If using somatic mode, you can generate a custom noise baseline file. The noise baseline file is built using normal samples that do not match to the subject the samples are from. The recommended number of normal samples is 50.
To generate a custom noise baseline file, use one of the following methods:
Use the DRAGEN Bio-IT Platform server. See the DRAGEN Bio-IT Platform Online Help for instructions.
Use DRAGEN Baseline Builder App on BaseSpace Sequence Hub. Use the BCL Convert pipeline in BaseSpace Sequence Hub Run Planning to generate FASTQ files. After the sequencing run is complete and 50 samples are available, input the FASTQ files into the DRAGEN Baseline Builder App.
For instructions to import noise baseline files to your instrument, refer to the system guide for your instrument NextSeq 2000 System Guide (document # 1000000109376)
Configure the settings for the analysis type selected for your run.
Use the following steps to configure Illumina DRAGEN BCL Convert analysis.
1- [Optional] Enter the following optional settings:
AdapterRead1: Adapter sequence for read 1. If using an Illumina library prep kit, leave the AdapterRead1 field empty.
AdapterRead2: Adapter sequence for read 2. If using an Illumina library prep kit, leave the AdapterRead2 field empty.
BarcodeMismatchesIndex1: The number of allowed mismatches between the first index read and index sequence. Allowed values are 0,1, or 2. The default value is 1. If a barcode is 6 bp, the recommended value is 0.
BarcodeMismatchIndex2: The number of allowed mismatches between the second index read and index sequence. Allowed values are 0,1, or 2. The default value is 1. If a barcode is 6 bp, the recommended value is 0.
OverrideCycles: String used to specify UMI cycles and mask out cycles of a read. The following values are allowed:
Y—Specifies a sequencing read.
I—Specifies an indexing read.
2- For Local mode, select whether to save a copy of your FASTQ files. FASTQ files are only generated if you select to keep FASTQ files.
3- For Local mode, select one of the following FASTQ output format options:
gzip - Save the FASTQ files in gzip format
DRAGEN - Save FASTQ files in ora format
4- Review and Complete the run configuration.
To save the run configuration for further editing, select Save as Draft. Draft runs appear in BaseSpace Sequence Hub planned runs list, but will not be available on the instrument until the draft runs are saved as planned.
To send the run configuration to your BaseSpace Sequence Hub account, select Save as Planned. Runs submitted to BaseSpace Sequence Hub appear in the planned runs list and are available for systems using Cloud mode or Hybrid mode.
To save the run configuration as a sample sheet in v2 file format, select Export. The sample sheet is required to initiate runs on systems using Local mode. This option is only available if Local was selected for analysis location.
Use the following steps to configure Illumina DRAGEN Enrichment analysis:
1- Select a reference genome. If using Nextera Flex for Enrichment with the Illumina Exome Panel, only hg19 and hs37d5 reference genomes are compatible with the DRAGEN Enrichment pipeline.
2- Select a *.bed file containing the regions you would like to target or upload a new custom file. Make sure the BED file's reference genome matches the reference genome selected in step 1. For a new custom BED file, use the following naming format: name_of_panel_versionNumber.referencegenome.bed
Use the following steps to configure Illumina DRAGEN Germline analysis:
1- Select a reference genome.
2- Select a map/align output format.
3- For Local mode, select whether to save a copy of your FASTQ files. FASTQ files are only generated if you select to keep FASTQ files.
4- For Local mode, select one of the following FASTQ output format options:
Use the following steps to configure Illumina DRAGEN RNA analysis:
1- Select a reference genome.
2- Select your map/align output format.
3- For Local mode, select whether to save a copy of your FASTQ files. FASTQ files are only generated if you select to keep FASTQ files.
4- For Local mode, select one of the following FASTQ output format options:
Use the following steps to configure Illumina DRAGEN Single Cell RNA analysis:
1- Select a reference genome.
2- [Optional] Upload a Gene Transfer Format (GTF) file for RNA Annotation file
Local mode — Select
Use the following steps to configure Illumina DRAGEN Amplicon analysis:
1- Select a reference genome.
2- Select a *.bed file containing the regions you would like to target or upload a new custom file. Make sure the BED file's reference genome matches the reference genome selected in step 1. For a new custom BED file, use the following naming format: name_of_panel_versionNumber.referencegenome.bed
Each element is separated by semicolons. The following is an example of OverrideCycles input: N1Y150;I8;I7N1;Y141U10
Local mode — Select Upload Custom File (Local) to upload for a single run or Upload Custom File (BaseSpace) for repeated use.
