Wisconsin Public Television Wednesday Nite at the Lab  High Throughput DNA Sequencing Jashua Hyman  Oct. 10,2012 UP784  cc >> Welcome, everyone, to Wednesday Nite at the Lab. I'm Tom Zinnen. I work here at the UW Madison Biotechnology Center. I also work for UW Extension Cooperative Extension, and on behalf of those folks and our other sponsors, Wisconsin Public Television, the Wisconsin Alumni Association, and the UW Madison Science Alliance, thanks for coming to Wednesday Nite at the Lab. We come to you every Wednesday night to get to share some of the great insights of science. And tonight is a remarkable story because tonight is the biological equivalent of one of the holy grails. Tonight, Josh Hyman is going to talk to us about this new next generation in sequencing and DNA, and it's a pretty amazing story because I think we're finally at this holy grail point, and that is the point where you can sequence all your DNA in one day for about 1K, for about $1,000. I think Josh will show that those figures are not quite there, but it's on the order of an MRI now. And think what that's going to do to change the relationship between you and your physician, and if you get your genome sequenced, what that might do as far as how much of that information will you want to share with your siblings, with your children, with your employer, with your insurer. So, the kinds of technologies that Josh is going to show tonight are really going to put the rubber to the road as far as these ethical conundrums that we've been talking about theoretically for the last 20 years. 

Now these are real nuts and bolts decisions that people are going to have to make. The other great thing about this talk, for those of you who are here in person, is afterward, Josh is going to give us a tour, and you'll be able to see at least three generations of DNA sequencing machines. And it's rather remarkable, up in Josh's lab on the second floor, there's a DNA x-ray film. This is about 20-22 years old. There's about 700 base pairs on that. That took about a week to do. That's 7 times 10 to the 2nd. He's got two machines in here that can do 7 times 10 to the 10th a day. So that's orders of magnitude, and that's the kind of boggling numbers that we have. This is a great saga of science, and I'm delighted that Josh Hyman is here to talk to us about high throughput DNA sequencing and its impact on the world and I would say on your world and on our world. 

Josh currently serves as director of the DNA Synthesis and Sequencing Facility here at UW Madison's Biotechnology Center. His past exploits, at least the ones I can tell you about, include designing a windshield and canopy systems for the F15-E fighter jet, building and operating a restaurant, and performing research for tomato and carrot breeding programs. Joshua's formal training includes a doctorate in genetics from Penn State University, an MBA from the University of Washington Seattle, and a BS in mechanical engineering from the University of Michigan. His wife finally said enough is enough, no more schooling. And I don't think she was fooling about that. >> She was not. [LAUGHTER] >> I think his wife said quite a few other things. [LAUGHTER] But I can't share those with you at this moment. Please join me in welcoming Josh Hyman to Wednesday Nite at the Lab. [APPLAUSE] >> Hopefully everybody can hear me that's in the room. 

Good. As Tom said, I run the DNA sequencing facility. We're just across the hallway. Literally, about 50 to 100 feet from here are all the machines. So we will get a chance to go in there, poke at them, look at them, look at some of the consumables that go on them and give you an idea of the size of the consumables, and then it sort of leaves you wondering how someone managed to engineer that. It is pretty incredible. Tonight, I'm going to talk somewhat about the sequencing technology and a great deal about its effect on us. And we're all sort of getting used to it now. We've gone to the point where in New York there's this van that goes around. It's the Who's Your Daddy van. 

[LAUGHTER] And, as you can imagine, they're performing a lot of paternity tests, and some of these are for people who don't want to be responsible for their children and other people want to be responsible for their children. They want to have visitation rights. And, in some cases, there are people that just want to know that they are related. There was one of the tests ran, this woman from I think it was Ohio came into town to visit her newly discovered father. She was probably in her 20s or 30s and had found out that she was adopted and located her father in New York, or at least who she thought was her father, and they were tested and found out, in fact, that they were father and daughter. So, it is not just for legal purposes. It goes well beyond that. And there are other things you can test for other than paternity. In the realm of the sort of, this is sort of the edge of where we are right now. They're looking for genes that effect social and physical attributes. 

