 Good day everyone and welcome to the human heredity in health in Africa or H3 Africa DNA day webinar. My name is Ebony Madden I am a program director for the H3 Africa ethical legal and societal implications program. And I would like to just welcome you to the H3 Africa DNA day. This is the second year that we are doing the H3 Africa DNA day presentation, and this is the first of two presentations that will happen this year. The second person presentation will be on Thursday, April 8, which is about two weeks from today. DNA days official celebration date is April 25, but the National Human Genome Research Institute celebrates it every year from January through May. DNA day is a global movement. It's to mobilize and energize and empower communities, educators and students to innovate collaborate and discover the promise of our shared humanity in connection to the natural world. So now I am very proud to present our presenter Catherine Skeepers. Catherine Skeepers has a master's in science and human genetics from the University of this father span, South Africa, as well as a master's in philosophy and computational biology from Cambridge in the UK. She also has a PhD in virology from Bitswater in South Africa. Her current position is a senior medical scientist at the Center for HIV and STIs at the National Institute for Communicable Diseases. And she has a joint appointment as a researcher for the antibody immunity research unit in the School of Pathology at the University of Bits. Dr. Skeepers is currently a co-investigator on a project within the Asia-Africa consortium that's focused on understanding antibody genetic diversity within South African populations and how that genetic diversity might impact antibody responses to HIV and other infections, such as SARS-CoV-2 responsible for COVID-19. Her presentation that she's going to give today is titled, Do You Know You Have 10 Billion Different Antibodies in Your Body? Just a little bit of shop keeping. The chat has been disabled. So whenever Catherine concludes her presentation, Ali Osgood, a program analyst with the National Human Genome Research Institute, she will moderate the Q&A. So please place all of your questions in the Q&A at the bottom of the screen. And I'll turn it over to Catherine. Thank you so much, Ebony. And again, thank you for this opportunity to give this talk. And yeah, thanks everyone for joining this DNA Day event. So as Ebony said, I'm based at the National Institute for Communicable Diseases. We are based in Johannesburg in South Africa. And I thought I'd just give you a little bit of background about the NICD before I started. So I'm based in a lab that is primarily focused on HIV vaccine discovery. But in the last year, we've been working on coronavirus and vaccine research for COVID-19. So the NICD has really been central to South Africa's COVID-19 response, understanding the number of cases that are happening in South Africa, looking at the different viruses that are circulating in South Africa and the efficacy of all the different vaccines against South African viral strains as well. So today I'm talking to you about antibodies, but it's going to be in the context of viruses. So viruses are these really tiny little bugs, if you can call them. So I thought I'd start by giving you an idea of how tiny these things are. So on this side of the scale on the spectrum here, an adult female is roughly around a meter in height. And all of the things that you can see on this side of the scale are things that you can see with your naked eye. All the way down to the human egg. When you start getting into this middle range here, these are plant cells, animal cells, bacteria. These are things that you'd have to see under a light microscope. Then a virus like the flu virus is on this side of the scale where you actually need something called an electron microscope to be able to see it in any sort of detail. So this is just put into perspective how tiny these little things are that's causing such chaos in our lives right now. So there are many, many different types of viruses. And in fact, there is estimated 380 trillion viruses that are living in and around us at any given time. Now, most of these viruses are not harmful and they can be helpful, much like good bacteria. So I'm sure you've heard about probiotics and how having certain bacteria in your gut is actually quite useful. So viruses are the same. So this is just another picture of a number of viruses that actually can cause harm. So while they are good ones, they're also bad ones as well. And within viruses, you can see that they are also different sizes of viruses. So for example, variola virus that causes smallpox and Ebola virus are fairly large, right down to a parvo virus, which causes disease in dogs. So this is what the rabies virus looks like and HIV is over here. There's other virus that I study. This virus here is the SARS virus, and it's closely related to the coronavirus that we're now dealing with that's causing COVID-19. So this is an electron microscope picture of what SARS-CoV-2 is, which is the virus, the coronavirus that's causing COVID-19. So it has these little red blobs all over the virus, and that's what gives the virus its name, the corona, because it looks like a crown. But it's not just pretty. These red blobs, we call them the spike protein. And this is how the virus actually attaches to ourselves and gets in and infects you. So if we zoom in to this spike protein and you see this green arm that sticks out. This is how the virus actually attaches to ourselves. It gains entry, and then that's how you get infected. But as I said, this talk is not really about viruses. It's about antibodies. And so what have antibodies got to do with this? Well, antibodies are our natural defense force against SARS coronavirus. So antibodies stick onto the virus and then it prevents it from attaching to yourselves and from getting in. In fact, antibodies protect us against many infections. So children are exposed to between 2000 and 6000 different