Hello everybody, welcome back to esoteric Atlanta. Of course, my name is Bryce I hope I can remember how to do this. It's been a while Since I filmed because I was traveling and a lot of the videos that you saw last week were pre-recorded videos My voice is kind of going out a little bit But nonetheless, we will we will continue continue for hits forth go forward with art with our work today It is Sunday I am pre-recording this for Monday for tomorrow and just a little side note before we get into the sixth tablet If you are somebody that is joining me on Sundays at sacred garden yoga for the primary series lead class If you're joining me on zoom that is We are going to start to work through the full primary series If you are brand new to yoga then this is obviously something that might be a little much much for you Just because it's primary series does not mean that it's Primary it's just the primary series because it's yoga to keep that physical therapy and we've been doing half primary series We're the first half for a very long time and we're starting to add in the second half of it Which is some advanced postures to like like behind-the-head posture all sorts of stuff So if that's something you're interested interested in I am going to put a link to my friend Morgan his His YouTube page us on the nurse and his full primary series You guys are probably familiar by now with his half primary series But he has a very beautiful full primary series that I'm going to put a link below so that you can see what the full primary series looks like Because we are going to start working the full primary series And this is why I tell you guys it takes about ten years to work through each series So don't beat yourself up with that being said. All right. I will also on top of that I will be putting a link to sacred garden yoga in case you're like, what is this? She's talking about it. No, I could practice with her every week. Yes You can practice with me every week. You can practice with me on Sunday mornings at sacred garden yoga I have a live class that happens there. The class is usually pretty packed But now we're offering zoom options so you can zoom in and practice with me and all my Local students on Sunday mornings that I would love to have you if if that's something you're interested in All right, so we're gonna get into tablet six today. I'm excited about this tablet I haven't read it yet, but it's titled the key of magic What I think we're gonna do this is what we did last time on Aquarius Rising Africa is I'm gonna read through first I'm gonna read through thoughts Writing and then we're gonna go back and look at Doriel's commentary. Once again, I am using Doriel's commentary There's many commentaries on the emerald tablets as always Please get your own copy if you can because it's super important that you have your own information And you're not just like going off of what I'm saying or what other people are saying But you actually have the resource yourself to come to your own inclusions and have your own perception of what thought is saying All right, so let's get started with Tablet number six the key of magic So this is thought speaking Hark ye o man to the wisdom of magic hark to the knowledge of powers forgotten Long long ago in a days of the first man Warfare began between darkness and light men than is now were filled with both darkness and light and while some Darkness held sway and others light field filled the soul and so again He's he's referring to what happened with the fall of Atlantis And if this is your first time listening to this series on my channel welcome So glad you're here, but you might want to go back and I will put all the other Videos of the emerald tablets down in the description box below you might want to start at the beginning Because each tablet is very fascinating because he kind of goes back and forth He talks about like what what happened was like what happened when Atlantis fell what what we were as Atlanteans and Then like tablet 3 he basically is teaching you like how to be a good person like don't gossip Don't do all these things keep your vibration high and now we're gonna go Now we're seems like we're leading into like also what we knew in Atlantis that we don't know now and as you guys know If you've been around the last table the fifth table of the Dweller O'bunall has been like my favorite so far because well It was so mysterious and I think we're starting to understand more stuff now because we know our geography isn't what they say it is So and what are you saying here with the darkness and the light? That's the power struggle, right? That's the friction. That's the polarity of third density as if you follow the law one You understand that polarity of The third density which brings us to the the density of choice. You have to make a choice I had somebody asked me in the comment section about eating meat like why is it okay that animals eat meat? But we're not supposed to well because animals are second density They're not in the realm of choice. You are you are a higher considered a higher not not higher consciousness in the in the Realm that you are any better than the animals No, miss do not mistake that you are not any better than the animals as far as consciousness You would just have a deeper understanding because you've harvested to third density So now you have to make choices animals are not in the density of choice Second density is not the density of choice, right? Next when they do get into third density when they do take on a human body, then they will be in choice as well So you humans everything you do is either for the light or for the dark, right? So there are laws of forgiveness for example if you're out walking around the field So you're gonna step on some bugs that you didn't know were there, right? You didn't kill the bug intentionally, right? So so that's a law of forgiveness.  You are actively choosing the darkness now This does not mean that you're going to graduate into service to self. You have to be like 96% Service to self and you have to be for the light not 51% service to others So you can have some fuck-ups here and there like, you know You can have you can have some some some mess subs But the more you start to understand choice the more you want to hopefully make better choices, right? What's what some what's gonna serve humanity to its highest consciousness, right? We also know for the law of one that martyrdom is a negative trait like to sacrifice yourself For for anything else is considered demonic, right? Which we know we see that with what they do and the Luciferian side is they're constantly doing sacrifices, right? And eating flesh and all that kind of stuff, which is what they do in the churches, too So that's all demonic. That's all satanic Which is if that's the path you want to go on then you're gonna pick to go on that path, right? but if you want to go into the light you're gonna focus on Your karma on Dharma on on being you know the one of the Bible verses that I like Do you want to others as you would have them do one to you if you were a cow?  Like put yourself in that shoes because you have the ability to do that, right? So that's that's the difference and yeah, so We know that with Atlantis when Atlantis fell it was a huge template of graduating to darkness and light like there's a huge light and dark happening polarity choice and we understand that our Ancestors are we when we were there ourselves that we understood more about life about consciousness about magic, right? that we are magic and This this friction started to get really intense between the darkness and light just like we're seeing right now in our world, right? And Atlantis the darkness actually from what I understand took over and that's why it flooded Yeah, because even in the law of one they'll tell you service to self only goes up to a certain density And then they have to start all over again at the bottom to eventually merge into the light because you can't you can't it all implodes on itself, right? You can't go that far and service to self. Yeah, so anyway Let me start that over again because that was super interesting Harky Oh man to the wisdom of magic Harky to the knowledge of powers forgotten Long long ago in the days of the first man warfare began between darkness and light Men that is now were filled with both darkness and light and while some darkness held sway and others light filled the soul I Age old is this warfare The eternal struggle between darkness and light Fiercely it is fought all through the ages using strange powers hidden to man Adept have there been filled with blackness Struggling always against the light, but others there are who filled with brightness have ever conquered the darkness of night Where ever he may be in all ages and planes surely you shall know the battle with night long ages ago The sons of the morning descending found the world filled with night There in that past time began the struggle the age old battle of darkness light and again We talked about this to the macro and the micro You know with the shadow work challenge you we all have a shadow side I posted in my community tab a couple days ago of a psychiatrist from a reality show who gave an excellent Breakdown commentary on shadow work and how necessary shadow work is if you don't tend to your shadow side If you don't acknowledge the sides of you that are low vibrational the jealousy the abandonment the betrayal trauma all that stuff If you try to hide that it's just going to intensify What we resist will persist and we'll get more powerful But when we're able to acknowledge our triggers and bring them to the light and work through them Then we brought them to the light and what's the one thing that can get rid of darkness? It's light You go into a dark room you flick the switch on it doesn't like it's not like it slowly gets light It immediately gets light, right? So that's why shadow work is so important That's why it's good to be triggered if you're triggered by something you people do this to me all the time They get all triggered and they get a comment section. They were real abusive in the comment section It's not my trigger. It's not my fault. You're triggered. It's you You know we see that in this truth or community Where we get all upset because our friends and our families are asleep We're not upset because they're asleep What's being triggered in us as parts of us that are still asleep that our souls telling us to pay attention to? Because everything is a mirror and so when he's talking about for some in the light They were able to walk forth towards the darkness and turn it to light because they did their own shadow work If you ignore it is not going anywhere. You're just gonna have to deal with it next life, right? That's that's all karma is karma is just cause and effect. It's just your work all right Many in that time were so filled with darkness that only fleably flamed the light from the night some there were month Some there were masters of darkness who sought to fill all with their darkness sought to draw others into their night Fiercely withstood they the masters of brightness fiercely they from the darkness of night sought they ever to tighten the feathers The chains that bite man to the darkness of night use they always dark magic Brought into man the power of darkness magic that enshroud man's soul with darkness We know all about black magic on this channel. Listen Listen, you do not want to mess with black magic. Okay? First of all any type of magic light or black has to come through you first as a conduit So it's never gonna work out for you It might work out for you for a hot second But you're constantly gonna be having to serve that right where light magic is not that way You can do something good. You can help others and then it's done, right? and the thing about Harvesting negative