Typer software version 4

PCR was performed using 10 ng cfDNA per plex according to manufacturer's instructions (Agena Bioscience, San Diego, CA). Reactions were incubated initially at 94^°C for 4 min. Forty-five cycles of PCR were performed at 94^°C for 30 s, 56^°C for 30 s, and 72^°C for 1min. The PCR was completed with a final incubation of 5 minutes at 72^°C. Thermocyling and incubation were performed in a GeneAmp PCR System 9700 (Thermo Fisher Scientific). Amplified products (5mL) were treated with shrimp alkaline phosphatase for 40 minutes at 37^°C, followed by denaturation for 10 minutes at 85^°C. Single-base extension was performed at 94^°C for 30 s, followed by 40 cycles at 94^°C for 5 s with five nested cycles of 52^°C for 5 s, then 80^°C for 5 s and incubation at 72^°C for 3 min. Streptavidin-coated magnetic beads were used to capture the amplicon. Beads with captured products were pelleted using a magnet and, suspended with 13mL of elution solution, and incubated at 95^°C for 5 minutes. Eluted products were conditioned with 5 mL (3 mg) of anion exchange resin slurry. Finally, the analyte was dispensed onto a Spectro CHIPArray solid support using a MassARRAY RS1000 Nano-dispenser. Data were acquired via matrix-assisted laser desorption/ionization time-of-flightmass spectrometry using the MassARRAY Analyzer. Data analysis were performed using Typer software version 4.0.26.74 (Agena Bioscience)

Data were analysed with the Typer software version 4.0.26.74 (Agena Bioscience, San Diego, CA, USA). The signal intensity for the mutant allele was normalized to the capture control peaks. An intensity value = 1 indicates that the peak intensity in the mutant allele is equal to the peak intensity of the average of the five capture control peaks. The capture control peaks are biotin-labelled, non-reactive oligonucleotides that are added to the extension reaction and used as an internal control for the streptavidin-bead capture and elution of the mutant extension product steps. Mutant allele calls were returned by an automated software report specific for the UltraSEEK™ Lung Panel. A signal-to-noise ratio ≥6 and a z-score ≥7 were considered significant. For allele calling, the reporter algorithm considered the instrument-specific baseline for each mutation assay. The assay-specific noise was assessed by analysing a cohort of wild-type samples and the mutant call significance was controlled by analysing commercial mutation controls as titration of the mutant allele frequencies down to the limit of detection of 0.1%.