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The Illumina MiSeq instrument integration provides preconfigured workflows that map to established lab protocols and steps used with the MiSeq instrument. The integration also supports the associated library prep kits, reagent kits, and assays used in these protocols.
The following sections explain how to validate the installation of the Illumina MiSeq Integration Package v8.3.0.
The validation involves the following processes:
Running samples through the Library Prep Validation v2.3.4 workflow. The workflow contains a single-step protocol that models the library prep required to produce normalized libraries. At the end of the step, the normalized libraries automatically advance to the MiSeq Sequencing v3.2 workflow.
Running normalized libraries through the MiSeq Sequencing v3.2 workflow. This process validates the following information:
Automated generation of a sample sheet for use with the MiSeq Control Software (MCS) and Local Run Manager (LRM).
Automated tracking of the MiSeq sequencing run and parsing of run statistics into Clarity LIMS, including the following information:
Run status and metrics of sequencing run
Sequencing run parameters
Real-Time Analysis (RTA) v1 run directory location and other run-specific information
The following steps prepare Clarity LIMS to run samples through the Library Prep Validation and MiSeq Sequencing v3.2 workflows.
In the Clarity LIMS Configuration area, select each workflow and change the Workflow Status toggle to Active.
On the Projects & Samples screen, create a project. Add samples to the project.
Assign the samples to the Library Prep Validation workflow.
This single-step protocol models the library prep required to produce normalized libraries that are ready for the MiSeq Sequencing v3.2 workflow.
Follow the steps in Library Prep Validation Protocol to run the Library Prep Validation workflow with the following:
Label Group = TruSeq HT Adapters v2 (D7-D5)
Sequencing Instrument = MiSeq
On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the MiSeq Sequencing v3.2 workflow, which is Library Pooling (MiSeq v3.2) step.
In Lab View, locate the MiSeq Sequencing v3.2 protocol. Samples are queued for the Library Pooling (MiSeq v3.2) step. Select the step to proceed to the Queue screen.
On the Queue screen, do as follows.
Add the samples to the Ice Bucket.
In the Add Control Samples panel, add the PhiX v3 control sample to the Ice Bucket.
Select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
The Validate Maximum Number of Samples automation script runs. The maximum number of samples allowed is 1536.
On the Pool Samples screen, do as follows.
Create a pool of samples by dragging samples into the Pool Creator.
Name the pool or accept the default name (Pool #1).
Select Place Samples.
On the Placement screen, do as follows.
Select the pool from the Samples to be Placed area and drag it to the container.
Select Record Details.
On the Record Details screen, select Next Steps.
On the Assign Next Steps screen, next step is automatically set to Denature and Dilute (MiSeq v3.2).
Select Finish Step.
In Lab View, locate the MiSeq Sequencing v3.2 protocol. The pooled samples are queued for the Denature and Dilute (MiSeq v3.2) step. Select the step to proceed to the Queue screen.
On the Queue screen, add the pool to the Ice Bucket, and then select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
The Validate Single Input automation script runs.
On the Placement screen, do as follows.
Scan the MiSeq reagent cartridge barcode into the MiSeq Reagent Cartridge field.
Place the pool of samples into the reagent cartridge.
Select Record Details.
On the Record Details screen, do as follows.
In Reagent Lot Tracking, select the reagent lot used in the step.
If the reagent lot is not listed, add/activate the lot on the Reagents and Controls screen.
Use the Preset drop-down list to help populate the fields in Step Details, as needed.
For more information on presets, refer to Using Step Details Presets. Workflow, Experiment Name, and Read 1 Cycles are required fields.
In the Sample Details table, enter the Final Loading Concentration. Select from the following preset options or enter a different value.
225 (for PCR-free workflows)
400 (for Nano workflows)
Select Validate Run Setup and Generate MiSeq SampleSheet.
Clarity LIMS generates the sample sheet and attaches it and a log file to placeholders in the Files area of the Record Details screen.
Download the files and validate the format and content.
Select Next Steps.
On the Assign Next Steps screen, samples are automatically assigned to the MiSeq Run (MiSeq v3.2) step.
Select Finish Step.
In Lab View, locate the MiSeq Sequencing v3.2 protocol. The pool of samples is queued for the MiSeq Run (MiSeq v3.2) step. Select the step to proceed to the Queue screen.
On the Queue screen, add the pool to the Ice Bucket, and then select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
On the Record Details screen, the fields are read-only.
For more information on adding instruments, refer to Add and Configure Instruments in the [Clarity LIMS (Clarity & LabLink Reference Guide) documentation](../../../clarity-lims/clarity-and-lablink.md).
When the run completes, the integration automatically performs the following actions:
Populates the fields.
Attaches files to the Illumina Run Report, Link to Run Folder, Run Parameters, and Run Info placeholders.
Populates the fields in the Sample Details table. For details, refer to MiSeq Integration v8.3.0 Configuration.
The Log File is attached after the next step for samples is assigned by the Next Step - Advance automation.
Select Next Steps.
On the Assign Next Steps screen, the next step is automatically set to Mark protocol as complete.
Select Finish Step.
Most of the custom fields in Step Details are analysis settings used by Local Run Manager (LRM) and are for advanced users only. The following fields are exceptions:
Workflow
Experiment Name
Description
Read 1 Cycles
Read 2 Cycles
Custom Primers
To use the default analysis settings, select the analysis module from the Preset drop-down list.
The default values populate the respective fields and indicate the following actions are needed:
Required — The field is required for the specific analysis module selected. Replace the word Required with a proper value. For example, the Genome Folder field should contain the directory to the genome folder.
None — The field is not applicable for the specific analysis module selected. Leave the field as is.
For a list of fields that are applicable for the analysis module selected, refer to the Applicable analysis fields for the selected Workflow field in Step Details.
This validation checks the following information:
The destination path is correctly configured.
The instrument computer can access and write to the destination path.
There are no syntax errors in the Clarity LIMS batch file.
Use the following steps to confirm that event files are created by the batch file in the destination path. The steps assume that the default configuration has been successfully imported.
In C:\Illumina\gls, double-click the batch file gls_event_mcs_rta_lrm.bat.
Confirm that an empty event file appears in the configured DESTINATION_PATH.
Manual execution of the batch file produces an output resembling the following example:
If an automation trigger does not appear to run the corresponding scripts, see the following topics:
Troubleshooting Automation Worker in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
Troubleshooting Automation in the Clarity LIMS (API & Database) documentation.
If an error occurs that does not provide direction on how to proceed, confirm the version of the installed MiSeq Integration Package. To confirm the version, run the
command from the server console.
If the error is related to data parsing, retrieving run results data, or report values not appearing as expected, review the MiseqIntegrator.log file. The file is located at
Additional troubleshooting information for this integration is provided in the Illumina Instrument Integrations FAQ.
If you are unable to resolve the issue, contact the Clarity LIMS support team. Supply the relevant information from the troubleshooting already performed.
