Welcome everybody, I'm Bernice Batu. Today, we will follow tutorials on the identification of microorganisms in a beer using nanopore sequencing. So the idea of this tutorial is to take data that has been sequenced from a beer and trying to identify which microorganisms we can find there. And for doing that, we will do it, we will have a bit of theory around that and then follow by practice and we will use the Galaxy platform for that. So let's start one tutorials from the Galaxy Training Network that you can access by going on training.galaxiproject.org that we found there in the... If you scroll down on the topic there on the left, you can find it in the metagenomics here. You should go click on metagenomics and then you have the tutorials called identification of microorganisms in a beer using nanopore sequencing. And then you would be redirecting there. The idea of this tutorial is to identify... to know how can we identify yeast strains in a beer sample that has been sequenced and how can we process metagenomic data sequence using nanopore. So at the end of the tutorials, we hope that you will be able to inspect metagenomic data, run some metagenomics tool, identify yeast species in a sequence beer sample using DNA and visualize the microbiome community in a beer sample. So let's start from really the beginnings of what is a microbiome because we will talk a lot about this in doing this with this tutorial. So microbiome is the collection of all the small living creatures in a sample, in a small environment. So these creatures are usually called microorganisms and they are everywhere. You can find them in your gut, in the soil, in vending machines, even inside beer. And most of these microorganisms are really good for us. For example, in the gut, most of them help you to digest the food, to fight against pathogens, etc. But some can make you ill. And all the microorganisms, usually you have ear about bacteria, but there can be also varices. It can be some small orchards, really small organisms that are in the orchard side. But most of the time, we hear mostly about bacteria. And these microorganisms come in really different shape and size, but they are also similar components. So all living organisms have a similar component, which is the DNA. It's like the blueprint of life. It encodes the shape, the size, and many other characteristic uniques to a species or to an organism. Because the DNA is specific to a species or to the organisms, reading the DNA can be used to identify which kind of species we have. So by comparing, for example, it's the same for humans or for different vertebras, or mammals even. So if you think about, you can use the DNA of the individuals to know if they are human or if they are a fish or something else. So then we're using, when we do, we can take a sample of somewhere like salt or beer, extract all the DNA from that, and try to, when we have the DNA, sequence it to get all the follow of the different small, yeah, the ATCG that make the string of DNA, special for each organisms. And using that, we can try to compare that information to DNA we know from other organisms and try to identify which kind of species we have inside a sample. And to do that, so it's part of a project that is called the Beer Decoded Project. It's the idea is to organize workshops around that idea of taking a beer, extract the DNA out of the beer, sequence it, doing the data analysis to identify all the yeast. And this tutorial is part of this project, but it can be used also outside that project to understand more how to identify microorganisms from metagenomics data. I use already the word metagenomics a lot. So metagenomics is the part of taking the DNA from a sample, sequencing it, so all this part is called metagenomics is when we take, we don't target a specific DNA from a specific organisms, but we take everything that is in sample, we take all the DNA and without knowing from where it come from, from which organisms it come from, we take everything, we sample, we extract everything, we sequence everything and then afterwards we try to organize the DNA in the different species, group things, et cetera, at least called metagenomics. So why are we interested in the beer? Because beer is alive, like a lot of other environments. We can call it an environment. So it's alive, it contains a lot of microorganisms, especially the yeast, which is usually the main things we know about beer. But so grain and water creates a sugar and liquid with it called the wort and usually the beer brewers add yeast to it and by eating the sugar, the yeast creates the alcohol and other components that give the beer a special flavor. Yeast, which is a really small microorganism, it's the one you can find usually, so you hear yeast about yeast in beer, but also, for example, in bread, in yogurt, in a lot of things. So the majority of beer used, so yeast is a microorganism, so it's huge, more precisely, it's a fungi in miscellaneous fungi, so it's a one cell, so it's a really small organism with one cell and it's a fungi. And the majority of beer use a yeast called Saccharomyces, which means in Greek means sugar fungus. And within that, there is two types of Saccharomyces that are mostly common. You use the Caccharomyces cerevisiae, which is the one that is used, for example, in hal-el, so in white beer, red beer, stout, umber, triple-season APA. So it's mostly, it's usually the one that is used, the most likely that it's been the early one used by accidents. And the other species we can find is Saccharomyces pastourianus, the one that is usually on the bottom of the tank when it's fermented. It's usually, it's a lager yeast that you can find in pilsner, lagers, bog and other one. It was initially used by Bavarian brewers 200 years ago. So yep, and it's commonly used yeast in terms of raw amount of beer produced around the world now. But the yeast is not specific to beer and you can find it everywhere and there is also some beer that are spontaneously fermented without being adding yeast or that use wild yeast and the microbiota around or microbiome around to do the fermentation. So what we did there is, during one of the beer-decoated workshops, we extracted yeast out of a bottle of Chime beer, so it's a Belgian beer. And we extracted the EDNA of this yeast. We sequenced it using a Minayan, which is a small sequencer that do use Nanopore sequencing for that. I will not go in details, but it's, it produced longer reads than the usual Luminar sequencing. And now we would like to identify the yeast species that has been sequenced here and identified the diversity of microorganisms, so what is called the microbiome community in the beer sample. And to do that, we will follow a few steps. So first check the quality of the data, then assign a taxonomic label to each of the sequences that have been for each of the DNA sequences, and then visualize the distribution of how many sequences have been found for the different species. So that is for doing that, we will use bioinformatics tools. And usually when you do bioinformatics, you need a computer science background or at least knowledge about the command line. But what we will do now is we will use Galaxy, which is an open source platform for data analysis that enables anyone to use bioinformatics tools using a geographical web interface and that can be accessed on any web browser. So you can open Firefox or Chrome and you can access it. So we will use Galaxy today to extract and visualize the community of yeast in the beer, sorry. Sorry. So the first things we need to do is to open Galaxy. So for that, so I already open it, but I will open again.  I will use the Galaxy server, European Galaxy server. So use galaxy.eu, but you can also use, use Galaxy.org, use Galaxy.org.au or no other, any other Galaxy server that you know of. The first things I recommend you to do is to login. So I'm already logged in here, but you can click on user there on the top and you can log in there. And another things we will just do before really starting the tutorials is visualizing the tutorials directly in the Galaxy interface. It will make the things a bit simpler. So please click on the hot here on the top. So once you open Galaxy and you're logged in, you click on the top, the hot to see Galaxy training materials here. Then the Galaxy training materials website would be loaded again. So you can scroll down until metagenomics. So go again to the tutorial. So metagenomics here and then identification of microorganisms in the BI using Nanopore sequencing.  So once you do that, so here you have Galaxy directly visible inside your Galaxy instance, you can get out by clicking outside here and come back to your tutorials by just clicking on the small hot here on the top. So once you open Galaxy, you need to create an account. So if you didn't, you need to log it in and then the third things you see in Galaxy is you have three panels for the one on the left. You have the tools, so a list of tools to get data so to manipulate your data. Here you can add a column, et cetera. You have a middle panel where you have some information is where the tools when we open the tools will be the interface to the tools will be loaded here. And on the right is called something called the history. It's where your data are stored there. So here you have the first time of Galaxy. You should not have, if it's the first time you should have nothing in your history here. If so, the first things you need to do is always create a new story. So a new, it's like a new folder where you will put your new data. It's a way to organize your data at that. So to create a new history, you click there on the top left, you get to create a new history. It will create a new history. And the first things you need to do is to give it a name to make it easy to find. So I will put it be here, microbiome tutorial here. So let's call that way. And then you have the tutorials, then you say that the history is empty. So you need to put your data there. So to get that, so we already create a new history. Let's go back to that. So you click on plus, create a new history, and then you click on the pencil on the left here to edit and then you can name it. Now let's get the data. So before we can begin in analysis, we need to put the data. And here we will put what is called a FASQ file. I will show you afterwards what is a FASQ file. But first we need to import the sequencing data. The different way you can do it is you can get your data from upload. So if you have the data directly on your computer, you can get the data from your computer to Galaxy. You can use a new URL, and here we will use the URL because we have the data stored somewhere else. So you can click here on the copy here and then you will be able to upload that. So let's copy here, go back to Galaxy. So click outside and click on the top left here in the tool section. You click on upload data. You click logo to pass and fetch data and you can pass here the link that has been copied before and you can click on start. It will become green and then it will start something on your history and you can close here. I do it again. So you click on the Galaxy here, you go there, you copy this part by just clicking here on the right copy. You can just click here.  