## 3 Results  
We initially evaluated the OLcfA performance using the Multiplex I cfDNA Reference Standard set-Horizon Discovery 780 (HD780). This commercial kit contains low allele frequencies variants (5%, 1% and 0.1%) at 8 hotspot genomic positions in genes _EGFR_, _KRAS_, _NRAS_ and _PIK3 CA_, covered by the Oncoming panel. Notably, 4 key alterations were investigated for _EGFR_ gene (Leu858 Arg, Thr790 Met, Glu746_Ala750 del, Val769_Asp770 insAlaSerVal), 1 for _KRAS_ (Gly12 Asp), 2 for _NRAS_ (Ala59 Thr, Gln61 Lys) and 1 for _PIK3 CA_ (Glu545 Lys) and results shown in Table 1.  
Each reference standard was tested by using 30 ng of cfDNA, as described by the manufacturer (ThermoFisher Scientific, [[https://assets.thermofisher.com/TFS-Assets/LSG/brochures/verification-oncoming-lung-cfdna-ion-s5-white-paper.pdf](https://assets.thermofisher.com/TFS-Assets/LSG/brochures/verification-oncoming-lung-cfdna-ion-s5-white-paper.pdf)]([https://assets.thermofisher.com/TFS-Assets/LSG/brochures/verification-oncoming-lung-cfdna-ion-s5-white-paper.pdf](https://assets.thermofisher.com/TFS-Assets/LSG/brochures/verification-oncoming-lung-cfdna-ion-s5-white-paper.pdf))). Additionally, we checked reproducibility in critical samples (i.e., those with cfDNA input <30 ng) and tested the HD780 reference standard at 0.1% MAF by using 20 ng of cfDNA in a duplicate experiment. Detailed data on MAFs are reported in Table 1. The overall variant-based sensitivity was 92.5%. Specifically, only in one case did the system fail to call a low frequency variant at 0.1% and it was relative to the hotspot Glu545 Lys in _PIK3 CA_ gene. In addition, two hotspots, i.e., the deletion Glu746_Ala750 del and the missense mutation Leu858 Arg were filtered out by the bioinformatics pipeline in the 20 ng cfDNA replicates at 0.1% MAF, because not enough molecular families had been reached to enable a call.  
Furthermore, reference cfDNA HD780 at 0% MAF, i.e., the standard characterized by the absence of low allele frequencies variants, was examined and no false positives were found at the genomic target regions analyzed, not even visualized on IGV. This indicates a 100% specificity of OLcfA and confirms its reliability to be used in a clinical setting.  
\begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \multicolumn{2}{c}{**Reference Standard**} & \multicolumn{4}{c}{_EGFR_} & \multicolumn{2}{c}{_KKAS_} & \multicolumn{2}{c}{_NRAS_} & \multicolumn{2}{c}{_PR3 CA_} \\ \hline Frequency & Input (pg) & Ch/\%4\_6\_, \(\Delta\)L/\%30 del & Leu858 Arg & Th/\%0 Met & Val/\%9\_Asp70 msAlServerVal & Gly12 Asp & Al49 Thr & Chto11 Lys & Chto54 Lys \\ \hline
0.1\% & 30 & 0.14\% & 0.09\% & 0.11\% & 0.10\% & 0.32\% & 0.07\% & 0.22\% & 0.08\% \\ \hline
0.1\% & 20 & 0.07\% & **0.06\% \({}^{\bullet}\)** & 0.07\% & 0.16\% & 0.18\% & 0.21\% & 0.31\% & **0.00\%, \({}^{\bf b}\)** \\ \cline{2-9}  & **0.03\%. \({}^{\bullet}\)** & 0.13\% & 0.19\% & 0.10\% & 0.21\% & 0.03\% & 0.10\% & 0.19\% \\ \hline
1\% & 30 & 0.91\% & 1.15\% & 0.91\% & 0.88\% & 1.16\% & 0.96\% & 0.98\% & 1.22\% \\ \hline
5\% & 30 & 5.81\% & 5.02\% & 5.46\% & 4.17\% & 7.36\% & 5.77\% & 6.80\% & 7.17\% \\ \hline \end{tabular}  
* variants filtered out by the bioinformatic pipeline in _EGFR_ gene. \({}^{\bf b}\) a failed call in _PKSCA_ gene.  
\end{table}
Table 1: Frequency variants detected in Multiplex cfDNA Reference Standard for each of the 8 hotspot variants occurring in 4 genes (_EGFR_, _KRAS_, _NRAS_ and _PIK3 CA_). In bold, variants filtered out by the bioinformatic pipeline in _EGFR_ gene and a failed call in _PIK3 CA_ gene.