cc  >> Nadia Dominguez: Thank you  for joining me for Wednesday  Nite at the Lab at the  UW-Madison Biotechnology Center.  Tonight we welcome  Dr. Josh Hyman, who actually  resides, during the day, right  here in this very building,  the Biotechnology Center  here at UW-Madison.  Dr. Josh Hyman has an  interesting route as to how he  got to the UW-Madison, and I  just wanted to share it with you  because I found it quite,  both humorous and interesting.  Josh got his BS in mechanical  engineering from the University  of Michigan at Ann Arbor.  He moved to St. Louis where  he began to work on the F-15E  fighter jet, but apparently that  was not exciting enough for him  so he moved out to Washington,  where he worked on his MBA  at the University of Washington  and then decided to get involved  in the seafood industry  in Seattle.  And after depleting the  Whitefish Fishery, he headed  back east to Pennsylvania  to pursue a career  in a very illustrious,  yet short-lived, career in the  garment label making industry.  [LAUGHTER]  After that short-lived venture,  he then opened a restaurant  in Pennsylvania, and finally  he landed himself back  in the arms of academia  where he earned his Master's  in horticulture, as well as his  PhD in genetics at Penn State.  So tonight please join me in  welcoming Dr. Josh Hyman  to Wednesday Nite at the Lab.  [APPLAUSE]  >> Josh Hyman: Thanks, Nadia.  What Nadia is not telling you is  that even when I was working  with McDonnell Douglas, I was  working with things that at  least had been alive for a  while. 

 On the F-15E project my task was  to improve the strength of the  windshield and canopy system.  So we ended up shooting chickens  at the windshield.  And that was how we tested it.  They were not alive, of course.  They were chickens in a bag, and  we had this large gun, chicken  gun that we'd use to shoot the  windshields.  At any rate, today we're going  to talk about sequencing, in  particular, DNA sequencing  because that's what our facility  handles, and the advancements in  DNA sequencing, but we're going  to start by talking a little bit  about why we do this, why we  care about DNA sequencing.  And, as most of you are aware  of, DNA is basically the  instruction set that our bodies  have as to what to do and how to  do it.  Modifications in the DNA affect  physical and, in some organisms,  especially humans, social  responses.  We can look at alcoholism,  propensity to take risks,  gambling, things like that, they  do have a basis in our genetic  code.  It can also give us insights  into how we're related both  between one group of people in  the world and another group of  people in the world. 

 Within our families, what  happens within our families and  things that show up in the  family tree, whether it happens  to be susceptibility to certain  cancers, allergies, those sorts  of things, and it allows us to  predict what's going to happen  with people, with plants, with  any organism.  But DNA is not the whole story,  obviously, our environment plays  a huge role.  Some things are fairly fixed,  eye color, things like that.  There's a certain range that  we're going to get, and most of  that is already coded into us.  And also everything here is  pretty much fixed.  We know we're going to have  fingers at the ends of our hands  and our eyes are pretty much  where they're supposed to be and  ears and everything, it's all  set.  But environment plays a huge  effect in most of what goes on  in our lives.  And that's made up of what we  call a genotype, an  environmental effect going back  and feeding into the phenotype  and phenotype is just our traits  that we display.  They can be social traits, they  can be physical traits but  they're traits we display.  In an animal, it's the traits  that particular animal has. 

 In plants, it's the same thing.  Plants can grow tall, they can  be short, they can be green,  they can take on all sorts of  traits.  And it's based on part of the  genetic code for every single  one of the traits.  And it isn't necessarily a  discrete region in the genetic  code.  It can be many regions  interacting.  And there can be multiple  pathways so even if you knock  out some of these regions, your  body will continue to do what  it's supposed to do.  And this is one of those things  that is a stumbling block.  For some people they think,  well, if I'm missing this,  what's going to happen?  Am I going to cease to exist?  Am I going to develop something  all of the sudden?  And the answer to that in many  cases is no.  That's not going to happen  because you do have alternate  pathways that are already set  up, and, as you'll see, we, as a  group here, are probably not  real uniform.  Every one of you has something  missing or added to your genes. 

 And you also have things added  on top of the DNA, as you'll  see.  Now the environment affects  expression of a lot of things.  Obviously, if you eat well, if  you get lots of vitamins, you  can grow taller, if you have a  really poor diet, you'll tend to  be shorter, your growth in some  way will be impeded.  If you start smoking early on,  you can actually fix your need  for nicotine and it will be much  harder to break that habit.  Whereas, they've found that  people start smoking when they  get quite a bit older have a  much easier time to kicking the  habit.  And it's not just will power,  it's actually fixing some  things, as you'll see.  And the other thing is  environment can induce  mutations.  And we can see that anytime we  get in contact with toxins.  Those things that happen above  the DNA are called epigenetic  effects, or the study is just  epigenetics.  And this is variation that's not  related to the DNA code  directly. 