And some of the physical attributes are fairly obvious, but a dancing gene, well, that's not quite so obvious. And it turns out, and I'm going to hop over, I hope it will pull it up here, but first we'll look at the gene for romance, and I'll see if it kicks in and hopefully you'll be able to see it. So, they were looking, not at humans, unfortunately. They're looking at voles and nematodes and flies. They found that there are these genes that are very important in courtship rituals. And other species have these genes, and if you inactivate these genes, the males are often not at all interested in the females. And it's a single gene which has quite a number of different forms to it, but it is, obviously, very effective in the mating process. So, these genes do exist. And then there is also the dance gene where it turns out, if this will pull it up again,  that the dancing gene is more of a social gene than an actual physical attribute gene. And it is people that tend to be social and to want to be out in the middle of these groups that they identified. 

Although, there was a study in Jamaica where they did find that certain men had genes that allowed them to move a little smoother. They could dance a little better, but they've only found it in this group in Jamaica. [LAUGHTER] I think the rest of us are kind of out of luck at the moment. And it goes beyond that. They've looked a lot at athletes, and there are slow twitch muscles and fast twitch muscles. And depending on the mix of certain alleles you have, you may be more prone to have some of the fast twitch muscles or you may be more prone to have more of the slow twitch muscles, the one that a power lifter would use versus the ones that a sprinter would use. And you can, in fact, find these. Does this really impact your running or your lifting? To a certain extent, but it doesn't effect it so much that you would make a decision on that. So parents that are going out and having their children tested, that's a little bit unrealistic to just test and then say, well, my child is going to be a great power lifter or my child is going to be a great hurdler or great swimmer or something like that. And then in the more serious realm, we have folks over in the United Kingdom, and they've been doing this for a while, they do prenatal tests, especially when there's in vitro fertilization. 

In fact, all of these are set up for in vitro fertilization. You do the tests before you start impregnating a woman. And you want to know whether or not the embryo that you're using is going to carry the alleles for certain diseases. And this is particularly important when there have been complications and there are what we call test tube babies and a man and a woman know that one or the other is carrying these disease alleles. If you have a family and you know that you've had Huntington's disease within the family and you don't want to pass that on to your children, you get tested. But even so, and this is giving you some idea of the things they're testing for, even so, this has caused some controversy over there. But, quite honestly, the people that are involved and have to make a decision are very, very serious about these things. They don't test for something that's of sort of dubious penetrance where it may happen, it's likely not to even if you have the disease allele. They don't test for things that are effected more by environment than by genetics. And many of our diseases are impacted much more by environment than by genetics. 

And we can see this quite well when we look at our society right now and say what's happening with type 2 diabetes. Going off the charts right now, that has very little to do with the DNA code itself. Although, there are susceptibility genes in there, we're pushing that way beyond where it should be within our society. We're just doing it to ourselves. So, they don't have to test for things like that. And, as we go along, we're testing for more and more things, and one of the reasons is, for one, we can identify more of these disease genes. We can do larger and larger tests with many, many people in the test, larger populations being used to find disease alleles, to find all sorts of things in the genome. And it's now possible, also, to test a single individual for many, many more things. Instead of using a single test and we used to just amplify a small fragment and then look for one of the disease alleles in it, now we can take thousands, millions of fragments at a time and look at them. And it's so fast. 

It's just incredible how much this has accelerated just in the last 5 to 10 years. And now we're also getting to the point where we're trying to move it into the clinic. Now, obviously in the UK they're doing this prenatal testing, but if you go to a hospital in most cities in the United States, they're not going to run many DNA tests. The doctors don't know about them. They don't know, even if there is a test out there, whether it will give them any useful information. So what you need to do is start teaching the doctors, first of all. Letting them know that this is possible, letting them know that there's the variations in the tests. Some tests are very accurate in letting you know what's going to happen. Some tests will tell you that there's a high probability of something happening, or maybe it's a lesser probability that something's affected more by environment than anything else. Now, that doesn't mean we shouldn't test some of these things if it's affected by environment because obviously if you have disease alleles and you can change your environment, why not do it? But here are two schools that are obviously starting, and these started just recently. 

I think this is the first year they've done this at both of these schools. So, University of Miami, what they do is with their incoming doctors they give them a chance to get a master's degree in genomic medicine. That means they're going to be exposed to the technology, the types of tests that can be run, and what those tests mean, what the significance of those tests are. And then Mount Sinai School of Medicine is giving the students, and there are 20 students signed up for this so far, but they're giving them a chance to sequence their own genome and start going through it and then looking at what they find and using that as a basis for determining when to tell people things, or rather, just to get a feel for what sort of information is out there in their own genome. Is it relevant to them? Did they really want to know? Because that's important. Do you want to know? There are some things we can do something about, and we want to know about it. There are other things that maybe we can't do anything about them, and is it worth knowing? Now, in this building, I've been probably the most heavily sequenced person. That's because I can't ask anybody else for DNA when we want to run evaluations. I'm not allowed to ask any of the people in my lab if I can use their DNA to run something on the machine. So, I've pretty much used my own DNA. 

