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Use Run Planning tool in BaseSpace Sequence Hub to create and configure your run settings.
If your instrument is configured for Cloud mode or Hybrid mode, submit the run configuration to your BaseSpace Sequence Hub account. The run becomes available in the planned runs control software of the instrument.
If your instrument is configured for Local mode, use Run Planning tool to create your sample sheet in v2 file format. Alternatively, create a sample sheet without BaseSpace Sequence Hub using a provided template.
If your instrument is not configured for Cloud mode integration, you can export the sample sheet in v2 file format. Placing the sample sheet in the instrument run folder and sequencing with Run Monitoring and Storage enabled will allow the specified analysis to launch automatically when the upload to BaseSpace Sequence Hub completes.
Planning a run in BaseSpace Sequence Hub is available for the following sequencing systems:
NextSeq 1000 and NextSeq 2000 Sequencing Systems - see
NovaSeq X Series Sequencing Systems - see
MiSeq i100 Series Sequencing Systems - see
Analysis Configuration Template is a template containing configuration/settings for a secondary analysis to allow planning a run on Clarity LIMS. Analysis Configuration Templates created on BaseSpace Sequence Hub can be retrieved and used in a workflow on Clarity Lab Information Management System (LIMS).
To use Analysis Configuration Template, please turn on the Advanced LIMS Run Planning from the User Account Settings page (Note that only Workgroup owner has the permission to change this setting). Once the setting is enabled, go to the Resources page by selecting Resources from the User Account menu and select the Analysis Configuration Templates tab.
In the Template Name field, enter a unique name of your preference to identify the template.
[Optional] In the Template Description field, enter a description for the template.
Select the Instrument Platform
Analysis Configuration Template is currently only supported for NovaSeq X Series and MiSeq i100 Series.
Select the analysis location.
Cloud - Analyze sequencing data using Illumina pipeline in the cloud.
Local - Analyze sequencing data on-instrument.
Select an analysis type and version from the Application dropdown.
Select the Reference Genome (only applicable if analysis type is not BCL Convert).
Enter other settings that are applicable for the selected analysis.
RNA Differential Expression settings is not supported in Analysis Configuration Template. Please setup the Differential Expression on BaseSpace Sequence Hub after creating the planned run on Clarity.
Select Save to save the template.
The saved template shall be available on Clarity LIMS. After a run is planned on Clarity LIMS, it can be opened on BaseSpace Sequence Hub Run Planning for further editing. Please note that sample and kit information are not editable.
To begin editing, click on the template name hyperlink
Update the settings
Select the Save button to save the changes or the Cancel button otherwise
To delete a template, select the trash button located at the right most column.
You can import settings from an analysis configuration template into a configuration in Run Planning.
Click on the "import from Analysis Configuration Template" menu below the Configuration title after selecting an Application version.
The Import from Analyis Configuration Template dialog will be displayed.
Select the pre-created template to use and click on Confirm button.
Upon confirmation, the Library prep kit, index adapter kit, barcode mismatches index and analysis settings from the template will be applied to the Configuration.
Note that existing values in the Configuration (if any) will be overwritten.
Complete the Configuration creation with the Samples data and other necessary information.
The Prep tab is deprecated and no longer available. For run planning and sequencer integration, see the Run Planning section.
On the Pools page, drag a library from the plate to a pool. For Nextera Rapid Capture libraries, assign samples from the same enrichment to 1 pool.
In the Pool ID field, enter a unique name for the pool.
[OptionalSelect Add Pool to add a pool.
[Optional] Use 1 of the following methods to pool libraries from multiple plates:
Select the Plate ID drop-down arrow to switch between plates.
Select Save & Continue Later, select the checkbox of each pool to merge, and then select Merge Pools.
Select Plan Run.
To help manage multiple pools, the color of each sample well matches the color of the pool the sample was added to.
The Prep tab is deprecated and no longer available. For run planning and sequencer integration, see the Run Planning section.
