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).