16S rRNA Gene Sequencing Protocol

Partial 16S rRNA gene sequences were amplified from extracted DNA using the primer pair Probio_Uni (5′-CCTACGGGRSGCAGCAG-3′) and Probio_Rev (5′-ATTACCGCGGCTGCT-3′) targeting the V3 region of the 16S rRNA gene sequence (37 (link)). Illumina adapter overhang nucleotide sequences were added to the partial 16S rRNA gene-specific amplicons, which were further processed by means of the 16S metagenomic sequencing library preparation protocol (part 15044223, rev. B; Illumina). Amplifications were carried out using a Verity thermocycler (Applied Biosystems). The integrity of the PCR amplicons was analyzed by electrophoresis on a 2200 TapeStation instrument (Agilent Technologies, USA). DNA products obtained following PCR-mediated amplification of the 16S rRNA gene sequences were purified by a magnetic purification step employing Agencourt AMPure XP DNA purification beads (Beckman Coulter Genomics GmbH, Bernried, Germany) in order to remove primer dimers. DNA concentration of the amplified sequence library was determined by a fluorometric Qubit quantification system (Life Technologies, USA). Amplicons were diluted to a concentration of 4 nM, and 5-μl quantities of each diluted DNA amplicon sample were mixed to prepare the pooled final library. Sequencing was performed using an Illumina MiSeq sequencer with MiSeq reagent kit v3 chemicals.