So welcome back everyone thanks for joining us for this special interview We do this every so often with one of this channel's longtime sponsors the CEO of double gold column plume. Thank you. Welcome, sir Hey, how are you? Yeah, good to be here Yeah, we like we like having you on every so often and then talking about this stuff I normally talk about media-centered stuff But I am really into pretty much any news and especially the wars going on right now. We got you know Russia and Ukraine we got Hamas and our Israel fighting Hamas and Gaza and Now I'm reading that we have very real trouble flaring up in the Red Sea Which means the Suez canal and I think most people know that it's a major shipping port It's actually the fourth busiest shipping lane in the world and so any disruption there is just gonna Intensify the economic troubles that we're already seeing so in that odious environment. What does that mean? For the dollar. I mean right now. We have gold surging to $2,130 and Bitcoin too for that matter So, you know, what does that mean for the dollar and for people watching? What does that mean for you know, if they're thinking about diversification or the retirement? Yeah, well first. I just wanted to say gold is in the 2030 range It hasn't broken 2100 yet. Oh, oh, yeah, it did break. Yeah, it did break over three weeks ago over the weekend It broke 2082 Which is the all-time high so it's it's definitely been flirting With all-time highs. I've been saying for a long time that 2000 sort of a psychological number out there and and You know, a lot of people are concerned about the economy I mean Harry Dent came out and said that he thinks we're gonna have the biggest collapse In the stock market that we've ever seen next year You know, he's a guy that's you know been right in a number of occasions in terms of predicting You know economic calamity and I think it's We're in such a strange time in that We have finally hit the point in the US economy where we So dependent on debt That we have to have money cheaper. Otherwise things don't move yet Cheap money also in a lot of ways makes it difficult for us to to grow and it also Intensifies us with all the the debt that we've had in this country and you know, we're right now our servicing our debt As a country cost us as much as our military budget. So it's Which is a massive number. I mean to think to think about that Kind of take a step back and say that we are we basically have a big credit card bill that Is more expensive than they cost to protect us as citizens So that's when you sort of run into some situations where you know, we we We always talk about being broke. I think it's those numbers really highlight the fact that it's It's gonna be difficult for us to continue Unless we see some dramatic changes with the government spending changes, right? Yeah, we're not seeing that we're actually seeing a lot of mass denial from the government and from the media just about the economy in general Yeah, yeah, I was at this weekend. I was at Charlie Kirk has a his turning point his nonprofit has a big Fast called American fast. I went to this weekend. They had a huge lineup of speakers Very interesting group and you know, you had you know Republicans and business people Patrick Beck David was there and yeah, I think the back and Tucker were there too, weren't they? So yeah, so that that's what I was gonna bring up the back You know, he's such an exciting speaker, I mean, he's just he was balancing off the stage their Phoenix and They asked him that he took a he did a Q&A and they asked him about the Fed What he would do and he said Thing you do is fire 80 or 90% of the staff because it's way over staff And then he said that he thinks if he said if he was president He would peg us back on the gold standard Tied to commodities, maybe not solely gold But maybe you have a basket of currencies which a lot of countries have talked about but that was his That was one of his remedies to try to our spending under control that was his one of his strategies, which I haven't heard a Presidential candidate say anything so bold In a long time, right, I mean, they're usually much safer In their language Yeah, and that also the funny part was that he was cussing a lot. So the next day Charlie Kirk Mentioned that he was getting a lot of emails because there was actually a lot of There was a lot of college kids there through turning point But there was also a lot of families that brought their kids like young kids So those parents were emailing saying You know, the fact was using the F word and he was kind of going off FU stuff. Yeah Yeah, yeah, and so, you know, all these parents were like trying to earmuff their kids is with Vex You know dropping f-bombs and he you know, he he laid into Someone from CNN I can't remember who he laid into but he kind of was kind of ranting about him But yeah, so it was yeah, so oh van Jones. Yeah, and Jones, right exactly Yeah, and then and then Tucker came out and spoke Yesterday so I caught actually was the last person I heard speak before it I flew out of town And and Tucker had a mad biggest crowd by far. I think it's a whole event And he was just talking about about morality and what's kind of happening in the world and You know where we're kind of going that we're going down kind of a scary path But but long story short, I think that the debt Which was a real constant conversation at this Event was that the debt is really out of control and that we need to you know pull back spending pretty dramatically a lot of people said it in many different ways So it is something that we need to talk about it And that's why voting is so important that you can actually vote for people that have the more sound sort of business practices and the idea of slowing things down and you know Obviously reducing the big government and moving in in a lot of different directions is really important And for people that that are watching that might not understand. Sorry to cut you off there Just like why is that a good thing? Why would we want to change over back? You know back to the gold standard? Well because you can't Put money on a thin air if you're on the gold standard you'd have to you have this has to be pegged You know for before we went off the gold standard every dollar that was out there had Gold behind it. So just couldn't print freely. So when Nixon took us took us off the gold standard You know completely in 1971 We've been able to Just print as much as we have wanted And without any recourse because we're the world's reserve currency. We have this flexibility We had it had to something Inflation would be lower some substantially lower Governments the government would have to have a budget which they they haven't balanced the budget I don't know how long it's been ten years. It's been very long. I think Clinton was our was Clinton. Is that the last time? Maybe yes So we we were you know, we're in a position now where our Our Facet and what we're spending is is ballooning and it's it's you know in a few very specific places It's health care, obviously Medicare and Medi-Cal Very expensive and people are living longer in government programs Debt as I mentioned our debt payments right now costs as much as our military budget. So you have the monies coming out and Very typical places they know where it's all coming out from but no one's Really ready to do anything to fix those issues Which was a lot which was discussed at this American fast was that you know people are being for a resolution and We can't just continue to Raise the debt ceiling every two months and continue to go down this path With people want more sound money principles and that's why it typically always comes back to gold and silver as the currency Because it is something that comes out of the ground and you you have to have enough of it to back it and so it creates some some Boundaries of spending we don't currently have that's why The gold standard or or having like a fractional backing of gold with our currency I don't think we could do it for dollars. I think there's too many now Having some kind of system that that restricted our our spending Would be much much better for for you and I and and everybody listening because We now As of inflation we have to a return on our investment to keep up. That's so high Keep our standard of living, right? It's not like it used to be You could you could make a smaller return a safer return now That's why you know crypto and all these things have come to the forefront is because people realize they need they need dramatic returns So they're taking bold risk and a lot of them lose unfortunately I don't know if you're big in a crypto, but I saw it at the FTX You know the big scandal with FTX and I knew this happen. They're trying to pay people back That have been held hostage by this But they want to pay them back in cash They don't want to pay them back and and I find it so I mean I knew when this went down this this is what they're gonna try to do but It's sort of a lines with oh, well, you know, we'll give you back cash and also with the other thing it does Is it all those people have to pay tax? For one of those people that get cash now they've converted it they're gonna have to pay tax So it's sort of a win for the government too. All right Ready to go in that dress what and why why couldn't they just give them back the crypto that they bought You know, that's the problem with these, you know, these situations is they just present an opportunity for the government to win And there's probably a lot of people to go. I don't want my cash I want the Bitcoin. I want the Salon or what I bought what I bought, right?  