 In other words, there's four  letters that make up your  genetic code.  And those can stay exactly the  same and yet your skin's on the  outside, your blood vessels are  on the inside and somehow that  came to be.  So something is regulating that  above the genetic code.  Things are turned on, things are  turned off.  And that is epigenetics.  That's the study of how these  things are regulated.  But what's interesting is that  what you have done before your  children are born has affected  your children.  You've passed things on.  Some of these modifications, the  code's the same, the DNA that  you passed on hasn't been  altered from the genes that you  got from your parents maybe, but  there's something above that  that's affecting it.  And I'll show you a video and  you'll get some idea of what's  going on. 

 And this will be explained in  the video.  So your DNA, and it's showing  here, is just this big coil.  But there's a small methyl group  that you can see up on this  screen, and that can actually  attach to one of the nucleotides  and these are generally in  places just before genes called  promoter regions and that's  where the enzymes come sit down  and move along and start  transcribing it into RNA and  then it becomes proteins and so  forth.  But they can land, they can bind  to those sites and either induce  or suppress expression.  And they usually end up blocking  something that represses  expression of another gene is  what usually happens.  So there's triggers that go  along and sometimes two or three  things are needed in pathway or  something is produced in some  other area of the genome that  represses that pathway.  So if you knock out this other  area, all of the sudden you've  allowed this whole cascade of  events to occur.  So it can turn things on and it  can turn things off.  And the other thing you have is  in the genome everything is not  just loose and sort of stringy.  It's compacted. 

 And those areas that are open  and accessible to the enzymes  that transcribe things and turn  it into RNA and then into  enzymes, proteins, those areas  that are open get expressed and  those areas that are coiled  tightly are less accessible.  So there are what are called  histone modifications that  occur.  And when things get coiled up  tightly, then you don't get as  much expression or you get no  expression.  >> The histones that you've  shown there are all the same  size, is that typical?  >> Hyman: There are units that  go together so you can have like  an area that's really coiled up  with many of these subunits  together.  Does that make sense?  >> Yes, so it doesn't really  limit the gene size?  >> Hyman: Right, that's true.  It doesn't limit the gene size.  One thing you have to remember  is that every organism has DNA,  but the amount of DNA doesn't  dictate how big we are, how many  genes we have or anything else.  There's a certain amount of what  they used to call junk DNA, and  it's still called junk DNA to a  certain extent, it's the stuff  we don't know what it does.  But it often has some reason for  being there and some of it is  just to help with this  regulation.  It doesn't necessarily code for  something that we would see or  find in the body but it serves a  purpose. 

 And so you can get something  like, say, the elephant which  has a little bit more DNA than  we have, and then you can get  something like fritillaria,  which is a plant that's about  that tall, that has roughly  40 times the DNA that we have.  So size has nothing to do with  the amount of DNA you have.  And I'm going to jump real  quick, it's going to say end  show, but what we're going to do  is shrink this down and let you  watch a video and I'm hoping  that it is loud enough so you  can hear everything.  And this is all about, this is a  great video.  It's off of NOVA.  It's about epigenetics and I've  shown it to a number of people.  And this is one I show to  everybody because it's  absolutely fascinating.  >> Do you ever notice that if  you get to know two identical  twins, they may look alike but  they're always subtly different.  >> I don't know if you can hear  that at all.  I can pause this a second. 

 >> As they get older those  differences get more pronounced.  Two people start out the same  but they're...  >> You can hear it?  Can anybody else hear this?  >> I can hear.  [INAUDIBLE]  >> There's music and that's  talking.  >> Do you ever notice that if  you get to know two identical  twins, they might look alike but  they're always subtly different.  >> Okay, let me try this.  >> 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 gray  hair.  >> No, no, I don't. 

 Identical twins have the same  DNA, the exact same genes.  >> Yeah.  >> And don't our genes make us  who we are?  >> Well, they do, yes, but  they're not the whole story.  Some researchers have discovered  a new bit of biology that can  work with our genes or against  them.  >> You're 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.  >> As a child, when I looked in  a mirror I'd 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. 

