Now that you have completed Part B, step 1, this section shows how to use primer walking to help improve sequence coverage and fill in gaps.

  1. In the Project Summary window, select Contig 5 (if it is not already selected).
  1. Choose Contig > Primer Walk to open the Primer Walking Setup dialog.
  1. In the upper left of the dialog, choose Both. This tells SeqMan Pro to look for primers near low coverage areas, as well as for primers at the ends of the contig.
  1. In the upper right of the dialog, leave the Minimum Coverage at 4, but choose On Each Strand. Click OK.

After primers have been calculated (~ 3 minutes), the Primer Walking Report opens automatically.

By default, primers are sorted in descending order by Score. This is a composite score combining weighted measures of desired location, Tm, assembly quality and mispriming, then scaled from 0 to 100. A higher score is preferred. For detailed information, including how to save the report, see Primer Walking Report in the online SeqMan Pro help.

  1. Within the Primer Walking Report, experiment with the following:

    • To show or hide columns - Right-click and choose Show/Hide Column, then check or uncheck boxes.

    • To move a column - Use the mouse to drag and drop it in the desired location.

    • To sort data in alphabetical or numerical order - Click on the column header that you wish to sort.

    • To open the Alignment View with the primer selected - Double-click on a row in the report.
  1. When you are done, individually close all the windows except the Project Summary window (De novo assembly.sqd).

Proceed to 3. Finding and adding features using BLAST.

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