Welcome to All Hands Update. I'm Petty Officer Tyrell Morris. The Center for Personal and Professional Development is conducting a fleet-wide review of Petty Officer Selectee Leadership courses and wants your feedback. The survey opened October 6th and focuses on the leadership skills and behaviors seen in and used by superior enlisted leaders. The purpose of the survey is to evaluate the current leadership courses' value and effectiveness. More than 750 sailors have participated in the survey. Surveys are open to all active or reserved component sailors E1-06. All feedback is confidential and sailors taking the survey will remain anonymous. Log on today and take the online survey if you want to let the Navy know your thoughts on Petty Officer Selectee Leadership courses.


Let's look at the artwork. Let's take a look at this. The Raven by Edgar Allan Poe. Bill Fingers the artwork, and this is from Mad Comics, number nine from 1954. Okay. This comic is sent to us as a care package by Max and Max. Thank you very much for the love. I hope you're enjoying this reading. Let's have a read through the second story of this comic, The Raven by Edgar Allan Poe. Classical type comics department. Once upon an evening, dreary, while we pondered weak and weary in the public library, on a comic story plot, while we, nodded nearly nappy, came at a tendon, a tappy, on our head so gently wrappy, spoke. That's all the time you've got. Oh, were we mad? We howled, we raved, and that's what this story is about. The Raven by Edgar Allan Poe.  Once upon a midnight dreary, while I pondered weak and weary, over many a quaint and curious volume of forgotten lore, while I nodded nearly napping, suddenly there came a tapping, as of someone gently wrapping, wrapping at my chamber door, this some visitor I muttered, tapping at my chamber door. Only this and nothing. Clunk clunk bash bam. Distinctly I remember it was in the bleak December, and each separate dying ember wrought its ghost upon the floor. Eagerly I wished the morrow, vainly I had sought to borrow, for my books surceased of sorrow, sorrow for the lost Lenore, for the rare and radiant maiden whom the angels named Lenore, nameless here for evermore. And the silken sad curtain, rustling of each purple curtain, thrilled me, filled me with fantastic terrors, never felt before. So that now, to still the beating of my heart, I stood repeating, clunk clunk clunk, to some visitor entering, and treating entrance at my chamber door, some late visitor, and treating entrance at my chamber door. This is it and nothing more. Presently my soul grew stronger, hesitating then no longer. Sir, said I, or madam, truly your forgiveness I implore. Back this I was napping, and so gently you came rapping, and so faintly you came tapping, tapping at my chamber door. That I scare was sure I heard you. Here I opened wide the door, darkness there and nothing more. Deep into that darkness peering, long I stood there, wondering, fearing, doubting, dreaming dreams, no mortal ever dared to dream before. But the silence was unbroken, and the stillness gave me, gave no token, and the only word there spoken was the whispered word Lenore. This I whispered, and an echo murmured back the word Lenore, merely this and nothing more. Back in the chamber turning, all my soul within me burning. Soon again I heard it tapping, somewhat louder than before. Surely, said I, surely that is something at my window lattice. Let me see then what threats is, threats is, and this mystery explore. Let me let my heart be still a moment, and this mystery explore. This the wind and nothing more. Open here I flung the shutter, wind with many a flirt and flutter, in their step a stately raven of the saintly days of yore. Not the least obedience, made he, not a minute stopped or stayed he, least obeisance made he. But with mine of Lord or lady perched above my chamber door, perched upon a dust of palace just above my chamber door, perched and sat and nothing more. Then this ebony bird be guilty, my sad fancy into smiling, by the grave and stern the quorum of the continents in lore. Though thy crest be shorn and shaven, though I said art sure no raven. Gasly grin an ancient raven wandering from the nightly shore. Tell me what thy lordly name is on the night's plutonian shore. Quoth the raven, nevermore. Much I marveled this ungainly foul to hear discourse so plainly. Though its answer little meaning, little relevancy bore, for we cannot help agreeing that no living human being ever yet was blessed with seeing bird above his chamber door, bird at least, bird or beast upon the sculptured bust above his chamber door, with such name as nevermore. But the raven sitting lonely on the placid dust spoke only that one word as if his soul in that one word he did outpour. Nothing further than he uttered, not a feather then he fluttered, till I scarcely more than muttered. Other friends have flown before, on the morrow he will never, he will leave me as my hopes have flown before. Then the bird said, nevermore. Startled at the stillness, broken by reply, so aptly spoken, doubtless said I. What it, what it utters is its only stalk and store caught from some unhappy master whom unmerciful disaster followed fast and followed faster till his song won burden bore till the dirgs of his hope that melancholy burden bore of never, nevermore. But the raven still beguilding my sad fancy into smiling. Straight I wheeled the cushion seat in front of bird and bust and door. Then upon the velvet sinking I betook myself to linking fancy onto fancy, thinking what this ominous bird of lore, what this grim ungainly ghastly gaunt and ominous bird of lore, meant in crock croaking nevermore. This I sat engaged in guessing but no syllable expressing to the foul whose fury I now burned into my bosom's lore. This and more I sat divining with my head at ease reclining on the cushion's velvet lining that the lamplight clotted oar but whose velvet violet lining with the lamplight gloating oar. She shall press, ah, nevermore. Then me thought the air grew denser, perfumed from an unseen censor, swung by saffrin, saffrin whose footfalls tinkled on the toughed floor. Rich I cried that God hath lent thee by these angels he hath sent thee. Respite, respite, an nap of napenth from the memories of Lenore. Quoth, oh quoth, this kind nap of napenth, and forget this lost Lenore. Quoth the raven, nevermore. Prophets, said I, think of evil, prophets still, if bird or devil. Whether tempter sent or tempest tossed, thee here ashore, desolate yet all, undaunted. On this desert I land enchanted. On this home by horror haunted, tell me truly I implore. Is there, is there balm in Gilead? Tell me, tell me I implore. Quoth the raven, nevermore. Prophet, said I, think of evil, prophets still, if bird or devil. By that heaven that bends above us, by that God we doth adore. Tell this soul with sorrow laden if, within the distant aiden, it shall clasp a sainted maiden whom the angels name Lenore. Clasp a rare and radiant maiden whom the angels name Lenore. Quoth the raven, nevermore. Be that word our sign of parting, bird or friend, I shrieked on starting, starting.  Leave my loneliness unbroken, quit the bust above my door, take the beak from out my heart, and take thy form from off my door. Quoth the raven, nevermore. And the raven, never flitting, still is sitting, still is sitting, on the pallid bust of palace just above my chamber door, and his eyes have all the seeming of a demon, demons that is dreaming. And the lamp light, o'er him streaming, throws his shadow on the floor, and my soul from out that shadow that lies floating on the floor shall be lifted, nevermore.


