NextSeq 500/550 Integration v2.3 Installation

The Illumina NextSeq 500/550 Integration v2.3 supports the integration between Clarity LIMS and the NextSeq 500/550 instrument. This document provides instructions for installing NextSeq 500/550 Integration v2.3. It also describes the components that are installed in the default configuration.

Illumina NextSeq 500/550 Integration v2.3 includes the following features and functionality:

• Added support for Oracle Linux. For compatibility, refer to NextSeq 500/550 Integration v2.3 Release Notes.
• A new NextSeq 500/550 v1.1 workflow that maps to lab protocols and instrument runs. This workflow includes a minor bug fix and has no additional changes from the previous version.
• Automated generation of the sample sheet. This sample sheet is used with the bcl2fastq2 v2.20.0 analysis software.
• Automated tracking of the NextSeq sequencing runs and parsing of run statistics into Clarity LIMS, which includes the following information:
– Sequencing run metrics
– Sequencing run parameters
– Real-Time Analysis v2 (RTA2) run directory location and other run-specific information
• Preconfigured Library Prep Validation v2.3.1 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 NextSeq 500/550 Sequencing v1.1 workflow. For more information, refer to NextSeq 500/550 Integration v2.3 Validation and Troubleshooting.

The integration is compatible with the following software:

• Clarity LIMS v6.2 and later
• Secret Util v1.0 and later (v1.2 is recommended)
• IPP v2.6 and later

This integration is not fully compatible with NextSeq 500/550 Integration v1.

• For details on installed protocols and steps, automations, generated and captured files, and rules and constraints, refer to NextSeq 500/550 v2.3 Configuration and User Interaction.
• For information on validating and troubleshooting the integration, refer to NextSeq 500/550 Integration v2.3 Validation and Troubleshooting.

NextSeq 500/550 Integration v2.3 has the following prerequisites:

• Mount run data network-attached storage (NAS) share
• Secret Util is installed
• IPP is installed

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, use Read/Write priviledges as the glsjboss user. The following data can be mounted to the NAS share:

• Run data (eg, \\network-storage\run_data\)
• Clarity LIMS-created events triggered by the End Run event of the Illumina sequencing run (eg, \\network-storage\illumina\gls_events\)

With Read access, the Clarity LIMS server reads the following information in individual sequencing run data folders:

• Run information metadata from <runFolderRoot>/RunInfo.xml and <runFolderRoot>/RunParameters.xml
• Run statistics from <runFolderRoot>/InterOp/*.bin

The Clarity LIMS server generates the following files and information locally and stores them in Clarity LIMS:

• Sample sheet (CSV file)
• Run report (PDF file)
• Run folder root link

The Clarity LIMS server copies and stores the following files from individual sequencing run data folders in Clarity LIMS:

• <runFolderRoot>/RunInfo.xml
• <runFolderRoot>/RunParameters.xml

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 (v6.2 or later)

The Clarity LIMS installation tooling configures this installation. No additional configuration is necessary.

Installation on an on-premise Clarity LIMS instance (v6.2 or later)

Install and configure Secret Util as follows.

1. Install the ClarityLIMS-SecretUtil RPM.
2. 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 Core Functionality documentation.

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.

3. 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=<password> <key>

For a custom API username, set the <key> to apiusers/{custom API username} (eg, apiusers/novaseq_user). For more information on Secret Utility configuration, refer to the Clarity LIMS Core Functionality documentation.

4. 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 <key>

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

NextSeq 500/550 Integration v2.3 depends on the configuration provided in IPP (v2.6 and later) for Clarity LIMS v6.2 and later.

If the base configuration is not installed, then install it on the Clarity LIMS server that is being used for the NextSeq integration. For details on IPP v2.6 installation and configuration, refer to the Illumina Preset Protocols documentation.

If you are upgrading the base configuration, make sure that the IPP package is compatible with the version of Clarity LIMS you are installing (in this case, IPP v2.6 for Clarity LIMS v6.2).

NextSeq 500/550 Integration v2.3 is distributed as the ClarityLIMS-Illumina-NextSeq-Package-v2 RPM package that must be installed on the Clarity LIMS server. This package installs the following items:

• Bash scripts used to run the service
• The nextseq-sequencing.jar file
• The configure_extensions_nextseq_sequencingservice.sh script
• Smoke test directories

When upgrading from an existing NextSeq 500/550 workflow, the following warning messages can display when the illumina-preset-protocls-installer.sh script is running:

• WARN Installer - Conflicting content:
• WARN Installer - Analyte UDFs: % Aligned R1
• WARN Installer - Analyte UDFs: % Aligned R2
• WARN Installer - Analyte UDFs: % Bases >=Q30 R1
• WARN Installer - Analyte UDFs: % Bases >=Q30 R2
• WARN Installer - Analyte UDFs: Cluster Density (K/mm^2) R1
• WARN Installer - Analyte UDFs: Cluster Density (K/mm^2) R2
• WARN Installer - Analyte UDFs: Clusters PF R1
• WARN Installer - Analyte UDFs: Clusters PF R2
• WARN Installer - Analyte UDFs: Clusters Raw R1
• WARN Installer - Analyte UDFs: Clusters Raw R2
• WARN Installer - Analyte UDFs: Intensity Cycle 1 R1
• WARN Installer - Analyte UDFs: Intensity Cycle 1 R2

These messages are due to configuration changes that have increased the precision of the listed fields from 1 to 2 so that they show more decimal places. It is safe to override these warnings.

The following installation steps are required for the installation of NextSeq 500/550 Integration v2.3.

The NextSeq RPM must be installed on the Clarity LIMS server. This is where the AI node or Automation Worker is installed. For LIMS v5.x and v6.x, the automations and sequencing service use the existing Automation Worker.

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

You must use the --enablerepo command line argument to enable the repo. For the repo file and the correct name to use, contact Illumina Support.

yum install ClarityLIMS-Illumina-NextSeq-Package-v2 --enablerepo=< repo name info from support >

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

After confirmation, you are prompted to install the NextSeq 500/550 workflow from the IPP RPM.

If you have not installed the SecretUtil RPM before, this RPM installation also installs it.

• For hosted installations, the Secret Util installation and configuration is handled by tooling.
• For on-premise installations, if the Secret Util package is not already installed or configured, you must configure the Secret Util first. For more information, refer to Prerequisite 2: Secret Util Installation.
4. Install the NextSeq 500/550 workflow as follows.
a. As the glsjboss user, run the following command to view the complete list of IPP workflows:

/opt/gls/clarity/config/illumina-preset-protocols-installer.sh -o list

b. Run the following command to install the NextSeq 500/550 Sequencing v1.1 workflow:

/opt/gls/clarity/config/illumina-preset-protocols-installer.sh -o install Illumina_Instruments.nextseq550-v1.1

c. [Optional] Run the following command to install the Library Prep Validation v2.3.1 workflow that is used to validate the NextSeq 500/550 Sequencing v1.1 workflow:

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

5. Run the following script to configure the service properties:

/opt/gls/clarity/config/configure_extensions_nextseq_sequencingservice-v2.sh

To enable capture and generation of files associated with the sequencing run, configure the following properties using the omxProps-ConfigTool utility. The property configuration for NextSeq 500/550 v2 has the suffix .v2 and is different from the setting for v1.

Configurable Database Properties

Property

Description

Default Value

nextseq.v2.seqservice.eventFileDirectory.1

A monitored network location used for event files.

/mnt/gls_events

nextseq.v2.seqservice.eventFileDirectory.99

The monitored smoke test location used for event files. This location is also used for the installation and upgrade of smoke tests.

/opt/gls/clarity/smoketests/nextseq/

monitored

nextseq.v2.seqservice.netPathPrefixSearch.1

The network directory prefix contained in the event file.

\\nas\network\run_data

nextseq.v2.seqservice.netPathPrefixSearch.99

The default benign smoke test search entry. This property is used for the installation and upgrade of smoke tests.

/

nextseq.v2.seqservice.netPathPrefixReplace.1

The mapped network directory mount name on the server used to access the run data directory.

/mnt/run_data

nextseq.v2.seqservice.netPathPrefixReplace.99

The default benign smoke test replace entry. This property is used for the installation and upgrade of smoke tests.

/

nextseq.v2.seqservice.eventFileDirectorySuffixes

A list of eventFileDirectory path entries used to monitor for event files. The value is one or more comma separated integers.

99

When installed, this property must be configured to point to 1 unless a validation test is being performed with the 99 version of the search, replace, and event file entries.

nextseq.v2.seqservice.netPathPrefixSearchReplaceSuffixes

A list of netPathPrefix search and replace entries for transforming Windows to Linux network paths. The value is one or more comma separated integers.

99

When installed, this property must be configured to point to 1 unless a validation test is being performed with the 99 version of the search, replace, and event file entries.
1. Run the following command to start the sequencing service:

systemctl start nextseq_seqservice-v2

In NextSeq Control Software (NCS) v4.0 the location of NextSeq.Configuration.xml RTA configuration file is changed. The new location is C:\Program Files\Illumina\RTA\2.11.3\Configs.

A new gls_events_ncs_rta.bat batch file is required to generate a valid EndRun event file. This file is available in the NextSeq 500/550 RPM at /opt/gls/clarity/extensions/Illumina_NextSeq/v2/InstrumentIntegrations.