antigens daily. Now an antigen is not necessarily something that is going to be harmful. It's anything that's farm. It can also be a bacteria. It can be a virus. It can be something that would give you an allergic reaction like pollen or a bee sting or nuts. And so antibodies, each of these antibodies are unique. So as I've shown them here inside this body is different colors represents them being different to each other. But in addition to them being unique, antibodies are also specific to particular antigens. So for example, this yellow antibody here would be specific to this yellow virus. So you can imagine if you have billions of different antibodies, it increases your chance of finding a match to any given antigen that you might be exposed to, whether it's bacteria or virus or whatever it is. So antibodies just really form part of a bigger immune system. So your immune system is made up of an innate immune system and adaptive immune system. And I'm not going to talk about this in detail, just to say that the innate immune system is made up of a bunch of different cells like a natural killer cell that kills and viruses and bacteria in different ways. I'll talk about them a little bit more a little bit later. But B cells are the ones that we really care about or that I'm going to care about in this particular talk, because B cells are the ones that make antibodies. And so in fact you can get different types of B cells. So plasma cells are the ones that actually make the antibodies and release them into your bloodstream. And then you get memory cells. And these are the ones that once you have found a match to any given virus or bacteria. These memory cells then stick around, they hang around in your blood, so that if you ever come into contact with that virus or bacteria again, you have some kind of protection. And so these memory B cells are really important for vaccine setting. So for COVID-19 vaccine, the idea is that we take that SARS coronavirus spike, those little red blobs that I showed you, and we put them in a vaccine in such a way that is safe to patients. So it doesn't cause an infection, and it's not harmful to the patients in any way. But it does prime the immune system to create a memory antibody response to the virus. So this is really the basis of how most vaccines work. You find the part of the pathogen that binds the human cell. Then you use that to prime your immune system to create a memory against that virus or bacteria or whatever pathogen it is. So that if you ever come into contact with the real thing, that you actually have some kind of protection. So antibodies are also important for other prevention methods as well as treatment. So if we use COVID-19 again as an example, the idea is if you have a patient that had COVID-19 and then they recovered, we then take blood from that patient and we can isolate their antibodies. Or we can isolate their sera, which is just a part of your blood that actually contains all your antibodies. And then we can use that as a prophylaxis, which is just a fancy word for prevention. And the idea is you give it to people that are at high risk of that particular infection. So in the case of COVID-19, it's for healthcare workers. And so you would give them the antibodies so that should they ever come into contact with the coronavirus, they actually are protected because they already have antibodies. The other side then is to use them as a therapy for patients that have coronavirus or are experiencing COVID-19 already. And the idea here is that the antibodies would then help fight the infection. So these kinds of approaches have been used for many diseases, including HIV and cancer. So what exactly is an antibody and how does it work? Well, an antibody is this Y shaped protein that I'm showing you here, the black parts on top of what we call the antigen binding portion. So these are the arms really that grab out the pathogen or the virus and they attach onto the virus and block it from attaching onto your cells. Then there's this part here that's called the FC portion, and it's responsible for effective function, which just means different ways to kill the pathogen. So you can think of this part of the antibody as a general in the military. So he can either recruit the Air Force or he can recruit the Navy. It depends on what kind of pathogen or enemy he has and where they're based. And all of these cells down here, these are those innate immune cells that I was talking about earlier, and they can kill the virus in different ways. And so this general decides which of these cells he's going to recruit depending on what kind of pathogen he wants and how he wants to kill. So how do we get so many different kinds of antibodies in our blood was all in the genes. So these antibodies that dark the black parts are called light chains and the gray parts are called heavy chains. And these two parts of the antibody are genetically different from each other. In fact, you need seven different kinds of genes to make a fully functional antibody. So in a light chain, you have a variable adjoining in a constant region gene. The heavy chains also have these types of genes, but they have an additional diversity gene. And so you need a combination of all of these genes to make your antibody. And so these genes are encoded on your DNA and your DNA is packaged very tightly into these little X and things that we call chromosomes. And that's how you found them in the nucleus of your cell. So if you think of each of these chromosomes as a neighborhood and the position in the chromosome as a physical location or physical address where you would find a gene. So the heavy chain genes are found on the neighborhood of chromosome 14 and they're physically located right at the end of that chromosome. And if this wasn't all complicated enough, we have two different light chains and they're on two different neighborhoods. So the Lambda light chains are found on chromosome 22 and Kappa on chromosome two. And these light chains are found somewhere in the middle