So yeah, it seems like that's never gonna happen It's gonna take a rogue kind of guy to do it Yeah, a Trump sort of in the Trump vein right someone Yeah, not afraid and doesn't need the money and doesn't need the you know, not doing it for To grow their wealth someone. That's like Please they can make some massive massive change and I don't know if he you know I don't know if he's the guy or whatever, but he's been saying some interesting things and you know, I think, you know, probably his biggest fault is that he's Not afraid to get into it with anybody and he's run to a lot of people wrong, which makes it sort of hard with politics to to get some bills passed but you know coming up on the end of the year we're in December now, you know, a lot of people are Trying to decide what to do and they're trying to predict I mean, you know, that's the thing with investments You got to look at what's gonna happen next year and what can happen for the next few years And I'll tell you my predictions for next year I think rates come down as the Fed has has discussed which means bank, you know returns to the banks are going to come down So I think the heyday of the 5% money is probably going away And you know, we're getting back to probably to 2 to 3% maybe lower than that which Any estimation is not going to keep up with inflation even 5% is not going to keep up so I think that's one of the things that happened I think they're going to open up the money supply a little bit next year because You know, the Fed wants to keep, you know, buying an office That's the smart move for them right to lower rates and hopefully open up the economy a little bit So I And you know, if you go back in history and you look at The year before or during the election year, there's always a lot of interesting things that happen With the Fed and how they control the economy How strong they're and how they can really dictate who's elected or not elected So I think that they are going to open up the money supply next year So I think things will kind of widen open which Which which will be great for a lot of industries real estate being one of them I think it'll it'll help obviously because you know, real estate's been Just dead, you know, there's no no transactions happening And there's no merger and acquisition transactions happening I was just reading that Mergers and acquisitions are a huge part of the economy these large companies Mergers and acquisitions this year were down 70% And you know some people would argue maybe that's a good thing, right? They don't want these big, you know companies becoming one big company But also some mergers and acquisitions and a good good amount of them. They actually improve The company they improve the product. They make the company stronger So this year because of interest rates and just the whole volatility of the market That whole sector has been dead. There's just no none of those large Transactions happening. So any like mergers any, you know loans or anything in that anyone in that world has been You know, basically brought down to their knees with these high interest rates So I think that I think things open up next year Um, and then it's like what's going to happen with these banks, right? Are banks going to survive a lot of banks are teetering right now Especially mid-range banks. Are they going to survive? Next year, you know, I think the only way they do is if you know loan productivity picks up and Just loans pick up and you know commercial loans pick up a little bit. So that'll probably help them Which I think having more mid-range banks is a benefit to all of us We don't want to have just these, you know massive banks that control the money So I think that's what we're going to see happen Uh in 2024 Do we get that massive pullback in the stock market? It's you know, it's hard to gauge You know, I know we've seen people say that it can have a massive Unless there's some kind of dramatic black swan event next year I don't see us dropping 50% of the stock market But you never know if there's you know, we had COVID, you know, there's all these things that can happen out there So I didn't ever really know What can once around the corner here? So What are you thinking about the ev market and how that's affecting Things right now special with precious metals Yeah, I mean, I think like, you know, you have so many new automakers And ev coming out there's I was watching an interview with the CEO of Rivian the the truck ev and You know, he was he was asked like is tesla a competitor and he said that there's so few ev manufacturers still that there's no real competitors Where they're all sort of helping grow that industry You know, and listen, I know people I love gas cars So I mean, I'm just gonna put that out there as much as I love silver I love I have an old 68 Pontiac GTL And I think I get like, you know, maybe four miles of the gallon on that I mean, you basically just drive from one gas station to the next Right. I mean I am obsessed with that car.  And I just I accidentally left that you know that door open So, you know, just for the record, I do And that's part of the charm of it That's part of the charm. Yeah the smell I remember when I I took my young kids on the car in the back and they're like dad, I smell again. Yeah, that's cool You're you smelling like gas is cool. That's why we have this car You know this car only goes 50 miles an hour or whatever it goes, you know, it looks it looks cool. You smell cool Um, so yeah, I mean, I think the EV market still you know still got momentum Obviously the cost of those cars has come down dramatically, which was the big problem Two big issues was the cost which has come down a lot. I mean even Tesla prices have dropped Then you know the length of of you know, how far they can get And you know lucid and a lot of these new ones are are breaking 400 miles on a charge I mean, I don't know how many people are are driving more than 400 miles That consistently. Yeah, maybe you're you're doing a road trip here there But I think for the most part they've they've worked out a lot of the kinks there So I think that market's going to continue to grow. That's great great for silver That's going to be really exciting and then on the catalytic converter side You know, the a lot of the companies are moving to platinum For usage they're moving away from palladium The four to f-150 Actually has about $2,000 in palladium in that vehicle The new lightning that everybody loves. So you're seeing a lot of these, um, you know besides gold These alt metals kind of like in the in the crypto biz silver Platinum palladium getting a lot of excitement So I think those things are are are really good and and I think just overall You know, you saw the alternative currency world really explode and continue to explode You saw bitcoin again, you know broke 43,000 it's pulled back a little bit But really, I mean, I think that the the reason you see people buy these items these You know bitcoin and these alternative currencies It's really a reaction to the us debt. That's really what they're Bitcoin doesn't do what it does unless that if the dollar's strong We're paying our bills. There's no bitcoin. I mean, it's it doesn't it doesn't hold water Um, but in this environment where people are just fed up Um, and they're looking for alternative ways to to to use their money You know the idea of having a money like bitcoin that has significantly outperform the dollar Is is appealing and so that's that's why you see these things come about. It's only because of reaction of of our overspending and on our big massive Government debt Yeah, and it's just it's an it's an overall distrust and the experts that you know keep saying these things And yet people they feel the effects every day, you know at the grocery or gas or wherever And you know, I that brings us to the fact that I you know, I get messages all the time right now in our discord There's people in there who were interested and the fact that you were going to be here. They wanted to ask questions I'm not going to take questions today, but it's really interesting Oh, you do want me to take questions Okay, I will all right. Well, I already have some questions. So that I got from them earlier I just didn't want people like coming in here and but we can do that if you want to it's it's up to you I'm okay to be a harassment question. Okay. That's cool. All right. Excellent. Cool. I I didn't want to put you on the spot But uh, we will see if those people are here. I think they are so um Basically we have all these people They they want to get into this stuff for all the reasons that we've talked about and