In this part of Tutorial 2, you will set up and run a reference-guided assembly using the SeqMan NGen wizard. The data used in this tutorial 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 30-45 minutes.

  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 1. Extract the .zip archive contents to any convenient location (e.g., your computer’s desktop).
  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 De novo assembly and 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 Add button, add the paired reads s_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 Read Options dialog, leave all settings at the default values and click Next.
  1. The length of the reference sequence is 4,686,137 bp. In the Assembly Options dialog, enter the rounded-up value of 4700000 (47 followed by five zeroes) next to Expected genome length. Click Next

  1. In the Assembly Output screen:

    1. Type “De novo 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 approximately 30-45 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: Analyzing results in SeqMan Pro.

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