so the people that want to go to a negative polarity if they can get you if they can manipulate your energy Then that makes them stronger and the negatives That is why I keep telling you guys 90% of the truthers that you watch on YouTube are part of the cabal They're purposely infiltrating you because they can feed off of you and That makes them stronger on the negative side So we have to have discernment so I have to do your own research So they don't fall for it alright Banded together and as order brothers of darkness they through the ages antagonists they to the children of men walk they always secret and hidden Found yet not found by the children of men forever. They walked and worked in darkness hiding from the light in darkness of night And what's a great way to hide in darkness? Infiltrate the light remember darkness can't create anything only the light can't so what does the darkness do it mimics the light? Or it steals from the light and verts it Again, we see that in our own community Silently secretly use they their power and slaving and binding the souls of men Exactly boom boom boom boom boom. I'm telling you guys these like truthers that you think are truthers You're out there parroting everything they say When you haven't done your own research and they're just binding your soul to the darkness and you're allowing it because you're Consenting and you're just repeating what they're saying and here they talked about Brothers of darkness There's a Luciferian brotherhood too Most of the 90% of truthers are part of it right Maybe about the French Revolution you guys like This is history repeating itself when the French Revolution started it started as a good thing the people against the 1% They wanted their their own sovereignty back Well, what happened was that the controllers got involved with the revolution and turned it around and ended up beheading The people that started the revolution and they ended up giving the country back to the controllers That's happening in this community, too Right, we talk about in Cale's Remind control, right? The controllers are here and we look at the other side of this the people on the left that the normies as they're called and We see them. Oh bless their hearts. They're being totally brainwashed by the media by mainstream media Well, so are you? It's just your mainstream media is now YouTube.  I don't care. They're infiltrators. I know that What do you think? Where's your research? Take your power back? Because you're just having your soul bind it over here in the YouTube land, which is another form of media Where you're being mind-controlled Just like what happened at the French Revolution Come on like wake up wake up, right? All right Unseen they come and unseen they go man and his ignorance calls them from below Dark in the way of the dark brothers travel dark with a darkness not of the night travel or earth They walk through man's dreams powers They have gained from the darkness around them to call other dwellers from out of their place in ways that are dark and unseen By man into man's mind space reach the dark brothers around it. They close the veil of their night There through its lifetime the soul dwells in bondage bound by the feathers the veil of the night Mighty are they in the hidden forbidden knowledge forbidden because it is one with the night Don't think that needs any commentary Hark you man enlist to my warning be free from the bondage of night Surrender not your soul to the brothers of darkness. Don't give your soul over to truthers Don't even quote me do we that's why I'm telling you get this is why I read this live to you guys It's right here. You can get your own copy Don't even quote me. I'm just researching like you should be research research for yourself Right Don't give your soul over To these people on YouTube that you don't know Who are paid by the three letter agency to derail you? Thank you thought thank you thought for for bringing this up keep thy face ever turned towards the light No, you not a man that your sorrow only has come through the veil of night. I Man heed ye my warning strive ever upward turn your soul towards the light the brothers of darkness seek for their brothers for those Who have traveled the pathway of light for well No, they that those who have traveled fall far towards the son of their pathway of light have great and yet greater power to Bind the darkness with the children of light black magic.  I'm not going to until I can They know things Don't think they don't know things. They know how to use things that you don't know how to use The only thing you can do is stand up for yourself and say I do not consent and I revoke any permission You think you have To use my wounds as an entry point You got to go and if they break that consent, then they've got a higher power They're gonna have to deal with far beyond this plane of existence Lisio man to he who comes to you but way in the balance of his words be of light for many They they are who walk in dark brightness yet are not the children of light, right? They're mimicking They're mimicking the light There are there are a lot of Divination people on the truth or community. I love divination, but they are not divining for the good They are mimicking the light to try to suck you in and spell past you into following them It's like the Pied Piper, right? Go follow the Pied Piper. That's what they're doing. That's what they're paid to do Easy it is to follow their pathway easy to follow the path they lead but yet Oh man heed my warning light comes only to him who strives Hard is the pathway that leads to wisdom. Hard is the pathway that leads to light. Yes. It takes effort. It takes I Mean, hello. Thank you thought like this is a huge part of this channel.  It's never been for my own entertainment I'm there to work. I'm up at two o'clock in the morning for Brahma, Martha.  You have to have friction. You have to have opposing forces You have to be uncomfortable. You will not progress in spirituality Until you get uncomfortable Until you feel that need to want to run and but you but instead of running you stay Right So thank you thought thank you for Reiterating everything we've been saying on this channel and Shanti has been saying and tomorrow has been saying all these true Spiritualists have been saying the fake spiritualists are out there telling you don't worry about it Sit back. It's just a movie. The white hats have control.  And by working on yourself, I don't mean getting manicures every day I mean allowing yourself to get triggered and then going in and leaning into that before you projected onto someone else so that you Could heal you so you can work. You're on your own karma. Yeah All right Many shall you find the stones in your pathway many the mountains to climb towards the light yet No, you oh man to him that comrade Free will be of a pathway of light follow you not the dark brothers ever always be a child of light for you For no ye. Oh man in the end light must conquer and darkness and night be banished from light Listen, oh man, and heed either wisdom even as darkness So is the light when the darkness is banished and all veils are rendered out there shall flash from the darkness the light Even exist among them dark brothers so there exists the brothers of light Antagonist they of the brothers of darkness seeking to free men from the night Powers have they mighty and potent know the law the planets obey Work they ever in harmony and order bring man's soul from its bondage of night secret and hidden walk They also know not are they to the children of men yet? Know that ever they walk with thee showing the way of the children of men for for half They fought the dark brothers conquered and conquering time without in yet always light shall be the end Master driving away the darkness of night That gives me chill bumps. I Man know ye this knowing always beside thee walk the children of light Master they have a sunpower ever unseen yet the guardians of men open to all is their pathway open To he who will walk in the light? Free are they of dark a mentee free of the halls where light reigns supreme sons Are they in lords of mourning children of light to shine among men like man? They are they and yet are unalike never divided were they in the past one had they been in oneness eternal Throughout all space at the beginning of time up. Did they come in oneness with the all one up from the first space? Formed and unformed kind of is again mirroring what the law of one says, right? And yeah, there are there are always beings around us. We know that they're always Guides trying to help us the difference between the children of light the children of darkness Is that the children of light very much respect consent children of darkness not so much, right? So you have to ask for their help Given to man have they secrets that shall guard and protect him from all harm He who would travel the path of the master free must be from the bondage of night Conquer must be the formless and shapeless conquer must he the phantom of fear knowing must he gain all the secrets Travel the pathway that leads through darkness yet ever before him keep the light of his goal Obstacles great shall he meet in the pathway yet press on to the light of the Sun Here you oh, man. The Sun is the symbol of the light that shines at the end of thy road Now to the I give the secrets how to meet the dark power meet and conquer the fear from the night Only by knowing can you conquer only by knowing can ye have light? That's giving me chill box you guys I This last week I got heavily attacked again like heavily And a lot of people had to step in and help because I was bumped and bruised and scratched and it was awful And I was at the Sarah garden this morning preparing for class and I was working on my journaling before people came in and Cindy's back garden the Shala kind of faces of that garden. She always has birds back there And they're usually like cardinals redbirds Bluebirds sometimes, but I walked back there and for the first time in like over ten years that I've worked there I saw an American gold gold finger. I think it's called. I had to look it up. It was a bright yellow bird I'd never seen anything like it so I immediately looked it up and then I looked up its spiritual meaning and it basically was like the abundance of the Sun and creativity and how the Sun brings light and It's crazy like I'm getting chill bumps reading this because I knew I was planning on doing this this afternoon when I got finished Teaching bird for Monday for Monday's reading There's obviously a dog outside. My dog is probably barking out, but the fact that there was a golden American gold finger outside bright yellow bird and they're they're talking about the light. I don't know It's not lost on me. I know I might sound weird coming out of my mouth verbalizing it, but I see you spirit. I see you Now I give unto thee the knowledge known to the masters the knowing that conquers all the dark fears use this the wisdom I give thee masters that shall be of the brothers of night When unto thee there comes a feeling drawing be nearer to the dark gate examine thy heart and find if the feeling thou hast has come from within If thou shalt find the darkness thine own thoughts and banish them forth in place of thy mind Sin through the body a wave of vibration irregular first and regular second Repeating time after time until free Start with the wave force in the brain center Directed in the ways from thine head to thy foot. We do this in yoga. We talk about feeling the vibe. I see you spirit But if thou findest that heart is not darkened be sure that a force is directed to thee Only by knowing can now overcome it only by wisdom can now hope to be free. Yes, knowledge is power and knowledge protects Knowledge brings wisdom and wisdom is power attain and ye shall have power or all Seek ye