Bacterial 16S rRNA Gene Amplicon Sequencing

Samples were prepared for Illumina Sequencing by 16S ribosomal RNA (rRNA) gene amplification of the bacterial community. The DNA was amplified for the hypervariable V3-V4 region with specific primers and further reamplified in a limited-cycle PCR reaction to add sequencing adapters and dual indexes. First PCR reactions were performed for each sample using KAPA HiFi HotStart PCR Kit according to manufacturer suggestions, 0.3 μM of each PCR primer: forward primer Bakt_341F 5′–CCTACGGGNGGCWGCAG-3′ and reverse primer Bakt_805R 5′–GACTACHVGGGTATCTAATCC-3′ [14 (link),15 (link)] and 12.5 ng of template DNA in a total volume of 25 μL. The PCR conditions involved a 3 min denaturation at 95 °C, followed by 25 cycles of 98 °C for 20 s, 55 °C for 30 s and 72 °C for 30 s, and a final extension at 72 °C for 5 min. Second PCR reactions added indexes and sequencing adapters to both ends of the amplified target region according to the manufacturer’s recommendations [16 ]. Negative PCR controls were included for all amplification procedures. PCR products were then one-step purified and normalized using SequalPrep 96-well plate kit (Thermo Fisher Scientific, Waltham, MA, USA) [17 (link)], pooled and pair-end sequenced in the Illumina MiSeq^® sequencer with the V3 chemistry, according to the manufacturer’s instructions (Illumina, San Diego, CA, USA) at Genoinseq (Cantanhede, Portugal).