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NextSeq 500/550

The documents in this section support the Illumina NextSeq 500/550 Integration.

NextSeq 500/550 v2.5.0

The Illumina NextSeq 500/550 Integration v2.5.0 includes the following features and functionality:

New workflow that maps to lab protocols and instrument runs. In NextSeq 500/550 Sequencing v1.3, the forward-orientation samplesheet generated in Dilute and Denature step is now compatible with NextSeq 500/550 Control Software Version 4.2 and Local Run Manager v4.0 for the following modules:
- Assembly Analysis
- DNA Enrichment Analysis
- GenerateFASTQ Analysis
- Library QC Analysis
- 16S Metagenomics Analysis
- PCR Amplicon
- Resequencing Analysis
- DNA Amplicon Analysis
- RNA Fusion Analysis
- Small RNA Analysis
Automated generation of the sample sheet. The reverse-complement 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 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 workflow. The versions of the Library Prep Validation workflow NextSeq 500/550 Sequencing workflow depends on the version of IPP installed (see table above). For more information, refer to NextSeq 500/550 Integration v2.5.0 User Interaction, Validation and Troubleshooting.

Release Notes

Last Updated: December 2024

Release Date: December 2024

These release notes describe the key changes to software components for the Clarity LIMS NextSeq 500/550 Integration Package v2.5.0.

Compatibility

Refer to Compatibility under Instruments & Integrations.

New Features & Updates

The NextSeq 500/550 v1.3 protocol now also generates forward orientation samplesheet that is compatible with NextSeq 500/550 Control Software Version 4.2 and Local Run Manager v4.0 for the following modules:
- Assembly Analysis
- DNA Enrichment Analysis
- GenerateFASTQ Analysis
- Library QC Analysis
- 16S Metagenomics Analysis
- PCR Amplicon
- Resequencing Analysis
- DNA Amplicon Analysis
- RNA Fusion Analysis
- Small RNA Analysis.

Note that sample sheet generated by the updated workflow is incompatible with NextSeq 500/550 Control Software version prior to v4.2 and Local Run Manager version prior to v4.0.

Reverse-complement sample sheet generated by NextSeq 500/550 v1.3 protocol is compatible with bcl2fastq2 v2.20.0 analysis software.

Defect Repairs

Removed unused fields Reagent Cartridge Part # and Flow Cell Version from run report.
In the run report, Genus LIMS Run ID is replaced with Clarity LIMS Run ID.

Installation

The Illumina NextSeq 500/550 Integration v2.5.0 supports the integration between Clarity LIMS and the NextSeq 500/550 instrument. This document provides instructions for installing NextSeq 500/550 Integration v2.5.0. It also describes the components that are installed in the default configuration.

Compatibility

Refer to Compatibility under Instruments & Integrations.

This integration is not fully compatible with NextSeq 500/550 Integration v1.

Supporting Documentation

For details on installed protocols and steps, automations, generated and captured files, and rules and constraints, refer to NextSeq 500/550 v2.5.0 Configuration.
For information on user interaction for each step, validating and troubleshooting the integration, refer to NextSeq 500/550 Integration v2.5.0 User Interaction, Validation and Troubleshooting.

Prerequisites

This integration has the following prerequisites:

Mount run data network-attached storage (NAS) share
Secret Util is installed
IPP is installed

Prerequisite 1: Mount Run Data NAS Share

Mounting the NAS share of run data is needed to capture and generate files associated with the sequencing run. To mount NAS shares that contain data from the Clarity LIMS server, use Read/Write priviledges as the glsjboss user. The following data can be mounted to the NAS share:

Run data (e.g., \\network-storage\run_data)
Clarity LIMS-created events triggered by the End Run event of the Illumina sequencing run (e.g., \\network-storage\illumina\gls_events)

With Read access, the Clarity LIMS server reads the following information in individual sequencing run data folders:

Run information metadata from these files

<runFolderRoot>/RunInfo.xml

<runFolderRoot>/runParameters.xml

Run statistics from
```
<runFolderRoot>/InterOp/*.bin
```

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

Sample sheet (CSV file)
Run report (PDF file)
Run folder root link

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

<runFolderRoot>/RunInfo.xml

<runFolderRoot>/runParameters.xml

Prerequisite 2: Secret Util Installation

If Clarity LIMS Secret Utility is not installed or configured, use the following information to configure Secret Utility:

Installation on a hosted system with Clarity LIMS (v6.2 or later)

The Clarity LIMS installation tooling configures this installation. No additional configuration is necessary.

