Module 5: Lauren McKnight - DNA Sequencing, Profiling and Genotyping Technologies

Biology. Biology. Biology. Biology. Alright, hello everyone and welcome to another episode of the HSE Biology Podcast. Today I am honored to introduce the first guest on the podcast and that is Lauren McNight from the Garvin Institute. Lauren thank you for joining me today. I really nice to be here. I was just going to congratulate you on your podcast. It's a really great service you're providing for the students and their teachers. Thank you so much I appreciate that. When I first started it was something meant just for the students but I know a lot of people are listening now and yeah they're giving me a lot of support so it's certainly good to feel supported in this industry. Look before we get into it today we're going to be talking all about sequencing and profiling. Do you want to just give us a quick rundown of your background in science and sort of how you ended up at the Garvin Institute? Yeah sure. So I did a science degree at Sydney Uni and they stayed on there to do both honors and a PhD in immunology. So a whole different section of the syllabus and I looked at the role of regulatory T cells in allergy. So that was a lot of fun. After that I did a fair bit of teaching at Western Sydney University and my favorite unit that I taught into was called scientific literacy and it got students to investigate a socio-scientific issue and what that means is an issue that is relevant for both science and society. So maybe it's a controversial issue, something where maybe the media portrays a few different perspectives and got students and really taught them to analyze evidence and formulate arguments and really think critically and scientifically about issues that really matter. And so that's what I'm really passionate about and when I went for my current role which is an educator role with the Kinghorn Center for Clinical Genomics. I found a colleague who shares that passion with me and so now in the context of genetics we're able to provide resources and things to help students explore those sorts of ideas in this context. Wonderful. Well that sounds like sort of you've got a lot of experience across multiple fields in the well at least in the biology syllabus terms which would be super useful. So I might have to get you back on to do the immune system as well by the sound of it. I'll have to dredge up some old knowledge for you. Look with your current role so you said you're an educator at the Garvin Institute. So what are you doing at the moment or kind of things do you get up to? So my role is as a project officer technically and the team that I'm working in is education and communication and so we do a wide variety of things. I've created some resources for HSE teachers. Our most recent project has been to create some resources for students who are undergoing a community genetic screening program. So we help students to make informed choices about whether they wanted to participate and being involved in a career screening activity and also our research study. So it's really about providing easy to understand information that helps to people to make informed choices for their own health care. Wonderful. Well look I think there's no more relevant time to be looking into the aspects of science that are you know permeating through society at the moment. Scientific literacy may be the most important factor at the moment, especially given everything that's happening with vaccinations and COVID. So maybe we'll talk a bit about where people can access more information on that at the end. So let's get into some of the content stuff now and I'd like to sort of go through if you could maybe just give us a quick overview of sequencing and profiling and I know that we spoke yesterday about a new term that isn't in the syllabus that you thought might or should be included in the future. Yeah that's right. So the syllabus term talks about DNA sequencing and DNA profiling but there's actually another really important technique that we use to look at DNA and that's called genotyping. So the thing that all three of these different techniques have in common is that we're looking at the DNA of an individual and so students might be aware that across our human genome we are about 99.9% similar to one another and so there's been a lot of studies and a lot of science around you know the human genome project and so you know when they sequenced and they found out the order of bases of all 6 billion bases in our genome that unlocked a huge amount of discovery but although that 0.1% of DNA that we don't share is relatively small compared to that which we do it's actually the most important and the most interesting. So because we have 6 billion bases 0.1% of that is still 3 to 5 million places along the genome where two unrelated people will differ. So the syllabus still refers to those mutations in the industry we tend to refer to them as variants because you know no one wants to be immune when you're talking to a patient you don't like to talk about mutations and also that term mutation seems to suggest that these things are new or unwanted when really variation is very important it's what makes us all unique and individual it's actually a wonderful thing and so these millions of variants that we have between us are really wonderful and some of them in fact actually a very small proportion of them will actually affect things like our health, our physical traits, our behaviors, those sorts of things and so when we're actually thinking about the differences between people it's those variants that we want to look at. It's also the parts about DNA that make our DNA unique to us so unless two people are identical twins DNA can be used to uniquely identify a person as well so they are the reasons that that this syllabus point so sequencing, profiling and we'll talk also about genotyping have really important implications for healthcare, for forensics and also for discovering what genes do. Wonderful thank you I talked about SMP's last week on the podcast and I think there's some of the variants you're speaking of and I did mention and I want to see your opinion on this I'm mentioned that an SMP a single nucleotide polymorphism occurs in at least one percent of the population and so I as well said last week that it's not really a mutation at that point it's a polymorphism you know it's it's large enough in the population was I correct in saying that again like it's more of a variant rather than a mutation. So you can have variants that are very very common so you know if it affects more than one percent of the population it's a polymorphism as you said you can also have variants that are very very rare you know that may be only ever been seen in one person and so I think that term mutation used to people weren't really sure what they were referring to was it just common ones or