Simultaneous absolute quantification and sequencing of fish environmental DNA in a mesocosm by quantitative sequencing technique

Tatsuhiko Hoshino,Ryohei Nakao,Toshifumi Minamoto,Hideyuki Doi

doi:10.1038/s41598-021-83318-6

Tatsuhiko Hoshino, Ryohei Nakao + Show 2 more

Open Access

https://doi.org/10.1038/s41598-021-83318-6

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Abstract

The combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

Highlights

Investigating biodiversity, population size, and time-course changes associated with environmental change is important for the conservation of biodiversity
We calculated the relative abundances of the sequences of each fish species in the sequence library obtained by high-throughput sequencing (HTS) using MiFish primers for comparison with the digital PCR (dPCR) and quantitative sequencing (qSeq) results
The abundance of Environmental DNA (eDNA) quantified by qSeq ranged from 9.8 × 102–5.4 × 105 copies, 8.2 × 102–5.8 × 105 copies, and 8.3 × 102–2.5 × 105 copies/L for O. latipes, H. neglectus, and M. anguillicaudatus, respectively (Fig. 3, left, middle row)

Summary

Introduction

Investigating biodiversity, population size, and time-course changes associated with environmental change is important for the conservation of biodiversity. The abundance of eDNA (i.e. the fish mitochondrial 12S rRNA gene copy number) in the extracted DNA from the aquarium experiments using two mock fish communities was quantified by dPCR and qSeq. We used qPCR to quantify the eDNA of only three fish species, Hemigrammocypris neglectus, Misgurnus anguillicaudatus, and Oryzias latipes, whereas qSeq was used to quantify the eDNA of all five fish species used in this study.

Results

Conclusion