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. www.exponent.com 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