Thank you, Ming. By the way, good morning, DevCon too. Ah, nice. I think developer conferences are always a little bit more fun than business-oriented conferences, right? The participation from the audience is always fantastic. Excellent. So my name is Manu. I'm from Autonomous. We're in the business of incorporating companies, but I use the analogy of the Starship USS Enterprise blueprint that I have on the top there to describe what we're doing. So we're building the warp drive, right, in that schema that you see there. The warp drive is what's going to fuel innovation, production, and the Starship is basically the startups, organizations, and so on. This is really the final frontier in automation. I think over the years, we've automated a lot of things, whether it's self-driving cars or completely fully functional robots, but where we haven't innovated is in the area of law, corporate law, legal processes, the way companies, organizations are governed, asset management, and so on. And this is kind of sad, and that's what we're trying to address. So before I go into how we do what we do and our design pattern, it's important to understand first why the charter company. So the charter company, as I mentioned, is the underappreciated engine of innovation and production. We all build amazing things only because we are organized. Without that organization, I don't think we will achieve the things that Ethereum has achieved, for instance. And it's legal. So the way we designed our design pattern on the autonomous plan, everything that you do on our dashboard is completely legal.  And honestly, I think it's the best use case for Ethereum smart contracts. I don't know why nobody else has done this before us, but I think it's the most straightforward use case that you can implement where you convert, in our case, we convert a certain section pertaining to corporate law into smart contracts, self-governing, self-executing, self-settling smart contracts. Hopefully, at some point, it will decentralize the courts and then lawyers become coders. That'll be interesting. So this is basically what we do. Now, there's been a lot of talk the last couple of days about security, especially yesterday, there was formal verification stuff. All that is amazing. I'm a technologist. I'm a developer. So I really appreciate the mathematics behind formal verification, but it's still complex stuff, right? It's not your average developer, average Joe developer that understands these things. So there's another tip I'm going to give developers, especially new developers. You don't have to design things from scratch. You don't have to create your own design patterns. You can seek inspiration from two locations. One is history, which is why I've got this timeline here, and the other one is nature. And you can learn lessons from there and design your smart contracts. And this is how we do things as well. And if you look at history and the blockchain and the chartered company evolution over time, it's sort of where we have arrived now with the blockchain chartered company as autonomous. So the first chartered company was somewhat in the 13th century. These were the traders, right? They went around the world. These were actually the chartered company itself. Then you had the Dutch East India Company. Then you had the Industrial Revolution. And since the Industrial Revolution until today, there has not been much innovation in the space of the corporation, right? A lot of the processes, policies are still the same until we came along, autonomous came along, and we put the company on the blockchain. We allow you to govern the company through a series of smart contracts, which I'll show you in a bit. Now what have we achieved? We have automated the corporate governance process. And why do we want to automate the corporate governance process? The cost of doing business today is so expensive because we have to hire additional compliance people now for new regulations. We've got policies that are more complex for us to comply to. So we try to reduce all of this friction. We try to save startups, especially money, and let them focus on the core building of the starship, right? So that's what we do. And it's not that we don't believe in the decentralized autonomous organization. We do. And if you see, we build everything towards a future of a decentralized autonomous organization. But what I believe, and if you see the headers there that I have, that last header shows you that for a truly decentralized autonomous organization, I think we need to go through some kind of socio-economic, political revolution before we're truly ready for an efficient decentralized autonomous organization. So basically what I'm trying to say here in summary is that when we design stuff, we go by the Secure by Design framework and the company, the corporation, the charter company, through its years of evolution has arrived at a certain state today and I think is Secure by Design. So we just convert the Secure by Design framework into a smart contract. How do we do this? Before I can even speak about the company smart contract, which is basically our main bread and butter, I want to talk about the identity smart contract. So again, going back to the energy of Star Trek, this is the dilithium crystals. I see a lot of blank faces. So I'm assuming you guys are not from the Star Trek generation. Okay. So dilithium crystal meaning that this is the fuel for everything else that we build and the central point of all interaction with the company smart contract. Now what is this? The identity in our case can be, it's probably slightly different from the U-Port guys or somebody else that's building an identity smart contract. We had to make this because we wanted it to work with our ultimate use case of the company smart contract. So in our case, the identity can be two things. First, persona, so a regular person, a natural individual. Second, a company, right? So it can be a collection of a company that's governed by a constitution, which is a collection of individuals. And it can also be a verification entity. So we said, you know, it's not enough for us to just allow the user to create an identity contract. We also need to verify this.  We want to do this in a decentralized manner as well. And we said, okay, then we've got to create verification entities. But the way we've built them, so autonomous in this case is a verification entity. And we, as a result of you having to, if you decide to incorporate a company with us, you will also create your identity and we will then have to verify this identity. This identity can have a layered approach. So we do a proof of identity type KYC where we ask for your proof of address, your passport copy and so on. But this can easily be extended. So you can have a layered approach where a bank comes into play. Or you could have a government agency, for instance, verifying that a bank is allowed to do a specific type of KYC. And that's what you see at the bottom there on the left. And more importantly, this identity smart contract also has got identity information, right? So it's a collection of an encrypted identity file that is selectively shared. So by default, you can assume that it's shared with the public addresses of the verification entities. And it has the stamps of these verification entities. So the KYC stuff I talked about, it's all in a collection of stamps, if you would. And it routes calls. So if you go to the identity smart contract and you say, hey, get me the KYC of this identity with a specific bank, then it's going to route that call to that bank and get you the up-to-date KYC state. Should you wish not to use the localized stamp? The secure key recovery mechanism in place as well that we've attached to the identity. So basically, it's not enough. And a lot of things we design, right? We understand at the end of the day, people are using the system and people are flawed. Systems might be perfect, but people always lose their keys. And we've had many, many people losing their keys. Actually, as we speak, I think even my developers are still recovering some client's key, I'm sure. So we've come up with a very secure way in which you can store your key and retrieve it. So even the retrieval mechanism that we've put in place ensures that after you've recovered, you can still continue to use the keys because they've not been compromised in any way during the recovery process. That's also something that we did. And obviously, you need to be able to replace it. So in summary, I think we've identified or we've solved, at least for our use case, we've solved privacy, recovery and compromise of identity, right? And with this, you lay the foundation then to be able to sign documents, hold assets and do whatever it is that you need to do otherwise in the real world, in the digital world. Now, this here is the main blueprint. This is what we've been working very hard for the last one year. Actually, I wrote my first smart contract right about the same time when DEF CON 1 was going on. And today, we've got a whole suite of them. It's interesting. So central to all of this is the company smart contract and what is the company smart contract? Itself is an identity. It's an identity that's controlled by the stakeholders of the company. So these can be directors, these can be shareholders, there can be a corporate secretary involved in there and they all do this by way of legal documents. So there are preset legal documents in the real world which you use to enact any action in the company smart contract. Whether it's a transfer of an asset, you want to add directors, any corporate action that you have to do is by way of the proper legal document and process. So we've also replicated real world legal processes. So there can be a multi-step process, there can be 10 documents that you need, let's say in the case of a funding round, and they might have a certain sequence and that 10 documents will have to be executed in the correct sequence, otherwise it will not work. I think I'm running out of time probably, but let me just quickly go through what else we are trying to do. Okay, so before that, so that's what we do. So we've got a collection here that's got a complete trace of legal documents, identities, transactions, everything. But what is cool is those two purple circles that you see. One is the decentralized exchange. The company itself could become a decentralized exchange, which is pretty interesting. And you can also do that by way of a synthetic equity framework as well. And we've also designed this already. So this is my synthetic equity smart contract. In summary, I'm not going to go through it, but it is a way to replicate the payoff that you would otherwise have by holding real equity via smart contract. So that's basically what this does. So that is something that we've designed, we haven't implemented yet, and this is what I mean when I say we're working towards a completely decentralized autonomous organization at some point. These are things that you would need. And of course, the company is a decentralized exchange. This is a very interesting slide too. I'm not going to go into the details, but in one line, your company is a decentralized exchange now. So this is also very powerful working towards this. And the team, if I may, just before I get cut off here, I think the team Han, our CEO, is right there. And the team that's always working hard with me on the weekends. Guys, stand up and take a bow please. Ladies and gentlemen, these are the guys that give up their weekends for me. Thank you very much.