Installation on an on-premise Clarity LIMS instance (v6.2 or later)

Install and configure Secret Util as follows.

Install the ClarityLIMS-SecretUtil RPM.
If Secret Util is not installed or configured, you must configure it before proceeding. As the glsjboss user, run the following script:
```
bash /opt/gls/clarity/config/pending/05_configure_claritylims_secretutil.sh
```
For more information on installing Secret Util with the Integration Module, refer to Install/Upgrade Secret Management for Integration Modules in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
ℹ 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.
If a custom API username is required, configure the username and password using Secret Util:
```
java -jar /opt/gls/clarity/tools/secretutil/secretutil.jar -n=INTEGRATION -u={password} {key}
```
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 Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
Use the following command to make sure that the password is saved correctly in Secret Util:
```
java -jar /opt/gls/clarity/tools/secretutil/secretutil.jar -n=INTEGRATION {key}
```

For more information on Secret Utility configuration, refer to the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

Prerequisite 3: IPP Installation

The NextSeq 500/550 Integration depends on the configuration provided in IPP. Refer to Compatibility under Instruments & Integrations for compatible versions of IPP and Clarity LIMS.

If the base configuration is not installed, then install it on the Clarity LIMS server that is being used for the NextSeq integration. For details on IPP installation and configuration, refer to the Illumina Preset Protocols documentation.

If you are upgrading the base configuration, make sure that the IPP package is compatible with the version of Clarity LIMS you are installing.

Installation

This integration 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:

WARN Installer - Conflicting content:
WARN Installer - Analyte UDFs: % Aligned R1
WARN Installer - Analyte UDFs: % Aligned R2
WARN Installer - Analyte UDFs: % Bases >=Q30 R1
WARN Installer - Analyte UDFs: % Bases >=Q30 R2
WARN Installer - Analyte UDFs: Cluster Density (K/mm^2) R1
WARN Installer - Analyte UDFs: Cluster Density (K/mm^2) R2
WARN Installer - Analyte UDFs: Clusters PF R1
WARN Installer - Analyte UDFs: Clusters PF R2
WARN Installer - Analyte UDFs: Clusters Raw R1
WARN Installer - Analyte UDFs: Clusters Raw R2
WARN Installer - Analyte UDFs: Intensity Cycle 1 R1
WARN Installer - Analyte UDFs: Intensity Cycle 1 R2

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 this integration.

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.

Install the RPM

On the Clarity LIMS server, log in as the root user.
Run the following yum command to install the RPM:
ℹ You must use the --enablerepo command line argument to enable the repo. For the repo file and the correct name to use, contact Illumina Support.
```
yum install ClarityLIMS-Illumina-NextSeq-Package-v2 --enablerepo=< repo name info from support >
```
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.
ℹ If you have not installed the SecretUtil RPM before, this RPM installation also installs it.
- 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.
1. As the glsjboss user, run the following command to view the complete list of IPP workflows:
  /opt/gls/clarity/config/illumina-preset-protocols-installer.sh -o list
2. Run the following command to install the NextSeq 500/550 Sequencing workflow:
  /opt/gls/clarity/config/illumina-preset-protocols-installer.sh -o install Illumina_Instruments.nextseq550-v1.3
3. [Optional] Run the following command to install the Library Prep Validation workflow that is used to validate the NextSeq 500/550 Sequencing workflow:
  /opt/gls/clarity/config/illumina-preset-protocols-installer.sh -o install Illumina_Instruments.Library-Prep-Validation-v2.3

Run the following script to configure the service properties:

/opt/gls/clarity/config/configure_extensions_nextseq_sequencingservice-v2.sh

Configure the Integration Properties

The integration properties can be configured to enable capture and generation of files associated with the sequencing run. Refer to Integration Properties Details for details.

