A horse with a speed of light, a cloud of dust, and a honey-hio silver, a lone ranger. A faithful Indian companion-tuttle, a daring resourceful mastrider of the Plains, let the people law and order in the early western United States. 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 loop beats of the Great War Silver. The lone ranger rides again once more. Let's go! Let's go! As Silas Kilby rode along the main street of Redhill, heading for his wrench a few miles to the south, he heard someone calling his name as he passed the speech to him. Silas! Silas Kilby! Oh, hey, I hope! I knew you was uptown, Silas. I'm gonna watch it for you the first time. Got a letter for you. Come on this day from St. Louis a while ago. A letter from St. Louis for me? Yep. Thanks. I can't think of anyone who'd be racking me from St. Louis since my daughter Sally came back from school. Well, uh, is she gonna open it? Time enough to open it when I get home. Come on! Get up there! Come on!   I didn't even hear you riding into the rain charge. Sorta snuck up on me, is that it, Kate? There isn't much you miss around here. Good to know I can get away with something once in a while. Silas! I snuck back a letter from Tonky. A letter? Yeah, came in on the stage from St. Louis today. I read it on the way out. Here it is. Let me see it. It says from your old friend Dick Barton. Who's Dick Barton? I've never heard of him. Just go ahead and read the letter, then you'll know. Just like a woman who reads the tale and first. Let's see. Dear Sy, it's been 25 years since I made my strike and came back east. I married a wealthy guy. And we have a son, Bill. When his grandmother died, she left the boy a sum of money in his own name. Bill's 20 now, and he thinks money will get him anything. Hmm. I lay out a talk to Bill and persuaded him to try ranching for a year. I told him if he hopes to inherit my fortune, he'll have to work and make a man of himself. Well, Bill, you know what? I don't know. Unfortunately, he'll have to work and make a man of himself. Dick Barton worked hard to get what he has. Stop interrupting, Sylas. Sy, I'm sending Bill out to you to work on your ranch. I'm hoping it'll make him see things in a different light. He'll arrive soon after this letter. Regards from your old friend Dick Barton. Yeah, Dick and I were close friends when we pioneered out this way. I took the ranch and he went prospecting. Found a gold mine that gave him a fortune. Guess if the boy's on his way, there's nothing to do but give him a job here. Of course we'll put him to work like Dick wants me to. If that don't make him come to his senses, they're nothing real. Maybe so, Sylas, but it's going to be mighty hard to my way of thinking. If he's used to having plenty of money whenever he wanted it. We'll do what we can with Dick's son and just hope for the best. Several days later, Sylas and his 18-year-old daughter, Sally, work it up the stage, people, for the arrival of the stagecoach from the east. The stage ought to be coming along any minute now. I wonder if Bill Barton will stay with us very long, Dan. Don't know about that. While he is at the ranch, he's going to work as hard as anyone else. I'll see to that. Here comes the stage now. I'm anxious to see what he looks like. Somebody's getting out now, Ben. Sure is a handsome young fella getting out. Do you think that could be Bill? I'll be for Mr. Kilby. Can you tell me where I'm? I'm Sylas Kilby. You must be Bill Barton. That's right. Glad to meet you, Mr. Kilby. Oh, who's the beautiful girl? Oh, this is my daughter, Sally. How do you do, Bill? My dad didn't tell me about this pleasant surprise. I thought Western girls were... Wow. Rough, tough and homely perhaps? Well, something like that. You know, pioneer stuff and all that sort of thing. You'll find real down-to-earth women out here, young fella. Not the namby panty-cide with paint all over the faces. Well, Sally doesn't need anything like that. In fact, I think I'm going to like it here. Well, we'll get your luggage to the buckboard and start for the ranch. Come on, dad. On the way back to the ranch, both Sally and her father soon realized that Bill Barton was going to be a difficult problem. Now, Bill, according to your father's wishes, I'm going to put you to work on my ranch. Working with the cowhands and learning all there is to know about ranching will do you good. Is that the kind of work I'm going to do, Mr. Kilby? Be just one of the ranch hands? Of course. What else do you expect?  No, but I don't think I'm going to like working with cattle. Well, what's wrong with it? As dad said, it'll do you good. And in my mind, there's no better way for a real man to make a living than to be a rancher. Well, maybe if you spent several years in the East, you'd change your mind, son. For your information, I did spend several years in the East. In St. Louis, where I was educated. Frankly, I was very happy to get back home to the West. What chance does a man have to make a name for himself? You know, to give his name before the public out here in this wild place. Son, let me tell you something. History will be full of the names of real men who've done their part helping the West grow. Men like Kit Carson, General Huston, Ben Austin, and Fremont. Son, I'll keep my promise to your dad. But I warn you, I expect you to do your part and work as hard as the others.  And then I'll have to write to your father that it was all a big mistake having you here. Get out there!  The re-roading silence, though, bill didn't seem concerned what had been said. Just before reaching the ranch, I saw an Indian running trail. It was Tuttle, companion of the Long Range here. Hey, look, coming down a trail, an Indian. Yeah, if you see plenty of them around here. Oh, I know that. Look at that horse he's riding now. That's a beauty. Stop the buck for a minute, will you? Before. I want to talk to that Indian. Well, all right. Oh, there. Oh, there. Why do you want to speak to that Indian? I thought you guessed. I want that paint horse. Don't be foolish. Well, I'll be done. Hey, hey there, Indian. Oh, what do you want? How much do you want for that horse of yours? Me not sell horse. I like that horse and I want to own him. I'll give you $500 for him right now. Oh, you're not for sale. He doesn't want to sell his horse. Let's drive on, Dave. Now, wait a minute. Look at him. I'll double the offer. Well, hold on, young fella. That's a lot of money. Well, Indian, what do you say? You keep money and me keep horse. Hang on. Hold on. Wait a minute. Hang on. He doesn't want your money?  Ritzkin doesn't realize the value of money. I suppose it doesn't occur to you that perhaps the Indian thinks more of his horse than he does of all the money you could offer him. Tommy Rott.  That's why you're wrong, Bill. I happen to know that's one of the smartest Indians around here. Like Sally says, he'd rather have his horse than all your money. Of course, you wouldn't understand a man feeling like that. Get up there.  In the week war on, Sally's opinion of Bill dropped lower. One morning, she listened to her father discussing him with her mother. Kate, did Bill leave with the ranch hands this morning to help with the roundup? Not that I know of, Silas. I think he's still sleeping. Oh, Sonic, that boy's about the laziest critter in the territory. Yesterday, I told him to take three of the horses to the blacksmith. Didn't he do it, Dad? The horses got new shoes all right, but instead of taking them to town himself, he gave one of the hands $10 to take them in. Is it good for the rest of the hands to have Bill doing things like that? I know it. If he keeps giving out money, the hands will be dissatisfied with what I'm paying him. I hate to disappoint my old friend Dick, but I have a mind to send Bill back east and call it quits. Wait a while. Give him a chance. Chance? He had his chance, and he's proved already he isn't worth the salt. Well, give him another week. That's all. Gotta get out of the range. When that good-for-nothing Bill gets up, tell him to come out there.  See you later. Late evening, Tuttle returned from town and stopped at the camp where the lone ranger was waiting. Horse-gun opener? Easy-scun. Easy-cunner. What's the news from town, Tuttle? Well, farmers from South have meet in a bank. They've had to get money to finish building dams. Banks say no.  The land is not producing for one of the irrigation. Have you dulled their cash to talk to a big damman? Not right. If the dam is completed, it will be the answer to their problem. But it will cost more than I thought. And he must hubby. Me see a fellow who tried by a spout the other day. Him talking to a girl outside bank. And what you've already told me, there's been a great deal of talk about Bill Barton. Isn't he staying at the killby ranch? Ah.  Me hear him. Them talking about farmers. And her kept plenty mad at young fellow. Oh, why? Well, them come out a bank. Damn near Hitchirac. And me put in supplies and settled banks. Me hear and talk. I feel terribly sorry for those farmers. They've mortgaged the land to pay for building a dam, but it isn't completed and their money's run out. The bank refuses to help. How much would it take, Sally? Oh, about $30,000, they say. Such a lot of money. You know what? It might be worth that much to me to make my name known throughout the territory. I'll give them money if they'll call it the Bill Barton dam. They look at me as sort of a hero. Bill Barton. I'll try to overlook the way you've acted, but what you've just said lowers my opinion if you were all the wiggins about them. Why? Well, you're not thinking of helping those farmers. You're helping only the publicity you'll get out of it. What's the difference? They'll get the dam, won't they? Yes. Yes, that's true. Oh, what a use of trying to talk to you. I've been hoping you'd get to like me. You see, I... It's no use, Bill. Go in there and act a good Samaritan. Pat yourself on the back and take the blow away, but I want no part of it. I'm going on home. There are some things your money can't buy. You'll find that out someday. Oh, girl, row down the town. Young Palatola. I see the girl's viewpoint, Tutto. I think in a way, Barton's trying to impress her. He's been born with plenty of money at his disposal. He had a lot to learn about people. I hope something finally brings him to his senses.  