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The documents in this section support the Illumina NextSeq 500/550 Integration.
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 NextSeq 500/550 Integration Package v2.4.0.
Refer to Compatibility under Instruments & Integrations.
Integration-related properties can now be accessed and updated via System Setting in Clarity v6.3. Refer to NextSeq 500/550 Integration v2.4.0 Configuration for configurable properties.
Fixed the SQL Injection (CVE-2024-1597) vulnerability.
Removed the unused Run Type custom field in Step Details milestone in the NextSeq 500/550 Run step.
Missing contents in the Instrument Details section of the run report.
This guide explains how to validate the installation of the Illumina NextSeq 500/550 Integration Package v2.4.0.
The validation process involves:
Validating the creation of event files.
Running samples through the Library Prep Validation v2.3.4 workflow. This workflow is installed by Illumina Preset Protocols (IPP) v2.9 and contains a single-step protocol that models the library prep required to produce normalized libraries. At the end of the step, the normalized libraries are automatically advanced to the NextSeq 500/550 Sequencing v1.2 workflow.
Running normalized libraries through the NextSeq 500/550 Sequencing v1.2 workflow. This validates the automated generation of a sample sheet for use with bcl2fastq2 v2.20.0 analysis software. It also validates the automated tracking of the NextSeq sequencing run and parsing of run statistics into Clarity LIMS, including:
Run status and metrics of sequencing run
Sequencing run parameters
Real Time Analysis v2 (RTA2) run directory location and other run specific information
The following steps set up Clarity LIMS in preparation for running samples through the Library Prep Validation v2.3.4 and NextSeq 500/550 Sequencing v1.2 workflows.
In the Clarity LIMS configuration area, activate the Library Prep Validation v2.3.4 and NextSeq 500/550 Sequencing v1.2 workflows.
On the Projects and Samples screen, create a project and add samples to it.
Assign your samples to the Library Prep Validation v2.3.4 workflow.
This single-step protocol models the library prep required to produce normalized libraries that are ready for the NextSeq 500/550 Sequencing v1.2 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 = NextSeq
On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the NextSeq 500/550 Sequencing v1.2 workflow, which is Library Pooling (NextSeq 500/550 v1.2) step.
In Lab View, locate the NextSeq 500/550 Sequencing v1.2 protocol. You will see your samples queued for the Library Pooling (NextSeq 500/550 v1.2) step.
Select the step to proceed to the Queue screen.
On the Queue screen, add samples 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.
On the Pool Samples screen, create a pool of samples as follows.
Drag samples into the Pool Creator.
Name the pool. You can also accept the default name Pool #1.
Select Place Samples.
On the Placement screen, move the pool to the container as follows.
Select the pool in the Samples to be Placed area on the left. Drag it over to the container on the right.
Select Record Details.
On the Record Details screen, select Next Steps.
On the Assign Next Steps screen, the next step is already set to Denature and Dilute (NextSeq 500/550 v1.2).
Select Finish Step.
In Lab View, locate the NextSeq 500/550 Sequencing v1.2 protocol. You will see your pooled samples queued for the Denature and Dilute (NextSeq 500/550 v1.2) step.
Select the step to proceed to the Queue screen.
On the Queue screen, add the pool to the Ice Bucket. Select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
On the Placement screen, move the pool to the reagent cartridge as follows.
Scan the NextSeq reagent cartridge barcode into the NextSeq Reagent Cartridge field.
Place the pool of samples into the reagent cartridge.
Select Record Details.
On the Record Details screen, create the sample sheet as follows.
Under Reagent Lot Tracking, select the reagent lot used in the step. You may need to add/activate the lot on the Reagents and Controls screen.
In the Step Details table, populate the fields as appropriate.
The Workflow and Read 1 Cycles are required fields.
In the Samplesheet Template drop-down list, the reverse complement template is selected by default. Do not change this value.
In the Sample Details table, enter the Final Loading Concentration. You may select preset 225 (for PCR-free workflows) or preset 400 (for Nano workflows). You can also enter a different value.
Select Generate bcl2fastq2 NextSeq 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 their format and content.
Select Next Steps.
On the Assign Next Steps screen, make sure samples are already assigned to the NextSeq 500/550 Run (NextSeq 500/550 v1.2) step.
Select Finish Step.
In Lab View, locate the NextSeq 500/550 Sequencing v1.2 protocol. You will see your pool of samples queued for the NextSeq 500/550 Run (NextSeq 500/550 v1.2) step.
Select the step to proceed to the Queue screen.
Add the pool to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
On the Record Details screen, the fields in the table are read-only. Once the run completes, the integration will automatically populate the fields.
The integration also attaches files to the Illumina Run Report, Link to Run Folder, Run Parameters, and Run Info placeholders. The Log File is attached once the next step for samples has been assigned by the Next Step - Advance automation. The integration also populates the fields in the Sample Details table. For details, refer to NextSeq 500/550 v2.4.0 Configuration.
For more detailed steps on adding instruments, refer to the Add and Configure Instruments section of the [Clarity LIMS (Clarity & LabLink Reference Guide) documentation](../../../clarity-lims/clarity-and-lablink.md).
Select Next Steps.
On the Assign Next Steps screen, make sure the next step is already set to Mark protocol as complete.
Select Finish Step.
Follow the steps below 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, and support for NextSeq Control Software (NCS) v4.0 has been configured.
This test validates that:
Your 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.
You can validate the gls_event_ncs_rta.bat batch file by executing it manually as follows.
Edit gls_event_ncs_rta.bat using any text editor (Notepad++ recommended).
By default, this file is installed to C:\Illumina\gls.
In the code line set DESTINATION_PATH=C:\Illumina\gls\Events, change C:\Illumina\gls\Events\ to the network path of the event files directory.
Remember to include the trailing "" at the end of the file path.
Save your changes.
Manually executing the batch file will create a dummy EndRun event file in the directory that you defined in the previous step.