On the Plan Run page, click the Select Instrument drop-down arrow, and select a sequencing system, either MiniSeq or NextSeq.
In the Name field, enter a name for the sequencing run.
[Optional] In the Reagent Barcode field, enter the barcode ID of the reagent kit used for the run. Entering the barcode ID links the reagent kit to the run.
[Optional] Select Use Custom Primer options:
R1 — Use custom primer for Read 1.
R2 — Use custom primer for Read 2.
Select a read type, either Single Read or Paired End.
Enter the number of cycles for each read in the sequencing run:
Read 1 Cycles — Enter a value up to 151 cycles.
Read 2 Cycles — Enter a value up to 151 cycles. This value is typically the same number of cycles as Read 1.
Review the indexing scheme for the run. To make changes, override the defaults as follows.
Select the Override default indexing scheme checkbox.
Select an indexing scheme:
Make sure that a pool is present.
Select 1 of the following buttons to continue:
Sequence — The run appears on the Planned Runs list with a status of Ready. The run becomes available from the control software of your sequencing system.
Save and Continue Later — The run appears on the Planned Runs list with a status of Planning. When you are ready to sequence, select the checkbox for the run and select Sequence. The run then becomes available from the control software of your sequencing system.
*Indexing is required when sequencing multiple libraries.
Dual Index — Performs a run with 2 index reads.
No Index — Performs a non-indexed run.
Enter the number of cycles for each index read*:
Index 1 Cycles — Enter the number of cycles required for the Index 1 (i7) primer.
Index 2 Cycles — Enter the number of cycles required for the Index 2 (i5) primer.
NeoPrep functionality has been deprecated and is no longer available.
In the Review Run Details page, review the run parameters.
If the parameters are acceptable, select Finish.
[Optional] Select the run setup details or library card mapping to view or print details.
Select Done to return to the NeoPrep Runs page.
The new run is listed with a status of Ready, which means the run is listed in the NeoPrep control software.
NeoPrep functionality has been deprecated and is no longer available.
In the Assign Samples to Wells page, set up each well as follows.
In the Sample ID field, select Select Sample,
In the Select a Biological Sample dialog box, select a sample, and then select Select.
In the Library ID field, enter a unique name for the library.
Select Next.
Select the Index drop-down arrow, and select a unique index to add to the sample.
If you selected mixed insert sizes on the previous page, select the Insert Size drop-down arrow, and select the insert size of the library.
The Prep tab is deprecated and no longer available. For run planning and sequencer integration, see the Run Planning section.
When prepping a library, select + Custom Library Prep Kit in the Library Prep Kit dropdown menu. The Custom Library Prep Kit Definition page opens.
Fill out the name of the custom prep. It has the following requirements:
Unique for your account.
Your custom library prep has been added to the library kit drop-down!
Less than or equal to 50 characters.
Select at least 1 of the supported read types.
Select at least 1 of the indexing strategies. Only selecting None is not allowed.
Fill out the default number of cycles.
Select template to download the index definition file template.
Fill out the Settings section the following way:
For single read only: no adapter (blank), or 1 adapter sequence for Read 1.
For paired-end: no adapter (blank), or 2 adapter sequences, 1 for Read 1 and 1 for Read 2.
Each adapter sequence meets the following criteria:
Sequence of A, T, C, or G character.
Length from 1 to 20 characters.
Fill out the Index1Sequences and Index2Sequences sections the following way:
For Single Index, with or without None: 1 to 100 Index 1 names
For each Index 1 name an associated Index 1 sequence
For Dual Index, with or without None and Single Index: 1 to 100 Index 1 names
For each Index 1 name an associated Index 1 sequence: 1 to 100 Index 2 names
For each Index 2 name an associated Index 2 sequence
Each index name meets the following criteria:
Unique within the file
Length from 1 to 8 characters alphanumeric, hyphen, or underscore characters.