I kind of Equate it to working out, you know a lot of people want to get into working out But it's intimidating because you don't really know what you know if you don't know what you're doing And so what would you recommend that people do? What's the first step? In working out always start with push-ups. That's always my first and pull-ups Start with the basics. No, um Well, that is true. That's a good idea though And I do I do it every day But um, no, I think like the basics is is getting educated in anything I think that that's so, you know, that's the big thing that we've always focused on is just You know, if you go out in the gold and silver space, you could look at it There's a million different ways to look at it. Some companies have thousands of coins of bars The stuff on their website. We don't do any of that. We basically sell between 10 and 15 different products Okay, um, because those are the products that people buy those are the ones that the central banks buy those are the ones that Institutions are buying, you know during um during the pandemic, you know, when it was really hard to get silver I was buying a thousand ounce silver bars and I was competing on purchases with with comics Because they needed the bars too. So we were actually buying the same bars for a lot of the same places so So that just the only reason I bring that up is to say that like I'm buying products that Banks are using to buy or the or the ETFs are using to back their ET Like we buy a lot of the same items because we know that those For time we're going to get you the best return because you're you're getting the most value the most bang for your buck And I think that's really important. So I think the first thing to do is like get educated call us We'll send in a free guy talk to somebody Ask a lot of really important questions because it is different than You know on your phone where you buy a stock or you buy, you know In those situations, you're just buying a fraction of a company As with us you're actually buying the whole thing that you're that you're the company, right? You're you're in charge of it. And I think that's just alone is sort of New concept for people is that they're the idea that they're owning it on their own. So I think that's the first thing To do and then just you know, I would start looking at the charts Researching you can cause and ask questions. I mean our staff is really well Uh reverse and like what's the trends and what's happened in metals? Um over the last 15 to 20 years So we can you know, you can always ask those questions like where was it?  Where was it 20? Tapping what's happened the last six months? I mean, these are all Relevant questions that I think that anybody should ask Um, so I think getting educated on those things is is really important And then just you know, I think when you're looking at a more of a safety play like Old and silver applied to palladium But you're balancing it with the other things that you have so you're looking at Um, your portfolio as a whole and you know, I got my house. I got a little stock market. I go like what's What what do I want it? What do I want in sort of a safety play and that's how you determine? um, how much you want to put into uh into gold and silver And you know, you know, obviously there's people out there. I met the the ex CEO of Overstock Um, who's a huge golden silver buff. We were talking about and he's got so much of his net worth Uh in golden silver and bitcoin And so he has more than most but he just believes the whole system the dollar everything's gonna collapse and that's where his heart is That's where he believes I think you know people other people are like sort of in the middle They like yeah, that could happen But if it doesn't happen, you know, where do I want to be too? So I think you have to figure out what your level is and then you know, find a number that you feel comfortable with buying and You know test this out and get it shipped and And then I think you'll find um And read our reviews and do all those things and I would do that with any company that you're gonna buy any product Go and check don't like go to the go to the better business bureau.  There are some bad actors out there and you want to be careful But I think you should do that with any any products before you make a purchase Yeah, absolutely, and you'll see and I've talked about noblegoals got five star ratings and good reviews all over the place But definitely you want to go check that out and like you said it is a balancing thing and you know I hear a lot from people asking You know, they don't necessarily have a ton of money to put into it But they want to try it out and they're wondering if you have sort of like, you know like hundred dollar bundles Or something like that a sort of entry level Ways of getting in to it Yeah, so so our our minimum cash purchase is 5000 Because we're we're sort of set up in a In a way that we're you know, if you're doing below that I would say find a local shop Like a coin or or dealer that can help you locally You know when you're dealing with a firm like us, you're gonna get the metal shift You're gonna have a concierge. You're gonna, you know, there's a lot of people behind a purchase so we typically focus on 5000 and above for cash And then for IRAs we we focus on 15 000 and above Okay, the reason we do that is because with the IRA they have to be stored in a depository You segregate a storage, but there is a fee pay 150 dollars per year We don't recommend anyone to do less than 15 000 because you're gonna have to store those metals for 150 bucks Which are gonna be responsible for so those are sort of our minimums set up We don't have any maximum on a purchase But yeah, anything above that is is sort of our our wheelhouse And you know, we we offer, you know, some incredible pricing It's a great even though gold and silver have been skyrocketing in terms of prices We have some unbelievable pricing on billion bars, billion coins There was an overreaction by A lot of the major mints last year. So they over minted So we have we have products at the lowest prices Near very near to the spot price the lowest we've seen in years To give you an idea during the pandemic silver eagles Our cost was like anywhere from eight to ten dollars above the spot price That that premiums way down. We're like, I think four or five dollars above the spot price now So that shrunk a lot bars. So it's it's a good even though price has gone up. It's still a very good time There's there we have a good inventory and you can get great some great pricing On on and silver's still 50% lower than where it was In 2011 so it's still still, you know, all time high for silver is 50. We're sitting in the, you know, 24 $25 range Okay, sounds good I wanted I want to bring some people in to talk. I'm trying to get them in here right now Um, but I did want to quickly ask you while we're waiting on that And this is another question I get I do talk about In the ads about that there is always risk and investment And they're just curious, you know with the volatility and all this what is noble goals approach? to risk management and are there any sort of processes in place to mitigate Any of those, you know potential downsides There's risk in any investment, right, you know, I think that's that's why you know It's important to be diversified um You have to If you buy a stock You know, it loses 50% of its value You know, that's it is what it is, you know, and and you know people always ask like what, you know, what can you do?  So there's not that much of it. So there is a limited supply Um, the biggest buyers of gold in the world are central banks like china Has been buying tons of gold It's even been reported this year specifically It's even reported that they have way more gold holdings than it's reported You know, because the royal gold council has like a list of how much how many tons each country has and There's a lot of reports that they have way more because they actually mine Internally too and they're not the best at Recording, if you know what I mean, right? So Yeah, right, they don't want to disclose. So I think that's You know a good good thing in any investment is you want like supply and demand Value um Fungibility like it's you can trade it anywhere in the world. You know, that's the cool thing about it is like ounce of gold here ounce of gold in Dubai ounce of gold in london, you know There's always value anywhere in the world. So that I think is great, you know, there's There's not always that No universal value of a product everywhere in the world, right? So um I think that gives it some some strength um, and and then I would just say that it's you know in Times like this where we have You know, we're in two wars here in the u.s. Maybe a third potentially you know people You know take tend to go to AFTI, you know during times like this Which is probably the only reason I would say is maybe that's right about next year is if we had that like black salon event Then I could see this, you know, uh You know, maybe a big pullback in the stock market But I think it's yeah, there's always downside risk into