If an automation trigger does not appear to run its corresponding scripts, refer to the following topics:
Troubleshooting Automation Worker in the Administration section of the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
Troubleshooting Automation in the Automation section of 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 Illumina NextSeq Integration Package by running the following 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 NextSeqIntegrator.log file. It is located at
Additional troubleshooting information for this integration is provided on the Illumina Instrument Integrations FAQ page.
If you are unable to resolve the issue, contact the Clarity LIMS Support team. Make sure you supply the relevant information from the troubleshooting that was already performed.
This guide explains how to validate the installation of the Illumina NextSeq 500/550 Integration Package v2.3.0.
The validation process involves:
Validating the creation of event files.
Running samples through the Library Prep Validation v2.3.1 workflow. This workflow is installed by Illumina Preset Protocols (IPP) v2.6 and contains a single-step protocol that models the library prep required to produce normalized libraries. At the end of the step, the normalized libraries are automatically advanced to the NextSeq 500/550 Sequencing v1.1 workflow.
Running normalized libraries through the NextSeq 500/550 Sequencing v1.1 workflow. This validates the automated generation of a sample sheet for use with bcl2fastq2 v2.20.0 analysis software. It also validates the automated tracking of the NextSeq sequencing run and parsing of run statistics into Clarity LIMS, including:
Run status and metrics of sequencing run
Sequencing run parameters
Real Time Analysis v2 (RTA2) run directory location and other run specific information
The following steps set up Clarity LIMS in preparation for running samples through the Library Prep Validation v2.3.1 and NextSeq 500/550 Sequencing v1.1 workflows.
In the Clarity LIMS configuration area, activate the Library Prep Validation v2.3.1 and NextSeq 500/550 Sequencing v1.1 workflows.
On the Projects and Samples screen, create a project and add samples to it.
Assign your samples to the Library Prep Validation v2.3.1 workflow.
This single-step protocol models the library prep required to produce normalized libraries that are ready for the NextSeq 500/550 Sequencing v1.1 workflow.
Label Group = TruSeq HT Adapters v2 (D7-D5)
Sequencing Instrument = NextSeq
On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the NextSeq 500/550 Sequencing v1.1 workflow, which is Library Pooling (NextSeq 500/550 v1.1) step.
In Lab View, locate the NextSeq 500/550 Sequencing v1.1 protocol. You will see your samples queued for the Library Pooling (NextSeq 500/550 v1.1) step.
Select the step to proceed to the Queue screen.
On the Queue screen, add samples 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.
On the Pool Samples screen, create a pool of samples as follows.
Drag samples into the Pool Creator.
Name the pool. You can also accept the default name Pool #1.
Select Place Samples.
On the Placement screen, move the pool to the container as follows.
Select the pool in the Samples to be Placed area on the left. Drag it over to the container on the right.
Select Record Details.
On the Record Details screen, select Next Steps.
On the Assign Next Steps screen, the next step is already set to Denature and Dilute (NextSeq 500/550 v1.1).
Select Finish Step.
In Lab View, locate the NextSeq 500/550 Sequencing v1.1 protocol. You will see your pooled samples queued for the Denature and Dilute (NextSeq 500/550 v1.1) step.
Select the step to proceed to the Queue screen.
On the Queue screen, add the pool to the Ice Bucket. Select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
On the Placement screen, move the pool to the reagent cartridge as follows.
Scan the NextSeq reagent cartridge barcode into the NextSeq Reagent Cartridge field.
Place the pool of samples into the reagent cartridge.
Select Record Details.
On the Record Details screen, create the sample sheet as follows.
Under Reagent Lot Tracking, select the reagent lot used in the step. You may need to add/activate the lot on the Reagents and Controls screen.
In the Step Details table, populate the fields as appropriate.
The Workflow and Read 1 Cycles are required fields.
In the Samplesheet Template drop-down list, the reverse complement template is selected by default. Do not change this value.
In the Sample Details table, enter the Final Loading Concentration. You may select preset 225 (for PCR-free workflows) or preset 400 (for Nano workflows). You can also enter a different value.
Select Generate bcl2fastq2 NextSeq 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 their format and content.
Select Next Steps.
On the Assign Next Steps screen, make sure samples are already assigned to the NextSeq 500/550 Run (NextSeq 500/550 v1.1) step.
Select Finish Step.
In Lab View, locate the NextSeq 500/550 Sequencing v1.1 protocol. You will see your pool of samples queued for the NextSeq 500/550 Run (NextSeq 500/550 v1.1) step.
Select the step to proceed to the Queue screen.
Add the pool to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
On the Record Details screen, the fields in the table are read-only. Once the run completes, the integration will automatically populate the fields.
For more detailed steps on adding instruments, refer to the Add and Configure Instruments section of the [Clarity LIMS (Clarity & LabLink Reference Guide) documentation](../../../clarity-lims/clarity-and-lablink.md).
Select Next Steps.
On the Assign Next Steps screen, make sure the next step is already set to Mark protocol as complete.
Select Finish Step.
Follow the steps below 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, and support for NextSeq Control Software (NCS) v4.0 has been configured.
This test validates that:
Your 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.
You can validate the gls_event_ncs_rta.bat batch file by executing it manually as follows.
Edit gls_event_ncs_rta.bat using any text editor (Notepad++ recommended).
By default, this file is installed to C:\Illumina\gls.
In the code line set DESTINATION_PATH=C:\Illumina\gls\Events, change C:\Illumina\gls\Events\ to the network path of the event files directory.
Remember to include the trailing "" at the end of the file path.
Save your changes.
Manually executing the batch file will create a dummy EndRun event file in the directory that you defined in the previous step.
If an automation trigger does not appear to run its corresponding scripts, refer to the following topics:
If an error occurs that does not provide direction on how to proceed, confirm the version of the installed Illumina NextSeq Integration Package by running the following 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 NextSeqIntegrator.log file. It is located at
Additional troubleshooting information for this integration is provided on the Illumina Instrument Integrations FAQ page.
If you are unable to resolve the issue, contact the Clarity LIMS Support team. Make sure you supply the relevant information from the troubleshooting that was already performed.