If the supported indexing strategy specifies Single Index, you can set up Default Layout By Well the following way:
Each well unique from A01 to H12
For each well, an associated index name exists in the specified Index1Sequences section
If the supported indexing strategy specifies Single Index or Dual Index, you can set up Default Layout By Column the following way:
Each column number unique from 1 to 12
For each column, an associated index name exists in the specified Index1Sequences section
If the supported indexing strategy specifies Dual Index, you can set up Default Layout By Row the following way:
Each row letter unique from A to H
For each row, an associated index name exists in the specified Index2Sequences section
Select the Choose.csv File button to select and upload your custom index file.
Select Create New Kit to complete the process.
Import Samples from CSV file is an alternative way to populate Sample table and sample-level analysis settings on the Configuration page.
Each CSV template is different depending on the Index Adapter Kit and number of Index Reads. To download a template, first make the appropriate selections in the Run Planning tool then use the Download Template link above the Sample Table.
Sequence of A, T, C, or G characters
Length from 1 to 20 characters
All index sequence lengths (Read 1 and Read 2) are equal
Index 1 sequences are unique within the file set of Index 1 sequences
Index 2 sequences are unique within the file set of Index 2 sequences
The CSV template only includes editable columns in the Sample Table and the sample-level analysis settings if applicable (e.g. for RNA Seq application with Differential Expression). Import will auto-populate the derived columns from the input data, e.g. Index names and sequences are derived from the respective Well Position.
For custom or standard kit with fixed plates:
Sample ID*, Well Position*, Project, and sample-level analysis settings (if applicable)
For custom or standard kit with fixed indexes:
If single index strategy is selected: Sample ID*, I7 Index* , Project, and sample-level analysis settings (if applicable)
If dual indexes strategy is selected: Sample ID*, I7 Index* , I5 Index*, Project, and sample-level analysis settings (if applicable)
For Not Specified kit:
If single index strategy is selected: Sample ID*, Index 1*, Project, and sample-level analysis settings (if applicable)
If dual indexes strategy is selected: Sample ID*, Index 1*, Index 2*, Project, and sample-level analysis settings (if applicable)
Columns marked with asterisks are mandatory.
Import will fail if an incorrect CSV template is used (columns do not match the Sample table).
All rows in the input CSV file will be imported.
Invalid inputs will result in blank cells in the Sample table.
Invalid well position or Index name when custom or standard kit is used
Incorrect data type, e.g. string input when a number is expected
Out-of-range value
Invalid option for a select or dropdown list field
If there are multiple rows defining the same Sample ID, the sample-level analysis settings must be consistent.
If different values are specified, the resulting sample-level analysis settings will be blank.
For instruments that support loading separate lanes, the CSV template also includes Lanes column.
Enter one or more lane numbers in this column. When multiple lane numbers are specified, separate the values by commas.
If a Lanes cell contains an invalid lane number, the resulting cell will be blank.
From the Runs page, select New Run, and then select Prep Tab.
From the Prep page, select NeoPrep.
From the NeoPrep Runs page, select Create New Run to open the Configure Run screen.
Select the Protocol drop-down arrow, and select a protocol, either TruSeq Nano DNA or TruSeq Stranded mRNA.
Select the Version drop-down arrow, and select a version of NeoPrep control software.
[Optional] In the Notes field, enter any notes.
In the Run Name field, enter a name for the run.
Select the Default Project field, select the project to configure for the run, and then select Confirm.
Select the checkbox for each process you want to run:
Prep Library—Requires preparation of libraries.
Quantify—Quantifies libraries after library prep is complete.
Normalize—Normalizes libraries after quantification is complete.
Complete the following fields as applicable. Read-only or unavailable fields are default selections for the run.
Select the Sample Count drop-down arrow, and select the number of samples to include in the run.
Select the Index Type drop-down arrow, and select the indexing scheme for the samples.
Select the Insert Size drop-down arrow, and select the insert size of the libraries. Mixed insert sizes are specified on the next page.
