Let us continue, we want to discuss applications of the Gram-Schmidt process. It follows immediately from the Gram-Schmidt procedure that the following result holds. So I will simply state this as a theorem and skip the proof. Every finite dimensional inner product space, every finite dimensional inner product space has an orthonormal basis. I will not write down the proof. The proof is it is a finite dimensional inner product space so it is a finite dimensional vector space so it has a basis consisting of finite many elements. Since it is a basis it is linearly independent apply Gram-Schmidt process to that to get an orthonormal set. The fact that these pans are the same proves that this is an orthonormal basis also, okay. So you can write down the proof on your own. I am more interested in applying this GS procedure to what is called as a best approximation problem, okay. Let us develop some machinery before stating this problem. So I have the following result which will be useful later. Let V be an inner product space. Let us call U as the set of all vectors U1 U2 etc Un. This is not necessarily a basis but an orthonormal set of V. Let V be inner product space and U be an orthonormal subset of V. Let us take an arbitrary element X and V, arbitrary but fixed element in V and define a vector U as a linear combination of these UJs in this particular manner. Define a vector U in this manner. For a fixed X then this vector U satisfies the following properties. Remember X is fixed. The first property is that X minus U this vector is perpendicular to span of U. The vector X minus U is perpendicular to span of U. What is the meaning? The meaning is that the inner product of X minus U with W equal to 0 for all W in span of U. It is orthogonal to every vector in the span of U. It is the first property. Property 2 is called Bessel's inequality which is the following inequality. The first one is an equation. What is norm U square? Norm U square is summation J equals 1 to n modulus of the inner product of X with UJ in the whole square and this does not exceed the norm of X. That inequality is Bessel's inequality. So there is a formula for norm of U square if you know the coefficients. If you know the numbers inner product X UJ and this number does not exceed the norm of X square. That is the second property. So norm U square less than or equal to norm X square is Bessel's inequality. Property 3. If U is a basis for V, U is already an orthonormal set. So it is an orthonormal basis. If you have that condition also then X equals summation J equals 1 to n X UJ UJ and norm X square in that case is summation J equal to 1 to n modulus X UJ the whole square. That is the view is a basis for V then X coincide with this little view that we started with. Once you realize that the other two are straightforward. This is called the Fourier expansion of X with respect to the basis that we started with. So this one is called Fourier expansion of X with respect to the basis that we started U1, U2, etc U n and this is sometimes called Perseval's identity. Let us look at a proof. First I must show that X minus U is orthogonal to span of U. U is this orthonormal set. So it is enough if I show that X minus U is orthogonal to each of these vectors. Span of U, anything in the span of U is a linear combination of these and so this is enough. So let us consider U with UK for a fixed K. We do not have the difference symbol then it is easy to understand. So U1, U2, U1 is orthonormal set.  So if you do not have the difference symbol then it is easy to understand. I will change that everywhere. U is a single element so how can I say it is basically. So I will replace U by V. I am just renaming the set U by script B proof. So for each UI coming from that set script B, I look at the inner product of U with UK. I have the definition for U. Summation J equals 1 to n inner product X U J UJ. I take the inner product of that with UK but this is the first term goes out with goes out as it is the first scalar. Then I look at the inner product of UJ with UK. J is a running index K is fixed and K runs between 1 and n. So when J is equal to K that is the only term which will remain all the other terms are 0 because it is an orthonormal set. Script B is an orthonormal set. So this is delta JK. So this takes a value 1 when J is equal to K and in that case it is just X with UK and J equals K. This number is 1 all other terms are 0 and so what we have proved is that inner product X minus U with UK is 0 for all K and so it follows that X minus U is perpendicular to span of B okay that proves the first part okay second bezel is inequality. Just use this relationship between the norm square and the inner product in the X band. This is summation J equals 1 to n mod X UJ UJ.  This is summation J equals 1 to n X with UJ summation J equals 1 to R equals 1 to n X with UR the whole thing goes with the conjugate I can apply the conjugate to each of the terms and then the rest is UJ UR. You can do it either way first look at this sum R equals 1 to n where J is fixed and then the summation is over J. So when J is fixed R takes a value J this will be 1 all other terms are 0. So when R is equal to J this will be X comma UJ with a conjugate summation J equals 1 to n X comma UJ into conjugate X comma UJ bar. See for the second sum R is running index and R takes a value J, J is fixed. Now this is simply complex number into its conjugate so modulus square okay so that is the formula for norm U square.  If you look at norm X minus U square it is a non-negative number so this is I have added something non-negative so that must be greater than or equal to norm U square.  Third is immediate once we prove that X is equal to U okay so let me I will prove that here itself I will prove that X is equal to U then it follows from what we proved in 2 that these two hold if I prove X equals U then we know the formula for U that I have written down this is a formula for U so if X is equal to U that first thing holds if X is equal to U from norm U square equal to summation modulus square this follows okay that is easy look at if let this be a basis for V then span of B is V span of B is V till now it is not a basis in 3 we are assuming it is a basis also the span of B is V now what does the first part say first part says X minus U is perpendicular to span of B so X minus U perpendicular to V which means X minus U inner product of that with V this is 0 for all V in B in particular for V equal to X minus U we get the following X minus U X minus U must be 0 but X minus X minus U is norm X minus U square okay you can even use the thing from the inner product so I will say this is 0 from the positive definiteness of the inner product it follows that X is equal to U and so the third part follows okay so if B is a basis then span B is V is what is being used this is just to develop machinery for a particular problem that I mentioned briefly the problem is the following often we consider the following situation given an inner product space and a fixed vector we seek a vector U in a subset W of V which approximates X which approximates X given a fixed X in an inner product space and a subset W among all the elements from that subset W I want to choose a particular vector U which satisfies the property that it will serve as an approximation for the X that I started with okay let me give one or two examples one example motivating example from analytical geometry suppose I am given a vector let us say 1 2 1 in R 3 I want to find I want to find a vector on this plane this is W, W is the set of all X in R 3 such that let us say X 1 minus X 2 plus X 3 is 0 for example we ask this question what is the length of the projection from a point on to a plane what is the distance of a point from a plane etc I have a point this is X this is my subset W in this case it is a subspace the point U can be thought of as an approximation to the point X 1 2 1 already belongs change the equation 1 2 1 is already there so let us say this is my equation X 1 plus X 2 plus X 3 is 0 the question is there a point U in W that will approximate X from analytical geometry of three dimensions we know that you need to just project X on to this plane that projection on to W will give the vector U that projection will give the vector U will give the vector U let us also observe in this case that the vector X minus U is perpendicular to the plane the vector X minus U is perpendicular to the plane okay remember what we proved in the first first part of the previous result so these are related X minus U is perpendicular to the plane because it is a projection okay this is one motivating example sometimes in the space of functions space of continuous function let us say C 0 1 we would be interested in approximating the exponential function in terms of just writing down as a linear combination of certain polynomials certain polynomials you look at the subspace spanned by those polynomials then I would