The TripleLock™ Advantage

Environmental DNA (eDNA) methods offer several advantages over conventional species survey methods, especially when carried out with the right amount of expertise at each step of the workflow. Such advantages include time and cost savings, especially when on-site detection is used. eDNA offers higher sensitivity and specificity reducing observer bias compared to conventional methods. eDNA also reduces disturbances to the species and its natural habitat.

Although eDNA science is relatively young, it is maturing rapidly, with many aspects of survey design, sampling methods, and laboratory analysis being accepted as best practices – and one day industry standards [1,2]. However, eDNA surveys that fail to comply with such a set of best practices can quickly fall into a trap of poor detection probability and erroneous results. While eDNA surveys can appear relatively simple to conduct, one cannot simply grab a water sample in a Nalgene bottle and go to a lab, hoping to achieve optimal detection probability or have high confidence in their results. Quality eDNA surveys are conducted with careful consideration of all aspects of survey design and sample collection and processing.

Here at Precision Biomonitoring Inc. we developed the TripleLock™ Platform which at its core is based on the widely accepted best practices for eDNA surveys, placing us at the forefront of the eDNA survey industry. Conducting robust eDNA work is especially important at this early stage if eDNA methods are to be accepted so that the advantages of eDNA can be employed for environmental assessments and conservation biology across the world. The focus of this blog will be to outline the advantages of the TripleLock™ Platform and how they compare to other methods in the industry.

Platform Core I: TripleLock™ qPCR Assays

The first core of the TripleLock™ platform consists of high-quality, rigorously validated qPCR assays for the detection of a target species. At Precision Biomonitoring Inc. we have conceived and developed a proprietary method for stringent assay development, which allows us to consistently design and validate species specific and highly sensitive qPCR assays. We developed our method meeting and exceeding The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. The MIQE guidelines outline several standards that should be adhered to for reliable and interpretable qPCR analyses, and by building our platform based on such rigorous standards we ensure replicable results while minimizing errors. Our TripleLock™ assays are also verified with highest level of international standards by a 3rd party ISO 17025 accredited laboratory. The advantages of following standardized methods are clear as they produce consistency in results. Assays need to conform to such rigorous standards and cannot be simply collected from different sources of literature and 3rd party reporting, since that would result in inconsistent standardization across the catalogue.

We take pride in developing our own assays in-house, as opposed to obtaining published assays in literature, as it allows us to optimize assays using our trusted methods and equipment. While the quality of assays in the eDNA literature may vary, one must remember that an assay consists of more than just a primer and probe set, it encompasses the exact type of qPCR reagents and instrumentation with which it was validated. This means that replicating the exact sensitivity of published assays on different equipment can entail extra costs and time, while also moving away from the MIQE standards if the published assay parameters can’t be replicated in vitro.

Additionally, our assays are designed with an internal positive control (IPC) to detect PCR inhibition, an absolute necessity to avoid false negative results. Many experts in eDNA science concur that checking for PCR inhibition is part of the foundation of good eDNA work [2].

Platform Core II: Optimal Survey Design

The second core of the TripleLock™ Platform is providing optimal survey designs to maximize the probability of detection for a target species. It is widely understood that the distribution of eDNA is not homogenous and depends on several variables including species ecology, water quality, pH, and turbidity, to name a few. It is for this reason that understanding the ecology of eDNA, and how best to sample for it, is so crucial. Our platform brings together optimal sampling designs, together with sophisticated sampling methods to conduct eDNA surveys with the utmost confidence.

We take into account the critical environmental variables, species biology and ecology, and site specific considerations that directly relate to the purpose of the study. At Precision Biomonitoring we use a proprietary method based on statistical analysis, including habitat occupancy modelling, to determine sampling optimality for a given eDNA survey. We are currently developing a software tool (patent pending) that will allow for customized survey designs based on the most up to date eDNA data available. This survey design tool adds another level of consistency and standardization to our workflow.

The foundation of a good sampling scheme is how the samples are collected to begin with. Our field sampling methods are far more advanced compared to other offerings. Precision Biomonitoring makes use of the ANDe™ sampling backpack, the first purpose-built aquatic eDNA sampling system [4]. The ANDe™ affords increased sampling versatility, allowing us to sample more effectively at depth, across transects, with increased throughput, and higher water volumes. For example, we can sample for species that typically reside on the bottom of a lake, at 30 feet deep, very easily using the ANDe™ system, which greatly increases the effectiveness of such a survey. This is something that cannot be done using small plastic bottles to sample surface water, a low fidelity practice offered by others in the industry. Our survey design methods along with our advanced sampling techniques let us conduct superior eDNA surveys compared to others in the eDNA industry.

