 that archive using Galaxy. The tool we'll be using today was designed to cater for researchers that want to upload SARS-CoV-2 sequences to the ENA, but it can also be used to submit other species sequences to the ENA. We will cover two examples. The first one will involve inputting the metadata using the tools interactive metadata forms and the second example will involve inputting the metadata using a pre-built template. There are two requirements for using the ENA upload tool. First, you need to be registered at ENA's women submission platform. If you do not have credentials yet, you can go to the registration page and sign up. I included the link to this in the description of the video. The second requirement is that you are registered with usegalaxy.eu. The first thing we need to do is to include our webbing credentials into the Galaxy user information. For this, go to the top menu, select user and preferences and then select manage information. Once there you can scroll down until you find the ENA webbing account details box and there you can put your webbing number and your passport and then save. And now the tool can submit sequences to the ENA. For the first example in this tutorial, we will submit a single sequence to the ENA and generate the metadata interactively using the tool. We begin by creating a new history and we upload the data. All the data that is used for this tutorial comes from the SRA and has been processed so that the human traces have been removed from them. We'll use FTP to upload the data and I've included the links to the Zanotto repository in the description of the video. While the file loads, you can start looking at the actual tool that we'll be using. It's under the send data menu. It's called the ENA upload tool. So the first thing we see is we have two options to execute either add new data, modify metadata, we'll leave it to add new data. The second box includes debugging features. The first one is an option to submit to the ENA test server and we will say yes to that because otherwise your reads and your raw data will be submitted to the public server and everyone will be able to see it. The second option is to print the metadata tables but not submit the datasets but we will leave this at no. Next, we come to the metadata section where we ask if we'd like to submit pre-generated table files or to interactively generate the metadata. For this exercise, we will choose to interactively generate the metadata and now we get to the metadata input forms. First question is are we submitting viral samples? We'll select yes and what this does it adds metadata fields to the sample metadata object. Maybe a brief note on the ENA metadata model. The ENA's metadata model has overarching studies which are populated with experiments and samples and runs are added to experiments. I've added the link to the metadata model so you can read more on it. In the ENA submission tool, there is a form for each metadata object and they are nested such that you are allowed to add samples to studies and experiments to samples and runs to experiments. For this example, we will have one study, one sample, one experiment and one run. For this example, we'll use mock metadata placeholders instead of real metadata. The last metadata object is the runs and this links the actual data files to the rest of the metadata and we select the file and finally we include our affiliation at the bottom. Click on execute and our data metadata is submitted to the ENA. There are four tables that are created, one for each metadata object and a submission metadata ticket or receipt that can be later used to input the metadata to other tools. Once the tool has submitted the reads and the metadata, we can go to the ENA test server to confirm that our submission was successful. I've included the link to the test server from the ENA in the description of the video. Once we log in, we can go to studies and confirm that our mock data has been successfully submitted, our mock sample has been successfully submitted and our mock experiment and run have been successfully submitted. In the second part of this training, we'll upload a larger data set to the ENA but this time we'll be using a metadata template to upload the metadata. First we're creating a new history to hold our data and metadata. You can find the metadata template at the bottom information of the ENA upload tool. You can then download the template and fill it with your own metadata. The template has four sheets, one for each of ENA's metadata objects, study, sample, experiment and run. It also has field descriptions indicating what is expected at that field and whether that field is mandatory or optional. And lastly, it has control vocabulary fields. For the sake of time, we will not fill this metadata template during the training but instead we'll upload a pre-filled template into Dallas. I'll include this link in the description of the video. You can now have a quick look at the metadata and we'll see there has one study with two samples and one experiment per sample and one run per experiment each containing a set of paired-end data. Next, we can upload these paired-end sequencing files. Again, use an FTP link that I include in the description of the video. After uploading, you can make a collection with the paired-end files and give it a name. Now that our data and metadata is ready, we can go back to the tool to submit it to the ENA. We'll leave the action to add new data and again submit to the ENA test server. We will leave the user generated metadata tables and select the Excel file with the field metadata template. We'll check yes for a viral sample and select the paired collection for our data inputs. Last thing to do is fill the affiliation center and submit to the ENA. Finally, we can go to the ENA test server to confirm that our submission was successful. And there we can see that our study was accepted. I hope you found this tutorial useful and you can use the tool to submit your data to the ENA in the future. Thank you for listening.