Cloud or Hybrid mode — Select Upload Custom File (BaseSpace). The custom BED file is only available in the Workgroup it was uploaded to.
3- Select either the germline or somatic variant caller
4- [Optional] If using the somatic variant caller, select a noise baseline file.
If using the DRAGEN Enrichment workflow in somatic mode, you can use a noise baseline file to filter out sequencing or systematic noise. You can download standard custom noise files from the or create a custom noise baseline file. Refer to for more information.
5- Select a map/align output format.
6- For Local mode, select whether to save a copy of your FASTQ files. FASTQ files are only generated if you select to keep FASTQ files.
7- For Local mode, select one of the following FASTQ output format options:
gzip - Save the FASTQ files in gzip format
DRAGEN - Save FASTQ files in ora format
8- Review and Complete the run configuration.
To save the run configuration for further editing, select Save as Draft. Draft runs appear in BaseSpace Sequence Hub planned runs list, but will not be available on the instrument until the draft runs are saved as planned.
To send the run configuration to your BaseSpace Sequence Hub account, select Save as Planned. Runs submitted to BaseSpace Sequence Hub appear in the planned runs list and are available for systems using Cloud mode or Hybrid mode.
To save the run configuration as a sample sheet in v2 file format, select Export. The sample sheet and secondary analysis supporting files are downloaded in a *.zip folder and are required to initiate runs on systems using Local mode. This option is only available if Local was selected for analysis location.
gzip - Save the FASTQ files in gzip format
DRAGEN - Save FASTQ files in ora format
5- Review and Complete the run configuration.
To save the run configuration for further editing, select Save as Draft. Draft runs appear in BaseSpace Sequence Hub planned runs list, but will not be available on the instrument until the draft runs are saved as planned.
To send the run configuration to your BaseSpace Sequence Hub account, select Save as Planned. Runs submitted to BaseSpace Sequence Hub appear in the planned runs list and are available for systems using Cloud mode or Hybrid mode.
To save the run configuration as a sample sheet in v2 file format, select Export. The sample sheet is required to initiate runs on systems using Local mode. This option is only available if Local was selected for analysis location.
gzip - Save the FASTQ files in gzip format
DRAGEN - Save FASTQ files in ora format
5- [Optional] Upload a Gene Transfer Format (GTF) file for RNA Annotation file
Local mode — Select Upload Custom File (Local) to upload for a single run or Upload Custom File (BaseSpace) for repeated use.
Cloud or Hybrid mode — Select Upload Custom File (BaseSpace). The GTF file is only available in the Workgroup it was uploaded to.
6- Select whether to enable differential expression.
7- If you enabled differential expression, select a control or comparison value for each sample. In each comparison group, any sample marked as control is compared with all samples marked as comparison. If the sample does not contain a control or comparison value, select na as the value.
8- Review and Complete the run configuration.
To save the run configuration for further editing, select Save as Draft. Draft runs appear in BaseSpace Sequence Hub planned runs list, but will not be available on the instrument until the draft runs are saved as planned.
To send the run configuration to your BaseSpace Sequence Hub account, select Save as Planned. Runs submitted to BaseSpace Sequence Hub appear in the planned runs list and are available for systems using Cloud mode or Hybrid mode.
To save the run configuration as a sample sheet in v2 file format, select Export. The sample sheet and secondary analysis supporting files are downloaded in a *.zip folder, and are required to initiate runs on systems using Local mode. This option is only available if Local was selected for analysis location.
Cloud or Hybrid mode — Select Upload Custom File (BaseSpace). The GTF file is only available in the Workgroup it was uploaded to.
3- Select your map/align output format.
4- For Local mode, select whether to save a copy of your FASTQ files. FASTQ files are only generated if you select to keep FASTQ files.
5- For Local mode, select one of the following FASTQ output format options:
gzip - Save the FASTQ files in gzip format
DRAGEN - Save FASTQ files in ora format
6- Select the configuration identical to your library prep kit type. For example, if you selected Single Cell RNA Library Kit 1 as your library prep kit, select Type 1 for the Configuration Type.
7- Select the barcode read.
