Amplicon Sequencing of 16S rDNA Regions

The 16S primers used in this study are shown in Supplemental Table 2. Six sets of primers for short amplicon sequencing were designed for 16S rDNA variable regions (V2, V3, V4, V6–7, V8, and V9), for 2D sequencing. The pooled PCR product was subjected to 2D DNA library preparation. Primers S-D-Bact-0008-c-S-20 and S-D-Bact-1391-a-A-17, which cover nearly the full length of 16S rDNA, were used for 1D sequencing^{13 (link)–15 (link)}. One nanogram of DNA was used for PCR. KAPA HiFi HotStart ReadyMix (KAPA Biosystems, MA, USA) and Agencourt AMPure XP system (Beckman Coulter Genomics, CA, USA) were used for PCR and PCR product purification, respectively. The PCR conditions are shown in Supplemental Table 3.