2.3 | DNA extraction and 16S rRNA gene sequencing
Total DNA was extracted from the gut contents of A. japonicas by
using a FastDNA SPIN Kit for Feces (MP Biomedicals, Santa Ana, CA, USA)
in accordance with the instructions provided by the manufacturer. The
extracted DNA was dissolved in 50 μL of TE buffer, quantified using a
NanoDrop spectrophotometer (NanoDrop, Thermo Scientific, USA) and stored
at -20°C prior to analysis. PCR amplification of the bacterial 16S rRNA
hypervariable V4-V5 regions was conducted using the universal primer set
515f (GTGCCAGCMGCCGCGGTAA) and 907r (CCGTCAATTCMTTTRAGTTT), with 5-bp
barcodes fused to the forward primer to allow sample multiplexing. The
purified PCR products with different barcodes were normalized in
equimolar amounts, then prepared using an NEB Next® Ultra™ DNA Library
Prep Kit for Illumina (NEB, USA) following the manufacturer’s protocol
and sequenced on an Illumina HiSeq platform.