Earl Glenn township Chevrolet shop form in here another addition of Tuck-tuck Today we're just gonna go over a quick winter time obviously we get storms and our weather's been real crazy over class a little bit here So just some tips and some information about you know that initial start after a big storm or just some Things you can do to help prevent some issues Happen to you in the future when you get a storm So one thing we get a lot of people coming in here and I know a lot of their shops do is after a snow storm You've done your you start your carry letting more up Well, you finish up in the driveway or you're getting ready to go out or you're gonna move in your apartment for the plow to come through You see it you know a liner or a bunch of drops of bright green or bright yellow Fluid on the ground or something underneath your car that looks like any freeze And that's what people associate it with they'll come in and say listen started my car this morning I think I got an antifreeze leak the green even check it. You can't find a leak anywhere and normally what the cause of that is They put zinc in a lot of the exhaust systems today. It's to help keep the pipe from rusting It's a rust inhibitor and when it gets hot it crystallizes And then of course from the salt and the water and the snow when it gets hot It runs off the exhaust and when it does it gathers these sink crystals and hits this bright white snow And it kind of looks green or really bright yellow and you usually can tell it's not any freeze Because you'll have a line of it Traveling in the direction of your exhaust. So if you see that that's what that is. It's not a big deal It's nothing to panic over obviously if you've got overheating issues or you've got a Warning light coming on saying that your overflow bottle is empty or there's antifreeze problem or coolant persistent problem That's another story all together, but if you see those little drops nothing else associated with it That's most likely what's going on there So we've got let's say we've gotten a storm you're going out you're getting you ready to clean your car Of course headlights tail lights Cleaning all that stuff off real important, especially nowadays a lot of the headlight bulbs and signal light bulbs and and And tail light bulbs are all LED LEDs tend not to produce as much heat as the old incandescent bulbs So they don't seem to melt themselves off as well So before a quick little brush and a little bit of remnants of snow on there if you drove after a little while You know it would melt itself off, but it doesn't want to do it anymore because of the temperature so another thing to keep in mind too is When you go to start your car you should always open the hood the car in front of the car is designed So that it's taking fresh air in while when the car is not moving there's wind blowing at it The fresh air is going to roll up underneath behind the grill in the bumper in around where your fan area is You get a bunch of snow in there all around your engine you start your car You move it you pull it back in that snow is now melted a bit Go back in the house now It's freezes up and some components of your vehicle could be frozen Which is hard on them obviously especially your fan your fans gonna have to be packed full of snow or potentially could be and You go to start your car It's idling out there and you're doing some shoveling or you waiting for it to warm up when you're out of start Fan goes to turn on and it can't and now you've got an overheating issue You could potentially blow a fuse for your fan overheat the engine or you can actually ruin your family or sometimes It'll the amperage draw enough before the fuses blow Especially an older fan will actually burn the fan out and those fan modules can be assemblies can be pretty expensive So that's another thing to kind of keep in mind Another common problem too after especially with our temperature fluctuating it has been over the last little while Temperature changes where we get a you know a lot of snow it melts and it freezes again Now you've got ice around your door rubbers ice in your door locks ice on your window rubbers You go through the drive-through and you're pressing that button repeatedly trying to get that window down And you know finally it breaks free and you get your order But what you're doing is you're taking life out of your motor and you're potentially damaging those seals and gaskets and stuff One thing to help prevent that is a silicone space spray We do put that on here It's something only takes a couple minutes You put that stuff on when it's dry and it helps the ups the ice and stuff from sticking to that So your windows will move a lot for yourself when you're coming on to winter time You're coming in for an all change or something or any other service And you want your windows or your door rubbers sprayed. It's a good thing to ask It could save you some money down the road could save you some wind noise issues Key lock same thing a lot of people now key fobs a lot of cars Don't even have a keyhole to use but some of the cars that still have keyholes You're using your key fob all the time to unlock and lock your doors You do that and now you've got a key that's sitting there not moving Could be two three years before you ever need your key and you come up to your car And you get a dead battery or something and you go to key fob and there's nothing or the key fob battery itself Is bad now you need to take your key out and actually put it in the car and unlock it or same thing as a trunk You always use your trunk button, but you got a trunk keyhole there You've never used before well in our climate.  There is some Graphite for locks that'll help keep that stuff moved up So just something else to mention when you come in the garage and you're like, you know Coming on wintertime maybe like to get my my hinges and my door rubbers and seals and my door locks I'll lubricated for the winter to keep all the salt and the ice and corrosion. That's probably a good idea So anyway, a few quick tips there Over the wintertime the Atlantic Canada here to keep your car running good and keep yourself from having issues That's been another addition to the tech talk and can't wait to do this with you. Thanks


And I did a podcast with a woman who is autistic and she was eight months pregnant and she talked about the pregnancy piece as an autistic woman and how that impacted her and how the healthcare system was not prepared to hear her needs and to hear the challenges she was having because of her neuro type and that's a whole other podcast probably. But you know that that's important too for those folks that do choose to get pregnant whether they're non-binary or female at birth you know you have to have a healthcare team that understands what you need. So


All right, Sabra trader is welcome on back here this They say anyway Tuesday Wednesday Thursday. We're losing track of our days here this wednesday september 29th Shortly after 2 30 p.m. Eastern time and of course this time the day at least welcome on back for our afternoon meeting Great to see all of our students and members alike here bonnie deb. I got a jeffrey here I got patrick mark got jeff b also we picked up some nice Change between cei here halted on the way down and also a couple of other trades We were calling out so far heading into the afternoon alongside them We have gmsw cat lean and a bunch of us otherwise great to see all of us here lory included so without further ado We have class coming up at 3 p.m. Eastern time the phase 2 stock course finishing things up with lesson number 4 We'll more or less do a different variation of our traders talk workshop in class And it's not to say answer just a variety of questions like a mishmash But it's really evaluating and studying and researching You know trades in full almost reverse engineering them from a to z and seeing where we could have taken the best entries What could have happened by jumping in at so-and-so price? So no that's really the best way for you to learn in hindsight at least when the markets are closed or during off hours So we'll be conducting that class at 3 p.m. Eastern time We have the the last hour of the market day left otherwise So what we'll do is go right from top to bottom we'll rescan the market for those of us not joining us in class Obviously cei will put on our list to begin Now on this trade cei to go Very slightly into this move Because we were watching this all from the morning today all from the afternoon I mean really since the market opened it made the slingshot and ran up Um, I'll tell you I personally missed out on the first half of this move at least from the morning I ended up coming back from class and it was chopping around at that point So I had no interest in the stock from there But man this ended up beginning to make a nice break back over resistance at 4 I jumped in right off the pop from 4 here got about 25 cents and then on this shake down I was watching it from the corner of my eye and like, you know, you see this shake and I think all right Well, this could be it. This could be just where it tanks Well for as big of a shake down as we had we had as fast of a pop back up That's a clue. That's a clue right there to me So I ended up actually just leaning right into the trade at four dollars and 23 cents And I rode in the chapboard right there. I said I'm looking for a bounce on this stock right away And I wasn't expecting this to make the move up to five as it almost did just before man This ended up running up so nicely. I left a ton of money on the table. I got uh 2000 shares in from 423 I took half out under 450 440 was my my close there. I thought it was gonna shake from that last from the consolidation here So I took half out ended up squeezing back up And then I took the second half the final thousand shares that I had out at 462 that was my exit price there So as you could see this thing just popped right above that price and kept going I thought 465 would have been a strong resistance level Uh, hey, I was wrong. I'll take it though. I mean, I'm just glad to have a piece of the pie But nonetheless as this stock kept going up and up and up you would hope to see it make the move up to five But obviously as you could see right now, this thing just had a hell of a shake One of our students came back to the trading room and just asking, you know, if it was halted right away Yes, you could just tell like obviously it's it's not trading here. But is there any news on it? Well, I didn't see any news as I wrote back to that student Let me just double check right now to see if there's anything that popped up since So I don't have anything here from benzinga pro. This this was just the volatility halt It just moved down too much too fast to the point where they ended up freezing trading for five minutes It's not five minutes, then it'll be 10 minutes got halted at well actually 225 right now So it really should have been opened up Uh, sometimes volatility halts can be 10 minutes long I don't know the structure as to why it's 10 minutes over five most volatility halts are, you know, flat five minutes But there are some that are 10 So we'll see what happens coming up at 235 and 54 seconds based on my time in sales right there That should be when this will reopen up We'll come back to it as we go through all right. We'll put it on our watch list to begin Uh, I always like to say this it's a semi joke, but semi truth. Uh, it's the ctu watch list Not the ctu trade list We're not forced to trade this stock if this reopens up in tanks You know, perhaps we could find a bounce area at some point But folks don't try and catch the following night if don't assume the support's gonna hold Because I mean I was writing to you folks in the trading room I was looking for support right off of 432 to 439 as that was a really strong top I wasn't expecting this halt and I wasn't expecting this fast of a drop But it's to say that if you try jumping in right off those prices that I called out You know, you essentially caught the falling knife You didn't wait for it to break under then back over this just kept falling to the point where it just got halted There was no opportunity to buy this stock based off the rules that we teach So at this point it wouldn't shock me if this ends up reopening up a lot lower and then perhaps bouncing back up from there Um, so we'll see we'll put this on our watch list to begin All right, so like I said, we're finishing up the phase two stock course coming up at 3 p.m Eastern time 305 so we'll go right from top to top to bottom here I got a call out coming up There's a stock that I saw I was like breaking a new high just a moment ago I'm sure we'll find it as we go from top to bottom The grom stock to begin the stock ended up making a huge run for us going into the early afternoon shortly after 12 30 Eastern time ended up running up like out of nowhere from 2 up to 6 You know got halted a couple of times on the way up got a halted once on the way down and You know if you follow me here in these afternoon meetings, you know what I say all the time for a stock that gets halted on the way down That's a rack that's game over So, you know this stock, I guess it made a little wiggle back up But then right back down so not much happened here one of us on youtube saying we both says it's suspended Well, yeah, it's halted from volatility. I mean, we'll see what happens actually Hell check in with it right now because the time is 2 36 Yep, there's the reopen Right on cue Actually, I don't know if this was the best idea because this is actually locking up my platform I could just tell based off my my cursor moving here. That's not good Oh boy There we go. All right. All right. Let's try and stay away from this stock at least during this meeting It's still going to be on our watch list. There's the reopen much lower and then the slingshot so far off of 4 That's uh to a good degree what I thought could have happened there.  