Fiery horse with a speed of light, a cloud of dust, and a hardy high old silver, the Lone Ranger. With his faithful Indian companion, Tonto, the masked rider of the planes led the fight for law and order in the early west in the United States. The stories of his strength and courage, his daring and resourcefulness, have come down to us through the generations and nowhere in the pages of history can one find a greater champion of justice. Return with us now to those thrilling days of yesteryear. From out of the past come the thundering hoofbeats of the great horse, Silver. The Lone Ranger rides again. Gold was found in the Black Hills, new communities sprang up almost overnight. Each day brought men with their families who had staked all on being among the lucky ones who struck it rich. Flimsy houses were thrown together, cafes and gambling places did a flourishing business. Prices of commodities went sky high and everyone talked in millions. The Lone Ranger and Tonto arrived in the Black Hills after a trip of many days from the country to the south. They headed toward Deadwood. We'll make camp somewhere near Cranesville, Tonto. That long way before town Deadwood. I know it. I want to talk to a few people in Cranesville. Maybe we won't have to go to Deadwood. Why that? We want to learn all we can about Clark Drexel. That's right. Cranesville is near one of the richest gold mines in the Black Hills. In fact, the town is named after the owner of the mine, old Eli Cranes. If Clark Drexel and his gold mine syndicate had any dealings in the Black Hills, well, Eli Cranes will know about them. I think we can learn as much from Cranes as we can in Deadwood City. And maybe find where Drexel do crooked work, huh? We've got to find something in the career of that man, Tonto. Some evidence of crime so he can be jailed. Yes, we know that Drexel was one of the organizers of the Black Arrow, but that fact can't be proved. The only thing we can do is find some other reason for jailing Clark Drexel, and that won't be an easy matter. Hey now, Kimosabe. Who's got a home? Look down there, Tonto, in the valley. Uh, their town. That's the town of Cranesville. We'll make camp here until morning, and we'll push on to town and have a talk with Eli Cranes. Uh, you wait. What is it? Me hear horse wagon come this way. Wagon? Needs mountains? Yes, you're right. That, Tonto, I hear it too. Ah, there come. From hill above. You see it? Yes. It's a fine place for a man to bring a wagon. I wonder why he didn't use packed news. Here, pull off the trail, Tonto. Give him as much room as we can. You'll need it. Uh, come scout, Tonto. That's it, boy. Easy now.  I want to keep going down hill. Masked. I'll shoot a purie's mask. Never mind who they are, Lodge. Lairpuff and git for hold. This is just the way we'd have planted. In a special order. Git! Maybe them from a local. You hear them? Yes, I heard them, Tonto. Why them take empty wagon down hill? I don't know. Why are they using a wagon at all? A hard trail for a pack mule. Dangerous for a wagon. And wagon empty. There's no town uphill from here. The only town close by is Cranesville. If they'd been going uphill, I would think they'd plan to bring ore down in their wagon. But this... You see the way one fella look at you? Yes, at my mask. I wonder what he meant by that statement. You remember what him say? Yes, he looked at me at the mask and said, never mind who they are, git for hold. This is the way we'd have it if we'd planned a special order. Home. Him say git for home. That mean down hill. Maybe Cranesville. You want a fella? Not tonight, Tonto. Our horses have had enough. We'll camp until morning. Then we'll ride to Cranesville. Eli Crane was an old man and a wealthy one. His gold mine was the richest in the Black Hills and his home the largest in the district. His son, Jim, lived with Eli as well as Jim's daughter, Molly. Molly was a pretty girl of 19. As she opened the door for her grandfather, his face showed worry and concern. Graham, you've been hurt. What happened? Oh, go on the door. It's my cousin infernal bad luck again. Come in. Let me wash your head. Oh, you poor dear. What happened? Don't get yourself excited. Just a little cut. Not near as bad as it looks.  This way, Graham. Oh, how did you do it? I didn't do it. You think I'm from local? Bastion mown head? I was in the gold crown cafe and a couple of liquor, swizzling saddlebombs got doggy and then shoot. It's a boy's wound. Just grazed me. Then a quarry. It's a killin' you. Oh, dog gone. It, Molly. It hurts when you wash it. You shouldn't be allowed outside the house. The trouble you've been into during the past weeks. It's my dog gone bad luck. If I'd had this kind of luck all my life, I'd never lived to be past 60. Easy with that there, biting, burning medicine now, Molly. This'll fix the wound. Now don't yell when it starts biting. No. Oh, that's like a red hot branded iron. That is the worst of it. Now you'll be all right. That scratch won't need a bandage. How's your arm? Oh, my arm's all right. But it's a nine day wonder. It wasn't busted from that fall I got when the bridge went down.  You sure must be a tough one, Grant. Wasn't being tough that saved my neck. Was grabbing on to a tree on the bank when the bridge caved in under me. So hang it all, Molly. I wonder if I'm jinxed and hoodooed lately. Seriously, Grandpa. Do you think all the things that have happened to you, the narrow escapes were, were really accidents? What else did they be? You haven't any enemies? Enemies? Of course not. Well, might be a few men I wouldn't call friends, but no enemies is what'll kill me. Now sit down and rest. Your head must take something frightful. Not too much. Sorry, Rodney Granton Town. Oh, then, then he's back from Deadwood City? Yeah. Did, did he see Pa there? Talk to him. I'm glad. Let me know that Pa's safe. Rodney said that Jim wanted him to tell us he'd be back here in a week or 10 days. Good. It was just a plump foolish notion of yours, Molly, that anything had happened to Jim. Oh, I suppose so. Turn nation, take it. You had me beginning to worry. Where'd you get the idea in the first place? I, I don't know, Grandpa. Why, I felt that something had happened to Pa. Just a woman's full hunt, I guess. I guess so. And Rodney Grant said he might drop in for a little while. Oh. He don't act over pleased about it. Why should I? All right, Molly. Here, if Pa thinks Rodney'd make a fine husband for you, Rodney thinks the same.  Rodney's educated, of course. And a good job, working from that big goldmines indicate. But you make up your own mind, honey. Don't let yourself be influenced. I'll answer it. It's probably Rodney now. Well, Miss Molly, good evening. Good evening, Mr. Grant. Won't you step in? Thanks. Well, Eli, how, what happened to your head? Got grazed by a bullet in the cafe just after you left. You don't say. Yep. Sit down, fillet. Thanks. You had an arrow escape, Eli. Had a lot of them the past week. Getting kind of tiresome. Oh, I told Molly that you'd talk with Jim. Oh, yes, sir. Thank you, Mr. Grant. It was kind of you to go all the way to Deadwood just because of my silly feeling that something had happened to Paul.   I was so prulish when that old prospector came in from his trip and told him seeing a dead man at the bottom of a ravine. I had the strangest feeling that it was Paul that he'd seen. I don't know why I felt that way, but I couldn't shake it off. But you needn't have any further concern, my dear. Your father's alive and well in Deadwood City. Does anyone know who the dead man is? No, but we will very soon. Two men went after the body. They should be back by this time. Probably some poor prospect.  You didn't say that you hired Lige and Hank to go after him. Oh, well, I didn't. Did you really? Nothing at all. Drexel's syndicate will be glad to pay the expenses. We have a great interest in all prospectors and miners, you know. What I hear that outfit you work for can well afford to help prospectors and miners and see him get a decent burial. Right you are, Eli. Well, that reminds me. Have you reconsidered my proposition? Nothing to reconsider. I won't sell my mine. Of course, it's up to you.  Paul says you do well to sell out. All of us could go and... I know what the mine's worth. And Drexel's syndicate don't come close to that price. Oh, we can't, you see, Eli. Many a time a mine will peter out suddenly. And of course, the way we operate, it costs a lot to bring and install a heavy machinery. When I'm dead and gone, Jim can do what he's a miner. While I'm here, I'm keeping the mine. No hard feelings about it? Of course not. None at all. Hey, Mr. Grant! What's happening outside? Mr. Grant! Mr. Grant, are you here? Well, that's Lydz, one of the men I sent to get the dead man. I left the word for him to report to me here. Hey, Mr. Grant! Oh, what is it? When are you here? It was stolen. Masked man, don't interrupt this. Well, wait a minute, Lydz. You come inside with me. The rest of you stay there. Here, Mr. Doug, I'm going to sing. Wait a minute here, Mr. Grant. We went up there with the prospector said. Did he go with you? No, he told me in Hank where he saw the dead man on the floor of the ravine. We found him all right. Was it anyone you knew? No, just no prospecting. What did you take the body? Well, that's just it.  Watch that! It's true, so help me, Lydz. Why? I don't know why. This is a fine thing to talk about in front of Miss Molly. You come outside and tell me the rest. Mr. Grant, I... Such things are not for you to hear. Out with you. Sure thing. Hey, Mr. Grant, what did you do? I'll talk to you later.  Oh, hello, Mr. Grant, let me tell you...  The one else knew that Jim Crane went to that section instead of the deadwood. He was making a secret trip. I told you just to make sure it was Jim, and if so, to come back and say you hadn't been able to find the body. But listen, if we'd have done that, the sheriff might have sent the man who reported it out there. The fool I saw, too, the old prospector left the community. He's miles away now. Yeah, but we said we couldn't find the corpse. The sheriff might have sent more men hunting it. You could have hidden it until we needed it. The sheriff still might have found it. We did hide it. We saw that masked man and his Indian friend. We figured it'd be a good idea to see if we're stolen. Now, the sheriff will scour these mountains for the masked man. What if he finds that man? Let him. The man's likely a crook anyhow. What does his word mean? Who'll believe him if he says he didn't steal the dead man?  