GeneWatch THE MAGAZINE OF THE COUNCIL FOR RESPONSIBLE GENETICS | ADVANCING THE PUBLIC INTEREST IN BIOTECHNOLOGY SINCE 1983
Volume 26 Number 5 | Nov-Dec 2013
Featuring: Rob DeSalle Science, Plain and Beautiful David Schindel on the Barcode of Wildlife project Jonathan Deeds on identifying mislabeled seafood ISSN 0740-9737
GeneWatch November-December 2013 Volume 26 Number 5 Editor and Designer: Samuel W. Anderson Editorial Committee: Jeremy Gruber, Sheldon Krimsky, Ruth Hubbard GeneWatch is published by the Council for Responsible Genetics (CRG), a national, nonprofit, taxexempt organization. Founded in 1983, CRG’s mission is to foster public debate on the social, ethical, and environmental implications of new genetic technologies. The views expressed herein do not necessarily represent the views of the staff or the CRG Board of Directors. Address 5 Upland Road, Suite 3 Cambridge, MA 02140 Phone 617.868.0870 Fax 617.491.5344 www.councilforresponsiblegenetics.org
board of directors
Sheldon Krimsky, PhD, Board Chair Tufts University Evan Balaban, PhD McGill University Paul Billings, MD, PhD Life Technologies Corporation Robert DeSalle, Phd American Museum of Natural History Robert Green, MD, MPH Harvard University
In Memoriam: Adrienne Asch The Council for Responsible Genetics is saddened by the passing of Adrienne Asch on November 19. A former member of the CRG Board of Directors, Adrienne was most recently director of the Center for Ethics at Yeshiva University and the Edward and Robin Milstein Professor of Bioethics as well as professor of epidemiology and population health and family and social medicine at Albert Einstein College of Medicine. A pioneer in disability studies, Adrienne was always trying to change the all too frequent belief that disability was tragedy rather than just another aspect of human life and maintained that the rights of disabled women should be as much a feminist concern as those of able-bodied ones. Her work was devoted to the ethical, political, psychological, and social implications of human reproduction and the family. She produced tremendous scholarship that stood at the nexus of bioethics, disability studies, reproductive rights and feminist theory. Her publications include two volumes of which she was a co-editor: Women with Disabilities: Essays in Psychology, Culture, and Politics (1988, with Michelle Fine) and Prenatal Testing and Disability Rights (2000, with Erik Parens). Adrienne was fiercely committed to defending the rights of all human beings, particularly the rights of children with disabilities and opposed the use of prenatal testing and abortion to select children free of disabilities. As she wrote in one of her frequent contributions to GeneWatch: My moral opposition to prenatal testing and selective abortion flows from the conviction that life with disability is worthwhile and the belief that a just society must appreciate and Continued on page 4
Jeremy Gruber, JD Council for Responsible Genetics
comments and submissions
Rayna Rapp, PhD New York University Patricia Williams, JD Columbia University staff
Jeremy Gruber, President and Executive Director Sheila Sinclair, Manager of Operations Samuel Anderson, Editor of GeneWatch Andrew Thibedeau, Senior Fellow Vani Kilakkathi, Fellow Cover Design Samuel W. Anderson Editorial & Creative Consultant Grace Twesigye Unless otherwise noted, all material in this publication is protected by copyright by the Council for Responsible Genetics. All rights reserved. GeneWatch 26,5 0740-973
Cover photo by Grace Twesigye
GeneWatch welcomes article submissions, comments and letters to the editor. Please email firstname.lastname@example.org if you would like to submit a letter or any other comments or queries, including proposals for article submissions.
founding members of the council for responsible genetics Ruth Hubbard • Jonathan King • Sheldon Krimsky Philip Bereano • Stuart Newman • Claire Nader • Liebe Cavalieri Barbara Rosenberg • Anthony Mazzocchi • Susan Wright Colin Gracey • Martha Herbert • Terri Goldberg November-December 2013
GeneWatch Vol. 26 No. 5
4 President’s Note: DNA Barcoding and Misplaced Priorities By Jeremy Gruber 5 DNA Barcoding Ready for Breakout It’s an eminently practical technology that hasn’t received much fanfare – but that may be about to change. By Mark Stoeckle 7 Science, Plain and Beautiful DNA barcoding may not be “sexy,” but it is beautiful in its simplicity and expediency. By Rob DeSalle 9 Citizen Science Easy enough for kids and amateur scientists to use, you might say DNA barcoding is inherently democratic. By Ellen Jorgensen 11 What’s the Holdup? Outside academia – and especially in highly regulated environments – DNA barcoding is only gradually replacing slower, less accurate, and more expensive methods. Which begs the question: Why? By Peter Christey 14 The Barcode of Wildlife Project DNA barcoding takes center stage in the international effort to combat wildlife trafficking. By David Schindel 17 Foiling Poachers With DNA Barcoding In South Africa, where poachers and smugglers continually adapt to stay a step ahead of environmental law enforcement, DNA barcoding is about to be a game changer. By Jacques du Toit 18 Identifying Commercial Seafood DNA barcoding is a valuable tool for FDA for the species identification of seafood. By Jonathan Deeds 19 Uncovering Herbal Fraud With DNA Barcoding How do you know your Gingko biloba is Ginkgo biloba? A new study shows that’s a very valid question (and answers it). By CRG Staff Image: S.W. Anderson
20 Monitoring Animal Products and Feeds With DNA Barcoding An interview with Haile Yancy, U.S. Food and Drug Administration. 22 Measuring the Health of Aquatic Ecosystems With DNA Barcoding The U.S. Environmental Protection Agency is finding DNA barcoding to be faster, more precise, and more accurate than previous methods of aquatic bioassessment. By Erik Pilgrim 23 Endnotes
Volume 26 Number 5
Memoriam, continued from p. 2 nurture the lives of all people, whatever the endowments they receive in the natural lottery. I hold these beliefs because there is abundant evidence that people with disabilities can thrive even in this less than welcoming society. Moreover, people with disabilities do not merely take from others, they contribute as well-to families, to friends, to the economy. They contribute neither in spite of nor because of their disabilities, but because along with their disabilities come other characteristics of personality, talent, and humanity that render people with disabilities full members of the human and moral community.
When recently asked what she saw as the most pressing bioethical problem today, Adrienne responded: There isn’t enough social justice discussion in bioethics, whether it’s about healthcare or about the equality of all people with their different characteristics. I’m looking for a society that respects the uniqueness and the contributions of every individual, and the capacity of each individual to contribute according to their abilities ... and to be provided for according to their needs. And that’s an old socialist-Marxist notion, but it’s the society I’m interested in creating. And I’d like a bioethics along with a feminism that was interested in creating that. I think that’s out of fashion but that’s really what I’m looking for.