Select the PCR Cycles drop-down arrow, and select the number of PCR cycles for the run.
Select Next.
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.
The Run Planning tool can be used to requeue planned analyses for supported Cloud applications.
Illumina DRAGEN Apps version 3 or higher
DRAGEN TruSight Oncology 500 Apps version 2.1 or higher
To initiate a requeue, navigate to the Run Summary page and select Requeue > Planned Run from the Status menu.
You can use the sample sheet from the selected run or select a new sample sheet file to be load. This will load the analysis configurations contained in the selected Sample Sheet into the Run Planning tool, and allow edits before requeuing the analysis.
Depending on the instrument platform and analysis specified, some fields may be non-editable, including Run Name and Library Tube ID. See the page for specific instructions on using the Run Planning tool.
For instruments that support multiple analysis configurations, existing configurations can be edited or deleted. New configurations can also be added before requeueing.
When all changes have been made (if desired), review the settings on the Run Review page and click "Requeue" to initiate the requeue.
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.
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.
NeoPrep functionality has been deprecated and is no longer available.
The library prep run setup for NeoPrep includes the following three parts:
Create and configure the run.
Assign samples to wells.
Review the run setup.
At any point during this process, you can select the Save button to save your work. Saved runs appear on the NeoPrep Runs page with a status of Planning. When you are ready to resume work, select the run name.
[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.
Local — Analyze sequencing data on-instrument. 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.
Index 2 — Enter the number of cycles for the Index 2 (i5) primer.
Read 2 — Enter the number of cycles for Read 2.
[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.
If applicable to your application, select a reference genome.
Select Next to configure secondary analysis settings.
The Prep tab is deprecated and no longer available. For run planning and sequencer integration, see the Run Planning section.
The MiniSeq, NextSeq, and NeoPrep systems provide an option to set up a run in BaseSpace Sequence Hub using the Prep tab.
When you complete run setup in BaseSpace Sequence Hub, the run becomes available in the control software of the instrument. For instructions to complete the run, see the system guide for your instrument:
MiniSeq System Guide (document # 1000000002695)
NextSeq 550 System Guide (document # 15069765)
NextSeq 500 System Guide (document # 15046563)
Select Create New Kit.
From the runs page, select New Run, and then select Prep Tab.
From the Prep page, select Biological Samples.
Select Import.
[Optional] Create a .csv file as follows.
Select the spreadsheet image to download a template.
Complete the following fields for each sample:
Sample ID—Enter a unique sample ID.
Select Choose .csv File.
Browse to the appropriate file and select Open. Information from the .csv file populates the Biological Samples page.
[Optional] Select additional samples as follows.
Select Save & Continue Later.
Select the checkbox for each sample you want to use.
Select Prep Libraries.
The Prep tab is deprecated and no longer available. For run planning and sequencer integration, see the Run Planning section.
Complete the following steps to create biological samples one at a time. For information about adding multiple samples, see .
Select the Prep tab, and then select Biological Samples.
Select Create.
Provide the following information:
BaseSpace Sequence Hub converts sample name underscores to dashes in the output FASTQ files. To avoid unexpected FASTQ file names, use only alphanumeric characters and dashes in sample names.
[Optional] Species—Enter the appropriate species.
Project—Enter the name of the project to save samples to. The selected project is the default project that contains biosample data output.
Nucleic Acid—Enter RNA or DNA.
Save the file.
Name—Enter a descriptive name for the biological sample.
[Optional] Species—Select a species from the drop-down list.
Project—Select Select Project to select or create a project to add your samples to. The selected project is the default project that contains biosample data output.
Nucleic Acid—Select the type of nucleic acid.
[Optional] Select additional samples as follows.
Select Save & Continue Later.
Select the checkbox for each sample you want to use.
Select Prep Libraries.
The Prep tab is deprecated and no longer available. For run planning and sequencer integration, see the Run Planning section.
