horse with a speed of light, a cloud of dust, and a high high old silver. The Lone Ranger! Before this exciting adventure, a word from our sponsor. General Mills, makers of Cheerios, the ready-to-eat Oats cereal that gives you go power, and Wheaties, the breakfast of champions present by special recording, the Lone Ranger! Hello friends, this is the Lone Ranger.  Oh, what is a country in every direction? How you, how you do it is a question, and here's one that the happy people have to pay. Wheaties, the Wheaties, and the do-do-do-do and a cable cage. You know, that's right. People in various parts of the country have different accents, perhaps, or dress a little differently, but the ones with plenty of drive, the go-getters, have one thing in common. They're careful about their diet. They see to it that they eat a good, honest breakfast every day, and a breakfast built around wheat couldn't be better for you. Wheat is real man food, so bear in mind.  With his faithful Indian companion, Tonto, the daring and resourceful mask rider of the plains led the fight for 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 hoofbeats of the great horse, Silver! The Lone Ranger rides again! Come on, Silver! That's going far! Are you Silver? Hurry! Cain Dolan, outlaw leader and his men, sprinkle their feet and drew guns as they heard hoofbeats approaching their hideout. Have your guns ready, men? Cain glanced through the window and said, Oh, it's all right. That's Jake, the Army's scout. Hi, Jake. Come on in. You men know Jake. Well, you got news for us? Yeah. An escort of six troopers is leaving Fort Laughton this afternoon and heading east along the Butterfield Trail to meet a big wagon train and escort it across the plains. There's a wagon load of Army rifles and ammunition they're bringing to Fort Laughton. The major's brother-in-law is wagon master. What if he plenty of cash in that wagon train? Including you, there are six of us, but we couldn't very well- Wait a minute, Cain. I figure you could get help from the Comanches. Chief Long put a jump at the chance if you offered to let them have the rifles and ammunition. The escort will be on the lookout for an attack and have the wagon stop at protected places. But if we could be sure of surprising them on the plain... There must be some way to... Wait a minute. If we showed up as the escort, we'd have complete control of things. Hey, how could we do that? Yeah, what you got in mind, Cain? We'd get a few along-foot braves to help us ambush the six troopers, take their uniforms and Army horses, and go on to meet the wagon train ourselves. Later that afternoon, Cain Dolan and his five followers, augmented by as many Indians, waited to ambush the Army escort. Chief Longfoot, you and your men take our horses to your village. We ride those with the Army brand on them. Then take your braves to the big plains and be ready to attack day after tomorrow at dawn. We'll see to it that the wagons break camp around the plains by then. Here comes the escort. Let them have it. The six troopers were taken entirely by chance. Some of them fell before they had a chance to defend themselves. And within a matter of minutes, all of them had fallen victims to the vicious and ruthless attack. They're all done for. Now we'll change clothes with them, drag them back into the brush, and leave on their horses to meet the wagons. All right, let's get busy. Soon the transfer of clothing was made, and the troopers, hidden behind the brush. As the outlaws prepared to mount the Army horses, came with them over, then chuckled. Some of the uniforms don't fit any too well, but nobody'll notice. Now remember, my uniform has the chevrons of a sergeant on the sleeves, so be sure to call me sergeant. Right, sergeant. We go back village now. Then we ride with 100 braves to big plains. Attack wagon. Good. Adios, chief. All right, let's go, men. It was almost sundown. It was almost sundown when the Lone Ranger and Tato reached the spot where the ambush had taken place. After finding bloodstains on the trail, they looked around and found the troopers, one of whom lived just long enough to tell what happened and to warn them of the attack on the wagon train. Then he died, and the masked man and Indian made graves for the six troopers. When the unpleasant task was completed, the Lone Ranger spoke. Tato, I'll give you a note to Major Belding at Fort Laughton. He'll remember you, I'm sure. Tell him outlaws, posing as troopers plan to lead the wagons into a trap. Ah, me tell Major, what happened here? And what you do? I'll remove my mask and disguise my features. Then posing as a rancher, I'll join the wagon train to protect the pioneers until help comes. The following morning, as the wagon train moved slowly along the trail, the wagon master, Seth Cushing, driving the lead wagon, suddenly spoke to his wife, Amy. Okay, me, riders coming for us. Yes, I see them, Seth. Well, I thunder their troopers, I can make out their uniform. So they must see them before my brother came in. I reckon so. The major roads that he'd send an escort to meet us and give us added protection across the big plains. Well, the sight of them makes me feel we're almost there, Seth. Our long journey is almost over. We still have quite a distance to go, Amy. But like you say, it does make you feel like it trips about over to see the men from your brother's fort. And here they come, we better stop and greet them. Stop the wagons! The outlaws were accepted as a military escort and took their place at the head of the wagon train. Some hours later, the Lone Ranger disguised as a rancher. Watched from hiding as the long line of prairie schooners headed by the uniformed horsemen followed the trail through the hills as they made their way toward the big plains beyond. After the last wagon had passed, the Lone Ranger started along the trail behind it. Come on, till there! A short time later, he overtook the wagons, passing them all until he rode alongside the one driven by the wagon master, Seth Cushing. Easy fella, easy! Howdy, mister. You reckon you're in charge of this wagon train, aren't you? That's right, stranger. Where'd you come from? Just happened to catch up to you. I'm heading back to the territory near Fort Laughton. All of my join your trains from here on in. Well, I reckon it's all right, mister. A smith will do. A smith, huh? Sure meet plenty by that name. I'm Seth Cushing. This is my wife. I'd be glad to meet you both. The Gweepe are quite important. They have a military escort. Yeah, I notice the uniformed hombres, ma'am. Oh, here comes the sergeant now. Easy for you. Everything all right back here, mister Cushing? Fine, fine, Sergeant. I'll meet Mr. Smith. He just caught up to the wagons. He's gonna travel the rest of the way with us. Howdy, sergeant. A smith, huh? Convenience name, mister. It'll do. Mr. Cushing, it's risky to let a stranger join the wagon train. We already told Smith he could join it. No, that's not it. Well, have it your way.  