nd that could only happen because of the results of the confidence the people have and this pattern Mr President has continued all over the months thereafter the month of March April May they have been increases in the VAT collection now you may want to say it may just be me that is not the case because I know some of you may have an answer for that but let us look at the other hardwares in this country you've heard them speak you've heard them say that they've been increased in the construction sector increasing the purchasing of building materials you have heard them say so the initial figures Mr President for cement which is a benchmark or a guide that the government of St. Lucia used to assess the construction sector in St. Lucia by the consumption of cement and how much cement is imported and purchased in this country initial figures are indicating that in this year Mr President that there are significant increases in the importation and consumption of cement in this country how can this happen if this economy is not growing how can this happen if this government is not on the right track to take in this economy to the place that it is supposed to be and most of all Mr President you've heard it from the entire chamber of commerce the chamber of commerce survey indicated that there is growth that there is increase in consumer spending in this country and as a result that there is the projected increase in jobs for our people in this country and it is not just only projected Mr President it is also a fact we have seen the statistics from the statistical department indicating that there is a reduction in the unemployment rate in this country there is a reduction in the unemployment rate in this country Mr President so it means by this government reducing that it has assisted in the growth and development of this country and it will ultimately and has started doing so increase the revenue collection for this government Mr President and so let me use this analogy to indicate what has happened before and what is happening Mr President this country in Creole okay so this open. So you'll make a tax, play tax. So you're gonna make a play tax. That's what he's been saying that you are going to convince the pulsa here and they don't have any use of the salmons. We can't take a few of them at all, because they're going to be too busy to pay for the day. It's not busy, we need a salmons. So you'll make pressure and let the pulsa here. You'll make pressure and let the pulsa here and try to take a few of them at all. And then you'll sell them. Who Just like the minister in the ministry of finance said. Okay.  So so Pulsa If you are sick, you have to go to the hospital and stay in the hospital. So, the website says that you have to go to the hospital. So, you have to play with it. If you try to do everything you can, you have to try to do everything. You have to do everything you can. And if you get sick, you have to go to the hospital to do everything. It's very difficult to go to the hospital. I would do everything with my cards. You have to go to the hospital. And if you go to the hospital, I would go to the medicine shop. It is difficult for you to go to the medicine shop, Mr. President. So this is the vision of this government. This is the vision of the minister of finance in this country, right? So we're growing the economy in that form. So let Nukai Viet here a lot. Let Nukai Viet here a lot. They will serve up with it. Let by guy will be there. Nukai Viet, guy many people are going to be there. Nukai Viet, guy many people are going to be there. Nukai Viet, kind toward pool. And then Nukai Sa, Nukai Sa, Nukai Sa, I said, Mr. President. So this is the way it is, this is the way it is, Mr. President. This is what we are doing in this country. We are reducing unemployment, we are creating confidence in the consumer in this country. And so we are creating confidence in the voter of this country, Mr. President. And so, I wish the leader for the opposition, business was sitting there, I don't know why he ran out. But I can say, Mr. President, that contrary to what he has said, I do not believe it. He has said, it looks like it is easy for you to win elections in this country, but it is difficult for you to govern. I think this is an insult to the people and the voters of our country. I believe that the people of this country know exactly what they are doing and they know who they are electing to govern this country. And I do not believe that they have made a mistake by electing this UWP government into office because we are delivering for the people of this country. And so, he used the analogy, he made it like that, only for the people of Kailah, because it is difficult for you to win elections in this country, because it is difficult for you to win elections in this country. So, what do you know? I know that government is a part of that. They do not want to win elections, they want to make people elect people. This is what I believe in the new intelligences here. I believe that because you're making the people elect you, what does it do?  If you think that you are not making the people elect you, if you do not make the people elect you, what does it do? The people have to fire you. And this is why, Mr. President, this is why the people of this country fired them. And I do not blame that woman that he used as that analogy. Okay, that woman is a smart woman. And that is why that woman left him. Because he's not producing and bringing to the house what is required for that woman. All right? He's not doing so. And because he's not doing so, that woman has left him. And that woman has gone for a better pasture. That woman has gone to a better man. He's from Vietnam. He's from Vietnam. That's correct. Mr. President, he's from Vietnam. She's not going back there. She's not going back there because she realized she's getting better from that new man. So he is hoping that come the next four years he will go close to the balcony and knock and say, Angela, come away. But Angela don't even want to see him by the balcony. This is the people of the country. This is how they'll be treated. Angela do not want to see him by the balcony because he did not deliver. And that this new man who's inside of the house sleeping well is working not even five days a week. He's working six days a week just like the Lord asked him to do. And he's producing for this country. And so my brother, Minister of Finance, I must tell you, continue this good job. Continue the work that has been done. Continue the good work for the people of this country.  They elect the people that they want to govern them. And Mr. President, this is exactly what has happened. When we borrow this $25 million, we are injecting capital into the economy. This in itself is a form of spending into the economy and growing the economy itself. They talk about we come here and we are only borrowing money and borrowing money. And that we are only adding. We said we were coming to balance the budget, but we're not balancing the budget. We're coming to borrow more money and more money and more money. But I'm asking them, why did we get to the point where we are today? We heard from the minister with responsibility for financing in the ministry, in the prime minister's office, said that the national interest on the national debt grew from $100 million to $178 million over their term. This is what they've done. This is the pressure they have created. So this becomes our problem now that we have to deal with. Where do we get the funds for us to be able to service those debts? So we have to grow. We have to do so. Risk has to be taken. But since we are headed in the right direction and we're headed in the proper channels, Mr. President, I remain confident that this government will take its people out of the indulgence. And in the days just like Noah and his Ark was able to rest on top of Mount Arorak, the day for this country is coming when this government will journey its people to safety. I thank you, Mr. President. Did the government business? Mr. President, I beg that this Senate be suspended for one hour so that we can have lunch. Honourable Senators, the question is that the Senate be suspended for the next hour. I now put a question as many as are of that opinion, see aye. As many as are of the country opinion, see no. I think the ayes have it. The ayes have it. The House is suspended. Well, that was the last presenter for the first half of the day sitting of the Senate. And you just heard from Senator Timothy Mangal, the morning session began with a contribution from Senator Fortuna Belrose, who reflected on some of the activities and events in celebration of Creole Heritage Month in October, and also Senator Mary Isaac, who reported on her recent participation in a conference on noncommunicable diseases. Most of the debate so far focused on the motions seeking parliamentary approval for the Minister for Finance to borrow just over 40 million dollars, most of which would go towards financing. The 2017-2018 budget and solutions disaster vulnerability reduction project. Well, this brings us to the end of the first session of today's setting of the Senate. And as you heard from the leader of government business, the Senate will resume at 3.15. So I invite you to stay tuned to the national television network for the resumption of today's setting of the Senate. Thank you.


This is a newly released LCD projector. It's the Gamma by Vust. It's by a company called Artly. I've reviewed Artly projectors in the past, so I'm pretty excited to try this one out. Before I go ahead and unbox this, just a bit about the pricing information. This is roughly retailing on Amazon right now around the 300 pound price mark, but they run voucher codes and discounts from time to time. Check out the description, hit the link, and see the latest pricing information, and also check out for any exclusive discount codes that I have for you guys. So you can get this cheaper than anyone else. Before I open this up, let's take a look at some of the key specifications of this projector. This is 1080p native, but it upscales to 4K absolutely fine. It has Wi-Fi casting, so you can mirror your iOS iPhone or your Android via mirror cast. One of the key things that I look for any projector before I purchase them are these two specifications here. Autofocus and Auto Keystone. This makes it so much easier for you to position your projector wherever you like for it to automatically adjust and get you the best picture angle with minimal effort. It doesn't mean it doesn't have any manual focus or keystone. If you want to correct it and adjust it slightly, then you do have the manual options inside the settings so you get best of both worlds. It has Bluetooth 5.0, so you can connect this to a soundbar or to your headphones and maintain a really solid connection. And it also supports 2.4G and 5G Wi-Fi. One of the things that's not listed on here, which I think is one of the key specifications about the GambleBerry Vust, is this includes Dolby Audio inside the speakers. Now to anyone that's not familiar, Dolby Audio is a 2D audio technology that makes subtle enhancements to whatever you're watching, be it a sporting event, movie or TV series. So that sounds generally better than normal Hi-Fi speakers that you generally get on projectors that you buy on Amazon. It automatically adds details so that you can hear better dialogue, for example. I don't want people to get confused with Dolby Atmos on the other hand because Dolby Atmos is a 3D immersive technology that allows a speaker or speaker system to trick your brain into thinking that the sound is coming from all around you. It essentially gives you the effect of feeling like you're watching a movie in the cinema with speakers positioned all around you even when they're not. But just having Dolby Audio inside these speakers, I think that is a very good option and I'm excited to see how good the sound quality is. As always with all of my projector reviews, how you guys see the demo and hear the demo on whatever screen you're watching this on will be slightly different to how I'm seeing it in person. So just bear that in mind and I always recommend that you do use headphones when you are listening to this video just so you can get the best audio quality and a sense of how this performs. Let's go ahead and unbox this. So you have this very nice two-tone light gray and dark gray carry case. Just on the side, you have yourself the remote control. Let's take a look how this looks. You've got the power button, the mute button, the focus adjustments, the source, menu directional and enter button there in the middle, back menu, home. You've got the keystone correction button there in the middle if you wanted to manually adjust that and the plus and minus to increase and decrease the volumes. It does require two AAA batteries. Let's go ahead and see what else is in the box. So you have a HDMI to HDMI cable to connect this to any other device such as an Apple TV or Amazon Fire Stick, whatever it may be. You've got your power adapter and then just on the side, you have yourself some cleaning