The property configuration for NextSeq 500/550 v2 has the suffix .v2 and is different from the setting for v1.

Start the Sequencing Service

Run the following command to start the sequencing service:

systemctl start nextseq_seqservice-v2

NextSeq Control Software v4.0 Configuration

In NextSeq Control Software (NCS) v4.0 the location of NextSeq.Configuration.xml RTA configuration file is changed. The new location is

C:\Program Files\Illumina\RTA\2.11.3\Configs

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

/opt/gls/clarity/extensions/Illumina_NextSeq/v2/InstrumentIntegrations

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
```
C:\Program Files\Illumina\RTA\2.11.3\Configs
```
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.
```
<PostProcessEventFile>C:\Illumina\gls\gls_event_ncs_rta.bat</PostProcessEventFile>
```
Save the configuration file.

Validate the file by running RTA manually. In the command prompt, run the following command:

"C:\Program Files\Illumina\RTA\2.11.3\RTA.exe" "." configFile="C:\Program Files\Illumina\RTA\2.11.3\Configs\NextSeq.Configuration.xml" Processing.WorkProviderTypes="OfflineBased" processedfolder="."

The following error message displays:

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

Validate the batch file by executing it manually as follows.
1. Using a text editor, edit the gls_events_ncs_rta.bat file.
2. 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.

Workflows, Protocols, and Steps Installed

The NextSeq 500/550 Integration works with the NextSeq 500/550 Sequencing workflow, which contains a single protocol with the same name. This protocol includes the following steps:

Library Pooling (NextSeq 500/550)
Denature & Dilute (NextSeq 500/550)
NextSeq 500/550 Run (NextSeq 500/550)

For descriptions of the protocol and the steps, refer to NextSeq 500/550 v2.5.0 Configuration. For instructions on using the Library Prep Validation workflow to validate the automated sample sheet generation, refer to NextSeq 500/550 Integration v2.5.0 User Interaction, Validation and Troubleshooting.

Instrument Software

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.

Instrument Integration

The instrument integration must be performed and maintained by Illumina Support. Illumina Support requires remote access to the instrument while it is idle.

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.

Configure Batch Files

When the instrument is running, the final destination for the run data is a network storage path. The software is configured with a network storage path root (e.g., \\network-storage\illumina). Each sequencing run generates a unique run ID, which is appended to create a unique data run directory (e.g., \\network-storage\illumina\110419_InstrumentName_0001_ARG1234567).

The Clarity LIMS batch files must be configured to write to a directory within the network storage path root. This directory is typically named gls_events, but the directory name can be different as long as no spaces are used.

Before configuring the batch files, do the following:

Back up the NextSeq.Configuration.xml configuration file to
```
C:\Illumina\RTA\Configs\
```
Make sure that the instrument is idle.
Shut down NCS.
Set up the directory structure as follows.
1. Create a directory (C:\Illumina\gls is recommended) on the local computer to hold the batch file.
  ⚠ For Windows 10, the folder must be under C:\Illumina instead of C:\Illumina\gls because of Windows software restriction policies. If the folder is not in that directory, the batch script does not run. For versions before Windows 10, C:\Illumina\gls is acceptable.
2. Create a directory (e.g., gls_events) on the NAS to hold the event files.

Configure the batch file

Determine the site or instrument specific network storage path root.
Change the DESTINATION_PATH line to the name of the event file directory in the gls_event_ncs_rta.bat batch file.
⚠ Make sure to include the trailing \ in the DESTINATION_PATH line. Refer to the following example:
```
set DESTINATION_PATH=\\network-storage\illumina\gls_events\
```
Copy the DESTINATION_PATH and paste it into the Windows Explorer address bar.
Make sure that the network location is accessible and opens from the instrument. The batch file needs to be deployed to the NextSeq 500/550 instrument. This file must be deployed to its own directory (C:\Illumina\gls is recommended) on the instrument computer so that it is not overwritten or removed during an instrument software update.