anything. Um, yeah, of course that's why That's what I was talking about 2000 is sort of that psychological number for a goal that I remember That broke a thousand in 2009 You know, there's people that you know, it was just kind of flirting around a thousand and then it just once it broke through You and it hit, you know 1900 and then pulled back to 12 and everybody said it was going to go back to a thousand And it just kind of never did it just kind of any time it would hit a really low number. You just see the massive buying That's typically a good sign Of of something that of an item that has inherent values that if it hits a really good number Or are buying it, you know pretty dramatically. So You know, you got it. You got a this is part of the messing It's it's like it in anything but I think we all have to make educated guesses about our investments And so this is kind of out of my wheelhouse But uh, one of our users here his name is riddle. He asks Do you think that bricks has the influence and practicality to replace the petrodollar? Considering the flattening chinese economy as well as western sanctions on russia It's that's been the big conversation. I think that's the idea was And they've wanted to do that. They've wanted to go away from petrodollar when they're making large transactions They are they are to some extent. I mean, they are doing less transactions in the dollar They're seeing them make transactions. I mean, obviously russia has been transacting in gold Very heavily for the last, you know year and a half Because they've they have sanctions against them and that's that's the big risk if you look at, you know Central bank holdings of us treasuries those have shrunk so dramatically over the last 10 years 10 years ago from today you had about It was about a 70 percent most central banks had up 70 to 72 percent Was in us treasuries Now we're below 60 we're like 59 and a half percent So you you are seeing people move away From the dollar In terms of, you know, these massive transactions Um, you know germany, uh, brought back a lot of their gold from the bank in new york They wanted to have it internally because they were worried about our debt So you are seeing people divest Over the last six months you've seen many heads of countries You know France's president many people saying yeah, we're divesting from the dollar. So yeah You can't have this thing move overnight and also I think at the end of the day Really isn't a better option out there that's been presented and until there is a better option Dollar will still have some value to it. Yeah in terms of countries because like we were saying like with china You know supposedly have a lot more gold than they say they do But because they're they're the way that they report to the world and it's so you can't trust What they're reporting, you know, we're not going to move to the end till there's more, you know A transparency about how things are going in that country and what's going on with that and how much debt they're in Because you know, there's a lot of estimations that they're in pretty dramatic debt because they've made a big real estate push Over the last few years and they've been heard by these higher interest rates of that the world's having also So I think until a better option is presented, you know, where the dollar will still be there but the big question will be In the next four or five years If china just says we want to call in some of this debt like we don't want to just take the interest Right, you know, we won't go without principal back And that's going to be the issue. That's the real issue that we're going to run into Because we don't have it Right, it looks like we actually have somebody here. What's up, ashley? Nothing, can you hear me well?  So I have some income that I'd like to contribute toward a retirement and mortgage and commercial back securities are crazy Real estate's crazy right now and everything looks too volatile and being undereducated about the subject I'm kind of looking for a safe place to make an investment Sure So you are you still working or you're no longer working? Yes. Yes, of course. I'm working. Yeah Oh, okay. Okay, and you have a 401k at your job or or No, sir. Um, I'm uh, I guess you would call me an independent contractor or a work from home person I only my income is meager. It's pretty good for my area, but it's meager compared to the national average I only bring a little over 4,000 a month But other than utilities, I have no debt my mortgage is paid off. That's amazing So I have some I'm I'm 44. So I have some income I have no retirement and I'm I'm looking for Something that I can contribute to that will provide me with some sort of meager retirement Yeah, absolutely. So what I would recommend is I mean, it sounds like from your situation You could be considered self-employed if you're a contractor what you may want to look into is starting a Individual retirement account like an IRA. You could either do a SEP IRA or traditional or Roth And and the reason you wouldn't want to do that is because those vehicles give you tax advantages You know, whether You want the tax advantages in the year that you put the money in or if you want them later on That would be a good way to to put some money aside that could grow a little bit easier because Being 44, you still, you know, have a long runway to Build a nice nest egg And the fact that you don't have any debt you can really start You know contributing and putting money away that can lead to to future growth So I would recommend looking into some kind of retirement vehicle That could get you and you know, you could buy you could buy gold and silver You could buy, you know, any other asset you want, but I think getting some money In a tax deferred vehicle would be Be very wise. Well, did you guys did you guys have anything else for them? I actually have to bow out because I have to go back to work But I just want to say thanks Collin for coming hanging out with the group. No, thank you. Yeah I appreciate you jumping on on your break and and You know, I hope, you know, this is informational. It was a lot of fun for me to answer questions I'm always happy to do it and you know, if any if we can ever do anything for you guys, you know We're we're always around and love this show and just appreciate, you know, coming on and talking about, you know Something fun and and something I love which is which is precious metals Well, we really appreciate it. I really appreciate it and uh, again, if anybody wants to give them a call It's<PHONE_NUMBER> And like I said, you can get the link direct link there and the description and pin comment of most of my videos Okay, thank you. Yes, and uh, Collin, uh, I appreciate you coming. Did you have anything else for us? It's great. It's fun. I'll talk to uh, have a safe happy Merry Christmas. Happy holidays, and I'll uh, I'll check you in 2024 See if any of my predictions are accurate. Yeah Well, I really appreciate it. Merry Christmas, Collin All right, thanks so much


Hello everybody and welcome back here to the channel. We're staring at the starting gate for the next round of ESTA we're going to Fuji so we're gonna be taking the next level racing Mazda racing energy innovations e36 comm car out for some shakedown laps here before qualifying the end of this week so make sure you follow me on all social media all the switch found in the description box below let's get to it so we are on the maza r5 for this round I've actually been getting quite comfortable with this wheel and how small it is so we're sticking with it because I've been super comfortable with it so we're gonna go take a look at the track first off we're gonna do our little track walk we have our very very long entry and then we have a initiation that's a little bit bumpy and then we do have a inner clip right there to an outer little touch and go into a nice long left-handed sweeper to a wall which if you miss it and you clip that wall that's going to be very deadly to another inner clip and then we got to stretch this out almost all the way out to here not trying to hit the rumbles as possible to another outer zone and then a hard uphill bumpy transition to a inner clip and a final touch and go so this track is a little bit tricky we're going to run it a couple times the first person then switch to the roof cam because for me I like to personally start running the tracks in first in third person because I get a sense of where my car is and I start getting a little muscle memory in place and then I start to get fully dialed in so we're gonna start singing second gear I believe this could be a fist gear track that is a really bumpy entry okay we're gonna come out touch and go transition over kind of monitor yep that wall is gonna be very deadly if you kid it we kind of nicked it but we didn't go fully into it stretching out all the way to those rumbles into the outer zone kind of hooking it back into this inner zone gotta stay in power there and then there so overall a bit of a challenging track I feel with the bombs going in the initiation and stuff like that it's a little bit more challenging we're gonna try going