Dr Shaun Wilkinson1,2
1Wilderlab Australia Pty. Ltd, , , 2Curtin University, ,
Abstract:
Environmental DNA metabarcoding involves dozens of interacting parameters, from sampling effort and filtration through PCR replication, marker selection and sequencing depth. Each can influence detection, yet exhaustive optimisation of every parameter and interaction is impractical. More importantly, maximising individual parameters does not necessarily maximise the utility of the overall workflow: additional effort may deliver diminishing improvements in detection while increasing cost, complexity and barriers to large-scale adoption.
Here, we use empirical data from a standardised, high-throughput eDNA metabarcoding workflow to examine five parameters that substantially influence sampling and analytical effort: water filtration volume, filter surface area, field sample replication, PCR replication and the number of vertebrate assays employed.
Across freshwater and estuarine applications, increasing filtration volume showed diminishing returns, with 1–2 L filtered through 7cm2 of 5µm membrane generally providing sufficient sampling effort within our workflow, while 2–4 L provided a practical operating range for coastal marine samples. Similarly, detection performance largely plateaued at six field replicates and four PCR replicates, with additional replication providing limited gains relative to the additional field, laboratory and sequencing effort. Finally, comparison of vertebrate marker combinations demonstrated that four complementary assays provided a strong balance between taxonomic coverage, detection performance and analytical effort.
As eDNA moves from research into routine environmental monitoring, we argue that standardisation should emphasise demonstrated performance, reproducibility and transparency rather than prescribing universally high levels of sampling or analytical effort. Methods should remain responsive to ongoing benchmarking and inter-laboratory comparison as technologies improve.