16S amplicon sequencing, milk metagenome, Raw milk sample, sample: B, Extraction kit: PR3, replicate: 3
500 ml of three raw bulk tank milk samples (raw milk) were collected in sterile collection tubes from three different farms (A, B, and C), examined to be free from contagious pathogens such as Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma bovis, according to the guidelines of National Mastitis Council. Samples were delivered to the laboratory at 4C, and frozen at -20C for metagenomic analysis. Bacterial DNA was extracted from the three raw milk samples by using (i) the protocol of Cremonesi and co-workers (protocol 1  PR1), the NucleoSpin Soil Kit (Macherey-Nagel, Dueren, Germany) (protocol 2  PR2), and the DNeasy PowerFood Microbial Kit (Qiagen, GmbH, Hilden, Germany) (protocol 3  PR3).  For PR1, a sample pre-treatment was used; five ml of milk sample were centrifuged at 500  g for 5 min at 4 C, the supernatant was discarded, and the pellet was resuspended with one ml of saline solution (NaCl 0.9%) and centrifuged at 500  g for 5 min at 4 C. The supernatant was discarded and, then, the bacterial DNA was extracted from the samples as described previously (8). Sample lysis was obtained with a combination of a chaotropic agent (guanidium thiocyanate) with silica particles. For the PR2 and PR3 based on commercial kits, the instructions from manufacturers were followed, starting from 500 l of milk sample.  Bacterial DNA was amplified using primers which target the V3-V4 hypervariable regions of the 16S rRNA gene. All PCR amplifications were performed in 25 l volumes per sample. A total of 12.5 l of Phusion High-Fidelity Master Mix 2 (ThermoFisher Scientific, Walthem, MA, USA) and 0.2 l of each primer (100 M) were added to 2 l of genomic DNA (5 ng/l). Samples were denatured at 98C for 30 s, followed by 25 cycles with a denaturing step at 98C for 30 s, annealing at 56C for 1 min and extension at 72C for 1 min, with a final extension at 72C for 7 min. Amplicons were cleaned with Agencourt AMPure XP (Beckman, Coulter Brea, CA, USA) and libraries were prepared following the 16S Metagenomic Sequencing Library Preparation Protocol (Illumina, San Diego, CA, USA). The libraries obtained were quantified by Real Time PCR with KAPA Library Quantification Kits (Kapa Biosystems, Inc., MA, USA), pooled in equimolar proportion and sequenced in one MiSeq (Illumina) run with 2250-base paired-end reads.