Cain quickly joined his men, and as they rode, he discussed the newcomer. You know, Jake, something about that fellow Smith I don't like. Yeah? What? I don't know. The way he looked at it. I saw him staring at the bullet hole in the breast of this uniformed jacket. You think he suspects something? I don't know. But to be on the safe side, I'm gonna see that he has an accident. A serious accident. Before the commensurate attack in the morning. We'll continue our lone ranger adventure in just a moment. G-man Jimmy is eight years old. He is strong and he is bold. He can capture outlaws cause he knows. From Cheerios, yes, he's got go-pops. That's Cheerios, all right. The nourishing old cereal that's shaped like little letter O's. The ready-to-eat cereal with a wonderful toasted old flavor. What's more, every delicious spoonful of Cheerios and milk is real muscle-building food. That's right, each spoonful contains vitamins, minerals and proteins your body needs. And these good things in a Cheerios breakfast do good things for your body. Help you have healthy nerves, good red blood, strong bones and muscles. Yes, Cheerios is made to give you real go-power. So try Cheerios, the famous old cereal that needs no cooking. And soon you'll hear people say... He's feeling his Cheerios. Now to continue. A wagon crane moves slowly westward and the rest of the afternoon passed without incident. At sundown, the wagon circled for the night at the edge of the plains. The lone ranger decided it was time to warn Seth Cushing. Mr. Cushing, I have a word with you. Sure. What is it, Smith? It's about those troopers, sir. Well, what about them? They're leading the wagons into a trap. I found out... Those troopers were sent to escort us by Amy's brother. Fact is, the sergeant came to me a while ago and warned me to keep an eye on you. I'm glad to let you ride with us, but I won't stand for anyone stirring up trouble. You'd just better forget whatever you started to say. Now I have work to do. The sergeant suggested we start an hour earlier than usual in the morning. Better turn in for the night. Kindlein's plan was evident to the lone ranger. He wanted to start the wagons early so they'd be moving in a line when the attack came, making it difficult to defend the train against the Indians. Determined to delay the wagons and try to keep them circled until help came. The lone ranger crawled through the shadows under the wagons and removed the pin bolts from some of the quiffle trees and threw them into the brush. Then he rolled into his blanket outside the circle beside one of the wagons. Later, King Dolan, standing watch with Jake, spoke in a low voice. I heard this Indian headband feather in my saddlebag. You take it with you. Crawling the shadows outside the circle. Use your knife on that stranger and drop the headband. When he's found in the morning, folks will think an Indian sneaked apart. Now here, be careful, but get him. Right. You'll never know what happened. The lone ranger lay awake staring into the darkness. Suddenly he heard the crack of a twig. A sound came from the darkness outside the circle of wagons. Quickly he slid from the road blackened and waited. Then, instantly the lone ranger splang grappling with a dark figure who had wielded the knife. Try to knife me up. I'll show you. Wait, wait, you made a mistake.  Begin with a light inside the circle. Hold it there. What's going on here? This man tried to knife me. Knife him. Why, he's one of the troopers. Why could he try to knife him? I saw an Indian creeping up under my followed. This guy there, I heard the blow. This ombre jumped me. I reckon the red skin got away. You're lying. I have a lantern, we'll take a look. Knife stuck into your blanket, all right. I don't see how you avoided it. Look there, an Indian headband with a feather stuck in it. My thunder, you're right. An Indian did try to sneak up on you. What's all the excitement? An Indian tried to knife Mr. Smith, Sergeant. He got away, but it looks very bad. If Indians are around here... We'll check thoroughly, Mr. Cushion, and be alert for indications that other Indians might be nearby. You just leave it out. Come on, Jake. We'll get our horses and look around for that red skin. Sure, Sergeant. The rest of you men might as well get back to your wagons.  Better move your blanket inside the wagon circle like everybody else. See you in the morning. Oh, Mr. Cushing, I... Hmm, how are you trying to convince him now? Before the sun rose, the camp became active again. The pioneers prepared to start the wagon crane on its way. Then they learned of the missing boats. They crowded around Seth with their complaints. The Lone Rangers stood listening. Now, wait a minute. Hold on a minute. We can't move the wagons till we get more boats to hold the whippo trees. Oh, hey, oh! What's the delay, Mr. Cushing? Seth quickly told Cain what the trouble was. Cain's eyes rested on the Lone Ranger. Then he spoke. Those thin boats were deliberately taken out during the night. Maybe Smith knows something about them. Why should I? Smith, you bear watching. Mr. Cushing, you better get more boats. Fix those wagons so we can get started. We lost a half hour already. The sun will soon be up. I may have some of my tool chest. We'll soon get the wagons rolling. Don't worry, Sergeant. Come on with me, man. Get up there. Come on. Mr. Smith. Oh, good morning, man. Why did you take those thin boats out? I saw you last night. To delay the wagons will keep them circled. You think there might be an attack? I tried to talk to your husband. Are you, Seth, told me? I don't know why, but I trust you. And I don't trust the sergeant, miss. I've been around soldiers long enough before I married the notice team. They couldn't have trained under my brother Major Belden. Believe me, Mrs. Cushing, I know they aren't troopers. You must convince your husband of that. And they're planning to have the Comanches attack. That's why they're so called sergeants anxious to have the wagons break camp and start over the plain. There was some way to find out for sure. Wait here, come to Sergeant. I'll ask him some questions. Oh, good morning, man. Miss, I want... Sergeant, tell me something. Of course, ma'am. I heard of the Major's accident. You know, when his horse fell, I'm worried about it. The Major's leg was badly injured, wasn't it? Well, yes, a mighty bad, but he's getting along all right now. Did Martha, the Major's wife, you know, arrive all right? His wife? Oh, yes, yes, ma'am. I'm so glad we'll soon be together again. Won't be long now, ma'am. I have to hurry things up so we can get started.  Well, ma'am? My brother never had an accident and he isn't married. Well, I found something bolted by Smith. Smith, if I saw you... You must listen. I just found out for certain the Sergeant is an imposter. Smith tried to tell me that... I asked questions about Frank. The Sergeant gave the wrong answer, Seth. Please listen to Mr. Smith. Well, Smith, what do you got to say? Briefly, the Lone Ranger told all that had happened. Seth explained... You mean they aren't troopers from the Major's fort? No, they aren't. They're outlaws.  Wait, wait. You talk different, mister. I don't savvy... Never mind that now. Those men plan to lead these wagons into a trap. By thunder, I'll tell everybody... No, wait. I suggest you delay the wagons, keep them in a circle, and place some of your best men near those outlaws. When and if the attack comes, have your men disarm them immediately. Tie them and put them into one of the wagons of the guard. Good idea. We'll take them by surprise. I sure hope that friend of yours gets here in time with the Regiment. In spite of Cain's urging, Seth managed to delay the departure of the wagons until after sunrise. The wagon master had talked quietly to some of the men, who, from then on, stayed close to Cain's followers. The Lone Ranger, mounted on Silver, managed to stay near Cain. Finally... Hey, look! Coming over the rise on the plane! 