 Welcome to MOOC course on Introduction to Proteogenomics. After understanding how one can sequence gene by using a Luminar platform, we will now move on to another NGS platform by Thermo Fisher. Our today's speaker is Dr. Atima Agrawal who has already explained this about how the thermo NGS platform works in her previous lecture. In today's lecture she will talk briefly about how the result files look and how this platform has increased the coverage. She will also talk about various applications of this NGS platform especially in the areas of metagenomics, genomics and multiplexing sequencing. She will also talk about thermo database of 16S RNA from more than 9000 organisms. Dr. Atima Agrawal will then talk about already available panels of well-studied targets which one could use directly their own research. Another colleague from Thermo, Mr. Hirsh will also join her and talk about the workflows how one could analyze the sequencing data. He will also talk about the torrent suite software to load the basic softwares and requisites required for using the sequencer. So, let us now welcome Dr. Atima and Mr. Hirsh from Thermo Fisher. We all know it is tumor sample is heterogeneous. So, what sample we are actually taking the DNA out whether that is representing the actual tumor position, tumor condition we do not know. So, liquid biopsy that ways is a better way of handling and more so because it is a non-invasive way. So, it becomes a very good prognostic marker. So, especially when you are dealing with lung cancer cases you are not allowed to do biopsy many times. So, after a point of time or at times there would be patients wherein you are just not having that flexibility of taking any biopsy. So, there these liquid biopsy solutions are coming very handy. This again is a panel which has some 22 genes or things like and these are all multiplexed in one cube and they are all getting amplified at one go. So, this is so, you generated so many million reads and these are the read lengths because it is a selfie DNA as it is by nature it is a fragmented DNA. You do not get DNA fragments more than 120 to 170 base pairs. So, you would like your primer pairs to be amplified very short regions. So, that you do not skip on that since we are barcoding many number of samples. So, this is just the statistics of how many bases every sample has produced and these are the read length histograms for every sample. Then it shows that now you were targeting problems for example, 52 genes within that whole 3GB human genome yeah. So, how well that is covered? So, there is no point of doing an NGS if out of those 52 genes you are not getting good data for 7 genes 8 genes. So, the whole point of doing an NGS is that you can do a multi biomarker study with very little amount of sample and all with the same efficiency which is which is kind of a very specific highlight for our system. So, this is what is being shown in this coverage analysis that how many reads were on target, what is the depth. So, in NGS we talk a lot about the depth like in Sanger what we used to do. So, supposedly you have an amplicon you would sequence it with a forward primer, you would sequence it with a reverse primer. So, that is what you are getting a 2x coverage that is all. In especially with samples with oncology samples, these samples being heterogeneous in nature there could be a normal lot of normal cells or wild type DNA fragments which are coming. So, you tend to sequence them at a larger depth. So, this is the depth which is shown at times it is in 100s 1000s also and this is how well that those 52 genes have been covered. So, how well all those 52 genes at one go have been covered. So, these statistics all have been will be generated once the run is over within 1 or 2 hours, all these statistics are already generated. And then you have an array of applications which can be done on the system, you can do RNA seek, you can do microbial viral or metagenomics applications wherein you are trying to sequence these genomes, you can do targeted sequencing. Now, these targeted sequencing is generally done by various kinds of panels. These panels can be targeting various kinds of inherited diseases, these panels can be targeting various tumors. So, various markers which are known to be relevant in various kinds of tumors. And then once you have the data we have softwares in place to give you very well annotated variants which can be directly reported. So, this is our metagenomics kit which sequences 7 hyper variable regions of the 16s RNA at one go and you can multiplex depending on the number of reads or amount of data which you want to generate per sample you can multiplex these samples. And finally, once you have amplified your ligate adapters then it goes on to chef or on to 1, 2 and ES and you sequence them and you analyze the data on ion reporter software wherein you are basically blasting these sequences to various databases. So, we have a curated micro seek ID which has full length 16s sequences more than 9 coming from more than 9000 organisms. This is the database which has been which is maintained by a thermo fisher then there is a curated gene based gene green genes database which has with this is a publicly available 16s data