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NovaSeq X Series

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 v1.2.1

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 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 Clarity LIMS Product Analytics Integration v1.3.1 Configuration.

Release Notes

Last Updated: November 2024

Release Date: July 2024

Document Version: 2

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 Calculat Volume automation functionality.

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:

NovaSeq X Series Integration v1.2.0 Configuration
- NovaSeq X Series Integration Package version has been updated from v1.2.0 to v1.2.1.
NovaSeq X Series Integration v1.2.0 User Interaction, Validation and Troubleshooting
- NovaSeq X Series Integration Package version has been updated from v1.2.0 to v1.2.1.

Revision History

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 NovaSeq X Series Integration v1.2.0 User Interaction, Validation and Troubleshooting.
[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 Clarity LIMS Product Analytics Integration v1.2.0 Configuration.

Configuration

The Illumina NovaSeq X Series Integration Package v1.2.0 supports the integration of Clarity LIMS to Illumina NovaSeq X series instruments.

For instructions on validating and troubleshooting the Illumina NovaSeq X Series Integration, refer to NovaSeq X Series Integration v1.2.0 User Interaction, Validation and Troubleshooting.

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 NovaSeq X Series Integration v1.2.0 User Interaction, Validation and Troubleshooting.

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 Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

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 Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

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.
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

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:

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.
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.

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 NovaSeq X Series Integration v1.2.0 User Interaction, Validation and Troubleshooting.

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.

Create a new Routing Script automation and update it as follows.

For Channel Name, enter limsserver.

For Command Line, enter the following script:

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \
\
--FIELD_NAME 'Sequencing Instrument' \
--FIELD_VALUE 'NovaSeq X Series' \
--WORKFLOW 'NovaSeq X Series v1.1' \
--STEP 'Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"

For Automation Use, select the custom library preparation workflow.

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 NovaSeq X Series Integration v1.2.0 User Interaction, Validation and Troubleshooting.

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.

/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 -exp 'submittedSamples.each { sample -> if ( !sample.name.matches( ::[A-Za-z0-9_-]+:: ) ) { fail ( ::Submitted sample :: + sample.name + :: should only have alphanumeric, dash, and underscore characters. Please rename and try again.:: ) } }' -log {compoundOutputFileLuid1}

Retrieves the list of ACTs that were created in BaseSpace Sequence Hub.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/SIS/SISServices/extensions/automation/novaseqxseries-automation.jar script:retrieve_act_list -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1}

Registers the start time of the step by publishing messages to CLPA through the ICA.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/step_started.groovy'

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).

Saves the details into a file that is available to download. This file is stored in the Analysis Configuration Metadata file placeholder.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/SIS/SISServices/extensions/automation/novaseqxseries-automation.jar script:retrieve_act_info -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -a '{udf:Analysis Configuration Template}'

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.

/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 -t false -h false -exp 'if (!input.hasValue(::Molarity (nM)::)) { fail(::The Molarity cannot be empty.::) ; } else if (input.::Molarity (nM):: <= 0) { fail(::The Molarity cannot be negative or zero.::) ; }' -log {compoundOutputFileLuid1}

Checks that all samples are indexed and for invalid indexes. The indexes applied are the same as the selected ACT.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/SIS/SISServices/extensions/automation/novaseqxseries-automation.jar script:validate_indexes -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -a '{udf:Analysis Configuration Template}'

Assigns the selected ACT to all samples in the step.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/SIS/SISServices/extensions/automation/novaseqxseries-automation.jar script:assign_act -i {stepURI:v2} -u {username} -p {password} -a '{udf:Analysis Configuration Template}' -l {compoundOutputFileLuid1}

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)).
```
nextStep = ::ADVANCE:
```

Automatically triggered on exit of the step, this automation registers completion time of the step by publishing messages to CLPA through ICA.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/step_completed.groovy'

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.

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.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/step_started.groovy'

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.
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).

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/SIS/SISServices/extensions/automation/novaseqxseries-automation.jar script:validate_physical_logical_configurations -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -m PerPool script:validate_analysis_config -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -m PerPool && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar script:validate_same_analyte_udf_value_for_all_inputs -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -f 'Run Mode' -perPool true

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.

if (!input.hasValue(::Molarity (nM)::)) { return; };

Uses Number of Samples in Pool to calculate the volumes needed for the 2 nM intermediate library pools.

