Configuration

The Illumina NovaSeq X Series On-Prem Integration Package v1.0.0 supports the integration of Clarity LIMS to Illumina NovaSeq X Series sequencer connected to the Illumina Run Manager platform on premise.

For instructions on validating and troubleshooting the Illumina NovaSeq X Series On-Premise Integration, refer to NovaSeq X Series On-Premise Integration v1.0.0 User Interaction, Validation and Troubleshooting.

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

Prerequisites and Assumptions

The NovaSeq X Series On-Premise Integration v1.0.0 requires NovaSeq X Control Software version v1.3 and above.

Analysis Configuration Template

The NovaSeq X Series On-Prem Sequencing v1.0 workflow uses analysis configuration templates (ACT) to configure secondary analysis for the planned runs. It is assumed that the required ACTs for your run has been pre-created using Illumina Run Manager UI. Please make sure that the index adapter kit (label group on Clarity) selected in the ACT has already been created on Clarity LIMS. The same label group shall be used in the library preparation step. Names of ACT must be unique. Please refer to the NovaSeq X Series On-Premise Integration v1.0.0 User Interaction, Validation and Troubleshooting for details on creating ACT on Illumina Run Manager User Interface (UI).

Clarity LIMS trims all leading and trailing spaces from the ACT names and ACTs created in Illumina Run Manager UI with names containing leading and/or trailing spaces may not be recognized properly on Clarity. Please ensure that the ACT do not have leading and/or trailing spaces.

Samples

It is assumed that samples that enter the NovaSeq X Series On-Prem Sequencing v1.0 workflow have gone through library preparation and quantification processes before they are assigned to the workflow:

• Samples have been accessioned into Clarity LIMS.

• Sample and library names must contain only alphanumeric, dash, or underscore characters.

• Samples have been run through QC and library prep.

• Samples have the Molarity (nM) global field set to a valid value (required for the 'Calculate Volumes' automation in the Make Bulk Pool step).

Workflows, Protocols, and Steps

The NovaSeq X Series On-Premise Integration Package v1.0.0 includes the following workflows:

• NovaSeq X Series On-Prem Sequencing v1.0

• 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 preparation steps required to advance samples to the NovaSeq X Series On-Prem Sequencing v1.0 workflow.

  ⚠ The label group (index adapter kit) used for library preparation must be the same as the index adapter kit specified in the ACT that is being used. For more information, refer to NovaSeq X Series On-Premise Integration v1.0.0 User Interaction, Validation and Troubleshooting.

• 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 On-Prem Sequencing v1.0 workflow (Assign Analysis Configuration Template (NovaSeq X Series On-Prem Sequencing v1.0)).

Steps:

1. Library Prep Validation v2.3.5

NovaSeq X Series On-Prem Sequencing v1.0 Workflow

Protocol 1: NovaSeq X Series On-Prem Sequencing v1.0

Purpose:

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

Steps:

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

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

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

2. Make Bulk Pool (NovaSeq X Series On-Prem Sequencing v1.0)

   • Samples are pooled and resuspension buffers and reagents are added with the help of the 'Calculate Volumes' automation.

   • ACT of pooled samples are validated with Validate Analysis Configurations automation to ensure that the secondary analysis configurations remain valid after pooling.

3. Dilute and Denature (NovaSeq X Series On-Prem Sequencing v1.0)

   • Samples from step 2 are denatured and diluted to the final loading concentration with the help of the 'Calculate Volumes' automation.

4. Load To Library Tube Strip (NovaSeq X Series On-Prem Sequencing v1.0)

   • The library pools from step 3 are now ready to be loaded to the NovaSeq X Series library tube strip.

   • Run and analysis information is validated.

   • Samplesheet is generated and planned run is created on NovaSeq X Illumina Run Manager.

5. AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0)

   • This is a fully-automated step that is started and completed automatically once the sequencing run is started and completed on the instrument side.

   • All the sequencing run metadata e.g. run configuration, primary run metrics etc. are recorded automatically.

   ⚠ Samples should be queued at the step. The user must not add samples to the Ice Bucket or start or complete the step. The integration will do this automatically.

6. AUTOMATED - Analysis Run (NovaSeq X Series On-Prem Sequencing v1.0)

   • This is a fully-automated step that is started and completed automatically once the analysis run is started and completed on DRAGEN on-board the instrument.

