 In the last lectures we started discussing about the revolution of next generation sequencing technologies and intention is to provide you the details of current leading technology platforms. In this slide we discussed about iron torrent technology and today Mr. Praveen Illawe will talk to you about a specific software called Oncomine knowledge based reporter used for the oncology studies. Many times when you are able to generate large data set or big data set from the you know these kind of big technology platforms like NGS or mass spectrometry it is really crucial to broaden your views and think about how to now use that data to think about at the systems level information. For example you have obtained now genomic information can you try to integrate that from the other data sets available you know from the other communities other databases can we try to integrate the you know DNA at the with the RNA level as well as protein level. So now when you have the information from genomics, transceptomics and proteomics you can try to integrate that and obtain some of the systems level information and some of these softwares which are available even commercially or even open access softwares they do provide you ability to integrate information from variety of data sets available from different databases. So I hope today's lecture is going to be useful for you to get understanding about one of such software and resource available for you to do a deep data analysis. So let us continue with this lecture. So I will take a step by step mode. I first did designing on AmpliSeq right, then I did the torrentsuit software where it decodes all the bases and provides you the sequences for it. It provides you alignment with the reference genome. It does the coverage analysis for you which looks for the regions which have been designed your interest, gene of interest and then take it further and do variant calling. So once you have done with the variant calling you just have variants with you you have just the SNPs giving you the change from A to T, C to T or just the deletions A is deleted or T is deleted but you still need to know something more about it where exactly this is happening, which gene it is happening, whether it is actually having deleterious effects or not, whether it is having any effects with the patients or not. So you need to know something more about that. So for doing that there is one more tool called as ion reporter software. So it has lots of information inbuilt into it. So this helps you to correlate your variant with the information that is already stored into databases and reporter software is available as cloud based. So you can register yourself and sign in and utilize it for your analysis. During variant calling we have a format called as VCF which is generated over here. So whatever variants that you call up these would be provided into a VCF format and these VCF formats could be easily uploaded on ion reporter software. Now this ion reporter software will help you to understand the variant positions, take it further and do certain statistical calculations. Have you heard about the protein evolutionary study parameters which are called as SIFT, polyfam, grantham. So these are certain parameters which are actually calculated when there is a protein change happening into a particular like you have a variation coming in and a protein change happening into it. So this values or these parameters also defines whether a particular change is deleterious to that protein or not or deleterious for this region or not. So these parameters helps you to also filter out whether these variants would be really deleterious or not. So the software ion reporter helps you to calculate those protein evolutions over here. With that it assigns databases from DBSNP, Cosmic, OMIM, DGV where it correlates with all the information that is related to your genetic disorders, cancer and various other genes that are oriented to it, genes transcripts that are related to it. So you must have heard about NCBI, NCBI is a database where you have lots of information stored whether it is gene, OMIM, DBSNP, so every information from there is correlated over here in ion reporter. So what happens over here? So you have torrentsuit software which helps you to plan your runs, run your data, generate your data, generate your variants and understand what is the coverage across those regions. You are getting results over there as variants, mutations. You take that further and put it into ion reporter. My ion reporter will help you in integrating, annotating and interpreting my results. So you will have more information to read about where you will understand your variants are really important or not, whether you want to keep it, whether you want to utilize it or you want to filter them out. So this tool comes in lots of help over here. The same thing that I was talking about. So you have got variants from torrentsuit software, from torrent variant caller and then you are bringing it to a biological meaning where you can understand what the variant does into your samples. With the same I will just take it forward with ion reporter software which helps you to understand what can be done over here. So I have a way for going forward with it. You sequence your data, you take your data, you can import it onto the ion reporter software, analyze them for whatever you want and take it further filtering based on your parameters available. So what happens filters may be of different types, it may be your coverage filters. You want to take variants which are at a very high coverage, say you want 1000 reads should be covering a particular region, then only you want to take those as a particular variant called variants, right. You have filters for SIFT polyfram grantham, the protein evolution parameters that play an important role over here. You can also look into databases like you have UCSC common SNPA databases which has information which are the common SNPs which are not affecting at all in any conditions to any of the proteins. So you want to filter them out and you have to take them out and keep it separately. So whatever remains back, you take the databases, you get the database information and can be downloaded or even shared with different people across, ok. Once