50 years! This is it, men! The gun doesn't take care of you. Even before the Comanches were within range, Cain and his men were on their way. On a few of the pioneers tied them, the rest gave their attention to fighting off the approaching Comanches. The pioneers, ready for the attack, because of the Lone Ranger's warning to Seth, fought valiantly. Now watch them! Old waste bullets! Make each cut count! The Comanches pressed forward, riding at a gallop around the outside of the wagons. Many on both sides fell wounded, and from all appearances, the Indians were rapidly gaining the advantage. Then... Look! Coming in from both sides! The Comanches! Look! Coming in from both sides! The Troopers! Troopers! The troopers, far off numbering the Indians, moved in from both sides with guns blazing. Those Comanches who weren't dead or wounded had no chance of escape, and before long, they were subdued and quickly disarmed. The battle had ended. Later, Tato and the Lone Ranger listened as the Major verified the story told by the Mask Man about the ambush of the escort. He finished by saying... Those killers would be taken back and tried for murder along with Chief Longfoot. I hate to think of the result if the outlaw's plan had gone through. Thanks to Smith, we found out in time. Smith? The gentleman on the way stayed in Frank. He tried to tell Seth last night, but Seth wouldn't listen to him. We owe our lives to Mr. Smith and ours for one and great work. Easy, Tato, easy boy. Thank you, Mrs. Cushing. We're all thankful to him and his friend Tato. Why do you call him Smith? That's the name he gave us. Of course, I noticed earlier that he changed his way of speaking. His drawl suddenly disappeared. It was used only as a convenience, Mrs. Cushing. Well, Tato and I are right on ahead, Major, and make certain there are no more Indians waiting to attack. Adios, everybody! Goodbye! Mr. Smith is such a fine man. Somehow, I trusted him from the very first. And his name isn't Smith, Amy, and you had every reason to trust him. He's the finest American I know, and one of whom the West is mighty proud. Who is he, Major? Yes, Frank, by heaven's sake, get to the point. Just who is that man? He usually wears a black mask, but the present, his features are disguised. Amy, you'll hear a lot more about him out here. He's the Lone Ranger. Copyrighted feature of the Lone Ranger Incorporated is produced by Kendall Campbell Muir Incorporated. The part of the Lone Ranger is played by Brace Beamer, your announcer, Fred Floyd. Listen to the Lone Ranger brought to you by special recording Mondays through Fridays at this same time.


Welcome to MOOC course on Introduction to Proteogenomics. This generation sequencing has really seen large applications especially in the clinical settings. It is a really good idea to catch up on use of new NGS platforms. In this slide we have invited industry experts to provide you the hands on session how to use the latest NGS technologies. One day Dr. Arthi Desai from Illumina will provide you a brief lecture on sequencing technology especially sequencing biosynthesis. She will also give you an introduction of how sequencing technology actually works. She will talk about two key concepts sequencing biosynthesis and paired end sequencing. She will show you how to open an account in base space account and we will proceed for the hands on session in the next lecture. So, let us welcome Dr. Arthi Desai from Illumina. Before we actually get started with the hands on session I wanted to show you guys a short video that recapitulates the Illumina sequencing technology. Mukesh did a great job of explaining all the platforms that we have and the key oncology applications and the panels that are currently available from Illumina. But we are not sure on you know whether the concept of reads, the read lengths, paired end sequencing, depth of sequencing if all of that is known to everybody. So, before we start the actual hands on session which is going to be short what we would like to do is you know just give you a brief understanding of how the Illumina sequencing technology actually works. And there are plenty of videos available on YouTube. We have just picked one of the you know the one that really quickly and easily demonstrates how the sequencing technology works. Sample preparation begins with extracted and purified DNA. The first step in next-tier sample preparation is tagmentation. During tagmentation transposums simultaneously fragment and tag the input DNA with adapters. Once the adapters have been ligated reduce cycle amplification as additional motifs such as the sequencing primer binding sites, indices and regions that are complementary to the flow cell oligos. Cycling is a process wherein each fragment molecule is isothermally amplified. The flow cell is a glass slide with lanes. Each lane is a channel coated with a lawn composed of two types of oligos. Hybridization is enabled by the first of the two types of oligos on the surface. This oligo is complementary to the adapter region on one of the fragment strands. A polymerase creates a complement of the hybridized fragment. The double-stranded molecule is denatured and the original template is washed away. The strands are clonally amplified through bridge amplification. In this process the strand folds over and the adapter region hybridizes to the second type of oligo on the flow cell. Polymerases generate the complementary strand forming a double-stranded bridge. This bridge is denatured resulting in two single-stranded copies of the molecule that are tethered to the flow cell. The process is then repeated over and over and occurs simultaneously for millions of clusters resulting in clonal amplification of all the fragments. After bridge amplification the reverse strands are cleaved and washed off, leaving only the forward strands. The three primings are blocked to prevent unwanted priming. Sequencing begins with the extension of the first sequencing primer to produce the first read. With each cycle four fluorescently tagged nucleotides compete for addition to the growing chain. Only one is incorporated based on the sequence of the template. After the addition of each nucleotide the clusters are excited by a light source and a characteristic fluorescent signal is emitted. This proprietary process is called sequencing by synthesis. The number of cycles determines the length of the read. The emission wavelength along with the signal intensity determine the base call. For a given cluster all identical strands are read simultaneously. Hundreds of millions of clusters are sequenced in a massively parallel process. This image represents a small fraction of the flow cell. After the completion of the first read the read product is washed away. In this step the index one read primer is introduced and hybridized to the template. The read is generated similar to the first read. After completion of the index read the read product is washed off and the three prime end of the template is deprotected. The template now folds over and binds the second oligo on the flow cell. Index two is read in the same manner as index one. Index two read product is washed off at the completion of this step. Polymerasis extend the second flow cell oligo forming a double-stranded bridge. This double-stranded DNA is then linearized and the three prime ends blocked. The original forward strand is cleaved off and washed away leaving the reverse strand. Read two begins with the introduction of the read two sequencing primer. As with read one the sequencing steps are repeated until the desired read length is achieved. The read two product is washed away. This entire process generates billions of reads representing all the fragments. Sequences from pooled sample libraries are separated based on the unique indices introduced during the sample preparation. For each sample reads with similar stretches of base calls are locally clustered. Paired and reverse reads are paired creating contiguous sequences. These contiguous sequences are aligned back to the reference genome for variant identification. The paired end information is used to resolve ambiguous alignments. Part of the takeaway of that video one is the chemistry that is used for generating data on the Illumina sequencers what is known as sequencing by synthesis. So as you saw we actually add one base at a time and record that base. So we are literally reading one base at a time which is why we have very high accuracy in our data set. The second one was the paired end synthesis. So we are essentially using the same fragment of DNA that we are using in our library prep to read it from two ends. We read it from the forward end and then we read it from the reverse end. So which is why a lot of the Illumina data that