were polymorphism mutations I prefer to talk about everything's a variant and then it's either common or rare it's new or old it's large or small so that term SNIP actually talks about two different things doesn't it it talks about it being a polymorphism so it's common but it also talks about it being small just a single nucleotide so that's actually a bit of a combo term the SNIP and you know it's an important combo term because we use these SNIPs all the time but when we're categorizing terms it can get a little bit confusing. For sure I love the way you refer to it as just a variant type and then smaller or larger I think that's a really nice way to put it so look you just spoke a little bit about why profiling and genotyping and sequencing are all important. One thing that I find when I go through this with my students is the process or each of the processes is quite difficult to understand from a non-scientific background and there's a few different techniques that we can that people can use in sequencing and profiling so the first thing I'd ask is are you able to give us a rundown of first of all sequencing as sort of as simply as you can and and then we'll go through the other ones and and see how they differ. Yeah sure so DNA sequencing really just refers to scientific methods that allow us to figure out the order of bases in a stretch of DNA so it's the sequence of these bases that actually encodes the information in the DNA right so it's really as extracting the the information it's extracting the DNA code it's really reading it and turning that into. a long string of ACTs and Gs. So there are a number of different techniques and they have evolved a lot over time, but anytime you're taking a sample of DNA and you're turning it into data that is ACTs and Gs in order, then you're sequencing. So you can sequence a single gene, you can sequence the entire genome and so and those anything in between and they all have different uses. So probably the gold standard at the moment is called next generation sequencing. I don't like terms that involve next generation because sure enough they'll be a next one and then what are we going to call it? So I like to call it massively parallel sequencing and that's another common term. So massively parallel sequencing, what it does is it actually uses sound waves to break the DNA up into short stretches. So not based on any sequence, it just basically shakes the DNA apart so that it's in short stretches of about 200 base pairs and then what it does is it sticks those little fragments to a glass slide and then it does this really clever thing where it actually replicates those fragments and as it goes it sticks each new bit to the slide and so you get a little cluster of identical fragments and what that does is it just makes it big enough so that the machine can actually interpret the signal. So instead of one bit of DNA, you now have a cluster of DNA so that you can so you can read it and the way it reads it it's called sequencing by synthesis. So it actually replicates the DNA right there on the slide using bases that are attached to fluorescent dies. So one step at a time it adds a new nucleotide and then it takes a photo and it washes away the dye and then it adds the next nucleotide takes a photo, washes away the dye and so that little cluster is going to start flashing colors in the order of the bases being added and so you imagine now you've actually done that not with one tiny fragment but hundreds of thousands of fragments all at once all attached to different spots on this glass slide and you've got to look up a video of it it's just gorgeous really all these little spots flashing color along with the sequence of that fragment and so these machines they then take those sequence of photos and turn it into data for each fragment and then it becomes a bioinformatics job. So bioinformatics means using computers to deal with biological data and so the machines put out this huge amounts of strings of letters and then it takes the very fast computers to line those strings up with the sequence that we already know of the human genome and then we can actually start to look for places where that person's DNA is different from the reference genome so the one that we have on file and so it really does come down to looking at differences. So you can you can store someone's whole genome in a data file but usually what we do is we reduce it to a list of variants and we store those because that's obviously a much much smaller file. Wow so first time for me hearing the technique that is used in the lab because the one that we teach and the one from our books is something called sang a sequencing which I'm sure you could explain as well and maybe I'll just talk about how I taught the kids that technique as well and you can tell me where it might be right or wrong but that's super interesting and it sounds like you know the dot point itself is all about looking at the use of technologies to determine inheritance patterns and I think so can you repeat what was the name of the technology what was it called again? Look all it massively parallel sequencing. Okay so yeah again never heard of that before but it sounds like a really nice way where you get that visual stimulus so students might be able to understand the idea of those flashing lights in a row and the clustering which increases that signal strength which then can be sort of calculated by a computer and you're given graphical representations of the data and then eventually those letter combinations that you're after so I think as a visual like for the first time hearing it that was really nice and it's something that I'll definitely be looking into and how new is this technology so this is something that yeah like I said I haven't heard of before how long have you been using this the gold standard? So I think 2008 the first machines came about and it's really great if you look up a graph of the cost of sequencing over time you can see it takes a very sharp turn down when this technology was developed so compared to sangha sequencing which is the technology you know the previous gold standard on which the human genome project was completed that was orders of magnitude more expensive than this new technique so this new technique has reduced the cost and the speed of whole genome sequencing to the point where we can actually sequence the genome of a patient to find out the cause of their genetic illness and so that actually then makes sequencing useful for medicine and so we can now in our institute we can sequence 40 human genomes in four days yeah so if you think the human genome project it took 13 years to sequence one genome and now we can do it within a week and do a whole bunch at a time and generate massive amounts of data that then need to be stored and managed and processed and so genomics has really moved away from being a bench science and