resource which has around over more than 400000 organisms after curation. So, these are some comments from some users we have plenty of them we have plenty of publications we have plenty of clinical trials going on. So, going to RNA applications again we have various solutions starting from targeted RNA sequencing. So, if you want to sequence certain specific transcripts or certain specific RNA within the within your sample you can design a custom panel if not that then we already have a readymade kit which is AmpliSeq transcriptome human gene expression or AmpliSeq transcriptome mouse gene expression kits. These are these gene expression kits are targeting more than 20,000 ref seek transcripts and the beauty is that you can get all this data with as little as 10 nanograms of RNA. So, this is one thing then one another way of planning a RNA seek experiment is that you just want to sequence anything and everything whatever RNA is there in your sample you want to sequence the mRNAs you want to sequence the long term coding RNAs you want to sequence small RNA. So, there what you do is you enrich your sample based on the fraction which you are interested in. So, supposedly you are interested in sequencing all the mRNAs which are lying in your sample. So, what you will you do is so now we all know that most all the mRNAs are adenylated. So, they have a atail. So, you will use the oligodete beads and fish out all these atail fragments and then prepare a library by almost similar way with what we have discussed. Now, if you are interested in the total transcriptome but not the ribosomal portion because we know that 90 percent of the cellular RNA will have ribosomal portion which is quite constant and you really do not want to waste your reads being mapped to ribosomal portion. So, there are kits from thermo fisher itself which something like ribo minus which will deplete this ribosomal fraction and now what you will have is all the RNA fragments coming from your sample which are apart from the ribosomal fragment fraction. So, you sequence all of that at one go. Then small RNA sequencing, small RNAs we know that they are generally in the range of 18 to 40 base pairs. So, how you enrich these small RNAs based on the size exclusions. So, there are certain bead based exclusions or column based exclusions which you do and you enrich these fragments and then again you ligate it with adapters and then you sequence this fraction and again since the system is quite versatile it will give you from anywhere from 2 million reads to 130 million reads you can plan all these experiments on a single instrument just by changing the consumable like by changing the either you use a 510 chip or a 540 chip or a 550 chip base. So, the box remains the same which is the main I would say main expenditure in terms of having an instrument it is just the consumables which are changing and which are giving you variable throughputs and which are allowing you to do multiple different kinds of experiments. We already have certain RNA panels which are there of the shelf. So, if you are working on any of these kind of pathways you can just take these panels and you are good to go and then coming to targeted sequencing applications. So, again as we discussed that some there are some specific challenges for in cancer research that I do not want to sequence only single nucleotide variations. I want to sequence all different kinds of aberrations which are going on in the genomic content which includes fusions which includes various kinds of CNVs and all. So, and then you want a very these panels need to be very specific they need to have that capacity that all the targets are being amplified with the equal efficiency. And then you at the same time you need to have various efficient LOD. So, as to not compromise on the sensitivity of the assay. So, how do we integrate all these things into one thing. So, we discussed about the power of advocacy technology. So, which is a kind of boon to cancer research wherein all the templates or all the targets are being amplified with equal efficiency. Then these are being templated on Ion Chef system sequenced on GeneStudio S5 and then Harsh is going to talk about Ion Reporter and OKR software briefly to tell you that how these variations are then annotated and then finally a report is given out. Very recently during like not very recently, but sometime during the beginning of this year we had come up with even better technology than AmpliSeq. This is AmpliSeq HD technology which is specifically designed for samples which are highly degraded and samples. So, these would be samples or DNA or RNA which is coming from FFP blocks or coming from liquid biopsy samples. So, this AmpliSeq HD technology really helps you in get not compromising on the LOD and still amplifying all the targets at the same efficiency. So, this is our various difference. So, basically with all these panels we are enabling full characterization of oncology samples. These are various panels which are targeted for solid tumor cancers. These are for human oncology, these are for liquid biopsy if you are working on liquid biopsy and we all know that immunoconology is being discussed a lot. This is basically the idea here is that