8- [Optional] Edit the number of bases in the barcodes and the UMI. The values are automatically filled based on the library prep kit and configuration type you selected.
9- Select the strand orientation.
10- [Optional] Select a file containing your barcode sequences or upload a new custom file.
11- If using an Advanced/Custom configuration type, enter values for the number of override cycles, the barcode position, and the UMI position.
12- Review and Complete the run configuration.
To save the run configuration for further editing, select Save as Draft. Draft runs appear in BaseSpace Sequence Hub planned runs list, but will not be available on the instrument until the draft runs are saved as planned.
To send the run configuration to your BaseSpace Sequence Hub account, select Save as Planned. Runs submitted to BaseSpace Sequence Hub appear in the planned runs list and are available for systems using Cloud mode or Hybrid mode.
To save the run configuration as a sample sheet in v2 file format, select Export. The sample sheet and secondary analysis supporting files are downloaded in a *.zip folder, and are required to initiate runs on systems using Local mode. This option is only available if Local was selected for analysis location.
Cloud or Hybrid mode — Select Upload Custom File (BaseSpace). The custom BED file is only available in the Workgroup it was uploaded to.
3- Select either the germline or somatic variant caller
4- Select a map/align output format.
5- For Local mode, select whether to save a copy of your FASTQ files. FASTQ files are only generated if you select to keep FASTQ files.
6- For Local mode, select one of the following FASTQ output format options:
gzip - Save the FASTQ files in gzip format
DRAGEN - Save FASTQ files in ora format
8- Review and Complete the run configuration.
To save the run configuration for further editing, select Save as Draft. Draft runs appear in BaseSpace Sequence Hub planned runs list, but will not be available on the instrument until the draft runs are saved as planned.
To send the run configuration to your BaseSpace Sequence Hub account, select Save as Planned. Runs submitted to BaseSpace Sequence Hub appear in the planned runs list and are available for systems using Cloud mode or Hybrid mode.
To save the run configuration as a sample sheet in v2 file format, select Export. The sample sheet and secondary analysis supporting files are downloaded in a *.zip folder and are required to initiate runs on systems using Local mode. This option is only available if Local was selected for analysis location.
Select the Runs tab, and then select the New Run drop-down.
Select Run Planning.
Run Settings wizard will be loaded.
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] In the Run Description field, enter a description of the current run. The run description can contain a maximum of 255 alphanumeric characters.
Select the Instrument Platform.
Select the analysis location. Depending on the selected instrument type, not all options may be available.
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.
[Optional] In the Library Tube ID field, optionally enter the library tube ID of the current run. The library tube id can contain a maximum of 255 alphanumeric characters.
Select Next
Configuration wizard will be loaded
Select an analysis type and version. For more information about secondary analyses, see DRAGEN Secondary Analysis Output Files on the system guide for your instrument or the BaseSpace Sequence Hub app documentation. If you selected DRAGEN Single Cell RNA analysis, see the NextSeq 1000/2000 Products Files page for information on third-party single cell RNA library prep kit compatibility.
For on-instrument analysis, the version selected must match the version of DRAGEN installed on the instrument. To confirm the version of DRAGEN installed on the instrument, see DRAGEN Workflow and License Updates on the system guide for your instrument.
[Optional] Set up 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.
[Optional] Set up 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 the following instrument settings. Depending on the library prep kit, recommended options are automatically selected. Some library prep kits have hard-coded number of indexes reads and read types, which cannot be changed.
Library prep kit
Index adapter kit
Enter sample information into the Sample Data spreadsheet using one of the following options. To group samples for data aggregation during downstream analysis, assign a name for the group in the Project column.
Select Import Samples and the type of the import source file in the dropdown, either CSV or Sample Sheet, and then select your source file. If CSV file is selected, make sure that your file follows the template that can be downloaded by selecting Download Template button.
If Sample Sheet is selected, make sure that your sample sheet meets the formatting requirements. If CSV is selected, make sure to use the correct template as the template can be different depending on the selected index adapter kit and index strategy. For more details, see .
Local - Analyze sequencing data on-instrument.
Select Create New Kit.
Read type
Number of sequencing cycles per read
If Not Specified is selected for library prep kit, enter the number of Index sequencing cycles to be used in the run.
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 well positions or indexes. If Not Specified is selected for the library prep kit, enter Index 2 (i5) sequences in the forward orientation.