of each of these chromosomes. So if I zoom in to the heavy chain locus on at the end of chromosome 14, I said that there are four different types of genes that make up these antibodies. Well, each of those types of genes have multiple versions. So for the V genes, you get upwards of 129 different V genes, 27 D genes, 9 J genes and 11 constant region genes. So when you're making an antibody, what happens is that you get a single version of each of these genes come together for the heavy chain and then for the light chain, and then they make the antigen binding portion. So you can think of this as like building with Lego blocks. So each of these different genes represent a different either shape or color of a Lego block, and then you add them all together to create one single antigen binding site. So you can imagine all the different combinations of either colors or shapes of genes that you might have or Lego blocks that you might have that allows your immune system to create all these different versions of antigen binding site that allows you to respond to different bacteria or viruses. So the African continent is generally poorly represented in genetic studies. So as an example. So this is a little bit outdated. Now this is based on 2016. And this is GWAS study, which is just a fancy word for a type of genetic study. So based on 35 million samples that were used for these types of studies, the black part here represents 81% of those of European ancestry. Some of this small part of the pie represents the Asian population and then this small part of pie represents everyone else. So if we zoom on to everyone else of those only 3% of those samples represent people from the African population so you can see, not only is Africa underrepresented in genetic studies but other populations as well. So because I'm based in Africa we're trying to contribute towards a greater understanding of African genetics. And so I am based in South Africa. South Africa is right at the tip of the African continent. And we look at, we study populations from Quasaluna Tull, which is just a province on the east coast of South Africa. And we look at two different locations. So Durban is right along the coastline of Quasaluna Tull and then Vullenglela is a rural area for the inland. And so we ask the question, do South Africans have unique antibody genes? And so to understand this we take blood from people from those different locations that I just described, then extract their DNA, and we zoom in to the heavy chain antibody genes. And so if I zoom in again, this is work done by one of our master students, Elaine Marsden, and I'm going to just focus on a small portion of the different versions of V genes that you get. So this little block here represents the sequencing that we did for one person called CAP-88. Each of these columns or blocks on the top here with numbers represent different genes and the numbers are the gene names. Now everybody has two copies of every chromosome, one there to get from your dad and one there to get from your mom. So each row here represents the different chromosomes. So the block, if it's gray, it means that this person has that gene. If it's white, it means that they don't have that gene. So you can see that this person CAP-88 has two copies of the gene 333 but no copies of the gene 433. And if you compare across the chromosomes, you can see that some cases you would have the gene on one chromosome but not the other. So this means that between your chromosomes, you can have very different antibody genes. And then if you compare a different person that we studied CAP-255, you can see that she doesn't have any of these genes here, but and then CAP-88 does. So not only can your genes differ between your chromosomes, they can differ between people. Now this is not something that is specific to South Africans. We've seen this in other populations as well. And this is work done by the collaborator of ours, Dr. Corey Watson, and he is based at the University of Louisville in the US. But now if we zoom into each of these genes and each of these colors represents different version of those genes, you can see that you can also get very different versions. And this can differ between chromosomes and between people. So the red colors here represent completely new versions or versions that have not been described before and until the South African population that we've had a look at. In fact, out of all the versions of these antibody genes that we've looked at in South Africa, almost half of them have not been described before. So this means that people can make very different antibody responses. But while people make very different antibody responses, we also see that people can make very similar or the same response against the same virus or bacteria. And this is really important when it comes to vaccine design. So in a case like COVID-19, where we're trying to test the same or very similar vaccines in a global setting, you want to know that that vaccine is actually going to be protected in the global setting. So up until now, I've been talking about changes in the binding parts of the part that grabs onto antibody. I mean, sorry, onto the virus. But that's not the only part of an antibody that can change. So you can also change the effector end of the antibody. So this is the general in the military. You can also change up your generals. So these are all the different types of generals that we get in antibodies. And so we call them isotypes. They're called antibody isotypes. So IgM and IgG antibodies are the first antibody response that you get against any pathogen. IgE antibodies respond to allergens. So those are the ones that give you an allergic reaction to either bee stings or pollen or nuts, for example. IgGs are the most abundant antibody that you find in your bloodstream. And they're really important for viral infections. IgA are also important for viral infections that they're interesting in that you can find them either as a single version or as a double version stuck together. In