equipments and microfiber cloth and then you also have the instruction manual to get up and running if you need to. And last but not least, let's take a look at the projector itself. Now I have to say, I really like the design of this projector. It's the first one I've seen that has this leather strap and rubber handle at the top making it easy for you to carry and position in different various locations around your home. Let's go ahead and take a look at some of the buttons. So you have yourself the power button just there at the top and if you wanted to use the buttons on the projector itself without the remote control you have your back button, the menu directional buttons up, down, left, right and an okay button just there on the right hand side. The front of the projector has this type of military green color to it which I think is quite nice. I've not seen any projector cutter like this. It does have a lens cap that you can remove like so. And you've got your infrared sensors there. Now you can mount this on the ceiling. So you do have four screws there that you can adjust with any bracket for ceiling mounts. You have a tiny little kickstand if you wanted to adjust the elevation of this and change the angle. You have speakers on both sides of the projector there and I'm also going to be testing the fan noise to see how silent this is. And then at the back, if you wanted to connect your headphones via the cable then you can do that directly in the headphone port there to HDMI ports, to USB ports and then the power port there. This doesn't have any Android TV on there so you do need to connect an HDMI source to watch content. So I'm going to be connecting my Apple TV to show you guys a demo on the quality. So let's go ahead and now set this up on my projector screen. It can go up to around 300 inches which is absolutely great but the ideal size I would say is around the 100, 120 inch point. So I'm going to be showcasing this up to 100 inches on my screen and then showcasing some sample videos to you as well as an audio sample via the Dolby Atmos YouTube channel. Okay, let's get the screen down and I've got a bit of daylight coming in because I wanted to also showcase how this looks in very well lit rooms. But I'm going to start off with first showing you the auto focus and auto keystone by moving the projector around the desk a little bit in various different positions, elevations, height levels, those kind of things. And then I'll run through some of the settings that you see inbuilt into this system and then we can go straight into the demos of the video quality and the audio quality itself. Okay, just powering this up for the first time. I'm standing next to the projector as well. So if you guys can hear any of the fan noise, you can hear the fan noise quite a bit. But once you do start playing something then it should disappear a little bit. As you can see, this is starting to auto focus itself without any involvement from my side. I haven't touched the projector yet. Once this is done, it will load up these settings. So I'm going to go ahead and get this started. I'm using the remote control. Now it's not positioned accurately onto the screen. So I'm now going to move the projector around a little bit just to make it larger and fit the screen a little bit more comfortably. You'll see it will start to auto keystone and auto focus itself. It's now doing automatic keystone correction. There we go. That was very quick and easy and that was perfect. If you press the keystone button on the remote control, you can turn automatic keystone off if it does perhaps maybe interrupt during a movie for example or anything like that. Or if you wanted to manually adjust the keystone then you can do that from here. And likewise you can turn automatic focus on and off from the settings of the projection here. You also have the option for digital zoom. If you click this, then you can adjust this by pressing the up and down buttons on the remote and zooming in accordingly to your liking. Now this is a pretty standard menu screen. I'm going to just run through some of these settings but you can connect to USB and then play movies, video files, music and photos. There's even an office suite here as well so if you wanted to showcase some documents and use this for business and do a presentation perhaps then you can also do that from here. If you got an iPhone, you can do iOScast. You simply click that and just follow the instructions. Make sure you are connected to the wifi and this should work absolutely fine. And likewise with MiraCast, you can connect your Android screen and mirror anything you want to showcase from your Android phone or tablets. Let's go ahead and check these settings quickly. You can connect to wifi with the wifi settings. You can go to Bluetooth and look for anything you want to pair. Maybe your headphones or a soundbar. Projection settings which is what I just showed you. You can find that here. You can get the system update. If you go into other settings, you can also change the language if you'd like to and you can schedule a shutdown if that's something you want to do automatically. So pretty standard settings. I'm going to go ahead and connect my Apple TV to the HDMI1 port and let's just jump in to one of the video demos. Also I'm going to be standing quite close to the projector. So listen out to how the audio quality is with the Dolby Digital Audio and then also listen out to how loud the projector fan might be and if you feel like that might interrupt your audio.  So there won't be any light source coming in. It will be very dark. And then I'll give you my review on how the video quality performs. So let's get straight into it. Okay, let's go ahead to HDMI1, turn on my Apple TV. One thing I just want to say is with projectors of this price range, I think the brightness level and the clarity in the screen that I'm seeing right now is brilliant. This is 400 ANSI lumens, which I've seen a lot of projectors, especially that are even slightly higher priced than this one with lower ANSI lumen intensity. I also have a 550 pound BenQ projector at home, which is only 300 ANSI lumens and this one is much cheaper and it's such stronger picture that I just can't fault the performance of this. Let's go ahead and actually see with this brightness level how the video quality comes in a couple of videos that I'm about to play to you guys. Okay, for me personally, that demo that you just saw, it was one of the clearest pictures I've seen using that same demo clip in a lot of the projectors that I reviewed in the past. The picture was clear. I tried to upscale it to 4K as well because that video was available in 4K and it looked great to be honest and just remember how I'm seeing it in person might be slightly differently compared to the screen that you're watching this video on, but it is super clear and although I have made it dark in this room, it just makes the video stand out so much with the vibrant colors, the sharp details, the deep blacks and I'm just gonna showcase one more clip very similar to that one and hopefully you guys can get a good clarity in the audio that comes from this as well as some dialogue in the background of the video. So let's go ahead and play. Brightest Self-Lit OLED, LG OLED EVO. Light up your world, LG OLED EVO. And one thing I also wanted to mention I only had the volume at 60. I didn't go all the way to 100 because at 60, that was loud enough for me to fill up my large bedroom here. If I wanted to go to 100, then I can imagine that'll be really loud and it warrants me not to have a soundbar connected via Bluetooth. Ultimately, it's your decision if you want to use Bluetooth to connect this to external speakers just to increase your audio experience to whatever you're watching. But just with the internal speakers, if you care more about clear dialogue and how loud the speaker is for you to hear it at a distance, then I think this does a very good job. Now on audio, I'm going to showcase this demo audio clip from Dolby Cinema. Of course, this isn't Dolby Atmos, this is Dolby Audio. So try to hear out to see the quality if you can try to use some headphones and let's go ahead and see how this performs. This is Dolby Cinema. It's where the most advanced cinematic technology ever experienced begins. This is Dolby Atmos. The number of speakers around you no longer matters because this is the world's first object-based cinematic audio. With powerful moving audio that transcends from channels, to moving around you with pinpoint accuracy, whether the soundscape sits in the middle of the scene, Hey guys, let's go home. Or captures the full extent of nature's fury. Remember this? It's just the beginning of Dolby Vision. Because what you thought was black, isn't it? This is black. This is contrast that reveals details deeper than any image you've seen on the screen. This is luminance that means the difference between white and pure energy. Energy that is about to reveal an entire universe of color. You've never experienced in the cinema. So there you have it guys. Hopefully that video and audio demo was useful for you guys. For me, personally, the clarity in the video quality, the contrast, the colors, the vibrancy, I can't find too many faults with this. I think this is a very decent projector, especially at this price range. You can see from this demo video that I'm playing right now, how clear it is, how much it actually lights up the room, which sometimes is quite difficult to do with projectors with lower brightness levels. I think this is great. This is a brand new projector that was recently released and they've done a very good job making sure they have all of the key specifications that anyone would need for a home cinema. And with that Dolby Cinema demo I just showed you, I had to put the audio up to 100 because the video itself on YouTube is actually quite low in volume. So if you really wanted to set up a permanent position for this, I think ideally you should have external speakers with it, but as an overall package to set up your first home cinema system, I think this is great. So hopefully that review was very useful. It gave you guys an indication of the capabilities of this projector. Do check out the link in the description below to find out more information and look at more specs and look at the latest pricing information. If you guys have any other specific questions about this projector, as always, drop a comment down below. I'll leave a playlist of all my projector reviews that I've done in the past in case you guys wanna check those out as well. Make sure to subscribe. I have new videos out all the time reviewing projectors and other cool tech gadgets and I will catch you guys at the next one. Take care.


We wanted to create an area that was wildlife friendly, that increased biodiversity for pollinators, but they also help people improve their own wellbeing through creating it. Through term time, we will help usually disengage youths, the type of children that don't fit in at school.  I started coming down here as a volunteer instead of coming to school C footage in the video is great because it is just the environment. I feel a lot happier now and my mental health is so much better since I started coming down here. One thing that can inspire your gang for Penches is giving me a lot of hope to be happy and to learn in a way that I feel comfortable and where I don't feel pressured to do well. I'd just like to say a big thank you to the National Lottery and to the National Lottery players for helping us to make all of this possible.


What makes for excellence in heart care? It starts with one of the most amazing teams of heart surgeons and cardiologists that I've seen assembled under one roof and the region's leading technology in a new $40 million facility. This is the nationally recognized Heart and Vascular Institute of Eastern Kentucky at Pikeville Medical Center. Pikeville will be celebrating its bicentennial in 2024 and in anticipation the Appalachian Center for the Arts is partnering with five Kentucky playwrights to create monologues of influential figures in the city's history. So we have found Kentucky playwrights, they're all Kentucky based, they're from all over the state, some from Louisville and they are coming together to each write a 20 to 30 minute monologue about five different people from Pikeville's history. The playwrights partnering with the app will spend 2023 researching and writing the monologues and local actors will be chosen to portray the characters in 2024. When you look at history, like if you read a history book, right? You see facts, you see, oh this person lived here, this person did this, they built this, started this business, but a lot of times in the history books you can't really get who they were personality wise and you can't really put a personality to them. So what theater is doing is it's helping bring life to these people that we see in our history books. For more information and updates about the Pikeville 200 program, you can visit the Appalachian Center for the Arts Facebook page. For Mountain Top News, I'm Kelsey Dean.


