9UNP0156SD_Josh Hyman_WordPress
"Inside the Latest Generation of DNA Sequencing Machines" April 16, 2008

- Welcome to our Plato class in frontiers of life science. Today we have josh Hyman at the biotech center and I will let him tell a little bit of his background, about he will give us the latest on DNA sequencing. So welcome, josh. 
- All right. I'm the director of the DNA sequencing facility, my background is in genetics, and quite a few other things. I also have a background in horticulture, mechanical engineering, and business. So, as my wife says, I have too many letters after my name as it is, I can't go back to school anymore. We'll be covering sequencing, both what used to account for sequencing, and it's how I learned to do sequencing, and very quickly, sort of tumble through last year's technology, quite literally, this year's technology and the technology we are going to see probably in about three or four more years. Things are changing very, very rapidly. 

I'm going to give you a sort of brief overview of some of the basics. Show you the technology itself, I'm hoping it doesn't take too long to get through that. If it gets too laborious for you, tell me and I’ll give you more of the reader's digest version. With my past, just so you are filled in a little bit, I worked at McDonnell Douglas on the 15e project designing windshield canopy systems. Went back, got a degree in business, worked in the seafood industry for a while, before striking out, and working on my own, actually with a family business, ransom restaurants, that sort of thing, and then decided what I really loved to do and what I was dissuaded from doing when I was in high school and college was going into horticulture, and genetics. So my wife wandered in one day when I was getting things ready for the garden and she said you know, we are here at Penn state, just get your horticulture degree. She regretted that for the next seven or eight years, but at any rate, ended up in again genetics and finally made our way up here and this was a, probably a choice of one of four or five places that my wife was willing to move to. She's originally from Michigan, Traverse City, Michigan, so this was close enough to home, but not so close that, you know -- it became like moving back in with the parents. All right. First of all last year's sequencing was all based on Sanger sequencing, and we are going to review that very, very quickly. 

There is another sequencing that was used originally back in the 70s and I think it may have even stretched into the 80s which was gilbert son sequencing, we are not going to cover that. So, sanger sequencing relies on having short fragments of DNA that you already have sort of focused in on, or collecting DNA and then amplifying very short fragments, but individual fragments so that's all you are dealing with at one time. And one of the ways we produce those, through a process called PCR. And I don't know if everybody here knows what PCR is, I’m going to cover it briefly, it's a way of amplifying one distinct stretch of DNA. Sometimes you end up with more than a stretch that you want, but your focus is on one stretch of DNA. So, DNA basics, DNA has four letters that make up the alphabet of the DNA. A, c, g and t. And these are what they represent. They are just four different molecules. Everything about you and everything about every bit of life out there is pretty much caused by the combination of those letters. 

We have a lot of letters in our book, in our instruction manual. We have three billion of those as was pointed out in a blurb I saw the other day, if you try to count or say every single one of those in order and you took one second to say each one, it would take you about 1200 years to say all the letters in your DNA code. It's typically double stranded, there are some organisms, viruses that don't have double stranded DNA. Each strand has a direction, a positive and negative way to go through this and we tend to call the 5 prime and 3 prime, where the bonds occur, and the other thing that we know is that there are pairings, bondings that occur in your DNA. So, add mean always pairs with the thigh mean. One strand going one way, the other strand going the other way, you read down one side of it and you might see that. Well, that tells us immediately that the other strand going in the other direction is a reverse complement, looks like that. G with a c, t with an a, so forth, all the way through. That allows us to duplicate something, read one strand and know what's on the other strand. If we read the strand that isn't expressed in the cell, isn't used for the code to tell the cell to produce certain RNA that then gets coded into peptides, protein, and everything we need. 

We can just go through one strand and know what it is. Here is how PCR works. We start out with a double strand of DNA like you have in all your cells. We heat it up to 96 degrees. That forces the two strands -- bonds break down at those high temperatures. When that occurs, a fix in a forward primer and reverse primer and these are really, really short strands of DNA that we can synthesize in the lab. Typically we used 20 to 30 base pairs, 20, 30 letters in a row that are all hooked together as one word, so to speak, and they'll have a target somewhere in the DNA that we know about and when we cool things down to 50 degrees, 55, 60, somewhere in that range, they will find their target and they'll stick together. They have a 5 prime and 3 prime end and they'll go from 5 prime to 3 prime, they'll extend that way, if we add something called po limb ricks. We usually use tack -- it comes from an organism and isolated just that enzyme that will extend and add nucleotide. So we add a mix of these single bases, single letters out there that are capable of being added in this process and they are hooked together, and extends out. 

So it starts down right where the primer is, and goes from this is the 5 prime into that primer, this is the 3 prime end, and it will just extend out and it will make a copy of your DNA. And it does it fast. We use, they say average about 25 base pairs per second. And we know that other will move much faster in your body. So, in fact all organisms. So we go through one step and as you notice we have actually shot beyond where that first primer site is. So if this primer were down here, if we saw reflection it would be right here. We have gone beyond that, extended out and beyond that. So, we go through another round and in the second round we have a fragment that starts here and extends out in that direction. We have a fragment that starts here and extends out in this direction. 

And this is important because in the next round of PCR, now our primer lays down right here. It can't go beyond this point. Now, we do have some of the original template in here, here and here. But we only have a certain number of copies of that original template. And each round of PCR, we are going to create more copies of these stretches. So that we start doubling the numbers of these short stretches, but as far as template go we always have the same number of copies of that really long stretch of DNA. So, this reaction here is going to occur linearly, every time we get, we run through one cycle of PCR , we are only going to start with that template, the original template and we are going to add one more copy where we can extend out and beyond this point. But in subsequent rounds of PCR we are going to increase the number of short fragments and so we keep increasing that template. And that occurs exponentially. We go from two copies to four copies to eight copies to 16, 32, 128, grows to be a very big number very quickly. 