Her sharp mind and her love of ideas will be sorely missed by her friends and colleagues. nnn
President’s Note: DNA Barcoding and Misplaced Priorities By Jeremy Gruber As a bioethical organization, the Council for Responsible Genetics often finds itself in the role of the critic. We identify and raise awareness of bad science and the excesses, misplaced priorities and foolhardiness attendant to reductionist approaches toward developing genetic science and technology. Rarely do we see the need to play the role of cheerleader among a loud and diverse chorus promoting biotechnology development at all costs. DNA barcoding is a critical exception. DNA barcoding is a simple, standardized way of identifying species from a small sample of DNA. In animals, for example, a set of about 650 base pairs is scanned, with unique results for each species. That “barcode” can then be searched against a reference library for a matching barcode, which will tell you what species your sample came from. DNA barcoding has almost limitless potential as both a conservation and consumer protection tool. It can be used to enhance protection of endangered species by aiding in the identification of bushmeat and other animal products. It can be used to conduct detailed biosurveys to identify what lives in a specific area in order to determine whether the ecosystem is in distress, or whether protected or invasive species are present. It can be used to identify falsely labeled consumer products, from sushi to herbal supplements to medicine. And it’s such a simple tool
that non-scientists can pursue it for both educational purposes and the common good. You would think such a fantastic tool, developed as a result of the revolution in our understanding of genetics, would receive regular attention in both major media and biotechnology-oriented publications. You’d be wrong. Indeed, in the realm of conservation genetics, most media and public attention has been drawn in recent months to discussions of resurrecting extinct and endangered species through cloning, an eyebrow-raising proposal that has succeeded in grabbing headlines and funding for conferences but which has dubious practical benefits in any near term for conservation. Meanwhile, every day around the world scientists are developing DNA barcoding technology, building reference libraries, and diversifying its applications. For these very reasons, this special issue of GeneWatch is devoted to getting our priorities straight by offering our readers a comprehensive picture of the benefits of this important technology and the challenges we face in promoting its broader acceptance and use. nnn Jeremy Gruber, JD, is President of the Council for Responsible Genetics.
DNA Barcoding Ready for Breakout It’s an eminently practical technology that hasn’t received much fanfare – but that may be about to change. By Mark Stoeckle
DNA barcoding is simple enough to be employed by high school students and versatile enough to help ferret out food fraud, rein in illicit trade in endangered timber, recognize disease-carrying mosquitos, reveal what tiny insects and big mammals eat, and speed discovery of new species on land and sea. DNA barcode similarities and differences among species help make plain the story of evolution. In its first decade DNA barcoding racked up these and many other successes. What lies ahead is expansion into routine use by consumers, educators, citizen scientists, DIY biologists, and regulatory agency enforcers. The roots of DNA barcoding lie deep in evolutionary study and the recognition that the history of life forms is written in their DNA. The manifest story began at the University of Guelph in 2003, when Paul Hebert and colleagues made an imaginative proposal: a universal DNA identification system for all macroscopic life.1 They focused on a 650 base pair region of mitochondrial cytochrome c oxidase I (COI), previously reported recoverable from diverse animal phyla by polymerase chain reaction using a single primer pair. According to the limited data then available, this region varied little within animal species but generally differed among even closely-related species, making it usually straightforward to match sequences to species names. Hebert and colleagues called this biological identifier a “DNA barcode” Volume 26 Number 5
by analogy to the Uniform Product Code (UPC) for commercial goods, and presciently proposed a universal reference library. Recognizing that the value of DNA barcoding lay in standardization, it was nonetheless evident at the outset that alternative targets or amplification strategies would be needed for certain groups, including plants and fungi, as COI differed little among species in the former and often contained introns (non-coding pieces of DNA) in the latter. In 2003, the Alfred P. Sloan Foundation sponsored workshops at Cold Spring Harbor Laboratory which explored making this vision a reality. Participants envisioned a DNAbased standardized identification system of great use to society and science: Organisms in any life stage could be unambiguously named from bits and pieces, including morphologic “look-alike” cryptic species, by experts and non-experts, at relatively low cost, and potentially via automated devices. The biggest challenge was how to build the library, which would involve sampling multi-millions of specimens, each of which had to be identified by experts and archived in museums or herbaria so they would be available to be re-examined. New database practices were needed to link sequence records to individual specimens. The need to organize a wide scientific community effort led to the Consortium for the Barcode of Life (CBOL), an international initiative GeneWatch 5
devoted to developing DNA barcoding as a global standard for the identification of biological species, inaugurated in April 2004 with support from the Alfred P. Sloan Foundation. As of 2013, the Consortium has over 200 member organizations from over 50 countries. Participants at CBOLsponsored workshops agreed on standard loci for animals, plants and fungi.2 The Gordon and Betty Moore Foundation provided crucial early funding that helped establish DNA barcoding as a scientific enterprise. In parallel to CBOL, major support from Genome Canada and the Ontario Genomics Institute helped launch the International Barcode of Life (iBOL) in 2010 to speed library building and promote regional networks and taxon and ecosystem campaigns. The University of Guelph built an online database and workbench, Barcode of Life Datasystems (BOLD). As of October 2013, BOLD contains over 2.5 million barcode records from over 190,000 named animal, plant, and fungal species. The barcoding initiative has highlighted how much we don’t know about macroscopic biodiversity. Many barcoded specimens have turned out to represent new species, and many more as yet undescribed species are represented in databases, awaiting formal names. The underlying challenge is the immense number of living species: about 2 million described among an estimated 8 million existing. For animal COI barcodes, BOLD recently instituted an automated Barcode Index Number (BIN) system that assigns numbers to clusters of closely related sequences.3 In well-studied groups, BINs usually correspond to species, suggesting this algorithm can also organize records of unidentified specimens into sets representing new taxa and speed formal descriptions. 6 GeneWatch
Although new sequencing technologies may supplant current Sanger standard, a decade of experience says the key elements — namely, agreed-upon standard gene region(s) and high quality sequence records from documented specimens — are irreplaceable. Much wider use of DNA barcoding in everyday applications awaits. Potential arenas include food and herbal product testing, education, and citizen naturalists. Consumers want to know more about what they eat: where it comes from, whether it is healthy for you, and whether it is produced in a way that is good for the environment, for example. At the same time, barcoding uncovers routine mislabeling of diverse foods — in such cases, forget worrying about where it came from, it is not even the species the label says it is! These include fish (1/3 of U.S. fish products are mislabeled), ground meat (horsemeat-in-hamburger scandal in Europe), olive oil, cheese, tea, and pet food, with costs to consumers and threats to the environment. Investigations to date suggest if a product is expensive and can’t be readily identified by appearance, it is at risk of mislabeling. A recent barcoding study of herbal products, which are morphologically unidentifiable even by experts, supports this point — the majority contained contaminants, substitutions, or fillers.4 Regulatory agencies are adopting this technology, perhaps a prelude to routine use by food and herbal product distributors, enabling certification, supplemented with consumer level testing by agencies or individuals. In education, DNA barcoding offers a relatively simple and widely applicable technology that allows students to design and carry out diverse investigations. Science is a process of discovery, but most high school
laboratory exercises have pre-determined outcomes. DNA barcoding enables students to make real discoveries and contribute to reference databases. Model projects in a few dozen schools, notably in NYC, California, and Ontario, have demonstrated the potential.5-7 These could serve as templates and be expanded to be a routine component of high school biology. Another plus is that comparing DNA barcode sequences to reference databases provides students a direct look at evolution. DNA barcoding democratizes access to knowledge about biodiversity. It can help citizen naturalists and DIY biologists make modern day voyages of bio-discovery in urban, rural, and wild environments. Just as GPS went from a cumbersome research tool to an everyday application, DNA barcoding has potential appeal to a large, diverse set of interested individuals. For wider commercial use, such as by food distributors, certified, affordable, and speedy barcode testing services are needed. Robust and rapid protocols for DNA extraction, amplification, and sequencing are already available, indicating the hurdles, if any, are in business aspects. For consumer, educational, and DIY use, easy-to-use inexpensive kits with mail-in sequencing services would likely boost demand. We look forward to DNA barcoding’s breakout applications. nnn Mark Stoeckle, MD, is Senior Research Associate in the Program for the Human Environment at The Rockefeller University. He has been involved in the DNA Barcoding Initiative since its beginnings in 2003.
