Recognizing false positives: synthetic oligonucleotide controls for environmental DNA surveillance

Abstract

Summary1. Environmental DNA (eDNA) is increasingly used for surveillance and detection of species of interest in aquatic and soil samples. 2. A significant risk associated with eDNA methods is potential false-positive results due to laboratory contamination. 3. To minimize and quantify this risk, we designed and validated a set of synthetic oligonucleotides for use as species-specific positive PCR controls for several high-profile aquatic invasive species. 4. The controls consist of species-specific sequences for the species of interest, with the addition of a synthetic insert containing recognition sites for several restriction enzymes. 5. Following PCR, the presence of the synthetic insert can be detected using gel electrophoresis, restriction enzyme digests or DNA sequencing. For quantitative PCR (qPCR), false positives in environmental samples can also be detected using a fluorescent probe designed to detect the synthetic insert. 6. The generation of synthetic controls is a cost-effective, reproducible method that increases the power and reliability of eDNA testing by eliminating misinterpretation of false-positive results from laboratory contamination.

Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

“…A problem arising from the use of these types of positive extraction control is that contaminant DNA from these controls can be difficult to distinguish from the amplified target DNA, potentially leading to false positive detection results. To address this issue, Wilson et al (2016) suggest using synthetic oligonucleotides as positive controls. Replacing tissue derived controls with distinguishable short synthetic ones seems like a promising approach, reducing the risk of sample contamination, but such an approach has yet to gain widespread use in empirical studies.…”

“…We suggest this because sequencing error and the amplification of contaminant DNA can provide false positive results, which, if not identified as such, have the potential to initiate a costly chain of events, such as an attempt to eliminate a biosecurity incursion of a falsely-detected unwanted organism (Wilson et al 2016). We emphasise that the presence or lack of DNA sequence from a particular species should not, by itself, be used as absolute evidence of the presence or absence of the species in that sample environment; additional analysis is recommended to confirm the presence of priority species, particularly species of conservation concern, pathogens, or possible new incursions.…”

Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

“…However, as tissue powder is easily electrically charged, direct transfer of powder into the 96-well plate should be avoided. environmental DNA (eDNA), mock samples and even synthesised DNA (Wilson, Wozney, & Smith, 2015) can be could be used as positive control. The tissue can then be incubated according to extraction protocol and the lysate safely transferred into the 96-well plate, to reduce the risk of cross-contamination.…”

“…For studies with a high contamination risk, e.g. environmental DNA (eDNA), mock samples and even synthesised DNA (Wilson, Wozney, & Smith, 2015) can be could be used as positive control. However, generating and thoroughly characterising these can be costly and time consuming.…”

Improving the containment of a freshwater invader using environmental DNA (eDNA) based monitoring

“…However, eDNA-based species monitoring is technically challenging and continued research is needed to ensure the implementation of this technology in standard monitoring surveys (Rees et al 2014a, b;Roussel et al 2015). Publications to date have focussed on improving our understanding of; eDNA production and degradation rates (Klymus et al 2014;Maruyama et al 2014;Strickler et al 2014), the fate of eDNA within the environment (Jane et al 2014;Turner et al 2014a, b), the relationship between species biomass and eDNA quantities (Doi et al 2015;Takahara et al 2012) and the collection and processing methods (McKee et al 2014;Minamoto et al 2016;Renshaw et al 2014;Wilson et al 2016). While eDNA-based monitoring has been successfully used in both lentic water bodies and perennially flowing rivers, no study to date has used eDNA-based monitoring in rivers with intermittent flow.…”