*2.2. Targeted Deep Sequencing and Data Analysis*

A panel covering the exons of 53 lung cancer-related genes (see Supplementary Table S1) was designed in-house to perform targeted sequencing. These genes were selected after a literature search based on the following criteria: (a) genes involved in lung cancer according to The Cancer Genome Atlas [4,5] and other, similar projects [6–10] or (b) genes frequently mutated in lung cancer according to the Catalogue Of Somatic Mutations In Cancer (COSMIC) database [11]. Ion AmpliSeq designer software (Thermo Fisher Scientific) was utilized for the primer composition, as previously reported [1,12,13]. An Ion AmpliSeq Library kit (Thermo Fisher Scientific) was utilized for the preparation of sequencing libraries. The library samples were bar-coded with an Ion Xpress Barcode Adapters kit (Thermo Fisher Scientific), purified using Agencourt AMPure XP reagent (Beckman Coulter, Tokyo, Japan) and subsequently quantified using an Ion Library Quantitation Kit (Thermo Fisher Scientific). The libraries were templated with an Ion PI Template OT2 200 Kit v3 (Thermo

Fisher Scientific). Sequencing was performed on Ion Proton (Ion Torrent) with an Ion PI Sequencing 200 Kit v3.

The sequence data were processed on standard Ion Torrent Suite Software. Raw signal data were measured using the Torrent Suite version 4.0. The pipeline consisted of signaling processing, base calling, quality score assignment, read alignment to the human genome 19 reference (hg19), mapping quality control and coverage analysis. After the data analysis, the annotation of single-nucleotide variants and indels (insertions and deletions) was performed on the Ion Reporter Server System (Thermo Fisher Scientific). Blood cell DNA extracted from the peripheral blood was used as a normal control to detect variants (Tumor-Normal pair analysis). Sequencing data were visually analyzed using an Integrative Genomics Viewer.
