 Cool. So, my name is Nicholas, I'm a VSD candidate at the Institute of Applied Sciences in Greece. This is the first time I'm presenting at the bioconducted conference and I represent Lee Netsport detecting south of to the energies in an educational loading municipal resporter. So, to begin with, let's start by what exactly is Lee Netsport? Lee Netsport is another package that aims to identify and assign different transcripts to the energies in wastewater samples based on the reported amino acid mutations. Having this in mind, I would like to begin on how the society came up. Here you can see the COVID-19 situation in Greece since the beginning of the pandemic. As you can see, we had a different number of cases depending on the period and different variants coming up again and again. Greece tried to use PCR based methods and clinical NTS sequences in order to monitor the whole situation. We quickly realized that these kinds of methods were not scalable in the whole country and especially when new variants arise like the old variants. So, we started thinking of alternative ways to track the virus and detecting south of to load in wastewater and identify its different variants in wastewater. It was an idea that came up. So, this is how Lee Netsport was born actually. A very quick overview of the output of the tool. The tool accepts the inputs, a list of PCF files, each one corresponding to a different sample and containing mutations. And the list of reference files, each one corresponding to south of to Lee Nets. Which is being used as a reference source. So, once the analysis is performed, Lee Netsport produces a table that quantifies the south of during NTS or in every sample that was given as input. Here is a more detailed overview of the overall workflow. As I already said, Lee Netsport takes these input VCF files. It should be noted here that apart from the nucleotide mutations, each VCF files be annotated with the amino acid mutations. Outbreak info and pangolin, mostly outbreak info, are being used as reference sources for every lineage of interest that there is a file containing the gene and the corresponding amino acid mutation. And once the analysis is performed, a table is produced, quantifying its lineage presence in every sample. Now, in order to succeed in this quantification, we need to find three metrics based on the island frequency. S island frequency will mean the number of reads, a particular amino acid mutation has occurred at a specific position by the total number of reads at this position. So, the first metric is the average island frequency that the characteristic mutations of lineage of interest have. You went back on mute, Klaus. I don't know why this is happening. We can hear you now. Okay, cool. Do you want me to go a step before the previous step or just right here? I think we heard up to here. Cool. Okay. So, as I said, we defined three metrics to quantify the lineage abundance. The first one is the average island frequency of the characteristic mutations of every lineage of interest. The second one is the average island frequency by excluding common mutations of the cure among the lineages that we gave as a reference to the two. And the second one, and the third one is the minimum island frequency of these unique mutations by excluding any zeta frequency mutations. We applied lineage spot in 14 wastewater samples across a six-month period in the southern Mickey. The two was applied in samples from December 2020 till May 2021. On the right, there is a heat map containing the characteristic mutations of the alpha variant. And on the left, you can see the average island frequency of the alpha variant during this period. In both cases, the results from lineage spot came in agreement with the related clinical data that we had during that time. In particular, Salimniki was dealing with the alpha variant and a small part of the delta variant during that time. This is a very quick overview of how to use lineage spot. Of course, you can download it through the pico-taphyl repository. You can simply call the lineage spot function by providing the folder containing the VCF file and the folder containing the reference files of the lineage of ingests. Lineage spot is kind of new, so there's still a lot of work that we would like to do. Our main focus is to integrate the package with the outbreak info, which is also a package under development by the outbreak info team. And of course, we would like to cooperate with other teams in Europe that work on wastewater surveillance. During this project, a different number of people have been well-coming from different backgrounds. The main work was conducted by the Institute of Climate Sciences and the Aristotle University of Salimniki. And finally, I should say that lineage spot has been published at scientific reports. You can read the paper online. And of course, it has been submitted to the pico-taphyl repository. Thank you all for your attention. Sorry for the inconvenience. Thank you for a very-