it's now very much a data science and so our institute is full of bioinformaticians and computer scientists that sounds wonderfully complex and something that is obviously better for humanity that we're going in that direction sequencing obviously gives us a lot more information than just genotyping or profiling that we'll talk about in a minute and I guess all those things then need to be yes calculated to figure out where the differences are and how they can mean certain different things so I look there's so much so much you can unpack in that but it's wonderful to know that you know we're moving in that direction of making things cheaper and easier to access health care for all so all right um with the DNA sequencing that I teach my students I'm just going to give you my quick flow chart that I use to teach the sangose sequencing method and you can tell me whether or not you think it may need things or take things away if it's too complicated or not enough and we'll go from there so you're ready to go sure sounds good all right so here's how I describe it step one DNA section of interest is copied using PCR so that's polymerase chain reaction basically making lots and lots of copies I tell my students the DNA sections that are copied are then placed into test tubes the test tubes contain a fluorescent nucleotide or a terminator base which tells the enzyme DNA polymerase to stop copying the DNA is then taken from the test tube and placed into a gel tube gel electrophoresis then takes place in the smaller pieces of DNA migrate faster than the longer pieces the gel can then be analyzed as a laser light will cause those bases that fluoresce to be to be recorded and the recorded bases will be in order ACTG whatever they flash so that's my little run run down of sangose sequencing how's that yeah yeah that sounds pretty good um so yeah a couple of take-home points there are that sangose sequencing can only be done on relatively short sections um so you know as technology went on and increased then those sections got longer um but you you could never do well it would take a very long time and it did take a very long time to sequence the whole genome um using sangose sequencing because you can only really do those short sections at a time so if you just want to examine the sequence of one gene sangose sequencing is still used and still effective um a couple of changes to the method that you described there so these days um all four bases would be put into the same tube and they would be labeled with different colors dies so that you can you can do all root four reactions at once and then now instead of doing it on those flat gels that you might have seen after that you might use after a PCR for example um they'll actually do it in these tiny little capillaries so they do it what's called capillary electrophoresis and so just like you said the different fragments will get sorted according to size and so because you've done so many copies you'll end up with a few copies that correspond to each position and so then when they get sorted by size you'll have a fragment for each position and it will be labeled with the base at that position so then you can read along the capillary using the um different lasers for the different dies and so you can get the same sequence out like that. And it results in those graphs that you might have seen where there are blips in different colors and it'll have the letters underneath. That's the typical readout for a sangha sequence. Good. Yeah, I have a little, I don't can't show you right now, but I do have a little model that I like to show students that does have those little blips and the letters that correspond with the colors. So again, I like talking about that one because it's very visual when you get into the laser light and the color representing each letter. I think kids understand that quite well. But I didn't know about that it only did sections which is a really good point. And I guess that leads me into the next one which is that why you might not use the say Oxford Nanopore which is another technology that's used to sequence is that a similar thing that it only does sequence like small sections and things like that or is it, am I completely off here? No, no. So Oxford Nanopore is sort of what we call emerging technology. So it's not gold standard, it's brand new. There's a few different. So Oxford Nanopore is one, but there are a few other companies and these are called long read sequencing techniques or technologies. The benefits of a long read technology is that we don't have these tiny, you know, 200 base pair sections that we have to line up. That can cause some difficulty, especially over the trickier areas of the genome. Long reads can be very beneficial, especially because then we know which strand the DNA came from. So we can see variants that might be on the maternal chromosome versus the paternal chromosome, for example, whereas when you're only doing short sections that can get a bit modeled up. And yeah, as I said, there are tricky areas of the genome, such as areas with lots of repeats where massively parallel sequencing can get a little bit confused. So Oxford Nanopore is really, really beneficial in that way. Another beauty of Nanopore is that the machines are tiny. I'm talking, well, I have an eclipse mince box here on my desk and the smallest machines of the Nanopore are not much bigger than that box. And so scientists can take a Nanopore machine out into the field. They can actually do their prep and prepare their DNA in the field and they can load it up onto a Nanopore machine. And they can get their readout in real time. They can watch that information coming out on the computer or actually now even on a smart phone. And they can just keep collecting data until they've got enough and then they can stop the reaction. So you can imagine the way that this opens up science and a whole bunch of new possibilities. That's why we don't use that currently for whole genome sequencing on its own is that it's a bit less established. I believe it's still a bit more expensive per gigabase. And so you can, you would just sort of run a lot of those reactions at a time. So the bigger versions of the machine have just a lot more of those little reactors in them. And so you can do large sections. But they're not really designed to be done on that scale. So usually we would do most of our sections with massively parallel sequencing and then some of those trickier sections. We might use the Nanopore. If you want to have a look at how Nanopore works, I'm not even going to try and describe it. But the videos of those are very visually appealing as well. Very cute little molecular motors involved. I have seen those videos and it's amazing those devices. They may be in schools one day. I mean, you never know if they're getting small enough. And yes, if we can get the price down, that's I guess the main