The following sections explain how to validate the installation of the Illumina MiSeq Integration Package v8.2.0.
The validation involves the following processes:
Running samples through the Library Prep Validation v2.3.1 workflow. The workflow contains a single-step protocol that models the library prep required to produce normalized libraries. At the end of the step, the normalized libraries automatically advance to the MiSeq Sequencing v3.2 workflow.
Running normalized libraries through the MiSeq Sequencing v3.2 workflow. This process validates the following information:
Automated generation of a sample sheet for use with the MiSeq Control Software (MCS) and Local Run Manager (LRM).
Automated tracking of the MiSeq sequencing run and parsing of run statistics into Clarity LIMS, including the following information:
Run status and metrics of sequencing run
Sequencing run parameters
Real-Time Analysis (RTA) v1 run directory location and other run-specific information
The following steps prepare Clarity LIMS to run samples through the Library Prep Validation and MiSeq Sequencing v3.2 workflows.
In the Clarity LIMS Configuration area, select each workflow and change the Workflow Status toggle to Active.
On the Projects & Samples screen, create a project. Add samples to the project.
Assign the samples to the Library Prep Validation workflow.
This single-step protocol models the library prep required to produce normalized libraries that are ready for the MiSeq Sequencing v3.2 workflow.
Label Group = TruSeq HT Adapters v2 (D7-D5)
Sequencing Instrument = MiSeq
On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the MiSeq Sequencing v3.2 workflow, which is Library Pooling (MiSeq v3.2) step.
In Lab View, locate the MiSeq Sequencing v3.2 protocol. Samples are queued for the Library Pooling (MiSeq v3.2) step. Select the step to proceed to the Queue screen.
On the Queue screen, do as follows.
Add the samples to the Ice Bucket.
In the Add Control Samples panel, add the PhiX v3 control sample to the Ice Bucket.
Select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
The Validate Maximum Number of Samples automation script runs. The maximum number of samples allowed is 1536.
On the Pool Samples screen, do as follows.
Create a pool of samples by dragging samples into the Pool Creator.
Name the pool or accept the default name (Pool #1).
Select Place Samples.
On the Placement screen, do as follows.
Select the pool from the Samples to be Placed area and drag it to the container.
Select Record Details.
On the Record Details screen, select Next Steps.
On the Assign Next Steps screen, next step is automatically set to Denature and Dilute (MiSeq v3.2).
Select Finish Step.
In Lab View, locate the MiSeq Sequencing v3.2 protocol. The pooled samples are queued for the Denature and Dilute (MiSeq v3.2) step. Select the step to proceed to the Queue screen.
On the Queue screen, add the pool to the Ice Bucket, and then select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
The Validate Single Input automation script runs.
On the Placement screen, do as follows.
Scan the MiSeq reagent cartridge barcode into the MiSeq Reagent Cartridge field.
Place the pool of samples into the reagent cartridge.
Select Record Details.
On the Record Details screen, do as follows.
In Reagent Lot Tracking, select the reagent lot used in the step.
If the reagent lot is not listed, add/activate the lot on the Reagents and Controls screen.
Use the Preset drop-down list to help populate the fields in Step Details, as needed.
In the Sample Details table, enter the Final Loading Concentration. Select from the following preset options or enter a different value.
225 (for PCR-free workflows)
400 (for Nano workflows)
Select Validate Run Setup and Generate MiSeq SampleSheet.
Clarity LIMS generates the sample sheet and attaches it and a log file to placeholders in the Files area of the Record Details screen.
Download the files and validate the format and content.
Select Next Steps.
On the Assign Next Steps screen, samples are automatically assigned to the MiSeq Run (MiSeq v3.2) step.
Select Finish Step.
In Lab View, locate the MiSeq Sequencing v3.2 protocol. The pool of samples is queued for the MiSeq Run (MiSeq v3.2) step. Select the step to proceed to the Queue screen.
On the Queue screen, add the pool to the Ice Bucket, and then select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
On the Record Details screen, the fields are read-only.
For more information on adding instruments, refer to Add and Configure Instruments in the [Clarity LIMS (Clarity & LabLink Reference Guide) documentation](../../../clarity-lims/clarity-and-lablink.md).
When the run completes, the integration automatically performs the following actions:
Populates the fields.
Attaches files to the Illumina Run Report, Link to Run Folder, Run Parameters, and Run Info placeholders.
The Log File is attached after the next step for samples is assigned by the Next Step - Advance automation.
Select Next Steps.
On the Assign Next Steps screen, the next step is automatically set to Mark protocol as complete.
Select Finish Step.
Most of the custom fields in Step Details are analysis settings used by Local Run Manager (LRM) and are for advanced users only. The following fields are exceptions:
Workflow
Experiment Name
Description
Read 1 Cycles
Read 2 Cycles
Custom Primers
To use the default analysis settings, select the analysis module from the Preset drop-down list.
The default values populate the respective fields and indicate the following actions are needed:
Required — The field is required for the specific analysis module selected. Replace the word Required with a proper value. For example, the Genome Folder field should contain the directory to the genome folder.
None — The field is not applicable for the specific analysis module selected. Leave the field as is.
For a list of fields that are applicable for the analysis module selected, refer to the Applicable analysis fields for the selected Workflow field in Step Details.
This validation checks the following information:
The destination path is correctly configured.
The instrument computer can access and write to the destination path.
There are no syntax errors in the Clarity LIMS batch file.
Use the following steps to confirm that event files are created by the batch file in the destination path. The steps assume that the default configuration has been successfully imported.
In C:\Illumina\gls, double-click the batch file gls_event_mcs_rta_lrm.bat.
Confirm that an empty event file appears in the configured DESTINATION_PATH.
Manual execution of the batch file produces an output resembling the following example:
If an automation trigger does not appear to run the corresponding scripts, see the following topics:
If an error occurs that does not provide direction on how to proceed, confirm the version of the installed MiSeq Integration Package. To confirm the version, run the
command from the server console.
If the error is related to data parsing, retrieving run results data, or report values not appearing as expected, review the MiseqIntegrator.log file. The file is located at
Additional troubleshooting information for this integration is provided in the Illumina Instrument Integrations FAQ.
If you are unable to resolve the issue, contact the Clarity LIMS support team. Supply the relevant information from the troubleshooting already performed.
The MiSeq Integration Package v8.3.0 only supports Clarity LIMS v6.2 and above.
The Illumina MiSeq Integration v8.3.0 includes the following features:
Preconfigured MiSeq Sequencing v3.2 workflow that maps to lab protocol steps and instrument runs.
Automated sample sheet generation for use with MiSeq Control Software (MCS) and Local Run Manager.
