16S rRNA gene sequencing protocol

The V3 to V4 region of the 16S rRNA gene was amplified using the primer set S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21 [23 (link)]. Indexed libraries were prepared by limited-cycle PCR using Nextera technology (Illumina, San Diego, CA, USA) and further cleaned up with VAHTS DNA Clean Beads (Vazyme, Red Maple Hi-tech Industry Park, Nanjing, China). The libraries were pooled at equimolar concentrations (4 nM), denatured, and diluted to 5 pM before loading onto the MiSeq Reagent kit V3 (Illumina). Sequencing on the MiSeq platform was performed by using a 2 × 300 bp paired-end protocol, according to the manufacturer’s instructions (Illumina). The raw sequence files were deposited in the Sequence Read Archive (SRA) under the project number PRJNA1054360.