16S amplicon sequencing, milk metagenome, UHT milk sample, PLUS mock community, Extraction kit: PR1, replicate: 7
25 l of ZymoBIOMICSTM Microbial Community Standard cells (ND6300, Zymo Research, EuroClone S.p.A., Milan, Italy) were added to 20 mL of ultra-high temperature commercial milk (UHT milk), collected from local market.After the mock-community spiking, the UHT milk was divided into more aliquots. The DNA was then extracted from seven replicates of this artificially contaminated UHT milk and from seven replicates of the same non-spiked milk by using the three DNA extraction procedures. Bacterial DNA was extracted from UHT milk sample with and without mock-community, 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.