Deploy the batch file.

Download the batch file.
Copy the file to the directory that you created on the instrument computer (e.g., C:\Illumina\gls).
From the Command Prompt, run the following command to list the contents of the directory:
```
dir C:\Illumina\gls\
```
Make sure that the name of the batch file does not contain any special or hidden characters.

Connect Batch Files to Instrument Events

Update the NextSeq Control Software (NCS) configuration files as follows.

Restart the computer and make sure that the instrument is off.
Using Task Manager, make sure that NCS is not auto-launched by Windows.
If NCS is being auto-launched, remove it from the auto-launch list and restart the computer.
Make a backup copy of the RTA configuration XML file and name it NextSeq.Configuration.xml.
Edit the file.
Save the edited file.
Restart the computer.
Turn the instrument on and launch NCS.
Open the configuration file and make sure that the changes were saved.

Connect to RTA End Run Event

Edit the NextSeq.Configuration.xml configuration file at
```
C:\Illumina\RTA\Configs\
```

Insert the following text inside the <Processing> tags:

<PostProcessEventFile>C:\Illumina\gls\gls_event_ncs_rta.bat</PostProcessEventFile>

Validate the RTA configuration changes as follows.

From the command prompt, run the following command:

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

If the validation is successful, the following warning message displays.
```
Error: No run info file found in input directory .\RunInfo.xml
```

If the configuration file has an error, the command returns specifics about the problem. Refer to the following example:

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

Restart the computer.
Turn on the instrument and start NCS.
Open the NextSeq.Configuration.xml configuration file and make sure that the changes were saved.

Validation

Sample Sheet Generation

For instructions on how to validate the automated sample sheet generation, refer to NextSeq 500/550 Integration v2.5.0 User Interaction, Validation and Troubleshooting.

Manual Invocation of Event Files

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

Filename (e.g., event-EndRun-07295667.txt)
cycleNumber (e.g., 318)
runFolder (e.g., "D:\Illumina\NextSeqTemp\230815_M99999_0028_FC1234567-ABCDE")
netFolder (e.g., \\network-folder\Run_Data\230815_M99999_0028_FC1234567-ABCDE)
readType (e.g., 4)
eventType (e.g., EndRun)
softwareType (e.g., NCS)
finishDate (e.g., 2023-08-15)

If an event file is triggered automatically during a NextSeq 500/550 run, then the completion event does not appear until the run has completed and RTA completes primary analysis.

For instructions on validating the creation of event files, refer to NextSeq 500/550 Integration v2.5.0 User Interaction, Validation and Troubleshooting.

Instrument Sequencing Run

The instrument sequencing run test validates that the Clarity LIMS batch file is connected properly and invoked on the instrument events. Before validating the batch file, make sure that you have the following prerequisites are met:

You have access to the NAS share.
The default configuration has been successfully imported.
Manual invocation of the event files has been validated. This validation checks for the following information:
- The DESTINATION_PATH is configured correctly.
- The instrument computer can access and write to the DESTINATION_PATH.
- There are no syntax errors in the Clarity LIMS batch script.

For more information on event file validation, refer to NextSeq 500/550 Integration v2.5.0 User Interaction, Validation and Troubleshooting.

The sequencing service processes and archives event files, which can cause validation issues while the service is running. You can make the following changes to avoid losing the event files that are you attempting to validate:

Modify the FINAL_EXTENSION value in the Clarity LIMS batch file so that the file extension is .test instead of .txt. The service only processes and archives TXT files. Make sure that you change FINAL_EXTENSION back to .txt after manual validation.
Monitor the NextSeqIntegrator.log file, which logs the file name and contents of each event file that is processed. The log file is at
```
/opt/gls/clarity/extensions/Illumina_NextSeq/v2/SequencingService
```

Validate the sequencing run as follows.

