REFERENCE

And so this is the Clone Wars, ladies and gentlemen, we are experiencing technical difficulties as we maneuver through this space. And it is with great trepidation that we move forward. We are starting to ask questions about people that we've been close to and starting to ask questions about people that we've worked with in the past. And it is because of incongruencies in behavior over time, things that I've observed and experienced over time, which, when accurately summarized, can only leave you with one conclusion and that is that some people are just not behaving like they are patriotic Americans who would like to save our sovereign nation. And I'm one of the Americans on the internet who's shaken a can on the internet, so to speak. And I'm a patriotic American, even though I'm dressed in a completely pro-Dutch outfit tonight. But that's just tough because my wife is Dutch and my Dutch grandma, my Dutch mother and law is in town. And so that's what you get.

Sorry, I'm starting so late. It is becoming exceedingly difficult to extract myself from the research and from the back channel communications that are currently going on. Thank you all for joining me very much. Thank you very much. Sorry, I'm so late.

I got to check this Huff post, I didn't see the Huff. I didn't see the Huff. So the idea is that we figured it out. It's Barrick who did it. It's his technology. It's his laboratory. It was his idea to use coronaviruses. And so it must have been him. With the goal of developing novel technologies, some of which are already licensed here at UND. See, they already licensed it all. Specifically focused on using those technologies. Oh man, and then they were going to use those technologies. Holy man. So you invented the phantom virus, hoping it would scare Eric away. That's right. And it worked. Till you guys showed up. You were afraid that we would find out who created the virus. So you beamed us into cyberspace. The prize would have been all mine, if it wasn't for us meddling kids!

…a faction of the US government and intelligence agencies that's still around in some form or another fighting with other factions of the US government and intelligence agencies for ultimate control and power over one of these many democratic systems that have been hijacked a long time ago by fiat currencies and central banks and it's time for all of us to come together under this banner of understanding that we've been lied to for quite some time that we're in a dark age that has been induced is it induced dark age.

So we're gonna learn some biology today I'm excited to watch a lecture with you and try to piece together how it is that they do this sequencing stuff because a lot of this narrative is based on sequences these doctors are dancing about the variance and that variance story is part of this mythology and dispelling this mythology is the central aspect of this battle versus good versus evil it's our kids who are gonna dissolve this man this mythology and either accept it as truth or see it as what it is they're either gonna accept IDs that we put under their skin or we're gonna stop it it's one or the other we need to be more people learning biology and we need to be usurping the people who should have known better no I didn't make it that was still a really bad transition I want to learn the history just like you it's a recent history though ladies and gentlemen there is three years of recent history that has the potential to be erased and we already are seeing it happen it's already being scrubbed so you got to wake up that the more deep history you go the more likely it's already scrubbed and so it's the relationships between people it is the contracts it's the companies it's the the political intertwining of these people that needs to be seen very soon we're not gonna be free to talk about this stuff so we've got to do it now we're gonna need to meet every day or every day that we can to learn the immunology to crack the window of the the very poor models that they are giving us on television and in social media we need to keep swinging because this dragon is far from dead you've been with me for a while you're at the top of the wave and if not you might be a skilled TV watcher that we can save by helping you stay focused on the biology not taking the bait on television and loving your neighbor we are in the midst of a shift in the way that we do things around here I'm not plugging it so much but we are still completely viewer supported completely viewer funded we are also helping Robert F Kennedy jr. and the Children's Health Defense behind the scenes with scientific advisement so there is some funding coming from there as well which makes us more stable takes away a little time but it makes us a lot more financially stable I'm the chief biologist of gig on biological and this is an independent biological news source puts you put together by one biologist it's a one-man band and so it is not a perfect production it is not a perfect set of content but it is an ever evolving understanding of what has happened over the last three years and that ever evolving understanding is starting to take a very big pivot and that's what we are starting to to talk about here on gig on old biological it is a an illusion it is only sustained through your active participation thank you very much to my best friend and wife for supporting this screen with all of her hard work and trust as well this is gig on biological high resistance low noise information we brought to you by biologist United Non-Compliance is the only way out.

I used to be a neurobiologist you can find me on the Pub meds and all that kind of stuff. I worked at the Freie Universität in Amsterdam the EPFL in Lausanne, the NTNU in Trondheim and the Erasmus Medical Center in Rotterdam before finally finding myself at the Department of Neurobiology and School Medicine of the University of Pittsburgh at the start of the pandemic, where I was trying to build a rig to do multiple whole cell patch clamp recordings to extend this meager set of publications and at the same time to scratch and itch about, about, about teaching I started a YouTube channel where I was doing neurobiology on my bike JC Journal Club on a bike and it, it it did okay. I got to around Journal Club number 22 this is actually yeah 22 bike rides about neuroscience and then I did coronavirus.

I did a coronavirus one here also and so you can see that I slowly kind of dived in and then eventually it completely took over my YouTube channel and my life and it's kind of been that way ever since letter to censor anyone on social media who may got covered ability of a lab in it can't be true the Lancet says it's not true 27 eminent scientists we got covered in in Vanity Fair and Newsweek and it was it was a quite an interesting time because some of the people in drastic were very happy to get covered but other people were noticeably upset that they completely accredited the whole organization to some people completely different including one guy with everything but CIA cufflinks and essentially stealing the story from us and yet fueling this Scooby-Doo episode of us being figuring out that oh it's drastic and drastic figured out that it was a lab leak thank goodness for these guys and so that narrative pretty much stuck with us from the middle of 2020 all the way through until four weeks ago I don't think that that's a very silly way of bringing it up I think I think actually that's a pretty good way of putting things up you know I made this as one of the first giga ohm hats already a few years ago you can see that it's like this different it's too too small for my head with it's like got no form but it's an orange one because I had always intended to wear and if you know something like today happened something like today happened so I have my orange giga ohm hat on at least I found it it took me a while to find it actually so here we are several years later you know we are all humans together on team human we are all still trapped in this matrix and this matrix is not to be it's not to be scoffed at this matrix is real I don't know if I can turn the screen on down here yet the matrix is real it is a it is a set of stories that has actually trapped both sides the sides that love the vaccines and believe the PBS news hour and and and think that either Fox News or you know MSNBC is more right than the other one all those people they all agree everybody in America and around the world agrees is probably a lab leak and so we should probably do something about those lab leaks shouldn't we it's all gone exactly as planned and I was part of creating that narrative I was part of telling that story of pushing that idea wow we figured it out guys let's high-five it's over we're gonna get them we're gonna take them into a lawsuit these guys are all going to jail because they made a virus that went all around the world it's a very incredible play that they have done on us and I believe the play is still being executed on all sides that we can see and that's why I think it's imperative that we stick together rather than try to claim that one of us knows and the other one doesn't which is what a lot of people are doing right now. I want you to know that I was also somebody who was into the spike protein I was also somebody who was into the into the idea that the spike protein was a toxin and so we had to be very careful of it and I have still not ruled that out but if there is spike protein it is produced when the RNA is in high purity and in in high fidelity and I even question whether or not they can do that you know what I found out a couple days ago that cure vac a company that prints RNA is Elon Musk is Tesla so I mean who is really Elon Musk buying Twitter and feigning to let everybody who's good back on it who hasn't let me back on and a bunch of other people back on who is Elon Musk who claims to be able to implant things in people's heads it's just such chicanery it's so it's such charlatanism, it's terrible.

And between now and Christmas we're gonna cover all of that stuff it's just gonna be a daily onslaught because we need to push this ball forward and there aren't very many kicking the ball in the direction it needs to go anymore so we're gonna need to fight against this. I'm trying to argue the PBS NewsHour and CNN and all these people are coordinated for a reason and it has nothing to do with a pandemic virus because most of them don't care aren't smart enough to know and the ones that are smart enough to know know that there is no pandemic coronavirus for the last three years, there might have been one for the first six months.

But these people are all charlatans, but they're all doing it for a reason and I'm making the argument very strongly that there is reason to believe that that reason is that as the natural forces of the of ecology and population biology and the carrying capacity of the earth start to level out the population of the human race, and then the peak population is reached and then it reached goes down to some other equilibrium. There is, there are few simulations which project far into the future and suggest that this will go on forever most simulations suggest that if there was no major measures taken we would still peak in between 9 and 10 billion, and then decrease rapidly because of the nature of the population pyramid that currently exists we aren't having enough kids to replace ourselves.

There's gonna be a decrease even if it comes back up in 500 years in the future and so any reasonable prediction that looks at any reasonable length of time says that we're about to peak we may already have peaked and then we're gonna level off and decrease and it might be a very precipitous decrease depending on how this all happens and Elon Musk has said as much right he's said as much and people have said oh wow he's weird or oh wow he knows or but the consequences of that are never actually spoken out loud and the consequences are very simple and that's why I use the go-game analogy every unique human genome and the phenotype medical data associated with that genome is an instance that the AI that the trans humanist people believe will eventually solve the problem of the human genome. that problem will need many instances of its existence expression blossoming and decay.

If an AI is ever gonna understand how the human genome generates a kid through development and adult and decay, then the only way an AI is ever gonna figure that out is regular genetic sampling over time from as many humans as possible, and since that's nowhere near in the realm of possibility right now, these people who can see ten to twenty years in the future can see that they have this opportunity to collect this data, they have already thought about what data it is that they really want, and it's not data from 60 year olds, it's data from young adults right now that if they change their mind right now, if they can get them on a system right now where you're gonna test all the time, you're gonna take shots regularly, you're gonna conform to our public health standards, then all the college-age children right now, all the college-age young adults are being brainwashed. That's why they're the only ones on the street you see wearing masks still, because their brainwashing has been extreme. They gave up this in order to go back to school, they wore one of these to go back to school, and they tested regularly to go back to school.