Russia will continue developing its nuclear triad of ballistic missiles, submarines and strategic bombers because such weapons are the main guarantee of its sovereignty. Defense Minister Sergei Shoygu said on Tuesday, January 10, we will continue to develop the nuclear triad and maintain its combat readiness since the nuclear shield has been and remains the main guarantor of sovereignty and territorial integrity of our state, Shoygu said. We will also increase the combat capabilities of the aerospace forces, both in terms of the work of fighters and bombers in areas where modern air defense systems are in operation and in terms of improving unmanned aerial vehicles.


All right, so, yeah, thanks for coming to our second infectious disease genomic epi workshop. As Anne mentioned, we started this last year sort of in response to demands from both public health and some academia interested in learning how to apply genomics in clinical and public health microbiology. Last year, we focused a lot on public health because most of us, as you heard, came from the public health side, but this year we added some more applications that are applicable to clinical microbiology as well, and also last year we focused a lot on bacteria, but with the events and technology and so on. This year we actually added some viral components as well. I don't think we're covering parasite quite yet, but a lot of the analysis still do apply, and my job here for the first module is to give a general introduction of the course material and the content and also the background to help everyone sort of get on the same page. So, feel free to stop me anytime if you have any questions. The lecture will probably take about 60, 70 minutes. Some interruption is totally fine, and so for the... It's a packed workshop, as Anne mentioned, and we have divided the workshop into seven different modules plus a keynote talk, and each of the module will be followed by a hands-on tutorial session except this first one and the keynote, so you'll actually get to practice what we discussed in the lecture material. I didn't list it here. We also will have a brief tutorial right after this about how to use the Amazon Web Services and how you can use that to analyze your data. So we set up Amazon Cloud Instance for every one of you, and tomorrow we will also show you how to access to another analysis platform that, collectively, the instructor created and worked on in the last few years, and it's a web-based platform that you can upload your data and do some analysis as well. In terms of the actual course content, for module two, you'll hear from Gary about how phylogenetic analysis and how to detect single nucleotide variants and how to use that for transmission studies and for other applications. Modules three, you'll hear from Dylan about molecular stop typing, mainly focused on gene-based or allelic-based molecular typing method, such as MRST, using whole genome sequence data.  And, Fiona, do you want to quickly introduce yourself? Hi, I'm Fiona Brigham, an active professor at South Korea University, very interested in mathematics, but also some lab training. And tomorrow, Brian, early, you'll hear from... What am I talking about? Andrew? I only got, like, four hours of sleep, and from Vancouver, this is, like, five in the morning. Anyway, from Andrew about the... And a lot of you mentioned about antimicrobial resistance being your research interest. So, Andrew will talk about the identification of AMR genes from whole genome data and some of the resources that will be available to you to do that. Module five on phylogenetic analysis. This lecture was typically developed by Rob Beacole, who developed Genghis, which is a tool that allows you to combine phylogenetic trees with geographic locations. We can make it this year, so Anna will cover the lecture and will also talk a bit about the visualization aspect. And module six, Gary will talk about emerging pathogen detection, and this is where the idea is you can search for potential pathogens in metagenomics data, and we'll introduce some viral applications in this case as well. Module seven, Anna will talk about data visualization, some of the principles behind it, some of the good practices behind data visualization, and we also have a hands-on tutorial on developing some of these visualizations yourself, and look at the data from different angles and different lengths. So the general learning objective for this workshop is to understand how genomic epidemiology can improve clinical and public health microbiology. You'll learn to process genomic sequence data using a variety of bioinformatics tools. The focus of the workshop is not so much on how you process the sequence data, because a lot of the tools are readily available, so we won't necessarily go into the details on how you will assemble and annotate your genomes, but rather focus more on the downstream once you have those... once you have a sequence process, how you can use for genomic IP investigation and other applications. And we also will talk about how you can interpret your genomic data in epidemiological contexts and perform several types of genic IP analysis, and that's sort of the meat of the workshop. And understand the fundamental data visualization, and as you can imagine, we're dealing with heterogeneous data sets are highly complex, so some of these visualization techniques can really improve your ability to interpret the data. And last but not least is the... this is a rapidly evolving field, so we also want you to go home with a sense that there are limitations and challenges still associated with genomic epidemiology analysis. And this is an active research area that people are working on, even though a lot of the work can now be operationalized as I'll show in a bit. So for this particular module, the learning objectives become familiar with high-throughput sequencing and its application in clinical and public health microbiology. Be familiar with the sequence data processing, so I'll go over the process briefly in this introduction. Be able to recognize the importance of metadata and sequence analysis and data integration. And lastly, I'll give a brief overview of the other modules. And as I was doing the introduction, I was saying that we'll talk a bit about my research. So one of the important message for the workshop is that sequence data cannot stand on its own. You need the metadata to facilitate the analysis, but metadata cannot stand on its own. Either, as you sometimes don't get the resolution that you need to make interpretation. So the importance of being able to bring the two together is actually what my lab is interested in. So half of my group worked on the sequence analysis aspect, and I'll mention about that in a bit. And the other half of my group actually works on data harmonization and data standardization approaches to improve the quality of metadata. So we live in an increasingly interconnected world. This is the commercial flight path of 2014 or so. And just like people and goods can travel around the globe rapidly, pathogens can be carried with these travelers, animals and goods quite rapidly and spread around the globe through international travels. And case to point is this study done a few years ago looking at the actual human waste from long-haul flights. So a Danish group collected samples from 18 different flights from three different continents and filtered through 400 liters of human waste and extracted the DNAs and sequenced the DNA using metagenomics approaches. And they then clustered the sample based on the microbiome profiles that they observed and also searched for antimicrobial resistant genes in the dataset. So what they found is actually that the samples do cluster with geographic locations. And as you can see here, the North American clusters forms its own distinct cluster or two clusters actually, whereas the northern, it's split into northern Asia and southern Asia form their distinct clusters as well based on the microbes found in these samples. And also by looking at the antimicrobial resistant genes, they found that certain types, certain samples, namely the ones from South Asia actually have higher proportions of antimicrobial resistant genes in the human waste or human expert. So it's a nice study just to show how you can do global surveillance by monitoring flights. And as you can imagine, if the pathogens are secreted in human waste, they're carried into the destination that these travelers go to as well. And another study, look at the emergence of infectious disease events. And EID events have defined newly evolved strains of pathogens in human, excluding re-emergent pathogens. So they, for example, did not count the reason re-emergent of Ebola outbreaks or Zika outbreaks, but they count the first instance of a particular pathogen or a new variant of that pathogen getting into the human population. So this is done over the last, this survey was done over the past six decades or so since the 1940s. And the pattern is clear that there's a steady increase of these emerging infectious disease events in the last few decades. And they attribute the increase in 1980s to HIV and related pandemics. But I don't know if they fully understood why there's a slight drop in 2000. But in any case, the increasing trend is quite consistent and it's dominated by zoonotic pathogens that are hopping over from animal populations into the human population. And in the study, they were also able to then identify global hotspots where these EID events occur. And perhaps not too surprisingly, South Asia is also one of these hotspots. So you have a combination of higher probability, I should say, of newly emerging infectious diseases coupled with increased antimicrobial resistance genes as well, creating kind of a perfect storm that might be of interest in. And that's a lot of the public health agencies and groups do closely monitor the region. So in a subsequent paper that's just published last year, I think, 2017, they also identify some of the risk factors associated with these emerging infectious disease events. And namely, being tropical forests where there's dense population, both human and animals, and rich species diversity and climate effects are all significant risk factors associated with the emergence of new infectious diseases. Now, so ODA is to really illustrate the importance of infectious disease in our modern society. And perhaps the outbreak a few years ago of Ebola in West Africa is still fresh in people's minds. Most daily Ebola outbreak in the history resulting in about 28,000 cases, reported cases, so the actual cases is likely to be higher and also 11,000 or so death over the span of three years or so. It has significant impact, impacted the global travel. Remember when at BCCC, even though there's no case in Canada, we still have to go through, or not me personally, because I'm a mathematician, we don't really go into the field. But the the technicians and the biosafety officers have to go through rigorous trainings in handling in case the Ebola case were detected in Canada. So it's quite a resource intensive exercise to prepare for potential outbreaks and again highlights the global natures of infectious diseases that you know infectious disease in one region, if it's significant enough, can actually have a global impact. So the Ebola genomes, what's different between this particular outbreak and the previous ones is that with the mature maturation of sequencing technologies, about 5% of the cases were sequenced and genomes of these Ebola viruses are available for analysis. And this is really one of the best examples of how genomic epidemiology really facilitate the research and the health care interventions of an outbreak. So this study is one of the earlier studies that look at just the 99 early isolates and what you use in just sequence data alone, but then later corroborated with the epidemiological evidence, they're able to show that the human exposure to nature, to a natural reservoir is likely to be a single one-time event, because there's only a, they're only detecting sort of one original case that's spread human to human, through human to human transmission. And this is likely due to the funeral practice and the lack of proper quarantine in these West African countries that have never seen Ebola virus, Ebola outbreaks before. And, but they also from the phylogenetic tree, they were