 everyone, even if you're watching this on YouTube later, or if you're watching on Twitch, welcome back to part number three of the lecture about primer design. So we just selected our SNP, right? So this is a SNP which is in cows and probably in sea cows, we don't know, but could be. So imagine that we want to PCR out this little piece of this gene. Then what we have to do is first search for the RSID. So let me show you Firefox. The slides will be available online. So you can just read the slides later and there it's described in detail, but I'm just going to show you guys how to do this, right? So we have our SNP, right? So the SNP is located here. So instead of directly going from here, I can just click on this variant name, right? So it's so slow today. Ensemble, loading, loading, loading. All right, let's just search for the SNPs. I just go search all species because SNPs are species specific. So when we search for the SNPs, for this one specific SNP that we're interested in, it's so weird that it continues loading here while actually it already figured out that there's one variant and that it's in cattle, but it doesn't live demos, never do live demos. That's just horrible. Bioinformatics is a lot of waiting for websites to do what you want. All right, let's search again. Ah, there it goes. All right, so here we have the information page of the SNPs, right? So here we see that it's a missense variant. It's located here at the forward strand, right? So we can just say genomic context. So when we click on genomic context, we see the SNP, right? And now we should, in theory, be able to go to export data, but it doesn't allow me to. So I'm just going to click on the primary assembly, then it should focus around the SNP. So let me actually go back and remember the exact position, right? So just copy it. So I'm just going to go to the primary assembly, and now I'm just going to say export data, right? So I want to export data. So I'm just going to say, well, I can select my location. So I'm just going to put in the exact position of the SNP. And then I'm going to say give me 500 or 600 base pairs in front and give me 600 base pairs in the back, right? So five flanking and three flanking. So five prime is in front and three prime is in the back, right? And then I'm not going to repeat mask it. I'm just going to say unmask the sequence because I want you guys to know that there's other tools which allow you to mask sequences. So we're just going to go next. And then we're just going to say, give me this format in text, right? So here I have the sequence, right? We know from FASTA sequence that every line is 600 base pairs, right? So we have a whole bunch of lines and we have one base pair here. And that is, of course, because the SNP is in the middle and I'm taking 600 in front and 600 in the back. So there should be 20 lines here in total. And then, of course, had the SNP needs to be in the middle, right? Of course, this is something that we kind of need to check, right? So what I normally do is when I export my sequence, right, I'm just going to paste it into Notepad++, which we see here. So if we go to Notepad++, then here is the sequence that I just copied, right? And now we need to find our SNP, right? So the SNP that we were looking at is an A to T SNP. So I'm in the middle of this sequence, there should be an A. So I'm just going to select and, oh, you guys can see that. So here in the bottom of Notepad++, it tells you how many you have selected, right? So here in theory, I am expecting the A to be, well, I select 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. So this is the SNP that we're interested in. So this is the base pair that some animals have an A and other animals have a T, right? So the SNP is indeed there. Alright, so then the next step, now we have our target sequence, right? So now having this target sequence, we can now actually start doing our primary design. Unfortunately, that is not true because when we did the export, and let me go back here, right? So when I said export the data, right, I told it that I wanted to export the data unmasked, right? But the mammalian genome, it consists of up to 50% of repeats. And of course, because a primer needs to be unique, we need to get rid of these repeated sequences, right? So these sequences which occur multiple times in the genome. So Ensemble can do this for you as well, but there's another tool called repeat masker, which I like a lot. So let me guys just show you the repeat masker website. And so we go to Firefox, here we have the repeat masker. So the only thing that I'm going to do is just take the sequence that we downloaded, put it in here, and then I'm just going to say, well, my DNA source is in this case, a other mammal, other than below. And I'm just going to say, well, use the default settings and then I'm just going to say submit the sequence. And then it takes a little bit of time to run the job. But in the end, what I will get is I will get the sequence back. It should not be too slow. Might be. Alright, so it's searching, searching, searching, searching, refresh it quite quickly. Alright, so here we have the annotation file is done. Search for it again. No repetitive sequences were detected. Oh, that's interesting. So let's make sure that we have some repetitive sequences, right? So let's just say export data. And I'm just going to use the mask here. So I'm going to say heart repeat masker of the location that we were interested in, which is, let me see. I forgot to forgot the exact location. So let me do this again. Let's go back to the snip, get the location of the snip, go forward again, export the data, waiting, waiting, waiting, waiting, waiting, so much waiting in bioinformatics. So I want to do here to here. And I say give me like 1500 base verse in the front 1500 base verse in the back, right? And instead of saying unmasked, I'm just going to run repeat masker inside of ensemble, which might take a longer time because ensemble is relatively slow at the moment. So it's doing the same steps as that repeat masker is doing. So it's taking the sequence that I exported, then looking to see if there are any repeats in there. And then masking out those repeats. So now when I go to text, then now it should show me here that there are repeats, right? So the repeats here are blocked out because it just says NNNN. So this region in the genome occurs not just once in the cow genome, but it occurs multiple times. And here you see that there are other regions as well, where we should not design our primers on, right? And here we see the same thing, little piece of sequence, which is repeated multiple times. So we don't want that in our primer. Alright, so I just take the sequence, right? And then I go to primer three for designing the primer. So I should have the link on the next slide. Yeah, that's perfectly fine. And let me see where the number three is. Here, there's the link. Alright, that's annoying cannot be found. Let's go this one. Alright, so this is how the primer design program looks like. It's just an online online system. So what you can do is you can just paste your sequence in here, right? And now, of course, we want to know where we want to design our primer, right? So we need to say what is our target, right? So since I exported 1500 base pairs in front 1500 base pairs in the back, I want to say that my target is now at 1501, right? And I want to say, well, I want to have at least like four or five base pairs surrounding that, right? So now when I say 1501 comma four, it means at target position 1501, but also the four base pairs in front and the four base pairs in the back, right? And I