16S rRNA Amplicon Sequencing Protocol

DNA was extracted from each newly collected sample using the bead-beating method [16 (link)], and the V1–V2 regions of the 16S rRNA gene were amplified using the following primers: 8F (5-AGA GTT TGA TYM TGG CTC AG-3) with the Ion Torrent adapter A and the sample-specific 8-base tag sequence and 338R (5-TGC TGC CTC CCG TAG GAG T-3) with the Ion Torrent trP1 adapter sequence. PCR amplification, purification, and quantification of each PCR amplicon was performed as previously described [17 (link)]. The purified PCR amplicons were pooled, and gel purification was performed using the Wizard SV Gel and PCR Clean-Up System (Promega, WI, USA). The DNA concentration was determined using a KAPA Library Quantification Kit (KAPA Biosystems, MA, USA), and the DNA was diluted for use as the template DNA in emulsion PCR. Emulsion PCR and enrichment of template-positive particles were performed using Ion PGM Template Hi-Q View OT2 Kit (Thermo Fisher Scientific) in Ion One Touch 2 system (Thermo Fisher Scientific). The enriched particle was loaded onto Ion 318 v2 chips (Thermo Fisher Scientific), and sequencing was performed on the Ion PGM (Thermo Fisher Scientific) using the Ion PGM Hi-Q view Sequencing Kit (Thermo Fisher Scientific).