like to write down the exponential function in that space so what is the approximation to that exponential function in that subspace this is one another question okay so let us pose this problem we need to understand this meaning approximation and then see how this problem can be solved okay see in the finite dimensional case this problem can be solved in the infinite dimensional case it is not there are examples where the problem does not have a solution okay but we will confine our attention to the finite dimensional case so let me rewrite this problem mathematically the problem is given given X and V we need to determine if possible U in W such that so it is an approximation, approximation is given in terms of norm, norm induced by an inner product given X and V to determine U such that norm of X minus U this particular U this norm should not exceed the norm of X minus W for all W this is the problem so it is not just an approximation it is a best approximation in that sense such a U is called a best approximation a best approximation to X it also depends on the subset W so it is a best approximation to X from W this will obviously change if I change W so I need to minimize this norm I need to minimize norm X minus W for all W and W to see whether there is a U in W that satisfies this condition this is called the error of the approximation norm X minus U is called error of the approximation. Now in many practical problems this norm is the two norm that is sum of squares or integrals of squares of functions so this is in many instances called the least squares approximation least squares approximation and this vector U sometimes is also referred to as a projection of X on to W there is a motivation coming from geometry the problem that we considered just now the vector U is a projection of X on to the subspace W sometimes U is called a projection of X okay that is another terminology in general as I told you in general this U may not exist it may not be unique okay but for in the finite dimensional situation the compute the existence is there the computation follows by using Gram-Schmidt process okay so let me prove that result next and go back to this problem and see how this problem can be solved by this method. So this proof is construct in the next theorem tells you how to construct the best approximation in case it is unique I have an inner product space and X is a fixed vector we need W to be a subspace let W be a subspace let U be a best approximation to the vector X from the subspace W see what this also means is that U belongs to W among all the vectors in W U is the one that minimizes norm X minus W U be a best approximation X from W then X minus U with I will use W this is equal to 0 X minus U must be perpendicular to W for all W in W the converse also holds that is if U is a vector in W that satisfies this condition then U must be a best approximation to X converse also holds okay let me just write down that is if U element of W satisfies okay that is a converse we also have the following I will call this one what we would like to have ideally if a best approximation exists then it must be unique that is always the case existence might be a problem but if it exists then it is unique in the case of an inner product in the case of a see this problem is posed as a problem involving norms the general norm linear space it may not exist but if it is a norm induced by the inner product in the general norm linear space it may not be unique but if it is a norm induced by an inner product if it exists then it is unique okay and second property second part tells you how to construct this U let this be U 1 U 2 etc U n be an orthonormal basis for W so this last part assumes that W is finite dimensional V can be infinite dimensional there is no condition on V we can be an possibly infinite dimensional inner product space but if W is finite dimensional subspace then the last part tells you the formula for the best approach then U is summation j equals 1 to n X U j U j is the unique best approximation again this should remind you of the formula for U that we defined earlier okay so if W is a finite dimensional subspace of V and if you have this orthonormal basis then the best approximation to X is given by this explicit formula okay you know X you can compute these numbers so this U can be determined maybe I will give an example we will take the same example and use this construction given in 2 and then solve the problem and then come to the proof of this result what is U that satisfies norm X minus U less than or equal to norm X minus W for all W in capital W probably we could look at the other methods of solving this problem for instance how would you solve this problem using techniques from analytical geometry of 3 dimensions do you remember yes any point alpha beta gamma then alpha then we find the equation of line X U is using direction percent 1 divided by 1 okay is a direction the issues okay that is not enough then U is perpendicular to the plane so use that and then solve this problem that is from analytical geometry can you use calculus because the minimization problem so how using the characterization X 1 plus X 2 plus X 3 0 by using Lagrange method you can find yeah you must use Lagrange multiplier method for function several variables to determine this minimum okay you try both those methods and also the method that comes from applied linear algebra okay what does this tell you this last part tells you that you must first construct an orthonormal basis for W okay let us consider a basis for W say this is the problem the problem is to determine U that minimizes this distance where X is this vector W is this subspace okay I want a basis orthonormal basis for W what is the dimension of W into 2 so I need to determine 2 vectors so let us call U 1 as 0 1 minus 1 and U 2 to be 1 0 minus 1 both these vectors belong to W and they are linearly independent one is not a multiple of the other okay I should not call it U 1 U 2 I will call it A 1 A 2 U 1 U 2 is a notation for an orthonormal basis A 1 A 2 is a basis so we need to apply Gram-Schmidt process for the first one for me U 1 is 1 by root 2 into 0 1 minus 1 again for convenience I am writing the vectors as row vectors this is U 1 and what is U 2 to determine U 2 I must use W 2 W 2 is U 2 minus sorry A 2 minus A 2 U 1 U 1 A 2 is 1 0 minus 1 minus A 2 with U 1 these two terms are 0 1 by root 2 there will be another 1 by root 2 that is 1 by 2 into U 1 0 1 minus 1 so that is 1 0 minus 1 by 2 minus 1 plus 1 by 2 minus 1 by 2 this is orthogonal to this and the norm of this so I can write this as I will take minus 1 by 2 outside then it is minus 2 1 1 so that I get 1 minus 1 by 2 minus 1 by 2 V 2 is what is the norm of this 1 by 4 2 by 3 root 2 by 3 into W 2 minus 1 by root 6 into minus 2 so this I should call it U 2 4 plus 1 plus 1 so norm is 1 this is orthogonal to this vector now determine U from the formula that is given here X is 1 2 1 right call U as summation so that is X X is 1 2 1 X comma U 1 X comma U 2 X comma U 1 this goes with minus 1 just 1 1 by root 2 into U 1 U 1 also has a 1 by root 2 U 2 goes with 1 by 6 in a product X with U 2 that is minus 2 1 1 minus 2 1 those 2 get cancelled I get just a 1 again into U 2 U 2 is minus 2 1 just check if my sign is correct minus so that is taken care of I should go with a minus 2 1 1 okay so that is minus 1 by 3 then 1 by 2 plus 1 by 6 2 by 3 minus 1 by 2 plus 1 by 6 minus 2 by 3 the last term is minus 1 by 2 plus 1 by 6 that makes it minus 1 by 2 plus 1 by 6 so this is 3 minus 1 by 3 so I can write this as 1 by 3 say minus 1 by 3 into 1 2 1 minus 2 1 this is my vector U so you please go back and check that this is the vector that you would get by the Lagrange multipliers method and analytic geometry okay.  W is finite dimension then you must exist suppose if you take a dimension W is infinite dimension then is it possible to exist or not? No you can give examples it may not exist but if it exists it is unique in an inner product space that is in a normal linear space induced by an inner product in a general normal linear space it may not exist in a general normal space it may exist but it may not be unique.


The Kabya State Government has approved the payment of the remaining balance of more than 456 million Naira to the West African Examinations Council WIEC for the 2022 West African Examinations Certificates Exam... The 2022 West African Schools Certificates Examinations. The Commissioner for Basic and Secondary Education in the State, Muhammadu Magawata Aliero, disclosed this while addressing newsmen in Benin Kabya, the state capital. The Commissioner recalled that the state government sponsored over 30,000 candidates for the 2022 WIEC at a registration fee of 18,000 Naira per candidate. Hello, hope you enjoyed the news. Please do subscribe to our YouTube channel and don't forget to hit the notification button so you get notified about fresh news updates.


