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NovaSeq X Series v1.3.0

The integration includes the following:

Preconfigured NovaSeq X Series Sequencing v1.2 workflow that maps to lab protocols and instrument runs.
Preconfigured protocols:
- Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2)
- Make Bulk Pool (NovaSeq X Series Sequencing v1.2)
- Dilute and Denature (NovaSeq X Series Sequencing v1.2)
- Load to Library Tube Strip (NovaSeq X Series Sequencing v1.2)
- AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2)
- AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.2)
Automatic validation of run setup information before sample sheet generation or planned run creation.
Automated generation of a samplesheet. The samplesheet will be used to start the sequencing run on NovaSeq X Series Control Software via Local run mode.
Automated creation of a planned run on Illumina Connected Analytics (ICA). The planned run created will be retrieved by NovaSeq X Series Control Software to start run via Cloud run mode.
Automated tracking of the NovaSeq X Series sequencing run and parsing of run statistics from BaseSpace Sequence Hub (BSSH) into the LIMS.
Automated tracking of the NovaSeq X Series analysis run and high level analysis summary from BaseSpace Sequence Hub (BSSH) into the LIMS.
Support for configuration of multiple secondary analyses with multiple library prep kit (LPK) and index adapter kit (IAK) for the planned run. The analysis are either performed by the ICA in Cloud mode or by DRAGEN on-premise in Local mode.
(Optional) Preconfigured Library Prep Validation v2.3.5 workflow used for validation purposes only. The workflow contains a single-step protocol that models the library prep workflow required to produce libraries tagged with index sequences. At the end of the step, these libraries are routed to NovaSeq X Series Sequencing v1.2 workflow.
(Optional) Clarity LIMS Product Analytics (CLPA) integration which makes Clarity data available to the ICA service. CLPA integration installation and configuration is required. Refer to Clarity LIMS Product Analytics Integration v1.4 Configuration for details on configuring and using CLPA.

Release Notes

Last Updated: December 2024

Release Date: December 2024

These release notes describe the key changes to software components for the Clarity LIMS NovaSeq X Series Integration Package v1.3.0.

Prerequisites

BaseSpace Sequence Hub account that has the Enterprise or Professional subscription

Compatibility

Refer to Compatibility under Instruments & Integrations.

New Features & Updates

Updates on automations:
- Register NovaSeq X RunCompleted and Register Step Completed
- Register Step Started and Register NovaSeq X Run and Register NovaSeq X Run Association and Register NovaSeq X RunStarted
- Validate Flowcell Inputs and Validate Analysis Configurations and Register Step Started
- Validate Reagent Labels and Apply Selected ACT to Samples and Set Next Step
- Validate Run Setup and Create Planned Run
- Validate Sample Names and Retrieve Analysis Configuration Template List and Register Step Started
Updates on configuration:
- Library 2-tube Strip and Library 8-tube Strip containers
- BP Aliquot Volume (ul) derived sample global field
In the Load to Library Tube Strip step,
- the master step field Cloud Run ID has been renamed to Planned Run ID.
- a new step field Instrument Type is added.
In the AUTOMATED - Sequencing Run step,
- the Derived Sample Generation configuration setting has been updated to Fixed with 1 Derived Sample generated.
- removed unused RTA Version step field.
- under Record Details milestone, the sample table is now expanded by default and run metrics in Sample Details table have been updated.
In the AUTOMATED - Analysis Run step,
- the Library Tube Strip Barcode hidden master step field has been replaced with the App Session ID hidden field.
- the Naming Convention under Step Type has been updated to {SubmittedSampleName}{AppliedReagentLabels}.
- under Record Details milestone, the sample table is now expanded by default.

Refer to NovaSeq X Series Integration v1.3.0 Configuration for details on changes in automations, configuration and run metrics. Manual upgrade instructions from NovaSeq X Series Sequencing v1.1 workflow to v1.2 workflow are provided in NovaSeq X Series Integration v1.3.0 Manual Upgrade.

Known Limitations

Tracks only the analysis configured with the planned run. Does not track analysis requeue or analysis triggered externally with the same planned run.
The Assign Analysis Configuration Template step does not support pooled libraries.
The integration does not support --bcl-sampleproject-subdirectories option of BCL Convert.

Known Issues

On the Make Bulk Pool step, the log displays a warning when the Calculate Volume automation is triggered and at least one pool consists of multiple inputs. This issue is caused by the output custom field being reset multiple times at the end of the automation. This issue does not affect the Calculate Volume automation functionality.
For BCL Convert only analysis, the high level analysis summary information is not available. Upon analysis successfully completed, an empty file is being attached at the AUTOMATED - Analysis Run step and the step is automatically completed.

Documentations

The NovaSeq X Series Integration v1.3.0 User Interaction, Validation and Troubleshooting guide follows exactly that of NovaSeq X Series Integration v1.2.0 with the following updates:

Version updates:
- NovaSeq X Series Integration Package version updated to v1.3.0.
- NovaSeq X Series Sequencing workflow version updated to v1.2.
- Library Prep Validation workflow version updated to v2.3.5.
Automation name update:
- Validate Flowcell Inputs and Validate Analysis Configurations and Register Step Started automation updated to Validate Flowcell Inputs, Analysis Configurations and Register Step Started
Updates of run metrics displayed in Sample Details table of AUTOMATED - Sequencing Run step. Refer to NovaSeq X Series Integration v1.3.0 Configuration for the updated metrics.
Update of analysis configuration supported. Refer to Set Up Secondary Analysis in NovaSeq X Series documentation for limits of the number of analysis application and reference genome combinations supported.