So finally what we'll end up with is just those very, very short copies. It can't go beyond this point, after a while. 
- You keep changing the degrees? 
- We cycle them. What we do is we start at a high temperature where we denatured it and then cool it down so the short primer can so it's short, it has a much lower melting temperature. We get down that that temperature and turns out most are most active at about 70, 72 degrees. Some of them that have been isolated or better off at 68 degrees, but that range. So, this starts to extend and then we ramp up the temperature and by that time this is bound to the template fairly well because it started to extend, so there's more of it to bind. And then it takes on. So after a while, obviously, we are creating a lot of these fairly short fragments. 

Okay. So that's PCR. So, how does that play into sequencing? Well, first of all, we need a lot of those short fragments because if we don't have a lot of them, we don't have a lot of target for our primer to attach to in the sequencing process. The sequencing process isn't an exponential process, it's a linear amplification which is what you will see. We can't sequence from both ends. That's like taking a book and trying to read it from both directions at the same time, back to front and front to back. You would have two letters at every location and you don't want that. So when we do sequencing, we add another batch of primers, dilute everything in the mix you just saw, and then we add another batch of primers but only add one primer too -- to it. We start on one and go the other way, and different reaction the other end and go back this way so we can read back to front if we want to, or we can read front to book. 

But we can't read in both directions at the same time, we'll just get confused. So, we need multiple copies of single stranded template DNA, we have just talked about. We need a primer. Now most of the time when we do this, we use the same primer, we just amplify this. We just happen to use only one of them. We need A which we talked about and a pool of normal. Sanger sequencing, new the nucleotide are the letters we will add one by one. A small portion of the nucleotides will be special ones. Those are ones that have been modified in a way that you can't add something afterwards. And we'll label those somehow. 

Okay. And the way they are modified is on one end of them, there is a slight change. Group has been altered. And because of that, these are molecules that look the same and will be added the same but you can't add anything beyond them. So, the reaction terminates. So you go so far and then it stops. And you go a little further and it stops. But you know we can make them so that it's a T or an A or whatever it happens to be. Now if you took one reaction and had a pool of normal knee tides and add to it, it would go out a certain distance and terminate with the t and might go out a different difference and terminate at the t. Wherever it terminates will have the t. 

Does that make sense? So you get a whole bunch of fragments of varying sizes, but at every one of these lengths you stop at a certain knee tied and it happens to be a t. Guess where we are talking about. Okay. That's what we have just discussed. They stop at random. So you have a mix of DNA lengths and then you put them in some matrix and let them travel through that matrix. Now, DNA has a charge to it. All you have to do is put a positive and negative charge across whatever matrix you happen to be using, and DNA will travel across that. And the small fragments will move faster, and large fragments will move slower. 

And since now we know we have labeled these things, we can take products from four different reactions, t reaction, a reaction, g reaction, g reaction, and I’ll get through this they'll move down a gel. So we have four different lanes on a gel. So we could actually read through this. And we read from the smallest fragments that have been moving fastest, on up. Okay. So, this is what you get, actually that's a picture of what you get. What you really get is this. And if you look on this and pass it around, this isn't what's called an auto rat. Took probably a graduate student maybe four days to produce that. And there aren't that many lanes on there. 

If you look on there, I think there are six lanes on that. But in each one of these you can see the separate reactions, four reactions in each one of these lanes. So somebody had to look at that and work their way up it. And as they worked their way up it, they read from the bottom on up, and they had to go okay, this is the c lane and this is the t lane, so they go c, t. This is the A, and then this is the g, a, gg, a, t, ccc, g, g, and they worked their way up it like that. So some graduate student gave it too an under grad who would take the ruler and go up it that way and record that by hand. That's how it had to be done. Now, you could label those with colors instead of putting a radioactive isotope on your you could put a color. What happens here is you had to label them with radioactive isotope, which was dangerous, and then you had to run the gel out, and open up the cassette, or the basically put the auto rad against the gel and expose that for a few days, and take that, which is, it's just film is all it is. Take that out, develop it, and then read it. 

So that's a fairly long process. But if you could label them with something that fluoresced with different colors, you could see it much more easily. Discriminate which one was which, and you could run them down a single lane. They would go down the one lane, and if you used a laser, you could actually make it fluoresce, run it through a prism and separate the colors out and detect it. And that's exactly what has happened. So, last year's piece of equipment for a mere $350,000 could do that. And it gave you this type of output because it just went lieu the compute -- through the computer on the machine and it would tell you here is your readout. Typically what you would get electronic and paper form is something like this that has all the different colored peaks on it but would go out hundreds of bases. This doesn't take all that long, it takes you about an hour or so to set up. A single plate of DNA that you have run these reactions in. 

You put it on a cycler, it ramps up the temperature up and down, and it's sending these out, and you do that and you can do 96 at a time. So instead of doing six at a time and taking four days, now I could probably do eight plates easily in a day. In fact, you have done eight plates in a day. And you wander over to the DNA sequencing facility, you drop it off with us, we put it on the machine, and you have done the clean-up and everything else, for a whopping $50, you could do an entire plate. Think of the labor savings and think of the time. Now, you have 800 reactions that you've done in a day. Versus six in a week. This is digital information. Easy to read, easy to get through. You can take it, throw it up against databases, check to see if it matches anything else, all that sort of thing. 

Figure out really what's going on with your organism. And the way it works, I’ll pass this around, is that within the guts of that machine that I just showed you, you have capillaries. They look like fine hairs but they are in fact glass tubes. And you have steel pins down here, they go into your sample. There's an electrical current that goes across from the outside of this, which is the steel piece, across that, and through the polymer in here and there's a little bottle at the other end and electricity can basically travel through that. And it's injected on that. It's not that the sample itself is put onto the end here, it's just that the fragments are moved across using a charge. So that's how they moved on to this and then it comes out of here, goes into a buffer, which of course allows the current to continue. Why don't you go ahead and take

a look at that, you'll notice at the very end of that array of capillaries there's a little window here where all the capillaries go through, and a laser scans that back and forth and then that is where we do all our reading, that's the window of all those fragments as they come through. 

