16 s metagenomic sequencing library preparation guide

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.

The sequencing library preparation was performed according to Illumina 16S Metagenomic Sequencing Library Preparation guide (part no. 15044223 Rev. B), with the exception of using Qiagen HotStar Master Mix (Qiagen, Hilden, Germany) for the amplicon PCR. The primers 16S-IlluF (5′-CCTACGGGNGGCWGCAG-3′) and 16S-IlluR (5′-GACTACHVGGGTATCTAATCC-3′) targeting the V3–V4 hypervariable regions were used to amplify a 550 bp fragment (32 (link)). The PCR amplification was performed for 25 cycles using an annealing temperature of 55°C. Purified PCR products were then loaded on an Illumina MiSeq and sequenced using the MiSeq 500-cycle V3 Kit (Illumina, San Diego, CA, USA).