the single version, you find them in the blood. In the double version, you find them in the mucosa, which is just a fancy word for, say, where you have mucus. So in your nose, your mouth and in your gut, for example. So you can switch out your antibodies through a process called class switch recombination. So we spoke about this VDJ. This is the Lego block of the antibody binding site. And so your IgM antibodies are the first that get made because they're the closest one to this Lego block. So when your antibody comes in contact with a virus, what happens is that you get chemicals getting released. They're called cytokines. This is your general that then shouts to say, actually, we need something more specific. And in this case, it might be an IgG1. You get an enzyme that then helps is called AID. It helps bring IgG1 closer to the Lego block. And then it's so these little ovals here with the lines they're called switch signals. So you go from an IgM switch to an IgG1 switch. Anything in between that gets cut out and you make an IgG1 antibody. So the important part here is that the Lego block stays the same. So you're still hanging on to that same virus. You're just making a more specific antibody response. So we're also interested in as to whether we see genetic differences in these different generals as well. And this is work done by some other master students of ours, Ty and Holly. So IgG antibodies are fairly well described. So we know a lot about them and we've seen lots of versions of them. So for IgG1, we've seen 14 different versions. And for IgG3, we've seen 29 different versions. But despite them being well described, we've described we've seen another two versions in IgG1 in South African population and another five IgG3s. So IgA antibodies are not as well described in general. And so we don't know as much about them. So we've only seen three versions of either IgA1 or IgA2. So within our South African population, we've seen another eight versions of IgA1 and five versions of IgA2. So what does this all mean? Well, remember I said that this part of the antibody is the general that recruits the different parts of the military. So IgG3 antibodies are a particularly powerful general. They can recruit a lot of cells and they are very effective at mediating different functions. So what we try and understand then is what do these different versions of these antibodies, how does it change their function? And this is work done by a postdoc in our lab, Simone Richardson. So what we do is we take an antibody, we keep the same antigen binding site, but then we make that as an IgG1 version and the different IgG3 versions that we've discovered. So we test the ability of these antibodies to recruit natural killer cells and to mediate cellular lysis. So this graph shows the ability of these antibodies to mediate the cellular lysis. So the higher the bars, the better they are doing this. So this antibody is the same whether it's an IgG1 or these two versions of IgG3 at mediating this cellular lysis, but at this version of IgG3 is worse. We test it against phagocytosis and in this case the IgG3 versions are much better than IgG1. And then when we compare trogocytosis, which is just the way nibbling away different cell membranes, you see that this version of IgG3 is much better than the others. So you can see that it's not a one size fits all situation. Some generals are better at mediating certain functions than others, but it is helpful when it comes to understanding antibody responses. So what does this mean in general? So I spoke about antibodies being used as therapeutics or in prevention. So we take antibodies and you can give them either as an IV or you can give them as an injection. If we know that that antibody is really potent or really good at responding to coronavirus, let's say coronavirus. So we take this antibody, we know that it's fairly good at responding to coronavirus, but if we know that there are changes somewhere in this top part here that would make it better. We can actually engineer antibodies. So we make those changes in the antibody before we use it as a therapeutic or prevented. And then we create a much more potent or much stronger response. But what we can also do is engineer the bottom part of the antibody. So instead of being a lone soldier trying to fight off this virus, we can change this bottom part to recruit far more military and be a lot more effective. So not only can we make it a stronger response, we can make it a broader response that's much more effective against any given pathogen. So going back to the coronavirus. So like antibodies, viruses can also change to avoid detection by the immune system. And we've seen this happen now with COVID. So if this pegged virus here represents the first virus, the first version of the SARS-CoV-2 that was described in China, it has now mutated across the world. And we're seeing different versions of the virus. So what this means is that all the vaccines that we have currently, the Oxford AstraZeneca, the Pfizer, the Moderna, Johnson & Johnson and Novavax vaccines are all based on this first version of the virus. That means we have really strong antibody responses. So when they were talking about efficacy, everything was 90% effective against the virus. And that was true for 90% efficacy against this first strain, really strong antibody responses. And we were all really excited. And then the virus mutated and now the responses are not as good. The good news is we can and most of these vaccines are being modified to look more like the newer versions of the virus. And we have already seen that antibodies against the later versions of the virus can then be really strong. So this is something that the field is generally working on. So I hope that I've now convinced you that antibodies are really important, that antibody genes are highly diverse and that our genes that make antibodies can be very different between individuals. So it's really important to understand antibody genetics on a global scale. So