The following supported Local Run Manager analysis modules:
Generate FASTQ
Library QC
Resequencing
DNA Enrichment
DNA Amplicon
Automated tracking of the sequencing run and the parsing of the following run statistics to Clarity LIMS:
Sequencing run metrics
Sequencing run parameters
Real-Time Analysis RTA v1 run directory location (and other run specific information)
Preconfigured Library Prep Validation workflow that is used for validation purposes only. This workflow contains a single step protocol that models the library prep required to produce normalized libraries that are ready for the MiSeq Sequencing v3.2 workflow. MiSeq Integration Package v8.3.0 works with Library Prep Validation v2.3.4, which is installed with Illumina Preset Protocols (IPP) v2.9. For details, refer to MiSeq Integration v8.3.0 User Interaction, Validation and Troubleshooting
MiSeq Integration v8.3.0 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.
The MiSeq Integration Package v8.2.0 only supports Clarity LIMS v6.2.
The Illumina MiSeq Integration v8.2.0 includes the following features:
Preconfigured MiSeq Sequencing v3.2 workflow that maps to lab protocol steps and instrument runs.
Automated sample sheet generation for use with MiSeq Control Software (MCS) and Local Run Manager.
The following supported Local Run Manager analysis modules:
Generate FASTQ
Library QC
Resequencing
DNA Enrichment
DNA Amplicon
Automated tracking of the sequencing run and the parsing of the following run statistics to Clarity LIMS:
Sequencing run metrics
Sequencing run parameters
Real-Time Analysis RTA v1 run directory location (and other run specific information)
Preconfigured Library Prep Validation workflow that is used for validation purposes only. This workflow contains a single step protocol that models the library prep required to produce normalized libraries that are ready for the MiSeq Sequencing v3.2 workflow. MiSeq Integration Package v8.2.0 works with Library Prep Validation v2.3.1, which is installed with Illumina Preset Protocols (IPP) v2.6. For details, refer to MiSeq Integration v8.2.0 User Interaction, Validation and Troubleshooting
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.
MiSeq Integration v8.2.0 includes the MiSeq v3.2 workflow upgrade. This upgrade is typically done through the API. To upgrade the workflow manually, create the Validate Single Input automation and update its trigger information as follows.
Before updating the script, make sure the MiSeq v3.1 workflow is installed.
In Clarity LIMS, under Configuration, select the Automation tab.
Select New Automation.
Create an automation called Validate Single Input.
In the Command Line field, add the following string:
Under Automation Use, enable this automation on the Denature and Dilute master step.
In Clarity LIMS, under Configuration, select the Lab Work tab.
Find the Denature and Dilute step.
In the Automation section, update the Trigger Location and Trigger Style for the Validate Single Input automation as follows.
Trigger Location — Step
Trigger Style — Automatic upon entry
The Illumina MiSeq Integration Package v8.2.0 supports the integration of Clarity LIMS to Illumina MiSeq Sequencing Systems.
The integration allows for automated tracking of an Illumina sequencing run in Clarity LIMS. This capability includes tracking sequencing run status, generating run report, and capturing and parsing run statistics. In addition, this integration provides automated generation of a sample sheet file for use with the MiSeq Control Software (MCS) and Local Run Manager (LRM).
For instructions on user interaction for each step, validating and troubleshooting the Illumina MiSeq Integration Package, refer to .
The MiSeq Sequencing v3.2 workflow is only compatible with MiSeq Integration Package v8.2.0.
MiSeq Integration v8.2.0 is only required for MiSeq Control Software (MCS) v4.0. For MCS v3.1 or earlier, do not upgrade to MiSeq Integration v8.2.0. This upgrade breaks the integration.
Before samples are assigned to the MiSeq Sequencing v3.2 workflow, make sure that the following prerequisites are completed:
Samples have been accessioned into Clarity LIMS.
Samples have been run through QC and library prep.
Samples have been normalized, and the value is captured in a field called Normalized Molarity (nM).
For more information on sample accessioning, refer to Sample Accessioning and Upload and Modify Samples in the .
Samples can be assigned to the MiSeq Sequencing v3.2 workflow automatically using a routing script or manually from the Projects & Samples dashboard. Refer to Assign and Process Samples in the .
The Illumina MiSeq Integration includes the MiSeq Sequencing v3.2 workflow, which contains a single protocol of the same name.
In this step, the lab scientist manually places libraries into pools in the Clarity LIMS Placement screen.
The following field is configured on the Library Pooling (MiSeq v3.2) master step. The field displays on the Record Details screen at run time.
Library Pooling (MiSeq v3.2) Master Step Field Configuration
The following table lists the global fields that are displayed on the Queue and Ice Bucket screens of the Library Pooling (MiSeq v3.2) step. Most fields display in expanded view only.
Global Field Configuration (Submitted Sample)
Global Field Configuration (Derived Sample)
In this step, pooled libraries are denatured and diluted, and then placed into the reagent cartridge loaded into the MiSeq instrument.
Most fields configured on the Denature and Dilute (MiSeq v3.2) step display on the Record Details screen in the Step Data table.
These fields are manually populated at run time. The values are then used to generate the sample sheet.
Denature & Dilute (MiSeq v3.2) Master Step Field Configuration
Groups of Defaults
The following table shows the global fields that are configured to display on the Queue, Ice Bucket, and Record Details screens of the Denature and Dilute (MiSeq v3.2) step:
Global Field Configuration (Submitted Sample)
Global Field Configuration (Derived Sample)
Placeholders for the following files are configured on the Record Details screen of the Denature and Dilute (MiSeq v3.2) step.
The following fields are configured on the MiSeq Run (MiSeq v3.2) step. These fields display on the Record Details screen at run time. The read-only fields are automatically populated at the end of the run.
MiSeq Run (MiSeq v3.2) Master Step Field Configuration
There are several sample and measurement global fields that are displayed on the Record Details screen of the MiSeq Run (MiSeq v3.2) step. These fields are autopopulated at the end of the sequencing run.
Placeholders for the following files are configured on the Record Details screen of the MiSeq Run (MiSeq v3.2) step:
Illumina Run Report (automatically attached)
Link to Run Folder (automatically attached)
Run Parameters (automatically attached)
Run Info (automatically attached)
Lab Tracking Form (manually uploaded)
Log File (automatically attached)
Sample sheet generation occurs in the Denature & Dilute (MiSeq v3.2) step. This step places samples on the container loaded in the system.
The default configuration provides only the Validate Run Setup and Generate MiSeq SampleSheet automation. This automation uses the Template File Generator (DriverFileGenerator.jar) and a template file to generate a CSV format file for use with the MiSeq Control Software (MCS).
The sample sheet content is determined by the fields on the Record Details screen of the step in the Step Data table. The values entered into these fields are used to populate the sample sheet.
To customize the template used to create the sample sheet, you can insert additional columns.
The following additional details are available:
The MiSeq Run (MiSeq v3.2) step records information for the flow cell lane and generates a report summarizing the results. In addition, run parameters, run info, and a link to the run folder are automatically captured.
