Manual Upgrade

NovaSeq X Series Integration Package v1.2.0 includes modifications to the existing NovaSeq X Series Sequencing workflows to support the following new features:

• PhiX control included in the Dilute and Denature step instead of the Make Bulk Pool step.

• Support for the new 1.5B and 25B flow cells.

• Analysis tracking for the status and high level analysis result summary through the AUTOMATED - Analysis Run step (this is done at the metaworkflow level).

  ⚠️ Analysis tracking is a new step in NovaSeq X Series protocol and cannot be added to a non-pending protocol or workflow. For analysis tracking at the metaworkflow level, do not proceed with the manual upgrade. Instead, install the NovaSeq X Series v1.2.0 Integration Package with the NovaSeq X Series Sequencing v1.1 protocol.

• The following workflow updates are included in NovaSeq X Series Integration Package v1.2.0:

  • AUTOMATED - Sequencing Run step: Renamed the Library Tube Barcode master step custom field. This field is now named Library Tube Strip Barcode.

  • Load to Library Tube Strip step: Removed the Output Folder master step custom field.

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

Use the following instructions to manually update the workflows.

Prerequisites

Illumina recommends upgrading to Illumina Preset Protocols (IPP) v2.7 to support the new features. If you do not have IPP v2.7 installed, do the following before starting the manual upgrade:

• Install ClarityLIMS-NGS-Package-v5 RPM v5.24.

• Replace the following files in /opt/gls/clarity/extensions/conf/driverfiletemplates:

NovaSeqXSeries_Bulk_Pool1.csv:

356B

NovaSeqXSeries_Bulk_Pool1.csv

NovaSeqXSeries_Bulk_Pool2.csv:

271B

NovaSeqXSeries_Bulk_Pool2.csv

NovaSeqXSeries_Dilute_Denature_Calculate_Volumes.csv:

672B

NovaSeqXSeries_Dilute_Denature_Calculate_Volumes.csv

Add New Containers

1. On the Configuration tab, select Consumables, and then select Containers.

2. Add the following information to enable the Library 2-tube Strip container that supports the 1.5B flow cell:

   ℹ️ No action is required for the 25B flow cell. The flow cell uses the same Library 8-tube Strip as the 10B flow cell.

   1. For Container Name, enter Library 2-tube Strip.

   2. For Rows, update the following fields:

      • Number: 2

      • Naming: Alphabetic

   3. For Columns, update the following fields:

      • Number: 1

      • Naming: Numeric

      • Start at: 1

   

Add New Master Step Fields

Enable PhiX control in the Dilute and Denature step as follows.

1. On the Configuration tab, select Custom Fields, and then select Master Step Fields.

2. Add the following master step fields for the Dilute and Denature (NovaSeq X Series Sequencing v1.x) step:

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

   Field Name	Field Type	Options	Additional Options and Dropdown Items
   PhiX Volume (ul)	Numeric	Read Only	Decimal places displayed: 1
   1–2% PhiX Spike-In	Toggle Switch	Required Field	Default: No

   

   

Modify Existing Master Step Fields

1. On the Configuration tab, select Custom Fields, and then select Master Step Fields.

2. Modify the following master step fields:

   Make Bulk Pool (NovaSeq X Series Sequencing v1.x) Master Step Field Modifications

   Field	Modification	Purpose/Notes
   Final Loading Concentration (pM)	Update Dropdown Items with the following options: 90 140 150 160 180	Only perform this modification if the new Dropdown Items are required. This modification is not required to enable support for the new flow cells or PhiX control in the Dilute and Denature (NovaSeq X Series Sequencing v1.x) step.
   Flowcell Type	Change Field Type from Text to Text Dropdown. Update Field Options as follows. Read Only = No Custom Entries = No Update Dropdown Items with the following options: 1.5B 10B 25B	This modification is required to support the new flow cells.

   

   

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

   Field	Modification	Purpose/Notes
   Output Folder	Remove the field.	If the step has been run previously and this field is used, it cannot be removed. This field does not affect the functionality of the workflow. This modification is not required to enable support for the new flow cells or PhiX control in the Dilute and Denature (NovaSeq X Series Sequencing v1.x) step.

   AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.x) Master Step Field Modifications

   Field	Modification	Purpose/Notes
   Library Tube Barcode	Rename the field from Library Tube Barcode to Library Tube Strip Barcode.	If the installed NovaSeq X Series workflow is v1.1 or earlier, this modification is not required to enable support for the new flow cells or PhiX control in the Dilute and Denature (NovaSeq X Series Sequencing v1.x) step. For NovaSeq X Series workflow v1.2, this modification is required. Run event handling fails without this modification.

   

Modify Existing Global Field

1. On the Configuration tab, select Custom Fields, and then select Global Fields.

2. For the NovaSeq X Flowcell Type field, update the Dropdown Items with the following values:

   ℹ️ A merge conflict is expected when upgrading from IPP v2.6 to v2.7 due to the changes in the NovaSeq X Flowcell Type field.

   • 1.5B

   • 10B

   • 25B

   

Add, Configure, and Modify Automations

Add Automation

1. On the Configuration tab, select Automation, and then select Step Automation.

2. On the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.x) step, add the following automation to enable new flow cell types:

   • Name: Validate Flowcell Inputs and Validate Analysis Configurations and Register Step Started

   • Channel Name: limsserver

   • Command line:

     bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} \
     script:validate_same_udf_value_for_analytes -f 'Run Mode' -f 'NovaSeq X Flowcell Type' \
     script:validate_selected_container \
     -fn 'NovaSeq X Flowcell Type' -fv '1.5B' -ct 'Library 2-tube Strip' \
     -fn 'NovaSeq X Flowcell Type' -fv '10B' -ct 'Library 8-tube Strip' \
     -fn 'NovaSeq X Flowcell Type' -fv '25B' -ct 'Library 8-tube Strip' && \
     /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/SIS/SISServices/extensions/automation/novaseqxseries-automation.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -m PerRun script:validate_analysis_config script:validate_physical_logical_configurations && \
     /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -s 'com/illumina/upa/scripts/common/step_started.groovy'"

Configure Automation Settings

1. On the Configuration tab, select Lab Work.

2. Configure the trigger location and style for the following automations:

   Load to Library Tube Strip (NovaSeq X Series Sequencing v1.x) Step Automation Configuration

   ℹ️ These configuration updates are required to enable the new flow cell types.

   Automation	Setting
   Validate Flowcell Inputs and Validate Analysis Configurations and Register Step Started	Trigger Location: Step Trigger Style: Automatic upon entry
   Validate Input Count and Validate Analysis Configurations and Register Step Started	Trigger Location: Not used

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

   ℹ️ This configuration update is required to enable PhiX control on the Make Bulk Pool (NovaSeq X Series Sequencing v1.x) step.

   Automation	Setting
   Calculate Volumes	Trigger Location: Record Details Trigger Style: Manual button

Modify Step Automations

1. On the Configuration tab, select Automation, and then select Step Automation.

2. Select the Calculate Volumes automation that is enabled on the Make Bulk Pool (NovaSeq X Series Sequencing v1.x) step.

3. Update the existing command line as follows.

   This modification is required to move the PhiX control from the Make Bulk Pool (NovaSeq X Series Sequencing v1.x) step to the Dilute and Denature (NovaSeq X Series Sequencing v1.x) step. Changes are underlined.

   bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} -log {compoundOutputFileLuid1} \
   script:evaluateDynamicExpression \
   -exp 'if (!input.hasValue(::Molarity (nM)::)) { return; }; if (output.hasValue(::Number of Samples in Pool::)) { output.::Number of Samples in Pool:: = output.::Number of Samples in Pool:: + 1; } else { output.::Number of Samples in Pool:: = 1; }' -t true \
   script:evaluateDynamicExpression \
   -exp 'output.::Bulk Pool Volume (ul):: = 100 * step.::Number of Lanes to Sequence::;
   output.::NovaSeq X Flowcell Type:: = step.::Flowcell Type::;
   output.::Final Loading Concentration (pM):: = step.::Final Loading Concentration (pM)::;
   input.::Per Sample Volume (ul):: = 2 * output.::Bulk Pool Volume (ul):: / input.::Molarity (nM):: / output.::Number of Samples in Pool::;
   output.::Total Sample Volume (ul):: = 0;' -t true \
   script:calculate_multipool_adjusted_per_sample_volume -t \
   script:evaluateDynamicExpression \
   -exp 'if (output.hasValue(::Total Sample Volume (ul)::)) { output.::Total Sample Volume (ul):: = output.::Total Sample Volume (ul):: + input.::Adjusted Per Sample Volume (ul)::; } else { output.::Total Sample Volume (ul):: = input.::Adjusted Per Sample Volume (ul)::; }' -t true \
   script:evaluateDynamicExpression \
   -exp 'if (output.::Total Sample Volume (ul):: > output.::Bulk Pool Volume (ul)::) { output.::RSB Volume (ul):: = 0 } else { output.::RSB Volume (ul):: = output.::Bulk Pool Volume (ul):: - output.::Total Sample Volume (ul):: }' -t true \
   && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} script:driver_file_generator \
   -t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeqXSeries_Bulk_Pool1.csv -o 1.csv \
   script:driver_file_generator \
   -t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeqXSeries_Bulk_Pool2.csv -o 2.csv \
   && cat 1.csv 2.csv > {compoundOutputFileLuid0}.csv \
   && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} -log {compoundOutputFileLuid1} \
   script:evaluateDynamicExpression \
   -exp 'output.::Number of Samples in Pool:: = ::::; output.::Total Sample Volume (ul):: = ::::; output.::Bulk Pool Volume (ul):: = ::::;' -t true \
   && echo 'Calculate Volume completed successfully'"

4. Select the Calculate Volumes automation that is enabled on the Dilute and Denature (NovaSeq X Series Sequencing v1.x) step.

5. Update the existing command line as follows.

   This modification is required to move the PhiX control from the Make Bulk Pool (NovaSeq X Series Sequencing v1.x) step to the Dilute and Denature (NovaSeq X Series Sequencing v1.x) step and to enable support for the 1.5B and 25B flow cells. Changes are underlined.

   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.'"

6. Select the Set Next Step automation that is enabled on the Dilute and Denature (NovaSeq X Series Sequencing v1.x) step.

7. Update the existing command line as follows.

   This modification is required to enable support for the 1.5B and 25B flow cells and future 8-tube library strips. Changes are underlined.

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

8. Select the Validate Library Tube Strip Barcode automation that is enabled on the Load to Library Tube Strip Barcode (NovaSeq X Series Sequencing v1.x) step.

9. Update the existing command line as follows.

   This modification is required to enable support for the 1.5B and 25B flow cells and future 8-tube library strips. Changes are underlined.

   bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:validate_output_containers -l {compoundOutputFileLuid1} -r 'Library 8-tube Strip:LC[0-9]{7}-L[A-Z]{1}1' -r 'Library 2-tube Strip:LC[0-9]{7}-L[A-Z]{1}2' -max 1"

Modify Existing Steps

Enable PhiX Control

1. On the Configuration tab, select Lab Work.

2. Select the Make Bulk Pool (NovaSeq X Series Sequencing v1.x) step.

3. For Control Types, remove PhiX v3.

4. For the Record Details milestone, navigate to Step Data and remove the following fields:

   • Final Loading Volume (ul)

   • % PhiX (2.0 nM) Spike-In

5. Select the Dilute and Denature (NovaSeq X Series v1.x) step.

6. Select the Record Details milestone.

7. Navigate to Step Data and add 1-2% PhiX Spike-In to Master Step Fields.

Enable New Flow Cell Types

1. On the Configuration tab, select Lab Work.

2. Select the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.x) step.

3. Select the Queue milestone.

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

5. Select the Ice Bucket milestone.

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

7. Select the Placement milestone.

8. Navigate to Destination Containers and add Library 2-tube Strip.