fourth gear and then shifting into fifth before we get into that outer wall section we do have a pretty good base tune on this car from other rounds so we may not do too much fine tuning so we're gonna start in first we're gonna do a fourth gear entry and kind of pop it into fifth oh yeah that wall is gonna be pretty uh pretty deadly if you smack into that so we're gonna go one more time so we are on the actual ESD public service so you know ground and temperatures and everything are correct I did like the fifth gear entry so we're just gonna go full send on fifth and uh and one more time in third person and we're gonna switch into my roof cam and stuff like that so second gear start so not too shabby definitely got to clean up a little bit we're using left foot braking I'm hoping not too much to the point where it's going to doc us massive lots of points because I'm not using it to slow down I'm kind of using it really just to keep my line which is something that I guess would be up to judges discretion on that one but we're trying to minimize if you notice we're only using handbrake on initiation and that's it we're not using handbrake any other time so we're definitely trying to utilize less handbrake and more just gentle taps to left foot brake just to kind of keep our angle and line and stuff like that so let's see what we can do in our roof cam have a different line there we may have came out of that that zone a little bit early there's a little bit wide there but not too shabby so we're gonna run one more run like that and then we're gonna go see if we can see replays the last two runs so definitely feel a little bit more confident in this track than other ones definitely like I said miss the last round due to um I lost my family but we're back behind the rig we're gunning for this we can try and qualify in for this round in 432 battles we've got to make sure that jitters don't get to us don't think we're allowed to touch rumbles there so we're gonna have to alter that slightly well that's a little bit wide and we came out of that zone really early kind of had to pump that handbrake to get the car to go where we wanted it to on that run so let's take a look at the replay of those runs oh in order to take a replay we're gonna have to forgot we're on an online session so we're gonna run one more time in a third just to kind of see where we're at and then we'll go take a look at those two first roof cam runs so this track is definitely challenging with the bumps on entry uh line heard a lot of people were using the bumps as like their initiation point so we're gonna try and just flick entry this as much possible without a handbrake and see what happens we'll send it I mean it is totally possible but I was on the right line that's totally possible to do but I feel like I would not be able to utilize that correctly so we're definitely going to just sustain what we've been already doing just a quick ebrake flick this is kind of a full bore track like pedal to the metal very minimal throttle you know just full commitment on throttle using brakes to gently tap it so let's go take a look at those replays and we'll be right back all right so we're gonna take a look at these two runs to kind of digress these a little bit in of course the uh judging cameras and cinematic cameras so we'll see kind of where we're at so initiation I'm not sure if we need to stay off the rumbles but that was actually quite decent pretty decent on that touch and go oof that was that uh that was that wall hit all right so let's take a look at this one because that wall hit was kind of uh kind of a hurt so I'm not sure I need to not initiate on the rumbles I feel I feel like that's going to be a problem just touch rumbles there which we need to address pretty deep in that zone and pretty decent just clicking the rumble there and nice and deep we did come out of that zone a little bit early earlier than I wanted to but we did able to bring that up fully into the final zone so overall that was not too terrible of a run the car is actually feeling quite great um I thought we were gonna have a little more tuning to do but we do have a slight tuning to do but not as much I believe we might tweak the gear ratio a little bit maybe not to run fifth gear maybe you run it in fourth um but fifth gear is not too bad starting in second because it's about the same amount of um you know gear change and things like that so see this I believe this was the other run we were in roof can't yeah it was because I quickly shift in the second so we take a look at this final run here initiating on the rumbles I need to double check on that I think we need to initiate off the rumbles pretty decent in that touch and go in that first zone there's that way hard about the same line through that zone and then this we kind of actually didn't straighten out too bad there's not much wavering in my wheels kind of very similar line overall not too bad we could be doing a lot worse for the first testing on this track so make sure you guys follow me on all social media all that's found in the description box below hopefully we can get into top 32 qualifying is this Friday so as always I think you guys are coming back and watching I'm EvilRabbit I'll see you guys on the track


But before we dive into that, we're going to review sort of a little bit of the background behind the next phase of the workshop which is really about alignment and visualization. So we've kind of set ourselves up with the data and the reference genome and the annotations to do some alignments and that's what we're going to do in the second lab tutorial. So we're just going to talk a little bit about the background of that before diving into that. So we're on module two now, which as I said is RNA-seq alignment and visualization. The learning objectives of this module are to why is this so small? Perform alignment or learn about alignment challenges and common questions, discuss a couple different alignment strategies. We're going to review very briefly bowtie top hat, which is the aligner we're using in this course in these particular lectures. We're going to introduce the BAM embed formats and do some or talk about some basic manipulation of BAMs. We're going to visualize RNA-seq alignments in the integrative genomics viewer and then we're going to do some BAM read counting and look at variance allele expression status in some real data. So there are a number of challenges with RNA-seq alignments. One of them is computational cost. So we've been playing with a data set that has, I'm not sure, maybe tens of thousands of reads in it, but your real data is going to have hundreds of millions of reads. So when you do your alignments, you'll see that even with this tiny data set, it takes a few minutes. It's a complete alignment. With real data, you can expect it to take hours or days. So there's definitely a real computational cost. Unlike genome alignments, RNA can and often does have splicing. So the alignments are spliced. So we're going to talk about the distinction between spliced versus unspliced alignments. Unfortunately, you probably can't just align your data once and be done with it. So what you're going to find is that, depending on what downstream processes you're trying to do, so for example fusion detection might require a different alignment strategy than expression estimation. So not only is it computational and computationally intense, but you find yourself actually having to go through alignment again with maybe slightly different parameter setting or with a different algorithm. So is Toppat the only mapper to consider for RNA-seq data? Obviously, the question to that is no. We're going to play with two just in this lecture alone, but there are lots of others. I've included a link there to a BioStar forum post. I think we have these elsewhere throughout the lectures, or if you just go to BioStars like Malachi talked about and search through the search interface for whatever you're interested in. There's quite a lot of content there discussing the issues of RNA-seq analysis. So really in broad terms, there are three RNA-seq mapping strategies. In the top left, there's the idea of de novo assembly. So you might do this if, for example, you don't have a reference genome, or you have a reference genome, but you don't have good gene annotations, or maybe you have gene annotations, but you want to identify novel transcripts. So isoforms for your genes that have not been observed yet or annotated yet. Even in human, which is probably the most well annotated genome with the, you know, the highest quality reference and best annotations, there are still new transcripts being discovered all the time. And then you have things like fusion transcripts, where there's basically an infinite number of possible transcripts that can resolve from different genes being fused together. And a de novo assembly might be a good way to detect those kinds of events. So here you're not starting with any preconceived notion of the genome or transcriptome. You're using an assembly-based approach to construct what you believe all the transcripts are in that population of human