Targeted NGS of cell-free DNA

NGS was carried out on all cfDNA samples. The Oncomine Solid Tumor DNA Kit was used to prepare libraries on 1.1–10 ng of cfDNA following the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). NGS was conducted using the Ion Chef Instrument and the Ion Personal Genome Machine (PGM) System (Thermo Fisher Scientific). Sequencing was performed using Ion 316 v2 BC chips containing eight cfDNA samples per chip. Primary data processing was carried out using the Torrent Suite Software (version 5.0.4) on a Torrent Server and the Ion AmpliSeq Colon and Lung Cancer panel v2 template (all Thermo Fisher Scientific). Variant calling was performed using the Ion Reporter Software (version 5.0) and the AmpliSeq CHPv2 peripheral/CTC/CF DNA single sample workflow (Thermo Fisher Scientific). Default settings were used except from the reference and hotspot BED files, which were supplied in the kit. Sequencing was considered successful if the mean sequencing depth was ⩾2000. If this criterion could not be met, the sample was disqualified. Called variants were only accepted if the allele frequency (AF) was ⩾1%. The Integrative Genomics Viewer (Broad Institute, Cambridge, MA, USA) was used for visualisation of variants (Thorvaldsdóttir et al, 2013 (link)).