This method is appropriate for the following:
Prepared libraries
Samples with assigned indexes If your samples do not have indexes assigned and are not in libraries, use .
On the Libraries page, select Import.
[Optional] Create a CSV file as follows.
Select the spreadsheet image to download a template.
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 .
Use the following instructions to plan a run for the MiSeq i100 Series systems in BaseSpace Sequence Hub.
Select the Runs tab, and then select the New Run drop-down.
Select Run Planning.
[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.
Enter the number of cycles performed in each read:
Read 1 — Enter the number of cycles for Read 1.
Index 1 — Enter the number of cycles for the Index 1 (i7) primer.
Index 2 — Enter the number of cycles for the Index 2 (i5) primer.
Read 2 — Enter the number of cycles for Read 2.
Select Next.
Select your analysis application. Please note that MiSeq i100 Series only supports one analysis per planned run.
[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.
For each sample, enter the following information.
Sample ID—The unique sample ID.
Name—A descriptive name for the biological sample.
[Optional] Species—The appropriate species.
[Optional] Project—The name of the project to save samples to. Although optional at this step, a project is required later to store the data.
NucleicAcid—The nucleid acid, either RNA or DNA.
Well—The plate well.
Index1Name—The Index 1 name.
Index1Sequence—The Index 1 sequence.
Index2Name—The Index 2 name.
Index2Sequence—The Index 2 sequence.
Save the file.
Select Choose .csv File.
Browse to and select the appropriate CSV file, and then select Open. The information from the file populates the Import Sample Libraries page.
[Optional] Select additional libraries as follows.
Select Save & Continue Later.
Select the checkbox of each library you want to use.
Select Pool Libraries.
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.
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.
The Prep tab is deprecated and no longer available. For run planning and sequencer integration, see the Run Planning section.
If your kit is not listed but is compatible with your sequencing system, set up a custom library prep kit or select a kit that uses the same index adapter set. For example, select Nextera Rapid Capture for TruSight Cardio.
In the Libraries page, select the Library Prep Kit drop-down arrow, and select a kit.
In the Plate ID field, enter a unique plate ID.
[Optional] In the Notes field, enter any notes. For Nextera Rapid Capture libraries, specify that the plate contains multiple enrichments.
Biological samples are assigned an index even if you are not performing indexed sequencing. During run setup, you can specify no indexing.
[Optional] Assign the sample to a different project.
Select the checkbox of each library to assign to a project.
Select Set Project.
In the Select Project dialog, select or create a project to assign libraries to, and then select Select.
Select Auto Prep to automatically assign each library to a well and index location. For Nextera Rapid Capture libraries, samples belonging to the same enrichment must be in the same row. Each plate has a maximum layout of 12 indexes by 8 indexes. If you require more wells or indexes, create multiple plates.
[Optional] To change indexes, select an index on the plate and select a new index from the drop-down list. NOTE: This option is not available for fixed layout plates.
[Optional] Move libraries to a different well as follows. Select the checkbox of the library to move. Drag the selected library from the Libraries area to the appropriate well.
Select Export to save your library prep settings in a .csv file. This file serves as a reference when preparing samples in the lab.
[Optional] Select additional plates as follows.
SelectSave and Continue Later to return to Libraries.
Select the checkbox of each library you want to use.
Select Pool Libraries.
Run Planning provides a list of Illumina index adapter kits used for sequencing. If your index adapter kit is not available, please follow the following instructions to create a custom one.
Creating a custom index adapter kit can be done from within Run Planning (when creating a Configuration) or from the Resources page (select Index Adapter Kit tab).
A custom Index kit can be configured in yaml or tsv.
The following are basic rules to follow when configuring in yaml
3 dashes indicates the start of the definition
Begin a comment line with '#' character
Each line is typically in the format of SettingName: SettingValue. Setting which value is a string has to be enclosed in double quotes. Other types like numeric or boolean do not require double quotes.