Science, Plain and Beautiful DNA barcoding may not be “sexy,” but it is beautiful in its simplicity and expediency. By Rob DeSalle
Image: S.W. Anderson
While doing a literature search for a manuscript recently, I came across an interesting web blog from Michael Eisen, a geneticist at UC Berkeley and a Howard Hughes Investigator. The article started with “I confess, I wrote the arsenic DNA paper.” For those of you not familiar with this story, the paper was published in Science in 2011, describing the discovery of a bacterial species that used arsenic instead of phosphorus in its nucleic acids. This bizarre finding received tons of press, but alas was shown to be incorrect. Dr. Eisen, Volume 26 Number 5
later in the web article, confesses that he didn’t actually write the Science paper, but rather uses it as an example of a disturbing trend in scientific publication. I confess that I have my own “I confess” story. I confess I wrote a paper in 1992 describing the sequencing of a tiny fragment of DNA from a termite entombed in thirty million year-old amber. The paper was published in Science and also gained a lot of public attention. While I still stand by this work, subsequent attempts to isolate DNA from amber preserved
specimens have failed miserably. The ancient DNA community stepped back and started to demand higher standards for ancient DNA work, and now the oldest specimen that has yielded DNA that the research community accepts is only 700,000 years old. The reason I am dredging up the arsenic DNA and amber insect stories is that they demonstrate a curious aspect of publishing and of public perception. After publishing the original Science paper, several better controlled experiments were done on the amber insect specimen GeneWatch 7
and these were published in lower visibility journals with no fanfare. I consider these subsequent papers orders of magnitude better than the original Science paper. Many scientists who have published in Nature or Science (the two preeminent science journals worldwide) will more than likely not point to their publications in those journals as their best science (but they will list them prominently on their CVs). Most scientists would agree that getting the public to understand our work is very difficult and frustrating. But it is perhaps one of the most important things we can do as scientists. And given the current attitude many United States citizens have toward science, it has become essential that the public understand science better. Granting agencies like the National Science Foundation require that a proposal have what is called a “Broader Impacts” section. These broader impacts are ways that researchers propose to disseminate their research to the general public. Even when the research is conducted at a museum (as mine is), it is still very difficult to push scientific work out into the public. Unless it is incredibly “sexy,” the news media usually ignores it. This means that many highly worthwhile scientific endeavors get overlooked or are invisible to the general public. One example of “hidden” basic science is the hard basic work that virologists have done in trying to understand infectious entities like HIV and HPV. The public more than likely is not interested in how these viruses replicate or how their genes are expressed, yet these subjects comprise the scientific background for medical advances against the viruses. Another example is the basic science that grew up around research on cancer. The National Cancer Institute was 8 GeneWatch
founded in 1937, and over the past seventy-five years this institute has funded an enormous number of proposals, many of them peripheral to cancer, but all of them informative to basic science. Yet another example is the basic genetic work that conservation biologists have accomplished in the last two decades characterizing variability in endangered and threatened species. While this work has aided in conservation policy, much of it goes unnoticed by the public. So, short of scientists hiring publicity agents (some do, while others have strong public information departments at their institutions), what is a scientist to do? One strategy is to keep chasing “sexy” stories. A sexy story will almost always make it into a high visibility journal. The problem is, what is considered sexy is up to the expertise of the editors at the high visibility journals, as Michael Eisen points out. A more sure way to accomplish this is to play the “broader impacts game” and put serious thought into how to disseminate information to the public. A somewhat neglected scientific endeavor in the “hidden” category of organismal biology is DNA barcoding, the topic discussed in this issue of GeneWatch. It makes sound basic science sense to catalog organisms. Sir Robert May once said: “Without taxonomy to give shape to the bricks, and systematics to tell us how to put them together, the house of biological science is a meaningless jumble.” This quote points to the essential importance of cataloguing biodiversity. The problem is, cataloguing organisms does not sound “sexy” to the public (unless of course the Loch Ness Monster is actually found, named, archived and catalogued). DNA barcoding wants to obtain a universal sequence from as many organisms as possible as a means to cataloguing
biodiversity. In the process, specimens are collected, archived and catalogued, producing an invaluable resource in and of itself. The barcode sequence is “icing on the cake.” DNA barcoders, though, have discovered an excellent way to disseminate the information and idea behind the endeavor. They use the approach to teach kids about biodiversity, genetics and biology. Because of its inherent simplicity and low cost, the approach is amenable to dissemination in this way. Cold Spring Harbor Laboratories and their DNA Learning Center have developed a program called the Urban Barcoding Project. The UBP, initially supported by the Sloan Foundation and currently supported by the Pinkerton Foundation, enlists high school students from the New York City area to characterize a group of organisms using barcoding. During the process, students learn a spectrum of basic biological principles and techniques. The high school kids take on projects covering everything from the basic biology of specimen identification to DNA sequencing and informatics. It is a wonderful example of citizen science. I attended the first annual student awards competition, and the number of lit lightbulbs above the kids’ heads was stunning. To these kids, DNA barcoding and biodiversity was incredibly sexy. Eleanor Roosevelt once said: “No matter how plain a woman may be, if truth and honesty are written across her face, she will be beautiful.” And so goes science. Plain, basic, in-thetrenches science like DNA barcoding is important and beautiful, and it needs to be made more accessible to the general public. nnn Rob DeSalle, PhD, is a Curator and Professor at the American Museum of Natural History and The Sackler Institute for Comparative Genomics. November-December 2013
Citizen Science Easy enough for kids and amateur scientists to use, you might say DNA barcoding is inherently democratic. By Ellen Jorgensen In 2008, I happened to see an intriguing news story about two New York City high school girls who had used a new DNA-based identification method to determine if their neighborhood sushi restaurants were selling mislabeled fish. That was my first encounter with the technique known as DNA barcoding. Since then, I have helped hundreds of amateur scientists use barcoding to question the identity of everything from ‘heirloom’ oranges to ‘beef ’ meatballs to the diversity of Alaskan plants. The idea of identifying species through a very short genetic sequence, rather like the manner in which a supermarket barcode identifies products, was first proposed in a 2003 paper by Dr. Paul Hebert, a researcher at the University of Guelph in Ontario, Canada. The beauty of barcoding is that even non-specialists can obtain barcodes from tiny amounts of tissue and conclusively identify a species. Compare this to standard taxonomic identification, which requires intact specimens (often impossible in situations where you want to know the identity of foodstuffs) and an expert able to distinguish subtle anatomical differences between closely-related species using morphological features like the shape and color of the organism’s parts. As the New York Times put it in their article about the abovementioned ‘SushiGate’ kids: “What may be most impressive about the experiment is the ease with which the students accomplished it. Although the testing technique is at Volume 26 Number 5
the forefront of research, the fact that anyone can take advantage of it by sending samples off to a laboratory meant the kind of investigative tools once restricted to Ph.D.’s and crime labs can move into the hands of curious diners and amateur scientists everywhere.” Readers of GeneWatch are probably more aware than most of the astounding rate at which DNA science in general is progressing. What they may not know is that there is a growing movement to democratize the technology, to put it into the hands of the public for the greater good. Professional scientists like myself have been inspired to found open, publicserving laboratories that are accessible to anyone who wants to pursue a safe and useful project. Genspace, which I co-founded and direct, is a
