PCR amplification of the V3–4 region of bacterial 16 S rRNA genes

16S rRNA genes were amplified by PCR with forward and reverse primers containing Illumina adapter sequences and unique dual indexes used to tag each PCR product³⁹, according to the 16S-protocol provided by Illumina. Briefly, PCR reactions were carried out in 25-μL reactions with 0.2 μM forward and reverse primers, with 12.5 ng template DNA and 12.5 μl of 2 × KAPA HiFi HotStart Ready Mix kit (KAPA Biosystems). Thermal cycling consisted of initial denaturation at 95 °C for 3 min followed by 25 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, followed by a final step of 72 °C for 5 min. The amplicons (464 bp product) were purified with Agencourt AMPureXP Kit (Beckman Coulter). A second PCR was thereafter performed to attach Illumina adapters and unique dual indexes to each sample, followed by a clean-up step with AmPureXP Kit (Beckman Coulter) (Table S3). PCR amplicons were visualized using 0.1% agarose gel electrophoresis. Negative extraction controls did not produce visible bands.