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

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.

output.::Bulk Pool Volume (ul):: = 100 * step.::Number of Lanes to Sequence::; output.::NovaSeq X Flowcell Type:: = step.::Flowcell Type::; output.::Final Loading Concentration (pM):: = step.::Final Loading Concentration (pM)::;

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.

input.::Per Sample Volume (ul):: = 2 * output.::Bulk Pool Volume (ul):: / input.::Molarity (nM):: / output.::Number of Samples in Pool::;output.::Total Sample Volume (ul):: = 0;' -t true

Calculates the adjusted per sample volume for the pools.
```
script:calculate_multipool_adjusted_per_sample_volume -t
```

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.

script:evaluateDynamicExpression \
-exp 'if (output.hasValue(::Total Sample Volume (ul)::)) { output.::Total Sample Volume (ul):: = output.::Total Sample Volume (ul):: + input.::Adjusted Per Sample Volume (ul)::; } else { output.::Total Sample Volume (ul):: = input.::Adjusted Per Sample Volume (ul)::; }' -t true \
script:evaluateDynamicExpression \
-exp 'if (output.::Total Sample Volume (ul):: > output.::Bulk Pool Volume (ul)::) { output.::RSB Volume (ul):: = 0 } else { output.::RSB Volume (ul):: = output.::Bulk Pool Volume (ul):: - output.::Total Sample Volume (ul):: }' -t true \

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.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeqXSeries_Bulk_Pool1.csv -o 1.csv \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeqXSeries_Bulk_Pool2.csv -o 2.csv \
&& cat 1.csv 2.csv > {compoundOutputFileLuid0}.csv \

Resets the Number of Samples in Pool, Total Sample Volume (ul), and Bulk Pool Volume (ul) to null before exiting the step.

-exp 'output.::Number of Samples in Pool:: = ::::; output.::Total Sample Volume (ul):: = ::::; output.::Bulk Pool Volume (ul):: = ::::;' -t true

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.
Sets the next step for samples to ADVANCE, which advances them to the Dilute and Denature (NovaSeq X Series Sequencing v1.1) step.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} -log {compoundOutputFileLuid1} \
script:evaluateDynamicExpression -exp 'if (!input.hasValue(::Molarity (nM)::)){ return; }; output.::Run Mode:: = input.::Run Mode::; nextStep = ::ADVANCE::;'"

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.
Registers the completion time of the step.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/pooling.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/step_completed.groovy'

This automation completes this action by publishing messages to CLPA through ICA.

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.

/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}

Registers the start time of the step by publishing messages to CLPA through ICA.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/step_started.groovy'

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.
Computes the BP Aliquot Volume (µl) and RSB Volume (µl) required for the dilution of pools to the required final loading concentration.

Sets the PhiX Volume (µl) and PhiX Concentration (µl) if there is a PhiX spike-in.

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} -log {compoundOutputFileLuid1} \
script:evaluateDynamicExpression \
-exp '
if (input.::NovaSeq X Flowcell Type:: == ::10B:: || input.::NovaSeq X Flowcell Type:: == ::1.5B::) {
    output.::NaOH Volume (ul):: = 8.5;
    output.::TT2 Volume (ul):: = 127.5;
    output.::BP Aliquot Volume (ul):: = input.::Final Loading Concentration (pM):: * 170 / (2 * 1000);
    output.::RSB Volume (ul):: = 34 - output.::BP Aliquot Volume (ul)::;
    if (step.::1-2% PhiX Spike-In::) { 
        output.::PhiX Volume (ul):: = 1; output.::PhiX Concentration (pM):: = 300; 
    }else { 
        output.::PhiX Volume (ul):: = ::::; output.::PhiX Concentration (pM):: = ::::; 
    };
} else {
    output.::NaOH Volume (ul):: = 14;
    output.::TT2 Volume (ul):: = 210;
    output.::BP Aliquot Volume (ul):: = input.::Final Loading Concentration (pM):: * 280 / (2 * 1000);
    output.::RSB Volume (ul):: = 56 - output.::BP Aliquot Volume (ul)::;
    if (step.::1-2% PhiX Spike-In::) { 
        output.::PhiX Volume (ul):: = 1.6; output.::PhiX Concentration (pM):: = 300; 
    }
    else { 
        output.::PhiX Volume (ul):: = ::::; output.::PhiX Concentration (pM):: = ::::; 
    };
}'

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.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeqXSeries_Dilute_Denature_Calculate_Volumes.csv -o {compoundOutputFileLuid0}.csv -q true -destLIMSID {compoundOutputFileLuid0} -l {compoundOutputFileLuid1} \
&& echo; echo 'Calculate Volumes completed successfully.'"