The following table describes the run information files, reports, placeholders, and links that Clarity LIMS automatically generates or captures during a sequencing run:
Run Information Generated or Captured by MiSeq Run (MiSeq v3.2) Step
The following list shows metadata that Clarity LIMS automatically captures from the Illumina sequencing software as part of a sequencing run. This information is gathered from various run result files and events.
Chemistry
Experiment Name (entered in software)
Finish Date (run completion date)
If the End Run event contains a date in the format YYYY-MM-DD, Finish Date is set to the date in the event file.
If the End Run event does not contain a date or the date is in the wrong format, Finish Date is set to the date when the event file is processed.
Flow Cell ID
Flow Cell Version
Index 1 Read Cycles (intended Index cycles)
Index 2 Read Cycles (intended Index cycles)
Output Folder (run folder root)
PR2 Bottle ID
Reagent Cartridge ID
Reagent Cartridge Part #
Read 1 Cycles
Read 2 Cycles
Run ID (the unique run ID)
Run Type
Status (current status of the sequencing run on the instrument)
Workflow
The following table lists the real-time analysis (RTA) primary analysis metrics Clarity LIMS automatically captures and records per read, for samples in each flow cell lane. These metrics are captured upon run completion and are stored as fields in the Sample Details table on the Record Details screen.
To see both per read and per lane metrics, expand the output.
RTA Primary Analysis Metrics Captured by MiSeq Run (MiSeq v3.2) Step
The sequencing service runs on the Clarity LIMS server. The service detects event files that instrument RTA produces as the run progresses. The event files let the service know where to find the run data.
As the run data are written out and the End Run event is detected, the following events occur:
The data are matched to the step based on the reagent cartridge ID that was entered or scanned on the Denature and Dilute (MiSeq v3.2) step.
Read-only field values on the Record Details screen are populated accordingly.
When the service has finished processing the end run event and updating the fields in Clarity LIMS, the sequencing service generates the report and attaches it to the step.
This integration requires components installed with the Illumina Preset Protocols (IPP).
The Illumina MiSeq Integration Package v8.2.0 RPM installs the scripts and files listed in the following table.
The properties installed in the Illumina MiSeq Integration v8.2.0 are described in the following section.
The integration also installs several additional properties, each with the 99 suffix appended to their name, that are not listed in the table. These properties are intended for use by the Clarity LIMS support team in automated validation tests.
Sequencing runs are matched using the reagent cartridge ID and the base name of the sequencing step, MiSeq Run (MiSeq v3.2).
Properties Installed with the Illumina MiSeq Integration Package
Reagent categories or label groups are installed with the IPP workflow configuration slices.
The MiSeq Reagent Kit is included in the Illumina MiSeq Integration.
The PhiX v3 control type is included in the Illumina MiSeq Integration.
The following container types are included in the Illumina MiSeq Integration:
MiSeq Reagent Cartridge
96 well plate
Tube
All one-dimensional container types with both numeric rows and numeric columns are supported.
To make sure that the Illumina instrument warranty remains valid, the instrument integration must be performed and maintained by the Clarity LIMS Support team. To perform this integration, the Support team requires remote access to the system while it is idle.
The following steps are performed by the Clarity LIMS Support team when configuring the sequencing for use with the Illumina MiSeq Integration.
Create a directory on the local computer to hold the batch files. These batch files write event files to the network attached storage (NAS) shares.
Create a directory on the NAS to hold the event files.
Modify Illumina software configuration files to call the batch files that create the event files.
Update sequencing service default properties to match the specifics of the installation.
The Illumina MiSeq Integration operates with the following constraints:
The reagent cartridge ID must be unique. There should not be multiple reagent cartridge containers in the system with the same name.
The reagent cartridge ID must be scanned as the reagent cartridge Container Name on the Denature & Dilute (MiSeq v3.2) step.
Last Updated: November 2024
Release Date: September 2023
Document Version: 2
These release notes describe the key changes to software components for the Clarity LIMS MiSeq Integration Package v8.2.0.
Refer to under Instruments & Integrations.
Upgraded Java and third-party dependency libraries.
Upgraded Groovy to v3.0.7.
The Denature and Dilute step now only allows a single pool to be added to the Ice Bucket when you enter the step.
Fixed the MiSeq integration service not starting automatically after a system reboot.
Fixed the SQL Injection (CVE-2022-31197) and Information Disclosure (CVE-2020-15522) vulnerabilities.
None
The Illumina MiSeq Integration Package v8.3.0 supports the integration of Clarity LIMS to MiSeq instruments.
MiSeq Integration v8.3.0 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.
MiSeq Integration v8.3.0 is compatible with the following software:
Clarity LIMS v6.2.0 and later
Secret Util v1.0 and later
IPP v2.9 and later
MCS v4.0/LRM v3.0 and later
Oracle Linux (for compatibility, refer to )
MiSeq Integration v8.3.0 has the following prerequisites:
Mount run data network-attached storage (NAS) share
IPP is installed
MiSeq Integration v8.3.0 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 v8.3.0.
On the Clarity LIMS server, log in as the root user.
Run the following yum command to install the RPM:
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.
Install the MiSeq workflow as follows.
As the glsjboss user, run the following command to view the complete list of IPP workflows:
Run the following command to install the MiSeq Sequencing v3.2 workflow configuration:
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).
[Optional] Run the following command to install the Library Prep Validation v2.3.4 workflow that is used to validate the MiSeq Sequencing v3.2 workflow:
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.
Run the following script to configure the service properties:
Run the following command to start the sequencing service:
MiSeq Integration v8.3.0 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)
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.
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.
Last Updated: November 2024
Release Date: July 2024
Document Version: 2
These release notes describe the key changes to software components for the Clarity LIMS MiSeq Integration Package v8.3.0.
Refer to under Instruments & Integrations.
Integration-related properties can now be accessed and updated via System Setting in Clarity v6.3. Refer to for configurable properties.
Fixed the SQL Injection (CVE-2024-1597) vulnerability.
None
Only one pool is allowed as an input.
If the MiSeq Run master step is configured to track instruments, select the applicable instrument from the Instrument Used dropdown on Record Details screen and select Save.
Make sure the Illumina Run Report has been attached before continuing.
Follow the steps in to run the Library Prep Validation workflow with the following:
Only one pool is allowed as an input.
For more information on presets, refer to . Workflow, Experiment Name, and Read 1 Cycles are required fields.
If the MiSeq Run master step is configured to track instruments, select the applicable instrument from the Instrument Used dropdown on Record Details screen and select Save.
Populates the fields in the Sample Details table. For details, refer to .
Make sure the Illumina Run Report has been attached before continuing.
Troubleshooting Automation Worker in the .
Troubleshooting Automation in the .
This automation validates the information entered on the Record Details screen, generates the sample sheet (refer to ), and attaches the sample sheet to the Denature and Dilute step.
The Submitted Sample field, Progress, (added from previous MiSeq workflow) is obsolete in MiSeq v3.2.