nonprofit community biolab located in Brooklyn, NY. We provide workspace, access to equipment, and mentorship in the biosciences. Genspace offers adult education courses, free public events such as open barcoding nights, low-cost lab space for inventors, and is a place for students to work on projects for science competitions. One of the best uses of community labs is the kind of DIY investigation that can tell you more about your environment, health, or food. Is that goat cheese made with cow’s milk? Bring it in and we’ll teach you to barcode it. Want to know if your soy milk is Roundup Ready? We can teach you to determine that too, it’s an even simpler protocol than barcoding. We want everyone to become more literate in the biosciences in order to join the discussion about GeneWatch 9
them from a position of knowledge as opposed to forming opinions based on ignorance and fear. And I strongly feel that the best way to learn is hands-on in the lab. Barcoding is a regular activity at Genspace. It’s a great way for amateurs to participate in real science. Although the DNA barcodes of most common species have been deposited into public databases, most of the millions of species on earth have not been barcoded yet. This gives the student or citizen scientist an opportunity to contribute to the growing public database of DNA barcodes. Genspace first began teaching barcoding as part of Cold Spring Harbor Laboratory’s 2011-2012 Urban Bar-
code Project, a science competition for high school students. Genspace worked closely with their Harlem DNA Lab and acted as its satellite site in Brooklyn for teacher training and open lab hours to mentor students in barcoding, a relationship that continues today. Our newest barcoding project focuses on the importance of identifying organisms to help monitor the biological effects of global climate change. Accelerating habitat destruction is particularly evident 10 GeneWatch
in the Alaskan landscape, where glaciers recede practically before our eyes and environmentalists attempt to preserve species diversity in the face of opposing economic interests. In Genspace’s Alaska Barcode Project, we invite the general public to monthly open nights where we teach them to barcode plant samples collected from remote locations in interior Alaska. The goal is twofold: to create a baseline survey of plants in particular areas such as the Skolai Valley in Wrangell-St. Elias National Park, and to add new identifying barcodes to the Barcode of Life Database to empower future amateur scientists to conduct similar surveys. Part of the DNA sequence of the chloroplast gene rbcL has been designated as one of the two barcode regions for plants (the matK gene is the other region but is not used at Genspace). Barcoding a specimen starts with extracting its DNA. You only need a small piece, the diameter of a pencil eraser, to get plenty of DNA for barcoding. In a tiny plastic tube, the sample is mixed with a few drops of a solution that disrupts the cellular structure and then ground into a paste using a little plastic pestle. The DNA is then absorbed onto silica, which is washed with salt-containing buffers until all other cellular components are gone. The clean DNA is eluted off the silica with water and the barcoding region amplified using a procedure called polymerase chain reaction (PCR). The amplification is
necessary to get enough material in the tube to send out for sequencing. PCR is a standard lab technique that has become mostly automated. Prepackaged mixtures of enzymes and reaction components such as the PCR primers that target the barcoding region can be bought cheaply in bulk. All one has to do is add a minute quantity of your DNA to the PCR mix and stick it into a preprogrammed machine. What comes out is ready to be sent off for sequencing at a feefor-service facility doing hundreds of sequencing reactions daily. The total cost for the whole procedure can be less than $20 per sample. Our barcoding nights have been very popular. They educate people and make them more informed about cutting-edge science. There is also a social component to the project where participants often engage in discussions about the promise and the repercussions of the technology. It wasn’t that long ago that major scientific contributions were made by curious amateurs, and science itself was less of a profession and more of a hobby. The popularity of our barcoding nights might be predictive of the resurgence of such citizen science, where a diverse cross-section of the general population are enthusiastic participants in scientific inquiry. It’s empowering to be able to use the latest breakthroughs to answer questions of importance to you. I can think of no better use of my time than to continue to facilitate this empowerment through my work at Genspace. And please do stop by and barcode something if you are in the neighborhood! nnn Ellen Jorgensen, PhD, is co-founder and President of Genspace, where she spearheads the Urban Barcode Project and other programs. She was an invited speaker at TEDGlobal 2012. November-December 2013
What’s the Holdup? Outside academia – and especially in highly regulated environments – DNA barcoding is only gradually replacing slower, less accurate, and more expensive methods. Which begs the question: Why? By Peter Christey DNA barcoding is an immensely powerful technology. It offers the ability to take any biological sample and with a relatively fast, cost-effective and accurate test, answer the simple question: What species is this? The capability to answer the species ID question in a simple manner is valuable in many arenas. • Customs and quarantine inspectors are interested in identifying biological materials they intercept at borders: Is this piece of meat from an endangered species? Is this larva from an insect that will devastate our national citrus harvest? Is this wood illegally logged? • Authorities responsible for monitoring water quality are interested in identifying species present in rivers, streams and lakes: Does the species profile indicate that the water is polluted or pure? • Companies and regulators involved in the medicinal plant business are interested in identifying plants used as raw materials: Is this dried plant I received from my supplier what he says it is? Is this plant material I am testing for a new product formulation what I think it is? Is this company labeling its product accurately? • Authorities responsible for monitoring the integrity of the food supply need to have robust species identification methods: Is the fish being sold what the vendor says it is? Is this minced beef Volume 26 Number 5
really all beef? Is this meat from an endangered species or illegally imported? These are all critical questions that need to be answered on a daily basis by regulatory, commercial and enforcement entities around the world. DNA barcoding uniquely offers a single, standardized approach to this problem, yet it is not utilized broadly. Instead a variety of manual, time-consuming and expensive approaches are used. The obvious question: Why is DNA barcoding not used broadly outside the academic community if it offers such benefits in speed, accuracy and cost in so many arenas? The answer is two parts: One factor is that many of the non-academic (applied) applications are governed by a legal umbrella that must be satisfied. The second important factor is that in these applied areas there are stakeholders, often with conflicting interests, that must be accounted for. The net result is that for barcoding approaches to be accepted and utilized broadly they must be implemented in a manner that is credible and satisfies the various stakeholders involved. One example is water quality testing. Currently, a commonly used measure of water quality is to take samples from a water reservoir, such as a river, and measure the frequency of different species that are typically found in that habitat. As different species have different tolerances for
low water quality, the species profile is an excellent indicator of the quality of the water and possible pollution. There are well-established indices, based on extensive historical data, that were created using manual collection and taxonomic classification of species. Replacing those manual methods with DNA barcoding would greatly streamline the process, and making the switch seems like an easy decision. However there are several stakeholders in this process who need to be satisfied. The regulators who use this data to monitor water quality need to be assured that the new methodology is sound. The entities subject to the regulators oversight (local authorities, companies that discharge into the reservoir) need to accept the new methodology as fair and robust, and need to be assured that it does not represent a change in standard against which they are measured. There may also be legal requirements written into local law that need to be satisfied. To implement a change in methodology from traditional approaches requires multiple development activities: standardized techniques for sample collection, storage and transport need to be developed and tested to ensure the integrity of the DNA is reliably maintained; standardized primer sets and DNA barcoding protocols need to be developed and tested; a DNA barcode database that reflects the local species profile needs to be GeneWatch 11
