title: shotgun metagenomic of Equus caballus: adult female feces
design: We weighed 0.2-g portions of 16 freshly thawed fecal samples into 2-ml bead beating tubes containing Qiagen 0.7-mm garnet beads, added 750 l of PowerBead Solution and 60 l of C1 buffer from Qiagens QIAamp PowerFecal DNA Kits to each tube, vortexed each tube for 2 seconds, and incubated tubes for 10 min at 60C. After incubation, we bead beat samples for 10 min on a Vortex-Genie 2 fitted with Qiagens Vortex Adapter (cat. No 13000-V1-24). All other extraction steps were completed as per Qiagen kit recommendations (single-tube extraction protocol). Final DNA extracts were eluted in 100 l of molecular grade water. DNA extracts were quantified using Quant-iT dsDNA Broad Range Assays. Extracts were sent to the University of Calgary Centre for Health Genomics and Informatics, where shotgun metagenomic libraries were created using iGenomx Riptide preparations (1:1 mixture of low GC and High GC primers), as per manufacturer specifications. We sequenced a total of 188 samples using an Illumina NovaSeq 6000 (300 cycle SP sequencing kit v1.5 2 lanes) to a target depth of 4.1M (150 bp) read pairs per sample.