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.

/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 -t false -h false -exp 'output.::Run Mode:: = input.::Run Mode::; nextStep = ::ADVANCE::' -log {compoundOutputFileLuid1}

Automatically triggered on exit of the step, this automation registers the completion time of the step by publishing messages to CLPA through ICA.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/step_completed.groovy'

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.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} \
script:validate_same_udf_value_for_analytes -f 'Run Mode' -f 'NovaSeq X Flowcell Type' \
script:validate_selected_container \
-fn 'NovaSeq X Flowcell Type' -fv '1.5B' -ct 'Library 2-tube Strip' \
-fn 'NovaSeq X Flowcell Type' -fv '10B' -ct 'Library 8-tube Strip' \
-fn 'NovaSeq X Flowcell Type' -fv '25B' -ct 'Library 8-tube Strip'

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).
```
/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/SIS/SISServices/extensions/automation/novaseqxseries-automation.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -m PerRun script:validate_analysis_config script:validate_physical_logical_configurations
```

Registers the start time of the step by publishing messages to CLPA through ICA.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/step_started.groovy'

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.

/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:validate_output_containers -l {compoundOutputFileLuid1} -r 'Library 8-tube Strip:LC[0-9]{7}-L[A-Z]1' -r 'Library 2-tube Strip:LC[0-9]{7}-L[A-Z]2' -max 1

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.

Run Name can only contain alphanumeric, dash, underscore, or period characters. Do not use spaces.

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::);}

Run Name cannot exceed 255 characters.

if (step.::Run Name::.length() > 255) { fail(::Run Name shall not 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.
```
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::); };
```

Generates the sample sheet and creates the planned run in ICA. The sample sheet is attached to the step.

/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}

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.

For more information on sample name character restrictions, refer to NovaSeq X Series Integration v1.2.0 User Interaction, Validation and Troubleshooting.

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.

output.::Run Mode:: = input.::Run Mode::; nextStep = ::ADVANCE::

Automatically triggered on exit of the step, this automation registers the completion time of the step by publishing messages to CLPA through ICA.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/step_completed.groovy'

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 v1.1) 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.

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.
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.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/step_started.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/novaseqxseries/register_novaseqxseries_run.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/novaseqxseries/novaseqxseries_associate_seqrun.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/novaseqxseries/novaseqxseries_seqrun_started.groovy'

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.
Registers the completion time of the step.

This automation completes these actions by publishing messages to CLPA through ICA.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/novaseqxseries/novaseqxseries_seqrun_completed.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/step_completed.groovy'

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
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

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.

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.

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.
- 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.

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.
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.

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.
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.
Start the run. Refer to information on starting a planned run in the NovaSeq X Plus Product Documentation on the NovaSeq X Plus Sequencing System support site.

Start a Sequencing Run in Cloud Run Mode

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.

Enabling Planned Run Generation for Samples Having Duplicate Name with Different Indexes

The library preparation workflow of the samples must be configured to ensure unique derived sample names 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
Resuspension Buffer (RSB)
TT2

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.
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.

User Interaction, Validation and Troubleshooting

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.
- 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 . For the adapter sequences for your library prep kits, refer to .

Analysis Configuration Template Creation in BaseSpace Sequence Hub

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.
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.
[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.
On the Ice Bucket screen, select Begin Work.
Create a pool of samples as follows.
1. On the Pooling screen, create a pool by dragging samples into the Pool Creator.
  If a control sample was added, it appears under the Sample List and can be pulled into the pool that needs the control.
1. Enter a name for your pool or accept the default name (Pool #1).
1. [Optional] If multiple pools are required, select the plus sign (+) next to Pool Creator to create a pool.
1. [Optional] To remove a pool, select the X in the top right corner of the pool.
2. 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.
On the Record Details screen, navigate to the Reagent Lot Tracking section to track the lot information used in the step.

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
  - 25B
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)
  - 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.
On the Record Details screen, perform the following actions:
1. In the Reagent Lot Tracking section, select from the active lots displayed in each drop-down list.
2. 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.
3. [Optional] In the Sample Details table, select the pool icon to view details of the working pool composition.
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.
1. Drag the pools into the Placed Samples area on the right.
2. Scan or type the barcode of the library tube strip into the container name field.
3. 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.
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
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.
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.
- Generates the sample sheet and attaches it to the placeholder in the Files area on the Record Details screen for all Run Mode types.
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.

