The integration includes the following functionality:
Preconfigured NovaSeq 6000 v2.3 workflow that maps to lab protocols and instrument runs.
Preconfigured NovaSeq Standard (NovaSeq 6000 v2.3) protocol that supports the loading of pooled libraries into a library tube.
Preconfigured NovaSeq Xp (NovaSeq 6000 v2.3) protocol that supports individual lane loading on the NovaSeq.
Automated generation of a sample sheet for use with bcl2fastq2 v2.20 analysis software.
Automatic generation of run recipe file (JSON format) that is automatically uploaded to the sequencing instrument and used to set up and initiate the run.
Automated tracking of the NovaSeq sequencing run and parsing of run statistics (per run per lane) into Clarity LIMS, including:
Progress and metrics of sequencing run
Per instrument sequencing runs (tracked as part of the Run ID field value)
Preconfigured NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) workflow used for validation purposes only. The workflow contains a single-step protocol that models the library prep required to produce normalized libraries that are ready for the NovaSeq 6000 v2.3 workflow. For details, refer to .
Sequencing run parameters
Real-Time Analysis v3 (RTA3) run directory location and other run-specific information
The Illumina NovaSeq 6000 Integration v2.6.0 supports the integration between Clarity LIMS and the NovaSeq 6000 instrument. This integration also provides support for Clarity LIMS v6.2.0 and includes updates to the technology stack and third party libraries and utilities.
Clarity LIMS is automatically stopped during installation of the RPM and will need to be restarted. The file-based and API-based integrations cannot run at the same time. When the API-based integration has finished installing, shut down Clarity LIMS and uninstall the file-based integration.
This document provides instructions for installing NovaSeq 6000 Integration v2.6.0. It also describes the components that are installed in the default configuration.
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
NovaSeq 6000 Integration v2.6.0 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, set up the following Write privileges as the glsai user:
Sample sheet directory — This directory is where the Prepare Files for NovaSeq script writes sample sheets.
Prerequisite 2: Secret Util Installation
The Clarity LIMS installation tooling configures the Secret Util installation. No additional configuration is necessary. For more information on Secret Utility configuration, refer to the .
Installation
NovaSeq 6000 Integration v2.6.0 supports both on-premise and cloud integrations. This integration is distributed as the following RPM packages:
BaseSpaceLIMS-novaseq-extensions
BaseSpaceLIMS-novaseq-sequencing-service (depends on BaseSpaceLIMS Automation Worker v1.x)
The BaseSpaceLIMS-novaseq-extensions RPM installs the following items:
Protocols and steps
Validation workflow
Integration properties that configure the service
The BaseSpaceLIMS-novaseq-sequencing-service RPM installs the following items:
Bash scripts to run the novaseq_seqservice through systemctl
novaseq-sequencing.jar
novaseq-remote-extensions.jar file that contains scripts that need access to the run folders on the NAS
On-Premise Installation
Use the following instructions to install the BaseSpaceLIMS-novaseq-extensions and BaseSpaceLIMS-novaseq-sequencing-service RPMs on the Clarity LIMS server.
Install the RPMs
On the Clarity LIMS server, log in as the root user.
Run the following yum commands to install the RPMs:
Import Workflow Configurations for NovaSeq 6000
As the glsjboss user, use the following command to run the configure_extensions_novaseq.sh script:
When prompted, enter the channel name for the Automation Worker and sequencing service.
Configure the Integration Properties
For more information on the configurable integration properties that enable capture and generation of files associated with the sequencing run, refer to .
The sample sheet, run recipe, and run output folders can all be configured independently and do not have to be on the same mount or share. For examples of valid configuration mount points and properties, refer to .
Start the Sequencing Service
Run the following command to start the sequencing service:
Cloud Installation
The BaseSpaceLIMS-novaseq-extensions RPM must be installed on the Clarity LIMS server. The BaseSpaceLIMS-novaseq-sequencing-service RPM can be installed remotely on another server within the network.
Specifications
The following hardware, operating system, and network specifications must be met to install the BaseSpaceLIMS-novaseq-sequencing-service RPM:
Hardware requirements:
Install the RPM
On the applicable server, log in as the root user.