Configuration

The Illumina NovaSeq X Series Integration Package v1.3.0 supports the integration of Clarity LIMS to Illumina NovaSeq X series instruments.

The configuration provided in this integration has been established to support NovaSeq X Series lab processes. Any configuration changes to protocols or workflows (including renaming protocols, steps, and fields) could break the process.

Prerequisites and Assumptions

The NovaSeq X Series Integration v1.3.0 requires a valid BaseSpace Sequence Hub account with an Enterprise or Professional subscription.
The required analysis configuration templates (ACTs), have been created on BaseSpace Sequence Hub.
⚠ ACT names created in BaseSpace Sequence Hub must not have leading or trailing spaces.
The index adapter kit configured in the ACT must be created as label group in Clarity LIMS and used in the library preparation step for indexing the samples.
Samples must go through the library preparation and quantification process before they enter the NovaSeq X Series Sequencing v1.2 workflow. It is assumed that the following steps have completed before samples are assigned to the workflow:
- Samples have been accessioned into Clarity LIMS.
- Samples have been run through QC and library prep.
- Samples have the Molarity (nM) global field set to a value.
  This value is required for the Calculate Volumes automation in the Make Bulk Pool step.
⚠ 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 v1.3.0 includes the following workflows:

NovaSeq X Series Sequencing v1.2
Library Prep Validation v2.3.5 (optional, but recommended for validation purposes)

The following describes the protocols and steps included in these workflows.

Library Prep Validation v2.3.5 Workflow

Protocol 1: Library Prep Validation v2.3.5

Purpose:

Included for validation purposes only, this protocol models the library prep steps required to advance samples to the NovaSeq X Series Sequencing v1.2 workflow.
⚠ 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.
The protocol contains a single step - Library Prep Validation v2.3.5. After this step, a routing script sends the samples to the first step of the NovaSeq X Series Sequencing v1.2 workflow (Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2)).

Steps:

Library Prep Validation v2.3.5

NovaSeq X Series Sequencing v1.2 Workflow

Protocol 1: NovaSeq X Series Sequencing v1.2

Purpose:

This protocol models the lab processes of run setup for starting a NovaSeq X Series sequencing and secondary analysis run.

Steps:

Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2)
- The secondary analysis of samples is configured using the ACT.
- The labels applied to the samples are validated against the selected ACT so that samples use valid labels.
Make Bulk Pool (NovaSeq X Series Sequencing v1.2)
- The Calculate Volume script assists with pooling samples and adding resuspension buffers and reagents.
- The Validate Analysis Configurations automation validates the ACT of pooled samples to make sure that the secondary analysis configurations remain valid after pooling.
Dilute and Denature (NovaSeq X Series Sequencing v1.2)
- The Calculate Volume script helps denature and dilute samples from step 2 to the final loading concentration.
Load To Library Tube Strip (NovaSeq X Series Sequencing v1.2)
- The library pools from step 3 are ready to be loaded to the NovaSeq X Series library tube strip.
- Validates run and analysis information.
- Generates the sample sheet and creates the planned run, depending on the run mode.
AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2)
- The step is fully automated and starts and completes automatically after the sequencing run is started and completed on the instrument side.
- The sequencing run metadata (e.g., run configuration, primary run metrics, and so on) are recorded automatically.
AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.2)
- The step is fully automated and starts and completes automatically after the analysis run is started and completed in ICA.
- The analysis run metadata (eg, status and high level analysis summary) are recorded automatically.

Validation Workflow

The Library Prep Validation v2.3.5 workflow allows for validation of the system after installation is complete. This workflow can be replaced by other custom library preparation workflows.

Configure a custom library preparation workflow as follows.

Select Configuration, then select Lab Work.
Add a Routing Script automation to the last Master Step of the library preparation workflow.
Select Configuration, then select Automation.

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 v1.2' \
--STEP 'Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"

For Automation Use, select the custom library preparation workflow.

Step 1: Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2)

The Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2) step uses ACTs to configure secondary analysis for samples. Each ACT contains details related to the secondary analysis (e.g., index adapter kit to use, reference genome setting, and so on). You can select an ACT from a list and assign samples to it. A planned run can have multiple ACTs and this step must be repeated for each ACT that is required for the run.

Validate Sample Names, Retrieve ACT List and Register Step Started Automation¹

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

Checks that submitted sample names contain only alphanumeric, dash, and underscore characters.