Maybe you are a slick operator, but me and Hank Savard, the way things are handled in this part of the country. Let the sheriff find that masked man. We'll handle things. Following morning, when the lone ranger approached the community, he was seen by a man who let out a wild yell. There he is, riding in the town. Hey, sheriff! Come on, Steve Boyce! Come on in! In an instant, men came running from the stores, the nearby houses and cafes. Get guns on him! Get him, bro! The sheriff wearing his star of office and his vest pushed in the foreground. Get off that horse, stranger. You're the gent we're looking for. What's the matter? Has everyone got sweat on them? That's our mask. Oh, yes. Well, let me talk to you alone, sheriff, and I think I can explain the mask. You'll explain a lot of things, and you'll do it right here or now. Here was a map in the prospectus pocket. I told you about that, sheriff. You attacked two men who were bringing a body of a prospector. You made off with a dead man. I what? Who told you that? I did. Here's my partner to back up what he said. The law acts fast in these parts, mister.  How can I tell when I don't know? You can't make us think you don't know. Now talk. The curtain falls on the first act of our Lone Ranger story. Before the next exciting scenes, please permit us to pause for just a few moments. Now to continue our story. The Lone Ranger remained in the saddle while the sheriff and many townsmen surrounded him with the demands for a confession of crime. Come along now. We'll take you to the jail and maybe see if that won't make you ready to talk. One minute, sheriff. I'm not going to jail. No, we'll see about that. Those men have lied. They saw me on the down trail. They saw an Indian with me. That's right, sheriff. I told you about the red skin. Their wagon was empty at that time. You stole the corpse to get a map that he had on him. Show him where his claim was at. The name of the Indian with me is Tullo. I wrote ahead of him in case I met someone who misunderstood the reason for my mask. Right now, Tullo is watching us. We'll arrest him, too. He's watching and ready to shoot any man who tries to stop me. I'm leaving here. Come on, silver. Hey, get him! Shoot him now! I'll get you! Shoot that gun! Don't shoot! I dropped my gun. There's the red skin.  Just come on! Get him, boys! Shoot him! Don't go on. They both got away. Here's any house, sheriff. We can watch for him. Fine pack we are. A dozen of us here and we'll let that masked man get away scot-free. But that horse, Cislin Bacon, did you see a traveler? Nothing like it. We'll hunt him down. We'll start more men out to look for him. You deputies come to my office and we'll organize so he'll be hunted down. Hey, you go tell Mr. Grant what's happened. It's all worked out for us, Ray. Paul in the town seized with excitement. Men rode through the hills looking for the masked man in the Indian but found no trace of either. One man in town followed events with more interest than anyone else. It was Eli Crane. These young upstarts never got around in the West, Molly. That's the trouble. Thunderation. I could tell them a few things about that masked man, the white horse in the Indian named Tunnel. You? You know him, Grandpa? No, but by Juniper I'd like to outlaw my eye, thief my foot, lige and hanker, the outen-outness no good's ever lived. The only thing I wish is that I knew why they lied. I would... Well, I'll be boiled in stale water. Grandpa, what? The masked man? Please don't be frightened. I had to slip in with the rear door. There he stands, Molly. Take a look at him. I am. They call that man the Lone Ranger. Mr, I want to take your hand and shake it. Thanks, Eli. Thanks. I've got to speak to you alone. Well, without Molly here? Yes, it's important. I'll go to my room, Grandpa. Thank you, Molly. You'll understand later. Very well. Eli, do you recognize this silver belt buckle? What? Where'd you get that? Do you recognize it? I gave it to my son when he was 21 years old. He's always wore it. This buckle brought me here. I, uh, I found it in the hills. Jim was in Deadwood. Now, Eli, he wasn't in Deadwood. The hills? Well, after Tonto and I got away from the sheriff's man, we followed the back tail of those two that lied about us. We found what they'd done with the man they took from the ravine. Born? Yes. This isn't a pleasant duty, Eli, but it may prevent further mischief. You know now. Jim is dead. It was Jim in the ravine. Yes. He took his belt. Brought it here? To make sure who he was. I, I hope to learn why those two hid the body. Was he murdered? No. He stood too close to the edge of a ravine. Ground caved in under his feet. I wonder. Don't call it all. I wonder what those two lied for. It was Rodney Grant that hired him. Grant? He's from a Drexel syndicate. Trying to buy my gold mine. I won't sell. Tell me some more, Eli. Tell me everything you can about Rodney Grant. Will you trust me that far? I'll tell everything I can think of.  For over an hour old Eli talked.  Then Molly was called into the room and the little conference continued for another hour. Later that night. Great Scott shooting an old Eli's house. Get him. Get him. There he goes. Sooner or later.  All right. All right, get the sheriff. Tell him to come at once. Tell him it's my grandfather. Shot? You had the shots. You saw that mask man. The mask man. Great gun. Kill the sheriff to come. Oh, do hurry. Wait here, man. I'll see Miss Molly. Sheriff, I'm waiting for you. Come in. Right with you, Miss Molly. Come in, sheriff, but come in alone. It wasn't until the following day that Rodney Grant heard the news. And he called a meeting of the men who were helping him. He grinned as he closed the door behind him. He certainly gave us a hand, boys. It's a good omen. Yeah, it'll take stock and good homage. You pay us some cash and it'll make us feel us better. Well, I can't give you all I promised just yet, boys, but it'll come, never fear. Don't tell us you're sure to cash money. Well, I had to pay out a lot, remember that. Several men who tried to put old Eli away had to be paid. Well, did they? They didn't do anything. The old men escaped everything they tried. Yeah, I didn't want them to be just satisfied and start talking. How much did it cost to hire that mask man? Yeah, that's what I've been wondering. How'd you get him lined up to do the shooting? I didn't have anything to do with the murder. That's a strange part of it. That's why I said that fate was good to us. Now what? Are you ready for us to make the next move? Yes. Can you two get out of town with your wagon without being seen? Sure thing. That won't be hard. And sure if we spend most of his time at the crane house. Very well. Get everything fixed then. We'll plan the fire for the night. We got the shack all fixed. It's between here and Deadwood. All right. Sure, it will be hard on poor Miss Mully. I'm sorry for... It'll be the best thing for in the long run. She thinks your father's still alive. Well, she's got to know some time. Now she can sell a mine and go east with a fortune. Maybe as my wife. I kind of knew too to handle the fire tonight. Don't slip up. I'll pay you some as I get more cash from my company. Send it, Walter. When they hear that I can buy the Eli Crane gold mine, I should say they will send it. All right then. Come on, Hank. We've got things to do. Well, there's nothing in the way, nothing at all. The girl will sell. No doubt of that. Hey, Mr. Grant. Well? The sheriff's over at the Crane place. He's going to read Eli's will. Well? You want to go over? He said he thought you might be interested in it. Oh, sure. Thanks. This is my big day. You can sit right there, Mr. Grant. Thank you. I won't waste time going through all the legal angles. I'll just tell what the will says. I hope, Sheriff, you're not displeasing Miss Mully by rushing things. After all a few days wait till Jim Crane gets home and Eli is buried. There's a reason for doing this now, Grant. As long as Mully doesn't object. Eli made this will some time ago. It leaves everything to his son, Jim. That's simple and clear. But if Jim dies before Eli, or if Jim marries again, then there's a cash settlement to Miss Mully the granddaughter. All the gold mine property goes to Jim's younger brother. Oh, younger brother, huh? Yes. He's an east. He's an operator in gold mines. He wouldn't sell to you, Grant. Well, as it stands now then, Jim Crane owns the property, doesn't he? That's about it.  Well, if there are no questions, Sheriff, I'd like to... I've got some questions. I'm coming in now, Sheriff. Talk to that ordinary polkaid. He likes cranes. Serious, dirty schemer. Look at the man you tried to murder. You're not dead. You don't go right, I ain't. Where's the Lone Ranger? Where is he, Sheriff? I see him coming. He's dubbing outside now. Good. He's on the porch. Come in.  Lige and Hank have gone. I was watching them. Good. Those two will tip Grant's hand. But I don't understand. You will. You tell them, Lone Ranger. Tell them weasel-faced skunk. You wanted to buy the Crane property for your boss. Clark Drexel. Of course, but I... I wouldn't sell. So you figured on having me killed off, knowing that Jim had sold. And you learned that Jim had been killed in a fall. But you didn't tell anyone. You looked up Eli's will. You found that with Jim dead before Eli, the property would go to another son. One who would not sell to you. I don't know what you're talking about. You will. You hired Lige and Hank to hide Jim's body until Eli was dead. And you planned on making everyone think that Jim died after his father. Well, this is ridiculous. Why should I? The law would then assume that Jim inherited the gold mine. And when he died, his daughter would be the heiress. She would then sell to you at Drexel's price. You scheme it that way, Grant. You can't deny it. Who can't deny it? I deny every word of it. You lied when you said you talked to Pa and Deadwood. You talked to him and Deadwood. How does it happen that I found this belt on the man Lige and Hank hidden in the mountains? I don't know anything about it. You're getting there. Don't do anything to my arm. Where are your cows, Grant? Uh, me find them. Them got wagon outside. Them talk plenty now. We didn't do no crime. Lige, look at me.  We didn't always scheme to kill... Hugh, the man's man. There's an old ranger, you coyote. Go on. Don't do nothing to us. We got Jim from that ravine. We only kept him where he wouldn't be found, that's all. What were you going to do next? Why, we was... Go on, speak. We was to take him to a hugger's cabin instead of fire tonight. So everyone would think he'd burned to death 24 hours after his father was killed. Who suggested that to you, Lige? Grant did. I'll get you for that. He did. He paid us. I'll tell more about him, sir. He hired a couple of breeds to start a fight and shoot Eli as if by accident. He hired the same breed to try and kill Eli by cutting bridge under him. Boy, you're funny. Sooner or later, I don't know what the law can do, but I know what I'm going to do. No, no, don't. Oh! There. Wrecking, I can still handle my fist. Oh. Grad, it looks like he's busted that chair in his form. You're not going to clean out, Eli.  Scheme to buy my gold mine. Molly, your pa wouldn't have sold it the price Grant was offering. No matter what he thought at first, he wouldn't have done it. No more than I would. Oh. When you inherit after I'm dead and gone, you can do what you want. I'll never sell, never. We cranes will always operate our mines. Some of us have got to stand by our lands. Yeah. If this Drexel syndicate gets all the mines, the way they're out to do it'll be bad. They'll set the prices for men's labor. That's right. They'll set prices for shipments. They'll have too much power. Now, this year country isn't based on giving one outfit all to say. We've got to have competition. Free enterprise. Men that run their own business. You're right. I understand, Grandpa. Boys, you can take Lige and Hank to jail. Between them, they can tote Rodney Grant. If there's no law to jail, Grant, then he'll be downright unlucky. Because if I know the men here, he'll leave town on a rail wearing hot-tarn feathers. Miss Molly, I'll attend to Jen. Thanks, Sheriff. Say, where's that mask man? Where's the Indian? What? They've gone. Well, get him. I want to thank him. There they are. Only horses outside. I want to thank him. You have just heard, is a copyrighted feature of the Lone Ranger Incorporated.