   • All the analysis run metadata e.g. status and high level analysis summary and demultiplexing result files are recorded automatically.

   ⚠ Samples should be queued at the step. The user must not add samples to the Ice Bucket or start or complete the step. The integration will do this 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. For details, refer to NovaSeq X Series On-Premise Integration v1.0.0 User Interaction, Validation and Troubleshooting.

[Optional] Configure a custom library preparation workflow. Routing of libraries from the custom workflow to the NovaSeq X Series On-Premise Sequencing protocol can be enabled in the master step with the routing script available in the NGS package.

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

In this step, secondary analyses are configured for the samples using ACTs. Each ACT contains details related to a particular secondary analysis (e.g., index adapter kit to use, reference genome setting). At this step, the user selects an ACT from a list of pre-constructed ACTs and assigns samples to it. A planned run can involve multiple ACTs and users have to repeat Step 1 for each ACT that is required for the planned run. For more information on creating ACTs and assigning samples to them, refer to NovaSeq X Series On-Premise Integration v1.0.0 User Interaction, Validation and Troubleshooting.

Validate Sample Names Automation

Automatically triggered on entry of the step, this automation validates that the 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}

1. Retrieve ACT List Automation

Triggered when you select '1. Retrieve ACT List' button on the Record Details screen, this automation retrieves the list of ACTS that has been created using Illumina Run Manager UI.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/novaseqxseries-onprem/automation/novaseqxseries-onprem-automation.jar script:retrieve_act_list -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -n 'INSTRUMENT_USED' -t true

2. Retrieve ACT Information Automation

Triggered when you select '2. Retrieve ACT Information' button on the Record Details screen, this automation:

• Retrieves the details of the selected ACT and populates the step UDFs (e.g., Analysis Version, Library Prep Kit, Index Adapter Kit, Reference Genome)

• Saves the details into a file stored in the Analysis Configuration Metadata file placeholder, which is available for user to download.

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

Apply Selected ACT to Samples and Set Next Step Automation

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

• Checks that the molarity of all samples are 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, indexes are the same as the selected ACT.

• Assign the ACT ID, ACT Name, Run Mode and Instrument ID to all the samples in the step.

  /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/novaseqxseries-onprem/automation/novaseqxseries-onprem-automation.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -a '{udf:Analysis Configuration Template}' -n 'INSTRUMENT_USED' --derivedSampleInstrumentIdUdf 'Instrument ID' script:validate_indexes script:assign_act

• Sets the next step for samples to ADVANCE, advancing them to the next step in the protocol - Make Bulk Pool (NovaSeq X Series On-Prem Sequencing v1.0).

  /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 'nextStep = ::ADVANCE::' -log {compoundOutputFileLuid1}

Master Step Fields

The following table lists the configuration details for custom fields that are defined on the Assign Analysis Configuration Template (NovaSeq X Series On-Prem Sequencing v1.0) step.

Assign Analysis Configuration Template (NovaSeq X Series On-Prem Sequencing v1.0) Master Step Field Configuration

Field Name	Field Type	Options
Analysis Configuration Template	Text Dropdown	Allow Custom Entries
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 lists the global custom fields that are configured to display on the Assign Analysis Configuration Template (NovaSeq X Series On-Prem Sequencing v1.0) step.

• Molarity (nM)

• ACT Name

Step 2: Make Bulk Pool (NovaSeq X Series On-Prem Sequencing v1.0)

Samples are pooled in the Make Bulk Pool (NovaSeq X Series On-Prem Sequencing v1.0) step. You can manually create working pools based on the final loading concentration required.

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. See here for more details.

• Pooled samples assigned with the same secondary analysis application must have the same analysis version (e.g. v3.8.4) and settings (e.g. Map/Align Output Format = CRAM).

/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 'Instrument ID' -f 'Run Mode' -v 'perPool' && \
/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.::Instrument ID:: = input.::Instrument ID::' -log {compoundOutputFileLuid1} && \
/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/novaseqxseries-onprem/automation/novaseqxseries-onprem-automation.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -m PerPool -n 'INPUT.Instrument ID' script:validate_analysis_config script:validate_physical_logical_configurations

Calculate Volumes Automation

Triggered when you select 'Calculate Volumes' on the Record Details screen, this automation completes the following actions:

• Checks to ensure all samples have molarity values assigned.

