Ion torrent proton instrument

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

Molecular barcode sequencing was carried out using the Ion Torrent Oncomine cfDNA Lung Assay (Thermo Fisher Scientific), which targets 11 cancer‐associated genes: ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, ROS1, and TP53. Library preparation was carried out using cfDNA (maximum of 10 ng) according to the manufacturer's instructions. 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 5.10 program (Thermo Fisher Scientific). Reads were aligned with the hg19 human reference genome, and potential mutations were called using a plug‐in cfDNA variant caller.

Using tissue DNA and ctDNA, amplicon-targeted sequencing was carried out with the Ion AmpliSeq™ Colon and Lung Cancer Research Panel (ver. 2; Thermo Fisher Scientific K.K., Tokyo, Japan), which targets 22 cancer-associated genes: KRAS, EGFR, BRAF, PIK3CA, AKT1, ERBB2, PTEN, NRAS, STK11, MAP2K1, ALK, DDR2, CTNNB1, MET, TP53, SMAD4, FBXW7, FGFR3, NOTCH1, ERBB4, FGFR1, and FGFR2. For library preparation, tissue DNA or ctDNA (up to 10 ng) was subjected to multiplex PCR amplification using the Ion AmpliSeq Library Kit 2.0 (Life Technologies, Carlsbad, CA) according to the manufacturer’s protocol. Purified libraries were pooled and then sequenced using an Ion Torrent Proton instrument, the Ion PI Hi-Q Chef Kit, and the Ion PI Chip Kit v3 (all from Life Technologies). DNA sequencing data were accessed through the Torrent Suite version 5.10 program (Life Technologies). Reads were aligned with the hg19 human reference genome, and potential mutations were identified using Variant Caller version 5.10, as previously described.¹⁹