Amplicon sequencing protocol for microbial community analysis

The PCR was composed of 12.5 μL of 2× KAPA HiFi HotStart ReadyMix (Kapa Biosystems), 200 nM 515F and 926R primers (Table 2), and 12.5 ng of template DNA in a 25 μL reaction volume. The PCR was performed with the following cycle conditions: 3 min at 95°C, followed by 25 cycles of 30 s at 95°C, 30 s at 55°C then 30 s at 72°C; and 5 min at 72°C. The final cycle ended with infinite hold at 4°C. Each reaction was generated in triplicates, then pooled and purified with SPRISelect magnetic beads (Beckman Coulter) with double size selection ratio of 0.85 to 0.56, according to the manufacturer’s instructions. The quality of the amplicons was validated with the Agilent 4200 TapeStation System, using the AgilentD1000 ScreenTape Assay (Agilent Technologies, Inc). Sequencing was performed with MiSeq Illumina 2 × 300 bp chemistry, and the sequence reads were analyzed through the DADA2 version 1.10.1 pipeline (67 (link), 68 (link)), performed in R version 3.5.3 (69 ) using Mac OS version 10.14.4.