For more information, refer to .
For details, refer to .
For a sample template that you can download and customize for the lab, refer to .
For instructions on customizing the template, refer to .
Do not change the base name. The sequencing service that captures sequencing run results expects this name. The base name is stored in the sequenceProcessBaseName property. If the name is changed without the property being updated, the reagent cartridge ID <-> sequencing step base name matching system fails. If you need to rename the step, for example to add a version number, modify the text before or after the base name portion. This part of the name is not used in the matching system. For example, change MiSeq Run (MiSeq v3.2) to Demo-MiSeq Run (MiSeq v3.2)-Updated.
Changes on miseq.v8.seqservice.sequenceProcessBaseName property take effect upon updates and do not require restart of the integration service. For all remaining properties, integration service has to be restarted for property changes to take effect.
You can configure support for multiple identical seqservice.netPathPrefixSearch property values. For more information, refer to .
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.
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.
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 .
The script creates the configuration properties in the database. For a list of the configured properties, refer to .
For more information on the configurable integration properties that enable capture and generation of files associated with the sequencing run, refer to .
For descriptions of the protocol and the steps, refer to . For instructions on using the Library Prep Validation v2.3.4 workflow to validate the automated sample sheet generation, refer to .
For more information on MCS, refer to the MiSeq documentation at .
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.
Make sure to include a trailing \ in the DESTINATION_PATH and DEFAULT_LOCAL_OUTPUT_PATH lines. Refer to the following examples:
Make sure that you rename the backup file. If you do not rename the file, it can cause the RTA not to work properly.
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 .
Field Name | Field Type | Field Constraints/Options | Preset Values/Additional Options and Drop-down Items |
Comment | Multiline Text |
Field Name | Field Type | Field Constraints/Options | Preset Values/Additional Options and Drop-Down Items |
Application | Text Dropdown | Custom Entries | Presets
|
Pooling | Text Dropdown | Custom Entries | Presets
|
Read Length | Text |
Sequencing Coverage | Text |
Sequencing Method | Text Dropdown | Custom Entries | Presets
|
Field Name | Field Type | Field Constraints/Options | Preset Values/Additional Options and Drop-Down Items |
Normalized Molarity (nM) | Numeric | Decimal places displayed = 2 |
Field Name | Field Type | Options | Additional Options and Drop-Down Items |
Chemistry | Text | Read Only |
Comment | Multiline Text |
Experiment Name | Text | Read Only |
Finish Date | Date | Read Only |
Flow Cell ID | Text | Read Only |
Flow Cell Version | Text | Read Only |
Index 1 Read Cycles | Numeric | Read Only | Decimal Places Displayed: 0 |
Index 2 Read Cycles | Numeric | Read Only | Decimal Places Displayed: 0 |
Output Folder | Text | Read Only |
PR2 Bottle ID | Text | Read Only |
Read 1 Cycles | Numeric | Read Only | Decimal Places Displayed: 0 |
Read 2 Cycles | Numeric | Read Only | Decimal Places Displayed: 0 |
Reagent Cartridge ID | Text | Read Only |
Reagent Cartridge Part # | Text | Read Only |
Run ID | Text | Read Only |
Run Type | Text | Read Only |
Status | Text | Read Only |
Workflow | Text | Read Only |
Item | Description |
Run Info Run Parameters |
|
Link to Run Folder |
|
Illumina Run Report |
|
Lab Tracking Form |
|
Per Read LIMS Field Name (Stored on derived sample/analyte input to the step) | Per Lane LIMS Field Name (Stored in measurement placeholders in the Sample Details table on the Record Details screen) |
% Aligned R1 | % Aligned R1 |
% Aligned R2 | % Aligned R2 |
% Bases >=Q30 R1 | % Bases >=Q30 R1 |
% Bases >=Q30 R2 | % Bases >=Q30 R2 |
% Error Rate R1 | % Error Rate R1 |
% Error Rate R2 | % Error Rate R2 |
% Phasing R1 |
% Phasing R2 |
% Prephasing R1 |
% Prephasing R2 |
%PF R1 |
%PF R2 |
Cluster Density (K/mm^2) R1 |
Cluster Density (K/mm^2) R2 |
Intensity Cycle 1 R1 | Intensity Cycle 1 R1 |
Intensity Cycle 1 R2 | Intensity Cycle 1 R2 |
Reads PF (M) R1 |
Reads PF (M) R2 |
Yield PF (Gb) R1 | Yield PF (Gb) R1 |
Yield PF (Gb) R2 | Yield PF (Gb) R2 |
Version | Changes |
2 |
|
1 |
|
Version | Changes |
2 |
|
1 |
|
Field Name | Field Type | Options | Additional Options and Drop-Down Items |
Adapter Read 1 | Text |
Adapter Read 2 | Text |
Aligner | Text Dropdown |
|
Aligner-Key | Text |
|
|
Aligner-Value | Text |
|
Annotation | Text Dropdown |
|
Applicable analysis fields for the selected Workflow | Multiline Text |
|
Comment | Multiline Text |
Custom Primers | Text Dropdown |
|
Description | Text |
Experiment Name | Text |
|
Export to gVCF | Text Dropdown |
|
Flag PCR Duplicates | Text Dropdown |
|
Genome Folder | Text |
Indel Realignment | Text Dropdown |
|
Indel Repeat Filter Cutoff | Text Dropdown |
|
Indel-Realignment-Key | Text |
|
|
Indel-Realignment-Value | Text |
|
|
Manifest | Text |
Manifest Padding | Text Dropdown |
|
Manifest-Section | Text |
|
|
Picard HS Metric Reporting | Text Dropdown |
|
Read 1 Cycles | Numeric Dropdown |
|
|
Read 2 Cycles | Numeric Dropdown |
|
|
Read Stitching | Text Dropdown |
|
Reverse Complement | Text Dropdown |
|
UMI - Read 1 Length | Numeric |
|
UMI - Read 1 Start From Cycle | Numeric |
|
UMI - Read 2 Length | Numeric |
|
UMI - Read 2 Start From Cycle | Numeric |
|
Validation Script 1 | Multiline Text |
|
|
Validation Script 2 | Multiline Text |
|
|
Variant Caller | Text Dropdown |
|
Variant Caller Depth Filter | Numeric |
|
Variant Quality Filter | Numeric |
|
Variant Frequency Percentage | Numeric |
|
Variant-Caller-Value | Text |
|
|
Workflow | Text Dropdown |
|
|
Field Name | Field Type | Field Constraints/Options | Preset Values/Additional Options and Drop-Down Items |
Read Length | Text |
Sequencing Method | Text Dropdown |
|
|
Field Name | Field Type | Field Constraints/Options | Preset Values/Additional Options and Drop-Down Items |
Final Loading Concentration | Numeric Dropdown |
|
|
Component | Location | Description |
configure_extensions_miseq_sequencingservice-v8.sh | /opt/gls/clarity/config/ | Script that installs the service properties in the database. |
log4j.xml | /opt/gls/clarity/extensions/Illumina_MiSeq/v8/SequencingService/conf | File containing the settings for logging the sequencing jar. |
miseq-sequencing-v8.jar | /opt/gls/clarity/extensions/Illumina_MiSeq/v8/SequencingService | Jar file containing API-based Clarity LIMS extensions used for capturing run results and report generation. |
InterOp libraries | /opt/gls/clarity/extensions/Illumina_MiSeq/v8/lib | Illumina shared library for parsing InterOp data files. |
MiSeqSamplesheetv3.csv | /opt/gls/clarity/extensions/conf/driverfiletemplates | Template file used for file generation. |
Property | Description | Default Value |
