21 KiB
21 KiB
COBRApy Comprehensive Workflows
This document provides detailed step-by-step workflows for common COBRApy tasks in metabolic modeling.
Workflow 1: Complete Knockout Study with Visualization
This workflow demonstrates how to perform a comprehensive gene knockout study and visualize the results.
import pandas as pd
import matplotlib.pyplot as plt
from cobra.io import load_model
from cobra.flux_analysis import single_gene_deletion, double_gene_deletion
# Step 1: Load model
model = load_model("ecoli")
print(f"Loaded model: {model.id}")
print(f"Model contains {len(model.reactions)} reactions, {len(model.metabolites)} metabolites, {len(model.genes)} genes")
# Step 2: Get baseline growth rate
baseline = model.slim_optimize()
print(f"Baseline growth rate: {baseline:.3f} /h")
# Step 3: Perform single gene deletions
print("Performing single gene deletions...")
single_results = single_gene_deletion(model)
# Step 4: Classify genes by impact
essential_genes = single_results[single_results["growth"] < 0.01]
severely_impaired = single_results[(single_results["growth"] >= 0.01) &
(single_results["growth"] < 0.5 * baseline)]
moderately_impaired = single_results[(single_results["growth"] >= 0.5 * baseline) &
(single_results["growth"] < 0.9 * baseline)]
neutral_genes = single_results[single_results["growth"] >= 0.9 * baseline]
print(f"\nSingle Deletion Results:")
print(f" Essential genes: {len(essential_genes)}")
print(f" Severely impaired: {len(severely_impaired)}")
print(f" Moderately impaired: {len(moderately_impaired)}")
print(f" Neutral genes: {len(neutral_genes)}")
# Step 5: Visualize distribution
fig, ax = plt.subplots(figsize=(10, 6))
single_results["growth"].hist(bins=50, ax=ax)
ax.axvline(baseline, color='r', linestyle='--', label='Baseline')
ax.set_xlabel("Growth rate (/h)")
ax.set_ylabel("Number of genes")
ax.set_title("Distribution of Growth Rates After Single Gene Deletions")
ax.legend()
plt.tight_layout()
plt.savefig("single_deletion_distribution.png", dpi=300)
# Step 6: Identify gene pairs for double deletions
# Focus on non-essential genes to find synthetic lethals
target_genes = single_results[single_results["growth"] >= 0.5 * baseline].index.tolist()
target_genes = [list(gene)[0] for gene in target_genes[:50]] # Limit for performance
print(f"\nPerforming double deletions on {len(target_genes)} genes...")
double_results = double_gene_deletion(
model,
gene_list1=target_genes,
processes=4
)
# Step 7: Find synthetic lethal pairs
synthetic_lethals = double_results[
(double_results["growth"] < 0.01) &
(single_results.loc[double_results.index.get_level_values(0)]["growth"].values >= 0.5 * baseline) &
(single_results.loc[double_results.index.get_level_values(1)]["growth"].values >= 0.5 * baseline)
]
print(f"Found {len(synthetic_lethals)} synthetic lethal gene pairs")
print("\nTop 10 synthetic lethal pairs:")
print(synthetic_lethals.head(10))
# Step 8: Export results
single_results.to_csv("single_gene_deletions.csv")
double_results.to_csv("double_gene_deletions.csv")
synthetic_lethals.to_csv("synthetic_lethals.csv")
Workflow 2: Media Design and Optimization
This workflow shows how to systematically design growth media and find minimal media compositions.
from cobra.io import load_model
from cobra.medium import minimal_medium
import pandas as pd
# Step 1: Load model and check current medium
model = load_model("ecoli")
current_medium = model.medium
print("Current medium composition:")
for exchange, bound in current_medium.items():
metabolite_id = exchange.replace("EX_", "").replace("_e", "")
print(f" {metabolite_id}: {bound:.2f} mmol/gDW/h")
# Step 2: Get baseline growth
baseline_growth = model.slim_optimize()
print(f"\nBaseline growth rate: {baseline_growth:.3f} /h")
# Step 3: Calculate minimal medium for different growth targets
growth_targets = [0.25, 0.5, 0.75, 1.0]
minimal_media = {}
for fraction in growth_targets:
target_growth = baseline_growth * fraction
print(f"\nCalculating minimal medium for {fraction*100:.0f}% growth ({target_growth:.3f} /h)...")
