Qubit 2.0 fluorometer dna high sensitivity assay kit

Metagenomic shotgun libraries were prepared using New England BioLabs Ultra FS II library kit (New England BioLabs, Ipswich, MA) and cleaned with a 1:1 ratio of Sera-Mag speedbeads after ligation (Rohland and Reich, 2012) . Following ligation, samples were tagged with iTru primers (Glenn et al., 2019) and quantified with a Qubit 2.0 Fluorometer DNA high sensitivity assay kit (Thermofisher, Waltham, MA). Samples were then cleaned with a 1:1 ratio of speedbeads and pooled into groups of 8-12 for enrichment or set aside for sequencing. A positive control of 2.5 ng/µL of E. coli and a negative control of molecular grade water were included.

Pooled libraries were then used to perform hybridization capture with biotinylated baits using a custom Arbor Biosciences myBaits kit (Arbor Biosciences, Ann Arbor, MI). The kit was used following manufacturer's protocol (v3-5.01) with 16-18-hour hybridization at 65°C for all samples. Following hybridization, Dynabeads M-280 Streptavidin magnetic beads (Life Technologies, Carlsbad, CA) were used for enrichment. A post-enrichment amplification was performed using Illumina P5/P7 primers and KAPA HiFi HotStart reagents. The cycling conditions were as follows: 98°C for 45s, followed by 16-28
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The copyright holder for this preprint (which this version posted July 21, 2021. ; https://doi.org/10.1101/2021.07.20.452950 doi: bioRxiv preprint cycles of 98°C for 20s, 60°C for 30s, and 72°C for 60s, and then a final extension of 72°C for five minutes. P5/P7 PCR consisted of 25 cycles for the resistance mock resistance community, 25 cycles for the built environmental samples, 18 for the poultry litter samples, and 18 cycles for the WWTP samples. PCR product was cleaned with Sera-Mag beads, quantified on a Qubit 2.0 fluorometer DNA high sensitivity assay kit (Thermofisher, Waltham, MA) and pooled in equimolar ratio for sequencing.