Okay, so module two is about the data. So you have seen with Mike what you will do at the end of the day when you receive the data, when you have generated your volume call and you want to find the clinical aspect of the work. So in order to really understand what you will have at the end of the day, it's important to understand how the data is generated and processed. So it's what we will see in that module and in the lab afterwards. So what are the objectives? I will give you a short introduction to what type of resource we use to do computing of this genomics data. To understand the kind of data we will use in genomics and to understand how we analyze them to how we map this read on this data on the genomes and what are the type of error you could face when you do this type of analysis and you will also learn about terminology and format. And in the lab we'll do the first step of data processing. So first, a small introduction on high-performance computing. So when we work in genomics, we cannot use traditional computer like your own laptop or your own personal computer. So what we need to have high-performance computer, which are called clusters, and it's a really a kind of pool of 100 or 1000 of different computers together you could use to do your analysis. Why we need that is because the size of the data is really big and the throughput of the data that is generated all over the world and especially here in Canada is really increasing every year. So building this high-performance computer is really costly. So you don't need to do that because there is an initiative in Canada which is for Compute Canada which built all this high-performance computer server in all over Canada. So in every province you can find at least one or several server and you can use the one of your province or the one of other provinces. So the idea of Compute Canada and of this server is to ask for an account and if you are a Canadian academy or a Canadian researcher, you will have access freely to this resources. So you ask for an account, they give you an access, then you will have an allocation every year which means allocation, you will have a complete time. So a number of cores you can use for a given of time and you have a storage space.  So it's really a simple way. How you can get an account, we'll not give you detail but you need to go and you have the style, you need to go to Compute Canada to apply and then you go to the contraption, so to the province contraption and you apply for a specific account in a specific cluster. Once you have your account, you can now log and do your analysis. What is important and when you do your analysis because usually you don't want to have your result as fast as possible is the important thing is the queuing time. Being on queue because a lot of people are using this resource, it's a shared resource, so being on queue could take, could use most of your time than the real analysis. So you have some parameters you need to play when you do, when you set up your jobs is to, what are the lengths of the job, what are the number of CPU you need and how you have already used your allocation. So this is a parameter you can use to try to avoid spending so much time on queue. So how busy is the server? You cannot control, but if the server almost full, you will have to wait for time. So we have this resources available through Compute Canada and we will use this resource during the practical after my talk. But what is interesting is that when you do genomic analysis, you don't want, you want to use this resource, but you want to have access to all the software that people are using and all the other resources. So in partnership with Compute Canada, the C2G have built a system based on the CMFS, so it's a CERN virtual machine file system, which is a way to provide to every HPC site a set of resources. So the idea is we take care of installing and maintaining the resources in one location and then every site will have the same resources available. So you could use a cluster in Ontario, in British Columbia, or in Quebec, you will have access to the same software, to the same data set in terms of genome references and so on. So we do that through an initiative, which we have called Genpipes, where we provide so the bioinformatics tool. So we provide more than 90 different tools for genomics. We provide genomic resources, so 20 builds for 14 spaces. And we also provide a Catern analysis pipeline, where you can just use the pipeline and you will see what we'll do today is part of the beginning of one of the pipeline, which is the NSIC pipeline, but we have a pipeline for cancer, for RNA-seq, for chip-seq, for methyl-seq, metagenomics, so all these kind of things are already set up for you if you want to use it and analyze your data. So it's a really, really big effort that we have done in partnership with Complete Canada, so any trees, so feel free to use it. Many of you, if you are not Canadian, you may not be able to use it. So if you're not Canadian, you could consider it to become Canadian, but it's not so easy. But other thing is that all this aspect of HPerformance computing also could be applied to other type of resources, like cloud computing. So if you're not Canadian and you want to use Amazon, things could be, analysis could be done the same way. And we are now working on a version of GenPype in a container that you can directly use in this type of resources, like cloud computing, where you will have access to the same type of resource in terms of software and genomic resources. OK, so now we have a better idea of where we should do this analysis. It's what the data and what the analysis we will do for genomics and is specific when you want to generate the variant. So what's the data? So most of you should know about Sengar sequencing as a usual traditional sequencing method. So here we talk about high-super data, so next generation sequencing data, and it's not a clone-based data as we did previously, but it's a cluster data. So what the main difference is that instead of generating hundreds of sequence at a time, we generate now hundreds of millions of seconds at a time. So it's another scale of analysis. So all of this is based on what we call for small re-jumpification. So when you do your library prep, you take your data fragment, you share it in small pieces of a given land that you have chosen, and all these pieces you put adapter at the two ends of your fragment, and you put that on the flow cell of the sequencer, and then you will do amplification. So the probes at the end of the fragment will fix probes on the flow cell, and the second probe will fix another probes, and you will create a bridge, and then you will have an amplification of your sequence, and then you will have two strand structures after the first amplification. Then you will relapse into single strand structures, and you will do the same again and again to generate a white round copy of your initial fragment. So at the end of the flow cell, you will end up with that type of profile where you will have a lot of clusters that represent the same initial fragment you have put in the flow cell. The sequencing will start from the top of your sequence, and at each cycle, each fragment will have a base which is labeled with a fluorescence that will be had to do the sequencing. So at each cycle, the machine will take a picture like this one. So it's a small part of the picture, and each of the cluster will generate a dot on the picture. And it will correspond to one base. So for the first cycle, here you will have a picture, another for the second, three, four, five, and you will be able to generate the corresponding sequence for your fragment. So if we take that cluster, we'll have yellow, blue, green, yellow, red, and it will be GCTGA. OK, everybody understand that? So how? We do that for every cluster at the time, for a million, hundreds of million clusters at the time, and we're able to generate each basis of each fragment for all the DNA fragments that have been put on the flow cell. So that's what the sequencer will output. So based on that, what we'll do, we'll take this and we'll process in order to generate a variant called, so to generate what are the specificity of each of your samples. OK, so in that module, we'll only see how we can start from the data we've got on the sequencer to what we call a BAM file. So it's an alignment file, so a high quality file that you can use to do variant coding. So this is a full pipeline. So in this module, we'll just focus on that part, which could be represent like that. So we'll have the FASQ file from the sequencer and we want to generate alignment-ready file to do variant calling. So variant call and what we do from the variant will be presented this afternoon in the module tree. So when I see a FASQ file, when I talk about FASQ file, it's what you receive usually from your center. So you will either receive one or two files per sample, depending on the design of your sequencing. But whatever the number of files you will receive, the format will be the same. So for each sequence, you will have four lines. The first one is the header that represents the name of the sequence and some positional information. Followed by the line where you have the real sequence itself here. Then you have a second header. So depending on which technology you are using, but you have either in the second header or just a plus sign. In the numerator plus sign. And then you will have a second set of sequence, which is the base quality sequence. So it's a score that is encoded as an ASCII characters that tell what the quality of each score that have been made for this sequence. So how it works, you take this character, you translate it into numbers with the ASCII code. And then this number gives you a probability of error. So it's what we call the base quality. So what that means, so the base quality, the number you have there, is a thread score. A thread score means minus 10 log base 10 of probability. And the probability in the case of base quality is a probability that you wrongly call the base. So for example, if we have a base quality of 20, we have 1% chance that the base we called for this sequence is wrong. So the higher the score is, the better you can trust. The better is your