Installation

Mounting the NAS share of run data are 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 privileges 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

MiSeq Integration Package v8.2.0 depends on the QC Protocols 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 MiSeq 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).

If you do not have QC Protocols, then install them as follows.

1. As a glsjboss user, run the following command to view the complete list of IPP workflows:

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

2. To install the dependent QC Protocols, run the following command:

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   /opt/gls/clarity/config/illumina-preset-protocols-installer.sh -o install QC_Protocols.qc-protocols

When the instrument is running, the final destination for the run data are 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. The batch file used for MiSeq Control Software v4.0 is gls_event_mcs_rta_lrm.bat.

Before configuring the batch files, do the following:

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

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   C:\Illumina\RTA\Configs\

2. Make sure that the instrument is idle.

3. Shut down MiSeq Control Software.

4. Set up the directory structure as follows.

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

   2. Create a directory (e.g., 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. Make the following changes to the gls_event_mcs_rta_lrm.bat batch file:

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   set DESTINATION_PATH=\\network-storage\illumina\gls_events\
   set DEFAULT_LOCAL_OUTPUT_PATH=D:\Illumina\MiSeqTemp\

   1. Change the DESTINATION_PATH line to the name of the event file directory.

   2. Change the DEFAULT_LOCAL_OUTPUT_PATH line to the name of the default local temp run folder directory.

      The default directory is

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      D:\Illumina\MiSeqTemp\

3. Copy the DESTINATION_PATH and paste it into the Windows Explorer address bar.

4. Make sure that the network location is accessible and that it opens from the instrument.

   The batch file must be deployed to the MiSeq 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. From the Attachment section, 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 MiSeq Control Software configuration files as follows

1. Reboot the instrument.

2. Make a backup copy of the RTA configuration XML file and name it MiSeq.Configuration.xml.

3. Edit the file to connect to the RTA End Run event as follows.

   1. Open the MiSeq.Configuration.xml file at

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      C:\Illumina\RTA\Configs\

   2. Update the following content within the <ProcessCompleteEventFile> tags:

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      <ProcessCompleteEventFile>C:\Illumina\gls\gls_event_mcs_rta_lrm.bat</ProcessCompleteEventFile>
4. Save the edited file.

5. Validate the file as follows.

   1. From C:\Illumina\RTA, create a backup of the RTA.exe.config file and name it Backup_RTA.exe.config.

   2. Open RTA.exe.config and change the DefaultConfigFile value to MiSeq.Configuration.xml.

   1. Save the changes and close the file.

   2. From the Command Prompt, run the following command:

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      C:\Illumina\RTA\RTA.exe "." processedfolder="."

   3. If the validation is successful, a warning message displays that says the output path cannot be placed into the run directory. Select OK.

   1. In the Command Prompt, make sure the following error message displays:

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      RunInfo.xml does not exist: System.ApplicationException: Error in the application.,0,0

   1. Press Ctrl + C to terminate the RTA.exe application that is still running.

      If the configuration file has an error, the command returns specifics about the problem. Refer to the following example:
6. After the validation is complete, go back to C:\Illumina\RTA and delete the current RTA.exe.config file.

7. Restore the backup file from a by renaming it RTA.exe.config.

8. Start MiSeq Control Software.

9. Open the MiSeq.Configuration.xml file and make sure that the changes were saved.

For instructions on how to validate the automated sample sheet generation, refer to MiSeq Integration v8.2.0 User Interaction, Validation and Troubleshooting.

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

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

• cycleNumber (e.g., 318)

• runFolder (e.g., "D:\Illumina\MiSeqTemp\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., MCS)

• finishDate (e.g., 2023-08-15)

If an event file is triggered automatically during a MiSeq 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 MiSeq Integration v8.2.0 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 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 and DEFAULT_LOCAL_OUTPUT_PATH are 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 MiSeq Integration v8.2.0 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 you are 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 MiSeqIntegrator.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_MiSeq/v8/SequencingService

Validate the sequencing run as follows

1. During the run, monitor the contents of the gls_events directory.

2. After the run is completed and the RTA completes primary analysis, make sure that a final EndRun event displays (e.g., event-EndRun-11043279.txt).