Platform Core III: On-site Detection

The third core of the TripleLock™ platform is on-site detection which enables us to obtain rapid results in the field. On-site detection brings together several components of our field sampling methods including the ANDe™ filtration system, as well as on-site DNA extraction and qPCR analysis. The advantages of on-site detection come when obtaining results is time sensitive and a decision is pending. By making use of the Biomeme eDNA sample prep kit and Biomeme Three9™, a portable qPCR device, we can obtain results in the field and communicate them rapidly, via built in data connectivity, to a decision maker. This means no waiting on samples to be transported and analysed back at a central lab. Another advantage of these methods is that by filtering, extracting, and eluting DNA into a pH and thermostable buffer, in the field we minimize the potential for sample degradation during transit, thus decreasing the possibility of false negatives.

Removing the potential for sample degradation is an inherent advantage of our field methods compared to methodologies offered by others. By quickly extracting DNA from a filter into a stable buffer we preserve those valuable fragments of eDNA for analysis.

There are several eDNA analysis companies that offer sampling “kits” consisting of plastic bottles which are used to grab a small surface sample which then must be shipped all the way back to a lab before any analysis can start. Using handheld plastic bottles places the technician right where they need to sample, causing disturbances in the water as well as increasing the potential for contaminant transmission by said technician between sampling points, not to mention contamination stemming from the wet exterior of a bottle. By using the ANDe™ system we enable our technicians to be up to 3.6 meters from the point where the sample is being taken, and contain our sample in an enclosed and sterile, single-use filter cartridge, greatly reducing the potential for contamination.

Besides the serious disadvantages that stem from being limited to surface samples due to using bottles, other disadvantages and concerns arise from transporting water samples back to a lab for analysis. Transporting water samples introduces significant opportunity for eDNA to degrade. Transporting water samples on ice can potentially decrease degradation but does not eliminate it. Additionally, delays in shipment, especially during warm weather, make keeping water samples cold logistically difficult and at times impossible. By extracting and analysing DNA in the field we remove the potential for DNA to decay, and eliminate these significant logistical challenges.

Additionally by minimizing DNA degradation we eliminate the need for superfluous, ambiguous, and costly additional qPCR tests for so called total viable eDNA. These tests are often based on plant/algal DNA. Plant/algal cells are surrounded by resilient cell walls that prevent osmotic lysis, but animal cells are not. In theory this means that more plant/algal DNA may be preserved inside cells caught on filters prior to eDNA extraction, while animal cells easily lose integrity and release DNA, making it less long-lived compared to plant based eDNA. Inferences about the integrity of a target animal species DNA that are based upon the results of a plant based test are likely suspect. Furthermore, tests for total eDNA of a family of organisms, all fish for example, are also superfluous. Target DNA can be in extremely low abundance compared to total eDNA, shown to be > 0.0004% of total eDNA in some cases [5]. In theory, tests for total fish or amphibian eDNA most likely don’t give pertinent information about the integrity of a single target species eDNA, especially in cases where samples are taken from an isolated location or depth where other organisms in a family may not inhabit. A theoretical example of such a case is when an anthropogenically constructed habitat, i.e. a remediated river, is home to only a select few pioneer or reintroduced species. In these cases, a test for total fish eDNA may be interpreted as a false negative due to low abundance, not low integrity.

Precision Biomonitoring’s TripleLock™ Platform brings together multiple aspects of molecular biology, environmental survey design and sampling, and leading innovations to produce gold standard eDNA surveys. The development of our platform is anchored in the best practices for eDNA surveys as well as standards such as the MIQE guidelines, making Precision Biomonitoring an industry leader for eDNA surveys.

References:

1.            Wilcox TM, Carim KJ, Young MK, McKelvey KS, Franklin TW, Schwartz MK. Comment: The Importance of Sound Methodology in Environmental DNA Sampling. North Am J Fish Manag. 2018; 1–5. doi:10.1002/nafm.10055

2.            Goldberg CS, Turner CR, Deiner K, Klymus KE, Thomsen PF, Murphy MA, et al. Critical considerations for the application of environmental DNA methods to detect aquatic species. Methods Ecol Evol. 2016;7: 1299–1307. doi:10.1111/2041-210X.12595

3.            Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clin Chem. 2009;55: 611–622. doi:10.1373/clinchem.2008.112797

4.            Thomas AC, Goldberg CS, Howard J, Nguyen PL, Seimon TA, Thomas AC. ANDe TM : A fully integrated environmental DNA sampling system. 2018;2018: 1379–1385. doi:10.1111/2041-210X.12994

5.            Turner CR, Barnes MA, Xu CCY, Jones SE, Jerde CL, Lodge DM. Particle size distribution and optimal capture of aqueous macrobial eDNA. Gilbert M, editor. Methods Ecol Evol. 2014;5: 676–684. doi:10.1111/2041-210X.12206