They took the positive test very seriously and isolated in order to stay in school. All of these things create a a psyche that is essentially different from those of us who never fell asleep or never got snapped into it, but once that grid is fit on your thinking, it's very hard to break out, it's an uncomfortable release. And we have only about six months or a year in order to adequately explain to these young people what has been done to them, very likely what is being done to them, and is planned to be done with them going forward. It's the last time American universities are gonna be this full, in five years or ten years there aren't gonna be this many university students anymore, there just aren't. People aren't having children, you can see it when you go around yeah there are kids, yeah there are kids all the schools are full sure they are but it's gonna be a precipitous drop, and when it does the whole economy is gonna change, the whole you know who do we need and where do we need them is gonna change, and they see all this coming they know about all this stuff this is not this is this is decades in the making and decades in the in the approaching. I'm losing my train of thought here and I'm sorry for that, but I really feel like this message is extremely important right now because it is a, it is a do not think about all cause mortality do not talk anything except for a novel coronavirus and antibodies and seroprevalences and vaccines, they work. This is what people are saying right now that are getting boosted. People that are getting promoted and not getting censored are saying these things. Don't think about all cause mortality. It's a novel virus likely made in a laboratory and leaked from Wuhan. And antibodies and seroprevalence do matter. One of the things I need to point out to you is that these guys that I was talking about yesterday, who purport to be experts in this stuff, never mentioned natural immunity never mentioned T cells versus B cells, never mentioned vitamin D, never mentioned obesity and chronic disease, never mentioned any of the vulnerabilities that contributed to the enormous increase in deaths classified as pneumonia. Unprecedented number of people died of what used to be a pretty curable situation for anybody of reasonable health, and none of this calculus is done by these people. Now suddenly everybody is just talking it is a it is a lab leak virus, definitively gain of function needs to be monitored, and controlled, and money needs to be spent in that direction in this direction because this is real. And I'm suggesting to you that although there are people

And I'm suggesting to you that although there are people who are saying that there is no virus, I think those people are just off, and I think that there is a possibility of uniting the no virus people with with people like CHD, without CHD having to say that there's no virus definitively, but rather to attack the the very specific points that it hasn't been isolated, the sequencing is bogus and boohooie, and the idea that a single virus is going around the world like this, and asymptomatically circulating with this much fidelity needs to be cross-referenced with what we knew before the pandemic, and be explained in a different way. And I think part of this is going to be understanding what sequencing is, so the just remember what the common narrative is: We've already tested it on millions of people, gain of function is real anybody in with eBay and a garage can do it we can vaccinate against a respiratory coronavirus antibodies our immunity and transfection is a state-of-the-art new methodology and here's what Tony Fauci said about three days ago as his last okay good afternoon everybody today I'm said about three days ago as his last…

Today I’m joined by White House COVID coordinator Dr. Ashish Jha and Chief Medical Advisor to the President and NIAID Dr. Director Dr. Fauci as you all know Dr. Fauci is retiring next month and I'm honored so honored to have him join me today one last time one more time at the podium and he's going to discuss the importance of getting your updated COVID vaccine shot ahead of the holidays for so many Americans throughout our fight with COVID Dr. Fauci has been a source of information and facts but Dr. Fauci's leadership and legacy stretch far beyond the past couple of years as you all have known him it it actually goes back even further as I was just stating whether it be HIV AIDS Ebola or COVID 19 for close to four decades and got malaria and Republican and Democratic presidents Dr. Fauci and our country is stronger and healthier because of his leadership. Today has returned again to tell you a bit more about the science related to this updated COVID shots that you've heard us all talk about and an important clear message you've heard from him before which is please get vaccinated, get your vaccine. So without further ado it is my pleasure to introduce and welcome Dr. Fauci for his last…

What is that is that? A limp limp hammer fist with this thumb up on top a limp… I don't know what that is.

…really very very kind introduction it's really a great pleasure to be back here with you again albeit I believe for the last time but as Kareem said I'm gonna spend the next just couple of minutes talking to you about the importance of getting an updated booster vaccine as we enter into the holiday season in the colder weeks and months of the late fall and early winter so let's just talk very briefly about what we know about the vaccines because we want to make our decisions really based on facts evidence and data are the vaccines safe that keeps coming up the answer is now overwhelmingly it should be off the table there have been 13 billion doses of a COVID-19 vaccine that have been distributed worldwide hundreds of millions in the United States and there's robust safety monitoring systems that are in place and clearly an extensive body of information clearly indicates that they're safe next are they effective I and I believe you are all aware of this if you look at the striking data overwhelmingly show the effectiveness of vaccines particularly in preventing severe illness and deaths and recent data that has come out…

Did you guys see the the recent piece in New England Journal of Medicine? I see you guys in the chat talking there hold on one second I think Eddie sent it to me and now I'm not gonna be able to find it I'm just gonna look it up on the internet but Eddie sent it to me a couple days ago it is a paper in New England Journal of Mail it's Fauci and it is never over and ain't over till it's over check this out look look look it's fantastic it's a perspective paper in New England Journal of Medicine and it's Anthony Fauci “It ain't over till it's over, but it's never over reemerging and he reemerging emerging and reemerging infectious disease” that's who we're talking about here just so you're aware I am seeing you in the chat it's pretty funny.

…cate that if in fact you were vaccinated and boosted compared to an unvaccinated person there's a 14 times lower risk of dying in the most recent BA four or five era compared to unvaccinated of testing positive compared to the unvaccinated individuals but then there's some issues that we have to deal with that are sometimes confusing to the American public if that's such great data why do you tell us that we should be for our own safety and that of our families in the community get a booster shot there are two issues that in some respects are unprecedented when it comes to infectious diseases and that is as good as the vaccine is and as good as post infection protection is the immunity and protection wanes over time let me put it into some perspective for you if you get vaccinated with measles or infected with measles the duration of protection is measured at a minimum in decades and likely for a lifetime that's just happens to unfortunately not be the case when you're dealing with coronavirus and particularly SARS-CoV-2 so you need to update the protection that we know is good protection next we have the complicating issue that we can't do anything about is that you have the emergence every several months now historically of variance remember Delta Omicron BA 45 BQ 1.1…

The “remember” remark there is actually quite extraordinary. It is and a request to recall something that you were told earlier, remember the evidence we gave you last year about these variants. You have to understand something that that that the quasi species swarm means that many of these variants are just a a hyperforced synthetic estimate of a sequence taken from a collection of fragments and one of the arguments made by dr. Bailey this guy from New Zealand along with his wife also Bailey I don't know I don't know what her name is anymore they one of their main arguments behind suggesting that these viruses are not what they say they are that there's no virus is that the sequencing is is bogus that what they do in the sequencing is is a forced in silico approximation of a sequence that is composed mostly of fragments that get assembled in silico, and that's not wrong, it's one of the things that I'm objecting to it's one of the things that I say and have said has rang a bell as to this being wrong the the NextStrain data, the Jitsi data is not possible. The map of point in Wuhan all around the world it's not how an RNA virus works, the fidelity isn't there to infect millions and millions of people mostly asymptomatic according to their to their narrative and then still extremely high fidelity copies that you can trace linearly around the world it's just absurd, and it rests on a gigantic presumption that there was nothing related to this virus before the pandemic, that there was nothing there before the pandemic that could explain why these tests are positive, why the majority of them are positive, why the majority of the people that are getting sick are not dying, that aren't even showing symptoms it is not because this virus is so weird and unprecedented and remarkable, it's because we're doing some things wrong, and we're representing them as though they are biologically sound, or ignoring the fact that we know they are not. This is what's happening here, and this insistence that no mistakes were made, that no incongruencies are there, that the assignment of PCR positivity to all deaths, and the charting them up as they are in the CDC numbers right now is no problem for the narrative that he's trying to sell you, which is absurd it's absolutely absurd. And yet here we are three years in, with the same guy at the head of NIAID for the last 40 years sustaining this immunology about B cells and antibodies and vaccines, B cells antibodies and vaccines, that's this guy's sales pitch for 40 years. He has been the red thread that has sustained this but, does that mean it's all him? Of course it doesn't. He's not able to do any of this work. He's the the pencil pushing yes man that allowed all of these black organizations in the background, DARPA, DTRA who's ever fighting for the money that he distributes, he's there they're they're lackey and as long as they like him he gets to keep his job as long as the pharmaceutical companies like him he gets to keep his job, he's like a diplomat more than anything else he's not a good doctor, he's not a super creative guy like he's the head of some Max Planck Institute or something, like that this is not somebody that we need to think of as a mastermind he is a he's a stooge. And his ideas about how things work with regard to to grants and things like that are not related to biology, they're related to how can I make what I know about science work with what I know about business, with what I've learned about how these this infighting between different intelligence agencies and different funding agencies works, how can I make that work to my career advantage for 40 years. That's who he is and so he's telling you some mythology about biology that he doesn't even really care about, that he's been lying about for 30-plus years, making excuses for. Well, this is how we did it, this is how we tried it we tested it and then after we tested it we did what the test told us to do, it was just following the science.

To blame him would be like blaming Trump. …that we're all hearing about and reading about and seeing you don't have that with (there are many smarter people behind him) you all familiar with there are no variants of measles. I was got infected with measles when I was a youngster because I'm old enough to not be getting the vaccine and then measles is the same measles that's circulating now in the developing world it doesn't change and that's the two major reasons for getting a booster the boosters bi-valent people get confused with that word what is bi-valent means there's two components one is the ancestral original vaccine that we all got and the other is the updated BA four or five. So then people ask appropriate questions do they really work what are the parameters to see if they work there are two parameters one is what the virus what the vaccine does in boosting an immune response we refer to that in the medical circles as immune correlates and then there's the real-world vaccine efficacy. If you look at the recent data that has now been coming out from the company…

So what I'm trying to suggest to you is is that Tony Fauci has been running the NIAID based on false immunological pretense that zero prevalence to an antigen is a correlate of immunity and. He is maybe even the author of this ridiculous idea, this concept of of having a proxy and then not really requiring that proxy to actually be connected to what you purported to be connected to, i.e. the immunity to a pathogen, the immune response to a pathogen. We are looking at a very severe situation where a whole generations tax dollars have been spent incorrectly given away incorrectly pursuing ideas that were probably known to be incorrect and emphasizing medical technologies they were probably known not to be advantageous to the kids they were given to. Maybe the vast majority of them didn't get hurt but we're in big trouble ladies and gentlemen, because we need people to come to our side quite quickly and it's a long ways away if they believe that this guy is worthy of a candle, if they believe that this guy is worthy of a bobblehead, I'm not gonna go get it but there's a bobblehead behind me right up there is a bobblehead of Tony.

…as well as academic investigators it is clear now despite initial bit of confusion that the BA four five bivalent booster what we refer to as the updated vaccine clearly induces a better response against BA four five and the sub lineages of BA four five then does the ancestral strain, so from a pure immunological standpoint it looks quite good clinical…

From a pure NIAID version of immunology and in vitro assays the antibodies that are produced after the bivalent infection are able to pull down the proteins the spike proteins of previous variants that were reported in their database better than the older variants, and so the in vitro data generated by this transfection can be applied to humans out on the street, because this is a correlate of immunity and that's how he used viral load measurements in AIDS patients to justify whether or not some random therapeutic was working, that some pharmaceutical company had on the shelf for a while as an anti-cancer drug and just decided you know we could give it to a lot more people if it worked for AIDS, well give it to us we'll test it, we'll test it we'll test poisons on people with a vax… with a virus that we say they have, and that we say that we can measure, who's we? Well me and Gallo and some other really nice people very trustworthy, we say that we can measure the virus and we'll put them on these poisonous drugs and see if the viral load goes down. If it does we'll blame the drugs, if it doesn't well you know it's a it's just it's a trial. Ladies and gentlemen we need to wake up. …efficacy data from the CDC (Christmas time is here) released in fact it already has been released it was supposed to be released at 11 years which is clinical efficacy data looking at real-world data of hundreds of thousands of people looking at the capability of the virus to protect against the real-world BA 45

See? He can't even speak anymore, it's looking at the virus for protecting? Obviously that's a little slip up a little flub up he meant the vaccine but it's a spell being cast it has been cast on our family and friends for three years these are not vaccines they're transfections even if this was a novel clone released in Wuhan you have previous immunity especially in the form of T cells which are focused on non-structural proteins, and those T cells likely have overlapping immune epitopes to conserved regions on these proteins that will help you fight off any infectious clone that might have been released in Wuhan, and certainly will help you fight it off better than seroprevalence in the blood when you really need seroprevalence in your mucus. Again a dr. Cole only claim for a very long time in 2020. Dr. Cole being one of the only immunologists to regularly teach T cell immunity and regularly teach that there are epitopes that are conserved across coronaviruses that make spike protein variants irrelevant.