first a place bound in darkness a place ye a circle about thee stand direct in the middle of that circle Use thy this formula and thou shalt be free raise thou thine hand to the dark space above thee Close thou thine eyes and draw on the light call the spirit of light through the space time Using these words and now shall be free Fill thou my body. Oh spirit of life Fill thou my body with spirit of light come from the flower That shines through the darkness Crawl from the halls where the seven lords rule Name them by name I the seven three four five and six seven eight nine By their names. I call them to aid me free me and save me from the darkness of night Untanas for toss Chaitel and Goyana her tall Simvata are doll and by their names. I implore thee free me from darkness and fill me with light And we know the said that's the chakras, right? So it's it's like they're having you. I was trying to picture this in my head And I probably will start doing this first thing tomorrow morning before my practice create a circle around you stand Close your eyes feel the light coming up asking for the light through Shoshuna Right back of the spine where the chakras line up through Shoshuna coming up through the pelvic floor up through Through the back up through the top head You guys start doing that too. Let me know what happens. Well, we'll run a little experiment No, ye Oman that when you have done this ye shall be free from the feathers that bind ye cast off the bondage of the brothers of Night see not the names have the power to free the vibrations that feathers that bind use them at knee to free Thou thine brother so that he too may come forth from the night Thou Oman aren't thy brother's helper. Let him not lie in the bondage of night Now unto thee I give my magic take it and dwell in the pathway of light light unto thee life unto thee Sun made out be on the cycle above All right, so that's what thought has to say. Let's go back and look at Doriel commentary. This is powerful stuff, eh? So Doriel says for Tablet six the key of magic in this tablet thought speaks of magic using the term to denote the usage of Developed powers in the warfare between the order and disorder. So order would be the light disorder would be the darkness This warfare has continued to the fall of man in the first cycle and will continue until the cosmic consciousness is ready to pass through Sunthal, so I think he says Sunthal or Sunthal SU NTL is not translated I have a feeling that means like the Ascension like if you remember you go back to the very first tablet thought is like Yo, I'm writing this for you guys now meaning us right now, right because we're about to ascend Yeah, we have to harvesting going to harvest are happening right now. We've got the negative harvest and the positive harvest That's why it's so crazy, right? So we're both sides are harvesting to go through Sunthal once we get to fourth density We will not be in a polarity of choice anymore. We will not be in a polarity of opposing forces anymore I don't know what that's gonna look like, but that's what the law of one says so that makes sense There are adepts who use the great powers the cosmos for destruction instead of construction For law operates either for good or evil positive or negative Those adepts who use cosmic force for destruction where the dark brothers black magicians who fought against the children of light They attempted to hold back and pull back those from the children of light We're trying to bring into the light still still doing that. They're still doing that. It's interesting If you guys remember years ago, we were going to the Dead Sea Scrolls the Essenes were called what what were the Essenes called? The children of light and what do we know the Essenes are? They're the Egyptians the priests and priests of Isis Isis back then was spelled ESSE so another connection The black brotherhood is the antithesis of the white brotherhood one destroys the other binds the black brotherhood has an Organization known today as the black Duke pass The adepts have their cellas and the white adepts have theirs They pattern their organization after that of the white lodge and often deceive men into thinking they are of the white lodge They help men to gain certain things and powers until they have them in their toils Yep, you know all about this Then when there is no escape, they clamp down they have certain specific powers developed such as opening The seventh dimension and calling in elementals to fulfill their purpose Remember demons are called upon by witches and warlocks of the black of the darkness Demons are not there by choice They were fallen angels that were taken and basically chained to do both the deans of dark ones So as I said before I don't it's not the spiritual world. I fear it's humans that I fear They have a power of mind control through a transference and hypnosis MK ultra mind control. We see that in a lot of truth. There's two There's some great videos on rumble about certain truthers and the words they use to try to hypnotize you and put you into a trance So thank you thought Through this they gain control of the mind and lead it to disorder if one surrenders to the black brotherhood and signs His name in their book. He is bound to them during that incarnation. Don't sign your name in old book guys Man's soul must not be bound if it desires to advance in light surrender to the dark forces entails the shutting off of light Only through darkness and disorder is man bound to the flesh. Therefore. He should become light and ordered. Yes That's what's in the oak sutras to like Man suffering comes from the idea that who man thinks he is is not really who he is We think we are our bodies. We think we are our identities in this world. That's just an avatar. That's just the ego That's the fault since itself.  That's how they get these people who were once good Get handled get sucked into the darkness because they put their egos. They blow their egos up, right?  That's it. That's it Once you once your body dies once Bryce dies, that's it Bryce is dead the night my soul moves into another avatar Which is gonna be totally different, right?   It's not gonna feel good. It's gonna be hard But that's the first step because then you start to understand that your body is amazing It's wonderful. It's beautiful. It has all these secrets and all these things about you and within it But it's not you. It's just a tool, right? That makes sense Okay The Black Brotherhood always tries to pull down the person who has gained development among the path of light. Yes Yes, but they have already developed powers. Yes. Yes. Well one says this to the Cassiopeia and saved this to That's why I say to all the black magicians out there that have been attacking me non-stop Why thank you you are proving me right because if everything I was saying was bullshit You would not attack me But the fact that you've put multiple death spells on me the fact that I have Pictures of bumps and bruises and scratches and people in my own personal life have seen this happen just proves That I'm on the right path that I know what I'm talking about So thank you Thank you For proving me rewrite Thank you for stealing all of my money Thank you for putting all those poverty spells to laugh. You see something about the darkness too Darkness doesn't understand love like true love or loyalty or anything like that, you know, they don't understand that And so when you are under heavy heavy annihilation or attack from a coven of the Dark Brotherhood and They're taking your money. They bring death spells on you all these things are happening And I can't I can't get into everything that's happened one day I'll write a book about it.   You're intimate life who are also of the light and Even though they've done stuff to me. That's horrific. My life really hasn't been altered Because I have people in my life that love me. I have amazing friends. I have an amazing family.  I'm never gonna be homeless. I'm never gonna be without because I have friends. I Understand love and so do they that's something the darkness doesn't know. I don't have to do black magic and spells To have people like me. I've never done that Never The people in my life, especially the ones that have been in my life since I was like in kindergarten Never had to do spell work. Are you kidding me?   We talked about the organic portals and more organic portals get sent your way Right for trying to derail you Just know that the more you the more you awaken the more you enlighten The more attacks you're gonna get It is for this reason of the person who was highly developed has to withstand more than the person of little or no development boom The more so I guess I just say thank you coven for showing people that I'm a highly developed You're doing me a favor. All right. I'm joking guys. Why me I'm not really joking, but that's a joke The more one has learned of light paradoxically the more he knows about Manipulation of disorder and the more valuable he is to the Black Brotherhood. Yep The development of reason and balance is necessary so that we can separate darkness from light order from disorder The words of those who come to us only through the overcoming obstacles and continual striving will the goal be obtained in Oppositions the Black Brotherhood stands the white lodge Striving constantly to free men from disorder and warding off the powers of the Black Brotherhood If the seeker has his real desire on light and not power The white lodge will stand between him and the Black Brotherhood for they have greater power than the Black Brotherhood Yet the Blacks are allowed. I don't like the way they will say the Black Brotherhood I don't like the way he worded that yet the Black Brotherhood are allowed to exist for they are a Form part of the darkness which man must overcome and rise above boom friction polarity. Yes They have their purpose. It provides you with friction, which is what you need. You need friction You can't you can't be of the light without friction that match has everything it needs in it to light But it cannot light until it is struck against the matchbook that my friends is friction You have to have friction So really the black the darkness the Black Brotherhood is being used Their tools basically for the light That sucks to be a tool The warfare between the forces of the Black Brotherhood the white lodge has continued since the beginning the masters and great Adepts of the white lodge use the power of the awakened son and man to guard and protect the children of light They who never lost their original oneness are also guardians of man who is their brother They are the custodians of secrets that push back the darkness and these are given to the ones who travel the way towards Mastership the ones who desire to be a master must learn mastery of the laws which regulate manifestation He must conquer fear and walk unafraid on the pathway of light as my teacher says life hearing Way fearing No fear is false evidence fearing real wife hearing The secrets of thought regarding the operations of the law of protection are offered only by knowing can you conquer I? Agree with that You must use the knowledge given otherwise it is useless many of the vibrations which seem negative are really from within your own self Not from outside conditions 100% that's your shadow side. That's your trigger Lack of mind balance often results in the arousing of such negative thoughts that we really feel as if it's outside entities or forces We're at work on us, and I will say one day again. I will show a lot of pictures one day If you're in the signal group after you've seen some of these pictures already when you are being presented with black magic attacks It's very evident It's very very very evident Okay, you have markings on you Your physical body will will sometimes look exhausted like it's it's very evident Okay, when it's your own shit most of the time.  