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.

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.

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

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.
1. 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.
1. 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:
1. In Lab View, find the step in the Recent Activities pane.
2. 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.

Validate Analysis Configuration Automation Check Fails

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.
1. Abort the step and remove samples with ACTs that have conflicting run modes from the Ice Bucket.
2. Make sure that all remaining samples in the Ice Bucket have ACTs with the same run modes.
3. Select Begin Work to continue the step.
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.
- 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.
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.
1. 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.
2. 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.

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).
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.
- Make Bulk Pool master step: Updated the dropdown items in the Final Loading Concentration (pM) 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:

NovaSeqXSeries_Bulk_Pool1.csv:

NovaSeqXSeries_Bulk_Pool2.csv:

NovaSeqXSeries_Dilute_Denature_Calculate_Volumes.csv:

Add New Containers

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:
1. For Container Name, enter Library 2-tube Strip.
2. For Rows, update the following fields:
  - Number: 2
  - Naming: Alphabetic
3. For Columns, update the following fields:
  - Number: 1
  - Naming: Numeric
  - Start at: 1

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
Load to Library Tube Strip (NovaSeq X Series Sequencing v1.x) Master Step Field Modifications
AUTOMATED - Sequencing Run (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:
- 1.5B
- 10B
- 25B

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
Channel Name: limsserver

Command line:

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} -l {compoundOutputFileLuid1} \
script:validate_same_udf_value_for_analytes -f 'Run Mode' -f 'NovaSeq X Flowcell Type' \
script:validate_selected_container \
-fn 'NovaSeq X Flowcell Type' -fv '1.5B' -ct 'Library 2-tube Strip' \
-fn 'NovaSeq X Flowcell Type' -fv '10B' -ct 'Library 8-tube Strip' \
-fn 'NovaSeq X Flowcell Type' -fv '25B' -ct 'Library 8-tube Strip' && \
/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/SIS/SISServices/extensions/automation/novaseqxseries-automation.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -m PerRun script:validate_analysis_config script:validate_physical_logical_configurations && \
/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/step_started.groovy'"

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
Dilute and Denature (NovaSeq X Series Sequencing v1.x) Step Automation Configuration