I have an agreement that I've signed that says, basically, if anything is found out, that's okay. I'm not going to worry about it. I haven't looked at any of that. I don't care. I know what happened to my dad. By my age, my dad had had some minor heart attacks. In, let's see, if I was on the same schedule as my dad, in about four or five months I'd be pulled off of the floor in a hotel room and have my heart restarted. Now, I've known about this for long enough that I've been very careful to take, luckily, statins and things like that to make sure my cholesterol stays low. So, I already knew. I knew what happened in my mom's family. 

I know what happened in my dad's family. You can pretty much look at that and know most of these things. It's when there are inherited diseases where you don't know if you got it or not and they're critical that it becomes more important. All right, so upstairs we have Sanger sequencers. These are machines that we used for about 10 years or so, and we're still using them. So now they've been working for about 15 to 17 years. But we started using these to do fairly high throughput single fragment reads. I'm going to differentiate between a single fragment and shotgun fragments. A single fragment is when we amplify something, we can actually go into a genome, a large set of chromosomes and genes and everything else, and we can use a couple of DNA tags that we can synthesize, also upstairs, and we can allow a process to occur with enzymes. And with those two tags facing each other, DNA is double stranded and we can make a copy in one direction and make a copy in the other direction starting at those points because if we heat everything up, the DNA will actually separate. 

And as it cools down, if we have these little tags, these little primers, they'll find their complement on the DNA strand and they'll extend if we have a polymerase enzyme in there. And it's just an enzymatic reaction that, with the right components, will make a duplicate copy of a chunk of DNA. And so we start it one direction, start it in the other direction at the same time, and go through cycle after cycle. What happens is a lot of those little chunks of DNA that we put in there, those primers, will start priming off of the piece that we created which has an end to it. And so we start amplifying a small chunk of DNA, and we can sequence just that small chunk of DNA. We have the machines upstairs. 700-800 bases of DNA. That's the way we've been doing it for a while. But you notice we've been doing one fragment at a time, one specific fragment that we have an idea of where it is and what it is in the genome. So, that we can do. 

And it produces about 700-800 bases, 800 letters of your 3 billion letters in your genome. And we can do 96 samples at a time. We have little plates, little plastic plates with wells in them, and they have 96 wells in them. We can do 12 to 13 plates in a day, and that gives us what we used to consider to be a fair amount of DNA sequencing. So, for every single machine, we had 1250 or so samples per day we could do, and this looked really good to us. 873,000 bases in a single day. And we did that five to six days a week. And then we say, well, if a single machine can generate that many bases in a day, how long does it take to completely sequence a human genome? And it turns out that if you do the math, if the human genome is three billion bases, it's going to take you about nine and a half years. But that assumes that we have every single fragment lined up end to end which is never the case. There are spots in the genome that we can't amplify that well. 

There are areas in the genome that still haven't been sequenced. They're just difficult to get to, or they're in what we call repeat regions. So, throughout the genome there are these regions where there's repetitive chunks of DNA, and we hit those repetitive chunks. They look the same throughout the genome. They could be anywhere. We can't know what's starting and ending that exactly other than by using some other techniques. But nine and a half years to sequence the human genome if we had it laid end to end. So, what do we do? We start using next gen sequencing. About seven years ago, next gen sequencing came into play. And what it does, it does millions of fragments at a time in parallel. 

So instead of doing one fragment and another fragment and another fragment, we just take the human genome and we break it into smaller pieces and we stick adapters on the end of every single one of those small pieces and then we put them on some substrate and they're fixed on that substrate and we let them amplify in their little spot on that substrate to make hundreds, thousands of copies of just that one little fragment in that one little spot. And then we use these machines to sequence them all at the same time. So, what that does is it really lowers the cost of sequencing. There are shorter reads right now, but it does lower the cost of sequencing significantly. How much does it lower it? Well, with Sanger sequencing, the ones we have upstairs, it's one to two dollars per sample depending on how little or how much of the reagents you use to do it, but it still works out to be about .15 to .3 cents per base. So, cheap but not anywhere near as cheap as next gen sequencing. So, if we look at the next two items on there, the single-base short-reads and the multi-base long-reads, those are two platforms that we have that are considered next gen sequencing. So at the very bottom, this was the first one that came in for next gen sequencing. This is a Roche system. That's the company that produces the machine. 