You go to upload data on the bottom, on the top left here. You go to pass and fetch on the bottom here and here you can pass the link that you just copied by right click and past or control C, control V, sorry. And you can click on start and you can close once you're done. And once it's done, when the data are uploaded, it will be available in Galaxy in your history and it will be green. And you see here you have the names of the data. Then you have some size which is 1.8 megabyte. It's a format is called FastQ and then you have some preview of the content of the data and it's just by, I click here. If I really want to see the full content of the data, I can click on the small icon here that is called displayed that will display in the center the full data sets. And here we can see that we have a FastQ file. So a FastQ file is a format to store sequencing information and their quality. So it's the things you can get directly from your sequencing after you sequence DNA. And the sequence of DNA. So you will have a lot, a lot of sequence of DNA and each sequence of DNA will be represented by four lines. The first line that will start with the art and will store some information about the sequence itself like the ID, the name of the sequence, the length of it when it has been starting, some flow cell information, the ID, et cetera. Then the second line will be the sequence of the DNA itself. So ATCG, a sequence of ATCG. The third line will be a plus. Usually you can have a similar information at the art or other information. And afterwards, it's a cryptic information and it's the same size at the DNA sequences. It means that for each base of DNA, so each ATCG that has been identified, we have a character that is identified here that encodes its quality. So how good this specific base of DNA has been identified? Is it, can we rely on that information or not? You can read a bit more about the FastQ formats here. We explain it a bit more. What is it? But I will not go too much in details. What is more information? It's like it's called, what we, the fourth line here is called a read. It's mean a succession of nucleotide for all fragmented the yeast of the beer in the FastQ format. And the first things we do usually when we have FastQ file is to check the quality. So transforming this information that looks cryptic into something that can be interpreted. So how good the quality of the sequencing has been done. So to do that, we will use a tool that is called FastQC. So to find the tools, you can either search here for FastQC and you click on FastQC here or the other way you can do if you use the thing, the tutorial that is embedded directly there, you can click on the FastQC here and it will load the date that, oh, it should normally, load the FastQC interface. I don't know why it doesn't do it but otherwise you click here. So it load the Galaxy, the tool interface. And then the first thing here we need to set up is some parameters or what information this bioinformatics tool needs to be able to run the program. And the first thing that it needs to do is to have the raw read data from the history. And you see it already selected the FastQ file. So it's correct. And then the other information we don't need. We don't have a contamination list and it's optional. We don't have adapter list. We don't have some models and the other things we keep it like it is like default parameters. And then we can click on run the tools really here. And you can see afterward that you have an help section and to help you understanding and even the citation tool to know how to cite these tools. So here what we want to do is to run the tools. You can click here or click on the top for the tool. And if you see now it say that the tools started and use the inputs. So this was as an input and we produce two outputs. That is already available in your history in gray. Gray means that it's waiting in the server to be run. Then when it will be run, then it will become yellow. Yeah, no. Yeah, yellow. That is mean ear like ear. It's running. And when it's done, when it's ready to be the tools is finished, then it will become green. Or if something bad and there is something that is not correct and the tools is failing, it become red. So here it's yellow. So it seems to run yet and we will wait until it's green to inspect the content. So it will create two data, two things. One is called red data, which is a tabular file where you have some metric that has been stored there. But the most important and most interesting content is this web page here. And you can click on display here. And it will create a report, a FASQC report here. Ah, sorry. If you want to visualize it in bigger because it doesn't take the whole space here, you can click on the left bottom left here to hide the panels of the tools here. So what FASQC created, it created a report where it said what are some basic statistics, the filenames, the type of file type, the type of encoding. So it's, yeah, it's a meaner. We don't mind. It's just the type of, there is different type of FASQC, FASQ file. The number of sequences. So we have 1,800, a bit more than 1,800 sequences. The lengths of the sequence that go from 130 to more than 2,000 base pair. So we have not all the reads of the same sequence and the GC percent. The second thing that is generated is this per base sequence quality which gives along the read aggregate. So look at all the sequences and try to identify if the sequences have a similar quality score over their length. So and create this box plot that give here in, for this base eight, for example, the mean quality score is around seven, this red bar here. But the score goes from three to, so 50% of the sequences are below between three and 12. And it creates a bit stable and there. And if you see here, you have some color codes like yield red below 20 above 20 and between 20 and 28 it's orange and above 28 it's green. It's mean that the quality is good and green. Okay, she's in orange and not so good in the bottom in the red area. In our case, what can we say if that globally the quality is not really good of the sequences, but it's known for Nanopore data that the quality is not really not really perfect. Yep, so it's exactly what is explained here. So yeah, you can, we have a dedicated rip. So fast QC generate another type, a lot of other reports and other graphs. I will not go in detail. So there is that you can check our dedicated tutorials for that. Now, what we want to do is to improve the quality of the data sets. And to do that, the first things we will do is we will call tool, take a tool that is called pro shop that where we'll remove some of the adapter that were added for the sequencing and potential camera. So when we do the sequencing, especially for the Nanopore, we had to put something on the bed beginning of each of the sequences to help the sequencing to go through the pores and being sequenced. And so we need to remove these adapters and other things we want to remove is camera. So the possibility that some, some sequence are together and they don't belong to species. And then those things we will do is to filter the sequencing with the local analysis score. So when the sequencing are becoming the global all, because here we have the, so here we have around each base, the quality score, but one of the things we can know is the means quality score per sequence. And if the quality score is below 10, the mean quality score of the sequence, we say, okay, this sequence is not really not good. So we will get rid of it one. And so it's what we will do with fast P. So the first thing we want to do is to use pro shop. I don't know. I don't understand why it doesn't work, but let's use the other way. So searching for the tool. So pro shop. And what we need to give it, it's the fast Q file. So the input data, the fast Q file, and we want the output data to be in fast Q file again, because we want to use that as an input again for fast P. The order we will keep like it is. And once pro shop is started, so you can see it's gray here, we can also already start the next one, even if this one is not finished. So we can say call for fast P. So it's a second tool we want to use. And then we will give it the output of pro shop. And you see it's already finding the good one. So we could select either the fast in their input, the input data or the pro shop. And what we want to do is to give it the pro shop output. What we want to do is disable adapter trimming options. We want to disable because we already do the adapter trimming with pro shop here. Yes. And after we want to filter options, we want filter options. We want a quality score of 10. Yeah. So you need to tape. So in filter option, if you click here, you expand, you have a lot of information and we want to have a quality score of 10. And we want in read modification, read modification option here. We want to disable polygy trail trimming. Then we can run it. So what it will do, it will say that once pro shop is finished, so here it become gray. It will wait until pro shop is finished to launch fast P afterwards. And then we can check how many sequences have been filtered and etc. Oh, yeah. We can already see in the pro shop. And can we have some information? Yeah, we don't know yet. We will stay check the fast P report to do that. And I will just pause until fast P is finished. Now here I can see some, by expanding here, I can give see something information about the number of read that were there before and after filtering, but a fast P and here is the fast Q file after quality check. And then you can also look at the HTML report and you can see, so the general information again, a bit similar to fast QC. So the number of read before filtering, so 1000 or around 1800, the number of base pair. And after filtering, we reduced remove around 8000 base pair reads and a lot of base pair also. What is the next question? I think it's this one. So you can see that the so around 8000 or 500. And what is the main mean length? So the mean length is 350 base pair before filtering and after filtering is 316. So not that big difference. So fast P already give you some information that there are filtering that has been made because of the quality. So now the next steps we want to do is really to identify the organisms that has been sequenced. And for that we want to do is what is called identify the taxon for each individual reads. So to which individual reads they belong. What is a taxon? What is a taxonomy? So taxonomy is the method that is used to name, define and classify group of biological organisms based on the share characteristic. It can be morphological characteristic but also a DNA data. It's based on the idea that the similarities become from shared on the ancestor, evolutionary ancestor. And so define groups of organisms are known as taxa. So taxa are given taxonomy rank and aggregated into different groups of hierarchy to create what is called a taxonomy hierarchy. And there is eight levels of the taxonomy from the top. So the really highest level is a domain and then we have Kingdom, Phelan, class order, family genus and species and below we can even have strains, et cetera. So let's look at, for example, the cats. So the cats are a species that is called Phelis catus and they belong to the genus Phelis and the family Phelidae and in the order Carnivora in the class Mamelia in the philum cordata and in the Kingdom Alimalia and in the domain Ocariota. I should have put the domain note on the top. The Panther, I don't know how to pronounce it. They're from the species Pantera par dus from the genus Pantera and the same family as the cats. And if you look at the dogs, dogs are from the genus Canis and then you have the dog, the Canus Familiaris or the wolf in the Canus Lupus. They belong to the same genus from the same family and the bear bear are in Ursus and with the Antarctic bear or the, I don't know how to pronounce