DNA Testing and the Next Generation of Environmental Forensics

Andrew M. Deines, Ph.D.; William L. Goodfellow, Jr., BCES; Karen J. Murray, Ph.D.

Historically, assessing species presence has relied on a time consuming and labor intensive process. Advances in genetic testing techniques provide a faster, cheaper, and more accurate taxonomic assessment, which can be used to answer a range of environmental and ecological questions. The rapid advancements and substantial reduction in cost for DNA sequencing in the field of human genomics has created an opportunity to use the same technologies and specific modifications for broad use in environmental and ecological studies – from baseline studies to risk and impact assessments.

In the last two decades the development of genetic sequencing technology and the expansion of the field of genomics (analysis of an organism’s entire genetic code) has unlocked information hidden within cells, transforming the world of medical diagnostics and providing avenues to additional scientific discovery. In the field of human genetics, researchers are nearing the goal of providing a human genome for $1000 using “next-generation” sequencing— what scientists call new high-throughput and high resolution genetic tools.

These same DNA sequencing methods are being applied to many aspects of environmental and ecological study. DNA sequencing technology reported in the academic literature has addressed important evolutionary and ecological questions, leading to the development of new fields of study such as environmental genetics and conservation genomics. The development of new techniques almost monthly creates a wealth of opportunities for environmental assessments. Rapid DNA sequencing allows for the exploration of the genetic make-up of individual animals, populations, and assemblages of species in astonishing detail. It also allows us to navigate broad landscapes filled with complex environmental mixtures of thousands of individual organisms of many different species to better assess the provision of ecosystem services in natural and degraded landscapes.

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Current Uses: Cutting Edge Identification of Species

Environmental assessments typically involve the

identification of species potentially at risk and focus on delineating the presence of target species. Identification of threatened, endangered, and charismatic species is often of special interest.

In the same way that a grocer’s scanner reads barcodes standardized across products to find their price, DNA sequencing can recognize standardized regions of an organism’s DNA and compares that sequence to a database (a “library”) of sequences recovered from known samples verified by taxonomic experts. Historically, the ability to identify species has been limited to highly trained and specialized taxonomic experts relying on physical characteristics of whole specimens to make identifications. For rare, cryptic, or complex species Figure 1. The central target of much genetic groups this process has been idiosyncratic, slow, and testing in the environmental fields is to detect expensive. the presence and/or the identity of species present in a habitat (inner ring), for many The presence of animals in a habitat can be assessed purposes such as invasive species control or by detecting traces of DNA they have left behind. GMO testing (second ring). Technology allows Identifiable traces of DNA from epithelial cells in these genetics tests to be performed on a feces, milt or scales, for example can be collected wide variety of sample types and mediums (third ring), and to target a large portion of and detected in water samples even if an actual the plant, animal, and microbial biodiversity organism is not captured. This type of detection is (outer circle). particularly useful if the species are hard to capture because they are rare, resistant to traditional capture techniques, or the habitat is difficult to sample effectively. In these cases DNA sequencing can provide a cheaper and more sensitive indicator of species presence than traditional ecological assessments. The information from this type of sampling can be used to identify boundaries of species distribution and as a screening tool to focus more traditional sampling.

Genetic testing can also be used to identify key indicator species of ecosystem health, such as stream invertebrates. In some cases, collected tissues from invertebrate samples may be necessary for chemical toxicity testing and therefore not available for traditional identification and preservation. As DNA sequencing requires only small amounts of tissue to identify organisms, these issues can be overcome using environmental genetic techniques.

www.exponent.com The collection of environmental DNA was an instrumental tool used to monitor the spread of invasive species such as Asian Carp from the Mississippi and Illinois Rivers into the Great Lakes (Jerde et al. 2011). Ballast water carried on board inter-continental shipping vessels is a major vector for the spread of invasive species between ports around the world, causing millions of dollars of environmental damage. Detection of invasive species in ballast water using DNA testing could prompt treatment before discharge and assuage the costs of unnecessary treatment as well as invasive species introduction.

