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 NovaSeq X Series Integration v1.1.0 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.

ℹ️ 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 NovaSeq X Series Integration v1.1.0 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 v1.0 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 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 v1.1.0 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:

1. 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:

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

   • The secondary analysis of samples is configured using the ACT.

   • The labels applied to the samples are validated against the selected ACT so that samples use valid labels.

2. 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.

3. 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.

4. 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.

5. 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

The Library Prep Validation v2.3.1 workflow allows for validation of the system after installation is complete. For details, refer to NovaSeq X Series Integration v1.1.0 User Interaction, Validation and Troubleshooting.

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

The Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0) step uses ACTs to configure secondary analysis for samples. Each ACT contains details related to the secondary analysis (eg, index adapter kit to use, reference genome setting, and so on). You can select an ACT from a list and assign samples to it. A planned run can have multiple ACTs and this step must be repeated for each ACT that is required for the run. For more information on creating ACTs and assigning samples to them, refer to NovaSeq X Series Integration v1.1.0 User Interaction, Validation and Troubleshooting.

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.

  /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 BSSH.

  /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 (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.

  /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 v1.0)).

  nextStep = ::ADVANCE:

Register Step Completed Automation

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

/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'

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

Field Name	Field Type	Options
Analysis Configuration Template	Text Dropdown	Required Field
Application	Text	Read Only
Application Version	Text	Read Only
Index Adapter Kit	Text	Read Only
Library Prep Kit	Text	Read Only
Reference Genome	Text	Read Only
Secondary Analysis Mode	Text	Read Only

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)

Field Name	Field Type	Options	Additional Options and Dropdown Items
Molarity (nM)	Numeric		Decimal places displayed = 2
ACT Name	Text	Read Only

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.

Register Step Started Automation

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

/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 (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).

/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) 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.

  if (step.::Flowcell Type:: == ::B3::) { 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.

  input.::Per Sample Volume (ul):: = 2 * output.::Bulk Pool Volume (ul):: / input.::Molarity (nM):: / output.::Number of Samples in 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).

  if (step.hasValue(::% PhiX (2.0nM) Spike-In::)) { step.::PhiX Volume (ul):: = step.::% PhiX (2.0nM) Spike-In:: / 100 * step.::Final Loading Volume (ul)::; };

• 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 v1.0) 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::;'

Register Pools & Register Step Completed Automation

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.

/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.

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

Field Name	Field Type	Options	Additional Options and Dropdown Items
% PhiX (2.5 nM) Spike-In	Numeric		Range = 0–100
Final Loading Concentration (pM)	Numeric Dropdown	Required Field Custom Entries
Final Loading Volume (ul)	Numeric	Required Field	Default = 200
Flowcell Type	Text	Required Field Read Only	Default = B3
Minimum Per Sample Volume (ul)	Numeric	Required Field	Decimal places displayed = 2 Default = 2
Number of Lanes to Sequence	Numeric	Required Field	Range = 1–10 Decimal places displayed = 0
PhiX Volume (ul)	Numeric	Read Only	Hidden Decimal places displayed = 1 Used for Calculate Volume script when there is PhiX spike-in. ⚠️ Do not remove, rename, or modify this field as it can cause the integration to break.

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)

Field Name	Field Type	Options	Additional Options and Dropdown Items
Final Loading Concentration (pM)	Numeric Dropdown	Required Field Custom Entries	Decimal places displayed = 0 Presets 225 400
RSB Volume (ul)	Numeric	Read Only	Decimal places displayed = 2
NovaSeq X Flowcell Type	Text	Required Field Read Only	Default = B3

Step 3: Dilute and Denature (NovaSeq X Series Sequencing v1.0)

The Dilute and Denature (NovaSeq X Series Sequencing v1.0) step allows you to dilute pooled samples with the addition of RSB.

The following automations are configured on this step:

• Validate Inputs Flowcell Type and Register Step Started²

• Calculate Volumes

• Set Next Step

• Register Step Completed¹

¹ These automations are required for CLPA support only.

² These automations are required for the NovaSeq X Series Sequencing v1.0 workflows and contain additional logic needed for CLPA support. If you would like to remove support for CLPA, contact Illumina Support.

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

Validate Inputs Flowcell Type and Register Step Started

Automatically triggered on entry to the step, this automation completes the following actions:

• Checks that the specified flow cell type is valid.

  /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 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.

  /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:: == ::B3::) { output.::NaOH Volume (ul):: = 10; output.::TT2 Volume (ul):: = 150; }; output.::BP Aliquot Volume (ul):: = input.::Final Loading Concentration (pM):: * 200 / (2 * 1000); output.::RSB Volume (ul):: = 40 - output.::BP Aliquot Volume (ul)::;'

• 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}

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.

/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}

Register Step Completed Automation

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

/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'

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)

Field Name	Field Type	Options	Additional Options and Dropdown Items
BP Aliquot Volume (ul)	Numeric	Read Only	Decimal places displayed = 0
NaOH Volume (ul)	Numeric	Read Only	Decimal places displayed = 2
RSB Volume (ul)	Numeric	Read Only	Decimal places displayed = 2
TT2 Volume (ul)	Numeric	Read Only	Decimal places displayed = 2

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.