All right. So this is locking me up here folks All right. My apologies on the foul language, but nonetheless. I'm having a tough time switching the screen here I might have to uh go rogue and just kind of uh Launch a second second workspace for us here in a moment. If this doesn't reload bear with me for about five to 15 to 20 seconds Perhaps It's my jeopardy theme music while we wait I'm launching up a second second workstation right now folks So if you could bear with me here for just a bit, I should have realized this also just walked right into cei locking me up here on my platform Up. Let's see. Okay. Move away. Oh, thank god. All right. Good. I was prepped up with the backup Platform ready to go. But nonetheless, here we are ev easy fl my apologies there on the way But easy fl making a nice afternoon move. I'm not going to jump back to cei. That's for sure At least here during this meeting But nonetheless this stock making a nice pop back up right now Obviously, it looks like a new stock new ipo dating back to you know, maybe a couple weeks ago perhaps We've done pretty well on recent ipos new listings perhaps and I was going to get to this anyway But the palt trade relatively new ticker and ended up just completely taking off today So i'm not expecting that uh easy fl stock to become exactly what p a palt became But if it can make a fraction of the type of run that that stock made today, that would be phenomenal So, yeah, the easy fl definitely, uh, we'll put on our watch list here so far It's a stock i'm not really that familiar with obviously is it's a new ticker, but we'll see All right, so getting back to palt, I guess right palt So let's talk briefly about this for a moment because this stock as soon as the market opened up Oh my god ended up completely taking off now I want to just jump back because I want to give a shout out to one of our members here in the trading room John s he was commenting on this yesterday and perhaps he knew something about the company in terms of what they do It's just to say when he'd called it out here I just didn't think it was worth watching at the time It was trading like low volume per minute very thin order book It was building the pennant so I was expecting the drop at first and you know it ended up just kind of dropping off heading into the close I guess a little pop after but man out of nowhere this stock as soon as the market opened up I guess you have four minutes after the market opened up This thing ended up popping halting and then running up from that point as you could see broke above resistance What appears to be off of seven dollars and 25 cents and from that point continued its move up So you know I know a bunch of us did really well on this trade John s included want to give him a shout out once more But I think he had picked up like five or six points on this trade all together Now I was in class teaching with most of us, you know after 10 30 eastern time from 10 30 to 11 30 eastern time We were in class So we were kind of studying the morning that was and not really trading live So it's unfortunate, but I ended up missing all of the run up but It's you know my entry in the afternoon just goes back to what we all talked about in class earlier today And that just has to do with preparation So long story short coming off of class. I saw the stock at 15 How am I doing the stock at 15? It went from you know six bucks all the way to 15 today I'm not going to chase it So I waited for the pullback and I waited for this to drop down It dropped down all the way down to the opening halt print even broke under that a bit But where this stock got halted heading into the late morning it reopened up right here at 10 dollars and 63 63 cents And that's the exact price level that I was calling out at the time 10 65 10 63 exactly This is my entry. I got in right from here average price at like 10 69 or so And ended up running it up. I left a lot of money on the table. I got out right at 11 50 It was on a double position, which I'm pretty thankful that it worked out that way but Man, this thing was just too volatile, right?  We caught the bounce inside our live trading room But if it doesn't continue to move up and if it moves back down Then we know what way more likely than not it's going to continue to go in So as you could see it slammed right below that price 10 63 10 61 the line and Obviously now just continuing to sink. So one of us on youtube asking Do you think palt could have a late rally left to it? Considering its volatility that would be the only thing that would lead me to believe that it can like just for a stock to make a 10-point move up then 7-point drop off if there's some sort of bounce Perhaps it can make a dollar move back up $2 move back up and and that could be worth your time for a nine ten dollar stock, right? Like so for the volatility, I love it But with this just breaking below that critical level I would only assume for that 10 63 price or 10 50 otherwise perhaps that 50 cent level to act as resistance So i'm not as keen to watch this right now rjf hitting into the close Um, I I always remember remember this there's there's one stock I ended up kind of uh dismissing briefly or very quickly in the afternoon meeting and right as we finished up It ended up making this giant run up and uh had a little egg in my face But you know what? I'm happy to have egg on my face when The unexpected happens meaning you know more often than not for a stock like this We're only expecting it to consolidate and sink or just not really provide as much for us So we'll focus on others at first if this ends up making a sharper bounce back up Then I would have no problem, you know for for us to call it out there at that point All right, so we keep going we'll kind of skim through now at this point really uh sdpi not much happening there Cheapy here em pretty flat That's what's too cheap fami. Ooh too cheap but making a really nice pop. Wow Well, there's another instance of me having a little egg in my face nice pop for 25 cents You know for as much as it's moving up. It's you know, I'm still only up five cents from that point So, you know just be careful on a trade like this But the part about this trade that I like is not only you know the trend up on the intraday chart But looking at the daily that that's a clear gap fill right so in a dream scenario This could fill most of the gap perhaps if not if not all of it, but uh Yeah, fami. I guess I'll slip on the back end of our watch list for right now Cheapy here prog got a bunch of cheapies here, which I like the eng trade. Wow back up to 314 I say backup because it moved up there earlier this morning. So I'm actually caught off guard by that right now This looks really nice. So I was waiting for this move to happen folks Not as if I was like, you know telling you that I mean I was more kind of inside my own head Dating back to yesterday morning pre-market This stock back on monday ended up closing right around three So I had it on my side list yesterday morning and it wasn't really one I focused on at all after the open but You know, I'd like to think for the teas that we had at three back on monday If this stock can make another crack and run at three we have more buying off of three Perhaps it could run so it did that earlier today and now it's making the much much better move. So Yeah, easily eng but we're gonna put this on the top end of our list here This one looks phenomenal also short above this craps out the other way, which I would not expect right now But uh, you know, you never know All right, I learned my lesson lesson on cei. I'm staying away from that thing for right now At least during this meeting That this one ended up being a much better short than it was along and I'm not fully surprised by that I got chopped up on this trade earlier today. I did all right on it at first But then I ended up trying to short it a couple of times I lost three cents than four cents then I just stopped trying to you know, take the trade at that point It was getting too choppy but shortly after I took those losses It ended up breaking this trend line that you could see our I'll put it up But you know, you could see this trend line essentially off the lows and once it broke that Be broke it ended up providing for a really nice drop off here a really good short on this trade So no where you hear where you hear me talk about a stock crapping out the other way You could short it if it's easy to borrow. I mean just look at this ncna Cannot be a better example. So that's where I say going back to e ng Which I don't think this would be a short you know short now given this you know spike up But you know, you never know just watch to see what happens perhaps off an iceberg level at 335 perhaps 22 000 share order on the Ask All right, let me zoom out on the five minute chart. How are the markets doing today and dropped off early on But made a really nice recovery. There we go. All right. That's cool We'll take that clean spark familiar name blue apron also a familiar name. We'll just check those out here If we're hopping over to the loser's list Apron was the other one. I was losing track of that last one there Uh blue apron little spotty not much going on All right, folks, we'll hop over to the loser's list right now actually really quickly We'll go to lucid lucid motors here first just to check this stock out This stock's been on our side list morning and afternoon watch lists over the last few days and No, as you could see ended up making a nice move bounced right off at 26 at the market open Actually goes to show how strong the level is it bounced off at a second time right before noon So ended up making a nice bounce from that point pulling back down to 27 as you could see All right, listen, this is definitely a tradable stock if you did well on this stock from today or you know The last couple of days this week then 100 you should put this stock on your personal watch list I'll put it on our side list right now and you know if we don't really find much more here at the end Then I would have no problem putting this on the main list All right, so we'll hop over to the loser's side bunch of stuff hard to borrow here to begin number nine edit is easy to borrow pretty flat You know what actually we'll just jump back up actually these stocks are down enough to where maybe if there's some sort of You know opportunity for a dead cat bounce who knows no idea What the hell is this thing g a g fa i? Hlbz I watched this this morning or at least wrote it on my notepad this