Run the following yum command to install the BaseSpaceLIMS-novaseq-extensions RPM:
Import Workflow Configurations for NovaSeq 6000
As the glsjboss user, use the following command to run the configure_extensions_novaseq.sh script:
When prompted, enter the channel name for the Automation Worker and sequencing service. The recommended channel name is remotens. For more information, refer to Update Channel Name.
Update Channel Name
The configure_extensions_novaseq.sh script prompts for a channel name and sets the name on the following preconfigured automations:
Prepare Files for NovaSeq (configured on Dilute and Denature (NovaSeq 6000 v2.3) and Load to Flowcell (NovaSeq 6000 v2.3) steps)
Configure the Integration Properties
For more information on the configurable integration properties that enable capture and generation of files associated with the sequencing run, refer to .
The sample sheet, run recipe, and run output folders can all be configured independently and do not have to be on the same mount or share. For examples of valid configuration mount points and properties, refer to .
Install Sequencing Service RPM on Remote Server
On the applicable server, log in as the root user.
Run the following yum command to install the RPM:
Set Up API Connection Properties File
Run the following script to set up the API connection properties file at /opt/gls/clarity/automation_worker/conf/api-access.properties:
The configure.sh script is provided by BaseSpaceLIMS-AutomationWorker.
Start the Sequencing Service
Run the following command to start the sequencing service:
Configurable Properties
For more information on the configurable integration properties that enable capture and generation of files associated with the sequencing run, refer to
The sample sheet, run recipe, and run output folders can all be configured independently and do not have to be on the same mount or share. For examples of valid configuration mount points and properties, refer to .
Workflows, Protocols, and Steps Installed
NovaSeq 6000 v2.3 Workflow
Protocol 1: Run Format (NovaSeq 6000 v2.3)
Steps:
Define Run Format (NovaSeq 6000 v2.3)
Protocol 2: NovaSeq Standard (NovaSeq 6000 v2.3)
Steps:
Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3)
Dilute and Denature (NovaSeq 6000 v2.3)
Protocol 3: NovaSeq Xp (NovaSeq 6000 v2.3)
Steps:
Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3)
Dilute, Denature & ExAmp (NovaSeq 6000 v2.3)
Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3)
For detailed descriptions of the steps and automations included in each protocol, and details on other components in the configuration, refer to .
For instructions on using the NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) protocol, refer to .
The configuration provided with this integration has been established to support NovaSeq 6000 lab processes. Any configuration changes to protocols or workflows, including renaming protocols, steps, and fields, could break processes.
Instrument Software
The NovaSeq instrument software includes the following components:
NovaSeq Control Software (NVCS) — Contains the user interface for setting up the sequencing run. Responsible for controlling the instrument and acquiring the images.
Real-Time Analysis v3 (RTA3) — Takes the images generated by the first module, processes, and analyzes them. Makes sure that data files are created and copied to the final destination folder.
Sequencing Analysis Viewer (SAV) — Displays the important quality metrics generated by the RTA3 software.
Instrument Integration Configuration
For the NovaSeq API integration to work, the NovaSeq instrument must be able to communicate with Clarity LIMS through the API. Complete the following steps to configure the NVCS and confirm that you can access Clarity LIMS from the instrument.
Launch NVCS and wait for the initialization process to complete.
On the NVCS Settings page, do the following:
Under Mode Selection, select File-Based.
Example Network Folder Structure and Database Properties
Instrument Control Software Configuration
Refer to the following examples for the configuration of the instrument control software:
The following examples assume the same configuration and mount points from the previous sections.
After completing the Dilute and Denature (NovaSeq 6000 v2.3) or Load to Flowcell (NovaSeq 6000 v2.3) step, an automated script copies the run recipe JSON file and sample sheet to the appropriate network locations. For example, a library tube or flow cell with the barcode AA12345-NNN would have the following directory content:
Run recipe directory — This directory is where the Prepare Files for NovaSeq script writes run recipe files in the JSON format.
Set up the Read privileges as the glsjboss and glsai users (or as the entire claritylims group) for the run output directory. This directory is where the sequencing service looks for run folders. The run folders must be in this directory. The service does not search sub-directories.
