Fecal samples were collected by the participants at home and frozen immediately. They were transported to the laboratory frozen and stored at − 80 °C until processing.
Bacterial DNA was extracted using a previously described repeated bead-beating method35 (link) with the following modifications for automated DNA purification: ca. 125 mg of fecal material were suspended in 1 ml of sterile ice-cold PBS, and 175 μl of fecal suspension was combined with 235 μl of RBB lysis buffer (500 mM NaCl, 50 mM Tris–HCl (pH 8.0), 50 mM EDTA, 4% SDS) in a bead-beating tube from the Ambion Magmax™ Total Nucleic Acid Isolation Kit (Life Technologies, Carlsbad, CA, USA). After repeated bead-beating, 200 μl of the supernatant was used for DNA extraction with a KingFisher™ Flex automated purification system (ThermoFisher Scientific, Waltham, MA, USA) using a MagMAX™ Pathogen High Vol. Duo program. DNA was quantified using a Quanti-iT™ Pico Green dsDNA assay (Invitrogen, San Diego, CA, USA) and 1 ng was used for V3–V4-region amplicon PCR of the 16S rRNA gene as previously described36 (link). Sequencing was carried out with Illumina HiSeq 2500 equipment in Rapid Run mode.
Bacterial DNA was extracted using a previously described repeated bead-beating method35 (link) with the following modifications for automated DNA purification: ca. 125 mg of fecal material were suspended in 1 ml of sterile ice-cold PBS, and 175 μl of fecal suspension was combined with 235 μl of RBB lysis buffer (500 mM NaCl, 50 mM Tris–HCl (pH 8.0), 50 mM EDTA, 4% SDS) in a bead-beating tube from the Ambion Magmax™ Total Nucleic Acid Isolation Kit (Life Technologies, Carlsbad, CA, USA). After repeated bead-beating, 200 μl of the supernatant was used for DNA extraction with a KingFisher™ Flex automated purification system (ThermoFisher Scientific, Waltham, MA, USA) using a MagMAX™ Pathogen High Vol. Duo program. DNA was quantified using a Quanti-iT™ Pico Green dsDNA assay (Invitrogen, San Diego, CA, USA) and 1 ng was used for V3–V4-region amplicon PCR of the 16S rRNA gene as previously described36 (link). Sequencing was carried out with Illumina HiSeq 2500 equipment in Rapid Run mode.
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