16S rRNA Microbiome Sequencing Protocol

Faecal and salivary microbiota compositions were profiled by sequencing the hypervariable V3–V4 regions of the 16S rRNA gene. Sequencing was performed by LifeSequencing S.L. (Valencia, Spain) on an Illumina MiSeq instrument (San Diego, California, USA). The V3–V4 region was PCR-amplified with universal primers S-D-Bact-0341-b-S-17 primer (forward 5′-CCTACGGGNGGCWGCAG-3′) and S-D-Bact-0785-a-A-21 primer (reverse 5′-GACTACHVGGGTATCTAATCC-3′) [20 (link)] designed for dual indexing following the Illumina 16S Metagenomic Sequencing Library Preparation protocol (Part # 15044223 Rev. B). In brief, PCR amplification was performed in two steps: (1) in a first step, the V3–V4 region was amplified with the addition of universal adaptors to the amplification products. All amplicons were purified (AMPure XP, Beckman, Danvers, MA) to remove short amplification products and quantified using the Quant-iT PicoGreen dsDNA kit (Invitrogen, Carlsbad, California, USA). (2) In the second PCR step, the amplicons from the first step were amplified by targeting the universal adapters and with the addition of sample specific indexes and sequencing adaptors. The final amplicons were purified (AMPure XP) and quantified using the Quant-iT PicoGreen ds DNA kit (Invitrogen). All samples were pooled in equal amounts and sequenced in a 300 bp paired-end mode.