Pitfalls during in silico prediction of primer specificity for eDNA surveillance

Abstract

While high efficiency and cost‐effectiveness are two merits of environmental DNA (eDNA) techniques for detecting aquatic organisms, the difficulty of designing species‐specific primers can result in significant expenditure of time and money. During the in silico stage of primer development, primer specificity is predicted with alignment techniques such as BLAST that is based on the number and position of the primer/nontarget template mismatches. However, we speculate that nonspecific amplification is influenced by additional parameters, which lead to inaccuracies of in silico prediction. We performed in vitro specificity tests for 38 species‐specific primers selected for seven fishes and six turtles, using single‐plex conventional PCR (cPCR). A subset of 12 primer pairs were further tested with SYBR Green‐based or TaqMan‐based single‐plex quantitative PCR (qPCR). We disentangle the relative importance of mismatch properties (types and positions), primer properties (length, GC content, and 3′ end stability), PCR conditions (template concentrations and annealing temperatures), and PCR technique (cPCR, TaqMan‐based, or SYBR Green‐based qPCR) in determining the occurrence of amplifications. We then compared the PCR outcomes with the specificity check under two stringency scenarios based on alignment (i.e., BLAST search). We conducted a total of 679 cPCR and 226 qPCR analyses, with 90% of the reactions tested with nontarget templates. Primer pairs predicted by Primer‐BLAST to be specific rarely showed such specificity during the in vitro testing. BLAST searches correctly predicted the outcomes of around 67% of cPCR and qPCR, but had low sensitivity in detection of nontarget amplification (29–57%). Primer specificity increased significantly with total number of mismatches and annealing temperature, but decreased with higher GC content in the primer sequence. Mismatches that consisted of A‐A, G‐A, and C‐C pairings exerted 56% stronger reduction in nonspecific amplification effects than other mismatches. To conclude, we show that the prediction of primer specificity based only on the number and position of mismatches can be misleading. Our findings can be applied to increase the efficiency of the in silico primer selection process to maintain the relatively high efficiency and cost‐effectiveness of eDNA techniques.

A validation scale to determine the readiness of environmental DNA assays for routine species monitoring

“…Targeted eDNA assay validation is a multi‐step process. It can be divided into in silico validation (i.e., computer‐based tests for primer specificity), in vitro validation (i.e., laboratory tests with reference tissue samples), and in situ validation (i.e., field tests with eDNA samples) (see Goldberg et al., 2016; Langlois et al., 2020; MacDonald & Sarre, 2017; So et al., 2020). Understanding the utility of an assay requires both knowledge of the context in which it has been designed, and a broader understanding of how it was developed.…”

“…Tests with varying PCR chemistry, reaction volume, primer/probe concentration, cycling conditions, and technical replication will ensure optimal, standardized, and error‐free target DNA amplification (Bustin et al., 2009; Goldberg et al., 2016; Wilcox et al., 2015). The assay must then be tested against closely related and co‐occurring nontarget taxa to ensure specificity, which is not automatically guaranteed after successful in silico testing (Goldberg et al., 2016; So et al., 2020). Ideally, tissue‐derived DNA samples from multiple individuals spanning a defined geographic area are tested to ensure the assay is robust to genetic variants of target and nontarget species.…”

A validation scale to determine the readiness of environmental DNA assays for routine species monitoring

“…laboratory tests with reference tissue samples) and in situ validation (i.e. field tests with eDNA samples) (see Goldberg et al, 2016; MacDonald & Sarre, 2017; Langlois et al, 2020; So, Fong, Lam, & Dudgeon, 2020). Understanding the utility of an assay requires both knowledge of the context in which it has been designed, and a broader understanding of how it was developed.…”

“…Tests with varying PCR chemistry, reaction volume, primer/probe concentration, cycling conditions and technical replication will ensure optimal, standardised and error-free target DNA amplification (Bustin et al, 2009; Wilcox, Carim, McKelvey, Young, & Schwartz, 2015; Goldberg et al, 2016). The assay must then be tested against closely related and co-occurring non-target taxa to ensure specificity, which is not automatically guaranteed after successful in silico testing (Goldberg et al, 2016; So et al, 2020). Ideally, tissue-derived DNA samples from multiple individuals spanning a defined geographic area are tested to ensure the assay is robust to genetic variants of target and non-target species.…”

Detecting and identifying Schistosoma infections in snails and aquatic habitats: A systematic review

“…Primer design often relies on in silico predictions to limit cross-reactivity, which may have pitfalls [ 25 ], underscoring the need for follow-up confirmation. Sanger or high-throughput amplicon sequencing with comparison to sequences in GenBank [ 26 , 27 ] will be particularly crucial during elimination and surveillance operations to ensure positive results that are recorded by a band, Ct value, or a color change in a tube definitively belong to the schistosome or snail species responsible for the positive sample.…”