Dneasy power food kit

Repeated Bead-Beating (RBB) combined with column-based purification was used to extract DNA from human and animal fecal samples according to protocol Q (IHMS_SOP 06 V2 - http://www.microbiome-standards.org/index.php?id=253) of the International Human Microbiome Standards consortium [20 (link)]. Bead-beating was done using the FastPrep™ Instrument (MP Biomedicals, Santa Ana (CA), USA) with 0.1 mm zirconium-silica beads (BioSpec Products, Bartlesville (OK), USA) to homogenize feces. DNA was finally purified by adapting to QIAamp DNA Stool Mini kit columns (Qiagen, Hilden, Germany).
Regarding the isolation of metagenomic DNA from processed food, 200 mg of sample was homogenized in 0.75 ml PBS (pH 7.2), and centrifuged for 3 min at 13,000 x g. Metagenomic DNA was subsequently isolated by the QIAGEN DNeasy Power Food Kit according to the manufacturer’s instructions for DNA isolation from solid food. For isolation of microbial DNA from water samples, 100 ml of collected water was filtered through 0.22 μm mixed cellulose esters membrane filters (Sartorius, Göttingen, Germany) to capture bacteria. One quarter of the filters were used for metagenomic DNA extraction using the QIAGEN DNeasy Power Water kit according to the manufacturer’s protocol.
Upon isolation, DNA concentrations were determined using the Quan-iT PicoGreen dsDNA assay (Invitrogen, Carlsbad (CA), USA).

DNA was extracted using the DNeasy PowerFood kit according to the manufacturer’s instructions (Qiagen, Germany), with an initial step of homogenization in a MagNALyzer (Roche, Switzerland). The DNA concentration was determined spectrophotometrically and fluorometrically, and DNA samples diluted to 5 ng/mL were used as a template in PCR with the eubacterial primers 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-MID-GT-CCTACGGGNGGCWGCAG-3′ and 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-MID-GT-GACTACHVGGGTATCTAATCC-3′, respectively, amplifying the V3/V4 variable region of 16S rRNA genes. Following amplification, the products were processed exactly as described previously [15 (link)]. Sequencing was performed using the MiSeq Reagent Kit v3 (600 cycle) and MiSeq apparatus according to the manufacturer’s instructions (Illumina, CA, USA). Raw reads were processed previously described [15 (link)].