Metagenomic Library Preparation and Quantification

Metagenomic libraries (mNGS) were prepared and quantified essentially as described [13 (link)]. Briefly, total nucleic acid was concentrated to 10 μl with RNA Clean and Concentrator-5 spin columns (Zymo Research, CA) and RNA was reverse transcribed with random primers using Superscript III (SSRTIII) 1^st Strand reagents (Life Technologies), followed by 2^nd strand synthesis with Sequenase V2.0 T7 DNA pol (Affymetrix). Double stranded DNA/cDNA was recovered with DNA Clean and Concentrator-5 spin columns (Zymo Research) and -barcoded with Nextera XT indices lacking 5’ biotin tags using 24 cycles of amplification (IDT, Coralville IA; Illumina, Carlsbad CA). Nextera libraries were purified with Agencourt AMPpure XP beads (Beckman Coulter) and quantified by a 2200 TapeStation (Agilent) and Qubit fluorometer (Life Technologies).