factor. The way I explain the Nanopore to the students, again, I use a very simplistic version then I'm sure what actually occurs. But I like to say that the DNA is fed in and these tiny pores are actually just enzymes and they just pull the DNA through. And as it's being pulled through, it crosses an electrical signal. And the different bases cause a different electrical signal to be given off. And then that's read as a different base and then calculated again by the computer, which then turns it into a string of letters. So that's how I like to describe it from the videos. I don't know if you have a different version of that, but that's not really. I wasn't willing to try it, but you've done a fantastic job. Good, good. Yeah. I spent a lot of time looking at those videos and they were helpful. So definitely check those out guys. I might put them on the page for people to check out as well, the particular useful ones. Oh, right. So what kind of things are you sequencing for at Garvin or what is it used for in general? Across the Garvin DNA sequencing is used for all sorts of medical research. There probably isn't a field that we study that we don't use sequencing in some aspect. A really new exciting field is called single cell genomics. And so particularly those who are studying their immune system will actually sort out cells according to the way they look. And then sequence, you can actually take one cell and sequence that and then look at the difference in the genes that are being expressed between different cells. So that's incredibly interesting. In our sensor, the Kinghorn Center for Clinical Genomics, we're really looking at ways to make this sort of DNA information useful in healthcare. So we have a lot of people looking at things like how to keep DNA information secure, but also useful. Lots of different bioinformatics. We have projects in all kinds of different disease types. Lots of different cancers. Developmental disorders, kidney disorders, heart disorders. You name it, we're using sequencing to look at the cause of the disease and importantly how the disease can be either detected or treated well. Good to hear that, you know, it's accessing so many different fields. And with the sequencing now, I don't know how the DNA is extracted in the first place, like the bit you're getting, but how much of it is human interaction and how much is automated, how much of the process is now up to the computers and robots? Yeah, so we do have our wonderful technicians overseeing every step of the process. But the good thing about using automated systems is that not only does your hand not get sore from all that perpeting, but you also reduce a lot of the human error that happens when your brain gets sore from too much perpeting. I can speak to that one from experience. So we use what we call liquid handling robotics. And so if you think of it as sort of a robot that can use 96 different perpets at the same time. And so I don't know if people are familiar with a 96-well plate, but it's a very standard piece of lab equipment. When you're working with small volumes, you just have a little plastic plate with 96 little wealth in it. And so we either use that or racks of small test tubes that are all bar coded and then the robots can move small amounts of liquid between all of those tubes and different washes or different chemicals that need to be added. So it's faster and it's quality controlled. But there's always human checks after every step. So we extract the DNA and then we check and we do a quality control and make sure that there's enough DNA and that it's of good enough quality and purity to proceed. Partly, that's so we make sure we get a good quality result. But it's also we don't want to go right through the process and then realize we've wasted a whole lot of expensive reagents. We want to make sure that we've got a good quality product before we go forward. So yeah, there's the DNA extraction, which really is just a swooped up version of the sort of strawberry DNA extraction that you might do in the classroom or the kitchen. It uses the same sort of technique. And then you know, the DNA is processed and depending on what you're doing with it, you might select certain parts of it that you're going to sequence. You might do some PCR to expand particular regions of interest. Really depends on the technology or the application, I should say. So for whole gene sequencing, we just extract a lot of DNA from our tissue sample and just process it as is. We don't need to amplify it and we don't want to introduce any sort of bias into the sample in that way. So we're just sequence as is. You can do something called whole exome sequencing. So you know, we know that only 2% of the gene, I'm actually genes, actually codes for proteins. And so most of what we know about variance is actually actually from within the exome, the protein coding part. And so we might just wanna pull out those parts and so we'll use certain probes to do that and just look at those sections of DNA. I was meant to be talking about automation though, sorry I don't know. - Okay, that's okay. - Oh look, you're on, like you kept coming back to it which is good. One thing that I do wanna know is like the start of the question is like you talk about, you know, getting the tissue sample, where do you get the sample from? Is it just from donated blood? Do you have like a certain cell that you like to target? Like, you know, part of the body skin cells or like, where do you get the first sample from? Where does that come from? - Yeah. So DNA can be extracted from all sorts of tissues. So if someone is having whole genome sequencing for diagnostic purposes often that will come from a blood sample, a lot of different screening, you know, we don't want that to be invasive and so that can be done on a cheek swab. Often we'll be sequencing tumor DNA so we actually take a little bit of tissue from someone's cance that we might sequence that and compare it to some of their regular cells and see what genetic changes are present in the canceous cells and that can actually help guide treatment. You can, as I said, you can take DNA from single cells from different parts of the body to do different experiments over at our partners at UNSW, they do a lot of sort of agricultural and ecological research and so they might take DNA from some koala poo or the microbes that they find in a swamp somewhere or wine. They're doing some great wine research over there. So really, anywhere that you can find DNA we'll find a way to pull it out and study it. - It's certainly incredible that you know, there's no limitation on the cell type you get and the access you get to that DNA, very interesting and I'm sure there's a lot more we could go into from getting that DNA out of the nucleus just by itself and separate from everything else, but I think we'll finish there with the sequencing talk. One more thing