Torrent suite v 5

Amplicon libraries were prepared using the Oncomine Lung Cell‐Free Total Nucleic Acid assay (Thermo Fisher Scientific) from 20 to 50 ng of total DNA/RNA isolated from BW samples. To identify the sample, each library was barcoded with a unique oligonucleotide identifier, according to the manufacturer's instructions. Libraries were pooled together in groups of 24/chip (Ion 540) and sequenced on the Ion S5 System (Thermo Fisher Scientific), achieving an average sequencing depth of 7000× (molecular coverage) and average reads number of 3 500 000 for sample. Sequencing raw‐data analysis was performed using torrent suite v. 5.10.1 and ion reporter 5.10.5 (Thermo Fisher Scientific). Briefly, low‐quality reads were removed, adapter sequences trimmed and samples sequence aligned against a reference genome (hg19) using the Torrent Mapping Alignment Program (Thermo Fisher, Carlsbad, CA, USA). Subsequently, the aligned BAM files were uploaded to Ion Reporter and processed using the ad hoc Oncomine TagSeq Lung v2 Liquid Biopsy w2.1 (Thermo Fisher)—Single Sample workflow. Each sample was analysed for mutations in the ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, ROS1, and TP53 genes and for ALK, ROS1, and RET gene fusions. Next‐generation sequencing (NGS) raw data are available in the European Nucleotide Archive (https://www.ebi.ac.uk/ena) under the accession number PRJEB38273.

The Variant Caller (VC) analysis for each samples was carried out using the Ion and Illumina informatics solution integrated by each specific NGS platform.
For Ion Torrent platforms, initial variant calling from the Ion AmpliSeq™ sequencing data was generated using Torrent Suite v.5.10.1 (ThermoFisher Scientific) with a plug-in VC program (VC v.5.10.1.20) with Generic - PGM (3xx) - Somatic - Low Stringency parameters. Moreover, Ion Reporter™ Software were used for variant annotation.
Illumina data was analyzed using BaseSpace (Illumina) to convert *.bcl files into FASTQ files, which contain base call and quality information for all reads passing filtering. DNA Amplicon App v.2.1.0 was used for alignment in the targeted regions (specified in a manifest file), or the Burrows Wheeler Aligner across the entire genome. We selected the option “Somatic Variant Caller” with a Variant Allele Frequency (VAF) threshold of 0.01 (Percentage) and a depth threshold of 10. The tertiary analysis was carried out using BaseSpace Variant Interpreter.
All identified variants were confirmed by the Integrative Genomics viewer (IGV) by visually examining mutations using Integrative Genomics Viewer software (http://www.broadinstitute.org/igv) [31 (link)].