It's about $8,000 for a single plate to run. But it produces about 600 million bases. Not quite a human genome but getting up there. And the cost is, roughly, a thousand-fold less per base. Then we look at the one in the middle, single-base short-reads, Illumina. We're getting down to, well, I don't even put it in these terms anymore. I always look at per million bases now. It's nine cents per million bases. So we went from .15 cents per single base to 9 cents per million bases, and that's where we're at right now with the technology we have. The throughput has obviously gone up quite a bit. 

If we look at the Illumina system right now, we're getting 210 billion bases in a single run on Illumina. Now, remember the human genome is three billion bases. So, obviously, we're getting many, many fold that in a single run, but we have a way to break up samples across a flow cell which goes on this system. So we don't use the whole thing for one human genome because that is more coverage than we need right at the moment. And with the Roche system, we're still getting pretty good coverage and pretty good output. We're looking at 600 million bases per run. So, we've gone from nine and a half years, roughly, to sequence a human genome if it were all linear with our Sanger instruments upstairs to about nine days, nine to 12 days to sequence it with the machines we have down here. To put this in perspective, it took almost 10 years to sequence the first human genome and it cost, roughly, $3 billion. We're now at 10 to 12 days and $5.5 thousand. Huge, huge change. 

So, why do we care? Why do we care that we can sequence all of this? Well, we've already touched on this a little bit, but let me go into some of the details. So DNA is sort of the base instruction set. It's sort of the how-to set. It's our instruction manual for how an organism develops and then runs. If we have any mutations in it, any modifications in it, it can affect physical and, in some cases, social responses. And it's not just in us, it's in all the organisms that we are looking at or that exist in the world. It can give us some insight into how we are related to other people. It can give us insight into how other organisms are related to one another. Plants, sometimes we're looking for certain attributes in plants, and we can find something that's closely related in the wild and bring it in, start breeding with one of our cultivator plants. And it allows us to look for medicine out in the field. 

If we know things are related and we're getting a medicine from one plant, maybe it's in some of the closely related plants. Maybe it's a better source in one of the closely related plants. And all this leads to us trying to predict outcomes. So we get information and we're trying to use it in some way to figure out what we can find out and what we can see into the future with what we're looking at, whether it's a related plant or a mutation in something, and does it cause disease or something like that. But it's only one part of the equation. This is, like I said before, some of these things will definitely tell you that there's going to be a change, and other times, environment plays a big impact. It's only one part of the equation because phenotype, what you see, the form, is a product of both the genes, genotype, and the environment, what we're exposed to. And phenotype can be physical, social, whatever. And, as I said, genotype is the genetic code. And it's part of your genes. 

So, genotype is often used to say you have a genotype for something. So you have the genes for blue eyes or something like that. This works in an interactive way because there are tags above the DNA code, and this field of study is called epigenetics, but there are tags above the DNA code that induce changes in expression because all your tissue is expressing different genes depending on which tissue it is. Obviously, your liver is different than your kidneys which are different than you eyes, all that material is different than your skin. So different genes are being turned off and turned on. And environment can induce mutations or affect gene expression. So this epigenetic is really not related. It's not part of the code. It's not part of those four letters that make up the alphabet of your DNA but something on top of that. It's variation that occurs within the body depending on what organ you're looking at and what layer of tissue you're looking at. 

So every single one of those layers will have a slightly different epigenetic profile. And it's variation that, surprisingly, like the DNA code, can be transferred from you to your children. So, they discovered this by looking at what was happening, and I don't know if it was Norway or one of the other countries in Europe where during the first World War there was rationing, and it affected, especially, children that were in, say, 8- to 15-year-old range. Their growth was severely stunted, and that impacted their children, their grandchildren, and their great-grandchildren. And they could follow that effect just from that group, follow it through, and then they started seeing it in other groups. Different effects depending on what they were exposed to. If we look at it, this is a depiction of it. There are more than these that we know about now, but the big ones that we talk about are methyl groups, they're basically small methyl molecules that attach to cytosines, mainly in adult humans. And there are alterations to histones which is, basically, what we're looking at is the coiling of DNA which keeps it from being available to be expressed. So a lot of your DNA is actually coiled and unavailable. 

