In this first part of Tutorial 7, you will set up and run the Sanger validation assembly in SeqMan NGen.

  1. Click to download the data folder data folder Then extract the contents to any convenient location (e.g., your computer’s desktop). The folder contains the following sequences:

    • Reference sequence NC_002695.gbk

    • A folder of 498 Sanger reads

    • A folder of paired-end Illumina reads with a depth of 32x
  1. Launch SeqMan NGen.
  1. In the Begin Project screen, click the image for Assemble on local computer or Assemble on the DNASTAR cloud.
  1. In the Choose Assembly Workflow screen, select Whole Genome. (Note that Exome and Gene Panel can also be used with this workflow). Click Next.

  1. In the Choose Assembly Type screen:

    1. Choose Sanger Validation.

    2. Use the Browse button to specify a location in which to save temporary files.

    3. Click Next.
  1. In the Input Reference Sequences screen:

    1. Add the reference sequence NC_002695.gbk by pressing the Add button, selecting the file and clicking Open. Alternatively, drag the file from your file explorer and drop it onto the large white space in the middle of the wizard screen.

    2. Uncheck the VCF and Targeted Regions checkboxes.

    3. Click Next.
  1. In the Input Sequence Files and Define Experiments or Individual Replicates screen:

    1. The drop-down menu at the top is used to specify the Read technology of the non-Sanger data. Choose Illumina. This choice automatically adds a checkmark to the Paired-end data box.

    2. Use the upper Add Folder button to add the entire folder of Illumina data. When prompted to specify an Insert Size for the pairs, leave the default of 500 and click OK.

    3. Use the lower Add Folder button to add the entire folder of Sanger data.

    4. Click Next. If you see a message titled “Check paired files,” click Yes.
  1. In the Assembly Options screen, check the Haploid box (since this is an E. coli project) and click Next.
  1. In the Assembly Output screen:

    1. Type “Sanger Validation” into the Project Name text box. This name will be assigned to all output files, including the finished assembly.

    2. Use the Browse button to specify a Project Folder for your assembly output files. For local users, an alternative way to select a location is to drag and drop a folder from the file explorer onto the Project Folder row.

    3. Click Next.
  1. In the “Your assembly is ready to begin” screen, press Start Assembly to begin the assembly. For this tutorial, assembly should take approximately 3-7 minutes.
  1. Wait until being informed that assembly has finished, then click Next.

  1. From the Project Report screen, click Launch in SeqMan Pro to view the assembly results in that application.
  1. Close the SeqMan NGen project by clicking the Finish button.

Proceed to Part B: Confirming non-Sanger reads in SeqMan Pro.

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