I want to mention is you called them exones. I've been calling them exons the whole time. Is it pronounced exones for the versus introns or is it introns? - No, no, let me clarify there. So within the anatomy of a gene you have an intron and an exon. - Okay, good, that's what I've been calling right. You're talking about something else there. - So we stick that suffix on the end of words to indicate that we're talking about all of the copies in the body. So you have the genome, which is all the DNA in the body. You have the exome, which is all the exons in the body. You have the proteome, which is the full range of proteins that are being expressed. You have the transcriptome, which is all the RNA copies. We just love sticking that home on the end of things to turn it into a big data science. - Well, that's good. It means I wasn't making a mistake on the initial term. I just assumed something about what you were saying and panicked a little bit in my head, but it's good to know that I've still got the term right. And I've learned a few new terms as well, exome. So one that I can certainly bring into the classroom. Apart from that, is there anything else you want to add about sequencing before we get into the profiling and genotyping? - I could maybe share some links or some images with you to share with people on the page, 'cause yeah, I think having that visual is really important. - Sounds really good. I think we'll compile a few different things and put them all in one post. Wonderful. Why don't we get into the profiling side of things now? And again, I'm gonna ask you to explain as best you can profiling. Like I've got again, a little flow chart that I like to use, but if you want to go for how it's actually done in the lab, and then we can talk about what it's used for and its importance as well. So, thank you. - So DNA profiling is by and large a forensic technique. So profiling creates what's sometimes called the DNA fingerprint, and it's often used in really similar ways that your fingerprints will be used. So to identify somebody, so it's used in criminology, but it's also used for things like identifying the victims of disasters. It used to be used in paternity testing. It is used in identifying relatives. Again, that's been used a lot in victim identification. So Australia maintains a DNA database, which is based on a particular type of variant. Again, we're talking about variants, okay? We're talking about differences between the DNA of people and this time it's about using that to identify them. And so we don't want to use parts of the genome that are gonna tell you something about your health. We don't want to use parts of the genome that are gonna tell you about the way you look. This is really about just finding random bits of DNA that we can use to tell people apart. And the random bits they use are called STRs. So these are called short tandem repeats and it's a type of variant that's quite common. It's actually 3% of the genome is made up of these STRs. And they're very polymorphic. That means that there's lots of different forms of these variants that exist throughout the population. So polymene many, morphic meaning form. So this is just an area of high variability. And so if we pick particular regions to look at, it gives us the best chance of being able to tell two people apart. So we look at a bunch of different STRs. The current database in Australia uses 18 different STRs. So it's gone from nine. I think the Americans use 13 in their coders. People have usually heard of coders because of the American crime shows. The Australian database, which is called the National Criminal Investigation DNA database. And yes, I do have that written down in front of me. It started off with nine and is now 18. And there's pushes around the world to move to a standard I set of 24. Anyway, we're ever expanding. And that just gives a better chance of telling between two people in the whole world, rather than say two people in Australia. And also, if there's a low-quality sample, the more different points you're looking at, the better chance you are that you'll get enough to basically get a conviction, which is often what it comes down to. So the technique that's used in DNA profiling is we just want to look at these STRs and these 18 particular STRs. And so we'll actually use PCR to amplify or make lots of copies of just those little bits. And so this is a really good time to think about how PCR works because you start off with two probes, one that matches up to the beginning of the sequence you're interested in and one that matches up to the end of the sequence you're interested in. And so when the TACA polymerase that we actually use in PCR, zips back and forth between those two probes will just make lots of copies of the section in between. Now, the cool thing about STRs, short-tanned and repeats is that we all have a different number of repeats. So maybe the repeat of interest is that it goes ACTA over and over, ACTA, ACTA. And I might have 43 repeats of that. And look, you might have 47. And so the section that has been amplified will be longer in you than it is in me. So if we look at 18 of these different STRs where you and I are gonna have two different, 'cause remember we've all got two copies of all of our genome. And so I might have a 42 and a 47 and what did you have you had a 47 and maybe a 49. Then if we look at that then across 18 different low-sci positions, then that's giving us a very unique and individual profile. That then the police can use to figure out that it was me that robbed that store and not you. That's wonderful, I suppose. It was my fault, but look, it sounds, it's pretty much exactly what I have written down which is really good to know. And I felt like I was following along nodding in agreement that that's yes, that's also how I understand it, which is good. So yeah, I've got a similar process to sequencing in that you use the PCR, but you're amplifying the genes or the regions that you need. And so yeah, we all have different lengths of those STRs. and then when we put them into a gel electrophoresis. basis, either Agriest Gel or we use, I don't know if you use the tubes again, the capillary tubes to separate them by length, but I know we get that patterning where they line up and again we'll put it some links up for the students, but those patterns will show you the indication of who was the father, who was the mother or who was the suspect and things like that. So the gel will once again pull the shorter strands down and the longer ones higher. I think that's how I've got it down anyway. Yeah, that's right. Look I'm not sure exactly the technology that they use these days, but looking at it as a gel electrophoresis is definitely used in a lot of different learning activities and that's a really great way to think about it and there's some great forensic puzzles out there that you can solve. For