that is there, once you have done that, the same thing that I was talking about, importing your data onto the system, analyzing them by various filters that are available and taking it further, reporting it further, ok. So what happens over here is, I have in ion reporter software, I will be detecting different things, not just this ion reporter software is not just for your variant analysis actually. There are lots of other tools that are also available in ion reporter, ok. We have something for the variant detection, that is one, ok. But we also have something called a 16S metagenomics with us, ok. This is something very different from what you do with the genomic level. This is something with the microbial content or microbial identifications that you do. So if you have bacterial samples or if you have certain samples that are coming for 16S sequencing, we have a kit which helps you to do that 16S sequencing onto the ion torrent system and then you can do a straightforward analysis in ion reporter over here. So differentiates your microbial content. As well as you have something called as aneuploidy. So you like to look into large deletions or like you like to look for the aneuploidies across your embryos, right. So you like to see whether your embryos, you must have seen about heard about IVF, right. So IVF they normally go forward with certain embryo screening where they like to know whether there are deletions or there are certain duplications happening into your data or not. At that moment this particular workflow would be very much helpful for you, ok. So not touching much about this rather than the other part of us. We have done the variant analysis. After that the same thing that I have shown you as IGV, visualizing your data, ok. So what I have visualized the region, gene region or that reads that a mapping. The same time same thing can be done over here onto the software itself, ok. So you can visualize the data over here. So if you see you are looking at particular variant, you can click at the variant and visualize it into IGV particularly, integrated genomic viewer. At the same time databases I have talked about, so it is DBSNP, Drug Bank, Cleanware, OMIM. So lots of things are available over here. So if you can play with them any time. Then the filtering is happening and you have a data security. Security as in since you have your own logins that are given to you or you can register your own logins which are encrypted. So there is nobody who is going to take away your data over there, yeah. So with this, this is about the AND report a part. So any questions still now, ok, yeah, novel gene, gene discovery as in since in RNA seek or as in so you already have certain free tools, right, available. So what happens over here in sequencing you can do the sequencing, right. You get generate your data for RNA seek and then take it further for different tools that are available. Like you must be knowing about if you have an idea about it, you have idea about cuff links. So that is one of the tool which is actually free, which is a command line based tool which helps you to define which are the new isoforms that could be generated for your RNA seek data, ok. So it helps you to know which are the regions which are getting aligned by your sequences and then defines whether it is an actually exon or not, which is actually fitting into your particular data or not, a particular gene or not. And then comes out into a particular transcripts for it and gives the different isoforms. So cuff links is a tool that helps you to do that, ok. We have a workflow which fits into this, but it does not has this particular workflow. It has to be done separately. We have a RNA seek plugin available, which does a human based alignment of the data and gives you the human based gene code based annotations are utilized. You get the gene information into it, as well as gene counts and transcript counts for it, ok. Then you can take it further and do differential expression. But when you are looking about particularly for those isoforms, we are looking more into the command line softwares that are available, freely available or the other commercial tools that are available. You have to do it separately, yeah, updating, right. So for the software that is online, it is already updated, ok. So whenever there is an update coming in, 6 or 3 to 6 months, that gets updated over there onto the cloud system that we have. So whenever I was talking about ion reporter, it already has certain databases which have been available over there, ok. No, no, nothing like that. So what happens, the data gets generated onto the S5 system or the instrument. Once the data is generated, you try to do the analysis, basic analysis at the system level itself. So we have a server with this, ok, which helps you to do the basic analysis, like aligning the data to the reference genome, then looking at the coverage analysis and then coming to a variant level, where you got, get all the variants into it, ok. So you have got the variants through the system itself, ok. Variants would be all the mutations that you are looking for. Now what happens, this mutation or variants that you are getting, we need to annotate them or we need to know more about them. So you are taking this and then we have a tool which is actually cloud based, ok, which is actually on website, ok. So we take this data particularly, we upload it onto this cloud and we can try to do the analysis. So it is not like a blast, it is not like a blast, but there is something similar to it. Yeah. So what happens over there, we have a tool called a steam map which does the alignment of the data to the, or mapping of the reads to the genome over here, ok. So when you are mapping that particular to genome, it is already done onto the system itself or the server that we have over here, ok, with the system itself. So it is locally done over there. Once you align it, whatever you get is in an alignment format and then we try to call the variants across it or the variations into it onto the server. So whatever variations you pick up, you can take it further and then put it further into the cloud system, ok. So that cloud will help you to correlate the positions that you have got and the database information that we have