The Illumina NovaSeq X Series integration provides preconfigured workflows that map to established lab protocols and steps used with NovaSeq X Series sequencing instruments. The integration also supports the associated library prep kits, reagent kits, and assays used in these protocols.
NovaSeq X Series v1.2.0
The integration includes the following features:
Preconfigured NovaSeq X Series Sequencing v1.1 workflow that maps to lab protocols and instrument runs.
The following steps in the NovaSeq X Series Sequencing v1.1 preconfigured protocol:
Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1)
Make Bulk Pool (NovaSeq X Series Sequencing v1.1)
Dilute and Denature (NovaSeq X Series Sequencing v1.1)
Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1)
AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1)
AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1)
Automatic validation of run setup information before sample sheet generation or planned run creation.
Automated generation of the sample sheet. This sample sheet is used to start the sequencing run on the NovaSeq X Series Control Software through Local run mode.
Automated creation of a planned run on Illumina Connected Analytics (ICA). The NovaSeq X Series Control Software retrieves the planned run for use with the Cloud run mode.
Automated tracking of the NovaSeq X Series sequencing run and parsing of run statistics from BaseSpace Sequence Hub to Clarity LIMS.
Automated tracking of the NovaSeq X Series analysis run and high level analysis summary from BaseSpace Sequence Hub to Clarity LIMS.
Support for configuration of multiple secondary analyses with multiple library prep kits (LPKs) and index adapter kits (IAKs) for the planned run. The analysis is either performed by ICA in Cloud mode or by DRAGEN on-board the instrument in Local mode.
[Optional] Preconfigured Library Prep Validation v2.3.2 workflow used for validation purposes only. The workflow contains a single-step protocol that models the library prep required to produce libraries that are ready for the NovaSeq X Series Sequencing v1.1 workflow. For more information, refer to .
[Optional] Clarity LIMS Product Analytics (CLPA) integration that can be used with ICA. The CLPA integration installation and configuration is required. For more information, refer to .
Preconfigured NovaSeq X Series Sequencing v1.1 workflow that maps to lab protocols and instrument runs.
The following steps in the NovaSeq X Series Sequencing v1.1 preconfigured protocol:
Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1)
Make Bulk Pool (NovaSeq X Series Sequencing v1.1)
Dilute and Denature (NovaSeq X Series Sequencing v1.1)
Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1)
AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1)
AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1)
Automatic validation of run setup information before sample sheet generation or planned run creation.
Automated generation of the sample sheet. This sample sheet is used to start the sequencing run on the NovaSeq X Series Control Software through Local run mode.
Automated creation of a planned run on Illumina Connected Analytics (ICA). The NovaSeq X Series Control Software retrieves the planned run for use with the Cloud run mode.
Automated tracking of the NovaSeq X Series sequencing run and parsing of run statistics from BaseSpace Sequence Hub to Clarity LIMS.
Automated tracking of the NovaSeq X Series analysis run and high level analysis summary from BaseSpace Sequence Hub to Clarity LIMS.
Support for configuration of multiple secondary analyses with multiple library prep kits (LPKs) and index adapter kits (IAKs) for the planned run. The analysis is either performed by ICA in Cloud mode or by DRAGEN on-board the instrument in Local mode.
[Optional] Preconfigured Library Prep Validation workflow used for validation purposes only. The workflow contains a single-step protocol that models the library prep required to produce libraries that are ready for the NovaSeq X Series Sequencing v1.1 workflow.
[Optional] Clarity LIMS Product Analytics (CLPA) integration that can be used with ICA. The CLPA integration installation and configuration is required. For more information, refer to .
Release Notes
Last Updated: November 2024
Release Date: September 2023
Document Version: 2
These release notes describe the key changes to software components for the Clarity LIMS NovaSeq X Series Integration Package v1.1.0.
Compatibility
Release Notes
Last Updated: December 2024
Release Date: February 2024
Document Version: 3
These release notes describe the key changes to software components for the Clarity LIMS NovaSeq X Series Integration Package v1.2.0.
Compatibility
Refer to Compatibility under Instruments & Integrations.
New Features
Updated Java and third-party dependency libraries.
Updated Groovy to v3.0.7.
Defects Fixed
Restricted the scope for the JWT token used in the integration.
Fixed issue where the SIS core service was not properly registered in systemctl.
Known Limitations
This integration does not track the analysis step. You must log in to BSSH to view the cloud analysis status and results. For local analysis, the secondary analysis results are located in the external storage configured in Illumina Run Manager.
The Local run mode does not support custom library prep and index adapter kits.
The Analysis Configuration Template (ACT) names must be unique.
The Assign Analysis Configuration Template step expects the input samples to be unpooled libraries.
Known Issues
On the Make Bulk Pool step, the log displays a warning when the Calculate Volume automation is triggered and at least one pool consists of multiple inputs. This issue is caused by the output custom field being reset multiple times at the end of the automation. This issue does not affect the Calculate Volume automation functionality.
Updated Compatibility section to reference Compatibility matrix table.
Updated Known Limitations section.
1
Initial release.
Refer to Compatibility under Instruments & Integrations.
New Features
Supports analysis tracking at the metaworkflow level for analysis status and high level analysis summary results.
Supports the new 1.5B and 25B flow cell types. The B3 flow cell has been renamed to the 10B flow cell.
Uses the SecureString type for improving the security of values stored in the AWS param store.
Includes the following improvements to the NovaSeq X Series Sequencing v1.1 protocol:
Removes the unused Output Folder step custom field from the Load to Library Tube Strip step.
Renames the Library Tube barcode step custom field to Library Tube Strip Barcode in the AUTOMATED - Sequencing Run step.
Defects Fixed
None
Known Limitations
The analysis step tracks only the first run, but not subsequent queued runs.
The Local run mode does not support custom library prep and index adapter kits.
The Assign Analysis Configuration Template step expects the input samples to be unpooled libraries.
Known Issues
On the Make Bulk Pool step, the log displays a warning when the Calculate Volume automation is triggered and at least one pool consists of multiple inputs. This issue is caused by the output custom field being reset multiple times at the end of the automation. This issue does not affect the Calculate Volume automation functionality.
For BCL Convert only analysis, the high level analysis summary file is not available. User will need to manually complete the AUTOMATED - Analysis Run step upon analysis completed.
Revision History
Version
Changes
3
Updated Known Issues section.
2
Updated Compatibility section to reference Compatibility matrix table.
These release notes describe the key changes to software components for the Clarity LIMS NovaSeq X Series Integration Package v1.2.1.
Prerequisites
BaseSpace Sequence Hub account that has the Enterprise or Professional subscription
Compatibility
Refer to Compatibility under Instruments & Integrations.
Defects Fixed
Fixed the issue of unable to generate sample sheet and planned run when any sample in the pools has been assigned QC flag prior to entering the Load to Library Tube Strip step.
Known Limitations
The analysis step tracks only the first run, but not subsequent queued runs.
The Assign Analysis Configuration Template step expects the input samples to be unpooled libraries.
Known Issues
On the Make Bulk Pool step, the log displays a warning when the Calculate Volume automation is triggered and at least one pool consists of multiple inputs. This issue is caused by the output custom field being reset multiple times at the end of the automation. This issue does not affect the Calculate Volume automation functionality.
For BCL Convert only analysis, the high level analysis summary file is not available. User will need to manually complete the AUTOMATED - Analysis Run step upon analysis completed.
Documentations
The NovaSeq X Series Integration v1.2.1 guides follows exactly that of NovaSeq X Series Integration v1.2.0 with the following updates:
If you do not want to use the latest workflow (NovaSeq X Series Sequencing v1.2) available in IPP v2.10.0, then update your existing workflow configuration manually using the following information as required.
1. Update Existing Containers
Library 2-tube Strip container
On the Configuration tab, select Consumables
Select Containers.
Select the existing Library 2-tube Strip container.
Library 8-tube Strip container
Repeat the update instructions of Library 2-tube Strip container above for the existing Library 8-tube Strip container.
2. Update Global Field (BP Aliquot Volume (ul))
The derived sample global field BP Aliquot Volume (ul) is used in Calculate Volumes automation in Dilute and Denature step.
On the configuration tab, select Custom Fields.
Select Global Fields.
Expand Derived Sample.
3. Update Automations
There is a minor update of Command Line for Validate Run Setup and Create Planned Run automation, as well as, renaming of several automations to improve readability and provide more clarity of the automation function.
Update Validate Run Setup and Create Planned Run Automation
Updates to the Command Line is to improve the error message.
On the configuration tab, select Automation.
Select Validate Run Setup and Create Planned Run automation.
Update the existing Command Line as follows. Changes are in red.
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \ bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \ script:evaluateDynamicExpression \ script:evaluateDynamicExpression \ -exp 'if (!step.::Run Name::.matches(::[a-zA-Z0-9-_]+[a-zA-Z0-9-_. ]*::)) { fail(::Run Name contains prohibited characters. Please check to make sure Run Name only contains alphanumeric characters, spaces, dashes
[Optional] Rename Automations
On the configuration tab, select Automation.
Rename the automations as specified in the table below.
4. Update Load to Library Tube Strip Step
Rename Cloud Run ID to Planned Run ID
On the configuration tab, select Custom Fields.
Select Master Step Fields
Expand Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1).
Add Instrument Type
New instrument type NovaSeq X is now supported.
On the configuration tab, select Custom Fields.
Select Master Step Fields.
Expand Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1).
On the configuration tab, select Lab Work.
Select Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1) step.
Under Record Details, add Instrument Type to Master Step Fields.
5. Update AUTOMATED - Sequencing Run Step
There are configuration updates on the AUTOMATED - Sequencing Run master step and step, as well as updates to the run metrics displayed.
Update Master Step Configuration
On the configuration tab, select Lab Work.
Select AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) master step.
Under Step Type, update Derived Sample Generation to Fixed with 1 Derived Sample generated.
Remove Unused RTA Version Master Step Field
On the configuration tab, select Custom Fields.
Select Master Step Fields.
Expand AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1).
[Optional] Expand Sample Table By Default
On the configuration tab, select Lab Work.
Select AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1) step.
Select Record Details milestone.
Update Run Metrics
Reflect Reads PF Metrics at Run Level
On the configuration tab, select Custom Fields.
Select Global Fields.
Add Reads PF field as specified in the image.
Reflect % PF Metrics at Run Level
On the configuration tab, select Custom Fields.
Select Global Fields.
Add % PF field as specified in the image.
Add % Occupied
On the configuration tab, select Custom Fields.
Select Global Fields.
Expand Container.
On the configuration tab, select Lab Work.
Select AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step.
Under Record Details, add % Occupied to Table Columns.
Remove Cluster Density R1 and Cluster Density R2
On the configuration tab, select Lab Work.
Select AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step.
Under Record Details, remove Cluster Density R1 and Cluster Density R2 from Table Columns.
On the configuration tab, select Custom Fields.
Select Global Fields.
Expand Container.
6. Update AUTOMATED - Analysis Run Master Step
Update Master Step Configuration
On the configuration tab, select Lab Work.
Select AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1) master step.
Under Step Type, update Naming Convention to {SubmittedSampleName} {AppliedReagentLabels}
Rename Library Tube Strip Barcode to App Session ID
On the configuration tab, select Custom Fields.
Select Master Step Fields.
Expand AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1).
The changes are due to updates on container configuration.
Upgrade to Clarity LIMS Product Analytics Integration v1.4.0
Restart the CLPA integration service
Update Naming of Rows to Numeric.
Update Start at of Rows to 1.
Select BP Aliquot Volume (ul).
Update Decimal Places Displayed from 0 to 1.
, periods
and underscores. Run Name must start with alphanumeric, a dash or an underscore::); }; if (step.::Run Name::.length() > 255) { fail(::Run Name shall not exceed 255 characters.::); }; if (step.::Index 2 Cycles:: > 0 && step.::Index 1 Cycles:: == 0) { fail(::Index 1 Cycles cannot be zero if Index 2 Cycles is non-zero::); };' \ -exp 'if (!step.::Run Name::.matches(::[a-zA-Z0-9-_]+[a-zA-Z0-9-_. ]*::)) { fail(::Run Name contains prohibited characters. Please check to make sure Run Name only contains alphanumeric characters, spaces, dashes, periods and underscores. Run Name must start with alphanumeric, a dash or an underscore::); }; if (step.::Run Name::.length() > 255) { fail(::Run Name shall not exceed 255 characters.::); }; if (step.::Index 2 Cycles:: > 0 && step.::Index 1 Cycles:: == 0) { fail(::Index 1 Cycles cannot be zero if Index 2 Cycles is non-zero::); };' \ -log {compoundOutputFileLuid1} \ -log {compoundOutputFileLuid1} \ -t true \ -t true \ && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/SIS/SISServices/extensions/automation/novaseqxseries-automation.jar script:generate_sample_sheet -i {stepURI:v2} -u {username} -p {password} -c {compoundOutputFileLuid0} -l {compoundOutputFileLuid1}"
Validate Sample Names, Retrieve ACT List and Register Step Started
Assign Analysis Configuration Template
Rename Cloud Run Id to Planned Run ID.
Add Instrument Type field as specified in the image.
Under Step Type, update Naming Convention to Lane {InputWellLocation:0,1}
Delete RTA Version.
Under Sample Table, update Sample Display Default to Expand.
Select Save.
On the configuration tab, select Lab Work.
Select AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step.
Under Record Details, remove Reads PF R1 and Reads PF R2 from Table Columns.
Add Reads PF to Table Columns.
Select Save.
On the configuration tab, select Lab Work.
Select AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step.
Under Record Details, remove %PF R1 and %PF R2 from Table Columns.
Add % PF to Table Columns.
Add % Occupied field as specified in the image.
Delete Cluster Density R1 and Cluster Density R2.
Rename Library Tube Strip Barcode to App Session ID.
Old Automation Name
New Automation Name
Used in Step
Register NovaSeq X RunCompleted and Register Step Completed
Register NovaSeq X Series Sequencing Run Completed
AUTOMATED - Sequencing Run
Register Step Started and Register NovaSeq X Run and Register NovaSeq X Run Association and Register NovaSeq X RunStarted
Register NovaSeq X Series Sequencing Run Started
AUTOMATED - Sequencing Run
Validate Reagent Labels and Apply Selected ACT to Samples and Set Next Step
Apply Selected ACT to Samples and Set Next Step
Assign Analysis Configuration Template
Validate Flowcell Inputs and Validate Analysis Configurations and Register Step Started
Validate Flowcell Inputs, Analysis Configurations and Register Step Started
Load to Library Tube Strip
Validate Sample Names and Retrieve Analysis Configuration Template List and Register Step Started
systemctl restart clarity_upa
Manual Upgrade
NovaSeq X Series Integration Package v1.2.0 includes modifications to the existing NovaSeq X Series Sequencing workflows to support the following new features:
PhiX control included in the Dilute and Denature step instead of the Make Bulk Pool step.
Support for the new 1.5B and 25B flow cells.
Analysis tracking for the status and high level analysis result summary through the AUTOMATED - Analysis Run step (this is done at the metaworkflow level).
âš Analysis tracking is a new step in NovaSeq X Series protocol and cannot be added to a non-pending protocol or workflow. For analysis tracking at the metaworkflow level, do not proceed with the manual upgrade. Instead, install the NovaSeq X Series v1.2.0 Integration Package with the NovaSeq X Series Sequencing v1.1 protocol.
The following workflow updates are included in NovaSeq X Series Integration Package v1.2.0:
AUTOMATED - Sequencing Run step: Renamed the Library Tube Barcode master step custom field. This field is now named Library Tube Strip Barcode.
Load to Library Tube Strip step: Removed the Output Folder master step custom field.
Use the following instructions to manually update the workflows.
Prerequisites
Illumina recommends upgrading to Illumina Preset Protocols (IPP) v2.7 to support the new features. If you do not have IPP v2.7 installed, do the following before starting the manual upgrade:
Install ClarityLIMS-NGS-Package-v5 RPM v5.24.
Replace the following files in /opt/gls/clarity/extensions/conf/driverfiletemplates:
On the Configuration tab, select Consumables, and then select Containers.
Add the following information to enable the Library 2-tube Strip container that supports the 1.5B flow cell:
ℹ No action is required for the 25B flow cell. The flow cell uses the same Library 8-tube Strip as the 10B flow cell.
Add New Master Step Fields
Enable PhiX control in the Dilute and Denature step as follows.
On the Configuration tab, select Custom Fields, and then select Master Step Fields.
Add the following master step fields for the Dilute and Denature (NovaSeq X Series Sequencing v1.x) step:
Dilute and Denature (NovaSeq X Series Sequencing v1.x) Master Step Field Configuration
Modify Existing Master Step Fields
On the Configuration tab, select Custom Fields, and then select Master Step Fields.
Modify the following master step fields:
Make Bulk Pool (NovaSeq X Series Sequencing v1.x) Master Step Field Modifications
Modify Existing Global Field
On the Configuration tab, select Custom Fields, and then select Global Fields.
For the NovaSeq X Flowcell Type field, update the Dropdown Items with the following values:
ℹ A merge conflict is expected when upgrading from IPP v2.6 to v2.7 due to the changes in the NovaSeq X Flowcell Type field.
Add, Configure, and Modify Automations
Add Automation
On the Configuration tab, select Automation, and then select Step Automation.
On the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.x) step, add the following automation to enable new flow cell types:
Name: Validate Flowcell Inputs and Validate Analysis Configurations and Register Step Started
Configure Automation Settings
On the Configuration tab, select Lab Work.
Configure the trigger location and style for the following automations:
Load to Library Tube Strip (NovaSeq X Series Sequencing v1.x) Step Automation Configuration
ℹ These configuration updates are required to enable the new flow cell types.
Modify Step Automations
On the Configuration tab, select Automation, and then select Step Automation.
Select the Calculate Volumes automation that is enabled on the Make Bulk Pool (NovaSeq X Series Sequencing v1.x) step.
Update the existing command line as follows.
This modification is required to move the PhiX control from the Make Bulk Pool (NovaSeq X Series Sequencing v1.x) step to the Dilute and Denature (NovaSeq X Series Sequencing v1.x) step. Changes are in red.
Modify Existing Steps
Enable PhiX Control
On the Configuration tab, select Lab Work.
Select the Make Bulk Pool (NovaSeq X Series Sequencing v1.x) step.
For Control Types, remove PhiX v3.
For the Record Details milestone, navigate to Step Data and remove the following fields:
Final Loading Volume (ul)
% PhiX (2.0 nM) Spike-In
Select the Dilute and Denature (NovaSeq X Series v1.x) step.
Select the Record Details milestone.
Navigate to Step Data and add 1-2% PhiX Spike-In to Master Step Fields.
Enable New Flow Cell Types
On the Configuration tab, select Lab Work.
Select the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.x) step.