miseq.v8.seqservice.sequenceProcessBaseName | Sequencing process type/master step base display name. Partial matching is used to look up the process type/master step. This property is added/updated by Illumina Preset Protocols (IPP) during the workflow installation. | MiSeq Run (MiSeq v3.2) |
miseq.v8.seqservice.eventFileDirectory.1 | A network location monitored for event files, e.g., /mnt/illumina/gls_events/ | /mnt/gls_events |
miseq.v8.seqservice.netPathPrefixSearch.1 | The network directory prefix contained in the event file. | \\nas\network\run_data |
miseq.v8.seqservice.netPathPrefixReplace.1 | The mapped network directory mount name on the server used to access the run data directory, e.g., /mnt/network/data | /mnt/run_data |
miseq.v8.seqservice.eventFileDirectorySuffixes | A list of eventFileDirectory path entries to monitor for event files. The value is one or more comma-separated integers. | 99 |
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 |
miseq.v8.seqservice.runReportViewsVersion | The current version of the Run Report views in the database. The value 0 represents the state before the views are created. This property is automatically updated by the run report. | 0 |
miseq.v8.seqservice.ignoreUnmatchedContainerIds | A flag indicating if event files that cannot be matched to reagent cartridges in Clarity LIMS should be archived after a certain period (true) or continually reprocessed (false). | false |
miseq.v8.seqservice.ignoreUnmatchedContainerIdsWaitDays | The number of days between when the event is created and the event file is archived. | 14 |
miseq.v8.seqservice.synchronizationPeriod | Invocation period in seconds. | 60 |
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.
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)
MiSeq Integration v8.2.0 has the following prerequisites:
Mount run data network-attached storage (NAS) share
IPP is installed
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.
On the Clarity LIMS server, log in as the root user.
Run the following yum command to install the RPM:
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.
Install the MiSeq workflow as follows.
As the glsjboss user, run the following command to view the complete list of IPP workflows:
Run the following command to install the MiSeq Sequencing v3.2 workflow configuration:
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).
[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:
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.
Run the following script to configure the service properties:
The script creates the configuration properties in the database. For a list of the configured properties, refer to 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
Run the following command to start the sequencing service:
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.
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.
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.
The Illumina MiSeq Integration Package v8.3.0 supports the integration of Clarity LIMS to Illumina MiSeq Sequencing Systems.
The integration allows for automated tracking of an Illumina sequencing run in Clarity LIMS. This capability includes tracking sequencing run status, generating run report, and capturing and parsing run statistics. In addition, this integration provides automated generation of a sample sheet file for use with the MiSeq Control Software (MCS) and Local Run Manager (LRM).
For instructions on user interaction for each step, validating and troubleshooting the Illumina MiSeq Integration Package, refer to MiSeq Integration v8.3.0 User Interaction, Validation and Troubleshooting.
The MiSeq Sequencing v3.2 workflow is compatible with MiSeq Integration Package v8.2.0 and v8.3.0.
MiSeq Integration v8.2.0 and above is only required for MiSeq Control Software (MCS) v4.0. For MCS v3.1 or earlier, do not upgrade to MiSeq Integration v8.2.0 or above. This upgrade breaks the integration.
Before samples are assigned to the MiSeq Sequencing v3.2 workflow, make sure that the following prerequisites are completed:
Samples have been accessioned into Clarity LIMS.
Samples have been run through QC and library prep.
Samples have been normalized, and the value is captured in a field called Normalized Molarity (nM).
For more information on sample accessioning, refer to Sample Accessioning and Upload and Modify Samples in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
Samples can be assigned to the MiSeq Sequencing v3.2 workflow automatically using a routing script or manually from the Projects & Samples dashboard. Refer to Assign and Process Samples in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
The Illumina MiSeq Integration includes the MiSeq Sequencing v3.2 workflow, which contains a single protocol of the same name.
In this step, the lab scientist manually places libraries into pools in the Clarity LIMS Placement screen.
The following field is configured on the Library Pooling (MiSeq v3.2) master step. The field displays on the Record Details screen at run time.
Library Pooling (MiSeq v3.2) Master Step Field Configuration
The following table lists the global fields that are displayed on the Queue and Ice Bucket screens of the Library Pooling (MiSeq v3.2) step. Most fields display in expanded view only.
Global Field Configuration (Submitted Sample)
Global Field Configuration (Derived Sample)
In this step, pooled libraries are denatured and diluted, and then placed into the reagent cartridge loaded into the MiSeq instrument.
Most fields configured on the Denature and Dilute (MiSeq v3.2) step display on the Record Details screen in the Step Data table.
These fields are manually populated at run time. The values are then used to generate the sample sheet.
Denature & Dilute (MiSeq v3.2) Master Step Field Configuration
Groups of Defaults
The following table shows the global fields that are configured to display on the Queue, Ice Bucket, and Record Details screens of the Denature and Dilute (MiSeq v3.2) step:
Global Field Configuration (Submitted Sample)
Global Field Configuration (Derived Sample)
Placeholders for the following files are configured on the Record Details screen of the Denature and Dilute (MiSeq v3.2) step.
The following fields are configured on the MiSeq Run (MiSeq v3.2) step. These fields display on the Record Details screen at run time. The read-only fields are automatically populated at the end of the run.
MiSeq Run (MiSeq v3.2) Master Step Field Configuration
There are several sample and measurement global fields that are displayed on the Record Details screen of the MiSeq Run (MiSeq v3.2) step. These fields are autopopulated at the end of the sequencing run.
For more information, refer to Sequencing Results Parsing.
Placeholders for the following files are configured on the Record Details screen of the MiSeq Run (MiSeq v3.2) step:
Illumina Run Report (automatically attached)
Link to Run Folder (automatically attached)
Run Parameters (automatically attached)
Run Info (automatically attached)
Lab Tracking Form (manually uploaded)
Log File (automatically attached)
For details, refer to Generated and Captured Files.
Sample sheet generation occurs in the Denature & Dilute (MiSeq v3.2) step. This step places samples on the container loaded in the system.