Andrew Huff and Charles Rixie and Kevin McCairn have never talked about that ever despite the fact that I've made it a central part of my stream every night for three years. T cell immunity previous immunity to previous coronaviruses aimed at the RNA dependent RNA polymerase and the end protein in the HeLa cases that are highly conserved proteins and only expressed in infected cells not present in virions, that's where your T cell immunity is aimed, that's where your T cell memory comes from, that's what you're sick for your whole childhood for, to build those T cells and if the spike protein injections do anything like express a nonspecific T cell epitope like like Geert Vanden Bossche says, then we have a problem because our T cell memory cells might be be at being activated in an unnatural way that will not allow them to transition back to memory but will instead just set them into apoptosis. If that's the case that could easily explain why a story about a gain-of-function virus being stable and going around the world is necessary. Because then these transfections can be expected to be effective in the imagination of the listener, these transfections could be expected to be effective in the imagination of the people, in the secret meetings in 2020, that's what we're dealing with here ladies and gentlemen, and anybody that's insisting that that narrative still holds now after three years, still holds now in spite of the fact of all we know about the numbers and how they were juiced and miscounted, how the tests were used and miscounted, how the people were mistreated for two and a half years, how Remdesivir simply doesn't work but still being applied in America, ladies and gentlemen we can't talk about gain-of-function virus then, we have to start at first principles and that is the response.

There's not a bumbled response on the part of Donald Trump. Donald Trump's not a virologist. This is a purposefully bundled… bungled response somehow or another and a gross over execution of a worst-case scenario plan with a level of vigor and commitment that was totally not justified. If people were really falling down in the streets and we couldn't clean up the bodies fast enough yeah, there's a whole different story. That's the story they told us on TV, that never happened. People like Richard Ebright that told us that if we go to put those college kids back to school in the fall of 2020 you'll be responsible for the murder of half a million adults. Guess what? That didn't happen and is anybody calling him to account? No he's still, he's still testifying in front of Congress because it's the narrative they want you to accept, that's why the Democrats the Republicans, the middle the left the right the anti-vaxxers the pro-vaxxers, they all think it's a lab leak and it's gain-of-function viruses, and that biology is real mission accomplished, and I'm telling you ladies and gentlemen, we see it now, those are the bricks and if we can teach our family and friends one by one that those are the bricks, that Mother Nature's coronaviruses can't do this and man-made coronaviruses can only do this if you make truckloads of them and carefully distribute them and orchestrate this on a worldwide level, if so or their tests just aren't very specific and applied on the background of previous SARS viruses and overlapping amplicons of other endemic coronaviruses, some of which aren't even charted because they're so rare, lead to us thinking that we go from 200 different causes of respiratory disease to one, again mission accomplished, simple story simple solution, simple simple immunology simple solution, simple cause simple solution, but none of these are, none of them are supported by what actually happened.

…that has been circulating. And we know that that is really quite good so we have immunological data and you have now clinical efficacy data everybody was asking the question where's the clinical efficacy data now it has come out with the CDC MMWR this morning so we know it's safe we know that it is effective so my message and my final message may be the final message I give you from this podium is that please for your own safety for that of your family get your updated COVID-19 shot as soon as you're eligible to protect yourself your family and your community I urge you to visit vaccines.gov to find the location where you can easily get an updated vaccine and please do it as soon as possible thank you. So I mean really what what can you say about that

So I mean really what what can you say about that. What can you say about that, if after three years this guy is still saying the same thing, safe and effective, we have the best vaccines. What can you even say about that? What does this mean? Where this brittle narrative has just bent only a little bit but still just it's the exact same story, with the exception of the fact that that guy's pretending that this isn't a lab leak, but he knows it's a lab leak, it's not a… it's not from a market and he knows that. And so we've got him figured out. No you don't. No you don't because the lab leak virus means that you think that there's a danger that isn't there, that means in five years from now your kids might think that there's a danger that's never there. Five years and now the kids of the college kids are gonna think that there's a dangerous virus out there. Do you see where we are? We need to wake up.

These college kids that were brainwashed by actually going back to school and we allowed them to go back to school to be coerced into testing, and to be coerced into taking the shot, we adults allowed that to happen the ones of us who didn't say just take your year off, do not go back to school. It's that simple. Why didn't we say that? That should have been a cardinal American value expressed in real time. Don't go back to those stupid universities, we already see it's November 2020, we've had six months, it doesn't really affect you healthy young adults. You can't require them to take a transfection before they go back, you can't require them to test. They didn't care, they didn't care, they did it anyway, we let them do it and they're still doing it now, testing vaccination proof of vaccination, masks. Come on guys, this is serious now.

And it's serious now because they're there after something much more. They're after your sovereignty. They want you to completely give up, they want you to complete… your kids to give up, they want your kids to say oh mom come on, you're just being silly, you're just listening to Donald Trump. Come on mom don't be so daft, that's what they want and in a lot of households they've already got it. It all started with this talk where I didn't even get to say everything I wanted to say.

…why don't we blow the system up? (you just can't forget) Just turn off the spigot on the system we have and then say hey everyone in the world should get this new vaccine we've been given to anyone yet, but there must be we grow vaccines mostly in eggs the way we did in 1947. In order to make the transition from getting out of the tried-and-true egg growing which we know gives us results that can be, you know, beneficial I mean we've done well with that, to something that has to be much better, you have to prove that this works and then you've got to go through all of the clinical trials phase ones phase twos phase three, and then show that this particular product is going to be good over a period of years. That alone, if it works perfectly is going to take a decade. There might be a need or even an urgent call for an entity of excitement out there that's completely disruptive, that's not beholden to bureaucratic strings and processes. So we really do have a problem of how the world perceives influenza, and it's going to be very difficult to change that unless you do it from within and say, I don't care what your perception is we're going to address the problem in a disruptive way, and in an iterative way, because you do need both. But it is not too crazy to think that an outbreak of a novel avian virus could occur in China somewhere, we could get the RNA sequence from that, beam it to a number of regional centers, if not local, if not even in your home at some point, and print those vaccines on their patch and self-administer.

Now the thing to understand about this is that lots of people including Elon Musk have been convinced that RNA in its final evolution as a technology will cure everything will be the tool and Mark Housatonic, Mark Kulacz has suggested that one of the main goals that they have is understanding the ribosome, and over the next couple days I'm going to show you some not-so-crazy evidence which suggests that he might not be so wrong about that. That that's that that's their current or has been one of their current focuses for a very long time behind the scenes, understanding that micro machine or nanomachine and we're gonna look at this shotgun sequencing tonight, and you're gonna see that as they try to shotgun sequence, these pathogens what they actually focus on our ribosomal subunits and ribosomal RNA as a way of figuring out, you know, what pathogen is present by what subunits of RNA are there or not there, and the sequences tend to be very specific and very collated by phylogeny. So this is a great video because it's right before the pandemic it has Rick Bright in it and a few other key people all talking about disrupting the system changing the system from within deliberately disrupting it in a way that can't be stopped by bureaucracy and all this other stuff and that's really what's happened with the shift from mRNA or from, you know, traditional flu vaccines every year, to now what they're doing which on TWIV yesterday includes a mRNA vaccine with all 20 hemagglutinine epitopes on it, all 20 represented in it, which is just an extraordinary it's an extraordinarily arrogant absurd manifestation of this immunomythology. I just can't even get over it. We're gonna probably get to that video at some point tonight too, it's gonna be a long stream so this is the the data that they'd ever show, this is the the the current hypothesis that I have I'm not gonna read it to you now because you can stop it and read it later. If you want to see that this is the Clone Wars and so we're talking about really battling about what clones really mean of clones versus the swarm about RNA and DNA sequencing and the real goals of this, because it can't be public health. We know that my assertion is TV algorithms have tried to convince you that Mother Nature has viruses and Mother Nature's viruses can be passage in cell culture or passage in animals in a laboratory to create a pandemic virus, and even worse people can stitch together different parts of coronavirus to make a novel coronavirus and we figured that out that's what Scooby-Doo is here, we figured it out through careful sleuthing that this virus came from a laboratory and this pandemic was caused by those people, and that's just biologically implausible now in my best estimation. And they are doing this to coerce us of individual sovereignty. It is not to save us from a virus, it is to permanently change our minds about how we think about human rights and the permissions that we have, and I believe that none of this is possible.

