[MUSIC] Hi, my name is Rene Hendriksen, I'm from the Technical University of Denmark, the National Food Institute. I'm here today to talk about the application of genomic tools, one technology takes it all. So, first of all we'll talk about the evolution of typing techniques, the toolbox of the Center of Genomic Epidemiology and the summary. So let's go down memory lane of sub-typing technologies. So we started, actually, in the 1920s with serotyping of salmonella, developed by Kauffmann-White. Later on, we had phage typing in the 40s, and then we moved up into the MLEF time, where we started with plasmid profiling REA, PFGE, and so forth. PFG is probably one of the more known molecular sub-typing techniques which is a pulsed-field gel electrophoresis. But many of them are known by their acronyms. So where are we today? We moved away, in the 2000s, with MLST, multilocus sequence typing and in 2010 we saw the first laboratory bench top whole genome sequencing machines on next generation sequencing. And this is what we will focus on in the future. This is the technique that would most likely replace all the other sub-typing techniques that we have seen in the past. So this is the old school, the current conventional global diagnostics that most laboratories probably are still using today. But others have moved forward using whole genome sequencing on next generation sequencing. But here, we of course start with a sample, in this case it could be a stool sample or feces sample. And from here we do the isolation of the bacteria for instance. And we here need select a media, and after we have isolated the bacteria, we'll move on with characterization and sub-typing of the bacteria. And this could take around a week, where the first part would take one or two days. And then if you want to enhance your data using molecular sub-typing techniques, like pulse-field gel electrophoresis, it could take actually up to several weeks, or months, depending how much that you want to do with your study. So for the new school using whole genome sequencing diagnostics it has changed a bit. So we'll still use the stool sample in the beginning and the isolation, that haven't changed, so that will take still one or two days. But then we have a new step, where we do the DNA preparation, and library preparation, and the whole genome sequencing on next generation sequencing as it also says. That would take one or two days, and then we have the novel thing where we would actually do the characterization of sub-typing using bioinformatics tools. So each of the different things that we want to investigate are actually replaced by smaller tools. And at the end you have the interpretation that might only take one day. So, all of this would really decrease the time for the whole procedure down to five or six days in total compared to what we have seen in the past where studies could track on for several weeks or even months. So, we believe that a next generation sequencing is a technology that takes all because it can be used on all different bacteria at the same time so you don't need one technology or test for just one bacteria. So, this is the novel part of it. So the big question is, when will whole genome sequencing or next generation sequencing replace all these other techniques? And that's of course the $100 million question. So currently today it would cost around 50 to 100 euros to sequence one bacterial strain. We believe that this would decrease to about 10 euros in the future, but let's see. Currently, the older technologies would be more expensive if you compile all of them to one cost compared to what it cost for next generation sequencing. For instance if we want to identify a bacteria it would cost around 10 euros, serotyping of a salmonella is 40 euros. And then some of the more advanced technologies like pulsed-field gel electrophoresis, 50 euros and MLST up to 250 euros. So, altogether this is quite expensive compared to next generation sequencing, if you take all of them together. So, this is the reason why we in 2010 we're granted a project. It's a prove of concept project that have been ongoing for six years with the aim to [COUGH] combine bioinformatics tools with global epidemiology in real-time. So the idea here was to develop a web-based solution, plug-and-play tools that can replace their conventional microbiology. So what is really important is to know what it is, so a species, but also how dangerous, so that could be violence. We also need to know if it's something we have see before like part of nosocomial outbreak. It could also be just one of occasion and for that we need some kind of phylogenetic tools. We also really need to know if the strain would be resistant or susceptible and that would be indicated by a resistance. So here we have a tool to identify resistant genes, all of these tools can be found on this website. So, ultimately idea, this is just the examples, but here we have three different food-borne pathogens and we still have for the conventional microbiology the isolation part and we'll simply replace it of course not with the isolation is the same, but the DNA preparation, library prep and the next generation sequencing. Then we have the characterization and the sub-typing part. And the idea is simple to replace that with bioinformatic plug-and-play tools that even researchers that have no bioinformatic skills can run. Interpretation of the data will remain the same. But the ideas that we would develop in the Center of Genomic Epidemiology, CGE, tools that can slowly replace all the different parts that was necessary to identify and characterize the different bacteria. The idea is not to fill in everything, because we have also developed a tool where scientists themself can develop what is needed for that specific analysis. So this is a overview of the CGE tools, the Center for Genomic Epidemiology. Just want to highlight a few of them. We have here, Resfinder, identifying resistant genes. We have here, SeqSero and SerotypeFinder that are used for serotyping of salmonella and e.coli. We have MLST finder, here that would allow you to detect the MLST type, the multilocus sequence type. But we also have here more for the phylogeny, the snip tree and the CSI phylogeny that are important to set up these phylogenetic trees and compare strains. The last one I want to mention today are the batch upload where you can actually upload several genomes at the same time and get the results of all the tools that you have here, otherwise the other one would run with only one bacteria or genome at the time. In this link you'll find all these tools available. Please use Firefox browser as that would be the best one for running the tools. I have now talked quite a lot about bacteria, but there are other applications and diagnostic using whole genome sequencing and next generation sequencing. For instance, we have human genomics, cancer genetics, personal medicine, functional genomics, structural genomics, metagenomics, identification of virus, bacteriophages, bioengineering, and I could continue forever. There's so many areas where whole genome sequencing can be applied. So in summary what we have learned is that next generation sequencing has reduced the time tremendously for the analysis down to 5 days, 6 days, if from sample to sequence results. We also have learned that next generation sequencing has really advanced. And the time is right, where we have analytic tools that are available for providing meaningful, low-cost, and timely data for detection of various interesting genes. They could be, for instance, be resistant genes. What is really important is that this bioinformatics tools would output plain language reports that would allow medical doctors, scientists, and other in the same area to interpret the result and not being a trained bioinformatic person. There is, of course, still a urgent need for moving into standardization of the methodology, so allowing us to measure the quality of the produced genomes. There is also a need to move on developing more tools to fill in the gaps that are showed. And then, of course, to expand the use of next generation sequencing as far down the public health area as possible, ultimately, at hospitals, clinics and so. So thank you for your attention, and I hope that you have learned something from this presentation. Thank you. [MUSIC]
