16S Amplicon Sequencing Protocol

The protocol of library preparation and sequencing has been described in detail elsewhere [29 (link)]. 16S barcoded amplicon libraries were generated using primers targeting the V3-V4 hypervariable region of the bacterial 16S rRNA gene and the Nextera XT index kit (Illumina, inc., San Diego, CA, USA), and their size and quality were verified using Agilent DNA 1000 Analysis kit (Agilent Technologies, Santa Clara, CA, USA) on the Agilent 2100 Bioanalyzer system (Model G2939B, Agilent Technologies, Santa Clara, CA, USA). Genomic libraries were quantified with a Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), normalized to a concentration equal to 4 nM, then pooled. The pooled library, and the adapter-ligated library PhiX v3 used as a control, were denatured and diluted to equal concentration (8 pM) and subsequently combined to obtain a PhiX concentration equal to 5% of the total volume. Combined 16S library and PhiX control were further denatured and sequenced on the MiSeq platform using MiSeq v3 Reagent Kit (Illumina).