Today we are going to talk about next generation sequencing technologies. Before we talk about this you know revolutionary technology platform, let me share a story with you. So, when I was a grad student like you, I was finishing my masters in year around 2000-2001, that time the various genome sequence projects were getting accomplished. It was you know big news that time, big breakthrough that time that the human genome project the first draft was completed. And many other model organism and their sequences were also getting completed at the same time. Just imagine that you know first time we were getting information that what you know makes it human, what our genetic composition is about, it was you know really big science breakthrough because first time we now know that how many genes are actually available to us which makes it a human which makes it so unique and different than other model organisms. Before that there were only speculation that you know what makes human and you know how many genes we may have, but the first time the evidence for genes and our genetic composition the genome was first time revealed right. It was definitely big revolution in the science field in general and differently in the biology and medicine area, but it is not end right, it is not finished that time. So many times a technology shows as a promise and then you know it fades away. But what has happened in this area of genomics that you know 2002 and 3 first time the draft human genome were released. But since then there has been tremendous advancement which has happened in this field of genomics reaching to where we are which we say as a next generation sequencing technology. A series of advancement happened for the first generation, second generation, third generation of regulation sequencing technology and I must admit that advancement made by engineers and technologist, big data scientist and biologist together has really brought where we are currently in our magnitude of doing sequencing based experiments. So what has changed from 2002 in 2018? Second thing which has changed is our speed, the speed at which we can now sequence a genome. Imagine the human genome projects took more than 10 years time to accomplish. Now within 2 days time or 48 hours time you can do sequencing of a given individual with the next generation sequencing technology that is humongous change. Additionally what took billion dollar to accomplish one human genome sequence now it takes may be only a thousand dollar to accomplish the same sequencing technology based work. So both cost wise as well as the speed wise as well as the accuracy wise the next generation sequencing moving from the Sanger sequencing based platform have made tremendous revolution. So I am sure now you are excited to know that what NGS technologies are which are the currently big industry player, big industry technologies which are leading this field and in this light we have invited a guest Dr. Atima Agarwal from Thermo Fisher Scientist who is manager a commercial service training and tech support at Thermo Fisher Scientific who will be taking this lecture and she will try to provide you the basics of next generation sequencing first and then give you little more detail and specs fix for one of the technologies of iron torrent. So let me welcome Dr. Atima Agarwal for her lecture on NGS based technologies and its applications. I am Atima Agarwal from Thermo Fisher I have been with this company since many years now so if you remember or if you have heard in pre 1990 era the sequencing used to be done on gels with radioactive labels yeah so and it used to be a very very cumbersome affair but then it had contributed a lot to the science in those terms that first human genome was being sequenced on these systems and there were many platforms many of these capillary electrophoresis platforms if you look into the pictures of Sanger Institute during that time in lines there were these kind of systems and they were generating sequences day and night and that's how the first human genome got sequenced and got assembled so those were a lot of like I would say that benchmarks and sequencing suddenly somewhere during 2004-2005 we started hearing about next generation sequencing. So if I were to talk about next generation sequencing it is more aptly called as massively parallel sequencing so with Sanger what you're doing is that either you are running it on 96 well plates or you're running it on strip tubes so one well is giving you one sequence yeah so one sequence which can which is a stretch of 80 gc and this can be as long as a thousand base pair 1100 base pairs and it can be as short as a 100 base pair fragment so the one well gives you one sequence now suddenly with massively parallel sequencing you can imagine what is happening is you have millions of wells and you have this sequencing per se happening in all those millions of wells and that is how you are generating a lot of data per run so that is massively parallel sequencing. So just to tell you that just because we are now nowadays talking a lot about NGS it is not that capillary sequencing has lost its charm or has lost its utility it is still a very user friendly technique for a lot of applications so for tech for things wherein you do not really require a lot of data things can be done simpler for a lot of microbial research or a lot of single gene based tests and all those things they can be still very well and very efficiently being done on capillary sequences till there this has been the gold standard technology for doing a lot of things like targeted so you have certain amplicons to be sequenced or you have a de novo organism to be sequenced to fill up those kind of assemblies then you have multiple there are multiple fragment analysis applications wherein you are not actually sequencing the fragment you are just looking into the size and based on the size polymorphisms you are telling you are giving certain scientific answers so this is about capillary sequencer coming to next generation sequencing so now as I mentioned that this is a technology wherein you have millions of wells so if you see that these are the chips which are used with iron technology and you have millions of wells on these chips so millions of wells so you can imagine that each well has a sequence and that is how you are generating millions of sequences in a run so the agenda how we have planned out the session for you is that we are going to talk about our new series of NGS platforms which is iron gene studio S5 systems the technology behind the gene studio S5 and some of the applications so this is the fastest sequencer which is available in terms of an NGS which can produce starting from once you have started your sequencing run it can produce data in as little as 3.5 hours from a sequencing run there are lots of different kinds of applications people want to work on NGS and lot of these samples are very challenging samples like when you extract DNA or RNA from FFP blocks when you are working with liquid biopsies like you do not you really are looking at cell free DNA yeah or you have very little RNA from a cell and still you want to study the whole transcriptome of the cell so our technology is such that it is compatible with as low as one nanogram of DNA or RNA and the backbone of this technology is AmpliSeq technology we will come to those details later so coming to the workflow now so how we are generating these millions of reads so you start with nucleic acid now this nucleic acid can be DNA can be RNA so where do you think you would be starting with the DNA what kind of experiments do you think you would be starting with the DNA on NGS DNA as a sample whole genome sequencing yes plasmids yes targeting resequencing so when you are not so targeted resequencing is basically when you do not want to sequence the whole genomes you just want to sequence some targeted regions of the genome that is targeted resequencing yeah so where do you think you would be using RNA as your starting material transcriptomes yeah again like targeted targeted trans you are looking at some targeted transcripts or you are looking at some targeted fusion events yeah anything else yeah RNAseq and RNAseq typically why would you do an RNAseq you want to sequence yeah so this DNA is transcribed into RNA and then hence there are more probabilities that this particular part of the genome is actually getting transcribed or further translated into a protein but that is just a possibility there are many long there are many non-coding RNAs also yeah like small RNAs yeah yeah which are yeah so which are there involved in lot of regulation activities of how the transcriptome is shaping within a cell yeah so people do a lot of different kinds of experiments now for your what you would look out for in your NGSS that all these experiments because the data requirement for all these experiments is very different yeah so when you are doing a genome it's a very straightforward calculation that you generally tend to generate a hundred x data so whatever is the genome size you multiply by hundred there are definitely ifs and buts around it will not go into those details but then the data requirements for these different kinds of experiments are very different yeah so that is where you need that flexibility that single NGSS system can cater to all those requirements so you started with your DNA or RNA yeah to begin with so you isolated DNA RNA from cells or from some culture isolates or whatever so now once you isolated this DNA and RNA the next step is that you prepare a library now library is basically a collection of fragments whether it is coming from DNA or coming from RNA which you finally want to sequence yeah so this can be an enriched portion yeah so for targeted sequencing you would need to enrich that those targets first and then only you can go for the sequencing for whole genome sequencing since your aim is to sequence whole genomes so you do not need any enrichment you will just extract DNA and you will go forward for library preparation so library preparation is basically collection of fragments whether DNA or RNA which you finally want to sequence yeah so what what is the basic aim in library preparation is