Amplification and Sequencing of 16S rRNA Gene

The V3-V4 region of the bacterial 16S rRNA gene was PCR-amplified using V3F primer (5′-CCTACGGGNGGCWGCAG-3′) and V4R primer (5′-GACTACHVGGGTATCTAATCC-3′), 5′-extended with adapter sequences 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′ and 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′, respectively, which are required for sequencing purposes (see below). PCR reactions were carried out in a Bio-Rad C1000 thermal cycler (Bio-Rad Laboratories, Veenendaal, The Netherlands) using a 50 μl total volume, consisting of 5 μl 10× KOD buffer (Toyobo, Japan), 3 μl MgSO4 (25 mM) 5 μl dNTPs (2 mM each), 1.5 μl V3F primer [10 μM (Eurogentec, Luik, Belgium)], 1.5 μl V4R primer [10 μM, (Eurogentec, Luik, Belgium)], 1.0 μl (0.02 U/μl) KOD hot start DNA polymerase (Toyobo, Japan) and 10 ng (minimum) of template DNA. PCR cycles were programmed with a single initiation cycle of 95°C for 2 min, followed by 25 amplification cycles encompassing denaturation at 95°C for 20 s, annealing at 55°C for 10 s, and elongation at 70°C for 15 s, and was completed by a single elongation step at 72°C for 5 min. Amplicon concentrations were measured by DeNovix DS-11 Spectrophotometer (DeNovix Inc., Wilmington, DE USA) and amplicon size (~550 bp) was verified by agarose (1%) gel electrophoresis. Subsequently, the amplicons were purified from the PCR reaction mixture using MSB Spin PCRapace (STRATEC Molecular, Germany).

Purified amplicons were subjected to extension PCR using barcoded Illumina universal index sequencing adapters prior to sequencing. The samples were sequenced (paired-end) using the Illumina MiSeq system (performed by BaseClear BV, Leiden, The Netherlands), generating FASTAQ sequence files by Illumina Casava pipeline version 1.8.3. Quality assessment was based on Illumina Chastity filtering and FASTQC quality control tool version 0.10.0. A BaseClear in-house filtering protocol was applied for removal of reads containing adapters and/or PhiX control signal.