  /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:validateSampleCount -min 8 -max 8

• 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).

  /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 PerRun script:validate_analysis_config -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -m PerRun && /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 false

• 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.

/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}-LC1' -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. If you are in Cloud run mode, the automation 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 v1.1.0 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 v1.0) step.

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

Register Step Completed Automation²

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

/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 v1.0) 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

Field Name	Field Type	Options	Additional Options and Dropdown Items
Cloud Run ID	Text	Read Only	Hidden Used for creating a planned run in ICA. ⚠️Do not remove, rename, or modify this field as it can cause the integration to break.
Index 1 Cycles	Numeric Dropdown	Required Field Custom Entries	Range = 0–20 Presets 0 6 8
Index 2 Cycles	Numeric Dropdown	Required Field Custom Entries	Range = 0–20 Presets 0 6 8
Output Folder	Text
Read 1 Cycles	Numeric Dropdown	Required Field Custom Entries	Range = 1–251 Presets 51 101 151
Read 2 Cycles	Numeric Dropdown	Required Field Custom Entries	Range = 0–251 Presets 51 101 151
Run Mode	Text Dropdown	Required Field	Presets Local Cloud
Run Name	Text	Required Field

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.

/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'

Register NovaSeq X RunCompleted & Register Step Completed Automation¹

Automatically triggered on exit of the step, this automation completes the following actions:

• Registers the sequencing run completion time, run status, and run metrics. This script requires the Cloud Run ID custom field from the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0) step.

• 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.

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

Field Name	Source File	Corresponding Field in Source File	Additional Information
Current Cycle	RunCompletionStatus.xml	CompletedReads	Visible on Record Details screen. Calculated based on the CompletedReads field.
Current Read	RunCompletionStatus.xml RunParameters.xml	CompletedReads PlannedReads	Visible on Record Details screen. Calculated based on the CompletedReads field in RunCompletionStatus.xml and PlannedReads field in RunParameters.xml.
Flow Cell Expiration Date	RunParameters.xml	ExpirationDate	Visible on Record Details screen. Corresponds to the value in the ExpirationDate field under the ConsumableInfo field for FlowCell Type.
Flow Cell ID	RunParameters.xml	SerialNumber	Visible on Record Details screen. Corresponds to the value in the SerialNumber field under the ConsumableInfo field for FlowCell Type.
Flow Cell Lot Number	RunParameters.xml	LotNumber	Visible on Record Details screen. Corresponds to the value in the LotNumber field under the ConsumableInfo field for FlowCell Type.
Flow Cell Part Number	RunParameters.xml	PartNumber	Visible on Record Details screen. Corresponds to the value of the PartNumber field under the ConsumableInfo field for FlowCell Type.
Flow Cell Side	RunParameters.xml	Side	Visible on Record Details screen.
Flow Cell Type	RunParameters.xml	FlowCellType	Visible on Record Details screen.
Instrument Control Software Version	RunParameters.xml	SystemSuiteVersion	Visible on Record Details screen.
Instrument ID	RunParameters.xml	InstrumentSerialNumber	Visible on Record Details screen.
Instrument Type	RunParameters.xml	InstrumentType	Visible on Record Details screen.
Output Folder	RunParameters.xml	OutputFolder	Visible on Record Details screen.
Library Tube Barcode	RunParameters.xml	SerialNumber	Visible on Record Details screen. Corresponds to the value of the SerialNumber field under the ConsumableInfo field for SampleTube Type.
Library Tube Lot Number	RunParameters.xml	LotNumber	Hidden Corresponds to the value of the LotNumber field under the ConsumableInfo field for SampleTube Type.
Run End Time	RunCompletionStatus.xml	RunEndTime	Hidden
Run Name	RunParameters.xml	ExperimentName	Visible on Record Details screen.

Other Clarity LIMS Master Step Fields

Field Name	Description	Additional Information
Run Status	Presets RunStarted RunCompletedSuccessfully RunAbortedByUser RunErroredOut Set by the integration service.	Visible on Record Details screen.
Sequencing Log	Set by the integration service during the sequencing run.	Visible on Record Details screen.
BaseSpace Run ID	Received from the message sent to the integration service.	Hidden
ICA Project ID	Received from the message sent to the integration service.	Hidden
Instrument Run ID	Received from the message sent to the integration service.	Hidden
Run Start Time	Received from the message sent to the integration service.	Hidden

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.

⚠️ 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

Configure the run mode. Select Cloud run storage options under Run Settings.

Start a Sequencing Run in Local Run or Cloud Run Modes

You can start the sequencing run in BSSH for both the Local and Cloud modes. Make sure that you log in using the account that was used during the NovaSeq X Series integration package setup. You must also import the sample sheet created from the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0) step for the Local Run Mode.

Start a Sequencing Run in Local Run Mode

Follow instructions to start a sequencing run with local secondary analysis in https://support-docs.illumina.com/IN/NovaSeqX/Content/IN/NovaSeqX/StartManualRun.htm#Start.

Start a Sequencing Run in Cloud Run Mode

Follow instructions to start a sequencing run with cloud secondary analysis in https://support-docs.illumina.com/IN/NovaSeqX/Content/IN/NovaSeqX/StartPlannedRun.htm.

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 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.