 Today you will hear again from Dr. Mukesh Jaiswal who is one of the application scientist works in the areas of next generation sequencing technologies. He will talk to you about whole exome sequencing kit for investigating rare diseases. This is probably going to be the last lecture on the NGS technology and its application and while today's lecture is not going to cover much of the basics of NGS, but it is definitely going to give you more information about possible applications from these platforms. So, let us continue on this lecture today and then we will try to conclude what we have learned out of these NGS based platform from basics to the applications. So, today we are going to talk about investigating the rare disease and its treatment with the Agilent solution. So, at this site, so I am going to cover about what are the rare disease and how basically it can be diagnosed by the NGS solution and how we can basically give the treatment to the patient. So, we have some solutions where basically you use Agilent solution for the diagnosis of rare disease and its treatment part. So, it is going to cover some background of rare disease and then I will talk about some part of CRISPR-Cas, how basically it can utilize for the treatment purpose. So, what are the rare disease? So, rare disease basically is a rare disease, it is affected 60 million people in US and Europe. So, it is a big number, it is quite a big number and 7000 rare disease basically known in till now and 80 percent reason of that is genetics, it is something wrong in their genetics. So, yes please. We are talking about 60 million affected in the US and Europe, is there any study called any? I am coming to the next slide. So, I am coming to the next slide, so this is like some word why I am telling. So, now I am going to the next slide we have, I have Indian data also and 50 percent affected children are basically affected with this disease. So, coming to Indian scenario, so ICMR the Indian Medical Council of Research launched this registry in 2017 and they said that around 70 million people in India is also suffering for this rare disease. So, it is a quite big number and that is why they launch a project ICMR registry where you can basically write a grant to them to work on the rare disease, what is the problem, how you can diagnose that thing, what is the treatment part of that. So, in 2017 they launched this project and so these are the key objectives of this ICMR registry. So, it is main objectives that to understand what is the problem of rare disease, what is the causation of that and how it can be, how this data can be utilized for the treatment of the rare disease. So, these are two main objective of the ICMR and so it launched and I think it is available for the grant application also. So, I am going to talk about some of the rare disease which is existing worldwide, it is worldwide. So, let us come in the first cystic fibrosis, this one. So, this is the disease basically where is the excessive mucus is deposit on the lung or pancreas which cause the respiratory failure and inability in the digestion part. And the median survival rate for this disease is 40 years, right. And the worldwide is 70,000 patients are known worldwide. Then leukodistrophy, this one and this is again the progressive disease affect the brain spinal cord and the nervous system. And this is basically children's basically affected with this disease and 5 to 10 year basically children's and around 60,000 children's are affected worldwide. Retinopigmentosa, this is another disease and it is affected, it causes the blindness and it basically the survival is like 40 year and around 100,000 or 15 million, 1.5 million worldwide patient are known for this disease. So, what is the cause of this? What is the cause of this? The cause of this basically sometime it is affected by the single gene or sometime it is affected by the multiple gene, right. So, if it is like the first one, cystic fibrosis is only the one gene that is CFTR, there are several mutational CFTR and then this transporter, transmembrane transporter is basically disturbed and that caused the cystic fibrosis. So, in this cystic fibrosis only one gene got affected, right. But if you see this, these two the multiple genes are affected. So, it is very difficult to identify when the multiple genes are affected. So, here like 30 genes are affected here and in this disease is 77, right. So, problem is that this caused the damaged neuron and here in the retinopigmentosa it is cone cell and the road cells are basically disturbed, right. So, these are the disease and they are multiple genes are basically involved there. Some more which is including the ICMR project basically. So, this is childhood ovarian cancer, ovarian cell carcinoma, andrometerosis and they are multiple genes basically involved in this disease also. So, this is also incorporated in ICMR registry. You can go in their website, you can basically look what are the rare diseases. The challenge is that is that diagnostic, how to diagnose this disease, right. So, if you go the regular process of diagnosis of this one, this one is pretty expensive and doctor basically takes at least 8 year to diagnose and they go like 40 different methods to diagnose this test. So, because the complicity of disease is not like one gene, it is like multiple gene they are affected, right. And they basically they take at least 7 year and 40 method to use for the diagnosis. So, that is the challenge, it is pretty expensive, it take time. But for a treatment purpose if you have early intervention of this disease, you know the cause of early, very early, then the very early diagnosis then you can do early intervention and then improve