min_medium = minimal_medium(
model,
target_growth,
minimize_components=True,
open_exchanges=True
)
minimal_media[fraction] = min_medium
print(f" Required components: {len(min_medium)}")
print(f" Components: {list(min_medium.index)}")
# Step 4: Compare media compositions
media_df = pd.DataFrame(minimal_media).fillna(0)
media_df.to_csv("minimal_media_comparison.csv")
# Step 5: Test aerobic vs anaerobic conditions
print("\n--- Aerobic vs Anaerobic Comparison ---")
# Aerobic
model_aerobic = model.copy()
aerobic_growth = model_aerobic.slim_optimize()
aerobic_medium = minimal_medium(model_aerobic, aerobic_growth * 0.9, minimize_components=True)
# Anaerobic
model_anaerobic = model.copy()
medium_anaerobic = model_anaerobic.medium
medium_anaerobic["EX_o2_e"] = 0.0
model_anaerobic.medium = medium_anaerobic
anaerobic_growth = model_anaerobic.slim_optimize()
anaerobic_medium = minimal_medium(model_anaerobic, anaerobic_growth * 0.9, minimize_components=True)
print(f"Aerobic growth: {aerobic_growth:.3f} /h (requires {len(aerobic_medium)} components)")
print(f"Anaerobic growth: {anaerobic_growth:.3f} /h (requires {len(anaerobic_medium)} components)")
# Step 6: Identify unique requirements
aerobic_only = set(aerobic_medium.index) - set(anaerobic_medium.index)
anaerobic_only = set(anaerobic_medium.index) - set(aerobic_medium.index)
shared = set(aerobic_medium.index) & set(anaerobic_medium.index)
print(f"\nShared components: {len(shared)}")
print(f"Aerobic-only: {aerobic_only}")
print(f"Anaerobic-only: {anaerobic_only}")
# Step 7: Test custom medium
print("\n--- Testing Custom Medium ---")
custom_medium = {
"EX_glc__D_e": 10.0, # Glucose
"EX_o2_e": 20.0, # Oxygen
"EX_nh4_e": 5.0, # Ammonium
"EX_pi_e": 5.0, # Phosphate
"EX_so4_e": 1.0, # Sulfate
}
with model:
model.medium = custom_medium
custom_growth = model.optimize().objective_value
print(f"Growth on custom medium: {custom_growth:.3f} /h")
# Check which nutrients are limiting
for exchange in custom_medium:
with model:
# Double the uptake rate
medium_test = model.medium
medium_test[exchange] *= 2
model.medium = medium_test
test_growth = model.optimize().objective_value
improvement = (test_growth - custom_growth) / custom_growth * 100
if improvement > 1:
print(f" {exchange}: +{improvement:.1f}% growth when doubled (LIMITING)")
Workflow 3: Flux Space Exploration with Sampling
This workflow demonstrates comprehensive flux space analysis using FVA and sampling.
from cobra.io import load_model
from cobra.flux_analysis import flux_variability_analysis
from cobra.sampling import sample
import pandas as pd
import matplotlib.pyplot as plt
import seaborn as sns
# Step 1: Load model
model = load_model("ecoli")
baseline = model.slim_optimize()
print(f"Baseline growth: {baseline:.3f} /h")
# Step 2: Perform FVA at optimal growth
print("\nPerforming FVA at optimal growth...")
fva_optimal = flux_variability_analysis(model, fraction_of_optimum=1.0)
# Step 3: Identify reactions with flexibility
fva_optimal["range"] = fva_optimal["maximum"] - fva_optimal["minimum"]
fva_optimal["relative_range"] = fva_optimal["range"] / (fva_optimal["maximum"].abs() + 1e-9)
flexible_reactions = fva_optimal[fva_optimal["range"] > 1.0].sort_values("range", ascending=False)
print(f"\nFound {len(flexible_reactions)} reactions with >1.0 mmol/gDW/h flexibility")
print("\nTop 10 most flexible reactions:")
print(flexible_reactions.head(10)[["minimum", "maximum", "range"]])
# Step 4: Perform FVA at suboptimal growth (90%)
print("\nPerforming FVA at 90% optimal growth...")
fva_suboptimal = flux_variability_analysis(model, fraction_of_optimum=0.9)
fva_suboptimal["range"] = fva_suboptimal["maximum"] - fva_suboptimal["minimum"]
# Step 5: Compare flexibility at different optimality levels
comparison = pd.DataFrame({
"range_100": fva_optimal["range"],
"range_90": fva_suboptimal["range"]
})
comparison["range_increase"] = comparison["range_90"] - comparison["range_100"]
print("\nReactions with largest increase in flexibility at suboptimality:")
print(comparison.sort_values("range_increase", ascending=False).head(10))
# Step 6: Perform flux sampling
print("\nPerforming flux sampling (1000 samples)...")
