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.
Follow the steps in 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.
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.
Add the samples to the Ice Bucket. In the Add Control Samples panel, add the PhiX v3 control sample to the Ice Bucket.
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.
Step 3: NextSeq 500/550 Run (NextSeq 500/550 v1.2)
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 .
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.
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 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.
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 .
Troubleshooting Automation in the Automation section of the .
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.
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.
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.
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.
âš Do not continue to step 5 and complete the step until the Illumina Run Report has been attached.
âš 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.
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
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 .
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.
Compatibility
Refer to 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 .
For information on user interaction for each step, validating and troubleshooting the integration, refer to .
Prerequisites
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
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)
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.
Prerequisite 3: IPP Installation
The NextSeq 500/550 Integration depends on the configuration provided in IPP. Refer to 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
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
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.
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.
Enter
Configure the Integration Properties
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.
Start the Sequencing Service
Run the following command to start the sequencing service:
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
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.
After manually executing the batch file, a dummy EndRun event file is created in the event files directory.
Workflows, Protocols, and Steps Installed
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)
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 .
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 .
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 . 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:
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.
Connect to RTA End Run Event
Edit the NextSeq.Configuration.xml configuration file at
Insert the following text inside the <Processing> tags:
Validation
Sample Sheet Generation
For instructions on how to validate the automated sample sheet generation, refer to .
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)
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.
Revision History
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
Run statistics from
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:
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:
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:
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 .
Use the following command to make sure that the password is saved correctly in Secret Util:
For more information on Secret Utility configuration, refer to the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
Smoke test directories
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.
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:
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.
Back up the NextSeq.Configuration.xml configuration file to
Make sure that the instrument is idle.
Shut down NCS.
Set up the directory structure as follows.
Create a directory (C:\Illumina\gls is recommended) on the local computer to hold the batch file.
âš 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.
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.
âš Make sure to include the trailing \ in the DESTINATION_PATH line. Refer to the following example:
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:
Make sure that the name of the batch file does not contain any special or hidden characters.
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.
Validate the RTA configuration changes as follows.
From the command prompt, run the following command:
If the validation is successful, the following warning message displays.
If the configuration file has an error, the command returns specifics about the problem. Refer to the following example:
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.
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.
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
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).
Version
Changes
3
Updated
Compatibility
Prerequisite 3
2
Corrected typo in Prerequisite 3 of Prerequisites section.
Error: No run info file found in input directory .\RunInfo.xml
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
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 .
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).
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 .
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.
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.
For more information, see the section below.
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)
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
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
This integration requires installation of the Illumina Preset Protocols (IPP). For details, refer to .
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
Refer to for the properties installed with Illumina NextSeq 500/550 Integration Package v2.4.0.
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.
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.
Template file used for file generation. In this integration, the reverse complement template is used by default.
Updates sequencing service default properties to match the specifics of the installation.
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
TruSeq mRNA sequencing
TruSeq DNA sequencing (large genome de novo)
TruSeq DNA sequencing (large genome re-seq)
TruSeq DNA sequencing (small genome de novo)
TruSeq DNA sequencing (small genome re-seq)
Nextera DNA sequencing
TruSeq Custom Amplicon sequencing
ChIP-sequencing
Exome sequencing
Mate pair sequencing
Small RNA sequencing
Pooling
Text Dropdown
Custom Entries
Presets
Yes
No
Read Length
Text
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
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-Down Items
Read Length
ℹ Displays on Queue & Ice Bucket screens
Text
Sequencing Method
ℹ Displays on Queue & Ice Bucket screens
Text Dropdown
Custom Entries
Presets
Single Read
Paired End Read
Indexed Single Read
Indexed Paired End Read
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-Down Items
Final Loading Concentration
ℹ Displays on Record Details screen
Numeric Dropdown
Required Field
Custom Entries
Presets
225
400
Decimal places displayed = 0
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
Item
Description
Run Info
Run Parameters
These XML files are captured automatically by Clarity LIMS from the instrument run folder. They include the key run parameters, many of which are parsed out into key step global custom fields.
Link to Run Folder
Automatically generated by Clarity LIMS, and is a link to the network run folder where the data that was captured from the instrument during the run is stored.
Illumina Run Report
Automatically generated by Clarity LIMS, this report provides key information about the run and the samples on the flow cell.
Information includes the flow cell ID, run directory location, and primary analysis metrics for the instrument, summarized per flow cell lane for the entire run, and individual reads if there are paired-end runs.
These metrics are compared against the instrument per lane averages, calculated using metrics from the last 5 sequencing runs. Any values outside of 1 standard deviation are highlighted.
Lab Tracking Form
This placeholder in Clarity LIMS allows you to attach a lab-specific tracking form to the step manually.
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
Files Installed
Location
Description
configure_extensions_nextseq_sequencingservice.sh
/opt/gls/clarity/config/
Script that installs the service properties in the database.
