Vector NTI

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Vector NTI
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Go Herd!
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Download your sequence and open the file

• Click your name on my web page on the class genes
  page
• http//mupfc.marshall.edu/murraye/good_bioinforma
  tics_genes.htm
• Save the file
• Open Windows Explore and right click the saved
  zip file.
• Select Extract Here
• You now should have two .abi files with a fish
  number. Remember your fish number, since Vector
  NTI will rename the files to their original abi
  names.

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Open Vector NTI Contig Express
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Import your sequences into contig express.

• Ignore any error messages from the program
• Once both of the sequence fragments have been
  imported into ContigExpress, select both of the
  fragments inside of the ContigExpress project
  window. Select Assemble Assemble Selected
  Fragments from the ContigExpress pull-down menu.
• The program tells you how many contigs were
  assembled and how long it took.
• Click on OK.

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Window should look like this
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What if it didnt assemble?

• It may have too many ambiguities, usually in one
  of the two sequences. You will need to open them
  one at a time and edit them, and then try them
  again.

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Editing your single sequence

• Add the first fragment as an abi file type. When
  it asks you to keep the original name, say no.

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You will notice junk at the beginning.
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Highlight Sequence and Cut (scissors)
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Now you see little red arrows for cut sequences.
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Do the same on the other side, all to the end of
the sequence. Use the slide to make the peaks
higher or lower.
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Edit Individual bases

• Use the cursor to place it after the edited
  section to the left in the box with the peaks.
  Use the small n at the top menu and select next
  ambiguous base.
• The box must be around the base you want to
  change- select the highest peak. DONT worry too
  much about being perfect- that is what the next
  step will check.

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Continue until you have resolved all the
ambiguities. Save the file with a next name.
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• Rename the Assembly by clicking on Assembly 1
  and then waiting a second and clicking again.
  Change the name to reflect the original fish
  number.
• Now double-click on your contig (i.e. Contig 1)
  in order to edit it. A contig editor window will
  open

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What you are seeing

• This window is broken up into three main panes.
• The upper left pane contains simple information
  about the contig, a textual annotated description
  of the contig, and a description of the fragments
  within the contig.
• The upper right pane presents a graphical
  representation of the contig and the way in which
  the fragments overlap in the contig. Also shown
  in the lower portion of this pane is the location
  of known discrepancies in the contig.
• These discrepancies represent nucleotides that
  differ between the two sequences, and will need
  to be resolved.
• The lower pane depicts the actual sequences
  themselves and depicts the fundamental details of
  the contig, showing the exact way in which the
  contig was constructed from the sequence
  fragments.
• Ambiguities and gaps are presented here in
  detail, and can be resolved manually in this
  lower pane.

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• The next step is to manually resolve all
  discrepancies in the contig.
• Prior to doing this, it is best to show the
  detailed chromatogram output from the automated
  sequencer.
• This can be done by right-clicking anywhere in
  the lower pane and selecting Show All
  chromatograms from the pop-up window.

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• Locate all ambiguity codes (depicted in red in
  the consensus sequence at the bottom of the lower
  pane, as a symbol underneath the consensus
  sequence and as green vertical lines in the
  graphics pane) and resolve the ambiguity
  character with the most likely alternative.
• In most cases, the most appropriate solution is
  simply to choose the unambiguous code from the
  other sequenced fragment in the same aligned
  column (where it overlaps).

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