DNA extraction

Genomic DNA was isolated from 5 g of feces using the Qiagen PowerMax Soil Kit (Qiagen Laboratories) according to manufacturer’s instructions, with two exceptions to increase yield. A large sample mass (i.e., 2 × 5 g) was used for DNA extraction with the intent of better potential representation of the resistome and microbiome communities in the fecal samples. First, samples were centrifuged for 5 min in the PowerMax bead tubes, as opposed to the recommended 3 min. Second, samples were eluted using 3 ml as opposed to the recommended 5 ml and were passed through the silica DNA filter twice. To increase DNA concentration, isolated DNA was precipitated with ethanol and 0.3 M sodium acetate, washed with 70% ethanol, and resuspended in 150 μl of PowerMax elution buffer. DNA was then quantified in duplicate using the Qubit 2.0 Fluorometer and dsDNA High Sensitivity Buffer and Reagent kit (Thermo Fischer Scientific) and a final concentration was calculated by averaging the two measurements. DNA was also assayed for quality (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀) using a NanoDrop 1000 Spectrophotometer (Thermo Fischer Scientific). If a sample failed to reach a target yield of 9 μg purified DNA per sample, it was extracted a second time and combined with the first. This large target for extracted DNA was necessary to allow multiple attempts for target-enriched library preparation using the manufacturer’s large input protocol (3 μg per attempt; Agilent, 2021). If there was not 5 g of sample remaining for the second extraction, sterile PBS was used to recover remaining feces off the transport tubes that samples were stored in. The volume of PBS used was dependent upon the weight of the remaining sample; more PBS was used for samples with less weight, in order to reach a total of 5 g (w/v) extraction volume.