Metagenomic Sequencing of Viral Nucleic Acids

Metagenomic next-generation sequencing was performed as previously described [6 (link),18 (link),25 (link),26 (link)]. In brief, samples were centrifuged at 10,000× g (4 °C) for 10 min to remove debris, then supernatants were transferred to a new tube and centrifuged further at 25,000× g (4 °C) for 3 h to pellet viruses. Nucleic acids were extracted from the resulting viral pellets using the QIAamp MinElute Virus Spin Kit (Qiagan, Germantown, MD, USA), and viral RNA was converted to double-stranded complementary DNA (cDNA) using the SuperScript IV First-Strand Synthesis System (Invitrogen, Waltham, MA, USA) and NEBNext^® Ultra™ II Non-Directional RNA Second Strand Synthesis (New England BioLabs, Ipswich, MA, USA). Samples then underwent purification using AmpureXP beads (Beckman Coulter, Indianapolis, IN, USA), and libraries were prepared using the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA) for sequencing on a MiSeq instrument using the MiSeq Reagent Kit, V3 chemistry, for 600 cycles (Illumina, USA).