Genetic testing is particularly useful for organisms that are not easily identified by sight, such as microbes. Traditional methods of identifying microbial communities have relied on growing the organisms in a lab and examining phenotypes, a procedure which is estimated to miss more than 99% of species, as many organisms are resistant to being cultured.i This method tends to repeatedly isolate the most easily grown organisms, the “weeds”, and is likely to underrepresent the microbes of interest in many environments.ii Microbial communities are essential to the bioremediation of crude oil and other organic compounds in surface and groundwater, are vital to the proliferation of many plant speciesiii and carry out important hydrogeochemical processes which control nutrient cycling though soils and water waterways, but may also be an indicator or cause of pollution and vector of disease. In disturbed sites, the absence of key microbial species may preclude attempts to remediate and reintroduce plantsiv.

Fecal coliform in waterways have long been assumed to be from human sources due to lack of proper wastewater management. However, genomic tools have facilitated the identification of fecal coliform from sources such as domestic animals, water fowl, and wildlife. These results demonstrate that genetic testing can reveal microbial species composition and indicate the efficacy of bioremediation as well as informing the development of management options which more accurately target sources of microbial contamination.

Potential Uses: Frontiers in Environmental Genetics

Research in environmental genetics is rapidly evolving and it is now possible to use genetic testing to identify most, if not all of the species present in a particular habitat. For instance, genetic testing may be used to collect baseline data about the species present in a site slated for development. These samples do not need to be analyzed immediately; they can be collected and stored for future analysis when necessary.

Genetic testing can also be used for environmental monitoring, since environmental DNA samples are typically smaller in volume with longer holding times than other tissue analyses, it is possible to collect and store samples over time (say each year) for a particular site, which can be used to establish temporal landscape changes. Because of the reduced number and volume of samples, genetic testing is ideal for remote areas, locations with security complications or regions that do not have adequate

www.exponent.com facilities to support large scale ecological sampling. Genetic testing can also limit disturbances that can occur in other types of environmental sampling.

Next-generation sequencing has also allowed assessment of DNA that belongs to organisms that have long been dead. In one study, researchers examined degraded ancient DNA in permafrost to reconstruct plant communities from the last glacial period. The information collected was compared to modern biodiversity and used to determine whether any taxonomic shifts may have occurred due to climate changev. Results with ancient DNA suggest that DNA sequencing may soon be able to reconstruct recent and/or historical biological communities to describe, post-hoc, baseline communities relative to an environmental disturbance.

The efficiency and costs of genetic testing diversity assessments are lower than similarly scaled assessments using traditional methods (Ji et al. 2013), the main benefits include: faster timelines, higher taxonomic resolution, and reduced bias in taxon sampling. The availability of verified libraries of reference samples of well described species and the selection of regions in species’ genomes robust to environmental conditions which may degrade DNA are some of the hurdles now being addressed by researchers (Taberlet et al. 2012).

Choosing the Right Tool

There are multiple techniques and imposing terms used for various DNA sequencing technologies such as: shotgun, Sanger, Illumina, 454, pyro, Miseq, MinION, and PacBio. In addition to DNA barcodes, and eDNA, environmental genetic analyses often examine raw genetic sequences as well as many other assays based on DNA sequencing: RAD-tags, microsatellites, DNA microarrays and Single Nucleotide Polymorphism genotyping are among some of the most popular. Each technique has its particular advantages and limitations and choosing the most appropriate requires navigating a complex and growing array of sampling methodology, laboratory techniques, and statistical possibilities in this rapidly diversifying field. In addition to the cost of a particular technique other considerations include: whether the sample is taken from live or dead organisms, or a complex mixture of different species per sample, whether interest lies in a particular species or many species, whether the species in question have been sequenced before, the number of samples, and cost per sample.