developed with controls and rigor that satisfies both the regulator and the regulated entities; studies demonstrating equivalence of results obtained from DNA barcoding vs. traditional approaches need to be performed. Funding this work, performing the various studies, demonstrating the benefit of change and gaining acceptance of the new methodology can take several years. A second example is the use of DNA barcoding for monitoring labeling of seafood in the U.S. The FDA facilitates programs related to the integrity of the U.S. seafood supply and have validated DNA barcoding for seafood identification. The FDA developed their own protocols and performed comprehensive validations of the methodology. They also have developed their own DNA barcode reference database under rigorous controls that satisfy the requirements of the environment in which they work.1 Itâ€™s easy to assume that the FDA methods are easily portable to another country. However, while the FDA has established an excellent example of forward-thinking with a new technology, another country establishing this technology for fish surveillance would need to do its own validations and testing to satisfy its own legal/ regulatory environment and stakeholders. It would also need to develop its own DNA barcode database to reflect the idiosyncrasies of the local fish supply. From these two examples we start to see why there are multiple steps that must be implemented before DNA barcoding can be used in regulatory or legal contexts: 1. Standardization: Standardized protocols for key parts of the process, including sample collection, sample transport, sample tracking, 12 GeneWatch
chain of custody and barcoding, may need to be established and validated. Standardization is often facilitated by the availability of commercial products manufactured to rigorous standards under modern quality control systems. 2. Comparison to current methods: In regulated environments, a full understanding of how barcoding methodologies compare to current methodologies needs to be established. 3. Demonstration of benefits: The benefit of moving to barcodebased methodologies needs to be accepted. 4. Availability of a reliable reference database: A robustly developed and managed reference database for DNA barcodes needs to be developed. The publicly available databases represent an extraordinarily valuable resource for the scientific community. As content is derived from many sources with differing degrees of reliability, these databases are unfortunately not acceptable for use in more regulated or legalistic contexts. 5. Certification: A means to determine that a laboratory is qualified
to perform such tests. 6. Optimization to meet local conditions: One size does not fit all! A robust and acceptable barcoding system for use in one country, city or jurisdiction cannot simply be cloned across the globe â€“ modification and development to address local needs will be required. The conditions outlined above are not unique to DNA barcoding. Two other examples offer models for what may be required for new technologies to expand to widespread use outside academia. The first is the use of DNA-based technologies for human identification in the forensic/ law enforcement arena. The environment for use of DNA technology for forensics has evolved over 20 years. Now in the U.S. there is a sophisticated system of controls to ensure the integrity of the system. Before a new DNA-based product can be used in this environment, the manufacturer must perform comprehensive validations to establish the performance parameters of the product. Guidelines for these validations are provided by an independent expert group. The product must be approved by the appropriate agencies before it can be used in conjunction with the national DNA profile database. Only certified labs may upload profiles into the database or search against the database. The database itself is subject to strict controls and regulations to ensure its integrity. A second model is the in vitro diagnostic model. Again we see similar themes. Before a new diagnostic test can be used, its protocol must be standardized and validated. The test must go through clinical trials to prove its utility and performance. A governing body must approve the test. The test can only be utilized in appropriately qualified laboratories. November-December 2013
Again, similar systems governing in vitro diagnostics exist in different countries, but each has evolved differently to meet local conditions. So, does this mean that it will be a long time before we see the widespread use of DNA barcoding technology? Fortunately the answer is no. The requirements above apply to highly regulated and/or legalistic environments. Barcoding is a powerful tool that will be applied in many areas where the requirements are not so high such as in educational programs, national park management, biodiversity monitoring and environmental impact assessment. Even in more stringent environments, barcoding may be used as a tool to guide investigations (but not used as evidence) and as a means of performing research. Now that the scientific foundation of barcoding has been firmly established in the academic community, we are seeing many creative uses of this technology. Use will grow as more people become familiar with the technology and DNA barcoding becomes more accessible through the development of commercial products and services and less complex instrumentation. The formal processes and structures required for regulated/legalistic environments will evolve in tandem – as for the biological ecosystems that DNA barcoding helps us understand, the DNA barcoding ecosystem itself will evolve and grow over time. nnn Peter Christey, PhD, is Vice President and General Manager for the capillary electrophoresis and 5500 DNA sequencing businesses at Life Technologies, Inc.
Launch of the California Genetic Privacy Network Information, Guidance, and Training on California Genetic Privacy Protections Consumers today are faced with almost daily risks to their genetic privacy. A tsunami of personal genetic data is being created as genetic testing increasingly becomes an integral part of medical research and health care. The vast amount of genetic data being generated raises serious medical privacy concerns. Many Californians are afraid that their genetic information will be used against them and are unwilling to participate in medical research or to be tested clinically, even when they are at substantial risk for serious disease. The public simply does not trust insurers, employers and other entities with incentives to improperly acquire and use genetic information. Despite the passage of several new laws to protect genetic privacy, many remain unaware of their privacy rights, of where they are protected and where they aren’t. It’s not difficult to ascertain why: there has never been a comprehensive public education program on genetic privacy. That is why the Council for Responsible Genetics and the Alliance for Human Biotechnology are pleased to announce the creation of the California Genetic Privacy Network; an ongoing project to educate Californians and the greater public about genetic privacy rights. The Network’s website will serve as a resource for Californian patients, consumers and other front line actors to have an informed understanding of their genetic privacy rights under California and federal law. The California Genetic Privacy Network also offers in person and online educational consultations. Check out the California Genetic Privacy Network website today at:
www.geneticprivacynetwork.org Volume 26 Number 5
The Barcode of Wildlife Project DNA barcoding takes center stage in the international effort to combat wildlife trafficking. By David Schindel
Wildlife crime has emerged in recent years as one of the top four international crimes, representing tens of billions of dollars per year and ranking alongside smuggling of drugs, weapons and human slaves. As the price of products made from endangered and protected species has increased, organized crime networks and terrorist organizations have entered this trans-boundary trade. These products include carved ivory, traditional medicines such as powdered rhino horn and lion bone, ‘bushmeat’ for consumption and religious rituals, exotic pets, leather goods and rare plants valued by landscape designers. To evade detection and prosecution, smugglers have learned how to make it virtually impossible to identify the species of origin by removing the diagnostic morphological features that experts can use to identify the contraband products. Only the DNA in the product can bear witness to the species of origin. Shortly after DNA barcoding for animals was proposed in Prof. Paul Hebert’s 2003 publication, the Alfred P. Sloan Foundation of New York sponsored two workshops to develop a roadmap for the development of barcoding as a global research tool. An important part of the roadmap was the establishment of the Consortium for the Barcode of Life (CBOL) through Sloan Foundation support to the Smithsonian Institution. CBOL officially opened its Secretariat Office 14 GeneWatch
in the Smithsonian’s National Museum of Natural History in September 2004 with the mission to support the development of DNA barcoding as a global standard for species identification. CBOL took on an ambitious program of work to build a coherent, collaborative community of practice, including:
• Facilitating the launch of several major international barcoding ‘campaigns’ such as the All Birds Barcoding Initiative and FISH-BOL; • Engaging the participation of developing countries through outreach workshops in southern, eastern and western Africa, Latin
Photo courtesy of the Kenya Wildlife Service.