able to show that the incursion of Ebola from Guinea into Sierra Leone might actually, even though the IP evidence suggests it's a single event, might actually have introduced two different strains of Ebola into the country. So as you can see, there's a split here of two distinct lineages, one here and one down here. And this type of detail information really helped the care workers trace the transmission and understand how these viruses are transmitted and the result was able to influence some of the policies and some of the interventions. However, as I mentioned, a lot of death occurred and it still took quite a few years to finally have the outbreak under control. So fast forward to 2018, some of you might know that there was there's currently another Ebola outbreak in the Democratic Republic of Congo. So what's different from, of this outbreak from the previous one is that DRC has already experienced multiple Ebola outbreaks before. So they're more aware of the symptoms to look out for and they're more aware of how to deal with the patients. And the death rate, the death toll currently stands around 50 or so. So it's much more well controlled outbreak. And other reasons for the outbreak being under control is that there's a faster mobilization of resources. So since the original Ebola outbreak, WHO and World Bank have set up some reserve funds to deal with public health emergencies. And their team put in place to be able to quickly mobilize to deal with these public health emergencies. They also have been able to use the previous outbreak as an opportunity to test out the Ebola vaccine that was actually developed at the National Microbiology Lab in Canada. And they were able to stockpile these vaccines cases for future outbreaks. And in this case, they were able to treat the high risk patients and certain not patient high risk individuals with these vaccines. And also experimental drugs and what developed and also health workers was mentioned able to be in position faster for this outbreak. But we really need to be constantly in alert to be aware of these potential infectious disease outbreaks that can be devastating if not not quickly managed. And hence the importance of a genomic epi based surveillance to monitor pathogens is what the community as a whole is working toward. So unlike Ebola outbreaks, Zika virus cause much milder symptoms. And it's often self the disease is self self eliminating. And most people who have been exposed to the disease developed immune response to it. And it has been an endemic in both Africa and Asia for Africa for much longer, but Asia for at least the last half a century or so before causing an outbreak between 2015 2016 in in America. And the outbreak resulted in a few thousand cases of microcephaly and microcephaly cases. And the likely reason is that the general population in America is naive to this virus. So when they are first exposed to the virus, there's more likely to to have complications and to develop in in the process to be developing immune response. My cost issue such as my microcephaly is in in in fetus. And so it's still an open question why microcephaly wasn't observed in Asia and in Africa to the same extent. But it's also possible some people are proposing based on the genomics data that was generated that may be a recent mutation of the virus has also increases virulence and might be dealing with a more virulent strength of Zika. And also to highlight because of the symptoms of Zika virus is similar to to some other fluffy viruses such as dengue virus. The syndromic base or surveillance are based on observing of symptoms can can actually be unreliable and you can miss cases. So zoological or genomic or molecular testings are needed to for confirmation. And again through the genomic analysis it was later on realized that the virus probably had been in circulation in America specifically in Brazil for at least a year or so before it was recognized as an outbreak. And such gap in in surveillance is likely to increase the magnitude of the outbreak. So as I mentioned genomic space surveillance moving forward can also can can reduce such gap and can improve our ability to to identify potential outbreaks. So another example here is a flight path of a different kind. So these are the the migratory bird flight path. And the reason I'm showing this is that these migratory birds are actually natural reservoirs for influenza viruses. And through these migratory pathways to influenza virus especially specifically avian influenza can actually spread around around the world. And also you notice that some of these flyways actually overlap each other. So it gives opportunities for device genomes to reshuffle and create new variants of the virus.  And the pandemic strains are typically hypervillain sorry hypervillain and typically arise from the mixing of human and animal food viruses. And these migratory birds such and that do have in their sort of process of being the the natural host of these influenza viruses have developed some resistance. So they don't typically get EO from the viruses. But then the viruses can be passed on to domestic poultry populations which are highly susceptible to these viruses at least to certain variants of these viruses. So also to related to human health occasionally the virus can can infect human can jump over to human. And typically when the country observed or at least in North America if we observe avian influenza calling of the birds the domestic poultry's population is the only solution right now. So in 2014-2015 there was a I guess a medium consider sort of a medium scale outbreak of avian influenza in North America. That it's believed that the Asian strain of H5 and H5N1 mixed with a North American strain in somewhere in Alaska. And in that process the highly virulent version of the H5 virus get carried down through these wild bird migratory passes along the coast into the US. And in BC the Fraser Valley is where we raise all our poultry where we have all our poultry operation. And as a result of the incursion of the H5 virus in the poultry population essentially 13 farms were affected and about a quarter million birds had to be destroyed and cost the industry about three hundred million dollars. The once it got into the US it scaled up and the estimate was direct cost to the direct damage to the the industry is about three billion dollars and directly translated to about 80 percent increase in eight prices in in the States. So these diseases can have a wide impact not just for human but also for trade and for economy. And in during that time genome BC got interested in how we can improve the surveillance of of these influenza viruses. So we received some funding together with the BCCC I mean see some funding with ministry together with ministry of agriculture to come up with better ways of doing influenza surveillance. So before I jump into that the current approach to do doing influenza surveillance for birds is through bird based testing. So both passive in other words testing dead birds and active capturing and testing of live birds or hunter killed birds for influenza viruses. As I mentioned because most of the wild wild waterfalls are not killed by influenza viruses so looking for dead waterfalls does not really improve your chance of detecting these viruses. And as a result the overall positive rate for detecting viruses in a bird is it's quite minimum it's approximately one percent or so. And for that particular 2015 outbreaks the actually we failed to detect these hypatogenic strains of influenza virus in the waterfowl before it got into the poultry population. So we developed an environmental genomic based surveillance approach and this is still ongoing work in my group and the process involved isolating and enriching for influenza genomic RNAs from wetland sediment samples and we go to wetlands where these wild waterfalls congregates and the viruses are excreted through their fecal material so the wetlines act as a their outdoor toilet essentially and this type of approach does not then depend on the capture of birds. However because the environment does dilute out the virus it is like finding a needle in a haystack so we have to develop both enrichment method and also improve bioinformatic method to detect these viruses in the environment. But as you can see here the punchline is the overall positive to raise is much higher and in the wetland samples it's about 25 percent positive and the farm samples of course were known to have the viruses the positive to raise is much higher and this is just from the environment around the farm not necessarily not direct sampling of the birds. And we are also able to detect the specific outbreak strains in our samples rather than just like a different strain so it's a proof of concept that show that genomic based approach can actually be used for environmental surveillance. So Andrew will talk a lot more about AMR so I'm just giving a quick example here where there should say many of the AMR genes encoded in mobile elements can move and move around from host to host so detecting of genes based on PCR without sort of the genomic background or the detection of or the identification of the specific bacteria without knowing whether the AMR genes there were not. So if you just identify based on culture or based on other method without knowing the AMR gene is there were not both insufficient to establish the transmission route for the patterns for these AMR gene AMR genes. So this study for example show that the so on this these columns here show the the AMR patterns and you can see the AMR patterns do not really correlate to the phylogenetic tree based on I think in this case MLST analysis really highlighting the importance of being able to identify both the genes and the the genomic background and whole genome sequencing of course it's an approach to to give you that information the caveat being the current sequencing methodology you still don't have it still requires some work to pull out both the plasmids and the genomic chromosomal background so it's still work in progress for these these type of application. But this is an interesting study highlighting how they did that. Now I mentioned quite a few studies so you might think that we're still not prime time yet we're still not operationalizing genomic epidemiology but in fact the public health England lab have been sequencing all their salmonella isolates since 2014 and have been using that to establish genomic based clustering and use that to to detect outbreaks so I gave some references down here but there's actually a paper that that summarized their experience I'll make that available through the through our course website. US FDA also have to establish a central centralized data repository and a little platform called Genome Tracker for both US and global partners and I think Fiona mentioned a bit about more about that in her talk and really highlighting that you can do genomic based surveillance in real-time or semi-real-time to help address an outbreak in this case foodborne diseases. So just before we get into the some of the technologies I'll first define that what I'm meant by genomic epidemiology I sort of keep it quite simple as just to say that it's the combination of whole genome sequence data from pathogens just to separate from separate out from the idea of whole genome sequencing of the host with the human and combining the sequence data with the epidemiological investigation to track the spread of infectious diseases and the epidemiological data provide the contextual information for genomic for interpretation of genomic sequence data and the genomic sequence data provide high resolution diagnostic information for helping to shape the direction of the epidemiological analysis investigation. So this is an improvement over the current clinical microbiology laboratory practice which required the lab to maintain a large number of different tests and different media to support the identification of different pathogens and also of course comes with this different diagnostic platforms machines you have to maintain so on and so forth. This sort of showing you some of the tests that are regularly performed in the public health diagnostic health microbiology lab and due to the regulatory and validation requirements sometimes developing a new essay or new tests it's a slow process and moreover some of these methods can take weeks