Illumina MiSeq sequencing and analysis

Amplicons were gel purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, CA, USA) according to the manufacturer’s instructions and quantified using QuantiFluor™-ST (Promega, USA). Purified amplicons were pooled in equal amounts and paired-end sequenced on the Illumina MiSeq platform at a read length of 2 × 300 bp (Majorbio, Shanghai)

Trimmed sequences were paired-end aligned using fastq-join and screened for quality using the following parameters: quality score ≥ 35 over a 50 nt window, no ambiguous bases, homopolymers ≤ 8 nt, and primer and barcode matching with 100% identity⁵³. Sequences were chimera checked using UCHIME software⁵⁴. The operational taxonomic units (OTUs) were picked at 97% sequence similarity and identified using the Ribosomal Database Project (RDP) classifier retrained with Greengenes (http://greengenes.secondgenomes). The NAST algorithm was used for alignment and Greengenes. Sequences were quality filtered and rarefied to 9,725 sequences per sample. OTU based analyses [sequencing coverage estimation, the Shannon-Wiener index (H), Chao and ACE richness estimators] were performed using the Quantitative Insights Into Microbial Ecology (QIIME) program⁵⁵.