README

This is a python implementation of DEXOM (Diversity-based enumeration of optimal context-specific metabolic networks)

The original project, which was developped in MATLAB, can be found here: https://github.com/MetExplore/dexom

Parts of the imat code were taken from the driven package for data-driven constraint-based analysis: https://github.com/opencobra/driven

API documentation is available here: https://dexom-python.readthedocs.io/en/latest/

Requirements

• Python 3.7+

• CPLEX 12.10+

Installing CPLEX

Free license (Trial version): this version is limited to 1000 variables and 1000 constraints, and is therefore not useable on larger models

Academic license: for this, you must sign up using an academic email address. - after logging in, you can access the download for “ILOG CPLEX Optimization Studio” - download version 12.10 or higher of the appropriate installer for your operating system - install the solver - update the PYTHONPATH environment variable by adding the directory containing the setup.py file appropriate for your OS and python version

alternatively, run python "C:\Program Files\IBM\ILOG\CPLEX_Studio1210\python\setup.py" install

Functions

These are the different functions which are available for context-specific metabolic subnetwork extraction

apply_gpr

The gpr_rules.py script can be used to transform gene expression data into reaction weights, for a limited selection of models.

It uses the gene identifiers and gene-protein-reaction rules present in the model to connect the genes and reactions.

By default, continuous gene expression values/weights will be transformed into continuous reaction weights. Using the --convert flag will instead create semi-quantitative reaction weights with values in {-1, 0, 1}. By default, the proportion of these three weights will be {25%, 50%, 25%}.

iMAT

imat.py contains a modified version of the iMAT algorithm as defined by (Shlomi et al. 2008).

The main inputs of this algorithm are a model file, which must be supplied in a cobrapy-compatible format (SBML, JSON or MAT), and a reaction_weight file in which each reaction is attributed a score.

These reaction weights must be determined prior to launching imat, using the GPR rules present in the metabolic model.

The remaining inputs of imat are: - epsilon: the activation threshold of reactions with weight > 0 - threshold: the activation threshold for unweighted reactions - timelimit: the solver time limit - feasibility: the solver feasbility tolerance - mipgaptol: the solver MIP gap tolerance - full: a bool parameter for switching between the partial & full-DEXOM implementation

note: the feasibility determines the solver’s capacity to return correct results. In particular, the relation epsilon > threshold > ub*feasibility is required (where ub is the maximal upper bound for reaction flux in the model)

By default, uses the create_new_partial_variables function. In this version, binary flux indicator variables are created for each reaction with a non-zero weight.

In the full-DEXOM implementation, binary flux indicator variables are created for every reaction in the model. This does not change the result of the imat function, but can be used for some of the enumeration methods below.

enum_functions

Four methods for enumerating context-specific networks are available: - rxn-enum.py contains reaction-enumeration - icut.py contains integer-cut - maxdist.py contains distance-maximization - div-enum.py contains diversity-enumeration

An explanation of these methods can be found in (Rodriguez-Mier et al. 2021).

Each of these methods can be used on its own. The same model and reaction_weights inputs must be provided as for the imat function.

Additional parameters for all 4 methods are: - prev_sol: a starting imat solution (if none is provided, a new one will be computed)

- obj_tol: a relative tolerance on the imat objective value for the optimality of the solutions

icut, maxiter, and div-enum also have two additional parameters: - maxiter: the maximum number of iterations to run - full: set to True to use the full-DEXOM implementation

As previously explained, the full-DEXOM implementation defines binary indicator variables for all reactions in the model. Although only the reactions with non-zero weights have an impact on the imat objective function, the distance maximization function which is used in maxdist and div-enum can utilize the binary indicators for all reactions. This increases the distance between the solutions, but requires significantly more computation time.

maxdist and div-enum also have one additional parameter:

- icut: if True, an icut constraint will be applied to prevent duplicate solutions

Parallelized DEXOM

The DEXOM algorithm is a combination of several network enumeration methods.

enumeration.py contains the write_batch_script1 function, which is used for creating a parallelization of DEXOM on a slurm computation cluster. The main inputs of this function are: - filenums: the number of parallel batches which should be launched on slurm - iters: the number of div-enum iterations per batch

Other inputs are used for personalizing the directories and filenames on the cluster.

After executing the script, the target directory should contain several bash files named file_0.sh, file_1.sh etc. depending on the filenum parameter that was provided.

In addition, there should be one runfiles.sh file. This file contains the commands to submit the other files as job batches on the slurm cluster.

The results of a DEXOM run can be evaluated with the following scripts:

- dexom_cluster_results.pycompiles and removes duplicate solutions from the results of a parallel DEXOM run.

- pathway_enrichment.py can be used to perform a pathway enrichment analysis using a one-sided hypergeometric test

- result_functions.py contains the plot_pca function, which performs Principal Component Analysis on the enumeration solutions

Examples

Toy models

The toy_models.py script contains code for generating some small metabolic models and reaction weights.

The toy_models folder contains some ready-to-use models and reaction weight files.

The main.py script contains a simple example of the DEXOM workflow using one of the toy models.

Recon 2.2

The recon2v2 folder contains the model and the differential gene expression data which was used to test this new implementation.

In order to produce reaction weights, you can call the gpr_rules script from the command line.

This will create a file named “pval_0-01_reactionweights.csv” in the recon2v2 folder:

python dexom_python/gpr_rules -m recon2v2/recon2v2_corrected.json -n recon2 -g recon2v2/pval_0-01_geneweights.csv -o recon2v2/pval_0-01_reactionweights

Then, call imat to produce a first context-specific subnetwork. This will create a file named “imat_solution.csv” in the recon2v2 folder:

python dexom_python/imat -m recon2v2/recon2v2_corrected.json -r recon2v2/pval_0-01_reactionweights.csv -o recon2v2/imat_solution

To run DEXOM on a slurm cluster, call the enumeration.py script to create the necessary batch files (here: 100 batches with 100 iterations). Be careful to use your own username after the -u input. This script assumes that you have cloned the dexom-python project into a work folder on the cluster, and that you have installed CPLEX v12.10 in the same work folder. Note that this step creates a file called “recon2v2_reactions_shuffled.csv”, which shows the order in which rxn-enum will call the reactions from the model.

python dexom_python/enum_functions/enumeration -m recon2v2/recon2v2_corrected.json -r recon2v2/pval_0-01_reactionweights.csv -p recon2v2/imat_solution.csv -o recon2v2/ -u mstingl -n 100 -i 100

Then, submit the job to the slurm cluster. Note that if you created the files on a Windows pc, you must use the command dos2unix runfiles.sh before sbatch runfiles.sh:

cd recon2v2/
sbatch runfiles.sh
cd -

After all jobs are completed, you can analyze the results using the following scripts:

python dexom_python/dexom_cluster_results -i recon2v2/ -o recon2v2/ -n 100
python dexom_python/pathway_enrichment -s recon2v2/all_dexom_sols.csv -m recon2v2/recon2v2_corrected.json -o recon2v2/
python dexom_python/result_functions -s recon2v2/all_dexom_sols.csv -o recon2v2/

The file all_dexom_sols.csv contains all unique solutions enumerated with DEXOM.

The file output.txt contains the average computation time per iteration and the proportion of duplicate solutions.

The .png files contain boxplots of the pathway enrichment tests as well as a 2D PCA plot of the binary solution vectors.