Installation

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

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  <runFolderRoot>/RunInfo.xml

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  <runFolderRoot>/runParameters.xml

• Run statistics from

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  <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:

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   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.

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

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   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} (e.g., apiusers/novaseq_user). For more information on Secret Utility configuration, refer to the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

4. Use the following command to make sure that the password is saved correctly in Secret Util:

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

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

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:

1. Back up the NextSeq.Configuration.xml configuration file to

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   C:\Illumina\RTA\Configs\

2. Make sure that the instrument is idle.

3. Shut down NCS.

4. Set up the directory structure as follows.

   1. Create a directory (C:\Illumina\gls is recommended) on the local computer to hold the batch file.

   2. Create a directory (e.g., gls_events) on the NAS to hold the event files.

Configure the batch file

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.

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   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.

1. Download the batch file.

2. Copy the file to the directory that you created on the instrument computer (e.g., C:\Illumina\gls).

3. From the Command Prompt, run the following command to list the contents of the directory:

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   dir C:\Illumina\gls\

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

Update the NextSeq Control Software (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.

1. Edit the NextSeq.Configuration.xml configuration file at

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   C:\Illumina\RTA\Configs\

2. Insert the following text inside the <Processing> tags:

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

3. Validate the RTA configuration changes as follows.

   From the command prompt, run the following command:

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

   • If the validation is successful, the following warning message displays.

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     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:

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     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.

For instructions on how to validate the automated sample sheet generation, refer to NextSeq 500/550 Integration v2.3.0 User Interaction, Validation and Troubleshooting.

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

• Filename (e.g., event-EndRun-07295667.txt)

• cycleNumber (e.g., 318)

• runFolder (e.g., "D:\Illumina\NextSeqTemp\230815_M99999_0028_FC1234567-ABCDE")

• netFolder (e.g., \\network-folder\Run_Data\230815_M99999_0028_FC1234567-ABCDE)

• readType (e.g., 4)

• eventType (e.g., EndRun)

• softwareType (e.g., NCS)

• finishDate (e.g., 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 v2.3.0 User Interaction, Validation and Troubleshooting.

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 v2.3.0 User Interaction, 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

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  /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 (e.g., event-EndRun-11043279.txt).