The Illumina NextSeq 500/550 Integration Package v2.4.0 supports the integration of Clarity LIMS to Illumina NextSeq 500 and 550 sequencing systems.
The integration allows for automated tracking of an Illumina sequencing run in Clarity LIMS, which includes tracking instrument 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 bcl2fastq2 v2.20.0 analysis software.
This document describes the integration between Clarity LIMS and the NextSeq system. It includes information about protocols and automations, configuration options, installed components, and rules and constraints.
For instructions on user interaction for each step, validating and troubleshooting the NextSeq 500/550 Integration Package, refer to .
It is assumed that samples enter the NextSeq 500/550 Sequencing v1.2 workflow as normalized libraries and have reagent labels attached.
That is, before they are assigned to the workflow:
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 Getting Started section of the .
You can assign samples to workflows automatically, using a routing script, or manually — from the Projects & Samples dashboard. Refer to Assign and Process Samples in the .
NextSeq Integration Package v2.4.0 includes the NextSeq 500/550 Sequencing v1.2 workflow, which contains a single protocol of the same name.
The NextSeq 500/550 Sequencing v1.2 protocol includes the following steps:
Library Pooling (NextSeq 500/550 v1.2)
Denature & Dilute (NextSeq 500/550 v1.2)
NextSeq 500/550 Run (NextSeq 500/550 v1.2)
The Library Pooling (NextSeq 500/550 v1.2) step is derived from the Library Pooling v1.0 master step. Libraries are placed into pools manually.
This field is configured on the Library Pooling (NextSeq 500/550 v1.2) step and displays on the Record Details screen at run time.
The following table shows field configuration details.
Library Pooling (NextSeq 500/550 v1.2) Master Step Field Configuration
The following table lists the global custom fields configured to display on the Queue and Ice Bucket screens of the Library Pooling (NextSeq 500/550 v1.2) step. Most of these fields show in the expanded view only.
Global Custom Field Configuration (Submitted Sample)
Global Custom Field Configuration (Derived Sample)
In this step, pooled libraries are denatured and diluted and placed into the reagent cartridge that is loaded into the NextSeq instrument.
There are 10 fields configured on the Denature and Dilute (NextSeq 500/550 v1.0) step. At run time, these fields display on the Record Details screen, in the Step Data table. The fields are manually populated. Their values are used to generate the sample sheet.
The following table lists field configuration details.
Denature and Dilute (NextSeq 500/550 v1.2) Master Step Field Configuration
Groups of Defaults
The following table lists the global fields that are configured to display on the Queue, Ice Bucket, and Record Details screens of the Denature and Dilute (NextSeq 500/550 v1.2) step.
Global Custom Field Configuration (Submitted Sample)
Global Custom Field Configuration (Derived Sample)
Placeholders for the following files are configured on the Record Details screen of the Denature and Dilute (NextSeq 500/550 v1.2) step
In this step, pooled samples are sequenced on the NextSeq 500/550 instrument and the run metrics are recorded in Clarity LIMS.
There are 16 fields configured on the NextSeq 500/550 Run (NextSeq 500/550 v1.2) step. These fields display on the Record Details screen at run time. Some of the field values must be completed manually, and the remaining fields are automatically populated at the end of the run.
The following table lists field configuration details.
NextSeq 500/550 Run (NextSeq 500/550 v1.2) Master Step Field Configuration
There are several sample and measurement global fields configured to display on the Record Details screen of the NextSeq 500/550 Run (NextSeq 500/550 v1.2) step. These fields are populated at the end of the sequencing run.
Placeholders for the following files are configured on the Record Details screen of the NextSeq 500/550 Run (NextSeq 500/550 v1.2) 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 on the step before the sequencing run Denature and Dilute (NextSeq 500/550 v1.2) step. Samples are placed on the container to be loaded in the instrument. The default configuration provides the Generate bcl2fastq2 NextSeq Samplesheet automation.
The Generate bcl2fastq2 NextSeq Samplesheet automation uses the Template File Generator (DriverFileGenerator.jar) and a template file to generate a CSV format file for use with bcl2fastq2 v2.20.0 analysis software. The sample sheet content is determined by the fields that display on the Record Details screen of the step (in the Step Data table) and the values entered into the fields. Templates can be customized to create the sample sheet. If additional columns are required by the lab, then they can be inserted.
The NextSeq 500/550 Run (NextSeq 500/550 v1.2) step records information for the flow cell lanes 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 lists the run information files, reports, placeholders, and links that Clarity LIMS automatically generates or capture during a sequencing run.
Run Information Generated or Captured by NextSeq 500/550 Run (NextSeq 500/550 v1.2) Step
The following list includes the 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
Flow Cell ID
Index 1 Read Cycles — Configured Index 1 length
Index 2 Read Cycles — Configured Index 2 length
Output Folder — Run folder root
PR2 Bottle ID
Reagent Cartridge ID
Read 1 Cycles - Configured Read 1 cycle
Read 2 Cycles - Configured Read 2 cycle
Run ID — The unique run ID
Run Type
Status — Displays the completed vs configured aggregated (i.e., Read 1 and Read 2) read cycles. Example: Cycle 10 of 100.
Workflow
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.
The following table lists the Real-Time Analysis v2 (RTA2) primary analysis metrics that Clarity LIMS automatically captures and records, per read, for samples in each flow cell lane. These metrics are captured after run completion and are stored as global custom fields in the Record Details screen Sample Details table. Per read and per lane metrics are viewable by expanding the output.
RTA Primary Analysis Metrics Captured by NextSeq 500/550 Run (NextSeq 500/550 v1.0) Step
The sequencing service runs on the Clarity LIMS server. The service detects event files that the instrument software (RTA2) produces as the run progresses, which tells the service where to find the run data. As the run data is written out and the End Run event is detected, the data is matched to the step. This matching is based on the reagent cartridge ID that was entered/scanned in the Denature and Dilute (NextSeq 500/550 v1.2) step. Read-only field values on the Record Details screen are populated accordingly. When 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.
