Illumina Sequencing of 16S rRNA

The V4-V5 region of 16S rRNA was amplified with the universal primers 515F: GTGCCAGCMGCCGCGG and 907R: CCGTCAATCMTTTRAGTTT (43). To identify each sample, a unique barcode was applied (44). Initial enzyme activation was at 95°C for 5 min followed by 35 amplification cycles of 30 s at 94°C, 35 s at 58°C and 30 s at 72°C (40). Sequencing of these barcoded amplicons was performed using an Illumina Hiseq 2500 platform (Novogene, Beijing, China).

Bioinformatic analyses was performed using USEARCH (10.0.240) (http://www.drive5.com/usearch/). Based on UPARSE clustering, reads were separated into Zero-Operational taxonomic units (ZOTUs). ZOTUs were set at 100% similarity level and chimeras filtered with UNOISE2 (45). ZOTUs represented by a single sequence were discarded. The Ribosomal Database Project, which uses the Greengenes database (Version 13.8, 16S rRNA gene database), was used to assign the relative abundance and taxonomic identity of ZOTUs (46, 47). The data generated in this study were deposited in the NCBI Sequence Read Archive (SRA) under the accession number PRJNA552669.