The V3-V4 region of the bacterial 16S ribosomal RNA genes was amplified by PCR using barcoded primers 341F 5′-CCTACGGGRSGCAGCAG-3′ and 806R 5′-GGACTACVVGGGTATCTAATC-3′ (Wang and Qian, 2009 (link)). PCRs were performed in a volume of 30 μl containing 15 μl 2 × KAPA Library Amplification ReadyMix, 1 μl each primer (10 μM), 50 ng template DNA, and ddH2O (Pereira et al., 2017 (link)).
Amplicons were extracted from 2% agarose gels, purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer's instructions, and quantified using Qubit®2.0 (Invitrogen, Waltham, MA USA). All quantified amplicons were pooled to equalize the concentrations for sequencing using the Illumina MiSeq PE250 (Illumina, Inc., San Diego, CA, USA). The paired-end reads (~250 bp in length) were overlapped at their 3′ ends for concatenation into original longer tags using PANDAseq (