The following table lists the scripts and files installed in the Illumina NextSeq 500/550 Integration Package v2.4.0 RPM.
Illumina NextSeq 500/550 Integration Package v2.4.0 Scripts and Files Installed
Reagent categories/label groups are installed with the IPP workflow configuration slices.
The NextSeq Reagent Kit is included in the NextSeq Integration.
The PhiX v3 control type is included in the NextSeq Integration.
The NextSeq Reagent Cartridge container type is included in the NextSeq Integration.
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 instrument while it is idle.
To configure the Illumina instrument for use with the Illumina NextSeq Integration, the Support team:
Creates a directory on the local computer to hold the batch files. These batch files write event files to the network attached storage (NAS) shares.
Creates a directory on the NAS to hold the event files.
Modifies Illumina software configuration files to call the batch files that create the event files.
Updates sequencing service default properties to match the specifics of the installation.
This integration operates with the following constraints:
The reagent cartridge ID must be unique. Avoid multiple reagent cartridge containers in the system with identical names.
The reagent cartridge ID must be scanned as the reagent cartridge Container Name on the Denature and Dilute (NextSeq 500/550 v1.2) step.
The Illumina NextSeq 500/550 Integration v2.3.0 includes the following features and functionality:
Added support for Oracle Linux. For compatibility, refer to .
A new NextSeq 500/550 v1.1 workflow that maps to lab protocols and instrument runs. This workflow includes a minor bug fix and has no additional changes from the previous version.
Automated generation of the sample sheet. This sample sheet is used with the bcl2fastq2 v2.20.0 analysis software.
Automated tracking of the NextSeq sequencing runs and parsing of run statistics into Clarity LIMS, which includes the following information:
Sequencing run metrics
Sequencing run parameters
Real-Time Analysis v2 (RTA2) run directory location and other run-specific information
Preconfigured Library Prep Validation v2.3.1 workflow used for validation purposes. This workflow contains a single-step protocol that models the library prep required to produce normalized libraries that are ready for the NextSeq 500/550 Sequencing v1.1 workflow. For more information, refer to .
The Illumina NextSeq 500/550 Integration Package v2.3.0 supports the integration of Clarity LIMS to Illumina NextSeq 500 and 550 sequencing systems.
The integration allows for automated tracking of an Illumina sequencing run in Clarity LIMS, which includes tracking instrument 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 bcl2fastq2 v2.20.0 analysis software.
This document describes the integration between Clarity LIMS and the NextSeq system. It includes information about protocols and automations, configuration options, installed components, and rules and constraints.
For instructions on user interaction for each step, validating and troubleshooting the NextSeq 500/550 Integration Package, refer to .
It is assumed that samples enter the NextSeq 500/550 Sequencing v1.1 workflow as normalized libraries and have reagent labels attached.
That is, before they are assigned to the workflow:
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 Getting Started section of the .
You can assign samples to workflows automatically, using a routing script, or manually — from the Projects & Samples dashboard. Refer to Assign and Process Samples in the .
NextSeq Integration Package v2.3.0 includes the NextSeq 500/550 Sequencing v1.1 workflow, which contains a single protocol of the same name.
The NextSeq 500/550 Sequencing v1.1 protocol includes the following steps:
Library Pooling (NextSeq 500/550 v1.1)
Denature & Dilute (NextSeq 500/550 v1.1)
NextSeq 500/550 Run (NextSeq 500/550 v1.1)
The Library Pooling (NextSeq 500/550 v1.1) step is derived from the Library Pooling v1.0 master step. Libraries are placed into pools manually.
This field is configured on the Library Pooling (NextSeq 500/550 v1.1) step and displays on the Record Details screen at run time.
The following table shows field configuration details.
Library Pooling (NextSeq 500/550 v1.1) Master Step Field Configuration
The following table lists the global custom fields configured to display on the Queue and Ice Bucket screens of the Library Pooling (NextSeq 500/550 v1.1) step. Most of these fields show in the expanded view only.
Global Custom Field Configuration (Submitted Sample)
Global Custom Field Configuration (Derived Sample)
In this step, pooled libraries are denatured and diluted and placed into the reagent cartridge that is loaded into the NextSeq instrument.
There are 10 fields configured on the Denature and Dilute (NextSeq 500/550 v1.0) step. At run time, these fields display on the Record Details screen, in the Step Data table. The fields are manually populated. Their values are used to generate the sample sheet.
The following table lists field configuration details.
Denature and Dilute (NextSeq 500/550 v1.1) Master Step Field Configuration
Groups of Defaults
The following table lists the global fields that are configured to display on the Queue, Ice Bucket, and Record Details screens of the Denature and Dilute (NextSeq 500/550 v1.1) step.
Global Custom Field Configuration (Submitted Sample)
Global Custom Field Configuration (Derived Sample)
Placeholders for the following files are configured on the Record Details screen of the Denature and Dilute (NextSeq 500/550 v1.1) step
In this step, pooled samples are sequenced on the NextSeq 500/550 instrument and the run metrics are recorded in Clarity LIMS.
There are 16 fields configured on the NextSeq 500/550 Run (NextSeq 500/550 v1.1) step. These fields display on the Record Details screen at run time. Some of the field values must be completed manually, and the remaining fields are automatically populated at the end of the run.
The following table lists field configuration details.
NextSeq 500/550 Run (NextSeq 500/550 v1.1) Master Step Field Configuration
There are several sample and measurement global fields configured to display on the Record Details screen of the NextSeq 500/550 Run (NextSeq 500/550 v1.1) step. These fields are populated at the end of the sequencing run.
Placeholders for the following files are configured on the Record Details screen of the NextSeq 500/550 Run (NextSeq 500/550 v1.1) 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 on the step before the sequencing run Denature and Dilute (NextSeq 500/550 v1.1) step. Samples are placed on the container to be loaded in the instrument. The default configuration provides the Generate bcl2fastq2 NextSeq Samplesheet automation.
