2.4 Library construction and sequencing
The PCR products showing the target bands were mixed in equal amounts, followed by electrophoresis in a 2% agarose gel and gel cutting. The PCR products were purified using a QIAquick Gel Extraction kit. Sixty nanograms of the purified PCR products was denatured at 95 °C and ligated with T4 DNA ligase at 37 °C to generate a single-stranded circular DNA library. Library replicates and library blanks were simultaneously included throughout the process. The concentrations and fragment size distributions of the libraries were checked on an Agilent 2100 Bioanalyzer. All libraries were subsequently pooled in equal amounts to generate DNA nanoballs (DNBs). Each DNB was pooled into 1 lane for sequencing. Sequencing was performed via 150 bp paired-end sequencing on the BGISEQ-500 high-throughput platform.
To obtain clean reads, the raw data were filtered to eliminate adapter contamination and reads of low quality. Then, paired-end reads were combined with tags based on overlaps using FLASH (Magoč and Salzberg, 2011). The tags were clustered into OTUs (operational taxonomic units) using USEARCH with a 97% threshold, and chimaera were filtered out using UCHIME (Rognes et al., 2016). All tags were mapped to each representative OTU sequence using USEARCH to obtain the OTU richness table. The taxonomic assignment of OTU sequences was mapped to the NT database downloaded from the National Center for Biotechnology Information (NCBI) GenBank database using the Blastn tool. Sequences were designated as belonging to a species if there was ≥ 96% sequence identity to the NT and NCBI database barcode across the entire length of the amplicon, if a sequence from at least one other species within the same genus was available for comparison (and < 96% identical). An OTU was categorized into another OTU if a sequence could not be assigned to a species. If a sequence could be assigned to several species (≥ 99% matching rate) and the species belonged to the same genus, the taxonomic resolution collapsed to the genus level.