Select the Queue milestone.
Select the Ice Bucket milestone.
Navigate to Sample Table and add the NovaSeqX Flowcell Type field to Column Headers.
Select the Placement milestone.
Make Bulk Pool master step: Updated the dropdown items in the Final Loading Concentration (pM) custom field.
For Container Name, enter Library 2-tube Strip.
For Rows, update the following fields:
Number: 2
Naming: Alphabetic
For Columns, update the following fields:
Number: 1
Naming: Numeric
Start at: 1
Additional Options and Dropdown Items
PhiX Volume (ul)
Numeric
Read Only
Decimal places displayed: 1
1–2% PhiX Spike-In
Toggle Switch
Required Field
Default: No
Final Loading Concentration (pM)
Update Dropdown Items with the following options:
90
140
Only perform this modification if the new Dropdown Items are required.
This modification is not required to enable support for the new flow cells or PhiX control in the Dilute and Denature (NovaSeq X Series Sequencing v1.x) step.
Flowcell Type
Change Field Type from Text to Text Dropdown.
Update Field Options as follows.
Read Only = No
This modification is required to support the new flow cells.
Load to Library Tube Strip (NovaSeq X Series Sequencing v1.x) Master Step Field Modifications
Field
Modification
Purpose/Notes
Output Folder
Remove the field.
If the step has been run previously and this field is used, it cannot be removed. This field does not affect the functionality of the workflow.
This modification is not required to enable support for the new flow cells or PhiX control in the Dilute and Denature (NovaSeq X Series Sequencing v1.x) step.
AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.x) Master Step Field Modifications
Field
Modification
Purpose/Notes
Library Tube Barcode
Rename the field from Library Tube Barcode to Library Tube Strip Barcode.
If the installed NovaSeq X Series workflow is v1.1 or earlier, this modification is not required to enable support for the new flow cells or PhiX control in the Dilute and Denature (NovaSeq X Series Sequencing v1.x) step.
For NovaSeq X Series workflow v1.2, this modification is required. Run event handling fails without this modification.
1.5B
10B
25B
Channel Name: limsserver
Command line:
Automation
Setting
Validate Flowcell Inputs and Validate Analysis Configurations and Register Step Started
Trigger Location: Step
Trigger Style: Automatic upon entry
Validate Input Count and Validate Analysis Configurations and Register Step Started
Trigger Location: Not used
Dilute and Denature (NovaSeq X Series Sequencing v1.x) Step Automation Configuration
ℹ This configuration update is required to enable PhiX control on the Make Bulk Pool (NovaSeq X Series Sequencing v1.x) step.
Select the Calculate Volumes automation that is enabled on the Dilute and Denature (NovaSeq X Series Sequencing v1.x) step.
Update the existing command line as follows.
This modification is required to move the PhiX control from the Make Bulk Pool (NovaSeq X Series Sequencing v1.x) step to the Dilute and Denature (NovaSeq X Series Sequencing v1.x) step and to enable support for the 1.5B and 25B flow cells. Changes are in red.
Select the Validate Library Tube Strip Barcode automation that is enabled on the Load to Library Tube Strip Barcode (NovaSeq X Series Sequencing v1.x) step.
Update the existing command line as follows.
This modification is required to enable support for the 1.5B and 25B flow cells and future 8-tube library strips. Changes are in red.
Preconfigured NovaSeq X Series Sequencing v1.2 workflow that maps to lab protocols and instrument runs.
Preconfigured protocols:
Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2)
Make Bulk Pool (NovaSeq X Series Sequencing v1.2)
Dilute and Denature (NovaSeq X Series Sequencing v1.2)
Load to Library Tube Strip (NovaSeq X Series Sequencing v1.2)
AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2)
AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.2)
Automatic validation of run setup information before sample sheet generation or planned run creation.
Automated generation of a samplesheet. The samplesheet will be used to start the sequencing run on NovaSeq X Series Control Software via Local run mode.
Automated creation of a planned run on Illumina Connected Analytics (ICA). The planned run created will be retrieved by NovaSeq X Series Control Software to start run via Cloud run mode.
Automated tracking of the NovaSeq X Series sequencing run and parsing of run statistics from BaseSpace Sequence Hub (BSSH) into the LIMS.
Automated tracking of the NovaSeq X Series analysis run and high level analysis summary from BaseSpace Sequence Hub (BSSH) into the LIMS.
Support for configuration of multiple secondary analyses with multiple library prep kit (LPK) and index adapter kit (IAK) for the planned run. The analysis are either performed by the ICA in Cloud mode or by DRAGEN on-premise in Local mode.
(Optional) Preconfigured Library Prep Validation v2.3.5 workflow used for validation purposes only. The workflow contains a single-step protocol that models the library prep workflow required to produce libraries tagged with index sequences. At the end of the step, these libraries are routed to NovaSeq X Series Sequencing v1.2 workflow.
(Optional) Clarity LIMS Product Analytics (CLPA) integration which makes Clarity data available to the ICA service. CLPA integration installation and configuration is required. Refer to for details on configuring and using CLPA.
User Interaction, Validation and Troubleshooting
Last Updated: December 2024
Release Date: September 2023
Document Version: 2
This section explains the user interaction for each step and how to validate the installation of the Illumina NovaSeq X Series Integration Package v1.1.0.
The validation process involves the following actions:
Running samples through the Library Prep Validation v2.3.1 workflow.
The workflow contains a single-step protocol that models the library prep required to produce libraries tagged with index sequences. At the end of the step, these libraries are routed to NovaSeq X Series Sequencing v1.0 workflow.
Running libraries through the NovaSeq X Series Sequencing v1.0 workflow validates the following items:
Successful sequential step advancement of samples through the steps of the workflow.
Automatic validation of run setup information before sample sheet generation or planned run creation.
Automated sample sheet generation. This sample sheet is used to start the sequencing run on the NovaSeq X Series Control Software through the Local run mode.
Automated creation of a planned run in Illumina Connected Analytics (ICA). The control software retrieves the planned run. The run is started through the Cloud run mode.
Automated tracking of the NovaSeq X Series sequencing run and parsing of run statistics into Clarity LIMS.
The validation steps assume that the following conditions have been met:
NovaSeq X Series Integration Package v1.1.0 is installed and you have imported the default Clarity LIMS configuration.
Analysis configuration templates (ACTs) are created in BaseSpace Sequence Hub for your run. Make sure that the index adapter kit label group is created in Clarity LIMS before selecting it in the ACT. This same label group is used in the Run Library Prep Validation v2.3.1 step. For more information on creating a reagent label group, refer to Add and Configure Labels and Label Groups in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation. For the adapter sequences for your library prep kits, refer to Illumina Adapter Sequences.
Analysis Configuration Template Creation in BaseSpace Sequence Hub
You must create the Analysis Configuration Templates (ACTs) that are required for configuring secondary analysis in the NovaSeq X Series Sequencing v1.0 workflows. Create and delete ACTs in BaseSpace Sequence Hub. For instructions, refer to the BaseSpace Sequence Hub Online Help on the Illumina support site.
Activate Workflow, Create Project, Add and Assign Samples
The following steps set up Clarity LIMS in preparation for running samples through the Library Prep Validation v2.3.1 and NovaSeq X Series Sequencing v1.0 workflows.
On the Configuration tab, under Workflows, activate both the Library Prep Validation v2.3.1 and NovaSeq X Series Sequencing v1.0 workflows.
On the Projects and Samples screen, create a project and add samples to it.
âš Only use alphanumeric, dash, or underscore characters in the submitted sample names. Invalid characters can cause a sample sheet validation failure in the Load to Library Tube Strip step.
Assign the samples to the Library Prep Validation workflow.
Library Prep Protocol
This single-step protocol models the library prep required to produce libraries tagged with index sequences that are ready for the NovaSeq X Series Sequencing v1.0 workflow.
Label Group = same as the Index Adapter Kit selected in the ACT that is being used
Sequencing Instrument = NovaSeq X Series
On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the NovaSeq X Series Sequencing v1.0 workflow, Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0).
Protocol: NovaSeq X Series Sequencing v1.0
The NovaSeq X Series Sequencing v1.0 protocol consists of the following steps:
Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0)
Make Bulk Pool (NovaSeq X Series Sequencing v1.0)
Dilute and Denature (NovaSeq X Series Sequencing v1.0)
Load To Library Tube Strip (NovaSeq X Series Sequencing v1.0)
AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0)
Each step contains a script to register the start time (upon step entry) and the time that the step is completed (upon step exit). This information is published to CLPA through ICA. These scripts are used only for CLPA support and can be incorporated as part of an automation that performs other functions.
Step 1: Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0)
In Lab View, locate the NovaSeq X Series Sequencing v1.0 protocol. The samples are queued for the Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0) step.
Add the samples to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work to start the Validate Sample Names and Retrieve Analysis Configuration Template List and Register Step Started automation.
The automation checks that the sample names do not use restricted characters and are within character limits and retrieves the list of ACTs that you can select. The automation also registers the start time of the step by publishing messages to CLPA through ICA. The part of the script for start time registration is used only for CLPA support.
Select the applicable ACT to assign to the samples. The index adapter kit specified by the ACT must correspond with the label group used in .
[Optional] Under Step Details, select Retrieve ACT Information to trigger the Retrieve ACT Information automation.
This automation retrieves ACT information (e.g., Library Prep Kit, Index Adapter Kit, Reference Genome, and so on) and populates the fields in Clarity LIMS. These details are saved to the ACTMetadata.csv file that you can download. If you are not sure of the analysis configuration before starting the sequencing and analysis run, refer to the details in the file.
Select Next Steps to assign the ACT to the samples.
This action triggers the Validate Reagent Labels and Apply Selected ACT to Samples and Set Next Step automation. This automation validates that the indexes applied to the libraries are valid for the selected ACT and assigns samples to it. All samples added to the Ice Bucket (mentioned above) are assigned to this ACT.
Select Finish Step.
To use multiple ACTs in a single run, repeat 2–7 for each ACT. For step 2, only add samples that are being assigned to that ACT into the Ice Bucket.
Step 2: Make Bulk Pool (NovaSeq X Series Sequencing v1.0)
In Lab View, locate the NovaSeq X Series Sequencing v1.0 protocol. The samples are queued for the Make Bulk Pool (NovaSeq X Series Sequencing v1.0) step.
Add the samples to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
Create a pool of samples as follows.
On the Pooling screen, create a pool by dragging samples into the Pool Creator.
Enter a name for your pool or accept the default name (Pool #1).
On the Record Details screen, navigate to the Reagent Lot Tracking section to track the lot information used in the step.
[Optional] Create a new lot. For more information, refer to Add and Configure Reagent Kits and Lots in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
In the Step Details area, complete the following required fields:
Number of Lanes to Sequence — Used in volume calculations, to make sure that the volumes are sufficient for the number of times the pool is sequenced. This field is applied to all pools in the step.
Final Loading Concentration (pM) — The final loading concentration of the pool in the flow cell.
Final Loading Volume (ul) — The final loading volume of the pool in the flow cell. This field is prepopulated with the configured default value (200 ul), but can be edited if more volume is required.
Minimum Per Sample Volume (ul) — The minimum volume for each sample. This field is prepopulated with the configured default value (2 ul) and can be edited. If the per sample volume is below the value set in this field, the Calculate Volumes script applies a rounding factor to affected samples so that the volume reaches the minimum volume.
% PhiX (2.0nM) Spike-In — The value entered in this field is used to calculate the volume of PhiX spike-in required.
Flowcell Type — This is a read-only field configured with the default value of B3.
Select Calculate Volumes to trigger the Calculate Volumes automation.
This automation calculates the volumes required for each library to form a pool that has the concentration and volume specified in the Step Details fields. The automation is applied to control samples when they are indexed and molarity is specified. It also generates the calculation file in a CSV format which is available for download under Files placeholder section below.
In the Sample Details table, select the pool next to the sample name to view details on the pool composition.
Select Next Steps to trigger the Set Next Step automation.
This automation sets the next step for samples to ADVANCE, which moves them to the Dilute and Denature (NovaSeq X Series Sequencing v1.0) step.
Select Finish Step.
Step 3: Dilute and Denature (NovaSeq X Series Sequencing v1.0)
In Lab View, locate the NovaSeq X Series Sequencing v1.0 protocol. The pool of samples queued for the Dilute and Denature (NovaSeq X Series Sequencing v1.0) displays.
Add the pool to the Ice Bucket and select View Ice Bucket.
[Optional] On the Ice Bucket screen, set the number of derivatives to create (placed into the library tube strip) and select Begin Work.
On entry to the step, the Validate Inputs Flowcell Type and Register Step Started automation is triggered. This automation makes sure that the configured flow cell type is valid. The automation also registers the start time of the step by publishing messages to CLPA through ICA. The portion of the script used for start time registration is used only for CLPA support.
On entry to the Record Details screen, the Calculate Volumes automation is triggered. This automation sets the following values:
BP Aliquot Volume (ul)
On the Record Details screen, the Reagent Lot Tracking section tracks the NaOH, Resuspension Buffer, and TT2 reagents used in the step. To add and activate reagent lots, refer to Add and Configure Reagent Kits and Lots in the .
On the Record Details screen, perform the following actions:
In the Reagent Lot Tracking section, select from the active lots displayed in each drop-down list.
In the Sample Details table, the BP aliquot, RSB, NaOH, and TT2 reagent volume values are auto populated and read-only.
In the Files area, download the Calculation File (CSV) and open it and view details on the RSB, NaOH, and TT2 volumes to add per working pool.
Select Next Steps.
Select Finish Step.
Step 4: Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0)
In Lab View, locate the NovaSeq X Series Sequencing v1.0 protocol. The pool of samples are queued for the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0) step.
Add the pools to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work to trigger the Validate Analysis Configurations and Register Step Started automation.
Validate Analysis Configurations automation performs the following basic checks on the analysis configuration for each pool:
Pooled samples are within the maximum configuration limit.
Pooled samples have the sample type of analysis (Cloud or Local).
Pooled samples that have the same secondary analysis also have the same analysis version and settings.
Register Step Started automation registers the start time of the step by publishing messages to CLPA through ICA. The portion of the script used for start time registration is used only for CLPA support.
On the Placement screen, do as follows.
Drag the pool into the Library 8-tube Strip in the Placed Samples area on the right.
Scan or type the barcode of the Library 8-tube Strip into the Library 8-tube Strip container name field.
The fields displayed on the Record Details screen are used to create the planned run and generate the sample sheet.
The analysis related information for the planned run is from the ACT associated with the samples and no further analysis configuration is required. Refer to the following table for details.
Fields Displayed on Record Details Screen of Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0) Step
Select Validate Run Setup and Create Planned Run to trigger the automation script. The script performs the following actions:
Validates the parameters entered on the Record Details screen.
Clarity consolidates sample/library information, run and analysis configuration details and send them to ICA to create a planned run and samplesheet for Cloud run mode.
Select Next Steps.
On the Assign Next Steps screen, the next step for the pooled samples is set to the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0) step.
Select Finish Step to advance the pooled samples to the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0) step. For more information on how to start the sequencing run for different run modes, refer to .
To use multiple ACTs in a single run, repeat steps 1–3 for each ACT. The pool of samples (for different ACTs) are then queued for the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0) step to be added to the Ice Bucket.
Step 5: AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0)
This protocol contains the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0) step. This step is fully automated.
Do not add samples to the Ice Bucket or start or complete the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0) step. The integration does this automatically.
The integration starts the step automatically and data from the run is parsed back into Clarity LIMS. User interaction is not required, but you can review the stages of the step in Clarity LIMS.
Review Run Data
Read summary metrics are recorded for the library pool. After the run is complete, open the step and review these metrics in the Step Details section and the Sample Details table.
Step Details Section
The following values populate the master step fields:
Run Name
Run Status
Output Folder
Current Read
Current Cycle
Library Tube Barcode
Flow Cell ID
Flow Cell Side
Flow Cell Type
Flow Cell Part Number
Flow Cell Lot Number
Flow Cell Expiration Date
Instrument ID
Instrument Type
Instrument Control Software Version
Sequencing Log
Sample Details Table
Summary metrics populate the following global custom fields:
% Bases >=Q30 R1
% Bases >=Q30 R2
% Error Rate R1
% Error Rate R2
Yield (Gb) R1
Yield (Gb) R2
Reads PF R1
Reads PF R2
%PF R1
%PF R2
% Aligned R1
% Aligned R2
% Phasing R1
% Phasing R2
% Prephasing R1
% Prephasing R2
Intensity Cycle 1 R1
Intensity Cycle 1 R2
Cluster Density R1
Cluster Density R2
Troubleshooting
If an automation trigger does not appear to run its corresponding scripts, refer to the Troubleshooting Automation section in the Clarity LIMS (API & Database) documentation.
Automated Step Does Not Start
If the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0) step does not start, Clarity LIMS is likely not receiving sequencing run events from BaseSpace Sequence Hub correctly. Check for the following issues:
Make sure that the NovaSeq X Control Software is configured properly as follows.
Before you start the run, in the NovaSeq X Control Software, select the appropriate BaseSpace Sequence Hub region in the Hosting location drop-down list.
Under Run Settings, check the boxes next to Cloud run monitoring and Cloud run storage.
If there are issues after modifying the configuration settings on the NovaSeq X Control Software, contact Illumina Support.
Automated Step Starts, But Does Not Complete
If the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0) step starts, but does not complete, do as follows.
Log in to Clarity LIMS and use one of the following methods to open the in-progress step:
In Lab View, find the step in the Recent Activities pane.
Search for the step in Clarity LIMS using the library tube strip barcode as the search term.
On the Record Details screen, the Sequencing Log multiline text field contains logging information.
If unable to reach the Record Details screen, or if the Sequencing Log field does not contain enough information to resolve the issue, contact Illumina Support. Provide the relevant information from the troubleshooting steps already performed.
The Validate Analysis Configuration automation check is in the Make Bulk Pool (upon pooling) and Load to Library Tube Strip (upon step entry) steps. If a failure happens, an error message displays and the step can be aborted, or you might be prevented from continuing the step. This check can fail for the following reasons:
The analysis configuration after library pooling or during the planned run creation exceeds the configuration limit.
Resolve this error as follows.
If the error occurs on the Make Bulk Pool step, reduce the number of analysis configurations for a pool. To reduce the number, reorganize the samples for the pooling step (eg, by pooling samples that have similar configurations together).
If the error occurs on the Load to Library Tube step, reduce the number of analysis configurations for the planned run by reorganizing the samples for multiple runs instead of a single run.
ACTs of samples in the same pool or planned run must have the same run mode (Local or Cloud).
Resolve this error as follows.
Abort the step and remove samples with ACTs that have conflicting run modes from the Ice Bucket.
ACTs of samples in the same pool or planned run that have the same secondary analysis must have the same analysis version.