And so since I've been putting this stuff out there, there's been quite a few people who have put the full-court press on this idea. One of them who's tripled down on it is peak prosperity's Chris Martinson. We used to be in regular contact on signal as well along with Brett Weinstein. Both of them I don't really have contact with any more, and most of that has to do with the fact that neither of them are very flexible about the idea that there is a virus and that it is a lab leak and that a virus and RNA coronavirus is capable of infecting tens of millions if not billions of people while maintaining the genetic fidelity that is described on NextStrain and Genset. And I don't believe that that is true. I was a part of DRASTIC. I was part of this group of people that thought we had figured out that Fauci's emails revealed what was really happening here that they were covering up the fact that they had previous technologies that they knew that some of these these early treatments would work and that they knew that these inhibitors would not they knew that these particular sequences were present the GP 120 the DC-SIGN one of the questions that I have is that Gallagher's and and Garry's fusion inhibitory antibodies and in fusion inhibition proteins that that that Charles tells us about are aimed at a part of the peptide… the spike protein of HIV which is homologous in coronaviruses and one of the interesting things is that the binding mechanism of coronavirus was discovered by Gallagher and Garry which was identified by Charles rightly so and they made this fusion inhibition small proteins and patented it and then later they actually found out that that same inhibitory protein idea would work on HIV viruses as well and it outlined a common mechanism of fusion protein working now the interesting thing that crosses my mind now is that could it be that GP 120 that sequence although it is in HIV and DC sign maybe even they could be highly conserved parts of a viral glycoprotein especially GP 120 if it has anything to do with the fusion mechanism that could actually be a quite conserved sequence that when looked at across coronaviruses might not be as rare as we say it is and I'm very curious as to what extent people have looked for it because they have looked for the fear and cleavage site they've looked for these things and said it's not there but there's an odd focus on the fear and cleavage site really focused on that fear and cleavage site and just casually mentioning the GP 120 which is a much longer sequence and if that sequence is truly not in any other coronaviruses that would be a much bigger find then but I don't hear anybody saying that that GP 120 sequences is not in other coronaviruses so I'm curious as to whether that's just something that somebody's not said yet or if it's actually something that we're kind of just ignoring that it is in some other coronaviruses unlike the the fear and cleavage site which apparently is not unlike the overlap with msh3 which is definitely new and in a patent and unlike the overlap with this sodium channel which is also you know interesting again I'm not ruling out the possibility that this is a synthetic clone I'm not ruling out the possibility that a clone leaked what I'm ruling out is the possibility that regardless of how a clone was leaked or released it could not produce a three-year long pandemic in the way that they have characterized it no way shape or how could that happen and I believe that we have been fooled into believing that's possible so that the potential for a pandemic is permanently codified in our history and permanently codified in our medical biology permanently codified in our system as a permanent possible emergency and I really believe that now is our last time to evaluate this and come to a conclusion that will save our grandchildren or in a half generation this will be as a new level of dark age and we have only this next five years or so to wake up our college-age kids only about five more years to wake them up and and then we're really in big trouble thank you so very much I gotta let it stop or it'll spray all over but it's okay it's had to be shaken thank you and so that's where we are the drink I ordered arrived and so yesterday we looked at this Kim comm thing where Andrew Huff and Charles Rixie the USMC WMD expert came on and spoke with this guy if you don't have a media that investigates and asks the right questions if you don't have you know the political independent investigations as well then you don't really get any answers with which means in the background everything just goes forward as usual whereas what we really need is a complete global ban of this type of development correct well I don't agree that entirely so I think it needs to be more limited and focus so live attenuated vaccines which is a type of gain-of-function they work if you don't advance the functionality on the ecological evolutionary timeline so I don't quite get that live attenuated vaccines are gain-of-function research except gain-of-function research is defined specifically by parameter changes that lead to more virulence more deadly more transmissibility if you're making a live attenuated vaccine it's by definition not gain-of-function yet somehow here he's trying to make the argument that playing with viruses should be allowed because playing with viruses is what allows us to make a live attenuated vaccines he's kind of confusing and I think he's confusing because he's pushing immunomythology on the future so if you do keep it within one step or within that you keep the genetics fairly similar to what is circulating naturally that is actually a good thing I've probably had if I look at my vaccination card I think I've had vaccine vaccinations against 38 different diseases and I know because Charles his work he probably has a similar plush or full vaccine vaccination card history or records so these are not all bad start doing this gain-of-function work where you're pushing things you know 10,000 20,000 30,000 years in the future and you're mixing what is he saying we're pushing viral evolution 30,000 years into the future he's also saying that I have like 38 vaccines so he's immune to 38 things that you aren't immune to apparently if you're never taking all your vaccines I guess if you subtract polio measles what is he immune to I mean he's immune to 30 more diseases than me and my kids is that really what he's saying out loud on this podcast and Charles likely is too come on guys you can't take these as serious people something is going wrong here and it's not just a simple they don't get it or they're not sharp something is purposefully being done here because otherwise they would be sort of more less disingenuously covering this animals which would never come into contact with nature because it's just logically implausible that these things would ever actually meet face-to-face like John Stewart said you know they did the pangolin kiss the turtle I mean that kind of thing doesn't happen in real life yeah but you you both agree that the United States because of the gain-of-function research at the Wuhan lab has a responsibility for this covert 19 outbreak correct absolutely both countries have responsibility I think the United States the United States portion of the responsibility is more egregious because everyone has been trained they you know if we're training the Chinese in these things we're obviously we are countries further along in our thought process our logic our technical ability it's really our responsibility to be good stewards of this type of research and then on top of it the just the systematic failures of the risk mitigate bio risk management process I mean it's just atrocious and I observed observed those folks firsthand while I worked in equal alliance and I discussed that in my book there you go he saw it while he was at EcoHealth Alliance it's obviously why he wrote the book he just had to do it all right so I have another question now if I were the leader of North Korea and I could somehow arrange a meeting with you and I could somehow convince you to come and work for me how much money would you need to build a lab where you can recreate what was done in Wuhan where you could create new viruses of the sort how much would this be well it depends how much risk you're willing to accept in your bio bio safety biosecurity protocols and facilities so you can actually do this probably you can start doing it for ten ten to twenty thousand dollars on the really cheap side but that would be with a lot of risk to your personnel of an escape to actually do this properly you'd probably want if I were gonna draft a budget for this probably three to four million dollars in capital cost to get started for something small plus your your operational budget would be another million are you writing in the chat that Kevin McCarran has a new amyloid identification website if that's true can you please put the the link in the chat for me I would really like that so at the cost of a single missile I could fund the creation of a bioweapon that can kill billions of people is that right absolutely the thing that's actually absolutely absolutely you can find the right combination of scientists the right combination of genes you can build that lab for about four million dollars and you can build a bioweapon that can kill billions one of my readers one of my readers pointed out that actually Kim comm doesn't sound like a guy who's hearing this for the first time Kim comm sounds like a guy who might even be reading from a script let me get this straight there was a virus that was combined in Wuhan and they are starting a new arms race and I could kill billions with just a few scientists is that right I'm starting to see right through Kim comm as well I mean keep in mind that this is still on Twitter it's not gone of course that's Elon Musk being all free speechy right free speechy more difficult to obtain is the scientific expertise knowledge and know-how and that's why the Chinese wanted to work with the United States if you can go buy crisper kits on Amazon right now and become a gene jockey in the garage they actually in the United States we've had bioterror domestic bioterror discussions around the biohacker in his garage he makes a mistake now the likelihood that they're gonna be able to perform gain a function work on something to make it so lethal transmissible and sustainable in the environment is pretty low because they don't have an interesting point he makes there what did he list transmissible virulent and sustainable in the environment so he's talking about aspects of what I think Mark could confirm are more likely to be virus like particles made in a laboratory because viruses aren't really actively working against or working to build UV resistance in case you're unaware of some of the implications of the viral swarm idea is that when you get infectious particles in your lungs and the vast majority of particles that they make are not infectious then those little ones that are infectious that small fraction that is infectious their primary job is not to go back out their primary job is to infect local cells that's the whole point of infection is to make lots of viral particles and you're an only way you do that is a local infection so coronaviruses aren't actively trying to evolve ways of becoming cysts and being stored in snow or or depositing in in dust particles and going across the ocean like fungal spores that's not what viruses do their idea when they get the chance is to pass from one cell to the cell right next to it because the vast majority of the particles they make are crap anyway and that's all the best they can hope for because they're small little fat baubles of RNA and the entire mucosal immune system is bent on destroying them the entire the entire complement system is meant to destroy them and this is something that the there are a few places where you can find people talking about this the complement system is thought to be primarily to control viruses and very small pathogens bacterial noise because it deposits on membranes and membranes that don't have the requisite proteins to neutralize the the complement system they're done viruses can't float around inside of your body forever just like floating around because if they bump into the complement system proteins they're over I don't think a lot of people understand that that viruses don't just float around all the time things aren't just allowed to circulate in your blood and so all this discussion about this without talking about the requisite immunology is absurd and they're doing it all the time now they're doing it because they I gotta go back because there's still a five more minutes I wanted to play of that I'm very sorry oh real public debate part is is that it's actually it's hard to gain me the knowledge necessary to to know how to do this but it's actually a lot cheaper than in like with the nuclear weapons there with nuclear weapons there's at least this hurdle of the cost of enriching in your uranium to get to the point where you could do a nuclear weapon was so immense that it created this this sort of bar the very few countries could go beyond but in this case if you have the scientists necessary you can pay scientists off for a lot less than you could because there's no enriching uranium that you have to do the so the input research in universities now wasn't there a story recently about a university have it so again this is a story about it's a story about people being able to do things that are cheaper and easier and and it's all better than nukes it's crazy this story they're telling and it has nothing it acknowledges none of the malfeasance none of the incongruence it's all just virus virus virus virus virus and Kim dot-com is also virus virus virus virus virus he has no other alternatives it's just blowing up in his mind oh my gosh here's comes it's gonna blow up in his mind accomplished that's most lethal version of the covid virus well that was slightly exaggerated it wasn't it was actually less lethal than covered but they're talking about the Boston University thing where they took the Omicron spike and put it on the original Wuhan virus and whoo that's really scary here was a perfect time for Andrew Huff or for Charles Rixie to say hey you know Jay’s got this idea and this is something you should understand that when they compared the original Wuhan virus with the Omicron spike to the Wuhan virus they started with two clones and then they infected those mice with infectious RNA generated from those two clones and so it was pure infectious RNA and so a few more mice died over here than died over there and so then they said that it was more lethal but since you're using infectious clones it doesn't really make much sense when trying to apply it to humans and the coronavirus swarm you see where we're going here why not take this opportunity because they don't want you to know that's that's exactly what's happening here referring to lethality in mice but the real key to that and what's ironic is three days after that there was another paper published by a friend of ours Alex Washburn and some fellow authors and so basically what that showed is that SARS-CoV-2 had the same chimeric construction as that Boston virus or as other viruses that they've been working with making chimeras out so correct me if I'm wrong all they have done is literally take the closest to the original COVID virus and combine it with the basically high retransmissible version that we have today and that combination led to you know 80 percent fatality in the humanized mice that they've tested it on is that right right but it doesn't do the fallacy isn't transfer exactly over but the bottom line is that they they basically combined the bottom line is they used an RNA infectious clone and it killed a 80% of the mice I bet they didn't even compare it to the original one they just did the the the new synthetic clone of Omicron spike plus Wuhan virus backbone it is exactly the kind of thing I've been describing for the last two weeks none of these papers are being done with wild type viruses with wild type swarm characteristics they're all being done by generating a cDNA clone and then producing infectious RNAs that they use to simulate and measure and and find viral infection and cytopathic effects this is really really the heart of their illusion and it has been codified in many papers codified in many grants codified by an insistence that seroprevalence is an appropriate correlate of immunity and all of this is nonsense it's really all nonsense it's all based on absolutely smoke and mirrors that has been sustained spike which is for decades has recently surpassed measles become the most infectious virus in history of humanity the most infectious virus in the history of humanity where does he come off saying this what is his motivation I can show you a twib episode from yesterday where dr. Peter Griffin says or whatever his name a Daniel Griffin Peter Griffin Daniel Griffin says that measles is much more infectious than COVID so that's supposed to be an expert but this is a guarantee the most infectious virus in human history I'm sorry but that level of hyperbole has to it has to really tweak you in the head right now because that is just a ridiculous statement it's a ridiculous statement just based on the fact that we have video from multiple health ministers around the world saying that well everybody the test positive is being counted as a COVID death so oh wait a minute Charles hold on maybe it's not as an infectious as you thought because so many of these tests were false pot absolutely no deviation from the brittle narrative that they are trying to throw from the very beginning I believe that's what's happening here this is a purposeful purposeful insistence uncertainty when there is none and they've combined those parts of it with they basically mixed the lethality of the first virus with that one and yes that's incredibly dangerous incredibly stupid and completely unnecessary so if I mean even that characterization right there if he was trying to really tell the story biologically plausibly the story he's trying to tell would at least require him not to say it like that that they combined the lethality with the transmissibility because the transmissibility is coded in the spike and the lethality is coded in the other 30 proteins Charles I'm sorry I am kind of upset and I'm I kind of feel like this is a crucial time and it is important for me to express how disappointed I am that after many months of giving people the benefit of the doubt many months just hoping that all of these people are good people and giving them the benefit of the doubt these people have regularly displayed behavior regularly displayed ideas regularly displayed a false sense of certainty which is incongruent with wanting to figure out what's really going on with wanting to really break free from the the the grip of these people if you follow their story to its logical end we will be in the grip of these people forever that's what I think and that's why I'm worried about this story do that in the university labs with some students what stops a foreign government that isn't friendly to the United States or the rest of the world to find someone like that and hire them and you know get them to work on on something that is even more lethal I mean theoretically you could take the spike framework from Omicron and merge it with Ebola what's stopping you from doing actually absolutely nothing and this is you know when people talk about the bio labs in Ukraine and that's been a hot button sorry just one second I want to copy this out of the thing so I can have it for later I'm just gonna put that here okay sorry here we go issue I guess for the past six or seven months the program behind that is called well the agency that runs is a sub agency the Department of Defense called the Defense Threat Reduction Agency and they have a program underneath that called the cooperative biologic biological engagement program and these these programs started really at the fall of the Soviet Union because in the US government people like Charles and I were asking the question well okay all these former Soviet republics now have independence where are they gonna happen with these nukes and these new clubs and these biological and chemical weapon labs that the Soviets used to have in places like Ukraine so the idea was that the US government would start building positive relationships with these laboratories and funding work there so one we can collect intelligence on their biosafety and biosecurity the capabilities of that laboratory then also have a positive relationship so they don't turn around and do something to fergus back to us well I think you know with with recent history it highlights the the sort of the weaknesses of that type of program so unless you have someone from you know at least from the United because I realized this is a