you will see will have two reads for every fragment. It is called R1 and R2. So read one and read two. And as was alluded to in the video what that gives you is it gives you again very high confidence in the data that you are generating primarily because the fragments are short. The fragments are about you know 150 to 200 base pairs and when you read them from both ends you have overlap. So the chances of you reading one base more than or rather twice every time you read it is very high. So you have again very high confidence in the data that you generate. And because again you are reading it from both ends it is very useful for certain applications like translocations or deletions or insertions and so on and so forth because the distance between the two reads is fixed. So every time you map it back to the reference genome if there is any deviation from this fixed length you can infer that there may be a structural variant in that particular region of the genome. So it is a very powerful way of sequencing genomic DNA and as you can imagine it is the leading provider of sequencing technology globally today more than 90% of the data that is available in public databases comes from Illumina technology and it is not only true for research but it is also very much true for clinical applications. We ended the video on the Base Space application so I wanted to take you guys to Base Space if I can. Base Space Sequence Hub is a luminous cloud based next generation sequencing platform that performs automated sample to result workflows for your lab. We recently released a number of new features designed to enhance your laboratory's efficiency including a new biosample centric data model that enables easy tracking of all biosample activity from lab preparation through analysis delivery, new automation and quality control features to streamline the efficiency and consistency of your workflows and an improved interface that helps you access your data and perform functions more quickly. The new biosample centric data model lets you easily track all biosample activity from lab preparation through sequencing to processing and uploading of data to the cloud and analyzing results. Biosamples support data aggregation which can be linked to multiple libraries, runs, recues, analyses and can have multiple data sets associated with them. FastQ data sets have replaced samples and are now stored inside of biosamples. Their existing samples have already been converted so that you can use the new file types as inputs when launching apps. Base Space Sequence Hub includes new features that allow you to automate analysis workflows, reducing the time it takes to process samples and eliminating costly errors. New features include automatic lane QC settings, automatic data aggregation, automatic app launches, automatic analysis QC settings and enhanced status tracking. The updated interface provides quick access to all your data from a single place while the new Action Toolbar contains new and improved app functions like recues, QC status changes, workflows and collaboration tools. Access your work groups and review your compute and storage usage from the account menu. Inline tooltips help you understand what's occurring with your data and the enhanced filters widget has been added to more places letting you get to the data you care about more quickly. The API, Base Base CLI and Base Mount Tools provide access to your data from a command line interface and have been improved to facilitate more advanced integration and automation. With these new enhancements, Base Base Sequence Hub takes your work from sample to result more quickly, more efficiently and with more control than ever before. To learn more about Base Base Sequence Hub and all of our new features, please visit www.alumina.com slash base base or contact us at techsupportatalumina.com. Okay, here it is. So Base Base is nothing but a cloud application that is developed by Illumina. So this is hosted on Amazon Web Services, AWS and it's a free application. In the sense anybody can access the application, some of the apps on Base Space are paid which means that you have to pay for using those apps, but primarily Base Space is freely available. What I want you guys to do is login to your Base Space account and refer to the handout that was given to you, but before we actually start doing the hands-on session according to the handout, I wanted to show one very interesting property of the Illumina data that is something that Mukesh again touched upon during his presentation that is the quality score, the very, very high quality data that is generated on the Illumina platform.  So those of you who have used Sanger sequencing will be aware that Fred score is a quality score that is assigned to a base call that is made by any sequencer and it is nothing but the confidence that the caller has in the base that it has called. So it is a probability. So when we say 99.99%, we are 99.99% sure that the base that we have called, let us say if we call it an A, it is going to be an A. So as Mukesh said that the error rate is going to be 1 in 1000. So we are going to be wrong 1 in every 1000 bases that are read. So you can imagine because the read lengths that we generate from our platforms are no more than 600 bases and our error rate is 1 in 1000. So the chances of there being an error in the data that we generate are very, very small. This is a IIT data. So we had run a project for one of the PIs here and you guys do not have access to this. So I am just going to show you the data because I really wanted to show you the quality of the data that gets generated. So on base space when you have some time and if you are interested there are multiple apps that are available. So apps are nothing but small widgets that are created either by Illumina or by third party, researchers, companies that are supporting data analysis on the Illumina platform and they are made available. So based on the application you want to or you are working on, you can choose the appropriate app and run the analysis. What I wanted to show you today was data that is generated from an application known as FastQC. So FastQC is an application, it is an open source application. I forget which university it came out of but this has been around for at least 8 or 9 years now very, very widely used to evaluate the quality of the data that is generated by sequencers. So the way to read this data is horizontally 1 to 99. So this is a 100 base pair read, 100 base read, right. We talked about the size of the fragment, the size of the read that you generate. So when we say read it means it is the contiguous output that is generated by the sequencer. So in this particular instance we are looking at a 100 base pair read data that is generated. So the Illumina platforms can generate as small as 36 base pair reads and as long as 600 base pair reads, okay. The Y axis or rather the vertical axis shows you the Q score, okay. So this is again a measure of the quality of the data that is generated on the Illumina sequencers and as you can see we are literally touching the ceiling of the scale that is available. So the Fred score ranges from Q10 to Q40, okay. So Q40 being 100% accuracy, Q10 is I think 1 in 10, 1 in 10 error rate. That is the way to interpret it. So you can see that for majority of the length of the read our Q scores are very, very well above Q30. That is essentially what that means is all the data that you are generating, practically all the data that you are generating is usable. You do not have to throw out or filter out any data because it is low quality and this is very, very critical in clinical applications primarily because you want to make sure that any data that you generate is of high quality, right. Because what are you looking for when you are generating sequence data? You are looking for differences from the reference genome, right. You are