samples = sample(model, n=1000, method="optgp", processes=4)
# Step 7: Analyze sampling results for key reactions
key_reactions = ["PFK", "FBA", "TPI", "GAPD", "PGK", "PGM", "ENO", "PYK"]
available_key_reactions = [r for r in key_reactions if r in samples.columns]
if available_key_reactions:
fig, axes = plt.subplots(2, 4, figsize=(16, 8))
axes = axes.flatten()
for idx, reaction_id in enumerate(available_key_reactions[:8]):
ax = axes[idx]
samples[reaction_id].hist(bins=30, ax=ax, alpha=0.7)
# Overlay FVA bounds
fva_min = fva_optimal.loc[reaction_id, "minimum"]
fva_max = fva_optimal.loc[reaction_id, "maximum"]
ax.axvline(fva_min, color='r', linestyle='--', label='FVA min')
ax.axvline(fva_max, color='r', linestyle='--', label='FVA max')
ax.set_xlabel("Flux (mmol/gDW/h)")
ax.set_ylabel("Frequency")
ax.set_title(reaction_id)
if idx == 0:
ax.legend()
plt.tight_layout()
plt.savefig("flux_distributions.png", dpi=300)
# Step 8: Calculate correlation between reactions
print("\nCalculating flux correlations...")
correlation_matrix = samples[available_key_reactions].corr()
fig, ax = plt.subplots(figsize=(10, 8))
sns.heatmap(correlation_matrix, annot=True, fmt=".2f", cmap="coolwarm",
center=0, ax=ax, square=True)
ax.set_title("Flux Correlations Between Key Glycolysis Reactions")
plt.tight_layout()
plt.savefig("flux_correlations.png", dpi=300)
# Step 9: Identify reaction modules (highly correlated groups)
print("\nHighly correlated reaction pairs (|r| > 0.9):")
for i in range(len(correlation_matrix)):
for j in range(i+1, len(correlation_matrix)):
corr = correlation_matrix.iloc[i, j]
if abs(corr) > 0.9:
print(f" {correlation_matrix.index[i]} <-> {correlation_matrix.columns[j]}: {corr:.3f}")
# Step 10: Export all results
fva_optimal.to_csv("fva_optimal.csv")
fva_suboptimal.to_csv("fva_suboptimal.csv")
samples.to_csv("flux_samples.csv")
correlation_matrix.to_csv("flux_correlations.csv")
Workflow 4: Production Strain Design
This workflow demonstrates how to design a production strain for a target metabolite.
from cobra.io import load_model
from cobra.flux_analysis import (
production_envelope,
flux_variability_analysis,
single_gene_deletion
)
import pandas as pd
import matplotlib.pyplot as plt
# Step 1: Define production target
TARGET_METABOLITE = "EX_ac_e" # Acetate production
CARBON_SOURCE = "EX_glc__D_e" # Glucose uptake
# Step 2: Load model
model = load_model("ecoli")
print(f"Designing strain for {TARGET_METABOLITE} production")
# Step 3: Calculate baseline production envelope
print("\nCalculating production envelope...")
envelope = production_envelope(
model,
reactions=[CARBON_SOURCE, TARGET_METABOLITE],
carbon_sources=CARBON_SOURCE
)
# Visualize production envelope
fig, ax = plt.subplots(figsize=(10, 6))
ax.plot(envelope[CARBON_SOURCE], envelope["mass_yield_maximum"], 'b-', label='Max yield')
ax.plot(envelope[CARBON_SOURCE], envelope["mass_yield_minimum"], 'r-', label='Min yield')
ax.set_xlabel(f"Glucose uptake (mmol/gDW/h)")
ax.set_ylabel(f"Acetate yield")
ax.set_title("Wild-type Production Envelope")
ax.legend()
ax.grid(True, alpha=0.3)
plt.tight_layout()
plt.savefig("production_envelope_wildtype.png", dpi=300)
# Step 4: Maximize production while maintaining growth
print("\nOptimizing for production...")
