Abstract
High‐throughput sequencing of environmental DNA (i.e., eDNA metabarcoding) has become an increasingly popular method for monitoring aquatic biodiversity. At present, such analyses require target‐specific primers to amplify DNA barcodes from co‐occurring species, and this initial amplification can introduce biases. Understanding the performance of different primers is thus recommended prior to undertaking any metabarcoding initiative. While multiple software programs are available to evaluate metabarcoding primers, all programs have their own strengths and weaknesses. Therefore, a robust in silico workflow for the evaluation of metabarcoding primers will benefit from the use of multiple programs. Furthermore, geographic differences in species biodiversity are likely to influence the performance of metabarcoding primers and further complicate the evaluation process. Here, an in silico workflow is presented that can be used to evaluate the performance of metabarcoding primers on an ecoregion scale. This workflow was used to evaluate the performance of published and newly developed eDNA metabarcoding primers for the freshwater fish biodiversity of the Murray–Darling Basin (Australia). To validate the in silico workflow, a subset of the primers, including one newly designed primer pair, were used in metabarcoding analyses of an artificial DNA community and eDNA samples. The results show that the in silico workflow allows for a robust evaluation of metabarcoding primers and can reveal important trade‐offs that need to be considered when selecting the most suitable primer. Additionally, a new primer pair was described and validated that allows for more robust taxonomic assignments and is less influenced by primer biases compared to commonly used fish metabarcoding primers.
Highlights
Obtaining accurate biodiversity estimates is critical for effective management of our natural resources (Dudgeon et al, 2006; Maxwell & Jennings, 2005)
Primers for environmental DNA (eDNA) metabarcoding should: (a) amplify a short DNA fragment to maximize the recovery of DNA from environmental samples, (b) amplify a barcode with sufficient taxonomic resolution to allow for robust species assignments, (c) be specific to the taxonomic group of interest to avoid amplification and subsequent sequencing of nontarget taxa, and (d) amplify DNA from all species of interest with equal efficiency to minimize primer biases (Clarke et al, 2017; Coissac, Riaz, & Puillandre, 2012; Elbrecht & Leese, 2017)
While commonly used barcoding primers allow for robust species identification, they are often unsuitable for eDNA metabarcoding applications
Summary
Obtaining accurate biodiversity estimates is critical for effective management of our natural resources (Dudgeon et al, 2006; Maxwell & Jennings, 2005). PCR amplification of small barcode sequences from environmental DNA (eDNA) combined with high- throughput sequencing (HTS) technologies, commonly referred to as eDNA metabarcoding, has become an increasingly popular tool. Reducing primer-template mismatches can minimize biases arising from the PCR amplification but may inadvertently decrease the specificity of the primers to the taxonomic group of interest as these primers are more likely to bind to highly conserved regions (Pinol, Mir, Gomez-Polo, & Agusti, 2015) In addition to these trade-offs, species biodiversity varies extensively between geographic regions (Abell et al, 2008; Olson et al, 2001; Spalding et al, 2007) which further complicates the selection process as the performance of metabarcoding primers will vary depending on the species composition at the sampling location. A subset of all primer pairs were used in metabarcoding analyses of an artificial DNA community and eDNA samples to evaluate the performance of the in silico workflow and validate one of the newly designed primer pair
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