transcripts. So that's one common approach. Probably the most common approach is what we're going to do or what we're going to focus the most on, which is aligning to a reference genome, but just being splice aware. So taking your short reads and acknowledging that they may not always align to one place in the genome, but there may be gaps and aligning like that to a reference genome. Another strategy is to align to a transcriptome. So where your reference isn't the genome, but it's a set of possible transcripts. So there you need to know what the transcripts are, and then you just align directly to them. This is probably the least common approach. So which strategy is best? Like I mentioned, a de novo assembly might be a good idea if you have a reference, don't have a reference genome for your species. If there are complex polymorphisms, mutations, haplotypes, etc. that might be missed by aligning to a reference genome. Aligning to the transcriptome, like I said, this isn't done as often anymore because we basically built splice aware aligners to avoid having to do this. We do sometimes recommend it if you have shorter reads, but most people don't have reads less than 50 base pairs these days. So most people are going to do this aligned to reference genome third strategy, which basically covers all other cases. Each of these involves different alignment or assembly tools. So this is a figure, we might have shown this already. It was in a review maybe a couple years ago basically showing when all of the different short read aligners came into use. And you can see there's a stack of, I don't know, maybe 40 or 50 different aligners broken down by application. So the ones in red are for RNA-seq specifically. You've got methylation, bisulfite sequencing, DNA, whole genome, exome type sequencing, and I can't actually read what's in green. MicroRNA, small RNA approaches. There are some algorithms that have been designed specifically for that. So a question is should I use a splice aware or unspliced mapper? As we talked about RNA-seq reads might span large introns. Or they will typically span introns. And the fragments being sequenced in RNA-seq represents messenger RNA, therefore the introns have been removed. So we're usually aligning these reads back to the reference genome, so you pretty much need to use a splice aware aligner. Again, if your reads are really short, you should use a splice aware aligner. And some examples are listed there, top hat, star, map splice. There's more options. So I think maybe in the early days this was a question, but for now I think it's pretty much resolved. The one that we're going to use is called bowtie top hat. I really wish I could see something on this screen. So bowtie top hat is one of these splice aware aligners. It requires a reference genome. If the way it works is it breaks the reads into little pieces, it uses the bowtie aligner to align the short pieces, and then it extends those alignments from these short alignments and dissolves the axon edges, which is why it's done. And that's kind of directly related. One of the questions we often hear is should I allow multi-mapped reads? So this is basically reads that align to more than one place with equal or near equal quality. So this could happen for a number of reasons. You could have let's say a gene family, repetitive regions, that sort of thing. In DNA analysis, it's fairly common to use a mapper that just randomly selects alignments from a series of equally good alignments. So if the alignment matches to multiple places equally good, it will actually just arbitrarily assign to one of them. That's less common in RNA analysis. But you might want to do that if you're doing variant calling. But you should probably allow multi-mapped reads for expression analysis with top hat-and-cuff links and certainly for fusion discovery, because those methods are starting with the assumption that some reads will map to multiple places and they've included in their models. They're taking a probabilistic approach and saying, okay, if this read maps to multiple places, I'm going to assign a probability that aligns to here versus here, and they basically take that into account. So you shouldn't exclude multi-mapped reads if you're using, for example, top hat-and-cuff links. So when you run bowtie top hat in a few minutes here, the output that you're going to get is a SAM file or a BAM file. SAM BAM files are basically the same thing. They stand for a sequence alignment map format file and BAM is just the binary or compressed version of the SAM file. And that's done for really space reasons. So these alignment files, as big as the raw sequence files are, the alignment files can get even bigger. And so to make it practical, to move these files around and store them cheaply, it's been necessary to compress them. So the BAM format was invented to basically make SAM more efficient. But unfortunately, that means that these compressed files require a little bit of extra special handling compared to plain text files. So you can't just open one and read it because it'll just be gibberish because it's been compressed. So that means you have to use separate or special tools to view the contents of a BAM file. Whereas a SAM file, you could technically just open it in a Word Editor. In many cases, the file would be so huge that it wouldn't be practical to open it in a Word Editor. But you could do things just straight manipulation of it, like add a command line without any sort of special tools. And there's a link to BIOS stars about how to convert BAM to SAM. This is a very common problem. You'll have a SAM file and you need it in BAM format or vice versa. And there are a lot of tools that help you go back and forth between those. Then I think we'll show a few examples during the exercises of how to do that. So this is an example of a BAM file. It's broken into two sections. So in the top section, you have a header. This you'll find at the top of every SAM file. It might be just a few lines or up to a few hundred lines. And it gives you information about the format or the version of the file, what kind of a liner was used, what regroups are contained in that file. All kinds of just general information about the data and the alignment that we've done. Nothing that's really specifically necessary. After that header, there's the name alignment section of the BAM file, where each line represents the alignment of one read from your data file that was aligned. So here I'm showing 10 alignments. Now we're going to zoom in on one of those and kind of break it down into its components. So it's basically, I think one line is sort of like here. A bunch of different fields of information and eventually the sequence evolves. Similar to a BAM file that we looked at before, but with more information because it's not just a sequence now, but it's how that sequence aligns to some part of the reference genome. You can kind of think of this like an alternative type of BLAST output. If you're familiar with BLAST, you've done, you know, taken a sequence, gone to NCBI BLAST and BLAST it and you get this alignment format. This is kind of like a more efficient representation of that kind of data. So you can follow the link to the SAM1 PDF to read about the specification. There's all kinds of details about how SAM BAM format works. Like I said, it's broken into two sections, the alignment and the header. It's compressed version of SAM. It's compressed using BGZF format. It's not really important. BAM files are often indexed. So just like we took our reference genome and we indexed it with bowtie, you often also index your alignment files to allow you to more quickly look up alignments in that BAM file. Because the BAM file is going to be huge. So you'll often basically sort that BAM file according to alignment position. So like chromosome 1, position 1 will come first and so on. And then you'll create this index file, which is a kind of a lookup file that allows other programs to very quickly go to the part of the BAM file where any specific alignment might be located. So it allows fast retrieval of alignments. So here's some example of the kinds of stuff you would find in that top header section. So this describes the source of the data, the reference sequence, the method of alignment, and so on. Each section begins with an apt symbol followed by a two-letter code, which tells you what kind of information it is. So for example, the SO code gives you the sorting order of alignments. The SQ refers to the reference sequence, RG to the root group, and so on. So you'll get information about the reference sequence name and length, so that could be something like chromosome 1 and it's out of the gate. The SQ sees that it's coming from and so on. And there's also information about the program. So this would be the program that was used to align your data. So it might say something along with it. And the version. Then in the main section, you had on one row 10 or 11 pieces of information separated by tabs. And the contents of that are listed here. So in the file, they're just listed one after another, but it's easier to read as a human has been