16S rRNA Amplification and Sequencing

The V3–V4 region of the 16S rRNA were targeted for amplification using specific primers 338F/806R with barcodes (Angebault et al., 2020 (link); Zhang et al., 2021 (link)). PCR amplification was performed with Phanta^® Max Super-Fidelity DNA polymerase (Vazyme, Nanjing, China) as described previously (Liu, Zhuang & Wang, 2021 (link)). Sequencing libraries were generated using NEB Next^® Ultra^™ II FS DNA PCR-free Library Prep Kit (New England Biolabs, Ipswich, MA, USA) and quantified by Qubit (Thermo Scientific, Waltham, MA, USA) and Q-PCR. After the library was qualified, paired-end sequencing was performed with a PE250 strategy via the NovaSeq6000 platform (Illumina Inc., San Diego, CA, USA) (Guo et al., 2023 (link)), which was conducted by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China).