In Part A of this tutorial, you sat up and ran a de novo RNA-Seq assembly using SeqMan NGen. It takes ~ 2-3 hours for the SeqMan NGen assembly to complete.

During the assembly process, the_de novo_ transcriptome assembly output is saved to a package called Transcript Project.Transcriptome. Any assembled transcripts with a database match exceeding the specified thresholds are termed “Identified Transcripts,” while assembled transcripts that did not have a database match are called “Novel Transcripts.” This part of the tutorial shows how to load the annotated transcripts into the SeqMan Pro application for downstream analysis.

  1. Wait until being informed that assembly has finished, then click Next.

  1. From the Project Report screen of SeqMan NGen, click Launch in SeqMan Pro to open the Transcript Project.Transcriptome package in that application. You may now close the SeqMan NGen project by clicking the Finish button.
  1. In SeqMan Pro, observe that the ensuing All Transcripts window contains two tabs. Each tab’s heading shows the total number of transcripts in the table, and the number currently selected. The tables in the two tabs support a wide variety of sortable columns which can be displayed or hidden, as desired.

The Identified Transcripts tab is active, by default. You should see over 1300 Total Identified Transcripts. Since you haven’t yet made any selections, the number of selected transcripts is zero.

  1. Click on the Novel Transcripts tab. You should see approximately 40-70 Total Novel Transcripts. This table lists the assembled contigs that did not have any match to the Transcript Annotation Database that met the search criteria thresholds and therefore, were not labeled with any match information. Note that this table contains only three columns.

  1. Return to the Identified Transcripts tab and experiment with the following:

    • To show or hide columns - Right-click and choose Show/Hide Column, then check or uncheck boxes. Each column is described in detail in the SeqMan Pro help.

    • To move a column - Use the mouse to drag and drop it in the desired location.

    • To sort data in alphabetical or numerical order - Click on the column header that you wish to sort. Note that the resulting groups are also shown in different colors to help visually differentiate between them.

  1. To open individual contigs in SeqMan Pro for visualization and editing, double-click on a row of interest to navigate to the corresponding contig assembly. The appropriate SQD document is loaded with the Alignment View of the selected contig displayed. All the usual visualization and editing tools in SeqMan Pro are available.
  1. To set stringency thresholds:

    1. Return to the Identified Transcripts tab and choose View > Sort.

    2. Set up the dialog to match the image below. To add the second row, click on the plus sign in the original row.

    3. Press Sort Now.
  1. To save the sub-set of transcripts that met the stringency thresholds:

    1. In the Identified Transcripts table, click the %Transcript Match header to sort the column in decreasing order.

    2. Select all rows where Transcript Match is > 99.0, noting that the Identity is also > 99 for those rows. In the header, you will see approximately 130-160 selected transcripts.

    3. Choose File > Save Selected Transcripts for. In the Save dialog, designate a name and location for the output file, and then click Save. Two files will be created, each with a different extension (.fas and .searchresults).
  1. (optional) To see what is contained in each of these file types, open the files in any suitable text editor.

Congratulations on completing Tutorial 4. For a complete list of our other available tutorials, click here.

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