base called and you can trust it. So usually what we receive is data. We do a lot of QC. One of the main important QC is to look at your base quality all around your sequence. This is a type of graph we generate where each box is represent one cycle. So all the first base of all read for that run, and you have the distribution of base quality for that cycle. So it's cycle, first cycle to cycle, third cycle, and so on until the end of your reads. So it's really important to look at that because having high quality is really important. If you have low quality, it will bias the mapping. It will bias the snip calling and all the other subsequent analysis you will do. So when we have this data, usually what we are doing, we have a look at the FASTQ. Usually we saw this pattern with decrease of quality at the end of the read. It's a normal pattern. So what we do, we usually trim the data. So what we mean by trimming, we filter read or part of the read that we don't want to keep analysis. So part of the read could be either we filter. So if we have read that are fragments that are short, we will have the sequence, your fragment. But at the end, we will start to sequence the adapter. So it is the sequence we add for the sequencing. So it's this technical sequence. So there's no genomic sequence. You don't want to have that in your analysis. So if we find them, we will remove from the sequence. Also, as we say, if the quality is not good, we'll start from the end of the read and check for quality. So the user will define which given threshold. And if we saw base under this threshold in terms of quality, we remove the basis. So at the end, you will end up with reads with different sizes. And what we do, we only keep a read that are longer than a given length. Because otherwise, the short read will not be really informative and may create some bias in your analysis. So it's the step of trimming. So after the trimming, what we can consider that what we have generated is the most high quality sequence. No, we want to do the alignment. So what the alignment means is to find the best location of your sequence on your reference genomes. I say best because as the reference is not complete, it may not be the real sequence. So you really want to find the best location. It seems easier to do that, but it's not so easy because, first, you need to do that for a million of sequence. And you need to find a location in a space that are a billion of letters, so it's not as easy. You cannot use a traditional algorithm like Blast. Otherwise, you will launch your analysis and you will come three months later. It will maybe finish or not. So we want something fast and accurate. And the issue is that some sequence will have many locations, so you will need to find which one is the real one or the best one. And you cannot only say I want perfect matches because the interest of the analysis is to find what is specific to your samples. So you want to tolerate not exact match to be able to catch the biological variation in your specific sample. But when you do that, you also let errors and technicality become part of the play. So you need to do not exact matching, but you will need, after that, to correct for possible error. So to do that, we use an algorithm which is called Brutal Widow Transformer. I will not explain it today, but it's the one that is not used by most of the mapper and will use the BWA mapper, which is one of the best mapper that are available for DNA-seq. If you use other type of data, of data, RNA, or GIP, you should use probably another mapper. When we do the mapping, it's really important to do. So when we do DNA, usually, it's happened quite often that we do several run of sequencing for one sample. So in that case, each run of sequencing should be aligned individually and then merged all together. So when you do that, you need to put a tag on each run of sequencing to be able to identify your read at the end. Because if you see something particular in your sample at the end of your analysis, you will be interested to go back and to see if this pattern is shared between all your experiments or if it's something that happened in only one experiment. In that case, it will be a batch effect or something like that. So it's really important to do that in order to decipher your signal. But also because a lot of tools that we use need to have these tags on the data. So when we do the alignment, we generate a file called another file, either a SAM or BAM. So the SAM is a text file. The BAM is a binary complex file. Now most of the tools use only BAMs because it's a way smaller in terms of space on your disk. And what's the format of this file? So for each sequence, you will have one line this time. The line will be like that. You will have the first column, which will be the name of your read, the same as in the FASTQ. You will have a score here, a flag that tells information about how the fragments have been mapped. So compared to what is made, compared to the strength, compared to all of this kind of information, we will see it more in detail during the practical. Then you have the position here, where as the sequence has been located. So the chromosome, the position on the chromosome, you have the score of your alignment. It's a thread score that tells you the quality of your alignment. You have a segar string, which tells you information about how your fragment have been matched to the reference. So it's really a kind of physical matching description. And then if you have a pardenary, so if you have two end of your fragments that have been sequenced for the same fragment, so that you have the mate information in terms of position and the inside side. Then you have the sequence in terms of bases, the sequence in terms of base quality. And usually, you've got extra flags that I will not describe here, because these flags are really aligner-dependent. So each aligner will generate its own flag. So when we have done that, we have generated the alignment. But alignment is a complex process. And when you do that, the aligner needs to choose some parameters like penalties and so on. So it's not a perfect job that has been done. There's no aligners that will do a perfect job. So what we need to do is to take this alignment and to refine it to have the high quality alignment file before doing the variant calling. So how we will refine the alignment, the first thing we will do is to do indelible alignment. So why we need to do that is because most of the aligner tend to favor to create mismatch between the sequence, the reference sequence, and the read sequence, instead of creating gaps. So if you are in a region where you have indels, you will quite often see this kind of patterns where you will accumulate mismatches on your reads in a really close proximity. So we know that we don't expect to see variant quite often in the genome around one variant every kilobases. Depending on the region, it could be less or more, but you don't expect to see like five or six variants in 100 bases. So what indelible alignment will do, it will look at your fragment and look at your reads on the genomes. They identify in the first place region where you think there may be an indel that have not been called. And then it will go back to this region, retake all the reads that are mapped on that region, and try to insert indel to see if that can improve the mapping. If you see that it don't have improved the mapping, it will let the read the same way. If you think that the indel could improve, it will realign the read. Now, in that case, in that region, you see that adding the indel make almost all the variants disappear. So probably all these variants were technical artifacts. So we're false positive if we don't have the indel realignment. Another type of refinement we want to do on the data is to mark or remove duplicates. So what are duplicates? Is duplicates is when you do your sequencing, you have your DNA fragment at the beginning, and what you want, your goal is to have one sequence per initial DNA fragment that you have generated at the library prep. But due to the way the library are generating, you could have several sequences that represent the same initial fragment. So you don't want to count that fragment several times. Because if there's an error at the beginning, then you will see this error that pop up in several fragments and you will see, oh, probably it's a variant. No, just an error that you see several times. So you really want to see only one copy. So where does this duplicate come from? It could come from in all Illumina flow cell. It could come from if you have a really large cluster, it could take this large cluster and consider it as one or several clusters at several clusters, two, three, four, five clusters. So it will generate several copies of the same data. In the new Illumina flow cell, where all the clusters are already defined, if you have empty cluster sequence can jump from one to the other cluster. It could come from PCR. So if you do a lot of PCR cycle, it could generate many copies of your data. So it's why now, many of the library kits that we use are called PCR-free to reduce, to remove all the PCR steps in order to remove the duplicates. Or it could be sister, so Camaric sequence that generate because of two sequence in the flow cell that merge together and that will propagate around a free cluster. So there are different ways to detect them. And here we'll look at the two different approaches during the practical. So it's really important to remove that because otherwise I say it could create false positive in your data. As I say previously, the base quality is really important when we do variant coding. It's usually based on a Bayesian approach which take different type of parameters in the model and one of the parameter is the base quality of the sequence at this location. So what we want, you want to have the real and more accurate base quality. And it had been shown that the vendor, the sequencer try to inflate the base that are generated by the machine to say, oh, we are good. And especially that this machine suffer from specific bias related to the genomic context. For example, which basis are you sequencing one after the other or by the position of your read by the position of the basis in your read. So the idea of the base for calibration is to take all your alignment to look at this known pattern of error to model it and then to correct for it to have a better representation of your quality. So when we have done that, we have variant coding ready alignment. So we are ready to do variant coding. But what is important to do also all along the process is to generate metrics. Really, metrics is really, really important. At each type of your analysis, you need to generate metrics. Why? Because if you have any issue at the end of your analysis, by looking at the metrics you have generated, you will be able to understand where the error come from and what's happening in your analysis. Otherwise you will have to restart one by one and see and check what I've been done. So metrics is really important. So there's plenty of tools that provide metrics and we'll see we'll generate it during the practical. If I have to pick up some of the metrics, the four important will be looking at trimming, looking at alignment. So trimming, as I say, the graph that we show alignment, alignment rate to understand if we are correctly aligning your read, if we are using the good references.  