Resolve this error as follows.
If the error occurs on the Make Bulk Pool step, pool the samples again. Make sure that samples with the same secondary analysis, but conflicting analysis versions, are in different pools.
ACTs of samples in the same pool or planned run that have the same secondary analysis must have the same analysis settings. The analysis setting fields can differ for a different secondary analysis and are configured in BaseSpace Sequence Hub when the ACT is created.
Resolve this error as follows.
If the error occurs on the Make Bulk Pool step, pool the samples again. Make sure that samples with the same secondary analysis, but conflicting analysis settings, are in different pools.
Revision History
Version
Changes
2
Updated Step 2: Make Bulk Pool (NovaSeq X Series Sequencing v1.1) to remove instructions on adding control. PhiX control should be added via the % PhiX (2.0nM) Spike-In step field.
1
Initial release.
NovaSeq X Series v1.1.0
The integration includes the following features:
Preconfigured NovaSeq X Series Sequencing v1.0 workflow that maps to lab protocols and instrument runs.
The following steps in the NovaSeq X Series Sequencing v1.0 preconfigured protocol:
Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0)
Make Bulk Pool (NovaSeq X Series Sequencing v1.0)
Dilute and Denature (NovaSeq X Series Sequencing v1.0)
Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0)
AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0)
Automatic validation of run setup information before sample sheet generation or planned run creation.
Automated generation of the sample sheet. This sample sheet is used to start the sequencing run on the NovaSeq X Series Control Software through Local run mode.
Automated creation of a planned run on Illumina Connected Analytics (ICA). The NovaSeq X Series Control Software retrieves the planned run for use with the Cloud run mode.
Automated tracking of the NovaSeq X Series sequencing run and parsing of run statistics into Clarity LIMS.
Support for configuration of multiple secondary analyses with multiple library prep kits (LPKs) and index adapter kits (IAKs) for the planned run. The analysis is either performed by ICA in Cloud mode or by DRAGEN on-board the instrument in Local mode.
[Optional] Preconfigured Library Prep Validation v2.3.1 workflow used for validation purposes only. The workflow contains a single-step protocol that models the library prep required to produce libraries that are ready for the NovaSeq X Series Sequencing v1.0 workflow. For more information, refer to .
[Optional] Clarity LIMS Product Analytics (CLPA) integration that can be used with ICA. The CLPA integration installation and configuration is required. For more information, refer to .
User Interaction, Validation and Troubleshooting
Last Updated: December 2024
Release Date: February 2024
Document Version: 2
This section explains how to validate the installation of the Illumina NovaSeq X Series Integration Package v1.2.0.
The validation process involves the following actions:
Running samples through the Library Prep Validation v2.3.2 workflow.
[Optional] If multiple pools are required, select the plus sign (+) next to Pool Creator to create a pool.
[Optional] To remove a pool, select the X in the top right corner of the pool.
Select Record Details to trigger the Validate Analysis Configurations automation.
This automation performs the following checks on the analysis configuration for each pool:
Pooled samples are within the maximum configuration limit.
Pooled samples have the sample type of analysis (Cloud or Local).
Pooled samples that have the same secondary analysis also have the same analysis version and settings.
RSB Volume (ul)
NaOH Volume (ul)
TT2 Volume (ul)
The automation also generates the calculation file (CSV) and attaches it to the step. This file contains information about the volume of RSB, NaOH, and TT2 to add per working pool.
[Optional] In the Sample Details table, select the pool icon to view details of the working pool composition.
The working pool number is appended to the bulk pool name so that you can identify which working pools are derived from the same bulk pool.
Select Record Details.
After exiting the Placement screen, the Validate Library Strip Tube Barcode automation makes sure that the library tube strip barcode conforms to the barcode mask LC[0-9]{7}-LC1.
The selected Run Mode must correspond to the ACT type.
Presets
Local¹
Cloud²
Read 1 Cycles
Presets
151
101
51
Read 2 Cycles
Presets
151
101
51
Index 1 Cycles
Presets
0
6
8
Index 2 Cycles
Presets
0
6
8
¹ The Run/Analysis configuration is imported manually to the NovaSeq X Series instrument through a sample sheet. The sample sheet generated on this step is downloaded and imported manually by the user on NovaSeq X Control Software to start the run. The downstream secondary analysis is done using the onboard DRAGEN module.
² The planned run configuration is downloaded to the NovaSeq X Series Instrument Control Software to start the run. The downstream secondary analysis is done on the cloud.
³ The custom value must correspond to the longest index sequence of the samples in the pools in the library tube strip. Otherwise, the planned run creation fails and an error message displays.
Clarity consolidates sample/library information, run and analysis configuration details and send them to ICA to generate samplesheet for Local run mode.
Make sure that all remaining samples in the Ice Bucket have ACTs with the same run modes.
Select Begin Work to continue the step.
If the error occurs on the Load to Library Tube step, reorganize the samples for the planned run. Make sure that samples with the same secondary analysis, but conflicting analysis versions, are in different runs.
If the error occurs on the Load to Library Tube Strip step, reorganize the samples for the planned run. Make sure that samples with the same secondary analysis, but conflicting analysis settings, are in different runs.
Field
Value
Run Name
Enter the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. No spaces.
The workflow contains a single-step protocol that models the library prep required to produce libraries tagged with index sequences. At the end of the step, these libraries are routed to NovaSeq X Series Sequencing v1.1 workflow.
Running libraries through the NovaSeq X Series Sequencing v1.1 workflow validates the following items:
Successful sequential step advancement of samples through the steps of the workflow.
Automatic validation of run setup information before sample sheet generation or planned run creation.
Automated sample sheet generation. This sample sheet is used to start the sequencing run on the NovaSeq X Series Control Software through the Local run mode.
Automated creation of a planned run in Illumina Connected Analytics ( ICA). The control software retrieves the planned run. The run is started through the Cloud run mode.
Automated tracking of the NovaSeq X Series sequencing run and parsing of run statistics from BaseSpace Sequence Hub to Clarity LIMS.
Automated tracking of the NovaSeq X Series analysis run and high level analysis summary from BaseSpace Sequence Hub to Clarity LIMS.
The validation steps assume that the following conditions have been met:
The NovaSeq X Series Integration v1.2.0 has a valid BaseSpace Sequence Hub account with an Enterprise or Professional subscription.
NovaSeq X Series Integration Package v1.2.0 is installed and you have imported the default Clarity LIMS configuration.
Analysis configuration templates (ACTs) are created in BaseSpace Sequence Hub (BaseSpace Sequence Hub) for your run. Make sure that the index adapter kit label group is created in Clarity LIMS before selecting it in the ACT. This same label group is used in the Run Library Prep Validation v2.3.2 step. For more information on creating a reagent label group, refer to Add and Configure Labels and Label Groups in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation. For the adapter sequences for your library prep kits, refer to Illumina Adapter Sequences.
Analysis Configuration Template Creation in BaseSpace Sequence Hub
You must create the Analysis Configuration Templates (ACTs) that are required for configuring secondary analysis in the NovaSeq X Series Sequencing v1.1 workflows. Create and delete ACTs in BaseSpace Sequence Hub. For instructions, refer to the BaseSpace Sequence Hub Online Help on the Illumina support site.
Activate Workflow, Create Project, Add and Assign Samples
The following steps set up Clarity LIMS in preparation for running samples through the Library Prep Validation v2.3.2 and NovaSeq X Series Sequencing v1.1 workflows.
On the Configuration tab, under Workflows, activate both the Library Prep Validation v2.3.2 and NovaSeq X Series Sequencing v1.1 workflows.
On the Projects and Samples screen, create a project and add samples to it.
âš Sample and library names must use only alphanumeric, dash, or underscore characters. Invalid characters can cause a sample sheet validation failure in the Load to Library Tube Strip step.
Assign the samples to the Library Prep Validation workflow.
Library Prep Protocol
This single-step protocol models the library prep required to produce libraries tagged with index sequences that are ready for the NovaSeq X Series Sequencing v1.1 workflow.
Label Group = same as the Index Adapter Kit selected in the ACT that is being used
Sequencing Instrument = NovaSeq X Series
On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the NovaSeq X Series Sequencing v1.1 workflow, Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1).
Protocol: NovaSeq X Series Sequencing v1.1
The NovaSeq X Series Sequencing v1.1 protocol consists of the following steps:
Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1)
Make Bulk Pool (NovaSeq X Series Sequencing v1.1)
Dilute and Denature (NovaSeq X Series Sequencing v1.1)
Load To Library Tube Strip (NovaSeq X Series Sequencing v1.1)
AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1)
AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1)
Each step contains a script to register the start time (upon step entry) and the time that the step is completed (upon step exit). This information is published to CLPA through ICA. These scripts are used only for CLPA support and can be incorporated as part of an automation that performs other functions.
Step 1: Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1)
In Lab View, locate the NovaSeq X Series Sequencing v1.1 protocol. The samples are queued for the Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1) step.
Add the samples to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work to start the Validate Sample Names and Retrieve Analysis Configuration Template List and Register Step Started automation.
The automation checks that the sample names do not use restricted characters and are within character limits and retrieves the list of ACTs that you can select. The automation also registers the start time of the step by publishing messages to CLPA through ICA. The part of the script for start time registration is used only for CLPA support.
Select the applicable ACT to assign to the samples. The index adapter kit specified by the ACT must correspond with the label group used in .
[Optional] In Step Details, select Retrieve ACT Information to trigger the Retrieve ACT Information automation.
This automation retrieves ACT information (e.g., Library Prep Kit, Index Adapter Kit, Reference Genome, and so on) and populates the fields in Clarity LIMS. These details are saved to the ACTMetadata.csv file that you can download. If you are not sure of the analysis configuration before starting the sequencing and analysis run, refer to the details in the file.
Select Next Steps to assign the ACT to the samples.
This action triggers the Validate Reagent Labels and Apply Selected ACT to Samples and Set Next Step automation. This automation validates that the indexes applied to the libraries are valid for the selected ACT and assigns samples to it. All samples added to the Ice Bucket (mentioned above) are assigned to this ACT.
Select Finish Step.
Step 2: Make Bulk Pool (NovaSeq X Series Sequencing v1.1)
In Lab View, locate the NovaSeq X Series Sequencing v1.1 protocol. The samples are queued for the Make Bulk Pool (NovaSeq X Series Sequencing v1.1) step.
Add the samples to the Ice Bucket and select View Ice Bucket.
ℹ If PhiX control sample is required for the run, the configuration setting shall be performed in the Step 3: Dilute and Denature with the 1–2% PhiX Spike-In toggle switch.
On the Ice Bucket screen, select Begin Work.
Create a pool of samples as follows.
On the Pooling screen, create a pool by dragging samples into the Pool Creator.
Enter a name for your pool or accept the default name (Pool #1).
On the Record Details screen, navigate to the Reagent Lot Tracking section to track the lot information used in the step.
[Optional] Create a new lot. For more information, refer to Add and Configure Reagent Kits and Lots in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
In the Step Details area, complete the following required fields:
Number of Lanes to Sequence — Used in volume calculations, to make sure that the volumes are sufficient for the number of times the pool is sequenced. This field is applied to all pools in the step.
Final Loading Concentration (pM) — The final loading concentration of the pool in the flow cell.
Minimum Per Sample Volume (ul) — The minimum volume for each sample. This field is prepopulated with the configured default value (2 µl) and can be edited. If the per sample volume is below the value set in this field, the Calculate Volumes script applies a rounding factor to affected samples so that the volume reaches the minimum volume.
Flowcell Type — The flow cell type that the run uses. This selection affects the computation of volumes in the Calculate Volumes automation that generates the calculation file. Select from the following options:
1.5B
10B
Select Calculate Volumes to trigger the Calculate Volumes automation.
This automation calculates the volumes required for each library to form a pool that has the concentration and volume specified in the Step Details field. It also generates the calculation file in a CSV format and attaches it to the step. Select the file name to download it.
In the Sample Details table, select the pool next to the sample name to view details on the pool composition.
Select Next Steps to trigger the Set Next Step automation.
This automation sets the next step for samples to ADVANCE, which moves them to the Dilute and Denature (NovaSeq X Series Sequencing v1.1) step.
Select Finish Step.
Step 3: Dilute and Denature (NovaSeq X Series Sequencing v1.1)
In Lab View, locate the NovaSeq X Series Sequencing v1.1 protocol. The pool of samples queued for the Dilute and Denature (NovaSeq X Series Sequencing v1.1) displays.
Add the pool to the Ice Bucket and select View Ice Bucket.
[Optional] On the Ice Bucket screen, set the number of derivatives to create (placed into the library tube strip) and select Begin Work.
On entry to the step, the Validate Inputs Flowcell Type and Register Step Started automation is triggered. This automation makes sure that the configured flow cell type is valid. The automation also registers the start time of the step by publishing messages to CLPA through ICA. The portion of the script used for start time registration is used only for CLPA support.
If PhiX is used for the run, navigate to the Record Details screen and set 1–2% PhiX Spike-In to Yes.
Select Calculate Volumes to trigger the Calculate Volume automation. This automation sets the following values:
BP Aliquot Volume (ul)
RSB Volume (ul)
NaOH Volume (ul)
TT2 Volume (ul)
On the Record Details screen, the Reagent Lot Tracking section tracks the NaOH, Resuspension Buffer, and TT2 reagents used in the step. To add and activate reagent lots, refer to Add and Configure Reagent Kits and Lots in the .
On the Record Details screen, perform the following actions:
In the Reagent Lot Tracking section, select from the active lots displayed in each drop-down list.
In the Sample Details table, make sure that the BP aliquot, RSB, NaOH, and TT2 reagent volume values are populated. The script sets these values and they cannot be edited.
In the Files area, select the Calculation File (CSV) to open it and view details on the volumes for each reagent and sample pool used for the dilute and denature process.
Select Next Steps.
Select Finish Step.
Step 4: Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1)
In Lab View, locate the NovaSeq X Series Sequencing v1.1 protocol. The pool of samples are queued for the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1) step.
Add the pools to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select one of the following destination container types:
Library 8-tube Strip
Library 2-tube Strip
Select Begin Work to trigger the Validate Flowcell Inputs and Validate Analysis Configurations and Register Step Started automations.
The Validate Flowcell Inputs automation ensures that the correct container is selected for the flow cell type and that the number of inputs is the same as the number of available wells on the library tube strip. The Validate Analysis Configurations automation is similar to the automation used in the Make Bulk Pool step.
The Register Step Started automation also registers the start time of the step by publishing messages to CLPA through ICA. The portion of the script used for start time registration is used only for CLPA support.
To use multiple ACTs in a single run, repeat Steps 1–4 for each ACT.
The pool of samples for each ACT is then queued for the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1) step to be added to the Ice Bucket.
On the Placement screen, do as follows.
Drag the pools into the Placed Samples area on the right.
Scan or type the barcode of the library tube strip into the container name field.
The fields displayed on the Record Details screen are used to create the planned run and generate the sample sheet.
The analysis related information for the planned run is from the ACT associated with the samples and no further analysis configuration is required. Refer to the following table for details.
Fields Displayed on Record Details Screen of Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1) Step
Select Validate Run Setup and Create Planned Run to trigger the automation script. The script performs the following actions:
Validates the parameters entered on the Record Details screen.
Creates the planned run and sends it to ICA when the Run Mode is Cloud.
Select Next Steps.
On the Assign Next Steps screen, the next step for the pooled samples is set to the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step.
Select Finish Step to advance the pooled samples to the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step. For more information on how to start the sequencing run for different run modes, refer to .
Step 5: AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1)
This protocol contains the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step. This step is fully automated.
Do not add samples to the Ice Bucket or start or complete the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step. The integration does this automatically.
The integration starts the step automatically and data from the run is parsed back into Clarity LIMS. User interaction is not required, but you can review the stages of the step in Clarity LIMS.
Review Run Data
Read summary metrics are recorded for the library pool. After the run is complete, open the step and review these metrics in the Step Details section and the Sample Details table.
Step Details Section
The following values populate the master step fields:
Run Name
Run Status
Output Folder
Current Read
Current Cycle
Library Tube Barcode
Flow Cell ID
Flow Cell Side
Flow Cell Type
Flow Cell Part Number
Flow Cell Lot Number
Flow Cell Expiration Date
Instrument ID
Instrument Type
Instrument Control Software Version
Sequencing Log
Sample Details Table
Summary metrics populate the following global custom fields:
% Bases >=Q30 R1
% Bases >=Q30 R2
% Error Rate R1
% Error Rate R2
Yield (Gb) R1
Yield (Gb) R2
Reads PF R1
Reads PF R2
%PF R1
%PF R2
% Aligned R1
% Aligned R2
% Phasing R1
% Phasing R2
% Prephasing R1
% Prephasing R2
Intensity Cycle 1 R1
Intensity Cycle 1 R2
Cluster Density R1
Cluster Density R2
Step 6: AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1)
This protocol contains the AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1) step. This step is fully automated.
Do not add samples to the Ice Bucket or start or complete the AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1) step. The integration does this automatically.
The integration starts the step automatically and data from the run is parsed back into Clarity LIMS. User interaction is not required, but you can review the stages of the step in Clarity LIMS. If the run analysis is successful, the integration completes the step automatically.
Review Analysis Data
After the analysis run is complete, open the step and review the following values in the Step Details section:
Analysis Status
Analysis Result Location
Log
After the analysis is complete, download the demultiplexing results and analysis results summary files from the file placeholders. Retrieve detailed analysis results from ICA.
The demultiplexing results file contains the following demultiplexing statistics for each sample:
Number of reads
Number of perfect index reads
Number of mismatch index reads
The analysis result summary file provides a summary of what analysis workflows were performed. This file also identifies the total number of samples analyzed and how many were completed or failed for each analysis.
At this point, the entire NovaSeq X Series Integration workflow is fully validated.
Troubleshooting
If an automation trigger does not appear to run its corresponding scripts, refer to the Troubleshooting Automation section in the Clarity LIMS (API & Database) documentation.
Automated Step Does Not Start
If the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step does not start, Clarity LIMS is likely not receiving sequencing run events from BaseSpace Sequence Hub correctly. Check for the following issues:
Make sure that the NovaSeq X Control Software is configured properly as follows.
Before you start the run, in the NovaSeq X Control Software, select the appropriate BaseSpace Sequence Hub region in the Hosting location drop-down list.
Under Run Settings, check the boxes next to Cloud run monitoring and Cloud run storage.
If there are issues after using the configuration settings on the NovaSeq X Control Software, contact Illumina Support.
Automated Step Starts, But Does Not Complete
If the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step starts, but does not complete, do as follows.
Log in to the default user account and use one of the following methods to open the in progress step in Clarity LIMS:
In Lab View, find the step in the Recent Activities pane.
Search for the step in Clarity LIMS using the library tube strip barcode as the search term.