Anybody -- everybody seen this? Okay. All right. Now, that's pretty nice. We can do 7 to 8 had -- to 800 bases per sample. We can do 96 samples at a time and 12 to 13 plates per day. So that gives us 873,600 bases per day that we can generate per machine. We can do that five to six days per week. We can load it up on a Friday night, it will run all day Saturday. But, the human genome is three billion base pairs long. 

So if I take that one machine, generating 873,600 base pairs per day, and I compare to the human genome, it's going to take nine and a half years to give me one x coverage, okay. So that means we would essentially create enough information to cover that genome one time over. Except that the fragments that were isolating most of the time overlap. So we need more than one x coverage for the entire genome, we want them to overlap because we have to line them up somehow. Sequence alignment is similar to taking all the works of Shakespeare and shopping them up into really, really short words or phrases and throwing them in a big pile. So if you can imagine taking 25 let ertion at a -- letters at a time or 50 letters at a time or 100 letters at a time out of all of Shakespeare’s works, he probably didn't have three billion letters, we can't count that high in a lifetime, you are expected to reassemble all of that into something that makes sense and that's what the computers do for us. They assemble all that. So we need overlap. So we don't want just one x coverage. We typically want ten to 30x coverage and if we have a new organism, 30 is more like it. 

So it's not nine and a half years, it's 30 times that. It's roughly 300 years it's going to take one machine to do all that. And I don't want to wait around for that, I don't think anybody else does. So, the way that it's been done in the past is they have these rooms, buildings filled with machines, hundreds of machines. So that instead of 300 years, you used 300 machines and do it in a year. Obviously that's kind of expensive, and it's a fairly slow process. Now remember what we have talked about so far, we have done fragments at a time, single ones at a time. Wouldn't it be nice if you could get a whole bunch of information at a time from all the different fragments that you could possibly create out of a single genome, one genome being all the chromosomes in a cell, and so we actually have quite a bit of information there. So, in a single genome, we want a complete coverage. We would like that. 

And there are other things that I’ll talk about later that we may want, information that's very, very important. But say we want to start with just sequencing a massive amount of DNA from a single organism. It would be nice to do it in parallel so instead of looking at a single region, we look at everything all at the same time. We take 100,000, a million, two million reads all at once. A read being a stretch of DNA we are looking at that's set together as one contiguous piece. Okay. So the way to do that is to chop everything up, and put adapters that we know the sequence of on the ends of every single one of those pieces. And get those ready to stick on a plate or some other surface and, you know, it looks big, these things are tiny. They are incredibly small. When you see these, you are looking at a chip that's about that long, about that wide, has eight channels on it and each channel can hold about, oh, four million to eight million reads, so we are looking at specs we can't see on something. 

Okay. So we put them down on this surface, and we do essentially a PCR reaction. Because on this surface we have primers that are attached right to the surface. And so this little chunk of DNA is denatured, so one strand goes floating off. The strand that remains there hooks over and finds a primer site. Because the way these are set up, they'll bind, each end will bind down to this substrate. But as soon as you denature it, both strands will have one end but not the other end that will bind so they'll sort of do that. So they come apart, but they can still bend over and find these other primers. So they are flip flopping around and they call this bridge amplification. And so they start to amplify them, and they are sparse enough so that you just get clusters forming. 

But each cluster is really amplification of one short strand. So now we are doing PCR of a single strand in one location, but we are doing millions of locations. So we have millions of strands. So we get all these strands and then there is one cycle that they don't tell you about in this literature, which is that one of these ends will cleave off and the other one won't, given a certain enzyme. I'm pretty sure that's how they are doing it. I have to sort of guess at it. So they cleave off essentially half of the strands because they only want to read one direction. At least that's the way they are showing it. And one of these adapters has a primer site on it that we are going to use for sequencing and it happens to be the adapter end that's floating freely up here. And so now you can start doing that sequencing reaction that we talked about earlier, except in this case instead of create and bunch of fragments, we are going to add a nuclear a tied and read it. 

And add another one and read it. So there are cycles that they go through here where that everything all the label nucleotides and one of them will be attached. Only one in this particular machinery, this top technology. So that one gets attached, everything else is washed off. All right. And then you take a picture of that slide and you take a lot of pictures. Because you've got a very, very small area you want to take it one time and you have to magnify that up and identify where all these spots are. And you have to know that you are taking exactly that area and you are matching it up as you go through this one cycle after another. So they do this and they keep going through these cycles where they image it, add another base, wash it all off, take another image. You keep doing that, until finally you can come back and look at one spot and go okay, we have all these images stacked up, and it's for this one little spot, it goes g, c, t, g, a. 

And that's this spot. Obviously there are other spots on here so we'll have different sequence. And you extend out. And you get a terrific amount of information that way. But these are very short reads. Remember, I said we got 700 plus bases on Sanger sequencing. This particular technology, the last run we did, at about 16 bases, we started to get an error rate that increased and increased and increased. So now we are really talking about short reads. Very, very short reads. Which are fine if you have an organism that isn't very complex. 

But anything remotely complex and it starts to break down and gets more and more difficult. So there is another technology, basically it's the same thing, adapters, we get single strands, they have an a end and b end being the adapters. Except in this case, I'm going to go through this pretty quickly, everything is done on beads instead of on a plate. And when they do, they mix it in with the beads. The whole mix has such a low concentration of DNA, what we are trying to do is get one single strand of fragment with one bead. And then they put this in oil, and blend it together violently enough so it creates this emulsion that has little beads surrounded by the liquid that contains everything else. And they do PCR in an emulsion. So each little glob u al, each little bead with the surrounding and everything else is all by itself floating around in this oil. And it's heating up and cooling down, heating up and cooling down, and so they amplify, just as they did on a plate, they amplify a whole bunch of strands but they are all the same strand on the bead. And this particular technology does it a little bit differently, and what they do -- put in a plate, they separate the beads out on this plate with holes in it, and these are tiny holes. 