  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 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 if any of the calculated volume is lesser than the value specified in Minimum Per Sample Volume (ul) step UDF.

  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}.cs

• 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 On-Prem Sequencing v1.0) step.

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

Master Step Fields

The following table lists configuration details for the custom fields that are defined on the Make Bulk Pool (NovaSeq X Series On-Prem Sequencing v1.0) step.

Make Bulk Pool (NovaSeq X Series On-Prem Sequencing v1.0) Master Step Field Configuration

Field Name	Field Type	Options	Additional Options and Dropdown Items
Final Loading Concentration (pM)	Numeric Dropdown	Required Field Allow Custom Entries	Dropdown Items 90 140 150 160 180
Flowcell Type	Text Dropdown	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

Global Fields

The following lists the global custom fields that are configured to display on the Make Bulk Pool (NovaSeq X Series On-Prem Sequencing v1.0) step.

• Final Loading Concentration (pM)

• RSB Volume (ul)

• NovaSeq X Flowcell Type

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

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

Validate Inputs Flowcell Type Automation

Automatically triggered on entry of the step, this automation checks the a NovaSeq X Flowcell Type has been assigned to each input.

/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}

Calculate Volumes Automation

Triggered when you select 'Calculate Volumes' on the Record Details screen, this automation completes the following actions based on the NovaSeq X Flowcell Type selected:

• Sets the NaOH Volume (µl) and TT2 Volume (µl) field values.

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

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

  bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} -log {compoundOutputFileLuid1} \
    script:evaluateDynamicExpression \
    -exp '
    if (input.::NovaSeq X Flowcell Type:: == ::10B:: || input.::NovaSeq X Flowcell Type:: == ::1.5B::) {
        output.::NaOH Volume (ul):: = 8.5;
        output.::TT2 Volume (ul):: = 127.5;
        output.::BP Aliquot Volume (ul):: = input.::Final Loading Concentration (pM):: * 170 / (2 * 1000);
        output.::RSB Volume (ul):: = 34 - output.::BP Aliquot Volume (ul)::;
        if (step.::1-2% PhiX Spike-In::) { 
            output.::PhiX Volume (ul):: = 1; output.::PhiX Concentration (pM):: = 300; 
        }else { 
            output.::PhiX Volume (ul):: = ::::; output.::PhiX Concentration (pM):: = ::::; 
        };
    } else {
        output.::NaOH Volume (ul):: = 14;
        output.::TT2 Volume (ul):: = 210;
        output.::BP Aliquot Volume (ul):: = input.::Final Loading Concentration (pM):: * 280 / (2 * 1000);
        output.::RSB Volume (ul):: = 56 - output.::BP Aliquot Volume (ul)::;
        if (step.::1-2% PhiX Spike-In::) { 
            output.::PhiX Volume (ul):: = 1.6; output.::PhiX Concentration (pM):: = 300; 
        }
        else { 
            output.::PhiX Volume (ul):: = ::::; output.::PhiX Concentration (pM):: = ::::; 
        };
    }'

• Generates a single CSV file containing 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, for user to download.

  /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, NovaSeq X Flowcell Type and Instrument ID from input to output.

• Sets the next step for samples to ADVANCE, advancing them to the Load to Library Tube Strip (NovaSeq X Series On-Prem Sequencing v1.0) step.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'output.::Run Mode:: = input.::Run Mode::; output.::NovaSeq X Flowcell Type:: = input.::NovaSeq X Flowcell Type::; output.::Instrument ID:: = input.::Instrument ID::; nextStep = ::ADVANCE::' -log {compoundOutputFileLuid1}

Master Step Fields

The following table lists configuration details for the custom fields that are defined on the Dilute and Denature (NovaSeq X Series On-Prem Sequencing v1.0) step.

Dilute and Denature (NovaSeq X Series On-Prem Sequencing v1.0) Master Step Field Configuration

Field Name	Field Type	Options	Additional Options and Dropdown Items
1-2% Spike-In	Numeric	Required Field	Decimal places displayed: 1
PhiX Volume (ul)	Numeric	Optional Field	Decimal places displayed: 1

Global Fields

The following lists the global custom fields that are configured to display on the Dilute and Denature (NovaSeq X Series On-Prem Sequencing v1.0) step.