that you are now these next generation sequences you we talked about that you are generating 200 base pair it reads you are generating 400 base pair reads you are generating 600 base pair reads so supposedly we take an example of whole genome sequencing now what you are going to do is with whole genome sequencing you want your aim is to sequence that genome as fast as possible yeah and with as bigger reads as possible so probably you will pick up a 400 or a 600 base pair chemistry and so what you need to do with the genome is you share this genome yeah you break this genome into smaller pieces so that can be done by enzymatic treatment or that can be done by sonication so once you broken down this genome now these are the fragments which you finally want to sequence but you do not know what is there so you need to have some adapters yeah or linkers which will help you facilitate sequencing these regions yeah so in library preparation what you are doing is you are enriching the region of interest and second is you are ligating it at both the ends you are ligating it with linkers these linkers are double stranded DNA fragments which are basically exploited so that the primer can then sit on these kind of fragments and then so you have your fragment of interest in between you link the adapters at both the ends and so why so that the now these adapter sequences you know because these are coming from a chemistry and we tell you that these are the adapter sequences and then the primer comes and sits on these adapters so that you can sequence this region in between yeah so that is basically so with library preparation what you are doing is you are enriching your region of interest and you are ligating these fragments to adapters yeah then comes template preparation so basically now these are the things which come which are specific to a technology so in template preparation what you are doing is that you are taking these library molecules and you are amplifying on to ion spheres so we are talking about ion torrent technology so we are using ion spheres as the mode these ion spheres get coated onto those or get loaded onto those chips and finally these ion spheres are carrying those regions of interest or those library molecules from which you are generating all those reads yeah so that process of taking these library molecules and amplifying them onto the ion sphere is called template preparation then comes sequencing now you have your ion spheres which are templated which have library molecules amplified onto them and now you want to sequence so you load these ion spheres onto the chip and then you generate the data so once you have loaded you initialize your sequencer which we discussed is going to take only 15 minutes and then your sequencing run goes for anywhere from 2.5 to 4.5 hours depending on the kind of read length you are targeting so talking about the instrumentation which is required in this workflow so we have so library preparation can be done manually can be done on ion chef there are some kinds some parts of library which can be done on ion chef yeah so this is an automated way of preparing libraries otherwise you have manual way of preparing libraries wherein you are using certain magnetic beads certain library preparation kits from us and the process generally for targeted sequencing or for whole genome sequencing the process is around 4 to 4.5 hours for manual and probably for RNSE it is a days protocol because you have to do certain levels of enrichment and you have to do more QCs in that so if you talk about manual preparing libraries manually so that is the kind of time frame otherwise you can use ion chef for preparing your targeted sequencing libraries automatically then comes the template preparation now as we discussed during template preparation you are loading these library molecules onto the ion spheres and then finally these ion spheres are loaded onto the chip now these loaded chips you take from here and you just load it in on this system and they are good to go for sequencing so just coming into few details on library preparation now how we go about library preparation is since you are targeting variations at the DNA level as well as at the RNA level you isolate DNA and RNA yeah then you reverse transcribe this RNA so now you have DNA or cDNA so you take this primer pool and you use this DNA and cDNA as your target all these genes yeah get amplified at one go and then since the primers are coming from conserved regions wherein you do not have any hotspots or any kind of variations to be looked into we partially digest these primer sequences and then we ligate adapters now when we say that you can do smaller experiments also so you have a 52 gene panel and you have some 12 samples to be done so what you are going to do is you index these 12 samples by 12 different barcodes now these barcodes so when we said that library preparation is all about enrichment of the targets and ligating adapters so when we are ligating these adapters you are using barcoded adapters to differentiate one sample from another yeah now this barcode is a 10 base pair unique sequence which is present in the adapter yeah which is which will finally help you to differentiate between different kinds of samples which you are loading on one chip so once you have once you have ligated these adapters you pull all these libraries now you are you are working with probably six samples on a chip so with based on the number of samples you plan to multiplex on a chip you are going to use the number of barcodes once you have done that now you want equal amount of data for all those libraries what you are going to do is in terms of their molarity concentration you will use equal molar concentration for all those libraries and make one pool of that but the aim is that when you are doing smaller in a sequencing for all these samples you are aiming that you generate equal amount of data for all these samples but because otherwise probably some of the samples have worked out pretty well and some will not so to ensure that you need to pool all these libraries in equimolar proportion so once you pool that after your library preparation step now you started with 12 samples or you started with 20 samples now that becomes one sample yeah and now how are you going to differentiate now because it is now one sample but you started with 20 samples so when you generate the data how are you going to differentiate yes we use barcoded adapters so the system is first thing it's going to use when it sequences it sequences the barcode it's barcode sequences and then it sequences your region of interest and then it makes the bins that okay this read belongs to this sample and it is in this bin once we are through with our library preparation then the next step is template preparation so in template preparation the aim is that you clonally amplify this so you have prepared you started with targeted sequencing or you started with whole genome sequencing or you started with an RNAseq experiment you have tons of target millions of target molecules which are to be sequenced so what we are aiming during template preparation is that one library molecule gets clonally amplified onto one ion sphere so this is the basic aim of this particular step and so this library molecule gets amplified onto this ion sphere and this is one clone which is getting amplified all over on the ion sphere then there could be some ion spheres which have not got amplified yeah so you do not want to load them on the chip so you negate all those ion spheres at the enrichment step yeah so what is happening is that we are using ion spheres which have an oligo which is linked to these so this oligo is complementary to one of the adapters which you used during library preparation so if we go back a few slides so we used two adapters P1 and one was barcoded adapter so and we mentioned earlier that we are basically exploiting these barcode these adapter sequences for us to enable these sequence to sequence these millions of fragments later on on one chip so we are using this particular P1 adapter sequence yeah for the library molecules to complementary go and bind onto the ion sphere so now if we go back to that slide so these ion spheres are loaded or are coated with oligos which are complementary to the P1 adapter so when these emulsions are being created you are basically making these water molecules which is surrounded by oil and you are trying to amplify these library molecules so when the this emulsion is being created this is a random distribution of these ion spheres with the library molecule and enough PCR components for it to amplify this library molecule onto this ion sphere so what is happening is a library molecule is coming it is getting denatured and it is sitting on the complementary sequence which is complementary to the P1 adapter and then it goes ahead and it sequences this part and that's how you sequence now this goes through a cycle of denaturation annealing and extension like your normal PCR but when while we are talking all this has been so you are not manually involved with all this process all this is being done by these systems either semi-automatedly or fully automatedly so then when when it gets denatured it then again binds to another oligo on that same bead and you then that's how you are generating you are clonally amplifying this particular molecule onto the ion sphere so what is if we go into the details what is happening is you have an emulsion oil then you have a library molecule you have some primers this is your ion sphere this library molecule gets denatured it gets bound to the ion sphere it gets extended again gets denatured now this template goes and binds to another oligo and finally you have this one library molecule clonally amplified onto this ion sphere yeah so now we said that there could be some molecules or some of those water droplets when the emulsion was being created they did not get a library molecule so they will not get amplified also but we do not want to take them on the chip because they are still going to occupy some space on the chip so why to lose on that data so what we are doing at the enrichment step is when we were doing amplification on the ion spheres one of