/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}'

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 v1.2)).
```
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 v1.2 workflows and contain additional logic needed for CLPA support. If you would like to remove support for CLPA, contact Illumina Support.

² These automations are required for CLPA support only.

All remaining automations are required for the NovaSeq X Series Sequencing v1.2 workflows.

Master Step Fields

The following table lists the configuration details for fields that are defined on the Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2) step.

Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2) Master Step Field Configuration

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 v1.2) step.

Global Field Configuration (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 v1.2)

Samples are pooled in the Make Bulk Pool (NovaSeq X Series Sequencing v1.2) step. You can manually create working pools based on the final loading concentration required.

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 v1.2) 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 v1.2 workflows.

Master Step Fields

The following table lists configuration details for the fields that are defined on the Make Bulk Pool (NovaSeq X Series Sequencing v1.2) step.

Make Bulk Pool (NovaSeq X Series Sequencing v1.2) Master Step Field Configuration

Field Name

Field Type

Options

Additional Options and Dropdown Items

Final Loading Concentration (pM)

Numeric Dropdown

Required Field
Custom Entries

Dropdown Items
- 90
- 140
- 150
- 160
- 180
Range from 0
Decimal places displayed: 0

Flowcell Type

Text

Required Field

Dropdown Items
- 1.5B
- 10B
- 25B

Minimum Per Sample Volume (ul)

Numeric

Required Field

Decimal places displayed: 2
Default: 2

Number of Lanes to Sequence

Numeric

Required Field

Range: 1–10
Decimal places displayed: 0

Global Fields

The following table lists the global custom fields that are configured to display on the Make Bulk Pool (NovaSeq X Series Sequencing v1.2) step.

Global Custom Fields Configuration (Derived Sample)

Field Name

Field Type

Options

Additional Options and Dropdown Items

Final Loading Concentration (pM)

Numeric Dropdown

Required Field
Custom Entries

Decimal places displayed: 0
Dropdown Items
- 225
- 400

RSB Volume (ul)

Numeric

Read Only

Decimal places displayed: 2

NovaSeq X Flowcell Type

Text Dropdown

Required Field
Read Only

Dropdown Items
- 1.5B
- 10B
- 25B

Step 3: Dilute and Denature (NovaSeq X Series Sequencing v1.2)

The Dilute and Denature (NovaSeq X Series Sequencing v1.2) step allows you to dilute pooled samples with the addition of RSB.

Validate Inputs Flowcell Type and Register Step Started¹

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

Checks that the specified flow cell type is valid.

⚠ This script is required for the NovaSeq X Series Sequencing v1.2 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.
ℹ The BP Aliquot Volume value is rounded up to the nearest µl. The value in the calculation file, however, is reflected with up to two decimal places.

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

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 v1.2) 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 v1.2 workflows.

Global Fields

The following table lists the global custom fields that are configured to display on the Dilute and Denature (NovaSeq X Series Sequencing v1.2) step.

Global Field Configuration (Derived Sample)

Field Name

Field Type

Options

Additional Options and Dropdown Items

BP Aliquot Volume (ul)

Numeric

Read Only

Decimal places displayed = 1

NaOH Volume (ul)

Numeric

Read Only

Decimal places displayed = 2

RSB Volume (ul)

Numeric

Read Only

Decimal places displayed = 2

TT2 Volume (ul)

Numeric

Read Only

Decimal places displayed = 2

Step 4: Load to Library Tube Strip (NovaSeq X Series Sequencing v1.2)

The Load to Library Tube Strip (NovaSeq X Series Sequencing v1.2) step allows you to scan the library tube strip barcode into Clarity LIMS. Then, you can place the working pools into the library tube strip used in the NovaSeq X Series run. This step also does the following actions:

Validates the run setup and analysis information.
Generates the sample sheet file.
Creates a planned run on Illumina Connected Analytics (ICA), depending on the selected run mode.

Validate Flowcell Inputs, Analysis Configurations and Register Step Started Automation¹

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

Checks that the selected destination container types on the Load to Library Tube Strip step matches the selected flow cell type of the samples. The 1.5B flow cell is compatible with the library 2-tube strip and the 10B and 25B flow cells are compatible with the library 8-tube strip.

/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 v1.2 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

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, periods 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.

Set Next Step Automation

Automatically triggered on exit of the Record Details screen, this automation completes the following actions:

Copies the Run Mode from input to output.
Sets the next step for samples to ADVANCE, advancing them to the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2) step.

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 v1.2 workflows.

Master Step Fields

The following table shows the master step fields that are configured on the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.2) step. These fields are required for sample sheet generation and planned run creation in ICA.

Load to Library Tube Strip (NovaSeq X Series Sequencing v1.2) Master Step Field Configuration

Field Name

Field Type

Options

Additional Options and Dropdown Items

Planned Run ID

Text

Read Only

Hidden

ℹ Used for creating a planned run in ICA.

Index 1 Cycles

Numeric Dropdown

Required Field
Custom Entries

Range = 0–20
Presets
- 0
- 6
- 8

Index 2 Cycles

Numeric Dropdown

Required Field
Custom Entries

Range = 0–20
Presets
- 0
- 6
- 8

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

Instrument Type

Text

Read Only

Step 5: AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2)

Do not add samples to the Ice Bucket or start and complete the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2) step. The integration does this automatically.

Data from the run is parsed back to Clarity LIMS. In this step, pooled samples in the library tube strip are sequenced on the NovaSeq X Series instrument.

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 Planned Run ID custom field from the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.2) 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 v1.2) 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 Strip Barcode