 The synchrony in your lives  constantly confronts you.  >> When I see my sister, I see  myself.  If she looks good, I think I  look pretty today.  But if she's 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, and you're literally  clones.  Better known as identical twins.  But now, imagine this,  one day your twin, your clone,  is diagnosed with cancer.  If you're 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 like Anna Marie to get a  dread disease while the other,  like Clotilde, 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  Anna Marie and Clotilde's,  despite the differences in their  color and size.  The human who studies them is  Duke University's Randy Jirtle.  >> So Randy, I see here you have  skinny mice and fat mice.  What have you done in this lab?  >> Well, these animals are  actually genetically identical.  >> The fat ones and the skinny  ones?  >> That's correct.  >> Because these are huge.  >> They're huge.  >> Can we weigh them to find  out?  >> Sure.  >> They can barely walk. 

 >> They can't walk too much,  they're not going to be running  very far.  So that's about 63 grams.  >> 63 grams.  >> Let's look at the other one.  >> So it's half the weight.  >> Right.  >> This gets even more called  mysterious when you realize that  these identical mice both have a  particular gene called --,  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?  Atkins?  No.  A tiny chemical tag of carbon  and hydrogen called a methyl  group has affixed to the -- gene  shutting it down. 

 Living creatures produce  millions of tags like these.  Some, like methyl groups, attach  to genes directly, inhibiting  their function.  Other types grab the proteins,  called histones, around which  genes coil and tighten or loosen  them to control gene expression.  Distinct methylation and histone  patterns exist in every cell  constituting a sort of second  genome, the epigenome.  >> Epigenetics literally  translates into just 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 epigenome would  be like the software that tells  the computer when to work and  how to work and how much.  >> In fact, it's the epigenome  that tells our cells what sort  of cells they should be: skin,  hair, heart.  You see, all these cells have  the same genes.  But their epigenomes silence the  unneeded ones to make cells  different from one another.  Epigenetic instructions pass on  as cells divide but they're not  necessarily permanent. 

 Researchers think they can  change, especially during  critical periods like puberty or  pregnancy.  Jirtle's mice reveal how the  epigenome can be altered.  To produce thin brown mice  instead of fat yellow ones,  he feeds pregnant mothers a diet  rich in methyl groups to form  the tags that can turn genes  off.  >> And I think you can see that  we dramatically shifted the coat  color and we get many, many more  brown animals.  >> And that matters because your  coat color is a tracer, is an  indicator of the fact that you  have turned off that gene.  >> That's right.  >> This epigenetic fix was also  inherited by the next generation  of mice regardless 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 like their mothers.  >> It seems to me this has  profound implications for our  health.  >> It does for human health. 

 If there are genes like this in  humans, basically what you eat  can affect your future  generations.  So you're 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're  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 so the  reason why we're showing you  Spanish twins.  In 2005 they participated in a  ground-breaking study in Madrid.  It's aim?  To show just how identical,  epigenetically, they are or  aren't.  >> One of the questions of twins  is if my twin has this disease,  I will have the same disease. 

 And genetics to last at this  high risk of developing the same  disease, but it's not really  sure they're going to have it  because our genes are just part  of the story.  Something has to regulate those  genes.  And part of the explanation is  epigenetics.  >> He wanted to see if the  twins' epigenomes might account  for their differences.  To find out, he and his team  collected cells from 40 pairs of  identical twins age 3 to 74.  Then, began the laborious  process of dissolving the cells  until all that was left were  wispy strands of DNA, the master  molecule that contains our  genes.  Next, researchers amplified  fragments of the DNA until the  genes themselves became  detectable.  Those that had been turned off  epigenetically appear as dark  pink bands on the gel.  Now, notice what happens when  the genes from a pair of twins  are cut out and overlapped.  The results are far from subtle. 

 Especially when you compare the  epigenomes of two sets of twins  that differ in age.  Here on the left is the  overlapped DNA of 6-year-old  Javier and Carlos.  The yellow indicates where their  gene expression is identical.  On the right is the DNA of  66-year-old Anna Marie and  Clotilde.  In contrast to the younger  twins, hardly any yellow shines  through.  Their epigenomes have changed  dramatically.  The study suggests that as twins  age, epigenetic differences  accumulate, especially when  their lifestyles differ.  >> One of the main findings of  the research is that epigenomes  can change the influence of what  we eat, of what we smoke, of  what we drink.  And this is one of the key  differences between epigenetics  and 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'd ever  want to hear.  >> When I went in and 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 epigenetics the reason?  Could the silencing of critical  genes turn normal cells into  cancerous ones?  It's scary to think that a few  misplaced tags can kill you, but  it's also good news because  we've traditionally viewed  cancer as a disease stemming  solely from broken genes, and  it's a lot harder to fix damaged  genes than to rearrange  epigenetic tags.  In fact, we already have a few  drugs that will work.  Recently, Sandra Shelby and Roy  Cantwell participated in one of  the first clinical trials using  epigenetic therapy.  >> The idea of epigenetic  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're  a human cell, you shouldn't be  behaving this way. 

