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.
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.
[Optional] Enter a description for the custom reference file.
Select Save.
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.
DRAGEN Server - Analyze sequencing data on a standalone DRAGEN server. When this option is selected, the planned run can only be exported to a sample sheet v2 file.
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 the number of cycles for Read 1.
Index 1 — Enter the number of cycles for the Index 1 (i7) primer. For a PhiX-only run, enter 0 in both index fields.
[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.
Index 2 — Enter the number of cycles for the Index 2 (i5) primer.
Read 2 — Enter the number of cycles for Read 2.
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.
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 steps to configure DRAGEN BCL Convert analysis.
[Optional] Enter a description for the configuration.
Select your library prep and index adapter kits.
Use the following steps to configure DRAGEN Enrichment analysis.
[Optional] Enter a description for the configuration.
Select your library prep and index adapter kits.
Enter the following optional settings.
Index Adapter Read 1 - Adapter sequence for Read 1. If using an Illumina library prep kit, you cannot modify this field.
Index Adapter Read 2 - Adapter sequence for Read 2. If using an Illumina library prep kit, you cannot modify this field.
Override Cycles - Specify UMI cycles and mask out cycles of a read. The values are automatically populated according to your library prep and index adapter kit information.
Use one of the following options to enter your sample information for the samples used in DRAGEN BCL Convert analysis.
Enter sample information in a *.csv file downloaded by selecting Download Template. To import the edited sample template, select Import Samples, and then select the CSV file.
Refer to Import Samples for more details.
Paste sample IDs and either index plate well positions or i7 and i5 indexes directly from an external file. Before pasting, enter the number of sample rows in the Rows field, and then select +. Sample IDs can contain up to 100 alphanumeric characters, hyphens, and underscores.
Fixed-layout index plates require entries for well position. Indexes that do not have a fixed layout require entries for i7 and i5 indexes. i5 indexes must be entered in the forward orientation.
Manually enter sample IDs and corresponding lane, well positions or indexes, barcode mismatches, and project. If Not Specified is selected for the library prep kit, enter Index 1 (i7) and Index 2 (i5) sequences in the forward orientation.
Select Next, and then review the run details.
[Optional] Perform any of the following actions:
To edit the run settings or configuration settings, select Edit next to the run or configuration.
To delete a configuration, select Delete next to the configuration, and then select Yes, delete.
To add another analysis configuration to the run, select Add another configuration.
To save the run, select one of the following options.
To edit the run details later, select Save as draft.
If storing data in the cloud, select Save as planned to finalize the run details and plan for sequencing.
If storing data locally, select Export to export a sample sheet v2 file.
Select a reference genome. If possible, use a reference genome with alt mask.
Select the germline or somatic variant type.
Select a variant calling workflow. If you select None, only alignment is performed. If you select All, the following variant calling workflows are performed.
Small variant caller
Structural variant caller
Copy number variant (CNV) caller (if panel of normals file is provided)
Select a *.bed file containing the regions you would like to target or upload a new custom file. A BED file is only required if performing small or all variant calling. Make sure that the reference genome for the BED file matches the reference genome selected.
[Optional] If using the somatic variant type, select a noise baseline file.
[Optional] If using the CNV caller, select a panel of normal files.
For instructions on importing BED file, noise baseline file or panel of normal file, refer to Import a Custom Reference File.
Enter the following optional settings.
Index Adapter Read 1 - Adapter sequence for Read 1. If using an Illumina library prep kit, you cannot modify this field.
Index Adapter Read 2 - Adapter sequence for Read 2. If using an Illumina library prep kit, you cannot modify this field.
Override Cycles - Specify UMI cycles and mask out cycles of a read. The values are automatically populated according to your library prep and index adapter kit information.
Use one of the following options to enter your sample information for the samples used in DRAGEN Enrichment analysis.
Enter sample information in a *.csv file downloaded by selecting Download Template. To import the edited sample template, select Import Samples, and then select the CSV file.
Refer to Import Samples for more details.
Paste sample IDs and either index plate well positions or i7 and i5 indexes directly from an external file. Before pasting, enter the number of sample rows in the Rows field, and then select +. Sample IDs can contain up to 100 alphanumeric characters, hyphens, and underscores.
Fixed-layout index plates require entries for well position. Indexes that do not have a fixed layout require entries for i7 and i5 indexes. i5 indexes must be entered in the forward orientation.
Manually enter sample IDs and corresponding lane, well positions or indexes, barcode mismatches, and project. If Not Specified is selected for the library prep kit, enter Index 1 (i7) and Index 2 (i5) sequences in the forward orientation.
Select a map/align output format.
The Analysis Settings section uses the settings in the first Enrichment configuration created for the sequencing run. To modify the settings, edit the first Enrichment configuration.
If storing data locally, select whether to save a copy of your FASTQ files. FASTQ files are only generated if you select to keep FASTQ files.
[Optional] Perform any of the following actions:
To edit the run settings or configuration settings, select Edit next to the run or configuration.
To delete a configuration, select Delete next to the configuration, and then select Yes, delete.
To add another analysis configuration to the run, select Add another configuration.
To save the run, select one of the following options.
To edit the run details later, select Save as draft.
If storing data in the cloud, select Save as planned to finalize the run details and plan for sequencing.
If storing data locally, select Export to export a sample sheet v2 file.
Select a reference genome. If possible, use a reference genome with alt mask.
