """
+    from tooluniverse import ToolUniverse
+    tu = ToolUniverse()
+
+    # Get KEGG compound IDs for metabolites
+    kegg_ids = []
+    for metabolite in metabolite_list:
+        # Convert HMDB ID to KEGG Compound ID
+        result = tu.run_one_function({
+            "name": "kegg_find_compound",
+            "arguments": {"query": metabolite}
+        })
+        if result and 'data' in result:
+            kegg_ids.append(result['data'][0]['entry_id'])
+
+    # Pathway enrichment
+    enrichment = tu.run_one_function({
+        "name": "kegg_enrich_pathway",
+        "arguments": {
+            "compound_list": ",".join(kegg_ids),
+            "organism": organism
+        }
+    })
+
+    return enrichment
+```
+
+**Pathway topology analysis**:
+```python
+def pathway_topology_analysis(metabolites, pathway_id):
+    """
+    Analyze pathway dysregulation considering topology.
+
+    Metabolites at key pathway positions (hubs, bottlenecks)
+    have more impact than peripheral metabolites.
+    """
+    # Load pathway structure from KEGG
+    # Calculate impact score based on:
+    # - Betweenness centrality (bottleneck metabolites)
+    # - Degree centrality (hub metabolites)
+    # - Pathway position (early vs late)
+
+    pass
+```
+
+### Phase 7: Multi-Omics Integration
+
+**Objective**: Integrate metabolomics with transcriptomics/proteomics.
+
+**Metabolite-enzyme correlation**:
+```python
+def correlate_metabolite_enzyme(metabolite_data, enzyme_expression):
+    """
+    Correlate metabolite levels with enzyme expression.
+
+    Expected correlations:
+    - Substrate + enzyme → negative correlation (consumption)
+    - Product + enzyme → positive correlation (production)
+    """
+    from scipy.stats import spearmanr
+
+    # For each metabolite-enzyme pair
+    correlations = {}
+
+    for metabolite in metabolite_data.columns:
+        # Find enzymes that produce/consume this metabolite
+        enzymes = find_metabolite_enzymes(metabolite)
+
+        for enzyme in enzymes:
+            if enzyme in enzyme_expression.index:
+                met_levels = metabolite_data[metabolite]
+                enz_expr = enzyme_expression.loc[enzyme]
+
+                r, p = spearmanr(met_levels, enz_expr)
+
+                correlations[f'{metabolite}_{enzyme}'] = {
+                    'r': r,
+                    'p': p,
+                    'relationship': 'product' if r > 0 else 'substrate'
+                }
+
+    return correlations
+```
+
+**Pathway-level integration**:
+```python
+def integrate_omics_pathway(metabolite_fc, gene_fc, pathway_id):
+    """
+    Integrate metabolite and gene fold changes at pathway level.
+
+    For each reaction:
+    - Check if metabolites are changed
+    - Check if enzymes are changed
+    - Score pathway dysregulation (combined evidence)
+    """
+    # Load pathway reactions
+    # For each reaction:
+    #   - Metabolite substrate/product changes
+    #   - Enzyme expression changes
+    #   - Concordance score
+
+    pathway_score = calculate_pathway_dysregulation(
+        metabolite_fc, gene_fc, pathway_id
+    )
+
+    return pathway_score
+```
+
+### Phase 8: Report Generation
+
+**Generate comprehensive metabolomics report**:
+
+```markdown
+# Metabolomics Analysis Report
+
+## Dataset Summary
+- **Platform**: LC-MS/MS (Orbitrap)
+- **Method**: Untargeted metabolomics
+- **Samples**: 40 (20 disease, 20 control)
+- **Metabolites Identified**: 324 (Level 1/2 confidence)
+- **Metabolites Quantified**: 298 (after QC)
+
+## Quality Control
+- **CV in QC samples**: 18% median (acceptable: <30%)
+- **Blank ratios**: All metabolites > 3x blank signal
+- **Missing values**: 8% average per metabolite
+- **Internal standard**: Recovery 95-105% across samples
+
+## Normalization
+- **Method**: Probabilistic Quotient Normalization (PQN)
+- **Transformation**: log2
+- **Batch correction**: Not required (single batch)
+
+## Exploratory Analysis
+- **PCA**: Clear separation between groups (PC1: 28%, PC2: 18%)
+- **PLS-DA**: Excellent discrimination (R2=0.89, Q2=0.75)
+- **Outliers**: 1 sample removed (technical failure)
+
+## Differential Metabolites
+- **Significant metabolites**: 87 (adj. p < 0.05, |log2FC| > 1)
+  - Increased: 52 metabolites
+  - Decreased: 35 metabolites
+
+### Top Increased Metabolites
+1. **Lactate** (log2FC=3.2, p=1e-12) - Glycolysis
+2. **Glutamine** (log2FC=2.8, p=1e-10) - Amino acid metabolism
+3. **Palmitate** (log2FC=2.5, p=1e-9) - Fatty acid synthesis
+
+### Top Decreased Metabolites
+1. **Citrate** (log2FC=-2.9, p=1e-11) - TCA cycle
+2. **ATP** (log2FC=-2.3, p=1e-9) - Energy metabolism
+3. **NAD+** (log2FC=-2.1, p=1e-8) - Redox balance
+
+## Pathway Enrichment
+### Top Dysregulated Pathways
+1. **Glycolysis/Gluconeogenesis** (p=1e-15)
+   - 12 metabolites: glucose, pyruvate, lactate, etc.