Three yaml templates are provided.
Non-fixed Layout: for non-fixed layout kit where any index can be selected for any sample.
Fixed Layout - Single Plate: for fixed layout kit with single plate, where each well has a defined index combination.
Fixed Layout - Multi Plate: for fixed layout kit with multi-plate, where each well has a defined index combination.
The supported values are "Dual", "Single", and "NoIndex". Create a list below this setting. Each list item should be preceded with a dash (-) character and enclosed in double quotes. Use two spaces for indentation. See .
i7Index1 Create a list of Index1 sequences below this setting. Each index should be in the format of IndexName: "IndexSequence". See .
i5Index2 Create a list of Index2 sequences below this setting. Each index should be in the format of IndexName: "IndexSequence". See .
The mapping should be defined in the format of "WellPosition/Index1Name-Index2Name" or "Plate-WellPosition/Index1Name-Index2Name". The allowed well positions are A01 - H12. If the kit requires well positions defined in different format, define FixedLayoutPositionKeyByIndexId: true. See , , .
The value is true or false. However, the usage is currently restricted to Instrument Platforms which allow multi-configuration. As each configuration only allows one Override Cycles, when setting up a run, samples with different index lengths should be separated into different configurations.
EnableCustomIndexCycles
The value is true or false. If the setting is set to false or if the setting is not defined, the Override Cycles used is Y;I;I;Y pattern.
This section contains custom BCL Convert settings. The settings will be included in the sample sheet generated by Run Planning.
TrimUMI Indicates if the UMI should be excluded from fastq files. The value is "0" or "1". Set to "0" if BCL Convert should still output UMI cycles to fastq files. See .
CreateFastqForIndexReads Indicates if the UMI in Index cycles should be trimmed or not. The value is "0" or "1". Set to "1" if BCL Convert should still output UMI cycles in Index to fastq files. Note that TrimUMI should also be set to "0".
Similar to yaml, three tsv templates are provided. Please note that currently .tsv file supports fewer custom kit settings (as compared to .yaml file).
Non-fixed Layout: for non-fixed layout kit where any index can be selected for any sample.
Fixed Layout - Single Plate: for fixed layout kit with single plate, where each well has a defined index combination.
Fixed Layout - Multi Plate: for fixed layout kit with multi-plate, where each well has a defined index combination.
A tsv file contains of three sections, namely [IndexKit], [Resources], [Indices], where each section contains rows of tab-separated values.
Each row in the Resources section consists of four columns: Name, Type, Format, and Value. It is used to define Adapter Read settings and the type of index kit (whether a fixed layout with single- or multi- plate or non fixed layout). In addition, the mappings of well positions and index names (only for a fixed layout kit) should be included in this section (see No 5 in the table below).
Index1 and Index2 sequences should be defined in this section. Each row consists of three columns: Name, Sequence, IndexReadNumber.
When a setting contains more complex information, it is usually defined in multiple lines. Ensure the right indentation to maintain the structure. Use two space characters instead of a tab character.
AdapterSequenceRead1
Adapter sequence for Read 1. Remove the line if it is not applicable.
AdapterSequenceRead2
Adapter sequence for Read 2. Remove the line if it is not applicable.
IndexSequences
i7Index1
A section of Index1 sequences. See .
IndexSequences
i7Index2
A section of Index2 sequences. See .
Settings
DefaultIndexStrategy
The default index strategy. It should be one of the strategy defined in the .
Settings
FixedLayout
Indicates if the kit has a fixed-layout (true) or not (false). See .
Settings
Multiplate
Indicates if a fixed-layout kit is single-plate (false) or multi-plate (true). See , .
Settings
FixedIndexPositions
A section containing mappings of well position to index names. It is only applicable for a fixed-layout kit. See .
Settings
AllowVariableLengthIndexSequences
Indicates if the kit can have index sequences with different lengths. See
Settings
EnableCustomIndexCycles
Indicates if the kit uses a custom Override Cycles. See .