During the run, monitor the contents of the gls_events directory.
After the run is complete and the RTA completes primary analysis, make sure that a final EndRun event displays (e.g., event-EndRun-11043279.txt).

User Interaction, Validation and Troubleshooting

Overview

This guide explains how to validate the installation of the Illumina NextSeq 500/550 Integration Package v2.5.0.

The version of Library Prep Validation and NextSeq 500/550 Sequencing workflows depends on the IPP version installed.

For IPP v2.10, the versions of Library Prep Validation and NextSeq 500/550 Sequencing are v2.3.5 and v1.3, respectively.

The validation process involves:

Validating the creation of event files.
Running samples through the Library Prep Validation workflow. This workflow is installed by Illumina Preset Protocols (IPP) 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 workflow.
Running normalized libraries through the NextSeq 500/550 Sequencing workflow. This validates the automated generation of a reverse-complement 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
ℹ The NextSeq 500/550 Sequencing v1.3 workflow also supports generation of both reverse-complement sample sheet and forward-orientation sample sheet. The latter is compatible with NextSeq Control Software v4.2 and Local Run Manager v4.0. Refer to NextSeq 500/550 Integration v2.5.0 Release Notes for the list of supported applications.

Activate Workflow, Create Project, Add and Assign Samples

The following steps set up Clarity LIMS in preparation for running samples through the Library Prep Validation and NextSeq 500/550 Sequencing workflows.

In the Clarity LIMS configuration area, activate the Library Prep Validation and NextSeq 500/550 Sequencing workflows.
On the Projects and Samples screen, create a project and add samples to it.
Assign your samples to the Library Prep Validation workflow.

Library Prep Validation Protocol

This single-step protocol models the library prep required to produce normalized libraries that are ready for the NextSeq 500/550 Sequencing 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 workflow, which is Library Pooling (NextSeq 500/550) step.

NextSeq 500/550 Sequencing Protocol

Step 1: Library Pooling (NextSeq 500/550)

In Lab View, locate the NextSeq 500/550 Sequencing protocol. You will see your samples queued for the Library Pooling (NextSeq 500/550) step.
Select the step to proceed to the Queue screen.
On the Queue screen, add samples as follows.
1. Add the samples to the Ice Bucket. In the Add Control Samples panel, add the PhiX v3 control sample to the Ice Bucket.
1. Select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
On the Pool Samples screen, create a pool of samples as follows.
1. Drag samples into the Pool Creator.
1. Name the pool. You can also accept the default name Pool #1.
1. Select Place Samples.
On the Placement screen, move the pool to the container as follows.
1. Select the pool in the Samples to be Placed area on the left. Drag it over to the container on the right.
1. 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.

Step 2: Denature and Dilute (NextSeq 500/550)

In Lab View, locate the NextSeq 500/550 Sequencing protocol. You will see your pooled samples queued for the Denature and Dilute (NextSeq 500/550) 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.
1. Scan the NextSeq reagent cartridge barcode into the NextSeq Reagent Cartridge field.
2. Place the pool of samples into the reagent cartridge.
1. Select Record Details.
On the Record Details screen, create the sample sheet as follows.
1. 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.
2. In the Step Details table, populate the fields as appropriate.
  - The Workflow, Experiment Name and Read 1 Cycles are required fields.
  - In the Samplesheet Template drop-down list, select the template type required.
    NextSeq_Samplesheet.csv for forward-orientiation sample sheet.
    NextSeq_Reverse_Complement_Samplesheet.csv for reverse-complement sample sheet.
3. 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.
4. Select Generate 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.
1. Download the files and validate their format and content.
2. Select Next Steps.
On the Assign Next Steps screen, make sure samples are already assigned to the NextSeq 500/550 Run (NextSeq 500/550) step.
Select Finish Step.

Step 3: NextSeq 500/550 Run (NextSeq 500/550)

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.

