Qiaxcel bioanalyzer

NGS libraries were prepared from plasma DNA and tumor and cell line gDNA. For patient plasma DNA, 112 ng DNA were used for library construction without additional fragmentation. For tumor, and cell line gDNA, 200–1,000 ng DNA was sheared prior to library construction with Ion Xpress™ Plus Fragment Library Kit for 150 bp fragments. The NGS libraries were constructed using the Ion Xpress Plus Fragment Library Kit (Life Technologies, New York, USA). The cleanup steps was using the Agencourt AMPure XP Kit (Beckman, USA). Ligation was performed for 15 min at 25 °C and 5 min at 72 °C using Ion Xpress Barcode Adapters. Single-step size selection was performed by adding 140 µL (1.4X) of Agencourt AMPure XP beads to enrich for ligated DNA fragments. The ligated fragments were then amplified using Herculase II Fusion DNA Polymerase (Agilent Technologies, USA) and 9–11 PCR cycles, depending on input DNA mass. Library purity and concentration was assessed by spectrophotometer (Qubit 2.0) and qPCR (KAPA Biosystems), respectively. Fragment length was determined on a QIAxcel Bioanalyzer using the QIAxcel DNA screening Kit (Qiagen).

The following materials were used in this study: QIAGEN QlAampDNA Mini Kit (Qiagen, Valencia, Germany); QIAGEN QlAampBlood Mini Kit (Qiagen, Valencia, Germany); Ion Xpress Plus Fragment Library Kit (Life Technologies, New York, USA); Agencourt AMPure XP Kit (Beckman, USA); H2O (Sigma-Aldrich, USA); Thermo NanoDrop 1000 (Thermo, MA, USA); SureSelect Target Enrichment System for Sequencing on Ion-Proton (Agilent Technologies, USA) ; SureSelect TE Reagent Kit, PTN (Agilent Technologies, USA); Herculase II Fusion DNA Polymerase kit (Agilent Technologies, USA); Ion OneTouch 2 System (Life Technologies, New York, USA); Qubit 2.0 (Life Technologies, New York, USA); Ion PI Template OT2 200 Kit v2 (Life Technologies, New York, USA); Ion PI Sequencing 200 Kit v2 (Life Technologies, New York, USA); ABI 3730xl Sequencing Machine (Life Technologies, New York, USA); and PCR machine (Life Technologies, New York, USA); QIAxcel Bioanalyzer (Qiagen, Valencia, Germany); ABI Ion-Proton Sequencing Machine (Life Technologies, New York, USA).