international audience but from the United States perspective unless we have somebody in that laboratory working with them on a daily basis how do you know what they're really up to and that was actually one of my concerns that I raised as an executive at you call clients on our work that we were doing with these foreign laboratories but isn't there another reason why the United States doesn't want to do this in their own labs because they are signatories to a number of treaties including own laws in the United States that prohibit this kind of stuff isn't it similar to what they did with Guantanamo where they just go into different jurisdictions where they can hold prisoners indefinitely something that wasn't possible in the United States or with the dark sides that they had where they tortured people they also outsource outsource that to other countries because they knew legally they could not do that in the United States isn't the same with these biolabs that they are funding internationally that they can do the kind of bioweapon research there that they are not permitted to do at home absolutely that's a really fun story isn't it and so it begs the question of had they always planned to have this ban and then to release the ban right before Trump's and and so then they could say that like oh see but the ban came out back and so then once the ban was lifted then they went right to work and then we had a pandemic and we must have been outsourcing it that's why we needed EcoHealth Alliance because we couldn't do it at home they prohibited it at home that's why Ralph Barrett couldn't do it he had to give it to the Chinese and it's so dangerous we wouldn't do it in America we do it other places like Ukraine and stuff and Russia had labs in Ukraine before we had labs in Ukraine so you know yeah yeah what a narrative what a story in fact that's why I have no doubt that that's part of it and it just imagine that's also I can almost guarantee that that's what's happened with all of the all the censorship and everything of social media which is assuming that the United States government and other governments are honest with their citizens which obviously it's probably not a good assumption just know that they still have like the five eyes agreements and things where they share intelligence and so they're basically they're basically using the intelligence there's a question in the chat about where is Kim comm famous from he is the guy who put this site up mega upload or mega download where people were sharing movies and then it got shut down for copyright and he's still in a lawsuit with the American government I think but he's mega rich and the way that he reads from a script here the way that he gets these ideas perfectly right on the first swing so let me get this straight we could have people people making lethal viruses that could kill billions of people I mean this is clearly a theater here whether or not they rehearsed it it doesn't matter this is a theater unless Kim comm sees my my stream and decides to have me on and listen to the fact that I think that this is all an elaborate hoax based on the fact that their worst-case scenario would be the release of a clone then I'm quite certain because it's staying on Twitter that it's probably more likely to be one of those things that we're gonna have to deal with that this is just more of the same nonsense trying to split us up on on false biology agencies to help censor things on social media because there's no way that Twitter was just doing that by themselves and so see now the censorship is over according to Charles there's no more censorship otherwise he would say that well one of the guys that still censored is me that I used to work with one of the guys that's still censored is Robert F. Kennedy that I used to work with but he's not saying any of those things and I want to suggest that that's that's not an oversight that is dubious because Charles has no reason to not still support Robert F. Kennedy jr. Robert F. Kennedy jr. thought he was worth six months of support and yet somehow or another he never mentions it and it bothers me a lot because I really think that that's the right team to be on right now I think that's the last banner left and it breaks my heart to say these words out loud but if Robert F. Kennedy jr. is already captured by the system we are in huge trouble ladies and gentlemen because there are no other banners that aren't pursuing this mythology the lawyer named Fleming is pursuing this mythology that it's a gain-of-function virus the gain-of-function viruses are real that they did this that they should go to jail for it and it's in my heart of hearts I believe that why they need to go to jail is because they lied about it they lied that it was possible they wasted millions if not billions of dollars setting up an entire set of research literature in publication to justify this pretense that coronaviruses have pandemic potential all of those papers are disingenuous that's what I believe no I mean absolutely there's a bigger effort from what some people call the deep state to make sure that topics like this are not really getting any traction in the public domain right and when we talk about intelligence agencies what I found quite irritating is when Biden got into office he basically gave this big speech where he said you know I'm going to ask my intelligence agencies to investigate the origin of COVID I want to have the answers and then a couple of months later he came back and said oh yeah they told me we will never know isn't that very convenient right so what I need to say what I need to say is that on August 27th when when the when that was published I already knew because I've been watching waiting to see what would happen I already knew about the diffuse proposal and we didn't release it for another month but I already knew about it and so I knew he knew and drastic knew I didn't know drastic science knew him and Yuri Dagan and all those real trustworthy people who are totally pro transfection were the ones who wrote the report up that he released and they held on to it for a month and then they released it when they thought it was the right time that report was published that the United States intelligence community was lying because I knew that the the diffuse proposal that pointed directly at the Equal Health Alliance and are you and our government was in their possession it had been found on their servers and they were lying directly to the American people about it I mean that is indisputable proof I mean Fauci was lying under oath in the Senate right when he was asked the questions about the funding of gain of functions is really obfuscation and misdirection and baloney and I'm not gonna make you watch it anymore listen to it anymore I know it took a long time I really think that we have been scooby dude into believing that this was a lab oriented our lab origin virus the lab and that tinkering of gain of function is responsible for this I even made what I think is kind of a mistake of writing an article for the Children's Health Defense and the Defender about this paper that Charles just cited where Alex and a couple other friends of his published the idea that the endonuclease foot footprint of a synthetic origin SARS-CoV-2 is present in the original sequence well that's not very surprising the original sequence is a synthetic virus it is a synthetic virus created by metagenomic sequencing and so after this long start I would like to get to that video and let's watch that video so that we can see where we really really are and what I need to do is go to YouTube and then I need to go just like this watch how you find it first I'll just get these out of the way so we have some things here watch how you find it you go you go nanopore sequencing and you go chow and the first guy that you get right here is the overview see that that dude is the man and here we're gonna flow those into the flow cell captured by these primers or these short DNA molecules on the surface are you ready here we go we're gonna learn sequencing right now our in 31 and we're gonna do this assistant professor at the University of California San Francisco and I'm gonna talk to you today about next-generation sequencing the outline of our talk today will cover traditional sequencing first and then we'll spend most of our time talking about Illumina sequencing by synthesis which most next-gen sequencing is being performed on today at the end of the talk also touched upon two other competing platforms from Oxford Nanopore and Pacific Bioscience so the human genome project really spurred the development of cheaper sequencing this was a 20-year effort that cost three billion dollars that was completed in the year 2001 and this lower the cost of sequencing a human genome to about 100 million dollars for a single genome and this use traditional Sanger sequencing before you go into Sanger sequencing I want to tell you a little bit about DNA just to bring everybody up to speed so what I have depicted over here is a sequence of DNA it's made of two different strands that run anti parallel so one strand runs in one direction the other runs in the opposite direction and your C's hybridized to G's and your A's hybridized to T's sometimes DNA is depicted as as an arrow shown here just to simplify images and we'll be using this quite a bit today now DNA is a double-trended molecule but it can be denatured and separated into individual strands and it can be re-natured back together or we can add on different sequences of DNA and hybridize those on such as this red piece of DNA over here that's not bound to this bottom template strand and DNA can be copied by polymerases and these DNA polymerases shown here in orange can bind to short pieces of DNA that are hybridized onto a template strand and they can polymerize DNA with building blocks to extend DNA into a newly synthesized strand and these building blocks of DNA's are called deoxyribonucleotide triphosphates and over here are the four different versions your A's, C's, G's and T's and these all share several common aspects first there's a triphosphate group that allow the growing strand to attach onto the building block they have a three prime hydroxyl group which is used to add on additional building blocks of DNA and they have four different bases attached to them so with traditional Sanger sequencing if you wanted to sequence a piece of DNA like this you would have to first denature it and add on a primer so that DNA polymerase can can bind on as depicted here and now instead of adding just the traditional DNA bases which would allow the polymerase to extend this DNA molecule we actually use fluorescent terminators and fluorescent terminators are very similar to the DNA building blocks depicted here but there are a couple differences first you might notice that there are different colors and so there's a different fluorescent group attached to each of the four bases a yellow for A a blue for G a red for C and a green for T additionally the three prime hydroxyl group that's present on the building blocks to allow DNA to continue extending is now terminated or removed this is why these are called fluorescent terminators they have fluorescent group and a terminator that prevents DNA polymerase from further extending the DNA strand and so in a test tube we actually have billions and billions of copies of this template and we put in the normal DNA building blocks and we spike in a low concentration of these fluorescent terminators what results is you have a bunch of fragments of newly synthesized DNA that are all different sizes because they randomly incorporated a fluorescent terminator and these molecules in the test tube are then put onto a DNA sequencer which will separate these and then allow us to determine the sequence what happens in the sequencer is these molecules are separated in size from largest to smallest the smallest ones come out of the sequencer first and get detected and then the next piece of DNA gets detected and the next and the trace that comes out of the DNA sequencer is a chromatogram down here at the bottom and if you follow the color changes as each of the different pieces of DNA come out of the sequencer you can build up your DNA sequence these machines can run up to 384 samples at a time and generate about 700 bases of sequence for each of those samples and you can sequence up to 1 million pieces of DNA or 1 million bases of DNA in a single day now this might sound like a lot but when you consider the human genome is actually 6 billion bases and to sequence a human genome you actually sample it many many times sometimes on the average of seven times this means that if you were to try to sequence a single human genome on one sequencing machine it would take about 100 years and so clearly the human genome project was completed in much under 100 years and this is made possible by factories of these sequencing instruments over here is a single sequencing instrument in this huge factory where we have sometimes dozens or even hundreds of these machines running 24 7 365 days a year so the human genome was was a really great undertaking generated a lot of useful data but it was only came from the genetic material of several people and so we still don't understand most of what this genome does you know we have the sequence but again it's only from a small subset but to really understand the function of the genome we need to sequence thousands to millions of different people to really sample the variety of genetic material present in the human population holy cow did you ever think I was gonna get it so right I just just play in this video because I knew it was about saying and somebody recommended him can you believe he just sequenced into he just segued into this the human genome is only based on a few people and we're gonna need millions more ladies and gentlemen accidental home run I'll take accidental home runs for 200 please Alex and it's obvious we can't do this with traditional singer sequencing so luckily over the past two decades the cost of sequence does you know we have the sequence but again it's only from a small subset but to really understand the function of the genome we need to sequence thousands to millions of different people to really sample the variety of genetic material present and it's obvious we can't do this with traditional sequencing I win luckily over the past two decades the cost of sequencing has dropped dramatically over here is a chart showing you how much it cost to sequence 1 million bases of DNA when the human genome project completed in 2001 it cost almost $10,000 today we're approaching one cent to sequence a million bases of DNA which represents a 1 million fold drop in the cost of sequencing and if you follow this chart you can see that there are several inflection now the question is are we really able to get off Moore's law here is this real or is this people claiming Theranos like sequencing ability is this are these technologies being exaggerated in their fidelity are they being exaggerated in their abilities in order to outsell each other in order to get stock prices to go up in order to get grant funding there's a very convenient little plot here but it makes me wonder whether on some fronts these sequencing technologies have been exaggerated or upon some applications their fidelity is exaggerated and that's where I think the difference between what he's talking about here and how it would be applied specifically to the rare RNA of a coronavirus in a sample full of RNA unrelated to it how they would do that points in 2007 2010 and 2015 work prices had some fairly steep drops and these were largely driven by new sequencing systems introduced by Illumina the dominant player in the next in sequencing markets on this slide are three different types of sequencers from Illumina they actually have several more but they really span the the scale of sequencing that's being offered on the left-hand side is aluminum I seek instruments in the middle is the high seek and on the right is the novice the newest high output sequencer from Illumina and if we compare the output of these machines and compared with the Sanger sequencing you guys remember Steve Gottlieb he's on the board of Illumina platforms we can see just how much more sequence we can generate from these instruments if you just look at how many reads you can get from a single run the my seek generates 30 million reads the high seat generates 3 billion reads the Nova seek generates 13 billion reads while the Sanger sequencing system generates about 400 reads and so it's a huge difference and if you just look at this in terms of how much sequence you can generate in a single day you can generate up to 4 trillion bases on the Nova seek in one single day compared to 1 million bases on the traditional Sanger instrument a little okay so let me just explain this to you a little bit to make sure that you understand what he's talking about the first thing is is that all those numbers are based on an unlimited quantity of pure DNA so if it's my genome you you have my genome and you've grown it in cDNA form and you've amplified it in bacteria and you've put it together in a way so that we can we can you know have enough of it and all those fragments to run on all those sequencers and get it all done and it'll take a long time to do my whole genome and they're never really doing that they're doing it from landmarks right doing it from landmarks and so if you do what it is to sequence the entire 6 billion base genome then it requires you to have a lot of high fidelity copies of all 6 billion bases what he's talking about here is sequencing you know this big piece of DNA then that big piece of DNA and then that big piece of DNA and then knowing how those big pieces of DNA are related in the whole and if you feed this all correctly then you can do that many sequences but it all requires clone DNA we're back to the clones again you need to be able to make cDNA and you need to be able to make large amounts of it so that you can produce all of these fluorescently terminated sequences so that you can produce these sequences that will bind to your primers and do all the things that they're going to show you in these next slides so understand that the assumption is that you have a lot of the DNA you want to sequence so that we can make more of it we can break it up we can tag it we can put all kinds of things on it so that we can run it through our sequencers and they can do their job but when we start with a virus we start with almost no RNA and then we make DNA from it and then we amplify that DNA and then we're gonna sequence it and however we get from there to there is the real the real bamboozlement because that sequence is not grown in a dish and then when you get enough of it you take a spoonful out and then you put it over here that sequence is generated in silico based on fragmentation sequencing of all the RNA present in the sample and then a screening on a background number one it's primers that are meant specifically for those coronavirus amplicons and over representation of them which which he will explain soon and then also the the database that that that those amplicons are compared to is completely isolated to SARS variants there's no possibility of getting a positive flu XYZ or RSV XYZ in any of these sequencing reactions because the way the sequencing reaction is done is that the fragments have to fit into the variant set and if you don't believe it you haven't done the requisite background research because they're not using nanopore sequencing for all of these sequences which were displayed as variants from the beginning they might be places in the world that's using nanopore sequencing for coronavirus clones now but they weren't doing it from patient samples they weren't doing it from patient samples they've never done it from patient samples in America it's all metagenomic sequencing metabiota proprietary software that used to pretty well work on the ribosomes of prokaryotes but not on viruses and that's the bamboozlement you see metabiota produced a real product that has been used incorrectly over here Moderna produced a real product that can do transfection and can do things in extreme situations but it's not an appropriate immunization but they want you to believe it is and so these these baits and switches these these this shell game that they're playing is going on all over and it includes the gain of function viruses but the sequencing is what this whole thing is based on so he's talking about he's talking about DNA and you got to remember the coronavirus starts as RNA when they try to pull it out of you and so this is not about what they're doing yet this is still about as much as DNA as you need you can do it then you can sequence this quickly the sequencing is the dominant player in the market it's an imaging based method and generates many many reads millions to billions of reads per run and from each of these