Installation

The Illumina NovaSeq 6000 Integration v2.5.0 supports the integration between Clarity LIMS and the NovaSeq 6000 instrument. This integration also provides support for Clarity LIMS v6.2.0 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 v2.5.0. It also describes the components that are installed in the default configuration.

Supporting Documentation

• For details on installed protocols and steps, automations, generated and captured files, and rules and constraints, refer to NovaSeq 6000 Integration v2.5.0 Configuration.

• For information on user interaction for each step, validating and troubleshooting the integration, refer to NovaSeq 6000 Integration v2.5.0 User Interaction, Validation and Troubleshooting.

Prerequisites

NovaSeq 6000 Integration v2.5.0 has the following prerequisites:

• Mount run data network-attached storage (NAS) share

• Secret Util is installed

• IPP is installed

Prerequisite 1: Mount Run Data NAS Share

Mounting the NAS share of run data is needed to capture and generate files associated with the sequencing run. To mount NAS shares that contain data from the Clarity LIMS server, set up the following Write privileges as the glsai user:

• Sample sheet directory — This directory is where the Prepare Files for NovaSeq script writes sample sheets.

• Run recipe directory — This directory is where the Prepare Files for NovaSeq script writes run recipe files in the JSON format.

Set up the Read privileges as the glsjboss and glsai users (or as the entire claritylims group) for the run output directory. This directory is where the sequencing service looks for run folders. The run folders must be in this directory. The service does not search sub-directories.

The sample sheet, run recipe, and run output directories can be configured independently and do not have to be on the same mount or share.

Prerequisite 2: Secret Util Installation

The Clarity LIMS installation tooling configures the Secret Util installation. No additional configuration is necessary. For more information on Secret Utility configuration, refer to the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

Installation

NovaSeq 6000 Integration v2.5.0 supports both on-premise and cloud integrations. This integration is distributed as the following RPM packages:

• BaseSpaceLIMS-novaseq-extensions

• BaseSpaceLIMS-novaseq-sequencing-service (depends on BaseSpaceLIMS Automation Worker v1.x)

The BaseSpaceLIMS-novaseq-extensions RPM installs the following items:

• Protocols and steps

• Validation workflow

• Database properties that configure the service

• novaseq-extensions.jar file that provides sample sheet generation and other utility scripts

The BaseSpaceLIMS-novaseq-sequencing-service RPM installs the following items:

• Bash scripts to run the novaseq_seqservice through systemctl

• novaseq-sequencing.jar

• novaseq-remote-extensions.jar file that contains scripts that need access to the run folders on the NAS

On-Premise Installation

Use the following instructions to install the BaseSpaceLIMS-novaseq-extensions and BaseSpaceLIMS-novaseq-sequencing-service RPMs on the Clarity LIMS server.

Install the RPMs

1. On the Clarity LIMS server, log in as the root user.

2. Run the following yum commands to install the RPMs:

   ℹ You must include the --enablerepo command line argument. Illumina Support provides the repo file and appropriate name to use.

   yum install BaseSpaceLIMS-novaseq-extensions --enablerepo=<< repo name info from support >>
   yum install BaseSpaceLIMS-novaseq-sequencing-service --enablerepo=<< repo name info from support >>

3. Enter y to confirm that you want to proceed with the RPM installations.

4. If Secret Util is not installed or configured, you must configure it before proceeding. As the glsjboss user, run the following script:

   bash /opt/gls/clarity/config/pending/05_configure_claritylims_secretutil.sh

   For more information on installing Secret Util with the Integration Module, refer to Install/Upgrade Secret Management for Integration Modules in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

   ℹ For the on-premise installation, the Illumina Vault server is not available for public access.

   In the configuration, make sure the system passwords are configured correctly in File mode.

5. [Optional] If you are upgrading from a previous integration, check for any usages of the Custom API Username in the current system as follows.