RunParameters.xml

SerialNumber

Visible on Record Details screen.
Corresponds to the value of the SerialNumber field under the ConsumableInfo field for SampleTube Type.

Library Tube Lot Number

RunParameters.xml

LotNumber

Hidden
Corresponds to the value of the LotNumber field under the ConsumableInfo field for SampleTube Type.

Run End Time

RunCompletionStatus.xml

RunEndTime

Hidden

Run Name

RunParameters.xml

ExperimentName

Visible on Record Details screen.

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
% PF
% Aligned R1
% Aligned R2
% Occupied
% Phasing R1
% Phasing R2
% Prephasing R1
% Prephasing R2
Intensity Cycle 1 R1
Intensity Cycle 1 R2

At the end of the step, the pools of samples are automatically removed from the step. The step completes automatically when Run Status is RunCompletedSuccessfully.

Step 6: AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.2)

Do not add samples to the Ice Bucket or start and complete the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2) step. The integration does this automatically.

Data from the analysis is parsed back to Clarity LIMS. In this step, the secondary analysis configured using the Analysis Configuration Template (ACT) is performed in Illumina Connected Analytics (ICA). There are no automations associated with this step.

Master Step Fields

The following tables show the master step fields that are configured on the AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.2) step.

AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.2) Master Step Field Information

Field Name

Description

Additional Information

Analysis Status

Presets
- Started
- Completed
- Failed
- NeedsAttention
- Stopped
- TimedOut
- CompletedWithErrors
Set by the integration service.

Visible on Record Details screen.

Analysis Result Location

Received from the message sent to the integration service.

Visible on Record Details screen.

App Session ID

Identified by the integration service using information from RunParameters.xml.

Hidden

How the Integration Works

The following information summarizes how the NovaSeq X Series integration works.

After the Validate Run Setup and Create Planned Run automation is triggered on the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.2) step, the run parameters entered in the Run Details screen are sent to Illumina Connected Analytics (ICA). The analysis configuration parameters from the selected ACTs are also sent to ICA. ICA validates the run and analysis configuration. If the validation fails, ICA sends an error message to Clarity LIMS. If the validation passes, ICA generates the sample sheet and sends it back to Clarity LIMS. ICA also creates the planned run based on the selected run mode (e.g., Local or Cloud).
- For the Local run mode, the sample sheet is generated and stored in Clarity LIMS. This sample sheet contains the run and analysis configuration information required to start the run on the NovaSeq X Series instrument.
- For the Cloud run mode, the planned run is created in ICA. The planned run contains the run and analysis configuration information required to start the run on the instrument. The sample sheet is generated and stored in Clarity LIMS for reference purposes.
When the sequencing run starts on the instrument, the NovaSeq X Series Control Software notifies BaseSpace Sequence Hub. BaseSpace Sequence Hub monitors the run and notifies Clarity LIMS via the integration service. The events are processed and the integration service retrieves the run information from BaseSpace Sequence Hub. This information is used to populate the custom fields in the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2) step.
Other run events follow the same information flow. When sequencing is complete, the control software uploads the sequencing run data (primary metrics) and associated files to ICA. Then, Clarity LIMS retrieves the primary metrics and uses them to populate the fields in the Sample Details table (e.g., % Error Rate R1). The custom fields (e.g., Run Status, Current Read, and so on) on the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2) step are updated using the run related information. If the sequencing run is successfully completed, the step automatically completes.
The integration tracks the analysis events and results in the AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.2) step. You must log in to BaseSpace Sequence Hub to retrieve the cloud analysis results. For local analysis, the secondary analysis results are in the external storage configured in Illumina Run Manager. The external storage information is found in the External Storage for Analysis Results configuration settings in Illumina Run Manager.

If a planned run with the same sample name and project name (case-insensitive) has been created previously, the sample sheet generated from the Validate Run Setup and Create Planned Run automation can reflect the original case of the previous sample name. This can cause validation errors for analysis configurations with sample-level settings. To resolve this issue, change the sample name or the project name on Clarity LIMS and run the automation again.

Start a Sequencing Run on NovaSeq X Series Instrument

The following sections outline the steps to start a Clarity LIMS cloud or local run on the NovaSeq X Series instrument.

Run Mode Configuration

In the NovaSeq X Series Control Software, log in to the instrument using an account that has permissions for configuring settings.
On the menu, select Settings, then select Cloud settings.
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 v1.2) step. Refer to information on creating a local run in the NovaSeq X Plus Product Documentation on the NovaSeq X Plus Sequencing System support site.
When the sample sheet is selected, the contents are validated. After the validation passes, select Next.
Select Save As Planned.
Run and analysis data is parsed and auto-populates in the list of runs. For more information on planning a NovaSeq X Series run, refer to the Plan a NovaSeq X Series Run section of the BaseSpace Sequence Hub Online Help at help.basespace.illumina.com and the NovaSeq X Plus Product Documentation on the NovaSeq X Plus Sequencing System support site.
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 v1.2) and AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.2) steps must not be manually started or completed. These steps are fully automated and the sequencing service does not update samples correctly if they have been manually started.
For the automated run to start successfully, you must select Validate Run Setup and Create Planned Run in the Load to Library Tube step.