 And we try to do that by  reactivating genes.  >> The results have been  incredible, and I didn't have  really any horrible side  effects.  >> I am in remission and going  in a 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 epigenome than our  genome, messing it up is easier  too.  We've got to get people thinking  more about what they do.  They have a responsibility for  their epigenome.  Their genome they inherit, but  their epigenome they  potentially can alter.  And particularly that of their  children.  And that brings in  responsibility. 

 But it also brings in hope.  You're not necessarily stuck  with this, you can alter this.  >> Hyman: So obviously there's  a lot of to learn yet.  And we're just now getting the  tools that allow us to sort of  investigate those things.  We've had some rather clumsy  tools in the past we've had to  work with, but a lot of that's  changing.  And now we have equipment that  allows us to look at whole  genomes.  We have equipment that allows us  very soon to start looking at  methylation.  I don't think we have anybody  working, necessarily, on histone  modification right now, on these  really high, high throughput  systems we're starting to see.  But we are at least getting to  the point where there is some  promise that we're going to have  equipment, which I'll talk about  in a little bit here, that  allows us to look at that  methylation across the entire  genome, and we'll be able to see  what's being switched on and  what's being switched off.  Now there is one thing that you  should keep in mind and that's  all your cells are a little bit  different throughout our body. 

 Obviously, you have different  tissue types and everything so  those modifications are  different in every cell type.  And some of the latest work that  they've done when they're  looking at histone modification  levels and their effect on gene  expression, they found it, they  found different levels of  expression, they expected to,  but they also noted that certain  histone modifications were  useful for predicting these gene  expression levels when you had  low CPG promoters, and those are  areas that aren't going to have  a lot of methylation, and then  other histone modifications were  good predictors when you had  high CPG.  And it's the C in the CPG that  gets methylated.  The other thing that can happen  to us, and this is not  epigenetics anymore, we're  talking about actual mutations  in the DNA, is we can have a lot  of different mutations, a lot of  different types of mutations,  and we tend to think of  ourselves as having the same DNA  that everybody else has.  But, in fact, we don't.  We have a lot of changes, as  you'll see.  They come in the form of single  changes to a nucleotide.  So a single base, a single  letter can change, say, from an  A to a G or something.  And it will change what's coded  in the RNA and in the enzymes,  proteins that come out the other  end of the whole process.  You can have an insertion or  deletion of one of these bases,  and that will change what's  coded for. 

 You can have a duplication and a  duplication is simply I have a  gene or a chunk of DNA and then  I'll get an exact copy of it, it  will just be copied over again.  You can have an inversion.  So if you think of DNA being  this long linear string, I can  get a piece flipped around.  I now the big difference between  a tomato and a potato is a very  large inversion, and that's one  of the most significant  differences they see.  And then you can also have a  translocation.  So you can have a chunk of DNA  that started out here in the  genome and went to here.  It went either to the same  chromosome or some other  location.  They looked at cancer cells and  when they're comparing cancer  cell lines, this is a particular  group, I can't remember what  this group was from, but when  they looked at their group of  cancer cell lines, they found  that there were two million of  these single SNPs, this single  nucleotides that switched from  one to another, 1,315 structural  variations.  And those can be duplications or  inversions, things like that.  Then there were 191,000 small  indels, so areas where they  added or subtracted a little  bit. 

 And 35 interchromosomal  translocations.  That means a chunk of DNA went  from this chromosome to that  chromosome.  It disappeared out of one and  appeared in another one.  Another group went through 24  breast cancer genomes.  And a genome is all your  chromosomes together.  So they looked at all these  different genomes and they found  a total of 239 interchromosomal  translocations.  When looking at autism, people  were looking at deletions and  duplication events.  So they either lost something or  they ended up getting more  copies of it.  And we generally refer to those  when we see genes where we  detect more copies or fewer  copies, we all those copy number  variance because if you have  more genes, you get more  expression of something.  Now how big were they?  Well, they were looking for copy  number variations in these 751  families that they initially  looked at, and they observed  five instances of a 593  kilobase, that's 593,000 bases,  half a million bases, gone. 

