Data analysis
First, all sequences were assembled and screened by length and quality
of reads using the Mother software package (v1.39.5) (Schloss et al.,
2009). The processed sequence reads were classified using the MiFish
pipeline
(http://mitofish.aori.u-tokyo.ac.jp/mifish/).
The parameters used in the MiFish pipeline analysis are described
elsewhere (Sato, Miya, Fukunaga, Sado, and Iwasaki, 2018). Subsequently,
representative sequences of individual operational taxonomic units
(OTUs) were matched to the sequence library for picking out sequences
clustered into individual OTUs using the Usearch program
(http://www.drive5.com/usearch/).
The random sequence tags (RST) at the end of sequences in the OTUs were
counted to quantify environmental DNA from each fish species as
described elsewhere (Hoshino and Hamada, 2017).