reads we can generate 300 to 600 bases of sequence it's really really accurate the error rate is roughly one in 1,000 bases and with the new machines we can actually sequence a human genome for a thousand dollars in less than 48 hours and the sequencing on these Illumina platforms happens in flow cells these are essentially microscope slides with channels on the inside what I have shown here is a my seek flow cell next to a standard Eppendorf tube that's used for a lot of lab work and these tubes are pretty small about one and a half inches in height and on this my seek flow cell we can generate about 1 to 30 million reads per run next up is the high seek flow cell you can see it's quite a bit larger than the my seek flow cell and this larger real estate allows us to generate more reads in a single run approaching 3 billion reads and lastly is the Nova seek flow cell which is even larger and on this flow cell we can generate 13 billion reads in a single run so you can't just put DNA into these flow cells and get sequences out you actually have to prepare your sample and so there are several steps that occur but basically what happens is you take your sample that you want to sequence which is DNA if you have something like RNA you can convert it with enzymes into DNA you take that DNA and then you have to add on these adapter sequences at the end so in blue our primer binding sites that allow the sequencing so a coronavirus genome is 30,000 nucleotides so what you're trusting them to do is to break the coronavirus genome into 600 base long segments install the primer and the capture and then sequence that insert and so at 600 base pair long inserts to optimize the Illumina sequencing you're trusting them to find what is the I mean you can divide 30,000 by 600 that's a lot of amplicons and so those 50 amplicons need to be sequenced but first they need to be amplified from the RNA that they find in your sample and your saliva in your nasal passages so a single copy of an in non infectious RNA missing some of the necessary proteins will not give you a whole sequence and the vast majority of the viral particles that are released in an infection are non infectious particles they're missing critical genes so they will be missing critical amplicons if you should get the reverse transcriptase to convert that RNA to DNA for PCR amplification but this guy's telling you that you can only sequence about a bay about 600 bases at a time which means that your amplicons that you will bring up from each SARS virus that you're purporting to sequence can only be 600 base pairs apart or it will be too long for Illumina sequencing which is the only way they're supposed to do it right now this is the full genome sequencing that doesn't use Sanger that's too slow if they use Sanger they even need more DNA and it needs to be more complete and so it's very hard to decide how they are doing that without metagenomic sequencing but here we go listen carefully action to occur this is similar to Sanger sequencing where we needed to have a primer bind for DNA plume race to move along the template DNA and then we also have these capture sequences in green and orange and these allow your sequencing sample to be captured onto the flow cell for sequencing once you have your DNA sample prepared it's a double-stranded molecule so this is happening right here and put into the flow cell and on the left hand side again is a picture of the high seek flow cell so there are eight channels and samples actually go into the glass slide there are eight different channels within there and inside of the slide are a mixture of short DNA molecules in orange and green and these orange green molecules will bind to both ends of your sequencing library so we denature our sample flow those into the flow cell and they can get captured by these primers or these short DNA molecules on the surface of the slide and once that occurs a DNA polymerase is added in the DNA building blocks introduced and we copy that template and so we have a newly synthesized strand that's now physically constrained to the bottom of the flow cell we wash out the original template strands and then allow the newly synthesized strand to now bind onto the other DNA sequence present on the surface in this case the orange piece and then we flow in some DNA polymerase and building blocks and we get another strand formed and we repeat this process many many times and in the end we get about a thousand copies within a cluster and remember that these thousand molecules all came from the same original template strands so they all have the same sequence we denature the strands and then we can select it this will make more sense when he shows the whole flow cell which has has many thousands of little dots that have been formed by that local PCR reaction building up similar similar transcripts in similar little dots and then on each of these dots this reaction will occur and the the colors can be resolved you'll hear cleave off one of the oligos or the primers in this case the green one and we've washed those away so now all the thousand molecules present are all the identical strand because we removed the other strand we flow in a sequencing primer and now the clustering process is complete and this is the instrument that forms clustering so first thing we do is we take a brand new flow cell again this is the size of a standard microscope slide and then we put it onto the stage of the instrument and the stage is a region of the instrument that can actually heat and cool to perform a lot of the enzymatic reactions next we load a blue plate of the reagents that perform the clustering and lastly a strip tube with the eight different samples that are going to be loaded onto that flow cell next a manifold is mounted on top of the flow cell and connected to the c-bots and in the back of the c-bot or this clustering instrument are a series of pumps that will now pull reagents from the blue reagent plates I find myself strip tube I find myself wondering how often that doesn't work or how often those tubes get clogged or how reliable this stuff is it's all very amazing how it looks but I guess Kevin McCarran and could tell us a little bit about that because he was involved in a lot of this early technology it may still be involved in it now and pass them through the flow cell and this is how reagents and liquids are delivered at this point the clustering procedure takes about three to four hours and once that's done we take that flow cell and move it on to the actual sequencer in this case a HiSeq instrument and on the HiSeq instrument we have a refrigerated compartment for all the sequencing reagents a lot of enzymes in there that need to be kept cool during the three to four day sequencing run above the refrigerator section it's a three to four day run so if any of those tubes get clogged or you know the refrigerator fails or I don't know what then the whole thing is lost I mean it's it's very impressive that pull reagents from that refrigerated compartment and send them to the flow cell which we will now load onto the stage and so that is the flow cell from the c-bot that just completed clustering this gets mounted onto a stage and locked in place and then we can begin the sequencing run and so just to keep in mind the clustering is that local PCR reaction where every one of those transcripts landed then it repeated it then it took away the other strand and then they fold it over and they doubled all locally until they had enough so that they could rinse that one away one side away and then they had only the ones bound to the green primer which means they're all the same sequence it's pretty clever that does is that will trigger the pumps to start flowing reagents from the refrigerator compartments into the flow cell and behind the flow cell is actually a really powerful microscope that is used to actually sequence each of those molecules of DNA and so remember that when we put the flow cell on to the sequencer this is what it looks like we have a lot of clusters that each have a sequencing primer bound to them and the chemistry for sequencing is very similar to the Sanger sequencing terminators that were used but the one difference is that these are reversible and so they're reversible in two ways so first these can get incorporated into the clusters by DNA polymerase and those clusters will light up in four different colors depending on which base gets incorporated a picture is taken and after that picture is taken we can actually remove these terminators and the fluorescent groups with some chemicals and what results is we regenerate the three prime hydroxyl groups so now that cluster can have another round of bases added to it and so this is what it looks like for a single molecule on this end over here we have our template strand bound to a sequencing primer and this allows DNA polymerase to bind and then add a base in this case is in yellow one so once this gets added a picture is taken and then we add chemicals to remove the fluorescent group and the terminator so that DNA polymerase can add a second base an image is taken chemistry is performed to remove that base and to regenerate the three prime hydroxyl and then we can add another base and so over time the cycle just repeats over and over again with multiple cycles of a base addition imaging and so each time those chemicals are flowing through the flow cell then flowing through the flow cell then flowing through the flow cell so it's not clear to me they must have like enzyme plus something go through and then the chemicals that strip it go through and then the enzyme comes back again it's a pretty ingenious little thing and then chemistry to remove the blocks and so if we look take a look at this depiction of five different cycles so each of these represents a different image taken and if we follow the top left and the bottom right clusters and see how those colors changes over time we can actually build the sequence so the top cluster the sequence is going to be AG CCT because it goes from yellow blue red red green and the bottom one is GT AAC and so again the power of the system is that you can sequence up to billions of sequences at the same time in parallel and this is what gives these instruments their throughput so some people ask me why can't we increase the amount of bases that we get in a single read by just repeating the chemistry over and over again do this a thousand two thousand ten thousand times and the main reason why is that the enzymes and chemistries aren't perfect so over here I have a cluster of one single cluster made out of the same template molecules but what happens is because chemistry isn't perfect some of the strands lag behind so this one is a yellow instead of a green and some clusters jump ahead so this one is now a red instead of a green because it hopped ahead and what happens is these errors aren't too bad but over time and many many cycles over the course of 100 200 300 cycles more and more strands start to lag and more start to jump ahead so that your true signal starts to disappear and gets weaker and weaker and this really limits a limit of sequencing to about 300 bases for for each read 300 bases for each read means that they need to break up a coronavirus genome of 3,000 bases into at least a hundred parts as I understand it so they need a hundred set of primers a hundred sets of primers in order to sequence the whole coronavirus genome and if any of those primers don't pick up an amplicon then it's up to them to decide with their meta sequencing data whether or not the genome is present and so they control all the data all the story is controlled by them because if they find something else it's not there the primers aren't meant to find it if there's something else in that sequence that their primers don't pick up and you're gonna hear him talk about this that it won't be there even if it is there and again the sequencing happens after the amplification occurs so if you're not using primers that will amplify the things that are present you're not gonna find it that's why they only find SARS and it could just be that SARS has always been back there so with the Illumina sequencer four different images are taken so how many dots there are each of the four color base see how many and those are all places where like this are not those are all places where that little PCR reaction occurred so these are all little clumps and they're all separate sequences that have been captured on the flow thing so it's pretty incredible very clear they're not perfect circles and they're very difficult to see by eye to figure out where one cluster starts and another begins you know one example is here let's say in the first cycle we have two clusters that are both G these just look like a single blob that's a single color and this is really hard for the sequencer to pick out but because we're sequencing many many sequences at a time in parallel chances are these aren't identical sequences and if we go through another cycle you know chances are that they'll have a difference so now this cluster on the right is an a while the cluster on the left is still a G but because of this the sequencer can now compare these images and determine where cluster A has a very pure signal where there's a mixture between cluster A and B and where cluster B has a pure signal and it'll decide to just take imaging information from just these clean areas where they have very pure signal so again this is four color chemistry from alumina this is what had been used for for many many years and it was very clear and obvious one color for each of the four bases but there are issues with this so the colors depicted here you know you can tell the colors are very very different however if you actually look at the emission spectra you can see there's significant overlap between the four colors and this requires the instruments to undergo a lot of color compensation and this can contribute to errors so a few years ago in Illumina introduced two color chemistry so instead of using four colors to represent four bases they're now using two colors to represent four bases in this case they're using red and green and the way this works is that you have T's which are green C's which are red A's which are actually a mix of both colors so you'll see them in both images and then G's actually have no color or no signal and so this is how you can encode four different bases with only two colors and so initially the quality of the two color chemistry wasn't as great as the four color chemistry but recent developments on the NovaSeq platform one of Illumina's newer sequencers that uses two colors the data quality actually rivals the four color instruments and so there's definitely been some improvements in this area the other benefit of two color sequencing is that these reagents are generally cheaper to make because you don't have to have as many colors and the instruments are also less expensive because you only have to capture two images instead of four so how far can you take this can you do sequencing with only a single color and the answer is yes a newer platform just released by Illumina uses a single color to encode four different bases and they do this by taking two images that are separated by a chemistry step in the middle so I'll walk you through this in the first image they add the four different bases and in this case the A's and T's have a color on there and again it's the same color the C's and G's don't have any color one thing to note is the A base has a cleavable linkage between the base and the color and the C base has a molecule that we can use to attach something later on so we take an image first so A's will have color and T's will have color C's and G's will not have any color next we go through a chemistry step that will cleave the green color from the A bases and it'll add a molecule that will bind to the C bases and make them now colored and so we take a second image so in the second image A's won't have any color because any that did had them cleaved off the G's still won't have any color but both the C's and T's will have color and what this looks like if you break this down is if you compare a single cluster and look at its color whether it's on or off between image one or two you can get four different bases outs so these are the different flavors of Illumina sequencing we started off with four color chemistry moved to two color chemistry and now we have one color chemistry all these chemistries are still used currently on existing platforms and each of them have have their benefits they're making money so now we're going to move over to the long read sequencers we're going to start with Oxford nanopore sequencing and with this sequencing technology it uses these nanopores which are extremely small pores with really small gaps in them so in this case 1.8 nanometers that are embedded in lipid membranes these lipid membranes act as a barrier that prevent currents from going back and forth so currents have to pass through the pore and the way the nanopore sequencer works is single-stranded DNA or even RNA in this case now are threaded through the pore and depending on which bases are in the pore to give a moment that changes the current that's read out and detectors can measure this change in current and over here is just a depiction of what this looks like so for instance you might have a similar one level of current for your T's a different ones for G's something else for C's and then another level of current for A's and you can just measure these current traces over time and build a sequence in reality the traces don't look this clean because in the pore they're actually up to six bases at a time present and with six bases times four different possible bases for your A's C's G's and T's this generates up to 4,000 different possible states and so it was really a technical tour de force to get this to work but today you can get this to work you can buy these systems and they do have high error rates anywhere from 10 to 15 percent so higher error rate means that you can't sequence viruses it means that if you have a very rare DNA and it's broken up and you need to sequence it you're not going to be able to make the statements that they're making for example that one amino acid was changed in the Wuhan virus and that allowed it to go all around the world like Paul Offit says like a few inserts like a few inserts were put in and now it was made super durable and super high fidelity and super long-sustaining and so the reason why I'm harping on nanopore is because nanopore thrown out as millipore was something that Kevin McCairn yelled about when he was on Tim Truth's stream and talking to Dr. Bailey about the sequencing reactions. Dr. Bailey is keenly aware that their meta sequencing is a sham and it is one of the primary boats that he stands on and I can't really find fault in his arguments I think the same things I've come to the same conclusions on my own that they can't sequence coronavirus like they say they can they're having you focus on the spike protein sequence because at least that's a sequence within the realm of reason but they're not showing you any of the other coronavirus genome because it's very rare that they can find all those amplicons they cannot amplify or sequence more than 300 at a time and in these longer base sequencing reactions that they're showing here the error rate is so high that you wouldn't really be describing anything that you didn't already know pretty well if you already know what you're looking for if you're already trying to decide between these two bacteria then this sequencing reaction is great and it's super useful in the field and it's awesome but if you're trying to characterize the point-wise mutations and the evolutionary steps that characterize the variance of a coronavirus as they go around the world for three years you can't and they didn't do it this way you can't and they didn't do it this way they did it with meta sequencing and meta sequencing is something entirely different than this and these errors tend to be biased but you can get really really long reads the current record is a two megabase or two million base read from a single piece of DNA you need to make that DNA that DNA needs to be made and you got a read of two million bases with 15% error rate where are the errors oh you don't know so then what again this is a little tiny tiny bit Theranos like look what we can do we figured out how a six thousand state electrophysiological ion channel could be used to make an electric electrophysiological trace that we can allow us to read two billion bases had an extremely high error rate oh that's really handy I guess in five years you'll get that error rate down to something more useful but right now it's just Theranos and this is in 2019 it's again as I mentioned you can now directly sequence RNA so you don't have to convert that to DNA and there are even possibilities of sequencing proteins in the future