looking for differences from the reference genome which can be in the form of single base variations, right. And what are errors in sequencing generally, they are single base variations, right. So you want to make sure that whatever variations you are calling, basis of which you may be taking some clinical decisions one have to be accurate, right. You have to be 100% sure that the base that you are calling as a variant is actually a variant, right. So this is where something like this becomes extremely critical and you can pick up any data. This is actually the data that we have generated here itself in-house or rather for IIT Bombay. So these are actually patient samples, these are tumor samples. So these are not even, you know, very, very well maintained cell lines or blood samples which is where you all pretty much expect to get high quality data. These are tumor samples. So again you can see that on real biological samples you get very high quality data. In today's lecture, we learnt about NGS technology platform, especially how Illumina chemistry works. Dr. Dixie also talked about the importance of Fred's code and how it gives the idea about our data quality. She also showed how a real data from biological sample actually looks like and how to read that data. So I hope now all of you have opened an account in base space which is available free. Please open an account and get ready for Dr. Arthidesa's next hands-on session which will be based on the base space account. In the next hands-on session, she will take you a journey where you can use various data sets from your own experiments or publicly available data sets, analyze them and make meaningful insights from their data. Thank you.


My name is Chris Gray. I'm married with three kids and a little Yorkie dog. I'm in a house full of ladies and I love it. I'm a project manager in the control center. The control center is a 24-7 shift environment where we're managing the power grid. We're doing monitoring, we're coordinating maintenance, emergency response, construction, a lot of different work across the state. All three of my children are adopted from China so we took three separate trips over there to get them so we just have a different connection just thinking abroad internationally. But I would say we've just very inclusive-minded and very open in thinking beyond just what you hear to really trying to put ourselves in somebody else's shoes. So I grew up in Houston, not in the best of neighborhoods so success for us in the 70s was getting out of that neighborhood but I feel like I never forgot where I came from and the fact that I had friends and cousins who were still back in that neighborhood. Focus Community Strategies is a nonprofit, it's a faith-based nonprofit that's been around for a little over 40 years. Currently I serve on the FCS board and have been a board member for about 10 years. We're a nonprofit but we have a very entrepreneurial mindset and so our key branches are mixed income housing so we do we have affordable and workforce housing and then economic development. You've seen that with our coffee shop next door where we hire folks from the community and then we have a community market here and there's other endeavors that we do that fit on that economic development piece and then our other key pillar is neighborhood engagement so we do different things that interact with youth different things that serve the community and then the last one is training. We try to take what we have done over the years and what we've learned over decades and help others do that in their community and so I think discovering this organization was just a way to reach back and take what I had gained and try to help another community. Last year when Rayshard Brooks was killed which was just right about a quarter mile from here right here in this community obviously that hit the local and national news so we're talking about just social justice and why that's important and I think it's important beyond just the fact that it's been a media thing and it is currently I think it it's a real thing that impacts real people. It's not an us and them it's us these are our these are co-workers these are our neighbors these are our friends and family that are impacted by these issues. When I joined George Power I was very excited about this whole idea of being a citizen where you serve and kind of understanding that we impact the community both directly through our work of providing electricity and we're scattered all over the state just what we represent and where we put our energy and what our foundation does I think all of that kind of comes together in a very powerful way.


The following is a production of New Mexico State University. When George and Lynn Dreer first saw this property, the front yard was an ugly duckling of a landscape. However, the current owner who was selling a house said, you've got to see the backyard. It is a real swan. George and Lynn have worked, and they have turned the front yard into a swan as well. George is going to tell us how he did that. George, there's no way that this landscape can be called an ugly duckling landscape now. What do your neighbors think of this? Well, I think if you go around Lakewood, you'll see that a lot of neighbors have adopted this. Certainly next door has made their garden look very similar to this. In fact, many people think that part of their garden is actually part of my garden, but it's really separate. So it's just an extension of this garden. And this is fairly water-conservative in here too, isn't it? It sure is. It won a zero-scape award from the Dallas Water Department back in 97, as I recall. And while there is a sprinkler system in the garden, we don't use it very much. In fact, I haven't used it. This is what we're filming here now in May, and I haven't had the system on since last September. So, here for the area, it's very well-adapted there. I notice the tree is mulching the landscape. That's right. So there's a lot of live oak leaves here. Live oak leaves, especially in the winters, when most of them fall. And we use that just as a mulch, rather than just sweeping it up and throwing it away. There's a lot of really interesting and well-adapted plants here. I see the yopon, which is a Texas native. Right. That's a dwarf version. Over here, you have a lamium, which is extremely good for dry shade. These crepe myrtles tend to take up most of the moisture, but as you can see, it looks very good, very lush, and it actually blooms in the springtime. It's colorful even when it's not in bloom. That's exactly right. I see the Texas sage here, which is a really good choice. The blue sage. And then I see a lot of annuals that you put in here. Right. You get a lot of color. The love and a mist. And I just let a few of them reseed, and that's it. I don't ever have to, you just plant them one time, and that's good. In fact, I don't really even plant them. I just throw the seed out. And they throw the seed out every year after that. That's it. And you've got a lot of ornamental grasses too. Yeah, I really like those because in the summertime, some of these plants go a little bit dormant because of the heat, and that ornamental grass tends to bulk up the garden and makes it look fuller. That's a good thing. And the crepe myrtles here. That's a nice thing to have. Yeah. Full bloom, short labor. Right. Now it's still beautiful, just with this beautiful bark. There's actually three of them here, and these are intermediate crepe myrtles, so this is about the mature height. Regulars can get up to 40 feet, but these are just going to top out at about 15 feet. This is adequate for this property. Yeah, it's perfect for this. The preceding was a production of New Mexico State University. The views and opinions in this program are those of the author, and do not necessarily represent the views and opinions of the NMSU Board of Regents.