morning It was down as much as it is now but Yeah, just no movement on the stock just very little volume being traded per minute All right, so talk about gap fills or at least you know making a dead cat bounce recovery esk type move Look at this alt from yesterday. How about that man? I mean this this stock got clobbered in pre market off of I think it was bad phase one study news And they announced it themselves. So they ended up saying that their phase one Trial for whatever drug they were making just didn't meet primary end points It was down like 30 early in pre market ended up making that full recovery and well obviously today dropped right back down And it remained shortable. So, you know, this was actually a pretty decent trade I called it out off 13 earlier expecting a bit of a dead cat bounce or a small pot from there I don't know maybe at this point you can get it, but it's trading such little volume per minute like maybe 2,000 shares per minute less than that less than that actually I'm gonna pass on this here too All right, folks, let me uh, let me know if there's anything else that you would want me to check out I gotta get to crypto here since today is wednesday. I want to make sure that we have enough time to talk about that I've gone this whole meeting without a sip of water. That's because I forgot to fill up my water bottle All right, so let's see what else we got cei in a halt at 366 says bill in the in the cyber group room Yeah, uh, not surprising, but yeah, I'm not gonna check it out right now because knowing my luck it will just kind of freeze up on me upon the reopen and Have to reload a new workspace. All right Nice scalp or great scalp says mark on the easy FL easy FL stock I came back down right now. That was from a few minutes ago perhaps mark's comment, but This one's not nearly looking as good now. That's for sure All right, uh, I'll tell you what this type of shake makes me want to like take this off the watch list at this stage really That's not good I kind of move this to the back end there like right there with the fam I guess All right, I'll tell you we'll add the lucid motors to our list Just as that's a really, you know volatile stock tradable stock And I say volatile in a good way because it has a pretty liquid order book You know a little pricier if you're you know Used to trading the cheaper stocks, but nonetheless, this is a this will be at least, you know very tradable for us heading into the close All right little crypto folks before we close out the show and talk just bitcoin Cardano and ethereum. That's all that's all we typically talk about here All right, so bitcoin first and we'll talk about cardano the cardano summit. They ended up having a huge uh multi-city multi-country Expo over the weekend they announced a bunch of stuff some some good things in the in the making for cardano, but uh to begin bitcoin Well, obviously it's been dropping off or at least it has been over the last 48 hours with the markets and indices falling Right now we're on a six hour graph so you could see I mean although it's like kind of near the lows here It's made a bit of a bounce so far Low so far in the six hour candles 41 just over 41 And now it's at 41 too wouldn't shock me if it keeps dropping off a little bit more here at this point But nonetheless, I mean I still feel like we're in great shape If anything with bitcoin one thing to keep an eye out for because as bullish as I am on bitcoin Let's talk about a couple of concerning things And really the one thing actually that I want to talk about It has to do with the moving averages because we talked about this on cardano and bitcoin back in early august And we killed it on a couple of really nice runs on cardano with that But we ended up seeing what is it the gold the death cross here, right between the 200 and 500 em is So everyone was freaking out about that like on cmbc fox news or death cross.  So, you know, that didn't really scare me that much But now at this point, we're kind of getting close to another one here. You could see the yellow 200 em a You know on my six hour graph. It's starting to kind of slip back down a bit You know, it wouldn't shock me if we have another death cross coming up in that You know having the second one there that could be a little more concerning in terms of a further drop That's purely technical analysis. That is nothing to do with any sort of, you know, fundamental news about bitcoin Or, you know, any sort of news about treasury yields and how the markets may look over the next three to four months But that is just a point of concern in terms of technical analysis But we'll see we'll see how things play out on ethereum really quickly One thing that I do like about ethereum is that I mean prior to the Bull run at least across the year so far this stack was holding right over the 500 em a extremely well And you know, of course here given how far it had dropped it doesn't shock me to see it break the 500 But it bounced back above it. It held over it so well after It goes to show at least on ethereum just food for thought like the 500 em a definitely is a really strong level I mean it's there now, you know If we happen to see crypto and the markets continue to slip off across this week Then of course ethereum will go with it But you know just if you're looking for an entry perhaps this could be a good opportunity right around the 500 All right Lastly cardano not as much on the cardano to usd chart that I want to talk about just more in terms of cardano They ended up announcing some major partnerships with dish tv with boost mobile They ended up announcing their own stablecoin Which is pretty interesting like by nance has their own stable coin I think it's called by nance coin But cardano, I think it's going to be called djed dj ed. It's in collaborate in collaboration with another crypto cheapy called kodi coti But that will be pretty interesting because that's going to accelerate the defi market for Cardano to the point where you're going to have all these new coins being readily available for traders. So I'll be pretty interesting But nonetheless the chart that I want to get to on cardano is the cardano to bitcoin because we were talking about this Like a week ago two weeks ago and we're and I don't have the lines up right now I'm not as prepared there I guess but We had a nice support level that we had called out over the last week to two weeks like practically like right here Based off of past charting where cardano made a nice bounce up Unfortunately, it's pulled back down a bit wouldn't shock me if we happen to see this slip back down Perhaps towards the 100 ema here on the daily All right, so that's all I got for you on on on crypto all together. I mean just a small drop I mean just today. I mean more of a drop from about 48 hours ago I told you folks I would get to that at least today, but nonetheless here we are now So folks if there's anything else that you would want me to check out up down left right More than 5% let me know you got class in about 12 minutes. I gotta make sure I'm good to go there All right going once going twice three times and you know, I'll check benzene approach just before we finish up Got a bunch of chat on cei. I appreciate you folks providing the commentary there for us I got two stocks. I'm gonna check out here actually just from my end before we finish up Nope, this one's not looking good. It's up 11 plus percent, but Just not much happening there This is exactly where I want to see tiva. All right, so tivo we're gonna put on our watch list It's only a 4% but this stock ended up making a really big pop yesterday afternoon I got it nowhere and ended up falling off of course But we we saw and called out this major iceberg order of 10 So here we go. It's right there at 10 now It started to fill 10 a bit, but the next time it goes up to 10 It wouldn't shock me if you happen to see a much bigger order pop up This is not a volatile setup here folks Like I don't think this would be like a setup that would get you 50 cents or a dollar Just you know, so don't try and get more than you can here on this trade But nonetheless either way as a shortable stack It makes a great setup in case if this falls breaks from 10 and then tanks Or otherwise provided this trend up if it busts over 10 and shows more signs of life from buying of buying at that point Then obviously it'd be a great call out for a long So i'll slip this on our list here too to finish things up the tivo stack All right from p.m here and then we'll close out for real, but uh thoughts on infi from uh p.m Now keep in mind now p.m Maybe a new attendee here. Maybe not but I just want to point out for any one of us on social media today facebook live or youtube All of our callouts this whole meeting is regarding. I mean outside of crypto this whole meeting is regarding day trading We're not swing trading. We're not investing. We're not doing options. So I just want you to know that first That's because I'll say on this stock your infi this provides no opportunity for us as a day trade It's just trading in penny range or a couple penny range here and there So you want to make sure that you're following something that is trading a lot of volume has a lot of momentum in its respective trend up or down Uh as crazy as cei is we'll keep that on our list But we got plenty of others otherwise the eng trade the lcid trade obviously tivo, which we just talked about And a couple of others. All right folks, but for all of us in social media, we're going to jump off right now We got class coming up in just about nine minutes time But otherwise make sure if you have yet to yet to have the chance to do so Just make sure that you like subscribe and hit the notification icon That is on our youtube channel that being youtube.com slash cyber trading you But otherwise if you're ready to join us live inside our trading room You could do so by going right down to the link on the banner below just getting yourself started right there Otherwise if you're not ready to do so yet It would rather just send over a quick question or email a trade in which you would want to cover with me Uh, feel more than more than free to do that. You could send over a quick email or trade right to my email josh at c2trading.com All right, i'm going to refill up on water because i'm as shocked as any that i went through this whole medium without like stuttering I did pretty good here. All right, so folks i'm going to get steppin I'm going to refill up water and get going inside the phase two stock course classroom I look forward to seeing most of us there Uh for those of us not trading or those of us not in class and we'll be trading heading heading it into the market close Uh, best of luck here and I know that rich will be doing commentary here for the next hour. Take care


Well it wouldn't be motor racing at the start of the event without a little bit of drama and inky the Evo's up on stands. We haven't even started mate. No it won't start that's a problem but it's not a real big drama just I don't know something's gone wrong in the gearing of the starter motor so it just means it won't start so we won't be stalling today and we'll get through tonight and then pull the gearbox out and fix it but I'll take it here over the main event any day so and it doesn't you know we're not out it's just a minor inconvenience nothing more than that. Hey you're looking any changes to the car since you went on the main event last year? A new engine actually yeah like the old engine was probably a little bit conservative and this one should be a little bit better so we'll see how it goes if the road's dry it should be good. All right good luck with that mate. Cheers mate. Not only with Glen Inkster having issues this morning, Lee Hoffa, Spide your car up on the jacks. What's up? There's always something up Jamie if you know this is motorsport so I was just something in the rear end knocking a little bit under acceleration first off the mark so we just put new high-grade axles in the rear so new CVs and they all went in okay so don't really know just see what happens and then there's always something going on and the electronic at the front end of the car that's all right some reason on the way down yesterday it started revving its head off with about 4,000 RPM idle so boys seem to fix that this morning. So a couple of scares already for a couple of the top teams I mean there's a set to Glen hundreds of hours of preparation going into this event and sometimes you just you just can't pick some of these things no you can't and that's motorsport you know you're ringing everything you can out of the machinery and you know it's something's got to give in it it's all on the limit you don't want to be over specking stuff because that's weight. Exactly it's not like they're competitive or anything are they? Yeah right yeah right all right good luck mate