The way they made these plates, took fiber optic cables, something like that, put them in a matrix, mashed them together and cut off a slice of that, and then etched out one side so they had the wells. So these are tiny wells. The beads go in with a lot of extra reagents, because at this point they have them out of that emulsion. And in this case, what they do, they add one of those nucleotides at a time. So it can only go out as far as that particular nucleotide can be incorporated. But unlike the other one, they can add a lot at one time. Because these won't terminate the reaction, the others had reversible terminators on them, so one got added and no more could get added, they had to wash that off. Change the chemistry a little bit of that last nucleotide put on there, and then have another one. So that's how they did it. And the other technology, but this particular technology they add all the as in a stretch at once, all the Ts in a stretch at once, all the cs or all the gs. 

And then they look at the intensity of the signal and say ah, this is a big signal so there were a lot of as added this time, in fact, it looks like there were 25 added, and here ten ts, one t, one g, one whatever it happened to be. So that's how they go through and they figure this one out. And this gives us much longer reads. In fact, the latest information from that company is we should be able to get up to 500 base pairs with that technology. So we are starting to approach what we were doing with Sanger sequencing. It doesn't give you as many reads, we are looking at about 100,000 reads, not 200 or 400 million reads. It will gave us base pairs and paired end reads. Paradigm reads means we take a read from this end and a separate read from that end, from a single chunk, and we happen to have a rough idea of how many bases are in between there. So instead of just reading off of one end and calling it quits, we take a longer fragment and read off both ends and we know they are attached now so they aren't just randomly placed somewhere in your genome. If we know where this one is, we have a pretty good idea where this one is. 

So we can take that information in a single genome much more easily. That's very powerful. The one that puts on multiple base -- bases at a time, give us 100, 250, 2007, projecting up to 500 bases at time. About 100,000 bases or 100,000, 100 million bases per run, this is projected to go up to 500 million. So this is producing less information than this, because this is already at a billion bases. And it's projected to go up to about four, five billion bases. So as you see, human genome is only three billion bases, we are approaching that. Passing that with some of the technologies. Sanger sequencing was a cheaper sample, $1 to $2 sample, you can get it lower than that if you were careful. But, 15 to 30 cents per base. 

Single base short reads, current technology, we are 1/50th of that already. Okay. But one run on the machine costs you $6,000. So there is the threshold to get over there. Now, the long reads that we were talking about, that machinery right now with the reagent's running, is about 12, $12,500 per run. You can assemble that a little easier than you can assemble the short reads. So that's not quite as good when you compare dollars per read or cents per read, okay. You have a ten-fold increase there. Now instead of nine and a half years to sequence the genome one time over, now we are down to nine days because these runs don't happen in an hour, they happen over, you know, either a day or three days or something like that. 

But, since we have short fragments that would give us that faster read or that amount of information, we are going to have to pair that up, with paired end reads or longer reads that we get off of the other technology with the beads. All right. So we have to use a combination of technologies to get there. All right. So, to sum it up, as far as the current technologies go right now parallel versus Sanger, parallel will get millions of fragments, it's very low cost per base. The random fragments, but they tend to be shorter reads. The Sanger sequencing is giving us individual reads to focus on, very low cost, but they are longer reads. But, there is an x next gen already out there. It's no longer next generation, but there is always someone out there trying to fill the need of all the researchers and health care industry, pharmaceutical, the whole bit, and they have already started producing equipment in labs that's capable of doing much more. We are talking about doing single molecule sequencing now. 

Not doing this amplification and putting it on beads or putting it on a plate or something like that. We are talking about taking a single molecule without altering it, without introducing any errors that could come along, and we are going to sequence those. Not only that, but we are going to do, I think there are thousands right now, but hope to do a million at a time. It will be extremely low cost. Extremely fast. And much longer reads. They have already tested it out to about 1500 base pairs. So roughly twice what Sanger sequencing can do. And they circularize the fragment of DNA so they can keep repeating one fragment over and over again and do a quality check on it. So, give me just a second here, I’ll stop this. 

We'll need the sound and I’ll show you their blurb. 
- Okay. This is on with your website. 
- Developing a transformative DNA sequencing technology that will enable researchers to answer questions important to human health care. We call it smart, single molecule real-time DNA sequencing, a breakthrough technology based on the natural process that occurs every time living cells divide. Prior to division, DNA is replicated by enzymes called DNA which essentially duplicate entire genomes in minutes by reading the DNA and is he sequentially building a complimentary strand with nucleotides. Smart sequencing engine by eaves dropping on it while it works to replicate DNA. This approach is enabled by two proprietary technologies. The first is foss fo linked. To visualize the activity, a different colored fluorescent label is attached to each of the four. 

A, c, g, and t. In contrast to other sequencing approaches, our link carry the fluorescent label on the rather the base. Through this innovation, it cleans away the fluorescent label as part of the incorporation process leaving behind a brand of DNA. This exploits including high speed, long read length and high fidelity. The second key technology is a visualization chamber called the zero mode wave guide, or zmw. Visualization of the individual molecules against the background while maintaining a high signal to noise. The zmw is a metallic chamber, approximately 70 in a no meters wide, illuminated through its glass support just 20. Diffuse in and out of the zmw in microseconds. When it encounters the correct, it takes several milliseconds to incorporate it, the label is excited emitting light captured by a sensitive detector. After incorporation, the label is clipped off and diffuses away. 

