## 3 Results
### Thr790 Met Detection and Torrent Variant Caller (TVC) Metrics Evaluation  
The _EGFR_ Thr790 Met variant was detected in 27/61 (44.3%) driver _EGFR_ mutated plasma samples and co-occurred mostly with exon 19 deletions in 17/27 (63%) and Leu858 Arg in 8/27 (29.6%) cases (Table S1). MAFs of Thr790 Met detected in mutated samples ranged between 0.06-17.67% (median 0.60%) and almost always were lower than those detected for driver mutation/s in the same patients (Table S1). No statistical significance was found between the starting NGS input of cfDNA and MAFs found for Thr790 Met (r\({}_{\text{s}}=0.33\); \(p=0.09\)) (Figure 6a), whereas a moderate correlation was reported when cfDNA amounts were related with allele molecular coverage of tagged DNA molecules containing the variant (r\({}_{\text{s}}=0.59\); \(p=0.001\)) (Figure 6b). Finally, the LoDs reached by Thr790 Met variant through the 27 samples (range 0.05-0.15%, median 0.10%) were correlated with the initial cfDNA amount and, as expected, a moderate negative association was found (r\({}_{\text{s}}=-0.66\); \(p=0.0002\)) (Figure 6c). It is intriguing that in general, the Oncomine NGS panel outperformed, reaching lower LoDs than expected. Figure 5: Boxplots representing the _EGFR_ mutational status behavior related to cfDNA input and abundance. Unmutated/mutated plasma samples were correlated with: (**a**) the cfDNA amount used for library preparation; (**b**) cfDNA abundance in plasma (cfDNA ng/plasma mL). Manual Review of Variant Caller Format (VCF) on Integrative Genomics Viewer (IGV) Evidenced Accuracy and TVC Pliggin Gaps  
In order to determine if variants found, including single nucleotide variants (SNVs), small insertions and deletions (InDels), were real events or artifacts as well as to exclude the presence of false negatives or misclassification of variant/s, VCF files were manually reviewed by using IGV software. In our experience, this visual inspection increased the confidence in SNV calls, and in all small canonical InDels in exons 19 and 20 of _EGFR_ gene reported in TSS by TVC. Again, importantly, no variant misclassification was pointed out. However, further investigation of VCFs allowed us to highlight some critical points of the bioinformatics pipeline predefined by ThermoFisher. In particular, it was noted that the TVC plugin failed to call the _EGFR_ complex mutations Lys745_Glu746 insIleProValAlaIleLys (exon 19) and Ala763_Tyr764 insPheGlnGluAla (exon 20) (Figure 7a), occurring in pt.25 and pt.35, respectively, and previously validated on tissue samples by Sanger sequencing (data not shown). In both cases, the variant falls outside the DNA target regions covered by the Browser Extensible Data (BED) file of the panel, but, only in the second case we were able to obtain the correct variant call, redoing the analysis after an adjustment in analysis configuration of TVC parameters JSON file (version 5.2), specifically within the TMAP string (Figure 7b). This update was made available to all users by manufacturer in the following versions of the TVC plugin. Figure 6: CfDNA input and TVC parameters from Thr790 Met positive samples. (**a**) No significant correlation between cfDNA and MAFs was reported (\(r_{\mathrm{s}}=0.33;p=0.09\)). Moderate correlations were found between the cfDNA input and: (**b**) the number of mutated DNA molecules (Allele Mol Cov) (\(r_{\mathrm{s}}=0.59;p=0.001\)); (**c**) the LoD of Thr790 Met variant (\(r_{\mathrm{s}}=-0.66;p=0.0002\)). The arrow indicates a peculiar case of a 0.05% LoD reached with only 12 ng of cfDNA input. Another case of false negative occurred in pt.60, who harbored a complex mutation in _EGFR_ exon 19, i.e., Arg748_Ala755 delinsThr. Even in this particular case, the variant was discovered in tissue by Sanger sequencing but found in the plasma only through the IGV visualization. However, despite fitting the amplicon, the variant was not called and thus a different modification of the TVC parameters, was required.