Ion ampliseq hifi master mix

Library preparations were conducted using the Ion AmpliSeq™ Cancer Hotspot Panel v.2 (CHPv2), Ion AmpliSeq Library Kit Plus, and Ion Xpress barcoded adapters (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). The panel covered hotspots of 50 tumor genes. However, only PIK3CA status was evaluated in this study, with 97 PIK3CA mutational hotspots analyzed. Multiplex PCR was conducted using a diluted 10 ng/μL genomic DNA with a premixed primer pool and Ion AmpliSeq HiFi master mix (Ion AmpliSeq Library Kit Plus, Thermo Fisher Scientific, Waltham, MA, USA). This was then followed by the partial digestion of the amplicons, where each sample was treated with FuPa reagent to partially digest the primer sequences and phosphorylate the amplicons. Following digestion, samples were briefly subjected to sequencing adapter ligation and were purified with the CleanNA reagent (CleanNA, Waddinxveen, the Netherlands). Post-purification, libraries were measured by quantitative PCR with real-time detection (qRT-PCR) using an Ion Library TaqMan™ Quantitation Kit (Thermo Fisher Scientific, Waltham, MA, USA) on the QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA), adhering to the manufacturer’s protocols.