The Generate bcl2fastq2 NextSeq Samplesheet automation uses the Template File Generator (DriverFileGenerator.jar) and a template file to generate a CSV format file for use with bcl2fastq2 v2.20.0 analysis software. The sample sheet content is determined by the fields that display on the Record Details screen of the step (in the Step Data table) and the values entered into the fields. Templates can be customized to create the sample sheet. If additional columns are required by the lab, then they can be inserted.
The NextSeq 500/550 Run (NextSeq 500/550 v1.1) step records information for the flow cell lanes 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 lists the run information files, reports, placeholders, and links that Clarity LIMS automatically generates or capture during a sequencing run.
Run Information Generated or Captured by NextSeq 500/550 Run (NextSeq 500/550 v1.1) Step
The following list includes the 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
Flow Cell ID
Index 1 Read Cycles — Configured Index 1 length
Index 2 Read Cycles — Configured Index 2 length
Output Folder — Run folder root
PR2 Bottle ID
Reagent Cartridge ID
Read 1 Cycles - Configured Read 1 cycle
Read 2 Cycles - Configured Read 2 cycle
Run ID — The unique run ID
Run Type
Status — Displays the completed vs configured aggregated (i.e., Read 1 and Read 2) read cycles. Example: Cycle 10 of 100.
Workflow
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.
The following table lists the Real-Time Analysis v2 (RTA2) primary analysis metrics that Clarity LIMS automatically captures and records, per read, for samples in each flow cell lane. These metrics are captured after run completion and are stored as global custom fields in the Record Details screen Sample Details table. Per read and per lane metrics are viewable by expanding the output.
RTA Primary Analysis Metrics Captured by NextSeq 500/550 Run (NextSeq 500/550 v1.0) Step
The sequencing service runs on the Clarity LIMS server. The service detects event files that the instrument software (RTA2) produces as the run progresses, which tells the service where to find the run data. As the run data is written out and the End Run event is detected, the data is matched to the step. This matching is based on the reagent cartridge ID that was entered/scanned in the Denature and Dilute (NextSeq 500/550 v1.1) step. Read-only field values on the Record Details screen are populated accordingly. When 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.
The following table lists the scripts and files installed in the Illumina NextSeq 500/550 Integration Package v2.3.0 RPM.
Illumina NextSeq 500/550 Integration Package v2.3.0 Scripts and Files Installed
The following table lists the properties installed with Illumina NextSeq 500/550 Integration Package v2.3.0.
Additional properties, each with the 99 suffix appended to their name, are also installed and intended for use by the Clarity LIMS Support team in automated validation tests. Those properties are not listed in the table.
Properties Installed with the Illumina NextSeq 500/550 Integration Package
¹ It is possible to configure support for multiple, identical seqservice.netPathPrefixSearch property values.
Reagent categories/label groups are installed with the IPP workflow configuration slices.
The NextSeq Reagent Kit is included in the NextSeq Integration.
The PhiX v3 control type is included in the NextSeq Integration.
The NextSeq Reagent Cartridge container type is included in the NextSeq Integration.
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 instrument while it is idle.
To configure the Illumina instrument for use with the Illumina NextSeq Integration, the Support team:
Creates a directory on the local computer to hold the batch files. These batch files write event files to the network attached storage (NAS) shares.
Creates a directory on the NAS to hold the event files.
Modifies Illumina software configuration files to call the batch files that create the event files.
Updates sequencing service default properties to match the specifics of the installation.
This integration operates with the following constraints:
The reagent cartridge ID must be unique. Avoid multiple reagent cartridge containers in the system with identical names.
The reagent cartridge ID must be scanned as the reagent cartridge Container Name on the Denature and Dilute (NextSeq 500/550 v1.1) step.
Last Updated: November 2024
Release Date: July 2024
Document Version: 2
The Illumina NextSeq 500/550 Integration v2.4.0 supports the integration between Clarity LIMS and the NextSeq 500/550 instrument. This document provides instructions for installing NextSeq 500/550 Integration v2.4.0. It also describes the components that are installed in the default configuration.
The integration is compatible with the following software:
Clarity LIMS v6.2.0 and later
Secret Util v1.0 and later (v1.2 is recommended)
IPP v2.6 and later (up to and including IPP v2.9)
This integration is not fully compatible with NextSeq 500/550 Integration v1.
For details on installed protocols and steps, automations, generated and captured files, and rules and constraints, refer to .
For information on user interaction for each step, validating and troubleshooting the integration, refer to .
NextSeq 500/550 Integration v2.4.0 has the following prerequisites:
Mount run data network-attached storage (NAS) share
Secret Util is installed
IPP is installed
NextSeq 500/550 Integration v2.4.0 is distributed as the ClarityLIMS-Illumina-NextSeq-Package-v2 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 nextseq-sequencing.jar file
The configure_extensions_nextseq_sequencingservice.sh script
Smoke test directories
When upgrading from an existing NextSeq 500/550 workflow, the following warning messages can display when the illumina-preset-protocls-installer.sh script is running:
These messages are due to configuration changes that have increased the precision of the listed fields from 1 to 2 so that they show more decimal places. It is safe to override these warnings.
The following installation steps are required for the installation of NextSeq 500/550 Integration v2.4.0.
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 NextSeq 500/550 workflow from the IPP RPM.
For hosted installations, the Secret Util installation and configuration is handled by tooling.
Install the NextSeq 500/550 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 NextSeq 500/550 Sequencing v1.2 workflow:
[Optional] Run the following command to install the Library Prep Validation v2.3.4 workflow that is used to validate the NextSeq 500/550 Sequencing v1.2 workflow:
Run the following script to configure the service properties:
Run the following command to start the sequencing service:
In NextSeq Control Software (NCS) v4.0 the location of NextSeq.Configuration.xml RTA configuration file is changed. The new location is
A new gls_events_ncs_rta.bat batch file is required to generate a valid EndRun event file. This file is available in the NextSeq 500/550 RPM at
Configuration
Download the latest batch file from the NextSeq 500/550 RPM and place it in a folder (e.g., C:\Illumina\gls). If necessary, remove the old batch file.