The whole process repeats, creating sequencing bursts of light. These are recorded, thus building the DNA sequence. Our smart sequencing harnesses the natural ability to synthesize ten or more bases per second over thousands of continuous incorporations, leading to high speed and long read sequencing. Additionally, our smart technology design allows simultaneous multiplexing of thousands of zmws in parallel, all concurrently replicating DNA in real-time. The data is assembled into whole genomes and analyzed by researchers. Our smart sequencing technology will ultimately translate nature's ability to replicate an entire genome in under an hour to an instrument that will in minutes for under $100. This unprecedented level of performance will completely redefine the field of again genetic analysis and enable new scientific advancements that will lead to improved health care worldwide. Smart DNA sequencing. 
- 500 to 600,000 is what they are projecting. 
[inaudible]

 Right now I think they are at 1,000 and want to get up to a million. 
[inaudible]they have to figure out, we are going to have another presentation from the web soon, so we probably can leave -- kay. Probably leave that going, because within about an minute or two we'll have another one. So they're shooting for a million wealth. Right now they are doing about five per second. They are shooting to get up to closer to 50. So they figured that they could do a human genome, once every two minutes. So they could do 30 full coverage within an hour for less than $100. 
[inaudible]

 
- Yeah. Probably. Except that these are very, very long fragments now. So, alignment is fast. 
- Easy to align longer fragments. 
- If you can create, you know, take this DNA and loop it over and make it circular again, then you can keep reading it so then you take these reads and you go okay, match up within that particular cell, that well, align those up, do the quality checks right then and there, it's done because you have 30 points to take from. You say well, two of the reads were wrong, the rest of them came out a certain way. This is how it is at this particular base. You have taken care of that. 

1500 bases is where they are starting. They are talking about going up to 10 to 20,000 bases per individual well. So they'll have these huge stretches, and that's extremely important as you'll see here in just a moment because it isn't just, you know, we have a human genome and we know what it is. We have human genomes. We have over six billion human genomes in this world right now and we are increasing. So, all that information is a little bit different from individual to individual. So it's -- with all that variation, now people want to know what's going on between individuals, what's causing diseases. Okay. To then we start looking at the uses of this information, and with Sanger, we have a few. We can a single region of interest for individual trait, bearing some importances related to disease resistance, nutrition, compare individuals within those small regions and things like that, and also aid parallel sequencing and finishing off assemblies. 

There is actually quite a bit more that we can do, but there's a tremendous amount that comes, that becomes possible once we have a huge amount of data per individual. Just make it a little easier. This is just a short list. I'm going to focus on a couple of these things and I have another presentation from the web that you guys can look at. And then we can sort of do question and answer, discuss a few things, see how you feel about having all this information available because I'm not so sure how I feel all the time. But obviously we can do some things very easily right now, we can sequence small genomes and many, many samples. And instead of going what was the difference between this bacteria and this bacteria, and grd u alley getting through it, now we can put it on the plate, resequence it, have the information assembled again and make comparisons. Why did we have to worry about this having much higher pathogeni st. -- and how can we stop that from occurring, things like that. So we can already do that to a certain extent. 

We can compare whole genome, say between ma maths and elephants, people have done that. We can do population studies where we take pools of cells, and we look for certain things occurring in those pools of cells, one pool versus another pool. We can look at gut microbes and find out what's in our guts. Because we aren't individuals. Okay. We are walking worlds, when it really comes down to it. Because if you didn't have gut microbes, your nutritional status would change drastically. All right. Sequencing of organisms such as squid, which have been very, very difficult to sequence prior tho this. Look for structural rearrangements and copy number variants, have a fairly large impact on your health. 

We are not the same and it isn't just one or another that's changing in between us. As we have sort of delved further and further into the human genome and compared different people, what we found out is there are fairly large chunks of DNA that are either present or missing in individuals, and some of those go into genes. So, we may not have all the genes that somebody else has, and we may have more genes than somebody, a third person has. May have a different mix of genes. It's not just that we have different variants of a single gene, but ours may be partially or drastically deactivated. We may have many numbers of a certain gene, because we do have genes that come in multiple copies, and they affect expression of a certain trait. So somebody may have ten copies, another person may have 25 copies. So we can look at that. We can do you'll see a little bit of precipitation because the DNA is not just one long strand, it's loose and floating. It coils up, it gets compressed, in regions that can't be accessed so those genes that are in there, they can't get expressed and other cases it relaxes and during development this goes back and forth a little bit and we get events also occurring which affect gene expression which is what we have been talking about, and here is another field that we'll discuss briefly. 

What we are used to when we talk about DNA and inheritance is thinking of this fixed alphabet, this fixed manual that we are passing on to our children. But that's not all we pass on. Because everything you've done in your life up to the point where you have children gets passed on. The effects that you've had on your DNA get passed on. It's not just mutation of a base here and base here, or chunk of DNA that gets spliced out randomly, it's not just that. There are events that occur. Okay. So this reflects back on the one thing that I was talking about where you get structural variation, you lose or you gain a part of DNA. But the other thing I want to talk about, the big thing I want to talk about is genetics. Genetic variation, it's not related directly to your DNA code, but it affects your DNA code. 

It's variation that can and does change over your lifetime. So what you are doing as a 10-year-old affects where you are at 20, affects where you are at 30, affects where you are at 40, all the way through. Things happen to your DNA and like I said, it's not just a mutation here and there that's occurring. And it is variation that you can pass on to your children, to your grandchildren, to your great grandchildren, as you will see. Okay. How does it happen. One of the things that happens is methal groups. They can be added and reduce expression of a given gene. All right? So these little groups can be added. 

The other thing is that you have these areas where I said it coils up, your DNA does. But there are factors that can attach to these tails and they can affect that coiling, so instead of having something that isn't available for transcription, translation, becoming a gene and expressing itself, all of a sudden it is available. So now we'll take a look at the other thing I wanted to show you. 
- You ever notice if you get to know two identical twins they might look alike but they are always some different. As they get older, those differences can get more pronounced. Two people start out the same but their appearance and their health can diverge. For instance, you have more grey hair. 
- An no, I don't. 
- Identical twins have the same DNA -- 
- We are getting some skipping here. 