In Lab View, locate the NextSeq 500/550 Sequencing protocol. You will see your pool of samples queued for the NextSeq 500/550 Run (NextSeq 500/550) 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 v2.5.0 Configuration.
⚠ 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.
For more detailed steps on adding instruments, refer to the Add and Configure Instruments section of the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
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.

Validating Creation of Event Files

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 or later 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.

Troubleshooting

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

rpm -qa | grep -i nextseq

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

/opt/gls/clarity/extensions/Illumina_NextSeq/v2/SequencingService

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.

Manual Upgrade

The NextSeq 500/550 Integration v2.5.0 integration package requires the NextSeq 500/550 v1.3 workflow available from IPP v2.10. If you do not want to upgrade through the IPP, you can manually upgrade the workflow configuration from NextSeq 500/550 v1.2 workflow to v1.3 workflow by following instructions in this guide to generate samplesheet compatible with LRM v4.0.

Generate NextSeq 500/550 Samplesheet Compatible with LRM v4.0

To support the generation of LRM v4.0-compatible samplesheet, you must modify the Denature and Dilute step, and replacement of template files.

Modify Denature and Dilute Step

Automation

From Configuration, select the Automation tab.
Rename the Generate bcl2fastq2 NextSeq Samplesheet automation to Generate NextSeq Samplesheet.

The default command line for the automation remains unchanged.

Master Step Fields

From Configuration, select the Custom Fields tab.
Select the Denature and Dilute Master Step.
Add the following master step fields:
Remove the following master step fields:
- Adapter
- Adapter Read 2
- Mask Adapter
- Mask Adapter Read 2
- SampleSheet Template
Update the following master step fields:

Group of Defaults

From Configuration, select the Custom Fields tab.
Select the Denature and Dilute Master Step.
Add the following Group of Defaults:
Update the following Group of Defaults:

Step File Placeholders

From Configuration, select the Lab Work tab.
Select the Denature and Dilute Master Step.
Rename the following Step File Placeholders under Record Details milestone:
- from bcl2fastq SampleSheet to Samplesheet
- from bcl2fastq SampleSheet Generation Log to Samplesheet Generation Log

Replace Template Files

Replace the following template files at /opt/gls/clarity/extensions/conf/driverfiletemplates/:

BCL2FASTQ_Reverse_Complement_Samplesheet.csv with NextSeq_ReverseComplement_Samplesheet.csv

BCL2FASTQ_Samplesheet.csv with NextSeq_Samplesheet.csv

Contact Illumina Support to replace the files if required.

NextSeq 500/550 v2.4.0

The Illumina NextSeq 500/550 Integration v2.4.0 includes the following features and functionality:

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

Release Notes

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.

Compatibility

Refer to Compatibility under Instruments & Integrations.

New Features

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.

Defect Repairs

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.

Known Issues

Missing contents in the Instrument Details section of the run report.

Revision History

Version

Changes

Updated Compatibility section to reference Compatibility matrix table.

Initial release.

Configuration

Last Updated: December 2024

Release Date: July 2024

Document Version: 2

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.

Prerequisites and Assumptions

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).

Workflows, Protocols, and Steps

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)

Step 1: Library Pooling (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.

Set Next Step - Advance Automation

This automation is automatically triggered on exit of the Record Details screen. The automation advances samples to the next step in the protocol.

The default command line is as follows.

Master Step Fields

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

Global Fields

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)

Step 2: Denature and Dilute (NextSeq 500/550 v1.2)

In this step, pooled libraries are denatured and diluted and placed into the reagent cartridge that is loaded into the NextSeq instrument.

Generate bcl2fastq2 NextSeq SampleSheet Automation

This automation is triggered by a button on the Record Details screen.

This automation generates the sample sheet and attaches it to the step. For details, see the following section on sample sheet generation.

The default command line is as follows.

Set Next Step - Advance Automation

This automation is automatically triggered on exit of the Record Details screen.

This automation advances samples to the next step in the protocol.

The default command line is as follows.

