Installation

The Illumina MiSeqDx Integration Package v1.10.0 supports MiSeqDx instruments running in Diagnostic mode. For MiSeqDx instruments running in Research mode, install the MiSeq Integration Package. For more information, refer to MiSeq Integration v8.2.0 Installation.

Compatibility

MiSeqDx Integration v1.10.0 is compatible with the following software:

• Clarity LIMS v6.2 and later

• Secret Util v1.0 and later

• IPP v2.6 and later

Prerequisites

MiSeqDx Integration v1.10.0 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

• Text and VCF files from the Alignment folder at

  <runFolderRoot>/Data/Intensities/BaseCalls/Alignment<#>/*.vcf

  ℹ CFTR-specific files with the extension cftr.vcf are ignored.

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

• Sample sheet (CSV file, e.g., SampleSheet.csv)

• Run report (PDF file, e.g., runreport.pdf)

• Run folder root link

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

• Applicable to all protocols:

  <runFolderRoot>/RunInfo.xml

  <runFolderRoot>/RunParameters.xml

  <runFolderRoot>/first_base_report.htm

  <runFolderRoot>/Data/Intensities/BaseCalls/Alignment<#>/*.vcf

• Additional file for CF 139-Variant assay:

  MiSeqDxCF139VariantAssay.txt

• Additional file for CF Clinical Sequencing assay:

  MiSeqDxCFClinicalSequencingAssay.txt

Prerequisite 2: IPP Installation

MiSeqDx Integration Package v1.10.0 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 MiSeqDx 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

MiSeqDx Integration v1.10.0 supports both on-premise and cloud integrations. This integration is distributed as the following RPM packages:

• BaseSpaceLIMS-miseqdx-extensions

• BaseSpaceLIMS-miseqdx-sequencing-service

The BaseSpaceLIMS-miseqdx-extensions RPM installs the following items:

• Protocols and workflows

• Database properties that configure the service

• Placement pattern files that determine reagent index assignment

• miseqdx-extensions.jar

• miseqdx-sequencing-report.jar

The BaseSpaceLIMS-miseqdx-sequencing-service RPM installs the following items:

• If not found, user configuration (the glsjboss user and the glsjdk8 and claritylims user groups)

• If not installed, Java 8

• Bash scripts to run the miseqdx_seqservice

• miseqdx-sequencing.jar

• The gls_events_mos_rta.bat batch file that configures RTA

• Smoke test directories

On-Premise Installation

Use the following instructions to install the BaseSpaceLIMS-miseqdx-extensions and BaseSpaceLIMS-miseqdx-sequencing-service RPMs on the Clarity LIMS server.

Install the RPMs

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

2. Run the following yum commands to install the RPMs:

   yum install BaseSpaceLIMS-miseqdx-extensions
   yum install BaseSpaceLIMS-miseqdx-sequencing-service

3. Enter y to confirm that you want to proceed with the RPM installations. After confirmation, the following scripts and files are installed:

   • configure_extensions_miseqdx_workflow.sh

   • configure_extensions_miseqdx_sequencingservice.sh

   • miseqdx-extensions.jar (contains the sample sheet generation and other scripts)

   • miseqdx-sequencing-report.jar (generates the sequencing run report)

   These files are installed at the following locations:

   /opt/gls/clarity/config/

   /opt/gls/clarity/extensions/miseqdx

   /opt/gls/clarity/extensions/miseqdx/SequencingService

Import Workflow Configurations for MiSeqDx

ℹ If you are upgrading from an earlier version of the MiSeqDx integration package and the system is configured with the configure_extensions_miseqdx_workflow.sh and configure_extensions_miseqdx_sequencingservice.sh scripts, refer to Installed Components.

1. When prompted by the RPM instructions to import the workflow configuration, run the following command as the glsjboss user:

   bash /opt/gls/clarity/config/configure_extensions_miseqdx_workflow.sh

2. When prompted by the RPM instructions to configure the sequencing service (which includes database properties with default values set for the integration), run the following command as the glsjboss user:

   bash /opt/gls/clarity/config/configure_extensions_miseqdx_sequencingservice.sh

Configure the Database Service Properties

For more information on the properties that must be configured, refer to Installed Components.