or months sometimes you have to send the samples away actually so we regularly send samples to the National Microbiology Lab if it's for specialized tests and that just a transportation process alone and getting on a queue could sometimes take weeks if not month depending on the test. So genomic sequencing of pathogens promise to simplify that workflow so instead of maintaining an array of different platforms you essentially have to maintain a sequencing platform and use and also you have to maintain or at least be able to carry out the bioinformatic analysis downstream of your sequence generation but many of the pathogens then can be sequenced and analyzed without having to to maintain a large array of other tests. So the benefit includes simplified workflows I mentioned and also in most cases you can guarantee a turnaround time if you're doing the sequencing and analysis in-house because you know how long the sequencing would take how long the analysis would take so it then depends on how quickly can you get the samples processed for sequencing it's a overtime issue achieved called saving by reducing the number of platforms and instruments that you need to to maintain and also sequencing itself is becoming cheaper and cheaper by the day the one of the really value added bonus for sequencing is that the the result of your sequences is actually comparable so it really doesn't matter where you generate a sequence by and large the sequence data are comparable and moreover that it's digitized format right so it's making sharing of the data easier as well and you can perform value added analysis value added research on the sequence data as I've shown in some of the the uh or in the Ebola and the and the Z card examples so some of the challenges include that the the results are harder to process interpreted whole genome sequencing for example give you a large amount of data so you do need specialized computer resources bioinformatic resources to process that information the there's also a rapid change in technology as I mentioned sometimes validating a test can take a long time so when you have a technology that's fast evolving how do you deal with the the accreditation or the validation of the platform so a few months ago I was at a conference so uscdc is just sort of talking about how they are going through this accreditation process for clear which is the clinical the body that maintains clinical accreditation in the US and detailing how labor intensive and time process time intensive it's to validate a genomic test for their foodborne disease program and the other issue is that if you're if you have low throughput then this per sample cost can be very high and often you need to accumulate enough samples and do batch processing to reduce the cost but many of the labs don't necessarily have the volume to sustain this high level of activity so it becomes again whether you centralize the the sequencing service where you can you will you do it distributed but then have to absorb the cost for the lower volume the cost due to lower volume so I talked for I guess for 30 minutes or so and well pause for a bit and just want to open up the the discussion to other people do you have any experience with with applying whole genome sequencing and are there any benefits and challenges that you have observed in your own work anyone enough samples yeah yeah so that's a common problem and and often sequencing can be done in a few days but you might be in the queue for a few weeks or a few months so one of the reasons why we're not a lot of time we're still not doing this in real time anyone any other thoughts on what are some of the potential challenges what benefits so how many people actually doing genomic sequencing of their samples and not doing analysis at the moment okay so about half you and what about the other half are you just receiving data and then want to do analysis yeah okay yeah so or we'll go into now the bioinformatics aspect a bit more oh actually before I do that so I just wanted to well introduce a bit of doubt about high throughput sequencing so the term high throughput sequencing had gradually sort of been becoming more popular to the next gen sequencing although you still see that a lot and part of the reason is I guess some it's a clarification between what's next gen sequencing versus what's sort of what's so-called third gen sequencing that's based on single molecular sequencing so the sort of by you by using high throughput sequencing it sort of creates a collective term that you don't have to worry about whether you're talking about Illumna sequencing or you're talking about Minayang sequencing or or Pac-Bio sequencing which are some of the third generation sequencers and sequencing data have many laboratory utilities can be used for diagnostics so you can do actually detail strain level identification can identify virulence genes and AMR genes from the sequence data alone you can carry out surveillance be it gym by gene type of surveillance or single nucleotide variant SNP type of surveillance you can even do in-circle serotyping so based on the genomics data predict the serotype and you can also use the sequence data to identify carbon number variants so and that the information can then be used for outbreak detection and and investigation so you can carry out trace back and you can carry out transmission route analysis and so on use the sequence data in combination of with the EPI investigation so the so I think most people probably are familiar with Illumna base sequencing that's sort of the current workhorse for generating a lot large amount of of shore reads from your your samples how many of you have heard of Minayang all of you great so I don't have to to talk I don't have to introduce it but this is a sort of a small form factor sequencer that gives you a longer reads but much lower fidelity in but it does promise to be portable and highly versatile so this graph shows the cost decrease in sequencing DNA and you can see an inflection point here that shows when next generation sequencers were introduced there's a rapid decrease in cost and also notice this is in log scale so so the drop is is I think it's about 10 to the 14th fold drop since 2001 in terms of pricing so right now it costs about point I guess that's one cent per megabase and of course show off and how many people are sequencing their genomes at one cent per megabase anyone no so so the caveat here is that this is a highly optimized highly batched solution so this data comes from the the the National Human Genomic Research Institute they sponsor some large-scale sequencing centers and this is the cost of these large-scale sequencing centers they're per megabase cost so what's important to take from this is really there's a significant drop in price but if you translate to a medium or small sized lab you're still looking at somewhere between 15 to 200 dollars per sample and roughly per genome or per roughly per genome if you're dealing with metagenomic samples the cost could be could be higher because you may need much higher coverage for the samples depending on your application so a quick summary a reminder of what pathogen genomes look like so for bacteria there's typically a single circular chromosome with some exceptions it's a haploid genome meaning there's only one copy of a of a given gene or one the leo I should say of a given gene in the in the genome and may have exocromosomal elements or plasmids that are carried around with with the the chromosome the genome sizes range from about 0.5 megabase to 100 sorry to about 10 megabase average especially for pathogens typically is about in the middle three to five megabase correspond to about 3000 to 5000 genes the viral genomes of course are typically smaller they can be both dnn RNA and the next gen sequencing platform typically only dealing with dna sequencing even though now you can do RNA direct sequencing on the minion but the efficiency is still much lower than dna base sequencing so often you're dealing with dc dna or reverse transcription of the RNA viruses before you can carry it out sequencing and the range is somewhere from one to two kb to one to two megabase which it's it's it's quite unusual to see such large genomes for viruses because they're usually quite compact and depend on the whole cellular mechanisms to to replicate uh i heard some of you are working with eukaryotic parasites uh which include fungi uh protests and and worms and they're usually few sorry i didn't quite type it out a few megabases to a few hundred megabases and usually have multiple chromosomes so um there are some some exceptions such as giardia or some of the east only about 12 megabases so they're about the size of a large bacterial genome with no introns but typically when you're dealing with eukaryotic parasites because of the more complex gene structures the downstream processing is it's more complex as well whereas bacteria and viruses were getting quite good at doing the uh gene annotations with with these pathogens and there are several dominant evolution driving forces that are changing the genomes uh i'm just listing some of them here such as the genome can be reduced over time so called the sort of specialized uh specialization where the organisms become specialized to a specific niche uh and uh as a result they come sort of lean and mean and then reduce the the access requirement for um maintaining a larger genome uh there's constant rearrangements in in most of the the microbial bacterial genomes and the gene duplication followed by uh diversification of gene functions it's another uh force of of change in the genomes and of course in in uh many microbes the lateral gene transfer is a it's a big driving force as well the acquisition of of non-parental or DNA material or non-material non-parental strains and recombination uh can quickly uh change the bacterial genomes and uh and because of all these driving forces we actually instead of sequencing a handful of genomes typically have to sequence hundreds if not thousands of genomes to really characterize a single pathogen species well okay so uh this is sort of outlining the whole genome shotgun sequencing approach and what i want to just highlight is that because uh there's no cloning stage and the uh the the DNA fragments are typically just uh quickly PCI amplified and sometimes the skip this step is even skipped before sequencing so you can uh streamline the process uh compared to the previous uh Sanger based approach and it's also highly parallel paralyzed so you generate a lot of data in the in the short period of time now moving into sequence data analysis so in the sequencing step especially on the um uh the next gen platforms such as Illumna the the DNA is broken down into smaller fragments that are sequenceable in size they're typically a few hundred uh base pairs long so you generate millions to billions of such short sequences uh short um uh fragments and you sequence them uh and so the typically the downstream process to before you can analyze the data is you have to put the fragments back together through a process called assembly and then you have to annotate the sequences to provide functional informations about the genes or about the the non-coding regions and then you can do compare the genomics to identify variants that are found in each of your genomes so the genome assembly uh the task is to reconstitute the whole genome if possible from fragments of DNA and there's two basic approach one is called the novel assembly essentially you're just finding overlaps in your fragments and stitch them together based on the overlap uh kind of like putting back a jigsaw puzzle finding the the overlapping edges uh the other is called mapping or some people call it references assembly although mapping is probably a better term for it what you're doing essentially is you're mapping your sequences to an existing related reference genome so keen to trying to essentially place each of these pieces back to the to using this reference picture place back to where they belong so of course a easier process but the challenge is that if your reference genome is diverse uh has diverged from your query genome where the genome that you're interested in then your mapping based approach is not going to give you a very accurate representation of your query genome in in addition to those challenges there are sequencing errors of on all platforms