Now why is that important Mark? Why is that important Mark Kulacz? Because what he's talking about is when he says sequencing RNA he's talking about sequencing ribosomes because the only way that that works is if you have sufficient copies of the RNA you want to sequence and the only way that's going to happen is if you go for something that's very abundant you know it's very abundant in all the cells of of a bacterial culture you know it's abundant in all prokaryotes freaking ribosomes and ribosomes contrary to popular belief are made of RNA they're not made of proteins there are protein RNA complex and so this Oxford Nanopore stuff this this using it to find all the all the parasites in a sample is very specifically aimed at the subunits of ribosomes not the whole genome of all the pathogens that are possible that would be ridiculous they aim it at the ribosome subunits that they expect to be there for all these different pathogens and then focus on the ones that are present in all of them and then screen those sequences and that's how they do it they're looking at ribosomal RNA that's why he's mentioning that RNA can be sequenced not because you can sequence an RNA virus they're not available in sufficient quantity not available and high enough fidelity but RNA is and RNA ribosomes are fighting we are winning nanopore system is that it's extremely portable you can see this scientist there it is right there doing some sequencing out in the field jungle somewhere in the world and so it really looks like wow they can do it right there in the jungle that of course they can sequence coronavirus no that's not true it's not true at all because sequencing coronavirus is very different than what she's doing there but she's talking about a sequencing ribosomal DNA so that they can figure out which ribosome variations are present and have a clue of what pathogen is in that sample you can't look for viruses like this it doesn't work like that don't let them fool you viral RNA is not available in sufficient quantity for them to use this like that see the foliage in the back so there's a laptop run to the sequencer and the sequencers actually the small device that the scientists pipetting into it's really this lies of a small remote control and it's powered by the computer that is attached to you so it means you can really take sequencing anywhere in the world these systems have even been taking up to the International Space Station for sequencing in space so the last technology I'm going to talk to you about today is specific biosequencing and this is another sequencing by synthesis method and it uses building blocks similar to traditional Sanger sequencing and Lumina sequencing but they're slightly different so down here I'm showing you an a base from the Pacific biosequencer and you'll notice first there's no block on the three prime hydroxyl end this means that once this gets incorporated another base can get incorporated right away and then the fluorescent group is actually attached to the phosphates and these phosphates actually are removed once a base is incorporated this means that this fluorescent group once it gets incorporated into a DNA strand will float away from the DNA strand so this means that we don't have to do separate chemistry to enable the reaction to proceed this happens in in real time and on the instrument there's a really really tiny array of wells into in a plate and these wells are only about a hundred nanometers in height at the bottom of each of these wells is a DNA polymerase and what happens is a templates molecule is bound to the polymerase and then it starts incorporating those modified bases and there's a camera at the bottom that's taking the video that's monitoring this reaction in real time so remember we have four different bases that have four different colors and these bases are flowing in and out of this well really really quickly and so you get a signal that's just kind of very noisy you don't really see anything happening but when the proper base is bound to the polymerase and matches the template it actually dwells there for a certain amount of time it's really a split second but that split second gets recorded by the video and you see this bump up and the fluorescent signal in this case blue which is the G base and so once the incorporation occurs the phosphates leave and the fluorescent group that has the phosphates also leaves and resulting in the signal dropping back down to this this background level and this will continue until the next base that's correct binds and you'll see another spike and so by looking at these spikes and signals across anywhere from one to eight million wells at a time we can build up DNA sequences and packed biosequencing generates long reads anywhere from one to two hundred kilobases in length so this is shorter than the nanopore sequencing but it's still much longer than a lumina sequencing it also has a high error rate similar to Oxford nanopore sequencing but its errors are random this is actually a good thing because the errors are random if you sample the same DNA molecule several times you could generate a very accurate consensus sequence and on the top…

