Protocol for thermodynamically optimal construction and analysis of metabolic networks using ThermOptCOBRA.

Thermodynamically infeasible cycles violate the second law of thermodynamics but are permitted by stoichiometric flux analysis. Here, we present a protocol for the construction and analysis of metabolic networks using ThermOptCOBRA, a MATLAB extension of the COBRA Toolbox. We describe steps for enumerating cycles (ThermOptEnumerator), determining feasible directions and blocked reactions (ThermOptCC), and building thermodynamically consistent context-specific models using omics data (ThermOptiCS). We then detail procedures for removing loops or sample loopless flux distributions (ThermOptFlux/ADSB-ThermOptFlux). For complete details on the use and execution of this protocol, please refer to Kumar and Bhatt.1.