NovaSeq 6000 Integration v3.4.0 Installation

The Illumina NovaSeq 6000 Integration v3.4.0 supports the integration between Clarity LIMS (v5.1 and later) and the NovaSeq 6000 instrument. This integration also provides support for Clarity LIMS v6 and includes updates to the technology stack and third party libraries and utilities.

Clarity LIMS is automatically stopped during installation of the RPM and will need to be restarted.

The file-based and API-based integrations cannot run at the same time. When the API-based integration has finished installing, shut down Clarity LIMS and uninstall the file-based integration.

This document provides instructions for installing NovaSeq 6000 Integration v3.4.0. It also describes the components that are installed in the default configuration.

• If you are installing the NovaSeq 6000 integration for the first time, follow the instructions provided in this document.
• If NovaSeq 6000 Integration v3.3.0 is already installed, with only minor changes to the default configuration, follow the instructions provided in this document and then duplicate the configuration changes in the v3.4.0 workflow.
• If NovaSeq 6000 Integration v3.3.0 is already installed, with significant changes to the default configuration, follow the instructions provided in NovaSeq 6000 Integration v3.4.0 Manual Upgrade.

Illumina NovaSeq 6000 Integration v3.4.0 includes the following features and functionality.

• Preconfigured NovaSeq 6000 v3.3 workflow that maps to lab protocols and instrument runs.
• The following preconfigured protocols:
– Run Format (NovaSeq 6000 v3.3)
– NovaSeq Standard (NovaSeq 6000 v3.3) (supports the loading of pooled libraries into a library tube)
– NovaSeq Xp (NovaSeq 6000 v3.3) (supports individual lane loading on the NovaSeq)
– AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.3)
• Automated generation of the v1 sample sheet. This sample sheet is used with either the bcl2fastq2 v2.20 analysis software or the v2 sample sheet. This file is automatically uploaded to the sequencing instrument through the Sequencer API and is used to set up the run.
• Automated validation of run setup information. This information is uploaded to the NovaSeq Control Software (NVCS) through the Sequencer API. It is used to create the run recipe and initiate the run.
• Automated tracking of the following NovaSeq sequencing runs and parsing of run statistics into Clarity LIMS:
– Progress and metrics of sequencing run
– Per-instrument sequencing runs (tracked as part of the Run ID field value)
– Sequencing run parameters
– Sequencing run data directory location
– Real-Time Analysis v3 (RTA3) version and other run-specific information
• [Optional] Preconfigured Library Prep Validation v2.0.4 workflow used for validation purposes. This workflow contains a single-step protocol that models the library prep required to produce normalized libraries that are ready for the NovaSeq 6000 v3.3 workflow.
• For details on installed protocols and steps, automations, generated and captured files, and rules and constraints, refer to NovaSeq 6000 Integration v3.4.0 Configuration and User Interaction.
• For information on validating and troubleshooting the integration, refer to NovaSeq 6000 Integration v3.4.0 Validation and Troubleshooting.
• For information on the NovaSeq 6000 instrument system, refer to the NovaSeq 6000 System Support Resources.

NovaSeq 6000 Integration v3.4.0 has the following prerequisites:

The Illumina NovaSeq Integration Package v3.4.0 depends on the configuration provided in Illumina Protocols (IPP) v2.3.

After the installation steps, install the following IPP configurations on the server used for the NovaSeq integration installation:

• QC Protocols Base configuration
• NovaSeq configuration
• Library Prep Validation configuration (optional, but recommended)

For details on IPP v2.3 installation and configuration, refer to the Illumina Preset Protocols documentation

The Illumina cloud hosted installation configures an additional include directive for /etc/httpd/conf.d/clarity.conf in the proxy configuration. Additional proxy configuration files for the Sequencer API service are expected to be placed in the /etc/httpd/conf.d/clarity folder.

If Clarity LIMS Secret Utility is not installed or configured, use the following information to configure Secret Utility:

• Installation on a hosted system with Clarity LIMS (v5.4 or later). The Clarity LIMS installation tooling configures this installation. No additional configuration is necessary.
• Installation on Clarity LIMS v5.3 (or other versions that are v5.1 and later). The installation includes the following components:
– Tenant DB Password
– Clarity DB Password
– FTP password
– LDAP manager password (if LDAP is enabled)
– LabLink database password (for Clarity LIMS v5.3 and later LabLink only)
– Reportingdatabase password (if installed)
– RabbitMQ password

For more information on Secret Utility configuration, refer to the Clarity LIMS Core Functionality documentation.

NovaSeq Integration v3.4.0 is distributed as the BaseSpaceLIMS-sequencer-api RPM package. This package must be installed on the Clarity LIMS server.

The BaseSpaceLIMS-sequencer-api RPM installs the following files and scripts:

•Sequencer API WAR file
•application.yml configuration file
•configure_sequencer_api_proxy.sh configuration script
•configure_sequencer_api_env.sh configuration script
• configure_sequencer_api_application.sh configuration script

The RPM installs the Sequencer API WAR file into the same Tomcat container as Clarity LIMS. The WAR file is at /opt/gls/clarity/tomcat/current/webapps/Illumina#Sequencer#v2##2.4.0.0.war. The version number can differ.