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.
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} -log {compoundOutputFileLuid1} \ script:evaluateDynamicExpression \ -exp 'if (!input.hasValue(::Molarity (nM)::)) { return; }; 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 \ script:evaluateDynamicExpression \ -exp 'output.::Bulk Pool Volume (ul):: = 100 * step.::Number of Lanes to Sequence::; output.::NovaSeq X Flowcell Type:: = step.::Flowcell Type::; output.::Final Loading Concentration (pM):: = step.::Final Loading Concentration (pM)::; input.::Per Sample Volume (ul):: = 2 * output.::Bulk Pool Volume (ul):: / input.::Molarity (nM):: / output.::Number of Samples in Pool::; output.::Total Sample Volume (ul):: = 0;' -t true \ script:calculate_multipool_adjusted_per_sample_volume -t \ script:evaluateDynamicExpression \ -exp 'if (output.hasValue(::Total Sample Volume (ul)::)) { output.::Total Sample Volume (ul):: = output.::Total Sample Volume (ul):: + input.::Adjusted Per Sample Volume (ul)::; } else { output.::Total Sample Volume (ul):: = input.::Adjusted Per Sample Volume (ul)::; }' -t true \ script:evaluateDynamicExpression \ -exp 'if (output.::Total Sample Volume (ul):: > output.::Bulk Pool Volume (ul)::) { output.::RSB Volume (ul):: = 0 } else { output.::RSB Volume (ul):: = output.::Bulk Pool Volume (ul):: - output.::Total Sample Volume (ul):: }' -t true \ && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} script:driver_file_generator \ -t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeqXSeries_Bulk_Pool1.csv -o 1.csv \ script:driver_file_generator \ -t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeqXSeries_Bulk_Pool2.csv -o 2.csv \ && cat 1.csv 2.csv > {compoundOutputFileLuid0}.csv \ && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} -log {compoundOutputFileLuid1} \ script:evaluateDynamicExpression \ -exp 'output.::Number of Samples in Pool:: = ::::; output.::Total Sample Volume (ul):: = ::::; output.::Bulk Pool Volume (ul):: = ::::;' -t true \ && echo 'Calculate Volume completed successfully'"
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.
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} -log {compoundOutputFileLuid1} \ script:evaluateDynamicExpression \ -exp ' if (input.::NovaSeq X Flowcell Type:: == ::10B:: || input.::NovaSeq X Flowcell Type:: == ::1.5B::) { output.::NaOH Volume (ul):: = 8.5; output.::TT2 Volume (ul):: = 127.5; output.::BP Aliquot Volume (ul):: = input.::Final Loading Concentration (pM):: * 170 / (2 * 1000); output.::RSB Volume (ul):: = 34 - output.::BP Aliquot Volume (ul)::; if (step.::1-2% PhiX Spike-In::) { output.::PhiX Volume (ul):: = 1; output.::PhiX Concentration (pM):: = 300; } else { output.::PhiX Volume (ul):: = ::::; output.::PhiX Concentration (pM):: = ::::; }; } else { output.::NaOH Volume (ul):: = 14; output.::TT2 Volume (ul):: = 210; output.::BP Aliquot Volume (ul):: = input.::Final Loading Concentration (pM):: * 280 / (2 * 1000); output.::RSB Volume (ul):: = 56 - output.::BP Aliquot Volume (ul)::; if (step.::1-2% PhiX Spike-In::) { output.::PhiX Volume (ul):: = 1.6; output.::PhiX Concentration (pM):: = 300; } else { output.::PhiX Volume (ul):: = ::::; output.::PhiX Concentration (pM):: = ::::; }; }' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} \ script:driver_file_generator \ -t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeqXSeries_Dilute_Denature_Calculate_Volumes.csv -o {compoundOutputFileLuid0}.csv -q true -destLIMSID {compoundOutputFileLuid0} -l {compoundOutputFileLuid1} \ && echo; echo 'Calculate Volumes completed successfully.'"
Select the Set Next Step 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 enable support for the 1.5B and 25B flow cells and future 8-tube library strips. 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} script:evaluateDynamicExpression -t false -h false -exp 'output.::Run Mode:: = input.::Run Mode::; output.::NovaSeq X Flowcell Type:: = input.::NovaSeq X Flowcell Type::; nextStep = ::ADVANCE::' -log {compoundOutputFileLuid1}"
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.
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:validate_output_containers -l {compoundOutputFileLuid1} -r 'Library 8-tube Strip:LC[0-9]{7}-L[A-Z]{1}1' -r 'Library 2-tube Strip:LC[0-9]{7}-L[A-Z]{1}2' -max 1"

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.
Navigate to Sample Table and add the NovaSeqX Flowcell Type field to Column Headers.

Select the Ice Bucket milestone.
Navigate to Sample Table and add the NovaSeqX Flowcell Type field to Column Headers.
Select the Placement milestone.
Navigate to Destination Containers and add Library 2-tube Strip.

Release Notes

Last Updated: November 2024

Release Date: February 2024

Document Version: 2

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

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.
Sample sheet and planned run generation will fail if any of the samples in the pools has been assigned QC flag in prior steps before entering the Load to Library Tube Strip step.

Revision History

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 .

Configuration

The Illumina NovaSeq X Series Integration Package v1.1.0 supports the integration of Clarity LIMS to Illumina NovaSeq X series instruments.

For user interactions for each step and instructions on validating and troubleshooting the Illumina NovaSeq X Series Integration, refer to .

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.
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

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:

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.

Validation Workflow

Step 1: Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0)

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
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 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.
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.

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).
Saves the details into a file that is available to download. This file is stored in the Analysis Configuration Metadata file placeholder.

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.
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)).

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
Register Pools & Register Step Completed¹

¹ These automations are required for CLPA support only.

All remaining automations are required for the NovaSeq X Series Sequencing v1.0 workflows.

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.
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).

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.
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.

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.
Sets the next step for samples to ADVANCE, which advances them to the Dilute and Denature (NovaSeq X Series Sequencing v1.0) step.

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.
Registers completion time of the step.

This automation completes this action by publishing messages to CLPA through ICA.

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.

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.
Registers the start time of the step by publishing messages to CLPA through ICA.

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.
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.

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.

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.
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.

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.
- 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.

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.

Automatically triggered on exit of the step, this automation registers the completion time of the step by publishing messages to CLPA through ICA.

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.
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.

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.
Registers the completion time of the step. This automation completes these actions by publishing messages to CLPA through ICA.

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
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

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.
- 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.

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

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

Start a Sequencing Run in Cloud Run Mode

Enabling Planned Run Generation for Samples Having Duplicate Name with Different Indexes

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
Resuspension Buffer (RSB)
TT2

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.
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.