• BP Aliquot Volume (ul)

• NaOH Volume (ul)

• RSB Volume (ul)

• TT2 Volume (ul)

Step 4: Load to Library Tube Strip (NovaSeq X Series On-Prem Sequencing v1.0)

In this step, the user scans the library tube strip barcode into the LIMS, and then manually places the working pools into the library tube strip to be used in the NovaSeq X Series run. In addition, this step validates the run setup and analysis information and generates samplesheet file and/or creates a planned run on instrument's Illumina Run Manager UI.

Validate Flowcell Inputs and Analysis Configurations Automation

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

• Checks that the selected destination container type at Load to Library Tube Strip step matches the selected flowcell type of the samples. 1.5B flowcell is compatible with Library 2-tube Strip while 10B and 25B flowcells are compatible with 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. See here for more details.

  • Samples in the same pools that have the same secondary analysis application must have the same analysis version (e.g. v3.8.4) and settings (e.g. Map/Align Output Format = CRAM).

  ⚠ This script is required for the NovaSeq X Series On-Prem Sequencing v1.0 workflow to function properly.

  /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/novaseqxseries-onprem/automation/novaseqxseries-onprem-automation.jar  -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -m PerRun -n 'INPUT.Instrument ID' script:validate_analysis_config script:validate_physical_logical_configurations

Validate Library Tube Strip Barcode Automation

Automatically triggered on exit of the Placement screen, this automation validates the library tube strip barcode to ensure it conforms to the barcode mask LC[0-9]{7}-L[A-Z]1 for Library 8-tube Strip and LC[0-9]{7}-L[A-Z]2 for 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

Triggered when you select 'Validate Run Setup and Create Planned Run' button 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 on Illumina Run Manager.

  • Run Name may only contain alphanumeric, dash, underscore or period characters. Spaces are not permitted.

    if (!step.::Run Name::.matches(::[a-zA-Z0-9-_]+[a-zA-Z0-9-_. ]*::)) { fail(::Run Name contains prohibited characters. Please check to make sure Run Name only contains alphanumeric characters, spaces, dashes and underscores. Run Name must start with alphanumeric, a dash or an underscore::); }

  • Run Name shall not 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 on Illumina Run Manager. The sample sheet is attached to the step.

  /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/novaseqxseries-onprem/automation/novaseqxseries-onprem-automation.jar  script:generate_sample_sheet -i {stepURI:v2} -u {username} -p {password} -c {compoundOutputFileLuid0} -l {compoundOutputFileLuid1} -n 'INPUT.Instrument ID'

Set Next Step Automation

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

• Sets the next step for samples to ADVANCE, advancing them to the AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0) step.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'nextStep = ::ADVANCE::' -log {compoundOutputFileLuid1}

Master Step Fields

The following table shows the master step fields that are configured on the Load to Library Tube Strip (NovaSeq X Series On-Prem Sequencing v1.0) step. These fields are required for sample sheet generation and planned run creation in Illumina Run Manager.

Load to Library Tube Strip (NovaSeq X Series On-Prem Sequencing v1.0) Master Step Field Configuration

Field Name	Field Type	Options	Additional Options and Dropdown Items
Index 1 Cycles	Numeric Dropdown	Required Field Allow Custom Entries	Range = 0–20 Presets 0 6 8
Index 2 Cycles	Numeric Dropdown	Required Field Allow Custom Entries	Range = 0–20 Presets 0 6 8
Instrument	Text	Read Only
Planned Run ID	Text	Read Only	Hidden
Read 1 Cycles	Numeric Dropdown	Required Field Allow Custom Entries	Range = 1–251 Presets 51 101 151
Read 2 Cycles	Numeric Dropdown	Required Field Allow Custom Entries	Range = 0–251 Presets 51 101 151
Run Mode	Text	Read Only	Default: Local
Run Name	Text	Required Field

Step 5: AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0)

Do not add samples to the Ice Bucket, start or complete the AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0) step. The integration will do this automatically.

In this step, the pooled samples in the library tube strip are sequenced on the NovaSeq X Series instrument.

There are no automation associated to this step.

