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Many studies have investigated the ability to identify species from environmental DNA (eDNA). However, even when individual species are identified, the accurate estimation of their abundances by traditional eDNA analyses has been still difficult. We previously developed a novel analytical method called HaCeD-Seq  (H aplotype  C ount from  eD NA), which focuses on the mitochondrial D-loop sequence. The D-loop is a rapidly evolving sequence and has been used to estimate the abundance of eel species in breeding water. In the current study, we have further improved this method by applying unique molecular identifier (UMI) tags, which eliminate the PCR and sequencing errors and extend the detection range by an order of magnitude. Based on this improved HaCeD-Seq pipeline, we computed the abundance of Pacific bluefin tuna ( Thunnus orientalis ) in aquarium tanks at the Tokyo Sea Life Park (Kasai, Tokyo, Japan). This tuna species is commercially important but is at high risk of resource depletion. With the developed UMI tag method, 90 out of 96 haplotypes (94%) were successfully detected from Pacific bluefin tuna eDNA. By contrast, only 29 out of 96 haplotypes (30%) were detected when UMI tags were not used. Our findings indicate the potential for conducting non-invasive fish stock surveys by sampling eDNA.

Estimation of tuna population by the improved analytical pipeline of unique molecular identifier-assisted HaCeD-Seq (haplotype count from eDNA)