# Set minimum growth constraint
MIN_GROWTH = 0.1 # Maintain at least 10% of max growth
with model:
# Change objective to product formation
model.objective = TARGET_METABOLITE
model.objective_direction = "max"
# Add growth constraint
growth_reaction = model.reactions.get_by_id(model.objective.name) if hasattr(model.objective, 'name') else list(model.objective.variables.keys())[0].name
max_growth = model.slim_optimize()
model.reactions.BIOMASS_Ecoli_core_w_GAM.lower_bound = MIN_GROWTH
with model:
model.objective = TARGET_METABOLITE
model.objective_direction = "max"
production_solution = model.optimize()
max_production = production_solution.objective_value
print(f"Maximum production: {max_production:.3f} mmol/gDW/h")
print(f"Growth rate: {production_solution.fluxes['BIOMASS_Ecoli_core_w_GAM']:.3f} /h")
# Step 5: Identify beneficial gene knockouts
print("\nScreening for beneficial knockouts...")
# Reset model
model.reactions.BIOMASS_Ecoli_core_w_GAM.lower_bound = MIN_GROWTH
model.objective = TARGET_METABOLITE
model.objective_direction = "max"
knockout_results = []
for gene in model.genes:
with model:
gene.knock_out()
try:
solution = model.optimize()
if solution.status == "optimal":
production = solution.objective_value
growth = solution.fluxes["BIOMASS_Ecoli_core_w_GAM"]
if production > max_production * 1.05: # >5% improvement
knockout_results.append({
"gene": gene.id,
"production": production,
"growth": growth,
"improvement": (production / max_production - 1) * 100
})
except:
continue
knockout_df = pd.DataFrame(knockout_results)
if len(knockout_df) > 0:
knockout_df = knockout_df.sort_values("improvement", ascending=False)
print(f"\nFound {len(knockout_df)} beneficial knockouts:")
print(knockout_df.head(10))
knockout_df.to_csv("beneficial_knockouts.csv", index=False)
else:
print("No beneficial single knockouts found")
# Step 6: Test combination of best knockouts
if len(knockout_df) > 0:
print("\nTesting knockout combinations...")
top_genes = knockout_df.head(3)["gene"].tolist()
with model:
for gene_id in top_genes:
model.genes.get_by_id(gene_id).knock_out()
solution = model.optimize()
if solution.status == "optimal":
combined_production = solution.objective_value
combined_growth = solution.fluxes["BIOMASS_Ecoli_core_w_GAM"]
combined_improvement = (combined_production / max_production - 1) * 100
print(f"\nCombined knockout results:")
print(f" Genes: {', '.join(top_genes)}")
print(f" Production: {combined_production:.3f} mmol/gDW/h")
print(f" Growth: {combined_growth:.3f} /h")
print(f" Improvement: {combined_improvement:.1f}%")
# Step 7: Analyze flux distribution in production strain
if len(knockout_df) > 0:
best_gene = knockout_df.iloc[0]["gene"]
with model:
model.genes.get_by_id(best_gene).knock_out()
solution = model.optimize()
# Get active pathways
active_fluxes = solution.fluxes[solution.fluxes.abs() > 0.1]
active_fluxes.to_csv(f"production_strain_fluxes_{best_gene}_knockout.csv")
print(f"\nActive reactions in production strain: {len(active_fluxes)}")
Workflow 5: Model Validation and Debugging
This workflow shows systematic approaches to validate and debug metabolic models.
from cobra.io import load_model, read_sbml_model
from cobra.flux_analysis import flux_variability_analysis
import pandas as pd
# Step 1: Load model
model = load_model("ecoli") # Or read_sbml_model("your_model.xml")
print(f"Model: {model.id}")
print(f"Reactions: {len(model.reactions)}")
print(f"Metabolites: {len(model.metabolites)}")
print(f"Genes: {len(model.genes)}")
# Step 2: Check model feasibility
print("\n--- Feasibility Check ---")
try:
objective_value = model.slim_optimize()
print(f"Model is feasible (objective: {objective_value:.3f})")
except:
print("Model is INFEASIBLE")
print("Troubleshooting steps:")
# Check for blocked reactions
from cobra.flux_analysis import find_blocked_reactions
blocked = find_blocked_reactions(model)
print(f" Blocked reactions: {len(blocked)}")
if len(blocked) > 0:
print(f" First 10 blocked: {list(blocked)[:10]}")
# Check medium
print(f"\n Current medium: {model.medium}")
# Try opening all exchanges
for reaction in model.exchanges:
reaction.lower_bound = -1000
try:
objective_value = model.slim_optimize()
print(f"\n Model feasible with open exchanges (objective: {objective_value:.3f})")
print(" Issue: Medium constraints too restrictive")
except:
print("\n Model still infeasible with open exchanges")
print(" Issue: Structural problem (missing reactions, mass imbalance, etc.)")
