In this part of Tutorial 8, 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). The folder contains the following sequences:

    • Reference sequence:

    • Unpaired sample data: three folders, each containing a single Illumina read file
  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, choose ChIP-Seq and press Next.

  1. In the Choose Assembly Type screen, select Reference based assembly. If no Temporary file location has been specified, use the Browse button to specify one. Then press Next.
  1. In the Input Reference Sequences screen, add the reference sequence by pressing the Add button, selecting the file, and then clicking Open. Press 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. Since these are unpaired reads, uncheck the box.

    2. Press the Add button to add replicate 1: FlhD_3xFLAG_rep1\ERR475429.fastq. In the Experiment column, type in D1.

    3. Press the Add button again to add replicate 2: FlhD_3xFLAG_rep2\ERR475430.fastq. In the Experiment column, type in D2.

    4. Press the Add button again to add the control: WT_rep1\ERR475434.fastq. In the Experiment column, type in WT.

    5. Click Next.
  1. In the Set Up Experiments screen, check the box to the right of WT, indicating that WT is the control. Press Next.
  1. In the Assembly and Signal Processing screen, select the radio button Do not calculate variants. Make sure that the Peak Detection Method is MACS, then click Next.
  1. In the Define Binding Proteins screen:

    1. Use the Known binding site motif drop-down menu to select Transcription Factor Database.

    2. Use the Organism drop-down menu to select Bacteria.

    3. Press Select and choose FlhCD_CS / FlhCD, then press OK.

    4. Press Next.
  1. In the Assembly Output screen:

    1. Type “ChIP-Seq workflow” 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.

    3. Press 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 ArrayStar to view the assembly results in that application.
  1. Close the SeqMan NGen project by clicking the Finish button.

Proceed to Part B: Analyzing the ChIP-Seq results in ArrayStar.

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