High-throughput sequencing of V3-V4 region of bacterial 16S rRNA diversity in soy-daddawa
The Illumina 16S metagenomics sequencing library preparation guide (available from https://www.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf). Briefly, the library preparation workflow entailed a first-stage PCR aimed at amplifying the V3-V4 region of the 16S rRNA with primer set 5`-CCTACGGGNGGCWGCAG-3` (forward) and 5`-GACTACHVGGGTATCTAATCC-3` (reverse) (primers contained Illumina forward and reverse overhang adapters attached to the 5`-end of forward and reverse primers, respectively) by using 2X KAPA HiFi HotStart readyMix. Thereafter, a PCR clean-up step was performed by using AMPure XP beads, followed by an index PCR step aimed at attaching dual indices and Illumina sequencing adapters to amplicons by using the Nextera XT Index kit. A second PCR clean-up step was performed and amplicons library subjected to library normalization, pooling and a final library denaturation prior to loading on the Illumina MiSeq platform (Illumina, San Diego, CA, USA). Following completion of 2x300 bp paired-end reads sequencing run, primary analyses of the reads, including removing indices were generated using the MiSeq reporter software.