 In these kids that were showing  signs of autism.  Another group they looked at,  five of the 512 children in this  other group, the children's  hospital in Boston group, who  are also being studied for the  same reasons, autism,  developmental delay, those  things, they found the exact  same deletion.  And another group of 300 they  found three.  So it seems to be about a 1%  chance in these groups that  they're going to have this  particular mutation.  But what's also interesting is  that in another group they found  a reciprocal duplication.  So instead of not having it,  they had two copies.  And the same thing was  occurring.  So if you have too much, you  have a problem, if you don't  have enough or you don't have it  at all, you also had a problem.  Now all of these things have  value.  They actually have a value to us  as people because, obviously, if  we know that there are these  mutations out there, if we know  we're missing something,  something can be done about it  in some cases or at least we can  use that information to predict  what's going to happen. 

 Right now, and this is from  February 2nd, there's a court  case between the ACLU and a  company called Myriad Genetics.  And Myriad Genetics has a test,  a genetic test for the BRCA1 or  BRCA2 genes which are indicators  of breast cancer.  So if someone has breast cancer  in your family and you want to  find out if you're willing to be  highly likely to get breast  cancer, then you get this test.  The only problem is that there  is one test out there and it's  from Myriad Genetics and it's a  $3,000 test.  That's a rather high price on a  test like this.  Of course, it's knowledge that  we want, that we really want if  someone shows up in our family  that has breast cancer, but  you're not going to go out and  get a second opinion.  They have the test, they have  the patent rights to the gene  and they're not exactly setting  up a lot of competitors with  themselves by letting other  people have the patent rights.  So the ACLU is fighting in court  right now to say, you can't  patent genes and you can't  patent mutations because they  exist and they've existed all  along and this is something  that's out in nature.  And so there's this battle going  back and forth.  Now in the biotechnology  community, there's the other  argument. 

 If with you don't have some  financial incentive to go out  and look for these things, some  of these tests wouldn't even  exist.  So there's some balance there  that has to be made.  But there is this battle going  on and these things are very  valuable both in social terms    and in financial terms.  So getting back to what we do  here, sequencing, how do we do  it?  Well, there have been two  methods, historically, of doing  this sort of standard sequencing  that we've done.  One of them is Maxam Gilbert,  we're not going to touch on  that.  The other one is the Sanger  method and that's what we use on  all our present day machines,  the modern machines that are  slowly being supplanted by the  next generation machines.  But we're going to talk about  the modern machines, the Sanger  sequencing technique.  And what it relies on is having  many, many fragments of your  particular region that you're  interested in.  It's a very targeted approach.  Now, human beings have about  three billion base pairs, three  billion letters in their genetic  code. 

 But with the Sanger method we  can focus on 700 to a thousand  of those at a time in a  particular region, and that's  it.  But it depends on having an  overabundance of that fragmented  DNA, and so it relies on a  method of amplification called  PCR or Polymerase Chain  Reaction, and all that's doing  is creating many, many copies of  that one region.  And just some DNA basics, the  code is made up of four letters  and it stands for, as you can  see, four different molecules,  adenine, thymine, guanine,  cytosine.  It's typically double-stranded.  And there is actually a  direction to it because the  molecules are oriented in a  certain way.  So you can say this is a five  prime end and this is a three  prime end.  And that has more to do with the  chemistry and how you look at a  molecule than anything else.  But they do have a five prime  and three prime end.  They are directional and  polymerases read them in one  direction.  They can actually go through and  a polymerase is something that  can duplicate that chunk of DNA. 

 And so in your DNA you get a  forward strand and a reverse  strand and they pair up.  As to Ts, Cs to Gs.  It's always that way unless  there's a mismatch in which they  don't really pair up, they're  actually spread apart a little  bit because they don't connect  together, and you can have  mismatches that occur but  there's generally a mutation  there that's created that  mismatch.  So you can take one strand and  know exactly what's on the other  strand.  So in PCR we depend on little  fragments called primers.  And you can see a little green  tag over there and a little red  tag over there.  So we've heated up our DNA, and  when you heat up DNA it spreads  apart.  You get it up to about close to  boiling point and it comes  apart.  So this double-stranded DNA all  of the sudden becomes two single  strands.  And you take these little chunks  of DNA that we can actually  synthesize upstairs, they're  called primers, and you put them  in the mix. 

 And as you cool things down,  they will find their target,  their perfect match.  The As to the Ts, the Cs to the  Gs.  And they'll have a three prime  end and a five prime end.  And at the three prime end,  you'll get a polymerase, and  that's up there, it's marked as  taq because it's --, that's the  organism it's from, but a taq  polymerase and that's going to  help extend the DNA.  And it will do that by  incorporating all of those  little molecules that we also  have floating around in there by  themselves, a separate T and C  and G and A, floating around in  that solution and it will grab  them, put them one by one and  attach them to the end of that  primer.  And so this one right here will  go out in that direction.  The bottom one will come back in  this direction and duplicate  what the opposite strand would  be.  So once again, it's making the  reverse complement to it.  And it does that a few times.  And what will happen, if you  think about it, the first time  it does it, it has this huge  template. 