it, the Chrisley bear, for example, year. And they all belong, all of them belong to the same order of Carnivora, Mamelia cordata animal. So the classification system domain begins with three domains that comprise all the possible living organisms and extinct form of life. So the bacteria and archaea are the most microscopic but they are the most widespread and then we have the domain of Ocaria which contains more complex organisms and including the us, the cats but also all the birds, all the plants are Ocaria. Ocariota, sorry. So when a new species is found they are usually assigned to a taxonomy, to a taxonomy hierarchy. For example, for the cats, if we found the cat now, we will pet the species, caters and pen the, in the F. caters, the genus Phyllis, et cetera, et cetera. We can also explore what is called the tree of life to see here, we can see the three domains so the Ocariota, the archaea and the bacteria and you can see here, here you see the difference so here you have the green plants you can scroll in, here you should have the fungi, the multicellular organisms here, insect, the cordata, so the cordata would be the vertebra. And then you should have, yeah, you can go really deep there. And the bacteria, which are the most abundant on earth, oops, you have, you should have Escherichia coli that you know, you probably hear in the news about Escherichia coli, you have, what else, a lot of different tuberculosis, bacteria also, et cetera. So what do we expect to find in our, in our, which microorganisms we expect to find in our data? So mostly yeast, so yeast are fungi, so Ocariota. And I think we can find also, so as I said, and all the caromises pastoreanus, so they are two species. And the type of beer we use is the albia, so we expect more caromises cerevisiae, but we can find the other one. And so the main expected organism is caromises avicii, and taxonomies will be domain fungi, ascomicota, et cetera, et cetera, until the species cerevisiae, the caromises cerevisiae. So to do the taxonomy, the classification is the idea is to assign an operational taxonomy unit, or UTU, it's mean a group of related individuals or a species that we can assign afterwards to a taxon, and then assign this UTU to each of the sequence. And to do that, we need to compare it to a reference database. And there is a lot of different tools to do that, and today we release Kraken. I will not go into details about how Kraken works, et cetera. What I can tell you is it's compared to a database and check if the sequence is similar to the one in the database and if these sequence belong to these organisms or not. So we will now use Kraken 2. So you can search for Kraken 2 to do that. So Kraken 2 here. I assign taxonomy clubbal to sequencing reads. And what we have, we have single data, and we take the input, the outputs of FASPI as input here. Then what we want to do is we want to print the names instead of just the taxonomic ID. So we want, usually when you get the output of Kraken you will get an ID and not knowing if it's the name of the species. Then what we want, we want to create a report. We want to print a report with aggregated cons in a file. And we want to select a database and for that we please scroll down and go to the pre-built refsec. So the one really on the bottom right now, that has been downloaded in 22. Yeah, this one. Yeah. And then you can launch the tool. Yeah. So again, you click on Kraken 2. Sorry, it takes a bit of time. Kraken single. In the file here you check, you be sure that the output selected here is FASPI. Then in create, you want to, you click on print scientific names instead of just tax ID here. Select here, you expand the create report section here. You click on print a report with aggregates, cloud and cons to file here. And the database you need to scroll down really a lot to get the plus PF one. But all the parameters that I told you are already listed here. So, and we use the plus PF because we want fungi and the plus PF contain the basic database plus also the protozero and the fungi. So it's being able to detect potentially bacteria but also fungi. And then the outputs of Kraken will be two files. One is the classification. So the classification will be a table where you see for each of the reads if it has been assigned to something or not and to what. And the report that aggregates all this information per DAXON and tell you, for these specific species we identify 800 reads or 200 reads or one reads, for example. So Kraken can take a few minutes to run. So I will also pause, make a break, a small break again. A report. So if we open the report that has been generated so the second file here, Kraken report, you can see that it's loading slowly but you have a five, six column. So the first column is a certain number like it's a percentage here. Then you have another column, there's column two, which is a number that would be the number of reads. Then the fourth column would be the level so the DAXONOMIC level. The level in the DAXONOMIC hierarchy here would be the DAXID and here the names of the DAXON. So if you see here, for example, if you go on this line here, oh, this one is maybe better. So you have here 56% of the read here has been assigned to OKAYOTAR, to the domain OKAYOT here. The DAXONOMIC ID is here. I don't remember what is the fourth column. A number of fragments I sent directly to this DAXON. So the number, the second is the number of the DAXON covered by the clade reader at this DAXON and here directly, directly, and OKAYOTAR takes it this year. And so here we can see that 38% of the read has been unclassified. So it means that 62 has been classified to a DAXON, a given DAXON, blah, blah, blah, year. So how many DAXON has been identified? To know that we need to identify, we need to check how many lines do we have in this file and if you expand here, you see you have 300 lines. So if we remove this line that of the unclassified, it means that we have 299 DAXON is identified. And so minus 2, OKAY. And then we have 62% of the read has been classified. The domain as I said has identified with a D year and which domain has been identified so what for that we can filter. So we can either search for D year or what we can do is using a tool that is called filter DNA data on any column using a single, simple expression. So you can search for filter, the file filter here. We use the report here. We want to filter on the report. We want to filter on the column 4 and that is a D. So we want to know when there is a D for which rows there is a D on the column 4. So it will run a filtering tool which sorry, I'm doing something in the parallel. It's a way of doing it and we will see how many percentage of the ink here. In parallel, we can also search for fungi. So if you go back to this file here and you search directly with control F with fungi and I find it. Fungi, I think it's a main F. Here, you see fungi. So in Kingdom Fungi, we have 25% of the read that has been assigned to fungi. I think it's what I saw. Sorry, what happened here? Oh, too long. I don't know what's going on. What happened here? Yeah. The filter takes time. We will go back to that. We can see that other taxon, the yeast has been identified. So for example, we see that we have a lot of homo sapiens, homo sapiens mean human that could come from maybe contamination when we did the DNA extraction or the sequencing. It can be also assignation. So Kraken has a bit of tendency to assign too quickly to something. So it could be a false positive there. But we also scroll down a lot. You have also some bacteria. So Escherichia coli that has been identified. But with really, really, really, really low percentage. So it means that it's maybe a contamination or false positive for Kraken. So one thing we can do is, for example, we could filter for the contamination by keeping, because here, for example, it's only one sequence that has been assigned to Escherichia coli. So we could set up, for example, a threshold of saying everything below five can be removed. It's probably a misassination. We will not do that. But you can try. And if you do that with a five, there is only 60 lines, 59 lines. So it's been 241 tax and has been removed because of low assignment rate. And then most of the reads that are assigned to humans still, so it's probably contamination either during the pre-production or more likely during DNA extraction. So now, okay, the filter is still growing. I think it's a bit slow on the server right now. But another thing we want to do is afterwards, because these things are still difficult to read. I mean, it's a lot of information, not in a really structured way. So you need to know that Saccharomyces belongs to Saccharomyces, but you know it's difficult to see that there is different species of Saccharomyces, Saccharomyces paradoxes or neios. But it's not that visible also from a hierarchical point of view. So is this one linked to what? And we want to also visualize the percentage more easily. So what we can do is using tools that is called, that helps to do that. And for this one, we will use a tool that is called Krakken, a Krona, sorry. And Krona will take the table and do that. But first, the Krona expect a certain input where the first table, the first column should be the number of read and the second column and the third column, etc., the different taxonomy level. So for that we need to prepare the reads. We need to convert the output of Krakken into something that Krona can process. So what you can do is searching for Krakken tool. Krakken tool is a set of, is a suite of tool just to clean to process the Krakken outputs. And here we want to convert the Krakken report file to Krona text file. So on the first one here you can click there and then you can say I want to use the report. Be careful on there and saying you want to use the report and not the classification on the output of Fitter. You want to use the report here and you can run the tool afterwards. So it will reformate reform make the table in a slightly different format. And once Krakken tool is run we should get a table that looks like this like column one is being the number of reads. Column two is for example the kingdom here afterwards we will get the philom the class, the order the family, the genus and the species. So that are the different columns so the seven level of taxonomy. And then this output can be given for Krona. So Krona you can search for it while the tools are waiting or running. I recommend you to use sorry let me check. The second one we have several of them we need to merge them so I recommend you to use the second one here should be the last one so it's here you can see all the version of the tool and we want the version at least 2.7.0 plus galaxy something so please be sure that you have this version here. The type of input that we have is a tabular and you need to select the output of Krakken tools and then you can launch it already and it really starts slowly to run. Once Krakken tool is finished so both of the tools are and it should generate something like this. I will make a break also again because it's still running and wait until it's done. Filter also first input of filter so where we try to identify which domains we have we can see we have a coyota archaea and bacteria but mostly archaea are really low and archaea it's really low it's 11 10 0.22 percent and bacteria it's around 3% and archaea and coyote are the most dominant there and Krakken tool really generated the similar file as we said so with the table that looks first with take a bit of time to load the first column being the number