you are harnessing the own body's immune system to fight against these cancers. Now when these kind of therapies are being used it is very important to know that whether a person is going to respond to these therapies or not. So, to make a well informed decision that whether these persons are going to respond to these therapies these are those various assays which will help you define that whether you are working with a active tumor microenvironment or a suppressive tumor microenvironment. So, basically I just spoke about AmpliSeq HD technology what it is enabling us to do is that you can use as low as 1 nanogram of sample. You can process multiple different kinds of samples together selfie DNA, FFP samples. This is a scalable technology which works for all different kinds of variations found at DNA and RNA level. And then you have a shorter sample prep time and the limit of detection of the panels which are used which are being designed using AmpliSeq HD technology at the back end is as low as 0.1 percent. And the beauty is that if at all you are not happy with the panels which are already there on shelf you can design these panels. So, Harsh is the core bioinformatic bioinformatician. So, he is going to briefly talk about the softwares how they work. I will briefly talk about the overview how we are positioned. I will talk about the AmpliSeq Designer software which is a tool for designing an essay. I will talk about the Torrent Suite software which comes along with the instrument. So, whenever you have any instrument in place in your lab this particular Torrent Suite software will be preloaded on that and it will do all the basic jobs for you. So, irrespective of whether you have S5 or S5 plus S5 prime or PGM the software are going to remain constant. Then we have an iron reporter software that is basically for annotating your variants, finding out the meaningful information about your mutations present in a sample. And then lastly on-common knowledge base reporter software for finding the relevant therapies or drugs available against a particular mutation. So, quickly so we will start with the AmpliSeq Designer it is mainly for a targeted sequencing. So, what you do is either you we already have a lot of panels available of the shelf. So, what you do is you go to AmpliSeq Designer see the list of panels available for you. If any panel is suitable for your requirement you can directly go ahead and order those panels if not then you create your own panel. Panel is basically your own essay. Then you sequence on the instrument. Now the machine comes with a software called Torrent Suite software. So, it will basically convert your raw signals into ATGC sequences. It will do the preliminary analysis it will align the data back to the human genome or any other organism that you are working with and it will find the mutation. Now once you have the mutations available you go to a software called Iron Reporter for annotating the mutations. Now this is the workflow is you design your essay you sequence your samples and then you finally report. Now to begin with the AmpliSeq Designer this is how the user interface looks like it is a web based portal the link is www.ampliSeq.com the registration is completely free it should not take more than 5 minutes for you to sign up there. Once you sign up for AmpliSeq Designer you are automatically signed up for the ThermoFisher cloud as well as the Iron Reporter software. So, you do not have to have multiple login credentials for ThermoFisher software is. When you login this is how you see. So, on the left hand side you have an AmpliSeq on the right hand side you have an AmpliSeq HD. On the left hand side you have on-demand panels made to order panels and ready to use panels. So, ready to use panels are the panels which are created by ThermoFisher. Made to order panel has basically two sections whether you can create your own panel or there are community panels which are designed by some other users and which are made publicly available for the other users and then third is on-demand panel. So, for on-demand panel we have our 5000 odd genes we have already designed the primers and you can choose which genes you want to sequence out of those 5000 odd genes. So, there are two categories there either you can select the genes by the disease research area or you can include your own gene list as well. So, let us say if you are beginning with an experiment where you do not know what are the genes associated with a particular disease then you simply go to the on-demand panel and then there are list of categories available you keep on further breaking it down till you it matches your requirement and then finally design a panel and then proceed further. But these are the basic on-common panels that we have available with us. So, if you use any of these panel we have a complete solution available you can go to our software and do the analysis for those samples. Now, the current challenge that we have is for especially for cancer samples is once you have identified the mutations of interest or driver mutation how do you actually go forward? Because the ultimate aim is to find a drug or therapy or find a cure for that or medicine for that. So, right now what we are doing