Settings
OverrideCycles
The custom pattern for the Override Cycles. See .
Settings
NumCyclesIndex1Override
Used to override the default Index1 cycles. See .
Settings
NumCyclesIndex2Override
Used to override the default Index2 cycles. See .
Settings
CustomBclConvertSettings
A section of custom BCL Convert settings. See .
NumCyclesIndex1Override The value should be a numeric value. If this setting is not defined, the number of Index1 cycles follows the number of bases in the Index1 sequences.
NumCyclesIndex2Override The value should be a numeric value. If this setting is not defined, the number of Index2 cycles follows the number of bases in the Index2 sequences. See example 7.
OverrideCycles
The value should be defined in this format: "Y{{Read1Length}};I{{Index1Length}};I{{Index2Length}};Y{{Read2Length}}?", where:
{{Read1Length}} is the number of cycles for Read1,
{{Read2Length}} is the number of cycles for Read2,
{{Index1Length}} is the number of cycles for Index1, and
{{Index2Length}} is the number of cycles for Index2.
If UMI is used, update the pattern accordingly.
E.g. if Read1 and Read2 cycles include 7 UMI cycles and 1 skipped-cycle: U7N1Y{{Read1Length-8}};I{{Index1Length}};I{{Index2Length}};U7N1Y{{Read2Length-8}}?"
E.g. if the kit is a single index kit, with UMI cycles instead of Index2: "Y50N{{Read1Length-50}};I8N{{Index1Length-8}};N{{Index2Length-16}}U16;Y50N{{Read2Length-50}}?". See
4.1
NoIndex: only allow No Index
4.2
SingleOnly: only allow Single Index
4.3
DualOnly: only allow Dual Indexes
4.4
NoAndSingle: allow No Index and Single Index; defaut is No Index
4.5
NoAndDual: allow No Index and Dual Indexes; default is No Index
4.6
SingleAndDual: allow Single Index and Dual Indexes; default is Single Index
4.7
All: allow No Index, Single Index and Dual Indexes; default is No Index
FixedLayout
FixedLayout
bool
Indicates if it is a fixed layout kit. Value is true or false.
4
Multiplate
Multiplate
bool
Indicates if it is a fixed layout kit with multi- or single- plate. Value is true or false.
5
{Well position name}
FixedIndexPosition
string
Index1 and Index2 names separated by a dash, e.g. D701-D501.
Name
Name of the kit. It is an internal name, which has to be unique within a domain.
DisplayName
Display name of the kit. It is used for the index kit display label in the Run Planning.
Organization
Organization name. It is informational and not used in planned run creation.
AllowedIndexStrategies
1
Name
Name of the kit. It is an internal name, which has to be unique within a domain.
2
DisplayName
Display name of the kit. It is used for the index kit display label in the Run Planning.
3
Description
Description of the kit. It is displayed below the index kit field when the kit is selected in the Run Planning.
4
IndexStrategy
The index strategies supported by the kit. See 4.1 - 4.7 for the supported values.
1
Adapter
Adapter
string
The Adapter sequence for Read 1.
2
AdapterRead2
AdapterRead2
string
The Adapter sequence for Read 2. Include this line only when applicable.
{Index name}
{Index sequence}
Value is 1 (for Index1) or 2 (for Index2)
The index strategies supported by the kit. See .