Manual Upgrade

NovaSeq X Series Integration Package v1.3.0 includes modifications to the existing NovaSeq X Series Workflows to support these changes:

Update existing containers (Library 2-tube Strip and Library 8-tube Strip)
Update global field (BP Aliquot Volume (ul))
Update existing automations
Update Load to Library Tube Strip master step global fields
Update AUTOMATED - Sequencing Run master step and step, including run metrics displayed
Update AUTOMATED - Analysis Run master step
Upgrade Clarity LIMS Product Analytics Integration (if enabled)

If you do not want to use the latest workflow (NovaSeq X Series Sequencing v1.2) available in IPP v2.10.0, then update your existing workflow configuration manually using the following information as required.

1. Update Existing Containers

Library 2-tube Strip container

On the Configuration tab, select Consumables
Select Containers.
Select the existing Library 2-tube Strip container.
Update Naming of Rows to Numeric.
Update Start at of Rows to 1.

Library 8-tube Strip container

Repeat the update instructions of Library 2-tube Strip container above for the existing Library 8-tube Strip container.

2. Update Global Field (BP Aliquot Volume (ul))

The derived sample global field BP Aliquot Volume (ul) is used in Calculate Volumes automation in Dilute and Denature step.

On the configuration tab, select Custom Fields.
Select Global Fields.
Expand Derived Sample.
Select BP Aliquot Volume (ul).
Update Decimal Places Displayed from 0 to 1.

3. Update Automations

There is a minor update of Command Line for Validate Run Setup and Create Planned Run automation, as well as, renaming of several automations to improve readability and provide more clarity of the automation function.

Update Validate Run Setup and Create Planned Run Automation

Updates to the Command Line is to improve the error message.

On the configuration tab, select Automation.
Select Validate Run Setup and Create Planned Run automation.
Update the existing Command Line as follows. Changes are in red.
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \ bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \ script:evaluateDynamicExpression \ script:evaluateDynamicExpression \ -exp 'if (!step.::Run Name::.matches(::[a-zA-Z0-9-_]+[a-zA-Z0-9-_. ]*::)) { fail(::Run Name contains prohibited characters. Please check to make sure Run Name only contains alphanumeric characters, spaces, dashes, periods and underscores. Run Name must start with alphanumeric, a dash or an underscore::); }; if (step.::Run Name::.length() > 255) { fail(::Run Name shall not exceed 255 characters.::); }; if (step.::Index 2 Cycles:: > 0 && step.::Index 1 Cycles:: == 0) { fail(::Index 1 Cycles cannot be zero if Index 2 Cycles is non-zero::); };' \ -exp 'if (!step.::Run Name::.matches(::[a-zA-Z0-9-_]+[a-zA-Z0-9-_. ]*::)) { fail(::Run Name contains prohibited characters. Please check to make sure Run Name only contains alphanumeric characters, spaces, dashes, periods and underscores. Run Name must start with alphanumeric, a dash or an underscore::); }; if (step.::Run Name::.length() > 255) { fail(::Run Name shall not exceed 255 characters.::); }; if (step.::Index 2 Cycles:: > 0 && step.::Index 1 Cycles:: == 0) { fail(::Index 1 Cycles cannot be zero if Index 2 Cycles is non-zero::); };' \ -log {compoundOutputFileLuid1} \ -log {compoundOutputFileLuid1} \ -t true \ -t true \ && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/SIS/SISServices/extensions/automation/novaseqxseries-automation.jar script:generate_sample_sheet -i {stepURI:v2} -u {username} -p {password} -c {compoundOutputFileLuid0} -l {compoundOutputFileLuid1}"

[Optional] Rename Automations

On the configuration tab, select Automation.
Rename the automations as specified in the table below.

Old Automation Name

New Automation Name

Used in Step

AUTOMATED - Sequencing Run

Register Step Started and Register NovaSeq X Run and Register NovaSeq X Run Association and Register NovaSeq X RunStarted

AUTOMATED - Sequencing Run

Validate Reagent Labels and Apply Selected ACT to Samples and Set Next Step

Apply Selected ACT to Samples and Set Next Step

Assign Analysis Configuration Template

Validate Flowcell Inputs and Validate Analysis Configurations and Register Step Started

Validate Flowcell Inputs, Analysis Configurations and Register Step Started

Load to Library Tube Strip

Validate Sample Names and Retrieve Analysis Configuration Template List and Register Step Started

Validate Sample Names, Retrieve ACT List and Register Step Started

Assign Analysis Configuration Template

4. Update Load to Library Tube Strip Step

Rename Cloud Run ID to Planned Run ID

On the configuration tab, select Custom Fields.
Select Master Step Fields
Expand Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1).
Rename Cloud Run Id to Planned Run ID.

Add Instrument Type

New instrument type NovaSeq X is now supported.

On the configuration tab, select Custom Fields.
Select Master Step Fields.
Expand Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1).
Add Instrument Type field as specified in the image.

On the configuration tab, select Lab Work.
Select Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1) step.
Under Record Details, add Instrument Type to Master Step Fields.

5. Update AUTOMATED - Sequencing Run Step

There are configuration updates on the AUTOMATED - Sequencing Run master step and step, as well as updates to the run metrics displayed.

Update Master Step Configuration

On the configuration tab, select Lab Work.
Select AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) master step.
Under Step Type, update Derived Sample Generation to Fixed with 1 Derived Sample generated.
Under Step Type, update Naming Convention to Lane {InputWellLocation:0,1}

Remove Unused RTA Version Master Step Field

On the configuration tab, select Custom Fields.
Select Master Step Fields.
Expand AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1).
Delete RTA Version.

[Optional] Expand Sample Table By Default

On the configuration tab, select Lab Work.
Select AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1) step.
Select Record Details milestone.
Under Sample Table, update Sample Display Default to Expand.

Update Run Metrics

Reflect Reads PF Metrics at Run Level

On the configuration tab, select Custom Fields.
Select Global Fields.
Add Reads PF field as specified in the image.
Select Save.
On the configuration tab, select Lab Work.
Select AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step.
Under Record Details, remove Reads PF R1 and Reads PF R2 from Table Columns.
Add Reads PF to Table Columns.

Reflect % PF Metrics at Run Level

On the configuration tab, select Custom Fields.
Select Global Fields.
