## 4 Discussion  
Liquid biopsy has emerged as a promising solution to overcome current limitations associated with tissue biopsies in precision medicine laboratories [18]. CfDNA represents the most stable genetic material and accessible source in the liquid biopsy field and its assessment allows the detection and monitoring of cancer-specific genomic alterations in blood [19]. Even if ctDNA analysis is not a replacement for histologic confirmation, it results in being clinically useful in the diagnostic approach. This molecular testing, due to its high specificity, non-invasive nature and fast turnaround time, can represent a valid tool at diagnosis or progression, even preceding tissue genotyping and can be used to guide treatment [20; 21]. Figure 7: Integrative Genomics Viewer (IGV) screenshots of Ala763_Tyr764 insPheGlnGluAla variant in _EGFR_ gene. (**a**) The figure shows the complex InDel in exon 20 of the _EGFR_ gene found at first by visualization with the IGV software. This is a rare insertion of 12 bp (purple braces) mapped outside the of the exon 20 not initially covered by the designed amplicon and consequently not called by the Torrent Variant Caller (TVC, red arrow). (**b**) The panel below depicts the results of the analysis after some bioinformatics changes to TVC parameters. As it can be noted, the blue bar of the amplicon was elongated to also cover the genomic position at the end of the intron where the mutation occurs (green arrow). Consequently, the hotspot Browser Extensible Data (BED) file was modified (green box) and the specific insertion was correctly reported in the TVC section of the TSS (screens on the right). Nevertheless, the analysis of ctDNA is challenging due to the mainly low representation of mutated tumor-derived DNA molecules in the plasma. Furthermore, although plasma of cancer patients contains more cfDNA compared to healthy individuals [17; 22], the proportion of ctDNA originating from the tumor is highly variable, ranging from \(<\)0.1% to \(>\)50% of total cfDNA [23; 24]. Therefore, methods which are able to detect small number of mutated molecules in an abundance of unmutated DNA fragments with high sensitivity and specificity are required. NGS methods have been revealing a promising approach to detect more sensitive and specific biomarkers for clinical utility. Considering NSCLC management, targeted therapies, like TKIs, require a sensitive and precise mutation profiling of the tumor to ensure identification of every patient who could potentially benefit from therapy. In particular, acquired resistance mutations of the \(EGFR\) gene, for example Thr790 Met occurring in up to 60% of NSCLC patients, represent a clinical challenge in the treatment [25; 26; 27; 28]. In this context the analysis of ctDNA has become a hopeful tool to verify the Thr790 Met presence in NSCLC patients, which developed a resistance to TKI therapy [29; 30]. Therefore, the most relevant requirements for an NGS panel to be used for liquid biopsy purposes are high levels of sensitivity and specificity to ensure true variant calling at very low frequencies. Technically, to overcome the drawbacks of PCR-based NGS (e.g., DNA polymerase errors, etc. ), the addition of UML, i.e., random nucleotide sequences barcoding each DNA molecule prior to PCR amplification, was introduced [5; 6; 7; 8; 9; 10]. This artifice allows us to distinguish reads amplified from the same original DNA molecule and to identify molecules containing true variants from false positives. In this study, we tested the feasibility of the OLcfA NGS panel UMI-based on a cohort of NSCLC patients (n = 76) and, mostly, we focused on pre- and analytical details from 92 plasma samples. We validated the assay on both wet bench and bioinformatics processes across a broad spectrum of validation parameters including sequencing performance, analytical sensitivity and specificity and LoD reached.