Start the Sequencing Service

Run the following command to start the sequencing service:

systemctl start miseq_seqservice-v8

Cloud Installation

The BaseSpaceLIMS-miseqdx-extensions RPM must be installed on the Clarity LIMS server. The BaseSpaceLIMS-miseqdx-sequencing-service RPM can be installed remotely on another server within the network.

Specifications

The following hardware, operating system, and network specifications must be met to install the BaseSpaceLIMS-miseqdx-sequencing-service RPM:

• Hardware requirements:

  • 64-bit processor (dual core 2.0 GHz)

  • OS requirements, plus at least an additional 512 MB RAM

  • A minimum of 5 GB of hard disk space

• Operating system requirements:

  • Oracle Linux (for compatibility, refer to MiSeqDx Integration v1.10.0 Release Notes)

• Network requirements:

  • SSL access to the Clarity LIMS server from the network

  • A mounted network folder where the sequencing runs are written

Install the RPM

1. On the applicable server, log in as the root user.

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

   yum install BaseSpaceLIMS-miseqdx-extensions

3. Enter y to confirm that you want to proceed with the RPM installations. After confirmation, the following scripts and files are installed:

   • configure_extensions_miseqdx_workflow.sh

   • configure_extensions_miseqdx_sequencingservice.sh

   • miseqdx-extensions.jar (contains the sample sheet generation and other scripts)

   • miseqdx-sequencing-report.jar (generates the sequencing run report)

   These files are installed at the following locations:

   /opt/gls/clarity/config/

   /opt/gls/clarity/extensions/miseqdx

   /opt/gls/clarity/extensions/miseqdx/SequencingService

Import Workflow Configurations for MiSeqDx

ℹ If you are upgrading from an earlier version of the MiSeqDx integration package and the system is configured with the configure_extensions_miseqdx_workflow.sh and configure_extensions_miseqdx_sequencingservice.sh scripts, refer to Installed Components.

1. When prompted by the RPM instructions to import the workflow configuration, run the following command as the glsjboss user:

   bash /opt/gls/clarity/config/configure_extensions_miseqdx_workflow.sh

2. When prompted by the RPM instructions to configure the sequencing service (which includes database properties with default values set for the integration), run the following command as the glsjboss user:

   bash /opt/gls/clarity/config/configure_extensions_miseqdx_sequencingservice.sh

Configure the Database Service Properties

For more information on the properties that must be configured, refer to Installed Components.

Install Sequencing Service RPM on Remote Server

1. On the applicable server, log in as the root user.

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

   yum install BaseSpaceLIMS-miseqdx-sequencing-service

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

Copy API Connection Properties File from the Clarity LIMS Server to the Remote Server

1. Make sure that the extensions RPM is installed on the Clarity LIMS server.

2. Run the following command to generate the integration.properties API connection properties file:

   java -jar /opt/gls/clarity/extensions/miseqdx/miseqdx-extensions.jar script:com.genologics.integrations.sequencing.generate_integration_property_file

3. Copy the integrations.properties file from the Clarity LIMS server to the following location on the remote server:

   /opt/gls/clarity/extensions/miseqdx/SequencingService/conf

Start the Sequencing Service

Run the following command to start the sequencing service:

systemctl start miseq_seqservice

Workflows, Protocols, and Steps Installed

MiSeqDx Integration v1.10.0 installs the following protocols:

• CF 139-Variant Assay Library Prep 1.2

• CF Clinical Sequencing Assay Library Prep 1.2

• Universal Kit Library Prep 1.2

• Illumina SBS MiSeqDx (CF 139-Variant Assay) 1.2

• Illumina SBS MiSeqDx (CF Clinical Sequencing Assay) 1.2

• Illumina SBS MiSeqDx (Universal Kit) 1.2

The integrations also install the following validation protocols that are included in a workflow with the same name:

• MiSeqDx Validation (CF 139-Variant Assay) 1.2

• MiSeqDx Validation (CF Clinical Sequencing Assay) 1.2

• MiSeqDx Validation (Universal Kit) 1.2

For descriptions of the protocol and the steps, refer to MiSeqDx Integration v1.10.0 Configuration. For instructions on user interaction for each step and using the MiSeqDx validation workflows to validate the automated sample sheet generation, refer to MiSeqDx Integration v1.10.0 User Interaction, Validation and Troubleshooting.

