This tutorial is written for those with a Cloud Assembly license, whether purchased or from a free trial. The sample is a single file of Brassica napus transcriptome data. (Brassica napus is known as “rapeseed,” and is the third largest source of cooking oil in the world). You don’t need to download any data to follow this tutorial, as it is provided in your Cloud Data Drive.

Note that the contigs that result from a RNA-Seq de novo transcriptome assembly can be used as the template sequence in a reference-guided RNA-Seq workflow.

To perform the assembly:

  1. Launch SeqMan NGen. If SeqMan NGen is already open as the result of setting up a previous assembly, close it and open it again before starting the tutorial.
  1. Launch SeqMan NGen.
  1. In the Begin Project screen, select Assembly on the DNASTAR cloud. At the prompt, enter your Email and the Password sent when you purchased or requested free trials of DNASTAR Cloud Assembly. Press Next.
  1. In the Cloud Assembly screen, click Next.
  1. In the Choose Assembly Workflow screen, select Transcriptome / RNA-Seq and click Next.
  1. In the Choose Assembly Type screen, select De novo assembly and click Next.
  1. In the Input rRNA or Other Contaminant Sequences screen, click Next.
  1. In the Input Sequence Files and Define Experiments or Individual Replicates screen:

    1. Press Cloud Demo Data.

    2. In the pop-up, choose the row beginning with “Brassica napus* and click Select.

    3. Back in the wizard, Select Illumina from the Read technology drop-down menu. Note that the Paired-end data box is checked automatically.

    4. Click Next.
  1. The Transcript Annotation Database screen lets you choose options related to the auto-annotation of assembled transcripts. In this screen:

    1. Leave the check mark in the box near the top.

    2. Click Download Database.

    3. In the pop-up, choose RefSeq Plant and click Select.

    4. Back in the wizard, click Next.
  1. In the Assembly Options screen, click Next.
  1. In the Assembly Output screen:

    1. Enter the project name Brassica.

    2. Press the Browse button to launch the DNASTAR Cloud Data Drive window:

      1. Within the Cloud Data Drive, press the Home tool ().

      2. If you see a folder named Assemblies, skip to Step iii. Otherwise, click the Create a new folder on the cloud tool on the top right. In the pop-up box, type in Assemblies and press Done.

      3. Single click on the Assemblies folder to select it, then press the green check mark in the bottom right corner of the DNASTAR Cloud Data Drive window.

    3. Back in the Assembly Output screen, press Next.
  1. In the “Your assembly is ready to begin” screen, press Start Assembly to begin the assembly.
  1. Assembly for this tutorial should take approximately 6 hours, and you can check the status of the assembly at any time. To begin another tutorial while this assembly is in progress, close the current instance of SeqMan NGen by pressing Next and Finish, then proceed to Cloud Assembly Tutorials 1, 3 or 6.
  1. When the Tutorial 5 assembly is complete, download the 5GB file Assemblies/Brassica/Brassica.Transcriptome.7z. Then double-click on the file to open it in SeqMan Pro for analysis. For ideas, see Tutorial 4, Part B.

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