The sample sheet, run recipe, and run output directories can be configured independently and do not have to be on the same mount or share.
novaseq-extensions.jar
file that provides sample sheet generation and other utility scripts
ℹ You must include the --enablerepo command line argument. Illumina Support provides the repo file and appropriate name to use.
Enter y to confirm that you want to proceed with the RPM installations.
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.
[Optional] If you are upgrading from a previous integration, check for any usages of the Custom API Username in the current system as follows.
Install ClarityLIMS-UpgradePreValidation.
Check for any custom API username requirements (e.g., novaseq_user).
Typically, the default API user name is apiuser.
Run the following script:
After the script is complete, check that a custom user is configured in the results.
If a custom API username is configured, a warning message displays.
If a custom API username is required, configure the username and password using Secret Util:
For a custom API username, set the 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:
64-bit processor (dual core 2.0 GHz)
OS requirements, plus at least an additional 2 GB RAM
Minimum requirements for remote Automation Worker (for more information, refer to the )
ℹ If you are running the sequencing service and Automation Worker on the same instance, make sure that the instance is running a compatible version of Oracle Linux. For compatibility, refer to .
Network requirements:
SSL access to the Clarity LIMS server from the network
The mounted folder where the run recipes are written
The mounted folder where the sample sheets are written
The mounted folder where the sequencing runs are written
ℹ You must include the --enablerepo command line argument. Illumina Support provides the repo file and appropriate name to use.
Enter y to confirm that you want to proceed with the RPM installation.
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.
[Optional] If a custom API username is required, configure the username and password as follows.
Run the following command using Secret Util:
For a custom API username, set the 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:
Read InterOp Metrics (configured on AUTOMATED NovaSeq Run (NovaSeq 6000 v2.3) step)
If you need to change the channel name, you must update it manually on each automation. The import script does not update this setting.
Enter y to confirm that you want to proceed with the RPM installation.
Load to Flowcell (NovaSeq 6000 v2.3)
NovaSeq recipes — Provides system operation instructions for use with NovaSeq 6000 reagent kits for SP, S1, S2, and S4 flow cells.
Universal Copy Service — Copies output files to destinations such as final destination folder and/or BaseSpace Sequence Hub (when the instrument is configured for use with BaseSpace Sequence Hub).
In the adjacent field, enter the network folder location that NVCS retrieves the JSON recipe from.
In the Output Folder field, enter the default output folder location.
Select Save to complete the configuration.
On the NVCS home page, you can now select Sequence to log into Clarity LIMS and start a run.
When the NovaSeq run starts, the instrument control software creates a new run folder named after the run ID. Then, the software copies the run recipe JSON file to a Clarity LIMS subfolder inside it. For example, a run ID of 170301_A001_AHHCC would have the following directory content:
The NovaSeq 6000 sequencing service detects the run folder based on the presence of the three files inside it and starts the AUTOMATED NovaSeq Run (NovaSeq 6000 v2.3) step automatically.
After the sequencing run has completed, the Universal Copy Service copies the run data and InterOp files to the network drive. Then, the service creates a CopyComplete.txt file in the run folder. At this point, the directory would have the following content:
The NovaSeq 6000 sequencing service detects the completed sequencing run based on the presence of the CopyComplete.txt file, executes InterOp metrics parsing for the run, and automatically completes the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step.
yum install BaseSpaceLIMS-novaseq-extensions --enablerepo=<< repo name info from support >>
yum install BaseSpaceLIMS-novaseq-sequencing-service --enablerepo=<< repo name info from support >>
yum install BaseSpaceLIMS-novaseq-extensions --enablerepo=<< repo name info from support >>
This guide explains how to validate the installation of the Illumina NovaSeq 6000 Integration Package v2.6.0. The validation process involves the following actions:
Running samples through the NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) workflow. The workflow contains a single-step protocol that models the library prep required to produce normalized libraries. At the end of the step, the normalized libraries are automatically advanced to the NovaSeq 6000 v2.3 workflow.
