Targeted Sequencing for GC Mutation

Mutation analysis was performed by targeted sequencing using the Ion Torrent platform (Thermo Fisher Scientific, Waltham, MA, USA) and a custom-designed sequencing gene panel (Ion AmpliSeq, Thermo Fisher Scientific) encompassing 525 amplicons covering coding regions of 58 GC related genes. The multiplex PCR based Ion AmpliSeq targeted sequencing technology (Thermo Fisher Scientific) was used for DNA library preparation and amplification of target regions using the Ion AmpliSeq Library Kit v2.0, as well as the specific GC sequencing panel consisting of four primer pools as described in detail previously [19 , 20 (link)]. Automated template preparation of the final libraries as well as chip loading (Ion 520, 530, or 540) was performed on an Ion Chef instrument and sequenced using an Ion S5XL instrument (Thermo Fisher Scientific). Data analysis was performed referring to Pfarr et al. [20 (link)] and ANNOVAR was used to annotate the sequence variants [21 (link)].