Next‐generation sequencing (NGS) of plasma was used to identify the mutations suitable for repeated measurements by droplet digital PCR (ddPCR) in the patients without available tumour tissue.
Sequencing libraries were prepared from cfDNA (2.8–9.7 ng) using the Oncomine™ Solid Tumour DNA kit (OST; Thermo Fisher Scientific, Watham, MA, USA) as described previously (Winther‐Larsen et al.,2017 ). In short, the Ion Chef™ Instrument (Thermo Fisher Scientific) was used for sample preparation, and the sequencing was conducted using the Ion Personal Genome Machine® (PGM™; Thermo Fisher Scientific, Watham, MA, USA) System. The Ion 316™ v2 BC chips were loaded with four samples. Variant calling was performed using the ion reporter Software (version 5.4; Thermo Fisher Scientific, Watham, MA, USA) and the AmpliSeq CHPv2 peripheral/CTC/CF DNA single sample workflow (Thermo Fisher Scientific). The samples were included whether the mean depth reached ≥ 2000. The variants were called if they were reported to COSMIC and the allele frequency ≥ 1%. The Integrative Genomics Viewer v.2.3.77 (Broad Institute, Cambridge, MA, USA) was used for the manual visualisation of variants (Robinson et al., 2011 ).
Sequencing libraries were prepared from cfDNA (2.8–9.7 ng) using the Oncomine™ Solid Tumour DNA kit (OST; Thermo Fisher Scientific, Watham, MA, USA) as described previously (Winther‐Larsen et al.,