Installation

The Illumina MiSeq Integration Package v8.2.0 supports the integration of Clarity LIMS to MiSeq instruments.

MiSeq Integration v8.2.0 runs as independent service and can be on the same server as other Clarity LIMS integrations. The integration is only required for MCS v4.0. If you are running MCS v3.0 or below, do not upgrade to this integration.

Compatibility

MiSeq Integration v8.2.0 is compatible with the following software:

• Clarity LIMS v6.2.0 and later

• Secret Util v1.0 and later

• IPP v2.6 and later

• MCS v4.0/LRM v3.0 and later

• Oracle Linux (for compatibility, refer to MiSeq Integration v8.2.0 Release Notes)

Prerequisites

MiSeq Integration v8.2.0 has the following prerequisites:

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

• IPP is installed

Prerequisite 1: Mount Run Data NAS Share

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

  <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: IPP Installation

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:

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

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

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

Installation

MiSeq Integration v8.2.0 is distributed as the ClarityLIMS-Illumina-MiSeq-Package-v8 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 miseq-sequencing.jar file

• The configure_extensions_miseq_sequencingservice.sh script

• Smoke test directories

The following installation steps are required for the installation of MiSeq Integration v8.2.0.

ℹ️ The MiSeq RPM must be installed on the Clarity LIMS server. This is the same location where the AI node or Automation Worker is installed. The automations and sequencing service use the existing Automation Worker.

Install the RPM

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

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

   ℹ️ 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-MiSeq-Package-v8 --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 MiSeq workflow from the IPP script.

4. Install the MiSeq workflow as follows.

   ℹ️ The following steps assume that the QC protocols from the IPP package have been installed. For more information, refer to Prerequisite 2: IPP Installation in Prerequisites.

   1. 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

   2. Run the following command to install the MiSeq Sequencing v3.2 workflow configuration:

      /opt/gls/clarity/config/illumina-preset-protocols-installer.sh -o install Illumina_Instruments.miseq-v3.2

      The installer does the following tasks:

      • Validates the import of the workflow and provides the Warning/Error details in STDOUT, which allows you proceed with the import or cancel it.

      • Automatically detects the version of Clarity LIMS and imports the applicable workflow configuration.

      • Creates or updates the miseq.v8.seqservice.sequenceProcessBaseName database property, which can be verified with the omxProps-ConfigTool utility. This property shows the sequencing master step base display name. It can also use partial matching to look up the master step by the sequencing service. The default value is MiSeq Run (MiSeq v3.2).

   3. [Optional] Run the following command to install the Library Prep Validation v2.3.1 workflow that is used to validate the MiSeq Sequencing v3.2 workflow:

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

      The installer does the following tasks:

      • Validates the import of the workflow and provides the Warning/Error details in STDOUT, which allows you proceed with the import or cancel it.

      • Automatically detects the version of Clarity LIMS and imports the applicable workflow configuration.

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

   /opt/gls/clarity/config/configure_extensions_miseq_sequencingservice-v8.sh

   The script creates the configuration properties in the database. For a list of the configured properties, refer to Configure the Database Service Properties.

Configure the Database Service Properties

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

Configurable Database Properties

Property	Description	Default Value
miseq.v8.seqservice.eventFileDirectory.1	A monitored network location used for event files.	/mnt/gls_events
miseq.v8.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/miseq/monitored
miseq.v8.seqservice.netPathPrefixSearch.1	The network directory prefix contained in the event file.	\\nas\network\run_data
miseq.v8.seqservice.netPathPrefixSearch.99	The default benign smoke test search entry. This property is used for the installation and upgrade of smoke tests.	/
miseq.v8.seqservice.netPathPrefixReplace.1	The mapped network directory mount name on the server used to access the run data directory.	/mnt/run_data
miseq.v8.seqservice.netPathPrefixReplace.99	The default benign smoke test replace entry. This property is used for the installation and upgrade of smoke tests.	/
miseq.v8.seqservice.eventFileDirectorySuffixes	A list of eventFileDirectory path entries used to monitor for event files. The value is one or more comma separated integers.	99 ℹ️When 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.
miseq.v8.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 ℹ️ 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.

Start the Sequencing Service

Run the following command to start the sequencing service:

systemctl start miseq_seqservice-v8

Workflows, Protocols, and Steps Installed

MiSeq Integration v8.2.0 works with the MiSeq Sequencing v3.2, which contains a single protocol with the same name. This protocol includes the following steps:

• Library Pooling (MiSeq v3.2)

• Denature and Dilute (MiSeq v3.2)

• MiSeq Run (MiSeq v3.2)

For descriptions of the protocol and the steps, refer to MiSeq Integration v8.2.0 Configuration. For instructions on using the Library Prep Validation v2.3.1 workflow to validate the automated sample sheet generation, refer to MiSeq Integration v8.2.0 Uesr Interaction, Validation and Troubleshooting.

Instrument Software

The MiSeq Control Software (MCS) is divided into the following modules:

• MCS — Controls the instrument operation, including various configuration settings. This software is installed and runs on the instrument.

• Local Run Manager — Analyzes sequencing data and integrates with MCS. This software can be accessed on the instrument through a web browser.

• Real-Time Analysis (RTA) — 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 MCS, refer to the MiSeq documentation at support.illumina.com.

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.

The support team 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 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

   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.

      ⚠️ 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.

   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:

   ℹ️ Make sure to include a trailing \ in the DESTINATION_PATH and DEFAULT_LOCAL_OUTPUT_PATH lines. Refer to the following examples:

   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

      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:

   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 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

      C:\Illumina\RTA\Configs\

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

      <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:

      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:

      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.

   ⚠️ Make sure that you rename the backup file. If you do not rename the file, it can cause the RTA not to work properly.

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.

Validation

Sample Sheet Generation

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

Manual Invocation of Event Files

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.

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 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

  /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).