Advantages and Limitations of Environmental DNA/RNA Tools
@Zerina Rahic
Aim of the paper
The aim of this manuscript is to review and assess the performance of eDNA/eRNA-based molecular approaches for deriving NIS-related biological information in the context of different stages of invasion and management applications.
Method 1.
Quantitative Polymerase Chain Reaction (qPCR, also known as real-time PCR) is an advancement on end-point PCR and is one of the most promising molecular tools for highly specific and sensitive detection of one or a few targets. It enables rapid turnaround and simultaneous analysis of multiple samples. Quantitative PCR assays rely on primers, or primers and a probe that have been designed to be specific for the target species. The amplification of this target can then be measured in real-time either through the use of intercalating dyes (Becker et al., 1996) or probe-based detection systems (Heid et al., 1996).
Biosecurity
From a biosecurity perspective, a potentially useful extension of the qPCR application is viability assessment for applications where determining the presence of a living organism is essential (e.g., assessing the success of applied treatment or a pest management programme; Darling and Frederick, 2017; Pochon et al., 2017). Nucleic acid intercalating dyes added to the sample before extraction (e.g., propidium monoazide [PMA]), that only penetrate damaged lipid membranes (i.e., dead cells), bind and covalently crosslink with double-stranded nucleic acids, inhibiting their extraction, and amplification (Nogva et al., 2003; Nocker et al., 2006). This approach is being used to distinguish between viable and dead cells of bacteria (Schnetzinger et al., 2013; Desneux et al., 2015). The so-called viability PCR has not yet been used for biosecurity applications and its applicability to assess viability of multicellular organisms is unknown. Substantial loss of DNA signal in viability PCR (Nocker et al., 2006) may cause false negative results in targeted surveys, e.g., failing to detect extracellular DNA of living organisms. Therefore, this method should be considered for application in combination with conventional end-point or qPCR, implying additional costs and effort.
Method 2.
Alternatively, to infer viability of the target organism(s), PCR tools can be applied to eRNA samples. Ribonucleic acid is a crucial component for protein synthesis, usually single stranded in a cell and transcribed from DNA by enzymes, i.e., produced in biologically active (living) organisms. Compared to eDNA, eRNA degrades more rapidly in the marine environment (typically hours to days, Thomsen et al., 2012b; Sassoubre et al., 2016), and is therefore considered a better proxy for detecting living biota. On the other hand, susceptibility of RNA makes it difficult to work with. Collection of RNA samples requires dedicated sampling protocols, more careful preservation, and storage. There is also additional processing time and costs associated with isolation and reverse transcription of RNA (Laroche et al., 2016), making it more expensive and challenging and thus a less attractive molecule to work with.
Method 3.
Inventory-Based Tools: Metabarcoding and Polymerase Chain Reaction Free High-Throughput Methods
The advent of high-throughput sequencing (HTS) has made it possible to produce enormous volumes of sequence data rapidly. Metabarcoding has become a well-established method for characterizing the biodiversity in different types of environmental samples (Chariton et al., 2010; Shokralla et al., 2012; Taberlet et al., 2012; Aylagas et al., 2014; de Vargas et al., 2015; Domaizon et al., 2017). It enables the identification of many taxa by matching short (typically 100–600 base pair) sequence reads obtained from HTS of PCR amplicons to reference sequences. Metabarcoding has proven to be very effective for characterizing marine communities and identifying potential pests (Pochon et al., 2013; Comtet et al., 2015; Zaiko et al., 2015c; Brown et al., 2016). However, there are some prerequisites required when applying metabarcoding for characterizing biotic assemblages and identifying potential NIS: (i) sufficient taxonomic resolution provided by the target gene, (ii) “universality” of the primers (i.e., capacity to amplify the target gene from a wide variety of taxa; see Geller et al., 2013), and (iii) availability of robust reference databases for reliable taxonomic assignments of obtained sequences (discussed in sections below).
Environmental RNA metabarcoding (targeting cDNA obtained through reverse-transcription PCR) is increasingly being used for characterizing viable assemblages associated with recent environmental change due to human marine activities (Pawlowski et al., 2014, 2016a; Dowle et al., 2015; Lejzerowicz et al., 2015; Pochon et al., 2015a; Visco et al., 2015; Laroche et al., 2016; Birrer et al., 2018), and is being considered for biosecurity applications (Pochon et al., 2017; Rey et al., 2018). However, there are uncertainties regarding potential biases associated with eRNA metabarcoding, resulting from e.g., overrepresentation of organisms with complex genomes and numerous copies of transcriptionally active marker genes (Gong et al., 2013), or a number of artifacts potentially occurring during RNA processing and PCR amplification (Laroche et al., 2017). Therefore, despite the potential of eRNA metabarcoding for differentiating living biodiversity, for example, in bilge (Pochon et al., 2017) or ballast water (Darling and Frederick, 2017; Rey et al., 2018), it remains challenging and requires further dedicated research and technological advancements to facilitate its uptake for biosecurity applications.
Future methods
There are several emerging and rapidly advancing PCR-free methods, such as shotgun sequencing (Wang et al., 2013), mitochondrial enrichment (Zhou et al., 2013), and gene enrichment (Mertes et al., 2011; Dowle et al., 2016). These have limited application for routine biosecurity surveys to date, as they require considerable sequencing and computing effort or additional laboratory processing which increases the associated cost. These methods have an important advantage though, since they overcome PCR-inherent biases which result in the preferential amplification of certain DNA (or cDNA) templates leading to the overrepresentation of some taxa and potentially missing others.
Overall
Despite the current limitations of eDNA-based techniques, they have a great potential for deriving biodiversity information and complementing marine biosecurity programmes worldwide. For example, even in the lack of quantitativeness, biodiversity information derived from molecular analyses (e.g., community metabarcoding or target species detection with qPCR) often surpasses that from conventional approaches (e.g., microscopy and morphological assessment) in terms of taxonomic resolution, precision, and sensitivity (Zaiko et al., 2016; Fletcher et al., 2017).
References
| (Author, year) | Link |
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| Anastasija Zaiko, Xavier Pochon, Eva Garcia-Vazquez, Sergej Olenin and Susanna A. Wood, 2018) | https://www.frontiersin.org/articles/10.3389/fmars.2018.00322/full |
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