5prime hotmastermix

PCR was performed on each fraction with detectable DNA. Each reaction tube contained 12 μl 5 Prime HotMaster mix (QuantaBio, Beverly, MA, USA), 1 μl (10 pg) barcoded 515F-Y forward primer (5′-GTGYCAGCMGCCGCGGTAA), 1 μl (10 pg) indexed 926R reverse primer (5′‐CCGYCAATTYMTTTRAGTTT), 1 ng DNA, and 10 μl molecular-grade water.
Thermocycling conditions were as follows: initial denaturation at 95°C for 3 min, 30 cycles of denaturation at 95°C for 45 s, annealing at 50°C for 45 s, and elongation at 68°C for 90 s, followed by a final elongation step at 68°C for 5 min.
PCR products from each fraction were cleaned using 1× Agencourt AMPure XP beads (Beckman Coulter), quantified with PicoGreen, and diluted to 1 ng/μl. A pool of 1 ng of each uniquely barcoded product was cleaned and concentrated again with 0.8× Agencourt AMPure XP beads.
The pooled amplicons were sequenced on an Illumina MiSeq instrument (University of California at Davis, USA) for 600 cycles. Each pool was also spiked with 1 ng of an even and a staggered mock community in order to assess the sequencing run quality (74 (link)). All expected OTUs were found in the observed mock communities and accounted in total for 99.5% of the reads, indicating that there was no unexpected bias in the sequencing run (75 (link)). Sequencing yield information can be found in Data set S1.

Total genomic DNA was extracted from fecal pellets from a subset of the mice in cohort 1 (chow = 13 mice from four separate cages over two experiments, WD = 13 mice from four separate cages over two experiments, LF/LF = 5 mice from two cages over two experiments) using the DNeasy PowerSoil Kit (Qiagen, Germantown, MD). Modifications to the standard protocol included a 10-min incubation at 65 °C immediately following the addition of the lysis buffer and the use of a bead mill homogenizer at 4.5 m/s for 1 min. The V4 variable region of the 16S rDNA gene was targeted for sequencing (515F: GTGCCAGCMGCCGCGGTAA, 806R: GGACTACHVGGGTWTCTAAT). The target DNA was amplified using 5Prime HotMaster Mix (Quantabio, Beverly, MA). Construction of primers and amplification procedures follow the Earth Microbiome Project guidelines (www.earthmicrobiome.org)^{66 (link)}. Amplified DNA was quantified in a PicoGreen (ThermoFisher Scientific) assay and equal quantities of DNA from each sample was pooled. The pooled DNA was sequenced using a V2 2 × 250 kit on the Illumina MiSeq platform (San Diego, CA) at the University of Colorado Anschutz Medical Campus Genomics and Microarray Core facility.

Total DNA was extracted using RapidWater DNA extraction kit (MoBio, Carlsbad, CA, United States) with minor modifications: at step 5 of the protocol, the PowerWater Beat Tube was heated at 65°C for 10 min and mechanical cell lysis was extended to 10 min for all samples. All other steps were performed following the manufacturer’s instructions. The V4 hypervariable region of the 16S rRNA gene was amplified by PCR, using the primer pairs 515F/806R (Caporaso et al., 2012 (link)), with the 5PRIME HotMasterMix (Quanta BIO, Beverly, MA, United States). The length of the amplicons was 253 bp. Negative controls were included during sampling and main wet-lab steps, including PCR blanks. Amplicons concentration, size range, and purity were measured using Agilent high sensitivity (HS) DNA kit on the Bioanalyzer 2,100 (Agilent Technologies Italia S.p.A, Milano, Italy). Based on the molarity estimated using Bioanalyzer, each PCR product was diluted before pooling. The final pool was purified using the Agen-court AMPure XP DNA purification kit (Beckman Coulter, Brea, CA, USA), following the manufacturer’s instructions. Amplicons were sequenced on an Illumina MiSeq platform with 2 × 300 bp paired-end protocol. The data are openly available in NCBI Sequence Read Archive with the accession number PRJNA837552.