   1. Install ClarityLIMS-UpgradePreValidation.

   2. Check for any custom API username requirements (e.g., novaseq_user).

      Typically, the default API user name is apiuser.

   3. Run the following script:

      /opt/gls/ClarityUpgradeValidation/bin/checkUsernameConfiguration.sh

   4. After the script is complete, check that a custom user is configured in the results.

      If a custom API username is configured, a warning message displays.

   5. If a custom API username is required, configure the username and password using Secret Util:

      java -jar /opt/gls/clarity/tools/secretutil/secretutil.jar -n=INTEGRATION -u= 

      For a custom API username, set the to apiusers/{custom API username} (e.g., apiusers/novaseq_user). For more information on Secret Utility configuration, refer to the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
   6. Use the following command to make sure that the password is saved correctly in Secret Util:

      java -jar /opt/gls/clarity/tools/secretutil/secretutil.jar -n=INTEGRATION 

Import Workflow Configurations for NovaSeq 6000

1. As the glsjboss user, use the following command to run the configure_extensions_novaseq.sh script:

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

2. When prompted, enter the channel name for the Automation Worker and sequencing service.

When upgrading from NovaSeq Integration v2.2.0 to v2.5.0, a conflict is expected during the workflow import with the Flowcell Type analyte custom field due to SP being added to the list of preset values. You can manually add SP as one of the values of the Flowcell Type custom field after you import the workflow.

Configure the Database Service Properties

To enable capture and generation of files associated with the sequencing run, configure the properties using the omxProps-ConfigTool utility.

Start the Sequencing Service

1. Run the following command to start the sequencing service:

   systemctl start novaseq_seqservice

Configurable Database Properties

Property	Description
novaseq.runSetupFolder	The directory where the Prepare Files for NovaSeq script writes run recipe files in the JSON format. This must be an existing folder and must be writable by the glsai user. For more information, refer to Prerequisites.
novaseq.sampleSheetPathPrefixSearch.SUFFIX	The directory where NVCS or analysis software looks for sample sheets. This directory is typically a Windows path or a network drive. The integration does not use this path directly, but the attachment filed in the run recipe file is determined by adding the sample sheet file name to this value.
novaseq.sampleSheetPathPrefixReplace.SUFFIX	The directory where the Prepare Files for NovaSeq script writes sample sheets. This must be an existing folder and must be writable by the glsai user. For more information, refer to Prerequisites.
novaseq.sampleSheetPathPrefixSearchReplaceSuffixes	This property must be set to the SUFFIX used in the novaseq.sampleSheetPathPrefixSearch.SUFFIX and novaseq.sampleSheetPathPrefixReplace.SUFFIX properties.
novaseq.seqservice.netPathPrefixSearch.SUFFIX	This property is the search prefix used to map the OutputRunFolder field in the RunParameters.xml file to the corresponding Linux directory. The OutputRunFolder is converted by replacing this string with the netPathPrefixReplace value. The value of this property is also in the output_folder field of the run recipe file.
novaseq.seqservice.netPathPrefixReplace.SUFFIX	The directory where the sequencing service looks for run folders. This must be an existing folder and must be writable by the glsai or glsjboss users, or by the claritylims group. For more information, refer to Prerequisites. The run folders must be in the configured directory. The service does not search sub-directories.
novaseq.seqservice.netPathPrefixSearchReplaceSuffixes	This property must be set to the single SUFFIX used in novaseq.seqservice.netPathPrefixSearch.SUFFIX and novaseq.seqservice.netPathPrefixReplace.SUFFIX.

This version of the integration only supports a single active search-replace pair of each type. The sampleSheetPathPrefixSearchReplaceSuffixes and netPathPrefixSearchReplaceSuffixes properties must contain a single number instead of a comma-separated list of numbers.

The sample sheet, run recipe, and run output folders can all be configured independently and do not have to be on the same mount or share. For examples of valid configuration mount points and properties, refer to Example Network Folder Structure and Database Properties.