for those position and can be correlated together for all the databases, ok. So there is an update happening on cloud every few months and there is nothing like license over there actually. What happens? You are given around 100 GB of space over, space onto the cloud, ok. So if you are taking the same system at your, as a local server, there is a two version out of it. One is iron reporter cloud version which is online which gives 100 GB free space. Once your space is full, you need to buy some more space. It's like a cloud system that you have. Otherwise it is like you have a local server also where if you already have the system, you can push the data to that local server, something like a Linux server and in that you can do the analysis further and do the same analysis that you are doing onto the cloud. Just that you need to update the server every three or six months, that's it, ok. So you have the database in both of them is available just that you need to upgrade them and utilize them with the data that you have, yeah, right. Over here we have everything onto the system itself and what we have done is it is like an appliance, it's like your toaster and kind of thing where you are just putting your data or doing sequencing and generating your data. You can't touch that particular system anywhere, you have to just access it through a web browser only. So nobody can access it externally unless there's some service person coming in and doing something out of it. So we have some data coming out, take it and do the analysis on cloud, ok. So at this stage if you have, like you have got the idea about the variants, right, now. What are the variants that are coming in? But even after doing so much of study what happens? Normally nowadays we require something where we, the results would be very quick, quick as in sense even if you have got the variants we need to get to know what is its effects and what is its counter effects happening across. And then what could be the drugs which could be available for such type of diseases or variants that you are getting, right. What you are doing over here is you are somewhere correlating your patients, samples information to the variants, getting meaningful information and then you are trying to generate the information what type of drugs could be utilized, right. You have a drug bank database that is correlated to this. But doing this would take some time, right, doing all the correlations would take some time and getting you the results out of it. At the same time we have developed something called as Oncomane Knowledge Base Reporter, ok. Oncomane Knowledge Base Reporter has a database called as the Oncomane Knowledge Base, ok. This Oncomane Knowledge Base even if you Google it you will get an idea, it has lots of information regarding to cancer, ok. This is specifically for the cancer related studies or else this is something like which will help you to correlate your cancer, cancerous variants to the drugs that are available in the market or into the clinical trials, ok. So you have certain databases, you have certain cancer driving drivers that are available which are verified and studied by the researchers over here. So same database we have generated over here called as Oncomane Knowledge Base Reporter Database. So this has all the curated studied information about genes, the targeted therapies or the drugs that are utilized for treating cancer, ok. And with that you have an interface called as Oncomane Knowledge Base Reporter Interface, ok. So now what happens over here? It has lots of information of your variants called as cancer or a driver information, ok. This driver information is correlated with different drugs information, ok. And the same thing is available as my design essays. So what I was talking earlier into AmpliSeq that I like to design my gene essays or gene regions target of interest and then sequence it to know which are the diagnostics variants that you are getting. The same thing is coming over here, you have certain essays which are available which can be utilized to detect which are the variants that are getting coming into your samples and the same samples are utilized to denote whether that variant that has been detected is having any clinical significance or not. And with the clinical significance whether it is correlated to any drugs or not, ok. So this is something that you have as a summary, you have something called as tumor sample, you take it further, prepare your library, you do your sequencing over here and then you do certain analysis, your certain analysis would be like you do the ion reporter analysis, understand the variant information, get the results from there. This ion reporter also has information regarding the driver variant, ok. So these driver variants are recorded over here in ion reporter as well as these could be pushed up into the tool called as Oncomine Knowledge Reporter. So once you push up this data to the Oncomine Knowledge Reporter, it will try to look for these driver information correlated with the different drugs available and then look into the clinical trials that have happened already over here, ok. So at this point if I take further, so you have something like this, you have ion reporter coming into play, you have genetic variants, you have a knowledge base reporter which has different guidelines, information regarding to your drugs, information regarding to a clinical trials, everything over here, you correlate them and finally generate a report for it, ok. And this would be very helpful when you are trying to give a drug to a particular patient, right. It would be very quick for you. So patient comes, you do the sequencing in one or two days, look for the drug that is actually having any effects, give the report to a doctor, he could let you know what could be the drugs utilized further for a particular treatment, ok. So this is how we can come to a place which would be very quick in responding to cancers, different types of cancers. So an example report for us where you have the variants, you can see here are the variants that we are looking at. There is a fusion, there is a C2T change that is available and there is a T2A change happening over here also, there is a protein change happening, V600E that is a protein change