 It goes on pretty much forever  as far as the polymerase goes,  and it can just keep going down  that and adding nucleotides.  But when you heat that up and  separate it again, one fragment  starts here and goes that way,  another fragment starts here and  goes that way.  So if you keep repeating this,  the primers will get on to  strands that, well, maybe some  of the original strand in which  case it goes on forever in one  direction, but if it's not, if  it's something that's just been  created in the last round of  extension, then it's going to  terminate out here.  So primer comes up and it starts  to go out and it hits the end.  So now it's only amplifying a  short or extending on a short  piece of DNA.  And if you keep doing this, what  you end up with is many, many  fragments of a very specified  size.  You've put a primer in here and  a primer in here.  They go out, the next round they  terminate and they terminate and  they keep terminating in  successive rounds so you have  all of them at the same size.  And as you see, as we go out we  start getting successively more  and more copies of these short  fragments, and, finally, you end  up with this overabundance of  that.  And you start with that. 

 And now we're going to start  doing something where we have  these short templates in there,  many copies so we can make a  really strong signal.  We have a suitable primer so  we're only going to use one of  those primers now because we're  going to sequence in one  direction only in our Sanger  method.  Because we can't read in both  directions at the same time,  we'd have two signals every  single time we wanted to look at  a nucleotide in a certain  position.  We need a DNA polymerase to  extend things and a pool of  normal nucleotides and then  we're going to add  dideoxynucleotides.  And all they do, and I'm just  going to sort of flip through  the next set of slides, but all  they do is they terminate our  reaction, our extension at a  certain point.  So if we went out to, say, the  hundredth position and it's a C  in our template, if we go out  that far and it's a C, the  dideoxynucleotide that ends up  there is going to be it's  compliment, it will be a G.  Does that make sense?  And if all we had were G  dideoxynucleotides in the mix,  it will always stop where a G  was or a C was in this case  because it's the reverse  complement.  So it would go out and it would  stop and find a C and by chance  we'd get a dideoxynucleotide  rather than a regular nucleotide  because the regular nucleotides  would just let them keep  extending.  So we put a very, very small  proportion of these dideoxys.  They're terminators. 

 So any fragments that say are  100 bases long, we know that  fragment ended in a G.  And in ones that say, 105,  that ended, and so forth.  And we'll know if it's a certain  length, and we only have one of  these terminators in there that  any fragment that's a certain  length is ending in the same  one.  So that's how we originally did  these things.  And I'm going to flip through  this because this just shows you  the difference in these two  where one's a dideoxy and one's  a regular one.  All right, so occasionally at  random these are taken up and  it's terminated.  So what we used to do is set all  four of these reactions up, one  with terminators that were A,  one with terminators that were  C, one with a G terminator and  one with T terminator, and we'd  run four separate reactions.  And then we would run them out  an a gel.  And a gel is just that, it's  like making Jell-O except it's  slightly different than the  Jell-O you'd eat at home.  But you basically pour it and it  firms up and you put your DNA in  there and you pull a charge  across that. 

 You have little wells on one  side and you have it in a  solution and you put a charge  across that.  And DNA has a charge on it so it  migrates through that gel.  And the small fragments migrate  very quickly, and the larger  fragments migrate more slowly.  So you can actually look at that  and stain it somehow, and the  way we used to stain it was we'd  radioactively label it and then  we put it against a piece of  film.  And it would sit in a case for a  few days and then we'd pull the  film off and we'd develop the  film and we'd get this, and I  don't know how well you can see  it, actually I'll flip through  here and you'll be able to see  one.  A gel pretty much migrates like  that.  And you can read up this gel and  know what your sequence is.  So an autorad, one of these, if  you get a slightly bigger  picture it's going to look at  that.  So you'd read up the gel and it  would just be like a ladder, you  would just go across and you'd  figure out which base was the  next one in line.  And what you really did was if  you were a graduate student or a  postdoc or somebody, you would  do this part of the work and get  it to this level, and then you'd  find an undergraduate, a poor  under graduate and you'd give  him a ruler and you'd tell him  to go up that thing and try and  figure out what the sequence  was. 

