Fig. 1
Diagram of adapter multiplex PCR (a) and high-resolution melting (b–e) in ALK fusion variant detection. Adapter multiplex PCR is a multistep amplification with adapter-primers and adapters as primers. The adapter-primers include a common adapter sequence (black wedge) at the 5′ end of gene specific primers (empty rectangle). Adapter-primers first target and enrich gene fragments including GAPDH and, if present ALK fusion fragments at a high annealing temperature. In later cycles, common adapters complete the final amplification at a low annealing temperature. Amplicons consist of a variable fusion partner fragment, a common ALK fragment and adapters on both ends. A high-resolution melting process (b) is run after amplification. Data are extracted from melting curve analysis with the instrument software (c). After exponential background removal and normalization (d), second derivative plots are calculated (e). Melting curves in b–e showed an ALK fusion positive sample (H3122, black line) and negative sample (A549, grey line). Calculations are shown in Supplementary Table 3. ALK fusion negative samples only display the GAPDH fragment (low temperature peak), while ALK fusion positive samples display both the GAPDH peak and the ALK fusion (high temperature peak)
