To illustrate the power of the NNAlign method to capture the binding motifs within unaligned quantitative peptide data, we applied the method to derive sequence logo representations of the 14 MHC class II HLA-DR molecules included the Wang dataset. NNAlign was trained with a binding motif length of 9 amino acids, Blosum encoding, including peptide length and flanking region length, and PFRs of 3 amino acids, homology clustering at threshold 0.8 using all data points, 20 hidden neurons and a 5-fold cross-validation without stopping at the best test set performance. These parameters were found to be optimal in the original NNalign paper for MHC class II binding prediction [20] , with the only difference that here we choose a single value for hidden layer size for a matter of prediction speed. Individual networks are aligned to a common register using the offset correction strategy previously described. The sequence logos obtained are shown in Figure 4 . The sequence logos reflect the overall consensus of the binding motifs for HLA-DR molecules, namely a prominent P1 anchor with strong amino acids preference towards hydrophobic Figure 2 . Identification of optimal motif length using the NNAlign method. Left panel: Histogram of the optimal motifs lengths for the 14 HLA-DR molecules in the Wang dataset as identified by the NNAlign method. Right panel: Predictive performance measured in terms of the root mean square error (RMSE) between observed and predicted values as a function of the motif length for the two molecules DRB1*0101 and DRB1*1501. NNAlign was trained using the same parameters settings described in Figure 4 . At each motif length are shown the mean and standard error of the mean RMSE as estimated by bootstrap sampling. For DRB1*0101 a single consistent optimal motif length of 9 amino acids is found. For DRB1*1501 all motif length 8-11 had statistically indistinguishable performance (paired t-test). doi:10.1371/journal.pone.0026781.g002 amino acids in general, and aromatic amino acids as F and Y in particular, and the presence of two or more additional anchors at P4, P6 and/or P9 each with a unique amino acid preference. Even though most of these motifs exhibit a strong preference for hydrophobic and neutral amino acids at most anchor positions, some dramatic deviations from this general pattern exist. Examples of this are the motifs of DRB1*0301 and DRB1*1101 molecules that have strong preferences for charged amino acids at P4 and P6, respectively.