150bp paired end sequencing

In total, peripheral blood, cancer, para-cancerous samples, and clinical data of three patients were collected for whole-exome sequencing (WES) in this study. NEBNext dsDNA Fragmentase (NEB, Ipswich, MA, USA) was used to extract and fragment genomic DNA from blood and cancer or para-cancer (normal tissues adjacent to cancer) tissues, followed by DNA end mending. After being detailed, end-repaired DNA segments were ligated with the NEBNext adaptor (NEB, Ipswich, MA, USA). Biotinylated RNA library baits and magnetic beads were coupled with the barcoded library to detect particular areas using the SureSelect Human All Exon V6 Kit (Agilent Technologies, Palo Alto, Calif.). On an Illumina X-ten system, the acquired sequences were amplified further for 150bp paired-end sequencing (Illumina, San Diego, CA, USA). Readings of high quality that passed the Illumina filter were kept for further processing.

RNA was extracted using a Quick-RNA MicroPrep Kit (ZYMO RESEARCH, R1050) according to the manufacturer’s instructions. Then, ribosomal RNA was removed using an NEBNext rRNA Depletion Kit (NEB, E6310). An NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, E7765s) was used to construct library according to the manufacturer’s instructions. The libraries were sequenced on a Hiseq X10 with 150 bp paired-end sequencing (Illumina).