want to have it pick a left primer and a right primer. If I already had a primer, I could just fill it in. But I'm in this case, we want to pick both the left and the right primer. How we we can give our product size range. So imagine that instead of having all of these, we want to have a fragment of DNA, which is between like 250 and 500 base pairs, right? So because we don't want the fragment to be too long, because then we're like paying a lot of money for sequencing that we don't need. And we just want to have like this little area surrounding the SNP being targeted. And then we just click pick primers, it's that easy. So then we get the output. So the output looks like this. So you just get the sequence back. Here you see the sequence that we targeted. So we targeted this a at 1500 and one, because some animals have an A, other animals have a T. It put the forward primer here. So here is the the way that they do forward primers. And here is, for example, the reverse primer, right? So now on the top, it has my primer sequences. It tells me that the melting temperature is for the first primer 59.88 degrees Celsius. And for the other primer, it's 59.9. So again, this should be within three degrees. It also tells me that if there are any hairpins possible, and all of these things, so that there's a lot of additional information. But it just picked the primer 20 base pairs long, starting here print 20 base pairs long, starting there, they are pointing towards each other. So these are the two primer sequences that I need to use. And then what I get is a product, which is 468 base pairs long. So I take these sequences, I fill them in on the order sheet for the primer company, because there's just companies that sell you primers. So I just say, give me this primer, give me that primer, and you just order them. Then two days later, the primer show up and you can then take your species of interest, which is which you want to test and then see if the DNA that you have came from a cow, from Sudan, a cow from Italy, or a cow from another region, right. And of course, here, we're only looking at a single snip. Of course, if you really want to make a determination, which species it is, you generally would look at like 10 snips in total, right, because you know that, well, the sequence for had the soap. So animals from Sudan generally have a T at this snip that we're currently looking at. But animals from the US also have a T, but at a different location in the genome, they have a G, right. So you use like 10 of these locations to figure out which exact species you are currently looking at. And in theory, you could use these primers multiple times, right, because a primer pair, you get a cup of primer, which you can use for probably like two to 300 reactions. And so you could, in theory, not just do this experiment for a single cow, but you can do it for like two to 300 cows easily. And then have for each of these cows, you would get to see if there's an A or a T here, right. So that is more or less how we create these matrices, which we saw during the assignment, right, because during the assignment, we had this genotype matrix. So here we have our marker. So PVV4 is actually just a snip in the genome. And some animals have a one, which means an A and other animals have a two, which is a T, right. And here you can see that we used 120 of these to characterize the whole genome for like 100 and something individuals. So in the end, for this experiment here, we bought around 120 primer pairs, which would cost us 120 times like, I think a primer currently is like five, six euros. So that's like 500 euros. And then have with this set of primers that we have, we can then amplify this piece of DNA or these different pieces of DNA and all of these different individuals. And then we can use sequencing to figure out if this animal had an A or a T there. Alright, so that's how you do it. So I made a small overview slide, which is the output of primer three. Let me show you guys that on the slide. So here we see the output right. So here it picked a certain primer. Here we had a longer region of interest. So that's with the stars or the stars denote which target which region you are targeting. And here it came up with a product which was 784. Of course, if you don't like the primers, you can say well, exclude a little bit or not. And generally, primer design is not about designing a primer for a single piece. Generally, you want to have like a universal primer, which targets multiple transcripts at the same time. Alright, so in summary, primer design, it's far better done by machines, you can do it yourself. But then you have to make sure that all these seven rules for primers are adhered to. There are many different programs available. I just gave you a little example of primer three, which you can find underneath this link. And that's kind of everything that I wanted to tell you guys about how to design primers and how to do less genotyping yourself if you wanted to do. Alright, so are there any questions for today? Gonna wait a little bit. Because there's always a little bit of delay on Twitch. But yeah, it's that easy. It's just using like the primer three online tool, just make sure that when you export sequences from ensemble that you have your thing that you're interested in in view, right? So to make sure that it's really the thing that you're looking at. And that's about it. It's not rocket science, like people have been doing this for almost 50 years now. And 1983, that's around 50 years old. And then there's like, also hundreds of different companies that will sell you primers and extracting DNA is something that is also just a standard laboratory procedure. Good. If there are no questions, then thank you for being here. Thank you guys, all nine of you for staying until the end. And I will see you guys again on the 6th of January. And I hope by then that we have actually a fixed date for the exam so that I can tell you guys when the exam will be and how it will be done. Because I've had a little bit of a fitty with the proofing bureau because the proofing bureau wants me to do the exam in person. And I'm not too fond of that. Not that I don't want to see you guys that that's not it. It's just that with the new Omicron variant and all of the other things. And it doesn't seem like a very smart idea to have an exam in person. When we could just do an online exam. But if you have any opinions on that, then just let me know. If you say, Well, I really want to do it in person, then we can do that. But I think that it would be just good to have like an online exam like we did last year, because I don't see any reason why we should sit all in a room with me pacing around seeing if you don't cheat. When you guys can just promise me to not cheat, and I can just monitor you guys online. And we can just do the exam like that. Good. So I skipped a couple of slides, because I just in the in the presentation, it's just not one, but it's like multiple. And there's a couple of other tools, like if you want to calculate melting temperature, there's also a calculator for that. So instead of having to do the formula yourself, you can have a computer do it for you. But that was the only thing that I wanted to show you guys today. So I hope that you're all more or less capable of designing primers now. And of course, the hard part is not designing a single primer for a single sequence, but doing things like multiplex primers, which is a lot harder. Good. Then I'm not seeing any questions in chat. So for the people on YouTube, thank you for watching and stop the recording then. So see you guys next time for lecture number 10.