Configuration

1. Download the latest batch file from the NextSeq 500/550 RPM and place it in a folder (eg, C:\Illumina\gls). If necessary, remove the old batch file.
2. Edit the NextSeq.Configuration.xml configuration file at C:\Program Files\Illumina\RTA\2.11.3\Configs.
3. If you cannot modify the file, then open it as an administrator. Insert the following text inside the <Processing> tag. If the batch file is not at C:\Illumina\gls, then change that text to the applicable path.

<PostProcessEventFile>C:\Illumina\gls\gls_event_ncs_rta.bat</PostProcessEventFile>

4. Save the configuration file.
5. Validate the file by running RTA manually. In the command prompt, run the following command:

"C:\Program Files\Illumina\RTA\2.11.3\RTA.exe" "." configFile="C:\Program Files\Illumina\RTA\2.11.3\Configs\NextSeq.Configuration.xml" Processing.WorkProviderTypes="OfflineBased" processedfolder="."

The following error message displays:

Error: No run info file found in input directory .\RunInfo.xml

6. Validate the batch file by executing it manually as follows.
a. Using a text editor, edit the gls_events_ncs_rta.bat file.
b. In the "set DESTINATION_PATH=C:\Illumina\gls\Events\" code line, change C:\Illumina\gls\Events\ to the network path of the event files directory. Make sure to include the backslash at the end of the file path.

After manually executing the batch file, a dummy EndRun event file is created in the event files directory.

NextSeq 500/550 Integration v2.3 works with the NextSeq 500/550 Sequencing v1.1 workflow, which contains a single protocol with the same name. This protocol includes the following steps:

• Library Pooling (NextSeq 500/550 v1.1)
• Denature & Dilute (NextSeq 500/550 v1.1)
• NextSeq 500/550 Run (NextSeq 500/550 v1.1)

For descriptions of the protocol and the steps, refer to NextSeq 500/550 v2.3 Configuration and User Interaction. For instructions on using the Library Prep Validation v2.3.1 workflow to validate the automated sample sheet generation, refer to NextSeq 500/550 Integration v2.3 Validation and Troubleshooting.

The NCS is divided into the following modules:

• NCS—Controls the instrument operation, including various configuration settings. This software is installed and runs on the instrument.
• Real-Time Analysis 2 (RTA2)—Performs image processing and base calling (primary analysis). The software makes sure that data files are created and copied to the final destination folder and is installed and runs on the instrument.

For more information on NCS, refer to the NextSeq 500/550 documentation at support.illumina.com.

The instrument integration must be performed and maintained by Illumina Support. Illumina Support requires remote access to the instrument while it is idle.

Illumina Support has created batch files that use custom scripts during the key events of a sequencing run. When these batch files are used, they read the event information and write it in a TXT event file at the same network share location that the instrument uses to write the run data. Another process running on the Clarity LIMS server receives the event files and takes the appropriate actions.

The sequencing service monitors the end of the run event. This event is used to capture key step data and files and parse run statistics for output custom fields.

For NCS v4.0 and later, refer to the configuration steps in NextSeq Control Software v4.0 Support Configuration. For NCS versions before v4.0, perform the following steps.

Configure Batch Files

When the instrument is running, the final destination for the run data is a network storage path. The software is configured with a network storage path root (eg, \\network-storage\illumina\). Each sequencing run generates a unique run ID, which is appended to create a unique data run directory (eg, \\network-storage\illumina\110419_InstrumentName_0001_ARG1234567).

The Clarity LIMS batch files must be configured to write to a directory within the network storage path root. This directory is typically named gls_events, but the directory name can be different as long as no spaces are used.

Before configuring the batch files, do the following:

1. Back up the NextSeq.Configuration.xml configuration file to C:\Illumina\RTA\Configs\.
2. Make sure that the instrument is idle.
3. Shut down NCS.
4. Set up the directory structure as follows.
a. Create a directory (C:\Illumina\gls is recommended) on the local computer to hold the batch file.

Because of Windows 10 software restriction policies, this folder must be under C:\Illumina. Otherwise, the script is prohibited from running.

b. Create a directory (eg, gls_events) on the NAS to hold the event files.

Configure the batch files as follows.

1. Determine the site or instrument specific network storage path root.
2. Change the DESTINATION_PATH line to the name of the event file directory in the gls_event_ncs_rta.bat batch file:

Make sure to include the trailing \ in the DESTINATION_PATH line. Refer to the following example:

set DESTINATION_PATH=\\network-storage\illumina\gls_events\

3. Copy the DESTINATION_PATH and paste it into the Windows Explorer address bar.
4. Make sure that the network location is accessible and opens from the instrument.