Okay. This is one of those things that I couldn't -- exact same genes. 
- Yeah. 
- Don't our genes make us who we are? 
- Well, they do, yes. But, they are not the whole story. Some researchers have discovered a new bit of biology that can work with our gene, or against them. You are heavier and I'm better looking. 
- Yeah, whatever. 
- Imagine, coming into the world with a person so like yourself that for a time you don't understand mirrors. 

- I looked in the mirror I would say that's my sister and my mother would say no, that's your reflection. 
- And even if you resist this cookie cutter existence, cultivate individual styles and abilities like cutting your hair differently, or running faster, uncanny similarities bond you together. Facial expressions. Body language. The way you laugh. Or dress for an interview, perhaps. When you hadn't a clue what your sister was going to wear. Sin cro any in your lives constantly confronts us. 
- When I see my sister, I see myself. She looks good, I think I look pretty today. 

But she is not wearing makeup, I say my god, I look horrible. 
- It's hardly surprising. Because you both come from the same egg. You have precisely the same genes. They are literally closed. Better known as identical twins. But now, imagine this. One day your twin, your clone, is diagnosed with cancer. If you are the other twin, what can you do? Except wait for the symptoms. 

- I have been told that I am high risk for cancer. That hangs over me. 
- And yet, it's not uncommon for a twin to get a dread disease while the other doesn't. But how can two people so alike be so unalike? Well, these mice may hold a clue. Their DNA is as identical as these, despite the differences in their color and size. The human that studies them is duke university. 
- So randy, I see here you have skinny mice and fat mice. What have you done in the lab? 
- They are identical. 

- The fat and skinny ones. 
- They are huge. 
- An they are huge. 
- Can we weigh them? 
- They cannot walk too much, not running too much. 63 grams. 
- 63 grams. 
- Let's look at the other one. 
- Half the weight. 
- Right. 

- This gets even more mysterious when you realize the identical moo

ifs mice have a particular gene. But in the yellow mouse it stays on all the time, causing obesity. 
- Just look at this. 
- So what accounts for the thin mouse? Exercise? Active? No. A tiny chemical tag of carbon and hydrogen has affixed to the gene, shutting it down. Living creatures possess millions of tags like these. 

Some, like methyl groups -- 
- it's buffering again. 
- Attach to genes directly inhibiting their function. Other types grab the proteins around which genes coil and tighten or loosen them to control gene expression. Distinct patterns exist in every cell, constituteing a sort of second genome. The epi genome. 
- Epi genetics literally translates to meaning above the genome. So if you would think, for example, of the genome as being like a computer, the hardware of a computer, the epi genome would be like the software that tells the computer when to work, how to work, and how much. 
- In fact, it's the epi genome that tells our cells what sort of cells they should be. Skin, hair, heart. 

All these cells have the same genes but the epi genomes silence the unneeded ones to make it different than other. They pass on as cells divide but are not necessarily permanent. Researchers think they can change, especially during critical periods, like puberty and pregnancy. The mice reveal how the epi genome could be altered. To produce thin, mice instead of fat ones, they -- tags turn the gene off. 
- I think you can see that we dramatically shifted the coat color and we get many, many more brown animals. 
- That matters because the coat color is a tracer, it's indicator. 
- That's correct. 
- Of the fact that you have turned off that gene. 
- That's right. 

- This epi genetic fix was also inherited by the next generation of mice, regardless of of what their mothers ate. And when an environmental toxin was added to the diet, instead of nutrients, more yellow babies were born. Doomed to grow fat and sick liar -- sick like their mothers. 
- It seems like this has profound implications for our health. 
- It does. Basically what you eat can affect your future generations. So you are not only what you eat, potentially what your mother ate, and possibly even what your grandparents ate. 
- So, how do you go to humans to do this experiment, when you have these mice and they are genetically identical on purpose. 
- That's right. 
- Who is your perfect lab human? 

- Well, then we look for identical humans, which are identical twins. 
- Twins. 
- And that brings us to the reason why we are showing you spanish twins. 2005 they participated in a ground breaking study in madrid. Its aim, to show just how identical epi genetically they are or aren't. 
- One of the twins is that if my twin has this disease,ly -- I will have the same disease and genetics have a high risk of developing the same disease but it's not really sure they are going to have it. Because our genes are just part of the story. Something has to relate the genes and part is the genetics. 
- Wanted to see if the twins epi genomes might have accounted for their differences. To find out, he and his team collected cells from 40 pairs of identical twins, age 3 to 74. 

Then, they had the laborious process of dissolving the cells until all it was left was the strands of DNA, the master molecule that contains our genes. Next, researchers amplified fragments of DNA until the genes themselves became did -- did he tech tibl. Those that had been turned off appeared as dark pink bands on the gel. Now, notice what happens when the genes from the pair of twins are cut out and overlapped. The results are far from some, especially when you compare the epi genomes of two sets of twins that differ in age. Here on the left is the overlapped DNA of 6-year-old xavier. The yellow indicates where the gene expression is identical. On the right is the DNA of the 66-year-old. In contrast to the younger twins, hardly any yellow shines through. Their epi genomes have changed dramatically. 

The study suggests that as twins age, epi genetic differences accumulate, especially when their lifestyles differ. 
- One of the main findings of research is that epi genomes can change what we eat, whether we smoke, whether we drink. And this is one of the key differences between genetics. 
- As the chemical tags that control our genes change, cells can become abnormal, triggering diseases like cancer. Take a disorder like mds. Cancer of the blood and bone marrow. It's not a diagnosis you would ever want to hear. 
- I went in, he started patting my hand and he was going your blood work does not look very good at all. And that I had mds leukemia. And that there was not a cure for it, and basically I had six months to live. 