of readers sign and then the different for each taxon so the same number of lines as before so we should have similar numbers for the same year until the species we have the different columns there but the most important and the most interesting is really this corona chart so corona how to interpret that it's an interactive thing so you can click on things and it will you can look at that here on the middle you have the road so the highest hierarchical level almost you go to the exterior of the circle the lowest taxonomy level we are so here we have the kingdom so here a coyote here bacteria here we have a lot of unclassified and if we click on the coyote here we can go to the different coyote we can see that here the really low abundance of a coyote and etc etc so we have a few questions there so what did the percentage of read assigned to homo sapiens so it's around inside the coyote so we can also go back here so 30% of the read has been assigned to homo sapiens it's what we see there and for archaea we can see archaea 0.08% of the read there so let's now investigate more in-depth so come back to the question so we want to really characterize the biomacrobium especially looking at the yeast there and the yeast doesn't really form a single taxonomic group they are part of the fungi but they have different phylum so they have the aschimicota where we can find the sarca homises and they have the basi yomica but most of the true yeast are really classified as sarca homises so what we can do is clicking here on the order sarca homicital here we can see the diversity of sarca homitalis that we have here what happened here something wrong I clicked on the wrong thing which these species have been identified and are the expected in beer so we can see in the species on the exterior here we have sarca homises serre vizae as expected we have some percentage of paradoxes but we have also some eubian news here and we have some other that like the candidate eublienesis and other sarca homicidal so if we click here so we have 6 species of things that have been identified from the sarca homises especially from the genus sarca homises so the servizae, the paradoxus and the eubianus so the servizae is the most in the one that is expected the paradoxus is the wild yeast that is close species to sarca homises serre vizae so it's expected to have there that these can be, have been misidentified the eubianus is likely it's a parent from the sarca homises pastorianus and maybe this read has been misidentified we have some from the cleavio remises genus and especially where is it so I cannot find it anymore but that was only one read so maybe it's not visible anymore and we have from trico and the eubianus but it's again really small quantity of reads so probably everything except the sarca homises serre vizae has probably misidentified read and then if we click on sarca homises here really we can see the percentage so 89% of the read in sarca homises has been identified for I've been assigned to sarca homises serre vizae and then we are 5% to paradoxes and 3% to eubianus and we can see that this is 20% of the of the reads, 25% of the reads 44% of the eukaryotic data etc and here we can see the levels air so it's what is written also in the tutorial here hmmm oh I think the number is 89 sorry but you can see here so that's why the things and another things we wanted to check and to look it's so the microbiome of several beers including the shime beer has been already investigated and specially targeted for fungus and for example it's the case in the paper from 2017 we could make some beer but only sequenced extracted the fungus and sequenced the DNA of the fungus so they couldn't identify potential some bacteria and try to see there is a combination of bacteria and things that could make the things that is something we didn't really look here but we could also look at the bacteria here and trying to see because we have a high diversity of bacteria here most of the things are 2% so it's mean probably 1 or 2 reads but for example this Alune cedar is 10% so it's mean a lot more than the other ones so maybe this one is also a bacteria that is useful for the brewing of the beer for that we may need to have more of that here but also and we have some close relatives of that one that could be misassigned and that become mostly from these species same maybe we have close cedar is also important some of the firmic critis but for that we will need more beer microbiome to do that but as I wanted to say here most of the case when the beer microbiome has been investigated they really targeted only the fungi and especially in this case they really targeted just the fungi and they identify for example for the chime so we have the chime here on the top and one triple on the ear and we can see that in this case they identify different species of yeast there they identify saccharomyces cereviziae but also mycatae which is usually used in they use also the cazactania martini it's from the same genus as saccharomyces family of saccharomyces from other type of saccharomyces the breta nusis that is somehow expected because it's typically used in the production of belgian beer and the chime beer is a belgian beer another the paradoxus as we identified in another wild yeast like this one another things of cazactania and other other yeast so if we try to make it in a structured way so where we have the phylum on the left to the species so saccharomyces cereviziae and different type of different species of saccharomyces here from another genus from the same family as saccharomyces the breta nusis and then some really other that are part of this the one we mention here on the top this different phila so this basidiomycy nicota phila there and if we look at the output of chacan that will be the easiest so the chacan tool output here to compare really we can really try to build a table of the species that is identified by chime and the other things and we can see that we can really compare the things and in saccharomyces cereviziae is identified in both of samples micatae is identified only in their data the paradoxus has been identified in both we identified the bianus that they didn't cazaxthania we didn't but we identified euclubirimises the bretanomises picia the dublenesis is something that we identified but they didn't etc etc so so some of the yeast that have been found in their data are really interesting especially the bretanomises proxylensis and we didn't why that is a good question did we not get enough reads to identify it that is something we probably need to redo a run of sequencing this data of this sequence and re-running the things maybe the databases was not good enough also maybe the data were not a good quality that could also explain for example that a lot of reads are wrongly assigned because the quality of the no-pose sequencing can make it less good to annotate the data to assign the reads to a specific sequence but globally we could really have an idea about the first idea of the diversity of beer microbiome with okiota, bacteria, we could identify archaea and different things we see that we have a contamination by homo sapiens that is definitely expected and a lot of other things and quite some diversity of saccharomyces and especially saccharomyces cerevisiae that is really expected in the beer so that is quite interesting something you can do afterwards is to share in your work if you want to share that with your colleagues for example you can share your history or extract a workflow that they can redo you can have a look also at that but globally to summarize what we did we take the data we check the quality of the data and clean the data before further processing to be sure that we can the quality work good enough to process that then we assign we assign the read to some taxon in the different taxonomic levels and then we could identify some yeast in that but we also identify contaminations possible contamination we then visualize the diversity the community of the microbiome community so all the different species or the different genus and their abundance that has been found in the data we did that with using different bioinformatics tools and galaxy make it easier to do it because of the galactic interface and to really summarize the biomarker brain is not just made of yeast as we say it's made of a lot of things and it can be quite complex and thank you for participating and if you have a bit of minutes to fill this feedback that would be awesome thanks a lot and I hope you learned something and you enjoyed these tutorials


This session, we are happy to introduce a terrific session called Open Simulator VR Commerce, a primer on contributing to the open-source GloBit money module. And our speaker today is Chris Colosi. Christopher Colosi runs GloBit, the most broadly used commercial service for OpenSim. He ran Second Life's Economy, Currency Exchange, and Web Marketplace for virtual goods after Lyndon Lab acquired Windmark Mark Interactive, a video game company he co-founded. Mr. Colosi has a BA in Computer Science from Harvard University. Welcome all, and hey, let's begin the session. Hello everyone, thank you all for joining us here today. Hopefully, you've heard of GloBit and hopefully most of you are users already, and you've come across a GloBit-enabled region. If you haven't, I recommend trying it out. Today, the focus we wanted to give was we recently released our plug-in for OpenSim as an open-source money module. Our repository is on GitHub, and we're hoping that the community will help us continue to improve, to maintain, to create a feature list. So today, I just wanted to give a quick kind of walk-through of the setup so anyone looking to do that will know where to start based on what they want to do. And I'll walk through just some high-level, I'm always available to answer questions. There's a lot more depth in our README file on our repository, but this will hopefully give a high-level intro for anyone looking to maybe improve the OpenSim money module, improve the kind of C-sharp generic component of the module, or port it to something. I know we have some people who've talked about web marketplaces, for instance. So GloBit is a whole website. What we're really focused on right now, if you've used it, you know that you can create an account. You've got your transaction history, your balance. But as far as the way the OpenSim system connects, GloBit has a RESTful API. That's an API that is hit via HTTP web requests. Some of those, not all of them that we make accessible, but a few of them up here, authorize, get balance submitting a user-to-user or a user-to-app transaction. So effectively, the money module for OpenSim is a tool so that our OpenSim worlds don't have to do all of the integration work themselves to make use of that. We effectively built a plugin that made it really simple for someone to tie the UI commerce functionality already built into OpenSim, your balance in the top right, buying an object in world, to effectively going through and making those calls on the GloBit website. This is a picture of our architecture effectively. Sorry. Adjusting my camera here so I can see my slides. The architecture to give you a high-level view of everything in our repository, all the files you might find in there, starting from the right, you have our website so effectively hitting our RESTful services. What communicates directly with that is a C-sharp client-side API. This is just something that is converting C-sharp functional calls to HTTP calls to web requests going to the GloBit