So I want to make that very clear like It's evident when it's a black magic attack Maybe one day I can get my boyfriend to come on and talk about what he's witness with me I don't know if I'll do that though, but Yeah, it's evident All right Apply the light from reason to the disbalance and find if it is from within or without It from within start a vibrational in the pineal and send it into their irregular waves to the body That is in the first wave through count one send another wave count three another wave count two and so on after doing this For a while send it through in regular waves and then manners and waves count two Wave two and so on if upon examination you find that it is an outside force You should go into a dark room or cave and draw a circle around yourself Not closing the circle until you're within it and follow the formula is given the formula is given is Serviceably for others as well as yourself the power given may be used alright So we thought talked about that and I will say it also kind of reminds me of the muna key rights Which I I should get Cindy on here soon The the what my friend who owns sacred garden yoga because she's done the muna key rights with me because she's a Peruvian descent So she's shamanistic as well. And so she's done the muna key rights. She's the only person In my life who's ever like spit on me because they have to drink the Florida water Then spit it on you to cleanse you and I was just like give it to the girl And so and then she's but she spat I'll ever man we both start laughing But anyway, she did the muna key rights by drawing the circle and going up my chakras And so maybe I can get her to come on and talk about that because that's Peruvian So it's interesting that all of these different cultures are basically saying the same thing these ancient cultures which obviously is Atlantean So anyway guys once again join us today at 10 o'clock over on a prayers rising Africa We're going to go over this on a live show if you can't catch us for the live show that obviously is is a Eastern standard time so check that with your local time to see the difference And if you can't join us for the live show You can always catch the replay and let me know your thoughts. Let's let's continue this conversation in the comment section Let me know what you think of that and what you're learning in your journey, right? All right. Don't don't quote anybody else. I Want to what you're learning. I don't care about the other truthers I mean the ones I film with are my friends so I care about them on a friendship level But I want to hear your research. I want to hear what you found to be true what your experience is I don't want you parroting what anybody else is saying. Okay, so let me know from you. All right, you guys love you guys Hope you're having a wonderful wonderful day, and we will talk to you soon. Bye everybody


Hello, this is Professor Steven Nashaband. I'm here to help you out a little bit with looking at the nodal structure on pi subsystems and conjugated polyenes. So that's quite a mouthful. And here I'm just let me kind of lay this out for you. So we have a hydrocarbon here.  It's all actually called deca-pentine. And what I want to show you first is you can see how it's a planar molecule. It's all flat like this. And if I want to look at the molecular orbitals on it, I go here and just going to start off at the bottom and kind of point out to you that they kind of come in two families, one which looks kind of like this, where all the electron density seems to be kind of in the plane, like that one and that one. But here we get our first instance of one that is going to have what we call pi symmetry. You can see it has reflection at symmetry across the plane of the molecule. And second, it doesn't have any transverse nodes, no nodes at all moving in the up and down direction here. And let's see if we can find some more. That was the first one. I'm still moving up, still moving up. Now this is the next one. This is the next pi subsystem molecular orbital. And you can see it has a node right down the middle. And this one has also pi subsystem and has two nodes and three nodes. Finally, we get up to the homo, which has four nodes and transverse nodes. And now we get up to the lumo, no electrons in this one. But you can see if there were electrons, there would be this many five nodes running transverse along the molecule. So the idea is that on all of these electrons are in this pi subsystem, electrons are definitely delocalized across the entire length of the molecule. And we have this idea of the more nodes in this transverse direction that sort of indicates that the electron is moving faster and faster in the horizontal direction. Or as I go down here in the structure, slower and slower because we're getting fewer and fewer nodes.


10th okay, so more or less concepts from class 10th okay, but is what the kinds of the kind of problems you are solving or you are dealing with okay, so the depth of understanding should be more okay and then of course it will not be exactly like what you have done in class 10, but then you know that series parallel connection and all that okay, so all that will be in this chapter, this chapter is a very easy chapter okay, there are not many concepts fine, so what happens is that students take it lightly, so they will have easy ones at the end also and that end never comes fine, so follow the when I'm doing this do this chapter completed it once you once if you complete this chapter this chapter is guarantee you marks, chapters like modern physics and this chapter so in fact they guarantee you marks fine, mechanics huge in fact so you can't just ignore entire mechanics but what I'm telling you you will never be able to master these topics fine, so it may happen that in order to master these topics you keep on spending more in mechanics how you should do it okay because these chapters you may not be able to solve a lot of questions in exam and just to master these topics which are very difficult you're leaving out so simple that you'll be able to complete it in one day or two days before the exam you have to give good and this chapter there is not much of a difference between you know the kind of problem you get in school kind of problem you get in J mains in fact advance also there will not be too much of variation but if you see the J advance question there's a huge gap fine so it will take a lot of time for you to master up to that level fine so so that you'll be able to understand all the guarantees this chapter guarantees the full marks okay so let's see how this chapter is affecting the section is very similar to kinetic theory of gas for electron motion inside the similarly electrons can move sim in a similar fashion inside a kind of energy fine okay we go into micro factors as in how current depends on how many free electrons it has okay and how current depends on with what velocity electron is moving so all that you'll study here okay next section okay no no not okay in fact the seventy to eighty percent of numerical will be section okay application of whatever concepts you have learned okay so you'll be two instruments fine not fine so you might right so we'll be okay and this everybody knows it's not a secret it's an open secret fine so this the person they'll pick person from the end a small section but they know that to casual student would not have studied this fine so we need till and no there's a lot more to it okay and in this that will study later okay okay so first section so when fine if so we'll be talking about that so during its motion up so you'll see that there is net net movement forward why because the battery is trying to push it now it is the movement is that also clear okay now then things will be very simple okay just like you have charged which is flowing suppose it revolves around a circle of find out the current due to this this so electron 2 pi r by v no


There are three main steps of PCR which are called as denaturation, second step is annealing and the third step is extension which is also called as elongation. So PCR consists of three main steps, first one denaturation in which the double standard DNA is converted into single standard. Second step is annealing where the primers they bind to the single standard DNA and the third step is extension which is also called as elongation. In this step DNA polymerase extends the newly strand that is complementary to the template strand. The first step in case of PCR is called as initialization step. So this step consists of heating the DNA which is double standard when we heat the DNA between 93 degree centigrade to 96 degree centigrade the double standard DNA is converted into single standard DNA. So usually during the first step which is called as initialization step at this step DNA or the tubes which are kept in the PCR machine they are kept for approximately 1 to 10 minutes. So this is the first step in case of PCR reaction. So once the initial denaturation has been done, so first regular cycling of the PCR starts and the first step is again denaturation. During the first cycling step of the PCR which is denaturation at this step DNA is kept at 93 degree centigrade but there can be some variation it can be 93, 94 or 95 degree centigrade for 20 to 45 seconds. So during this step the double standard DNA separates into single standard DNA. So this step is actually when we have to convert double standard DNA into single standard DNA and that step is called as denaturation of the DNA. The second step of the PCR is called as annealing. At this step which is usually at the temperature of 40 degree centigrade to 65 degree centigrade there can be variation between the annealing of different primers at some of the primers they can bind at 40, 45 and usually maximum temperature of the annealing is kept up to 65 or in some cases it can be 67. So we can say that there is some variation in case of annealing temperatures. So annealing temperature because during the previous step the DNA which was double standard has been converted into single standard during this step of annealing the primers which are also called as oligonucleotides they will bind to the single standard DNA. This is the third step of the PCR which is called as extension. In some literature or in some books it is called as elongation. So elongation is the step where DNA polymerase adds the nucleotides and new strand is formed that is actually complementary strand of the template DNA. The polymerase has the optimum activity between 70 degree centigrade to 80 degree centigrade but most of the time or in most of the cases of PCR commonly or commonly 72 degree centigrade is kept at which there is an optimum activity of the DNA polymerase. So this is the third step of the PCR which is called as extension. Extension is done with the help of DNA polymerase and mostly the temperature is kept 72 degree centigrade. This is the last or the final extension during the PCR because sometimes there are unfinished strands of the DNA so a final extension is given that can be one that can be one to seven minutes so that if there is any unfinished products that can be finished. So during extension DNA polymerase that have added the DNTPs to the newly synthesized strand so extension is the third step and once extension is done there is the final elongation or the final extension. At this extension 70 to 74 degree centigrade it can be for 5 to 15 minutes but in most of the cases it is performed at 72 degree centigrade for 5 to 10 minutes. Final whole temperature this is the last step of the PCR that is for a short term storage of the amplified product because the PCR has been finished once there is a final elongation so at the end for a short term storage of the amplified product this step is added which is called as final whole temperature.