Edit the NextSeq.Configuration.xml configuration file at
If you cannot modify the file, then open it as an administrator. Insert the following text inside the <Processing> tag. If the batch file is not at C:\Illumina\gls, then change that text to the applicable path.
Save the configuration file.
Validate the file by running RTA manually. In the command prompt, run the following command:
The following error message displays:
Validate the batch file by executing it manually as follows.
Using a text editor, edit the gls_events_ncs_rta.bat file.
In the "set DESTINATION_PATH=C:\Illumina\gls\Events" code line, change C:\Illumina\gls\Events\ to the network path of the event files directory. Make sure to include the backslash at the end of the file path.
After manually executing the batch file, a dummy EndRun event file is created in the event files directory.
NextSeq 500/550 Integration v2.4.0 works with the NextSeq 500/550 Sequencing v1.2 workflow, which contains a single protocol with the same name. This protocol includes the following steps:
Library Pooling (NextSeq 500/550 v1.2)
Denature & Dilute (NextSeq 500/550 v1.2)
NextSeq 500/550 Run (NextSeq 500/550 v1.2)
The NextSeq Control Software (NCS) is divided into the following modules:
NCS — Controls the instrument operation, including various configuration settings. This software is installed and runs on the instrument.
Real-Time Analysis 2 (RTA2) — 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.
Illumina Support 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: September 2023
Document Version: 2
These release notes describe the key changes to software components for the Clarity LIMS NextSeq 500/550 Integration Package v2.3.0.
Refer to under Instruments & Integrations.
Updates Java and third-party dependency libraries.
Updates Groovy to v3.0.7.
Fixed SQL Injection (CVE-2022-31197) and Information Disclosure (CVE-2020-15522) vulnerabilities.
The Run Type custom field is not updated under Step Details in the NextSeq 500/550 Run step.
Missing contents in the Instrument Details section of the run report.
When the sequencing run is finished, the integration generates a run report file. The Instrument Details section is left blank by default, unless the NextSeq 500/500 Run master step is configured to track instruments created in Clarity LIMS. For more information, refer to Add and Configure Instruments from the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
Do not continue to step 5 and complete the step until the Illumina Run Report has been attached.
If the NextSeq 500/550 Run master step is configured to track instruments, select the appropriate instrument from the Instrument Used drop-down on Record Details screen, then select Save.
Follow the steps in to run the Library Prep Validation workflow with the following:
When the sequencing run is finished, the integration generates a run report file. The Instrument Details section is left blank by default, unless the NextSeq 500/500 Run master step is configured to track instruments created in Clarity LIMS. For more information, refer to Add and Configure Instruments from the .
The integration also attaches files to the Illumina Run Report, Link to Run Folder, Run Parameters, and Run Info placeholders. The Log File is attached once the next step for samples has been assigned by the Next Step - Advance automation. The integration also populates the fields in the Sample Details table. For details, refer to .
Do not continue to step 5 and complete the step until the Illumina Run Report has been attached.
If the NextSeq 500/550 Run master step is configured to track instruments, select the appropriate instrument from the Instrument Used drop-down on Record Details screen, then select Save.
Troubleshooting Automation Worker in the Administration section of the .
Troubleshooting Automation in the Automation section of the .
For more information, see the section below.
This integration requires installation of the Illumina Preset Protocols (IPP). For details, refer to .
Refer to for the properties installed with Illumina NextSeq 500/550 Integration Package v2.4.0.
For more information, see the section below.
This integration requires installation of the Illumina Preset Protocols (IPP). For details, refer to .
Sequencing runs are matched using the reagent cartridge ID and the sequencing steps base name. The base name is NextSeq 500/550 Run (NextSeq 500/550 v1.1). Do not change the base name. The name is expected by the sequencing service that captures instrument run results. The base name is stored in the sequenceProcessBaseName property. If this name is changed without the property being updated, the 'reagent cartridge ID <-> sequencing step base name' matching system fails. If necessary, modify the step name by editing or adding text after the base name portion, as it is not used in the matching system. For example, change NextSeq 500/550 Run (NextSeq 500/550 v1.1) to NextSeq 500/550 Run (NextSeq 500/550 v1.1) v2.
Changes on nextseq.v2.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.
For more information on installing Secret Util with the Integration Module, refer to Install/Upgrade Secret Management for Integration Modules in the .
For the on-premise installation, the Illumina Vault server is not available for public access. In the configuration, make sure the system passwords are configured correctly in File mode.
For a custom API username, set the {key} to apiusers/{custom API username} (e.g., apiusers/novaseq_user). For more information on Secret Utility configuration, refer to the .
For more information on Secret Utility configuration, refer to the .
The NextSeq RPM must be installed on the Clarity LIMS server. This is 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.
If you have not installed the SecretUtil RPM before, this RPM installation also installs it.
For on-premise installations, if the Secret Util package is not already installed or configured, you must configure the Secret Util first. For more information, refer to Prerequisite 2: Secret Util Installation in .
The integration properties can be configured to enable capture and generation of files associated with the sequencing run. Refer to for details.
The property configuration for NextSeq 500/550 v2 has the suffix .v2 and is different from the setting for v1.
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 NCS, refer to the NextSeq 500/550 documentation at .
For NextSeq Control Software (NCS) v4.0 and later, refer to the configuration steps in . For NCS versions before v4.0, perform the following steps.
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 the trailing \ in the DESTINATION_PATH line. Refer to the following example:
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 .
Version
Changes
2
Updated Compatibility section to reference Compatibility matrix table.
1
Initial release.