Listen, Joe, did you see that tender hood that was in town with the killby girl? Yeah, what about him, Dusty? I found out in some of the hands of the killby spread that he's got a lot of money. Well, what's he doing working at Killby's? His old man sent him out of here to make a man out of him. What I hear, he don't kill himself doing much work. What do you got on your mind? I notice he comes in the town alone most every day when he's supposed to be riding the range. He always flashes a big roll of cash at the cafe. Well, tomorrow when he leaves town, we'll follow him. And we'll jump him and take that cash. Hey, that'll be risky. I heard some of the killby cowpaw saying that in spite of being a tender hood, he's an expert in shooting the rats and quick on the draw. Look at his old man talking. In that case, we'll plug him from ambush. A curtain falls on the first act of our Lone Ranger adventure. Before the next exciting scenes, please permit us to pause for just a few moments. Continue. The following day, the Lone Ranger and Toto were preparing to ride to town. Yeah, I mean, since... How about the blacksmith's shop? From down the trail came a puppy. I had to investigate. He said he'd be plenty fun. What's the limit? What's the count? Two men on the trail. There's a third man lying in the ground. Must have been an ambush. They're riding their way past. They'll stop and help the wounded, man. We'll tell them later. What's the hold, you said? Easy for that. Hello, it's Bill Buckman. Easy fellow, easy. Have some water. You're max. We're here to help you. Did you get a look at the two men who did this? Yes. They were going to rob me. They shot at me from down the trail. Tried to force me to go with them. I attempted to get away, and they shot me. It's a shoulder wound. Painful, but not serious. Our camp is nearby. We'll take you there. A short time later in the Lone Ranger's camp, Bill Barton, his shoulder expertly vended. Lay on a bed of blankets and watched as the Lone Ranger approached with a cup. They thought roughly good. I don't understand all this. Who are you? Does it matter? Here, I'll help you set up. Thanks. If you hadn't come to my aid, they might have killed me. I'm glad we were nearby, Barton. You know my name? Yes, I've heard of you. After you get me back to the Kilby Ranch, I'll see to it that you're well paid for what you did for me. I have plenty of money. We, uh, we don't want money. We were glad to be able to help you. But if I only knew your name, I'd tell people... Well, you have a lot to learn. Here's the problem. Thanks. Why did you say that, mister? Say what? I have a lot to learn. Sally Kilby said the same thing. You like her, don't you? I do. She's the finest girl I've ever met, but she doesn't like me at all. Well, perhaps you could change her opinion of you. Have you tried? I have tried, but it's no use.  That's likely one of the things that turned her against you. What do you mean? Have you ever in your life done anything for anyone without thinking of what it would bring you? Or without trying to impress someone? Well, I... Have you? No. No, I guess I haven't. Just because I am rich, everybody... Money is only a means to an end, Bill. A means by which those who have it can do good. But when you use it only to get what you want, it warps your character and turns people from you. Hey, now you're talking just like my father. Your money hasn't turns how like you'll be against you. But you have. Think it over, Bill. Maybe it will do some good. Now you better take the rest of your broth before it gets cold. While Dusty and Joe had doubled back and written to Tom, they grew rain in front of the cafe.  Listen, everybody. Where's the sheriff? Here I am, boys. What's up? Tell him, Dusty. We came up to the trail of the cellar here. So an owl hoot wearing a mask and riding to what stadium? It was with an Indian. Yeah. It held up that tent of photos out of the Kilby spread. The rich fellow. They must have heard it about his money. What happened to it? Now put it down, everybody. Quiet down. What happened, Dusty? Well, we tried in a fear, but they drove us off. They put the young Barton and then took him away with them. One of you men ride out and tells Cy Kilby. Resty hit letter. We'll go out with Dusty and Joe and have him show us where it happened. Then we'll trail those owl hoots. Let's go. It was almost dust. At the Long Rangers camp, Bill Barton was sleeping. And the masked man was preparing to leave to trail the two gunmen. Suddenly, Kato stood up, listening. What is it, Kato? Many horses are coming. Yes, I hear them. Then come and hear, Kato. We'll leave, Kato. Oh, I have your guns ready. We'll stay and find out why they come here. The sheriff is bringing a posse. Those guns won't do you much good, mister. We're 10-D or two. If there's a big gun play, you'll get the worst of it. We could have laughed. But we stayed to see that Barton gets proper attention. Here, there's Barton lying over there. Barton's been wounded, but he'll be all right. We heard about what you did, stranger. We got him cold. Lay it on the high net corpse, baby. Now, wait a minute. You're making a big mistake.  What do you mean? You were seen holding up Bill Barton. We trailed you here. You shot Barton in the back, and he's probably dead. He didn't have sense enough to get rid of the body before he died. Bill Barton isn't dead. The only ones who could have known he was shot from behind would be the men who really did it. Those men must have reported this to you. What's going on? What's going on? What's so soon? Talk to Barton. Well, son, tell me about these two old hoots. They didn't shoot them, Sheriff. I can describe the two men who did it. Oh, my God! Stop! Up the mess, man! Bug me! The low ranger's keen eyes had seen Dusty level his gun in Bill's direction and had quickly fired to save Bill's life. What's this about? Those two men just leaving better ones. Stop them! I don't have anyone. One of those men tried to shoot Bill Barton before he could talk. Wake up! Dusty and Joe was trying to get away. Sheriff, they reported to you because they thought Barton was dead. They shot him from ambush. They scared them off before they had a chance to rob him. Barton? Are they the men who ambush you? Yes, they're the ones. The masked men are the Indians' Satan. Take Dusty and Joe back to town with this and put them in jail till you press the charges against them. We'll take Bill Barton back to the killby ranch, Sheriff. Good. I'm sorry we come here like this. What's all right? You're wearing that mask. We're not outlawers, Sheriff. Well, nothing against you. We'll be going. Come along, man. Get going, you two. Get the killby ranch. Sally, with her mother and father, voted to appear from the posse. Soon they heard horses stopping outside. Someone's coming out. Oh, Dad, I hope you... Well, soon all that's happened, Sally. I didn't know this would affect you so much. Sir, yes, there's lots of men can't understand. Go open the door. Hey, what's this? We have brought Bill Barton home. A masked man? Oh, it's you, the Indian. They're friends, Mr. Cubick. I know, they helped me once a few months ago. Come on here. Are you all right, Bill? Yes, Mr. Cubick. I'm wounded in the shoulder, but it's not serious. Maybe you... Maybe you went a little too far trying to get publicity. Well, what an urge to come over here. It's all right. Sally thinks I'm no good. Maybe she'll change her mind about me soon. I'm sure she will, Bill. Well, we'll go now, but I'll meet you in town tomorrow afternoon. Are you... What is all this, Bill? How come you let the mask man?  The following evening, there was a knock at the door of the Killby rental house. I'll see who it is, Dad. Good evening, Mr. Killby. The mask man. I brought a note for your father. A note for Dad? Yes. Can I hear you say there's a note for me? Yes, Dad. Here it is. What is it, Dad? Well, listen to this. Dear Mr. Killby, since you are one of the investors in the dam, this is to inform you that sufficient funds have been placed in our hands to finish the construction. Work will be continued at once. Very truly yours, John Warren, President, Weston Construction Company. Oh, Dad, that's wonderful. Who put the money, Mr., you know? Someone who wishes to remain anonymous. Oh, Dad and I have tried so long to help the others raise the money to finish the project, but it just seemed hopeless. But now, somehow, someone comes along and put up the entire mod. I'm curious to know who it is. Perhaps he'll tell you someday. Oh, by the way, Bill Barton is waiting outside. Shall I tell him to come in now? Bill? Yes. Tell him to come in. Good. Goodbye, Mr. Goodbye. Oh, Dad, I think Bill is the man who put up that money. Hello, Sally. Come in, Bill. Son, this note just came from Construction Company saying enough money is available to go ahead with the dam. What do you know about it, Mr. Killby? Let's say it was someone who wanted to help, but doesn't want to be known. We can guess who he is. Yeah, and it's a mighty fine thing, too. Bill, I'm sorry for some of the things I've said. I deserved hearing them, Sally. Mr. Killby, from now on you'll find me right on the job. I'm going to learn ranching from the ground up.  But what about this sudden change, Bill? I found a friend, a real friend, who showed me the value of self-respect. He pointed out that with extra money to spend, I could help the future of the West by bringing in pure stock and breeding fine herds. A dream of every cattleman. It sounds wonderful. My new friend said that by breeding and raising improved stock here in the West, World Market would be open to us. And that the raising of fine livestock would become one of the leading industries of the future in America. Your friend is sure a man of fine vision, son. Yes, he is. Sally, I want you to help me show off as it can be done. I'll need you to remind me now and then about my masked friend if I start slipping. Oh, Bill. But now on is my friend too. Someday we'll call him our friend, Sally. He gave you something to think about that time, Sally. Oh, dad, please. Bill, who is your friend? And why does he wear a mask? He wears the mask so we can do good for others without being known. He's a man who has a great faith in the West and in America. He is the lone ranger.