You want, for example, 30x or you want 100 if you do exams and you want to be sure that you receive what you have asked for. And also, exercise, so when you do med pair, it's important to have the control for the size of your fragment because for, if you want to do structural value and coding as an exercise, it is really important in the detection of structural value. Okay, so... Is that your question of the trimming? Yes. Which is sometimes... Yes, I can comment on that. So now that we saw that many of you had any sequencer, so first the question was, is the trimming optional? No, I'm not. So if you have a really high quality data with really base quality, you could not do the trimming part. If you use mapper that supports what we call the soft clip. So the soft clip is if you find a sequence that is not in the genomes, it will mark this part of the sequence of your reads and take it, this is not part of the genomes, like adapters, it will mark them directly on the reads and so you won't need to remove it at the beginning of the trimming. So it's true that now if you have really good quality, you can skip this part, but not all the mapper supports that, so that's why I will show it, but if you have BWA support it, so if you have really good quality, all the base quality is on the roof, on top of the roof, and you have really small level of adapter, you could go without trimming. Okay, so in conclusion, if you want to do that, so if you want to do data processing, just need to one is that you will need a lot of mathematical and informatic skills to do that. If you don't want to do it, you will probably find bioinformatician to do it for you, but it's important that you understand what they are doing on your data, because each time we do analysis, we make choice, and if we make a choice, there is a cost to pay, I mean a cost, I mean you have a counterpart of your choice, and you need to understand the choice that have been made and what's that mean at the end on your samples, so because some variant could be missing because of this choice, some pattern should be probably due to this choice, so you need to understand exactly how your data have been generated. My main message is also matrix, matrix, matrix, that's really, really important. If I want to summarize also, try to always generate the best quality data at each step before going to the next steps, and this is a general comment, the major challenge now for doing this data processing is not the methodology, because now a lot of methodology are really there, except for really new type of analysis, but for general processing, all the methodology is there, all the tools are there, which is more challenging now is the compute resources and the storage capacity, so it's why I use the introduction on computer data because this really, you will have, like you will need like a lot of CPU to do the analysis, and if you do world genome sequencing, one sample could take more than a terabyte during the processing per sample, so if you have a hundred of samples, you need to have a hundred terabyte of space, so it's really challenging. Okay, so that's it, do you have any questions? Yeah. In the metrics, is there one problem that is more common than the other? There's so many problems, you can face it. One problem that you can see, for example, which is really easy and is, for example, you don't choose the right reference genomes, so here it's common because most of us will work on humans, so we have the right one, but if you then want to work on mouse, or if you think you will work with human, it's sampled, and then you align your genomes, and you see that your alignment rate is low, it should be okay, probably I don't use, or probably there's a mix up on the data. But even in human, a lot of times, as we've discussed before, the actual assemblies, so if you map one data set on one assembly, and then you use a different data set, there was map using a different human assembly, and that's a very common mistake. Yeah, for long-term projects, where we have some projects that start years ago where the last build was not the same, so it's a good issue, so I would not say that there's one matrix you can use, but what the main important thing you need to do, and it's related to what Guillaume is saying, is to harmonize your analysis all along your project. So, because if you change, if you run, for example, 10 samples on one pipeline, and then you say, oh, this new pipeline arrives, this new assembler, this new violent color, and then you use the other with a new pipeline, you will create technical artifact, and you will create this kind of batch effect that then you can perhaps take as biological signals. So really, when you do a large project with several samples, try to use the same kits, try to use the same sequencing technologies, the same pipeline, everything should be harmonized. Otherwise, you will have, we know that each pipeline, its kit, generate its own false positives that you could then interpret as a bogey called violent. Which is, I would add, so I think that the sequencers have been getting better, but there's some metrics that really have to be a sample itself, or the quality of the sample, like duplicate rate, so watching the duplicate rate is important, and marking duplicate says you're going to be doing, but there's some, I mean, sometimes there's problems with the sequencing, but often there's problems with the sample itself, and some of these metrics really have to come with the quality of the beginning that we need. I would say one of really important steps is also, yeah, the base of a calibration, because it's one that removes a lot of false positives, and it's, again, it's a pity because the tool that we used to do that, which is GTK, which is a broad tool, has no decrypted these tools, because they have no generate variant colors that do it directly at the variant calling. But we need to do it because we don't, we use this tool for variant calling for single nucleated variant calling, but if we want to do structural variant, all the type of analysis, you don't have this, you need to have this down file with this indel, really realign, already done. So for me, it's a really important part, and when I talk to people with GTK, I always say, why do you remove them? We're not, there's not only your tools that can be used, so I say there's no one particular metric, it's really a set of metrics.


Live from Copenhagen, Denmark, it's theCUBE. Covering Nutanix.NEXT 2019, brought to you by Nutanix. Welcome back everyone to theCUBE's live coverage of .NEXT Copenhagen Nutanix. I'm your host Rebecca Knight, co-hosting alongside of Stu Miniman. We're joined by Craig Routledge. He is the Vice President HPE Greenlight Sales. Thank you so much for coming on theCUBE. Yeah, good afternoon. So why don't you start by telling our viewers a little bit about HPE and Greenlight? So HPE is the part of the old HPE empire that's focused specifically on the hybrid computing world. So in the data centers, the hybrid cloud world and at the edge, and we're providing technology and services to our customers and our channel partners and we'll continue to do so. And the thing, the announcement that we've made this week here in Copenhagen is the announcement of Greenlight for Nutanix. So I'm not sure how much of our audience is very familiar with it. I've got plenty of experience with the various HPE cloud initiatives over the years, but this is at its core as a service, my understanding and help us understand where that fits in a customer's overall kind of cloud portfolio. Yeah, if you kind of take it back to the, go back to real basics, actually almost before cloud, customers access HPE technology and infrastructure through capital purchase, through leasing, in some cases through subscription models as the industry calls them. And then Greenlight was born about nine years ago in fact to help customers match cost to revenue. So it's a pay-per-use model and that's where it was originally born before really the cloud was almost, it's kind of around but not really in any scale. And then over the years, we've adapted Greenlight to be the private cloud solution for many direct customers, then for channel customers, for service providers and partners. And now we've added the Nutanix partnership as well, which we've been announced as being ready for sale today on stage, which was great timing by my engineering team, I'm pleased to say.  So it's gradually evolving, but it's we're not just doing the private world. We're increasingly working in an environment where the equipment is installed in a colo, but it's still dedicated to that customer. It's not a shared service. And we're also increasingly through our analytics portal connecting to the public cloud world. So we've announced we have significant partnerships with the public cloud providers and we can meter and monitor the customer's usage in that solution. So we can provide a single tool sets that gives them the private cloud usage and who's using it and connect them to the public cloud world so they can get the same functionality in the public cloud. So they could see how much our marketing department are spending on compute and storage and networking and virtualization, et cetera. And it's a very different way for customers to think about it. And in many ways, it should become more natural. If I got it right, it heard you say it's, you know, by the VM or the container or by a certain flash increment. Maybe explain that a little bit.  Ah, well, it's interesting. You make an interesting point there, actually. It is about customers buying workloads. And in the same way you buy a film on Netflix or you buy a series or you might choose to buy episode two, three, five, 10 and 12, but not the whole book effectively, not the whole library. And you buy that by the unit of measure. So in Netflix, it's a video or something. In the, in the GreenLake world, it's by virtual machine at the VRAM level effectively. It could be by container. So it's at the actual Kubernetes container level. It can be at the gigabyte of high-performing storage level. So we've disconnected the linkage between infrastructure. So the customer doesn't choose that infrastructure. The customer gives us a workload and then we specify how that workload is designed. We have some recommended architectures. And we're just about to launch the second iteration of our quoting tool so that the customer can get a quote on their smartphone or our salespeople in the pilot stages will be able to produce a quote on the, now when that moves into operation, it's our service team that are monitoring the customer's usage. 