16S rRNA PCR and Sequencing

16S rRNA genes of distinct regions were amplified using specific primers with the barcode. All PCR reactions were carried out with Phusion High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, United States).

Polymerase chain reaction products were mixed with the same volume of 1 × loading buffer (contained SYB green) and separated by electrophoresis on 2% agarose gel. PCR products were mixed in equidensity ratios. Then, mixture PCR products were purified with GeneJET Gel Extraction Kit (Thermo Scientific, United States).

Sequencing libraries were generated using Ion Plus Fragment Library Kit 48 rxns (Thermo Scientific) following manufacturer’s recommendations. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific). The library was sequenced on an Ion S5TM XL platform, and 400-bp/600-bp single-end reads were generated.