Freshwater eDNA 12S quantitative metabarcoding

Samplings were conducted in three tributaries (T1, T2, and T3) flow into the Kamitani Valley locates on the upper reaches of the Yura River in the Ashiu Forest Research Station, Kyoto University, Kyoto, Japan. Filtered 500 ml stream water at the lowest site of each tributary using a combination of a pore size 0.45 um Sterivex filter cartridge. Five artificially designed and synthetic internal standard DNAs, similar but not identical to the region of any existing fish mitochondrial 12S rRNAs, were included in the library preparation process to estimate the number of fish DNA copies.