it's not helpful if you're trying to look at a pandemic response if you only know what antibody responses are for a tiny proportion of the population. So it's really important to study antibody genetics in all populations. And understanding these genetic differences can help us improve antibodies either for prevention and treatment against diseases. So I just also want to acknowledge that I'm part of a really big lab and actually this photo is outdated, we're much bigger now. But this is a really big collaborative response between our lab that makes all of this work possible. I want to particularly shout out to all of the master's students and postdocs that have been involved in this work, as well as obviously Lynn Morris and Penny Moore that hit up our lab. Also collaborators at the Watson Laboratory at the University of Louisville. I also particularly want to acknowledge Caprisa. They are the group that coordinates all the different participants that are willing and prepared and do give us samples for us to examine. And then of course, various funders that also make all of this work possible and of course H3Africa that helps us coordinate all of this work as well. And with that, I will stop sharing and then I'll hand over to you, Ali. Thank you Catherine for that informative presentation. And thank you for sharing your time and expertise with us today. So it seems like we have some questions coming in the first one. Someone asked how does this preventative method for COVID-19 compared to PrEP. And they asked this earlier on in the presentation. Okay, so I'm going to assume that PrEP by PrEP, they mean either Tanafava being used for prevention for HIV or the use of antibodies against HIV. So PrEP in the sense of Tanafava is so what happens is that we're using a drug that we use to treat people for HIV. We give it to them before they become infected with HIV and it actually prevents them from getting infected. So the idea there is to use a drug for prevention rather than treatment. And we've also now finished the AMP study and what this was is a broadly what we call in the HIV field is a broadly neutralizing antibody. What that means is just an antibody that's really, really good. It's like a superman antibody against multiple strains of HIV. And we showed that if you give that antibody to somebody before they become infected with HIV, you can actually prevent HIV infection. And so this was the first proof of concept that we can actually use antibodies to prevent infection against viral infections, particularly in HIV. So the idea is the same here in terms of COVID where you're using an antibody as a prevention to coronavirus infection. So again, we're looking at, so right now we're looking at antibodies that were potent against the first string of the virus, but we're starting to see that people that were infected with newer strains of the coronavirus actually have a much better antibody response and they seem to have kind of a broad response against the newer variants and earlier variants of coronavirus. And so it's potential that we could then use those antibodies that are really potent and use them at prevention. And this is not going to be something that would be used instead of a vaccine. It's kind of a placeholder until we can vaccinate everyone. So you're using very quickly an antibody, get that out to people before we can actually get people to make the only million responses to a vaccine. So it's a very similar kind of idea. Thank you. And another question. Actually, these two questions sort of go together. What tools are proving most useful in expanding our understanding of antibodies. And sort of along with that, what are some of the innovations that you've seen due to the discovery of COVID-19. Okay, so tools, I'm assuming they're meaning experimental tools. So I mean, the field has moved incredibly quickly in response to COVID-19. So the idea of now having a vaccine 18 months later was almost unheard of before COVID-19. It used to take us years to get a vaccine that would be rolled out, or even into a phase three trial. So some of the tools, I would say, in terms of not necessarily laboratory tools, but one of the things that has really been incredibly instrumental is how we think about vaccine design. And what happened before is you would take an idea of a vaccine, you were tested in the lab, see that it's actually generating some kind of response that you expected to. You do some animal models, and then you start doing phased efficacy trials and safety trials. And then with COVID is we've piggybacked on technology that we've used for HIV and we've used for other viruses like Ebola and Zika virus. We just modify those vaccines to be specific for coronavirus rather than Zika or Ebola. And then we use that technology so we're not starting from scratch. So adopting technology that we've used for other viruses but applied to coronavirus. And then we've applied a strategic model of doing parallel safety and efficacy trials. So while there would be a pause between a phase one and a phase two trial, we don't pause, we just go straight into the next trial. That's allowed us to fast track things. So that's in terms of vaccine design. One of the things in terms of technologies is sequencing technology has also improved massively. So where before we would do something called Sanger sequencing that allow you to sequence a single gene for one person. We can now do a single gene for multiple or we can do multiple genes for multiple people on one sequencer very quickly. These are just sequencing technologies that we call next generation sequencing. That's helped us improve our understanding of antibody genetics massively. So sequencing technologies, other technologies that allow you to sort yourselves. So the technology is being massive progress, I would say, to help us understand antibody genes in general and antibody responses to different pathogens. I hope that answered the question. Thanks. And another question. Someone asked how do you engineer antibodies in the lab? So that's