The Illumina NextSeq 500/550 Integration v2.5.0 supports the integration between Clarity LIMS and the NextSeq 500/550 instrument. This document provides instructions for installing NextSeq 500/550 Integration v2.5.0. It also describes the components that are installed in the default configuration.
Compatibility
Refer to under Instruments & Integrations.
This integration is not fully compatible with NextSeq 500/550 Integration v1.
Supporting Documentation
For details on installed protocols and steps, automations, generated and captured files, and rules and constraints, refer to .
For information on user interaction for each step, validating and troubleshooting the integration, refer to .
Prerequisites
This integration 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, use Read/Write priviledges as the glsjboss user. The following data can be mounted to the NAS share:
Run data (e.g., \\network-storage\run_data)
Clarity LIMS-created events triggered by the End Run event of the Illumina sequencing run (e.g., \\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 these files
<runFolderRoot>/RunInfo.xml
<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
Prerequisite 2: Secret Util Installation
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.
Install the ClarityLIMS-SecretUtil RPM.
If Secret Util is not installed or configured, you must configure it before proceeding. As the glsjboss user, run the following script:
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.
If a custom API username is required, configure the username and password using Secret Util:
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 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.
Installation
This integration 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:
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 this integration.
The NextSeq RPM must be installed on the Clarity LIMS server. This is where the AI node or Automation Worker is installed. The automations and sequencing service use the existing Automation Worker.
Install the RPM
On the Clarity LIMS server, log in as the root user.
Run the following yum command to install the RPM:
ℹ 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 >
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.
ℹ 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.
Install the NextSeq 500/550 workflow as follows.
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
Run the following command to install the NextSeq 500/550 Sequencing workflow:
Download the latest batch file from the NextSeq 500/550 RPM and place it in a folder (e.g., C:\Illumina\gls). If necessary, remove the old batch file.
Edit the NextSeq.Configuration.xml configuration file at
C:\Program Files\Illumina\RTA\2.11.3\Configs
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.
Error: No run info file found in input directory .\RunInfo.xml
Validate the batch file by executing it manually as follows.
Using a text editor, edit the gls_events_ncs_rta.bat file.
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.
Workflows, Protocols, and Steps Installed
The NextSeq 500/550 Integration works with the NextSeq 500/550 Sequencing workflow, which contains a single protocol with the same name. This protocol includes the following steps:
Library Pooling (NextSeq 500/550)
Denature & Dilute (NextSeq 500/550)
NextSeq 500/550 Run (NextSeq 500/550)
Instrument Software
The NextSeq Control Software (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.
Instrument Integration
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.
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 (e.g., \\network-storage\illumina). Each sequencing run generates a unique run ID, which is appended to create a unique data run directory (e.g., \\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:
Back up the NextSeq.Configuration.xml configuration file to
C:\Illumina\RTA\Configs\
Make sure that the instrument is idle.
Shut down NCS.
Set up the directory structure as follows.
Create a directory (C:\Illumina\gls is recommended) on the local computer to hold the batch file.
⚠ For Windows 10, the folder must be under C:\Illumina instead of C:\Illumina\gls because of Windows software restriction policies. If the folder is not in that directory, the batch script does not run. For versions before Windows 10, C:\Illumina\gls is acceptable.
Create a directory (e.g., gls_events) on the NAS to hold the event files.
Configure the batch file
Determine the site or instrument specific network storage path root.
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\
Copy the DESTINATION_PATH and paste it into the Windows Explorer address bar.
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.
Download the batch file.
Copy the file to the directory that you created on the instrument computer (e.g., C:\Illumina\gls).
From the Command Prompt, run the following command to list the contents of the directory:
dir C:\Illumina\gls\
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 NextSeq Control Software (NCS) configuration files as follows.
Restart the computer and make sure that the instrument is off.
Using Task Manager, make sure that NCS is not auto-launched by Windows.
If NCS is being auto-launched, remove it from the auto-launch list and restart the computer.
Make a backup copy of the RTA configuration XML file and name it NextSeq.Configuration.xml.
Edit the file.
Save the edited file.
Restart the computer.
Turn the instrument on and launch NCS.
Open the configuration file and make sure that the changes were saved.
Connect to RTA End Run Event
Edit the NextSeq.Configuration.xml configuration file at
C:\Illumina\RTA\Configs\
Insert the following text inside the <Processing> tags:
If the validation is successful, the following warning message displays.
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
Restart the computer.
Turn on the instrument and start NCS.
Open the NextSeq.Configuration.xml configuration file and make sure that the changes were saved.
Validation
Sample Sheet Generation
Manual Invocation of Event Files
The manual invocation of an event file produces an output that contains the following information:
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.
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.
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
During the run, monitor the contents of the gls_events directory.
After the run is complete and the RTA completes primary analysis, make sure that a final EndRun event displays (e.g., event-EndRun-11043279.txt).
For more information on installing Secret Util with the Integration Module, refer to Install/Upgrade Secret Management for Integration Modules in the .
For a custom API username, set the {key} to apiusers/{custom API username} (e.g., apiusers/novaseq_user). For more information on Secret Utility configuration, refer to the .
For more information on Secret Utility configuration, refer to the .
The NextSeq 500/550 Integration depends on the configuration provided in IPP. Refer to under Instruments & Integrations for compatible versions of IPP and Clarity LIMS.
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 in .
The integration properties can be configured to enable capture and generation of files associated with the sequencing run. Refer to for details.
For descriptions of the protocol and the steps, refer to . For instructions on using the Library Prep Validation workflow to validate the automated sample sheet generation, refer to .
For more information on NCS, refer to the NextSeq 500/550 documentation at .
For NextSeq Control Software (NCS) v4.0 and later, refer to the configuration steps in . For NCS versions before v4.0, perform the following steps.
For instructions on how to validate the automated sample sheet generation, refer to .
For instructions on validating the creation of event files, refer to .
For more information on event file validation, refer to .
