Bacterial 16S rRNA Gene Amplification

The primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′) (Munyaka et al., 2015 (link)) were used to amplify the V3-V4 hypervariable region of the bacterial 16S rRNA gene. An 8 bp barcode sequence was added at the 5′ end of each upstream and downstream primers to distinguish between different samples. Amplicons were separated on 2% agarose gels, purified using the AxyPrep DNA Gel Extraction Kit according to the manufacturer’s instructions, and quantified using QuantiFluor-ST. The purified amplicons were pooled in equimolar concentrations and paired-end sequenced (2 × 250) on the Illumina platform according to standard protocols. PCR reaction mixture (25 μL) contained 12.5 μL 2xTaq Plus Master Mix, 3 μL BSA (2 ng/μL), 1 μL Forward Primer (5 μM), 1 μL Reverse Primer (5 μM), 2 μL template DNA, and 5.5 μL ddH₂O. Reaction parameters were as follows: pre-denaturation at 95°C for 5 min followed by 28 cycles of denaturation at 95°C for 45 s, annealing at 55°C for 50 s, and extension at 72°C for 45 s, and a final step of extension at 72°C for 10 min. The PCR products were detected by 1% agarose gel electrophoresis to determine the size of the amplified target bands, which were then purified using the Agencourt AMPure XP Nucleic Acid Purification Kit.