We performed a nested PCR to partially amplify the genes of interest for detection of putative mutations associated to macrolide (region V of 23S rRNA gene) and fluoroquinolone (parC and gyrA genes) resistance. Mutations investigated are reported in Table S1. The primer sequences (Pitt et al., 2018 (link)) used for nested PCR are summarized in Table S2. PCR reactions were performed using 5Prime Hot Master Mix (Quantabio, Beverly, MA, USA) according to manufacturer’s instructions and amplifications were carried out using AB™ Applied Biosystems GENEAMP 9700 PCR System, as shown in Table S3 (Pitt et al., 2018 (link)). Obtained amplicons were verified by electrophoresis on 2% agarose gel followed by GelRed-staining. Amplicons were visualized by VWR™ GenoSmart 2 and sequenced by Sanger method (BMR Genomics, Padua, Italy). The resulting electropherograms were visualized and analyzed using Sequence Scanner Software v2.0 software. All samples were analyzed by Sanger sequencing.

Stool samples were stored at −80°C until DNA extraction. Genomic DNA was extracted using the MoBio PowerSoil DNA isolation kit (Qiagen, Venlo, Limburg, NL). 16S rRNA genes were amplified via PCR in triplicate using barcoded PCR primers with 5PRIME HotMasterMix (Quantabio, Beverly, MA, USA) for each sample according to the Earth Microbiome Project (EMP) protocol (57 (link)). DNA extracted in CO was sent to AZ to be sequenced together with the AZ samples. Additionally, 9 samples from CO were sent to AZ for extraction as a control by employing the same isolation kit and protocols as those used in CO. Next-generation sequencing of the V4 region of the 16S rRNA gene region was performed on extracted DNA on the Illumina MiSeq platform (two MiSeq runs).

We extracted bacterial DNA from swabs using DNeasy PowerSoil DNA isolation kits (Qiagen), consistent with previous work in this field^{22 (link)–24 (link)}. Extractions were carried out in 14 batches of 24 (23 uropygial gland swabs plus one swab-only negative control per batch, including swabs used as part of separate studies; details in^{22 (link),24 (link)}). We amplified the V4 region of the bacterial 16S rRNA gene using the universal primers F518^{77 (link)} and R806^{78 (link)}. Each primer included an Illumina MiSeq adaptor sequence, four randomized nucleotides, and a unique ‘barcode’ of eight nucleotides. We performed PCR in a total volume of 25 µL, including 10 µL of 5PRIME HotMasterMix (Quantabio), 0.2 µM of each primer, and 2 µL of DNA template ( $\overline{\text{x}}$ concentration = 0.1 ng/µL, range = 0.01–0.12 ng/µL, measured using a Qubit fluorometer). The thermocycling profile was: 2 min at 94 °C; 35 cycles of 45 s at 94 °C, 60 s at 50 °C, and 90 s at 72 °C; and a 10 min final extension at 72 °C.

Bacterial colonies were subcultured on the same media as used for primary growth. Crude extract of bacterial DNA was prepared as previously described (Scholz et al., 2014 (link)). 16S rRNA gene amplification was performed using the B4 and B5 primers that amplify the V1–V3 region (Mikkelsen et al., 2000 (link)). A PCR reaction mixture of 25 μl containing 8 μl sterile PCR grade water, 2 μl primer mix (5 μmol each of B4 and B5 primers), 5 μl of the 1:100 diluted DNA, and 10 μl of 5Prime Hotmaster mix (Quanta Bio) was prepared. The following thermocycling scheme was used: 94°C for 5 min (1 cycle), 94°C for 1 min, 60°C for 1 min (30 cycles), 72°C for 2 min, and 72°C for 8 min (1 cycle). The PCR products were verified on agarose gels. Sequencing of the PCR products using the B4 and B5 primers was done at Eurofins Genomics (Ebersberg, Germany), and sequence comparison with the NR database at NCBI was done using blastn. A sequence identity >99% of the amplicon sequence with a database entry led to species assignment.