It's no secret that edge computing can create transformational opportunities for your business, like manufacturers using AI-powered apps to discover and solve problems in near real-time, or retail stores, applying intelligent video analytics to track inventory and drive sales, and clinicians receiving instant insights to make life-saving decisions. Industries everywhere are utilizing edge computing for better outcomes. But just as every use case is unique, every edge location is too. Locations can vary in physical size or available power and cooling capabilities. They can also vary by network connectivity that can be slow, unreliable, or intermittent between core and edge sites. So how can you overcome these variations? With Red Hat OpenShift, Red Hat OpenShift allows you to operate consistently, no matter your size, location, or environment, through three topologies that can help address your architecture's specific needs. They include three node clusters with built-in high availability in a smaller OpenShift footprint for sites that require continuous operations where connectivity to remote sites can be unreliable. Remote worker nodes that separate Kubernetes cluster management from worker nodes so that the smaller worker nodes can be placed in space-constrained environments and single nodes that combine the cluster management and worker node into one server, ideal for low bandwidth, intermittent, disconnected, or space-constrained locations. All three topologies provide support for even the most demanding workloads, including GPU-enabled nodes and AIML-powered intelligent apps that enable inferencing and real-time decisions at the edge. They can be mixed and matched as needed and all can be managed centrally and consistently through Red Hat Advanced Cluster Management for Kubernetes. With Red Hat OpenShift, you get support for thousands of nodes, clusters, and locations, and the dependable features and functionality that Kubernetes is known for. And when you bring it together with Red Hat's broad portfolio and Red Hat Marketplace of Ecosystem Technologies, we make it easier to design, deploy, and manage applications consistently across all sites. Flexible, scalable, consistent, that's what you get with Red Hat OpenShift at the edge. Learn more. Contact us today.


studying Othello. Here's how you can analyze that three scene one. The scene opens the morning after the drunken fight between Cassio and Rodrigo and these stage directions show that Cassio has hired some musicians who are going to go and play music to Othello and Desdemona. Cassio speaks using this in part to send this to the musicians telling them to play really well and he's going to pay them and he asks them to say their greetings to Othello. Cassio then speaks using alliteration to give the clown some money. He asks the clown to go and check if Amelia is awake and if she is awake to tell her that Cassio is waiting for her outside because he needs her help. As Cassio is waiting around Iago enters and he asks this question acting surprised as to why Cassio is awake so early and Cassio admits to Iago that he hasn't slept at all since the fight and since he got fired. Cassio then tells Iago that he's sent a messenger to go and speak to Amelia who's Desdemona's lady in waiting as he hopes that Amelia who is Iago's wife will help him have access to Desdemona so that he can speak to her and ask for her help in influencing Othello to give him his job back. Iago continues to pretend to be so concerned for Cassio. He tells Cassio that when he finally gets a meeting with Desdemona he's going to figure out a way of distracting Othello in order for Cassio to speak to Desdemona and ask for her help in influencing him. Cassio is really grateful to Iago for his help. When Iago leaves Cassio speaks using this hyperbole sentence to say that he's literally never met a person that's more kind or helpful than Iago. This is a great example of dramatic irony because Iago is the direct opposite. Iago is quite cruel, quite cunning and manipulative. When immediate Iago's wife finds Cassio she tells him that she's really sorry about what's happened and she speaks using this declarative sentence to say that Desdemona is already standing up for him. She speaks using sibilance to tell Cassio that Desdemona has already been telling Othello that he made a mistake firing Cassio. She is already standing up for him. Emilia then speaks using repetition to say that Othello actually loves Cassio. However his hands are tied because during the fight the Cypriot governor was hurt and now he had to fire Cassio to make an example of him. Cassio is heartened to hear this and he speaks in this imperative sentence asking Emilia if she can figure out a way of getting him to speak to Desdemona alone so that he can make his case to Desdemona that his firing was something that Othello should reverse. Emilia is very sympathetic towards Cassio and she speaks using repetition of these pronouns to say that she will definitely help him have some time alone with Desdemona so that he can speak to her.


So, my name is Guillaume Vogue, so as I mentioned this morning a little bit, I work at Miguel at the Genome Center and I also lead the Canadian Center for Computational Genomics that basically is a platform to do bioinformatics analysis and service as part of Genome Canada. So again, as mentioned by Anne this morning, so this is a new workshop, you know, we set it up with Mike really starting with sort of a basic introduction about the types of things that, you know, where variant calling in particular can be useful. Mathieu and my module are a bit more technical, sort of going through the steps of really variant calling and variant indentation and you'll see that at the end of the module three, Mike will actually come back and sort of then once you've generated these variants you'll go back and link back to his introduction on how you can actually then really use that to interpret disease and phenotype. So again, hopefully that whole overall flow will make sense. So my module really follows on Mathieu's module, so Mathieu was talking about variant calling and mapping and I'll be talking about variant indentation. So going back to the sort of the overview of what the data processing for calling variant looks like. So again, Mathieu covered the initial step with the data processing. Now we're going to be going over in module three, the variant discovery and variant annotation. So the specific objectives of this module is to, I guess that's the team for today, identification of disease causing mutation, understanding. So I've included some slides, we haven't talked about this yet, but what are the limits and the challenges with this and, you know, should you be doing exome and whole genomes, I'll be covering that. And then I'll go into the more technical part of the VCF and really these files with the variants and how do you actually filter and prioritize the variants that you get. So, you know, first of all, what's the overall objective here is to identify disease causing mutation and Mike touched on that a little bit, but so the terminology is, I think, important here. So you've got, you know, pathogenic mutation, which is what we're trying to identify that contribute mechanistically to the disease. And again, Mike touched on that, it's not necessarily fully penetrant, but it, you know, but that's, so that's, you know, the objective is to identify these. You can have variants that are going to be implicated in the disease. So that's when there's evidence that's consistent with them having a pathogenic role with a certain level of confidence. We've talked about associated variants that are significantly enriched, but there's really this whole range damaging mutations that would alter normal levels of the gene and then finally deleterious. So you really have these different sort of types of mutations that you might be looking for. So how do you actually identify variants that are implicated in human disease? So this paper that I have the, you know, the reference at the bottom, I think it's quite interesting in providing, this is just a subset of some of the recommendations, I guess, to, you know, really associate variants to the disease. So there's some general guidelines, really sort of, you know, you have to, you know, an observation you'll have to actually compute the probability of observing that by chance. This is related in part to, like the GWAS, if you're doing multiple hypothesis tests, you have to, you know, really calculate the probability of observing that by chance. An important guideline, and that will be sort of, I'll go back to that, but taking advantage of public data sets is obviously very important. So knowing whether it's a rare variant or something that's common in the population is a, you know, a very important aspect in implicating variants in human disease. So a number of guidelines, again, I highlighted a few of the key ones I thought. So what's the evidence for that gene to really be implicated? Is this a new report, but only do that when you can actually see, and again, Mike had a slide on that, you see the same variant in multiple individual, comparing the distribution between, hopefully, a match control cohort. So reporting what's the evidence for that variant to be, to be pathogenic. So recognizing the strong evidence that a variant is deleterious is not necessarily enough to implicate as a causal role. Ideally, you also want to be able to have sort of orthogonal experimental validation that actually confirms that that variant has an actual damaging impact. So, and then finally, it's important to highlight the actionable findings. But you also want to be reporting the additional findings at the same time. So again, so this is just sort of a snapshot, but I really recommend looking at that particular paper that provides guidelines as an example. So moving on to the next topic here, which is to identify and understand some of the limit and the challenges when you're trying to do this variant calling and annotation. So here's another, I think, recent and great review. And so I highlighted some key components. There's a big difference between, you know, so we talked about that a little bit in terms of applying these technologies in the research context versus applying them in a clinical context. So there's, you know, there's great potential to apply these technologies in, you know, really for diagnosis and in the clinic. But, you know, there needs to be some adjustments in how these tools are applied once it's done in a clinical setting. And Carl in the module at the end of the day will go back to that point in terms of some regulations and things like that. But there's also sort of more, well, some basic principles where when you're, so the quote that I'm highlighting here I think is quite interesting. So, you know, most of the algorithms that we're going to be using here were developed for discovery. So, you know, if you're missing a variant in exome sequencing, you know, at some level you're missing an opportunity for a discovery. And that's, you know, it's unfortunate, but it's not necessarily the end of the world. But once you're applying these methods in a clinical setting, making an inaccurate diagnosis because you're not picking up a variant is a whole utter ballgame. So related to that, it's quite interesting to think about. So once we start talking about applying these sequencing technologies in the clinic, you know, one of the first questions, I guess both in the research and in the clinical setting is whether to do exome or whole genome sequencing to detect these variants. And in this review, I think they do a great job of sort of showing and highlighting some of the differences and the implication of those differences. So here at the top you have, in the example, in a coding region, so this is, this is, this would be, for instance, an exome of the coverage you get from whole genome sequencing, from a standard exome capture sequencing, and