In vitro validation of NetMHCpan predictions. Selected peptides (supplemental Table 24) were synthesized and tested for binding to 4 MHCI molecules using an ELISA-based peptide-exchange assay. The threshold for predicted binding was set as described for Figure 5A. For experimental binding validation, the threshold was set at 2 standard deviations (2SD) from the mean absorbance value for 3 positive-control peptides (colored in red). Peptides above this threshold were considered strong binders. Of the 20 predicted strong binders, 15 exhibited binding values above the threshold (75.0%). Of the 15 predicted weak binders, 8 were above the threshold (53.3%). Color coding: green, CALR-derived peptides; yellow rectangle, MPL-derived peptides; magenta, SF3B1-derived peptides. Abbreviations are listed in supplemental Table 24.