is and this biomarkers are increasing day by day right. So, there has to be some software or there has to be a mechanism where you have a database of all the driver mutations and keep them updated and then utilize in an efficient manner. Now, we have acquired a company called company along back and we have created a strong database of on-common knowledge base. So, it has all the details about the genes associated with targeted therapies and labels and guidelines right. Now, we have a developed a software called on-common knowledge base that will basically annotate a variant in your particular sample and it will say whether the particular mutation is driver mutation or not. Now, with the use of both on-common knowledge base and on-common reporter database we have created our on-common panel. So, the on-common panel has the list of genes for which we have therapies available or some information available in clinical trials or FDA or SNO or NCCN. So, then when you sequence, so you take your tumor sample or cell-free sample or your blood sample you prepare your library you do sequencing and then you finally go to the analysis part. Now, in analysis you do the basic analysis on torrent sweet software. So, torrent sweet software will produce a BAM file which is a standard output file for any aligned data. Then we have a software called ion reporter. Now, you can do the variant calling on the torrent sweet software or you can do the variant calling on ion reporter software that is a choice which user has. So, variant caller software will take the BAM file and it will produce the VCF file which is a standard variant file format. And then once you have a VCF file you put into a software called on-common knowledge base reporter and it will give you a PDF report with all the therapies available for that particular target. So, this is the information about the torrent sweet software same way regarding the ion reporter and now this is basically the on-common knowledge base reporter software. So, on an on-common knowledge base reporter we have basically labels and guidelines which includes USFDA, USNCCN, ESMO and EMA and then we also have global clinical trials data available in that. So, this software we routinely upgrade as and when the new therapies and new labels become available we update the database so that user has the most updated database available for that analysis. Now, so you have the VCF file from ion reporter, you have the on-common knowledge base report with a software you put them together and then you finally generate a report. Now, this is an example how the report looks like. So, when you put the VCF file into the software this is how it looks like. So, this is the mutation it is a B rough how many therapies are available in FDA, how many of them are available in NCCN, how many are available in EMA, ESMO and clinical trials. Now, if you go scroll down further this is how it looks like there are lot of therapies available for different mutations or driver mutations. Now, if you scroll further you will have a detailed view of that. So, under USFDA this is how it looks like for B rough V 600D mutation this is the information available under NCCN it is represented as this and the clinical trials it is represented like this. So, for some of the yeah it is last place. So, at the end for some of the clinical trials you also have the contact details available at which centers these clinical researches are sorry clinical trials are going on. So, this is the this is the report which normally every clinicians wants to look at or the researches wants to look at who are working in the diagnostic area. So, this is the entire entire informatic workflow in a brief for ION. If you have any other specific questions regarding any specific applications we will be happy to answer if you have 2-3 more minutes. Sir, there is a doubt in the workflow, you said that when there is base parent, hydrogen ions should be released. Yeah. Hydrogen ions are not released when DNA base is released. They do, they do because there is a bond being formed between the OH and H and that is how a phosphatidized bond is being formed a good habit in some of this life. A phosphatidized bond is being formed and then that is how a hydrogen ion is released it does. So, you said that for mRNA sequencing you repeat the value of RNA. You enrich the adenylated portion of the RNA. So, all the RNA fragments so, because mRNAs these are the codein RNAs yeah. So, and these are going to have 8 days. So, what you do generally people do is they take the oligodetic column or they take oligodetic and they just fish out all these RNA fragments which have an 80. So, that is all now what the sample which you are sequencing with only your mRNAs. Today in conclusion, I hope you learnt about thermo NGS platform which has multiplexing capability and high efficiency. The empty seek has capability to sequence from low amount of DNA even up to the 1 nanogram of sample. The LOD of sequencing could be up to 0.1 percent. We also heard the ion reporter software which helps in the annotation of the genes and analyzing the relation among the genes. The next supplementary video will be on another application of genomic analysis using droplet PCR. Thank you.