Hediga, this is Alexis who has had digestive issues for the past seven years now. Stomach pain hasn't been able to like eat properly, everything has always hurt. She said as she was praying and though God was just moving in place, she said she felt this heat come upon her. She said she even still feels it right now. She said like a stomach is shrinking. I felt it shrinking. My stomach, I felt it shrinking. Like I'm always bloated, always, and I just felt it going like this. How long have you dealt with that bloating? It's been a long time. More than seven years. More than seven years. And as you were worshiping, you felt that going down? A hand like was pushing on my stomach and my stomach just went like this. It's his power. It's God's power. And during the worship, you were just worshiping the Lord and she's in a whole different place right now. I told you he's on the platform with me. If you're able, I have to come down here. What are you feeling right now? That's a creative miracle. She literally felt the bloating go down after seven years of dealing with that. Say thank you Jesus. Touch her in Jesus name. Let her receive the fullness of your power. Thank you for watching EncounterTV. Don't forget to subscribe and click the notification bell. Also help us spread the gospel of Jesus Christ and the power of the Holy Spirit. Make a one-time donation or become a monthly supporter by clicking on the donate link now.


beginning with Anthony Rizzo. Anthony, another tough night for the offense. Just what do you think is going on overall with this offense right now? Just grinding. We're grinding as a unit. It's something you just got to flush in and come back in tomorrow and get back in the saddle and keep working. There's no other way to put it. We're just, we're all continuing to grind. And the work's right. It's just obviously right now in the field isn't coming through. But this group is, I think, too powerful, one through nine at all times to hang our heads too much. Do you get the sense that guys are pressing or over-anxious at the plate at all? I don't think so, watching the at-bats. I just think that this is just part of the season. It's a 162 game season, and you're going to have really, really good weeks, really bad weeks. And in between, it's about grinding. So when things are going good, it's easy, and everyone loves you. And when they're going bad, you never think you're going to get up. It's just about staying in that middle and continuing to work and put your best foot forward. We'll take the next question from Brendan Cuddy. Anthony, watch Garrett Cole walk off that mountain in the fourth inning. What's going through your head? Just obviously concern about him and his health. You never want to see anyone have to come out of the game in any circumstance, and especially our horse. But he's in good spirits talking to him, so that's good. And hopefully, it's a fast recovery for him. And hopefully, it's not something that keeps him out too long.


Mark chapter 14 verses 32 through 42 we're going to be looking today at the oil press you'll see why I chose to entitle our study the oil press in just a moment now as is normal for me as you who have attended here for a while will know I'm going to give to you some background remind you of some things and I'll lead to these verses and so we'll begin by looking at verse 32 in Mark chapter 14 verse 32 then they came to a place which was named Gethsemane and he said to his disciples sit here while I while I pray so as we've been looking at this chapter this is the last night that Jesus will spend and he's ministering to his men he had shared the Passover with them and he's continuing to instruct them now I mentioned to you that on the Passover meal in the Passover meal a question will be asked of the host Exodus chapter 12 verses 26 and 27 it shall be when your children say to you what do you mean by this service that you shall say it is a Passover sacrifice of the Lord who passed over the houses of the children of Israel in Egypt when he struck the Egyptians and delivered our households while that night Jesus spent time teaching his men what Passover meant and his disciples are asking the question asking questions that night and in reality the questions that they're asking of him are going to relate to what is the significance of the Passover and so the disciples are there asking questions and Jesus as a host is actually going to answer the most important question that they might have in John's Gospel chapter 13 verse 33 he said little children I shall be with you a little while longer you will seek me and as I said to the Jews where I am going you cannot come so now I say to you notice how he said little children so during that that celebration there would be a question of the children children would ask the host Jesus was the host he would explain the meaning of Passover and that's what's taking place now as you go through the Gospels Matthew Mark and Luke all gives an abbreviated rendition of what took place actually all the Gauss Gospel accounts would be abbreviated but theirs are more so than John's because when you look at the Gospel of John the Gospel of John actually gives us more information you can see that information in chapters 13 through 17 so let me give you kind of a flow of what was taking place that Mark Matthew and Luke did not speak concerning because in chapters 13 through 17 we see that Jesus gave teachings to his men he taught his men to be servants he revealed to them that one of them would betray him he gave them what he called a new commandment the commandment was to love one another as he loved them he revealed to Peter that Peter would along with the others deny him he told them that he went before them to prepare a place for them he told Philip that to see him was to see the father he taught them about the importance of prayer he told them that love for him would be demonstrated by obedience to him on that night he gave the most extensive message on the Holy Spirit that he would give he also told them that he was the true vine he exhorted them to abide in him he told them that he was giving them his peace he said if they did what he commanded them then no longer would what he called them servants but would call them friends he told them that they would be persecuted because the world rejected him in John 1632 and 33 he said indeed the hour is coming yes has now come that you will be scattered each to his own and believe me alone and yet I am not alone because the father is with me these things I have spoken to you that in me you may have peace in the world you will have tribulation but be of good cheer I have overcome the world so when he got to chapter 17 Jesus prays the chapter is filled with his closing prayer as he prayed to his father he said to him that he had been faithful to complete his mission then he prayed for his men that he was going to send out that they might evangelize then he prayed for us those who would come to faith through their message and so by the time you get to chapter 18 verses 1 and 2 well those verses begin by saying when Jesus had spoken these words he went out with his disciples over the Brook Kidron where there was a garden which he and his disciples entered and Judas who betrayed him also knew the place for Jesus often met there with his disciples so with that introduction we can begin our study by first looking at verse 32 in verse 32 it says they came to a place which was named Gethsemane and he said to his disciples sit here while I pray now Gethsemane was an enclosed plot it's also referred to as an estate it was a garden could have been called an orchard when you're looking at a map it was beyond what is of Israel it's beyond what is called the Brook Kidron it's at the foot of the Mount of Olives about three quarters of a mile from the eastern wall of Jerusalem we've been to this this site many times every time we've gone to Israel we have gone to the Garden of Gethsemane I'll give a very similar Bible study there not as long as you'll have here but I give a similar study and there you are on on Gethsemane in an enclosed garden looking at the eastern gate you can see as you're looking to the west you can see the you'll see the eastern gate that has been bricked over it is actually lower than than the ground level because over the centuries it obviously the it has accumulated ground and all but right in front of the eastern gate and we'll point it out to you there's a graveyard there's a Muslim cemetery there they built with an attempt to keep Messiah for coming in the eastern gate and so you'll see that while you're in that particular place and we we have a Bible study there and we look at this particular study and this is a place where Jesus would often spend time Luke tells us in chapter 22 39 coming out he went to the Mount of Olives as he was accustomed and his disciples also followed him now this garden was actually owned more than likely by one of his disciples and it would be lent to him for his use it had become over time a familiar spot for the men they would come there they would actually camp out there and be ministered to by the Lord and they came there often and since they did so they would have had fond memories of this place the word gethsemane means oil press it is place where olives were crushed to produce the oil and this is where Jesus is going to agonize in prayer that's why it's referred to as the oil press in Isaiah 53 verse 3 the prophet Isaiah writing over 700 years before Christ speaking of Messiah said this he said he is despised and rejected by men a man of sorrows and acquainted with grief and we hit as it were our faces from him he was despised and we did not esteem him this is where the Lord Jesus Christ is going to be agonizing we'll see that in detail in just a moment but this is where Jesus is in his own personal oil press somebody said this there's no record in scripture of Jesus laughing but there are numerous accounts of his grieving his sadness and even his weeping he wept at the grave of Lazarus he wept over Jerusalem at the time of his triumphal entry and Jesus knew sorrow upon sorrow and grief upon grief as no other man who has ever lived but the sorrow he experienced in the gardener gethsemane on the last night before his crucifixion seemed to be the accumulation of all the sorrows he had ever known and so Jesus is entering into the wine press and according to verse 32 as they enter the garden Jesus says sit here while I pray and so what he does is he stations eight of his apostles remember Judas has already left there's only eleven remaining so he stations eight of them at the gate or at the entrance of the garden that's going to secure his privacy because he knows Judas is soon going to be there and so he stations them there and in verse 32 it says he said to his disciples sit here while I pray verse 33 and he took Peter James and John with him and he began to be troubled and deeply distressed Peter James and John now why these three now we know that when you look at the scripture you're going to see that there are numbers very often associated with Christ you'll you'll see multitudes used as it speaks of the multitudes who would come in and listen to him teach you you also would see how he appointed 70 so there were what are called by theologians the 70 there were 70 really unnamed but then you get to the 12 then you'll sometimes have the three the two and then sometimes it's just the one Peter James and John though are singled out here why is that now Judas has already left to betray him that leaves 11 eight of them have been stationed at the gate that way they could interact with or at least be aware of when Judas came in as he will but Jesus takes three of them Peter James and John very often they speak of Peter James and John as being what is called his inner circle and it is true that there are those that will work and operate with you in ministry very often that become very close to you because they operate with you quite often they get to know you better than the others that's true but why would he bring Peter James and John at this time well remember with me that each of them had vowed to him unshakable faith they had vowed this before him they had said that they were going to be true to him in every way remember James