from a sort of augmented or targeted special exome capture kits. So some of the newer generation of capture. So you see that, you know, well, first of all, you've got irregular coverage, even from whole genome sequencing, some parts are not necessarily covered as much as others. In some case, with some exome capture technology, you might have even bigger gaps. So this is in the coding region. Once you move to sort of a genomic region that includes coding and non-coding, you see again that whole genome coverage is not completely uniform such that you still have some regions that have relatively low coverage, at least here, you're covering most of the genome, but the coverage is not uniform. And again, that contrasts quite a bit with the exome, which, as you can see here, as expected, sort of covers very specific regions of the genome that are coding. So what does that translate to, and especially in the clinical context? So, so this is quite, so maybe we start with, we're looking over here. So these are the 56 most actionable genes from the American College of Medical Genetics guidelines. So these 56 genes are the ones that systematically should be, so if variants are detected in these genes, they should be reported. And what this plots are showing is that some of the bases, coding bases of these genes are not necessarily very well covered. Well, if you take, you look, for instance, at this exome array. So they did this experiment to see, you know, what the number of bases that are not covered for these 56 genes at a certain, you know, coverage level and a certain quality. So you see that there's quite a lot of variability. So this shows that even, you know, there are blind spots basically in these very important genes that you won't be able to call any variants in those, using that particular platform. That's also true if you're using whole genome sequencing, although this is a little bit better. And in some ways, it might be that these more targeted exome, in this case, have a slightly better coverage. But again, it's, I think it's useful to be aware that, you know, the choice of technology and whole genome sequencing is not necessarily, it's great for discovery, but it might, you know, not be the most appropriate thing depending on the types of questions that you're asking. So this is, I think, something important to keep in mind as you're choosing which platform to use and really thinking about the types of questions that you're gonna have. So this is in terms of the actual coverage. So there's lots of other challenges that are associated with using these next generation sequencing technologies for variant calling. So Matsuri again touched on that a bit, but there's, again, blind spots because these reads are short reads. So that has an impact in terms of, you know, whether you're able to map them uniquely on the genome. So you've got regions where you're mapping to the reference genome. So what this is one that's, I think, quite interesting. The reference itself that we're using to map the reads is a genome, right? I mean, it's not quite the genome of one individual, but it still contains variants that are actually rare. In some case, might actually be, you know, disease variants. So if the reference genome contains a disease variant, you're going to not be calling that the variant in your dataset because it's going to, you know, your data is going to look just like the reference at that location. So you might be missing some variants that were already also present in the reference genome. Long repeats and highly polymorphic regions. This is when the short reads are not great at mapping into these regions and where you also are going to have and make some mistakes. There's some more. I'll get back to this one in more detail, but this is about the fact that variant calling for single nucleotide variants is increasing. You know, it's pretty accurate and we have good pipelines and good algorithms to do that. But as you go towards other types of structural variants, that can be sometimes more challenging. There's additional challenges that are at some level a bit more technical, but the way that the variants are encoded in the VCF files and we'll get back to that. Sometimes there's some ambiguity in terms of the variants that you're reporting and might lead to mistake as well. But the key thing I wanted to expand on here is these structural variants here and other types of variants. So in the context of the module that we're going to do and the practical that we're going to do, we're really going to be focusing on detecting these point mutations. But especially with whole genome sequence data, you really have, in theory, the ability to detect a whole range of variants. So whether it's short indels, copy numbered alterations with deletions or duplications, and then translocation, foreign DNA and so on. So there's a range of variants that you can detect. So as I said, the focus of the pipeline that we'll present today are really around the single nucleotide variants, which tend to be the ones that, you know, where the benchmarking has been done and we have a good confidence in the calls that are made out of the data. But as you go towards some of these other types of structural variants that or regions in low complexity, for instance, there's clearly a lower accuracy of calling these variants once you're in those types of events.  In some case, you know, you won't find any very good hit, and it might be that it's through some of these other types of events that actually is associated with the disease.  But it might also be worth applying some of these other tools to the data to pick up these other variants as well. So related to that, another challenge, and now we're getting closer to the variant annotation. So this is a project that I was part of where we did the sequencing at the genome center. And so we sequence, so this is some of you are working in cancer. So you might be already aware of this. But so this is sequencing 100 kidney tumors. And what I'm showing you here are, so every square represents a thousand mutation that we found. So you see that in total we found more than half a million mutations from this data. So the challenge to identify, you know, within that giant list of mutation, which ones maybe are associated with the disease, can be quite daunting. And so this is from whole genome sequence data. The obvious thing, of course, is to focus on the coding mutations. So that's a very small subset of all the mutations that we detect. Most of, so again, it's funny because you use the whole genome sequencing and then in the end, most of what you use is really a very small subset of the mutations that you detect. Those are the one that we know a little bit how to annotate, how to predict the potential impact. And that's what we'll do as well today. But there's potentially some damaging mutation in the non-coding regions of the genome as well. And that's some of the things that we'll talk about tomorrow. So that's, I guess, another sort of ongoing area of research is how to actually annotate and prioritize and filter not just the coding mutation, but also the non-coding mutations that we detect. Okay, so that was my sort of my introduction in terms of what our objective is, what we want to do, and then some of the challenges. But now we're going to sort of continue on what Matthew started to present and continue with the workflow of annotating variants. So again, the overview of the workflow is that you do some data processing. You want to be mapping, and that's what we're going to do, assuming that the cluster is working, how you actually map the data, call the variants, and then annotate the variants. So before I talk about the variant annotation, I wanted to add a little bit to some of the things that Matthew talked about before lunch. So Matthew talked, so just on the variant calling still. So Matthew talked about, so you map the reads, and then there's a number of things that you can do to actually improve the variant calling. You can do local realignment to improve the variant calling. You remove the duplicates. You can correct the quality scores. Another interesting thing to improve variant calling is to actually use a family or a population structure to actually improve the calling. So this especially is important if you don't have extremely high coverage. Instead of calling the variants separately in the samples, you might actually want to call variants using the information that you have on the pedigree or on the population. So here's sort of an overview of this. So again, on the right side you have the basic calling, re-mapping, realignment, and so on, and then you do single sample. One at a time you call the variants, and that's going to produce a list of variants. But you can actually, and depending on whether you have a cohort or you have, again, families, you can also call the variants with that information in mind, do multiple sample calling. That actually has the potential in some case, and I didn't put the slides. I showed that, but in certain cases, especially if you have low sequencing, as I said, it might actually help you improve the quality of the calls quite a bit. And just to give you sort of a, I guess I do have the slides coming up, but to give you an idea of how that works, so suppose that you have two haplotypes in your population, so in the population there's a limited number of haplotypes in a given region that are in that population. So if you have information about this, that there's really a haplotype blue and a haplotype red, so if after that you observe reads from an individual that have these letters, you can probably guess what the end is without really looking at it. So again, you can use information about haplotypes in the population and about other sample to sort of correct or do a better job. But if you see an error in a read, you'll be able to detect it more easily. So again, I don't go into the detail, but I was just to give you an idea of the fact that in the context of variant calling, sometimes it's useful to either use information from the population or use other sample. And this is, I was not sure if I had that plot, but this shows from the initial papers from GATK, how much this can help improve the genotype accuracy, especially for in this low range here of, you know, rare. So but again, that's that was just sort of an aside. Yeah. I've seen one of the ways that you improve the accuracy by getting rid of duplicates. Yes. But duplicate reads don't really change the call. It would throw off your VAM or something like that. That's right. But it's just to get cleaner data or. Well, so the problem, so suppose you have an error and then that error is duplicated, right? So if it says, so you're going to think that you saw it five times and then in, you know, you're going to weight it too strongly, but if these are all exactly the same read, that's where it makes a difference. So how do you tell the difference between a duplicate and actually two independent reads? So it's usually based on the fact that it's exactly the same start and end. So if they have exactly the same sequence, those reads get pulled out. They get marked as duplicate and then they just get counted once in this calculation of whether, you know, because you're waiting the observation, right? So it just gets us a weight of one in this case. And then typically if you have relatively high coverage, you won't trust these, you know, single read that are saying that there's a change here. So, so again, so putting in this relates well to the, to the presentation by Mike, but taking information about the pedigree and calling and then looking at the segregation of these variants actually helps you quite a bit identify the good candidates. If you see, you know, if there's a very strong phenotype in the kid and that variant is found in the parent, you know, it's unlikely that that's really the cause of the disease. Okay. So this was sort of my add on to the module two on variant calling. So in terms of what types of files and what's the size of the files that you're dealing with. So you're starting, well, in the practical, Mathieu was kind enough not to start with the full file, sequence file, which would have been very big. But, and then you apply and that's what we'll do in the practical.  