the quality of the life. So, that is this is a challenge, but if it take a lot of like 8 year to diagnose only it would be difficult, right. So, that is why it is very important to diagnose the disease in very early stage. So, right now if it is if you see this rare disease now 2012 when the exam panel just started, it was only 130 gene. Now 2017 is more than 200k mutations are known for the rare disease. Basically, this is because of the more advancement of the exam panels. So, it is you need to extract the DNA from the patient, right. And then go for library preparation, library preparation and then target enrichment after seeing the library is basically perfectly fine, you can sequence and go for data analysis this kind of one workflow basically you can use our exam panel for the diagnosis of the rare disease. So, the challenges is always there, but I would talk about why the Agilent's exam panel is more better actually in sense because we make RNA Bates and it is it is oligo Bates basically and these are these are because we make the RNA Bates they have the better RNA DNA hybridization and these are the high fidelity bases which we make by the inject technology we make this this Bates by the oligo. So, this is our high fidelity bases if you see the error rate in the base in the probes basically is very low in Agilent it is like 1 or 2 error basically in 1 kB for others have lots of. So, we have we have the high fidelity probes basically which are biodegraded it is used for them making the libraries. So, this is the different ways you can make the exam libraries. So, starting with always with the genomic DNA right we have three different ways to make the libraries for the exam sequencing one is xt, xt2 and qxt, xt is basically is this one if you have a different patients 8 patients right you can make a individual library from each patients independent capture and you can pull while while sequencing right when you go for sequencing you can pull this sample and go for one sequencing run. So, that is the xt preparation of the exam preparation another if you want to do the some comparative study you can bar code the patient sample and pull itself 8 to 16 in one pool you can follow by the capture and go for sequencing. So, if you can compare between the patients also that is xt2 and another is library is based on enzymatic sharing. So, you can use transposition enzyme for the to make the libraries basically transposition enzyme then hybridization followed by the sequencing this is the fastest way you can make the libraries for the exam sequencing ok. So, performance for all these method to make the exam library are is pretty good and they get they get very good coverage more than 95 percent coverage all the methods this is the three pillars basically echelon works on basically performance of the exam library contents and the flexibility you work from decades to improve the performance content and the flexibility of the kids basically. So, if you see more of most of the rare disease basically now studied by this panel is called a clinical this is exam panel and this exam panel contains the all the axon region and also the the panels are not the intronic part of the region this means which basically is associated with the inherited disorder. We have the latest exam panel v7 this is mentioned v6 but we have now v7 and that basically cover the all the translation and the clinical research panel it cover whole exam. Another we have the very small panel of focus exam basically is covered a disease is a decision. But most of the most of the rare disease basically which I talked before it is basically used for the clinical research panel. So, if you see the contents performance of this one with this clinical research exam panel they have like a 5000 gene is basically it deeply covered with the disease associated with the clinical exam and the challenge is that when you when you when you go for the diagnosis of rare disease the most of the mutations are present in the GC that region to make the library for the GC that region is always challenge. So, but in with our clinical exam panel the performance in the GC region is very good it is very uniform preparation of the library when you go for the GC preparation. So, content basically what is the content on the for the for the for the for the probes basically it is basically designed by the Dr. Madhuri Hegde from Emory University and they make the basically the probes which is covered all the disease associated is an exon and entronic part which covers maximum rare disease parts. For example, this is the liquid distrofit right and the this pathogenic variant that is GC2 this gene basically if you see compare because this is the GC region and if you this compare with the other vendor you do not see any coverage there is no no coverage for this gene and if you see the clinical exam panel we covered this part also to able to read the 5 prime UTR variants with this disease. So, in the disease this is the kind of pathogenic variant and you can easily get it by the clinical exam panel. Another red net is pigmentosa if you see two other vendors this CFD1 gene is not covered in this part because this is entronic part we are exonic part entronic part are not covered right. But if you see our one is is fairly covered that part that means is detection of that mutation is is very easy on that then if you see this region again is well covered by this this this is these are all non-coding region these are well covered with the with our clinical exam and most of the pathogenic variant basically in for rare disease is present on non-coding region. So, it is is is fairly covered with the clinical exam panel. So, if you see overall in in the