The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified using universal bacterial primers. Forward Primer (5′ GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C 3′)(Integrated DNA Technologies) and Reverse Primer (5′ TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG 3′) (4 nmol Ultramer® DNA Oligo 55 bases : Integrated DNA Technologies)47 (link). Library preparation was performed according to the standard instructions of the 16S Metagenomic Sequencing Library Preparation protocol (IlluminaTM, Inc., San Diego, CA, United States). The size of the amplicons were then visualised using the 4200 TapeStation (Agilent Technologies). Two negative controls were used in the amplicon preparation step. The extraction negative control as well as no template control were used in the initial amplification step. No amplification was observed in either of the negative controls.
Libraries were then sequenced on the MiSeq platform using MiSeq Reagent kit v3 (Illumina) and paired-end 2 × 300 bp sequencing was performed at the Sequencing Core Facility, National Institute for Communicable Diseases, South Africa.
Libraries were then sequenced on the MiSeq platform using MiSeq Reagent kit v3 (Illumina) and paired-end 2 × 300 bp sequencing was performed at the Sequencing Core Facility, National Institute for Communicable Diseases, South Africa.
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