service, making those connections, handling the communication to pull the responses back in, processing that, turning objects in C-sharp into JSON code that's going through. The client-side API is made use of from a functional helper layer to the left. That layer is basically taking whatever information we've got from the platform and grabbing the objects that are necessary, converting some amount of information to what the client-side API needs. It's handling certain callbacks that come in from asynchronous web calls. It's effectively a layer that's just making that client-side API simpler to use. Above that, you have the object layer. This is just a set of classes, basically. What we're sending to the GloBit web services are JSON code and C-sharp is an object-oriented language. On our end, we're creating objects. These are transactions, subscriptions, users within the system. That just makes that easier to store, kind of containerizes the components that we're making use of. Those attached to a database layer, that is where we're storing everything behind the scenes. Traditionally, if you wanted to do this integration yourself with our service, you would have had to store things like an OAuth token that comes back in. You might have to store some information about a transaction, at least through processing, if not permanently. This object layer is storing all of that in a database layer. Then, ultimately, moving further to the left, you've got the platform glue layer. This is what really integrates with OpenSim. That is truly the OpenSim money module piece. Effectively, the GloBit money module, as it stands in that repository, is really two things together. We may separate these at one point, but one is a generic C-sharp API. That could be used by other platforms, or someone looking to do a web marketplace integration. Most of that wouldn't change if they can use a C-sharp API. On the left, the platform glue layer, a little bit of the functional helper layer, I probably won't get into those details today, would change. Maybe a little bit of the database layer. I know I don't have that highlighted for simplicity here, but that purple piece on the left is really what the OpenSim money module is. That is really the piece that is deeply tied to how OpenSim makes use of our C-sharp API to hit the GloBit services. Quickly, this slide is just giving you an idea of what those classes are. If you're going into a repository, if you're looking to modify the C-sharp API and improve it, you're going to be dealing with the GloBit API wrapper or the API. If you're looking to improve the object or the database layers, you've got that. If you're looking to add functionality to OpenSim, let's say I think we haven't done group join fees yet, we've had people request that. Say someone wanted to build the group join fee transaction flow. Most of that is going to happen within the GloBit money module. That's the piece where that is. If you're going to change and add configuration values to how an OpenSim world can change something about how GloBit works, that's going to happen primarily in the GloBit money module. It just gives you a layout of the classes. I quickly want to walk through just what is happening behind the scenes from a high level that is kind of vital. There's more to this. You'll see the GloBit money module class is rather large. Effectively, that is a shared region module for anyone who's done some OpenSim coding. That gets loaded for every region. It gets loaded once for a SIM process. There's an add region call for every region that gets added to that SIM. You'll see a section at the top of that file that kind of handles the general instantiation. We use GloBit.ini as where we store our config values, and effectively, these files need to read in that Iini file and set things up. You'll see an I region module interface at the top, and I shared region module interface. Those are OpenSim interfaces that we are consuming, and those functions are getting called, and we're using those to set up the API. These are the primary and maybe most important pieces of setting up the API. We're creating a GloBit API wrapper class. That's that functional helper layer that I mentioned. That, by the way, behind the scenes, if you looked at that class, is creating a GloBit API and initializing the databases, basically, so that our database implementations know where to write. We're registering three rather important web hooks, callbacks that the GloBit system needs to be able to hit. These get registered on this end, and transactions, auth functions will send those URLs to GloBit to say, hey, this is how you contact our system. Chris, some of the audience is asking about your slides. Are these available anywhere if they wanted to get a copy of them? I can certainly make that available. They're not at the moment, but I'm happy to do that. Maybe I can get them up on our dev site and share out a link. What's the best way to get that information out after the conference to people? I think we send out a post conference email as well, a follow-up, but yeah, if you can send it to Learlobal, that would be great. Also, remember, I think you have an expo booth, right? Yes, so I'll go check that out. You could place it there after the fact as well, and then folks would know where to go to get it. I want to get through a couple more slides, and then open up for questions that people have, and I know we don't have a lot of time, so I'll do this pretty quickly. I mentioned users. We create a user for every avatar on a grid, on an app effectively. That's either called a global user or an app user in our system. The global user object class.get will create or retrieve one of those. You can do that before you've authorized that person with global, but that just generates in our system. Authorization is an OAuth 2 handshake behind the scenes. That is a protocol that lots of different companies use. In our system, that goes through an authorized function, gets called in the API wrapper. That effectively calls this interface function that the money module has to implement that just says, hey, load this for a user. In the case of OpenSim, we send it in IM because we can't just launch a browser. In the case of a web app, you might just redirect URL if you know that user wanted to go into that flow. Behind the scenes, there's an OAuth complete function that comes back in from the server and exchanges an access token that gets stored in the database. In the case specifically of OpenSim, we use our getUserBalance function to do this. We don't directly call authorized. I don't think anywhere in the money module, but you could if you knew, hey, this is where I want to authorize a user. Another flow, just to walk people through a transaction flow quickly. We implement in the money module the iMoneyModule interface. We also register a handful of OpenSim events. So different transactions come in through the money module interface or through registered events. For any individual transaction, we create a transaction type enum. We check for some potential failures before we submit it. We build an OpenSim transaction description map, which effectively is a bunch of additional information that we want to make available to the users in their transaction history. And you can look through any of the existing transactions to really get an example of how this works. We build a global transaction in the system and submit that to Globit through a function in the API helper. And then we get three callbacks, generally two callbacks that will come back in and enact and consume. And that enact is where the OpenSim Money Module or anyone implementing a system with a different money module has to handle delivery. If it's a by object, it's deliver that object. If it was by a land pass in the case of OpenSim, it would be, hey, give this person access to this parcel for this amount of time. That has to return true or false back to our system so that we know and then we handle the transaction. We cancel it and give someone their money back and then alert the system as well. And we suggest that you message the user through this process. So that walks you through a couple of the flows quickly. I know that's brief for tech. Our repository, if you've got things you want, features you want, bugs you want fixed, I highly suggest people file issues. We've had a few people do that already. If you're going to work on it and help us improve it, clone or fork the repository within your clone or fork, create a feature branch, do your editing, and then submit a pull request to us via GitHub. We'll get that. The issue gives people a chance to discuss design maybe before you get into it or someone else. Pick up what you want. Doing a pull request gives us the opportunity to review it to discuss and hopefully pull your improvements back in. Like I said, there's a lot more information in the Read Me. I suggest people take a look and I will open up for questions with whatever time we have left. And I hope that was a useful little primer for people. Yeah, it's a complex topic but an important one. Thanks for the work you're doing on it. So audience, do we have any questions about this? Looks like somebody's asking about Bitcoin or Bitcoin. We got one here from Commander X. This is a general question about Globit. Bitcoin functionality on the website has been locked down for some time now. My question is why and when can we expect to be able to enter and exit the Globit economy with Bitcoin again? Sure. We implemented a Bitcoin exchange using Coinbase a while back. One of the challenges you run into with third parties is sometimes they change their APIs. Coinbase, I believe, changed that one with very little warning. So we just haven't had the time to redo that implementation. We don't have a ton of users demanding it. So we hope to get back to that but it's been more important to focus on some other problems people want fixed. It's just a matter of redoing that implementation. There was no product choice to remove that. Same thing goes for Amazon payments. We had implemented Amazon payments. Amazon basically removed a payment API they had. They wanted people to use a new API. We haven't had the time to redo that integration. So that's why you'll see Amazon grayed out but we still have PayPal and credit cards. Well, I look forward to trying to use it. I haven't honestly used Globit yet myself. I've used the Kitely. But I'm not sure if yours is part of the Kitely if that's something separate. But the outfit I'm wearing right now is a samurai. I was purchased through that with options to either wear it just on the Kitely grid or to wear it hyper-griddable to all grids that are on the hyper grid. So that was better things to open simulator than you had in the past. But it's nice to see yours coming out and being able to do so much. Yeah, our goal is to just make it as simple as possible to be usable everywhere. We'd love to get everyone onto a single currency. We think it'll help all of OpenSim as a whole. And if we can make that currency usable across other platforms as well, great for everyone using and the stability and the functionality they'll get. So we are separate from Kitely. They've done a lot of great as well. I don't really have time to talk about the differentiation there. But that's fine. In fact, we need to wrap up as it is. One thing, if you do have a link for the repository, if you could post that in the chat for folks, if not, if you could get it to Lear Lobo later, that would be great. I will post it in right now. Thanks, Chris. Great presentation you gave. Really terrific.