a good question. So what we do, so there are different ways that we make antibodies. So you essentially you take a sequence of the antibody. And if we have, so we know what the sequence is, so let's say we have the baby Superman version of an antibody. Okay, we know what that sequence looks like. Then we see in our genetic studies that there are some mutations that makes this antibody actually attached either better to the virus, or there's a mutation in the general part of the antibody that allows it to recruit better cells. What we do is we make those changes in the sequences, and then you use a method is called cloning. I know that's going to sound really scary because when people say cloning you think about cloning a sheep or cloning a person. We're not talking about macro cloning. We're talking about cloning in a very small cellular scale. And that allows you to just, we take bacteria and we get the bacteria to kind of make the antibodies for us. So you just, you change the sequence of the antibody, and then you just clone it out and get the bacteria to kind of make the antibody for you. That's a very crude explanation of it. That's a helpful explanation too. Someone asked what types of mutations can weaken a virus. So there are mutations. So when we talk about mutations, the virus can mutate and an antibody can mutate. So we're seeing now mutations that are happening in Corona virus. So all this co evolution. We study it quite a lot in HIV because HIV is a chronic viral infection. But we see it now also in a way with Corona virus. So what happens is when you create an antibody response, you, your antibodies are then attaching to the virus. So the viral virus, the virus also wants to get away from that response. So it can also change its DNA to get away from those responses. So we see some changes now in the spike. I showed those red blobs of the spike protein. So we see now Corona virus can actually make changes in that spike protein that now so the antibodies that we did have against the virus actually can't attach anymore. So that's one way how a virus can get away from antibodies. But then of course our immune system doesn't just sit and wait or just get infected. We continue to fight. And so our antibodies then make mutations in their binding site that allows it to still attach or to attach again to the new version of the virus. And this is how we call evolve in a way with the virus. So the virus changes and the antibody responds. The virus responds by changing and antibody response. So how this goes. And sometimes though viruses make mutations so viruses mutate all the time. And sometimes those mutations mean that the virus is not as effective anymore at infecting cells but that would be short lived and the virus. You know that version of a virus wouldn't stick around. So generally the viruses that are predominating would have some kind of immune. Effect or would be have a benefit for it to either it's infects better or it can escape from antibody responses better. Yeah. And someone asked, or they said earlier in your presentation you mentioned memory cells. Do those live, or are they around forever in the human body for someone's entire lifetime. Is a is a very good question. And so your memory response does change depending on what you get exposed to. So memory cells that you had early on in your life wouldn't necessarily the exact amount of time that it would stay in your system that I do not know. But probably somebody does know that on an answer to that. But your memory response can change so you're the memory cells that you have in your system can change depending on what you've been exposed to. And so I you know if you got the flu last week, or a couple of months ago, a particular type of flu or let's say you came across a completely strange type of bacteria, you might have a memory response to that now that you didn't have before. So your memory does change. And, but how long that those cells stick around for that's a good question and I shall go look at it. And someone also asked what genomic sequencing technologies are being used to detect the genetic variants associated with antibodies. So do they mean viral variants or antibody. So I'll talk about both. So, and, okay, so the sequencing technologies that we're using to understand the viral variants. So they are multiple technologies, the one is an Illumina my seek so I spoke about next generation sequencing. So this is a sequencing platform that allows you to sequence lots and lots of reads of multiple samples at the same time. So we predominantly use Illumina next seek. And it's a really high throughput sequencing platform, and that gives you a lot of depth. And so, another technology that we use is an iron talent. And it's also an next generation sequencing. Genexus is the type so like a next seek for Illumina Genexus is a type of iron talent. And that's also really good for sequencing viral variants. It's really quick. And, and it gets really good coverage as well of the of the virus in terms of antibody sequencing we try different approaches for this. So we can do an Amplicon sequencing approach which means that you just design primers to amplify just one gene, and then that those kinds of applicants for multiple people we can put on Illumina my seek. So an Illumina my seek is a short read technology that only gives you a maximum 600 base pairs and through a paired and sequencing. We also do a full length sequencing either of an Amplicon of constant region genes you can do that on a pack bio. It's also a next generation sequencer but it allows you to do really really long read so most next generation sequence a short read and pack bio is long read and we use that for long Amplicon sequences so for constant region genes. We also do in collaboration with Corey Watson's lab we do IGH capture. So when I showed the different chromosome 14. He then basically captures that whole region of chromosome 14 for the entire heavy chain. So all of those different regions D genes J genes. We sequence that on one