So those regions, those genes will not be expressed. Now, that being said, there are a lot of things that go into the environment. It isn't just what we do and what we drink and what we eat, but it's the neighboring cells. As I said before, we have layers of cells, so those neighboring cells are part of the environment and so is the microbiome. So what is this good for? We can study those microbiomes. What is the microbiome? The microbiome is that collection of cells, microbes, that are living with us, on us, in our environment, in the lakes, in soil, those sorts of places. And our bodies, to give you an idea of the balance here that we're looking at, the microbes outnumber our cells 10 to 1. So, for every single one of your cells, there's a whole slew of cells that are living with you that are not yours, that are not controlled by your genetic code. In fact, the genes in your microbiome outnumber your genes a hundred to one. And they've been very important. 

They have co-evolved with us in many ways. Food that we eat could often not be digested without the microbiome. If you're missing it, you simply wouldn't be able to digest most of your food. It's everywhere on your body, even in your lungs. Though one study looked at lungs of healthy volunteers, and in an area the size of your fingernail, they found 128 species, and there's about 200, or rather 2,000 microbes just in that little area alone in part of your lungs. So that gives you an idea that you're pretty much coated with microbes. And they vary. So if you looked at the person to the left or the right of you, chances are you only share 13% of the microbes that they have on their hands. So your hands are not the same as theirs with regard to microbes. And, depending on how you operate in public, only 17% of the species living on one of your hands is identical to the species living on the other hand because we're right-handed or we're left-handed, and we tend to go to the doorknobs and everything else with that one hand all the time. 

We can affect the microbiome. We affect it in the food we eat and in the exercise we get and all those things, but just with regard to the food we eat, we've looked at changing the diet. What happens to the microbiome? Well, there are microbes that will exist when we have a lot of sugars and a lot of fats, and there are microbes that have to chew up fibrous food. And, as it turns out, this can be shifted within a very short time period. In this case, it shifted within a single day. So, all of those genes that were present in the microbes when you were having a western diet were changed, were altered as soon as you switched over to a diet that was, say, heavier in just plant-based foods and not starch plant-based food. Not wheat but rather whole legumes in the entire vegetable, not just a ground up portion of it. We also affect it when we go to the hospital and we give birth. Babies born vaginally were quite different than babies born through Cesarean sections. The microbes were quite a bit different. 

And, in fact, the microbes on children that were born through Cesarean section were pretty much the microbes that are on our skin. And it does have an impact because if you're born through Cesarean section, you're more likely to get skin infections. You're more susceptible to MRSA, multiple drug resistant staph infections. It can also affect us. So, in this case, this study, they looked at germ-free mice. So what they do is they take mice and they put them in sterile conditions and they raise them in sterile conditions and they basically make sure that there's nothing in their guts. No microbes. Clean them out. Raise them. Take the progeny of those. 

They're perfectly clean. There's nothing there. They inoculate one set with microbes from other mice. Okay? So they aren't germ-free mice that they're inoculating from. They're inoculating another set of these germ-free mice with human microbes. And you'd think this would have some effect but maybe not a huge effect. Well, it turns out that with regard to their immune system, it has a huge effect because the mice that are getting mouse microbes, their immune systems developed properly. The mice that are getting the human microbes, they didn't get proper development in their immune systems. And so here you have a quick schematic. You take human gut bacteria. 

You put it into germ-free mice. You get very few T cells. They just aren't activated. You take rat microbes. You put it into mice. You'd think that would work. Same kind of diet, that sort of thing. Still doesn't work. You take mouse microbes. You put it into a mouse. 

You get lots of T cells. You get tremendous amount of immune development. And you can see that this is very, very specific because the rat microbes didn't help with the mice. It affects us in many ways. The studies I've looked at so far show that there's some correlation between our gut microbes, the balance that we have, and heart attack risk, obesity, malnutrition. I talked earlier about these microbes co-evolving with us. Well it turns out that milk, mother's milk, can't be easily digested by children without gut microbes. We're not built, actually, to absorb mother's milk. We've evolved. We've managed to live long enough and be inoculated with all these microbes, and they work for us and with us. 

