Methodology for fish biodiversity monitoring with environmental DNA metabarcoding: The primers, databases and bioinformatic pipelines

Environmental DNA (eDNA) originates from cellular material shed by organisms into aquatic or terrestrial environments and can be sampled and monitored using metabarcoding technology, which is revolutionizing fish biodiversity monitoring. Several reviews concerning fish eDNA have focused on standard sampling methods and its applications, though a systematic review focused on marker genes, databases, and bioinformatic pipelines has not yet been published. Here, we present a comprehensive literature review of studies applying metabarcoding technology to fish eDNA for the purpose of fish biodiversity monitoring. We systematically provide the available universal primers used to amplify barcoding sequences from fish eDNA, and then discuss reference barcoding databases, relevant bioinformatic analyses, as well as developed pipelines. The performances of universal primers and their relevant reference databases are summarized. Combined use of multiple primer pairs targeted for more than one gene marker (e.g., 12S, 16S, Cytb, COI), and use of both local and public databases are recommended as approaches to improve the sensitivity and reliability of fish eDNA analyses. We also compare the effectiveness of eDNA metabarcoding to traditional approaches for monitoring fish biodiversity and highlight challenges and future perspectives associated with this new tool. Ultimately, we advocate for greater incorporation of eDNA analysis into fish biodiversity assessments to assist environmental managers. 1. Here we first present a comprehensive literature analysis of fish eDNA studying within multi-species and community monitoring by metabarcoding technology. 2. We systematically summarized performances of universal primers and their relevant barcoding reference databases, as well as the related bioinformatic pipelines. 3. We summarized the comparison between the effectiveness of eDNA metabarcoding and traditional survey on monitoring fish biodiversity and highlighted the challenges and future perspectives about the eDNA metabarcoding technology.