16S rRNA Amplification and Sequencing

Partial 16S rRNA gene sequences were amplified from extracted DNA using primer pair Probio_Uni (5′-CCTACGGGRSGCAGCAG-3′)/Probio_Rev (5′-ATTACCGCGGCTGCT-3′), which targets the V3 region of the 16S rRNA gene sequence^{34 (link)}. Illumina adapter overhang nucleotide sequences were then added to the partial 16S rRNA gene-specific amplicons, which in turn were further processed by employing the 16S Metagenomic Sequencing Library Preparation Protocol (Part #15044223 Rev. B – Illumina; see also below). Amplifications were carried out using a Verity Thermocycler (Applied Biosystems). The integrity of the PCR amplicons was analysed by electrophoresis on a 2200 TapeStation Instrument (Agilent Technologies, USA).