The 16S V3-V4 hypervariable gene region (ca. 460bp) was polymerase chain reaction (PCR) amplified using the 16S_341F and 16S_805R primer pair for paired-end Illumina MiSeq sequencing (2x250bp) for DNA extracted from abalone (Haliotis midae) gut.
Prior to next-generation sequencing of the 16S V3-V4 hypervariable region, the Next Generation Sequencing Facility (NGSF) at the University of the Western Cape (UWC) performed post-PCR validation, PCR purification, indexing and library preparation for a total of 30 samples. Briefly, AMPure XP (Beckman Coulter) were used to purify and remove free primer dimer species from PCR products, as per manufacturers instructions. The Nextera XT Index Kit was used to attach dual indices and Illumina sequencing adaptors to the purified PCR products. This was followed by another PCR purification step using AMPure XP beads. Final library quantification and normalisation was performed, whereafter normalised libraries were pooled. Lastly, the pooled libraries were used for paired-end sequencing (2x250bp) on the Illumina MiSeq sequencer, as per manufacturers instructions.