We're going to be using VATK. There's alternative ways you can also call the variants. So you go from a very big file that contains all of these map reads to something that's much more manageable, manageable, sorry, which is the VCF files, which actually has the call. These are per patient. So yeah, so one patient, this is whole genome sequencing again, but that would be the size per patient. So if you have a big cohort of patient, this adds up to quite a bit. So that's why, you know, learning how to use clusters like Compute Canada is useful. By the time you get to the VCF, these are files that you can more easily load onto these, you know, web services, a bit like what you'll do with Mike at the end here. So what do these variant files look like? So again, Matthew talked about the FASTQs and the BAM. If you look at what the VCF files look like. So this stands for variant call format. It makes a lot of sense. So you've got a lot of, so header lines which provide information on what was run, what reference files were used, and various parameters of the variant caller that you use. So this is header information. And then the key information is embedded in all of these rows. So each row corresponding at this point to a variant. So you've got the chromosome position, you know, the ID of the, so the reference base, so what is usually seen in the reference genome at that position, what's actually observed, a quality score associated no longer with the read, but with the call itself. So that, you know, we're just talking about the number of reads that support that variant and so on. So all of that gets converted into a quality score associated with the variant call. And then additional info fields that contain information about the reads that we're supporting, that we're supporting the call. So again, so we'll go over that and you'll go over that in the practical with Matthew some more. So that's the basic file. And again, this is at least more manageable in size. These are all the positions that are different in your genome relative to the reference genome. So, but as I said, so you'll have, you potentially have in a given project, you know, a very big file, lots of number of variants from this. So the next step is really to sort of filter and annotate these, this file with the variant. So how do you filter the variant? So you can do some manual filtering based on, you know, how many reads and what's the score and things like that. But if you haven't done this before, it's a bit challenging to know exactly, you know, where, where do you draw the line? So do you take, so in here, if you have quality score of three, you know, is this a variant that you should pay attention to, right? So how do you know which scores and which, which variant to filter on? So, so there's, there's nice tool that actually actually using data and I'll show you how that works that actually use the data itself and known calls to do a better rank order of this based on the likelihood of being real. So, so we won't be doing that today, this part. But just so that you know that that, that can be done. So, so what you do is you actually take data that's, that's a good proxy either for false negative. So you, so you want to know, so, so there's high quality data like the HapMap project that actually reports known variants in the population from the HapMap project. So you can use that to see whether you're actually missing some, some variants in your call and you don't want to be missing out too many of what are known to be, you know, reasonable variants. So, and then you can use other data set like, well, it's a bit scary to be using dvSNP in this context, but it's known that in particular in, you know, there's a lot of variants that are mistake as part of dvSNP that we're included. So you can include that as a set of potentially false positive. And again, in the GATK package, what they've done and they've shown is that you can use these set of good calls and not so good calls to actually train the parameters and better rank, better rank the variants. So this is sort of, again, you're not going into the details of how that worked, but you can use that feature to actually recalibrate the variant score using, you know, good quality, you know, known variants and known false positive. You use that to recalibrate the score of the variant calling algorithm. So this is one of the feature that actually is also included in the framework that's being used for calling variants. So I'm coming towards the end now of what I wanted to present before we could get into the practical of actually doing all of that. So now the variant annotation and prioritization. So so far, all we've done is map the reads, call the variants, sort of clean them up and filter what look, you know, sort of rank them in terms of how likely they're good based on various properties of number of reads and so on. But now the other key step is really to annotate and prioritize them. So, well, so I've touched on some of these annotations already. So quality and confidence score is obviously a criteria that you want to be looking at when you're looking at these variants. So what was the quality of the call? If there are reads that are in low confidence regions, you want to annotate that. So this is annotations about the quality and the confidence. Another important thing, and again, talked about that a little bit, so you want to be able to report whether the particular variant is found in DBCIP has been observed before because, you know, if it's in 20% of the population, it's unlikely that this is the rare, you know, de novo mutation that you're looking for. So having, and I'll get back over some of these database of previously observed variants, but previously, you know, the annotation of whether there's been observed before is quite critical. So you have then this is in population, mainly, then you've got specific disease databases that you might be able to use to know whether a particular variant has been associated or observed in a clinical setting. So this is ClinVar. Again, I'll go over these databases a little bit. I should say that these lists are not comprehensive, but this gives you example of some of the important annotation you want to be adding to your variants. And then the last category of annotation is these variant effect predictor, and Mike touched on that this morning, especially for coding mutations. There's lots and lots of tools. There's lots of tools that can predict the impact of the variant on the gene. So whether it's likely damaging, and all of these are, for the most part, just putative effects, but it's still quite, you know, if it's in search of stop code on versus a slice change versus a synonymous change and so on. You also, as I said, for, you know, the majority, if you're doing all genome sequencing, the majority will be non-coding variants. The annotation there of the effect is less well-defined, but we'll talk about that part a little bit more tomorrow. So going back here, so I've talked hopefully at some level about these confidence scores. I'll talk now a little bit more about these databases that can be used. So separate from 1000 Genome and DBSNP, one resource that was initiated as the exact database and has now become the genome aggregation database, so our Nomad. So this is a great tool where basically a great database where people that are sequencing as part of various projects are submitting their VCF from exome and whole genome to be aggregated into this database. So again, you know, it's a great way. So as of right now, it contains data or the variant calls from over 100,000 exome over 15,000 genome. So this is a great resource because again, if you're sequencing a new patient and you want to know whether that has been observed before as a way of prioritizing, this is a very useful resource because you're able to say, you know, if it's, you know, you choose your own threshold of what you're comfortable with and maybe you don't set it to zero, but if you expect something that shouldn't be found too frequently because it's specific to that disease or that patient, you might set some filters and frequency in this database. So again, this is sort of really at large, any exome and whole genome sequence data. And then, but so here, the only information you get is on the frequency of that variant in this particular database. Yeah. These are people with benign disease. That's right. So or in many cases, just population, right? So, yeah, so that's a good question. So, I mean, again, here, so if it's a different platform, you might not have a call in that database because it didn't cover that particular region. So you can't really use it for being sure that it's not there, right? So but again, if it's been observed in that database, at least you know that it's been there, but it's not fully homogeneous. So it's different platform and different, different variant calling in some ways, right? So it's not a fully homogeneous data set, but at least if it has been observed before, you know, you know, but you can't use it to say it's never, you know, it's not been observed. So this is to give you confidence that that variant is real. Typically here, we're using it to say it's not been observed before. So if you're looking for something that's not been observed before, you know, that's a useful resource. That's typically the way these database are used. In cancer genomics, just to think if it's a benign polymorphism, especially if it's greater than one. That's right. That's right. That's right. That's right. But again, if it's found in 10%, yeah, it's unlikely to be very damaging. Okay. So these are really database for providing frequency information of variants. Then you have more targeted databases that are collecting information about variants that have been associated with disease and with supporting information. So this would be used sort of in the reverse way where you actually want to find, you know, something that's not too common in the population and then has been previously associated with a disease in a different context. You would probably want to increase, you know, that pushes up that variant in your list probably because it's already been associated with a disease. So this is true at the level of sometimes, so variants, specific variants. So this would be ClinVar. And in cancer, you have other databases more like Cosmic. At some level, there's also now a database through ICGC that really has also a frequency of different variants as opposed as observed in different types of cancer. So, but overall these, you know, this type of annotation of a frequency in population and of, you know, previously been observed and been associated with disease is obviously these are all fields that you want to fill in and add to your variant list. So the last category that I had after the database, if you remember, were these effect predictions. So another type of annotation that you want to add to your variants is predicted effects. So, you know, separate from presence or absence in various database, can you predict what that variant is doing to that gene? So here, so Mike talked a bit about polyphen too. I mean, there's a total of 87 different tools as reported on this particular website that do this. So there's lots and lots of different ways you can do that. And, you know, people have different opinion about what works, you know, better or worse. I don't think there's a single tool to do that annotation that's necessarily better than the others. It's good to sometimes have, and if you can sort of have annotation from a few of these in parallel to predict the impact of the variant. So now a bit of a challenge is so I've already