clinical test panel we cover all the clean variants pathogenic regions and it cover mostly like 98 percent regions are covered and others basically has low coverage. So, if you think about when you when when the doctor going to diagnose this Lycodistrophy it take eight years right by the normal method and the average test basically they do around 20-30 tests to do to diagnose for this one it take eight years right and the cost is is goes up like it is like 20000 dollar right but when you do one simple test our clinical exam panel easily identify this mutation and that is basically for the Lycodistrophy and it is very cost effective right and so it is is just going to cost like like 15000 rupees right. So, one test cost like 15000 rupees for exam panel because covers the intronic path exam path definitely it going to detect that thing but but detection rate is faster right and it take less time. So, doctor start their intervention much earlier with that like suppose this is our container we we does not make this containing vacuum we do the research and we make we make the probe spot cover all those reason right but sometime when you did some you experiment right and and some part basically you think that this is the part maybe pathogenic part and it is missing right and and you want to incorporate that part in your panel. So, it is very flexible we can customize the panel according to your requirement also. Suppose any any gene and it is not basically the intronic part is not covered and if you want interested I want to cover this part also we can basically add this panel and incorporate in your panel. So, that is our flexibility. So, we work on three parts performance and the contents always optimize year by year and then flexibility if you want to add more right. So, it is very simple workflow that I I told it start from the library preparation then we make the targeted panel by the probes right and then data analysis and reporting whole workflow basically takes three to four days and and is easy to identify the kind of challenges for a diagnosis of rare disease right. So, now this part is kind of over like if you if you if you get some kind of mutations right in in any disease not only in rare disease first cancer right and it is multiple mutation and you want to solve this problem for a treatment purpose. So, we have a tools called as the CRISPR-Cas where basically you can do do the gene editing right and to to to to solve to fix that gene for the for the for the treatment purpose. But this is very early we launch some libraries for the treatment, but it is very early stage. Let us start with that what is a CRISPR-Cas this allow you basically to allow the mutation to correct maybe or gene editing. So, it is based on the guide RNA right. So, this is the guide RNA this one and this is the site of recognition and this is the Casman enzyme. When this become active they attach and it goes to the PAM site like this if it is identical or hybridized on that like this it is identical with that it creates a nick right. This Casman enzyme when this guide RNA is specific to that it create a nick. Now, it allows you three possibilities. One possibility is that just leave like this and body repair body system our cell system basically go for non-homologous joining and cause the knockout of that gene right. It creates a knockout. Second is the homologous recombination if you want to do the gene editing and use some half homologous sequence you can incorporate this homologous sequence in that right. Third part this is the deactivated Casman enzyme. This yellow color part it might be activated or deactivated depending upon the activator it can induce the gene expression or reduce the gene expression. So, it allows whatever mutations basically you got from the exam panel right. You can basically try to correct or you can do the gene editing for their treatment purpose. So, that's whole story exam panel and what's their follow up. So, we are working on the functional genomics where basically we can try to edit this genes in a large scale like and it might be any knockdown knockout and knock-in anything and try to edit their genes to solve the problems. So, this is the if you study the functional genomics so very first prospect is knockout right. If it is knockout means guide and it breaks that one it makes the truncated protein right. So, if your protein not going to work is truncated protein. Knock-in means is basically it going to add some tag on that on the protein. Turn-off means if it is a high expression this is the this is the repressor fusion. If it is turn-off means the lower expression of the gene this is the genomic mutagenesis. Here basically it's a site especially mutation can create by the CRISPR-Cas. Suppose you got some mutation and you want to solve that mutation right you can change a base by base by the mutagenesis right. So, this way you can correct that SNPs you got right you can correct that part. So, this allows you to site specific changes with the Cas9 and if you want to do some genes are basically lowly expressed in sub-degies you can basically induce the expression to the higher level right. So, it can induce the gene expression. So, you can do multiple function by the Cas9 you can induce the gene you can you can repress the gene expression or you can do the site specific changes in the gene right. That's allow you five different possibilities to do. So, now the question is that suppose you like insistive fibrosis right you work on single gene and there may be one or two mutations you got with the exam panel right and you want to fix that problem right. So, there's two ways one way means if one gene and few mutations so basically you're going to use four