User Interaction, Validation and Troubleshooting

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 . For the adapter sequences for your library prep kits, refer to .

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 .

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.
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)

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.
[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.
You can also add control samples by selecting them from the Available control samples drop-down list and selecting Add.
On the Ice Bucket screen, select Begin Work.
Create a pool of samples as follows.
1. On the Pooling screen, create a pool by dragging samples into the Pool Creator.
  If a control sample was added, it appears under the Sample List and can be pulled into the pool that needs the control.
1. Enter a name for your pool or accept the default name (Pool #1).
1. [Optional] If multiple pools are required, select the plus sign (+) next to Pool Creator to create a pool.
1. [Optional] To remove a pool, select the X in the top right corner of the pool.
2. 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.
On the Record Details screen, navigate to the Reagent Lot Tracking section to track the lot information used in the step.

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)
  - 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.
On the Record Details screen, perform the following actions:
1. In the Reagent Lot Tracking section, select from the active lots displayed in each drop-down list.
2. In the Sample Details table, the BP aliquot, RSB, NaOH, and TT2 reagent volume values are auto populated and read-only.
3. [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.
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.
1. Drag the pool into the Library 8-tube Strip in the Placed Samples area on the right.
2. Scan or type the barcode of the Library 8-tube Strip into the Library 8-tube Strip container name field.
1. 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 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
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.
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.
- Clarity consolidates sample/library information, run and analysis configuration details and send them to ICA to generate samplesheet for Local 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.
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.

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

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.
1. 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.
1. 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:
1. In Lab View, find the step in the Recent Activities pane.
2. 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.

Validate Analysis Configuration Automation Check Fails

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.
1. Abort the step and remove samples with ACTs that have conflicting run modes from the Ice Bucket.
2. Make sure that all remaining samples in the Ice Bucket have ACTs with the same run modes.
3. Select Begin Work to continue the step.
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.
- 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.
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.
- 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.

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

Refer to 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.

Revision History

Configuration

The Illumina NovaSeq X Series Integration Package v1.2.0 supports the integration of Clarity LIMS to Illumina NovaSeq X series instruments.

For instructions on validating and troubleshooting the Illumina NovaSeq X Series Integration, refer to NovaSeq X Series Integration v1.2.0 User Interaction, Validation and Troubleshooting.

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

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 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.

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.
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 Integration v1.2.0 User Interaction, Validation and Troubleshooting.
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

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:

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.
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.
Do not add samples to the Ice Bucket or start or complete the step. The integration does this automatically.

Validation Workflow

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.

Create a new Routing Script automation and update it as follows.

For Channel Name, enter limsserver.

For Command Line, enter the following script:

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \
\
--FIELD_NAME 'Sequencing Instrument' \
--FIELD_VALUE 'NovaSeq X Series' \
--WORKFLOW 'NovaSeq X Series v1.1' \
--STEP 'Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"

For Automation Use, select the custom library preparation workflow.

Step 1: Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1)

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.

/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 -exp 'submittedSamples.each { sample -> if ( !sample.name.matches( ::[A-Za-z0-9_-]+:: ) ) { fail ( ::Submitted sample :: + sample.name + :: should only have alphanumeric, dash, and underscore characters. Please rename and try again.:: ) } }' -log {compoundOutputFileLuid1}

Retrieves the list of ACTs that were created in BaseSpace Sequence Hub.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/SIS/SISServices/extensions/automation/novaseqxseries-automation.jar script:retrieve_act_list -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1}

Registers the start time of the step by publishing messages to CLPA through the ICA.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/step_started.groovy'

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).

Saves the details into a file that is available to download. This file is stored in the Analysis Configuration Metadata file placeholder.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/SIS/SISServices/extensions/automation/novaseqxseries-automation.jar script:retrieve_act_info -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -a '{udf:Analysis Configuration Template}'

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.

/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 -t false -h false -exp 'if (!input.hasValue(::Molarity (nM)::)) { fail(::The Molarity cannot be empty.::) ; } else if (input.::Molarity (nM):: <= 0) { fail(::The Molarity cannot be negative or zero.::) ; }' -log {compoundOutputFileLuid1}

Checks that all samples are indexed and for invalid indexes. The indexes applied are the same as the selected ACT.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/SIS/SISServices/extensions/automation/novaseqxseries-automation.jar script:validate_indexes -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -a '{udf:Analysis Configuration Template}'

Assigns the selected ACT to all samples in the step.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/SIS/SISServices/extensions/automation/novaseqxseries-automation.jar script:assign_act -i {stepURI:v2} -u {username} -p {password} -a '{udf:Analysis Configuration Template}' -l {compoundOutputFileLuid1}

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)).
```
nextStep = ::ADVANCE:
```

Automatically triggered on exit of the step, this automation registers completion time of the step by publishing messages to CLPA through ICA.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/step_completed.groovy'

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.

