16S rRNA Amplicon Sequencing Protocol

The extracted DNA amplification and sequencing followed the method described by MacPherson et al. (55 (link)). The sequencing library was prepared according to Illumina’s “16 S Metagenomic Sequencing Library Preparation Guide” (Part # 15044223 Rev. B) with the exception of the use of Qiagen HotStar MasterMix for the first polymerase chain reactions (PCR) (amplicon PCR) and halving reagent volumes for the second PCR. The 16S V3–V4 hypervariable regions were amplified based on the following primers (without the overhang adapter sequence): 5′-CCTACGGGNGGCWGCAG-3′ (forward) and 5′- GACTACHVGGGTATCTAATCC-3′ (reverse), generating a fragment of around 460 bp. The first PCR (amplicon PCR) was carried out for 25 cycles with annealing temperature of 55^°C. Diluted pooled samples were loaded on an Illumina MiSeq system and sequenced using a 500-cycle (paired-end sequencing configuration of 2 × 250 bp) MiSeq Reagent Kit v3.