### Free energy of annealing

The free energy of annealing determines the equilibrium of a reaction. For negative values, which is the case for primer annealing, reactions occur spontaneously. The smaller the free energy, the higher the concentration of primer that is actually bound to the template sequences. Note that the free energy of annealing may call too many coverage events since it does not specifically take into account the impact of 3' mismatches on the interaction with the polymerase.