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.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/step_started.groovy'

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.
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).

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/SIS/SISServices/extensions/automation/novaseqxseries-automation.jar script:validate_physical_logical_configurations -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -m PerPool script:validate_analysis_config -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -m PerPool && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar script:validate_same_analyte_udf_value_for_all_inputs -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -f 'Run Mode' -perPool true

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.

if (!input.hasValue(::Molarity (nM)::)) { return; };

Uses Number of Samples in Pool to calculate the volumes needed for the 2 nM intermediate library pools.

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

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.

output.::Bulk Pool Volume (ul):: = 100 * step.::Number of Lanes to Sequence::; output.::NovaSeq X Flowcell Type:: = step.::Flowcell Type::; output.::Final Loading Concentration (pM):: = step.::Final Loading Concentration (pM)::;

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.

input.::Per Sample Volume (ul):: = 2 * output.::Bulk Pool Volume (ul):: / input.::Molarity (nM):: / output.::Number of Samples in Pool::;output.::Total Sample Volume (ul):: = 0;' -t true

Calculates the adjusted per sample volume for the pools.
```
script:calculate_multipool_adjusted_per_sample_volume -t
```

script:evaluateDynamicExpression \
-exp 'if (output.hasValue(::Total Sample Volume (ul)::)) { output.::Total Sample Volume (ul):: = output.::Total Sample Volume (ul):: + input.::Adjusted Per Sample Volume (ul)::; } else { output.::Total Sample Volume (ul):: = input.::Adjusted Per Sample Volume (ul)::; }' -t true \
script:evaluateDynamicExpression \
-exp 'if (output.::Total Sample Volume (ul):: > output.::Bulk Pool Volume (ul)::) { output.::RSB Volume (ul):: = 0 } else { output.::RSB Volume (ul):: = output.::Bulk Pool Volume (ul):: - output.::Total Sample Volume (ul):: }' -t true \

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeqXSeries_Bulk_Pool1.csv -o 1.csv \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeqXSeries_Bulk_Pool2.csv -o 2.csv \
&& cat 1.csv 2.csv > {compoundOutputFileLuid0}.csv \

Resets the Number of Samples in Pool, Total Sample Volume (ul), and Bulk Pool Volume (ul) to null before exiting the step.

-exp 'output.::Number of Samples in Pool:: = ::::; output.::Total Sample Volume (ul):: = ::::; output.::Bulk Pool Volume (ul):: = ::::;' -t true

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.
Sets the next step for samples to ADVANCE, which advances them to the Dilute and Denature (NovaSeq X Series Sequencing v1.1) step.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} -log {compoundOutputFileLuid1} \
script:evaluateDynamicExpression -exp 'if (!input.hasValue(::Molarity (nM)::)){ return; }; output.::Run Mode:: = input.::Run Mode::; nextStep = ::ADVANCE::;'"

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.
Registers the completion time of the step.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/pooling.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/step_completed.groovy'

This automation completes this action by publishing messages to CLPA through ICA.

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.

This script is required for the NovaSeq X Series Sequencing v1.1 workflow to function properly.

/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}

Registers the start time of the step by publishing messages to CLPA through ICA.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/step_started.groovy'

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.
Computes the BP Aliquot Volume (µl) and RSB Volume (µl) required for the dilution of pools to the required final loading concentration.
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.

