Qiasymphony instrument

For molecular characterization, DNA was isolated from 1 × 10⁶ tumor cell pellet in the sixth passage using silica column from Biopur Mini Spin Plus 250 Extraction kit (Biopur), as described by the manufacturer. Blood total DNA was automatically isolated from buffy coat of patient through Mini Kit DNA and Qiasymphony instrument (both from Qiagen) at BCH Biobank Department, according to the manufacturer. This technology combines silica-based extraction with magnetic particles purification. The protocol Blood 200 with an elution volume of 50 µL was used. Tumor DNA was isolated from a biopsy sample obtained from the patient through the Mini Kit DNA and Qiasymphony instrument (QIAgen). For tumor-isolated DNA, macrodissection was performed by an experienced pathologist and up to 25 mg of the sample containing at least 60% tumor area and up to 20% necrosis area were fragmented and submitted to digestion and homogenization procedures, according to the manufacturer’s instructions. The protocol Tissue 200 with an elution volume of 50 µL was used.
All DNA samples were quantified by both NanoDrop® 2000 (Thermo Scientific) and Qubit® 2.0 Fluorometer (Life Technologies) and were then stored at −20 °C for further genetic analysis. Blood DNA was used as reference to distinguish somatic from germline mutations.

To evaluate potential impacts of cfDNA extraction procedures on total extraction yield and the detection of mutant alleles, plasma samples of a KRAS c.38G > A (10% VAF) positive CRC patient were processed using different manual protocols from various vendors: A Jena PME free-circulating DNA extraction kit (spin-based, carrier RNA: optional) (Analytik Jena, Jena, Germany), QIAamp Circulating NA Kit (vacuum-based, carrier RNA: yes) (Qiagen, Hilden, Germany), and Zymo Quick cfDNA serum and plasma kit (spin-based, carrier RNA: no) (Zymo Research, Irvine, CA, United States). In addition, cfDNA of matched plasma samples from CRC patients (n = 15) was extracted in parallel using the manual protocol by Zymo Research and an automated procedure on a QIAsymphony instrument (Qiagen) using the PAXcircDNA_STA_2400 protocol (Qiagen). All extractions were performed using 1–3 ml of plasma according to the manufacturer’s protocols. For all kits, cfDNA was eluted into 60 μL ddH2O (or TE buffer), quantified by a β-globin–specific qPCR in comparison to a serial dilution of a reference DNA with a known quantity on a 7,500 Real-Time PCR System (Applied Biosystems, Foster City, CA, United States), and stored at −20°C until downstream processing for NGS. All cfDNA concentrations are presented in ng mL⁻¹ plasma to adjust for different volumes of starting material.

In the accredited routine diagnostics laboratory of the Max von Pettenkofer Institute, the following RT-qPCR assays were used and quantified as described previously [19 (link)] using the respective formulas: the nucleocapsid (N1) reaction (Center for Disease Control (CDC) protocol [31 ] ( $x={\mathrm{e}}^{\frac{(y-48.597)}{-1.461}}$ ) on a LightCycler 480 system, the Roche Cobas SARS-CoV-2 E-Gen reaction on a Cobas 6800 system ( $x={\mathrm{e}}^{\frac{(y-44.576)}{-1.401}}$ ) or the Xpert Xpress SARS-CoV-2 ( $x={\mathrm{e}}^{\frac{(y-50.859)}{-1.887}}$ ), Xpert Xpress SARS-CoV-2/Flu/RSV ( $x={\mathrm{e}}^{\frac{(y-45.904)}{-1.5}}$ ) and Xpert Xpress CoV-2/Flu/RSVplus ( $x={\mathrm{e}}^{\frac{(y-46.747)}{-1.501}}$ ) run on a GeneXpert System, with x equals Geq per ml and y equals the Ct/Cq value. For nucleic acid extraction, the QIAsymphony DSP Virus/Pathogen Kit was used with the QIAsymphony instrument from QIAGEN GmbH. In general, the calculations for quantification do not take into account variability between separate RT-qPCR runs. However, since this variability applies to all study groups, they do not affect the interpretation of the results in this study.