The default configuration provides only the Validate Run Setup and Generate MiSeq SampleSheet automation. This automation uses the Template File Generator (DriverFileGenerator.jar) and a template file to generate a CSV format file for use with the MiSeq Control Software (MCS).
The sample sheet content is determined by the fields on the Record Details screen of the step in the Step Data table. The values entered into these fields are used to populate the sample sheet.
To customize the template used to create the sample sheet, you can insert additional columns.
The following additional details are available:
For a sample template that you can download and customize for the lab, refer to Illumina Instrument Sample Sheets (NGS v5.17 & later).
For instructions on customizing the template, refer to Creating Template Files.
The MiSeq Run (MiSeq v3.2) step records information for the flow cell lane and generates a report summarizing the results. In addition, run parameters, run info, and a link to the run folder are automatically captured.
The following table describes the run information files, reports, placeholders, and links that Clarity LIMS automatically generates or captures during a sequencing run:
Run Information Generated or Captured by MiSeq Run (MiSeq v3.2) Step
The following list shows metadata that Clarity LIMS automatically captures from the Illumina sequencing software as part of a sequencing run. This information is gathered from various run result files and events.
Chemistry
Experiment Name (entered in software)
Finish Date (run completion date)
If the End Run event contains a date in the format YYYY-MM-DD, Finish Date is set to the date in the event file.
If the End Run event does not contain a date or the date is in the wrong format, Finish Date is set to the date when the event file is processed.
Flow Cell ID
Flow Cell Version
Index 1 Read Cycles (intended Index cycles)
Index 2 Read Cycles (intended Index cycles)
Output Folder (run folder root)
PR2 Bottle ID
Reagent Cartridge ID
Reagent Cartridge Part #
Read 1 Cycles
Read 2 Cycles
Run ID (the unique run ID)
Run Type
Status (current status of the sequencing run on the instrument)
Workflow
The following table lists the real-time analysis (RTA) primary analysis metrics Clarity LIMS automatically captures and records per read, for samples in each flow cell lane. These metrics are captured upon run completion and are stored as fields in the Sample Details table on the Record Details screen.
To see both per read and per lane metrics, expand the output.
RTA Primary Analysis Metrics Captured by MiSeq Run (MiSeq v3.2) Step
The sequencing service runs on the Clarity LIMS server. The service detects event files that instrument RTA produces as the run progresses. The event files let the service know where to find the run data.
As the run data are written out and the End Run event is detected, the following events occur:
The data are matched to the step based on the reagent cartridge ID that was entered or scanned on the Denature and Dilute (MiSeq v3.2) step.
Read-only field values on the Record Details screen are populated accordingly.
When the service has finished processing the end run event and updating the fields in Clarity LIMS, the sequencing service generates the report and attaches it to the step.
This integration requires components installed with the Illumina Preset Protocols (IPP).
The Illumina MiSeq Integration Package v8.3.0 RPM installs the scripts and files listed in the following table.
Refer to Integration Properties Details for the properties installed with Illumina MiSeq Integration v8.3.0.
Reagent categories or label groups are installed with the IPP workflow configuration slices.
The MiSeq Reagent Kit is included in the Illumina MiSeq Integration.
The PhiX v3 control type is included in the Illumina MiSeq Integration.
The following container types are included in the Illumina MiSeq Integration:
MiSeq Reagent Cartridge
96 well plate
Tube
All one-dimensional container types with both numeric rows and numeric columns are supported.
To make sure that the Illumina instrument warranty remains valid, the instrument integration must be performed and maintained by the Clarity LIMS Support team. To perform this integration, the Support team requires remote access to the system while it is idle.
The following steps are performed by the Clarity LIMS Support team when configuring the sequencing for use with the Illumina MiSeq Integration.
Create a directory on the local computer to hold the batch files. These batch files write event files to the network attached storage (NAS) shares.
Create a directory on the NAS to hold the event files.
Modify Illumina software configuration files to call the batch files that create the event files.
Update sequencing service default properties to match the specifics of the installation.
The Illumina MiSeq Integration operates with the following constraints:
The reagent cartridge ID must be unique. There should not be multiple reagent cartridge containers in the system with the same name.
The reagent cartridge ID must be scanned as the reagent cartridge Container Name on the Denature & Dilute (MiSeq v3.2) step.
Used for sample sheet generation
Used for sample sheet generation
Select a value from the drop-down list in the upper-right corner (below NEXT STEPS).
Used for sample sheet generation
Used for sample sheet generation
Used for sample sheet generation
Used for automation
Used for automation
Used for sample sheet generation
Displays on Queue & Ice Bucket screens
Displays on Queue & Ice Bucket screens
Displays on Record Details screen
Installed by IPP
Configured on install to point to 1
Configured on install to point to 1
To prevent the gls_events file directory from becoming cluttered, it is recommended that the value of this property is set to true
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.
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.
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.
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.
Make sure to include a trailing \ in the DESTINATION_PATH and DEFAULT_LOCAL_OUTPUT_PATH lines. Refer to the following examples:
Make sure that you rename the backup file. If you do not rename the file, it can cause the RTA not to work properly.
The Submitted Sample field, Progress, (added from previous MiSeq workflow) is obsolete in MiSeq v3.2.
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-down Items
Comment
Multiline Text
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-Down Items
Application
Text Dropdown
Custom Entries
Presets
TruSeq mRNA sequencing
TruSeq DNA sequencing (large genome de novo)
TruSeq DNA sequencing (large genome re-seq)
TruSeq DNA sequencing (small genome de novo)
TruSeq DNA sequencing (small genome re-seq)
Nextera DNA sequencing
TruSeq Custom Amplicon sequencing
ChIP-sequencing
Exome sequencing
Mate pair sequencing
Small RNA sequencing
Pooling
Text Dropdown
Custom Entries
Presets
Yes
No
Read Length
Text
Sequencing Coverage
Text
Sequencing Method
Text Dropdown
Custom Entries
Presets
Single Read
Paired End Read
Indexed Single Read
Indexed Paired End Read
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-Down Items
Normalized Molarity (nM)
Numeric
Decimal places displayed = 2
Field Name
Field Type
Options
Additional Options and Drop-Down Items
Chemistry
Text
Read Only
Comment
Multiline Text
Experiment Name
Text
Read Only
Finish Date
Date
Read Only
Flow Cell ID
Text
Read Only
Flow Cell Version
Text
Read Only
Index 1 Read Cycles
Numeric
Read Only
Decimal Places Displayed: 0
Index 2 Read Cycles
Numeric
Read Only
Decimal Places Displayed: 0
Output Folder
Text
Read Only
PR2 Bottle ID
Text
Read Only
Read 1 Cycles
Numeric
Read Only
Decimal Places Displayed: 0
Read 2 Cycles
Numeric
Read Only
Decimal Places Displayed: 0
Reagent Cartridge ID
Text
Read Only
Reagent Cartridge Part #
Text
Read Only
Run ID
Text
Read Only
Run Type
Text
Read Only
Status
Text
Read Only
Workflow
Text
Read Only
Item
Description
Run Info Run Parameters
These XML files are automatically captured by Clarity LIMS from the run folder of the system. They include the key run parameters that are parsed out into step custom fields in Clarity LIMS.