the primer was bitonylated so now all the amplified ion spheres are bitonylated we are using strepidine coated beads to fish all the amplified molecules and rest all molecules remain in the solution yeah and then we just go through so this is primarily your ion sphere this will have these molecules all over because we clonally amplified so you use strepidine coated beads and this strepidine coated beads are magnetic so with the help of a magnet the all these are fished out and then you have only the beads which are amplified which are finally to be loaded on the chip so this is the enrichment part of the template preparation then what is happening at the sequencing level now I showed you that chip that chip has millions of wells and each well is working is generating a sequence so your so this is your chip it has millions of wells and if we are now to consider that this is one well this one well has one ion sphere which is loaded with one kind of template so what what is happening during sequencing is that when we are when ion chef is preparing the sample for the sequencing it is already adding the primer and polymerase to the chip itself now what else it needs for extension dntps yeah so what the system does is it is adding dntps sequentially on to the chip and then naturally we know that whenever a dntp gets incorporated or gets polymerized into a growing chain of dna there is a there is a bond formed between the phosphate group and the hydroxyl group yeah and this phosphodiester bond when formed this releases a hydrogen ion yeah so now we coated these library molecules on to this ion sphere so this same even so supposedly the first base here is an A yeah so you you had flown dttp so it will have a complement it is a complementary nucleotide it will bind and it will release hydrogen ions and these hydrogen ions get converted into a voltage signal which is finally being captured so this ion basically is bringing these release of so many hydrogen ions in one well are bringing up a change in pH for that particular well yeah and when the pH is being changed for that particular well that change in pH is being recorded as a change in voltage so basically you are converting a chemical information into a digital information and that is how you are saying that a base a signal has been received and a base has been called now supposedly now we said that T is your or A is your first base and the system had flown a dgtp so now that is G is not going to bind to A yeah so what will happen is there will be no binding no hydrogen ion release so no no change in pH no signal detected by the system so we will just see this by a cartoon so considering that this is your one ion sphere these are your adapters yeah this is the barcoded adapter and this is your adapter which you used for ligating these sequences onto the ion sphere then what is happening is your primer is coming in binding here you add the dntp sequentially now what will happen is this will bind you will have a change in pH which will be recorded by the system and the system knows that it is flowing a dgtp at this particular time and that is why it can associate this signal with an A yeah so we are not using any fluorescently labeled dntp so this is a simple chemistry now what will happen in the next so we had flown in an A and the next sequence next target base is G they are not complementary so it will not bind it will get washed off then again so likewise you will build up the sequence now what will happen at this step supposedly we are flowing a dgtp what will happen sorry yeah since there are two A's in a stretch and there's nothing stopping it it's a growing chain yeah so you will have double the signal yeah so there are double molecules double number of hydrogen ion release which will bring up a double change in the voltage and that is how it is being recorded as signal which is double and that is why you can tell that there were two consecutive A's in the sequence so then this by adding the dntp sequentially finally the system is being is creating a sequence which is finally being read as an ionogram so this ionogram is again being read by the sequencer this has to be read from left to right and for the number of bases so whether you get a signal or not that will define the sequence of the dntps the intensity of the signal will define how many of those dnt how many of those A's or T's were in a row so hope you got a glimpse of next generation sequencing technologies which has really revolutionized the field of zero max you were given the basics and some possible applications using this technology platform and one specific technology of ion torrent was also illustrated and discussed these concepts will be again covered in more detail in the following lectures when we have invited some more industry experts representing different technology platforms who will also provide you the basics and the current status of these technologies so see you in the next lecture thank you


So, that's why I chose Cal. It was this spirit, the spirit of Berkeley in the 60s, the spirit of Mario Savio, and the members of this free speech movement that basically sold me there on the whole idea of becoming student activists, becoming major game players and changers in the world. And so I just wanted to celebrate, I'm thrilled to celebrate the 50th anniversary of the FSM with UC Berkeley and I just wanted to thank those members and the new members who are going to join with me in part of this movement, the ones who will inspire the rest of the world for change and social progress and justice. So, go Bears!


What's happening? It's Shane here. And today we're going to be talking about the most regretted college majors, right? So these are college degrees that people go to university, they spend all that time and money, probably go $40,000 in debt in order to get the degree, and then they regret it. Now, of course, there are going to be ones on this list, there's going to be people down in the comments section that say, I got this degree and it was the best thing that I ever did. But overall, on average, these are the degrees that are the most regretted out of all of them. And I'm basically going to be going over for, you know, the reasons, at least in my opinion, why that is. And it's so difficult to do research on the right college degree for you, because a lot of the time, people have this preconceived notion in their head that a degree is going to lead to a job. And that job is going to be a certain way. But the reality is completely different. But by the time they figure that out, it's too late. That's why I made this channel. That's why I make all these educational videos for you guys. And also I am getting amazing results from my College 101 course that you can find down in the description below. I have a very limited time coupon on that. It's probably the cheapest that it's ever going to be at this point. So before we get into it, make sure to gently tap the like button, hit the subscribe button, ring the notification bell, and comment down below any thoughts, comments, criticisms, etc. that you have on the video. And let's jump right in. All right. So number five on the list is going to be communications. This was a zip recruiter survey and they found that 27% of people who got a communications degree really regretted it. Right. And the biggest problem with communications, very obvious. It's just too general. Like there's so many different types of communicating. You can communicate with someone over the phone. You can give a speech to someone. You can communicate via writing. You can communicate via making YouTube videos. All of these different forms and mediums of communication are going to be completely different. Now, if you became an expert on one of those things, like starting a YouTube channel, making YouTube videos, hey, you can make a living from that. You can make a lot more than a living from that. But the problem with communications is just too general. Hiring managers, business owners are not going to know how you are going to add value to their company. And so many of these people probably graduated and they weren't able to find a job and then they, you know, were stuck with all that student loan debt. And that can be very disappointing. Next one on the list, number four is going to be social sciences and law. According to Zip Recruiter, 29% of them regretted it. And the reasons because it was too general, impractical, and it's hard to find a job without further study. Right. So I've said this many times before, you know, a lot of the social sciences and liberal arts degrees, they are going to be very difficult for you to find a job in that field. Many people, I would say most people end up graduating with a degree. They can't find a job in anything that has anything remotely to do with the subject of the degree. And so they either go back to grad school to get a master's or a doctorate, which is very expensive. It takes a lot of time, or they just settle for a job that doesn't really have anything to do with what they originally wanted to do. Now I think that social sciences, liberal arts teach you a lot of soft skills that can be very valuable, formulating argument, critical thinking, being able to give speeches, being able to write a paper. These are all things that are going to help you in many different facets of your life. But when it comes to the financial side of things, you really have to plan things out if you decide to get one of these degrees. Number three on the list is sadly going to be education. 31% of people regretted getting an education degree. And of course, they would get an education degree in order to become a teacher. Now, one of the positive things about getting these degrees is they have very, very low unemployment rates. So you are pretty much guaranteed to get a job. But unfortunately, the jobs don't tend to pay very well. And they also tend to be very high stress. And then there's limited job opportunities. A lot of the time people will end up going back, getting a master's, even a doctorate sometimes, because normal teachers just don't get paid all that much. Next one on the list, number two is probably going to come as a surprise to many of you. But that is biological and physical sciences. 