Metagenomic Next-Generation Sequencing Protocol

Metagenomic next-generation sequencing (mNGS) was performed as previously described [17 ,56 (link)]. Briefly, 18 μL of extracted RNA was treated with Turbo DNAse (ThermoFisher, Waltham, MA). First strand cDNA synthesis was completed using SuperScript IV (ThermoFisher) and random hexamers (Invitrogen, Carlsbad, CA) followed by second strand synthesis by Sequenase version 2.0 (ThermoFisher). The resulting cDNA was purified using either the DNA Clean & Concentrator kit (Zymo, Irvine, California) or 1.6× volumes of AMPure XP beads (Beckman Coulter, Brea, CA). Library preparation was performed using the Nextera XT Kit (Illumina, San Diego, CA). Libraries were cleaned with 0.7× or 0.75× volumes of Ampure beads (Beckman Coulter), quantified using either the Qubit dsDNA HS assay (ThermoFisher) or Quant-iT dsDNA HS assay (ThermoFisher), quality checked by Bioanalyzer or TapeStation (Agilent, Santa Clara, CA), pooled, and sequenced on 1 × 75 bp runs on an Illumina NextSeq or 1 × 101 bp runs on an Illumina NovaSeq.