capture on pack bio so again utilizing along sorry about that that's a it's an alarm telling me I need to go fetch my children. Sorry, so yeah so that it also utilizes the pack by a long read sequencing that we do for IGH capture. And I think those are the main technologies that we use so it's Illumina my seek next seek and pack bio sequencing for viruses and antibodies. Thank you. Another question someone asked how representative is the IMGT database, which is a reference database for African populations considering the high genetic diversity in these populations and on the continent. The good news is that most of the sequence sequences that we have generated from South Africans is currently in IMGT. The bad news is that in general, and I am GT and any other antibody reference database is really lacking not just for African sequences but any population other European or Caucasian individuals so this is something that we're trying to address on a global scale. So it's not just Africa you can think about Latin America is poorly underrepresented as well on IMGT but on any antibody genomics reference database and any genomics database in general so this is something that the scientific community as a whole needs needs to address. And going off of that, thinking broadly how could researchers build trust with communities in order to increase representation and studies like these. So this is one of the things that H3 Africa is really great with and something that they have been working on. It's really the idea of community engagement. So the last thing you want to do is just have a bunch of scientists going I have this great idea. Why don't you give me some of your blood, and I'm going to test these things and then you know the person knows has no ideas to why you're doing these things. And you know never hears from you again. So what we want is really to engage in the community and allow them, you know to understand what it is that we're trying to do, get them on board with the research, it's really really important to have in any kind of research, scientific in or clinical trial research is to have a community engagement you want the people that you're trying to do research with to be part of the process and and only by making them part of the process does it all work you know for everyone's benefit. We can't have, you know, scientists sitting in, in their labs going, let's just take blood from people. And the people don't know why we're doing things so community engagement is really really important in H3 Africa is that's one of the main components of what they do. Thank you. It seems like switching directions a little bit. And then going back to tools. They were curious what data science tools your lab is using to analyze the genetic data that is collected. Okay. So, and again I'm going to talk about this in terms of antibody repertoire as well as viral sequencing. So, and because I have a background in in bioinformatics or computation of biology, and it's okay for me to do command line analysis but there are a lot of people that want to be able to analyze their own data that can't, or don't have understanding of doing command line. And so one of the tools that we use galaxy. And so there is a, it's called use galaxy.org but there's various versions of galaxy. And that's an online web based tool, and you don't need to be a bioinformatician to be able to use this you basically log into a website. And there are various tools that you can use workflow so I use this a lot for COVID viral sequencing analysis. And there's a COVID 19 vaccine or sorry COVID 19 project, a galaxy project. And what happens is that you have bioinformaticians in the background developing these tools and then they make it available on the galaxy website so that anybody can then it is literally you just click a button that you press play, and it analyzes all the things for you so you have a team of computational biologists in the background that generate these tools and you can then analyze your data so galaxies really powerful in that regard for people that don't have computational biology background. In terms of antibody genetics so we do a combination of in-house analysis tools so when it's expressed antibody repertoire we use things like incantation pipelines we use sonar pipeline so these are all freely available pipelines that you can find on GitHub. And they're all command line based tools. There are other things like TIGA and PARTIS and these are tools that take expressed antibody repertoires and then predict what your germline would be. So for germline antibody sequencing again we do command line tools and we have a combination of in-house pipelines that we've written up in R but we use things like FastX toolkit SAM tools. These are all free tools that are available on GitHub. So if you're comfortable with command line then GitHub is the place that you want to be looking at for these kinds of tools. If you're not comfortable with command line there are loads of things available on Galaxy anybody can register for use galaxy.org. And you can use the tools then you can also just shout out or you know connect with some of the developers on Galaxy or on GitHub. And they can also direct you to you know the right analysis tools so in short I guess we use a combination of freely available and in-house things that we write up ourselves. Thank you. Another question someone had was how did you identify the new antibody alleles de novo assembly considering that these new sequences are not in the IMGT database. Great. Good question. What we do right now is that you use a database of sequences that we know are there. So we pull down all the sequences that are available on IMGT. We pull down sequences that are available also on IG PDB so IG PDB for those that don't know it's an immunoglobulin polymorphism database. And you will find sequences in there and that are not sometimes not on IMGT for various reasons that they might be short or they come from an expressed antibody repertoire whatever and we pull down all the