What if you guys remember in the famous movie I think Tom Hanks was in it Apollo 13 The Apollo 13 Mission got into real problems, and they thought they may lose the astronauts and The spacecraft because they had a problem that meant that they couldn't re-enter the Earth's atmosphere And it was a major problem And there's a scene in the movie which is true to what happened in life On that mission Where the ground control in Houston the NASA Engineers just had to say to them listen We can't help you from here You have to use what's on board to be able to figure out how to get back home and they literally used a bit of rubber band and duct tape and plastic from a plastic bottle To rig something up that was able to help them to deal with some kind of Air leak problem inside the capsule they were in I'm telling you this to tell you that when the NASA Engineers on Earth said to them you got to use what you have on board. I believe that is true for us in life that That NASA was too far away to come and help them Wasn't practical to wait in space until someone else flew up there to help them And all NASA could say was they got to use what you got on board And I want to say to you guys today some of you are waiting for help a waiting for Flight control waiting for NASA to come and help you out of a problem. They're not coming You have to learn to use what's on board and life is about Being resourceful Not always having all the resources that you need to figure out and get out of a fix and When no one can help you this is when you discover how resourceful you can be human beings are the most resourceful species On the planet, or we wouldn't still be here So I want to encourage you guys to Discover how resourceful you are and can be right now Rather than sit and waiting for NASA as it were whoever that is in your life To come to you and fix you use what you have on board on board in your life. You have Ideas you have emotional intelligence You have relationships you have friendships you have things you've learned you have wisdom your things you've been through You know more than you think you know and you'll never find out what that is if NASA always keeps coming to rescue you as it were so Use what you have on board today guys You're gonna find it doesn't look glamorous. It's not Expensive it doesn't look to be a nice solution. It's not sophisticated or fancy doesn't need to be Just use what you have on board today You're gonna find it will get you through more scrapes than out of more fixes Than NASA could ever have helped you with anyway. So today, whatever you're going through Stop looking outside and up and around and look within use what you have on board Learn to be resourceful Even though you think your answer is I need more resources. You don't Love you guys


While the United States is being distracted by yet another false flag operation in the Middle East, other countries are under their own psychological warfare operations enacted upon them by their own government. It's interesting how the average person doesn't look at what's going on outside their own country as it could never happen to them. We are already divided, very different, which is why the tyrants in charge will bring us globalism as a solution. Everyone's miserable besides the elite. Australia, for instance, is comparable to a prison or at least house arrest. NSW and ICT lockdown restrictions update to Sydney, regional NSW and Canberra rules explain. Lockdown extended for greater Sydney, including a curfew and exercise limit. Outdoor mask rules mandated for the entire style. More retail closes and permits system for authorized worker. ICT went into a seven-day lockdown from 5 p.m. Thursday. He has the fullest of what you can and can't do in NSW on the ICT. From Monday, August 23rd, areas of concern will be under curfew from 9 p.m. to 5 a.m. Exercise there will be limited to one hour a day. Face masks will also be mandatory outdoors except when exercising when you leave home across the state. Garden centers, office supply stores, hardware stores and pet supply stores must close except for click and collect in those areas. Child care workers and disability support workers who live or work the areas of concern must have their first injection by August 30th. Authorized workers who work outside their area can only work if they get their first injection by August 30th or if they are tested. From next Saturday, authorized workers from the areas of concern or those entering to work will also need a permit. Everyone must stay at home unless they have reasonable excuse to leave. They also cannot have visitors in their home from outside their household, including family and friends. People can still have one visitor at one time to fulfill carers or responsibilities or provide care or assistance or for compassionate reasons, including where two people are in a relationship but do not live together. All hospitality venues must be close to the public, including pubs, restaurants and cafes, except for takeaway. Every employer across NSW must require their employees to work from home unless it is not reasonably practicable. It is a reasonable excuse to leave your home for work, but only if it is not practicable to work from home. This sounds wild. I hope some of my Australian viewers can chime in if this is true. And it seems like based on the population of the state, how much control the government has already is how far they're pushing it. And Australia, I guess, is back to the prison island. And for those of you that don't know, and to my knowledge, Australia was originally an island where they sent prisoners, I think in the early 1900s as like an isolation or like an exile kind of thing.  Australia is giving $5,000 fines on the spot for breaching these rules. You know, while Aussies are locked in their homes, their students are being held hostage for their HSC, which is a high school credit exam in Australia. To my understanding, they're equivalent of the GED, General Education Deployment in the United States. And this is actually the reason I'm making this video. This is kind of like the straw that broke the camel's back. Like this is crazy. New South Wales Health and Education are doing everything to back you in and make sure that you can go safely to your your HSC exams by October. But what we really need you to do is to go for gold next week. We have about 24,000 of you who can get along to the Kudos Bank Arena next week. And the way you do that is that there will be a link come out to you. There will be messages come out to you with a letter that will explain what you are required to do. But what you are required to do is just keep safe, basically. Get along to the Kudos Bank Arena. You can get there by your own car or by public transport. Maybe your mum and dad can drop you. If mum or dad or one of your family drop you, make sure they stay outside the arena because we don't want too many people. Obviously, milling around inside the arena, make some arrangements for after you've got your your access to that golden opportunity of the vaccination to wait outside for you. And to mums and dads, I just want to say and to the young people themselves that if you if you need to make a call because perhaps there's an issue that you're not quite certain about or that the email that comes out to you, you still got questions, then you can ring 1-800-922-886. I'll just repeat that number, 1-800-922-886. This is your big chance to really go for gold and to star next week. You're getting an opportunity that so many others haven't yet managed to achieve and it's there for you and it's there for you to really light up your future by having your safety and security looked after so you can sit your exams come October. I want to stress that to the mums and dads, your children will be well looked after inside when they arrive. They'll be literally ushered. We'll have nurses, we'll have some of the youth command, we'll have some of the young police from the various operations within police there just to guide them through as to where they go. They'll be looked after every moment inside the stadium and they'll be well cared for so mums and dads feel secure. We've got about 24,000 students we're aiming to get through next week. The messages will come out this week and the opportunity is there, but really you've got to take it, graft it with both hands. What's more sinister that they're targeting students or not allowing their parents inside with their own children. And to even get them there in the first place, they're basically barring students off from being able to participate in society. High school and college are seen as mandatory by most people, especially parents, to make any progress in life. And as soon as exclusion on that level is brought up, people are willing to do things they wouldn't normally do. I mean, even now with my successful business and YouTube channel, my parents are still telling me to go to school. It's insane how ingrained it is in the average person that you need college to be successful, yet the people in charge control those institutions. And they're just, you know, it would really be interesting to know who actually owns all these colleges and who's pocketing the $50,000 tuitions, but we'll probably never know. France is being a little more straightforward than that. In his address to the nation on July 12th, Macron said that from August people would be required to produce health passes to visit cafes, restaurants, shopping centers, hospitals, retirement homes and social institutions, as well as for long distance trips on flights, trains or buses. Though Macron has said that people will need health passes to enter shipping centers from August, a statement by the French Health Minister, Olivier Varan, suggests that rules would be different for supermarkets. A recent report by Connexions carries a statement by Varan. We are talking about big shopping centers, not supermarkets. They really like mincing their words, huh? Oh, we only meant that you wouldn't be able to go to big supermarkets. That's the larger one. There are all the other French men and women, the millions of you who have not yet received an injection. Depending on how the situation develops, we will no doubt have to ask ourselves the question of mandatory vaccination for all French people. I am choosing to be confident and I solemnly call on all our unvaccinated fellow citizens to get vaccinated as soon as possible. So the vaccination will be mandatory for health care workers. There was after a consensus with other political forces that have to do it by September 15th or they will be at risk of losing their jobs and their salaries for everyone else. It's not mandatory yet, but your life will be much more difficult if you don't get the job. That was part of the announcement yesterday. But President Macron basically saying we're going to put a restriction on the non-vaccinated people rather than everybody. And so that means that we use the health pass, which is this app on your phone showing your vaccination status or recent negative tests for more socializing areas. Man, these people really need to be lined up in front of a ditch. Not even passive aggressive. They're straight up telling people their lives will be more difficult. We, as the government, are going to make your lives difficult if you don't listen to us. One interesting effect is that almost a million people yesterday booked an appointment for vaccination online after the President's address and certainly some positive effect there. A million people booking is an interesting effect.  But we will see long term what it means. Of course, some people criticizing this move by the president, but there's a general consensus among political forces that this is a way forward. Hold on. Let me repeat that. General consensus among political forces that this is the way forward.    How easily is the average person willing to listen? And how far do they have to go? We know the game is already rigged. They already have a huge advantage. They have secrets about life that if the average person had, it would be a much more even playing field. And that's the opposite of what they want. So they need to take everything if they have ninety nine point nine percent of the planet in their control, they want ninety nine point nine, nine, nine. They want a hundred percent. They don't want any chance of anyone outside of them having a chance. So that's really it. I mean, we talked about the social credit system. We've been talking about this stuff. Maybe I'll talk about Canada next week. But, you know, since they control the media and all social media platforms, it seems like we as the silent majority aren't the majority. But go figure. So thank you guys for joining me today. If you could please drop a like on the video. Leave a comment down below. Subscribe so that YouTube can unsubscribe you next week. And be sure to check that notification bell so they don't notify you of my videos. Therefore, you can go to FrankTasherFound.com to support me through all of my businesses. Thanks again, guys, and I'll see you tomorrow.


