1. GeneWise and Artemis Exercises

2. 2 1. Spliced Alignment using GeneWise Click on the GeneWise hyperlink on the course links page, http://www.tigr.org/tdb/e2k1/ttg/course_link.shtml. http://www.tigr.org/tdb/e2k1/ttg/course_link.shtml Select “interactive” from the drop down menu for Results. Check “Pretty alignment” if unchecked. Output can have all options checked. Peptide sequence: In the box that says Sequence 1, click Browse to upload the query file “rps4.pep” located in the Data folder in the Annotation_course/Sept_23 folder on the flash drive. DNA sequence: In the box that says Sequence 2, click Browse to upload the bac file “rps4.bac” located in the same folder as the rps4.pep file. Click Run. It will take at least 5 minutes, so please be patient! Analyze the output. notice the spliced alignment - 5 exons and 4 introns.

3. 3 When to use/trust GeneWise GeneWise based annotation in the absence of Fl-cDNA or ESTs GeneWise prediction is simply the one encoding the longest CDS sequence based on homology and works the best if annotation of closely related species are available. Ensembl style annotation

4. 4 2. Artemis Click Artemis on the course links page From the Sanger website, click LAUNCH ARTEMIS Click OK for Open with Java(TM) Web Start Launcher Click Run Click File; select Open and load the file artemis_genbank_file.txt from the Data folder in Annotation_course/Sept_23 on the flash drive. (please make Files of Type = “All files” to see this file) You are opening the annotation file (GenBank) of chromosome1 of the Cryptococcus neoformans genome. Click Yes to the warnings.

5. 5 Artemis basics Frame lines and features Scroll bars – horizontal and vertical Feature list Start and Stop codons Main Menu We will explore some basic functions in Artemis including Goto, Edit, Create and View. All the functions that you see on the main menu (top left) of the window are also accessible by right-clicking a gene or a feature like an exon.

6. 6 Artemis (contd.) Navigate to a gene and edit the start site: On the main menu, click: Goto; select: Navigator Select: Goto Feature With Gene Name. Type: CNA00570 in the text box. Click: Goto The gene CNA00570 is on the reverse strand. Zoom in to the start site. There is another potential start site upstream of the current start site. Click this upstream start site to see its coordinates [complement (162075..162077)] To change the start site: Right-click on the first exon. Select Edit and then select: Selected Features in Editor. This will open up a new window. In the text box for Location, replace 161984 with 162077. Click Apply. Click Ok

7. 7 Artemis (contd.) Navigate to a gene and add 5’UTR feature: Click on: Goto drop down menu. Select: Navigator Select: Goto Feature With Gene Name. Type: CNA00330 in the text box. Click: Goto Coordinates for CDS: 107173..110857; Coordinates for mRNA: 107107..110956 Coordinates for 5’ UTR: 107107..107172 To add the 5’ UTR feature: Right-click on the first exon of the mRNA for CNA00330 Select Create and then select: New Feature. This will open up a new window. From the Key drop-down menu, select: 5’UTR. In the text box for Location, replace existing range with 107107..107172 In the large text box, type: /note=added by XYZ Click Apply. Click Ok

8. 8 Artemis (contd.) View overlapping CDS features On the main menu, click: View Select: Feature Filters Select: Overlapping CDS Features. This will open up a new window which lists the coordinates of overlapping CDSs. There are multiple CDSs in the range ~60000 to ~64000. Double-click on the CDS with start coordinate 60562 This will bring up the gene CNA00200. For this gene there is 1 genomic sequence. How many sequences for mRNA? For CDS? (look in the feature list) Click each of these to see how they differ from each other. Do all the mRNAs have EST support? What can you conclude about CNA00200?

9. 9 Model a gene in Artemis Model the rps4 gene in Artemis using output from a GeneWise alignment (run on the command line in Unix) Launch Artemis (as before) Click File, click Open and load the file rps4.bac from the Data folder in Annotation_course/Sept_23 on the flash drive. (please make File of Type = “All files” to see this file) Click File, click Read an Entry and load the file rps4_genewise.gff from the same folder. You will see the GeneWise alignment of rps4.pep on the genomic sequence of rps4.bac (exons and introns) coordinates: 2521 – 6626 (feature = match)

10. 10 Model a gene (contd.) On the forward frame line, select the approximate range of the match region (left-click, hold and drag to select region) Click Create, click Feature From Base Range. This will open up a new window. Key should be CDS and Location should be ~2000..7000. Click Apply. Click Ok. Edit this CDS to get the correct coordinates for the exons, introns and start and stop sites. Click on the CDS feature (blue), click Edit. Click Selected Features in Editor. This will open up the editing window. In the text box for Location, type the coordinates for the five exons (from the feature list) as shown join(2521..2993,3141..4323,4410..4724,4831..5715,5832..6629) In the large text box, type /product=rps4. Hit Enter. Type /note=genewise alignment. Click Apply, click Ok.