In this part of Tutorial 4, you will use SeqMan NGen to de novo assemble and annotate the RNA-Seq data.

  1. Click to download the data folder data folder T4_De_Novo_RNA-Seq.zip. Then extract the contents to any convenient location (e.g., your computer’s desktop). The paired-end sample sequences are named Yeast_RNASeq_1Mreads_1.fastq and Yeast_RNASeq_1Mreads_2.fastq.
  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 De novo assembly and click Next.

  1. In the Input rRNA or Other Contaminant Sequences screen, click Next.
  1. In the Input Sequence Files screen:

    1. Set the Read technology to Illumina, which will automatically add a checkmark to the paired-end data box.

    2. Add the Yeast_RNASeq_1Mreads_1.fastq and Yeast_RNASeq_1Mreads_2.fastq files by pressing the Add button, selecting the files, and then clicking Open. Alternatively, drag the files from your file explorer and drop it onto the large white space in the middle of the wizard screen.

    3. When prompted to Set Pair Information, keep the default Insert size of 500 bp and click OK.

    4. Click Next.
  1. In the Transcript Annotation Database screen, leave Use database to annotate transcripts checked.
  1. From the Transcript Annotation Database of the SeqMan NGen wizard, click the Download Database button. Choose RefSeq Fungi and press Select. Choose a location in which to save the database. When download is complete, press OK. Then click Next.

  1. In the Assembly Options screen, click Next.
  1. In the Assembly Output screen:

    1. Type “Transcript Project” 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.

While the assembly is in progress, proceed to Part B: Viewing Transcripts in SeqMan Pro.

Need more help with this?
Contact DNASTAR

Thanks for your feedback.