informing the summit and the Government's response to the challenges that we have heard that are not just in school but also in our communities? I am happy to engage with the Scottish violence reduction unit. I know that it does some really good work, and I think that the member makes a very important point in relation to how that is encompassed with our response at the summit. I wanted to come back to a number of points that were made by Alex Rowley, and he talked about some of the points that have been raised by our teaching trade unions. Of course, I declare interest as a former member of the EIS. I recognise that this is not something that has happened post Covid. This is an issue that the unions have been campaigning on over a number of years now. It is important that we get it right, and we recognise it in Government. I hope that members have heard from me today that that is the approach that I will take, and certainly in my engagements with trade unions thus far, they have been supportive of that additionally. I hope that I am sure that it spoke of the importance of adverse childhood experiences, and I think that I alluded to some of that in my initial contribution, recognising that some young people in our school are traumatised, and we need to make sure that there is support in place to recognise that. I think that that is really important because we know that, in relation to exclusions, for example, children coming from disadvantaged backgrounds are far more likely to find themselves excluded, and that is detrimental to their progression. We need to be mindful of that, too. I thought that Carole Mullen made an excellent contribution in relation to the role that teachers have in their classrooms and the care that they have for their young people. The worry and care that sits alongside that, I very much would recognise that. She is right to also point to the cross-portfolio nature of challenge in relation to mental health. We have provided funding in relation to school councillors, but I am very keen to work with my health colleagues on that issue more broadly because I recognise the need for us to look across portfolios out of our silos and government to better support our young people. Cabinet Secretary, could I ask you please to conclude? Yes, I am mindful of time. I just want to thank members for their contributions this afternoon. In general, it has been a very positive debate. It is important that we get a response in government right. The summit will seek to bring together partners to do just that and also to work with our teachers who are at the chalkface, and I think that it will be central to delivering the solutions that we need to see in responding to behaviour in our schools. Thank you, Cabinet Secretary. I now call on Megan Gallacher to close to wind up on behalf of the Scottish Conservatives up to eight minutes. We have heard the harrowing stories from across the chamber about the increasing levels of violence in our schools. The first time I heard about the true extent of the problem was when I took part in a panel event with the NAW-UST. The union spoke openly about how the education workforce are adversely affected by a minority of pupils who challenge authority, use threatening behaviour, abusive language and, in some instances, physical violence. No one should go to work feeling unsafe. When I was putting together some words to say during today's debate, I found myself returning to the same question. How did things get so bad? The testimonies that we have heard today from across the chamber are a damning assessment of Scotland's education system. Colleagues have approached teachers directly so that they can share their stories and it shows that we have a serious problem. It is not our voices that need to be heard. It is the voices of education professionals who need this Government's support. However, I will say to Ross Greer that if he thinks that raising concerns on behalf of parents and young people is sloping up a culture war, then I think that he needs to maybe re-evaluate what it means to be an MSP. I am grateful to the intervention. Without wishing to assume the experience of others, I am pretty sure that I have more experience than most members in this room of being the pupil left bloodied by an attack at school. I am just interested in what made the Conservatives so uncomfortable when I was raising the experiences of LGBTQ young people who faced violence at school. That was not the case at all. We were referring to concerns raised on behalf of parents and young people and I do not think that Mr Greer should misconstrue what we were trying to point out at all. Voices are powerful and Murdoff Racer quoted the experiences of one teacher. He said that the teacher had seen people being sworn at, spat at, punched, scratched and bitten, tables thrown and colleagues who have had multiple trips to the hospital. However, the part of Murdoff Racer's contribution that should shame this Government is when the teacher said that getting it right for every child is an absolute joke. That is not coming from an MSP in this chamber but from one of Scotland's educators. There is something fundamentally wrong with our education system, and it is clear that our teachers have had enough. Rachael Hamilton pointed out. I wonder if she agrees with me that the Scottish Government's response so far has been reactive and scrambling to deal with symptoms. That is only one side of the coin. The consideration should be given to how we tackle these issues before they become a crisis, like investing in preschool activity, which includes a healthy breakfast so tackling hunger, mental and poor physical health, and tackling diseases and poor behaviour. I thank Brian Whittle for his intervention, and I couldn't agree more. Prevention is key, and we need to look at other ways in which we can tackle the issues that we see in our schools. Rachael Hamilton pointed out that our teachers have succeeded not because of this SNP Government but in spite of the reforms. We need to find solutions to the unacceptable level of violence that we see in our school. Last week, when I raised violence in our schools as part of a long list of SNP Government failings, and if the cabinet secretary is looking for somewhere to start, then this is the place to start by making our schools a safe space for teachers to teach and pupils to learn, because, as we have heard so frequently today, this isn't a new problem. Teachers have been raising this problem for years, and the unacceptable violence culture, as pan Duncan Glancy rightly raised, has been allowed to grow. As we have seen, we get to see a cabinet secretary of this Government do something about the increasing violence in our schools. However, I have taken quite a few today, so if you could forgive me, I would like to continue on. It was reassuring that the majority of the speeches today approached this issue with good intention. In June 2022, I raised the issue of violence in our classrooms and, again, concerns by the NAW-UST union. One union representative said that it is as if they, meaning the Scottish Government, really do not want to know the scale of the problem. I also asked the Scottish Government at that time to accept that cuts to council and education budgets were putting our teachers at risk, or the inaction, of course, of reducing classroom sizes, as mentioned earlier. Shona Robison, who was the cabinet secretary responding at that time, then announced that research into school behaviours had been cancelled due to Covid, with no confirmation that the research would be reinstated later. Although the advisory group of relationships and behaviours in schools met