to five different guide harness not more than that right. So, for that one you can make the guide harness in your lab. So, suppose this is this is the target basically you're going to you going to make a guide RNA for that one only. So, this is the target panel target target you want just add the T7 promoter on that right. If you add the T7 promoter on that and go for so the kids basically what they have they have a T7 primers and go for in vitro transcription to make a guide RNA for in vitro transcription you make a guide RNA which is the reverse complementary to the target which is going to be reverse complementary to the target. So, this is the kind of guide RNA this is this is the red part basically is very specific to the target you to the part where you interested for and this is the backbones minimum backbone and this when this going to bind on the target gene this this targeted part in the presence of Cas9 this gene basically cleaved into part. If you verify this one in the presence of Cas9 this gene basically cleave off right. So, this allow three two three five different possibility again basically going to maximum time you going to knock down the gene right. So, if it is your genes in numbers as one one or two gene and you want to use the you can make a guide in your lab also right. But mostly it's not a case it's not a case there are multiple multiple genes are going to is involved for the for any complex genetic genetic right like written pigment was a 77 gene is involved and mutations is like more than thousand mutation is basically involved there right. So, what we do we make a guide RNA for that for that panel right and this is totally custom custom thing is we don't make we some make some catalog but we some totally depend on the users. So, we make a guide RNA on a slide we cleave that one from the slide do the PCR amplification and packed on the viral particle noise ready to transfect in the cell system that much guide RNA basically which is for that mutation right it going to ready for transfection and you can use for the therapeutic opportunity for this. So, this is the this is. So, the target cells are actually cell culture. Yeah, cell culture. So, it's a viral particle these are these are cells basically to transfect directly into there. Transfect with this guide RNA they already have a cast-nane enzyme. So, it create the means cast-nane is there right. So, it going to cleave that part of the gene or is depending on what what you designed basically. So, those cells will be cleaved. Yeah. Then how will they go? So, see I told you this is in very this is very early stage again to do this thing you need to do lots of screening lots of engines work to identify it's going to work or not. So, this is just a thought and guide RNA is we can synthesize for you but again the protocols and how are you going to translate to the actually patient is very is in very early stage. If you select 10-20 gene you can make in your lab but it is more than 1000 gene in high scale then we can make. I mean it's for the high scale not otherwise you can just make in a lab just just your any target at T7 yes specific target just add the T7 promoter on that use T7 primer and do in vitro transcription that's inside the guide RNA. So, it's very straightforward protocol but yeah again you cannot make the 1000 guide RNA for multiple gene right. In that approach you can use this these guide RNA strategy and try to solve the problems but yeah I would tell you this is this is not easy it's very very hard right it's very hard because if you see this is going to be like thousands of the guide RNA and going to translate in the cells right now you're going to go give some treatment by drugs right and then you need to go for screening protocols day by day and again you going to verify this this thing going to be work by the validated or not by the NGS but of course this is the thought and you can basically try to solve that problems. So, in that contest echelon is happy to collaborate with the people who are interested for to make a guide RNAs right and we already had some some guide RNAs of different disease like if you have the cancer panels you already have the these are the genes and these are the guide RNA we make already have some and they are multiple multiple panel like mitochondria, gene expression, protein membrane these are the panels we already made we already cataloged the panel you can use that one but again the how it going to work and how it going to screen it is little difficult task. So, in summary means you can use the NGS application to identify the causation what's the cause of that and and I give the little brief idea how the CRISPR-Cas can be best solution for the drug validation and personal genomics. So, this is this is something you can use the NGS application to identify the problems and CRISPR can might be used for for the treatment part right. So, now I'm covering a five five five slide from the IVF segment. So, in reproductive medicine the most challenging part in the IVF is that a aneuploid in the embryos whenever couples go for the IVF and they do the in vitro fertilization and 70 percent are the embryos are basically aneuploid right and so what doctor do at least in India what doctor do they they look the good looking embryos identify it and basically implant like three to four embryo sometimes two sometimes three depends. So, the challenge is that when doctor do this thing if the good looking image aneuploid not going to implant so your IVF cycles fail if it is two embryos are good so both going to implant it going to give twins if it is three good embryo it going to go three kids right. So, it's a challenge you get either one two or three there's no