+   - Direction: Increased flux to lactate (Warburg effect)
+2. **TCA Cycle** (p=1e-12)
+   - 8 metabolites: citrate, succinate, malate, etc.
+   - Direction: Decreased activity
+3. **Glutaminolysis** (p=1e-10)
+   - 6 metabolites: glutamine, glutamate, α-KG, etc.
+   - Direction: Increased glutamine consumption
+
+## Multi-Omics Integration
+### Metabolite-Enzyme Correlations
+- **LDHA (lactate dehydrogenase)**
+  - Expression: 3.5-fold increased (RNA + protein)
+  - Lactate: 3.2-fold increased
+  - Correlation: r=0.85 (p<0.001) - Concordant upregulation
+- **IDH1 (isocitrate dehydrogenase)**
+  - Expression: 2.1-fold decreased
+  - Citrate: 2.9-fold decreased
+  - Correlation: r=0.78 (p<0.001) - TCA cycle suppression
+
+### Metabolic Phenotype
+Integration with RNA-seq and proteomics reveals:
+- **Warburg effect**: Shift from oxidative to glycolytic metabolism
+- **Glutamine addiction**: Increased glutaminolysis for anaplerosis
+- **Redox imbalance**: Decreased NAD+/NADH ratio, oxidative stress
+
+## Biomarker Discovery
+### Top 10 Metabolites for Classification
+Random Forest model (10-fold CV):
+- **AUC**: 0.96 ± 0.03
+- **Accuracy**: 92%
+
+**Biomarker Panel**:
+1. Lactate
+2. Glutamine
+3. Citrate
+4. ATP
+5. Palmitate
+6. Pyruvate
+7. Succinate
+8. NAD+
+9. α-ketoglutarate
+10. Glucose-6-phosphate
+
+## Biological Interpretation
+Metabolomics reveals fundamental metabolic reprogramming in disease state:
+
+1. **Glycolytic switch**: Increased glycolysis with lactate accumulation despite oxygen availability (Warburg effect), driven by LDHA upregulation.
+
+2. **TCA cycle suppression**: Decreased citrate and TCA intermediates, consistent with IDH1 downregulation. Shunts carbon to biosynthesis.
+
+3. **Glutamine dependence**: Elevated glutamine consumption and glutaminolysis provides alternative carbon source for anaplerosis and NADPH for biosynthesis.
+
+4. **Biosynthetic activation**: Increased palmitate indicates active fatty acid synthesis, supporting membrane production for proliferation.
+
+5. **Energy stress**: Despite active glycolysis, ATP levels are decreased, suggesting high energy demand outpacing production.
+
+This metabolic signature is characteristic of proliferative, biosynthetically active cells typical of cancer or activated immune cells.
+
+## Clinical Relevance
+- **Therapeutic targets**: LDHA inhibitors, glutaminase inhibitors to disrupt metabolic dependencies
+- **Biomarkers**: Lactate/citrate ratio as metabolic activity marker
+- **Drug response**: Metabolic phenotype may predict sensitivity to metabolic inhibitors
+```
+
+---
+
+## Integration with ToolUniverse
+
+| Skill | Used For | Phase |
+|-------|----------|-------|
+| `tooluniverse-gene-enrichment` | Pathway enrichment | Phase 6 |
+| `tooluniverse-rnaseq-deseq2` | Enzyme expression for integration | Phase 7 |
+| `tooluniverse-proteomics-analysis` | Protein levels for integration | Phase 7 |
+| `tooluniverse-multi-omics-integration` | Comprehensive integration | Phase 7 |
+
+---
+
+## Quantified Minimums
+
+| Component | Requirement |
+|-----------|-------------|
+| Metabolites | At least 50 identified metabolites |
+| Replicates | At least 3 per condition |
+| QC | CV < 30% in QC samples, blank subtraction |
+| Statistical test | t-test or Wilcoxon with FDR correction |
+| Pathway analysis | MSEA with KEGG or Reactome |
+| Report | QC, differential metabolites, pathways, visualizations |
+
+---
+
+## Limitations
+
+- **Identification**: Many features remain unidentified (Level 4)
+- **Coverage**: Cannot detect all metabolites (depends on method)
+- **Quantification**: Relative abundance (not absolute concentration without standards)
+- **Isomers**: Difficult to distinguish structural isomers
+- **Ion suppression**: Matrix effects can affect quantification
+- **Dynamic range**: Limited compared to targeted methods
+
+---
+
+## References
+
+**Methods**:
+- MetaboAnalyst: https://doi.org/10.1093/nar/gkab382
+- XCMS: https://doi.org/10.1021/ac051437y
+- MSEA: https://doi.org/10.1186/1471-2105-11-395
+
+**Databases**:
+- HMDB: https://hmdb.ca
+- KEGG Compound: https://www.genome.jp/kegg/compound/
+- Reactome: https://reactome.org