This protocol describes the process for generating a high-quality genome-scale metabolic reconstruction. It outlines the steps necessary to build such a reconstruction, including the creation of a draft reconstruction, manual refinement, and conversion into a mathematical model. The process involves genome annotation, identification of metabolic functions, and manual curation of reactions and genes. It also includes the evaluation of the reconstruction's quality, debugging, and validation. The protocol emphasizes the importance of using organism-specific information and experimental data to ensure the accuracy and predictive power of the model. It also discusses the challenges of reconstructing metabolic networks for complex organisms and the need for manual evaluation to address issues such as incorrect reaction directionality, missing functions, and incorrect gene annotations. The protocol also covers the conversion of the reconstruction into a mathematical format for computational analysis, including the use of constraint-based reconstruction and analysis (COBRA) methods. It highlights the importance of testing the model's ability to simulate growth, secretion, and other physiological functions, as well as the need for iterative refinement to improve the model's accuracy. The protocol also discusses the use of various data sources and databases to support the reconstruction process and the importance of ensuring that the model accurately reflects the organism's metabolic capabilities. Finally, it emphasizes the need for careful validation and testing to ensure that the model can be used for predictive purposes and to compare metabolic reconstructions between different organisms.This protocol describes the process for generating a high-quality genome-scale metabolic reconstruction. It outlines the steps necessary to build such a reconstruction, including the creation of a draft reconstruction, manual refinement, and conversion into a mathematical model. The process involves genome annotation, identification of metabolic functions, and manual curation of reactions and genes. It also includes the evaluation of the reconstruction's quality, debugging, and validation. The protocol emphasizes the importance of using organism-specific information and experimental data to ensure the accuracy and predictive power of the model. It also discusses the challenges of reconstructing metabolic networks for complex organisms and the need for manual evaluation to address issues such as incorrect reaction directionality, missing functions, and incorrect gene annotations. The protocol also covers the conversion of the reconstruction into a mathematical format for computational analysis, including the use of constraint-based reconstruction and analysis (COBRA) methods. It highlights the importance of testing the model's ability to simulate growth, secretion, and other physiological functions, as well as the need for iterative refinement to improve the model's accuracy. The protocol also discusses the use of various data sources and databases to support the reconstruction process and the importance of ensuring that the model accurately reflects the organism's metabolic capabilities. Finally, it emphasizes the need for careful validation and testing to ensure that the model can be used for predictive purposes and to compare metabolic reconstructions between different organisms.