sequences that we can get hold of. We then use a customized version of BLAST and we BLAST our sequences against those so we just look to see what is the closest relationship that we can find to any of the known sequences. Sometimes you get a hundred percent hits so the sequences that you see are already described. Sometimes you don't and it would be a single nucleotide or a few nucleotide differences and then we then call that within a certain percentage of identity you call it a new allele. However, there is using that approach when you just do a single amplicon you can't determine what whether something is just a new version of a gene or if it's a new gene. So this is where sequencing the whole chromosome is really powerful. So you can actually locate the position of the different genes on the chromosome and you can see then if you are dealing with an entirely new gene versus a new allele. But then again this you have the same problem what are you comparing it to. So what we do for the capture is again we pull down as much information as we can from all the sequences that we do have. We create kind of a mixed customized reference so it's mixed up of different sequences that we have and we do our best guess of what we can you know assembly assemble based on what we already know. But the more sequences we get from more populations the better our references going to be so this is why it's really important. And also it's important to understand what your own germline reference would be because when you're looking at your expressed antibody repertoire so that's after you've put all the Lego blocks together. And if you sequence that if you're going to try and figure out what is what is those individual blocks look like you need to have a really good reference for that as well. So it's really important to sequence as many people as we can to get a better idea of what the reference is going to be so that we can know which genes are really important for a particular response to particular virus or bacteria or whatever it is. Interesting. Thank you. And I think we have time for about two more questions. Someone asked, does CRISPR have a role in your research. Yes, I do. So we work with CRISPR. So, I guess, let me take a step. So there is CRISPR. And then there's CRISPR. Are they talking about CRISPR in terms of the experimental. Do you have the spelling of CRISPR? So in terms of like cutting things out and changing up the genes using CRISPR-Cas technology, is that what they mean for CRISPR? That's what I assume. Okay, so that's what they mean. Okay, so we don't use CRISPR for that in our way of changing up antibody genes or changing up viruses. We use something that's similar, but it's not the same technology that we use for that. All right. And then somewhat, just do you have any advice for people who want to be scientists? And from your perspective, where do you see opportunities in this field? So, I guess this is a general audience that we're talking to. So I would say to students that are interested in being scientists. If you, this is going to be, you know, like the typical stay in school, don't do drugs. But, you know, if you want to be a scientist and you're interested in this is continue to pursue your studies in school. So I should say that I first discovered genetics in grade eight. And since then, I also had a really fantastic biology teacher at the time that presented genetics to me. And from that get go, I decided I want to be a doctor in genetics. And so here we are. I don't want to say how many years later, but this is what I'm doing. So if you have, you know, if you have this desire to pursue this is just to keep going. It's a long haul. There's a lot of studying that goes involved. But if this is something that you're passionate about, then, you know, reach out to people. And, you know, people are, you know, welcome to reach out to me. If this is something that you're interested in doing. So find labs find people in areas that you are in that are doing these things and start shadowing those people start asking them questions and find out, you know, who's doing what. And COVID, you know, is is a great way to kind of get involved in science. Everybody around the world is is involved in this and trying to understand this. So if you're interested in science figure out who are the people that are responding to COVID and see if there's something that you could do it could be anything on any level, even as a student, you know, in school or in university. And so it would be for me it if you have a desire to do that, keep pursuing that and find out who the key people in your area are and align yourself with that. So one of the main, I think things for me that has been has contributed to my career this far has been aligning myself and working with Lynn Morris and Penny more so when I, I was in the UK first working there and wanted to come back to South Africa to do a PhD. I found out that Lynn was working on HIV doing amazing work and so I just emailed her to say hey I'm interested in doing a PhD would you take me. And so it's about finding key people that you know are doing amazing research and finding out if they are willing to work with you and aligning yourself with with with collaborators that do great science so as a scientist. You need to understand that it's not going to be about you it's a team effort. So you need to be prepared to collaborate with people but you also need to figure out who the right people are and get the right mentors to start off with and then those mentors will then introduce you to great collaborators but those are the main things continue to focus on your passion, but also understand that you're going to need keep key players in this it's not about you it's about a team. Well, thank you so much. And thank you for joining us today and for celebrating the DNA day season. And thank you to everyone who attended the next H3 Africa DNA Day event is on Thursday, April 8. And have a great rest of your day or evening, depending on where in the world you are.