Master Step Fields

The following tables show the master step fields that are configured on the AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0) step.

Clarity LIMS Master Step Field Information

Field Name	Description
Current Cycle	Current completed cycle count.
Current Read	Current completed reads count.
Flow Cell Expiration Date	Flow Cell expiration data.
Flow Cell ID	Serial number of the Flow Cell.
Flow Cell Lot Number	Flow Cell lot number.
Flow Cell Part Number	Flow Cell part number.
Flow Cell Side	Flow Cell side. Value can be A or B.
Flow Cell Type	Flow Cell Type. Value can be 1.5B, 10B or 25B.
Instrument Control Software Version	Version of the instrument control software.
Instrument ID	Unique identification for the instrument.
Instrument Type	Type of the instrument, either NovaSeqX or NovaSeqXPlus.
Library Tube Strip Barcode	Library Tube Strip barcode.
Output Folder	Output folder path for the sequencing run data.
Run Name	Run name user configured for the sequencing run.
Run Status	Status of the sequencing run.
Sequencing Log	Log messages by the Illumina Run Manager integration while handling the sequencing event from instrument.

All the fields above are Read-Only Text field, except Sequencing Log is a Read-Only Multiline Text.

Global Fields

The following global custom fields are used to capture the run metrics in Clarity LIMS:

• % Aligned R1

• % Aligned R2

• % Bases >=Q30 R1

• % Bases >=Q30 R2

• % Error Rate R1

• % Error Rate R2

• % Occupied

• % Phasing R1

• % Phasing R2

• % Prephasing R1

• % Prephasing R2

• % PF

• Intensity Cycle 1 R1

• Intensity Cycle 1 R2

• Reads PF

• Yield (Gb) R1

• Yield (Gb) R2

At the end of the step, the pools of samples are automatically route to next step.

Step 6: AUTOMATED - Analysis Run (NovaSeq X Series On-Prem Sequencing v1.0)

Do not add samples to the Ice Bucket or start and complete the AUTOMATED - Analysis Run (NovaSeq X Series On-Prem Analysis v1.0) 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 on DRAGEN on premise.

There are no automation associated to this step.

Master Step Fields

The following tables show the master step fields that are configured on the AUTOMATED - Analysis Run (NovaSeq X Series On-Prem Sequencing v1.0) step.

AUTOMATED - Analysis Run (NovaSeq X Series On-Prem Sequencing v1.0) Master Step Field Information

Field Name	Description
Analysis Result Location	Analysis files final copy out location.
Analysis Run ID	Run ID associate with the Analysis run. Hidden on Record Details screen by default.
Analysis Status	Status for the the analysis run.
Log	Log messages by the Illumina Run Manager integration while handling the analysis event from instrument.

All the fields above are Read-Only Text field, except Log is a Read-Only Multiline Text.

How the Integration Works

The following information summarizes how the NovaSeq X Series On-Premise Integration works.

• After the 'Validate Run Setup and Create Planned Run' automation is triggered on the Load to Library Tube Strip (NovaSeq X Series On-Prem Sequencing v1.0) step, the run parameters entered in the Run Details screen and the sample informations are sent to Illumina Run Manager. Illumina Run Manager validates the run and analysis configuration before the planned run is created, the automation also generates the sample sheet and attaches it to the step. In the event of incomplete information or misconfiguration on the planned run, an error will be displayed on the automation banner and details of the error will be logged in the automation log file.

• When the sequencing run starts on the instrument, the NovaSeq X Series Control Software notifies the Illumina Run Manager integration. The events are processed and the integration service retrieves the run information from NovaSeq X Illumina Run Manager. This information is used to populate the custom fields in the AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0) step.

• Other run events follow the same information flow. When sequencing is complete, the control software uploads the sequencing run data (primary metrics). Then, Illumina Run Manager integration 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 On-Prem Sequencing v1.0) 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 On-Prem Sequencing v1.0) step. The secondary analysis results are in the external storage configured in Illumina Run Manager. The external storage information is found in the External Storage for Analysis Results configuration settings in Illumina Run Manager.

If a planned run with the same sample name and project name (case-insensitive) has been created previously in Illumina Run Manager, 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.

Configuration of Instrument Setting

The integration requires secondary analysis files to be present on the instrument for proper functioning of the AUTOMATED - Analysis Run step.