Link to Run Folder
This link is the path to the network run folder where the data that was captured from the system during the run is stored. The link is automatically generated by Clarity LIMS.
Illumina Run Report
This report provides key information about the run and the samples on the flow cell. The report is automatically generated by Clarity LIMS.
Information includes the flow cell ID, run directory location, and primary analysis metrics for the sequencing run. Information is summarized per flow cell lane for the entire run and individual reads for paired-end runs.
These metrics are compared against the per lane averages of the sequencing run. The per lane averages are calculated using metrics from the last five sequencing runs. Any values outside of one standard deviation are highlighted.
Lab Tracking Form
This item in Clarity LIMS allows you to manually attach a lab-specific tracking form to the step.
Per Read LIMS Field Name (Stored on derived sample/analyte input to the step)
Per Lane LIMS Field Name (Stored in measurement placeholders in the Sample Details table on the Record Details screen)
% Aligned R1
% Aligned R1
% Aligned R2
% Aligned R2
% Bases >=Q30 R1
% Bases >=Q30 R1
% Bases >=Q30 R2
% Bases >=Q30 R2
% Error Rate R1
% Error Rate R1
% Error Rate R2
% Error Rate R2
% Phasing R1
% Phasing R2
% Prephasing R1
% Prephasing R2
%PF R1
%PF R2
Cluster Density (K/mm^2) R1
Cluster Density (K/mm^2) R2
Intensity Cycle 1 R1
Intensity Cycle 1 R1
Intensity Cycle 1 R2
Intensity Cycle 1 R2
Reads PF (M) R1
Reads PF (M) R2
Yield PF (Gb) R1
Yield PF (Gb) R1
Yield PF (Gb) R2
Yield PF (Gb) R2
Property
Description
Default Value
miseq.v8.seqservice.eventFileDirectory.1
A monitored network location used for event files.
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.
miseq.v8.seqservice.netPathPrefixSearch.1
The network directory prefix contained in the event file.
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.
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
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
Field Name
Field Type
Options
Additional Options and Drop-Down Items
Adapter Read 1
Text
Adapter Read 2
Text
Aligner
Text Dropdown
Presets
None (default)
BWA-MEM
BWA-Backtrack Legacy
BWA
TruSeq Amplicon Aligner
Aligner-Key
Text
Read Only
Hidden
Aligner-Value
Text
Read Only
Annotation
Text Dropdown
Presets
None (default)
RefSeq
Ensembl
Applicable analysis fields for the selected Workflow
Multiline Text
Read Only
Comment
Multiline Text
Custom Primers
Text Dropdown
Presets
None (Default)
Read 1
Index
Read 2
Read 1, Index
Read 1, Index, Read 2
Read 1, Read 2
Index, Read 2
Description
Text
Experiment Name
Text
Required Field
Export to gVCF
Text Dropdown
Presets
None (default)
Yes
No
Flag PCR Duplicates
Text Dropdown
Presets
None (default)
Yes
No
Genome Folder
Text
Indel Realignment
Text Dropdown
Presets
None (default)
Yes
No
Indel Repeat Filter Cutoff
Text Dropdown
Presets
None (default)
Yes
No
Indel-Realignment-Key
Text
Read Only
Hidden
Indel-Realignment-Value
Text
Read Only
Hidden
Manifest
Text
Manifest Padding
Text Dropdown
Presets
None (default)
0
50
100
150
200
250
Manifest-Section
Text
Read Only
Hidden
Picard HS Metric Reporting
Text Dropdown
Presets
None (default)
Yes
No
Read 1 Cycles
Numeric Dropdown
Required Field
Custom Entries
Presets
251 (Default)
151
101
76
51
Range = 1–1000
Decimal places displayed = 0
Read 2 Cycles
Numeric Dropdown
Required Field
Custom Entries
Presets
251 (Default)
151
101
76
51
Range = 0–1000
Decimal places displayed = 0
Read Stitching
Text Dropdown
Presets
None (default)
Yes
No
Reverse Complement
Text Dropdown
Presets
None (default)
Yes
No
UMI - Read 1 Length
Numeric
Minimum value: 1
UMI - Read 1 Start From Cycle
Numeric
Minimum value: 1
UMI - Read 2 Length
Numeric
Minimum value: 1
UMI - Read 2 Start From Cycle
Numeric
Minimum value: 1
Validation Script 1
Multiline Text
Required Field
Read Only
Hidden
Validation Script 2
Multiline Text
Required Field
Read Only
Hidden
Variant Caller
Text Dropdown
Presets
None (Default)
GATK
Germline
Somatic
Starling
Variant Caller Depth Filter
Numeric
Minimum value: 10
Maximum value: 10000
Variant Quality Filter
Numeric
Minimum value: 2
Maximum value: 1000
Variant Frequency Percentage
Numeric
Minimum value: 0.05
Maximum value: 100
Decimal places displayed = 2
Variant-Caller-Value
Text
Read Only
Hidden
Workflow
Text Dropdown
Required Field
Presets
GenerateFASTQ
LibraryQC
Resequencing
DNA Enrichment
DNA Amplicon
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-Down Items
Read Length
Text
Sequencing Method
Text Dropdown
Custom Entries
Presets
Single Read
Paired End Read
Indexed Single Read
Indexed Paired End Read
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-Down Items
Final Loading Concentration
Numeric Dropdown
Required Field
Custom Entries
Presets
225
400
Decimal places displayed = 0
Component
Location
Description
configure_extensions_miseq_sequencingservice-v8.sh
/opt/gls/clarity/config/
Script that installs the service properties in the database.
log4j.xml
/opt/gls/clarity/extensions/Illumina_MiSeq/v8/SequencingService/conf
File containing the settings for logging the sequencing jar.
miseq-sequencing-v8.jar
/opt/gls/clarity/extensions/Illumina_MiSeq/v8/SequencingService
Jar file containing API-based Clarity LIMS extensions used for capturing run results and report generation.
InterOp libraries
/opt/gls/clarity/extensions/Illumina_MiSeq/v8/lib
Illumina shared library for parsing InterOp data files.
MiSeqSamplesheetv3.csv
/opt/gls/clarity/extensions/conf/driverfiletemplates
Template file used for file generation.
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.
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.
Used for sample sheet generation
Used for sample sheet generation
Select a value from the drop-down list in the upper-right corner (below NEXT STEPS).
Used for sample sheet generation
Used for sample sheet generation
Used for sample sheet generation
Used for automation
Used for automation
Used for sample sheet generation
Displays on Queue & Ice Bucket screens
Displays on Queue & Ice Bucket screens
Displays on Record Details screen
Installed by IPP
