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.