GeneWise and Artemis Exercises

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GeneWise and ArtemisExercises
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1. Spliced Alignment using GeneWise

• Click on the GeneWise hyperlink on the course
  links page, http//www.tigr.org/tdb/e2k1/ttg/cours
  e_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.

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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

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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.

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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.

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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

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Artemis (contd.)

• Navigate to a gene and add 5UTR
  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 5UTR.
• In the text box for Location, replace existing
  range with 107107..107172
• In the large text box, type /noteadded by XYZ
• Click Apply. Click Ok

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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?

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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)

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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 /productrps4. Hit
  Enter.
• Type /notegenewise alignment.
• Click Apply, click Ok.