mentioned the fact that, you know, you want to be annotating your variant with databases, you want to predict their impact. So you have to run all of these different tools separately. So thankfully not, because there's a number of tools, including the one that you're going to be using in the practical that actually aggregate different types of annotation. And so you only have to run one tool to do this annotation and itself it will go to the various appropriate database and will actually predict impact using also sort of a collection or an ensemble of effect prediction algorithms. So again, there's quite a number of tools that do this in a different way. You know, in some cases they will claim to prioritize the variants even better. You know, I wouldn't say that there's just one way of doing this. Again, at some level it's convenience to be able to run one tool that's going to aggregate annotation and effect prediction from a number of tools will be good. So again, so we'll try one of them that actually aggregates as you'll see a few different types of annotation. Finally, so it's again from the same resource, they were pointing to a few tools not just to annotate but now to prioritize in the context of cancer in particular, there's a number of tools that are interesting that actually will prioritize genes based on the different, you know, a number of mutations that they're getting and so on relative to what you would expect by chance. So again, there's here again, whether it's based on the number of mutations per gene relative to what it's expected or whether you have, you know, an enrichment for mutation specific pathways. So you have again, sort of a number of tools that can do that for different purposes. So the tool and the approach that we'll be using in the practical is a tool that was developed by Pablo Singolani that used to work at the genome center but is a tool that's really well integrated with GATK and is used quite heavily. So, you know, so using SNPF, you can annotate using reference genome, you can calculate the effects of whether it's a, you know, if it's a coding mutation, whether it has a, you know, what type of effect it has and then it has basic prioritization into, you know, potentially high or moderate or low impact on the gene where it's detected. So this is again, we'll go over that in the practical in more detail. So just to continue with this overview and this workflow. So we started with the very big files with the alignments. We now have, you know, you'll generate the raw VCF file that basically just has good quality calls with quality scores. And so what you want to do after that is annotate this file and get a filtered and annotated list of variants still in the VCF format but now having, including a lot more information in the various other columns in the various fields. So just, so two little things before I end. One of the tools that we won't be using in the practical but that we use to actually navigate these VCF, especially once you've generated this annotated VCF for one patient, you might actually have multiple VCF with this annotation. And so one strategy is to load these various VCF into a tool. So for instance, Jim and I, and that actually allows you to navigate through this VCF and ask specific question to sort of sort, you know, a variance that are found, you know, that match the expectation based on the information you have on the pedigree and so on. So this is not something that we'll do, but it's another tool that I recommend once you actually have generated the VCF, especially if you have multiple patients or pedigree or family information and you want to sort of filter your VCF using additional criteria, that's, you know, maybe a tool also to explore and look at. So a final note before we move on to the practical, go backwards to the practical, is one thing that we won't have time to cover, but that's extremely important, is visualization of these data sets. So I didn't say what this tool was. So this is the integrative genome viewer, IGV browser, which is a tool that allows you to load both your BAMs and your VCFs, such that you can really, because at some level you're generating all of these files, but you don't have a way of checking, you know, what actually is coming out. So it's very important and useful and necessary to, because you might have a mistake in your pipeline somewhere, it's very useful to load your raw data, the raw BAM in this case, not the raw FASQ, but the BAM ones that reads are aligned. This is what you see here in gray. These are all the reads that have been mapped to the genome. We're looking at a particular region, and all the matches are shown in gray, and then you've got the mismatch that actually are, well, there's different views, but in this particular view, when there's a mismatch, you actually see the, you see the letter that said that specific position. So from this, it's quite clear that you've got some errors in your read at the rate that's expected, while the variant that we're looking at really stands out as being very clear. But once you get to the stage of having your VCF and having your variant that you think is most interesting, it's very good and necessary to go back to look at the evidence, basically, that we're in the reads for that variant, because by looking at it, you might see that maybe there was a mistake in one of the processing steps, or that the region is messier than you would expect it, but it's just quite useful and important to look at that. So that's the end of this module. So happy to take some questions, and if not, after that we'll go back and actually finish all these steps from the beginning. Going back to this point of the fact that there are regions of the genome that are not covered, if you have a gene that you suspect or you were looking for mutation potentially in that gene and nothing comes up in the VCF, it's also good, again, to go and visualize that data. There might not be coverage on parts of that gene or part of that exon that might explain why you're not seeing anything. So it's really, even though we're not covering that in this workshop because we didn't have enough time, there's other workshops that you could attend that cover the visualization, but it's really quite important to do that. Right? That's right. Yes. So the basis for this is not uniformly covered? So it's a combination of regions that are not mappable. So if they're pseudogenes or duplicated regions, you might have ambiguity in terms of where the reads come from. There's also like a depletion of GC rich regions in the sequencing. So just for technical reasons, those regions don't get sequenced nearly as much. And so in the whole exome capture data, a lot of time or even in the whole genome for that matter, the first exon, which tends to be GC rich, is frequently less covered. So if you don't have enough coverage, you end up having no calls. So that's another thing. So assume that you have a trio, and you see a mutation in the kid that you think is a de novo mutation because it's not called in the two parents. It's probably good to go check to see if there's any reads that still supported that in the parents because it might be under the cutoff of being a call as a high quality variant in the parent, but it might still have been there. So if you didn't do this three-way calling, you might have missed it in the parent and might still be there. So it's good to go and actually check whether there's coverage on what you think is a de novo mutation. So do you think? Yeah, so long-requencing will overcome the issue of maybe having a more uniform coverage and definitely going into these, you know, going into these harder types of repeats where, you know, so if you have, again, mobile insertions of L1 that are too long for the short reads, you can't detect them, but with the long reads you will be. The problem with the wrong reads is that it's still quite expensive for doing a deep enough human coverage. But at some point, that will be useful. With the longer reads, you also get a higher error rate as well. That's right. So sort of a ying-ying as you're introducing a different type of error. But the error rate, I think, is coming along. And again, as soon as you have multiple of these long reads, you can correct the errors quite easily. I think the cost is a bigger challenge with, like, right now, whole genome with Illumina is in the range of, you know, $1,500 or something like that while whole genome with Pykebio is $20,000 or something. So it's... The other approach is the things like the 10x where you have information about long fragments and then still do the short read. So that's... But these are, I think, still in sort of research and exploration. Is there people who try to... not expensive as a long read but not short read? That's $150 is too short but I don't need a 10k read. Yeah, no, it's a good question. I mean, to be fair, with $150, especially paired in, there's not much of the genome that's not mappable, right?  So how do you weigh that in? I mean, I went back and forth, but again, going back to this, how do you determine what's enough, right? So you're missing out just, you know, 500 nucleotide in these genes. Is that a problem or not? And one thing this doesn't show is whether or not the bases which are missed on subsequent assays are the same bases, just one table. You're missing 100 bases. So I think this is the average of multiple samples. So this is pretty systematic what you're missing. So it's going to be because of repeats, because of lack of capture. So it's consistent. Well, it's consistent within technology, right? So it's not going to be consistent. So that's one of the challenges if you're using these control cohorts and you have a new assay that's looking into regions or essentially it's not covered in the other one, you know, going back to your question in the back, that's going to be an issue. But it's definitely consistent within assay and within... But there's, you know, and again, you have dropouts because of the assay and you have dropouts because of the analysis pipeline that you're using. So you need to match both of those things to really be able to... How long does it take for once you press the button on it? It's bad on the other end. So Matthew, it would be better for me to answer that, but... So, David, so for general, say it's Exxon, it's a question of our route or if we go for World Genome, it's a question of the concern, which is how complicated the two are how complicated. It's weeks, few weeks to to run the pipeline. But we have some we have some way we're part of one project where we analyze the tumor in three days. We need some trade just to focus on it. But in a case, you know... Usually weeks for World Genome. Then you have to add a prior example. It's a better... which I cannot go into running the whole genome. But it is a limitation of the whole genome sequencing. So I think, as Matthew mentioned, we're part of the project where it's used in the clinical context and then waiting two weeks to get that answer is an issue. So we actually have to, you know, sort of trick and use only a subset of the whole genome data to get an answer faster where we set of mapping all the reads to the whole genome. We only map them to the exome to the coding regions which is where or even to a subset of actionable regions such that we get an answer faster and then we wait for the full answer two weeks later. But the time it takes can be an issue with the whole genome sequencing for sure.  So, you know, for some application it makes sense to instead of mapping to the whole genome which takes time you map just to the genes.


What's going on guys it's your boy the E.P.J here today with another Destiny video and in today's video we have top 5 trials of Osiris plays of the week I want to thank everybody for submitting and the support I've received over the past months of Destiny really appreciate the support thanks for stopping by as always and I'll catch you on the next one.