On the Record Details screen, the Sequencing Log multiline text field contains logging information.
If unable to reach the Record Details screen, or if the Sequencing Log field does not contain enough information to resolve the issue, contact Illumina Support. Provide the relevant information from the troubleshooting steps already performed.
Error When Creating Planned Runs
If you receive an error when creating a planned run, check the log message in the Load to Library Tube Strip step to identify the issue. If you cannot correct the issue, contact Illumina Support.
Incompatible Analyses in a Planned Run
Only compatible analysis versions should be combined in a single planned run. When incompatible analysis versions are combined, an error log message displays. An example of the error log message is shown below.
To resolve this error, check the ACTs that were used for the run and only select the ACTs that are compatible with the planned run.
The Validate Analysis Configuration automation check is in the Make Bulk Pool (upon pooling) and Load to Library Tube Strip (upon step entry) steps. If a failure happens, an error message displays and the step can be aborted, or you might be prevented from continuing the step. For NovaSeq X Series Integration v1.2, the configuration limits for the Cloud and Local modes are as follows.
Cloud — Maximum of seven applications and genome configurations (and, if needed, the BCL Convert Only application). Cloud mode also allows up to eight different library prep kits for each application and genome configuration.
Local — Maximum of three applications and genome configurations (and, if needed, the BCL Convert Only application). Local mode also allows up to eight different library prep kits for each application and genome configuration.
This check can fail for the following reasons:
The analysis configuration after library pooling or during the planned run creation exceeds the configuration limit.
Resolve this error as follows.
If the error occurs on the Make Bulk Pool step, reduce the number of analysis configurations for a pool. To reduce the number, reorganize the samples for the pooling step (eg, by pooling samples that have similar configurations together).
If the error occurs on the Load to Library Tube step, reduce the number of analysis configurations for the planned run by reorganizing the samples for multiple runs instead of a single run.
ACTs of samples in the same pool or planned run must have the same run mode (Local or Cloud).
Resolve this error as follows.
Abort the step and remove samples with ACTs that have conflicting run modes from the Ice Bucket.
ACTs of samples in the same pool or planned run that have the same secondary analysis must have the same analysis version.
Resolve this error as follows.
If the error occurs on the Make Bulk Pool step, pool the samples again. Make sure that samples with the same secondary analysis, but conflicting analysis versions, are in different pools.
ACTs of samples in the same pool or planned run that have the same secondary analysis must have the same analysis settings. The analysis setting fields can differ for a different secondary analysis and are configured in BaseSpace Sequence Hub when the ACT is created.
Resolve this error as follows.
If the error occurs on the Make Bulk Pool step, pool the samples again. Make sure that samples with the same secondary analysis, but conflicting analysis settings, are in different pools.
Revision History
Version
Changes
2
Updated Step 2: Make Bulk Pool (NovaSeq X Series Sequencing v1.1)
1
Initial release.
Type a custom value
Type a custom value
Type a custom value³
Type a custom value³
[Optional] If multiple pools are required, select the plus sign (+) next to Pool Creator to create a pool.
[Optional] To remove a pool, select the X in the top right corner of the pool.
Select Record Details to trigger the Validate Analysis Configurations automation.
This automation performs the following checks on the analysis configuration for each pool:
Pooled samples are within the maximum configuration limit.
Pooled samples have the sample type of analysis (Cloud or Local).
Pooled samples that have the same secondary analysis also have the same analysis version and settings.
25B
PhiX Volume (ul) and Concentration (pM) (if there is a PhiX spike-in)
The automation also generates the calculation file (CSV) and attaches it to the step. This file contains information about the volume of RSB, NaOH, and TT2 to add per working pool. If there is a PhiX spike-in, the file also contains information on the PhiX volume and concentration.
[Optional] In the Sample Details table, select the pool icon to view details of the working pool composition.
Select Record Details.
After exiting the Placement screen, the Validate Library Strip Tube Barcode automation makes sure that the library tube strip barcode conforms to the barcode mask. For the Library 8-tube Strip, the barcode mask is LC[0-9]{7}-L[A-Z]{1}1 and an error message displays if the barcode does not match. For the Library 2-tube Strip, the barcode mask is LC[0-9]{7}-L[A-Z]{1}2.
Presets
Local¹
Cloud²
âš The selected Run Mode must correspond to the ACT type.
Read 1 Cycles
Presets
151
101
51
Read 2 Cycles
Presets
151
101
51
Index 1 Cycles
Presets
0
6
8
Index 2 Cycles
Presets
0
6
8
¹ The Local run mode generates the sample sheet. The Run/Analysis configuration is imported manually to the NovaSeq X Series instrument through the sample sheet. The sample sheet is downloaded and imported manually by the user on NovaSeq X Control Software to start the run. The downstream secondary analysis is done using the onboard DRAGEN module.
² The Cloud run mode sends the Run/Analysis configuration to ICA. This configuration is downloaded to the NovaSeq X Series instrument through ICA to start the run. The downstream secondary analysis is done in the cloud.
³ The custom value must correspond to the longest index sequence of the samples in the pools in the library tube strip. Otherwise, the planned run creation fails and an error message displays.
Generates the sample sheet and attaches it to the placeholder in the Files area on the Record Details screen for all Run Mode types.
Make sure that all remaining samples in the Ice Bucket have ACTs with the same run modes.
Select Begin Work to continue the step.
If the error occurs on the Load to Library Tube step, reorganize the samples for the planned run. Make sure that samples with the same secondary analysis, but conflicting analysis versions, are in different runs.
If the error occurs on the Load to Library Tube step, reorganize the samples for the planned run. Make sure that samples with the same secondary analysis, but conflicting analysis settings, are in different runs.
Field
Description
Run Name
Enter the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. No spaces.
The configuration provided in this integration has been established to support NovaSeq X Series lab processes. Any configuration changes to protocols or workflows (including renaming protocols, steps, and fields) could break the process.
The Illumina Universal Sample Identifier is a global field that is imported with the Library Prep Validation v2.3.1 and NovaSeq X Series Sequencing v1.0 workflows. Illumina Universal Sample Identifier is a text field that is reserved for CLPA support. The field is optional and not required for this integration.
Prerequisites and Assumptions
Analysis Configuration Template
The NovaSeq X Series Sequencing v1.0 workflow uses analysis configuration templates (ACTs) to configure secondary analysis for planned runs. Required ACTs must be created using BSSH. The ACT names must be unique. The index adapter kit (the label group in Clarity LIMS) selected in the ACT is created in Clarity LIMS. The same label group must be used in the library preparation step. For more information, refer to .
Make sure that ACT names created in BaseSpace Sequence Hub do not have leading or trailing spaces. Otherwise, Clarity LIMS can have issues with recognizing the ACT names.
Samples
Samples must go through the library preparation and quantification process before they enter the NovaSeq X Series Sequencing v1.0 workflow. It is assumed that the following steps have completed before samples are assigned to the workflow:
Samples have been accessioned into Clarity LIMS.
Samples have been run through QC and library prep.
Samples have the Molarity (nM) global field set to a value. This value is required for the Calculate Volumes automation in the Make Bulk Pool step. For more information on sample accessioning, refer to Sample Accessioning and Upload and Modify Samples in the Getting Started section of the .
You can assign samples to workflows automatically, using a routing script, or manually—from the Projects & Samples dashboard. Refer to Assign and Process Samples in the .
Workflows, Protocols, and Steps
The NovaSeq X Series Integration Package v1.1.0 includes the following workflows:
Library Prep Validation v2.3.1 (optional, but recommended for validation purposes)
NovaSeq X Series Sequencing v1.0
The following describes the protocols and steps included in these workflows.
Library Prep Validation v2.3.1 Workflow
Protocol 1: Library Prep Validation v2.3.1
Purpose:
Included for validation purposes only, this protocol models the library prep steps required to advance samples to the NovaSeq X Series Sequencing v1.0 workflow.
NovaSeq X Series Sequencing v1.0 Workflow
Protocol 1: NovaSeq X Series Sequencing v1.0
Purpose:
This protocol models the lab processes of run setup for starting a NovaSeq X Series sequencing run.
Steps:
Validation Workflow
The Library Prep Validation v2.3.1 workflow allows for validation of the system after installation is complete. For details, refer to .
Step 1: Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0)
The Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0) step uses ACTs to configure secondary analysis for samples. Each ACT contains details related to the secondary analysis (eg, index adapter kit to use, reference genome setting, and so on). You can select an ACT from a list and assign samples to it. A planned run can have multiple ACTs and this step must be repeated for each ACT that is required for the run. For more information on creating ACTs and assigning samples to them, refer to .
The following automations are configured on this step:
Validate Sample Names and Retrieve Analysis Configuration Template List and Register Step Started²
Retrieve ACT Information
Validate Reagent Labels and Apply Selected ACT to Samples and Set Next Step
¹ These automations are required for CLPA support only.
² These automations are required for the NovaSeq X Series Sequencing v1.0 workflows and contain additional logic needed for CLPA support. If you would like to remove support for CLPA, contact Illumina Support.
All remaining automations are required for the NovaSeq X Series Sequencing v1.0 workflows.
Validate Sample Names and Retrieve Analysis Configuration Template List and Register Step Started Automation
Automatically triggered on entry to the step, this automation completes the following actions:
Checks that submitted sample names contain only alphanumeric, dash, and underscore characters.
Retrieve ACT Information Automation
Automatically triggered when you select Retrieve ACT Information on the Record Details screen, this automation completes the following actions:
Retrieves the details of the selected ACT and populates the step custom fields (eg, Analysis Version, Library Prep Kit, and so on).
Validate Reagent Labels and Apply Selected ACT to Samples and Set Next Step Automation
Automatically triggered on exit of the Record Details screen, this automation completes the following actions:
Checks that the molarity of all samples is specified.
Register Step Completed Automation
Automatically triggered on exit of the step, this automation registers completion time of the step by publishing messages to CLPA through ICA.
Master Step Fields
The following table lists the configuration details for fields that are defined on the Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0) step.
Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0) Master Step Field Configuration
Global Fields
The following table lists the global custom fields that are configured to display on the Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0) step.
Global Field Configuration (Configured on Derived Sample)
Step 2: Make Bulk Pool (NovaSeq X Series Sequencing v1.0)
Samples are pooled in the Make Bulk Pool (NovaSeq X Series Sequencing v1.0) step. You can manually create working pools based on the final loading concentration required.
The following automations are configured on this step:
Register Step Started¹
Validate Analysis Configurations
Calculate Volumes
Set Next Step
¹ These automations are required for CLPA support only.
All remaining automations are required for the NovaSeq X Series Sequencing v1.0 workflows.
Register Step Started Automation
Automatically triggered on entry to the step, this automation registers the start time of the step by publishing messages to CLPA through ICA.
Validate Analysis Configurations Automation
Automatically triggered on exit of the Pooling screen, this automation performs analysis configuration validity and checks each pool for the following characteristics:
Pooled samples are within the maximum configuration limit.
Calculate Volumes Automation
Automatically triggered when you select Calculate Volumes on the Record Details screen, this automation completes the following actions:
Proceeds if the sample has molarity values.
Uses Number of Samples in Pool to calculate the volumes needed for the 2 nM intermediate library pools.
Set Next Step Automation
Automatically triggered on exit of the Record Details screen, this automation completes the following actions:
Automatically triggered on exit of the step, this automation completes the following actions:
Registers the pool and library information used by the Clarity LIMS workflow. Samples in the pool are assumed to have gone through library prep.
Master Step Fields
The following table lists configuration details for the fields that are defined on the Make Bulk Pool (NovaSeq X Series Sequencing v1.0) step.
Make Bulk Pool (NovaSeq X Series Sequencing v1.0) Master Step Field Configuration
Global Fields
The following table lists the global custom fields that are configured to display on the Make Bulk Pool (NovaSeq X Series Sequencing v1.0) step.
Global Custom Fields Configuration (Configured on Derived Sample)
Step 3: Dilute and Denature (NovaSeq X Series Sequencing v1.0)
The Dilute and Denature (NovaSeq X Series Sequencing v1.0) step allows you to dilute pooled samples with the addition of RSB.
The following automations are configured on this step:
Validate Inputs Flowcell Type and Register Step Started²
Calculate Volumes
Set Next Step
Register Step Completed¹
¹ These automations are required for CLPA support only.
² These automations are required for the NovaSeq X Series Sequencing v1.0 workflows and contain additional logic needed for CLPA support. If you would like to remove support for CLPA, contact Illumina Support.
All remaining automations are required for the NovaSeq X Series Sequencing v1.0 workflows.
Validate Inputs Flowcell Type and Register Step Started
Automatically triggered on entry to the step, this automation completes the following actions:
Checks that the specified flow cell type is valid.
Calculate Volumes Automation
Automatically triggered when you enter the Record Details screen, this automation completes the following actions:
Checks that the NovaSeq X Flowcell Type is B3 and sets NaOH Volume (ul) and TT2 Volume (ul) field values to 10 and 150.
Set Next Step Automation
Automatically triggered on exit of the Record Details screen, this automation completes the following actions:
Copies the Run Mode from input to output.
Sets the next step for samples to ADVANCE, advancing them to the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0) step.
Register Step Completed Automation
Automatically triggered on exit of the step, this automation registers the completion time of the step by publishing messages to CLPA through ICA.
Global Fields
The following table lists the global custom fields that are configured to display on the Dilute and Denature (NovaSeq X Series Sequencing v1.0) step.
Global Field Configuration (Configured on Derived Sample)
Step 4: Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0)
The Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0) step allows you to scan the library tube strip barcode into Clarity LIMS. Then, you can place the working pools into the library tube strip used in the NovaSeq X Series run. This step also does the following actions:
Validates the run setup and analysis information.
Generates the sample sheet file.
Creates a planned run on ICA, depending on the selected run mode.
Validate Input Count and Validate Analysis Configurations and Register Step Started¹
Automatically triggered on entry to the step, this automation completes the following actions:
Checks that the number of input pools is the same as the number of tubes in the library tube strip.
Validate Library Tube Strip Barcode Automation
Automatically triggered on the Placement screen, this automation validates the library tube strip barcode to make sure that it conforms to the barcode mask LC[0-9]{7}-LC1.
Validate Run Setup and Create Planned Run Automation
Automatically triggered after a selection on the Record Details screen, this automation completes the following actions:
Validates the parameters entered on the Record Details screen. These parameters are used to set up the run, generate the sample sheet file, and create the planned run in ICA, depending on the run mode.
Set Next Step Automation
Automatically triggered on exit of the Record Details screen, this automation completes the following actions:
Copies the Run Mode from input to output.
Sets the next step for samples to ADVANCE, advancing them to the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0) step.
Register Step Completed Automation²
Automatically triggered on exit of the step, this automation registers the completion time of the step by publishing messages to CLPA through ICA.
¹ These automations are required for the NovaSeq X Series Sequencing v1.0 workflows and contain additional logic needed for CLPA support. If you would like to remove support for CLPA, contact Illumina Support.
² These automations are required for CLPA support only.
All remaining automations are required for the NovaSeq X Series Sequencing v1.0 workflows.
Master Step Fields
The following table shows the master step fields that are configured on the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0) step. These fields are required for sample sheet generation and planned run creation in ICA.
Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0) Master Step Field Configurationn
Step 5: AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0)
The AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0) step is fully automated. The integration starts the step, adds samples to the Ice Bucket, and completes the step. Data from the run is parsed back to Clarity LIMS. In this step, pooled samples in the library tube strip are sequenced on the NovaSeq X Series instrument.
Register Step Started & Register NovaSeq X Run & Register NovaSeq X Run Association & Register NovaSeq X RunStarted Automation¹
Automatically triggered at the beginning of the step, this automation completes the following actions:
Registers the start time of the step.
Register NovaSeq X RunCompleted & Register Step Completed Automation¹
Automatically triggered on exit of the step, this automation completes the following actions:
Registers the sequencing run completion time, run status, and run metrics. This script requires the Cloud Run ID custom field from the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0) step.
¹ These automations are required for CLPA support only.
All remaining automations are required for the NovaSeq X Series Sequencing v1.0 workflows.
Master Step Fields
The following tables show the master step fields that are configured on the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0) step.
Clarity LIMS Master Step Field Information
Other Clarity LIMS Master Step Fields
Global Fields
The following global custom fields are used to capture the run metrics in Clarity LIMS:
% Bases >=Q30 R1
% Bases >=Q30 R2
% Error Rate R1
% Error Rate R2
At the end of the step, the pools of samples are automatically removed from the step. The step completes automatically when Run Status is RunCompletedSuccessfully.
How the Integration Works
The following information summarizes how the NovaSeq X Series integration works.
After the Validate Run Setup and Create Planned Run automation is triggered on the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0) step, the run parameters entered in the Run Details screen are sent to ICA. The analysis configuration parameters from the selected ACTs are also sent to ICA. ICA validates the run and analysis configuration. If the validation fails, ICA sends an error message to Clarity LIMS. If the validation passes, ICA generates the sample sheet and sends it back to Clarity LIMS. ICA also creates the planned run based on the selected run mode (eg, Local or Cloud).
For the Local run mode, the sample sheet is generated and stored in Clarity LIMS. This sample sheet contains the run and analysis configuration information required to start the run on the NovaSeq X Series instrument.
If a planned run with the same sample name and project name (case-insensitive) has been created previously in ICA, the sample sheet generated from the Validate Run Setup and Create Planned Run automation can reflect the original case of the previous sample name. This can cause validation errors for analysis configurations with sample-level settings. To resolve this issue, change the sample name or the project name on Clarity LIMS and run the automation again.
Start a Sequencing Run on NovaSeq X Series Instrument
The following sections outline the steps to start a Clarity LIMS cloud or local run on the NovaSeq X Series instrument.
Run Mode Configuration
. Select Cloud run storage options under Run Settings.
Start a Sequencing Run in Local Run or Cloud Run Modes
You can start the sequencing run in BSSH for both the Local and Cloud modes. Make sure that you log in using the account that was used during the NovaSeq X Series integration package setup. You must also import the sample sheet created from the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0) step for the Local Run Mode.
Start a Sequencing Run in Local Run Mode
Follow instructions to start a sequencing run with local secondary analysis in .
Start a Sequencing Run in Cloud Run Mode
Follow instructions to start a sequencing run with cloud secondary analysis in .
Enabling Planned Run Generation for Samples Having Duplicate Name with Different Indexes
The library preparation workflow of the samples must be before routing the samples through the library preparation workflow.
Components Installed
The following sections describe the components (files, properties, reagent categories / label groups, reagent kits, and containers) that are installed by default as part of this integration.
Reagent Kits
Buffer Cartridge
Lyophilization Cartridge
NaOH
Reagent Cartridge
Container Types
Library 8-Tube Strip
Tube This integration supports the library 8-tube strip with the barcode in the LC[0-9]{7}-LC1 format (eg, LC1234567-LC1).