Master Step Fields

At run time, the master step 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

Resequencing

Read 1 Cycles = 251
Workflow = Resequencing

Custom Amplicon

Read 1 Cycles = 251
Workflow = Custom Amplicon

PCR Amplicon

Read 1 Cycles = 251
Workflow = PCR Amplicon

Enrichment

Read 1 Cycles = 251
Workflow = Enrichment

Library QC

Read 1 Cycles = 251
Workflow = Library QC

Metagenomics

Read 1 Cycles = 251
Workflow = Metagenomics

GenerateFastQ

Read 1 Cycles = 251
Workflow = GenerateFastQ

Assembly

Read 1 Cycles = 251
Workflow = Assembly

Global Fields

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)

Step File Placeholders

Placeholders for the following files are configured on the Record Details screen of the Denature and Dilute (NextSeq 500/550 v1.2) step

Lab Tracking Form

Manually uploaded
This form in Clarity LIMS allows for manually attaching a lab-specific tracking form to the step.

bcl2fastq SampleSheet

Automatically attached
This CSV file is automatically generated by Clarity LIMS for use with the bcl2fastq2 v2.20.0 analysis software. It can be opened as a text file or as an MS Excel spreadsheet.

bcl2fastq SampleSheet Generation Log

Automatically attached
Automatically generated by Clarity LIMS, this log file captures any errors that Clarity LIMS can encounter when generating the sample sheet.

Log File

Automatically attached
Automatically generated by Clarity LIMS, this log file captures the status of the EvaluateDynamicExpression script that is launched by the Set Next Step - Advance automation.

Step 3: NextSeq 500/550 Run (NextSeq 500/550 v1.2)

In this step, pooled samples are sequenced on the NextSeq 500/550 instrument and the run metrics are recorded in Clarity LIMS.

Set Next Step - Advance Automation

This automation is automatically triggered on exit of the Record Details screen. The automation advances samples to the next step in the protocol.

The default command line is as follows.

Master Step Fields

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

Global Fields

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.

Step File Placeholders

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

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.

Sequencing Results Parsing

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.

Generated and Captured Files

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

Metadata

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.

Primary Analysis Metrics

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 v1.2) 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.

Components Installed

Scripts and Files Installed

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

Properties Installed

Reagent Categories/Label Groups Installed

Reagent categories/label groups are installed with the IPP workflow configuration slices.

The NextSeq Reagent Kit is included in the NextSeq Integration.

Control Types Installed

The PhiX v3 control type is included in the NextSeq Integration.

Container Types Installed

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.

Instrument Integration

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.

Rules and Constraints

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.

Revision History

User Interaction, Validation and Troubleshooting

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

Activate Workflow, Create Project, Add and Assign Samples

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.

Library Prep Validation v2.3.4 Protocol

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.

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.

NextSeq 500/550 Sequencing v1.2 Protocol

Step 1: Library Pooling (NextSeq 500/550 v1.2)

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.
1. Add the samples to the Ice Bucket. In the Add Control Samples panel, add the PhiX v3 control sample to the Ice Bucket.
1. Select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
On the Pool Samples screen, create a pool of samples as follows.
1. Drag samples into the Pool Creator.
1. Name the pool. You can also accept the default name Pool #1.
1. Select Place Samples.
On the Placement screen, move the pool to the container as follows.
1. Select the pool in the Samples to be Placed area on the left. Drag it over to the container on the right.
1. 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.

Step 2: Denature and Dilute (NextSeq 500/550 v1.2)

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.
1. Scan the NextSeq reagent cartridge barcode into the NextSeq Reagent Cartridge field.
2. Place the pool of samples into the reagent cartridge.
1. Select Record Details.
On the Record Details screen, create the sample sheet as follows.
1. 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.
2. 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.
1. 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.
2. 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.
1. Download the files and validate their format and content.
2. 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.

Step 3: NextSeq 500/550 Run (NextSeq 500/550 v1.2)

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.
⚠ 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.
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.

Validating Creation of Event Files

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.

Troubleshooting

If an automation trigger does not appear to run its corresponding scripts, refer to the following topics:

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.