Running normalized libraries through the NovaSeq 6000 v2.3 workflow. This process validates the following details:
Successful routing of samples from the Run Format (NovaSeq 6000 v2.3) step to the NovaSeq Standard (NovaSeq 6000 v2.3) or NovaSeq Xp (NovaSeq 6000 v2.3) step.
Automated generation of a sample sheet for use with bcl2fastq2 v2.20 analysis software.
Automated generation of a run recipe file (JSON file) with the library tube or flow cell barcode as the name (eg, NV1234567-LIB.json or H1234DRXX.json). This file is automatically uploaded to the sequencing instrument and used to set up and initiate the run.
Automated tracking of the NovaSeq sequencing run and parsing of run statistics (per run per lane) into Clarity LIMS.
Before executing the validation steps, make sure that you have installed the Illumina NovaSeq 6000 Integration Package v2.6.0 and have imported the default Clarity LIMS configuration.
Activate Workflow, Create Project, Add and Assign Samples
The following steps set up Clarity LIMS in preparation for running samples through the NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) and NovaSeq 6000 v2.3 workflows.
In the Clarity LIMS Configuration area, activate the NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) and NovaSeq 6000 v2.3 workflows.
On the Projects and Samples screen, create a project and add samples to it.
Assign the samples to the NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) workflow.
This single-step protocol models the library prep required to produce normalized libraries that are ready for the NovaSeq 6000 v2.3 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 = NovaSeq
On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the NovaSeq 6000 v2.3 workflow—Define Run Format (NovaSeq 6000 v2.3). This is the only step in Protocol 1: Run Format (NovaSeq 6000 v2.3).
Protocol 1: Run Format (NovaSeq 6000 v2.3)
This protocol includes the Define Run Format (NovaSeq 6000 v2.3) step. The step allows for the assignment of per sample values for Loading Workflow Type, Normalized Molarity, Flowcell Type, and Final Loading Concentration (pM). At the end of the step, samples are routed to the NovaSeq Standard or NovaSeq Xp protocol, according to the selected Loading Workflow Type.
Step 1: Run Define Run Format (NovaSeq 6000 v2.3)
In Lab View, locate the Run Format (NovaSeq 6000 v2.3) protocol. The samples are queued for the Define Run Format (NovaSeq 6000 v2.3) step.
Add the samples to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
Protocol 2: NovaSeq Standard (NovaSeq 6000 v2.3)
In this protocol, samples are pooled and added to the library tube in preparation for the NovaSeq run. The protocol contains two steps:
Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3)
Dilute and Denature (NovaSeq 6000 v2.3)
Step 1: Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3)
In Lab View, locate the NovaSeq Standard (NovaSeq 6000 v2.3) protocol. The samples are queued for the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3) step.
On the Queue screen, add samples of the same flow cell type to the Ice Bucket and select Begin Work.
On the Pooling screen, create a pool by dragging samples into the Pool Creator.
At the end of this step, the pool of samples automatically advances to the Dilute and Denature (NovaSeq 6000 v2.3) step.
Step 2: Dilute and Denature (NovaSeq 6000 v2.3)
In Lab View, locate the NovaSeq Standard (NovaSeq 6000 v2.3) protocol. The pool of samples is queued for the Dilute and Denature (NovaSeq 6000 v2.3) step.
Add the samples to the Ice Bucket and select Begin Work.
At the beginning of the step, the Validate Single Input automation is triggered. This automation checks that there is only one container input to the step.
On the Placement screen, drag the pool into the library tube in the Placed Samples area.
On exit from the step, the Routing Script automation is triggered and samples are routed to Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3).
In Lab View, the pool of samples is queued for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step.
Do not add samples to the Ice Bucket or start the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step. The integration does this action automatically.
Protocol 3: NovaSeq Xp (NovaSeq 6000 v2.3)
In this protocol, samples are pooled and added to lanes on the NovaSeq flow cell. The flow cell type is determined by the option selected in the Define Run Format (NovaSeq 6000 v2.3) step.
The protocol contains three steps:
Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3)
Dilute, Denature & ExAmp (NovaSeq 6000 v2.3)
Load to Flowcell (NovaSeq 6000 v2.3)
Step 1: Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3)
In Lab View, locate the NovaSeq Xp (NovaSeq 6000 v2.3) protocol. The samples are queued for the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3) step.