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} -log {compoundOutputFileLuid1} \
script:evaluateDynamicExpression \
-exp '
if (input.::NovaSeq X Flowcell Type:: == ::10B:: || input.::NovaSeq X Flowcell Type:: == ::1.5B::) {
    output.::NaOH Volume (ul):: = 8.5;
    output.::TT2 Volume (ul):: = 127.5;
    output.::BP Aliquot Volume (ul):: = input.::Final Loading Concentration (pM):: * 170 / (2 * 1000);
    output.::RSB Volume (ul):: = 34 - output.::BP Aliquot Volume (ul)::;
    if (step.::1-2% PhiX Spike-In::) { 
        output.::PhiX Volume (ul):: = 1; output.::PhiX Concentration (pM):: = 300; 
    }else { 
        output.::PhiX Volume (ul):: = ::::; output.::PhiX Concentration (pM):: = ::::; 
    };
} else {
    output.::NaOH Volume (ul):: = 14;
    output.::TT2 Volume (ul):: = 210;
    output.::BP Aliquot Volume (ul):: = input.::Final Loading Concentration (pM):: * 280 / (2 * 1000);
    output.::RSB Volume (ul):: = 56 - output.::BP Aliquot Volume (ul)::;
    if (step.::1-2% PhiX Spike-In::) { 
        output.::PhiX Volume (ul):: = 1.6; output.::PhiX Concentration (pM):: = 300; 
    }
    else { 
        output.::PhiX Volume (ul):: = ::::; output.::PhiX Concentration (pM):: = ::::; 
    };
}'

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeqXSeries_Dilute_Denature_Calculate_Volumes.csv -o {compoundOutputFileLuid0}.csv -q true -destLIMSID {compoundOutputFileLuid0} -l {compoundOutputFileLuid1} \
&& echo; echo 'Calculate Volumes completed successfully.'"

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.

/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 -t false -h false -exp 'output.::Run Mode:: = input.::Run Mode::; nextStep = ::ADVANCE::' -log {compoundOutputFileLuid1}

Automatically triggered on exit of the step, this automation registers the completion time of the step by publishing messages to CLPA through ICA.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/step_completed.groovy'

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)

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:

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} \
script:validate_same_udf_value_for_analytes -f 'Run Mode' -f 'NovaSeq X Flowcell Type' \
script:validate_selected_container \
-fn 'NovaSeq X Flowcell Type' -fv '1.5B' -ct 'Library 2-tube Strip' \
-fn 'NovaSeq X Flowcell Type' -fv '10B' -ct 'Library 8-tube Strip' \
-fn 'NovaSeq X Flowcell Type' -fv '25B' -ct 'Library 8-tube Strip'

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).
This script is required for the NovaSeq X Series Sequencing v1.1 workflow to function properly.
```
/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/SIS/SISServices/extensions/automation/novaseqxseries-automation.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -m PerRun script:validate_analysis_config script:validate_physical_logical_configurations
```

Registers the start time of the step by publishing messages to CLPA through ICA.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/step_started.groovy'

Validate Library Tube Strip Barcode Automation

/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:validate_output_containers -l {compoundOutputFileLuid1} -r 'Library 8-tube Strip:LC[0-9]{7}-L[A-Z]1' -r 'Library 2-tube Strip:LC[0-9]{7}-L[A-Z]2' -max 1

Validate Run Setup and Create Planned Run Automation

Automatically triggered after a selection on the Record Details screen, this automation completes the following actions:

Run Name can only contain alphanumeric, dash, underscore, or period characters. Do not use spaces.

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::);}

Run Name cannot exceed 255 characters.

if (step.::Run Name::.length() > 255) { fail(::Run Name shall not 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.
```
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::); };
```

Generates the sample sheet and creates the planned run in ICA. The sample sheet is attached to the step.

/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}

For more information on sample name character restrictions, refer to NovaSeq X Series Integration v1.2.0 User Interaction, Validation and Troubleshooting.

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.

output.::Run Mode:: = input.::Run Mode::; nextStep = ::ADVANCE::

Automatically triggered on exit of the step, this automation registers the completion time of the step by publishing messages to CLPA through ICA.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/step_completed.groovy'

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

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)

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.
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.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/step_started.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/novaseqxseries/register_novaseqxseries_run.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/novaseqxseries/novaseqxseries_associate_seqrun.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/novaseqxseries/novaseqxseries_seqrun_started.groovy'

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.
Registers the completion time of the step.

This automation completes these actions by publishing messages to CLPA through ICA.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/novaseqxseries/novaseqxseries_seqrun_completed.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/step_completed.groovy'

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
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

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)

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.
- 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.

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.
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.

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.
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.
Start the run. Refer to information on starting a planned run in the NovaSeq X Plus Product Documentation on the NovaSeq X Plus Sequencing System support site.

Start a Sequencing Run in Cloud Run Mode

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.

Enabling Planned Run Generation for Samples Having Duplicate Name with Different Indexes

The library preparation workflow of the samples must be configured to ensure unique derived sample names 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
Resuspension Buffer (RSB)
TT2

Container Types

Library 2-Tube Strip
Library 8-Tube Strip
Tube

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.
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.