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 Dropdown 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 Dropdown Items |
Normalized Molarity (nM) | Numeric | Decimal places displayed = 2 |
Field Name | Field Type | Field Constraints/Options | Preset Values/Additional Options and Drop-Down Items |
Adapter | Text |
Adapter Read 2 | Text |
Experiment Name | Text |
Mask Adapter | Text |
Mask Adapter Read 2 | Text |
Read 1 Cycles | Numeric Dropdown |
|
|
Read 2 Cycles | Numeric Dropdown |
|
|
SampleSheet Template | Text Dropdown |
|
Workflow | Text Dropdown |
|
|
Field Name | Field Type | Field Constraints/Options | Preset Value/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 |
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 |
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 Clarity LIMS Field Name (stored on derived sample/analyte input to the step) | Per Lane Clarity LIMS Field Name (stored in measurement placeholders in Record Details screens Sample Details table) |
% 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 |
Files Installed | Location | Description |
configure_extensions_nextseq_sequencingservice.sh | /opt/gls/clarity/config/ | Script that installs the service properties in the database. |
log4j2.xml | /opt/gls/clarity/extensions/Illumina_NextSeq/v2/SequencingService/conf | File containing the settings for the sequencing jar logging. |
nextseq-sequencing.jar | /opt/gls/clarity/extensions/Illumina_NextSeq/v2/SequencingService | Jar file containing API-based Clarity LIMS extensions used for capturing run results and report generation. |
InterOp libraries | opt/gls/clarity/extensions/Illumina_NextSeq/v2/lib | Shared library for parsing InterOp data files. |
Installed from IPP
| /opt/gls/clarity/extensions/conf/driverfiletemplates | Template file used for file generation. In this integration, the reverse complement template is used by default. |
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 Dropdown 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 Dropdown Items |
Normalized Molarity (nM) | Numeric | Decimal places displayed = 2 |
Field Name | Field Type | Field Constraints/Options | Preset Values/Additional Options and Drop-Down Items |
Adapter | Text |
Adapter Read 2 | Text |
Experiment Name | Text |
Mask Adapter | Text |
Mask Adapter Read 2 | Text |
Read 1 Cycles | Numeric Dropdown |
|
|
Read 2 Cycles | Numeric Dropdown |
|
|
SampleSheet Template | Text Dropdown |
|
Workflow | Text Dropdown |
|
|
Field Name | Field Type | Field Constraints/Options | Preset Value/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 |
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 |
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 Clarity LIMS Field Name (stored on derived sample/analyte input to the step) | Per Lane Clarity LIMS Field Name (stored in measurement placeholders in Record Details screens Sample Details table) |
% 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 |
Files Installed | Location | Description |
configure_extensions_nextseq_sequencingservice.sh | /opt/gls/clarity/config/ | Script that installs the service properties in the database. |
log4j2.xml | /opt/gls/clarity/extensions/Illumina_NextSeq/v2/SequencingService/conf | File containing the settings for the sequencing jar logging. |
nextseq-sequencing.jar | /opt/gls/clarity/extensions/Illumina_NextSeq/v2/SequencingService | Jar file containing API-based Clarity LIMS extensions used for capturing run results and report generation. |
InterOp libraries | opt/gls/clarity/extensions/Illumina_NextSeq/v2/lib | Shared library for parsing InterOp data files. |
Installed from IPP
| /opt/gls/clarity/extensions/conf/driverfiletemplates | Template file used for file generation. In this integration, the reverse complement template is used by default. |
Version | Changes |
2 |
|
1 |
|
Version | Changes |
2 |
|
1 |
|
The Illumina NextSeq 500/550 Integration v2.4.0 includes the following features and functionality:
Added support for Oracle Linux. For compatibility, refer to NextSeq 500/550 Integration v2.4.0 Release Notes.
A new NextSeq 500/550 v1.2 workflow that maps to lab protocols and instrument runs. This workflow includes a minor bug fix and has no additional changes from the previous version.
Automated generation of the sample sheet. This sample sheet is used with the bcl2fastq2 v2.20.0 analysis software.
Automated tracking of the NextSeq sequencing runs and parsing of run statistics into Clarity LIMS, which includes the following information:
Sequencing run metrics
Sequencing run parameters
Real-Time Analysis v2 (RTA2) run directory location and other run-specific information
Preconfigured Library Prep Validation v2.3.3 workflow used for validation purposes. This workflow contains a single-step protocol that models the library prep required to produce normalized libraries that are ready for the NextSeq 500/550 Sequencing v1.2 workflow. For more information, refer to NextSeq 500/550 Integration v2.4.0 User Interaction, Validation and Troubleshooting.
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 |
|
|
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 |
|
|
Property | Description | Default Value |
nextseq.v2.seqservice.sequenceProcessBaseName | Sequencing process type / master step base display name. Partial matching is used to look up the process type / master step. | NextSeq 500/550 Run (NextSeq 500/550 v1.1) |
nextseq.v2.seqservice.eventFileDirectory.1 | A network location monitored for event files (e.g., /mnt/illumina/gls_events/) | /mnt/gls_events |
nextseq.v2.seqservice.netPathPrefixSearch.1¹ | The network directory prefix contained in the event file - most likely in Windows format. | \\nas\network\run_data |
nextseq.v2.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 |
nextseq.v2.seqservice.eventFileDirectorySuffixes | A list of eventFileDirectory path entries to monitor for event files. The value is one or more comma-separated integers. | 99 |
nextseq.v2.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 |
nextseq.v2.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 |
nextseq.v2.seqservice.ignoreUnmatchedContainerIds | A flag indicating if event files that cannot be matched to reagent cartridges in Clarity LIMS should be archived after a certain time (true), or continually reprocessed (false). | false |
nextseq.v2.seqservice.ignoreUnmatchedContainerIdsWaitDays | The number of days between when the event is created and the event file is archived. | 14 |
nextseq.v2.seqservice.synchronizationPeriod | Invocation period in seconds. | 60 |
The Illumina NextSeq 500/550 Integration v2.3.0 supports the integration between Clarity LIMS and the NextSeq 500/550 instrument. This document provides instructions for installing NextSeq 500/550 Integration v2.3.0. It also describes the components that are installed in the default configuration.