It's time got a hat just for this watch through I have to be sort of on brand so like friend for Naruto or porto I have the headband I had to have something for this and I don't think a straw hat would work very well So I thought this was cute. What's going on guys welcome back to the channel My name is Jack the bus or I should know be today We were back with some more one-piece episode seven and eight episode seven is called epic showdown swordsman Zoro versus Acrobat I'm not really sure last episode. We sort of set up the fight against a buggy the clown I don't know if they're like confronting him again, but they're going with the mayor this town this brave brave man And they're gonna take on buggy the clown see what goes down exactly I hope it's not another dramatic sad backstory that we got like with the dog in the last episode because I'm gonna be honest It cuts in my heart We're gonna get started if you knew the channel drop a sub hit that people with bells from a seed future episodes Drop a fat like on this one. Let's just hop right into it Oh, we went back kind of far actually. Oh, it's just it has to show this to me again So she's gonna be Navigator was that her job? They must go rescue the mayor. He can't die for this He doesn't deserve to honestly one of the only people that stayed back behind to defend the town at least try to The showdown begins So goofy, bro At least she's open about it I thought he would be touched that they would want to help him if it's more a dignity thing Don't you will get rolled by captain buggy.  I have a feeling is just gonna bounce off or something.   That was just a regular one So even if he had fired it when Luffy was in the cage, it wouldn't matter Who knows?  Did he use them to defend himself? Oh my god. That's shame was buggy.  I I hate it. It feels like there's just no threat to him at all at least yet It's gonna be interesting to see the first that like actually matches him I'm like buggy kind of does since he ate a double fruit too, but still not really. I don't feel worried at all Oh, that's the was he called in the title You really can't say much if the captain used to literal people who cares about Richie. I just care about the dog Scared of him. This guy actually looks a little bit more intimidating like even buggy. I don't know why He's actually just getting thrown around by both parties though. This guy looks kind of scary Oh, he's the acrobat. That's right And Zoro fighting fights him I guess it makes sense because it's like a circus Swords versus swords Sly you that's a that's a bold old statement. He is still injured though Backing up Open arsonist.  What is that? Oh my god? Wait Zoro's actually getting kind of beat up here It's not very he's already injured. I guess Zoro took the fight Hey, dude, listen listen, I wouldn't have been surprised if these guys fight dirty You can call it anything you want as long as it looks cool. I Kind of like this guy's whole style more than buggy low-key. It's just unique Hiding his power level. I feel like Luffy's just kind of yeah, he's just chillin I can't tell if he's entertained by it or just doesn't care I feel like it would upset him to see Zoro get hurt since he's the first member of the crew No, I mean he maybe There you go. Oh No Or maybe Luffy just trusted Zoro enough to handle it himself. That's why he doesn't care He has to be the best The need to miss an excuse Light injury literal stab wound there he goes. It just doesn't seem effective to hold it in his mouth What she has ulterior motives here I mean listen, can you blame her? You said you're doing the crew though. Wait, didn't she he's not gonna stop her Okay, what is this dude? I Just randomness They're bill different Yeah, but it'll be fun. Come on.  It's this buggy. Just watching the fight No one's gonna just be there to defend his stuff Meet mom my man's fighting for his life You can't just call everything these names Oh buggy wants to fight Luffy. No way you start partying What is this? No No manners to have some morals I guess he didn't really anticipate anyone breaking in so the only ones down, but I thought he would see her like sneak away I don't know she's blowing a hole in it.  What was her goal like a million dollars something like that? She's gonna have her own fight gotta earn it Okay, this dude looks like very side character villain and he's hungover too. We're still drunk Time to capitalize Top-tier acting. I Do like how she uses that like expands just like multiple pieces Okay, easy mode Oh the map Oh the chart. Yeah, she cares more about that than the treasure honestly Gotta pop off Zora What's next dude? This man says like these whole absurdly long names for simple like movements Tony Geary Now it's the 1v1 the big bad boss I mean if she actually got Luffy and Zora's how they could get all of it She thinking back to them all the mayor Become the greatest pirate of all time This first bump in the road he has to take care of So he knows He's like made a name for himself Gonna have to take him down to find out If he's fighting him to find out more about where shanks is he's gonna fight with even more passion.  I think we've seen What would he not be able to They're just figuring out each other's skills I feel like defense wise he has a lot of mechanics, but I Guess offense wise you can just throw himself at him But you can do a capitate and we can probably just reattach it.  Oh my god, that was beautiful That's not their fight at least they understand they're smart enough to understand That's very meta. It does make sense that they wouldn't be knocked out that long Come on Luffy Oh my oh, he did cut the hat. Oh No, I didn't see Luffy this man. Yeah for real. Oh Buggy, I would be scared if I were you brother Can he fix it so dirty dude? Oh, oh, he found a weak point. That's gonna tear him apart Oh Dude Luffy is about to lose it. I like how it like I said how the shows already set up these characters for us to like be hopeful to see Gosh Such surface-level thinking they're so over it. Oh God you messed up This by my side Oh gosh, I mean it could have been when buggy was a different man too. Oh God hi, I'm buggy.  She thinks that he's had some morals. Oh He betrayed him. I wouldn't have been surprised. I'm surprised buggy's actually willing to talk to Luffy You see me Dun-dun-dun On to the next what is episode eight called who is Victor double fruit power showdown? I let us get into it as they say My god it goes back so far That was Zoro's best moment yet though only Gary those six see people told me to avoid one piece Titles cuz they can spoil it literally like I'm not gonna look away Time for some lore, baby. Oh you got outsmarted Oh, they should have yeah had like one-on-one beef That dude that like knocked them on the head is part of of shanks crew now, I think that looks like a one of them I'm gonna plunder harder Yeah, see you can already tell there were differences between the two about how they viewed being pirates Although they were comrades This is the treasure I'm guessing that shanks stole My man has not changed even remotely or maybe this is how he got the Devil fruit. Maybe they both found a double fruit. That's how shanks came into possession of that one No, because I was much later on Although sharing a moment Dang, they actually were friends although they were just kind of on the same ship Yeah But if shanks actually did still his treasure that's like something like buggy would say pirates do I guess cuz they were friends So is that the one that eventually Luffy Okay, that's that's the one that he ate, okay makes sense It's worth that much and that's how he got the powers Just throw him in the water see if you can swim Wait, how would he swap it out? How do you make a fake one? Oh, I see dang you explain I'm selling it.  I knew shanks wasn't a bad guy buggy is such a goofster, man He literally jumped in to save him too, and so that's how he lost the treasure dude. You have to be joking I love the low-res shots of Luffy so much. Oh Oh my gosh, it's your fault. He did have a little bit of a glow up. I like the skull and crossbones and whatnot Popping off Just go for the legs wait, he's actually just gonna start like ripping the legs apart Oh I think about it like he would still feel it because it's still his body. It's just spread into different places Oh, so he leaves their little encampment and goes. Oh It makes a lot more sense the town people should go and help Taking them down fight for your town Oh Fight for your your place. Okay. I was wondering like where everyone went I figured they didn't just straight up hop off the island and go somewhere else got a hard to just pick up and move Luffy kind of just seems sad about his hat. Just start hitting his head with all the treasure Understandable not anymore.    Oh It all ripped. Oh man. I mean at least he has to chart of the grand line still In his honor How is that stabbed into the brick? I'm sorry, is he a ghost? What was that? She looked at Luffy and I think you've made a fatal miscalculation buggy.  Thank you. Oh Man, oh, no, it's just got sent to the next dimension. Oh, he even got the twinkle in the sky. Oh damn. That's it. Buggy's defeated You're free. What do you mean seem like 90% of you hated him?  Can you fix it? Still wearable. Wow. I'm surprised not more torn up about it. Oh, she saw how passionate he was She's joining the crew still Yes, good taste That was her dream 10 million Shoot yeah, she's giving it to have dang that's huge The first step in his journey become the greatest pirate Come on It'll be fun. Yeah, she was a navigator. Okay The mayor's just laying over there all the towns people are gonna come check it out and see the buggy's gone Well consider yourself saved. Oh No, it looks like they knocked him out No My man likes so much social skills You're gonna have to dip quick It's funny how genuine he is though Time to dip At least she got away with some of the treasure if only the mayor woke up to explain things But at this point it's too late. He said their pirates. They're just gonna see pirates is bad I feel like that is a good sign true There's no way to explain what would actually went down here. He's there to guard them now Yeah, she knows what went down here. She was she knows all do not disobey Thank you I like how it wraps together with shoo shoo having something else or someone else to defend in a sort of way That's pretty wholesome actually this buggy ship. Are they just gonna steal buggies. That's a nice little boat.  What even is your face? It's like a pointy on the sides Clearly like I said, there's no way he can explain what went down here Is he gonna tell them that they were actually like fighting for them. Oh I guess he would be kind of upset that he knocked them out But still they got rid of buggy It's a long story. I believe you would want some last words. Yeah Good and bad. They are they yoyking buggies. I wouldn't blame them. It's huge. Oh They're just taking both of those Till you showed up. Oh, that's sweet pirates aren't all so bad. He's got his town back feels good, man a bag of gold oh My I thought she left it on purpose. Oh My god Hey, that's fair. He can't swim nami You're gonna go yoyking from that poor mare well one way or another we have another member of the crew Put it in the words Zoro hold up Oh the next member and so a saga sort of ends in a way It seems like I guess the end of the entire buggy part I mean he literally like I said knocked him into the next Dimension and now we have another member of our crew joined the navigator nami and it looks like they're already teasing I believe that's another member of the crew like I said I've seen like shots of the crew before and I have like a general idea of what a majority of them look like I think there's only a couple more. I'm not really sure But nonetheless, I really enjoyed those fights. I actually really liked a Zoro's fight with the what was it? What was he called? Oh with the acrobat Kabaji actually really liked a lot really showed I guess Zoro sort of resolve I mean we already know Luffy is really strong and we saw Luffy pop off here Especially whenever the hat was was sort of I guess not really destroyed but tarnished a little bit but we really saw what Zoro could do especially under pressure like that and what he's willing to do to become the Greatest swordsman because that's really it for episode seven and eight of one piece Let me know we thought of the episode down in the comments below and as always I hope you guys have a wonderful rest of your day as per usual and I catch you in the next one his pace


