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Ion PGM Dr. Gordon Moore Environment Grant 

Dr. Mitchell Sogin, Senior Scientist and Director of the Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Sogin Laboratory.

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Dr. Sogin proposes using Ion technology to more accurately and rapidly identify both the source and extent of water contamination for the same cost as culture-based methods.

The Sogin Laboratory employs comparative phylogenetic studies of genes and genomes to define patterns of evolution that gave rise to contemporary biodiversity. The Sogin team is especially interested in discerning how the eukaryotic cell was invented as well as the identity of microbial groups that were ancestral to animals, plants, and fungi. Phylogenetic inferences based upon comparisons of ribosomal RNAs have discovered new evolutionary assemblages that are as genetically diverse and complex as plants, fungi, and animals.

Question & Answer

Your proposal is to use DNA sequencing to monitor water quality. How big of an issue is clean water in the US?

Dr. Mitchell Sogin: There are 40 million people who get their drinking water supply from the Great Lakes. The difficulty is that sometimes you get so much surface water coming into the sewage systems that they overflow, releasing sewage into the Great Lakes. The EPA estimates that nearly 140 communities on the Great Lakes that release more than 40 billion gallons of untreated sewage each year. That's pretty amazing. To detect these kinds of spills, water quality monitoring is done through standard microbiological methodologies, so called indicator organisms, such as E. coli. E. coli is present in the intestinal tract of all of us. It's not a very abundant organism in our intestinal tract, but it's easy to grow in a culture plate. The problem is that in using this approach, we're not detecting the presence or absence of potential pathogens, just the indicator organism.

The second problem is just because you see an elevated coliform, or indicator count, in the assay doesn't mean that we have a sewage release that involves human fecal microbial communities being released to the environment. In fact these organisms can show up as a result of bird populations, beaver populations, any number of animal populations have the same kind of organisms that on a cultivation basis are not readily distinguished from E. coli, the indicator organism we use. So that has a large economic impact in the way we manage our water supplies and, as I said before, it doesn't really tell us about actual pathogens that are being released. In 2005, nationwide 30% of coastal beaches reported at least one closing or advisory due to fecal pollution. Often these advisories warn the presene of dangerous microbes and it estimated that 12-40% of cases of gastroenteritis in the United States may be due to waterborne

Can you give me a sense of how widespread that type of water problem is?

Dr. Mitchell Sogin: It's prevalent enough that the U.S. Government is now funding entities called the Centers for Oceans and Human Health and this is a joint venture by NOAA, National Institutes of Health and most aggressively, the National Science Foundation. And so there are half a dozen of these centers spread across the country that are studying this problem and trying to apply new technologies to monitor and come up with better ways of doing pollution monitoring. And by the way it's not just human sewage, it's also agricultural sewage and run-off, so the EPA is also investing resources. It's going to become a worse issue as time goes on and that is predicted on the basis of what we've seen in terms of shifts in climate due to climate change, global warming, etc. Changing weather patterns with increased number of storm events will lead to a larger number of combined sewage overflows.
Why is sequencing a better solution than traditional culture based solutions?
Dr. Mitchell Sogin: So the culture based solution traditionally relies upon two or three different organisms that they try to culture and from that they extrapolate whether this represents a potential sewage release event. The idea of using sequencing is to look for a suite of organisms that together provide a much more complex signal of sewage pollution. So instead of just monitoring levels of E. coli or Bacteroides, we would be monitoring a large number of different sequences that come from various microbial populations that include different Bacteroides, different enterobacteria, different Firmicutes that normally occur in association with human fecal communities.

There's a lot of DNA sequence data coming out for fecal microbial communities and as a result we get an inventory, a complex signature, not just the different kinds of organisms, but also the relative abundance of the different kinds of organisms that live inside of human fecal material. We can then use these complex microbial profiles by comparing them to DNA sequence profiles of microbial communities in fresh water, or potentially contaminated fresh water, or as we've already done influent into sewage processing plants. The idea there is that you would be able to look for commonalities in the catalog of organisms you see in human fecal populations and then see if they are present in fresh water after sewage release might impact the recreational or drinking water. There are a lot of advantages to doing this. Not only do you have a complex signature, but now you can get an idea about the persistence, the survivability of many of these different microorganisms that may come from sewage entering fresh water supplies. And we can even specifically identify candidate organisms that might cause disease. What you don't know from cultivation of a single indicator organism or even molecular probes for targeted indicator organisms such as Bacteroides is whether the detected organism actually came from sewage or if it came from some other point source like a horse defecating in a stream or from birds or whatever the source might be from other animals.