So, that seems to imply that for Oxford nanopore sequencing it's not random and I guess that's because Oxford nanopore sequencing gets affected by the three-dimensional organization of the RNA or DNA that's going through the whole and so if it's folded up with with G quadruplexes or other other tertiary forms it might not flow at the same rate or it might not flow at all and so that could be part of the problem remember they're using single-stranded DNA single-stranded RNA so the the possibility for hairpin complexes and all this binding to itself stuff is a real issue which they're showing you here.

…is a model of what a packed bio-library looks like. There's a DNA sequence, and the adapters in green actually cause the DNA become this dumbbell shape. So it's one circular structure. And when a polymerase binds it starts a sequence and actually go around and around this molecule, many many times, generating a series of reads that are all attached together, that came from the same identical template. So for instance, if we have a mutation present in a sample and want to detect this this mutation should be present in all the copies, and so if we match up all of the different reads together they came from the same molecule we can see that the true mutation is actually detected, we see that in every single read, and then other random errors that show up don't match up with each other. And this allows us to generate a very accurate consensus that has an accuracy of anywhere from one to a thousand to one in ten thousand actually, so even exceeding raw Illumina sequencing accuracy. So why do long reads? Because there are certain downsides they're harder to repair in general they cost more but there are certain benefits and one example is if you want to assemble a new genome that hasn't been sequenced before and so this is kind of like solving a puzzle that's been either split into ten thousand pieces or into four pieces Illumina sequencing in this is analogous to the ten thousand pieces because you have many many short reads that you have to stitch together and this can be very very difficult however we have very long reads and not that many of them it's really easy to align them to rebuild this new genome that you want to assemble it's also useful if you want to identify structural variations so for instance in a lot of human cancer samples there aren't just single base changes and mutations there are some that are structural variations where there are huge chunks of DNA that have been moved to different rare areas of the genome here or there or flipped around and this can be very difficult to detect by short read Illumina sequencing but if you have really long read sequencing you can span these changes and really easily identify the structural variations lastly you might want to identify where mutations come from for instance in humans we get one set of our chromosomes from mom one set from dad and there can be mutations on either one so nice mutations might be on the same set of chromosomes some might be on either one and to be able to do this with Illumina sequencing again is very difficult because the short reads make it hard to determine which chromosome they came from but with long read sequencing it's much easier to do so so there are lots of applications for next-generation sequencing I'm going to talk about a couple examples one is prenatal testing this used to be done using a very invasive amniocentesis sampling which had certain rates of complications associated with it but with next-generation sequencing we can actually take a blood sample from the mom and nowhere near the fetus and actually sequence all the DNA that's present in the blood and the reason why this works is that fetal DNA actually makes its way into the mother's blood stream and so we can use that to detect any type of chromosomal abnormalities in the fetus and the same thing happens with cancer and transplant rejection patients so in cancer the cancer cells are constantly shedding their DNA out into the bloodstream and because the chance your genome is going to be slightly different than the normal genome we can identify those through sequencing and same thing with transplant rejection if a donated heart for instance is undergoing rejection its cells are dying and shedding DNA and those donor DNA sequences are very different from the recipients DNA sequences and we can detect that by sequencing that material another example of using next-gen sequencing is to detect pathogens pathogens have their own set of DNA and RNA that are very different from human sequences and so if there's a pathogen present in a human sample we can sequence this human sample ignore all the reads or the sequences that are human and ask what's left and typically if there's a pathogen present we'll be able to detect its sequence in that sample very specifically is referring to a ribosomal sequence there not a whole genome sequence but a signal that they're looking for with the primers that they aim at right it can't amplify all genomes present in a sample because you don't have the requisite primer knowledge to know how to amplify it all and so you either get lucky with the DNA that's present or you have to chemically amplify it and if you have to chemically amplify it you need to have some knowledge about it and lastly in the context of cancer treatment cancer again is driven by mutations and there are lots of different therapies for cancer but they're very specific for certain cancers and so by doing next generation sequencing on cancer samples we can determine the best types of treatments for patients and improve their outcomes so the future of sequencing is very exciting currently it's a thousand dollars to sequence a genome but in the next five or ten years this cost will probably come down to a hundred dollars and it'll be interesting to see what happens once this cost is reached for instance will genome sequence just become a routine part of your medical record and if so this would be a great resource for researchers trying to study genetic diseases a great resource for researchers studying genetic disease is a great way to keep track of you and all of your family and friends forever a great feast of data to feed into our systemic AI that we think in five or ten years when the sequencing becomes better and the data becomes sharper and we're able to sample from everybody because everybody has a real-time sampling device in their skin then we'll be able to do this what do you want I know and I'm still streaming so I can't come up I already told mama by the phone I already told mama I was doing it just tell her I'm almost done so we're really in a situation where where it is a bit of a Theranos thing that these people have been told behind the scenes it's gonna get better it's gonna go from a thousand to a hundred ten cents the Chinese have been telling us that we can make fake we can make synthetic bacteria soon we're gonna make synthetic genomes and synthetic organisms and we're gonna make better genes than you have in your genome well there's only one way they can do that is if they sample all the genes of all the people on the planet right now and then try to figure out using an AI which combination of those genes is better or which form of those genes which which actual type of those genes is the best an AI optimization of the human genome would require six trillion genomes might as well start with nine billion right now because there aren't gonna be any more he's telling you right now it'd be so great when we just told you a half an hour ago that we need more genomes we only have a few we need millions if we're gonna really understand it but at the same point you have to understand and figure out ways of protecting patients genetic information if all of this is out there but it's gonna be very exciting to see where what new applications are developed for next-gen sequencing as the technology continues to develop and mature develop and mature we have shut down the world based on the idea that it's already mature that it's already high fidelity that it already works on coronaviruses this is before the pandemic and it's before the pandemic and it shows you where it was at the start of the pandemic they're talking about sequencing DNA here and they're talking about using nanopore sequencing to sequence RNA but how in the world would they do that with RNA in your nose in a virus think about this for a minute ladies and gentlemen think about this for a minute please because I'm having the same problems you are is that about we have been believing that we figured this out globally from acute lung injuries came from a laboratory arrogance and we've been fooled into believing the phantom virus hoping it would scare Eric away that's right and it worked till you guys showed up you were afraid that we would find out who created the virus so you beamed us into cyberspace and what they want to do now is convince people like me like you like Robert F. Kennedy jr. the gain-of-function virus research is real that it has the potential for pandemics and that we're gonna have to permanently alter our societies in order to take this new thread into count forever that's the plan that's what they want you always knew that was the case but have you really it's so clear to see now why this is bamboozlement why gain-of-function viruses aren't the real danger if you want to understand a little bit about gain-of-function or gain-of-purity please look at this rounding the earth discussion me and Matt had a pretty good first take on it I don't think I've progressed that far since again this this whole stream or this series of streams for the last couple days has been inspired originally by this cover band that tried to cover my biology in a hotel room and went south from there at this time Charles Rixie was still working for Robert F. Kennedy jr. and it was at that weekend where that stuff all went south so this is what this is what they told us they've told us that that the RNA genome of the coronavirus is assembled around end proteins and then it goes into this endosomic vesicle where on the inside spike protein is expressed and then this assembly results in the assembly of new virons and then when this endosome binds with the outside membrane of the cell these new virons are released there is no there are no papers describing this process there are no stepwise analyses of how the entire genome is assembled like this in pieces and then put accurately into packages all we know is that Robert Malone says most of the time this doesn't work most of the time they're not infectious because they don't get all the genes they need then how does this happen how are coronaviruses assembled if if if if we don't really know are we just making an assumption is there machinery already there for making something called exosomes and this infectious RNA just takes that machinery over ladies and gentlemen they make these beautiful cartoons with cartoon animation they show them in secret meetings and they pretend that they have this high fidelity control and observation grid and they know what's going on and they do not they know that coronavirus is almost intractable they know that coronaviruses are almost impossible to sequence and they know that the only way that they can really feign working with them is to create infectious RNA clone versions of them and make videos like this one and so that's how we got here with the RNA versus DNA sequencing that's the part of the Clone Wars that we are on right now and I hope that this has been useful I'm gonna stream every day that I can now that grandma's here because I really think it's important that we keep the pressure on and we keep moving the ball down the field sorry that the USA lost today it was really a lot of fun for grandma and I and Fyrla and the boys to cheer on Holland and I think Holland is gonna do really well they look really deep and really tough and I think they looked really really strong today although America had lots of chances and it could have very easily been three to three and gone into to to overtime or whatever I mean it it's unfortunate too you could see the difference in the size of the players they were like grown men that have been playing you know professional international football for years playing for Holland and then a bunch of really young dudes that weren't really physically strong enough on top yet to to push around these 30 year old men from the Netherlands and so and not only that but the Netherlands is deep and so they substituted in the middle after halftime and you didn't even notice that they had changed players and so it's pretty it's a pretty amazing accomplishment by the young American team but I gotta say up Holland I'm really happy they won thanks very much guys for joining me on the Clone Wars episode tonight it's two and a half hours long I hope it wasn't too long I will see you tomorrow probably around 10 a.m. thanks guys for joining me I'll see you soon you