Figure 3.

Sequencing of amplified tumor cDNA (amplicon‐seq) by Sanger sequencing as well as next‐generation sequencing (NGS) to determine the presence of mutation sequences in the tumor RNA. (a) Variant allele frequencies (VAF) at each mutation site obtained by amplicon‐seq (NGS) were categorized as positive or negative by amplicon‐seq (Sanger) and shown in a beeswarm plot. (b) A receiver operator characteristic curve was drawn to determine the cut‐off value of the VAF obtained by amplicon‐seq (NGS) for the detection of mutant mRNA expression. The results of Sanger sequencing for detection of mutant mRNA expression were accurately paralleled by NGS when the cut‐off value of VAF was set at ≥0.039 (sensitivity, 0.966; specificity, 0.96). AUC, area under the curve.