Control Types
PhiX v3
Rules and Constraints
The workflow configuration contains several validation checks. To make sure that the calculations work properly, it is important that you do not disable any of this validation logic. The validation checks determine the following information:
Which samples, and how many, can enter each step together.
Which samples, and how many, can be pooled together.
The protocol contains a single step - Library Prep Validation v2.3.1. After this step, a routing script sends the samples to the first step of the NovaSeq X Series Sequencing v1.0 workflow (Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0)).
Steps:
Library Prep Validation v2.3.1
Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0)
The secondary analysis of samples is configured using the ACT.
The labels applied to the samples are validated against the selected ACT so that samples use valid labels.
Make Bulk Pool (NovaSeq X Series Sequencing v1.0)
The Calculate Volumes script assists with pooling samples and adding resuspension buffers and reagents.
The Validate Analysis Configurations automation validates the ACT of pooled samples to make sure that the secondary analysis configurations remain valid after pooling.
Dilute and Denature (NovaSeq X Series Sequencing v1.0)
The Calculate Volumes script helps denature and dilute samples from step 2 to the final loading concentration.
Load To Library Tube Strip (NovaSeq X Series Sequencing v1.0)
The library pools from step 3 are ready to be loaded to the NovaSeq X Series library tube strip.
Validates run and analysis information.
Generates the sample sheet and creates the planned run, depending on the run mode.
AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0)
The step is fully automated and starts and completes automatically after the sequencing run is started and completed on the instrument side.
The sequencing run metadata (eg, run configuration, primary run metrics, and so on) are recorded automatically.
The integration does this step automatically. Do not add samples to the Ice Bucket or start or complete the step.
Register Step Completed¹
Retrieves the list of ACTs that were created in BSSH.
Registers the start time of the step by publishing messages to CLPA through the ICA.
Saves the details into a file that is available to download. This file is stored in the Analysis Configuration Metadata file placeholder.
Checks that all samples are indexed and for invalid indexes. The indexes applied are the same as the selected ACT.
Assigns the selected ACT to all samples in the step.
Sets the next step for samples to ADVANCE, which advances them to the next step in the protocol (Make Bulk Pool (NovaSeq X Series Sequencing v1.0)).
Text
Read Only
Reference Genome
Text
Read Only
Secondary Analysis Mode
Text
Read Only
Register Pools & Register Step Completed¹
Pooled samples have the same type of analysis (eg, Cloud or Local).
Pooled samples that have the same secondary analysis also have the same analysis version (eg, v3.8.4) and analysis settings (eg, Map/Align Output Format = CRAM).
Uses the Bulk Pool Volume (ul) field and Number of Lanes to Sequence (x100) for the B3 flow cell type to calculate volumes needed for the 2 nM intermediate library pools.
Copies the Final Loading Concentration (pM) and Flowcell Type from the step inputs to the step outputs.
Calculates the per sample volume required for each library to make a 2 nM intermediate library pool.
If PhiX is used for a planned run (eg, a value for % PhiX (2.0 nM) Spike-In is specified), sets the PhiX Volume (ul).
Calculates the adjusted per sample volume for the pools.
Uses the Total Sample Volume (ul) to calculate the RSB volume (ul) required to top up the pools that are needed to create the 2 nM intermediate library pools. The RSB volume is saved to the file in the Calculation File placeholder in the Files section.
Uses the NovaSeqXSeries_Bulk_Pool1.csv and NovaSeqXSeries_Bulk_Pool2.csv template files to generate a single CSV file. This file contains information about the pools and the samples that they contain. The generated file is stored in the Calculation File placeholder in the Files section.
Resets the Number of Samples in Pool, Total Sample Volume (ul), and Bulk Pool Volume (ul) to null before exiting the step.
Sets the next step for samples to ADVANCE, which advances them to the Dilute and Denature (NovaSeq X Series Sequencing v1.0) step.
Registers completion time of the step.
This automation completes this action by publishing messages to CLPA through ICA.
Flowcell Type
Text
Required Field
Read Only
Default = B3
Minimum Per Sample Volume (ul)
Numeric
Required Field
Decimal places displayed = 2
Default = 2
Number of Lanes to Sequence
Numeric
Required Field
Range = 1–10
Decimal places displayed = 0
PhiX Volume (ul)
Numeric
Read Only
Hidden
Decimal places displayed = 1
Used for Calculate Volume script when there is PhiX spike-in.
âš Do not remove, rename, or modify this field as it can cause the integration to break.
Registers the start time of the step by publishing messages to CLPA through ICA.
Computes BP Aliquot Volume (ul) and RSB Volume (ul) required for the dilution of pools to the required final loading concentration.
Generates a single CSV file that contains information about the reagents volume required to dilute the working pools. The generated file is stored in the Calculation File placeholder in the Files section.
TT2 Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Performs basic checks on the secondary analysis configuration of the samples in the same planned run. The following checks are included in this script:
Secondary analysis configuration of samples in a planned run is within maximum configuration limit.
Samples in a planned run have the same type of analysis (eg, Cloud or Local).
Samples in the same pools that have the same secondary analysis (eg, Illumina DRAGEN Enrichment) also have the same analysis settings (eg, Map/Align Output Format = CRAM) and the same analysis version (eg, v3.8.4).
Registers the start time of the step by publishing messages to CLPA through ICA.
Run Name can only contain alphanumeric, dash, underscore, or period characters. Do not use spaces.
Run Name cannot exceed 255 characters.
Checks the Index 1 Cycles and Index 2 Cycles field values. If Index 2 Cycles is greater than 0, the Index 1 Cycles value must be greater than 0 or an error can occur.
Generates the sample sheet. If you are in Cloud run mode, the automation creates the planned run in ICA. The sample sheet is attached to the step.
There is a backend validation of the sample sheet content in ICA. This validation makes sure that the sample sheet is valid to set up a Local mode run and that it is ready for use with DRAGEN applications.
Registers the NovaSeq X Series sequencing run configuration.
Registers the association of the sequencing run with the samples by linking the Instrument Run ID with the sample ID.
Registers the time that the sequencing run started. This automation completes these actions by publishing messages to CLPA through ICA.
Registers the completion time of the step. This automation completes these actions by publishing messages to CLPA through ICA.
Flow Cell ID
RunParameters.xml
SerialNumber
Visible on Record Details screen.
Corresponds to the value in the SerialNumber field under the ConsumableInfo field for FlowCell Type.
Flow Cell Lot Number
RunParameters.xml
LotNumber
Visible on Record Details screen.
Corresponds to the value in the LotNumber field under the ConsumableInfo field for FlowCell Type.
Flow Cell Part Number
RunParameters.xml
PartNumber
Visible on Record Details screen.
Corresponds to the value of the PartNumber field under the ConsumableInfo field for FlowCell Type.
Flow Cell Side
RunParameters.xml
Side
Visible on Record Details screen.
Flow Cell Type
RunParameters.xml
FlowCellType
Visible on Record Details screen.
Instrument Control Software Version
RunParameters.xml
SystemSuiteVersion
Visible on Record Details screen.
Instrument ID
RunParameters.xml
InstrumentSerialNumber
Visible on Record Details screen.
Instrument Type
RunParameters.xml
InstrumentType
Visible on Record Details screen.
Output Folder
RunParameters.xml
OutputFolder
Visible on Record Details screen.
Library Tube Barcode
RunParameters.xml
SerialNumber
Visible on Record Details screen.
Corresponds to the value of the SerialNumber field under the ConsumableInfo field for SampleTube Type.
Library Tube Lot Number
RunParameters.xml
LotNumber
Hidden
Corresponds to the value of the LotNumber field under the ConsumableInfo field for SampleTube Type.
Run End Time
RunCompletionStatus.xml
RunEndTime
Hidden
Run Name
RunParameters.xml
ExperimentName
Visible on Record Details screen.
Received from the message sent to the integration service.
Hidden
Run Start Time
Received from the message sent to the integration service.
Hidden
Yield (Gb) R1
Yield (Gb) R2
Reads PF R1
Reads PF R2
%PF R1
%PF R2
% Aligned R1
% Aligned R2
% Phasing R1
% Phasing R2
% Prephasing R1
% Prephasing R2
Intensity Cycle 1 R1
Intensity Cycle 1 R2
Cluster Density R1
Cluster Density R2
For the Cloud run mode, the planned run is created in ICA. The planned run contains the run and analysis configuration information required to start the run on the instrument. The sample sheet is generated and stored in Clarity LIMS for reference purposes.
When the sequencing run starts on the instrument, the NovaSeq X Series Control Software notifies BSSH. BSSH monitors the run and uses the Stratus Integration Service (SIS) to notify Clarity LIMS. The events are processed and the integration service retrieves the run information from BSSH. This information is used to populate the custom fields in the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0) step.
Other run events follow the same information flow. When sequencing is complete, the control software uploads the sequencing run data (primary metrics) and associated files to ICA. Then, Clarity LIMS retrieves the primary metrics and uses them to populate the fields in the Sample Details table (eg, % Error Rate R1). The custom fields (eg, Run Status, Current Read, and so on) on the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0) step are updated using the run related information. If the sequencing run is successfully completed, the step automatically completes.
The analysis results are not tracked. You must log in to BSSH to retrieve the cloud analysis results. For local analysis, the secondary analysis results are found in the external storage configured in Illumina Run Manager. The external storage information is found in the External Storage for Analysis Results configuration settings in Illumina Run Manager.
Resuspension Buffer (RSB)
TT2
All submitted samples must have an associated secondary analysis that is configured using the analysis configuration template (ACT). The ACT must be configured on BSSH before starting the Assign Analysis Configuration Template step. The ACT names must be unique.
The library tube strip barcode must be unique. There must not be multiple library tube strip containers with the same name in the system.
Reagent labels, or indexes, must be unique.
One library pool can only contain one library or control with no label/index.
The AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0) step must not be manually started or completed. This step is fully automated and the sequencing service does not update samples correctly if they have been manually started.
For the automated run to start successfully, you must select Validate Run Setup and Create Planned Run in the Load to Library Tube step.
Field Name
Field Type
Options
Analysis Configuration Template
Text Dropdown
Required Field
Application
Text
Read Only
Application Version
Text
Read Only
Index Adapter Kit
Text
Read Only
Field Name
Field Type
Options
Additional Options and Dropdown Items
Molarity (nM)
Numeric
Decimal places displayed = 2
ACT Name
Text
Read Only
Field Name
Field Type
Options
Additional Options and Dropdown Items
% PhiX (2.5 nM) Spike-In
Numeric
Range = 0–100
Final Loading Concentration (pM)
Numeric Dropdown
Required Field
Custom Entries
Final Loading Volume (ul)
Numeric
Required Field
Default = 200
Field Name
Field Type
Options
Additional Options and Dropdown Items
Final Loading Concentration (pM)
Numeric Dropdown
Required Field
Custom Entries
Decimal places displayed = 0
Presets
225
400
RSB Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
NovaSeq X Flowcell Type
Text
Required Field
Read Only
Default = B3
Field Name
Field Type
Options
Additional Options and Dropdown Items
BP Aliquot Volume (ul)
Numeric
Read Only
Decimal places displayed = 0
NaOH Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
RSB Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Field Name
Field Type
Options
Additional Options and Dropdown Items
Cloud Run ID
Text
Read Only
Hidden
Used for creating a planned run in ICA.
âš Do not remove, rename, or modify this field as it can cause the integration to break.
Index 1 Cycles
Numeric Dropdown
Required Field
Custom Entries
Range = 0–20
Presets
0
6
8
Index 2 Cycles
Numeric Dropdown
Required Field
Custom Entries
Range = 0–20
Presets
0
6
8
Field Name
Source File
Corresponding Field in Source File
Additional Information
Current Cycle
RunCompletionStatus.xml
CompletedReads
Visible on Record Details screen.
Calculated based on the CompletedReads field.
Current Read
RunCompletionStatus.xml
RunParameters.xml
CompletedReads
PlannedReads
Visible on Record Details screen.
Calculated based on the CompletedReads field in RunCompletionStatus.xml and PlannedReads field in RunParameters.xml.
Flow Cell Expiration Date
RunParameters.xml
ExpirationDate
Visible on Record Details screen.
Corresponds to the value in the ExpirationDate field under the ConsumableInfo field for FlowCell Type.
Field Name
Description
Additional Information
Run Status
Presets
RunStarted
RunCompletedSuccessfully
RunAbortedByUser
RunErroredOut
Set by the integration service.
Visible on Record Details screen.
Sequencing Log
Set by the integration service during the sequencing run.
Visible on Record Details screen.
BaseSpace Run ID
Received from the message sent to the integration service.
Hidden
ICA Project ID
Received from the message sent to the integration service.
if (!input.hasValue(::Molarity (nM)::)) { return; };
/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -h false -exp 'if (!input.hasValue(::NovaSeq X Flowcell Type::)){fail(::Invalid Flowcell Type custom field value. Please make sure all input samples have a value assigned for Flowcell Type custom field.::)}' -log {compoundOutputFileLuid1}
if (output.hasValue(::Number of Samples in Pool::)) { output.::Number of Samples in Pool:: = output.::Number of Samples in Pool:: + 1; } else { output.::Number of Samples in Pool:: = 1; }' -t true
if (!step.::Run Name::.matches(::[a-zA-Z0-9-_]+[a-zA-Z0-9-_. ]*::)) { fail(::Run Name contains prohibited characters. Please check to make sure Run Name only contains alphanumeric characters, spaces, dashes and underscores. Run Name must start with alphanumeric, a dash or an underscore::);}
These release notes describe the key changes to software components for the Clarity LIMS NovaSeq X Series Integration Package v1.3.0.
Prerequisites
BaseSpace Sequence Hub account that has the Enterprise or Professional subscription
Compatibility
Refer to under Instruments & Integrations.
New Features & Updates
Updates on automations:
Register NovaSeq X RunCompleted and Register Step Completed
Register Step Started and Register NovaSeq X Run and Register NovaSeq X Run Association and Register NovaSeq X RunStarted
Refer to for details on changes in automations, configuration and run metrics. Manual upgrade instructions from NovaSeq X Series Sequencing v1.1 workflow to v1.2 workflow are provided in .
Known Limitations
Tracks only the analysis configured with the planned run. Does not track analysis requeue or analysis triggered externally with the same planned run.
The Assign Analysis Configuration Template step does not support pooled libraries.
The integration does not support --bcl-sampleproject-subdirectories option of BCL Convert.
Known Issues
On the Make Bulk Pool step, the log displays a warning when the Calculate Volume automation is triggered and at least one pool consists of multiple inputs. This issue is caused by the output custom field being reset multiple times at the end of the automation. This issue does not affect the Calculate Volume automation functionality.
For BCL Convert only analysis, the high level analysis summary information is not available. Upon analysis successfully completed, an empty file is being attached at the AUTOMATED - Analysis Run step and the step is automatically completed.
Documentations
The NovaSeq X Series Integration v1.3.0 User Interaction, Validation and Troubleshooting guide follows exactly that of with the following updates:
Version updates:
NovaSeq X Series Integration Package version updated to v1.3.0.
NovaSeq X Series Sequencing workflow version updated to v1.2.
Validate Flowcell Inputs and Validate Analysis Configurations and Register Step Started
Validate Reagent Labels and Apply Selected ACT to Samples and Set Next Step
Validate Run Setup and Create Planned Run
Validate Sample Names and Retrieve Analysis Configuration Template List and Register Step Started
Updates on configuration:
Library 2-tube Strip and Library 8-tube Strip containers
BP Aliquot Volume (ul) derived sample global field
In the Load to Library Tube Strip step,
the master step field Cloud Run ID has been renamed to Planned Run ID.
a new step field Instrument Type is added.
In the AUTOMATED - Sequencing Run step,
the Derived Sample Generation configuration setting has been updated to Fixed with 1 Derived Sample generated.
removed unused RTA Version step field.
under Record Details milestone, the sample table is now expanded by default and run metrics in Sample Details table have been updated.
In the AUTOMATED - Analysis Run step,
the Library Tube Strip Barcode hidden master step field has been replaced with the App Session ID hidden field.
the Naming Convention under Step Type has been updated to {SubmittedSampleName}{AppliedReagentLabels}.
under Record Details milestone, the sample table is now expanded by default.
Library Prep Validation workflow version updated to v2.3.5.
Automation name update:
Validate Flowcell Inputs and Validate Analysis Configurations and Register Step Started automation updated to Validate Flowcell Inputs, Analysis Configurations and Register Step Started
The Illumina NovaSeq X Series Integration Package v1.3.0 supports the integration of Clarity LIMS to Illumina NovaSeq X series instruments.
The configuration provided in this integration has been established to support NovaSeq X Series lab processes. Any configuration changes to protocols or workflows (including renaming protocols, steps, and fields) could break the process.
Prerequisites and Assumptions
The NovaSeq X Series Integration v1.3.0 requires a valid BaseSpace Sequence Hub account with an Enterprise or Professional subscription.
The required analysis configuration templates (ACTs), have been created on BaseSpace Sequence Hub.
âš ACT names created in BaseSpace Sequence Hub must not have leading or trailing spaces.
Workflows, Protocols, and Steps
The NovaSeq X Series Integration Package v1.3.0 includes the following workflows:
NovaSeq X Series Sequencing v1.2
Library Prep Validation v2.3.5 (optional, but recommended for validation purposes)
The following describes the protocols and steps included in these workflows.
Library Prep Validation v2.3.5 Workflow
Protocol 1: Library Prep Validation v2.3.5
Purpose:
Included for validation purposes only, this protocol models the library prep steps required to advance samples to the NovaSeq X Series Sequencing v1.2 workflow.
âš The label group (index adapter kit) used for library preparation must be the same as the index adapter kit specified in the ACT that is being used.
NovaSeq X Series Sequencing v1.2 Workflow
Protocol 1: NovaSeq X Series Sequencing v1.2
Purpose:
This protocol models the lab processes of run setup for starting a NovaSeq X Series sequencing and secondary analysis run.
Steps:
Validation Workflow
The Library Prep Validation v2.3.5 workflow allows for validation of the system after installation is complete. This workflow can be replaced by other custom library preparation workflows.
Configure a custom library preparation workflow as follows.
Select Configuration, then select Lab Work.
Add a Routing Script automation to the last Master Step of the library preparation workflow.
Select Configuration, then select Automation.
Step 1: Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2)
The Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2) step uses ACTs to configure secondary analysis for samples. Each ACT contains details related to the secondary analysis (e.g., index adapter kit to use, reference genome setting, and so on). You can select an ACT from a list and assign samples to it. A planned run can have multiple ACTs and this step must be repeated for each ACT that is required for the run.