Add % PF field as specified in the image.
Select Save.
On the configuration tab, select Lab Work.
Select AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step.
Under Record Details, remove %PF R1 and %PF R2 from Table Columns.
Add % PF to Table Columns.

Add % Occupied

On the configuration tab, select Custom Fields.
Select Global Fields.
Expand Container.
Add % Occupied field as specified in the image.

On the configuration tab, select Lab Work.
Select AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step.
Under Record Details, add % Occupied to Table Columns.

Remove Cluster Density R1 and Cluster Density R2

On the configuration tab, select Lab Work.
Select AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step.
Under Record Details, remove Cluster Density R1 and Cluster Density R2 from Table Columns.

On the configuration tab, select Custom Fields.
Select Global Fields.
Expand Container.
Delete Cluster Density R1 and Cluster Density R2.

6. Update AUTOMATED - Analysis Run Master Step

Update Master Step Configuration

On the configuration tab, select Lab Work.
Select AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1) master step.
Under Step Type, update Naming Convention to {SubmittedSampleName} {AppliedReagentLabels}

Rename Library Tube Strip Barcode to App Session ID

On the configuration tab, select Custom Fields.
Select Master Step Fields.
Expand AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1).
Rename Library Tube Strip Barcode to App Session ID.

7. Upgrade Clarity LIMS Product Analytics Integration (if enabled)

The changes are due to updates on container configuration.

Upgrade to Clarity LIMS Product Analytics Integration v1.4.0
Restart the CLPA integration service
```
systemctl restart clarity_upa
```

Configuration

The Illumina NovaSeq X Series Integration Package v1.3.0 supports the integration of Clarity LIMS to Illumina NovaSeq X series instruments.

Prerequisites and Assumptions

The NovaSeq X Series Integration v1.3.0 requires a valid BaseSpace Sequence Hub account with an Enterprise or Professional subscription.
The required analysis configuration templates (ACTs), have been created on BaseSpace Sequence Hub.
⚠ ACT names created in BaseSpace Sequence Hub must not have leading or trailing spaces.
The index adapter kit configured in the ACT must be created as label group in Clarity LIMS and used in the library preparation step for indexing the samples.
Samples must go through the library preparation and quantification process before they enter the NovaSeq X Series Sequencing v1.2 workflow. It is assumed that the following steps have completed before samples are assigned to the workflow:
- Samples have been accessioned into Clarity LIMS.
- Samples have been run through QC and library prep.
- Samples have the Molarity (nM) global field set to a value.
  This value is required for the Calculate Volumes automation in the Make Bulk Pool step.
⚠ 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 v1.3.0 includes the following workflows:

NovaSeq X Series Sequencing v1.2
Library Prep Validation v2.3.5 (optional, but recommended for validation purposes)

The following describes the protocols and steps included in these workflows.

Library Prep Validation v2.3.5 Workflow

Protocol 1: Library Prep Validation v2.3.5

Purpose:

Included for validation purposes only, this protocol models the library prep steps required to advance samples to the NovaSeq X Series Sequencing v1.2 workflow.
⚠ 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.
The protocol contains a single step - Library Prep Validation v2.3.5. After this step, a routing script sends the samples to the first step of the NovaSeq X Series Sequencing v1.2 workflow (Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2)).

Steps:

Library Prep Validation v2.3.5

NovaSeq X Series Sequencing v1.2 Workflow

Protocol 1: NovaSeq X Series Sequencing v1.2

Purpose:

This protocol models the lab processes of run setup for starting a NovaSeq X Series sequencing and secondary analysis run.

Steps:

Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2)
- The secondary analysis of samples is configured using the ACT.
- The labels applied to the samples are validated against the selected ACT so that samples use valid labels.
Make Bulk Pool (NovaSeq X Series Sequencing v1.2)
- The Calculate Volume script assists with pooling samples and adding resuspension buffers and reagents.
- The Validate Analysis Configurations automation validates the ACT of pooled samples to make sure that the secondary analysis configurations remain valid after pooling.
Dilute and Denature (NovaSeq X Series Sequencing v1.2)
- The Calculate Volume script helps denature and dilute samples from step 2 to the final loading concentration.
Load To Library Tube Strip (NovaSeq X Series Sequencing v1.2)
- The library pools from step 3 are ready to be loaded to the NovaSeq X Series library tube strip.
- Validates run and analysis information.
- Generates the sample sheet and creates the planned run, depending on the run mode.
AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2)
- The step is fully automated and starts and completes automatically after the sequencing run is started and completed on the instrument side.
- The sequencing run metadata (e.g., run configuration, primary run metrics, and so on) are recorded automatically.
AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.2)
- The step is fully automated and starts and completes automatically after the analysis run is started and completed in ICA.
- The analysis run metadata (eg, status and high level analysis summary) are recorded automatically.

Validation Workflow

The Library Prep Validation v2.3.5 workflow allows for validation of the system after installation is complete. This workflow can be replaced by other custom library preparation workflows.

Configure a custom library preparation workflow as follows.

Select Configuration, then select Lab Work.
Add a Routing Script automation to the last Master Step of the library preparation workflow.
Select Configuration, then select Automation.

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 v1.2' \
--STEP 'Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"

For Automation Use, select the custom library preparation workflow.

Step 1: Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2)

Validate Sample Names, Retrieve ACT List and Register Step Started Automation¹

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

Checks that submitted sample names contain only alphanumeric, dash, and underscore characters.

/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}'

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 v1.2)).
```
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 v1.2 workflows.

