Variant validation using High Resolution Melt analysis. A, B: HRM on 6-fish pools identifies a single pool (green line) with distinct denaturation kinetics. Each line represents a single six-fish pool. The X axis is temperature and Y axis is the fluorescent difference compared to the wildtype melt curve. C → T mutations (A) cause a larger temperature shift than T → A mutations (B). C, D: HRM on single fish of the six-fish pool identified above. Each line represents one fish (2 alleles).