Instrument Software

The instrument software is divided into the following modules:

• MiSeqDx Operating Software (MOS) — Controls the instrument operation, including various configuration settings. This software is installed and runs on the instrument.

• MiSeqDx Reporting Software (MRS) — Performs the following secondary analysis functions:

  • Demultiplexing

  • Alignment

  • Variant calling

  • Report generation

  The specific functions that are supported vary by the kit. This software is installed on or off the instrument.

• 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.

• Illumina User Management (IUM) — Contains a user database file that is used with the MiSeqDx instrument. This file controls user passwords and privileges for MOS.

For more information on the MiSeqDx software, refer to the MiSeqDx documentation at support.illumina.com.

Instrument Integration

Illumina provides a supported mechanism for using custom scripts on key events during a sequencing run. The Clarity LIMS support team has created batch files that plugs into these events. When the batch file is used, it reads the event information and writes 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 server where the sequencing service RPM is installed receives the event files and takes the appropriate actions.

The sequencing service monitors the following events (the actual event names may be different):

• End Run — This event is used to update the sequencing steps in Clarity LIMS, captures key process data and files, and parses run statistics for output custom fields.

• Begin Secondary Analysis — Indicates that secondary analysis in the MRS has started so that the sequencing service can start to monitor for results. After secondary analysis is complete, the VCF files are uploaded to Clarity LIMS.

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.

To avoid inadvertently removing or overwriting the batch file when updating the instrument software, the batch file can be placed in its own directory on the instrument computer.

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 MOS.

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 file as follows

1. Determine the site or instrument specific network storage path root.

2. Change the DESTINATION_PATH line to use the storage path root and the name of the event file directory.

   ℹ 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 that it opens from the instrument.

Deploy the batch file as follows

1. On the server where the sequencing service RPM is installed, copy the batch file from /opt/gls/clarity/extensions/miseqdx/InstrumentIntegrations to C:\Illumina\gls on the local computer.

2. If necessary, create the gls_events_mos_rta.bat batch file in C:\Illumina\gls. For more information, contact Illumina Support.

3. From the command prompt, list the contents on the C:\Illumina\gls directory using the following command:

   dir C:\Illumina\gls\

   Make sure that the name of the batch file you created does not contain any special (hidden) characters.

Configure RTA

Update the MOS configuration files as follows.

1. Turn off the instrument and restart the instrument computer.

2. Using Task Manager, make sure that MOS is not auto-launched by Windows.

3. [Optional] If MOS is auto-launched, remove it from the auto-launch list and restart the computer.

4. 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 <RTAConfiguration> tags:

      <ProcessCompleteEventFile>C:\Illumina\gls\gls_event_mos_rta.bat</ProcessCompleteEventFile>

5. Save and close the edited file.

6. Validate the file as follows.

   1. Open MiSeq.Configuration.xml configuration file in Explorer to perform some of the XML validation.

   2. To further validate the file, run the following command from the Command Prompt:

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

      If the validation is successful, the following message displays:

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

      If the configuration files contain an error, the command returns specifics about the problem. The following example shows an error that occurs when a <GLS> key is added to the file at line 83:

      Error: While loading Configuration "c:\illumina\rta\configs\MiSeq.Configuration.xml"
      There is an error in XML document <83, 5>
      Error loading xml file: Unknown node 'GLS' at line 83 pos 5

7. Start MOS.

8. 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 MiSeqDx Integration v1.10.0 User Interaction, Validation and Troubleshooting.

Manual Invocation of Event Files

For instructions on validating the creation of event files, refer to MiSeqDx Integration v1.10.0 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 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 file.

For more information on event file validation, refer to MiSeqDx Integration v1.10.0 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 MiSeqDxIntegrator.log file, which logs the file name and contents of each event file that is processed.

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).