 And this took five to six days.  And there are only six lanes on  here.  So it was fairly laborious.  Now, if you had them colored,  you'd have something like this.  It would be color coded.  And it would be a little bit  easier to read.  But you could also have a  machine do it.  And that's exactly what they  started doing.  They started putting molecules  that would fluoresce and label  these things.  They would run down a capillary,  a small, very fine capillary,  and this is an example of a set  of capillaries we use upstairs,  and it takes 96 samples at a  time, injects them, and these  are metal pins, and they draw a  current across and injects those  fragments onto these capillaries  that are filled with a polymer  and we draw a current across  here and they travel through  this and they essentially run  through the whole system, the  small fragments come out first  and whatever molecule they have  on attached to them, whatever  fluorescent label they have  attached to them gets picked up. 

 And then that light is separated  out and they detect which wave  length is coming through at a  time.  And for a mere $350,000 you,  too, can own one of these.  The nice thing is that what you  get is a machine readable code  that instead of you having to go  through this and work your way  up this tree to figure out  exactly which nucleotide, which  base is where, you can just get  something like this that comes  out.  And I can set up a plate like  that in half an hour, run the  reaction, drop it off, and the  next day I have 96 reactions.  The machines we have will run  about 13 plates in a day.  That's 1300 reactions.  We have three of them so we can  do about almost 4,000 reactions  in a day.  And that's far more efficient  than having one person dedicated  a whole week to getting six  reactions and having to read it  and decode it.  So that's allowed us to do quite  a bit.  And for the past five to 10  years that's what's been going  on in slightly better and better  forms. 

 So what you end up with is this  fairly lengthy read out, and it  can be digitized and of course  read by computers.  As opposed to this which gave  you about half as much  information per lane.  So, obviously, we're quite a bit  more efficient at doing that.  Now, if you look at what we're  doing, one of these machines  upstairs can do about 700 or 800  bases for every single one of  these samples that's in a well.  So a certain length.  We can do 96 samples at a time,  12 to 13 plates in a day, it  works out to roughly 873,000  bases per day.  And we can run it five to six  days per week.  But the human genome is 3  billion bases long.  So if one machine can generate  that amount in a day, how long  does it take to sequence a human  genome?  Well, if you had the one machine  and you were doing each fragment  sequentially, it would still  take you nine and a half years  with unique fragments.  But, typically, we have to  overlap the fragments. 

 And because DNA has a lot of  duplications, translocations and  other things in it, we may find  out that we're amplifying and  reading the wrong regions.  And it's a little harder to  assemble certain regions so we  get gaps.  So, obviously, it took a lot of  effort to sequence the first  human.  And they had many of these  machines, hundreds of these  machines working, and it cost  hundreds of millions of dollars  to do this kind of work.  So what we'd like to be able to  do is do an entire genome  quickly because we'd get all the  information all at once and we  could make decisions based on  that.  So enter next-gen sequencing.  So instead of doing single  fragments, 700 to a thousand  bases long, we'd like to do  random fragments but do millions  of them at a time.  So if we could do all of these  at one time, you wouldn't have  to sort of focus on one area,  you could go across the entire  genome and say, let's just do it  randomly and we'll take it all  out after we're done and we'll  try to assemble it.  That's a bit like saying, I'm  going to take all of  Shakespeare's works, chopping it  into 25-letter fragments,  throwing them into a pile and  trying to make sense out of a  thousand copies of Shakespeare's  work.  It's going to take you a little  while to assemble it. 

 And since you don't know how  it's supposed to read in the  first place, you're going to  spend a lot of time trying to  match everything up an put it in  order, but it's what we're doing  now.  And the way we do it is we,  essentially, just sheer the DNA.  So it's in these long strands  and we take it and we sheer it  and make short strands of it and  then we put adapters on the end  of it.  So these little chunks of DNA  that we know, we know the code  of it, and we can stick them on  the end, it's called ligation  but we essentially attach them  to the ends of these fragments.  And there's a way we can do it  so we know we have two different  adapters, adapter A and adapter  B, on there, and I could get  into a lot of detail but I'm  going to try to skip over that.  And then we put that on a very,  very small slide.  So what I have in my hand is a  slide we use on the machines  down the hall.  It's a next-generation  sequencer, it's the Illumina  Genome Analyzer 2X.  And all of our sequencing is  done on a little slide.  And this actually takes eight  different samples. 