happening over here, amino acid. And the same time there are different clinical trials and drugs affiliations available. So if the drug has been studied some place and has been giving a go for the utilization, so everything is given over here. So at the same, this is something like a summary which gives you idea whether it is going into a clinical trial or not, whether it is a US FDA approved or not, ok. At the same time you can get more information about more drugs into it. A different variant that you are looking at, V600E, so these are the various therapies that are available, drug therapies. So who have given which approval, which stages is approved, global clinical variants in which stage it has been approved. These details are coming into this particular report, particularly. At the same time what I like to know is in detail, in depth, if I am looking for a particular drug, I am concentrating on that what study it has been done across those particular drug. So that studies are coming over here, ok. So what type of drug effects are happening over here. So those information comes in complete details for each and every therapy that you are looking at, ok. This finally comes to a level where you can finalize it and give a report to the doctor, ok. So based on my different information or different approvals that we have got, this report comes in. So once this report is here, you can utilize the same report, ok, goes to the patient and you can directly assign drugs over there, particularly or there could be a discussion between a clinician and a person and the doctor who wants to give that drug and take it further. So in general as in since somebody who is using the ion reporter, sorry, who is using the oncomand reporter are making their own formats of reporting this particular variance, this particular data. So they are doing what is, if they are targeting a particular, say they are targeting a particular cancer as such, they have studied the cancer properly, they have already done certain diagnosis study. They come to a point where they have all these drug information, they just take out which is the drugs that are really required and provide the details to the clinician or to the doctor as such. So like the information of the clinician is what the research part is, but the clinicians do not experience or the clinicians do not want to implement it because it is not in the standard medical person, it is not in the standard. So that is what the person who are working on this are giving into a standard format. They should be in the WHO accepted protocols. Right. Of surgery. Right. The clinicians are also responsible. Right, right. You are there. So they do not want to change or degrade from the protocol. Okay. So has this. So this has been integrated, this has been done, like these are the approvals that they have got through the USFDS. The drug, whatever information we are giving is based on the approvals that are there available like USFDA or the European, I do not get that name. Yeah. Esmo. Esmo. So they have utilized the same information over here to summarize and give it to it. So how to represent it, it is all about the person who is going to use the NGS system. They plan it how to give it as a format or as a like what you say approval. So they try to work on that and then give it. At there being instances when Thurmu has approached the clinicians and are trying to make some changes in their approvals. No idea about it. No idea about it. We do not have an idea about it because whatever we are going. It is not applied in your clinics because of this. Actually that is why we have brought this particular tool. Like this particular software which helps you to get the information in such a way that it could be acceptable. So we are working on this. We are participating in those meetings and making them aware about all these tools. And then this is something to do beyond our approach. So we are making an interview, making an awareness. We are doing that. There will be some tools available. So now for you to implement these drugs or to comment on it. It is up to them. But then yes in terms of bringing that awareness we are already working with. Yeah. So any questions about this? I hope so you have got an idea or a feel how the data analysis happens in NGS. The only thing over here was to take you through the entire workflow. How we start with the analysis. How we like to look into the particular gene designs. Come to a level where you do the sequencing for the gene designs. And then get it annotated through ion reporters. It is a tool that is really available. And then looking for something like a quick response. There is Oncomine Knowledge Based Reporter which helps you to do that. Okay. Okay. So thank you very much for your time. So today's lecture you got a good understanding about one of the useful resource which is Oncomine Knowledge Based Reporter. From where you can get a lot of information for the cancer research. And again you know while we are really getting biased towards cancer or clinical applications. But there are similar kind of resources available for your model organism of interest as well. It is really important for you to dig deeper and know your available resources which many times now made publicly available. And anybody can use those resources. And if you have access to them then a wealth of information could be integrated from variety of data sources. So this database also contains various curated information about cancer, its variant forms, different type of drug and therapies impact. And you know it can be really useful for you to now start adding information on top of a data from the curation. And see that you know whether you can build now some hypothesis which could be actionable hypothesis now to take forward for your experiments. So in this lecture you have learnt more about you know how to do variant data analysis obtained from NGS platform which could be correlated with the patient data to study cancer. We will continue more about NGS platforms I am sure is one of the revolutionary technology and exciting areas to discuss. Arnold also try to bring more application orientation and application scientist to speak to you about latest developments happening in the NGS based platforms. Thank you.