DOI: 10.5061/dryad.41ns1rnb7
title: Data from: Pitfalls during in silico prediction of primer specificity for eDNA surveillance

description: 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 are based on the number and position of the
 primer/non-target template mismatches. However, we speculate that
 non-specific 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 singleplex conventional PCR (cPCR). A subset
 of 12 primer pairs were further tested with SYBR Green-based or
 TaqMan-based singleplex 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 non-target 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 non-target 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 of non-specific amplification effects than other
 mismatches. To conclude, we show that 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.
description: For the details of the methodology, please refer to So et al.
 (2020) 
description: The data is the additional supporting information for the
 Manuscript "Pitfalls during in silico prediction of primer
 specificity for eDNA surveillance" published in Ecosphere by So
 <em>et al.</em> (in press). The dataset contains three excel
 files: (1) Table S1 summarizes the genetic sequences retrieved from
 GenBank used for primer development; (2) Table S2 provide the overview of
 the number of independent replicates for each species during the
 <i>in vitro</i> cPCR and qPCR validation on primer specificity
 in two separate spreadsheets; and (3) Table S3 provides the results of the
 <i>in vitro</i> primer specificity validation using cPCR (n
 =679) and qPCR (n = 226) and the corresponding Primer-BLAST
 prediction. Main manuscript: So
 Y.K.K., Fong J.J., Lam I.P.Y. & Dudgeon D. (accepted). Pitfalls
 during in silico prediction of primer specificity for eDNA surveillance.
 <i>Ecosphere</i>.