sure and I did have down in my notes that it was 13 STRs and I'm super happy you've told me now that it's 18 in Australia. So I think the numbers was something ridiculous. It was like I think a few, maybe a hundred million or something for 13 STRs like the likelihood of matching up with someone else and so it was so slim but knowing they're moving up to 24 is also pretty incredible, but I guess like you said you want to have the most likely incidents that you are not the one to blame or you are the one to blame to say those things. Some of the stuff that I was refreshing up on before we had our chat was really fascinating. So obviously the standard uses to match the crime scene to the suspect, but there's all sorts of other things, so kinship. So they might look at identifying the relatives of somebody, particularly in this is used in disaster recovery and so they can actually look and see the cousin because we're looking at shared variants across different locus. Well, say then you can identify cousins and things. There was another one that I've forgotten now. That's right. They're all. They use they will can also look at mitochondria to find about people's maternal lineage and they look at the wirecromb assume to find out about people's paternal lineage. So yeah, they're sort of variations of forensic genetics. Wonderful. Look, I'm glad you brought up mitochondria because it's something that I think I've brought up in a previous podcast. The fact that the mitochondria only comes from mums over during fertilization and it's a good use for tracking lineage. I think recently this year they were talking about tracking the sort of first humans, not you know, by definition, but genetically speaking using that mitochondrial DNA because it's always passed down via the female. So it was a good way to track it. So definitely something that's called a talk about. Yeah, that's right. And there's also diseases that can be caused by harmful variants in mitochondrial DNA. And so our institute's actually done some pioneering work in diagnosing mitochondrial disease based on sequencing of mitochondrial DNA. It's really fascinating. It really is. Yeah, circular rings of DNA similar to plasmas I suppose, but obviously probably more complex and I think with more bases. So yeah, because I've talked about plasmas a bit before being in the bacteria that you guys also use to hijack and makes more of the genes of interest, but I have talked about that previously. Yeah. All right. So is there anything else you sort of want to add about profiling that you find interesting? So I think something really important to keep in mind is that if you have your whole genome sequenced, those STRs are going to be in there. And so by definition, your whole genome sequence includes information that can be used to identify you. It could be used forensically and this could be a really good thing if you are looking for a loved one in a disaster situation or it could be a bit of a tricky thing. And so there's lots of different legal and ethical implications about genomic data. And I guess the encouraging news is that there's lots of different research going into how to make DNA information both useful but also safe and ethical and very, very private and secure. Certainly an interesting talking point and one that I think, you know, the generation that are currently learning about this stuff are going to have to do with as the robots and the computers sort of slowly start to make decisions that are important. It will need a human with those ethical considerations in charge. So I like the fact that you spoke about the regions they choose for profiling are those that are non-informative to the people that are recognizing it because they could lead to, you know, understanding certain things about the person that may make them less likely to be insured in the future or give them an indication of a disease they're likely to get. So yeah, it's all a little bit scary at times, especially for students to hear about this. But as you said, it's important that we have the discussion and come up with the considerations so that we have frameworks in place to ensure everyone is safe and the DNA is mapped in a way that they don't feel intruded upon. So I mean, that really great ethical discussion to have in the classroom could be should the police be able to look at a suspect's DNA to find out things like their height, their eye colour, their skin colour? What about their ethnic heritage? These are things that our DNA can give us some indication of. Is that ethical? If it leads to an increase in crime solved, is it a good thing? If it decreases people's privacy, then is it a bad thing? And who's going to make the decision? I mean, in a country like Australia, are we the lucky ones that have more opinion and more consideration with these factors? And then you have other countries that might have totalitarian regimes that decide that you have to do it and these sort of things. So again, it's going to be a country kind of thing. But I definitely feel blessed to live in a country like Australia when we talk about these things in the future. And we could talk about the more recent one way the government were asking people to sign up to have your healthcare records on file permanently across all health things in Australia. Obviously aiming to improve health, but one of those things that people need to consider or needed to consider when you had the option of whether or not you wanted to sign up. Yeah, absolutely. And you know, that's really just a prelude to these types of discussions happening about genetic information. So to make genetic information truly useful for healthcare, we actually need scientists to have access to really large cohorts. Some people are very happy to donate their DNA to science. Other people are very much not, but they would like to be able to use it for their own healthcare. And so some of the research that our institute is doing is looking at ways where people can have full control over who has access to their DNA. You know, they might say, "Hey, I'd love to contribute to a research program for breast cancer because it's touch my family." But I'm actually not okay with contributing to Alzheimer's research because I'd rather leave that as an unknown. So we're looking at ways where people can actually control exactly who has access to their DNA, which clinicians, which research projects, and which parts of the DNA they're willing to share. Very interesting. I heard something about that recently. It was a decentralized database that was, again, giving patients access to their own DNA and allowing them to say yes or no to certain things as you just represented there. You're talking about whether or not they want it to use in certain research. But the idea is that the data is their own and nobody can access it. You know, they own their own information and only they can give