• Creating the BARCODE data standard for high-quality data records in GenBank, the European Nucleotide Archive and the DNA Data Bank of Japan; • Convening Working Groups that would conduct research leading to selection of the standard barcode region for plants, fungi and protists;
America, east Asia, China and India; • Representing DNA barcoding to international organizations such as the Convention on Biological Diversity (CBD), the Convention on the International Trade of Endangered Species (CITES), the Food and Agriculture Organization (FAO) and November-December 2013
the Convention on Phytosanitary Measures (CPM); • Creating a social network for the barcoding community and an informational website to explain barcoding to diverse audiences; • Promoting the adoption of barcoding by US governmental agencies such as the Department of Agriculture, Environmental Protection Agency, Food and Drug Administration, and the National Oceanic and Atmospheric Administration; and • Organizing international barcoding conferences held in London (2005), Taiwan (2007), Mexico City (2009) and Adelaide, South Australia (2011). At the Adelaide conference, CBOL’s Executive Committee decided that CBOL’s original mission had been accomplished to a great degree but a new challenge had emerged. Government agencies and private companies had not yet started to formally adopt and invest in barcoding. The problem seemed to reside in the lack of a public reference database of sufficient reliability. Despite the success of the BARCODE data standard in GenBank, barcoding was still viewed as a research tool for taxonomy, not the basis for regulatory or legal affairs. The Executive Committee decided that CBOL should become directly involved in a large and ambitious project to meet and overcome this challenge in one of five areas of application: • Protecting endangered species, • Enforcing truthful labeling of food in the marketplace, • Testing water quality, • Regulating labeling of medicinal plants and herbal remedies, and • Preventing the international introduction of agricultural pest species. Volume 26 Number 5
In July 2012, CBOL was approached by Google Giving with the opportunity to submit a project proposal involving species protection. In October 2012, the Smithsonian received a US$3 million Global Impact Award Barcode to CBOL for the Barcode of Wildlife Project (BWP). The project is structured as a partnership between CBOL and six partner countries. South Africa, Kenya, Nigeria and Mexico began participating immediately and countries in Asia and South America will be selected soon to fill the remaining slots. BWP has three goals: • Create a high-quality public reference database with standardized ‘DNA barcodes’ for the endangered species that are the highest priority to the partner countries. The project hopes to include 50,000 data records from 10,000 species, including 2,000 protected species and 8,000 closely related and look-alike species; • Enable each partner countries to identify crime exhibits by comparing their DNA barcodes with the reference library; and • Support efforts by partner countries to adopt DNA barcoding as a standard, sustainable tool for the investigation and prosecution of wildlife crime. Each partner country assembles a National Project Committee (NPC) of about 10 representatives of enforcement agencies, prosecutors, and academic researchers in biodiversity science. This mix of people is critical because the project’s success relies on participation of both the providers and users of barcode data. Each NPC is co-chaired by an enforcement official and a leading biodiversity research scientist from
that partner country. CBOL has developed a project roadmap with four phases: 1. Planning and assessment of partner country capabilities for DNA research and forensic science, exploration of legal standards for use of wildlife DNA in prosecution, and selection of priority endangered species. The four participating partner countries have completed this phase and the priority species on which they will focus can be seen on the Endangered Species Viewer; 2. Training of researchers, enforcement officials, forensic lab technicians, prosecutors and others; 3. Construction of reference DNA barcode library and testing of forensic lab capabilities to identify species with DNA barcodes; and 4. Implementation, investigation of crimes using DNA barcodes, and prosecutions. The project’s goal is to prepare and empower each partner country to conduct barcoding within its borders, without relying on the export of any biological samples to foreign labs. Simply put, BWP wants to import technology and capability, not to export specimens. Over the course of the project, partner countries will receive support for: • Specimen sourcing: CBOL will coordinate construction of the barcode reference library which will be based on voucher specimens from around the world. This budget category covers the cost of technician salary for tissue subsampling, shipment of samples to processing labs, honoraria for taxonomists who will verify species identifications, and management of data and metadata. • Processing specimens for GeneWatch 15
construction of the reference barcode library and forensic application: Specimen processing will be done in-country for some partner countries but others without molecular labs will need to export samples, at least initially. This amount will cover lab technician salary, reagents and consumables and data management. Funds are not available for lab facilities or equipment, though CBOL is willing to work with partner countries to seek additional funding for capacity-building. Partner countries will be expected to provide access to laboratory facilities during the project. If the project proves successful, CBOL and Google hope that partner countries will make barcoding a normal part of their budgets for CITES enforcement after the grant ends. • Planning, training, and outreach: This category includes the costs of planning meetings, training of technicians, researchers and enforcement officials, outreach to related organizations such as the CITES Secretariat, IUCN, Interpol, the Convention on Biological Diversity, and potential donors. Partner countries will be expected to cover staff salaries for people receiving training, and the costs related to the work of the National Project Committee between planning meetings in which CBOL is directly involved. Photo courtesy of the Kenya Wildlife Service.
The following websites provide useful background information about CBOL and BWP: • Information about the Barcode of Wildlife Project: www.barcodeofwildlife.org • Information about DNA barcoding: www.barcodeoflife.org
• Information about the Consortium for the Barcode of Life: http://www.barcodeoflife.org/ content/about/what-cbol nnn
David Schindel, PhD, is the Executive Secretary of the Consortium for the Barcode of Life, a project hosted by the Smithsonian Institution’s Museum of Natural History.
Foiling Poachers With DNA Barcoding In South Africa, where poachers and smugglers continually adapt to stay a step ahead of environmental law enforcement, DNA barcoding is about to be a game changer. By Jacques
“As fast as one thing evolves, another evolves to keep up with it…” This quotation aptly describes the “cat and mouse” relationship between environmental law enforcement officials and criminals whose activities threaten the survival of so many endangered species. As law enforcement (driven by the need to conserve our natural resources for the benefit of future generations) improves its networks, strategies and technology to effectively tackle contraventions of environmental laws, so too does the criminal element adapt to changing circumstances, allowing them to pursue their objective of personal enrichment. In South Africa, one of the evolutionary steps that has the potential to swing the pendulum in favor of environmental law enforcement (represented by the Environmental Management Inspectorate) is the application of DNA barcoding in the environmental criminal investigative and forensic field. By no means a new science within the academic world, the application of DNA barcoding is set to become a “a first step on the moon” moment for Environmental Management Inspectors hungrily awaiting its conversion into a practical law enforcement tool that will allow them to extend their reach to criminals previously beyond the scope of identification. The use of DNA analysis has Volume 26 Number 5
successfully been used in the identification of species pursuant to a criminal investigation. However, the scope of its application has been very narrow, due to the significant cost and time implications to undertake this analysis; as well as the limited number of species for which these tests could produce effective results. The ability to quickly, consistently and accurately identify suspicious environmental commodities has always been a stumbling block for Environmental Management Inspectors, preventing them from entering the next critical phase of criminal investigations. The DNA Barcoding Project has the ability to provide the green light for these law enforcement officials to proceed. Examples of its application to environmental contraband include: • Cycads that have been stripped of their leaves and roots being smuggled from the wild to plush suburban homes; • Endangered plants that are either ground into a powder or from which oils are extracted; • Look-alike species that are smuggled under the protection of a permit; • Lion bones that have been cooked and cleaned.
Lion paw bones. Image provided by the Department of Environmental Affairs, South Africa.
in the DNA Barcoding Project will keep our Environmental Management Inspectors one step ahead of offenders — and the criminals on their toes. nnn Jacques du Toit is Deputy-Director of EMI Capacity Development at South Africa’s Department of Environmental Affairs.
It is hoped that the coming together of the scientific, academic and law enforcement disciplines GeneWatch 17
Identifying Commercial Seafood DNA barcoding is a valuable tool for FDA for the species identification of seafood. By Jonathan Deeds
DNA sequencing provides public health officials with an important tool which can be used to help ensure that seafood products are safe and properly labeled. When the FDA is investigating either a foodborne illness outbreak linked to a seafood product or a seafood mislabeling case, the Agency wants to have a high degree of certainty when confirming the identity of the product in question – no small task, given that there are over 1,700 species of fish and shellfish potentially found in the U.S. marketplace. In almost all cases, DNA sequencing can provide that certainty. The benefits of using DNA to identify species aren’t just limited to public health officials; it can be an invaluable tool for industry when it comes to ensuring that the proper food safety measures are in place during processing and that the product is properly labeled. Historically, regulatory seafood species identifications either relied on visual examination of the external characteristics of the product, which are often removed during processing, or crude protein profiling methods that were sometimes difficult to interpret or did not work on heavily processed products due to protein degradation. Despite its limitations, protein based seafood identification methods were the regulatory standard for many years. Techniques involving DNA were sometimes used in high profile cases but its routine use 18 GeneWatch
was restricted by the cost and technical skill required to perform the analyses. As a result, it was not typically utilized during regulatory investigations. In 2003, a series of publications by a research group at the University of Guelph in Ontario Canada first highlighted the concept of “DNA Barcoding” for the FDA. This use of short fragments of DNA, generated
and analyzed under a standardized set of conditions, seemed like an ideal alternative to the protein based methods of the day. Advances in sequencing technology and techniques, which reduced both the cost and expertise required to perform these tests, allowed the FDA to modernize many of its food analysis laboratories around the country to include DNA testing. Working with many of the pioneers of DNA Barcoding, including the
Canadian Center for DNA Barcoding and the Smithsonian Institution, the FDA has developed a standardized protocol for DNA Barcode generation allowing the Agency to fully replace the older protein based methods in its regulatory investigations. An early example of FDA’s use of DNA analysis of seafood was during a 2007 investigation of several foodborne illness outbreaks in Illinois, California, and New Jersey. The illnesses were linked to fish that had been illegally imported into the U.S. from China as “headless monkfish” and sold in several Korean retail establishments and restaurants as “Bok.” DNA analysis revealed that the fish were in fact a species of pufferfish that was not allowed for import. The meat of that particular species of fish is known to be highly contaminated with the pufferfish toxin tetrodotoxin, making safe preparation of the species impossible. DNA barcoding is now used regularly to confirm the species of seafood associated with foodborne illness outbreaks. This has already resulted in a better understanding of food safety hazards associated with specific species of seafood, which in turn helps FDA to further refine its guidance for controlling these hazards and hopefully prevent outbreaks from occurring. Use of this technology need not be limited to regulatory officials. Proper species identification by the seafood processing industry is essential November-December 2013
Uncovering Herbal Fraud With DNA Barcoding How do you know your Gingko biloba is Ginkgo biloba? A new study shows that’s a very valid question (and answers it). By CRG because the controls necessary to ensure the safety of seafood during processing are determined by the species of seafood being processed. Proper labeling of seafood is also dependent on knowing the species of seafood. To assist the seafood industry in labeling their products in a manner that is truthful and not misleading, FDA has published a guidance document called “The Seafood List.” It contains a list of seafood species potentially found in U.S. commerce, the acceptable market names for each species, and guidance for developing acceptable market names for new species. With the increased globalization of the seafood trade, new species are always being introduced into the U.S. marketplace. If there is ever any question as to the identity of the fish a processor receives, DNA sequencing can provide the answer and help ensure that it is safely processed and properly labeled. As the technology progresses and sequencing equipment becomes smaller, more affordable, and more transportable, its use by regulators and industry members will likely increase further. The ability to perform this analysis outside of centralized laboratories and directly at the point of importation or processing will further ensure the safety and accurate labeling of seafood in the U.S. nnn Jonathan Deeds, PhD, is a Research Biologist at the U.S. FDA Center for Food Safety and Applied Nutrition.
