• All Files
Home/
Set Analysis Parameters

Related articles

Set Analysis Parameters

1. Open RNA Amplicon from BaseSpace Sequence Hub as follows.
  1. Select the Apps tab, and then select RNA Amplicon.
  2. From the Version drop-down list, select 2.0.0.
  3. Select Launch Application.
2. To override the default analysis name, enter a preferred analysis name in the Analysis Name field.

The default is the app name with the date and time the session was started.

3. Select Select Project(s).
4. Select a project to store app results to, and then select Select.
5. From the Targeted Amplicons drop-down list, select a panel of targeted amplicons representative of the selected samples.
6. If you selected Custom Panels from Targeted Amplicons, select Select Dataset File(s), and then select a manifest file. If the manifest file you want to use is not listed, import the file as follows.
  1. Open a new browser window and visit the BaseSpace Sequence Hub home page.
  2. Select Projects from the My Data tab.
  3. Select the project you want to upload the manifest file to.
  4. Select File | Upload | Files | Manifest.
  5. Follow the onscreen instructions to add the Custom Amplicon manifest file (*.txt) to the project. Make sure that the reference genome is specified in the header of the manifest file.
  6. Return to the RNA Amplicon app and repeat step 6 to select the newly added manifest file.

The labels of this field and button in the software interface might be different from the labels in this guide, depending on the BaseSpace Sequence Hub mode. In New mode, all data that is associated with the selected biosample is used in the analysis.

7. If you are using the AmpliSeq ERCC RNA Spike-In Mix for Illumina or the AmpliSeq ERCC RNA Companion Panel for Illumina, set the ERCC Analysis option.

When enabled, ERCC targets are merged into the input manifest for analysis. The gene name and amplicon sequence are checked to make sure that each ERCC target appears only once in the merged manifest.

8. For Sample Grouping, select from one of the following methods:
• Named Group(s)–Samples are analyzed in multiple, user-specified groups. Pairwise differential expression will be performed between all pairs of groups. Continue to step 9.
• Single Group–Samples are analyzed in one group. Continue to step 10.
9. From the Grouped Biosamples section, identify a sample group as follows.
  1. Enter a preferred name in the Label field.
  2. Select Select Biosample(s).
  3. Select the checkbox of each biosample you want to analyze, and then select Select.

You can set up multiple groups of samples. The samples within each group are treated as replicates in the differential expression analysis. Select Add a New Row to add additional groups to the analysis.

10. From the Ungrouped Biosamples section, identify a sample group as follows.
  1. Select Select Biosample(s).
  2. Select the checkbox of each biosample you want to analyze, and then select Select.

Advanced Settings

1. To enable gene-based normalization, select the Enable Gene-Based Normalization checkbox and enter a list of gene names in the Gene Name(s) field.

Make sure that you separate gene names with a semicolon. DESeq2, by default, uses all the amplicon targets to perform library-size normalization. When this option is enabled, only the amplicon targets targeting the provided genes are used for normalization.

2. Select Launch Application to start the analysis.

When analysis is complete, the status of the app session is automatically updated and you receive a confirmation email.