that you have to deal with so the assemblers have to be able to tolerate the sequencing errors and also one of the major challenges for shore read based assembly is that repetitive regions sometimes span or are bigger than each read so when you're trying to piece them back together two distinct repetitive regions could be collapsed into a single region because the there's no not enough information in your sequences to span the entire repetitive region uh this is by and large addressable by using uh say minnayang or oxford nanopore sorry or pack bio that will allow you to generate longer sequences hopefully span beyond the the repetitive region so this is quickly summarizing some of the common platforms are still in in use today for sequencing in the type of error and error rate and i just want to highlight that ilumna has comparatively uh very low error rate and uh it's typically a random error so it's sub substitution errors that happens randomly much easier to to uh to correct if you generate enough depth coverage so by taking the consensus then on average you'll get the right base rather than the wrong base so the consensus approach can easily correct for substitution errors uh the third generation sequencers still have greater than 10 percent error rate so one in 10 base that you get from from the sequencer will be incorrect and on top of that the third generation sequencers have at least minnayang i should say have uh errors that are more uh that are harder to correct they're based on uh complications due to the signal processing process so uh because of that uh there are systematic errors that are sometimes harder to to to uh to fix and again by sequencing to high cop depth coverage you can fix some of the problem but often a good practice is actually you will combine the minnayang data with the ilumna data and use the high quality ilumna data to correct uh the sequencing errors in the minnayang data but effectively that doubles will triples your cost per genome if you if you do it that way okay so after assembly uh there are often still gaps in the genome so you are not getting the one piece uh in uh genome back um so we call that each of these fragments can take these contiguous sequences a contek so i just wanted to introduce the the term there so we're all on the same page and the context is likely to represent in in most of the cases 97 plus of the genome but you're still likely to miss some of the repetitive regions and um and the process to close the gaps or what's called a finishing approach it's often time consuming and labor intensive because the in the old way of doing it is actually design PCR primers and walk the gap to try to close it uh the new way of as i mentioned is how you use longer reads to try to span these repetitive regions and um some of the well-studied pathogens is now quite easy to uh to get the full genome if you combine both sequencing uh technologies right so the next topic is on genome annotation so you get a string of ATC and G's back from your sequencing providers and there's really not much information attached to or there's no without the annotation process there's not much information attached to the the sequence so the goal of annotation is to identify features such as genes or non-coding regions such as different RNA molecules or RNA genes in the in the genome so typically want to find out the functions of the the features and also the location of where these features are in in your chromosomal or in your genomic sequence okay so uh the location of coding genes at least for bacteria and viruses are easier to to detect then and to figure out then um you carry out and the sequences can often be identified through this up initial gene prediction approach and this algorithm essentially works on the fact that the coding regions have different uh base frequencies compared to non-coding regions and if you train the software to recognize these words or these different coding frequencies it can actually pretty reliably predict these regions for you in in the in the context so this is a fairly well resolved problem in the sensitivity at least for bacterial genomes is somewhere in the 97 98 percent of the genes can be detected this way so this sort of quickly summarized the the annotation process you have a a contact that you carry out some regional annotation and detect both the protein coding genes and the non-coding genes such as RNA tRNA RNA tRNA and other potentially small RNAs and in promotion sort of promoter regions so on and so forth and the protein coding genes can be detected through that up initial gene prediction method as I mentioned and you then can annotate the functions of these genes by comparing to a reference database of known uh genes of known functions and this can be done automatically as as what you'll see in in the workshop and the manual annotation can be done as an option to improve the quality of your annotation so the most common way to do functional prediction is through sequence similarity search so I mentioned this is assuming that the genes that have sequence similarity are derived from the same ancestral gene and therefore they have similar functions so the most well-known similarity search tool is called BLAST so how many people have used BLAST before just wanted to make sure so everyone okay so the so you know how it works basically you're taking your sequence search against a database of sequences that have already annotation assigned to it so what you have to keep in mind is that that process creates what's called transitive annotation in other words you are you haven't done any sort of biochemical or functional characterization of your gene you're simply determining the function of that gene or protein based on the similarity of that genome protein to a known protein with gene so that process can often create issues such as that your your gene may be a may have actually have a different function than what's in the database due to a small mutation but it's still BLAST 2 99 percent identity but and this is quite common in some of the AMR determinants that small mutations can can have a huge effect on the function of the gene so have to be careful when you're carrying out transitive annotations okay so I think I can skip this slide but maybe just to say that it is one of the most cited bioinformatic tools and the reason is so popular is that there's actually a statistical framework that was described around the BLAST search so it's easier to predict the behavior of BLAST search results and to interpret the BLAST search result compared to some of the more heuristic approaches that are that does not have a statistical framework around it and it then becomes harder to to interpret some of the the results of some of other tools so the different versions of BLAST searches different combination of nucleotide and protein searches sure people are familiar with that now so in terms of interpreting the BLAST search results the big as I mentioned it's based on the statistical framework so there's the concept of expected value and the expected value essentially is the number that the number of well just read the number of BLAST alignments was given scores that I expected to be seen simply due to chance so does everyone know whether does anyone can anyone put that in a different word or does that sentence make sense to to everyone yeah show show of how many people can understand what that sentence most half of you okay so I just I like so analogy like typically give is just if you walk on the street and see someone that look like you what's the probability like that someone that look like you is actually related to you right so if you have a like maybe some of you have a face that's much more more common and aside from the fact that all humans are related to a certain degree and certainly all proteins are related to a certain degree we want we do want to figure out which ones are more closely related and which ones are which one looks similar just by chance so the E value gives a statistical based interpretation of the likelihood of finding a match purely due to chance in other words two unrelated sequences have similarities purely by chance so the rule sum is that the BLAST alignment with expectation expected score greater than 0.1 percent sorry not percent forget what I just said greater than 0.01 this is I say it's a number not a percent should typically not be trusted unless your sequences is really short and you have high identity to to the BLAST database that you're using so the gray zone is 0.01 to about to about one this you have to interpret accordingly as I mentioned if your sequences is very short and because the the expected value and scores are related but the scores are based on factors such as the alignment lens and or expect values adjusted based on the complexity of the sequences and the probability of finding a match in the database so a combination of percent identity which would be reflected in the score and expected value should can both be taken into consideration when you interpret the the results so there are quite a few automated annotation system that use BLAST and other type of similarity search tools that potentially a lot much a lot faster but BLAST is still considered the gold standard in terms of sensitivity but if you have to search thousands sometimes you know a metagenomic data say with hundreds of thousands of genes they potentially need a faster method than BLAST to search and I think Fiona will mention some of that in her talk but the idea of finding similarities of your query sequence in the database record is the same and in this workshop we will actually use this PROCA which is built into analysis platform that I'll talk a bit to do automated annotation of your genome but others are available as well that you can look into so once you have the the genomes you can do compare the genomic analysis and the goal of such analysis is to identify genomic variants that can be correlated to phenotypic or characteristic features of the an organism so for example might be interested in identifying a more virulent strain of a pathogen so by looking for certain toxins or by looking for non-virulence factors or looking for antibiotic resistant genes and we can also use the variants to track transmissions of the pathogen as I mentioned already. At a high level the variants can occur at three different levels one is regional this is due to for example horizontal gene transfer acquiring a genomic island or recombination events or reshuffling of of of viral genomes and so on so this can result in strain specific regions or segments of genome that are different from strain to strain and gene based analysis typically focus on strain specific genes so by doing gene by gene comparison looking at the allylic differences and this is the topic of module three this afternoon and as a single nucleotide based analysis looking for variations at the at the base level and this will be covered in the next session so comparative genomics is not a new concept ever since the first genome was published in 95 soon after that comparative analysis was already carried out and this actually highlights some of the consistent observations of microbial genomes that really improved our understanding of bacterial evolution and as you can see in this comparison just two strains of H. pylori that strain specific genes typically cluster in so-called hypervariable regions and sometimes go genomic islands as you can see there's denoted by these blue regions and this observation as I mentioned occurred over and over in other genomes as well and leading to a lot of studies about genomic evolution by horizontal gene transfer and so on and I think Phil now would also talk more about that in her lecture and extending comparative genomics to the concept of pen genome so the large number of genomic variations means that you typically have the sequence and large number of genomes to really understand the bacterial species and some species might never have enough genomes that can allow you to thoroughly characterize the species so the term was first coined in 2005 based on the study of six ruby strap genomes and what they define pen genome is the theoretical entire gene set of a species and they break down into two broad categories the core genome which are consistently found in members of that species and the strain specific or accessory genes that are found sporadically