In this first part of Tutorial 1, you will set up and run a reference-guided assembly using the SeqMan NGen wizard.

  1. Click to download the data folder data folder Then extract the contents to any convenient location (e.g., your computer’s desktop). Neither step is necessary if you already downloaded this data set while following Tutorial 2.

    • The reference sequence is DH10B_NC010473.gbk.

    • The sample sequences are s_1_1_sequence.txt and s_1_2_sequence.txt. These are 100bp paired-end Illumina sequences from E. coli. Sequences were trimmed from 1000x coverage to 32x coverage, allowing this tutorial assembly to finish in 2-5 minutes.
  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 and press Next.

  1. In the Choose Assembly Type screen, select Reference based assembly – normal workflows and click Next.

  1. In the Input Reference Sequences screen:

    1. Add the reference sequence DH10B_NC010473.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.

      p(banner tip){margin-left:2em}.Note: If a reference sequence had not been provided with the tutorial data, you could have downloaded an E. coli genome here using the Download NCBI Genomes button.

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

    1. Set the Read technology to Illumina. This causes the Paired-end data box to be checked.

    2. Using the procedure described in the previous step, add the paired reads__1_1_sequence.txt_ and s_1_2_sequence.txt.

    3. In the Set Pair Information pop-up, keep the default Insert size of 500 and click OK.

    4. Click Next.
  1. In the Assembly Options dialog, select High layout stringency (since this is a whole genome), check Haploid (since this is a bacterial genome), and then click Next.

  1. In the Assembly Output screen:

    1. Type “Templated assembly” 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 under 5 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.

You may now close the SeqMan NGen project by clicking the Finish button.

Proceed to Part B: Analyzing assembly results in SeqMan Pro.

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