Fecal materials were obtained from the individual pig by rectal stimulation on days 0 (before oxytetracycline treatment), 8 (after the oxytetracycline treatment), and 21 (two weeks after the withdrawal of oxytetracycline), and stored in sterile containers at –20 °C until processed. The total DNA was extracted from the fecal samples by the QIAamp PowerFecal Kit (Qiagen, Crawley, West Sussex, UK) following the manufacturer’s instructions with some modifications recommended by Hart et al.45 (link). The final DNA were eluted in 100 μL of 10 mM Tris buffer (pH 8) after being incubated for 5 min for maximum elution efficiency. A Qubit fluorometer (Qubit 3, Invitrogen) was used to determine the total DNA concentration, and purity was assessed via the 260/280 and 260/230 absorbance ratios using a spectrophotometre (NanoDrop® ND-1000). The samples were sent for DNA sequencing to the Teagasc Food Research Centre, Ireland. Paired-end sequencing libraries were prepared from the extracted DNA using the Illumina Nextera XT Library Preparation Kit (Illumina Inc., San Diego, CA) followed by sequencing on the Illumina NextSeq 500 platform using high-output chemistry (2 × 150 bp) according to the manufacturer’s instructions.
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