Cloud Installation

The BaseSpaceLIMS-novaseq-extensions RPM must be installed on the Clarity LIMS server. The BaseSpaceLIMS-novaseq-sequencing-service RPM can be installed remotely on another server within the network.

Specifications

The following hardware, operating system, and network specifications must be met to install the BaseSpaceLIMS-novaseq-sequencing-service RPM:

• Hardware requirements:

  • 64-bit processor (dual core 2.0 GHz)

  • OS requirements, plus at least an additional 2 GB RAM

  • A minimum of 5 GB of hard disk space

• Operating system requirements:

  • Clarity LIMS v6.2 or later

  • Oracle Linux (for compatibility, refer to NovaSeq 6000 Integration v2.5.0 Release Notes)
  • Minimum requirements for remote Automation Worker (for more information, refer to the Clarity LIMS (Clarity & LabLink Reference Guide) documentation)

  ℹ If you are running the sequencing service and Automation Worker on the same instance, make sure that the instance is running a compatible version of Oracle Linux. For compatibility, refer to NovaSeq 6000 Integration v2.5.0 Release Notes.

• Network requirements:

  • SSL access to the Clarity LIMS server from the network

  • The mounted folder where the run recipes are written

  • The mounted folder where the sample sheets are written

  • The mounted folder where the sequencing runs are written

Install the RPM

1. On the applicable server, log in as the root user.

2. Run the following yum command to install the BaseSpaceLIMS-novaseq-extensions RPM:

   ℹ You must include the --enablerepo command line argument. Illumina Support provides the repo file and appropriate name to use.

   yum install BaseSpaceLIMS-novaseq-extensions --enablerepo=<< repo name info from support >>

3. Enter y to confirm that you want to proceed with the RPM installation.

4. If Secret Util is not installed or configured, you must configure it before proceeding. As the glsjboss user, run the following script:

   bash /opt/gls/clarity/config/pending/05_configure_claritylims_secretutil.sh

   For more information on installing Secret Util with the Integration Module, refer to Install/Upgrade Secret Management for Integration Modules in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
5. [Optional] If a custom API username is required, configure the username and password as follows.

   1. Run the following command using Secret Util:

      java -jar /opt/gls/clarity/tools/secretutil/secretutil.jar -n=INTEGRATION -u= 

      For a custom API username, set the to apiusers/{custom API username} (e.g., apiusers/novaseq_user). For more information on Secret Utility configuration, refer to the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
   2. Use the following command to make sure that the password is saved correctly in Secret Util:

      java -jar /opt/gls/clarity/tools/secretutil/secretutil.jar -n=INTEGRATION 

Import Workflow Configurations for NovaSeq 6000

1. As the glsjboss user, use the following command to run the configure_extensions_novaseq.sh script:

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

2. When prompted, enter the channel name for the Automation Worker and sequencing service. The recommended channel name is remotens. For more information, refer to Update Channel Name.

When upgrading from NovaSeq Integration v2.2.0 to v2.5.0, a conflict is expected during the workflow import with the Flowcell Type analyte custom field due to SP being added to the list of preset values. You can manually add SP as one of the values of the Flowcell Type custom field after you import the workflow.

Update Channel Name

The configure_extensions_novaseq.sh script prompts for a channel name and sets the name on the following preconfigured automations:

• Prepare Files for NovaSeq (configured on Dilute and Denature (NovaSeq 6000 v2.3) and Load to Flowcell (NovaSeq 6000 v2.3) steps)

• Read InterOp Metrics (configured on AUTOMATED NovaSeq Run (NovaSeq 6000 v2.3) step)

If you need to change the channel name, you must update it manually on each automation. The import script does not update this setting.

Configure the Database Service Properties

To enable capture and generation of files associated with the sequencing run, configure the properties using the omxProps-ConfigTool utility.