Selecting regions of DNA to analyze from the billions in every individual is difficult and depends on the question to be answered; areas need to be sufficiently different in order to tell species apart and there is often debate among experts about the degree of differences that constitutes unique species. Entirely different regions are required to identify individuals, populations or parentage and different regions within a single individual may give different results. In the case of next generation sequencing millions of base pairs can be accomplished in a few hours yielding extremely large datasets.

www.exponent.com Scientists are left with the task of creating order out of jig-saw puzzle chaos. Parsing complex mixtures of species from such datasets requires specific bioinformatics expertise (i.e., advanced computing power and customized programming skills). Therefore, given the scope of each project, experience handling large datasets may also be an important factor.

Once vast stores of genetic information are produced, it is often useless without the means to interpret it and this may mean comparison to known samples in a “reference library.” Some reference libraries can be found in the peer reviewed literature or in comprehensive sequence databases such as GenBankvi Barcode of Life Data System (BOLD)vii. Moreover, once the sequences have been identified to species, additional statistical analysis play major roles in understanding the relationships between species and environmental variables of interest. The most important component of finding the appropriate genetic method, as in any scientific study, is having a clearly defined question. Incorrect assumptions could mean using the wrong approach and risking time and money, or worse yet, unknowingly ending up with the wrong answer. Ultimately the design and interpretation of genetic studies must be made in collaboration with a trained biologist that understands the scope of the problem to be addressed and is abreast of the best techniques available to deliver an answer while avoiding major challenges.

Next Steps

Although these emerging techniques play a large role in academic research, the rapid progression of these techniques means they don’t always benefit from standardized methodology and have not made the leap into everyday use in environmental assessments. Producing reliable results that can be used for decision making will depend on the ability of laboratories and organizations (e.g., the American Society for Testing and Materials- ASTM) to create standardized methods with the development of inter-and intra-laboratory variability estimates. This is especially problematic when scaling up to projects that involve collecting large amounts of samples. Although there are an increasing number of commercial labs equipped with both the technology and experience to do this kind of work. The growing capacity for rapid and cost-effective analysis of genetic data is allowing environmental genetics to play an increasing role in environmental risk and damage assessments, particularly in terms of identifying unknown species and determining the presence or absence of rare and difficult to sample species. Clients may wish to undertake pilot studies to evaluate the feasibility and usefulness of these emerging tools. The environmental genetics team at Exponent consists of scientists that have used these tools as part of their assessments of biological diversity to address environmental issues. Their expertise provides the capacity to assist in the design, implementation, and interpretation of environmental genetic studies.

www.exponent.com For More Information, Contact:

Andrew M. Deines, Ph.D. Senior Scientist 425-519-8753 | [email protected] www.exponent.com/andew_deines

William L. Goodfellow, Jr., BCES Principal Scientist & Practice Director 717-793-2791 | [email protected] www.exponent.com\william_goodfellow

Karen J. Murray, Ph.D. Managing Scientist 978-461-4610 | [email protected] www.exponent.com/karen_murray

i Staley JT, Konopka A. Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annual Reviews in Microbiology 1985; 39(1):321–346. ii Hugenholtz. Exploring prokaryotic diversity in the genomic era. Genome Biol 2002; 3(2):1-0003. iii Jeffries et al. The contribution of arbuscular mycorrhizal fungi in the sustainable maintenance of plant health and fertility. Biol Fertility Soils 2002; 37: 1-16. iv Anderson. Microbial ecophysiological indicators to assess soil quality. Agriculture, Ecosystems and the Environment 2003; 98:285-293. v SØNSTEBØ, JH, et al. Using next-generation sequencing for molecular reconstruction of past Arctic vegetation and climate. Molecular Ecology Resources 2010; 10:1009–1018. doi: 10.1111/j.1755-0998.2010.02855.x. vi http://www.ncbi.nlm.nih.gov/genbank/ vii http://www.barcodinglife.org/

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