16S rRNA gene amplicon sequencing of raw milk from Holstein cow which experienced K. pneumoniae clinical mastitis
The V4 region of the 16S rRNA gene was amplified via PCR using F548 and R806 primer pair and HotStarTaq Plus (QIAGEN, Germany). PCR reaction included a denaturation stage (95C, 5 minutes), 35 cycles of amplification (95, 30 seconds; 50, 30 seconds; 72, 1 minute), and an extension stage (72C, 10 minutes), followed by holding samples at 4C. Amplicons were purified using Agencourt AMPure XP (Beckman Coulter Inc., USA), then quantified using Quant-iT dsDNA Assay Kit (Invitrogen Inc., USA), as per the manufacturers protocol. Amplicons with sufficient concentration were normalized to a final concentration of 1.5ng/L using DNA/RNA-free water, then 2L of each normalized sample was pooled together prior to sequencing. The pooled library was sequenced using the MiSeq and a 500 cycle V2 MiSeq reagent kit (Illumina Inc., USA).