Bacterial 16S rRNA Gene Amplification and Sequencing

The bacterial communities for each sample was processed as described previously^{22 (link)}. In brief, the V3-V4 region of the bacterial 16S rRNA gene was amplified using the S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21^{29 (link)} primer pair containing Nextera library prep kit adapters. Approximately 100 ng of genomic template DNA was used in duplicate PCRs, each consisting of 35 cycles. Negative PCR controls were included in all PCR reactions as well as elute from the negative extraction control, which yielded no detectable products. Duplicate PCR products were pooled to a final volume of 50 µL and purified using Agencourt AMPure magnetic beads (Beckman Coulter Inc., USA) according to the manufacturer’s instructions. Purified PCR products were quantitatively assessed with Qubit dsDNA high-sensitivity kits (Life Technologies, New Zealand) and standardised to ~ 5 ng per sample. The purified products were submitted to the Auckland Genomics Centre for library preparation and sequencing on the Illumina MiSeq 2 × 300 base pair platform with pair end reads. Raw sequence reads are stored on a publicly available database (NCBI) under BioProject number PRJNA638970.