Superscript 3 ssrtiii 1st strand reagents

mNGS libraries were prepared as described [30 (link)]. Briefly, total nucleic acid from PBV+ sputum was converted to cDNA with random primers and Superscript III (SSRTIII) 1st Strand reagents (Life Technologies, Carlsbad, CA, USA), followed by 2nd strand synthesis with Sequenase V2.0 T7 DNA pol (Affymetrix, Santa Clara, CA, USA). Nextera XT was used to create barcoded, metagenomic NGS libraries which were multiplexed (n = 8) and sequenced on 3 MiSeq runs, with those requiring additional throughput re-sequenced on a HiSeq instrument.

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).