able to warm up. So the first thing that's listed is the Q name, which is the query template name. So that's usually the read that was aligned. There's a flag. So a flag is a way of representing all kinds of information about that alignment. And we're going to go through what the flags are and how they work in a minute. The R name is the reference sequence name. So this would be, the Q name is the name of the read, the sequence that you're aligning, and the R name is the name of the thing that you're aligning to. So in this case, it says 1 for couple of more. Position is the position of the alignment. The last most one-day position alignment. So here's alignment starting at position 11, 16, 3 on the final one. You'll then have a map Q information, which is the mapping quality. So that's a kind of score for over the whole read. How good did it align to the genome? So here the mapping quality is 12, so it's three of this. There's then a cigar string that's just called cigar string. That's another way to represent the alignment. And we're going to go through a specific example of cigar also in a minute. You then have the R max and P max, which are the reference name and position of the main. So remember, a lot of times you've got paired data. So here it's saying P max equals, that's a symbol saying that the main pair of this read is actually aligned to the same chromosome. But it doesn't necessarily have to be. It could be that one read of a pair of max chromosomes on one, one the other read of that pair of max chromosomes on two, and then that would be indicated here two. And then P max, same thing, is telling you the position. So here the equals is indicating that they're both in front of the one, and you're seeing the two positions, 1163 and 11740, basically they're 100 bases between read one. T, length, is the observed template length. That's an estimate of the size of the fragment based on the distance between those positions for each pair. And then you have your sequence of quality spring. This is just like what you saw in the Fast and Furious. The sequence of your read and then the quality of each base call. So remember, the second thing is a flag. So this is a really kind of confusing concept. Oh, I have a pointer. How am I? You can read all about flags at this URL. There's an 11-bit wise flag describing the alignment. These are stored as a binary string of length 11 instead of a column of 11. It's basically a really efficient way to say something about a whole bunch of different properties all in one number. So, for example, the value of one indicates the flag is set to something besides zero. So here you've got 11 positions and each of these can be either zero or one. And all those combinations of zeros or ones add up to 2047 different possible codes. And each of those codes maps to a single number, which you can put in your file and it basically maps back to this complicated code, which tells you something about all these different properties. There's a website that helps you kind of understand these. Actually, this might be the website. So just to go through like, is that going to work? Yeah. So just like it says, it tries to explain SAM flags in plain English. So the number one, which corresponds probably to 1-0-0-0-0-0-0 for that string of 11, if we have it explained, it means that the read is paired. There's another string of zeros or ones, 11 long, that corresponds to the number two. That means the read is mapped in a proper pair and so on. So you can pick any number from one to 2137 or whatever it was, and it'll give you some combination of these properties. So you can give a lot of information. Basically, any of these binary states can be set and described by this one single number. So the BAM format is really all about efficiency, right? Like giving you all this information. Otherwise, you'd have to have like a different field in the BAM file for each of these. You'd have to have a mate is unmapped, yes, no, and read is a PCR or optical duplicate, yes, no, and so on. So if you see those flags and you want to know, after a while you sort of start to know a few of them, but it allows you to basically like filter your data. You can say, I want all of the reads that are not duplicates and have a read pair mapped. You can look up the code for that, and then you can quickly filter your file for everything that has that code. To make it even more confusing in the specification, they don't list the simple number, like one, or this binary string, but they list it in hexadecimal format, which is like a third way of representing this single concept. So cigar strings, that's the other thing that we kind of put aside for the moment in the BAM file. So number six is the cigar string, which tells you something about the alignment. Yeah, so when you do your primary alignments, those will all be set to not duplicate or unknown or something, and then when you do run a separate tool like Picard mark duplicates, it'll go back and it'll edit the BAM file and change those flags wherever appropriate. I don't know. I haven't noticed one, but I wouldn't be surprised if there was. That does mark duplicates basically during alignment. I mean, the problem is you need to do all the alignments and then go back through the file, and actually the marked duplicates, they even usually do a two-pass strategy, where they go through all of the alignments, look for duplicates, and then they go back through it again and choose the best read out of all the duplicates to remain and mark everything else as a duplicate. So incorporating that into your primary alignment would maybe make the alignment slower, and they do it on the fly, but they still do it by taking the alignment and piping it through another step, right? Like it's still a separate tool, but it's done. It's right. Yeah, exactly. And they don't do that two-pass strategy to make this possible. So the cigar string, there's a number of different symbols. Here's an example of a cigar string. It's basically just a string of numbers and letters that in a very compact way tells you a lot about the alignment. The numbers represent numbers of basis, and the letters represent alignment states or statuses. So it tells you basically which basis aligns the reference, either a match or a mismatch, which are deleted, inserted, represent introns, and so on. So in this example, you have 100 base pair read consisting of 81 bases that match to the reference genome, then 859 bases that are spliced or skipped, and then another 19 bases that match. So that's a relatively simple cigar string indicating basically a perfect match of 81 bases, an intron, or some gap, and then another 19 bases of perfect matching. Does that kind of make sense? So you can see by using like this combination of symbols, you can describe basically an arbitrarily complex alignment for your one read. And then other software can look at that and say, like, again, you can apply filters and say, give me only reads that have no mismatches, or only reads that have no gaps, or only reads that do have gaps, and maybe those are all junctions, things like that. So understanding these cigar strings and flags and the layout of a BAM format is really important, especially when you're troubleshooting things and you're trying to understand what's gone wrong with your alignment or your data quality or so on. Another really common format is the bed format. When you're working with BAM files, it's really common to want to focus on a subset of the reference genome, let's say all the exons of a particular gene. And the bed file is just a really simple way of specifying that. So a lot of BAM manipulation tools accept regions of interest in the bed format. So you have your BAM file, which has all your reads in it and all your alignments, and then you have this other bed file that's, say, in a really simple example, just one gene with the positions laid out for all the exons. And if you wanted to extract, let's say, all the alignments that aligned to exons of your gene, you could use a tool like bed tools, which we asked you to try to install as extra practice, to basically intersect that BAM file with that bed file to extract just the part of the BAM file that relates to that one gene or the exons of that gene. The bed format is not compressed, it's just a simple text, plain text, tab separated that has chromosome name, start position, and end position. One thing you have to watch out for is that coordinates in bed format are zero based, whereas coordinates in a lot of other applications are one based. Does anyone know what I mean by the zero base versus one base distinction? So the idea is like, just to give a really simple example, if you have a reference, let's say chromosome one, it's not real, but let's say it starts with sequence like this. In the zero based convention, you number between bases. So you don't number the bases directly, but you say this is position zero, this is position one, this is position two, this is position three, this is position four, and that's a zero based format. One base, you're numbering directly on the bases, one, two, three, four. So this matters because if I want to