Welcome everybody to the second session, which will be on the EPD, the eukaryotic promoter database. That's a relatively old resource, more than 30 years old. So I take the liberty to start with kind of more philosophical remarks. Obviously if you want to develop a database for something, you should know what is your target. And promoters are a difficult case in this respect, because it's an ill-defined term, a little bit like genes, which maybe have created more confusion than clarifications. So we probably should go back to the origins. It was introduced by Schafko and Mono, who defined it as a DNA sequence located upstream of the gene, to which RNA polymerase binds and initiates transcription. So, you know, one question you could ask is whether this was a discovery or an invention, and another question you can ask whether such DNA sequences actually exist, and they do certainly exist in the case of the Locco Perron in Nikolai. But the problem is, when people started to analyze eukaryotic promoters, on the one hand they thought promoters would be something very interesting in this case, but it also became clear that such things do not exist in eukaryotes. Specifically, so the green, what is in green, that is actually applies to eukaryotic promoters as well, and what is red is false or only partially true. So in Nikolai, promoters are directly recognized by RNA polymerase in eukaryotes. So in the case and located upstream sometimes, but in the case, for instance, of tRNA genes, the promoters are said to be downstream. But again, I'm now referring to a specific sequence motif that is located downstream, not necessarily to eukaryotic promoter in the modern sense. So the only definition that somehow works for eukaryotes and which we applied in EPD when EPD was created is that promoter is a site of a short DNA region where poll 2 initiates gene transcription. And note that I specified that it should initiate gene transcription and not just transcription, because RNA polymerase can also initiate transcripts which do not give rise to RNA or protein products. So this is a theoretical definition. I mean, it's something that we imagine is happening in the nucleus. But if you want to compile a database, you need an operational definition, based on experimental data. And here the definition is, it is a cluster of mRNA 5-prime ends mapped to the genome. Note also, these thoughts about the definition basically have created a situation where many people ask me, so why do you call it a promoter database? It is a transcription start site database. I think it would make sense. It would make things easier if nobody had introduced the term promoter for eukaryotes. But given the background, we call it promoters. And this is basically also reflected by the newly coined term promoter arm, which is used to describe, to refer to the entirety of all transcription initiation sites of a genome. Now, I should say gene is also an ambiguous term. I wanted to remark that we recently extended EPD new to certain classes of non-coding RNAs. But most of EPD really relates to a protein coding gene. Let me give a brief historical overview. I started about how many years ago, almost 40 years ago, when a compilation of about 60 eukaryotic promoters was published in a review article by Breistner and Schambau. And so because I was interested at that time in promoters, I typed in this compilation into a computer, a big one about the size of a refrigerator, which had six kilobases of memory. And then I continued to update this collection with new promoters that I found reported in papers. So in 1986, we compiled, I started a PhD at the Weizmann Institute in Israel at that time. We compiled a list of 168 promoters in a paperinucleic acids research. And then I had the idea that maybe it would be better to publish this in machine-readable form. And I contacted people of the MBL Data Library and they were very happy to put my text file onto their magnetic tapes. So then later in, so that's basically how it became a kind of database in the modern sense. Although content-wise it was of course not different from the table that we published on paper. So in 1997, we introduced the WebCyber server. Then in 2005 to 2007, we, that means mostly Giovanna, developed the SSA Web Interface and the Gipsy server. Somewhat later, we created the MGA, which is a structured, a more structured data backend. And then it became necessary to completely redesign EPD. So it changed the basic structure and also the maintenance procedure. I will mostly show you what you see today is EPD new. And in 2020, EPD new covers 15 model organisms from diverse eukaryotes. We have problems switching. Okay. So the guiding principles of the old EPD database, which basically have not changed in its essence, but in the details very much. So promoter evidence, we accepted only experimental evidence, but under some under defined rules, also homology-inferred promoters. So this we don't do anymore in EPD new. Now an important assumption is that kept five prime ends of eukaryotic RNAs actually are created by transcriptional initiation, not by internal cleavage of an RNA. This is debatable. This has been debated. But if we don't make this assumption, we just have no data to make a database. So I won't comment much on this question. The primary data source were journal articles. The experiments that were used at that time to characterize promoters are called RNA sequencing, nucleus protection, primary extension, is the exception of RNA sequences. The other ones are not used anymore. What is important to underline is that the contents is based on a critical analysis of data shown, and that means mostly pictures, and not just on conclusions reported by the authors. So it was a lot of work to read all these articles. The promoters, that's something very unusual at that time. We didn't represent promoters as sequences, although most people thought of promoters as sequences. We represented them by pointers to positions in sequences. And the purpose was comparative analysis of promoter analysis. EPD was also used as training and test sets for promoter prediction algorithms. And we thought it would be a nice resource for bench biologists who are just interested in specific promoters of their preferred genes. But I think we were not so successful in publicizing EPD with bench biologists. So just an example of the old experiments. You see the picture. This is a primary extension experiment. People use a primer inside somewhere in the upstream regions of the RNA and then reverse transcript this primer and put the product on a gel. And you can, with the parallel sequencing lane, you can send a sequencing lane. You can guess, you can map the transcription initiation side to the sequence. In this case, it's actually quite sharp. What you may not realize is that at that time, one promoter was worth a paper. So in order to compile the 170 or 180 promoters in our first publications, we had to read that many papers. So this is slightly repetitive. So my personal motivation to create EPD was discovery and characterization of promoter motifs. And then other people's interests were more, especially computational biologists, were more to use it as a training and tested for promoter prediction. And what I want to say at this point is that actually the signal search analysis package, which Giovanna introduced this morning, was developed in parallel and highly synergistically with EPD. So basically, if I wanted to characterize promoter motifs, I needed a data resource of promoters and I needed a software tool to find the motifs. Motif discovery in promoters was so obvious as one could think. In a typical motif discovery situation, you have DNA sequences with a beginning and an end. And you know that these sequences have some function or they have some biochemical activity. And then you try to find a motif that is overrepresented within these sequences. But with promoters, you don't know where the promoters start and where the promoters end. And this has not changed since then. A promoter is an object which is mostly defined by a kind of center position, the TSS, or the major TSS, but which has very fuzzy borders. So that's why we basically, SSA has a different design from most other motif discovery programs. So to set up the problem of finding promoter motifs in a rigorous way, I defined promoter motifs as the generate sequence patterns, which can be represented by consensus sequences, possibly with mismatches allowed or a position weight matrix or maybe something more advanced. And the second element in the definition is that these motifs must occur at a fixed or constrained distance from the transcription start site. And finally, they should be overrepresented in this region relative to the start site in a statistically significant way. So that means the litmus test for promoter motifs is that they are locally overrepresented at a certain distance from the TSS and this overrepresentation is statistically significant or maybe just intuitively convincing by a motif occurrence profile as shown in the lower right corner of this slide, which is from a publication from 1990. So this figure was actually produced with an earlier version, which produced somewhat different pictures. So actually, this was the main use of EPD. This is also maybe what made it popular and which also called attention to my work. So in 1990, we published matrix descriptions of four major vertebrate RNA-POL2 promoter elements which we derived at that time from 500 to unrelated promoter sequences and you see the sequence logos of these motifs below. Now, since I've changed and we really have to understand that the very biological research on transcriptional regulation and promoters in particular has completely changed. When I started EPD, the data were produced by small labs, everything, all data. The technologies that were available were targeted at individual genes and usually research groups were also interested in a specific gene and the data were published in journal articles, piece by piece. And then in the middle of the late 90s, the development started, which one could call the high throughput biology revolution, the two factors which contributed to this development on the one hand, the invention of high throughput technologies which allowed, it was very applicable, which produced data in large amounts and some of them were applicable on a genome-wide scale. And the second factor was the human genome project which publicized the global approach. So you should go global. And associated with this was that the data from high throughput biology, they were no longer really accessible through journals, maybe conclusions, statistical analysis results were published in journals, but the data as such were only available online in computer readable form. So this basically made the traditional data collection procedure by EPD ineffective, mainly reading papers. So in recent times, the most significant technological advance in gene regulation was the introduction of so-called next generation sequencing technologies. They obviously not any more next generations. They are more like previous generation, but the term has remained. And these assays, they produce huge amount of data, they are all applied genome-wise and among the NGS-based assays which contributed most to our understanding of promoters or cage, which is a technology for quantitative single-based CSS mapping, gypsy for mapping in vivo protein DNA interactions and M&A seek, which allows the precise localization of nucleosomes. So just a slide on CAP analysis of gene expression cage. On the left side, it's schematic outline how