0.1 mm zirconium silica beads

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).

The DNA used for the 16S amplicon sequencing was extracted using QIAamp DNA Stool Mini Kit (QIAGEN, Venlo, The Netherlands) with an added bead beating treatment as the first step. Bead beating was performed with 0.1 mm zirconium/silica beads (Biospec Products, Bartlesville, OK, USA), 2 × 45 s with setting 5 using the MP FastPrep‐24 (MP Biomedicals, Irvine, CA, USA). Of the five samples used for amplicon sequencing, the first two were extracted from the original material while the latter three corresponded to three size fractions, selected by gravity precipitation. Since the SFB content of these size fractions was not significantly larger than for the full sample, all later DNA extractions were performed on unfractionated material. For the shotgun sequencing, two replicates were extracted with QIAamp DNA Stool Mini Kit, one with QIAamp Fast DNA Stool Mini Kit, and one with QIAamp DNA Microbiome Kit, according to instructions from the manufacturer (QIAGEN, Venlo, The Netherlands).

Genomic DNA (gDNA) was extracted from mouse faeces using the ReliaPrep gDNA Tissue Miniprep System (Promega, USA), after mechanical disruption of microbial cell walls. Briefly, 50 mg of faecal samples were suspended in 720 μL of lysis buffer solution (320 μL of PBS + 400 μL of CLD) in a screw cap micro-tube (Sarstedt, Germany) together with 0.3 g of 0.1 mm zirconium/silica beads (Biospec, USA), followed by disruption in a mini-beadbeater-16 (Biospec, USA) for 2 min and centrifuged at 14,000 × g for 3 min at room temperature. The supernatant was thoroughly mixed with 250 μL of Binding Buffer (BBA) and placed on a binding column. For further cleaning and eluting the manufacturer’s instructions were followed.