ITS1-2 sequencing of human intestinal microbiome
Fecal samples were collected in collection tubes containing 9 mL DNA/RNA ShieldTM (Zymo Research Corp, Irvine, CA, USA) and stored at 4C. After homogenizing the samples through vortexing, ~1 mL was transferred to a clean 2 mL tube, centrifuged for 5 min at 10,000 x g, and the supernatant was removed. The average weight of the resulting pellets was 125 mg, which was subsequently resuspended in 750 L bead solution from the DNeasy PowerLyzer PowerSoil Kit (Qiagen GmbH, Hilden, Germany). The DNA isolation was performed according to the manufacturers protocol, with the following adaptations: two rounds of bead beating (1 min and 4,000 rpm, PowerLyzerTM, Mo Bio Laboratories, Inc., Carlsbad, CA, USA) followed by 5 min incubation on ice, subsequently, bead beating tubes were centrifuged for 5 min. After the addition of Solution C6 (elution buffer), the spin columns were incubated at room temperature for 5 min before centrifugation. Paired-end (2 x 300 bp) sequencing of the isolated DNA (V3-V4 16S rRNA for bacteria and ITS1 and ITS2 for fungi) was performed at Applied Biological Materials Inc., Richmond, B.C., Canada, using the MiSeq platform (Illumina, San Diego, CA, USA).