Procedure to correct false negative (FN) gene essentiality predictions. For each reaction associated with a FN gene, we first examined whether the reaction required the presence of both the FN gene and one or more TN genes. If this was not the case, we attempted to correct the FN prediction by blocking the functions of isozyme(s). Next, we examined whether the reaction was in a dead-end pathway, i.e., a pathway containing metabolites that cannot be produced, metabolites that cannot be consumed, or both. If this was the case, we added (I) metabolite uptakes or (II) metabolites to the biomass objective function. The last attempt was to correct the FN prediction by suppressing one or more reactions (using the optimization model developed by Kumar and Maranas ). When a modification was successful, as determined by the criteria shown in Figure 4, we recorded and collected it in a set of possible modifications. RXN, reaction; TN, true negative; TP, true positive; Y, yes; N, no.