and John had asked for permission through their mother to be seated one at the left hand one at the right when he entered into his kingdom remember that and Jesus had spoken to them and said to them they really didn't understand what they were asking can you drink of the cup I will drink of Jesus said to them and they said yes we are able so they had stated that they had unshakable faith in Christ we're able to go through what you go through of course we can we want places of honor now the apostle Peter had just told Jesus though I'll deny you I will go to prison with you I will even die with you and we looked at that recently how that that Peter had made that comment so you have Peter you have James you have John all three of these men had said that they had unshakable faith in Jesus Christ they had said that they could take whatever he went through and Peter went so far as to say though all will deny you I never will and so that he had also presented himself as somebody that would never forsake Christ and so this is something called shallow self-confidence and that self-confidence put them in great danger of personal failure why is that because a false estimation of our own strength will always lead to a fall take heed where the man thinks that he's standing let him take heed less he should fall Proverbs 16 18 says pride goes before destruction a hotty spirit before a fall they had shallow self-confidence we will die for you we can take whatever you take I can drink of that cup they've all said that now these three men were leaders among the apostles but Jesus is about to teach them a lesson that they'll one day be teaching others and the lesson is very simple it's something we can learn to face temptation we should do so through prayer and dependence on God you see notice in verse 33 how it says he took Peter James and John with him and he began to be troubled and deeply distressed he began to be troubled that word troubled in the original language means to be struck with terror it speaks of being alarmed the word distressed well that's a word that speaks of being in great anguish it also is a word that can be translated with the word depressed distressed is the strongest of three Greek words in the New Testament that describes depression why would Jesus be described as being in anguish and severe depression when you consider it you need to think that on one level it would be easy to understand why he would be Judas betrayed him his disciples will soon forsake him Peter will deny him three times he's about to be unjustly accused tried beaten cursed mocked he soon will bear the sin of the world through the torture of crucifixion but to understand this better we need to remember how he spoke of his death and he spoke of it often and when he would speak of it he didn't show fear nor did he show concern about dying as you think about the Gospels and you think about his ministry from the beginning he made it clear that he was going to give up his life John 2 19 says destroyed this temple in three days I will raise it up later on in John chapter 10 verse 17 he said therefore my father loves me because I laid down my life that I may take it again in the gospel of Luke chapter 12 verse 50 I have a baptism to be baptized with and how distressed I am until it is accomplished even that night John 17 verse 1 records how Jesus spoke these words lifted up his eyes to heaven and said father the hour has come glorify your son that your son also may glorify you the hour has come it's time he's prepared he's ready he's going to yield up his life so seeing all of this why would he now be troubled and deeply distressed while his sorrow and anxiety is his anticipation of his separation from his father and that will occur when he becomes the sin offering and fellowship for the first time in eternity will be broken and so he's speaking to his men and notice again in verse 34 he said to them my soul is exceedingly sorrowful even to death stay here and watch it when he said my soul is exceedingly sorrowful that's another way of saying I am consumed with grief one commentator said that he was saying if I did not already have another way to die this would kill me he's overwhelmed with the fact that he will soon pay the penalty for our sins now ultimately we know that that was the reason Christ came into the world we know that God had predetermined that Jesus would become the sacrifice for sin he's the Lamb of God who takes away the sin of the world in the book of Acts in chapter 2 verse 23 we read that Jesus was delivered up according to the definite plan and foreknowledge of God that's how he's going to provide salvation for all who are going to be saved Jesus was well aware of this and Jesus embraced this plan in John 12 27 now my soul is troubled what shall I say father saved me from this hour but for this purpose I came to this hour in Romans 4 Paul wrote it in this way in verse 25 he said Jesus was delivered up because of our offenses and later in chapter 5 of Romans in verses 6 through 8 he said when we were still without strength in due time Christ died for the ungodly for scarcely for a righteous man will one die yet perhaps for a good man someone would even dare to die but God demonstrates his own love toward us and that while we were still sinners Christ died for us he describes us as without strength ungodly and sinners and yet Christ died for us so Jesus is preparing himself for that notice in verse 34 he says to Peter James and John stay here and watch now the word watch is a word that can be used in a military way it means to be on the alert stay awake it means don't fall asleep in Matthew 26 38 Matthew says that he said my soul is overwhelmed with sorrow to the point of death stay here and keep watch with me now no doubt they could sense the agony that Jesus was going through just looking at him the way he looked listening to him as he spoke the tone of his voice the words that he was choosing to use all of this was combining to make them aware of the agony he's going through they could sense his agony they could sense his anguish but they couldn't comprehend it now as he's speaking to them this reveals to us his humanity Jesus desired his men to be there with him he wanted them to sympathize with him and even be a support and it's sad when you realize that in the ministry of Christ with his men you read your Gospels you see the conversations that are recorded he didn't ask for much on a personal level yet in this particular case when he said watch and pray stay with me they failed they had said they would die with them they couldn't even stay awake with him now in verse 35 it says he went a little farther he fell on the ground he prayed that if it were possible the hour might pass from him the garden is dark but Jesus goes further further in for greater privacy he and his man had gone into the garden he had left eight Peter James and John had gone with him a distance further and now Jesus separates himself a bit Luke 22 41 tells us these are out 30 to 50 yards away from the the other eight he now moves a distance from Peter James and John and he's praying fervently notice how it says he fell on the ground and he prayed in verse 35 he prays if it were possible the hour this appointed time if it were possible this hour might might pass from him so what does he do he prays and he prays if there's another way may there be another way but there was no other way I remember the first time my wife and I went into the garden of Gethsemane it was in 1983 we went on a on a familiarization tour with my pastor Chuck Smith and so we went into Israel I brought my daughter Anna who was three months old at the time and she came with us and we went on this trip and as we went on the trip with our pastor we saw so many sites but I do remember this particular location the first time that we ever entered into this region called the Garden of Gethsemane I still remember it very well there are stones just all through the pathway there and pastor Chuck his son Chuck Jr. had given a message and and Chuck came up after his son had shared a very very beautiful message and Chuck walked up and he and he closed out that time and I'll never forget what he said he said Jesus said if there be any other way let this cup pass from me and then Chuck simply said it like this and it's riveted in my heart ever ever since he said it he said but there is no other way there is no other way there was only this one way from all eternity God had made a plan and that plan was so that we sinful people that we are might receive salvation he had made a plan he's going to give his only son to suffer sins penalty his son is going to be used to rescue fallen mankind in Hebrews 10 verse 10 it says it like this the writer writes by that will we have been sanctified through the offering of the body of Jesus Christ once for all it doesn't require many sacrifices see the Jewish way of of expiation of sin was to constant sacrifice and they would have on the day of atonement that that major sacrifice but the writer of Hebrews reminds us that that in the sacrifice there's a re a reminder because they have to be repeated yearly he was pointing out yearly they would have this sacrifice yearly as a constant flow and a constant reminder of man's sinfulness but it was preparing man for that one final sacrifice the Lamb of God Jesus Christ who would take away sin one time for all time he doesn't have to be crucified a second or third or fourth time it's that one time that was sufficient the blood of Jesus Christ's son cleanses us from all sin and it doesn't have to be repeated and so Jesus is going to the cross for that reason Jesus there though is saying is there another way there is no other way notice in verse 36 how he said father all things are possible for you take this cup from me he says Abba father Abba is the Hebrew Aramaic Hebrew way of saying daddy father would be something that the Greeks would use Abba father is a combination of the two Abba is the common word that a child will use when referring to their daddy when you go to Israel to this day you will hear this the little Jewish children will be behind their daddy I've heard it many times and you'll hear them Abba Abba and it's it's not a real formal it's more of a it's more of the word daddy it's more of a tender it's an endearing word and so Jesus is saying that he's saying daddy to his father all things are possible for you that reveals something about our relationship to God because we have been brought into his family through his son Jesus is referred to in Scripture as the only begotten of the father we are adopted we are not the only begotten we are adopted we're adopted into the family of God it's a different process we enter in through faith by receiving the gift that is given to us but we are adopted children chosen by God I remember hearing a story of a little girl who adopted and she she was it was a family gathering and all and and one of her cousins was talking to her and her cousin said to her you were adopted but I was born to my family but you you're adopted and she was saying it to try and hurt her her cousin but the little girl who was adopted said you know my mom and my dad have already told me that that I was in a home in and that that one day my mama said that she and my daddy came in and they looked at all the children that were there and they they chose me and my my my mama says that we chose you and we have loved you but you are our baby we chose you to be ours so she said you know my mama and my daddy they chose me but your parents they're stuck with you we were chosen yeah we were chosen the Lord chose us and we've been adopted into his family and Romans 8 15 it says you didn't receive the spirit of bondage again to fear you receive the spirit of adoption by whom we cry out Abba father Galatians 4 6 because you are sons God is set sent forth the spirit of his son into your hearts crying out Abba father before you really understood the love of God it's very possible that you looked at God as just a judge of some sort holy and and he is righteous and he is but distant but distant and perhaps you had a fear of him to the point that you tried to guard your thoughts and guard your actions tried to be good and all of that and and he just got stress filled that's how it was for