The WAR file is deployed automatically when Clarity LIMS launches. Because the RPM installs a new WAR file, Clarity LIMS is automatically stopped during installation of the RPM. Restart Clarity LIMS before installing the NovaSeq and Library Prep Validation workflow configuration from the IPP.

The application.yml configuration file is installed at /opt/gls/clarity/extensions/sequencer-api/application.yml. As part of the installation process, the file is configured with one of the configuration scripts.

For a complete list of all properties included in the application.yml file, refer to NovaSeq 6000 Integration v3.4.0 Configuration and User Interaction

The scripts in the following table are installed at /opt/gls/clarity/config/folder. During installation, the RPM runs these scripts as they are needed.

Script Name

User

Description

configure_sequencer_api_proxy.sh

root

Adds configuration to the proxy to allow communication with the Sequencer API after Tomcat has been started.

Run this script in the following scenarios:

• When the Sequencer API is first installed.
• When reconfiguring the Clarity LIMS proxy. This process happens until a version of Clarity LIMS that includes the necessary configuration files from /etc/httpd/clarity/*.conf is available.

Not required for Illumina cloud hosted installations.

configure_sequencer_api_env.sh

root

Adds Secret Management setting into the Clarity LIMS Tomcat configuration.

Run this script when the Sequencer API is first installed on a Secret Util-enabled Clarity LIMS.

configure_sequencer_api_application.sh

glsjboss

• Configures the installed application.yml file.
• Sets the Clarity LIMS URL and the key to use when signing access tokens.

Run this script when the Sequencer API is first installed.

If you run the script multiple times, the script reconfigures each setting to its default. The only exception is the signing key. For this setting, you are asked if you want a reset. If the reset is done, all previously issued tokens are no longer valid.

NovaSeq 6000 Integration v3.4.0 supports RedHat Enterprise Linux and CentOS up to v7.9. Use v7.9 due to mitigate security risks. CentOS v6 is not supported.

If running the sequencing service and Automation Worker on the same instance, the instance must also be running a supported version of the Linux operating system.

Install the base configuration of the QC Protocols from IPP as follows.

1. On the server being used to install the integration, log in as the root user.
2.Use the following yum command to install the IPP RPM:

yum install BaseSpaceLIMS-Illumina-Preset-Protocols --enablerepo=<< repo name info from support >>

The --enablerepo command-line argument must be included to enable the repo. The IlluminaSupport team can provide you with the repo file with the appropriate naming convention.

3.Enter y to confirm that you want to proceed with the RPM and package installation.
4.Make sure that NGS Extension Package v5.21 is installed.

If this package is not installed, you will be instructed to install it or upgrade the existing package.

5.As the glsjboss user, install the QC_Protocols.qc-protocols configuration from the following location:

/opt/gls/clarity/config/illumina-preset-protocols-installer.sh -o install QC_Protocols.qc-protocols.

For information on IPP installer commands, parameters, and options, refer to Illumina Preset Protocols Integration v2.3 Installation and Configuration in the Illumina Preset Protocols documentation.

Install the Sequencer API as follows.

1. On the Clarity LIMS server, log in as the root user.
2.Use the following yum command to install the RPM:

yum install BaseSpaceLIMS-sequencer-api --enablerepo=< repo name info from support >

The --enablerepo command-line argument must be included to enable the repo. The IllIllumina Support team can provide you with the repo file with the appropriate naming convention.

3.Enter y to confirm that you want to proceed with the RPM installation.
4.If necessary, install the Secret Utility Package.

For more information on this package, refer to Prerequisite 3: Secret Utility Configuration.

5.As the glsjboss user, run configure_sequencer_api_application.sh from the following location:

bash /opt/gls/clarity/config/configure_sequencer_api_application.sh

6.Enter values for the following questions:

You can also accept the default values in the application.yml configuration file by pressing Enter. For more information about the application.yml properties, refer to NovaSeq 6000 Integration v3.4.0 Configuration and User Interaction.

•Would you like to use the apiuser user for communication with Clarity? [Y]
•Enter required value for Token Expiry (hours) [88]
7.Update the Clarity LIMS proxy for the Sequencer API as follows.
•For an on-premise system, run the following command:

bash /opt/gls/clarity/config/configure_sequencer_api_proxy.sh

•For an Illumina cloud hosted system, make sure that the proxy has been updated as follows. Place the sequencer-api.include file in /etc/httpd/conf.d/clarity.

