Library preparation, iSeq sequencing, and bioinformatics
We performed eDNA metabarcoding using seawater samples to assess the differences in marine fish communities inferred by eDNA between BAC treatments. Each 12 µL of first-round PCR contained 1 μL template DNA, a final 300 nM concentration of MiFish-U primers, which amplify approximately 170 bp fragments of mitochondrial 12S rRNA regions from teleost fish (Miya et al., 2015), in 2 × KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, USA). The thermal conditions of the first PCR were as follows: 3 min at 95 °C, 40 cycles of 20 s at 98 °C, 15 s at 65 °C, and 15 s at 72 °C, followed by 5 min at 72 °C. PCR for eDNA samples and negative controls (1 μL of pure water instead of template DNA) was performed in eight replicates. After the first PCR, eight replicates from each sample were pooled and purified using the SPRIselect Reagent Kit (Beckman Coulter, Brea, CA, USA) according to the manufacturer’s instructions. We then quantified the total DNA concentrations of the purified PCR products using a Qubit dsDNA HS assay kit and a Qubit fluorometer 3.0 (Thermo Fisher Scientific) and diluted them to 0.1 ng/μL.
Each 12 µL of second-round PCR contained adapter and 8-bp index sequences for high-throughput sequencing added to the first PCR products, as well as 1 μL template DNA and a final concentration of 300 nM for each forward and reverse primer in 2 × KAPA HiFi HotStart ReadyMix. The thermal conditions of the second PCR were as follows: 3 min at 95 °C, 12 cycles of 20 s at 98 °C, and 20 s at 72 °C, followed by 5 min at 72 °C. After pooling all second PCR products, we selected the product size (approximately 370 bp) of the library sample by electrophoresis using E-Gel SizeSelect 2% (Thermo Fisher Scientific) with the E-Gel Precast Agarose Electrophoresis System (Thermo Fisher Scientific), which was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The library sample was then sequenced using an Illumina iSeq with 2 × 150 bp paired-end kits (Illumina, San Diego, USA). We performed data preprocessing and analyses of iSeq raw reads using USEARCH v10.0.240 (Edgar, 2010) according to the method described by Sakata et al. (2020a). We discarded all reads from seawater samples corresponding to (i) freshwater fish, regarding it as contamination from the rivers flowing into Maizuru Bay, and (ii) some bony fish, which were regarded as contamination of domestic wastewater (detailed information can be seen in Appendix S2).