me I tried to be very good as a child and my mom and dad had pictures of me and my brother we'd be at at different little house parties and all and we have pictures of my brother and me sitting there like you know two little little angels and all and and because I wanted to be good because I'd heard some things by going to to catechism classes that that you need to be good for God to accept you and I tried to be good until I was 15 at the age of 15 I I said I'm tired of being good and not having fun and that's how I thought and so that's when I got into alcohol and drugs I didn't know that God loved me I thought I had to do something to make him love me I thought I had to be good I had to do the right things I had to think the right thoughts and all of that and at a certain early age I came to realize that none of that was possible I didn't realize that God came to save sinners I didn't know that I didn't understand that that's why Paul could say that he said he was saving sinners and Paul went on to say of whom I am chief and so I came to realize the grace of God and so instead of just speaking of him as and as he is almighty God I could actually have a tenderness as I as I speak to him and the word Abba it comes into play I've said this before in the past I'll just say it briefly my father all my life from the time that I can remember speaking to him until the last conversation I ever had with him I never called my father dad I called him daddy because that was our relationship it was tender it was a it was a love that a son had for a father so I finally get it I finally could understand that though I don't refer to him in that way perhaps I would if I'd simply use the name Abba that would be saying the same thing I still speak to him as my father kind of like the Greeks would have but he's my daddy there's a tenderness involved in that relationship and that's what you're seeing here forgive me the emotions just stirred up as I think of this all things are possible for you take this cup away from me all things are possible father many years ago Abraham who is called Abram and Sarai who later was called Sarah well Sarah many years ago centuries ago was unable to conceive but in a miraculous way Abraham at the age of 100 and Sarah at the age of 90 well they were given strength to conceive and she bore a child and that child was named Isaac Isaac in Hebrew is translated laughter because she had laughed in unbelief when God had said this time next year your wife will have a son Sarah listening through the tent had overheard the conversation Abraham was having with with God she laughed shall I at my age have pleasure the pleasure of having a child and that's where the Lord spoke to her why did you laugh I didn't laugh oh yeah you did liar pants on fire you lied you will have a son his name shall be Isaac laughter every time you call his name you'll remember the laughter that you had when I gave you that promise that even though they had another son through a handmaiden Hagar God spoke to Abram and said to him take your son your only son now wait a minute you have Ishmael too no Ishmael is a son of the flesh Isaac is a son of promise take your son laughter and offer him to me in a place that I'll appoint for you the scripture tells us in Genesis 22 that Abram settled his donkey took with him servants and so the question is asked I see the wood and and the the implements for sacrifice but I don't see the offering and then Abram said God will provide himself a lamb which was a prophetic word concerning Messiah who is to come we all know the story how that Abram placed his son on the altar he just tells us he knew that he had received him when it was impossible and therefore God could deliver him back it was a hope of resurrection he trusted God this was the son of promise when he raised his hand to sacrifice his son God said stop and now I see now I see your faith is real and there was a Ram that was provided with this was caught in a thicket and that Ram was offered and so Jesus says all things are possible is there another way is there any way you could do the same for me is there another way for this to occur but he do the answer no there's no other way he says in verse 36 take this cup away from me this cup that he's speaking of represents God's wrath is his judgment it symbolizes the suffering that Jesus will soon endure on the cross the cup speaks of God's wrath in Psalm 75 verse 8 it says in the hand of the Lord is a cup full of foaming wine mixed with spices he pours it out and all the wicked of the earth drink it down to its very dregs Isaiah 51 17 awake awake rise up oh Jerusalem you who have drunk from the hand of the Lord the cup of his wrath you who have drained to its dregs the goblet that makes men stagger take this cup from me but he goes on in verse 36 nevertheless not as I will but as you will I'm totally submitted to your plan of redemption the psalmist said it like this in reference to Messiah Psalm 40 verse 8 I delight to do your will oh my God your law is within my heart and John 6 38 Jesus said it like this he said I have come down from heaven not to do my own will but the will of him who sent me I'm willing to do this I'm willing to be the lamb I'm willing to die somebody said that Jesus disarmed death by burying its shaft in his own heart well Luke the physician gives us greater insight into what's taking place you see he was a doctor and he speaks of what Jesus endured in Luke 22 43 and 44 there appeared an angel into him from heaven strengthening him being in agony he prayed more earnestly his sweat was as it were great drops of blood falling down to the ground he was in such stress and agony that he was pouring out blood in sweat in answer to his prayer God dispatched an angel the minister to him Luke tells us that the angel strengthened him the angel invigorated him writer of Hebrews in chapter 5 verse 7 says during the days of Jesus's life on earth he offered up prayers and petitions with fervent cries and tears to the one who could save him from death and he was heard because of his reverent submission while he's praying and he's being cared for well in verse 37 he came and found him sleepy said to Peter Simon are you sleeping could you not watch one hour watch and pray lest you enter into temptation the spirit indeed is willing but the flesh is weak and again he went away and prayed and spoke the same words so he speaks to the apostle Peter in verse 37 notice that notice that he refers to him by the name Simon Simon is his given name John 141 and 42 says that he had changed his name it says he first found his own brother Simon and said to him we have found Messiah which has translated the Christ and he brought him to Jesus now when Jesus looked at him he said you are Simon the son of Jonah you shall be called Cephas which is translated a stone well in verse 37 he found all three of them sleeping but notice he spoke first to Simon now this is the one who had said though all forsake you I will never fall away so he's the first one who's being addressed and Luke says gives us insight into why they were asleep in Luke 22 45 when he rose from prayer he went back to the disciples he found them asleep they were exhausted from sorrow some of us in this room understand what that means that the burden that you're carrying is so grave so deep so heavy that sometimes you actually get sleepy and that's what was taking place with them the sorrow affected them they're experiencing deep sorrow and that was used by the way by the enemy to tempt them to sleep making them unprepared and that's why in verse 38 Jesus said watch and pray let you enter into temptation the spirit truly is willing the flesh is weak watch and pray be alert be alert about the devil's plans pray for God's help in first Peter 5 8 it says be sober be vigilant because your adversary the devil walks about like a roaring lion seeking whom he may devour in first Peter 4 7 he went on to say the end of all things is at hand therefore be serious and watchful in your prayers you see you might be physically awake but you're spiritually asleep spiritual vigilance is not occasional it's constant the enemy the enemy never rests the enemy never sleeps that's why 1st Thessalonians 5 17 is important pray without ceasing well verses 39 and 40 again he went away and prayed and spoke the same words when he returned he found them asleep again for their eyes were heavy they didn't know what to answer him they were weighed down with sleep they weren't praying well Jesus was separated in prayer as men were sleeping on the job 1st Thessalonians 5 6 says therefore let us not sleep as others do let us watch and be sober he warned them yet the disciples were still not listening they yielded to their natural appetites and they were spiritually unprepared they were not heeding his word we sang a moment ago prior to the teaching a song and I noted it it says I believe every word you said and I suspect that that the men would have said the same thing I believe every word you have said we the church just saying that I believe every word you said but the fact is beginning with me sometimes I wonder because during that song in the first service especially I was thinking of it Lord do I really believe everything you said do I really act and live as if I believe every word you said you see they listen to so many sermons they even gave their own sermons they they saw so many works they even performed their own works they were there celebrating in a way that was beyond everybody they were so close and so deep with them they weren't the 70 they weren't just the 12 they were now the 11 Peter James and John were the three they had a close relationship James and John were actually related to Christ they were cousins so there was a deep connection and yet do I really believe every word that you said because Jesus has said before the the rooster crows to this night you will deny me three times I would go to prison for you I'd even die for you is what the response was so said the rest of them now they weren't listening to what he had to say and Jesus is concerned for them the circumstances leading to his arrest and and all is is going to overwhelm them not only will Jesus be taken but his men are also going to become targets and that was why that night Jesus had prayed in John 17 verse 11 he had prayed for them Holy Father keep through your name those whom you have given me he had prayed for them but he also commanded them to pray for themselves seek in the Lord and rely on his help will provide security like it says in Psalm 17 verse 5 uphold my steps in your paths that my footsteps may not slip Psalm 130 verse 5 I wait for the Lord my soul waits in his word I do hope well verse 41 says he came the third time and said to them are you still sleeping and resting it's enough the hour has come behold the Son of man is being betrayed into the hands of sinners rise let us let us be going behold see my betrayer is at hand Judas is approaching him at that moment so he says to him it's enough the time has come my betrayer has arrived you didn't prepare yourselves by prayer and now you're going to reap the results when he said in verse 42 rise let us be going see my betrayer is at hand notice with me that Jesus went out to meet the enemy he didn't have them seeking to find him he sought them out because he was ready through prayer and and dedication to the will of the Father he was ready to meet the enemy head on but his men weren't we're gonna see that next time we get together his men were not all this self-confidence all these words that they spoke to the Messiah to their their master all the time that they spent with them imagine that for a moment the walking the hours the camping out next to him the amazement when they would see him open the eyes of the blind the amazement when when Peter walked on water when they saw him raise the dead on no less than three occasions raised the dead they heard words that were so articulate so eloquent so deep sometimes they even had to ask him what do you mean by that can you please explain it to us we don't get it we've been with you in the seminary for so long we still don't get it Jesus and so he'd explained to them in a personal way John was so close to this man that even that night he had placed his head on the bosom