Sequencing 16S rRNA Bacterial DNA

Total bacterial DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) and then quantified using NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and 1% agarose gel electrophoresis. The V3–V4 region of 16S rRNA genes was amplified using the following primers: 338F forward primer (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R reverse primer (5′-GGACTACHVGGGTWTCTAAT-3′) by thermocycler PCR system (GeneAmp 9700; Applied Biosystems, Carlsbad, CA, USA) as described previously (Lin et al., 2018 (link)). The purified amplicons were sequenced using the Illumina Miseq PE300 platform (Illumina, San Diego, CA, USA) according to the standard protocol.