Optimization of signal peptide threshold prediction parameters searching for the lowest number of Mixed groups. Several combinations of the three parameters used for signal peptide prediction in PlasmoDB were tested in search of the optimal setting. (A) The first analysis was all possible values of NN-Sum combining with D-Score and HMM probability ranging from 0.05 to 1.0 (intervals of 0.05). (B) Graphic representation of this combination is showed for the optimal threshold of NN-Sum = 4. The area registering the lower numbers of Mixed groups (dotted rectangle) for a refined search using intervals of 0.01 units (C). The lowest number of Mixed groups (465) was achieved by resetting thresholds to: NN-Sum = 4; D-Score = 0.48 and HMM probability = 0.87; 0.90; 0.9 (indicated by the red arrows).