Master Step Fields

The following table lists the configuration details for fields that are defined on the Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2) step.

Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.2) Master Step Field Configuration

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 v1.2) step.

Global Field Configuration (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 v1.2)

Samples are pooled in the Make Bulk Pool (NovaSeq X Series Sequencing v1.2) step. You can manually create working pools based on the final loading concentration required.

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 v1.2) 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 v1.2 workflows.

Master Step Fields

The following table lists configuration details for the fields that are defined on the Make Bulk Pool (NovaSeq X Series Sequencing v1.2) step.

Make Bulk Pool (NovaSeq X Series Sequencing v1.2) Master Step Field Configuration

Field Name

Field Type

Options

Additional Options and Dropdown Items

Final Loading Concentration (pM)

Numeric Dropdown

Required Field
Custom Entries

Dropdown Items
- 90
- 140
- 150
- 160
- 180
Range from 0
Decimal places displayed: 0

Flowcell Type

Text

Required Field

Dropdown Items
- 1.5B
- 10B
- 25B

Minimum Per Sample Volume (ul)

Numeric

Required Field

Decimal places displayed: 2
Default: 2

Number of Lanes to Sequence

Numeric

Required Field

Range: 1–10
Decimal places displayed: 0

Global Fields

The following table lists the global custom fields that are configured to display on the Make Bulk Pool (NovaSeq X Series Sequencing v1.2) step.

Global Custom Fields Configuration (Derived Sample)

Field Name

Field Type

Options

Additional Options and Dropdown Items

Final Loading Concentration (pM)

Numeric Dropdown

Required Field
Custom Entries

Decimal places displayed: 0
Dropdown Items
- 225
- 400

RSB Volume (ul)

Numeric

Read Only

Decimal places displayed: 2

NovaSeq X Flowcell Type

Text Dropdown

Required Field
Read Only

Dropdown Items
- 1.5B
- 10B
- 25B

Step 3: Dilute and Denature (NovaSeq X Series Sequencing v1.2)

The Dilute and Denature (NovaSeq X Series Sequencing v1.2) step allows you to dilute pooled samples with the addition of RSB.

Validate Inputs Flowcell Type and Register Step Started¹

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

Checks that the specified flow cell type is valid.

⚠ This script is required for the NovaSeq X Series Sequencing v1.2 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.
ℹ The BP Aliquot Volume value is rounded up to the nearest µl. The value in the calculation file, however, is reflected with up to two decimal places.

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

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 v1.2) 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 v1.2 workflows.

Global Fields

The following table lists the global custom fields that are configured to display on the Dilute and Denature (NovaSeq X Series Sequencing v1.2) step.

Global Field Configuration (Derived Sample)

Field Name

Field Type

Options

Additional Options and Dropdown Items

BP Aliquot Volume (ul)

Numeric

Read Only

Decimal places displayed = 1

NaOH Volume (ul)

Numeric

Read Only

Decimal places displayed = 2

RSB Volume (ul)

Numeric

Read Only

Decimal places displayed = 2

TT2 Volume (ul)

Numeric

Read Only

Decimal places displayed = 2

Step 4: Load to Library Tube Strip (NovaSeq X Series Sequencing v1.2)

Validates the run setup and analysis information.
Generates the sample sheet file.
Creates a planned run on Illumina Connected Analytics (ICA), depending on the selected run mode.

Validate Flowcell Inputs, Analysis Configurations and Register Step Started Automation¹

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

/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 v1.2 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, periods 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}

Set Next Step Automation

Automatically triggered on exit of the Record Details screen, this automation completes the following actions:

Copies the Run Mode from input to output.
Sets the next step for samples to ADVANCE, advancing them to the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2) step.

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 v1.2 workflows.