35% of people regretted this major. Now, here's the thing about biological and physical sciences. I've done many, many videos on here about them. When it comes to STEM degrees, science, technology, engineering, mathematics, science degrees are the weakest out of those by far, you know, technology, engineering, mathematics, when it comes to the numbers, at least the financials, they are much more solid. But with that being said, science is still relatively good. It's not bad. But I think that people's perception of how good it is versus how it is in the real world is just completely off. And so people end up being extremely disappointed when they get a biology degree, a chemistry degree, etc. And they realize that, you know, the jobs that I'm looking at aren't going to pay me very well. Or in some cases, I'm going to have to go back to school in order to get a master's or a doctorate just to be able to get a job. And that's the truth for many of these science related degrees in order to get employed in the field. If you're actually wanting to do the subject that you studied, you're probably going to have to get a master's or a doctorate in many cases. But if you do get a master's or a doctorate, you're going to be good to go. So it's definitely kind of a double edged sword. There's some positives and some negatives there. I think the reason so many people regretted this one, in my opinion, is just because of the fact that they didn't expect it. Next one on the list, number one is going to be English and foreign languages. The regret with this one was about 42%. And to put that in perspective, guys, the least regretted degree was computer science and mathematics at about 12.7%. So 42% versus 12%. That's almost four times more people that regretted the degree. And the main reasons for this is that it's impractical and there were limited job opportunities. Right. So I remember, by far the most controversial video on my channel, which is the most useless degrees video, got over 2 million views now. I remember I put language studies and stuff like that as one of the most useless types of degrees. And I made a joke about how you can get Rosetta Stone or you can just go and learn it on your own, something along those lines. But I understand I got a lot of flack in the comments for that. People were saying, oh no, in order to be a train translator, you have to do this and that and blah, blah, blah. Well, there you go. Okay, the most regretted type of degree, right? So yes, there is certain cases, very specific cases where becoming a translator maybe going to college, getting that degree would be worth it. But for many, many people out there, they find that it isn't worth it because it's just impractical. If you are going to get one of these types of degrees, you know, because you're passionate about it or something like that, consider maybe double majoring in a different degree. That might help you out. Something that's a little more practical, something that is going to be able to get your foot in the door when it comes to landing a job. Or maybe considering pursuing that subject on the side, starting a YouTube channel, for instance, would be an awesome side project that you could do. But yeah, I hope you guys enjoyed the video. Check out my other videos right here. I made them just for you. Go ahead, gently tap the like button, hit that subscribe button, ring the notification bell and comment down below any thoughts, comments, criticisms, etc. that you have on the video. And I will see you guys next time.


Hello friends, let's discuss the following question. It says solve the following system of inequalities graphically. The given system of inequalities are x greater than equal to 3 and y greater than equal to 2. Let us now proceed on with the solution. The first inequality given to us is x greater than equal to 3 and its corresponding equation of line is x is equal to 3. So let us now draw the line x is equal to 3. These are the coordinate x's, this is xx's, this is yx's and here we have x is equal to 3. So we draw the line x is equal to 3 which is parallel to yx's. Now we have to identify the region for the inequality greater than x greater than equal to 3. For that we take any point not lying on the line x is equal to 3 and we'll see whether that point satisfies this inequality or not. If that point satisfies this inequality we'll shade the region which contains that point and if that point doesn't satisfy this inequality we'll shade the region which doesn't contain that point and generally we take that point to be 0 0 as we see that the point 0 0 does not lie on the line x is equal to 3. So x is equal to 0 and the inequality becomes 0 greater than equal to 3 which is not true. That means the point 0 0 does not satisfy the inequality x greater than equal to 3. So we'll shade the region which doesn't contain the point 0 0 for the inequality x greater than equal to 3. Now this is the point 0 0 and we have to shade the region which doesn't contain the point 0 0. So this is the region which doesn't contain the point 0 0, so we shade this region. Now the second inequality given to us is y y greater than equal to 2 and its corresponding equation of line is y is equal to 2. So let us now draw the line y is equal to 2. Here we have y is equal to 2. So let us draw the line y is equal to 2. Now again we have to identify the region for the inequality y greater than equal to 2 and we know that the 0.00 does not lie on the line y is equal to 2. So we take the 0.00 and we will see that whether this point satisfies this inequality or not. So here y is equal to 2. So the inequality y is equal to 0. So the inequality becomes 0 greater than equal to 2 which is not true. That means the 0.00 does not satisfy the inequality y greater than equal to 2. That means we need to shade the region which does not contain the 0.00 for the inequality y greater than equal to 2. Now this is the region which does not contain the 0.00. So we need to shade this region for the inequality y greater than equal to 2. So the region in dark grey is the required solution region which is common to both the regions but both the inequalities contains the sign greater than equal to. That is x greater than equal to 3 and y greater than equal to 2. That means the lines x is equal to 3 and y is equal to 2 are also included in the solution region. So we need to darken the line x is equal to 3 and y is equal to 2. So let us darken the lines to show that the lines are included in the solution region for the given inequalities. This is the required solution region for the given inequalities and this completes the question. Bye for now. Take care. Have a good day.


good to see you again. So I prepared some samples, some loops yesterday lunchtime. I actually had a different plan for them but I'm feeling the need to chill out so I'm going to try something a bit more relaxed with them and we'll just see where it takes us. I think I'm going to experiment with just letting these samples and loops just play with the envelope hold on forever and maybe use stepless tracks a little bit to manipulate the sound while it's going. Evening David. So let's start playing with that a little bit. As always if you have ideas or want to see things and just let me know in the chat or not as we go sample on this next track but I'm going to alter the loop length so that it's so it doesn't loop perfectly with the other one. What's the trick? Yes, sorry I do usually try and get that out of the way. It's Diplomatico this evening which is a rum I think I mentioned before probably my favorite or joint favorite rum in Santa Teresa which is also a Venezuelan rum very nice. Excited to see a Digitax stream, Digitax stream. Me too, it's as I've said many times before probably my favorite instrument. Let's get this other piano sample that I've prepared earlier looping on this other track and then we'll start playing with them. So this one's a bit more chordal trying to write it. I'm doing reverse because so I think I'm going to take Heimbach's half speed because half speed is best speed. So it's in shot really. I can put it further away if it makes people anxious. This device sadly, same reason I haven't tried Andrew Han's yet either which looks really good as well. So what I think I'm going to do start the sequence of going this evening is putting down stepless tricks that alter the sound which is going to drop it down to half speed. I had to pick just one feature because I think what I really like about the Digitax is so to talk about just one feature is tricky I think. But I guess ultimately my favorite feature of the Digitax is the workflowed parameter locking I guess which is also I guess one of my favorite features of the Digitone as well. Sometimes I superior my thumbs to my nipples and pretend that I'm a T-Rex. You've got to do what you've got to do in 2020 frankly. So I've done some tricks for it. Switches to playing it forwards that might be fun. Okay so let's take a similar approach. The tempo is forward. The samples I think were recorded at 80 or so I've hafted four two pitches because then I'll set a step probability so it's not always stepless tricks as well so we don't cut things. It's diplomatic not whisky this evening. The BPM likes to go so slow. Yes. How important is it to have these samples in sync to the tempo for what I'm doing not at all. Potentially allowing the samples to run over each other with the fact that I've got the tempo set this slow. The times on the Digitax are always a function of the tempo. So by setting the tempo I've got it set where I've got it set. In a case with this kind of music the Digitone for this sort of stuff I think are essentially a waste to have a single track that's dedicated purely to vinyl crack. We could try and ground it with some sort of there's no harm in trying. I'll just see if there's steps. And some of those will actually switch it back to forwards. Just going to call characteristics of them are being changed by the sequence but they're not actually being with my streams and I'll take it as a government. OBS is running her just a shelving EQ just for a bit more top end bottom end and a limiter. So you'll be hearing that dangerous backs plugins being run.


