To perform a secondary analysis, an Application may require certain types of files, such as:
AuxCnvPanelOfNormalsFile - for Enrichment workflow
AuxNoiseBaselineFile - for Enrichment workflow
BedFile - for Enrichment workflow
RnaGeneAnnotationFile - for RNA workflow
Reference files for NovaSeq X analysis are managed from Resources page.
Select Resources from the user menu on the top right of Sequence Hub page
Select Reference Files tab to see the list of reference files available for use in the run planning.
Both standard and custom files are included in the list.
Import a Custom Reference File
Select Import Custom Reference File to upload a custom file.
After file upload is completed,
Select the correct File Type. Run Planning tool will associate the new custom file with the Application based on the selected File Type.
Select one or more reference genome(s) that should be associated with the file.
[Optional] Enter a description for the custom reference file.
Select Save.
Edit a Custom Reference File
To edit a custom reference file's metadata, go to the listing page and select the file. Update the information on the Edit page and save it upon completion.
To update the file content, select Import Custom Reference File from the listing page and upload the new file.
Delete a Custom Reference File
To delete a custom reference file, go to the listing page, and select the delete icon beside the file.
NovaSeq X Series systems allow you to perform multiple DRAGEN analyzes in a single sequencing run. Before setting up secondary analysis, make sure you have installed the appropriate DRAGEN application on your instrument. For more information on installing DRAGEN applications, refer to the NovaSeq X Series Product Documentation.
If storing data in the cloud, you can create up to seven analysis application and reference genome combinations with an additional BCL Convert-only application. If storing data locally, you can create up to three analysis application and reference genome combinations with an additional BCL Convert-only application. For each combination, you can use up to eight configurations using a different library prep kit, index adapter kit, or configuration settings for an analysis application and reference genome combination already used.
The following combinations are included in the seven or three configuration limit:
The same analysis application and application version with a different reference genome
The same reference genome with a different application or application version
A different application or application version with a different reference genome
Use the following instructions to plan a run for the NovaSeq X series systems in BaseSpace Sequence Hub.
Select the Runs tab, and then select the New Run drop-down.
Select Run Planning.
In the Run Name field, enter a unique name of your preference to identify the current run. The run name can contain a maximum of 255 alphanumeric characters, spaces, dashes, and underscores.
[Optional] Enter a description for the run. The run description can contain a maximum of 255 characters.
Select your sequencing system as the instrument platform.
Select one of the following analysis locations.
BaseSpace — Analyze sequencing data in the cloud.
Local — Analyze sequencing data on-instrument. When this option is selected, the planned run can only be exported to a sample sheet v2 file.
If analyzing data locally, select one of the following FASTQ output format options: The output format is not available for cloud storage and only applicable if you select to keep FASTQ files when setting up secondary analysis.
gzip — Save the FASTQ files in gzip format.
DRAGEN — Save FASTQ files in ora format. See DRAGEN Compression for more details.
Enter the number of cycles performed in each read: If using multiple analysis configurations, use the longest read length required by the configuration. When setting up a configuration, override automatically trims the length based on the recommended lengths for the selected library prep kit.
Read 1 — Enter up to 151 cycles.
Index 1 — Enter the number of cycles for the Index 1 (i7) primer. For a PhiX-only run, enter 0 in both index fields.
Index 2 — Enter the number of cycles for the Index 2 (i5) primer.
Read 2 — Enter up to 151 cycles. This value is typically the same as the Read 1 value.
[Optional] Enter the ID for your library tube. The library tube ID is located on the label of your library tube strip.
Select Next.
Please take note of the following when setting up a configuration.
Instrument Platform and Analysis location in Run Settings page are not editable once a Configuration is created.
Application version cannot be changed once a Configuration is saved. You need to delete the configuration and create a new one instead.
Select your analysis application.
[Optional] Enter a description for the configuration.
Select a library prep kit or add a new custom library prep kit as follows.
Select Add Custom Library Prep Kit under the Library Prep Kit dropdown.
Enter the name, read types, default read cycles, and compatible index adapter kits for your custom library prep kit.
Select Create New Kit.
Select an index adapter kit or add a new a custom index kits as follows. If you are using more than one library, the libraries must have the same index read lengths.
Select Add Custom Index Adapter Kit under the Index Adapter Kit dropdown.
Select a template type and enter the kit name, adapter sequences, index strategies, and index sequences. Make sure the second index (i5) adapter sequences are in forward orientation.
Select Create New Kit.
If applicable to your application, select a reference genome.
Select Next to configure secondary analysis settings.