The following instrument setting must be disabled:

• Permanently delete secondary analysis files from the instrument after they are transferred to the external storage and/or cloud.

Start a Sequencing Run on NovaSeq X Series Instrument

To start a planned run, please refer to NovaSeq X - Start a Planned Run.

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.

Global Fields

Container Global Fields

Field Name	Field Type	Settings
% Aligned R1	Numeric	Read Only Decimal Places Displayed: 2
% Aligned R2	Numeric	Read Only Decimal Places Displayed: 2
% Bases >=Q30 R1	Numeric	Read Only Decimal Places Displayed: 2
% Bases >=Q30 R2	Numeric	Read Only Decimal Places Displayed: 2
% Error Rate R1	Numeric	Read Only Decimal Places Displayed: 2
% Error Rate R2	Numeric	Read Only Decimal Places Displayed: 2
% Occupied	Numeric	Read Only Decimal Places Displayed: 2
% PF	Numeric	Read Only Decimal Places Displayed: 2
% Phasing R1	Numeric	Read Only Decimal Places Displayed: 3
% Phasing R2	Numeric	Read Only Decimal Places Displayed: 3
% Prephasing R1	Numeric	Read Only Decimal Places Displayed: 3
% Prephasing R2	Numeric	Read Only Decimal Places Displayed: 3
Intensity Cycle 1 R1	Numeric	Read Only
Intensity Cycle 1 R2	Numeric	Read Only
Reads PF	Numeric	Read Only Decimal Places Displayed: 2
Yield (Gb) R1	Numeric	Read Only Decimal Places Displayed: 2
Yield (Gb) R2	Numeric	Read Only Decimal Places Displayed: 2

Derived Sample Global Fields

Field Name	Field Type	Settings
ACT ID	Text	Read Only
ACT Name	Text	Read Only
Adjusted Per Sample Volume (ul)	Numeric	Read Only Decimal Places Displayed: 2
BP Aliquot Volume (ul)	Numeric	Read Only Decimal Places Displayed: 1
Bulk Pool Volume (ul)	Numeric	Read Only Decimal Places Displayed: 2
Final Loading Concentration (pM)	Numeric Dropdown	Required Preset: 225 400 Allow custom entries
Instrument ID	Text	Read Only
Molarity (nM)	Numeric	Decimal Places Displayed: 2
NovaSeq X Flowcell Type	Text Dropdown	Required Preset: 1.5B 10B 25B
Number of Samples in Pool	Numeric
Per Sample Volume (ul)	Numeric	Read Only Decimal Places Displayed: 2
PhiX Concentration (pM)	Numeric
PhiX Volume (ul)	Numeric	Decimal Places Displayed: 2
RSB Volume (ul)	Numeric	Read Only Decimal Places Displayed: 2
Run Mode	Text	Read Only
Total Sample Volume (ul)	Numeric	Read Only Decimal Places Displayed: 2
TT2 Volume (ul)	Numeric	Read Only Decimal Places Displayed: 2

Reagent Kits

• Buffer Cartridge

• Lyophilization Cartridge

• NaOH

• Reagent Cartridge

• Resuspension Buffer (RSB)

• TT2

Container Types

• Library 2-Tube Strip

• Library 8-Tube Strip

• Tube

Instrument Types

• NovaSeq X Series

Supported Library Tube Strip barcode formats by the integration

• Library 2-Tube Strip - LC[0-9]{7}-L[A-Z]2

• Library 8-Tube Strip - LC[0-9]{7}-L[A-Z]1

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 NovaSeq X Illumina Run Manager before starting the Assign Analysis Configuration Template step. The ACT names must be unique.

• Assign Analysis Configuration Template step does not support pool library as input.

• The library tube strip barcode must be unique. There must not be multiple library tube strip containers with the same name in the Clarity LIMS 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 On-Prem Sequencing v1.0) and AUTOMATED - Analysis Run (NovaSeq X Series On-Prem Sequencing v1.0) 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:

  1. select Validate Run Setup and Create Planned Run in the Load to Library Tube Strip step and the automation must be ran successfully.

  2. complete the Load to Library Tube Strip step. The libraries must be queued at the AUTOMATED - Sequencing Run step.