# Step 3: Check mass and charge balance
print("\n--- Mass and Charge Balance Check ---")
unbalanced_reactions = []
for reaction in model.reactions:
try:
balance = reaction.check_mass_balance()
if balance:
unbalanced_reactions.append({
"reaction": reaction.id,
"imbalance": balance
})
except:
pass
if unbalanced_reactions:
print(f"Found {len(unbalanced_reactions)} unbalanced reactions:")
for item in unbalanced_reactions[:10]:
print(f" {item['reaction']}: {item['imbalance']}")
else:
print("All reactions are mass balanced")
# Step 4: Identify dead-end metabolites
print("\n--- Dead-end Metabolite Check ---")
dead_end_metabolites = []
for metabolite in model.metabolites:
producing_reactions = [r for r in metabolite.reactions
if r.metabolites[metabolite] > 0]
consuming_reactions = [r for r in metabolite.reactions
if r.metabolites[metabolite] < 0]
if len(producing_reactions) == 0 or len(consuming_reactions) == 0:
dead_end_metabolites.append({
"metabolite": metabolite.id,
"producers": len(producing_reactions),
"consumers": len(consuming_reactions)
})
if dead_end_metabolites:
print(f"Found {len(dead_end_metabolites)} dead-end metabolites:")
for item in dead_end_metabolites[:10]:
print(f" {item['metabolite']}: {item['producers']} producers, {item['consumers']} consumers")
else:
print("No dead-end metabolites found")
# Step 5: Check for duplicate reactions
print("\n--- Duplicate Reaction Check ---")
reaction_equations = {}
duplicates = []
for reaction in model.reactions:
equation = reaction.build_reaction_string()
if equation in reaction_equations:
duplicates.append({
"reaction1": reaction_equations[equation],
"reaction2": reaction.id,
"equation": equation
})
else:
reaction_equations[equation] = reaction.id
if duplicates:
print(f"Found {len(duplicates)} duplicate reaction pairs:")
for item in duplicates[:10]:
print(f" {item['reaction1']} == {item['reaction2']}")
else:
print("No duplicate reactions found")
# Step 6: Identify orphan genes
print("\n--- Orphan Gene Check ---")
orphan_genes = [gene for gene in model.genes if len(gene.reactions) == 0]
if orphan_genes:
print(f"Found {len(orphan_genes)} orphan genes (not associated with reactions):")
print(f" First 10: {[g.id for g in orphan_genes[:10]]}")
else:
print("No orphan genes found")
# Step 7: Check for thermodynamically infeasible loops
print("\n--- Thermodynamic Loop Check ---")
fva_loopless = flux_variability_analysis(model, loopless=True)
fva_standard = flux_variability_analysis(model)
loop_reactions = []
for reaction_id in fva_standard.index:
standard_range = fva_standard.loc[reaction_id, "maximum"] - fva_standard.loc[reaction_id, "minimum"]
loopless_range = fva_loopless.loc[reaction_id, "maximum"] - fva_loopless.loc[reaction_id, "minimum"]
if standard_range > loopless_range + 0.1:
loop_reactions.append({
"reaction": reaction_id,
"standard_range": standard_range,
"loopless_range": loopless_range
})
if loop_reactions:
print(f"Found {len(loop_reactions)} reactions potentially involved in loops:")
loop_df = pd.DataFrame(loop_reactions).sort_values("standard_range", ascending=False)
print(loop_df.head(10))
else:
print("No thermodynamically infeasible loops detected")
# Step 8: Generate validation report
print("\n--- Generating Validation Report ---")
validation_report = {
"model_id": model.id,
"feasible": objective_value if 'objective_value' in locals() else None,
"n_reactions": len(model.reactions),
"n_metabolites": len(model.metabolites),
"n_genes": len(model.genes),
"n_unbalanced": len(unbalanced_reactions),
"n_dead_ends": len(dead_end_metabolites),
"n_duplicates": len(duplicates),
"n_orphan_genes": len(orphan_genes),
"n_loop_reactions": len(loop_reactions)
}
validation_df = pd.DataFrame([validation_report])
validation_df.to_csv("model_validation_report.csv", index=False)
print("Validation report saved to model_validation_report.csv")
These workflows provide comprehensive templates for common COBRApy tasks. Adapt them as needed for specific research questions and models.