of Christ and had spoken to him as the one who loved he could he had his head on the chest of Jesus listening to the heartbeat of God think about that think about that you listen to the heartbeat of God and yet Jesus said watch and pray lest you enter into temptation the spirit indeed is willing but the flesh is weak be ready your enemy is prowling like a roaring lion seeking whom he may devour that's why in hindsight Peter could say you need to pray you need to be you need to be alert and by the way I'll close with this don't think that the enemy isn't after you because he is now I'm not trying to encourage you to paranoia so that you're walking every step turning around to see if he's going to be there so you see listen when you gave your heart to Jesus Christ before you were on the enemy side now you're on God's side do you think he's neutral about you he hates you the depth that God has love for you Satan has hatred for you he steals he kills he destroys his great pleasure is in the destruction he's a murderer Jesus said from the beginning he's a liar from the beginning and he's still prowling for you watch and pray listen the problems we're having in the United States as bad as they are with some of the things that we see going on and the great concern all of us share the battle is not just with flesh and blood we are not going to elect righteousness does that mean that we should not pursue it does that mean we shouldn't do our best to bring people into office who who best reflect the righteousness that we'd love to see America have no it doesn't mean that at all we should vote and we should be involved and we should do all of those things of course but the answer is not going to be electing somebody because even the best person who loves the Lord can be tempted by the power that is offered in offices that's why we pray for those who are in elected positions that's why we pray for them because the temptation to power is the first temptation that Eve herself was deceived by you shall be like God knowing the difference between good and evil the temptation to power to knowledge to inside information to control it's still with us the weapons of our warfare are not carnal but our mighty and God to the pulling down of strongholds we we are actually in a spiritual war and that's why the weapons of our warfare are not carnal but our mighty and God so we have the our loins are dirt with truth we have the breast plate of righteousness we have a helmet of salvation our feet are shot with the preparation of the gospel of peace we have the sword of the spirit and we have the shield of faith and that's what we use in these battles and that's why Jesus said we need to be awake and alert because our enemy desires to destroy us so instead of me yielding to the carnal desire to try and somehow control my own destiny I asked God to go before me that he might be my shield and my great strength that he might give to us victory in Christ and that's how it works and so Jesus is speaking and saying to his men watch and pray you have the desire but you don't have the power beyond the alert your enemy wants to destroy you they fell asleep on the job but Jesus didn't he went out to meet the enemy but as we'll see they fled I don't want to be one who runs I want to stand as a busy in the position of the victor because of what Jesus Christ has done for me because that's what Paul said having done all stand stand as a victor be prepared for the war but remember the last page of the Bible where it states we won in Jesus Christ never forget that we win we win and


So, good morning everyone, I'm Mathieu Bourgett, today I will present you an introduction to what we used to do data analysis of NGS data, so what tool we use, what type of computing structure we use and the data. So, I will do the presentation for this module and the lab for this module and the lab for the next module, the module tree. So, you will see that the two labs are really connected. So, today, the learning objective of this module is to introduce you computing resources that you can use to do NGS analysis to understand the type of data and what we do with the data to know, to learn about the terminology and the format of the file we will meet and to do that while looking at how we analyze the NSAC data. So, first I will present to you how we use HPC servers. So, HPC stands for high performance computing server. So, we are using HPC server because when we analyze NGS data due to the size of the data and to the volume of the data we generate for the kind of project we are working on, the traditional computer you can use is not enough power to do your analysis.  So, what we need is to have a really high level of computing resources to do your analysis. So, it is why in Canada we have the chance to have Compute Canada that provides this HPC center. So, what are HPC centers? It is a set of clusters. What are the clusters? It is like a grouping of hundreds or thousands of individual computers in the same place. So, not really an individual computer but just the computing resources like an individual computer. So, each computer is called a node and in each node we have a processor which is called CPU. So, in Canada we are lucky we have Compute Canada. So, it is a national platform which tries to merge all the consortia for the different province which provides the HPC resources. So, depending on which province you are coming from you will go to usually one of the other consortia. So, if you want to use Compute Canada to do your analysis and you need to have a Compute Canada account and why you need to go to have this account is because the idea behind Compute Canada is instead of the government, instead of financing directly researchers to build their own small cluster, they prefer to give the money to one group, so Compute Canada and to reduce all the management of this cluster. So, if you want to use the resources you will need to go and be part of the consortium to have your account and when you have your account it will, when you have your account you will have free resources. So, free resources need you will have a storage space so a place where you can put your data. So, it's a limited space but you will you could ask for extension every year if you are the site of the data, as a quantity of data you have growth and you will have a Compute Time allocation. So, what you mean by Compute Time allocation? It means that you will have a number of curly year use time. So, a bit complicated to understand but the idea behind you is it gives you a set of time where you can use the computer with high priority and once you have used all your allocation you can still use their resources but you will have lower priority compared to people that didn't have use all their allocation. So, just to have a fair use of everybody that is part of the consortium. So, when you are on Compute Canada servers and what you do you just launch your, what you want to do is to launch your job and when this job are execute then it will use your allocation. So, how you get your account? You just need to go to Compute Canada then what you need is when you have your account in Compute Canada you need to ask an account on the local provincial consortium you want to to be part of and in this consortium you will choose which HPC server you need to be running your analysis. When you have your set of everything and you could access to your server you will go to the server and you will log and you will arrive on the logging node. So, there's two types of nodes there's logging node working node. So, logging node is when you arrive on the server and it's just an entry point for everybody and the role of this, when you have this node is just to allow you to submit your job to a scheduler. So, a scheduler is a software that takes your jobs, finds the available resources on the graph of the cluster and match what you ask to what is available to make your job going through the good computing resources that match what is required for your job. So, when you go there be careful, don't, some people, some new user try to launch their job directly on logging nodes which is really bad because it's a really limited resources that is for everybody. So, if you run your job there then your sysadmin will try to be on your back and say stop doing that you block everybody so it's a kind of a general rule. In the case of the lab practical today, so we will work on one HP server in Quebec which is called Mammut but we won't be on the logging node it's directly set up that you will arrive and everybody will be on one big working node so we will directly launch our job which is so not exactly the way you will do it if you will be on the on a real HPC server. So, as we will don't see that during the practical, when you launch your jobs as I say you will specify the task you want to do to the scheduler and the scheduler will match your task to the computing resources so you will have a time where you will wait that the scheduler is able to find the good resources for your jobs which is the queuing time and this time will depend really on how what you set as is needed for your job so it depends on the number of CPU that's the length of your jobs and how loaded is the cluster. So, you can try to play with this parameter to reduce the job length the queue length sorry because sometimes if you are really short jobs you can spend more time in queue than really running your job so it's kind of a game to find the best the most optimal way to submit your job. So, that's a good thing you have this resources where you can launch your job but what you can do with this resources if you don't have any tools or resources you can use to do your analysis. So, C2G in partnership with Compute Canada we are working on a system which is called CBNFS which stands for CERN virtual machine file system which is the idea is to have one location where we maintain all the tools and resources at one location and it's sprayed between the different not all but a lot of cluster in Compute Canada where you will have whatever the cluster you use you will have access to the same tools that have been installed the same way the same type of resources I mean the same type of genome reference sequence and everything. So, the idea is to reduce the management for users so you don't need to install the genome you don't need to install the tools you just ask people and we will ask for you. Yes? Good question, is Compute Canada only available for people who are in Canada? Yes, I will talk in the in the in the two slides about that. Okay. But yes, so the question was is a Compute Canada is available for for Canadian only? Okay. Yeah. So, as I said in the CMFS we have a set of bioinformatics tools so most of tools I think now it's more than 90 but every time somebody asks us to install a new tool we will install the first test the tools see if the tool is interesting for the community and if yes we will install the tools so all the community will have access to the tools we're also providing resources in many so many built and for many spaces and we also provide a standardize part time to do a classical type of NGS analysis. So, all this resources are used through a set of module system so you mean when you arrive to your to the to the HPC server you have nothing in your environment but you have access through a module available you can have the list of all the resources that are available and you can load the tool you need to your environment and this will only set up your environment to use this tool so you can treat for five tools at the same time what how many you want and everything will be set up and you will you will be able to to use the tool so the question what to do if you are not Canadian so the first first show is to can become Canadian but no more than what is it yeah so you have to that most of what I present here is a really common concepts that are shared by most of the HPC server and most of the other computing server like cloud and other type of instance the only differences if you use this other type of instance or this other type of servers is that you won't have all the tool and pipeline that have been pre-installed for for you with a partnership with computer so you will probably have to to to install the tools on your own but if you