For the validation, we performed Sanger sequencing for the 20 variants on 14 selected genes, i.e., five genes harboring frequent ICC mutations (KRAS, TP53, APC, IDH1, and PTPN3), six cancer-related genes in TARGET database (RB1, MAP2K1, MED12, NF1, RAF1, and XPO1) and selected genes in the remaining categories (BAP1, ROBO1 and SMAD7) (Supplementary Figure S1). The gene discussed is RB1; the disease is intrahepatic cholangiocarcinoma.