Quan it picogreen dsdna assay

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).

Pooled and purified PCR amplified products were quantified using the Quan-it PicoGreen dsDNA assay (Invitrogen). GS-FLX Titanium kits were used for Rapid Library Preparation and Rapid Library MID Adaptor addition (Roche, Branford, CT). 500ng of each sample was nebulized, end repaired, and ligated with 454 library adaptors and MIDs. Fragments between 600–900bp were selected for and purified using AMPure beads. Library quality was assessed using the Agilent High Sensitivity DNA Bioanalyzer kit and chip (Santa Clara, CA), and the quantity of DNA was measured using the Quan-it PicoGreen dsDNA assay. Library concentrations were calculated using the online Roche Rapid Library Quantitation calculator. Each DNA library was diluted to a working stock of 1x10⁷ molecules/μl in TE buffer. Libraries generated from multiple samples (each with distinct sequence tags) were mixed at equimolar ratios. Emulsion PCR (Roche) was performed on the combined libraries using a ratio of 2–3 DNA molecules per bead. PCR-positive beads (~10–20% of emulsion PCR products) were then selectively enriched. Four million enriched beads were loaded onto a 454 picotiter plate and pyrosequences were generated using the 454 GS FLX system. Median number of pyrosequences generated per gene region for the subjects in this study is given in S3–S5 Tables.