Install Sequencing Service RPM on Remote Server

1. On the applicable server, log in as the root user.

2. Run the following yum command to install the RPM:

   yum install BaseSpaceLIMS-novaseq-sequencing-service

3. Enter y to confirm that you want to proceed with the RPM installation.

Set Up API Connection Properties File

1. Run the following script to set up the API connection properties file at /opt/gls/clarity/automation_worker/conf/api-access.properties:

   bash /opt/gls/clarity/automation_worker/node/bin/configure.sh

The configure.sh script is provided by BaseSpaceLIMS-AutomationWorker.

Start the Sequencing Service

1. Run the following command to start the sequencing service:

   systemctl start novaseq_seqservice

Configurable Database Properties

Property	Description
novaseq.runSetupFolder	The directory where the Prepare Files for NovaSeq script writes run recipe files in the JSON format. This must be an existing folder and must be writable by the glsai user. For more information, refer to Prerequisites.
novaseq.sampleSheetPathPrefixSearch.SUFFIX	The directory where NVCS or analysis software looks for sample sheets. This directory is typically a Windows path or a network drive. The integration does not use this path directly, but the attachment filed in the run recipe file is determined by adding the sample sheet file name to this value.
novaseq.sampleSheetPathPrefixReplace.SUFFIX	The directory where the Prepare Files for NovaSeq script writes sample sheets. This must be an existing folder and must be writable by the glsai user. For more information, refer to Prerequisites.
novaseq.sampleSheetPathPrefixSearchReplaceSuffixes	This property must be set to the SUFFIX used in the novaseq.sampleSheetPathPrefixSearch.SUFFIX and novaseq.sampleSheetPathPrefixReplace.SUFFIX properties.
novaseq.seqservice.netPathPrefixSearch.SUFFIX	This property is the search prefix used to map the OutputRunFolder field in the RunParameters.xml file to the corresponding Linux directory. The OutputRunFolder is converted by replacing this string with the netPathPrefixReplace value. The value of this property is also in the output_folder field of the run recipe file.
novaseq.seqservice.netPathPrefixReplace.SUFFIX	The directory where the sequencing service looks for run folders. This must be an existing folder and must be writable by the glsai or glsjboss users, or by the claritylims group. For more information, refer to Prerequisites. The run folders must be in the configured directory. The service does not search sub-directories.
novaseq.seqservice.netPathPrefixSearchReplaceSuffixes	This property must be set to the single SUFFIX used in novaseq.seqservice.netPathPrefixSearch.SUFFIX and novaseq.seqservice.netPathPrefixReplace.SUFFIX.

This version of the integration only supports a single active search-replace pair of each type. The sampleSheetPathPrefixSearchReplaceSuffixes and netPathPrefixSearchReplaceSuffixes properties must contain a single number instead of a comma-separated list of numbers.

The sample sheet, run recipe, and run output folders can all be configured independently and do not have to be on the same mount or share. For examples of valid configuration mount points and properties, refer to Example Network Folder Structure and Database Properties.

Workflows, Protocols, and Steps Installed

NovaSeq 6000 v2.3 Workflow

Protocol 1: Run Format (NovaSeq 6000 v2.3)

Steps:

1. Define Run Format (NovaSeq 6000 v2.3)

Protocol 2: NovaSeq Standard (NovaSeq 6000 v2.3)

Steps:

1. Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3)

2. Dilute and Denature (NovaSeq 6000 v2.3)

Protocol 3: NovaSeq Xp (NovaSeq 6000 v2.3)

Steps:

1. Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3)

2. Dilute, Denature & ExAmp (NovaSeq 6000 v2.3)

3. Load to Flowcell (NovaSeq 6000 v2.3)

Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3)

Steps:

1. AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3)

NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) Workflow

Protocol: NovaSeq Validation Library Prep (NovaSeq 6000 v2.3)

Steps:

1. NovaSeq Validation Library Prep (NovaSeq 6000 v2.3)

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 v2.5.0 Configuration.

For instructions on using the NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) protocol, refer to NovaSeq 6000 Integration v2.5.0 User Interaction, Validation and Troubleshooting.