[Optional] Select an RNA annotation file.
For instructions on importing BED file, noise baseline file or panel of normal file, refer to Import a Custom Reference File.
[Optional] Select Yes to enable down-sampling.
If down-sampling, select the number of reads to down-sample.
Enter the following optional settings.
Index Adapter Read 1 - Adapter sequence for Read 1. If using an Illumina library prep kit, you cannot modify this field.
Index Adapter Read 2 - Adapter sequence for Read 2. If using an Illumina library prep kit, you cannot modify this field.
Override Cycles - Specify UMI cycles and mask out cycles of a read. The values are automatically populated according to your library prep and index adapter kit information.
Use one of the following options to enter your sample information for the samples used in DRAGEN RNA analysis.
Enter sample information in a *.csv file downloaded by selecting Download Template. To import the edited sample template, select Import Samples, and then select the CSV file.
Refer to Import Samples for more details.
Paste sample IDs and either index plate well positions or i7 and i5 indexes directly from an external file. Before pasting, enter the number of sample rows in the Rows field, and then select +. Sample IDs can contain up to 100 alphanumeric characters, hyphens, and underscores.
Fixed-layout index plates require entries for well position. Indexes that do not have a fixed layout require entries for i7 and i5 indexes. i5 indexes must be entered in the forward orientation.
Manually enter sample IDs and corresponding lane, well positions or indexes, barcode mismatches, and project. If Not Specified is selected for the library prep kit, enter Index 1 (i7) and Index 2 (i5) sequences in the forward orientation.
Select a map/align output format.
The Analysis Settings section uses the settings in the first RNA configuration created for the sequencing run. To modify the settings, edit the first RNA configuration.
If storing data locally, select whether to save a copy of your FASTQ files. FASTQ files are only generated if you select to keep FASTQ files.
Select the pipeline mode to perform. The pipeline mode determines the output files generated. The full pipeline output includes gene fusion detection and gene expression quantification.
If performing the full pipeline mode, select whether to enable differential expression.
If you enabled differential expression, select a control or comparison value for each sample. Manually select the information or download the import samples template, modify the analysis comparison group, and then import the edited template. Use the following guidelines when selecting analysis comparison values:
If the sample does not contain a control or comparison value, select NA as the value or leave the value blank.
In each analysis group, any sample marked as control is compared with all samples marked as comparison.
Each analysis group must have 2–15 control samples and 2–15 comparison samples.
Analysis groups should not be reused between RNA analysis configurations with different reference genomes or RNA gene annotation files.
Select Next, and then review the run details.
[Optional] Perform any of the following actions:
To edit the run settings or configuration settings, select Edit next to the run or configuration.
To delete a configuration, select Delete next to the configuration, and then select Yes, delete.
To add another analysis configuration to the run, select Add another configuration.
To save the run, select one of the following options.
To edit the run details later, select Save as draft.
If storing data in the cloud, select Save as planned to finalize the run details and plan for sequencing.
If storing data locally, select Export to export a sample sheet v2 file.
Select a reference genome. If possible, use a reference genome with alt mask.
Select a variant calling workflow. If you select None, only alignment is performed. If you select All, the following variant calling workflows are performed.
Small variant caller
Structural variant caller
Copy number variant (CNV) caller
Repeat expansion detection
CYP2D6 caller
Regions of homozygosity (ROH) caller
Enter the following optional settings.
Index Adapter Read 1 - Adapter sequence for Read 1. If using an Illumina library prep kit, you cannot modify this field.
Index Adapter Read 2 - Adapter sequence for Read 2. If using an Illumina library prep kit, you cannot modify this field.
Override Cycles - Specify UMI cycles and mask out cycles of a read. The values are automatically populated according to your library prep and index adapter kit information.
Use one of the following options to enter your sample information for the samples used in DRAGEN RNA analysis.
Enter sample information in a *.csv file downloaded by selecting Download Template. To import the edited sample template, select Import Samples, and then select the CSV file.
Refer to Import Samples for more details.
Paste sample IDs and either index plate well positions or i7 and i5 indexes directly from an external file. Before pasting, enter the number of sample rows in the Rows field, and then select +. Sample IDs can contain up to 100 alphanumeric characters, hyphens, and underscores.
Fixed-layout index plates require entries for well position. Indexes that do not have a fixed layout require entries for i7 and i5 indexes. i5 indexes must be entered in the forward orientation.
Manually enter sample IDs and corresponding lane, well positions or indexes, barcode mismatches, and project. If Not Specified is selected for the library prep kit, enter Index 1 (i7) and Index 2 (i5) sequences in the forward orientation.
Select a map/align output format.
The Analysis Settings section uses the settings in the first Germline configuration created for the sequencing run. To modify the settings, edit the first Germline configuration.
If storing data locally, select whether to save a copy of your FASTQ files. FASTQ files are only generated if you select to keep FASTQ files.
Select Next, and then review the run details.
[Optional] Perform any of the following actions:
To edit the run settings or configuration settings, select Edit next to the run or configuration.
To delete a configuration, select Delete next to the configuration, and then select Yes, delete.
To add another analysis configuration to the run, select Add another configuration.
To save the run, select one of the following options.
To edit the run details later, select Save as draft.
If storing data in the cloud, select Save as planned to finalize the run details and plan for sequencing.
If storing data locally, select Export to export a sample sheet v2 file.