The nasopharyngeal microbiota of beef cattle before and after transport to a feedlot

Holman, Devin B.; Timsit, Edouard; Amat, Samat; Abbott, D. Wade; Buret, Andre G.; Alexander, Trevor W.

DNA extraction, PCR amplification, and sequencing of the 16S rRNA gene Total DNA was extracted from NP swabs using a Qiagen DNeasy Tissue kit (Qiagen Inc., Mississauga, ON, Canada) as previously described [1] . 16S rRNA gene sequence libraries were generated using a two-step PCR protocol. The first PCR step amplified the V4 region of the 16S rRNA gene using the universal bacterial and archaeal primers 515-F (GTGCCAGCMGCCGCGGTAA) and 806-R (GGACTACVSGGGTATCTAAT) [14] . The second PCR step was used to add a unique 10-bp barcode at the 5' end of each amplicon as well as to add Illumina (Illumina, San Diego, CA, USA) adapter sequences. All PCR amplification and sequencing steps were carried out at Genome Quebec (Montreal, QC, Canada). The 16S rRNA gene amplicons were quantified using a Quant-iT PicoGreen dsDNA assay kit (Invitrogen, Burlington, ON, Canada), pooled in equimolar ratios, and then purified with AMPure XP beads (Beckman Coulter, Mississauga, ON, Canada). Sequencing of 16S rRNA gene amplicons was carried out using an Illumina MiSeq (2 × 250) and the MiSeq Reagent Kit v2 (500 cycles; Illumina) according to manufacturer's instructions.