want to do that you can go on your on your website where we have a bunch of script for every tools we maintain we we provide an install script so and everyone wants to reinstall this tool somewhere someone else can just take our script and run it and it will go fetch a source on the internet and and install the script correctly okay so now we have seen where we should do analysis now what we do with the data and what type of data we are what we are talking about so we are talking about next generation sequencing data so this data are really evolution of an old technology which was the cold clone-based sequencing so in the older technology if you have a clone you put your your dna fragment and your sequence and you sequence around like 196 sequence at a time here are the ideas to extend this techniques to be able to sequence millions of fragments at the same time so how it works so you have this sequencer and the sequencer just instead of doing like a standard sequencing where is is measuring that how base is integrated is just a picture of each cycle and the picture generate a cluster of your results which represent every base incorporation in each fragment so how it works so there's many there's many technology to do the sequencing I decided to focus only on the Illumina technology because it's more used actually and probably will stay the more used for the for the for the longer time so the idea of the Illumina technology is to do bridge amplification so what you do you have your dna you have your dna you share your dna to a given fragment size usually around 4 to 500 base pair you add your adapters and so you have this set of thick of a short fragment and you load your so you have this specific adapter here at each end of the of the fragment and then you load a big set of of a fragment on your flow cell so the flow cell is what you will put in your your sequencer and in the flow cell you will have amplification of your of your sequence so the adapter you have you will put at the at each end of the fragment will match some free probes that are put on the flow cells so the molecule will bind the probes and they will be amplified to generate a two strand molecule so you will generate a complementary molecules and then the two two strand structure will be break and you will have a copy of your molecule the reverse copy of your molecule that will be attached to the flow cells so you will have other you will have then other probes that are free so your molecule with the the probe at the other end will match three probes in the close proximity of the other one you you you have generated and you will have the bridge amplification so the amplification of the of the reverse of the first copy so the copy of your of your initial fragment you will do amplification and you will break these two strand structure and you will have two copies in two directions which are linked to your to your flow cell and what you will do you will repeat this this process multiple time and you will end up in your flow cell with a cluster of molecules which contain two set of two set of molecules so the same molecules but in the two direction then you will start to do your amplification so you will start with one adapter so one of the two molecules direction you will you will match your adapter and then you will incorporate your the amplification the the the corresponding bases to your to your to your molecules while the bases you will incorporate contain a few recent few recent and as it's at each base incorporation you will take a picture of the flow cell and for each cluster you will see the same fluorescent that will that will flash and you will be able to see the the cluster as one dot on your flow cells like this one so if you go to the for for example at the first one you will if i if we take this cluster we will see a yellow yellow dots and the blue dots and the green dots yellow dots red dots and and it's how we're able for this molecule to understand what are the sequence because each color is linked with one specific basis so the the trick is what doing the sequencer is takes is takes this picture and then is able to for each cluster based on the position on the on the flow cell to regenerate the sequence of your of your fragment yes each cluster it's bi-directional right yeah so so although if we see it here so we'll start with one so when we do so usually we do Persian reads so we have one read that starts with adapters so the sequencing will start only with the molecule in one direction at the beginning because the there will be adapters that is complement the adapter the two and are different so the molecule that are in one direction you add first specific adapter so you will only go with in one direction and then you will clean the model of the molecule of your cluster and then you read the other type of adapters that will read the second attack in the other direction and then you will be able to not do not mix your signal with the two set of molecules sorry no it's just so the question is is the bidirectional sequencing is an error reduction technique no it's just because the quality of the sequencing decreased with time so you can go from one extremity to the other to the from one end to the other end in one shot otherwise the quality of the sequencing at the at the end of the molecule will be will be bad and also the molecule will be too long so so the sequencer won't be able to sequence this size of reads so the idea is really to to to sequence one extremity and the other extremity to have like the two copies of the of the like the two extremity of the of the of the dnaf original dnaf fragment to be able to locate the two fragments and to try to find some specific variation due to this fragment so it's it's more because we cannot go one in one shot over all the all the all the fragment first because the size of the fragment is too long and second because the quality will will drop down so another technique if you if we think about like bag bio or nanopore all these kind of new techniques which is longer reads are more doing one molecules but with longer reads and go straight but it's a totally different way of doing sequencing so if you're interested we can discuss about that at the end of the next break so now when we when you design your ngs experiment what you what type of parameter you need to take into account mainly the read length so the length of your reads so it depends on your size of your size of the molecule of size molecule the type of library so will you do only one direction two direction depending on on your on your type library for example if you use if you're interested with uh smaller any analysis you don't need to do two direction because your molecule is only like uh 30 best spell long so you just need to go straight to one molecule what type of error you will face depending on the sequencing technologies bag bio is more uh sorry lina is more like substitution bag bio for example is more like indels so depending on what you're interested in it's it's interesting to look at different technology uh also how you can how many reads you can generate and can you put several samples in the same lane because usually one length couldn't be too much for your experiment so you you want to to reduce the cost by putting several um uh sample in the same lane so the cost and the return turn around time is also some parameters but it should not be the parameters that uh lead your choice about how you design your experiment most of the time when people come to see us and they have designed their their analysis based on cost and turn around time they don't have made the good choice of how to answer their biological question so we have this ngs and so we have this sequence and what we want to do in the case as presented by uh mike in the previous lab we want to uh find the variants that are related to some specific diseases uh or some specific phenotypes so the what we want is to start from the sequence we have generated by the machine to uh so to the to the list of variants specific to the into uh or uh sample so this is the pipeline we use to do that and in in this lab we will focus on so this is a full full pipeline to do that so in this lab we will focus on the first part of the pipeline in lab three we'll focus on the second part uh uh but the idea of this really uh lab two is to provide you um the step to arrive to a file that is ready to do a variant calling so as we say here we start from the fastq file which i will explain which is a the format of value you will have from your sequencer and you want to have to uh to a file so data that are ready to do variant calling so what are the fastq file so this is the file you will receive from your uh center it's a set of sequence that represents if you are uh single one uh one uh read of each uh DNA molecule or two read of each DNA molecule if you are paired uh so these two sequence represent the two one or two and of your original DNA molecule for each molecules so if you up if you open these kind of uh molecules uh the format will be like that will have full line for each uh for each sequence the first line will be as the either of your of your uh of your sequence of your sequence which contains the name of the sequence and in the name you have the location on your flow cell so the name is really based on the experiment and your machines and everything and the location of your of your sequence and at the end here you will have an indication if you are on the read one on or on read two so all the read one will be together all the read two will be together the second line is really the sequence itself the third line is a second possibility for an either so most of Illumina uh format won't we won't use we want this second either but some other format could have the like kind of um duplication of the first either here and then you have a quality so the quality is okay I need to go fast so the quality of your of the of the sequence uh is um um ASCII character so ASCII can be translated to a numerical character so ASCII character that uh tells the quality of your of your data so what is the quality the quality is a threat score so when you convert your ASCII to a numerical value you obtain this what you call the best quality and this best best quality is turned for minus 10 log by 10 of the probability and the probability is the probability that the base you have sequence is not the good one so if we talk about the best quality of 20 we have one percent chance that the base that we are looking at is wrong so what we do with the data usually we have this this fast queue what we do usually we QC this fast queue so what we do we look at the base quality for each cycle for all the sequences and we're not sure that the quality is uh is enough so more than usually 20 or 30 uh in in general if you have low quality uh usually it could affect your analysis afterwards what we do also we usually look at the sequence the base content sorry I think you have the mean and the medium yeah yeah it's because your your cluster when you incorporate the bases all the sequence of your cluster do not have the same rate of incorporation so you will have some some molecule that will be in advance some molecule that will be delayed that will uh play on the quality of your of your cluster so the type of QC we do we look the base content we'll look also if we find the adapter so if your molecule are short you will find the sequence a non-genomic sequence that you have and find at the end of your molecules we also look the duplication rate so I may need time a molecule if we have some molecule that has been artificially um duplicated we also do kind of this kind of QC where we take random reads and just at last a set of random room for your fast queue to the to the end of databases to just ensure that what you have sequence correspond to what you expect in terms of spacey of uh so if you sequence human and you have this result of mouse you say oh I got a problem with my sequence so when we have done generate this matrix and we are okay with the quality uh what we do we usually do trimming if the quality is not good it's not good enough so uh what's the what the the goal of doing trimming is to remove the possible adapter that could be at your end of your uh it's extremely so when