Hello, welcome back to my channel and a very special welcome if you're new here. My name is Jen and I'm a certified weight loss and nutrition coach.  So I have been sick all week with a cold. So I apologize in advance if my voice is raspy, if I sound congested, my nose is so sore from blowing it. I literally used a box of Kleenex in one day. That is how this cold has impacted me. I have been pretty sick with this cold for almost an entire week. So I apologize in advance for the raspiness, but I definitely wanted to hop on and share with you how my week went overall, my weigh-in for the week and talk with you about this week's Weight Watchers workshop topic. I do a weigh-in video every single Friday. So if you're excited, give this video a thumbs up, subscribe, turn your bell on, because like I said, I do a weigh-in every Friday and I actually upload five videos every single week. Check out the description box down below where you will find nutrition coaching. I do offer personalized to you macros and calories. This is how I have lost and maintained a 140 pound weight loss. Highly recommend having your macros and calories done, as well as one-on-one coaching for accountability to ask questions or talk with me directly. Links and discounts to my favorite things and my Facebook group come over and join us. We would love to have you are all down in that description box. So let's hop into my week, my weigh-in in the Weight Watchers workshop topic. But like I mentioned, I have been pretty sick with the cold. I came back from my trip to Atlanta on Tuesday and by the time I got home from bootcamp on Friday morning, I wasn't feeling very well. And that has translated into this Friday and still not feeling very well. What was interesting is I felt like it got worse before it started to get better. I know that there's quite a few colds going around. For me, it's been mainly congestion. Like I said, blowing my nose constantly. I don't have a cough. I have a bit of a headache. I did have a sore throat for a short period. That went away. Luckily, I haven't had a fever but I just haven't been feeling very well overall. I've been very, very fatigued and tired.   I'm hoping that I'm at the tail end of the cold because besides just not feeling well overall, I also wasn't able to get in all my workouts this week. I took Saturday and Sunday off because that's when my cold first came into play and I was not feeling very well and I really wanted to rest up in hopes that I could go to boot camp on Monday. Now I did go to boot camp Monday morning and I made it through my workout but it was tough. It was tough to make it through my workout. I went back to boot camp on Wednesday. By Thursday, I was back to my normal workouts but I didn't, I don't feel like I got as much out of my workouts just because lack of energy and just really truly not feeling very well. It's also really impacted my sleep because I'm so congested that I have to breathe only through my mouth. I can't even breathe through my nose that I wake up multiple times during the night to blow my nose, to try to relieve some of the congestion. It's been a lot. It's been a lot since I've been back from my trip and I have not been sick and I don't know how long. Definitely not since we lived in Arizona. I have to say I'm pretty bummed out to have a cold. Nobody has time for a cold. I have a lot going on and in fact I'm doing a 5K with my boot camp group tomorrow so I'm really hoping that I wake up feeling better and can make it through this 5K. It's a super fun Valentine's Day wine themed 5K. I'll definitely post pictures on Instagram and in my Facebook groups. Fingers crossed that I wake up tomorrow morning less congested and can make it through this 5K. Because I'm so congested it's affected my taste buds as well. I can't really taste a lot of food so I've been not enjoying my food this week unless it's super spicy or super flavorful. I have a hard time even tasting it in general. I've been kind of hit and miss with tracking my food. I'm always mindful of what I'm eating but I haven't been the best with tracking my food. I've been really still focused on protein but because I hadn't tracked I wasn't sure if I hit my protein goal. Sometimes life happens and our weight loss journey has to go on the back burner and unfortunately for me I'm rounding out my eight week cut. I'm actually starting week eight today of my cut. So it's not the ideal time to be sick. It's not the ideal time to not track my food but like I said, sometimes life happens and I have to worry about taking care of my health more than I have to worry about tracking my food. We'll share my weigh-in of course for the week with you and as a reminder last week I actually gained a pound post my vacation so we're gonna talk about how that translated into my weigh-in this week and the fact that I haven't tracked my food maybe haven't eaten my normal foods this week and haven't gotten in all my workouts. We're gonna see how that impacted my weigh-in but before we do let's quickly chat about this week's Weight Watchers Workshop topic. That is a new way to respond to setbacks. Now this topic is perfect because this whole week has been one big utter setback for me. One big utter setback for me. It's been one thing after another with not feeling well and not tracking my food and not be able to taste my food and not be able to exercise. It's been multiple setbacks and it can be really discouraging when we encounter so many setbacks. They're totally normal but it's hard not to beat ourself up and it's hard to get away from the setback and get back into a healthy routine. So here's some things that we can try that are new when we encounter a setback. Number one, imagine your friend fills down because she went over her point or calorie budget. What would you say to her? What tone would you use? Number two, now imagine you feel down after doing the exact same thing. What would you say to yourself and what tone would you use? Number three, is there a difference between your two responses? If so, why? How would it sound if you talk to yourself like a friend? Would you ever lecture a friend who says they went off track or tell them they're a failure or they're never gonna lose weight, they're never gonna reach their goals? You probably wouldn't say that. So why would you say that to yourself? We need to make sure that we're talking to ourselves just like we would talk to a friend. We tend to be our friend's biggest cheerleader so why not be your biggest cheerleader? We talk badly to ourselves when we think negative thoughts, when we bring those negative thoughts into the universe, it can really derail our overall progress. When something happens in our life, for example, getting sick and we're like, well, now I can't work out, my food doesn't taste good, I'm not gonna track my food, forget it, I'm just gonna throw in the towel. Would you tell your friend that if your friend came to you and said, hey, I'm not feeling very well this week? No, you would help her navigate through that to still be successful in her weight loss goals. Thinking about how you talk to yourself moving forward can really help you overcome setbacks in a more positive, effective way. Give yourself grace, treat yourself like you would treat a friend. As always, I have three really good fast facts from Weight Watchers for you. Number one, research shows that tough love makes you less likely to reach your goals. Number two, consistently replacing self-critical thoughts with self-compassion helps you learn to treat yourself more kindly. And number three, being kind to yourself as you handle challenges has been shown to increase your motivation to keep going. Treat yourself like you would treat a friend. Be your biggest cheerleader. I promise you, and I promise you from personal experience, I promise you from helping so many of you reach your health and weight loss goals, talking to yourself in a positive way, reinforcing good behavior, learning from setbacks and bringing them into things and bringing those into things that help you be successful is really important to not only lose your weight, but to keep it off long-term. Give yourself grace and talk to yourself just like you would talk to a friend. Like I said, this topic is perfect timing because I have been beating myself up a little bit this week. I've been beating myself up because I had a bit of a game last week. I've been beating myself up because I haven't been able to work out. I've been beating myself up because I haven't been eating like normal. Honestly, I haven't been on the scale very much this week. I'm a daily wearer, but I just haven't been up to it because I just, I have been so focused on trying to get better that I really had to put my cut and my weight loss journey on the back burner.  It's okay to do that when you encounter a setback. What you have to do is once you start feeling better or once you've navigated that setback, just get back on track and in the process, talk to yourself in a positive way so that when you overcome that setback, you can get right back to reaching your weight loss goals.  I have not cared at all what the scale says. I have not cared about maintaining a cut status. I have not even cared or thought about my cut at all. I've been really just focused on improving my health so that I can get back to my normal routine. But I did decide to go ahead and step on the scale this morning just to kind of have a starting point moving into this next week in hopes that I'm feeling back to normal. And when I got on the scale today, I weigh exactly the same as I did last week. So I maintain my weight, which means that that pound of weight gain was most likely a true weight gain from my vacation. And I'm okay with that. I'm okay gaining a pound. I enjoyed myself on my vacation. I haven't been my normal self this last week.  I'm still down overall during my cut period and really truly a pound is nothing to me. Once I get back to normal, I should be able to get right back to my normal weight. And like I said, I maintenance is a range, not a number. And I'm well within my maintenance range. So truly with everything going on, I'm thrilled. I'm thrilled with having a maintenance. I'm okay with having a one pound gain overall from my vacation.  I'm honestly feeling really good about my body. I'm feeling really good about my health and my fitness.  I still was able to get in my full workout. I'm still proud of myself for getting in my workouts and not letting not feeling well derail me overall. So I'm okay with where I am. I'm okay with the maintenance. I'm looking forward to this next week and hopefully feeling better. And of course in next week's weigh in, I will be giving you the final results of my cut. I'll be inserting some pictures because even though the scale hasn't changed a lot, you may be shocked at the photos from the beginning of my cut to now. You know, I always say that the scale is really the least important thing to me anyway. The scale is the least important measurement of success for me. The photos, the measurements, the way my clothes fit, my health, my endurance, my athletic ability definitely outshines any number on the scale. So I'm excited to share with you the end of my cut and what I'm going to be doing moving forward. I wanna hear from you. How was your week? Did you gain? Did you lose? Do you talk to yourself in a positive way or do you talk to yourself in a negative way? And maybe these tips and tricks will help you treat yourself a little bit more like a friend. I'm definitely going to be utilizing these tips for myself.  Check out that description box down below for nutrition coaching, links and discounts to my favorite things, and come join our Facebook group. We'd love to have you. Happy Friday friends. Here's to a super fun weekend and I'll see you in tomorrow's first week haul. Bye.