3
AllowedIndexStrategies:
- "Dual"
- "Single"
Settings:
DefaultIndexStrategy: "Dual"IndexSequences: #Enter index sequences for both Index1 and Index2
i7Index1: #Index1
D701: "ATTACTCG" #<IndexName>: <IndexSequence>
D702: "TCCGGAGA"
D703: "CGCTCATT"
i5Index2: #Index2
D501: "TATAGCCT" #<IndexName>: <IndexSequence>
D502: "ATAGAGGC"
D503: "CCTATCCT"IndexSequences: #Enter index sequences for both Index1 and Index2
i7Index1: #Index1
D701: "ATTACTCG" #<IndexName>: <IndexSequence>
D702: "TCCGGAGA"
D703: "CGCTCATT"
i5Index2: #Index2
D501: "TATAGCCT" #<IndexName>: <IndexSequence>
D502: "ATAGAGGC"
D503: "CCTATCCT"
# Settings for non-fixed-layout kit with dual-index
Settings:
FixedLayout: false
Multiplate: false# Settings for fixed-layout kit with single plate
Settings:
FixedLayout: true
Multiplate: false
FixedIndexPositions: #Format: '- "WellPosition/Index1Name-Index2Name"'
- "A01/D701-D501"
- "B01/D702-D502"
- "H01/D703-D503"
- "H12/D704-D504"# Settings for single-plate fixed-layout kit, where well positions do not follow A01 - H12 format.
Settings:
FixedLayout: true
Multiplate: false
FixedLayoutPositionKeyByIndexId: true
FixedIndexPositions: #Format: '- "WellPosition/Index1Name-Index2Name"'
- "UDP0001/UDP0001-UDP0001"
- "UDP0002/UDP0002-UDP0002"# Settings for multi-plate fixed-layout kit
Settings:
FixedLayout: true
Multiplate: true
FixedIndexPositions: #Format: '- "Plate-WellPosition/Index1Name-Index2Name"'
- "A-A01/D701-D501"
- "C-B01/D703-D502"# Settings for non fixed-layout kit using single-index, with UMI in-place of Index2 :
# - NumCyclesIndex2Override is set to 16
# - Custom OverrideCycles is defined
# - CustomBclConvertSettings is defined so that fastq will be output for index reads.
IndexSequences:
i7Index1:
SI_NA_A1_1: "AAACGGCG"
SI_NA_A1_2: "CCTACCAT"
SI_NA_A1_3: "GGCGTTTC"
Settings:
FixedLayout: false
Multiplate: false
EnableCustomIndexCycles: true
NumCyclesIndex2Override: "16"
OverrideCycles: "Y50N{{Read1Length-50}};I8N{{Index1Length-8}};N{{Index2Length-16}}U16;Y50N{{Read2Length-50}}?"
CustomBclConvertSettings:
TrimUMI: "0"
CreateFastqForIndexReads: "1"# Settings for single-plate fixed-layout kit using dual-index, with UMI in Read1 :
# - Custom OverrideCycles is defined
# - CustomBclConvertSettings is defined so that fastq will be output for reads.
IndexSequences:
i7Index1:
SI_NT_A1: "ATTTACCGCA"
SI_NT_A2: "TTGTCGTAGA"
SI_NT_A3: "AGTCCTGCGG"
i5Index2:
SI_NT_A1: "GACAATAAAG"
SI_NT_A2: "CAATGTAGCA"
SI_NT_A3: "TGACACAAGT"
Settings:
FixedLayout: true
Multiplate: false
FixedIndexPositions:
- "A01/SI_NT_A1-SI_NT_A1"
- "A02/SI_NT_A2-SI_NT_A2"
- "A03/SI_NT_A3-SI_NT_A3"
OverrideCycles: "U28N{{Read1Length-28}};I10N{{Index1Length-10}};N{{Index2Length-10}}I10;Y90N{{Read2Length-90}}?"
CustomBclConvertSettings:
TrimUMI: "0"[Resources]
Name Type Format Value
Adapter Adapter string ACCTCCCCGTGA
AdapterRead2 AdapterRead2 string ACCTCCCCGTGA
FixedLayout FixedLayout bool true
Multiplate Multiplate bool false
A01 FixedIndexPosition string D701-D501
B01 FixedIndexPosition string D702-D502[Indices]
Name Sequence IndexReadNumber
D701 ATTACTCG 1
D702 TCCGGAGA 1
D501 TATAGCCT 2
D502 ATAGAGGC 2If 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)
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.