A subset of fecal samples from 20 total male cotton rats (10 from each species), taken at days 34 and 111 within the first cohort of cotton rats, underwent whole-metagenomic shotgun sequencing. From the same stool samples, genomic DNA was extracted using the Qiagen DNeasy PowerSoil Kit (Cat No./ID: 12888–100) by following the manufacturer’s protocol (skipping the optional 4 °C incubations). In addition, a negative sample (which did not contain any template but was otherwise processed the same as the rest of the samples) and a positive control (ZymoBIOMICS Microbial Community Standard) were processed in parallel with samples and sequenced. Samples were normalized to 75 ng/ μL in 1X TE prior to library construction. Metagenomic libraries were prepared using the NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® following the manufacturer’s protocol for inputs ≤100 ng. Samples were fragmented at 37 °C for 12 min to yield a fragment size of 200–450 bp. NEBNext Multiplex Adaptors were diluted 10-fold. NEBNext Multiplex Oligos for Illumina (Set 1, NEB #E7335) were used for PCR enrichment of adaptor-ligated DNA, and 5 cycles of PCR were run. Library quality was assessed on an Agilent 2100 Bioanalyzer System using the Agilent High Sensitivity DNA Kit (5067–4626). Samples were sequenced via the NovaSeq 6000 2 × 150 platform for Illumina at the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core, aiming for 40 million reads per sample.

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