Optimization of environmental DNA extraction and amplification methods for metabarcoding of deep-sea fish

Analyses of environmental DNA (eDNA) from macroorganisms in aquatic environments have greatly advanced in recent years. In particular, eDNA metabarcoding of fish using universal PCR primers has been reported in various waters. Although pumped deep-sea water was used for eDNA metabarcoding of deep-sea fish, conventional methods only resulted in small amounts of extracted eDNA and subsequent few or no PCR amplicons. To optimize eDNA metabarcoding of deep-sea fish from pumped deep-sea water, we modified conventional procedures of eDNA extraction and PCR amplification. Here, we propose a modified eDNA extraction method, in which a filter used for eDNA sampling was shredded and incubated in microtubes for efficient lysis of eDNA sources. Total eDNA yield extracted using the modified protocol was approximately six-fold higher than that extracted by the conventional protocol. The PCR enzyme Platinum SuperFi II DNA Polymerase successfully amplified a target region of fish universal primers (MiFish) from trace amounts of eDNA extracted from pumped deep-sea water and suppressed nonspecific amplifications more effectively than the enzyme used in conventional methods. Approximately 93% of the sequence reads acquired by next generation sequencing of these amplicons were derived from fish. The improved procedure presented here provided effective eDNA metabarcoding of deep-sea fish. • A modified eDNA extraction protocol, in which a filter was shredded and incubated in microtubes, increased eDNA yields extracted from pumped deep-sea water over the conventional method. • The PCR enzyme Platinum SuperFi II DNA polymerase improved the amplification efficiency of trace amounts of MiFish objectives in eDNA extracted from pumped deep-sea water with suppressing nonspecific amplifications. • The use of Platinum SuperFi II DNA polymerase for eDNA metabarcoding using MiFish primers resulted in the acquisition of abundant sequence reads of deep-sea fish through next generation sequencing.