The number of fast alchemical transitions between the two physical end-states is an important parameter in the NEQ approach. Such transitions need to be initiated from equilibrium conformations at each end-state and these conformations must be representative of the true conformational landscape in that state. Further, the number of transitions needs to be sufficiently large to ensure that the distributions of work values obtained in the forward and reverse directions overlap. In general, only a single or a handful replicas have been used by most practitioners and around 100 transitions were run in each direction.