They can work against us, but they also work with us. There's also been some correlations between the microbe balance in autism, the microbe balance in asthma. If we look at disease disorders, imbalances, there are things that live within us that at low levels don't really have an effect, but, however, at high levels, Clostridium difficile can kill you. Basically, you get everything else wiped out and Clostridium difficile starts to grow and you get diarrhea. You get such severe diarrhea that if it's not treated, you can die of dehydration in very short order. Now, the cure for that isn't exactly wonderful, but it works. It's actually very effective. So, 90% of the patients that are given these fecal transplants, they are cured probably within a few days. From what I read, they get a fecal transplant, it's usually from somebody close to them on the same diet in the same environment or a relative of theirs who's going to have a fairly close match to them as far as genetics and environment. And it tends to work in days. 

And, as it turns out, they said 90% but on a second inoculation I think they were up to 97%. These people are cured. They would die. These people are dying. These are thousands of people that are dying every year, and we really don't hear much about it, but that's what they're dying from. They're just getting incredibly sick and dying from dehydration in hospitals. And this is working. There is a problem with transporting some of these things back and forth, and I think this was a sister of one of the patients. She was so worried because it couldn't be frozen, everything had to be alive and fully functional when it got there. And she had to put on little ice packs, cold packs, and then use it as carry-on luggage, her sample, or at least the sample she was taking. 

She may not have been the relative, but she had to take the sample and she was so worried that at the airport they'd start opening up her luggage and this would be exposed. We can also use all these DNA advances, all this new technology to study gene mutations and expression in cancer cells. And there are a lot of mutations in cancer cells. We look at different mutations. Single nucleotide variations, that's when one of those letters changes to another one, and it can change the amino acid chain that's produced by that and cause issues. There are also structural variations, some of them fairly small, where parts are gained or lost in the genome. And there are also interchromosomal translocations where whole chunks of a chromosome get moved to another chromosome. And some of these are very, very prevalent. So, looking at 24 different breast cancer genomes, there were 239 interchromosomal translocations. I mean, basically, the genome is just going to pot. 

It's getting shifted around. There are so many mutations cropping up. It generally follows a pattern for different types of cancer. And you can actually detect what type of cancer it is, and you can also get an idea of the therapy that's better suited to that type of cancer. And so that's a direction we're moving in. We can target specific genes. We know that cancer is affecting certain genes so we can target those genes instead of doing the whole genome at a time and spending all that money on it. There are ways to use probes that will attach to certain parts of the genome, and I said before how we can denature the DNA and let our probes in to that. And then they'll have specific molecules that we've attached to those probes that can be pulled out with magnets. And then we can denature them again, and we can get these strands that we're most interested in, and we can also barcode because on our adapters that we can put on those, we can put in a little chunk of DNA that has a DNA code that is specific to that sample. 

And it will read the barcode first, and we know it's from patient A, and so that way we can take 20 patients and put them all together and sequence those genes we're interested in. And there are a lot of genes we're interested in. This is one set from a single group that's looking at all of these different genes, and I don't know what all of these are. Some of them we know. There's BRCA1 and BRCA2 up there. Those are the breast cancer genes, but there are so many genes up here that I'm not at all familiar with. They're just letters and numbers to me, but they all designate genes within the genome. And we can look at all of them. We can look at these. We can look at 500 more of these at a time and look for mutations and then get a better idea of what to do when we start looking at therapies. 

We can also study rare mutations. Rare mutations could be a single nucleotide changing in the genome. It may be affecting a gene of interest to us. I think that was, most of you know by now that the little boy who was in, close to Milwaukee, maybe Waukesha was where he was, and he had an intestinal disorder, and it was causing major problems. When you have these, they're critical problems. And they tested the family, and they looked for this type of variation and other variations as well. And they narrowed it down to, I think, two variants. Two variations. Just single bases like this. And that's what's causing the problem. 

So they treated him. He's doing much better. So, you can do that. You can look for an insertion or deletion. Instead of just a change in the base, you can look for whether a base has been dropped out or has been added which can really throw off everything downstream from there when you're looking at the amino acid chain that's produced. You can look for duplication. So sometimes you get copies of the DNA that are then inserted back into the chromosome. So, you get extra copies of genes. Or you lose a copy of a gene. You can look for inversions where the DNA in part of the chromosome is flipped around. 