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When you buy medicine, you probably don’t question whether it actually contains the active ingredients listed on the bottle. If you live in one of the world’s more-developed countries, your government probably has a stringent enough drug approval process to prevent egregiously mislabeled medications from reaching the shelves, or at least to pull them from the shelves if mislabeling is identified. But what if you take herbal supplements? Who makes sure the ground-up leaves inside an Echinacea pill are really Echinacea and not, for example, an invasive weed which has been known to cause allergies and skin rashes? If you have a nut allergy, how do you know your Ginkgo biloba doesn’t have, say, black walnut mixed in with it? Herbal supplements are subject to much less regulatory oversight, and until recently, it would have been very difficult (and often practically impossible) to crack open a pill and determine which species were inside. But now, with the advent of DNA barcoding, researchers or regulators can do just that – and so far, it’s not looking good for herbals. A study published in the journal BMC Medicine this fall used DNA barcoding to test the authenticity of 44 herbal supplements from 12 companies. To do this, the lab (at University of Guelph in Canada) created an herbal barcode library for reference, then matched DNA barcodes from the supplements to those in the reference library. The researchers were
able to collect enough DNA from 91% of the herbal products to determine which species were inside. They found that 59% of the products included DNA barcodes from species not listed on the label, and only two of the twelve companies had products which didn’t include fillers, substitutes, or contaminants. Some of the examples are astonishing. In addition to the Echinacea and Gingko biloba examples above (yes, those were both real), the researchers found two products labeled St. John’s wort that contained no St. John’s wort (one instead contained Alexandrian senna, a laxative); capsules that were supposed to contain dandelion also included grass, or contained no dandelion and instead only wheat and something in the banana family. Fully 9% of the products turned out to contain no trace of the herb listed on the label, only fillers and substitutes. The University of Guelph laboratory that conducted this research is working on standardized testing procedures and a DNA barcode library for commercial herbal species which herbal supplement companies could use to accurately and inexpensively authenticate their products. Given the results so far, that might become standard practice in the herbal supplements industry sooner rather than later. nnn See the full study at http://www.biomedcentral.com/ 1741-7015/11/222. GeneWatch 19
Monitoring Animal Products and Feeds With DNA Barcoding interview with
Haile Yancy, U.S. Food
Haile Yancy, PhD, is a senior research biologist at the U.S. Food and Drug Administration’s Center for Veterinary Medicine.
GeneWatch: Can you tell me a bit about how DNA barcoding relates to your work at FDA? How was it first used for monitoring animal products or feeds? Haile Yancy: We started out using it for preventing mad cow disease. The issue with mad cow disease is that ruminant material – bovine, sheep, or goat – is not allowed to be put into animal feeds and fed back to cows, because that’s how the disease spreads. So we didn’t use DNA barcoding per se to develop those assays, but we used those DNA sequences that are unique in barcoding and designed species-specific primers which we use to make sure that no ruminant material is in animal feeds. More recently, the Chicago office asked for our help identifying potentially mislabeled game meat and potentially mislabeled pet foods. These concerns are similar to the concerns with seafood mislabeling, where someone sells one product but labels it as something else so you can charge a higher price. With pet foods, there was concern about, for example, someone charging a higher price for gourmet duck pet food – was it actually duck meat? It’s the same thing for the game meats. FDA has jurisdiction over game meats, and they wanted to make sure that if a company is selling, let’s say, bear steaks – which are 20 GeneWatch
legally sold – are they actually bear steaks? I’m trying to imagine someone testing a piece of dry dog food. Where are the samples collected to make sure you have enough to get a DNA barcode? We can extract DNA from really any material now. If it’s wet dog food, dry dog food … we’ve actually done a lot of work with pet jerky, too. It’s been on the news recently, there’s concern about dogs getting sick from pet jerky – we did the testing of the jerky to make sure it is what’s on the label. So really any material we have, we can extract DNA from. But when we talk about animal feed that has been rendered, it can be very difficult to create a full-length barcode. So my colleague Yolanda Jones is not only validating full-length barcodes, but also looking at what they call “mini barcodes.” Whereas regular barcodes are about 650 base pairs, minis are only 100 base pairs. The materials we look at have sometimes been degraded, whether by cooking or rendering or just sitting out on a dock somewhere, which makes it very difficult to create an amplified product of 650 base pairs, but you can still create a product of 100 base pairs and use the same approach and still identify what that species is. If you weren’t using DNA barcoding, what’s the alternative? For example, how else would you identify ruminant material in feeds?
Currently, the gold standard for making sure there’s no ruminant material in animal feeds is microscopy. You have a microscopist who actually looks at the particles in the feed to determine whether it contains animal material. The problem with that is if you find, for example, a hair in the feed, it doesn’t tell you if this hair is from a cow, a sheep, or a pig. So although microscopy is the gold standard, it has a very difficult time identifying species. So you have to fall back on these molecular methods, because if there’s hair there, we need November-December 2013
to determine where it’s from. That’s what’s important: Not just determining whether there’s animal material in the feed, but is this material from a ruminant species? Are there other uses of DNA barcoding at FDA currently in use or planned besides the ones you have mentioned? We recently had a group that was looking at prevention of infectious diseases transmitted to humans by bushmeat. People had been bringing back exotic meats, or bushmeat, from other countries. There’s an executive order that the President just signed to combat wildlife trafficking, and one of the ways we’re doing that is putting together a national smuggling prevention network to prevent the illegal importation of bushmeat into the United States. Part of that effort is to identify which species are brought into the U.S. as bushmeat, so part of what FDA has been tasked with is creating a DNA barcode database to use for this. If you have a piece of meat come in, it may not have the distinguishing characteristics that tell you “this is a monkey” or “this is an alligator;” so what we’ll do is barcode that, match it against the database, and once you figure out what kind of bushmeat that is, you can associate it with risk. Specific bushmeats carry a specific risk – for example, primates can carry herpes virus or anthrax virus. It’s very difficult to establish risk without understanding which species it is. We’re part of a group working on this that includes the FDA, the U.S. Department of Agriculture, Customs and Border Protection, the Center for Disease Control, Fish and Wildlife Service, and hopefully including state and local governments.