 So there are eight lanes on  here.  And we're taking these little  teeny, tiny fragments that we  put adapters on and we're  flowing them through these  lanes.  And by the time we get done,  we're going to do this  amplification like the PCR we  talked about earlier but we're  going to do it right on this  slide because it has the  complements to those adapters  right on it.  So you get one strand extending  and then you denature things and  they stand up again and then you  let it amplify again and when it  does that and when you cool it  down the ends fold over or they  find their complementary primer  and then that's extended.  But at any rate, you create  little clusters of the same  fragment, monoclonal clusters of  the same fragment.  So you've got all these  fragments but they're spread way  out.  I mean, in their terms, they're  spread way out.  In our terms, they're on a  teeny, tiny little slide, but in  their terms, they're spread way  out so they're really not too  close to one another, hopefully.  So when they amplify, you get  many, many copies of one  fragment in one little dot on  this.  And you can say, how many dots  can you get on there?  For each one of these lanes we  take 120 pictures. 

 And remember there's eight  lanes.  So we're taking 120 pictures of  that lane.  And at each one of those we get  a couple hundred thousand  clusters.  So we get roughly 10 million  clusters to 20 million clusters  on each one of those lanes, and  each cluster, remember, is an  individual fragment that's just  been amplified so you have many  copies of it.  And so at the end what we do is  we make them all single-stranded  again, we heat them up and we  cleave off half of them because  we have a little enzyme that  will cleave from one side and  leave the other side the way  it's supposed to be just hanging  there off the slide.  So we have all the clusters now  facing in the same direction.  The DNA fragment is oriented in  the same direction with the A  side here and the B side there  with the adapters.  Then we put a little primer in  and we start sequencing down  this.  And in this particular system we  do this base by base.  So we add all four nucleotides  and they're all labeled but  they're all blocked, they can't  extend beyond one nucleotide at  a time. 

 We wash them across the slide.  They fluoresce when you hit them  with a laser.  We shoot two different lasers  across with two different  filters so we can pick up the  fluorescence from each one of  these separate fluorophores that  are attached to these  nucleotides, with As being with  one fluorophore, Gs with second  fluorophores, Cs third and T is  fourth fluorophore.  So we can pick up what's on a  single cluster.  And we can say, oh, this  particular nucleotide, this  particular letter was added  next.  And then we deblock all of them  and we wash another round of  fluorescently labeled  nucleotides on there and add  that one.  And so with this particular  system it adds one nucleotide to  every single one of these  strands, it takes a picture of  them and tells us which  nucleotide was added to each  distinct cluster, washes that  away and then repeats the  process.  And we can do, right now, about  75 to 80 bases in each  direction.  In other words, we can read down  in this direction on a cluster  and then we can flip all those  end to end and read from the  other end giving us about 150 to  160 bases off of each cluster  and there are 10 million  clusters in each one of these  lanes.  So it's obviously producing a  lot more information than this  system did. 

 And right now, and I'm going to  flip forward pretty quickly  because we're sort of going  short on time, we're dropping  the cost of doing sequencing  down a thousand-fold.  And we've gone from about nine  and a half years to sequence the  human genome to about nine days  to sequence the genome with  one-fold coverage.  But this has actually gotten a  lot better lately.  In fact, and I think I'm going  to stop here pretty soon, there  is a machine that they just  announced advancements to that  when it hits the market really  soon, like next Wednesday,  maybe, I don't know, but really  soon, it's supposed to be able  to do a hundred bases right now  and by the end of this year,  sorry, a hundred billion bases  and 300 billion bases by  midyear.  The machine we have right now  down the hall was slated to  produce 90 billion bases in a 10  and a half day run right now.  But they had to push that out a  little bit.  So we're expecting to hit those  levels in about three to six  months.  So if you have a human genome  that's three billion bases and  you can generate 90 billion  bases in 10 days, that means it  takes 10 days to give you a  30-fold coverage of the human  genome, and that's sort of  considered what's needed to  sequence the human genome.  And it used to take hundreds of  machines and millions upon  millions of dollars to do, and  we're quickly driving that down  to roughly the $6,000 to $10,000  level.  And the advancements in the  next-next-generation equipment  is slated over the next five  years to be able to do that for  hundreds of dollars when you go  into a hospital or some place  like that where they need  information about your entire  genome. 

 Now the only problem I have with  that is that in some cases  that's going to be used for my  benefit.  Absolutely.  But GINA, which is the law that  governors how this information  is shared and used, was passed  about a year or two ago, and  basically it's supposed to  protect us from having insurance  companies looking at that and  going, well, we're not going to  insure you because you're too  high a risk.  Or a business saying we're not  going to hire you because you  obviously don't have the  aptitude because it's in your  genome.  You can't do this.  And we don't want to get in  those situations where they see  a propensity for some disorder,  whatever it happens to be, and  have that hamper your ability to  get a job, get insurance, do  whatever you want to do in life.  And I think I'm going to stop  there, and if you have  questions, go ahead and fire  away.  [APPLAUSE]