So that's what you miss when you are looking at indicator organisms. Sequencing simply gives you more information about the composition of microbial communities released into a water supply if an event occurs. And because it's so much richer in information content, you have a much better chance of being able to identify whether or not it comes from a point source such as animal population versus a real human sewage impact.

That seems like a big advantage. Why doesn't everyone use sequencing?

Dr. Mitchell Sogin: When we only had available capillary sequencing and it would cost us a dollar a sequence to get information, it really constrained very severely how many sequences we could collect. And each one of these sequences, serves as a proxy for the occurrence of the micro organism in a community. If you're looking at microbial populations where maybe only five or ten percent of the organisms might come from a sewage release and the rest were organisms that were indigenously occurring in the water supply, which is a reasonable estimate, then you really can't hope to get enough information by just sequencing a thousand molecules, it's just not going to give you statically reliable data given the enormous population sizes of the microbial communities. So along comes some of the earlier next-gen sequencing technologies, and instead of paying a $1,000 to get a thousand sequences, you might pay $10,000.00 to get out on the order of 200,000, to 300,000 sequences. Now you're getting enough information about different taxa so that you can get real, very, very detailed microbial profiles, that is compositional profiles, which is what we're after. But you're still talking about a very expensive experiment that you're never really going to do on an every day basis if you want to adapt the technology for water quality monitoring. We typically could generate 300,000 sequences that might be enough for looking at five samples. So that would cost about $2,000.00 a sample.

What this Ion Torrent system offers now for the first time I think is the possibility of actually incorporating massive parallel sequencing into a laboratory that might routinely monitor water quality. The way those numbers work is if you're able to generate a half million sequences with your device, that's more than enough sequence to analyze 20 samples. So now we're talking someplace between $25.00 and $50.00 an assay for each sample we survey. So we've knocked down the cost by an order of magnitude. Moreover, that cost is comparable, in fact it's even a little bit less, than it would cost to do a cultivation-based assay for indictor organisms and yet it's so much more rich information. So that's what the big change is.

How would this be adopted, not just in the U.S., but in developing countries where water seems to be just a massive, massive problem?

Dr. Mitchell Sogin: I don't have a clear answer for you right now. One could argue that one of the offshoots of this technology is that it's going to allow us to build much better DNA or micro arrays that are hand held, that could now be applied, but it's going to take a lot of research before we get to that point. On the other hand, maybe the Ion Torrent technology, will one day be reduced to a field deployable instrument. And the idea of using semi conductors to sequence I think fits the idea of field deployable instruments of this nature. That would have a transformative impact on molecular microbial ecology.

What would a field deployable instrument look like to you? What would it need to have?

Dr. Mitchell Sogin: It could be a water monitoring station, it could be someone who carries the instrument in a backpack and it might be as small as a 6 x 6 inch box. I don't know how much these technologies can be miniaturized. I can tell you that some of the most difficult environments to do continuous monitoring include the oceans. And yet today instrumentation developed at MBARI (Monterey Bay Aquarium Research Institute) is being marketed for operation on a moored platforms out in the ocean where they collect samples, extract DNA and then interrogate DNA samples using a micro array. I can imagine the mating of miniaturized Ion Torrent technology to such a sample collection and processing capability currently being deployed on moored platforms in order to obtain real time data about microbial populations any place in the oceans, but most importantly in coastal areas.

If your proposal works as planned, how will it change people's lives five years down the road?

Dr. Mitchell Sogin: Well, number one, I think that they will be better protected. Number two, they won't be as inconvenienced as they often are today with false alarms regarding beach closures. And number three, I would hope that the technology could be developed in a way that the response time of knowing what the answer is would be fast enough that it would be almost real time. Now that's going to take field deployable instruments for sure. Current technology requires us to monitor a beach, go back to a lab, do cultivation- based assays, or in some cases perform molecular-based assays and it takes one or more days to get the answer. By that time, through natural processes, that beach may no longer be impacted. So we really need to have a more rapid response because with today's cultivation technology we don't even know that it is necessary to close the beach until the next day. Because of lack of real time response, people may be exposed. On the other hand, in some cases, contaminated recreational and drinking water supplies may change by the next day. Currents or diffusion processes may carry away dangerous or indicator organisms. In other words the source of pollution may have disappeared. It's all a matter of how quickly we get an answer and right now we don't get an answer fast enough. 24 hours doesn't work.

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