Set up NovaSeq X Series Secondary Analysis
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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.
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 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.
Use the following steps to configure DRAGEN Enrichment analysis.
[Optional] Enter a description for the configuration.
Select your library prep and index adapter kits.
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 .
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 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.
Use the following steps to configure DRAGEN RNA analysis.
[Optional] Enter a description for the configuration.
Select your library prep and index adapter kits.
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 .
[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 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.
Use the following steps to configure DRAGEN Germline analysis.
[Optional] Enter a description for the configuration.
Select your library prep and index adapter kits.
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 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.