tell you to go and look up a base in zero base, I have to say to find this t, go to chromosome one, one dash two, for example, or you need to know a convention to always look at the position after the number that I say, something like that. Whereas with one base, you just go to two. It's kind of, this one is probably a little bit more human and intuitive. You just number things and this t corresponds to two. Zero base has certain advantages in other situations though. So they're both, I would say almost equally used and there's always a total mix of them no matter what you're doing. And so you're always having to think like, am I in zero base or am I in one base? Is this deletion or insertion represented in zero base or one base? If it is, how do I convert it from one to the other? If you search for this topic in bio stars, you'll see I have problems with this all the time. So I like made myself a tutorial and put it in bio stars to remind myself how to go between zero and one base. And you can find it there. But just remember that bed files are zero base, BAM files are one base. So manipulating salmon BAM files and bed files, there's lots of tools. We're using Sam tools a few places in this set of tutorials. We're also using Picard to mark duplicates. We don't use BAM tools, but it's a common popular alternative to Sam tools. There's a lot of the same things, but on BAM files instead of Sam files, although they both work on both salmon BAM. So bed files probably the most popular and common is bed tools, which was the thing we asked you to try and install. But there's also bed ops and others. So how should I sort my Sam or BAM file? So sorting is something you also come across all the time. As soon as you start to try to do something with your BAM file, whether it's align it or put it through a fusion detection algorithm or whatever, almost the first thing that will happen is you'll get an error saying it's not sorted properly because it expects it to be sorted one way versus another way. This is often done by position. So you put the alignments for chromosome one, position one first, and so on. That's usually for performance reasons. So when you sort it by position, you can index it, and then it's just easier to get to arbitrary positions in a huge BAM file. But there are other applications where you want things read sorted, and the usual reason for that is if you read sort, then the read one and read two of the pairs come one after the other because they have exactly the same name except for that one slash one or slash two. So it'll be like read A pair one, read A pair two, read B pair one, read B pair two, and so on. So those are the two most common kinds of sorting, and you'll find you have to sort one way or the other just depending on what you're trying to do. The common way to, as a human, visualize alignments is in one of these viewers. So the IGV browser is the one that we use most commonly. I think it's probably the most popular, but there are other alternatives. This is the equivalent of the UCSC genome browser for next-gen sequence data. So has everyone used the UCSC browser? Are most people familiar with that? Ensemble browser? The NCBI browser? These are all just different kinds of genome browsers. And in those, you can get a representation of your alignments and you could load them into, for example, the UCSC browser and see maybe you've done it before with like, I don't know, a set of primers that you designed. You want to make sure that they map to the, you know, part of the genome that you expected. So you're visualizing them in UCSC genome browser. Same idea, but it can handle these huge data files.  That's happened to me before because you're just using too much of their bandwidth. Second of all, it would take forever. It just wouldn't be practical. So the integrative genomics viewer is a good alternative. You can fire it up. We're going to do this later and load in your BAM file. It usually needs to be indexed and you need to have your reference genome. And here what we're showing is a single chromosome. So you've got an ideogram. You can select the region here. The viewer position. So the portion of the genome that we're at is marked here with this little red mark. So we're looking at just this little sliver of the genome. You have a track. This thin track along the top gives you an idea of coverage. So basically in your alignments, how many reads are stacking up in each of these positions. So you can see spikes of coverage, which are occurring over exons. And the reason for that is that this is RNA-seq alignment. And we're mostly just getting reads aligning to the transcribed portions of this gene. Each of these little marks here or bars indicates a single read and where it aligned. So you can see there are some reads that align just within an exon. And there are others that have this little thin line between that indicates they're mapping from one exon to another exon. So those are reads that are actually spanning an exon exon junction. The bottom track is a gene track. So you can compare your alignments to what's known about this locus in the genome that you're studying. Has anyone used the IGV before? Yeah. Sorry, which track? Splicing signs. Yeah. You can put almost anything into IGV just like you can with the UCC browser. And in a similar way, you would make like for example a bed file that represents known junctions or splice sites or genes that aren't annotated or any arbitrary definitions you want.  Any other questions? So we're going to check this out in the lab tutorial. There are some alternatives like I mentioned. There's a couple bios to our posts talking about the alternatives. These are ones that we know of, Artemis, BamView, Chipster, Gbrows2, GenoViewer, MagicViewer, Savants, Tablet, and TView. The only ones I've used other than IGV or Savant, and it's very similar. It's a very good product as well. Subtle differences really you could use either I think and be fairly happy. So here I'm just using a screenshot of a view in IGV to illustrate the concept of variant allele expression. So I don't know if you can make it out very easily, but in IGV the reads here are color blue and basically the way it works is this is a reference you know indicated at the bottom. You only see bases indicated at color when they're different from the reference. So pretty much everywhere here you can see the alignments correspond perfectly to the reference you know, but you've got this stack here of differences. Now those could be errors like these three guys here are probably just random errors, but the fact that you've got basically like 50 percent of the reads representing a T here instead of the reference C probably indicates this is a real variant. So a variant of C to T is observed in 12 out of 25 of the reads covering that position. And from that you can actually calculate a variant allele frequency. So 12 out of 25 is 48 percent. So about 50 percent of the reads are supporting one allele and the other 50 percent are supporting the other allele. This makes sense for a heterozygous variant with no allele-specific expression. So again I'm actually showing are any C alignments here. It's possible that for whatever reason one allele is expressed preferentially or maybe the other allele is deleted in which case you might see 100 percent of reads supporting the T. So you can learn information about the variant allele frequency and you can also just visualize variants in IDV. And we're going to do some examples of that as well. But a lot of times we don't want to have to look in IDV to calculate a variant allele frequency. But we do want to know whether there are variants, what the read counts supporting those variants are, what the variant allele frequency is. So we're going to introduce you to a simple tool called BAM recount which basically for any arbitrary position that you give it will go and just count. So if you say tell me what's happening at position whatever 1,000,223 or whatever the position is it'll go there and just count and say there's zero C's, well in this case 13 C's, 12 T's, zero G's and zero A's. So that's the last part of that tutorial. It's a BAM recounting. So just to kind of remind you where we're at in this whole workflow we're going to do module two which is really the start of it which is getting our RNA seek reads and performing the read alignment. And we're going to use bowtie and top hat to do that. So do we have a break schedule now Michelle? Okay. So we might as well get started.


I think one of my favorite fan-goal bats coming into the football season is the LA Rams plus 500 to win the NFC championship game. Not the Super Bowl, the AFC is stacked, but just the NFC. Green Bay losing Devonte Adams doesn't have the firepower. This is Brady's last year in Tampa. Will they have the determination and focus of the Rams? I get a great coach, I get a great quarterback with Super Bowl winning confidence. We don't know what the Niners are doing at quarterback. Really think Dallas is in that space? Philadelphia made the playoffs, but I don't think they stack up with the Rams. Plus 500 to win the NFC? I'll take my chance as I like it.