you sequence a read if the read is too short you will have the adapter at the end so you want to remove this adapter because it's not part of your dynamic data you want also to remove uh the base that are under uh uh level of base quality because you don't want to uh to to introduce error in your data and so when we do that after after also cleaning the sequence so removing some bases if the read are too short we discard the reads because if you read that too short the mapping will be uh will be a crap so we don't want really short reads so we keep only a larger read so to do that we do we use traumatic but there's many of tools you can use so after doing uh trimming we do alignment so alignment is what you do is you read in when they are they are clean so you you have two choices either you have a reference sequence usually you do alignment so the the idea of the alignment is to find the best location of your reads onto your reference sequence if you don't have if you don't have any reference sequence you do you generally do assembly where you build this contentious reference sequence and then you do your alignment of on your contentious sequence so when we do read mapping it's a bit challenging because you need to locate a million of short fragments in a really large space of possibilities because the size of your genomes and it's challenging first because uh you will have many reads that have possible uh location and also you don't want to have a perfect alignment you want to tolerate mismatch or gaps in your in your in your look in your mapping because you want to identify variants if you only uh tolerate only perfect match you won't just will keep the what is what is exactly the same as the reference and that you don't need it in fact so there's many uh many um algorithm and methods to do that we use uh the bureau wheeler transform um uh approach which is implemented in the bwa uh aligner but there's also many other aligner you can use so bwa is a really good aligner it's one of the best aligners dedicated to dna but if you know like botai is one for that is really used for RNA so depending on what type of data you will probably have to change the liner when you have when you align your aligner it's really really important to include a kind of tags for each library you have generated that and each lane of sequencing uh you have generated the idea of that is to hide identifier to your uh to your experiment because sometimes you will merge several lanes of sequencing together to generate a final uh data set and you want to be able to try back where you read come from for the different uh experiment if you have any issue you can able to then to look if the issue is only um specific to one experiment or to the world biological sample and also because you will see that the users many tools that will use that need this information when you do your alignment you generate a set of file which is called uh SAM or BAM so it's the data as is the the data type we used to store alignment so SAM is a text format and BAM is a binary format so almost nobody uses SAM because it's too big the size of the BAM file could be between 10 and 500 gigabytes so you can imagine if we uncompress that it's really much larger so it's why nobody use uh SAM file almost so for each alignment in your in your uh in your BAM file you will have uh this one liner one line big line which will give this information so we have the same read name and in the FASTQ a flag so it's a bitwise flag so it's kind of way to encode how your mapping have gone so we'll see that in the in the in the practical you've got the two positions so chromosome and position on the chromosome of your reads of the starting of your reads the quality of your alignment the quite is the same as the base quite is a freight score but it instead of going on the error possibility of error of your basis possibility of error on your mapping so the same approach a sigastring will tell you how your read is is not the position of the paired and reads if you have paired and reads and then you will have the exercise between the two read and then you have your sequence in nucleotide and your sequence in base quality and then you will have extra flag which which are uh specific to each aligner yes is this the ungapped it is it only for ungapped what do you mean by ungapped it means that the read map to the present genome could second it's impossible to one half maybe one position yeah yeah the read could be split into uh i'm not sure so the read could be so the question is is the alignment reporting only ungapped uh mapping no the the read could be split in case the read is split one part of the read will be uh tagged as primary alignment because for stat you don't want to count the two a part of your read as a same read so one will be marked as a primary alignment but the other will be uh linked to the other so you could have really like gap and distance especially when you do RNA you expect to have read that map from one example to the other example so you need your read to be split into the two locations so when you generate your uh reads uh the next thing to do before doing variant calling is to refine your alignment because uh alignment is really something tricky uh aligner any none of the aligner is perfect so when you choose an aligner you you choose some constraint of what how you will do the alignment so you need to correct for this constraint to have the best the best alignment you can so the first thing you do is to do indel rearrangement so why we do that is because most of the aligner tend to prefer having mismatch instead of having gap so when you have indels they tend to do not uh insert the indel in the alignment and to install instead several snip fake snip around the indel so what you want to do is to go over this possible indel and to realign uh things that there's possibly there's a possibility of indel and to uh realign the reads to be sure that you don't create this fake uh snip so usually you have this kind of pattern before alignment after alignment you create the indel and also fake snip disappear also as i say uh you have a lot of uh you in your data you could face having some duplicates so the same several copies of the same original dinar fragment and you want to count only one copy of your of each original molecules because if you have for example a sequencing error at the beginning of your of your first step of your library and you have several several copies of these uh molecules with an error you will it will appear for you as a snip if you don't count that is just the same copy of the same fragment what you want is to see biological fragment biological variation in different original copy dinar copy so to do that we uh we remove the so the duplicates and we keep only one read per uh fragment also uh the sequencer uh the sequencer usually tend to inflate the data the quality of the data and especially some of the quality is biased by a specific genomic context or position in the read so what we need we need to correct to have a more homogeneous distribution of the base quality so we do a best recalibration so when you do that you have set of really like high quality alignment file and you are ready to do your variant calling but the one thing I want to add that which is really really important to do is that each step of your analysis you need to generate matrix it's really important because it's where you will be able to see when you have something that goes wrong and to understand what goes wrong so matrix at each step is really important so as I say we should correct the matrix at each step and there's a lot of tools you can use to do that so matrix at trimming alignment the ups of coverage inside a lot of matrix so the next the next the the next step of the pattern will be present in module three so in conclusion if you want to do ngs analysis it will require you to do a lot of entomatic skills to just learn the pipeline and render analysis and also a lot of mathematical skills to understand what you are doing because it's usually good to understand what you're doing just don't click on the button and trust what the software do really important as I say previously matrix matrix matrix the more matrix you will have the more the more confident you will have in your data important when you generate alignment element is not perfect refine the quality of your alignment and as I say which is go with the mathematics you need to have a good knowledge of what you are doing how the data generate to understand what the artifact you can face at the end of your analysis and the last thing is the more challenging actually for the doing ngs analysis at a large scale is not to do the analysis but is more to have the sufficient amount of storage and the sufficient amount of resources to do the analysis thank you any question yeah is this a process that you intervene with in each step along the way or is this press button you know you upload your fast file and you press button and you wait some variable like the time that pops your up so no so the question is is it a process that you do it manually or you do it like automatically in this like a pipeline way so no for like this kind of standard analysis at the center we have developed our own pipeline so we press a button and we hope everything will be fine until the end which is not which not often the case so there's always like a like a cluster the cluster can like pull it off or or a file could be too large for what the setting we use in the pipeline so sometimes we have to redo and go it manually but we in many cases it goes all along the process and we just need to like monitor how the process is going monitor when the stats is coming out of the of the of the pipeline to see okay we are at this step we saw we saw the matrix the matrix looks good let's continue to run the pipeline if we saw that the matrix is not good we stop and try to understand what's happened to the to the data but it's at the beginning we do it we were doing it manually but by the time we're now doing it since i like five between five and ten years of experiment to do that kind of analysis we have automated all the process so if you go in the pipeline in the pipeline I present on the beginning on the on the software all this pipeline is automated you can just like not one click because you have to set up the pipeline set up your file for you but you just will need to set up and then launch the pipeline and the job will be displayed will all dependency or job will be taken into account and you just need to attend them to receive the email that your job is is finished yeah how do you determine what are good quality metrics is that something you develop in house standards or so so there's no gold standard that have been published as okay this is the standard matrix but they're kind of standard but it's really upon each time you have a new technology so each time Illumina release a new version of their chemistry we need to reevaluate that and to see is or standard is still okay and if there's no set of metrics we need to add or no the result of quality we need to adjust so it's why there's no gold standard because the technologies change so so often that the standard will have to adjust to the to each new change but there's some kind of rule of thumb that we know is an experiment for example when we sequence human we expect that due to the quality of the reference genome we expect that the the percentage of freeds that are aligned to the to the genomes is around 95 percent of the read if you start to see lower level of read alignment like 1980 you start to see those probably there's all other type of DNA in my experiment you know this is this kind of of matrix but there's no like really gold standard that have been set in stone saying this is the matrix you need to to out it's more like kind of experiment but for example for the quality for for when we look at base quality we use 30 as a standard but if we want to do more less stringent because we want to generate more we can we can adjust the results so for example when you do dna you go around 30 but sometimes when you want to do assembly you don't want to have so much variation so much error so you can pop up to 35 or more so you need to adjust depending on what you are doing