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That’s pretty expansive! Yes, it’s a fairly new group of individuals, but we’ve got the wind behind our back with the presidential executive order. Some of these diseases are very nasty, and it’s something we hope can be prevented with all these agencies working together. We’re working very quickly to develop a method of barcoding so that when someone confiscates material at the border, they will be able to know what it is and make sure the risk is minimized. Are you using existing reference databases for this, like the Barcode of Life? Yes – one of the things we recognized very early is that it would be virtually impossible for the FDA by themselves to get the materials to build these reference databases. I’ve been working with Barcode of Life for over 12 years now. Their goal is to prevent the illegal importation of endangered species, so what we’ve done is piggyback on that, because if you look at the list for bushmeats and the list for endangered species, there is an overlap of common species. We don’t have the infrastructure to go to Africa and actually get samples of lions and elephants, so we have access to their specimens to help populate our database. Do you see any new uses for DNA barcoding in the pipeline or farther down the road? Because the technology is there to determine species of origin, you can see a shift in making sure, both on the producer and consumer side, that consumers get what they pay for – not only for economic reasons, but also for safety.
One thing we’ve been working on is concerns about heparin (an anticoagulant) being made from nonporcine material, specifically from cow material. The method we’ve developed for testing this is actually a modification of our assay for finding bovine material in feeds. That’s the safety aspect, but there’s also a religious aspect, because if you don’t eat pigs you’re concerned about heparin being made from porcine material, so you want the opposite – you want to make sure your heparin products are derived from cow rather than pig material. I see a lot of other uses for this for everyday things that we take for granted. For instance, if you’ve read the papers, there have been issues with food supplements being mislabeled. I can see DNA barcoding being used more and more as two things happen: Firstly, developing more efficient ways of extracting DNA and being able to sequence it; and secondly, making sure that the cost goes down. As those happen, you’ll see a large application. And consumers are becoming more and more aware of things being substituted and mislabeled – like the grocery store that found out some of the meat it was selling was not beef, but horse. More and more consumers are asking: Am I buying what I think I’m buying? And it matters from both an economic and a safety point of view. It’s way off in the future, but one of the long-term things we’re trying to work on, with Barcode of Life, is a handheld device that can do all these things. We’re heading in that direction. nnn
Measuring the Health of Aquatic Ecosystems With DNA Barcoding The U.S. Environmental Protection Agency is finding DNA barcoding to be faster, more precise, and more accurate than previous methods of aquatic bioassessment. By Erik Pilgrim At the U.S. EPA, several projects in the Office of Research and Development have been investigating the use of DNA barcoding for identifying various aquatic organisms to species. One of the main areas of research is in determining the utility of DNA barcoding for environmental bioassessment â€” evaluating the environmental health and condition of a site based on the organisms found living there. Standard bioassessment relies on identification of biota, typically benthic invertebrates, using morphological characters. These morphological IDs are done by highly trained taxonomists, which can take many months or even a year to complete. Our work at EPA, along with collaborators outside the agency, seeks to capitalize on the potential speed and cost savings associated with identifying these benthic organisms through DNA barcode data. The use of DNA barcoding for environmental bioassessment potentially has several significant advantages: 1) Identification by high-throughput molecular techniques is considerably faster than current morphological techniques, with samples being processed in weeks to 1-2 months, as opposed to 6-18 months. 2) With continued cost declines 22 GeneWatch
for molecular work, DNA barcoding for bioassessment is comparable in cost or even cheaper than standard morphological methods. 3) Identifications based on DNA barcodes are at the species level, but morphological identifications, because many benthic organisms are small or are juvenile life stages, are often at the family or genus level, especially for freshwater ecosystems
where accurate bioassessment is the most critical. 4) DNA sequence identifications are not subjective in that no matter how many times the same sequence is queried against the database, the identification will always be the same, whereas morphological IDs performed by different taxonomists or laboratories are known to raise
disagreements over IDs. 5) DNA-based identification can be applied to all biota, including groups normally omitted from standard bioassessment because of their small size (less than 0.5 mm) or extreme difficulty in identification (e.g nematodes). Current and past EPA research into DNA barcoding has shown several interesting results. Our previous research has shown that DNA barcoding of benthic invertebrates at a large scale is feasible, and that the largest impediment to successful DNA work is in proper preservation of samples as opposed to any of the molecular genetic techniques. Our current collaborative work with research groups like the Southern California Coastal Water Research Project (SCCWRP) and Stroud Water Research Center has helped develop new protocols for the collection and storage of benthic invertebrates for molecular genetic workflows, and has shown that DNA barcoding identification of freshwater benthic invertebrates provides more information than standard IDs and that this information has value for assessing environmental condition. Ongoing research at EPA has moved into the use of Next Generation DNA Sequencing for bulk processing of samples. The development November-December 2013
of Next Generation Sequencing techniques for environmental bioassessment has the potential to speed aquatic community identification and lower costs even more through DNA extraction, PCR, and DNA sequencing of bulk, unprocessed benthic samples. Current research investigates these NGS applications for stream, lake, and coastal marine samples. Our lab is working to develop techniques and workflows to generate the most useful data in the most cost-effective manner while also working to be user friendly for end users and decision makers. Toward these goals, we continue to collaborate with non-agency research groups such as SCCWRP, as well as maintaining strong relationships with international researchers in this field such as the Canadian Centre for DNA Barcoding and CSIRO–Australia. Several challenges lie ahead for the development of DNA barcoding for bioassessment. Our first challenge is to foster better communication between molecular genetic researchers and environmental scientists, to ensure that the work is collaborative instead of confrontational. Second, the data generated through DNAbased identification is different from standard bioassessment, and molecular geneticists and environmental researchers need to work together to determine the most appropriate uses and analyses of this new data. Lastly, this molecular work will generate much more data than has ever been possible for bioassessment, so methods must be developed to handle such large amounts of data while providing the most useful outputs for making determinations of environmental health. This is an exciting time for both DNA barcoding and environmental bioassessment. These molecular Volume 26 Number 5
genetic applications could be a transformative technology for the field of bioassessment. Our ultimate goal is to provide better information in a timely, cost-effective manner, in order to provide decision makers with best methods for assessing aquatic environmental health and condition. nnn
From the Council for Responsible Genetics on the 30th Anniversary of GeneWatch magazine:
Erik Pilgrim, PhD, is a Research Scientist at the U.S. Environmental Protection Agency, where he applies DNA barcoding to bioassessment and invasive species monitoring and detection.
Endnotes Stoeckle, p. 5 1. Hebert PDN, Cywinska A, Ball SL, et al (2003) Proc R Soc Lond B 270:313. 2. More specifically, those loci are: Animals, 5’ COI (2005); plants, rbcL/ matK (2009); and fungi, ITS (2012). 3. Ratnasingham S, Hebert PDN (2013) PLoS ONE 8:e66213. 4. Newmaster SG, Grguric M, Shanmughanandhan D, et al. (2013) BMC Medicine 11:222 5. http://www.urbanbarcodeproject.org 6. Santschi L, Hanner RH, Ratnasingham S, et al (2013) PLoS Biol 11:e1001471 7. http://malaiseprogram.ca Christey, p. 11 1. Further detail can be found on the FDA website at: http://www.fda. gov/Food/FoodScienceResearch/ DNASeafoodIdentification/ default.htms
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DNA Barcoding: The Quiet Revolution