Ion pi hi q chef kit

Amplicon sequencing was carried out using Ion AmpliSeq Colon and Lung Cancer Panel v2 (CLv2; Thermo Fisher Scientific), which targets 22 cancer‐associated genes: AKT1, ALK, BRAF, CTNNB1, DDR2, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FGFR3, KRAS, MAP2K1, MET, NOTCH1, NRAS, PIK3CA, PTEN, SMAD4, STK11, and TP53. For library preparation, cfDNA (maximum of 10 ng) was subjected to multiplex PCR amplification using the Ion AmpliSeq Library Kit 2.0 (Thermo Fisher Scientific) according to the manufacturer's protocol. Purified libraries were pooled and then sequenced with an Ion Torrent Proton instrument, the Ion PI Hi‐Q Chef Kit, and the Ion PI Chip Kit v3 (all from Thermo Fisher Scientific). DNA sequencing data were accessed through the Torrent Suite version 5.0 program (Thermo Fisher Scientific). Reads were aligned with the hg19 human reference genome, and potential mutations were called using Variant Call Format version 5.0, as previously described.12 For the detection of copy number gain, the read depth of each target region was divided by the average depth of the normal DNA (Promega, Madison, WI, USA) to adjust for the bias of PCR amplification. The adjusted read depth was log2‐transformed, and the median log2 value per gene was used for the copy number analysis. The log2 ratio cut‐off value for the copy number gain was set at 1.25 based on a previous study.20, 21

Routine molecular screening was performed at diagnosis for all patients on tumor biopsies. DNAs were extracted on a Maxwell 16 Forensic Instrument (Promega, France) using Maxwell 16 FFPE Plus LEV DNA Purification Kit (Promega, France) for FFPE samples. Quantification was done by Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, France). Colon and Lung Cancer Panel V2 libraries were prepared using the Ion Ampliseq library preparation kit v2 from 30 ng of tumor DNA. Libraries were normalized (Ion Library Equalizer Kit), pooled, processed on a Ion Chef System for template preparation and chip loading (Ion PI Hi-Q Chef Kit, Ion PI Chip Kit v3, Thermo Fisher Scientific), and sequenced on a Ion Proton System.