- Was epi genetics the reason? Could the silenceing turn normal cells into cancer ones? It's scary to think a few misplaced tags can kill you. But it's also good news because we have traditionally viewed cancer as a disease stemming solely from broken genes and it's a lot harder to fix damaged genes than to rearrange epi genetic tags. In fact, we already have a few drugs that will work. Recently sandra shelby and roy participated in one of the first clinical trials using epi genetic therapy. 
- The idea of epi genetic therapy is to stay away from killing the cell, rather what we are trying to do is diplomacy, trying to change the instructions of the cancer cells. Reminding the cell, hey, you are a human cell, you shouldn't be behaving this way, and we try to do that by reactivateing genes. 
- The results have been incredible. And I didn't have really any horrible side effects. 

- I am in remission. And going in the plus direction is a whole lot better than the minus direction. 
- In fact, half the patients in the trial are now in remission. But while it may be easier to fix our epi genome than our genome, messing it up is easier, too. 
- We have to get people thinking more about what they do, they have a responsibility for their epi genome. Their genome they inherit. But their epi genome they potentially can alter. And particularly that of their children. And that brings in responsibility. But it also brings in hope. 

You are not necessarily stuck with this. You can alter this. 
- Okay. So this has been fairly enlightening for me because this is something that has just come up in the past, I don't know how many years, five years or so, where it's -- it's really reached that critical point where people are paying attention to it in the numbers that should be. And the nice thing about our newest technology, I didn't have much more in the way of slides after that. Is that we can now take an entire genome and look at it. We cannot do anything to it and sequence it. I mean, in four years it looks like for $100 what if it's $500, it still doesn't matter, it's a number that is reasonable. We could take that whole genome and look at it and sequence it and put it all together. And we can also go back through and treat it with bisulfite which takes all the nonmeth lated sites and converts them back to. 

We can do a reaction and change the nonand then we can take your sequence. And before bisulfite treatment and after and say look. All of these are methlated. Now we know exactly where the events are in your entire genome and we can start studying these things and putting them together and saying these events affect these disorders, or, you know, these personality traits or whatever it happens to be. So now we have a lot more information and it's obviously going to take a lot of computer power and research to put all the pieces together. But it's phenomenally powerful when we can look across the whole genome instead of going is it this gene, we are going to amplify these 2000 base pairs out of the three billion and we are hoping we find something. A lot of times it isn't even just one gene. It's affected by many genes at the same time. And as you see in the literature, many times they'll say we found a gene that indicates that you have an increased incidence of a cancer or, you know, an ailment of the liver or whatever it happens to be. But it only accounts for 5% of it. 

It may be in the pathway that affects some basic process, and it's only one element in that pathway. And there could be multiple pathways that can get to a final product. All these things play into it. So being able to look genome wide is extremely important and being able to do it in somewhat economic manner makes a huge difference because instead of $100,000, it might be $100. And it might be that we can go in, have a sample taken, a week later have an answer. And say this is the treatment you need. Guess what, you are going to react badly to this drug, don't take that drug, take this one. Guess what, you are not going to react at all to this drug. Take that one. Al these things will be possible. 

And so I'm hoping it goes mainly in the right direction, not too often in the wrong direction, but there are other things we have to worry about because as we find out more and more about our genomes, we are going to start seeing psychological differences between people and saying okay, these genes or these events indicate certain things, these other genes indicate these other things, and I don't want employers and insurers taking that information and saying well, now we are going to divide everybody up and guess what, we are not going to hire you because the chance that you are going to become, you know, a klep tomaniac and start stealing everything, well, it's just too great for us to take on. When in fact, the person's raised in a good household, never even thought about stealing anything and they know the difference between right and wrong and they are careful in their lives. So, there's a lot of information I don't want just out there that's being freely used. But on the other hand, there's a terrific amount that has to do with our health, that has to do with plant growth, algae growth in lakes, you name it. If you can think of anything in the world that's going on, there may be a way to alter what's going on in our favor, in the favor of the animals and plants and everything else that we have to care for. So -- 
- Your body has several trillion cells, I don't know how many, and patterns will drive the genetics aren't necessarily related. Is that what you get from between the 4-year-old twins and the 60-year-old twins? How many times do you have to do the DNA sequencing of even one person to get the variety driving genetics? 
- Organ specific. 

It's like looking at a limb limbch node and picking up -- we are a mosaic. What's over here is not necessarily what's over here. 
- Very few people emphasize that. 
- I think more and more of that information -- 
[inaudible]
 
- have to do ten or 50 or 100 samples from one person in order to get an accurate view of what's going on genetically. 
- Yeah, you may. But what if you're only interested in certain disorders at a time. And right now how many times do you visit a doctor with this, that or the other and one person, you know, it's just a tool and it's not going to answer everything, obviously. 

It's going to answer a lot of things if we are looking at something fairly specific. 
- Have they found out that epi genetics is more impacting on some people than on others, on some twins than others, depending upon their lifestyle? 
- The study I first saw just to give you an idea, the study I first saw about epi genetics was, there was an old study on people that had gone through first or second world war in norway, and they all had gone through this rationing period where they were all short of food. So they didn't grow. And they had come from fairly tall ancestors. And all of a sudden their children didn't grow. And their children didn't grow. So you can have an entire population affected or you can have different events that occur in your life that are going to occur you more or less. It's not a bad thing. It's what makes you who you are and it controls expression in different cells and obviously you need that. 

If you didn't have any math laition anywhere in your body, I think you would be in a lot of trouble because you wouldn't have that control of a gene expression. And that's one of the cells' way of controlling which genes are turned off -- turned on and turned off. We only need certain genes turned on in a particular cell. 
- So kind of interested in that idea of passing on, you know, to generations. So so far do they think that the important period is probably during pregnancy? 
- That would be anything leading up to pregnancy. 
- Out there in the population and see things that are happening with children that are from birth. And so I mean, I just -- you know, allergy to peanuts, for example. Obviously somewhere something has changed because those are not things that most of -- they get it very early know, you know, children know but you don't realize. So somewhere there was a change that's now causing this thing to be probably passed on, right? 

