title: Baseline Plasma cell-free DNA and RNA-Seq of Homo sapiens: Adult Female
design: Circulating Nucleic Acids (cfTNA; i.e., DNA and RNA) were isolated from 4 mL of plasma using the MagMAX Cell-Free Total Nucleic Acid Isolation Kit (ThermoFisher Scientific, Waltham, MA, USA), followed by quantification with a Qubit 3TM Fluorometer (ThermoFisher Scientific). Targeted NGS was performed using the Oncomine Lung cfTNA Research Assay able of identifying gene variants (i.e., single nucleotide substitutions and short indels) in ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, ROS1, and TP53; gene fusions in ALK, RET, ROS; MET exon 14 skipping and the copy number of MET. Manual library preparation was performed after the cDNA reverse transcriptase step starting from 20 ng to 50 ng of cfTNA as detailed in manufacturer protocol (Oncomine CellFree Research Assay MAN0017065). Three to six diluted (60pM) libraries were pooled and used for template preparation on Ion Chef System and sequenced on Ion 520 and/or 530 chips (ThermoFisher Scientific). Run sequencing was performed on the Ion Torrent GeneStudioTM S5. To detect a gene variant with a VAF of 0.1%, a Median Read Coverage (MedReadCov) and Median Molecular Coverage (MedMolCov) starting from 25,000 and 2,500 were needed, respectively. The output BAM and VCF files were initially analyzed on Ion Torrent Suite v.5.16.1 and then annotated Ion Reporter v.5.18 (ThermoFisher Scientific).