Is it really there? Addressing inadequate sampling and false detection in environmental DNA metabarcoding

The use of environmental DNA (eDNA) for biodiversity assessment has garnered significant attention due to its cost-effectiveness, rapidity, accuracy, and non-invasiveness in acquiring data on organisms in various habitats. Despite its numerous advantages over traditional methods, there remains a pressing need for improved protocol standardization. This review synthesizes potential strategies to address false detection in eDNA metabarcoding, including inadequate sampling and limited persistence of eDNA, contamination, primer biases, inhibition of DNA amplification, differentiation of eDNA from deceased organisms, incomplete databases, and sequencing errors or poor-quality sequences. Best practices such as collecting 1 to 2 liters of surface water, replicating polymerase chain reactions (PCR), using multiple genetic markers and primers to mitigate PCR primer biases, and implementing stringent contamination controls at each analysis step are recommended. While no universal recommendations currently exist for the diverse applications of eDNA metabarcoding in species detection, adopting these measures can enhance the reliability and accuracy of results. Despite the present limitations in sequence databases and the necessity for improved primer quality and analysis pipelines, the eDNA approach is anticipated to evolve and become more standardized. This review provides practical guidance on eDNA technology for researchers, consultants, and managers, facilitating its effective use in biodiversity assessments.