The integration is compatible with the following software:
Clarity LIMS v6.2.0 and later
Secret Util v1.0 and later (v1.2 is recommended)
IPP v2.6 and later
This integration is not fully compatible with NextSeq 500/550 Integration v1.
For details on installed protocols and steps, automations, generated and captured files, and rules and constraints, refer to NextSeq 500/550 v2.3.0 Configuration.
For information on user interaction for each step, validating and troubleshooting the integration, refer to NextSeq 500/550 Integration v2.3.0 User Interaction, Validation and Troubleshooting.
NextSeq 500/550 Integration v2.3.0 has the following prerequisites:
Mount run data network-attached storage (NAS) share
Secret Util is installed
IPP is installed
NextSeq 500/550 Integration v2.3.0 is distributed as the ClarityLIMS-Illumina-NextSeq-Package-v2 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 nextseq-sequencing.jar file
The configure_extensions_nextseq_sequencingservice.sh script
Smoke test directories
When upgrading from an existing NextSeq 500/550 workflow, the following warning messages can display when the illumina-preset-protocls-installer.sh script is running:
These messages are due to configuration changes that have increased the precision of the listed fields from 1 to 2 so that they show more decimal places. It is safe to override these warnings.
The following installation steps are required for the installation of NextSeq 500/550 Integration v2.3.0.
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 NextSeq 500/550 workflow from the IPP RPM.
For hosted installations, the Secret Util installation and configuration is handled by tooling.
For on-premise installations, if the Secret Util package is not already installed or configured, you must configure the Secret Util first. For more information, refer to Prerequisite 2: Secret Util Installation in Prerequisites.
Install the NextSeq 500/550 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 NextSeq 500/550 Sequencing v1.1 workflow:
[Optional] Run the following command to install the Library Prep Validation v2.3.1 workflow that is used to validate the NextSeq 500/550 Sequencing v1.1 workflow:
Run the following script to configure the service properties:
To enable capture and generation of files associated with the sequencing run, configure the following properties using the omxProps-ConfigTool utility. The property configuration for NextSeq 500/550 v2 has the suffix .v2 and is different from the setting for v1.
Configurable Database Properties
Run the following command to start the sequencing service:
In NextSeq Control Software (NCS) v4.0 the location of NextSeq.Configuration.xml RTA configuration file is changed. The new location is
A new gls_events_ncs_rta.bat batch file is required to generate a valid EndRun event file. This file is available in the NextSeq 500/550 RPM at
Configuration
Download the latest batch file from the NextSeq 500/550 RPM and place it in a folder (e.g., C:\Illumina\gls). If necessary, remove the old batch file.
Edit the NextSeq.Configuration.xml configuration file at
If you cannot modify the file, then open it as an administrator. Insert the following text inside the <Processing> tag. If the batch file is not at C:\Illumina\gls, then change that text to the applicable path.
Save the configuration file.
Validate the file by running RTA manually. In the command prompt, run the following command:
The following error message displays:
Validate the batch file by executing it manually as follows.
Using a text editor, edit the gls_events_ncs_rta.bat file.
In the "set DESTINATION_PATH=C:\Illumina\gls\Events" code line, change C:\Illumina\gls\Events\ to the network path of the event files directory. Make sure to include the backslash at the end of the file path.
After manually executing the batch file, a dummy EndRun event file is created in the event files directory.
NextSeq 500/550 Integration v2.3.0 works with the NextSeq 500/550 Sequencing v1.1 workflow, which contains a single protocol with the same name. This protocol includes the following steps:
Library Pooling (NextSeq 500/550 v1.1)
Denature & Dilute (NextSeq 500/550 v1.1)
NextSeq 500/550 Run (NextSeq 500/550 v1.1)
For descriptions of the protocol and the steps, refer to NextSeq 500/550 v2.3.0 Configuration. For instructions on using the Library Prep Validation v2.3.1 workflow to validate the automated sample sheet generation, refer to NextSeq 500/550 Integration v2.3.0 User Interaction, Validation and Troubleshooting.
The NextSeq Control Software (NCS) is divided into the following modules:
NCS — Controls the instrument operation, including various configuration settings. This software is installed and runs on the instrument.
Real-Time Analysis 2 (RTA2) — 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 NCS, refer to the NextSeq 500/550 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.
Illumina Support 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.
For NextSeq Control Software (NCS) v4.0 and later, refer to the configuration steps in NextSeq Control Software v4.0 Configuration. For NCS versions before v4.0, perform the following steps.
Displays on Queue & Ice Bucket screens
Displays on Queue & Ice Bucket screens
Displays on Record Details screen
Displays on Queue & Ice Bucket screens
Displays on Queue & Ice Bucket screens
Displays on Record Details screen
Installed from the 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.
For the on-premise installation, the Illumina Vault server is not available for public access. In the configuration, make sure the system passwords are configured correctly in File mode.
The NextSeq RPM must be installed on the Clarity LIMS server. This is 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.
If you have not installed the SecretUtil RPM before, this RPM installation also installs it.
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 the trailing \ in the DESTINATION_PATH line. Refer to the following example:
Property
Description
Default Value
nextseq.v2.seqservice.eventFileDirectory.1
A monitored network location used for event files.
/mnt/gls_events
nextseq.v2.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/nextseq/monitored
nextseq.v2.seqservice.netPathPrefixSearch.1
The network directory prefix contained in the event file.
\\nas\network\run_data
nextseq.v2.seqservice.netPathPrefixSearch.99
The default benign smoke test search entry. This property is used for the installation and upgrade of smoke tests.
/
nextseq.v2.seqservice.netPathPrefixReplace.1
The mapped network directory mount name on the server used to access the run data directory.
/mnt/run_data
nextseq.v2.seqservice.netPathPrefixReplace.99
The default benign smoke test replace entry. This property is used for the installation and upgrade of smoke tests.
/
nextseq.v2.seqservice.eventFileDirectorySuffixes
A list of eventFileDirectory path entries used to monitor for event files. The value is one or more comma separated integers.
99
nextseq.v2.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
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.