Master Step Fields

Load to Library Tube Strip (NovaSeq X Series Sequencing v1.2) Master Step Field Configuration

Field Name

Field Type

Options

Additional Options and Dropdown Items

Planned Run ID

Text

Read Only

Hidden

ℹ Used for creating a planned run in ICA.

Index 1 Cycles

Numeric Dropdown

Required Field
Custom Entries

Range = 0–20
Presets
- 0
- 6
- 8

Index 2 Cycles

Numeric Dropdown

Required Field
Custom Entries

Range = 0–20
Presets
- 0
- 6
- 8

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

Instrument Type

Text

Read Only

Step 5: AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2)

Do not add samples to the Ice Bucket or start and complete the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2) step. The integration does this automatically.

Data from the run is parsed back to Clarity LIMS. In this step, pooled samples in the library tube strip are sequenced on the NovaSeq X Series instrument.

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 Planned Run ID custom field from the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.2) 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 v1.2) 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 Strip Barcode

RunParameters.xml

SerialNumber

Visible on Record Details screen.
Corresponds to the value of the SerialNumber field under the ConsumableInfo field for SampleTube Type.

Library Tube Lot Number

RunParameters.xml

LotNumber

Hidden
Corresponds to the value of the LotNumber field under the ConsumableInfo field for SampleTube Type.

Run End Time

RunCompletionStatus.xml

RunEndTime

Hidden

Run Name

RunParameters.xml

ExperimentName

Visible on Record Details screen.

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
% PF
% Aligned R1
% Aligned R2
% Occupied
% Phasing R1
% Phasing R2
% Prephasing R1
% Prephasing R2
Intensity Cycle 1 R1
Intensity Cycle 1 R2

At the end of the step, the pools of samples are automatically removed from the step. The step completes automatically when Run Status is RunCompletedSuccessfully.

Step 6: AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.2)

Do not add samples to the Ice Bucket or start and complete the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2) step. The integration does this automatically.

Master Step Fields

The following tables show the master step fields that are configured on the AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.2) step.

AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.2) Master Step Field Information

Field Name

Description

Additional Information

Analysis Status

Presets
- Started
- Completed
- Failed
- NeedsAttention
- Stopped
- TimedOut
- CompletedWithErrors
Set by the integration service.

Visible on Record Details screen.

Analysis Result Location

Received from the message sent to the integration service.

Visible on Record Details screen.

App Session ID

Identified by the integration service using information from RunParameters.xml.

Hidden

How the Integration Works

The following information summarizes how the NovaSeq X Series integration works.

After the Validate Run Setup and Create Planned Run automation is triggered on the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.2) step, the run parameters entered in the Run Details screen are sent to Illumina Connected Analytics (ICA). The analysis configuration parameters from the selected ACTs are also sent to ICA. ICA validates the run and analysis configuration. If the validation fails, ICA sends an error message to Clarity LIMS. If the validation passes, ICA generates the sample sheet and sends it back to Clarity LIMS. ICA also creates the planned run based on the selected run mode (e.g., Local or Cloud).
- For the Local run mode, the sample sheet is generated and stored in Clarity LIMS. This sample sheet contains the run and analysis configuration information required to start the run on the NovaSeq X Series instrument.
- For the Cloud run mode, the planned run is created in ICA. The planned run contains the run and analysis configuration information required to start the run on the instrument. The sample sheet is generated and stored in Clarity LIMS for reference purposes.
When the sequencing run starts on the instrument, the NovaSeq X Series Control Software notifies BaseSpace Sequence Hub. BaseSpace Sequence Hub monitors the run and notifies Clarity LIMS via the integration service. The events are processed and the integration service retrieves the run information from BaseSpace Sequence Hub. This information is used to populate the custom fields in the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2) step.
Other run events follow the same information flow. When sequencing is complete, the control software uploads the sequencing run data (primary metrics) and associated files to ICA. Then, Clarity LIMS retrieves the primary metrics and uses them to populate the fields in the Sample Details table (e.g., % Error Rate R1). The custom fields (e.g., Run Status, Current Read, and so on) on the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.2) step are updated using the run related information. If the sequencing run is successfully completed, the step automatically completes.
The integration tracks the analysis events and results in the AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.2) step. You must log in to BaseSpace Sequence Hub to retrieve the cloud analysis results. For local analysis, the secondary analysis results are in the external storage configured in Illumina Run Manager. The external storage information is found in the External Storage for Analysis Results configuration settings in Illumina Run Manager.

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 v1.2) step. Refer to information on creating a local run in the NovaSeq X Plus Product Documentation on the NovaSeq X Plus Sequencing System support site.
When the sample sheet is selected, the contents are validated. After the validation passes, select Next.
Select Save As Planned.
Run and analysis data is parsed and auto-populates in the list of runs. For more information on planning a NovaSeq X Series run, refer to the Plan a NovaSeq X Series Run section of the BaseSpace Sequence Hub Online Help at help.basespace.illumina.com and the NovaSeq X Plus Product Documentation on the NovaSeq X Plus Sequencing System support site.
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 v1.2) and AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.2) steps must not be manually started or completed. These steps are fully automated and the sequencing service does not update samples correctly if they have been manually started.
For the automated run to start successfully, you must select Validate Run Setup and Create Planned Run in the Load to Library Tube step.