16S rRNA metagenomic sequencing of bovine respiratory microbiota
Total nucleic acids were extracted from NP swabs as described previously (Holman et al., 2017). In DNA from all swabs, the V4 region of the 16S rRNA gene was amplified using primers 515-F (5-GTGYCAGCMGCCGCGGTAA-3) and 806-R (5-GGACTACNVGGGTWTCTAAT-3) (Holman, Yang and Alexander, 2019). The amplicon was sequenced on a MiSeq instrument (PE250, with 25,000 read coverage, Genome Quebec, Montreal, Quebec, Canada) with the MiSeq Reagent Kit v2.