16S rRNA Gene Amplification and Sequencing

The V3-V4 region of the 16S rRNA gene was amplified by using the following primers: forward primer: 5’- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG -3’ and reverse primer: 5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC -3’. The libraries were constructed according to the protocol (https://support.illumina.com/downloads/16s_metagenomic_sequencing_library_preparation.html) for MiSeq 16S Metagenomic Sequencing Library Preparation (Illumina, San Diego, CA, USA) with the Phanta Max Master Mix Kit (Vazyme, Jiangsu, China). As previously described (Ye et al., 2016 (link)), we used three tubes of sterile water as negative controls during the library construction process. No DNA products were detected in the negative controls when evaluated by the E-gel electrophoresis system (Life Technologies). The libraries were then sequenced on a MiSeq platform (Illumina) to generate 2 × 300-bp paired-end reads. Each sample was sequenced for one time.