the technology works. The essential part is that a method to separate messenger RNAs from degradation products based on the presence of a CAP structure, then a linker is added to the CAP structure and from this linker, the sequences of the five prime ends of bona fide complete transcripts are generated. Initially, there is a technology called Sage, but today, obviously, with any of the popular short-treat sequencing technology, these reads are then mapped to the genome. So what is important about cage is that it is a quantitative method to characterize RNA. So in a way, there are some substitutes for other types of gene expression essays like RNA seek or microarrays. However, the advantage that it gives you expression values, promoter-specific expression values, as you probably know, many eukaryotic promoters have multiple. Eukaryotic genes have multiple promoters, and these promoters often are regulated in tissue-specific manners. And as a side product, this technology also provides a detailed picture of the transcription initiation pattern in the promoter regions, which may tell you something about the specific molecular mechanisms by which transcription is initiated and regulated. Now, cage is not the only massively parallel five-prime-end sequencing technology. Similar essays have been invented. TSS seek is based on an oligocapping method to sequence kept RNA. More interesting is grow-capped. This is a method which sequence kept nuclear run-on transcripts. This technology has some importance for exotic organisms where actually the kept end of messenger RNAs do not derive from the promoter, but from a spliced needle that is spliced onto the five-prime region of the gene. This is true for C. elegans, for instance. So without grow-cap, there will not be EPD for C. elegans. Now, just to show you what kept does, you see here the initiation patterns of two promoters revealed by cage. On top, you see a narrow promoter where most transcripts actually initiate at exactly the same position. And below, there is a more broad promoter where the transcription initiation pattern is quite messy. I would say, please have a look at the numbers at the scale on the right, on the left side, especially in the upper part. So the peak represents 83,000 individual cage tags. So that is pretty high coverage. Now you can extrapolate it to maybe 30,000 promoters of the genome. And you realize how much information you get with a single cage experiment. And don't forget, cage is applied to tissue samples. So the cage resources, they offer that level of resolution for hundreds of the different tissue samples. MNA-seq is also an old technology, basically, which has been used for some time, which allows to separate nucleosomes. So MNA-seq is a particular, as shown on the left side, is a particular nucleus, which does not attack DNA that is reptile nucleosomes, but then precisely cuts at the borders of the nucleosome. So MNA-seq is used basically as gypsum. It just cuts out, it produces nucleosomal fragments. The picture on the right is wrong in the sense that the overhanging green fragments are far too long in MNA-seq. They would be very close to the protein DNA complex. But I also introduced, show this picture because MNA-seq can be combined with this gypsy for histone marks and in which case it produces a high resolution mapping of subsets of nucleosomes which carry a certain mark. Now MNA-seq led to a surprise discovery, namely that the promoters have a very specific nucleosome architecture. Left you see the picture that was obtained for yeast and on the right side, a similar picture for humans, which we published some time ago. So you should... The black curve shows the distribution obtained after... Well, the red and the green curves basically show the distribution obtained with the five prime and the three prime tags. So the red curve marks the beginning of a nucleosome and the green curve, the end of the nucleosome. At that time we did not yet shift the tags. So NGS has really changed our views of promoters. Some new insights gained cage, revealed that not only promoters but also enhancers are transcribed by polymerase II. And Chipsic allowed us to identify promoter-specific histone marks like H3K4ME3 and to investigate the function of histone marks in a more systematic manner. And finally, MNAC has revealed that active promoters are nucleosome-free. This was suspected but not really demonstrated and that they are flanked by position nucleosomes on the downstream side. Now, I should say something about position nucleosomes. Maybe let's go back to the previous figure. It wasn't clear, it's still not entirely clear in general whether nucleosomes in different cells or on sister chromosomes within the same cells actually occupy the same positions or whether they randomly fill up the chromosomes and just at leaving spaces between them of variable lengths of 10 to 50 base pairs. Now, what today is clear that in a large part of the genome, nucleosomes are completely randomly positioned, maybe 80%, maybe 60%. I'm speaking of human, not of yeast. And if the nucleosomes would be randomly positioned, I mean, in any cell or in any chromosome, obviously the nucleosome occupies a very specific site at any given time. But if they would occupy different positions in different cells, then we would see a completely flat pattern in this plot. And so these peaks, they actually mean that for a large number of promoters, at least, the nucleosomes occur at precisely the same positions relative to the TSS. And this is specifically the case in the downstream regions. So in response to the high school put biology revolution, the time has come where we came to the conclusion that we need to completely redesign EPD. This doesn't necessarily mean that we would not stick to the basic principles, like that we rely exclusively on experimental data, so we wouldn't use sequence-based predictions, or that we base the data contents on quality-controlled data and the like. But it meant that we would completely change the procedure to generate the data and also choose new formats and new ways to link the promoters to experimental data. So that's why in the years 2001 to 2002 we completely redesigned EPD from scratch. And so that means that during a short time period we had basically two databases in parallel, which were used, but in the meantime EPD new has completely superseded EPD and so we call it again EPD. Here the design principle. So since we didn't have a large labor force at our disposal, so EPD new was designed to be a light resource that would be cost-effective in terms of maintenance. Unlike the old EPD database, we focused on model organisms, so we decided that has to do with the fact that now EPD is based on data obtained with technologies that characterize the genome at once, so that was not the case before. So that means that there are relatively few organisms for which we have genome-wide data. And then we switched to automatic extraction of promoter information from high school data. But that obviously is dangerous.  And so we complemented the automatic procedures with stringent quality control of the input data and the stringent quality control of the input data is still largely done in an intuitive and manual manner. So we also do visual and automatic checks of the output database, and if they don't look okay, so then we change the extraction procedure. We have an entry viewer which is now mostly based on the UCSC genome browser, but in this viewer we add homemade custom tracks. Promotor selection and analysis tools are offered by independent resources, namely the gypsum and SSI servers. And information on promoter structure is not anymore hard-coded in the database itself. It's delivered by on-the-fly integration tools accessing the MGA repository. And we also, for the web server mostly, we made it more interoperable with external resources through direct navigation buttons. So the goals are in a way very similar. We still, we now can see there's two major objectives. One is to produce an accurate SS collection for a selected number of model organisms, and if I probably should have said high quality rather than accurate, so high quality in terms of comprehensiveness, higher enrichment in true promoters, whatever that means, and higher accuracy in the TSS mapping itself. The target users are computational biologists, and they are also geoscientific research researchers. Now the second main part of EPD is now a viewer for viewing promoters as such and related chromatin data. And the viewer is meant to provide a useful data selection of cage, chip, sec, M&As, seek data and additional data, so to provide an optimal data display in the UCSC genome browser. The target users for the viewer are more wet lab biologists interested in specific genes. Now, and third, we deploy promoter selection and analysis tools via accessory resources. There's still a search page where promoters can be selected on the basis of hard-coded information in the EPD database, but otherwise we delegate the selection and analysis tools to the gypsum and SSA server, which obviously can be applied to other genomic features than promoters. So in particular, we can select promoters on the basis of motif content, chromatin state, and basically any type of additional genomic information. And still we offer promoter element data for download from the mass genome annotation data repository. The target users for promoter selection and analysis tools are, of course, everybody, experimental biologists and computational biologists. Now, this is the most important slide of my talk. It's a busy slide and it explains in principle everything you should, you have to know about EPD new in order to understand what it is. So it all starts with external data from, for instance, from GEO, from Ensoble, from EVI. And in particular, we need as input cage data or similar data and gene annotation catalogs like the GenCode gene catalog. Now, these data, they go to quality evaluation control and some never make it into the MGA data repository. I listed here EPD, MGA data repository, but the EPD is not really necessary because most of the data in the MGA repository are somehow relevant to promoters. Now, from there, we do a second selection step to select the input data for the EPD assembly pipeline. This is the computational pipeline that takes some data as input and in the end creates a catalog of promoters with consensus or DSS positions. So the output of the EPD assembly pipeline is then evaluated. We have a motif-based evaluation and we do also manual checks. So what usually happens is that after the first trial, we are not happy. So the quality control steps provide feedback and sometimes the feedback prompts us to remove a data set from the EPD source data because we realize that it is of bad quality or it has some other disadvantages and sometimes we may add some additional data. But most of the fine-tuning then goes into the assembly in the computational processing pipeline and we repeat these cycles typically two to three times and then we tend to be happy or we don't see any improvement in terms of quality control. So I'm now going to say a little bit more on the elements shown on this slide. The first element is the MGA repository. So Javan already told many things about it so this may duplicate. First, it is an FDP accessible directory it is hierarchically organized at three levels genome assembly, series and samples. All data are in SGA format and for each series there is an HTML documentation file and then this describes the source data, the samples and also the reformatting procedures that we use to generate our productive version of the data. The MGA repository is accessible from EPD it's accessible from SSA it's accessible also from the chip six over and additional tools we have developed. The interface, that's an important point. The interface with the web servers is achieved through machine-readable data description files so each sub-directory in the MGA repository has two data description files one that describes the series and one that describes all the samples and the menus you see both in the chip six over and on the SSA server they are automatically generated by reading these data description files which are in the MGA repositories and