<Location "/Illumina/Sequencer/v2">

ProxyPass ajp://localhost:9009/Illumina/Sequencer/v2

ProxyPassReverse ajp://localhost:9009/Illumina/Sequencer/v2

</Location>

8.Update the Clarity LIMS Tomcat configuration as follows.
a. Make sure that the Secret Util is installed and configured. For more information, refer to Prerequisite 3: Secret Utility Configuration.
b. Run the following command to add the Secret Utility configuration to Clarity LIMS:

bash /opt/gls/clarity/config/configure_sequencer_api_env.sh

This script modifies /opt/gls/clarity/tomcat/current/bin/setenv.sh for Clarity LIMS Tomcat to load the Secret Utility environment variable file (/etc/profile.d/claritysecret.sh) during startup.

c. Use the following command to Start Clarity LIMS:

/opt/gls/clarity/bin/run_clarity.sh start

Install the NovaSeq and Library Prep workflow configuration from IPP as follows.

1. As the glsjboss user, run the following command to install the NovaSeq 6000 v3.3 workflow configuration:

bash /opt/gls/clarity/config/illumina-preset-protocols-installer.sh -o install Illumina_Instruments.novaseq-v3.3

The installer validates the import of the workflow and provides the Warning/Error details in STDOUT, which allows you to either proceed with the import or cancel it.

2.[Optional] Run the following command to install the Library Prep Validation v2.0.4 workflow:

/opt/gls/clarity/config/illumina-preset-protocols-installer.sh -o install Illumina_Instruments.LibraryPrepValidation-v2.0

This step is optional, but recommended. The Library Prep Validation v2.0.4 workflow allows you to validate the NovaSeq 6000 v3.3 workflow.

Make sure that the Sequencer API is working as follows.

1. Visit https://<< Clarity LIMS URL >>/Illumina/Sequencer/v2/sequencing-run/login. This page returns the following URL string:

https://<< Clarity LIMS URL >>/Illumina/Sequencer/v2/oauth/authorize?client_id=illumina_sequencer&amp;redirect_uri=../sequencer_login&amp;response_type=token

2.Copy and paste the URL string into the browser. The Clarity LIMS Sign In page opens.
3.Enter sign in credentials.
4.Select Sign In. A Successfully Authenticated message displays.

The installer script configures the application.yml configuration properties by prompting for values. You can also configure these properties by editing the application.yml file, and then restarting Clarity LIMS. Configuring these properties is optional.

For more information, refer to NovaSeq 6000 Integration v3.4.0 Configuration and User Interaction.

NovaSeq 6000 Integration v3.4.0 includes the workflows, protocols, and steps listed in the following table.

Package

Workflow

Protocols

Steps

NovaSeq 6000 Integration v3.4.0

NovaSeq 6000 v3.1

Run Format (NovaSeq 6000 v3.3)

1. Define Run Format (NovaSeq 6000 v3.3)

NovaSeq Standard (NovaSeq 6000 v3.3)

1. Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.3)
2.Dilute and Denature (NovaSeq 6000 v3.3)

NovaSeq Xp (NovaSeq 6000 v3.3)

1. Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.3)
2.Dilute, Denature, and ExAmp (NovaSeq 6000 v3.3)
3.Load to Flowcell (NovaSeq 6000 v3.3)

AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.3)

1. AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.3)

IPP v2.0

Library Prep Validation v2.0.4

Library Prep Validation v2.0.4

1. Library Prep Validation v2.0.4

For detailed descriptions of the steps and automations included in each protocol, and details on other components in the configuration, refer to NovaSeq 6000 Integration v3.4.0 Configuration and User Interaction.

For instructions on using the Library Prep Validation v2.0.4 protocol, refer to NovaSeq 6000 Integration v3.4.0 Validation and Troubleshooting.

The NovaSeq instrument software includes the following components:

•NovaSeq Control Software (NVCS)—Contains the user interface for setting up the sequencing run. Responsible for controlling the instrument and acquiring the images.
•Real-Time Analysis v3 (RTA3)—Takes the images generated by the first module, processes, and analyzes them. Makes sure that data files are created and copied to the final destination folder.
•Sequencing Analysis Viewer (SAV)—Displays the important quality metrics generated by the RTA3 software.
•NovaSeq recipes—Provides system operation instructions for use with NovaSeq 6000 reagent kits for SP, S1, S2, and S4 flow cells.
•Universal Copy Service—Copies output files to destinations such as final destination folder and/or BaseSpace Sequence Hub (when the instrument is configured for use with BaseSpace Sequence Hub).

For the NovaSeq API integration to work, the NovaSeq instrument must be able to communicate with Clarity LIMS through the API. Complete the following steps to configure the NVCS and confirm that you can access Clarity LIMS from the instrument.

1. Launch NVCS and wait for the initialization process to complete.
2.On the NVCS Settings page, do the following:
a. Under Mode Selection, select Server-Based.
b. In the adjacent field, enter the Clarity LIMS URL (eg, https://customerserver.claritylims.com).
c. [Optional] In the Output Folder field, enter the default output folder location. You can also specify the output folder location when setting up a run in NVCS.
3.Select Save to complete the configuration.
4.On the NVCS home page, you can now select Sequence to log into Clarity LIMS and start a run.