access to another person. And this means that the economic benefits and the health and the safety and all the privacy benefits stay with them as well. So definitely something I heard recently and I thought was super interesting. It sounds like you guys are well in the way to getting that done as well. Just wondering if it's going to be controlled by a central party, a central authority and then is that going to have security risks as well in the future. So lots to talk about there. We better move on before we make this podcast a two hour one. I could talk to you all day about this stuff. But let's get into the last one which is Geno typing one that I hadn't heard of before. Do you want to give us a rundown of geno typing and sort of how it works and what it's used for, please? Yeah, sure. So geno typing sometimes referred to as a SNCC chip because that's a fun little rhyming term. So these are the types of analyses that they will do if you send your DNA off to a company like 23 and me or ancestry.com. And so some people actually think that they're having their whole genome sequenced when they do this. But for about $100, that's just not quite feasible. What a SNCC chip is. It's a little bit similar to DNA profiling in that what they do is they're just looking at certain variants across the genome. Just variants of interest and these are just SNPs. So single nucleotide polymorphisms. So they're common variants and there's lots of variability and we're just looking at one base page. change at a time. The difference here is that, well, the current version of the 23-an-machip looks at about 600,000 SNPs at once. The technology, again, it uses a glass slide. It has pre-printed probes. So the DNA of the individual is extracted and broken up. And then fluorescence is used to tell whether that probe has been bound to with complementary base pair speaking. So from this one glass slide, you can find out about multiple, so, thousands, hundreds of thousands of variants at once. And so these variants are not necessarily ones that are going to cause changes in disease risk or things, but they're used as markers. So, you know, we know about linkage, you know, that different low-sided close together, so different places along the genome that are close together are going to be inherited together, generally speaking. The closer they are, the less likely that they'll get reshuffled during fertilization. So these markers can then be used to give an indication of that person's, I guess, genotype, phenotype relationships. So these SNPs can be used for ancestry because these markers are passed down in the generations, and so depending on which regions your ancestors lived, you'll have different combinations of certain variants. They can be used for associations with complex traits. So for example, things like your risk of heart disease, there's not just one gene of interest here, like there might be for genetic disorders, but instead there's actually, again, hundreds of thousands of places along the genome where small differences are going to each contribute a tiny little bit of difference. And so these sorts of markers can be used to indicate someone's risk. They're certainly used to study genetic risk and look at the different genetic factors that contribute to a lot of diseases. Those sorts of studies are called genome-wide association studies, and they're fascinating, but again, we won't go into those too much. They can also be used to pick out certain variants of interest, and so these genotyping chips can be used to find out if people are genetic carriers of certain genetic disorders, and so that can then help people with family planning. So lots of different uses for these genotyping chips, and I guess they sort of fall in between sequencing and profiling, but they're very common and they're very powerful tool that scientists use. And again, they're probably, at the moment, the ones that are of most relevance. I do hope most of your students will never have the need to make use of DNA profiling in their life, but certainly every time they decide what they're going to buy dad for father's day, I'm sure that one of these companies will pop up and add, and they're going to be making real life genetic decisions that are going to have relevance for their family. There's a whole bunch of episodes of Jerry Springer about people that have found out family secrets by these different companies, so all sorts of interesting things. Yeah, I'm wondering if the kids have seen Jerry Springer if they're not, they can go and look it up for them, and they're all talking about that, but I definitely understand. It was a staple. I think when you're younger for laughing at ridiculous things in America, but let's move on from that and get into some of the talk about other things you're doing at Garvin, and actually before we get into that, one thing I find interesting about SMPs is that how can they give us an indication of a disease if they're in a non-variant part of the genome? Is it just coming down to statistics and data? It just seems that more people with this SMP seem to have this disease, or is there some sort of, is it having some sort of effect on the expression of that genotype? Is it causing an effect? Okay, so SNPs absolutely can be causative variants. So the variant associated with sickle cell anemia, for example, technically qualifies as a SNP in some populations. It's that common that it's actually a polymorphism. Obviously, the disorder itself is not that common, but whether you're a carrier, because the SNP only has to occur in that heterozygous form to be counted. So single nucleotide variants absolutely can cause disorders, even if they are outside of coding regions. This is because they're both known and unknown regulatory features of non-coding DNA. So some that are a better understood are things like promoter regions. So these are areas of the genome just before genes that help to control whether a gene is switched on or switched off. But there are other ones that were only just beginning to understand things like regulatory RNA, where a piece of DNA will be transcribed, but never translated. And it's actually the RNA itself that has function. It might go along and interfere with the expression of another gene, for example. And there's also so many fascinating things that they're discovering about non-coding DNA, like how important it is for the genome to be organized within the nucleus. And how non-coding DNA actually helps to get the coding parts in the right spot, to get little islands of function within the nucleus during the interface, absolutely fascinating stuff. So yes, SNPs in non-coding regions could be functional. They could actually make some small contribution to a phenotype. They could also, so these genome-wide association studies, they use SNPs more as markers. So there might be a causative variant, and it might be what we call an endel, a