if you are curious what they look like then you just have to go can go to the FDP site and look at these data description files. Note that the MGA provides the source data for EPD and that is mostly caged data and other high throughput transcription start site mapping data but it also provides the complementary functional genomics data for promoter analysis and for promoter selection. Now just a very brief overview over the MGA it contains primary data, gypsy, chromatin accessibility DNA methylation, not much for the moment and transcription profiling data we call it now transcription profiling because it contains only data that are related to the transcriptional process and which are collinear with the genome so we exclude RNA-seq data that are produced from spliced cytoplasmic RNA then it contains also derived data so we consider gypsy peaks for instance published peak lists like the peak list from encode and genome annotations such as promoters, splice junctions and the like and we also have a very interesting and diverse section called sequence intrinsic these are features that are computed from genome sequences only they don't need any additional experiments in addition to the genome sequence those include cross species conservation scores genome variation data like SNPs for instance so let me say a few words on deep in the assembly pipeline so the goal is to identify promoters and promoters are defined not as single transcription start site but as transcription initiation regions and underlying is a certain assumption about the size of the promoters so we think that a promoter can be as large as about 100 base pairs or maybe 150 base pairs that's maybe what corresponds to the largest transcription start site clusters we observe clusters that are clearly associated with the gene and for these clusters we try to select the most representative single initiation site often we choose the one which is most used but in general we take the one that is most used but usage is in a specific way averaged overall tissues that are available so that's how it works so this is an old slide which refers to version 3 of EPD-New now we are at version 6 but the principle is the same so the input data is a gene catalog at that time we use two CSC known genes and primary TSS data these are cage libraries in general the primary TSS data they undergo a read mapping state and then we do kind of peak calling and identify clusters of transcription initiation sites the UCSC gene catalog is filtered based on some criteria so for instance we still treat the basically filter for protein coding genes the recent collections for non-coding RNA promoters are based on a separate pipeline these filters for that kind of genes and we also eliminate pseudo genes or genes which are incomplete or where the coding region where the initiation codon is missing or other strange things basically we map the peak the cage type peaks to genes by proximity many of the peaks fall through either because they are not near any gene or because they are just a little bit too far away from a gene we require a different range of about 100 base pairs relative to a gene start annotated in the gene catalog so this leads to a validated list of transcription start sites for each individual cage library and then we basically do a kind of similar procedure for the TSS obtained from each samples we now have many we probably have more than 1000 samples so we merge this and we take not the promoter which has the highest number of cage tags in total but the one which was selected as the major promoter in the highest number of samples and that leads to a new EPD promoter collections which contains the reference TSS site for genes sorry so now we go to the evaluation so I will this slide shows you the primary data for the current version of EPD so we now use Gencode as the gene catalog we also use a new data type called Rampage this is a technology which is based on paradigm sequencing and it's actually meant to directly associate the capped 5 prime end with transcript which to which the 3 prime end of the same fragment matches as you can see on the tags the number of tags are pretty astronomical I mean we have now nearly 40 billion individual cage tags now to motif the evaluation I will show just motif based evaluation so we introduce this procedure about 15 years ago so the principle is that we know actually that specific motifs are over represented at characteristic distances from the TSS these motifs are for instance the Tata box or the initiator side and then we say that the height of a motif peak is indicated of promoter enrichment in the collection of course if I say enrichment we target something like 95 or 98% and the width of a motif peak is indicated of the precision of the TSS mapping so the result of this evaluation procedure is a statistical quality measure for a promoter collection as a whole it is not a method that would allow you to decide whether an individual promoter is actually a true or a false one so in that sense it's not an evaluation procedure for promoters it's an evaluation procedure for the computational pipeline that extracts promoters from high throughput data and unfortunately we don't know the absolute enrichment figure because we only get relative values but of course the visual checks we do they indicate enrichment is very high there are some caveats with this procedure especially if we compare promoter collections between different organisms in the first EPD release published in 1986 70% of all promoters contain a Tata box and today we are down at 10% so why this is the case it's the case because initial sets were biased by strong promoters at that time you could only characterize a promoter if you were able to extract tons of messenger RNAs this is the case for genes like beta-globin so messenger in a cell is encode for beta-globin also for hormones insulin or valvoli and the like so this means that one has to pay attention an enrichment in a motif also may mean that we favor for instance highly expressed promoters which we do not want so this is an example below is the peak the signal occurrence profile again generated is OPOF for an EPD for the current EPD release for comparison is the same figure obtained for gene starts from a gene catalog so you see a very sharp peak indicating that the mapping is precise but as said before the number of promoters which contain a tartar box is not extremely high actually this is for an older release it has increased a little bit now note that this is a screenshot from the EPD website the EPD website posts the quality this type of quality report for each data collection now the second important element of EPD is the EPD promoter viewer so the entry point for the user is a single HVML page with essential information and links so the contents of the page is text-based annotations genome coordinates, gene symbols and the like a picture of the genomic contextual feature this is a picture downloaded from UCSC a link to UCSC for dynamic visualisation and hyperlinks to other resources for instance works with Regulom now the graphical interface is UCSC genome browser based it has EPD supplied custom tracks for instance for display of cage data and it also provides a customised view so the implementation is as a public truck hub so this is a structure that has been introduced by UCSC a structured and annotated collection of homemade data tracks in standard genome browser track formats like big week and big bed now a truck hub can be temporarily connected to the UCSC genome browser by various mechanisms directly from the browser menu or automatically via HTML links the EPD viewer is also based on a configuration and annotation file in text format and all data are really downloadable via a URL so the viewer configuration file it's called a session in UCSC terminology triggers the connection of the browser to external public or private truck hubs it defines which tracks are displayed or hidden it defines also how the tracks should be displayed and in what order and the session files they can be manually uploaded or automatically via a URL so I think now it's time to go to a demonstration on the web server so let me try to switch to another screen okay so this is the EPD home page so if you want to access the viewer you click on an organism and then on this on this page you see some summary information for instance you have 29,000 genes promoters for 16,000 genes and you have a picture on this side now this is the UCSC snapshot of the UCSC view you can click this picture and then you are in the UCSC browser so this shows a selection of tracks some of the tracks are hosted by UCSC all those come from the EPD track hub if you scroll down you see the EPD viewer hub and you'll see the menu of the different components so let me go through the tracks you see on top is the notifications so what you see in this example is that you have positioned an H3K4 ME3 labeled nucleosomes on this side on both sides so that's probably because we have two promoters which direct transcription in different directions so then we have tracks this is a histone modification tracks specifically H3K2 H3K27 acetylation which is supposed to reflect the activity of the promoters but also of enhancements if you are interested in what's going on you can change of course the display mode and instead of transparent you choose non and now you see the different tracks separately you see that this promoter appears to be highly active in the UVAC cell so if you go further down we see actually the cage provides a single base resolution this is actually the distribution if you combine all the cage libraries and here you have a tissue specific view in dense display for healer cells which look pretty much the same we have three collections we have encode cage, encode rampage and we have phantom 5 for phantom 5 we have one of the tracks now if you are interested in tissue specific cage usage then you can display more tracks so for instance you can activate all last tracks and you should keep it dense and then you will see lots of tracks so that's not what I wanted you reset the results first by mistake you reset ok so let's try I don't want full and I went to activate ok let's try again so this takes a little bit of time because there are hundreds of libraries but actually not too much because the browser only accesses the data of a particular region but I still don't see it scroll down maybe ok ok it put it ok sorry I put it at the end all the data you can choose your sample and you also get an impression where it is strongly expressed but I would say in this case it's largely a constitutive promoter you see additional transcription start sites in some tissues and you can explore them individually so ok so what else can you do you can also zoom in on the initiation patterns to get so now you see how the the transcription starts are actually distributed and you see that they are actually not so not so sharp there are several sites they tend to be the same in different collections although there are some differences between encoded and phantom but the major site is always the same and this is the site which is indicated by this EPD icon which shows 10 base pairs of the RNA by a thick bar and then 50 base pairs of the upstream region by a thinner line ok then you can also you have DNA sensitive clusters and you have additional data I will now switch I will go back I will go back to this a promoter database home page as you can see we have additional viewers for instance this was the viewer for genome assembly 38 but we also have a viewer for for HG 19 now because I want to look at the same promoter I click again in this promoter and I use the menu which is below the picture you will also see that I have the choice between the UCSC server and the European mirror now usually the data transfer is faster with the European mirror so if you are based in Europe I suggest that you use the European mirror now let's look at the viewer for HG 19 because there are some tracks which I like better in the HG 19 viewer but I said it's faster than UCSC well it's not so fast I don't know but ok so the picture is the same more or less the same what we have in the European viewer is a chip seek track which I now I'm going to expand