And you can look for gross translocations. We talked about earlier a big chunk of a chromosome inserting itself somewhere else. You can use these to track things, to look for super bugs, in this case. So here's a New York City hospital, and this is in 2011. They had a woman there that had multiple drug resistant pneumonia. She was transferred to the NIH clinic that handles these very critical cases. And three weeks after her release, she was fine. She got better. Somebody else started getting infected within that clinic. And they had been very, very careful to sanitize everything to make sure that gloves were used, they're disposed of, they're burned. 

Everything is very carefully run, but other people were becoming infected. Now, they had swabbed this lady while she was there, and they took swabs from different parts of her body because, as it turns out, when you have something and it becomes resistant, it is becoming resistant in you. And so you could have some areas in your body where you have the original strain and some areas in your body where you have a drug resistant strain that's starting to spread very quickly. Well, some of the cases within the clinic displayed additional drug resistance. So it wasn't just to the drugs that her strain was resistant to, but there were additional drugs that were ineffective. And they started looking at the people that were being affected. In the end, there were 18 people infected in the NIH clinic. Eleven people died, six directly from pneumonia. So, what they wanted to know was, were all the patients infected from patient number one? Did it follow some path through the clinic, or were these new infections? People were coming in and then leaving later on, and were they bringing something in? If it was within the clinic, how was it being transferred? You want to know in a clinic where it's traveling to. You want to know if it's on the equipment. 

You want to know if it's on people. What's going on within the clinic to spread it? And what can you do to stop it once you know that? And what can you do to reduce drug resistance? All those questions needed to be answered. So they took these different isolates from this woman and they grew them out, and they took them from her throat, her groin, BAL, and I was with some doctors and they just told me what BAL was but I think it's further down. >> [INAUDIBLE] >> What does that mean? >> They basically run some fluid into your lungs, pull it out, and take a cell sample. >> Okay. So they looked at all these different locations, and they also tested the other patients that were around her and that were getting sick as well. They looked at everything in the rooms. The ventilators, sinks, drains, all of those things were swabbed and tested. And they sequenced every single one of the isolates that they found. So they grow them out, they start with a Petri dish and grow something out, and apparently when they initially started the Petri dish, the medium in that was not working very well. 

They had to switch media to something else that worked a little bit better. And when they looked at all of that, what they found was that there certain things, and you can see where it says BAL, groin, throat, and urine, urine, urine. Urine was the original. Okay? That was the original isolate. And what you're seeing here is sort of a chart of where all the variants are, the black squares being variants. And they managed to find that, in fact, some of the variants were occurring with this woman. And since this was fairly specific to this woman, they tracked it to all of the other groups. They found three groups in the clinic. And they determined from movement within the clinic and from this information how things spread. So they had the first patient, that was her, the second patient to get sick, a third patient to get sick who they thought was infected from the second patient, but, in fact, it wasn't that way. 

It was a separate infection. Actually, the third patient that came down sick was directly infected by this woman and passed it to a different person who happened to be the fifth person who showed symptoms who passed it to the second person who showed symptoms. So some people were asymptomatic for a while, and then all of the sudden they said, oh, my gosh, here we have another person who's infected. But the asymptomatic people were spreading it. And they also found it on a ventilator that they had sanitized twice. They were absolutely sure that the ventilator was clean, but they thought, well, we'll swab it one more time. They swabbed it one more time, lo and behold, there was still pneumonia on it. So, what they determined was that you have to have this very extensive and frequent surveillance going on. You have to have the right media to use. You have to know where these people are going, where they've been. 

You have to assign nurses and doctors specifically to those people, and, while they're with them, they don't get to visit any other patients. There are people that are set up specifically for these certain regions there. And they talk about having ongoing effective surveillance before the outbreaks occur. So, you should swab things periodically and try and grow things out even if there hasn't been somebody in there. How important is this? The last patient of those 18 left in either January or February of this year. Mid-summer, a 10- or 12-year-old boy went into the NIH clinic, came down with pneumonia with one of these isolates. It was still there after all that time. They'd been very, very careful, but this little boy got it, and he is the seventh victim that died directly from this pneumonia. They also found out from that that pneumonia is very, very stable in the environment. And you have to verify that after the cleaning process that everything is, in fact, clean. 

All right, there are many other things that we use this for. We're getting more and more into epigenetic studies to find out how these switches are turned on and turned off, how long they last, those sorts of things. We're coming up on 8 o'clock right now. This is pretty much what I have today, and so I'll say thank you very much for your time and attention. [APPLAUSE]