Validate Sample Names, Retrieve ACT List and Register Step Started Automation¹
Automatically triggered on entry to the step, this automation completes the following actions:
Checks that submitted sample names contain only alphanumeric, dash, and underscore characters.
Retrieve ACT Information Automation
Automatically triggered when you select Retrieve ACT Information on the Record Details screen, this automation completes the following actions:
Retrieves the details of the selected ACT and populates the step custom fields (e.g., Analysis Version, Library Prep Kit, and so on).
Apply Selected ACT to Samples and Set Next Step Automation
Automatically triggered on exit of the Record Details screen, this automation completes the following actions:
Checks that the molarity of all samples is specified.
Register Step Completed Automation²
Automatically triggered on exit of the step, this automation registers completion time of the step by publishing messages to CLPA through ICA.
¹ These automations are required for the NovaSeq X Series Sequencing v1.2 workflows and contain additional logic needed for CLPA support. If you would like to remove support for CLPA, contact Illumina Support.
² These automations are required for CLPA support only.
All remaining automations are required for the NovaSeq X Series Sequencing v1.2 workflows.
Master Step Fields
The following table lists the configuration details for fields that are defined on the Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2) step.
Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2) Master Step Field Configuration
Global Fields
The following table lists the global custom fields that are configured to display on the Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2) step.
Global Field Configuration (Derived Sample)
Step 2: Make Bulk Pool (NovaSeq X Series Sequencing v1.2)
Samples are pooled in the Make Bulk Pool (NovaSeq X Series Sequencing v1.2) step. You can manually create working pools based on the final loading concentration required.
Register Step Started Automation¹
Automatically triggered on entry to the step, this automation registers the start time of the step. This automation completes this action by publishing messages to CLPA through ICA.
Validate Analysis Configurations Automation
Automatically triggered on exit of the Pooling screen, this automation performs analysis configuration validity and checks each pool for the following characteristics:
Pooled samples are within the maximum configuration limit.
Calculate Volumes Automation
Automatically triggered when you select Calculate Volumes on the Record Details screen, this automation completes the following actions:
Proceeds if the sample has molarity values.
Uses Number of Samples in Pool to calculate the volumes needed for the 2 nM intermediate library pools.
Set Next Step Automation
Automatically triggered on exit of the Record Details screen, this automation completes the following actions:
Proceeds only if the sample has a molarity value.
Copies the Run Mode from input to output.
Register Pools and Register Step Completed Automation¹
Automatically triggered on exit of the step, this automation completes the following actions:
Registers the pool and library information used by the Clarity LIMS workflow. Samples in the pool are assumed to have gone through library preparation.
¹These automations are required for CLPA support only.
All remaining automations are required for the NovaSeq X Series Sequencing v1.2 workflows.
Master Step Fields
The following table lists configuration details for the fields that are defined on the Make Bulk Pool (NovaSeq X Series Sequencing v1.2) step.
Make Bulk Pool (NovaSeq X Series Sequencing v1.2) Master Step Field Configuration
Global Fields
The following table lists the global custom fields that are configured to display on the Make Bulk Pool (NovaSeq X Series Sequencing v1.2) step.
Global Custom Fields Configuration (Derived Sample)
Step 3: Dilute and Denature (NovaSeq X Series Sequencing v1.2)
The Dilute and Denature (NovaSeq X Series Sequencing v1.2) step allows you to dilute pooled samples with the addition of RSB.
Validate Inputs Flowcell Type and Register Step Started¹
Automatically triggered on entry to the step, this automation completes the following actions:
Checks that the specified flow cell type is valid.
âš This script is required for the NovaSeq X Series Sequencing v1.2 workflow to function properly.
Calculate Volumes Automation
Automatically triggered when you select Calculate Volumes on the Record Details screen, this automation completes the following actions based on the NovaSeq X Flowcell Type selected:
Sets the NaOH Volume (µl) and TT2 Volume (µl) field values.
Set Next Step Automation
Automatically triggered on exit of the Record Details screen, this automation completes the following actions:
Copies the Run Mode from input to output.
Sets the next step for samples to ADVANCE, advancing them to the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.2) step.
Register Step Completed Automation²
Automatically triggered on exit of the step, this automation registers the completion time of the step by publishing messages to CLPA through ICA.
¹ These automations are required for the NovaSeq X Series Sequencing v1.2 workflows and contain additional logic needed for CLPA support. If you would like to remove support for CLPA, contact Illumina Support.
² These automations are required for CLPA support only.
All remaining automations are required for the NovaSeq X Series Sequencing v1.2 workflows.
Global Fields
The following table lists the global custom fields that are configured to display on the Dilute and Denature (NovaSeq X Series Sequencing v1.2) step.
Global Field Configuration (Derived Sample)
Step 4: Load to Library Tube Strip (NovaSeq X Series Sequencing v1.2)
The Load to Library Tube Strip (NovaSeq X Series Sequencing v1.2) step allows you to scan the library tube strip barcode into Clarity LIMS. Then, you can place the working pools into the library tube strip used in the NovaSeq X Series run. This step also does the following actions:
Validates the run setup and analysis information.
Generates the sample sheet file.
Creates a planned run on Illumina Connected Analytics (ICA), depending on the selected run mode.
Validate Flowcell Inputs, Analysis Configurations and Register Step Started Automation¹
Automatically triggered on entry to the step, this automation completes the following actions:
Checks that the selected destination container types on the Load to Library Tube Strip step matches the selected flow cell type of the samples. The 1.5B flow cell is compatible with the library 2-tube strip and the 10B and 25B flow cells are compatible with the library 8-tube strip.
Validate Library Tube Strip Barcode Automation
Automatically triggered on the Placement screen, this automation validates the library tube strip barcode to make sure that it conforms to the barcode mask LC[0-9]{7}-LC1 for the library 8-tube strip and LC[0-9]{7}-L[A-Z]2 for the library 2-tube strip.
Validate Run Setup and Create Planned Run Automation
Automatically triggered after a selection on the Record Details screen, this automation completes the following actions:
Validates the parameters entered on the Record Details screen. These parameters are used to set up the run, generate the sample sheet file, and create the planned run in ICA, depending on the run mode.
Set Next Step Automation
Automatically triggered on exit of the Record Details screen, this automation completes the following actions:
Copies the Run Mode from input to output.
Sets the next step for samples to ADVANCE, advancing them to the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2) step.
Register Step Completed Automation²
Automatically triggered on exit of the step, this automation registers the completion time of the step by publishing messages to CLPA through ICA.
¹ These automations are required for the NovaSeq X Series Sequencing v1.2 workflows and contain additional logic needed for CLPA support. If you would like to remove support for CLPA, contact Illumina Support.
² These automations are required for CLPA support only.
All remaining automations are required for the NovaSeq X Series Sequencing v1.2 workflows.
Master Step Fields
The following table shows the master step fields that are configured on the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.2) step. These fields are required for sample sheet generation and planned run creation in ICA.
Load to Library Tube Strip (NovaSeq X Series Sequencing v1.2) Master Step Field Configuration
Step 5: AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2)
Do not add samples to the Ice Bucket or start and complete the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2) step. The integration does this automatically.
Data from the run is parsed back to Clarity LIMS. In this step, pooled samples in the library tube strip are sequenced on the NovaSeq X Series instrument.
Register NovaSeq X Series Sequencing Run Started Automation¹
Automatically triggered at the beginning of the step, this automation completes the following actions:
Registers the start time of the step.
Register NovaSeq X Series Sequencing Run Completed Automation¹
Automatically triggered on exit of the step, this automation completes the following actions:
Registers the sequencing run completion time, run status, and run metrics. This script requires the Planned Run ID custom field from the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.2) step.
¹ These automations are required for CLPA support only.
Master Step Fields
The following tables show the master step fields that are configured on the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2) step.
Clarity LIMS Master Step Field Information
Other Clarity LIMS Master Step Fields
Global Fields
The following global custom fields are used to capture the run metrics in Clarity LIMS:
% Bases >=Q30 R1
% Bases >=Q30 R2
% Error Rate R1
% Error Rate R2
At the end of the step, the pools of samples are automatically removed from the step. The step completes automatically when Run Status is RunCompletedSuccessfully.
Step 6: AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.2)
Do not add samples to the Ice Bucket or start and complete the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2) step. The integration does this automatically.
Data from the analysis is parsed back to Clarity LIMS. In this step, the secondary analysis configured using the Analysis Configuration Template (ACT) is performed in Illumina Connected Analytics (ICA). There are no automations associated with this step.
Master Step Fields
The following tables show the master step fields that are configured on the AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.2) step.
AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.2) Master Step Field Information
How the Integration Works
The following information summarizes how the NovaSeq X Series integration works.
After the Validate Run Setup and Create Planned Run automation is triggered on the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.2) step, the run parameters entered in the Run Details screen are sent to Illumina Connected Analytics (ICA). The analysis configuration parameters from the selected ACTs are also sent to ICA. ICA validates the run and analysis configuration. If the validation fails, ICA sends an error message to Clarity LIMS. If the validation passes, ICA generates the sample sheet and sends it back to Clarity LIMS. ICA also creates the planned run based on the selected run mode (e.g., Local or Cloud).
For the Local run mode, the sample sheet is generated and stored in Clarity LIMS. This sample sheet contains the run and analysis configuration information required to start the run on the NovaSeq X Series instrument.
If a planned run with the same sample name and project name (case-insensitive) has been created previously, the sample sheet generated from the Validate Run Setup and Create Planned Run automation can reflect the original case of the previous sample name. This can cause validation errors for analysis configurations with sample-level settings. To resolve this issue, change the sample name or the project name on Clarity LIMS and run the automation again.
Start a Sequencing Run on NovaSeq X Series Instrument
The following sections outline the steps to start a Clarity LIMS cloud or local run on the NovaSeq X Series instrument.
Run Mode Configuration
In the NovaSeq X Series Control Software, log in to the instrument using an account that has permissions for configuring settings.
On the menu, select Settings, then select Cloud settings.
Start a Sequencing Run in Local Run or Cloud Run Modes
Start a Sequencing Run in Local Run Mode
Log in with the BaseSpace Sequence Hub account and select the workgroup used during the NovaSeq X Series integration package installation.
Enabling Planned Run Generation for Samples Having Duplicate Name with Different Indexes
The library preparation workflow of the samples must be before routing the samples through the library preparation workflow.
Components Installed
The following sections describe the components (files, properties, reagent categories / label groups, reagent kits, and containers) that are installed by default as part of this integration.
Reagent Kits
Buffer Cartridge
Lyophilization Cartridge
NaOH
Reagent Cartridge
Container Types
Library 2-Tube Strip
Library 8-Tube Strip
Tube
This integration supports the library 8-tube strip with the barcode in the LC[0-9]{7}-L[A-Z]1 format. The integration also supports the library 2-tube strip with the barcode in the LC[0-9]{7}-L[A-Z]2 format.
Control Types
PhiX v3
Rules and Constraints
The workflow configuration contains several validation checks. To make sure that the calculations work properly, it is important that you do not disable any of this validation logic. The validation checks determine the following information:
Which samples, and how many, can enter each step together.
Which samples, and how many, can be pooled together.
The index adapter kit configured in the ACT must be created as label group in Clarity LIMS and used in the library preparation step for indexing the samples.
Samples must go through the library preparation and quantification process before they enter the NovaSeq X Series Sequencing v1.2 workflow. It is assumed that the following steps have completed before samples are assigned to the workflow:
Samples have been accessioned into Clarity LIMS.
Samples have been run through QC and library prep.
Samples have the Molarity (nM) global field set to a value.
This value is required for the Calculate Volumes automation in the Make Bulk Pool step.
âš Sample and library names must use only alphanumeric, dash, or underscore characters.
For more information on sample accessioning, refer to Sample Accessioning and Upload and Modify Samples in the Getting Started section of the .
You can assign samples to workflows automatically, using a routing script, or manually—from the Projects & Samples dashboard. Refer to Assign and Process Samples in the .
The protocol contains a single step - Library Prep Validation v2.3.5. After this step, a routing script sends the samples to the first step of the NovaSeq X Series Sequencing v1.2 workflow (Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2)).
Steps:
Library Prep Validation v2.3.5
Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2)
The secondary analysis of samples is configured using the ACT.
The labels applied to the samples are validated against the selected ACT so that samples use valid labels.
Make Bulk Pool (NovaSeq X Series Sequencing v1.2)
The Calculate Volume script assists with pooling samples and adding resuspension buffers and reagents.
The Validate Analysis Configurations automation validates the ACT of pooled samples to make sure that the secondary analysis configurations remain valid after pooling.
Dilute and Denature (NovaSeq X Series Sequencing v1.2)
The Calculate Volume script helps denature and dilute samples from step 2 to the final loading concentration.
Load To Library Tube Strip (NovaSeq X Series Sequencing v1.2)
The library pools from step 3 are ready to be loaded to the NovaSeq X Series library tube strip.
Validates run and analysis information.
Generates the sample sheet and creates the planned run, depending on the run mode.
AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2)
The step is fully automated and starts and completes automatically after the sequencing run is started and completed on the instrument side.
The sequencing run metadata (e.g., run configuration, primary run metrics, and so on) are recorded automatically.
AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.2)
The step is fully automated and starts and completes automatically after the analysis run is started and completed in ICA.
The analysis run metadata (eg, status and high level analysis summary) are recorded automatically.
Create a new Routing Script automation and update it as follows.
For Channel Name, enter limsserver.
For Command Line, enter the following script:
For Automation Use, select the custom library preparation workflow.
Retrieves the list of ACTs that were created in BaseSpace Sequence Hub.
Registers the start time of the step by publishing messages to CLPA through the ICA.
Saves the details into a file that is available to download. This file is stored in the Analysis Configuration Metadata file placeholder.
Checks that all samples are indexed and for invalid indexes. The indexes applied are the same as the selected ACT.
Assigns the selected ACT to all samples in the step.
Sets the next step for samples to ADVANCE, which advances them to the next step in the protocol (Make Bulk Pool (NovaSeq X Series Sequencing v1.2)).
Text
Read Only
Reference Genome
Text
Read Only
Secondary Analysis Mode
Text
Read Only
Pooled samples have the same type of analysis (e.g., Cloud or Local).
Pooled samples that have the same secondary analysis also have the same analysis version (e.g., v3.8.4) and analysis settings (e.g., Map/Align Output Format = CRAM).
Uses the Bulk Pool Volume (ul) field and Number of Lanes to Sequence (x100) to calculate volumes needed for the 2 nM intermediate library pools.
Copies the Final Loading Concentration (pM) and Flowcell Type from the step inputs to the step outputs.
Calculates the per sample volume required for each library to make a 2 nM intermediate library pool and sets the total sample volume to zero.
Calculates the adjusted per sample volume for the pools.
Uses the Total Sample Volume (ul) to calculate the RSB volume (ul) required to top up the pools that are needed to create the 2 nM intermediate library pools. The RSB volume is saved to the file in the Calculation File placeholder in the Files section.
Uses the NovaSeqXSeries_Bulk_Pool1.csv and NovaSeqXSeries_Bulk_Pool2.csv template files to generate a single CSV file. This file contains information about the pools and the samples that they contain. The generated file is stored in the Calculation File placeholder in the Files section.
Resets the Number of Samples in Pool, Total Sample Volume (ul), and Bulk Pool Volume (ul) to null before exiting the step.
Sets the next step for samples to ADVANCE, which advances them to the Dilute and Denature (NovaSeq X Series Sequencing v1.2) step.
Registers the completion time of the step.
This automation completes this action by publishing messages to CLPA through ICA.
Number of Lanes to Sequence
Numeric
Required Field
Range: 1–10
Decimal places displayed: 0
Registers the start time of the step by publishing messages to CLPA through ICA.
Computes the BP Aliquot Volume (µl) and RSB Volume (µl) required for the dilution of pools to the required final loading concentration.
ℹ The BP Aliquot Volume value is rounded up to the nearest µl. The value in the calculation file, however, is reflected with up to two decimal places.
Sets the PhiX Volume (µl) and PhiX Concentration (µl) if there is a PhiX spike-in.
Generates a single CSV file that contains information about the reagents volume required to dilute the working pools. The generated file is stored in the Calculation File placeholder in the Files section.
TT2 Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Performs basic checks on the secondary analysis configuration of the samples in the same planned run. The following checks are included in this script:
Secondary analysis configuration of samples in a planned run is within maximum configuration limit.
Samples in a planned run have the same type of analysis (e.g., Cloud or Local).
Samples in the same pools that have the same secondary analysis (e.g., Illumina DRAGEN Enrichment) also have the same analysis settings (e.g., Map/Align Output Format = CRAM) and the same analysis version (e.g., v3.8.4).
âš This script is required for the NovaSeq X Series Sequencing v1.2 workflow to function properly.
Registers the start time of the step by publishing messages to CLPA through ICA.
Run Name can only contain alphanumeric, dash, underscore, or period characters. Do not use spaces.
Run Name cannot exceed 255 characters.
Checks the Index 1 Cycles and Index 2 Cycles field values. If Index 2 Cycles is greater than 0, the Index 1 Cycles value must be greater than 0 or an error can occur.
Generates the sample sheet and creates the planned run in ICA. The sample sheet is attached to the step.
There is a backend validation of the sample sheet content in ICA. This validation makes sure that the sample sheet is valid to set up a Local mode run and that it is ready for use with DRAGEN applications.
Read 1 Cycles
Numeric Dropdown
Required Field
Custom Entries
Range = 1–251
Presets
51
Read 2 Cycles
Numeric Dropdown
Required Field
Custom Entries
Range = 0–251
Presets
51
Run Mode
Text Dropdown
Required Field
Presets
Local
Cloud
Run Name
Text
Required Field
Instrument Type
Text
Read Only
Registers the NovaSeq X Series sequencing run configuration.
Registers the association of the sequencing run with the samples by linking the Instrument Run ID with the sample ID.
Registers the time that the sequencing run started.
This automation completes these actions by publishing messages to CLPA through ICA.
Registers the completion time of the step.
This automation completes these actions by publishing messages to CLPA through ICA.
Flow Cell ID
RunParameters.xml
SerialNumber
Visible on Record Details screen.
Corresponds to the value in the SerialNumber field under the ConsumableInfo field for FlowCell Type.
Flow Cell Lot Number
RunParameters.xml
LotNumber
Visible on Record Details screen.
Corresponds to the value in the LotNumber field under the ConsumableInfo field for FlowCell Type.
Flow Cell Part Number
RunParameters.xml
PartNumber
Visible on Record Details screen.
Corresponds to the value of the PartNumber field under the ConsumableInfo field for FlowCell Type.
Flow Cell Side
RunParameters.xml
Side
Visible on Record Details screen.
Flow Cell Type
RunParameters.xml
FlowCellType
Visible on Record Details screen.
Instrument Control Software Version
RunParameters.xml
SystemSuiteVersion
Visible on Record Details screen.
