We just discussed how DNA is copied. Now, how does a sequencer eavesdrop on this copying process in order to sequence many, many templates simultaneously? That's what we'll see here. So, let's say we're sequencing your genome and you've been kind enough to provide us with a blood sample. And in that blood sample there is a bunch of cells, each with a copy of your genome. So, in the laboratory we can extract that DNA and then we can chop it up into little snippets. And we can also make that DNA single stranded so we can turn it into short single stranded templates. And then finally we can take those short single stranded templates and then deposit them onto some sort of flat surface like a slide. So, these single stranded snippets, these templates, are now scattered randomly across the surface of a slide. So, only one template is show here in this picture, so you have to imagine that this slide goes way out in every direction and there's many, many template strands attached to this slide. So these templates are the molecules we would like to sequence. We are going to get one sequencing read per template on this slide. So, here is a Lego version of what we just saw. So, this is this Lego slide in gray and there are three template strands and even though we see only a few templates in this diagram, of course, as we say, there are many, many templates attached to this slide. This slide is very big and you should imagine millions or billions of these template strands scattered across it. So the important part is what we do next. We're going to add some DNA polymerase, which you'll recall, DNA polymerase is that molecule, that little machine that helps us incorporate complementary bases and turn a single-stranded DNA template into a double-stranded piece of DNA. So we add some DNA polymerase, and we also add some bases, which are the raw material that we need to synthesise these complementary strands. The DNA polymerase takes a base and sticks it in its appropriate place next to the template strand. Now, since we've thrown in this DNA polymerase and we've thrown in these bases, you might think that what would happen next is that the DNA polymerase would just go ahead and make all these single-stranded templates double-stranded. But there's something a little bit different in this example. These bases are terminated, they have a little piece attached to them which is called a terminator and in our Lego metaphor you can think of that terminator as like a little flat Lego piece that goes on top, and it prevents another Lego brick from being stacked on top. So because of this terminator, when the polymerase goes to build the complimentary strand it will actually only add one complimentary base for each of the templates, and then it'll stop. Because of the terminator, it won't be able to add any more bases after the first one. So, after we add the polymerase and the terminated bases, and we give these reactions some time to happen, then we get a picture that looks like this. Again, because the bases are terminated, the polymerase adds only a single terminated complimentary base to each template strain. So, the next thing that we do is to basically take a photo. So imagine a camera that's pointed down at the slide from above. It snaps a photo, and another important point is that the terminators are engineered to glow a particular color, so where the color corresponds to, the base. So maybe the terminated C here glows orange, so this C glows orange, and this C glows orange, and the terminated G here glows red, etc. So that when we take our photo, the camera's going to pick up light being emitted by the terminators, and so that's why you get this particular photo that you see up in the upper right. We see three little bits of light coming from these three different terminators, orange here corresponding to orange here, red here corresponding to red here, and orange here corresponding to orange here. As you can imagine, this photo is important data for us. This is valuable data to have because it tells us which base was added to each template strand, but we only have one base so far. So what do we have to do next? Well next, we remove the terminators so there is a way that you can cleave off the terminators from these bases so that they are no longer blocking us from adding on more bases. And then we basically repeat, we repeat this entire process. We're going to add the polymerases again. We're going to add the terminated bases again. And again, the polymerase will add one new terminated, complimentary base to each of the templates. And then again we can snap a photo, and now the photo's going to look like this. Again, this green here corresponds to this green in the photo, this blue here corresponds to this blue in the photo, and then this orange corresponds to this orange. And we just continue to iterate this process. So at the end of the day we get a series of photos, here's the next one, and here's the next one, here's the next one, here's the next one. [COUGH] One per sequencing cycle. We use the term sequencing cycle to describe one round of the process that I just described. So that at the end of the day we have a series of photographs that look like this, one per cycle. So if you look at these pictures, look at these photos, but look at just a particular template, so, for example, we can look at just the template that's in the lower left corner, Then you can read off the series of colors that were observed for that particular template. So if we concentrate on this lower left template and we follow that template through all of the six photographs that we have here, we see a series of color's. Orange, green, blue, green, red, red. And then if we take the corresponding bases and take the compliments of those bases, then we get the sequence of our template strand like you can see in the picture at the bottom. So if we want to sequence, billions of templates at once, we can do this. For all the billions of dots that would be found on that photograph, one dot per template strand. So to sum up some key facts about this technique are, first of all we can put billions of these single stranded templates all on a single slide. The fact that we can photograph all these strands at once is also very important, because that's what makes the process massively parallel. And then finally, the terminators keep the polymerisation reactions, the complimentary strand building reactions in sync, and then give us time to snap our photograph, which is the data that we use to infer the sequence of the templates.
