DNA Sequencing of Bovine Feces Time 168 hr
Fecal DNA was extracted using ZymoBIOMICS DNA Miniprep Kit (Zymo Research, Catalog Number D4300, Irvine, CA). Genomic DNA (gDNA) was submitted to the North Carolina State Genomic Sciences Laboratory (Raleigh, NC, USA) for Illumina NGS library construction and sequencing. Prior to library preparation, the isolated DNA template was quantified, and quality was assessed using an Agilent 2200 Tapestation High Molecular Weight DNA assay (Agilent Technologies, USA). Library construction was performed using an Illumina TruSeq Nano Library kit with provided protocol. Briefly, the gDNA was fragmented using a Covaris S220 Ultrasonicator (Covaris, USA) and purified using sample purification beads included in the TruSeq kit. The fragments were then end-repaired, followed by 350 bp insert size-selection using sequential bead isolation steps. After adapter ligation, the library was enriched by PCR amplification. The amplified library was checked for quality and final concentration using the Agilent 2200 Tapestation (D1000) before sequencing on an Illumina NovaSeq Sequencer utilizing a S4 150 x2 PE flow cell (Illumina, USA). Raw base call file (bcl) were then de- multiplexed by sample into discrete .fastq files for data analysis.