last December, the then cabinet secretary did not attend something that Willie Rennie raised in his contribution. However, I do not think that the advisory board has met since, and that was something that I did want to ask the cabinet secretary earlier on, because it is important that we, as MSPs, know exactly what the groups are discussing in order for us to be able to take the matters on and raise them fully within our respective roles. Stephen Kerr also pointed out that parents are worried about their children's safety and prospects. Pam Gossel spoke about bullying and the untold damage that this will have on a child's mental health and ability to learn. That reiterates the importance of bringing everyone together to tackle this problem. However, I want to know and understand further how the Government can understand the scale of the problem when they have not collected, let alone published data, on violent incidents in our schools since 2016. That is seven years ago. We can hazard a guess that some of the causes—yes, certainly, cabinet secretary. I have responded on this point a number of times in the chamber. Today, of course, research should have been carried out in 2020. It could not be carried out in 2020 because of the lockdown, but it is going ahead. I hope that that gives the member our reassurance in relation to the reason why it was not able to be carried out, because we were on lockdown and children were not in school. Yes. I accept that point, but it does not mean that it is acceptable that it has taken seven years where nothing has happened in that time. The Conservative motion today is one of concern and solution. I am pleased that the SNP has supported our principles and action points, and it is good to see that they have finally accepted the Conservative education policy. Although I would say that changing a couple of words and trying to call it amendment might be a bit of a stretch, however, the Scottish Conservatives will be supporting the SNP's amendment this evening. That is cross-party working at its best. I do hope that the Scottish Government will agree to our action points outlined by Stephen Kerr earlier on, particularly the first three, the summit that must meet urgently, a statement in Parliament on the outcomes of the summit and an action plan to tackle violence and disruption in our school. This Government cannot be allowed to get away with any more years of doing nothing. I welcome the cabinet secretary's warm words of working to tackle this issue, but until we see the results, it is just another way that the Scottish Conservatives will need to hold the Scottish Government to account to end the violence in our schools. Thank you, Ms Gallacher. That concludes the debate on ending violence in Scottish schools, and it is now time to move on to the next item of business. The next item of business is consideration of motion 9109 in the name of Marie Todd on powers of attorney bill UK legislation. I call on Marie Todd to move the motion. The question on this motion will be put at decision time. The next item of business is consideration of business motion 9147 in the name of George Adam on behalf of the parliamentary bureau setting out a business programme. I call on George Adam minister to move the motion. Thank you very much, Presiding Officer, and moved. Thank you minister. No member has asked to speak on the motion. The question is that motion 9147 be agreed. Are we all agreed? Yes. Are we all agreed? Yes. The motion is therefore agreed. The next item of business is consideration of business motion 9148 in the name of George Adam on behalf of the parliamentary bureau on stage 2 timetable. I call on George Adam minister to move the motion. Thank you, Presiding Officer, and moved. Thank you minister. No member has asked to speak against the motion. Therefore, the question is that motion 9148 be agreed. Are we all agreed? Yes. The motion is therefore agreed. The next item of business is consideration of two parliamentary bureau motions. I ask George Adam minister on behalf of the parliamentary bureau to move motions 9149 on approval of SSI and 9150 on parliamentary recess dates. Thank you minister. The question on these motions will be put at decision time to which we have now come. The first question to be put and in this regard I would remind members that if the amendment in the name of Jenny Gilruth is agreed to, the amendment in the name of Cam Duncan Glancy will fall by way of preemption. The first question is that amendment 9126.3 in the name of Jenny Gilruth, which seeks to amend motion 9126 in the name of Stephen Kerr on ending violence in Scottish schools be agreed. Are we all agreed? Yes. We are not all agreed and therefore we will move to vote and there will be a short suspension to allow members to access the digital voting system.


FX has just unveiled the first official teaser for season 10 of American Horror Story, which is called Double Feature. And the teaser is packed with a bunch of hints to dissect, and with less than a month until the season premieres, trust me, there is a lot to discuss. But before I jump into my breakdown of the teaser, I wanted to take a moment to thank my channel members. You guys make these videos possible, and your support is greatly appreciated. Alright, so one of the major reveals of this teaser was the subtitles of the two parts of this season. Part 1 will be called Red Tide, while Part 2 will be called Death Valley. Let's first talk about everything the teaser let us know about Part 1, Red Tide. First off, let's talk about what the title means. Red Tide is a term that commonly refers to discoloration of the sea surface, commonly caused by microorganisms, sewage runoff, among other things. It can be caused by natural causes, or be influenced by human activity. And red tides often have toxins that are harmful when exposed to marine life. Basically, where a red tide comes, dead fish could be washing up for up to two weeks afterwards. Okay, so with the subtitle of Red Tide and its literal meaning in mind, I think I have a small theory based on what we already know about the season so far. One of the most perplexing characters we've learned about is the chemist, who is being played by Angelica Ross in the Red Tide part of Double Feature. And I definitely have a hunch that this character will have a hand in the terror that is about to ensue in Provincetown. This is purely speculation, but I'm going to predict that the chemist is an actual scientist who is conducting experiments on the people of Provincetown. So with the name Red Tide in mind, perhaps she is slipping some chemical toxins into the sea, and either these chemicals are turning beachgoers into these siren-like sea monsters, or perhaps her experiments are disturbing or altering the sea life that is already below the surface. Could the chemist be responsible for these siren creatures coming ashore? Let me know what you guys think in the comments below, but let's get back to the teaser. In the teaser, we get another look at these creatures, who were first spotted frolicking around Provincetown back when Double Feature was filming there in about March this year. The teaser, though, shows us these creatures in action, and they seem to be at least semi-aquatic. Now, I don't know if these are sirens. Definitely, they are not a classic image of a siren, but a lot of people online are referring to them as such, and they could very well end up being AHS's version of sirens. So for the sake of not overcomplicating things, I'm just going to call them sirens, but just now, I don't know what they are. One stray shot in the teaser shows us a siren skull next to a human skull, and it's been