On the Queue screen, add samples of the same Flowcell Type to the Ice Bucket and select Begin Work.
On the Pooling screen, create a pool by dragging samples into the Pool Creator.
At the end of this step, the pool of samples automatically advances to the Dilute, Denature & ExAmp (NovaSeq 6000 v2.3) step.
In Lab View, locate the NovaSeq Xp (NovaSeq 6000 v2.3) protocol. The pool of samples is queued for the Dilute, Denature & ExAmp (NovaSeq 6000 v2.3) step. Add the pool to the Ice Bucket.
On the Ice Bucket screen, set the number of derivatives to create (they are placed into the flow cell lanes) and select Begin Work.
The Validate Inputs Flowcell Type automation checks that there is only one container input to the step.
On entry to the Record Details screen, the Calculate Volumes automation is triggered. This automation sets the following values based on the selected Flowcell Type:
Step 3: Run Load to Flowcell (NovaSeq 6000 v2.3)
On the Ice Bucket screen in the Container Options panel, select the appropriate flow cell type from the Destination Container drop-down list and select Begin Work.
On the Placement screen, drag the pools from the left of the screen over into the Placed Samples area on the right.
Scan or type the barcode of the flow cell into the Flow Cell field.
At this point in the workflow, the user interaction ends. The flow cell is queued for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step.
Proceed to Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3).
Do not add samples to the Ice Bucket or start the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step. The integration does this action automatically.
Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3)
This protocol contains a single fully automated step - AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3).
The integration starts the step automatically and data from the run is parsed back into Clarity LIMS. No user interaction is required. However, you can open and review the various stages of the step in Clarity LIMS.
Refer to Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) in for how the integration works, and for details on the automations.
Troubleshooting
If an automation trigger does not appear to run its corresponding scripts, see the following sections in the Clarity LIMS documentation:
Troubleshooting Automated Worker in the .
Troubleshooting Automation in the .
If an error occurs that does not provide direction on how to proceed, complete the following steps:
Confirm the version of the installed Illumina NovaSeq Integration Package by running the command as follows:
For an on-premise deployment, run the command on Clarity LIMS server command line. This command retrieves the version of both RPMs installed.
For a cloud hosted deployment, run the command on the local server installation. This command retrieves the version of the remote extensions RPM installed.
On the Record Details screen, specify the Sample Details.
Loading Workflow Type — Select NovaSeq Standard or NovaSeq Xp from the drop-down list.
Normalized Molarity — Enter values or use values copied from the previous step.
Populate the following fields:
ℹ Values can vary across samples.
Flowcell Type — Select SP, S1, S2, or S4.
Final Loading Concentration (pM) — Select from the two preset options—225 (for PCR-free workflows) or 400 (for Nano workflows)—or enter a different value.
Select Next Steps to trigger the Set Next Steps automation, which does the following actions:
Sets the value of the next step (for all samples) to Remove from workflow. The Routing Script automation expects this value and requires it to advance samples to the next step successfully.
Calculates the Minimum Molarity.
Checks Normalized Molarity value. For samples with no Normalized Molarity value (eg, an empty value, not including 0), the automation generates an error message stating that the field cannot be empty.
Compares the Normalized Molarity value of each sample with the Minimum Molarity value.
On the Assign Next Steps screen, review the Sample Details table and make sure that the Next Step for all samples is prepopulated with Remove from workflow.
Next Step must be set to Remove from workflow, regardless of the Loading Workflow Type.
âš If the Next Step value is not set to Remove from workflow, the routing script automation cannot advance to the next step.
Review the Warning field entries for the samples. If the field indicates that the Normalized Molarity is too low, continue with one of the following options:
Return to the Record Details screen and adjust the Normalized Molarity value so that it equals or exceeds the Minimum Molarity value. Set the Loading Workflow Type to NovaSeq Standard or NovaSeq Xp, as applicable.
Alternatively, complete the protocol without correcting the Normalized Molarity value. In this case, the samples in question are removed from the Clarity LIMS workflow.