Earl Campbell Eddie George Give any George that's back up the middle 35 has room 40. He's out of here 50 45 40 35 30 25 20 15 10 5 So 68 yards Eddie George Touchdown Chris Johnson sweet left Johnson turns the corner dives touch down Titans Derek Henry Mariota Gives it to Henry Henry bounces it outside five ten fifteen stiff arm 40 All great running backs All for the same NFL Organization this week's Titans at 25 presented by Bud Light features, Tennessee's Fabulous for plus Amy Wells sits down with a money hooker as he settles in as one of the team's leaders You're watching Titans all-access and it starts right now But there he is that you only bulldozer Derek Henry that Chris more can he catch it? Will Leviss Leviss to Hopkins Welcome to the bet MGM studio and Titans all-access. I'm Mike Keith glad you're with us A money hooker safety for the Tennessee Titans signed a contract extension in September of 2022 and has become One of the top leaders on this football team Our Amy Wells had a chance to sit down with a money hooker and talk about his developing leadership style Team chemistry and a lot more in this week's Nissan insider To steal himself a money hooker with his second take away of the first half a money hooker another season You're five tell me about the coaching staff that you've been working with a little bit that I know there's some new faces There's some new people that you guys have gotten to experience. What have you liked about that? Um, I started with a DV coach coach Harris. I mean he's an exciting guy He loves the ball ex-player, so he knows the game He knows what it's like to to be in certain situations, so he's very relatable in that aspect I'm in coach low. I'm also seeing her how she works with the other guys the D linemen and The way that she just has energy and fire, you know, it gets guys pumped up. You're the youngest of what four brothers I know two older sisters and older brother having those relationships having a lot of siblings that you're maneuvering How did that experience help you be a part of a team and develop some leadership skills? Since I'm the youngest I was able to see you know my oldest siblings my sisters go through You know the same type of team aspect they had their struggles in different types of ways So I you know listen to my dad on his advice to them Then you know I was just like four or five years old growing up and then my brother comes along He's you know, he's a basketball player. He was a star in high school and in college So seeing how he was a leader of all the teams that he was on You know that just helped me kind of pick apart each aspect and what to learn from and what to apply to my life Does that make you comfortable within a locker room setting like having people around who have that camaraderie in that bond? Yeah, I mean, I think so I mean because we want to have a family atmosphere here You know, we want to have that connection. So when it comes to game day I know I can look at Christian and he knows what's going on So kind of like that that brother that sibling feel having that on the field in the I guess action of a game When things are moving so quickly it has to be so important that you can almost Anticipate not only the thoughts but also the movements of the people around you. Yeah, it's very important I mean, that's what we're doing now. We're trying to build that chemistry So then when it comes to get down to the two minutes or end of the game and the crowds loud You might not be able to hear the call across the field But everyone trusts each other and know that you know that you're in the right position What are things that you guys can do to build that chemistry because that's got to be something that happens all season Yeah, definitely all year long. I mean it happens in practice. It happens in a locker room Also happens when you're just eating lunch sitting down having a conversation Getting to know guys because the more you know them personable level and that way, you know They can trust you a little more. Is that something that you have to? Communicate maybe the younger guys to rookies to things like that like you've got to invest in these people Yeah, you got it. You do got to invest in these people I mean that's something that I try and preach like you can't just you know sit back and be be individual You got to be able to to put yourself out there and be involved in that way other guys can recognize that And they they trust that you want to be here and you want to know you love football Later on in this edition of Titans all-access coach Dave McGinnis joins me to go Beneath the surface powered by Microsoft to look at the quarterback the Titans will face this weekend in Jacksonville Trevor Lawrence But up next a look at a special relationship Continuing to build over 25 years the men and women of Fort Campbell Coming to Ascension St. Thomas Sports Park for another edition of the military Bowl. We'll show it to you next on Titans all-access This is stadium in 60 a quick update on the Titans new stadium presented by Nissan The new stadium will be built on a giant parking lot to the east of Nissan Stadium So in between the current stadium and the highway Titans president and CEO Burke Nihill gets more specific If you if you were to pull up a Google map right now and look at at the stadium site from above You'll see that there's a stadium shaped parking lot between the current stadium and the and the highway Which we have learned was was not an accident There was there was always a thought that maybe there would be a second stadium built and they wanted to preserve a space for it But now in this 2.0 version of the stadium You get to take those things that you loved about that central location about you know Just being a great place to watch football And you get to add to it these other elements that really no other stadium in the NFL has Where there's going to be this neighborhood that develops around it There's green spaces and open spaces that you're just going to change the game day Environment but also make it really fun to be in around the stadium you know in the middle of March Because it's just going to be this amazing part of town and we're so proud and honored and blessed to be a part of it For the latest news visit Titans new stadium calm It's time for the Hughes and Coleman decision of the week the decision to sign rookie Eric Garrett to the active roster has proven to be an excellent one Garrett has already made 10 tackles as the Titans nickel back in the last three games plus He has 62 yards on 10-punt returns Promoting Eric Garrett from the practice squad to the active roster is the decision of the week presented by Hughes and Coleman Welcome back to the bet MGM studio and Titans all-access You hear a lot about salute to service and it's vitally important all over the National Football League in Tennessee maybe even more so because of our relationship with the service men and women from the 101st airborne at Fort Campbell We invited them to come to our complex Ascension st. Thomas Sports Park for a little fellowship and some flag football The military Bowl is what it's called and it is this week's listen up with Duncan We're here today with the Tito's military Bowl to kick off salute to service month At a sense of st. Thomas Sports Park real excited to have a hundred and twenty five Active-duty soldiers from the 101st airborne from Fort Campbell. We've been doing this about five years. It's a great event Highly competitive as you can imagine It's truly a blessing to be here and to put up for the army is just amazing It was a great experience Man, I've been playing football for 20 years and I can remember so coming here and show my skills and you know Snagging on people, you know, I haven't everybody laughed having a good time. It's a great time Honestly the team effort I got the opportunity to play quarterback for 531st and we came out here to you know With one mission and I was to win the championship Collectively as a team came out and did that so this is one of our favorite things that we do all year It's a great kickoff to salute to service month. Our defense played extremely well in the championship In the modern era of the National Football League a lot of teams can claim that they've had two great running backs Maybe even three great running backs But none can match the Tennessee Titans organization having four all-time great running backs in the modern era Titans at 25 make sure you know exactly who they are and why it makes the Titans stand out when Titans all access continues after this You look at our running back Roll Campbell set the tone then you have Eddie George Then you got Chris Johnson They got Derek Henry. Those are our pillars as usual Keith Bullock is right, but let's take it a step further Can you name another NFL franchise that has four running backs as accomplished as these four a Lot of NFL teams can