Qiamp ultrasens virus kit

Viral DNA was extracted from cell culture supernatant to perform the sequencing by Illumina platform using a QIAmp UltraSens Virus Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. DNA quantification was performed using an Epoch microplate spectrophotometer (BioTek, Winooski, VT, USA) and a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. The whole sequence of 58 Sardinian viruses was obtained through Next Generation Sequencing (NGS). Viral DNA libraries were prepared using the Illumina Nextera XT DNA Sample preparation protocol (Illumina Inc., San Diego, CA, USA) and Nextera DNA Flex Library Prep kit (Illumina). Thirteen samples were sequenced with a HiSeq 2500 Instrument (Illumina) generating paired-end reads 2 × 150; the NGS of 44 samples was obtained using Novaseq 6000 (Illumina) generating paired-end reads 2 × 150 (at CRS4, Pula, Italy; and at AMES Group, Instrumental Polydiagnostic Center Srl, Naples, Italy, respectively). The sequences of the B602L (bases 96,322–97,938) and the EP402R genes (bases 68,928–70,112) were confirmed by Sanger sequencing using the primers and the methods described previously by Sanna et al. [29 (link)].