Installation

The Illumina NovaSeq 6000 Integration v3.6.0 supports the integration between Clarity LIMS (v6.2 and later) and the NovaSeq 6000 instrument. This integration also includes updates to the technology stack and third party libraries and utilities. The key changes from v3.5.1 are as follows.

• Updated the preconfigured NovaSeq 6000 workflow to include Clarity LIMS Product Analytics (CLPA) automation scripts by default.

• Compatibility with Oracle Linux. For compatibility, refer to NovaSeq 6000 Integration v3.6.0 Release Notes.

• 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 v3.6.0. 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 v3.5.1 is already installed, with only minor changes to the default configuration, follow the instructions provided in this document and then duplicate the configuration changes from the v3.5.1 workflow.

• If there have been significant changes to the default configuration, follow the instructions provided in NovaSeq 6000 Integration v3.6.0 Manual Upgrade.

Supporting Documentation

• For details on installed protocols and steps, automations, generated and captured files, and rules and constraints, refer to NovaSeq 6000 Integration v3.6.0 Configuration.

• For information on validating and troubleshooting the integration, refer to NovaSeq 6000 Integration v3.6.0 User Interaction, Validation and Troubleshooting.

• For information on the NovaSeq 6000 instrument system, refer to the NovaSeq 6000 System Support Resources.

Prerequisites

NovaSeq 6000 Integration v3.6.0 has the following prerequisites:

Prerequisite 1: Clarity LIMS v6.2 and above

This integration requires Clarity LIMS v6.2 and above and uses the Clarity LIMS Secret Utility. The Secret Utility is installed and configured by the installation tooling for Clarity LIMS v6.2 and above. No additional configuration is required.

For more information on Secret Utility configuration, refer to the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

Prerequisite 2: Illumina Preset Protocols (IPP) v2.6

The integration package depends on the configuration provided in Illumina Protocols (IPP) v2.6.

The installation procedure provided in this document includes steps that install the following IPP configurations on the server used for the NovaSeq 6000 integration installation:

• NovaSeq configuration

• Library Prep Validation configuration (optional, but recommended)

For details on IPP v2.6 installation and configuration, refer to the Illumina Preset Protocols documentation.

Prerequisite 3: Proxy Update

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.

Sequencer API RPM Installation Components

NovaSeq Integration v3.6.0 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

Sequencer API WAR

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##2.6.0.0.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.

Configuration File

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 v3.6.0 Configuration.

Configuration Scripts

The scripts in the following table are installed at the /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. ℹ Not required for Illumina cloud hosted installations.
configure_sequencer_api_env.sh	root	Adds Secret Management setting into the Clarity LIMS Tomcat configuration.
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.

Installation Steps

If running the sequencing service and Automation Worker on the same instance, the instance must also be running a compatible version of Oracle Linux. For compatibility, refer to NovaSeq 6000 Integration v3.6.0 Release Notes.

Install the IPP Package

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 Illumina 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 and package installation.

4. Make sure that NGS Extension Package v5.23.0 is installed.

   If this package is not installed, install it or upgrade the existing package.

For information on IPP installer commands, parameters, and options, refer to the Illumina Preset Protocols documentation.

Install Sequencer API

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 Illumina 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 1: 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

1. 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 v3.6.0 Configuration.

   • Would you like to use the apiuser user for communication with Clarity? [Y]

   • Enter required value for Token Expiry (hours) [88]

2. 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.

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

3. Update the Clarity LIMS Tomcat configuration as follows.

   1. Make sure that the Secret Util is installed and configured. For more information, refer to Prerequisite 1: Secret Utility Configuration.
   2. Run the following command to add the Secret Utility configuration to Clarity LIMS:

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

      ℹ 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.

   3. Use the following command to Start Clarity LIMS:

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

Install NovaSeq and Library Prep Validation Workflow Configuration

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 v3.8 workflow configuration:

   bash /opt/gls/clarity/config/illumina-preset-protocols-installer.sh -o install Illumina_Instruments.novaseq-v3.8

   ℹ 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 v2.3.1 workflow:

   /opt/gls/clarity/config/illumina-preset-protocols-installer.sh -o install Illumina_Instruments.LibraryPrepValidation-v2.3

   This step is optional, but recommended. The Library Prep Validation v2.3.1 workflow allows you to validate the NovaSeq 6000 v3.8 workflow.

Check Sequencer API

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&redirect_uri=../sequencer_login&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.

[Optional] Configure application.yml Properties

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 v3.6.0 Configuration.

Workflows, Protocols, and Steps Installed

NovaSeq 6000 Integration v3.6.0 includes the workflows, protocols, and steps listed in the following table. The workflows are found in IPP v2.6.0 package.

NovaSeq 6000 v3.8 Workflow

Protocol 1: Run Format (NovaSeq 6000 v3.8)

Steps:

1. Define Run Format (NovaSeq 6000 v3.8)

Protocol 2: NovaSeq Standard (NovaSeq 6000 v3.8)

Steps:

1. Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8)

2. Dilute and Denature (NovaSeq 6000 v3.8)

Protocol 3: NovaSeq Xp (NovaSeq 6000 v3.8)

Steps:

1. Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8)

2. Dilute, Denature & ExAmp (NovaSeq 6000 v3.8)

3. Load to Flowcell (NovaSeq 6000 v3.8)

Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8)

Steps:

1. AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8)

Library Prep Validation v2.3.1 Workflow

Protocol: Library Prep Validation v2.3.1

Steps:

1. Library Prep Validation v2.3.1

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 v3.6.0 Configuration.

For instructions on using the Library Prep Validation v2.3.1 protocol, refer to NovaSeq 6000 Integration v3.6.0 User Interaction, Validation and Troubleshooting.

The configuration provided in this integration has been established to support NovaSeq 6000 lab processes. Any configuration changes to protocols or workflows, including renaming protocols, steps, and fields, could break process.

Instrument Software

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).

Instrument Integration Configuration

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:

   1. Under Mode Selection, select Server-Based.

   2. In the adjacent field, enter the Clarity LIMS URL (e.g., https://customerserver.claritylims.com).

   3. [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.