The batch file needs to be deployed to the NextSeq 500/550 instrument. This file must be deployed to its own directory ( C:\Illumina\gls is recommended) on the instrument computer so that it is not overwritten or removed during an instrument software update.

Deploy the batch file as follows.

1. Download the batch file.
2. Copy the file to the directory that you created on the instrument computer (eg, C:\Illumina\gls).
3. From the Command Prompt, run the following command to list the contents of the directory:

dir C:\Illumina\gls\

4. Make sure that the name of the batch file does not contain any special or hidden characters.

Connect Batch Files to Instrument Events

Update the NCS configuration files as follows.

1. Restart the computer and make sure that the instrument is off.
2. Using Task Manager, make sure that NCS is not auto-launched by Windows.
3. If NCS is being auto-launched, remove it from the auto-launch list and restart the computer.
4. Make a backup copy of the RTA configuration XML file and name it NextSeq.Configuration.xml.
5. Edit the file.
6. Save the edited file.
7. Restart the computer.
8. Turn the instrument on and launch NCS.
9. Open the configuration file and make sure that the changes were saved.

Connect to RTA End Run Event

1. Edit the NextSeq.Configuration.xml configuration file at C:\Illumina\RTA\Configs\.
2. Insert the following text inside the <Processing> tags:

<PostProcessEventFile>C:\Illumina\gls\gls_event_ncs_rta.bat</PostProcessEventFile>

3. Validate the RTA configuration changes as follows.
c. From the command prompt, run the following command:

C:\Illumina\RTA\RTA.exe "." configFile="C:\Illumina\RTA\Configs\NextSeq.Configuration.xml"

Processing.WorkProviderTypes="OfflineBased" processedfolder="."

d. If the validation is successful, the following warning message displays. Select OK.

Error: No run info file found in input directory .\RunInfo.xml

If the configuration file has an error, the command returns specifics about the problem. Refer to the following example:

Error: While loading Configuration "c:\illumina\rta\configs\NextSeq.Configuration.xml"

There is an error in XML document <83, 5>

Error loading xml file: Unknown node 'GLS' at line 83 pos 5

4. Restart the computer.
5. Turn on the instrument and start NCS.
6. Open the NextSeq.Configuration.xml configuration file and make sure that the changes were saved.

Sample Sheet Generation

For instructions on how to validate the automated sample sheet generation, refer to NextSeq 500/550 Integration v2.3 Validation and Troubleshooting.

Manual Invocation of Event Files

The manual invocation of an event file produces an output that contains the following information:

• Filename (eg, event-EndRun-07295667.txt)
• cycleNumber (eg, 318)
• runFolder (eg, "D:\Illumina\NextSeqTemp\230815_M99999_0028_FC1234567-ABCDE")
• netFolder (eg, \\network-folder\Run_Data\230815_M99999_0028_FC1234567-ABCDE)
• readType (eg, 4)
• eventType (eg, EndRun)
• softwareType (eg, NCS)
• finishDate (eg, 2023-08-15)

If an event file is triggered automatically during a NextSeq 500/550 run, then the completion event does not appear until the run has completed and RTA completes primary analysis.

For instructions on validating the creation of event files, refer to NextSeq 500/550 Integration v2.3 Validation and Troubleshooting.

Instrument Sequencing Run

The instrument sequencing run test validates that the Clarity LIMS batch file is connected properly and invoked on the instrument events. Before validating the batch file, make sure that you have the following prerequisites are met:

• You have access to the NAS share.
• The default configuration has been successfully imported.
• Manual invocation of the event files has been validated. This validation checks for the following information:
– The DESTINATION_PATH is configured correctly.
– The instrument computer can access and write to the DESTINATION_PATH.
– There are no syntax errors in the Clarity LIMS batch script.

For more information on event file validation, refer to NextSeq 500/550 Integration v2.3 Validation and Troubleshooting.

The sequencing service processes and archives event files, which can cause validation issues while the service is running. You can make the following changes to avoid losing the event files that are you attempting to validate:

• Modify the FINAL_EXTENSION value in the Clarity LIMS batch file so that the file extension is .test instead of .txt. The service only processes and archives TXT files. Make sure that you change FINAL_EXTENSION back to .txt after manual validation.
• Monitor the NextSeqIntegrator.log file, which logs the file name and contents of each event file that is processed. The log file is at /opt/gls/clarity/extensions/Illumina_NextSeq/v2/SequencingService.

Validate the sequencing run as follows.

1. During the run, monitor the contents of the gls_events directory.
2. After the run is complete and the RTA completes primary analysis, make sure that a final EndRun event displays (eg, event-EndRun-11043279.txt).