small insertion or a deletion that changes the way a gene functions. And our SNP chip might not pick up on that exact endel, but a couple of hundred base pairs away. There's a SNP that we look at often. And so those two things are inherited together so often that it will give us a good idea of the genetic contribution to that disease. Incredible. Incredible that we have this precision to work on a molecule. We still can't really even see. And yeah, now we're getting to the point where it's becoming cheaper and effective. And eventually in the future, the 23 and the 30-year-old species of gene, they're going to be able to get a lot of things out of the gene. So we're going to have to be able to get a lot of things out of the gene, and we're going to have to be able to get a lot of things out of the gene. So we're going to have to get a lot of things out of the gene. So we're going to have to be able to get a lot of things out of the gene. So we're going to have to be able to get a lot of things out of the gene and we're going to have to be able to get a lot of things out of the gene. So we're going to be able to get a lot of things out of the gene. So we're going to have to be able to get a lot of things out of the that way. to do this safely securely and sort of doing up a lot of modeling and stuff to suggest you know what might be the economic benefits, what are some of the ethical and access requirements involved. And so that's really, really interesting and moving forward like you said, you know, won't be long before everyone has their genome sequenced and we need to be able to do that well. Some other really exciting things happening at the Garvin, we've got the Australian Parkinson's mission. So like we said, there's things like these genome-wide association studies, but also looking at the contribution of rare variants to a disease that's really not very well understood, but incredibly debilitating like Parkinson's. So there's lots of different layers to that research project and a lot of experts coming together and using lots of different tools to try and unravel this problem of Parkinson's disease. Amazing. Yeah, no, my auntie is affected by Parkinson's, unfortunately, and she was lucky enough to have brain surgery that actually fixed a considerable portion of her shaking and things like that. I don't know exactly how what they did, but again, it's all leading back to those genetics and the predispositions and things like that. So super important research and one that I'm sure will affect many people in the future for the positive. So really powerful stuff there. And other one I was thinking, when you're talking about lifelong sequencing, the using DNA for the life, I know in 2018 that HSC was talking about Alzheimer's, and I thought as soon as you started talking about that, I thought that would be one of the things that that would be useful for in the future, looking at the genetics around Alzheimer's and the development because it is again so debilitating and so common in the population as people get older. So there are some variants that are known to influence the risk of Alzheimer's. There's a gene called, well, it's abbreviated to apotee. And so when people participate in some genomic research, they get offered the question, would you like to know about Alzheimer's risk? And because it's a disorder that we can't fully prevent yet, people's opinions on this are really mixed. That really comes down to the individuals' values and their tendency towards anxiety, rather, you know, over planning or, you know, it's a really interesting question if you did have an increased risk of Alzheimer's. It wouldn't be a definite, we couldn't tell you if you were definitely going to get it or which age you were definitely going to come down with it. Would you want to know, would that help you with your planning for your life or would it just make you feel worried the whole time? So these are questions that are here and now and really interesting. Powerful questions too, because once you've heard the answer, you can't sort of unhear it and as relevant as families as well. That's very true. You know, there's a few different scenarios floating around with the gene for hunting tins. Sorry, I should say the gene. So you never say the gene for because the gene is the good thing that we want to have functioning and it's actually the variant that's the problem. So the harmful variant that causes hunting tins disease, you know, there are grandchildren who want to find out if they're going to, if they've inherited their grandparents disorder, but they actually can't find that out without revealing the genotype of the parent. And so there've been lawsuits around whether the child has the right to know if that's going to then inform the parent as well. Incredible. Yeah, it's again coming back to that. Would you want to know and then what implications would that have on hunting insurance and things as well? So lots to discuss there, but the overall aim of science in these processes is to improve the quality of life for those who receive it in the future. And I think that's something that I always try and reiterate with my students like this progress is going to happen and it's our job and your job, I say to them it's your job to make sure that it's done right because just like a computer started off in a room, it's now in your hand. And so will the power of DNA sequencing and the power of manipulating genomes in the future. So look, this has been a really wonderful chat and I really do want to have you back on again. So I'm hoping you say yes to that at some point when I ask you. But before we go, is there sort of anything else you want to add and working people, working people find you? How do they sort of, how do they get in touch with you? Yeah. So, um, teachers, I would encourage you to go and check out some of the race sources that we have put together. So if you go to galvan.org.au/kccg-teaches, all that stuff is there. Students probably won't find it quite as interesting, although there is a page with some stimulus activities and so some of the videos and things that we've put together that we might share on on Looks page. But yeah, I am always happy to get questions from teachers and students alike. Putting that out there in public might get me overwhelmed. So I apologise if I'm delayed, but answering questions and getting those aha moments is, you know, it's gold to me. So please do feel free to get in touch and I'll have Luke share my email address as well. Wonderful. Look, thank you so much again for joining me today. I really think that, you know, if anyone listened to the very end, you're going to get a lot out of out of the podcast and a lot out of the information that Lauren had. So yeah, thanks again and hopefully we get to chat again soon about all things immunology and maybe some ethics again. All right, I'll see you later, everybody.