## Materials and Methods  
### Patients, Plasma Collection and Panel Sequencing  
Ninety-two plasma samples from 76 advanced NSCLC post-TKI patients were collected and tested for \(EGFR\) Thr790 Met mutation. The present study was approved by the Ethics Committee of Liguria Region (Italy) (P.R.273 REG2016) and conducted according to the principle of the Declaration of Helsinki. For each patient included in the study a written informed consent was obtained. Procedures for molecular tagging-based NGS testing are described in details in reference 16 and here briefly retraced and displayed in Figure 1.  
Plasma samples were isolated from 12-18 mL of peripheral blood collected into EDTA-containing tubes and up to 4 mL processed for cfDNA extraction using the MagMAX(tm) Cell-Free DNA Isolation Kit (ThermoFisher Scientific, Waltham, MA, USA), followed by quantification with a Qubit 3 TM Fluorometer (ThermoFisher Scientific). Genomic profiling of samples by targeted NGS was performed by using the above cited OLcfA, covering DNA target regions containing hotspots variants relevant in pathogenetic and resistance molecular mechanisms in NSCLC (169 key hotspot mutations across 11 genes such as _ALK_, _BRAF_, _EGFR_, _ERBB2_, _KRAS_, _MAP2 KI_, _MET_, _NRAS_, _PIK3 CA_, _ROS1_ and _TP53_). This NGS assay applies UMI to improve the sensitivity by decreasing the amount of sequencing artifacts. With the recommended input of 20 ng cfDNA, the use of UMI enables a limit of detection (LoD) as low as 0.1%. However, a range between 1-50 ng of cfDNA amount can be used with consequent higher LoD reached as indicated by the manufacturer ([[http://tools.thermofisher.com/content/sfs/brochures/ngs-analysis-mutations-cfdna-app-note.pdf](http://tools.thermofisher.com/content/sfs/brochures/ngs-analysis-mutations-cfdna-app-note.pdf)]([http://tools.thermofisher.com/content/sfs/brochures/ngs-analysis-mutations-cfdna-app-note.pdf](http://tools.thermofisher.com/content/sfs/brochures/ngs-analysis-mutations-cfdna-app-note.pdf))). Oncomine library quantities were determined by qPCR using the Ion Library TaqMan Quantitation Kit (ThermoFisher Scientific) and then diluted to a final concentration of 50-60 pmol/L. Next, sample barcoded libraries were pooled together for template preparation on the Ion Chef(tm) Instrument and loaded onto a Ion 520 chip (Ion 520 tm Chip Kit, ThermoFisher Scientific). The chip was sequenced on a Ion GeneStudio(tm) S5 System (ThermoFisher Scientific), and the unaligned BAM (Binary Alignment Map) files generated by the sequencer were mapped against the human reference genome (GRCh37/hg19) using the Torrent Mapping Alignment Program (TMAP), with default parameters and Ion Torrent Suite(tm) Software (TSS, version 5.10). Analysis and annotation of variants were locally carried out with Torrent Variant Caller (TVC, version 5.10) plugin using preconfigured parameter settings for liquid biopsy application. NGS data of library performance and variant calling quality were considered and presented for all plasma samples analyzed. The manufacturer recommends a Median Read Coverage (MedReadCov) \(>\)25,000 and Median Molecular Coverage (MedMolCov) \(>\)2500 to detect a variant with a MAF of 0.1%. The output variant caller format (VCF) files from each sequenced sample were reviewed for further visual analysis on the Integrative Genomics Viewer (IGV, version 2.6, Broad Institute). VCF files were uploaded on IGV and the wild type/mutated molecular alleles aligned to the reference genome hg19 (GRCh37). Reproducibility of the Oncomine Lung cfDNA panel was already evaluated in our previous paper [16]. Specifically, a high sensitivity tool such as the dPCR was tested on 26 cfDNA samples: 10 T790M-negative and 16 T790M-positive, with a 100% of concordance in both samples demonstrated. Figure 1: Flowchart of the complete workflow for circulating tumor DNA (ctDNA) next-generation sequencing (NGS) analysis.