long theorized that the season will have something to do with body modification. So I wonder if the body modification will have something to do with humans becoming sirens? I don't know, I'm just curious as to where the body modification will play into the season, if it does. Based on the still that was released earlier, we know that they are able to walk on land, but I'm going to guess the sea is where these creatures originate. One shot of the siren swimming underwater is inner cut with a shot of a great white shark. So maybe this is just drawing visual comparison between their teeth, but this could also be a Jaws reference, as it wouldn't be surprising for the season to draw some inspiration from the 1975 horror blockbuster, which also filmed partially in Massachusetts. And who knows, maybe this is further proof that the season may touch on the cold case of the Lady of the Dunes, which also has a connection to Jaws. I did a video about how this Provincetown true crime story could play into season 10, so make sure you guys check that out if you're interested and haven't already. But next, we see a shot of one of these sirens under a frozen body of water. This is in line with the red tide part of this season taking place in the winter time in Provincetown, Massachusetts. This is all but confirmed, and this teaser is just further proof that it is taking place in winter. The winter season is also referenced in this shot of a snowy road, which looks very Massachusetts, and it is then contrasted with this sandy desert road, which brings us to part two, Death Valley. The subtitle Death Valley is likely referring to the desert and national park located near the California and Nevada border. This makes sense as Nevada has been a rumored location for the second part of the season. This area also has a lot of notoriety for its UFO sightings, and not too far into Nevada you'll find Area 51. So this subtitle and general location makes perfect sense if part two is going into full on alien abduction territory. So let's talk about the aliens. These guys are the same ones we've seen in two of the posters that have been released for double featured so far. But is this what our extraterrestrials will look like in the actual season? Well, I wouldn't assume so. However, given that the sirens featured in the teaser look just like the ones from an officially released still, maybe the teaser aliens are our final design after all. I don't hate how they look. They're very clearly making them look like the classic caricature image of extraterrestrials, and the teaser itself seems to be taking inspiration from the 1947 faked alien autopsy video. Only in the teaser, it's the aliens who are conducting an autopsy of the sirens. Now, I wouldn't hate if these are our aliens. However, I think this design might be a bit too on the nose for AHS. And if this is how our aliens actually end up looking in the season, I may be suspicious that they aren't aliens at all and instead some sort of hoax, just like the alien autopsy video referenced. It's also worth noting that these aliens look nothing like the aliens from Asylum. However, I don't expect all aliens to look the same. So it's definitely not a problem that they look different. The teaser also features a slide with the words two worlds collide, which I am hesitant to take as complete confirmation that the two parts of the season will be connected.   I mean, it's they're definitely trying to point us in that direction. So I am definitely leaning towards the side that these two parts are connected. The teaser ends with the alien and the siren fighting it out with the closing shot being a beached alien. We also see the siren biting the alien's neck, which may be a hint that these creatures have some vampiric tendencies as well. I don't take this duel as a hint that our sirens and our aliens will ever meet in season 10, but rather that forces of both of their respective half seasons will be connected. I will say I do think it's very clever to have these two horror creatures, figures being the figureheads, basically, for the two parts of the season. I think it's very clever marketing campaign they've got going on. But anyway, some other stray visuals that I can't completely make sense of are the black pills, which we've also seen in one of the posters. I'll take these as another reference to the chemist, though. There is also a shot of a deer and, aside from deers being a common motif in horror films, I'm not sure what to make of that. Then there's red light bulbs that appear throughout the teaser. Not sure what all that could mean, but let me know if you guys have any thoughts. And then finally, to me, the most out of place shot in the teaser. We've got a meat violin. I just I just have no idea what this means, and I don't know if it's worth trying to understand. I don't know if it has a meaning, but I'll leave that to you guys. If you guys want to dissect the meat violin, which is a sentence I would have never guessed I would be saying. All right, well, there are all of my thoughts and theories for this first teaser of AHS Double Feature. Make sure you tell me all of your thoughts in the comments below, as it is always great to hear from you guys. Give this video a like if you liked it. Subscribe for more AHS coverage and I'll see you next time.


This is TFNN, the Tiger Financial News Network, news update. Good morning everyone, Basel Chapman here for the 10 o'clock Tiger Financial News Network update. We're looking at the Dow of 221 at 34,690. Look at this roll over. We've got different, this is really typical of the market for months now. When one sector takes a breather, the other one takes over. Yesterday the Dow was lagging and all the other sectors were doing really well, especially the semiconductors and of course we'll talk about that in my show coming up, Tiger Technicians Hour and we got the Dow at 34,675. So it's touched the pink nine-period moving average for the first time in about a week and we'll see if it can hold that. You've got the S&P which did very well yesterday. It was above the pink and the 14-period moving average. Now it's still above, it's up 19 at 44.55. Is this a turn of absolute consequence? Is this the low for a move that last weeks or is it a low? At this point I still think it's a low, not the low. We're looking at the QQQ in the X100. Big red candle. I spiked up to 372.74. It's at 369.57, up 50 cents right now. Got a little carried away but the chart actually is starting to improve. IWM Russell 2000, playing a little catch-up today up 22 cents and 186.01, still not a very good looking chart. Gold. Gold is trading down almost four at 1944, the fabulous session yesterday. I'm saying to gold, you know, I like the pattern. You need to prove something now because silver's taking the lead. It's taking a little bit of a breather today. Silver's down just a little bit but that kind of rotation. Yeah, again, you've got this rotation in the metals where one sector's doing a little bit better than the other. Look at crude oil. Crude oil had a nice rally earlier this morning. Now it's at 27 cents and 78.61. That ninth period moving average went negative but the price is still holding pretty well when you think about it. So we've got the TLT which is bonds. Let's see what happens tomorrow. I think it's more Jackson Hole with Powell. TLT's down 18, ticks at 95, 36. We'll see what happens. Did I do the dollar? Yes, I did the dollar. Dollar's acting very well. We've got 44 ticks at 183.84. I don't think you can count the dollar out yet. I'll be back for the Target Technicians Hour and thanks to those who participated in my webinar last night. I'll be back in a moment.