no full or pack is fine so now we chose chip seek peaks from encode and the color meaning dark black means a strong peak and light gray means a weak peak you also see these green parts these green areas correspond to a sequence motif from the factor database so we can look what kind of interestingly what we find here is we find CT CF sites with motifs and we find Elk 4 which is a member of the AIDS family this is not a very strong peak by the way but Elk has a you see Elk is another member of the AIDS family which is strong and if you click on this icon then we get the summary from encode but this peak has been found in four different cell types and you see that in this one it's relatively weak but it's a very strong peak in the other three cell types including the lymphoblastoid cell line GM12878 which has been extensively characterized by encode you then also see the sequence motif and have some documentation not surprisingly we see somewhere a peak for RNA polymerase but I know ok here so we can also look at this one and here we see obviously it's found in a lot of cell types and you can maybe assume that the coverage by RNA polymerase may also give you some indication about the activity of the promoters now further below is the sequence conservation profile so this is derived from cross species comparisons I guess these strong areas they correspond to exons but there are also some spikes in the promoter region so it's always interesting to check whether some of these short regions which are conserved exactly coincide with transcription factor binding sites so let's zoom in on a short region so what you can see here is that apparently there is a transcription factor binding site it is for L4 for L1 and for L1 and for GABPE now these are all transcription factors of the AIDS family and they have virtually identical binding specificity so the motifs are basically the same but obviously these are different proteins and so different members of the elk family may recognize the same sequence motif in the same promoter and what is really nice here is the precise correspondence of the elk motif and the conservation peak you can assume that this peak is probably also conserved but now going back going back to these motifs so if you are interested in which cell type elk binds to this motif you click on this and here are the cell types we saw before so it's not many actually and maybe GABPE is binding this motif in other cell types let's see not necessarily because these two we saw before I think Kela was not present but so that would indicate that the same motif is recognized by different members of the same transcription factor family in the same cells now I didn't want to give a lecture about it's one family member the motifs of these proteins and the role in promoters I just wanted you to show how you can dig in on biology using the EPD viewer I now obviously you can do all these things without going through EPD so we see our mission to provide you with a useful subset of tracks that we automatically activate through a different file and which may be helpful to you in the sense that you will get to this use to this view as a starting point where you can find interesting information the UCSC default view is not tuned to a particular user community so it contains a broader mixture of tracks and it is then more difficult for ordinary users to find those which are particularly interesting to their domain so we see our role as kind of guides to interesting genome information for people who are interested in promoters there is note that we also have a cell type specific viewer we plan to add more cell type specific viewer in the future for the lymphoblastoid cell line at GM 12 878 so here you have histone marks only for this cell type and obviously you have the cage data you have all cage data then you have you have dns1 data and the dns1 data there is something interesting it's this single base resolution track which is available from UCSC directly in principle this gives shows you the fine structure of the promoter region so wherever you have high signal the DNA is probably not occupied by a protein complex and where you have low signal it may be occupied so black is highly accessible and low signal is protected and you could speculate I don't know whether it's true that here you have a kind of valley and this may be correspond to the area where the pre initiation complex is assembled on the promoter and it may coincide with the polymerase one peaks okay let me switch back to the slide collection okay this is just the slides for the promoter viewer you have seen this examples of interesting promoters so this is a case where we have tissue specific promoters in particular there is a strong promoter which appears in two cancer cell lines it is shown on the sorry on this side and this promoter is absent in most other cell types so this is related repetitive elements in principle contain promoter regions they are typically inactivated in some ways but in cancer cells sometimes they become active again so this is recorded as a promoter in EPD as an alternative promoter of the main promoter so this is also a quite interesting case so this is from zebrafish so it's a time course it's cage data for a time course and what you see is that in the psychotic samples we have to know that in early development messenger RNA transcription starts at a later stage the initial during the initial cell division events protein synthesis relies on maternal messengers so the maternal messengers they apparently start here there is a tartar box or kind of a tartar box near near this side and then at later stages transcription initiation switches to this place so this is just a summary of the promoter analysis tools I think I can more or less keep this slide you have seen chip core, you have seen chip extract and we also have seen opoff and then these tools or related tools can be used as promoter subset selection tools you will see how you can do it in an exercise this afternoon but basically you can use all the data in the MGA repository to promote the selection to select subsets which are enriched in a certain feature or subsets that are depleted in a certain feature, you can do this with chip core followed by a feature selection menu, but also find them, it's a program from the SSA patch, it works in a similar way as opoff but rather than plotting a profile of motif occurrences it selects promoters that contain a certain motif and these tools can be used in a cascade so again this is partly redundant so for instance here we look at histone modifications in EPD promoters so this is the input form we have seen this already with Giovanna we select EPD, EPD is available from both the SSA and chip 6-hour menus then we select histone modification from this data set and this produces this picture the feature selection tool appears on the output page so you could use, this is not a good example for not the best example to illustrate it so with this tool you can select the promoters which have between minus 500 and 500 more than 100 mapped tags, so this would be the more strongly labeled promoters you can also just enter in the select top and rich depleted reference features you can say you want to select 1000 top and rich features and you can switch it, you can switch to depleted feature selection which allows you to select promoters that are weekly covered by this histone mark or it could also be a transcription factor side, now if you use this tool then if you use this tool then you get a new page where you have the promoter that has been selected and you can do whatever you want with the promoters that are highly enriched in H3K4M3 you can use chip extract to look at the heterogeneity which is hidden behind a general aggregation plot and you can see if you extract the tags for individual promoters and you cluster them by K-means then you see very distinct classes of promoters, promoters that have strongly positioned H3K4M3 3 label nucleosomes on the downstream sites, promoters that have such nucleosomes on both sides and this number of promoters which still do not have this mark now you can also look at nucleosome data and this is just a picture you obtain if you analyze the nucleosome positions in yeast relative to transcription actually no, it is not yeast it is fission yeast S-pombi and you can use SSA to look at the enrichment of a specific transcription factor binding sites in human promoters so that's how you fill out the form of the exercise in the afternoon and that is the picture you get as a result note that you can require to do the same analysis for shuffled sequences shuffling is regional it means the computer takes each sequence cuts it into 10 base pair windows and changes the order of the bases in each window this preserves, is supposed to preserve the base composition, and this is sometimes important because promoters are highly G-series and G-series transcription factor binding motifs may only be overrepresented because they are G-series but here this is not the case the shuffled sequences show a flat distribution and a via binding site are highly enriched some concluding remarks so EPD together with the MGA chip seek and SSA is actually can be viewed as a data integration hub data analysis platform so for instance if you're interested in the promoter or in the group of promoters you may ask questions like in which tissue are they active what sequence motifs do occur how are nuclear zones arranged and whether they are SNPs near the TSS and these answers can basically be obtained under fly accessing data that are in the MGA repository or by running tools or by do by underfly motif analysis in genome sequences and I think this design has a major important over a static conventional database the individual resources can be asynchronously maintained so we don't have if we have a new data set chip seek data set we don't have to add this to EPD we add it simply to the MGA repository and then it will automatically be linked to EPD and analysable via EPD we can use software that is useful for other purposes that also relates to the independence maintenance argument so we have not necessarily developed chip seek for EPD but it is very useful for EPD and that's why we think there's no point in developing a more specialised tool for EPD the underfly combination or assembly of information about promoters also makes the information more traceable to the primary data it offers a lot of flexibility to the end user and it's label efficient at the developers end we also try to make EPD open science and so the general principle is that all components should be open sourced and publicly accessible the MGA repository is an open FTP accessible directory the source URLs and are given for all the scripts that were available that have been used for data reformating the web applications they post you may not have noticed it a link to the script that has been run on the server so if something funny occurs you may be able to trace it back you can also copy the script to your computer and run the same analysis then with the command line tools we also try to provide the numerical data for all the graphics so that you can make nicer graphics and in EPD we try to provide a precise description of the assembly pipelines it's not yet reproducible files and of course we link to all the source data and with this I come to an end of my talk and I want to say thank you to all the people who have worked so hard on these tools Reida was our quality control manager Giovanna did almost everything on the access tools for which I have no photo unfortunately have been the main EPD developers and Roman Grover was a PhD student which had his own project but also helped on many occasions to make our resources better now these are not the people who created the EPD in the first place even though these are the people who developed the EPD new but this will not have been possible with the former team members Claude Bonner, Vivian Prat, Thomas Junier and Christoph Schmid who also did a tremendous job they created the first web interface and started to develop the automatic procedures so I'm not sure whether we have five minutes time for questions Patricia or what do you think I think we have time for questions let's get started