there's no control on that right. So, we are discussing with the IVF cleaning and working on a single embryo transfer paradigm means check the embryos they are good enough they are aeuploid embryo not aneuploid and only one single aeuploid embryo is basically go for the further IVF cycle and for the implantation. So, we are discussing this thing with single embryo transplant doctors basically do they do the blastocyst culture and they don't do they do the biopsy and frozen on the embryo but they skip this part PGS pre-implantation genetic in India and after what if frozen this embryo taken and go for the IVF. So, they use three to four embryos and directly implant what we are talking to doctor do the PGS identify the good embryos once good embryo and implant process for the implantation. So, the couples get going to get only one kid right. So, when when we talk about the problems if they with the pregnancy goes with the two twins there's a lots of problem with the pre-term labor pre-claims here and it's high rate of the paedal death in the twins right. So, that's why we talk to doctor go for this test means you can go for frozen with that do the PGS. PGS means you just go for the screening of all 24 chromosome identify all the chromosomes are good enough identify the uploid embryo and then go for the IVF. So, how basically they do by transferring the one embryo basically it also costs effective for the couples also it's five type less pain basically to grow the kids right. So, how they do this test is basically done by the biopsy of the embryo. So, this is the embryo and this is the this is the blastocyst and they take the one cell of the embryo only one cell of the embryo. So, this is one cell they collected. So, when this biopsy is done one cell is collected from the IVF embryologist and for that because one cell has a very low amount of DNA you cannot do anything with that. So, we do we do the whole genome amplification to increase the quantity of DNA right and we label with this DNA with the with the Psy 3 and Psy 5 type right and hybridize on a micro reslide and go for the analysis right. If there is some mutations this is the mutations if some mutation on the chromosome number 3 or chromosome 20 22 or deletion at chromosome number 21 it shows that this embryos are aneuploidy and do not process for the for the IVF right. So, you can identify this by adding the PGS test you can basically identify the aneuploidy in 8 hours and and after do this basically the success rate of the IVF basically increase when they do not do any test the success rate going to be 80% with this single embryo transfer paradigm right because she identified the all the chromosome ways that there is a problem if it is a deletion do not process for the IVF. So, this is the smart ART right before to doing process for the aneuploidy just do the PGS validate this embryo good enough and then go for the IVF site right. Thank you. Alright so I'm sure after listening today's application based lecture you must have found this very interesting and you saw that how the whole exome sequencing kit can be used for diagnosis of rare diseases and how the results can be used to choose the right treatment. I'm sure this is just one of the success stories of many things which can be done on various type of NGS based platforms. Dr. Jaiswal also briefly gave you an idea about CRISPR-Cas technology which is one of the much talked about gene editing technology is available and I hope you have enjoyed not only today's lecture but also the series of lecture which we had in the last couple of days and week about NGS technology platforms and this is one of the revolutionary technology which is really transforming the way we have seen the medicine and clinics are really you know getting revolutionized with much faster pace of assays coming to the clinics for the patient care. So your understanding and your knowledge about these applications and these novel technology platforms are definitely going to be very useful and I must say there is a wealth of data available now from various type of genome sequencing projects if you know what you're looking for you can do a lot of data analysis from yourself. I'll give you one instance one example the cancer genome at least TCGA is one of the good resource for looking at the you know patients cancer data available and while they published that work couple of years ago in science and nature series of papers published but what is more important when they made data publicly available for thousands of patients genome data then their meta data analysis from that data many people have looked at very specific type of questions what is the impact of given genes in this patient survival for example or looking at a specific pathways and you know maybe hundreds of papers are actually published just by looking at data alone not by generating data right. So what I want to convey you is that you need not to generate the patient derived genome sequence data just for the sake of looking at everything biologically you if you are interested you can just go download these data use many of the publicly available software and resources analyze in your own manner and then probably you can get some very meaningful and new information even possible just by looking at these data for addressing certain questions. So I hope some of these exposures what we are trying to provide you is going to really make you more comfortable and also make you more enthusiastic and motivated to really take lead forward. Well I'll thank you to stop today's lecture but we will have more exciting things to continue in the next lectures as well when we talk about a new resource for you which is human protein atlas. Thank you.