in in the different strains of of that species so the idea is that the core would be more stable and the strength strength specific or the lifestyle genes would would be more fluid and in over time so the pen genome calculation then is extrapolation of the observed observed number of genes based on your limited number of genomes to the theoretical number of genes that are required to fully capture the pen genome of the species and as I mentioned some organisms you'll not you'll not saturate its gene content so that's called an open pen genome so you can see some of these that would these lines will never reach at least at least not in the reasonable time reaching zero meaning that you'll continue to discover new genes as you sequence more genomes of that given species so this is sort of as you add one additional genomes how many new how many new genes are you discovering so the number goes down and for close pen genomes the number can go down very quickly so in in the case of anthrax the the number of genomes need to sequence to characterize that organism is is quite low and partly it's a because it's a it's a highly it's a a newly invert in emerged pathogen relatively speaking still like a few millennium you know years old but but the the number of genomes required to describe it is it's trackable and this as you mentioned that this concept helped the microbiologists to predict how species will evolve over time whether it has an open pen genome and and have a lot of horizontal gene transfer events where in a lot of genomic changes where it has a close pen genome meaning that it's a more stable genome and this information is helpful in terms of interpreting your genomic epi results as well so as I mentioned pretty much everyone in the in the in the faculty faculty is part of this era that consortium and a few years ago Gary Fiona and I wrote a grant to genome Canada to build a analysis platform for genomic epidemiology that's that's tailored to public health agencies and public health workers who lock the tools to process to process genomic data and the idea is to have a free open source standard compliant and high quality genomic epidemiology analysis platform to support real-time disease outbreak investigations and the core functions of the platform include managing of the users managing of the projects and streams but also building workflows that allow you to characterize these sequences and there's also some visualization tools built in to allow you to visualize the trees and looking at the looking at the analytical results it's still an active project in in development and a lot of the core platform development happened in in at the NML in Gary's group but Fiona's group also hosts a public instance at this assignment phraser university that will create actually account for all of you and you'll use some of that functionalities tomorrow in our integrated assignment so I mentioned the goal is to build a platform that's that's easy enough to use for non-bioinformatic specialists so mentioned it's a it's a partnership of a lot of between both provincial and provincial and federal public health agencies but also with a lot of academic partners and the slides it's slightly out of date but if you go to our website there's a long list of partners involved and the project teams what's unique about the area that partnerships are the project teams such as Gary and I actually embedded in the together in the user organization so we try to build tools that can tailor to to the public health microbiology and take feedback from the users to improve the process but as I mentioned we're opening up this platform actually has been open the whole time but we're making it more readily available through this public instance so there's a few so others can can use it but also more importantly if you're able to contribute to the to the development we're also happy to to engage you for that as well so the website as I mentioned is area.ca where you can download install your own copy you can use a public instance or you can read up on the documentation and so on okay so it's designed to be user-friendly you'll see this interface tomorrow basically once you loaded your data you just have to click this big red button to to launch analysis there's not much configuration needed to to do fairly complex to to run fairly complex workflow and you'll also have you'll also be able to combine the metadata with the with your sequence analysis results okay we're coming up to the hour so I'll just go through this last bit fairly quickly so switching gear into the the metadata component so there are many players involved in infectious disease surveillance and outbreak investigation however because of privacy and confidentiality concerns the information tend to be aggregated at the the information collected at the the front line labs or the front line agency investigation by the investigators typically are aggregated and lost as we move from the the front line labs to the more centralized the board centralized resource but the bound for mac expertise on the other hand typically start developed in a more resource rich environments such as a national lab and it's slow to propagate down to the the front line lab and that's probably why the era that platform was developed to try to equilibrate this process but also it highlights the importance of how we can share the information across different players so also to mention that different uh agencies have different um analysis platforms and this is like trying to of you know fit a plug into the wall when you have uh different uh standards right so this shit yeah so there's a lot of retrofitting of of data into the various standards and and dealing with when we deal with data harmonization issues and that's to say that the contextual information is often collected in institutional specific format there's different codes or acronym used in different systems there's our terminologies that describe the same concept but have different names and there's different units of measurements different um here's an example of different severity grade used by the american hospital association versus the uk uh the nhs and as you can see the uk have much uh more finer gradients compared to the the aha aha uh standard so how do you fit all these descriptors in uh a single uh gradient and it can be challenging and certainly difficult for software to understand such nuance another issue of course is metadata often quite dirty people are typing in the information there's a lot of spelling errors uh there's synonyms used uh there's issues with the semantics uh used such as grammatically incorrect terms and so so uh uh the um for example here is the word cloud describing of different terms describing um essentially feces so the bigger the more common terms are in larger fonts than the smaller ones but you can see it's it's quite a mess so um ontology is um the idea of ontology is that you can use it as a as a universal converter that allow better inter interoperability and better harmonization of the data set through a mapping process so ontology is a mechanism to study to specify a body of knowledge to to specify an area of study and essentially involves standardized well-defined hierarchy of terms that have a relationship describing their how these terms are related to each other there's definitions for each term and also each of the terms has a unique universal id so you're no longer bound by the actual words used to describe the the concept whether there's an universal id assigned to each of the concept so it allows you to use different synonyms for the same concept and uh the terms are expressed in formats are both human and computer readable so um you so through the through this approach you can improve the uh interoperability of of data sets and this is the area of research that are quite active in in my group uh so the idea is for example you can uh take these terms and then start mapping them to existing or uh to ontologies and that the different terms can be mapped to the same id or uh or even the same word if they have two different usage could be mapped to different id but you're essentially expressing these terms in a in a more um systematic way and a more formalized way so and also so I mentioned each of the terms then have a clear definition and the different ontologies are meant to be interoperable too so you can reuse terms from one ontology in in another ontology so so uh my group was in in collaboration with with Fiona and Andrew uh had in the last few years developed a application ontology that's specifically useful for uh genomic epidemiology investigations and so we try to come through epi lab and genomic and clinical data type uh data types and data fields and come up with consistent descriptors for for these terms and and uh use these terms to describe then use the terms to describe the activities because we're interested in foodborne infections we also started a kind of interesting project that tried to come up with food descriptors from farm to fork activity so for food products for food processing uh steps and for preservation method and of course this has applications beyond just infectious disease application so we have uh partners are interested in using this for to describe their nutritional studies we have partners interested in using this to uh uh describe the the food web for environmental studies and so on so again the idea of being able to develop a knowledge framework that has wide applications and help to bring these different applications uh the data from these different applications in a harmonized way it's a it's a um interesting area for for research so this requires to develop an ecosystem uh which uh i shall i'll skip this slide but the idea is that uh the ontology self if no one uses it it's not useful so we need to develop tools that would enable people uh to reuse the ontology so uh one of the tools that we developed is called genomic epidemiology entity mart and this is trying to address the problem that people typically develop their data specifications in silos resulting in slightly incompatible specifications even though they're working in the same area so the idea is to be able to come up with a platform that allow people to publish their data specification but also to reuse ontology terms when they build their data specification so the different data specification can be um more interoperable to each other but it also doesn't force people to adopt the same data specification the other function built in is to improve the data consistencies by enforcing some data value constraints directly as part of your data specification so you cannot type in random or erroneous text in in or information in the field in a data field and to make a user friendly we create a web interface that allow people to shop for terms and develop their data specification so there's a link there it's still work in progress as well but we're hoping to wrap this up later this year the other issue that we try to deal with is that there are a lot of inconsistent terms in existing databases so but then to manually map them would be a labor-intensive process so we try to develop an automated way method to map the database short phrases that you found in the databases so the two different ontological terms and in the process it will clean up the inconsistencies in in these terms and identify and map the corresponding uh the the terms to the corresponding ontology um so this sort of outline all the steps that uh the system or the yeah the system that we call lexmapper go through to clean up the data so just to summarize the idea is to be able to draw your metadata descriptors from a body of ontologies that then can be published in genes and will clean that by lexmapper that then so you have a consistent descriptors for your metadata and then you bring that together with standardized sequence analysis workflow and both of that is critical for downstream uh genomic epidemiology analysis here i put here that but it could be any of the of analysis platform that you use the idea is still the same that you want to have both clean or well processed sequence data and metadata for your analysis um i think i actually did cover most of the the modules in my introduction so i'll skip given the time i'll skip these um so this is just highlighting each of the modules uh okay sorry it took longer than than expected but any questions