In this first part of Tutorial 3, you will learn how to set up the project in SeqMan NGen and run the assembly. To perform the assembly in Part A, you must download 4 GB of data and then wait about 1-3 hours for the assembly. A much shorter option is to simply read this section, then proceed to Part B, where you can download a 4 MB data set and perform the entire downstream analysis in ArrayStar.

  1. If you plan to run the assembly, click to download the data folder data folder T3_Templated_RNA-Seq.zip. Then extract the contents to any convenient location (e.g., your computer’s desktop). The folder contents consist of:

    • The E. coli reference sequence: MG1655_U00096.3.gb

    • The sample sequences: six folders beginning with flhC, flhD or WT.
  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 Transcriptome / RNA-Seq and press Next.

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

  1. In the Input Reference Sequences screen:

    1. Press the Add button.



    2. Select the E. coli reference sequence MG1655_U00096.3.gb and then click Open. Alternatively, drag the file from your file explorer and drop it onto the large white space in the middle of the wizard screen.

      Note that if a reference sequence had not been provided with the tutorial data, you could have downloaded the E. coli genome using the Download NCBI Genomes button.

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

    1. Set the Read technology to Illumina, and uncheck the paired-end data box.

    2. Check the Run Multi-sample data as separate assemblies box.

    3. Check the Samples have replicates box. When you do so, note that the “Experiment” column header below changes to “Individual Replicate.”



    4. Use the Add Folder button six times, each time adding one of the six sample folders from the tutorial data folder..

    5. One at a time, name each of the six replicates by clicking on “ENTER NAME.” Type in the names flhC_del_1, flhC_del_2, flhD_del_1, flhD_del_2, wt_1 and wt_2, as appropriate for that row.

    6. Click Next.
  1. In the Group Individual Replicates into Replicate Sets screen:

    1. Select the two flhC_del replicates and click on the Group Selected button. In the dialog, name the set “flhC_del” and click OK.

    2. Do the same for the two flhD_del replicates, naming the set “flhD_del.”

    3. Do the same for the two wt replicates, naming the set “wt.”

    4. Click Next.
  1. In the Set Up Experiments screen, specify the wildtype samples as the control. To do this, check the Is Control box to the right of “wt,” then click Next.
  1. In the Assembly Options screen, keep the default Variant detection mode of Do not calculate variants and the default Normalization method of DESeq2. Click Next.

  1. In the Assembly Output screen:

    1. Type “Templated RNA-Seq” 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.
  1. Wait until being informed that assembly has finished, then click Next.

  1. From the Project Report screen, click Launch in ArrayStar.
  1. Within ArrayStar, use File > Save Project to save the project as Templated_RNA-Seq.astar.

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

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

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