24 by seven, we own the metering and the management technologies. So we can snapshot the customer's usage and their infrastructure environment as often as we need to. So on Black Friday, you can guarantee we snapshot every retail customer in our portfolio at least every 30 minutes. And if there's a financial services crisis as various presidents pick a fight with a global trade war, share prices bounce up and down and dollars go in different directions and the RMB goes, you need to meter the usage very rapidly, very accurately and very often. And that's what our metering technology does. Now the service part of this is not only do we kind of make sure that's all running 24 by seven, our part of our service is to do the capacity management for the customer. So we take that responsibility off them. And if they, if we think they're, if the portal is telling us and the intelligence built into the portal and into the experienced service managers saying, you need an upgrade, we need to upgrade this piece, then we can produce a change control note, one or two days, sign it and then we can get some more infrastructure capacity rolled in of the chosen architecture for that customer. Just describing what you were saying about the retailers on Black Friday and then watching the currencies fluctuate based on whatever our world leaders are tweeting about. How has this in your mind changed the way the business world works today? Just the fact that we can see all this information, this real-time data, I mean, wow. It's changed the speed and I think it's the change of speed at which companies like ourselves have got to operate. And I think it's changed the speed at which the IT teams inside the end customers have got to operate. And they get, I think they've probably got the harder job than we have. But we, an IT manager in an organization these days not only has to watch those macro factors, the dollar going up and down, Britain's finally sorts out its position on Brexit and we won't go into that one. And the IT team have got to look at that and see the impacts on the business. But they've also got to cope with a very rapidly changing environment and a whole user base. I mean, I don't know if any of you, I presume you have to download the app on your smartphones, you press it and if it doesn't download in three seconds you're going something wrong here. And that level of expectation in terms of the delivery of new application requests is inherent in the user base now, particularly the younger people that are coming through in the wave of early stage employees in our customers. They expect instant gratification almost. They want a new app, didn't, they were a bit vague about how they want it to run but they want it today now and they want to pay a low volume price per click basis. So that's kind of, you know, we're partly reacting to many of those trends and part of our solution is in fact, we provide a, if we think the initial size is, let's just say we need 500 VMs or we need 400 Nutanix, Greenlate Licenses, Nutanix and Greenlate Licenses, we always provide a buffer and in the early stage, let's just say it's 20% buffer and that gives the customer some overflow room. So not only will we provision above their utilization but we'll add a buffer to de-risk it for them at our risk and that's to make sure that the service is seamless and that's something that IT departments are not used to it but it matches the expectation of the internal, I call them the IT consumers really in business or out or customers of a bank.  I had a chastening experience on Sunday. My bank, the app is online on Sunday but they don't move money on other weekends and I went, what? That was a bit of a stunning, and so my expectation is fueled by this kind of instant society that we live in. So it's orderable now? Yes, it's orderable now. We finished it yesterday. Available within 30 days. I mean we think we'll have it available by the end of the month for delivery. Great, and from a customer standpoint, will the customer be asking for Nutanix GreenLake? How does it, how do you give them the decision tree or is it a customer saying I wanted Nutanix? We have some people that are far more technically oriented than I am, technically literate than I am. We have some pre-sales specialists inside the Nutanix team and inside the HP teams and we have some sizing tools as well to help us. So if the customer comes to us and says they want a particular workload, because we've expanded the choice, if they're talking to HP, we'll look at what's the right solution. And if it's Nutanix, then we use the Nutanix pre-sales teams to help us. And that seems to be a very popular message in the marketplace and is resonating very well. So we're helping the customer make a choice and obviously in an indirect motion, the partners will be helping the customer make that choice and then coming us to, they'll specify the technology solution and they'll come to us with a specification. We'll turn that into a detailed specification and a detailed cost and contract. So just Greenlake's been around for nine years now. Is this the first HCI based offering in the Greenlake portfolio? No, we've been working on the, we've had Greenlake on SimpliVity, which is the HP owned HCI solution, two and a half, maybe three years now. And very successfully it's working very well in a few large cases. But it works at a different level with different scaling parameters. And so this is actually the Nutanix partnership and the reason why the two CEOs were excited was this gives the customer another choice. And it gives them another choice other than the default virtualization engine which everybody uses. And it also brings in the Nutanix expertise end user computing, they call it VDI as I would call it, but that expertise and in the database world it bring their expertise in that space is very valuable to us. So it augments our portfolio and it brings two solutions to them, not just the Greenlake solution, pay-per-use solution, but it also brings the proven HP server technology into their appliance portfolio. And the alignment on the optionality is really what is also driving this. Yes, and it is, we both genuinely believe in customer choice, in options. If the customer's only going to have one choice, A, you only give the customer one choice and you might win or might lose, but you're going to have a resentful customer. If the customer says they want to go with HP and we only give them one choice or can only give them one choice, doesn't make for a long-term relationship. And certainly I think both companies, HP clearly, we believe in having lifetime, we value a customer for its lifetime relationship with us. So it's very important that we offer the customer choice, then narrow down to the right solution, refine that solution and draw it up into a contract. Excellent, so it's the right choice. Yeah. Craig Routledge, you are now a CUBE alum. Thank you so much for coming on theCUBE. Thank you very much, thank you. I'm Rebecca Knight for Stu Miniman. Stay tuned for more of theCUBE's live coverage from .NEXT.


So, this script has actually been written for nearly a week, but I didn't feel like I could shoot it because I was just missing something. This microphone. I had to buy this microphone. When Gerard Carmichael came out to the audience at City Winery on January 16th, I'm not sure how many of us realized what had happened. I didn't realize it until months later, April 1st in fact when most of the world found out, because it wasn't presented to us like it was new information, just another part of a joke about his issues communicating with his parents. When I bought a ticket to see his first live performance in two years, a work in progress tentatively titled Alone, I didn't really know anything about him as a person. I had seen the Bo Burnham directed Eight as well as the special he directed for Drew Michael, but that was as far as it went. And in Eight there's really not that much about himself, though it does end on an interesting note and one that seems to have formed the basis for Alone.  That moment never happened to him, but it did happen to his dad.  We were promised a work in progress and we sure got it. Carmichael had a notebook that he'd refer to at times and there was no real structure and certainly no satisfying conclusion, but that hour was captivating nonetheless. It was also shockingly personal. Eight put you mostly at a distance, he talks about his thoughts, only sometimes his feelings. And when he's talking about, say, getting and then getting rid of a dog because it didn't make him feel as warm as he'd hoped, it's hard to know if any of that is true. His first tape special, Love at the Store, was much the same. It's a very specific type of observational comedy, but it's observational nonetheless. And so this time, observation is replaced with experience. It gets personal immediately and stays there. He talks about his family, his feelings, his sexuality, his family's feelings about his sexuality, but I didn't realize at the time how much that was new. When I recounted some of the jokes to my girlfriend later that night, I took it for granted that everyone else knew he was gay and that the only reason I didn't was because I never bothered looking at his Wikipedia page. Well, did I know I could have been the one to add that fun fact to his bio? In retrospect, though, that revelation is likely a big part of why this special has seemingly the fastest turnaround time in comedy history. Hello, by the way, and welcome to the week air of view. My name is Alec. And today I am talking about Gerard Carmichael's new HBO special, Nathaniel. For clarity, Nathaniel is the final version of the show that I saw less than three months ago. Most of the material in Nathaniel was there in January, but it has been stripped to its core. Setups were changed, ideas were excised. You may have realized, for example, that his dad's comment about how mean he is didn't end up on HBO. And in general, it became more focused. Like, alone was arguably a comedy show. But Nathaniel is comedy with an asterisk.  And not just because Bo Burnham serves as director and editor, but you may not be shocked to learn that virtually no part of Carmichael's life experience tracks onto mine, so I connected to Nathaniel more intellectually and artistically than I did emotionally. I'm sure a lot of people felt that way about inside. It's okay. But Nathaniel is exactly the opposite of inside. Inside is highly polished and carefully crafted. There is no improvisation, no mistakes, and authentic point is made inauthentically. And for people like me, that is great. And I have an hour of my own talking about why. But Nathaniel is pure authenticity.  