The configuration provided with 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 processes.

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 File-Based.

   2. In the adjacent field, enter the network folder location that NVCS retrieves the JSON recipe from.

   3. In the Output Folder field, enter the default output folder location.

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.

Example Network Folder Structure and Database Properties

Instrument Control Software Configuration

Refer to the following examples for the configuration of the instrument control software:

• Sample sheet directory: \\nas\novaseq\sample_sheets

• JSON run recipes directory: \nas\novaseq\run_setup

• Output directory: \nas\novaseq\runs

Linux Server Mount Points

Refer to the following examples for the Linux server mount points on either the Clarity LIMS or sequencing service server:

• Sample sheet directory: /mnt/novaseq_sample_sheets

• JSON run recipes directory: /mnt/novaseq_run_setup

• Output directory: /mnt/novaseq_runs

Clarity LIMS Configuration Properties

Refer to the following examples for the Clarity LIMS configuration properties:

• novaseq.runSetupFolder: /mnt/novaseq_run_setup

• novaseq.sampleSheetPathPrefixSearchReplaceSuffixes: 1

• novaseq.sampleSheetPathPrefixSearch.1: \\nas\novaseq\sample_sheets

• novaseq.sampleSheetPathPrefixReplace.1: /mnt/novaseq_sample_sheets

• novaseq.seqservice.netPathPrefixSearchReplaceSuffixes: 1

• novaseq.seqservice.netPathPrefixSearch.1: \\nas\novaseq\runs

• novaseq.seqservice.netPathPrefixReplace.1: /mnt/novaseq_runs

Network Folder Structure and Contents

The following examples assume the same configuration and mount points from the previous sections.

After completing the Dilute and Denature (NovaSeq 6000 v2.3) or Load to Flowcell (NovaSeq 6000 v2.3) step, an automated script copies the run recipe JSON file and sample sheet to the appropriate network locations. For example, a library tube or flow cell with the barcode AA12345-NNN would have the following directory content:

• /mnt/novaseq_run_setup/AA12345-NNN.json (run recipe file)

• /mnt/novaseq_sample_sheets/AA12345-NNN.csv (sample sheet)

When the NovaSeq run starts, the instrument control software creates a new run folder named after the run ID. Then, the software copies the run recipe JSON file to a Clarity LIMS subfolder inside it. For example, a run ID of 170301_A001_AHHCC would have the following directory content:

• /mnt/novaseq_run_setup/AA12345-NNN.json

• /mnt/novaseq_sample_sheets/AA12345-NNN.csv

• /mnt/novaseq_runs/170301_A001_AHHCC/LIMS/AA12345-NNN.json

• /mnt/novaseq_runs/170301_A001_AHHCC/RunInfo.xml

• /mnt/novaseq_runs/170301_A001_AHHCC/RunParameters.xml

The NovaSeq 6000 sequencing service detects the run folder based on the presence of the three files inside it and starts the AUTOMATED NovaSeq Run (NovaSeq 6000 v2.3) step automatically.

After the sequencing run has completed, the Universal Copy Service copies the run data and InterOp files to the network drive. Then, the service creates a CopyComplete.txt file in the run folder. At this point, the directory would have the following content:

• /mnt/novaseq_run_setup/AA12345-NNN.json

• /mnt/novaseq_sample_sheets/AA12345-NNN.csv

• /mnt/novaseq_runs/170301_A001_AHHCC/LIMS/AA12345-NNN.json

• /mnt/novaseq_runs/170301_A001_AHHCC/RunInfo.xml

• /mnt/novaseq_runs/170301_A001_AHHCC/RunParameters.xml

• /mnt/novaseq_runs/170301_A001_AHHCC/InterOp

• /mnt/novaseq_runs/170301_A001_AHHCC/CopyComplete.txt

The NovaSeq 6000 sequencing service detects the completed sequencing run based on the presence of the CopyComplete.txt file, executes InterOp metrics parsing for the run, and automatically completes the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step.