So that, so it would be earlier, so something would have to be with the mother either before pregnancy or during pregnancy possibly would cause this mutation, this change. What about, something again like autism as again another thing that we are seeing. And they are figuring out some sort of genetic something. Could that again fall into this category of something that happens, because certainly there's more of it now than there was in the past. 
- A lot more, a lot more. 
- Either it's being report add -- reported a whole lot more. I was going through the 60s, 70s, into the 80s, we just -- you just never heard of these things. I didn't know any. I finally met in college people with food allergies and before that I didn't know anything with any severe food allergies. 
- And so I always thought, for example, autism might be something that's genetic and a reaction to something in the environment where you get a certain, you know, component and then it doesn't really happen unless something like a blast -- it could be this thing, too, as well, right? 

It could be that there is some kind of a change back and then we are seeing the result of that in future genetic changes? 
- And it could be, we'll just have to wait and see where the research takes us on these things. But yeah, there could be something that's occurred during one of our generations that's increased the incidence of peanut allergy, autism or whatever else we are dealing with. And we are passing it on. You know, once we have it, then we have increased risk for the next generation and the next generation. So you see the numbers increase. The same as having a mutation that gets passed on. But it's, we are seeing so many kids come through now with these sort of things that it doesn't beg the question. And I don't know, honestly, I don't know if it is or isn't. But could it be? 

Very well could be. 
- The video clip where they were treating these elderly people with cancer, what were they treating? 
- I don't know. I was very curious when I saw that. 
- Experiment or something -- half of them went into remission. Is that absolute math laition? 
- Sounded like it was math laition that they were treating. But my question as soon as I saw it was well that's great, but I don't want to demeth late all the sites. So how are they targeting the sites that they are interested in? There must be some way, it must be somewhere in that drug, there must be target specificity. 

Because you obviously don't want to take every single cell in your body and strip all the methyl groups off of all the DNA because then you have just lost all the regulation that your body has incorporated as it's been growing and that helps make skin cells skin cells, and you know, liver cells liver cells. Because the DNA is there in every single cell but only certain genes are getting expressed and that's what you don't think of too often and they should. Somehow the body is regulating that and somehow it has to do with neighboring cells and it's getting signals from them and it knows what's next to it through these signals and the other part is it's just turned certain genes off. 
- Then to figure out how the positive attitude turns the genes on and off and helps in the recovery from health, illness. 
- Yeah. All sorts of things that could be going on in your body that we are affecting. Both in what we do and then how we think. Our attitudes towards life. 
- What is the specific bioscience, is that a corporation? 
- You can look it up on the web pretty easily. 

It's, i've heard they are going public, but yes, it is. Corporation. 
- With a university? 
- As far as I know it's all privately funded. And there are people that, you'll have to look and verify this on the web, but there were people that were up at cornell together and some of them were looking at this zero mode wave guide, they were basically just looking at putting things down in a small enough hole and then being able to read it from the bottom through glass and they figured out that they could punch holes in aluminum much easier than other substrates, and you know, use that information, they started trying to figure out few things and then they sort of dispersed and one of them had a realization they could could you please -- couple it with something else and the ideas came together and called everybody together and said what if we use that technology, what about that that someone else discovered. These are all groups, and when you see the groups form, they are groups of people that have run into each other at various times or worked on various things and they are sharing ideas, and somebody comes up with that idea of links two of the technologies together and then they all start talking and they pool people from all these different areas and it's just incredible what they manage to do. To think that you could even punch a hole that small in something that you can get a single enzyme and anchor it somehow, that's the other thing. They have taken this molecule and they have, they are trying to get just a single molecule and I know they have had trouble doing that, sometimes they get multiple molecules in there which will just give them garbage, but to get a single molecule anchored down at the bottom of that tiny well. 
- Any protein molecule? 
- We are just talking about the molecule that's used to do PCR , that enzyme, and they have one of them anchored down in the bottom of that well. 

- I don't know much about that. That varieties? 
- There are different enzymes in the body. Some will repair and all they do is go in and repair short stretches of DNA. Others are used more for replication, so when your cells are dividing you need to have another set of DNA so you can make the new cell. And there are, you would have to go back and review it and I haven't looked at it in years, but there's, they have divided them up and even within one group or one purpose there are different that will do slightly different things. I mean, there are a lot of good things that can come out of this and there are some bad things. And I'm hoping that they get gena passed through congress, and that's a again -- genetic, basic nondiscrimination act. So, that would disallow companies from using that information for hiring purposes, for insurance purposes, because there are some things we just can't do anything about. I mean, there is a certain level of discrimination anyway, rates are set according to how high my father's cholesterol is and how high mine s you know. 

Things like that. And if I can get mine down, they lower my rates. So there are certain things I can't do anything about, and genetics is one of them. Epi genetics, yeah, maybe I can do something about that. 
- When did epi genetics come in as a term? 
- I'm not sure. I don't know if it had been used -- 
- This year? 
- I'm not sure if it had been around for a long time. 
- Any environmental impact on your DNA display or DNA -- 

- I think it encompasses everything that contributes to gene expression, that is not directly en coded in the DNA. So anything that's going to affect the coiling and the meth laition events and all these other things that will affect your gene expression. So anything on that second layer. But -- you know, I haven't come across a definition. It's always the same, you know. Every site that I have looked at for a definition of epi genetics. In fact, I have never come across the same definition for a single word of everything that i've looked at through google or whatever it happens to be. 
- 1942. So this is back to that first study. Was it back to that first study or very close to it where they were coming through and looking at people or, or I guess other organisms? 

- 1953. 
- The field of genetics has been around longer than the understanding of the structure of DNA. So -- so we are -- we are going back to the 1800s there. So, you know, we are starting with darwin, going through mendell and progressing up through, with some understanding of how traits get passed on or the fact, the very fact that they do get passed on and we are not just, you know, what they originally thought was, you know, we were just a blending of our parents, like two colors of paint and threw it together and there you had it, a different color. But we are realized after a while that it wasn't that way. Two short people could have a tall child, that sort of thing. 
- Thank you very much. 
- You are welcome.