This is especially true of the last 15 or so minutes when the pre-written material is largely dropped as he struggles with how to connect with his true audience. It's not the people in that room. It's not even us, your average home viewer, though you would be forgiven for thinking it might be. It's a really cool video by Comedy Without Errors that breaks down its production and specifically notes hot takes that were written by people who attended taping and were deeply confused by what they had seen because Carmichael would repeat jokes at different points in order to get it exactly right or aim certain jokes directly at the camera instead of engaging with the audience. He was making a show less for people at the Masonic Temple than for the much, much larger group of people who would be watching it later. Nathaniel is being performed for just a handful of people and really maybe just one. That final 15 minutes is for his mom. And when you realize that, suddenly the entire show feels like it is. Alone featured other family members much more prominently than Nathaniel does. We still get some things about his father and brother, but it is so much more focused on his mother. And nearly every cut that was made to the content of the show emphasized that. The one real counter example is in the highly shortened bit about his brother where he mentions how much more money he makes. That was originally in the context of discussing his mother's relative feelings about her sons. Much of this recent trend of comedy but is it, though, results in what feels like a rhetorical therapy session.  Perhaps it's what they wished they had said to their therapist on that first session. But therapy isn't scripted or polished. There's a lot of silence as someone grasps for the right words to articulate their feelings, occasionally prompted by a question that hopefully pushes them towards enlightenment. Which is to say that the last section of Nathaniel is therapy. And I think it's probably worth noting that Carmichael is in therapy himself. I don't believe he mentions it here, but one of the jokes they got cut was about how he spent more money on therapists than sneakers and how that seemed like a step in the right direction. This is all stuff that he's definitely talked through before. And frankly, he's probably been asked the same questions on a couch that the audience asks him in that chair. The main difference is that his mother is going to hear his answers now. And so when he says that he feels he may need to cut her out of his life because it's likely too late for her to change, that's for her. She will hear it. He knows she'll hear it, but he says it because he needs her to know it and it's probably easier for him to say it when he doesn't have to look at her. I've gone back and forth on the audience participation aspect of the show. It's part of Carmichael's thing. He always invites the audience to speak and right at the start of Nathaniel, he makes clear that he needs everyone to feel comfortable and even like family if it's going to work. There's a thoughtfulness to the way that he has these interactions. He rarely claps back. He takes the time to mull it over. And sometimes he makes a joke and other times he's serious. He's also not having a real conversation with them. They speak, but he's not interested in the people doing the talking. And to be clear, that's fine. I'm not going to throw stones from a glass house. Plus, crowd work really ain't my thing. Sure, there was a real wild story I heard at Taylor Tomlinson's performance at the Beacon Theater on the night that Nathaniel hit HBO that ended with literally bowing at the altar of this woman's pettiness asterisk.  So that wasn't great. This homophobia in the audience at Blue Note Jazz Club, just as there was the city winery, you can hear it in the reaction, some of which Carmichael calls out and some of which he just lets go. At my show, someone killed the night with it. He had finished with what he knew he wanted to say and started looking through that notebook to see if there was anything else worth bringing up. But then a woman near the front shouted that she could totally switch him. Most of us groaned. If you didn't close the notebook, looked at her, said he would give her his dad's number and then walked off stage. And like it's good joke, a perfect callback to much of what he had talked about with us and some of what he talked about with Blue Note. But the context in which he felt compelled to make that joke. Not great. Homophobia and its relationship to masculinity looms large in Nathaniel. He talks about kissing his boyfriend, but feeling compelled to say no homo in the process. How his women friends have been incredibly supportive while the men in his life haven't. And it makes me wonder a bit about the solid chunk of love at the store when Carmichael talks about how he would come out if he were gay, which of course he said he wasn't. But if he was, you know, here's some thoughts on it. He tells us now that he never intended to come out. And that reminded me a little bit of that letter Lil Nas X wrote to his younger self about how he promised that he'd never come out, but why it's important that he did. But Carmichael was in the closet much longer. He wonders aloud if it was too long, if he missed the chance for acceptance from the people in his life who now see him as a liar. Apparently not thinking about how a statement like, I feel like I got tricked into having a gay best friend might justify not coming out in the first place. Khadija Embo has a video about how coming out isn't and shouldn't necessarily be a thing for everyone, especially those who are marginalized in other ways. But that is something that I don't get nor want to have an opinion on. But he's not really here to unpack anyone's damage but his own. And it takes time. Carmichael has this tick where he rubs the back of his head and neck when he's thinking. He does it in every standup I've seen. And it seems to be his way of filling silence with motion instead of sound. And since Nathaniel features so much silence, there's a lot of that motion. The silence is deafening, really. It's probably why so many people feel like they have to speak, especially in that final section, because silence is uncomfortable. But we, as the viewers at home, have an even more intimate vantage point. You don't realize that comedy rarely features true closeups until you spend most of a special staring at one. Close enough that the camera has to move every time Carmichael does to keep his face in frame. You really feel like you can see the gears turning in the clockwork of his mind and it emphasizes that authenticity.  I don't know. As with previous Burnham collaborations, the shots are long, too. Many stories play out mostly or entirely without edits, only cutting when the subject is changed. And I wonder if this will have more of an impact than it did because of the way it seems to have hit the zeitgeist. I wanna believe that it could create a ripple effect, pushing comedians to think harder about the venues in which they perform their specials and the way that they use cameras and editing to help tell their stories. Because there is so much more that can be done than basically anyone who wasn't involved in this specific production seems to take advantage of. I'm grateful that I got to see how this show changed in such a short time, that I got to hear more details about his parents' sex tape and his willingness to die for his nieces, even if it would ruin his entire family's financial security. There were a lot of great jokes that didn't make it into Nathaniel. But I appreciate the show even more because I see how he sacrificed guaranteed laughs for thematic cohesion. This is not the funniest version of the show.  Thank you so much for watching. Thank you particularly to my patrons, my mom, my cat, Cat Saracota, Benjamin Schiff, Anthony Cole, Elliot Fowler, Greg Lucina, Kojo, Phil Bates, Willow, I'm the sword, Maddie Zimmerman, Claire Bear, Tatled Indice, Andrew Madison Design, and the folks who'd rather be read than said. If you liked this video, great. If not, don't care. If you want to see more, please subscribe. Hope to see you in the next one.


There are thousands of phytochemicals that will never make it onto the side of a cereal box, but may play a role in reducing the risk of chronic diseases. And that's just the ones we know about. We know whole plant foods have consistently been found to be protective, and so it's reasonable for scientists to try to find the magic bullet-active ingredient to sell it in a pill, but pills simply cannot mimic this balanced natural combination of phytochemicals present in fruits and vegetables. When isolated out, compounds may lose their activity or behave differently. The antioxidant and anti-cancer activities of plant foods is thought to derive from the additive or synergistic effects, meaning the whole may be greater than the sum of its parts. This helps explain why a pill simply cannot replace the complex interaction of phytochemicals present in whole plant foods. As T. Colin Campbell has pointed out, more than 100 trials overwhelmingly show no long-term benefit of vitamin supplements, along with worrisome findings that certain supplements may even increase risk for diabetes, heart disease, and cancer. Supplementation with fish oil appears useless or worse, yet the science doesn't seem to matter. People continue to buy it. The public desire for quick fixes through pills is overwhelming, especially when money can be made. Each plant has thousands of different phytochemicals, and each plant has a whole different phytonutrient profile, and so there may be synergistic effects when consuming different foods together as well. Just like eating beta-carotene in carrot form is more beneficial than in pill form, because of all the other compounds in the carrot that may synergize with the beta-carotene. When you dip that carrot in hummus, all of a sudden you have the thousands of carrot compounds mixing with the thousands of chickpea compounds. And that's what this study was all about. What happens if you mix different fruits with different vegetables with different beans? Combining foods between different categories did indeed increase the likelihood of synergy. For example, here's the antioxidant power of raspberries alone, and here's the antioxidant activity of azuki beans alone. So if there was strictly an additive effect, the expected combination would come up to here. But the observed antioxidant power of the combination came out more than either eaten alone. What about anti-cancer effects? What if you repeated this study, but this time tried dripping different combination of foods on breast cancer cells growing in a petri dish? Sometimes you can get the same synergistic effects. Here's what grapes can do to breast cancer cells, suppressing their growth about 30%, but onions worked even better, cutting breast cancer growth in half. Now if you added half of each together, you'd assume you'd get somewhere in the middle between the two. They'd average each other out. But instead, the researchers got this, suppressing cancer cell growth by up to like 70%. So the whole plus the whole was greater than the sum of the whole parts. So do they recommend people eat a variety of foods? Maybe add some raisins along with some chopped red onion to your next salad? Where's the money in that? No, the reason they were investigating the different types of interactions was to identify mixtures that could ultimately lead to the development of something like grape-flavored funyons or something.


