Manual Upgrade

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

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

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

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

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

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

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

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

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

Use the following instructions to manually update the workflows.

Prerequisites

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

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

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

NovaSeqXSeries_Bulk_Pool1.csv:

NovaSeqXSeries_Bulk_Pool2.csv:

NovaSeqXSeries_Dilute_Denature_Calculate_Volumes.csv:

Add New Containers

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:

   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

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

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

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

Modify Existing Global Field

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:

   • 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:

     Copy

     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

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

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 in red.

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

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 in red.

   bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} -log {compoundOutputFileLuid1} \ script:evaluateDynamicExpression \ -exp ' if (input.::NovaSeq X Flowcell Type:: == ::10B:: || input.::NovaSeq X Flowcell Type:: == ::1.5B::) { output.::NaOH Volume (ul):: = 8.5; output.::TT2 Volume (ul):: = 127.5; output.::BP Aliquot Volume (ul):: = input.::Final Loading Concentration (pM):: * 170 / (2 * 1000); output.::RSB Volume (ul):: = 34 - output.::BP Aliquot Volume (ul)::; if (step.::1-2% PhiX Spike-In::) { output.::PhiX Volume (ul):: = 1; output.::PhiX Concentration (pM):: = 300; } else { output.::PhiX Volume (ul):: = ::::; output.::PhiX Concentration (pM):: = ::::; }; } else { output.::NaOH Volume (ul):: = 14; output.::TT2 Volume (ul):: = 210; output.::BP Aliquot Volume (ul):: = input.::Final Loading Concentration (pM):: * 280 / (2 * 1000); output.::RSB Volume (ul):: = 56 - output.::BP Aliquot Volume (ul)::; if (step.::1-2% PhiX Spike-In::) { output.::PhiX Volume (ul):: = 1.6; output.::PhiX Concentration (pM):: = 300; } else { output.::PhiX Volume (ul):: = ::::; output.::PhiX Concentration (pM):: = ::::; }; }' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} \ script:driver_file_generator \ -t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeqXSeries_Dilute_Denature_Calculate_Volumes.csv -o {compoundOutputFileLuid0}.csv -q true -destLIMSID {compoundOutputFileLuid0} -l {compoundOutputFileLuid1} \ && echo; echo 'Calculate Volumes completed successfully.'"

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 in red.

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

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 in red.

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

Modify Existing Steps

Enable PhiX Control

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.

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

   • Final Loading Volume (ul)

   • % PhiX (2.0 nM) Spike-In

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

2. Select the Record Details milestone.

3. 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.

1. Select the Ice Bucket milestone.

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

3. Select the Placement milestone.

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