Instrument ID
RunParameters.xml
InstrumentSerialNumber
Visible on Record Details screen.
Instrument Type
RunParameters.xml
InstrumentType
Visible on Record Details screen.
Output Folder
RunParameters.xml
OutputFolder
Visible on Record Details screen.
Library Tube Strip Barcode
RunParameters.xml
SerialNumber
Visible on Record Details screen.
Corresponds to the value of the SerialNumber field under the ConsumableInfo field for SampleTube Type.
Library Tube Lot Number
RunParameters.xml
LotNumber
Hidden
Corresponds to the value of the LotNumber field under the ConsumableInfo field for SampleTube Type.
Run End Time
RunCompletionStatus.xml
RunEndTime
Hidden
Run Name
RunParameters.xml
ExperimentName
Visible on Record Details screen.
Received from the message sent to the integration service.
Hidden
Run Start Time
Received from the message sent to the integration service.
Hidden
Yield (Gb) R1
Yield (Gb) R2
Reads PF
% PF
% Aligned R1
% Aligned R2
% Occupied
% Phasing R1
% Phasing R2
% Prephasing R1
% Prephasing R2
Intensity Cycle 1 R1
Intensity Cycle 1 R2
For the Cloud run mode, the planned run is created in ICA. The planned run contains the run and analysis configuration information required to start the run on the instrument. The sample sheet is generated and stored in Clarity LIMS for reference purposes.
When the sequencing run starts on the instrument, the NovaSeq X Series Control Software notifies BaseSpace Sequence Hub. BaseSpace Sequence Hub monitors the run and notifies Clarity LIMS via the integration service. The events are processed and the integration service retrieves the run information from BaseSpace Sequence Hub. This information is used to populate the custom fields in the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2) step.
Other run events follow the same information flow. When sequencing is complete, the control software uploads the sequencing run data (primary metrics) and associated files to ICA. Then, Clarity LIMS retrieves the primary metrics and uses them to populate the fields in the Sample Details table (e.g., % Error Rate R1). The custom fields (e.g., Run Status, Current Read, and so on) on the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2) step are updated using the run related information. If the sequencing run is successfully completed, the step automatically completes.
The integration tracks the analysis events and results in the AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.2) step. You must log in to BaseSpace Sequence Hub to retrieve the cloud analysis results. For local analysis, the secondary analysis results are in the external storage configured in Illumina Run Manager. The external storage information is found in the External Storage for Analysis Results configuration settings in Illumina Run Manager.
In Run Settings, select the Cloud run storage checkbox.
For Hosting location, select the applicable location from the drop-down options.
[Optional] For Private domain name, enter the domain name according to the workgroup that was configured with the NovaSeq X Series integration package.
Select Save.
Import the sample sheet created in the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.2) step. Refer to information on creating a local run in the NovaSeq X Plus Product Documentation on the NovaSeq X Plus Sequencing System support site.
When the sample sheet is selected, the contents are validated. After the validation passes, select Next.
Select Save As Planned.
Run and analysis data is parsed and auto-populates in the list of runs. For more information on planning a NovaSeq X Series run, refer to the Plan a NovaSeq X Series Run section of the BaseSpace Sequence Hub Online Help at help.basespace.illumina.com and the NovaSeq X Plus Product Documentation on the NovaSeq X Plus Sequencing System support site.
To start a sequencing run in cloud run mode, refer to information on starting a planned run in the NovaSeq X Plus Product Documentation on the NovaSeq X Plus Sequencing System support site.
Resuspension Buffer (RSB)
TT2
All submitted samples must have an associated secondary analysis that is configured using the analysis configuration template (ACT). The ACT must be configured on BaseSpace Sequence Hub before starting the Assign Analysis Configuration Template step. The ACT names must be unique.
The library tube strip barcode must be unique. There must not be multiple library tube strip containers with the same name in the system.
Reagent labels, or indexes, must be unique.
One library pool can only contain one library or control with no label/index.
The AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2) and AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.2) steps must not be manually started or completed. These steps are fully automated and the sequencing service does not update samples correctly if they have been manually started.
For the automated run to start successfully, you must select Validate Run Setup and Create Planned Run in the Load to Library Tube step.
Field Name
Field Type
Options
Analysis Configuration Template
Text Dropdown
Required Field
Application
Text
Read Only
Application Version
Text
Read Only
Index Adapter Kit
Text
Read Only
Field Name
Field Type
Options
Additional Options and Dropdown Items
Molarity (nM)
Numeric
Decimal places displayed = 2
ACT Name
Text
Read Only
Field Name
Field Type
Options
Additional Options and Dropdown Items
Final Loading Concentration (pM)
Numeric Dropdown
Required Field
Custom Entries
Dropdown Items
90
140
150
160
180
Range from 0
Decimal places displayed: 0
Flowcell Type
Text
Required Field
Dropdown Items
1.5B
10B
25B
Minimum Per Sample Volume (ul)
Numeric
Required Field
Decimal places displayed: 2
Default: 2
Field Name
Field Type
Options
Additional Options and Dropdown Items
Final Loading Concentration (pM)
Numeric Dropdown
Required Field
Custom Entries
Decimal places displayed: 0
Dropdown Items
225
400
RSB Volume (ul)
Numeric
Read Only
Decimal places displayed: 2
NovaSeq X Flowcell Type
Text Dropdown
Required Field
Read Only
Dropdown Items
1.5B
10B
25B
Field Name
Field Type
Options
Additional Options and Dropdown Items
BP Aliquot Volume (ul)
Numeric
Read Only
Decimal places displayed = 1
NaOH Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
RSB Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Field Name
Field Type
Options
Additional Options and Dropdown Items
Planned Run ID
Text
Read Only
Hidden
ℹ Used for creating a planned run in ICA.
Index 1 Cycles
Numeric Dropdown
Required Field
Custom Entries
Range = 0–20
Presets
0
6
8
Index 2 Cycles
Numeric Dropdown
Required Field
Custom Entries
Range = 0–20
Presets
0
6
8
Field Name
Source File
Corresponding Field in Source File
Additional Information
Current Cycle
RunCompletionStatus.xml
CompletedReads
Visible on Record Details screen.
Calculated based on the CompletedReads field.
Current Read
RunCompletionStatus.xml
RunParameters.xml
CompletedReads
PlannedReads
Visible on Record Details screen.
Calculated based on the CompletedReads field in RunCompletionStatus.xml and PlannedReads field in RunParameters.xml.
Flow Cell Expiration Date
RunParameters.xml
ExpirationDate
Visible on Record Details screen.
Corresponds to the value in the ExpirationDate field under the ConsumableInfo field for FlowCell Type.
Field Name
Description
Additional Information
Run Status
Presets
RunStarted
RunCompletedSuccessfully
RunAbortedByUser
RunErroredOut
Set by the integration service.
Visible on Record Details screen.
Sequencing Log
Set by the integration service during the sequencing run.
Visible on Record Details screen.
BaseSpace Run ID
Received from the message sent to the integration service.
Hidden
ICA Project ID
Received from the message sent to the integration service.
Hidden
Field Name
Description
Additional Information
Analysis Status
Presets
Started
Completed
Failed
NeedsAttention
Stopped
TimedOut
CompletedWithErrors
Set by the integration service.
Visible on Record Details screen.
Analysis Result Location
Received from the message sent to the integration service.
Visible on Record Details screen.
App Session ID
Identified by the integration service using information from RunParameters.xml.
if (!input.hasValue(::Molarity (nM)::)) { return; };
/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -h false -exp 'if (!input.hasValue(::NovaSeq X Flowcell Type::)){fail(::Invalid Flowcell Type custom field value. Please make sure all input samples have a value assigned for Flowcell Type custom field.::)}' -log {compoundOutputFileLuid1}
if (output.hasValue(::Number of Samples in Pool::)) { output.::Number of Samples in Pool:: = output.::Number of Samples in Pool:: + 1; } else { output.::Number of Samples in Pool:: = 1; }' -t true
if (!step.::Run Name::.matches(::[a-zA-Z0-9-_]+[a-zA-Z0-9-_. ]*::)) { fail(::Run Name contains prohibited characters. Please check to make sure Run Name only contains alphanumeric characters, spaces, dashes, periods and underscores. Run Name must start with alphanumeric, a dash or an underscore::);}
The configuration provided in this integration has been established to support NovaSeq X Series lab processes. Any configuration changes to protocols or workflows (including renaming protocols, steps, and fields) could break the process.
Prerequisites and Assumptions
The NovaSeq X Series Integration v1.2.0 requires a valid BaseSpace Sequence Hub account with an Enterprise or Professional subscription.
Analysis Configuration Template
The NovaSeq X Series Sequencing v1.1 workflow uses analysis configuration templates (ACTs) to configure secondary analysis for planned runs. Required ACTs must be created using BaseSpace Sequence Hub. The ACT names must be unique. The index adapter kit (the label group in Clarity LIMS) selected in the ACT is created in Clarity LIMS. The same label group must be used in the library preparation step. For more information, refer to .
Make sure that ACT names created in BaseSpace Sequence Hub do not have leading or trailing spaces. Otherwise, Clarity LIMS can have issues with recognizing the ACT names.
Samples
Samples must go through the library preparation and quantification process before they enter the NovaSeq X Series Sequencing v1.1 workflow. It is assumed that the following steps have completed before samples are assigned to the workflow:
Samples have been accessioned into Clarity LIMS.
Samples have been run through QC and library prep.
Samples have the Molarity (nM) global field set to a value.
This value is required for the Calculate Volumes automation in the Make Bulk Pool step.
Sample and library names must use only alphanumeric, dash, or underscore characters.
For more information on sample accessioning, refer to Sample Accessioning and Upload and Modify Samples in the Getting Started section of the .
You can assign samples to workflows automatically, using a routing script, or manually—from the Projects & Samples dashboard. Refer to Assign and Process Samples in the .
Workflows, Protocols, and Steps
The NovaSeq X Series Integration Package v1.2 includes the following workflows:
NovaSeq X Series Sequencing v1.1
Library Prep Validation v2.3.2 (optional, but recommended for validation purposes)
The following describes the protocols and steps included in these workflows.
Library Prep Validation v2.3.2 Workflow
Protocol 1: Library Prep Validation v2.3.2
Purpose:
Included for validation purposes only, this protocol models the library prep steps required to advance samples to the NovaSeq X Series Sequencing v1.1 workflow.
âš The label group (index adapter kit) used for library preparation must be the same as the index adapter kit specified in the ACT that is being used. For more information, refer to
NovaSeq X Series Sequencing v1.1 Workflow
Protocol 1: NovaSeq X Series Sequencing v1.1
Purpose:
This protocol models the lab processes of run setup for starting a NovaSeq X Series sequencing and secondary analysis run.
Steps:
Validation Workflow
The Library Prep Validation v2.3.2 workflow allows for validation of the system after installation is complete. This workflow can be replaced by other custom library preparation workflows. For details, refer to .
Configure a custom library preparation workflow as follows.
Select Configuration, then select Lab Work.
Add a Routing Script automation to the last Master Step of the library preparation workflow.
Select Configuration, then select Automation.
Step 1: Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1)
The Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1) step uses ACTs to configure secondary analysis for samples. Each ACT contains details related to the secondary analysis (e.g., index adapter kit to use, reference genome setting, and so on). You can select an ACT from a list and assign samples to it. A planned run can have multiple ACTs and this step must be repeated for each ACT that is required for the run. For more information on creating ACTs and assigning samples to them, refer to .
Validate Sample Names and Retrieve Analysis Configuration Template List and Register Step Started Automation¹
Automatically triggered on entry to the step, this automation completes the following actions:
Checks that submitted sample names contain only alphanumeric, dash, and underscore characters.
Retrieve ACT Information Automation
Automatically triggered when you select Retrieve ACT Information on the Record Details screen, this automation completes the following actions:
Retrieves the details of the selected ACT and populates the step custom fields (e.g., Analysis Version, Library Prep Kit, and so on).
Validate Reagent Labels and Apply Selected ACT to Samples and Set Next Step Automation
Automatically triggered on exit of the Record Details screen, this automation completes the following actions:
Checks that the molarity of all samples is specified.
Register Step Completed Automation²
Automatically triggered on exit of the step, this automation registers completion time of the step by publishing messages to CLPA through ICA.
¹ These automations are required for the NovaSeq X Series Sequencing v1.1 workflows and contain additional logic needed for CLPA support. If you would like to remove support for CLPA, contact Illumina Support.
² These automations are required for CLPA support only.
All remaining automations are required for the NovaSeq X Series Sequencing v1.1 workflows.
Master Step Fields
The following table lists the configuration details for fields that are defined on the Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1) step.
Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1) Master Step Field Configuration
Global Fields
The following table lists the global custom fields that are configured to display on the Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1) step.
Global Field Configuration (Derived Sample)
Step 2: Make Bulk Pool (NovaSeq X Series Sequencing v1.1)
Samples are pooled in the Make Bulk Pool (NovaSeq X Series Sequencing v1.1) step. You can manually create working pools based on the final loading concentration required.
Register Step Started Automation¹
Automatically triggered on entry to the step, this automation registers the start time of the step. This automation completes this action by publishing messages to CLPA through ICA.
Validate Analysis Configurations Automation
Automatically triggered on exit of the Pooling screen, this automation performs analysis configuration validity and checks each pool for the following characteristics:
Pooled samples are within the maximum configuration limit.
Calculate Volumes Automation
Automatically triggered when you select Calculate Volumes on the Record Details screen, this automation completes the following actions:
Proceeds if the sample has molarity values.
Uses Number of Samples in Pool to calculate the volumes needed for the 2 nM intermediate library pools.
Set Next Step Automation
Automatically triggered on exit of the Record Details screen, this automation completes the following actions:
Automatically triggered on exit of the step, this automation completes the following actions:
Registers the pool and library information used by the Clarity LIMS workflow. Samples in the pool are assumed to have gone through library preparation.
¹These automations are required for CLPA support only.
All remaining automations are required for the NovaSeq X Series Sequencing v1.1 workflows.
Master Step Fields
The following table lists configuration details for the fields that are defined on the Make Bulk Pool (NovaSeq X Series Sequencing v1.1) step.
Make Bulk Pool (NovaSeq X Series Sequencing v1.1) Master Step Field Configuration
Global Fields
The following table lists the global custom fields that are configured to display on the Make Bulk Pool (NovaSeq X Series Sequencing v1.1) step.
Global Custom Fields Configuration (Derived Sample)
Step 3: Dilute and Denature (NovaSeq X Series Sequencing v1.1)
The Dilute and Denature (NovaSeq X Series Sequencing v1.1) step allows you to dilute pooled samples with the addition of RSB.
Validate Inputs Flowcell Type and Register Step Started¹
Automatically triggered on entry to the step, this automation completes the following actions:
Checks that the specified flow cell type is valid.
âš This script is required for the NovaSeq X Series Sequencing v1.1 workflow to function properly.
Calculate Volumes Automation
Automatically triggered when you select Calculate Volumes on the Record Details screen, this automation completes the following actions based on the NovaSeq X Flowcell Type selected:
Sets the NaOH Volume (µl) and TT2 Volume (µl) field values.
Set Next Step Automation
Automatically triggered on exit of the Record Details screen, this automation completes the following actions:
Copies the Run Mode from input to output.
Sets the next step for samples to ADVANCE, advancing them to the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1) step.
Register Step Completed Automation²
Automatically triggered on exit of the step, this automation registers the completion time of the step by publishing messages to CLPA through ICA.
¹ These automations are required for the NovaSeq X Series Sequencing v1.1 workflows and contain additional logic needed for CLPA support. If you would like to remove support for CLPA, contact Illumina Support.
² These automations are required for CLPA support only.
All remaining automations are required for the NovaSeq X Series Sequencing v1.1 workflows.
Global Fields
The following table lists the global custom fields that are configured to display on the Dilute and Denature (NovaSeq X Series Sequencing v1.1) step.
Global Field Configuration (Derived Sample)
Step 4: Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1)
The Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1) step allows you to scan the library tube strip barcode into Clarity LIMS. Then, you can place the working pools into the library tube strip used in the NovaSeq X Series run. This step also does the following actions:
Validates the run setup and analysis information.
Generates the sample sheet file.
Creates a planned run on Illumina Connected Analytics (ICA), depending on the selected run mode.
Validate Flowcell Inputs and Validate Analysis Configurations and Register Step Started Automation¹
Automatically triggered on entry to the step, this automation completes the following actions:
Checks that the selected destination container types on the Load to Library Tube Strip step matches the selected flow cell type of the samples. The 1.5B flow cell is compatible with the library 2-tube strip and the 10B and 25B flow cells are compatible with the library 8-tube strip.
Validate Library Tube Strip Barcode Automation
Automatically triggered on the Placement screen, this automation validates the library tube strip barcode to make sure that it conforms to the barcode mask LC[0-9]{7}-LC1 for the library 8-tube strip and LC[0-9]{7}-L[A-Z]2 for the library 2-tube strip.
Validate Run Setup and Create Planned Run Automation
Automatically triggered after a selection on the Record Details screen, this automation completes the following actions:
Validates the parameters entered on the Record Details screen. These parameters are used to set up the run, generate the sample sheet file, and create the planned run in ICA, depending on the run mode.
Set Next Step Automation
Automatically triggered on exit of the Record Details screen, this automation completes the following actions:
Copies the Run Mode from input to output.
Sets the next step for samples to ADVANCE, advancing them to the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step.
Register Step Completed Automation²
Automatically triggered on exit of the step, this automation registers the completion time of the step by publishing messages to CLPA through ICA.
¹ These automations are required for the NovaSeq X Series Sequencing v1.1 workflows and contain additional logic needed for CLPA support. If you would like to remove support for CLPA, contact Illumina Support.
² These automations are required for CLPA support only.
All remaining automations are required for the NovaSeq X Series Sequencing v1.1 workflows.
Master Step Fields
The following table shows the master step fields that are configured on the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1) step. These fields are required for sample sheet generation and planned run creation in ICA.
Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1) Master Step Field Configuration
Step 5: AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1)
The AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step is fully automated. The integration starts the step, adds samples to the Ice Bucket, and completes the step. Data from the run is parsed back to Clarity LIMS. In this step, pooled samples in the library tube strip are sequenced on the NovaSeq X Series instrument.
Do not add samples to the Ice Bucket or start and complete the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step. The integration does this automatically.
Register Step Started & Register NovaSeq X Run & Register NovaSeq X Run Association & Register NovaSeq X RunStarted Automation¹
Automatically triggered at the beginning of the step, this automation completes the following actions:
Registers the start time of the step.
Register NovaSeq X RunCompleted & Register Step Completed Automation¹
Automatically triggered on exit of the step, this automation completes the following actions:
Registers the sequencing run completion time, run status, and run metrics. This script requires the Cloud Run ID custom field from the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1) step.