claim to or maybe three but four Can any team match the Tyler Rose? 27 CJ 2k and of course the King The franchise had good backs before Earl Campbell But Campbell's first four years in the league were like nothing since Jim Brown from 1978 through 1981 Campbell averaged over a hundred yards rushing per game 4.6 yards per carry and scored 55 touchdowns I think the girl Sheer all Played like a linebacker played the running back position You could feel his tape you can watch it feel it internally you had a Physically action when we watched Earl Campbell whether it was with a cringe worthy. It was The pain that you could feel that he was inflicting on people so That's that's what I think of when I think of when you think of Eddie George you think of incredibly consistent In eight years with the franchise. He never missed a game 71,000 yard seasons 74 touchdowns In Houston or Memphis or Nashville? Eddie George set the standard of excellence Man, he's he's a like hard nose. He wanted he wanted a pressure He want to come in there and he gonna run hard He been doing the same thing since he was at Ohio State Coming to the lead him and Ray Lewis having those great battles and I just used to love watch him play and play with him On dreamcats. I used to run run people over on the dreamcats game and stuff like that But yeah, he's a definitely a great player Chris Johnson was definitely a great player CJ had over a thousand yards rushing in each of his six seasons with the Titans Johnson had six ruds of 80 yards or more and of course he rushed for over 2,000 yards in 2009 CJ 2k I Felt like he touched the ball He was gonna score every time man. That's unbelievable at the NFL level So yeah, he was special like that. I'll never forget How quickly you knew that Some one of his handoffs was gonna result in a touchdown, you know some box You know get through the first wave then you've got to do a lot of work to still get to the end zone Chris Johnson If he had any crease at all, you knew he was gone Derek Henry has a 2,000 yard season of his own that came in 2020. He's got the 1,000-yard seasons. He's got the long touchdown runs. He has embarrassed Tacklers the king has tomorrow Campbell in it with the physicality. He looks a lot like Eddie George Only bigger and while not as fast he has CJ 2k's breakaway ability Campbell is like a superhero, you know Watching him and hearing all the great stories about him growing up and then going to Eddie somebody I looked up to got to watch play and Maira very dearly me and Chris Johnson who just brought the game to another level whenever he's touched foot to this organization the electric season that he had and all the great highlight moments, you know Something that you know, we can't can't compare to but there's a lot of great tradition about step Whenever I step on the field, that's the standard and something that you know, hopefully I can live up to one day These four backs have had the ability to carry the franchise in the biggest moments You could turn and hand each the football Understanding that they would use their unique skill set to help win the game Earl Campbell Eddie George Chris Johnson Derek Henry No other NFL franchise can claim four backs in the last 50 years who were as impactful They are this organization's fabulous for Derek Henry has had a lot of great games back in his hometown of Jacksonville That's where the Titans go next and up next on Titans all-access We go beneath the surface powered by Microsoft with coach Dave McGinnis to take a look at the Jaguars top weapon That the Titans must slow that's next on Titans all-access Wait mate mayfield under pressure hit Tear Tart gets it back outside the 40 yard line Titans all-access continues from the bet MGM studio look who's here coach Dave McGinnis from Titans radio welcome Great to be here ready to go to Jacksonville. Yes. I'm ready to go Jacksonville I'm ready to go over there to the telestrator first. All right Well, let's talk before we go to the telescope about the guy you're going to telestrate Anyway, Trevor Lawrence, we know him. This will be the fifth time we've seen him What are the Titans expect to get when they play against Trevor Lawrence? Well, first of all number one pick in the draft and for a reason this guy is a franchise quarterback The guy can do it all he can reach all aspects of the field. He's a smart guy. He can really move he can run So he's a he's a problem. He's a problem in the cylinder. He's a problem on the move He's got some really good explosive players to throw it to and what they've done now They will use a lot of different formations to get those receivers out for him But if you don't if you're not really really conscious about your rush lanes, Mike He can really hurt you with his legs. All right Let's see how we can move to the telestrator as promised coach Dave McGinnis going to show us some Trevor Lawrence here Right here, right? That would be him. All right The thing the thing we want to look at to start right here is this is an empty set You heard me talk about you heard me talk about that the weapons that he has Travis Etienne Okay, this guy right here does a lot more than just be a running back He's very dangerous in the screen game, but he can also move out and run individual routes He has moved out now to numbers plus splits anytime. I say numbers plus here are the numbers you move that way It's numbers plus. Okay, if you move this way, it's numbers minus. All right, so there's numbers plus splits He's gonna move out.  All right, they move out here. All right We see we see him the way the the way the Pittsburgh has been deployed. This is a man-to-man defense now They're deployed man-to-man He has got him. All right as he's looking out here. He's got three receivers. This is a three This is a three to split you got three receivers up here two down here But here's the matchup. He's looking for when he came out of the huddle Let's let it run he points for protection as they look at they get a four-man rush watch the move here It's over with it's over with that's Etienne to the house as a wide receiver on the back end Let's just watch it from the end zone. You can see watch him point He points as they start to move you're gonna get a four-man rush right here the safety stop it Mike The safety right here has moved to the middle of the field and once he knows the safety has moved to the middle of the field He is throwing to this side immediately because the safety has moved over here to top these receivers So it's a one-on-one to the top side. Let's take a look touch down Etienne I mean that was not a good matchup for the Steelers It was a great matchup for the Jaguars and Etienne can really catch. Oh, I mean he's a fabulous fabulous player Now what we're going to look at this is a what we call a trip speed formation It's trip speed because you've got three receivers up here to the top right here We've got three receivers up here to the top. You've got a close. This is closed back here We call this close because the tight end is in tight So you've got a trip speed formation up here to the right close formation Watch what he does now when he gets a little bit of extra room with his legs. Take a look at it, Mike Let's let all everybody take a look and see what he does here It comes he sees it pressure Gone now.  Thank you, Mike Keith. You're wonderful doing this. I try.  That's coming up next when Titans all-access continues Welcome back to Titans all-access from the bet MGM studio time now for my seat geek keys To a Titans victory in Jacksonville key number one. Do not let Josh Allen have a day I'm not talking about the Buffalo quarterback. I'm talking about the pass rusher for the Jacksonville Jaguars He's gotten better every year. He's been in the NFL Already nine sacks this season for Josh Allen The Titans must find him and block him and not let him dominate the day key number two Slow Evan Ingram and Christian Kirk on third downs Those are the two leading receivers for the Jacksonville Jaguars They also have 12 catches a piece on third downs That's where Trevor Lawrence likes to go with the ball most of all to those two players The Titans must contain Christian Kirk and Evan Ingram on third downs and finally Don't let Trevor Lawrence run Trevor Lawrence is six six two hundred twenty pounds, but he can certainly move He too is outstanding on third down the Titans must keep him in the pocket and not let him escape Because if he does he often goes for more than just five or ten yards. He can take it 25 to 30 yards Don't let Trevor Lawrence run on Sunday Titans in the Jaguars this Sunday from Everbank Stadium in Jacksonville We're on the air with Titans countdown at 11 a.m. Central time Kickoff set for 1202 central on your Titans radio station and in Nashville. That's our flagship 1045 the zone for our outstanding staff. I'm Mike Keith thanking you for watching and we'll see you next time