Skip to 0 minutes and 5 seconds Hello everyone, my name is Dr. Anna Protasio. I work at the Wellcome Sanger Institute. Today, we are going to learn how to create our own files in Artemis. For this purpose, we are going to use the example of pathogenicity islands. We are going to isolate these regions and save their sequence and annotation to a separate file. From the Artemis software, lets navigate to the File Manager. I’m in the desktop. I have the St.dna and St.tab files. I’m going to open first the St.dna, and on top of that I’m going to Read An Entry, which is going to be the St.tab.
Skip to 0 minutes and 51 seconds I’m going to ignore the warnings for now. So, here we have the Artemis window with the sequence and the notation. For this example, I am going to use the pathogenicity island called SBI7, which is located in the nucleotide 4, 4, 0, 9, 5, 11. We are going to use the Navigation tool to get to this position. And this is located under the Go To Navigator. In the Go To base, I’m going to type 4, 4, 0, 9, 5, 11 and hit Go To, and here I am positioned at the beginning of the pathogenicity island, which is this very large region here. And zoom out a little bit. You notice that the view gets a little bit busy.
Skip to 1 minute and 49 seconds So, I am going to turn off the Stop Codons, like that and this becomes much clearer. I can slide a little bit to centre the pathogenicity island in the centre of my screen. Perhaps, I need to zoom out a little bit more because it’s quite a large region, and here its contained within the whole sequence. In order to select this region, I’m going to click here at the beginning of the sectors and I’m going to drag the mouse all the way to the right until the end. And I can un-click now, and from here I’ll go to Edit. and then I select the option that says Subsequence (and Features).
Skip to 2 minutes and 35 seconds This will open a second window in which we have only the sequence that we have selected and the annotation that is contained in it. To do some good housekeeping, I’m going to minimise the other window. I’m going to keep the new window on top, Open it up, and I’m going to save these two files. First, to do that, I need to right-click on the first entry, which at this point has no name. File - Save An Entry As - New File and we will select ‘no name’ at the top, and I am going to call it SPI7.dna.
Skip to 3 minutes and 27 seconds So, that will save my sequence. In order to save the annotation, which is saved in the second entry, I’m going to right-click on the ‘no name’ entry and you’ll see that the highlight moves;
Skip to 3 minutes and 41 seconds and here I’ll repeat the same actions: File - Save An Entry As - New File. And this time the ‘no name’, and we’re going to call it the same, SPI7. But instead of dna I’m going to call it tab, and that’s saved. So, now all these files are saved and they are a copy of the region of the pathogenicity island found in the St.dna file. In the next step of this exercise, we are going to transfer just a selection of genes to an even new entry. I will just pull here to the right.
Skip to 4 minutes and 18 seconds And we will see that some of the genes that are part of the pathogenicity island are coloured in pink, and this is because they are part of a bacteriophage. Then I’ll turn off the Stop Codons again. So, if I want to move them all at the same time to a new entry, I could potentially select them one at a time, but that is going to take me quite a long time. I can use the fact that they are all of the same colour. So they have been annotated to have the colour pink, which is encoded in just a number, 12.
Skip to 4 minutes and 57 seconds I can select all these features automatically by choosing the tab Select and then Feature Selector, and in here, I want to keep the entries that have a Key equal to CDS (CoDing Sequence) and a Qualifier, which is going to be colour, and the text containing in that Qualifier needs to be 12. I can view these genes, and here we have a list of all the genes that have that particular characteristic. You can see as I click here in the list, I can see them highlighted in the main Artemis window. I can now select them all at the same time and see that they are all highlighted, and I can now move them to a new entry.
Skip to 5 minutes and 42 seconds In order to create a new entry, I’ll go to Create - New Entry, and that is there. It’s being created. It has no name and I can now right-click anywhere in the annotation where the genes have been highlighted, and I can go to Edit and then Copy Selected Features to the ‘no name’ entry. To make sure that these Features have been moved, I can turn off the SPI7.tab entry, and we can see that the only ones remaining are the ones that I just moved. This will allow me to manipulate this file without compromising the original file. I will now save this entry to an EMBL file, so I can keep it somewhere safe.
Skip to 6 minutes and 29 seconds For that, make sure that the entry that you want to save is highlighted, as it happens now. Then you go to File - Save On Entry As - Choose EMBL Format and then choose ‘no name’. See that it automatically gives you the option of adding the .embl extension at the end. I’m going to call it, test1.embl, and I am going to Save it. With this, we’ve come to the conclusion of this video, where we’ve learnt how to isolate a defined region of the genome for future analysis. We also learned how to save the sequence and the annotation. Don’t forget to give us your feedback in the comments area. My name is Dr.
Skip to 7 minutes and 10 seconds Anna Protasio and I will see you very soon.
Navigating Artemis using the example of pathogenicity islands with Dr. Anna Protasio
In this Step, we will use Artemis. We will isolate a region of interest in the Salmonella typhi genome. We will analyse this region and save it in various file formats.
In order to reinforce our learning, there is an exercise for you to try before or after watching the video.
We will use the genome (St.dna) and annotation (St.tab) introduced in earlier steps. If you need to download these files, you can do so now: ftp://ftp.sanger.ac.uk/pub/resources/coursesandconferences/Online_Courses/Course3/data/S_typhi.dna and ftp://ftp.sanger.ac.uk/pub/resources/coursesandconferences/Online_Courses/Course3/data/S_typhi.tab.
You may need to copy and paste the links in your internet browser and download the files using your browser. We recommend use of Chrome or Firefox browsers for downloading data files.
Open the St.dna file and load the annotation from the St.tab file. Use the Navigation to go to position 4409511. The region we are looking at is defined as a Salmonella pathogenicity island (SPI). SPI-7, or the major Vi pathogenicity island, is ~134 kb in length and contains ~30 kb of integrated bacteriophage. The region you should be looking at is shown below and is a classical example of a Salmonella pathogenicity island (SPI).
The definitions of what constitutes a pathogenicity island are quite diverse. However, below is a list of characteristics which are commonly seen within these regions, as described by Hacker et al., 1997 in their review Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution. https://onlinelibrary.wiley.com/doi/epdf/10.1046/j.1365-2958.1997.3101672.x
- Often inserted alongside stable RNAs
- Atypical G+C contents.
- Carry virulence-related functions
- Often carry genes encoding transposase or integrase-like proteins
- Unstable and capable of changing location in the genome (jumping DNA)
- Of limited phylogenetic distribution
Pathogenicity islands are important genomic regions. They contain genes that contribute to the virulence of the bacterium. These genes can have a direct effect by making bacteria more virulent or by controlling the expression of genes found outside the pathogenicity islands.
In order to study this pathogenicity island in depth, we will isolate the region that contains its sequence and its annotation.
Watch the screencast above to learn how to do these steps and learn more about pathogenicity islands.
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