¹ These automations are required for CLPA support only.
Master Step Fields
The following tables show the master step fields that are configured on the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step.
Clarity LIMS Master Step Field Information
Other Clarity LIMS Master Step Fields
Global Fields
The following global custom fields are used to capture the run metrics in Clarity LIMS:
% Bases >=Q30 R1
% Bases >=Q30 R2
% Error Rate R1
% Error Rate R2
At the end of the step, the pools of samples are automatically removed from the step. The step completes automatically when Run Status is RunCompletedSuccessfully.
Step 6: AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1)
The AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1) step is fully automated. The integration starts and completes the step automatically. Data from the analysis is parsed back to Clarity LIMS. In this step, the secondary analysis configured using the Analysis Configuration Template (ACT) is performed in Illumina Connected Analytics (ICA). There are no automations associated with this step.
Do not add samples to the Ice Bucket or start and complete the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step. The integration does this automatically.
Master Step Fields
The following tables show the master step fields that are configured on the AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1) step.
Do not modify the names of the master step fields. Modifications cause the integration to break.
AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1) Master Step Field Information
How the Integration Works
The following information summarizes how the NovaSeq X Series integration works.
After the Validate Run Setup and Create Planned Run automation is triggered on the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1) step, the run parameters entered in the Run Details screen are sent to Illumina Connected Analytics (ICA). The analysis configuration parameters from the selected ACTs are also sent to ICA. ICA validates the run and analysis configuration. If the validation fails, ICA sends an error message to Clarity LIMS. If the validation passes, ICA generates the sample sheet and sends it back to Clarity LIMS. ICA also creates the planned run based on the selected run mode (e.g., Local or Cloud).
For the Local run mode, the sample sheet is generated and stored in Clarity LIMS. This sample sheet contains the run and analysis configuration information required to start the run on the NovaSeq X Series instrument.
If a planned run with the same sample name and project name (case-insensitive) has been created previously in ICA, the sample sheet generated from the Validate Run Setup and Create Planned Run automation can reflect the original case of the previous sample name. This can cause validation errors for analysis configurations with sample-level settings. To resolve this issue, change the sample name or the project name on Clarity LIMS and run the automation again.
Start a Sequencing Run on NovaSeq X Series Instrument
The following sections outline the steps to start a Clarity LIMS cloud or local run on the NovaSeq X Series instrument.
Run Mode Configuration
In the NovaSeq X Series Control Software, log in to the instrument using an account that has permissions for configuring settings.
On the menu, select Settings, then select Cloud settings.
Start a Sequencing Run in Local Run or Cloud Run Modes
Start a Sequencing Run in Local Run Mode
Log in with the BaseSpace Sequence Hub account and select the workgroup used during the NovaSeq X Series integration package installation.
Enabling Planned Run Generation for Samples Having Duplicate Name with Different Indexes
The library preparation workflow of the samples must be before routing the samples through the library preparation workflow.
Components Installed
The following sections describe the components (files, properties, reagent categories / label groups, reagent kits, and containers) that are installed by default as part of this integration.
Reagent Kits
Buffer Cartridge
Lyophilization Cartridge
NaOH
Reagent Cartridge
Container Types
Library 2-Tube Strip
Library 8-Tube Strip
Tube
This integration supports the library 8-tube strip with the barcode in the LC[0-9]{7}-L[A-Z]1 format. The integration also supports the library 2-tube strip with the barcode in the LC[0-9]{7}-L[A-Z]2 format.
Control Types
PhiX v3
Rules and Constraints
The workflow configuration contains several validation checks. To make sure that the calculations work properly, it is important that you do not disable any of this validation logic. The validation checks determine the following information:
Which samples, and how many, can enter each step together.
Which samples, and how many, can be pooled together.
.
The protocol contains a single step - Library Prep Validation v2.3.2. After this step, a routing script sends the samples to the first step of the NovaSeq X Series Sequencing v1.1 workflow (Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1)).
Steps:
Library Prep Validation v2.3.2
Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1)
The secondary analysis of samples is configured using the ACT.
The labels applied to the samples are validated against the selected ACT so that samples use valid labels.
Make Bulk Pool (NovaSeq X Series Sequencing v1.1)
The Calculate Volume script assists with pooling samples and adding resuspension buffers and reagents.
The Validate Analysis Configurations automation validates the ACT of pooled samples to make sure that the secondary analysis configurations remain valid after pooling.
Dilute and Denature (NovaSeq X Series Sequencing v1.1)
The Calculate Volume script helps denature and dilute samples from step 2 to the final loading concentration.
Load To Library Tube Strip (NovaSeq X Series Sequencing v1.1)
The library pools from step 3 are ready to be loaded to the NovaSeq X Series library tube strip.
Validates run and analysis information.
Generates the sample sheet and creates the planned run, depending on the run mode.
AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1)
The step is fully automated and starts and completes automatically after the sequencing run is started and completed on the instrument side.
The sequencing run metadata (eg, run configuration, primary run metrics, and so on) are recorded automatically.
âš Do not add samples to the Ice Bucket or start or complete the step. The integration does this automatically.
AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1)
The step is fully automated and starts and completes automatically after the analysis run is started and completed in ICA.
The analysis run metadata (eg, status and high level analysis summary) are recorded automatically.
âš Do not add samples to the Ice Bucket or start or complete the step. The integration does this automatically.
Create a new Routing Script automation and update it as follows.
For Channel Name, enter limsserver.
For Command Line, enter the following script:
For Automation Use, select the custom library preparation workflow.
Retrieves the list of ACTs that were created in BaseSpace Sequence Hub.
Registers the start time of the step by publishing messages to CLPA through the ICA.
Saves the details into a file that is available to download. This file is stored in the Analysis Configuration Metadata file placeholder.
Checks that all samples are indexed and for invalid indexes. The indexes applied are the same as the selected ACT.
Assigns the selected ACT to all samples in the step.
Sets the next step for samples to ADVANCE, which advances them to the next step in the protocol (Make Bulk Pool (NovaSeq X Series Sequencing v1.1)).
Text
Read Only
Reference Genome
Text
Read Only
Secondary Analysis Mode
Text
Read Only
Pooled samples have the same type of analysis (e.g., Cloud or Local).
Pooled samples that have the same secondary analysis also have the same analysis version (e.g., v3.8.4) and analysis settings (e.g., Map/Align Output Format = CRAM).
Uses the Bulk Pool Volume (ul) field and Number of Lanes to Sequence (x100) to calculate volumes needed for the 2 nM intermediate library pools.
Copies the Final Loading Concentration (pM) and Flowcell Type from the step inputs to the step outputs.
Calculates the per sample volume required for each library to make a 2 nM intermediate library pool and sets the total sample volume to zero.
Calculates the adjusted per sample volume for the pools.
Uses the Total Sample Volume (ul) to calculate the RSB volume (ul) required to top up the pools that are needed to create the 2 nM intermediate library pools. The RSB volume is saved to the file in the Calculation File placeholder in the Files section.
Uses the NovaSeqXSeries_Bulk_Pool1.csv and NovaSeqXSeries_Bulk_Pool2.csv template files to generate a single CSV file. This file contains information about the pools and the samples that they contain. The generated file is stored in the Calculation File placeholder in the Files section.
Resets the Number of Samples in Pool, Total Sample Volume (ul), and Bulk Pool Volume (ul) to null before exiting the step.
Sets the next step for samples to ADVANCE, which advances them to the Dilute and Denature (NovaSeq X Series Sequencing v1.1) step.
Registers the completion time of the step.
This automation completes this action by publishing messages to CLPA through ICA.
Number of Lanes to Sequence
Numeric
Required Field
Range: 1–10
Decimal places displayed: 0
Registers the start time of the step by publishing messages to CLPA through ICA.
Computes the BP Aliquot Volume (µl) and RSB Volume (µl) required for the dilution of pools to the required final loading concentration.
ℹ The BP Aliquot Volume value is rounded up to the nearest µl. The value in the calculation file, however, is reflected with up to two decimal places.
Sets the PhiX Volume (µl) and PhiX Concentration (µl) if there is a PhiX spike-in.
Generates a single CSV file that contains information about the reagents volume required to dilute the working pools. The generated file is stored in the Calculation File placeholder in the Files section.
TT2 Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Performs basic checks on the secondary analysis configuration of the samples in the same planned run. The following checks are included in this script:
Secondary analysis configuration of samples in a planned run is within maximum configuration limit.
Samples in a planned run have the same type of analysis (e.g., Cloud or Local).
Samples in the same pools that have the same secondary analysis (e.g., Illumina DRAGEN Enrichment) also have the same analysis settings (e.g., Map/Align Output Format = CRAM) and the same analysis version (e.g., v3.8.4).
âš This script is required for the NovaSeq X Series Sequencing v1.1 workflow to function properly.
Registers the start time of the step by publishing messages to CLPA through ICA.
Run Name can only contain alphanumeric, dash, underscore, or period characters. Do not use spaces.
Run Name cannot exceed 255 characters.
Checks the Index 1 Cycles and Index 2 Cycles field values. If Index 2 Cycles is greater than 0, the Index 1 Cycles value must be greater than 0 or an error can occur.
Generates the sample sheet and creates the planned run in ICA. The sample sheet is attached to the step.
There is a backend validation of the sample sheet content in ICA. This validation makes sure that the sample sheet is valid to set up a Local mode run and that it is ready for use with DRAGEN applications.
Registers the NovaSeq X Series sequencing run configuration.
Registers the association of the sequencing run with the samples by linking the Instrument Run ID with the sample ID.
Registers the time that the sequencing run started.
This automation completes these actions by publishing messages to CLPA through ICA.
Registers the completion time of the step.
This automation completes these actions by publishing messages to CLPA through ICA.
Flow Cell ID
RunParameters.xml
SerialNumber
Visible on Record Details screen.
Corresponds to the value in the SerialNumber field under the ConsumableInfo field for FlowCell Type.
Flow Cell Lot Number
RunParameters.xml
LotNumber
Visible on Record Details screen.
Corresponds to the value in the LotNumber field under the ConsumableInfo field for FlowCell Type.
Flow Cell Part Number
RunParameters.xml
PartNumber
Visible on Record Details screen.
Corresponds to the value of the PartNumber field under the ConsumableInfo field for FlowCell Type.
Flow Cell Side
RunParameters.xml
Side
Visible on Record Details screen.
Flow Cell Type
RunParameters.xml
FlowCellType
Visible on Record Details screen.
Instrument Control Software Version
RunParameters.xml
SystemSuiteVersion
Visible on Record Details screen.
Instrument ID
RunParameters.xml
InstrumentSerialNumber
Visible on Record Details screen.
Instrument Type
RunParameters.xml
InstrumentType
Visible on Record Details screen.
Output Folder
RunParameters.xml
OutputFolder
Visible on Record Details screen.
Library Tube Strip Barcode
RunParameters.xml
SerialNumber
Visible on Record Details screen.
Corresponds to the value of the SerialNumber field under the ConsumableInfo field for SampleTube Type.
Library Tube Lot Number
RunParameters.xml
LotNumber
Hidden
Corresponds to the value of the LotNumber field under the ConsumableInfo field for SampleTube Type.
Run End Time
RunCompletionStatus.xml
RunEndTime
Hidden
Run Name
RunParameters.xml
ExperimentName
Visible on Record Details screen.
RTA Version
Not used
Not used
Hidden
Received from the message sent to the integration service.
Hidden
Run Start Time
Received from the message sent to the integration service.
Hidden
Yield (Gb) R1
Yield (Gb) R2
Reads PF R1
Reads PF R2
%PF R1
%PF R2
% Aligned R1
% Aligned R2
% Phasing R1
% Phasing R2
% Prephasing R1
% Prephasing R2
Intensity Cycle 1 R1
Intensity Cycle 1 R2
Cluster Density R1
Cluster Density R2
For the Cloud run mode, the planned run is created in ICA. The planned run contains the run and analysis configuration information required to start the run on the instrument. The sample sheet is generated and stored in Clarity LIMS for reference purposes.
When the sequencing run starts on the instrument, the NovaSeq X Series Control Software notifies BaseSpace Sequence Hub. BaseSpace Sequence Hub monitors the run and notifies Clarity LIMS via the integration service. The events are processed and the integration service retrieves the run information from BaseSpace Sequence Hub. This information is used to populate the custom fields in the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step.
Other run events follow the same information flow. When sequencing is complete, the control software uploads the sequencing run data (primary metrics) and associated files to ICA. Then, Clarity LIMS retrieves the primary metrics and uses them to populate the fields in the Sample Details table (e.g., % Error Rate R1). The custom fields (e.g., Run Status, Current Read, and so on) on the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step are updated using the run related information. If the sequencing run is successfully completed, the step automatically completes.
The integration tracks the analysis events and results in the AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1) step. You must log in to BaseSpace Sequence Hub to retrieve the cloud analysis results. For local analysis, the secondary analysis results are in the external storage configured in Illumina Run Manager. The external storage information is found in the External Storage for Analysis Results configuration settings in Illumina Run Manager.
In Run Settings, select the Cloud run storage checkbox.
For Hosting location, select the applicable location from the drop-down options.
[Optional] For Private domain name, enter the domain name according to the workgroup that was configured with the NovaSeq X Series integration package.
Select Save.
Import the sample sheet created in the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1) step. Refer to information on creating a local run in the NovaSeq X Plus Product Documentation on the NovaSeq X Plus Sequencing System support site.
When the sample sheet is selected, the contents are validated. After the validation passes, select Next.
Select Save As Planned.
Run and analysis data is parsed and auto-populates in the list of runs. For more information on planning a NovaSeq X Series run, refer to the Plan a NovaSeq X Series Run section of the BaseSpace Sequence Hub Online Help at help.basespace.illumina.com and the NovaSeq X Plus Product Documentation on the NovaSeq X Plus Sequencing System support site.
To start a sequencing run in cloud run mode, refer to information on starting a planned run in the NovaSeq X Plus Product Documentation on the NovaSeq X Plus Sequencing System support site.
Resuspension Buffer (RSB)
TT2
All submitted samples must have an associated secondary analysis that is configured using the analysis configuration template (ACT). The ACT must be configured on BaseSpace Sequence Hub before starting the Assign Analysis Configuration Template step. The ACT names must be unique.
The library tube strip barcode must be unique. There must not be multiple library tube strip containers with the same name in the system.
Reagent labels, or indexes, must be unique.
One library pool can only contain one library or control with no label/index.
The AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) and AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1) steps must not be manually started or completed. These steps are fully automated and the sequencing service does not update samples correctly if they have been manually started.
For the automated run to start successfully, you must select Validate Run Setup and Create Planned Run in the Load to Library Tube step.
Field Name
Field Type
Options
Analysis Configuration Template
Text Dropdown
Required Field
Application
Text
Read Only
Application Version
Text
Read Only
Index Adapter Kit
Text
Read Only
Field Name
Field Type
Options
Additional Options and Dropdown Items
Molarity (nM)
Numeric
Decimal places displayed = 2
ACT Name
Text
Read Only
Field Name
Field Type
Options
Additional Options and Dropdown Items
Final Loading Concentration (pM)
Numeric Dropdown
Required Field
Custom Entries
Dropdown Items
90
140
150
160
180
Range from 0
Decimal places displayed: 0
Flowcell Type
Text
Required Field
Dropdown Items
1.5B
10B
25B
Minimum Per Sample Volume (ul)
Numeric
Required Field
Decimal places displayed: 2
Default: 2
Field Name
Field Type
Options
Additional Options and Dropdown Items
Final Loading Concentration (pM)
Numeric Dropdown
Required Field
Custom Entries
Decimal places displayed: 0
Dropdown Items
225
400
RSB Volume (ul)
Numeric
Read Only
Decimal places displayed: 2
NovaSeq X Flowcell Type
Text Dropdown
Required Field
Read Only
Dropdown Items
1.5B
10B
25B
Field Name
Field Type
Options
Additional Options and Dropdown Items
BP Aliquot Volume (ul)
Numeric
Read Only
Decimal places displayed = 0
NaOH Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
RSB Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Field Name
Field Type
Options
Additional Options and Dropdown Items
Cloud Run ID
Text
Read Only
Hidden
ℹ Used for creating a planned run in ICA.
Index 1 Cycles
Numeric Dropdown
Required Field
Custom Entries
Range = 0–20
Presets
0
6
8
Index 2 Cycles
Numeric Dropdown
Required Field
Custom Entries
Range = 0–20
Presets
0
6
8
Field Name
Source File
Corresponding Field in Source File
Additional Information
Current Cycle
RunCompletionStatus.xml
CompletedReads
Visible on Record Details screen.
Calculated based on the CompletedReads field.
Current Read
RunCompletionStatus.xml
RunParameters.xml
CompletedReads
PlannedReads
Visible on Record Details screen.
Calculated based on the CompletedReads field in RunCompletionStatus.xml and PlannedReads field in RunParameters.xml.
Flow Cell Expiration Date
RunParameters.xml
ExpirationDate
Visible on Record Details screen.
Corresponds to the value in the ExpirationDate field under the ConsumableInfo field for FlowCell Type.
Field Name
Description
Additional Information
Run Status
Presets
RunStarted
RunCompletedSuccessfully
RunAbortedByUser
RunErroredOut
Set by the integration service.
Visible on Record Details screen.
Sequencing Log
Set by the integration service during the sequencing run.
Visible on Record Details screen.
BaseSpace Run ID
Received from the message sent to the integration service.
Hidden
ICA Project ID
Received from the message sent to the integration service.
Hidden
Field Name
Description
Additional Information
Analysis Status
Presets
Started
Completed
Failed
NeedsAttention
Stopped
TimedOut
CompletedWithErrors
Set by the integration service.
Visible on Record Details screen.
Analysis Result Location
Received from the message sent to the integration service.
Visible on Record Details screen.
Library Tube Strip Barcode
Identified by the integration service using information from RunParameters.xml.
if (!input.hasValue(::Molarity (nM)::)) { return; };
/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -h false -exp 'if (!input.hasValue(::NovaSeq X Flowcell Type::)){fail(::Invalid Flowcell Type custom field value. Please make sure all input samples have a value assigned for Flowcell Type custom field.::)}' -log {compoundOutputFileLuid1}
if (output.hasValue(::Number of Samples in Pool::)) { output.::Number of Samples in Pool:: = output.::Number of Samples in Pool:: + 1; } else { output.::Number of Samples in Pool:: = 1; }' -t true
if (!step.::Run Name::.matches(::[a-zA-Z0-9-_]+[a-zA-Z0-9-_. ]*::)) { fail(::Run Name contains prohibited characters. Please check to make sure Run Name only contains alphanumeric characters, spaces, dashes and underscores. Run Name must start with alphanumeric, a dash or an underscore::);}