Hey there friends, how's it going? My name is Kevin and today we're playing Ru-ru-ruin-ar-ki-chish. I left school, okay?  I was joking the first time when I was like making up a name and then the second time I actually went, wait, what actually is that word? And we're going to be basically an evil overlord who's looking over all the villagers and we cause chaos. So basically, Sims. I do not need a tutorial to ruin people's lives. Oh wait, I'm going on to level one and I've realized it's not my fault that I can't pronounce things. They're just bad at spelling. Look, that's how they spell one.  I'm looking at all the powers you can get and there's a locus swarm, earthquakes, and I'm really drawn to landmine and all the things. Okay, I'll do a locus swarm as well. You know, I want to appear somewhat godly. I don't want to just do guerrilla warfare. Okay, I'm ready to torture here, villagers. Let's see, before I click play, let's see if I can cast some spells. What the hell is going on in here? Oh, she just has red hair. I thought she was bleeding. Somebody get this man in ambulance! Okay, this person is armed. I'm going to afflict them with psychopathy. Choose victim profile. You're going to go after the males.  All right, I'll affect one other person. Scarecrow dude over here. I'm trying to escape real life here by playing video games. Let's do kleptomania. Steal some stuff.  So I can use some of this currency here. What's it called? Mana.  Yeah, I can use mana to trigger a flaw immediately. Trigger flaw psychopath. Yes. Look, there's a man right there. Carolizile. Okay, play. Apparently that immediate effect is not actually immediate. It has a timer of four hours, but that's okay. That's like no time at all in this world. Sea's object. Yeah, I'm going to put this guy's guitar or whatever in this guy's house, so hopefully it causes a bit of chaos. What do you mean you didn't steal my loot? What? It just floated in there and then I assume they'll kill each other. Wake up from your murder nap. I mentioned going for a nap and you wake up in your psychopath. You just have the urge to kill. I'm stuck piling all these stolen items in the other dude's house, so it really looks like it's just this guy went on a bit of a spree. He didn't even realize he's so tired he came home and he's like, two beds? Nah, that has always been like that. All right, I'm wasting all my powers here. I should probably stop. I'm doubling up all his items. The primary way of getting more mana is by finding chaos orbs produced by villagers whenever they perform criminal acts. Okay, is this guy stealing stuff? Wait, he's attacking that desk. Is this his own stuff? I think he's taking back his things, yeah. You know what? Good on you. You stand up for yourself. I think he's just killing them, but he's not even looking to take them back. This guy's sleeping through the whole thing. Oh yeah, he was attacking them. Carl started attacking bed. Reason, suspicious object. Look at it. It's just sitting there.  What the? What? A spider is taking Kelby away. Kelby is going to the temple. I don't think Kelby's going to the temple. I think she's being kidnapped and brought to the temple. I didn't even do that, but you know what? I'll take the credit. Oh wait, is this my temple? Ah, good. They got them into a web. Perfect. And now they are eating Kelby. Great. How could I deny my children fresh meat? I've got a prison here. How can I get them to the prison?  Maybe I can put them in trial for a hate crime, but then again, they'd be tried as a minor. Do you get it? Because they're in a cave mining other dead nevermind. Honestly, I don't feel like I need to get involved.  All right, I've planted the guitar in there. I'm just going to put them right next to the guitar. Look at that. They've stolen your guitar. Villages are prone to making suspicious assumptions, especially when they're in a bad or critical mood. I literally lifted them from their bed and put them in there. They've now assumed that it was stolen. Well, I suppose he would, wouldn't you? Now she's going about destroying everything. Well, if I can't have it, no one can.  Fantastic. Bridget is fleeing from her crimes. I mean, the person broke into your house, accused you of stealing their stuff and then attacked you. I think you can claim self-defense, but you know what? Exile yourself to the desert. That really helps me out. It's just sleeping on the ground. Can I encourage the spiders to do another job for me? There we go. Oh God, I don't know if you can beat Bridget. She's a very good KD so far. Wait, Bridget is the psychopath. No wonder she's so strong. There we go. I triggered the thing. She's on the hunt. I think she's actually poisoned by the spider as well. Oh God, she's gone right in for him. Oh no, she forgot and started eating. Wait, what is this guy carrying? Casey is going to drop Matthew in the Kenton prison. Why? What did he do? He's a criminal apparently.  Oh yes, he's the kleptomaniac. Okay, yes. Sorry about that. I couldn't set you up to fail. Matthew just broke out of prison. He's going to steal something immediately. Is Bridget dead? I think Bridget may have died. All right, time for an act of God, I think. Casey's sleeping on the ground. Perfect victim. Let's go with Meteor. Drop it right on Casey. Fectus guy in particular. Oh my God, he lived. Fair play. He's surrendering. He's surrendering himself to God. Now he's a believer. I'm going to put a landmine in the prison. Now I just need Matthew to steal something so that they have a reason to go there. Oh, they're putting out the fire. Of course, the sand is on fire.  It's flammable. It gets everywhere. I'm seizing all the town's guitars and putting them in Matthew's house.  It won't be much of a stretch to believe he's stolen them all. And now he's fighting a cat or something. He's just not a very nice guy. He can play a mean tune on the guitar though. The legend says he can play five at once, which is why he's collected them all. Karen is daydreaming. Oh, I'm talking to the manager. I get my way just by complaining constantly and screaming. Oh, what a great daydream. Oh God, the village is just going nuts. They're not arresting Matthew. They're just killing him.  You're taking away the gift of music, Matthew. Right. I'll be a bit more direct. I'll put a landmine outside the only exit to Karen's house. There we go. They've so much health. She just lived through a landmine. There's only three of the villages remaining. I don't even know what happened to bridges. Uh, you might not want to go to bed, Karen. Got hit by another landmine, but again, she's fine. Right. Locust swarm time. Let's go. Can I do multiple ones? Oh, I can. And you know what? Spin up those locusts good. A tornado is coming in. Okay. Well, that killed Matthew. The poor guy didn't really have a chance. And now Oakley is getting cornered by goblins. He's just trying to run around the outskirts of the map. Now he escaped and he went to sleep. I'm going to drop a meteor on him as he sleeps. There we go. That should be him dead. I think I have enough powers to just continuously drop meteors now. Honestly, I think you just built this on a burial ground or something. The whole village is getting destroyed by every natural disaster known to man. Okay.  Wait, is he still alive? Oh my God. Put a landmine on his body and locusts. Come on. Come on. Die, please. And a meteor and a tornado. Come on. You got to be dead now. Thank you. All right. I'm immediately going to start off by building a prison like right outside their village. You're building too close to a village. Are you sure you want to?  They might be a bit suspicious. So this is just risen from the ground. All right. Let me seize someone. Since this idea just dawned on me. Dawn, I think I'll take you. And now we torture. This structure allows the player to brainwash villagers. Oh, yes. Build that next to the prison. It's the game's getting past because I'm building it pretty much in the village. Like brainwash. Perfect. Oh God, I think they killed Dawn. She died from the torture. That's not what I wanted. Dawn is screaming for help. John was successfully brainwashed and has become a cultist. Great. He is subscribed. Your threat level has reached maximum and has triggered a counterattack. Several hostile villagers are on their way. What? Where are you even attacking? The prison is it? All right. Splash water on them. Splash water on them. They just got rid of my prison.  All right. John is in the cult. He's happy out. How about you transform into an abomination?  No, just poison your neighbor. Poison Misty. She's blessed apparently and I don't like that. Oh God. Oh, she's pissed. And John is happy. Oh wait. Oh no. They're all attacking John. Let him transform. He just lit Misty on fire. Who's blessed now? John is fleeing from his crimes. Where did he go? All right. Let me just start a plague. Oh, John is back. Oh, I turned him into an abomination. Great. No way, John. Where are you going? All right. I'll seize him and just put him right into Daisy's house. There we go. Go on, John. Get her. Oh God. The John was not a good abomination. Good Lord. They're all fighting each other now. Honestly, if I just let them to it, I think they're just going to kill each other by themselves. I still want more cultists though.  Being brainwashed. Come on. Get brainwashed. Join the cult. It's great. I'm building walls inside his house, so he's just strapped. The poor guy and I'm going to splash water on you. What are you going to do?  They got rid of it. Like, you know what? Things have been going kind of bad since that cultist temple moved into the neighborhood. Plus, the property prices have plummeted. I don't think this is your real issue anymore.  I've given them all different flaws, like psychopath, hot-headed. One is a werewolf. I can't even remember which one. Now, I'm just spawning demons. I don't even know what they're doing. But oh God. Oh, I just spawned a monster under their bed. Poor Daisy didn't have a chance. This is a much more direct way of attacking the villagers. I like after I destroy their house, their solution is just sleep in the sand. Even though there's only three people remaining, the rest are just empty houses. You could just stay there. Oh, here come the spiders. If anyone wants to find out what's going on, all you have to do is follow to spiders. Wait, she was successfully brainwashing as becoming a cultist. Then why is she knocking down the whole thing? Go poison the neighbor. Quick, yeah, go, go, go. Stop knocking down the temple. Booby trap neighbor. Yeah, get them. That doesn't look like booby trapping. That just looks like eating. She's getting carried away. What is going on? She couldn't find an appropriate object of booby trap.  She's going burying my cultist. Do not bury her. It's the only villager left alive. I'm going to give you paralysis.  All right, got a nice week. Now let's build another prison. You destroyed my previous one. In you go into the prison. I torture them to death.  And you know what's more? Splash some water on her. Splash some water. There you go. She's happy. Why? You just got tortured and you're paralyzed. Splash water. There we go. I locked her in this box.  She just transformed into something. Mildred is going to the monster lair. I mean, I think I made her a werewolf all right, but I thought it'd be big monster, not just little wolf when I agitated. Wait, where is he going now? The Anc of Anibus is acting strangely. The hell is that? Okay, whatever happened, the villagers died. I guess I did it. That was a quirky little game. I enjoyed that. I enjoyed the torturing and the killing especially. But I hope you enjoyed the video. This was a fun little game. I might play it again when the game fully releases. But yeah, I hope you enjoyed the video. I appreciate you're watching as always. If you want to watch more of me, I post every day. I also stream over a Twitch if you want to check that out. Link in the description. But I won't ramble. I'll just say thanks for watching and I hope to see you next time. Bye for now.