Select Finish Step.
The Routing Script automation is triggered if:
Samples with a Loading Workflow Type of Remove from workflow (ie, where Normalized Molarity value is lower than the Minimum Molarity) are removed from the Clarity LIMS workflow.
Samples with a Loading Workflow Type of NovaSeq Standard are routed to the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3) step. This step is the first of two steps in Protocol 2: NovaSeq Standard (NovaSeq 6000 v2.3).
Samples with a Loading Workflow Type of NovaSeq Xp are routed to the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3) step. This step is the first of three steps in Protocol 3: NovaSeq Xp (NovaSeq 6000 v2.3).
âš Only one pool must be created.
Type a name for the pool or accept the default name (Pool #1).
Select Record Details.
On exit from the Pooling screen, the Validate Inputs Flowcell Type and Single Pool is triggered. The automation checks the following information:
All samples in the pool have been assigned the same Flowcell Type.
Only one pool has been created.
On the Record Details screen in the Step Details area, complete the fields as follows.
Enter the value for the Number of Flowcells to Sequence field.
This value is used in volume calculations to make sure that the volumes are sufficient for the number of times the pool is sequenced.
Enter the value for the Minimum Per Sample Volume (ul) field.
The value in this field is used to calculate how much of each sample is included in the pool. The field is prepopulated with the configured default value (5 µl), but the value can be edited if necessary.
If necessary, enter the value for the % PhiX (2.5 nM) Spike-In field.
The value in this field is used to calculate the volume of PhiX v3 control to be included in the pool for the given percentage of spike-in. The field is optional and can be edited.
ℹ Assuming the default Minimum Per Sample Volume (ul) value of 5, for a given batch:
If the smallest Per Sample Volume (ul) value is less than 5, the LIMS automatically assigns a value of 5 to the sample's Adjusted Per Sample Volume (ul) field.
Clarity LIMS then adjusts the Adjusted Per Sample Volume (ul) field value for all other samples in the batch, based on the ratio used to increase the lowest value to 5.
In the Sample Details table, select the pool icon to view details on the pool composition.
Select Calculate Volumes to trigger the Calculate Volumes automation. This automation performs calculations based on the selected Flowcell Type, then generates and attaches the Calculation File (CSV) to the step. This file contains volume information about the pool and the individual samples that it contains.
Select Next Steps to trigger the Set Next Steps automation.
This automation sets the next step for samples to ADVANCE, advancing them to the Dilute and Denature (NovaSeq 6000 v2.3) step in the protocol.
Select Finish Step.
Scan or type the barcode of the library tube into the Library Tube field.
Select Record Details.
On exit of the Placement screen, the Validate Library Tube Barcode automation checks that the library tube barcode conforms to the barcode mask [A-Z]{2}[0-9]{7}-[A-Z]{3}. If not, an error message displays.
On the Record Details screen, the Reagent Lot Tracking section tracks the NaOH, Resuspension Buffer, and Tris-HCI reagents used in the step. To add and activate reagent lots, refer to Add and Configure Reagent Kits and Lots in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
In the Reagent Lot Tracking section, select from the active lots displayed in each drop-down list. The fields displayed in the Step Details section are used to generate the sample sheet and run recipe files. Some of these fields are autopopulated and some must be completed manually. Refer to the following table for details.
Fields Displayed on Record Details Screen of Dilute and Denature (NovaSeq 6000 v2.3) Step
Field
Value
Experiment Name
Enter the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. No spaces.
BaseSpace Sequence Hub Configuration
Presets
Not Used
Run Monitoring Only
Run Monitoring and Storage
On the Record Details screen, select Generate Sample Sheet & Run Recipe. This selection triggers the automation script: The sample sheet and JSON files are generated and attached to the placeholders in the Files area of the Record Details screen. The Sample Sheet Path field is populated with the path to the sample sheet file.
Select Next Steps. This selection triggers the Prepare Files for NovaSeq automation, which does the following:
Copies the sample sheet and run recipe files to the location specified during installation. The NovaSeq instrument software uses these files to set up the run.
Sets the value of the next step to Remove from workflow. The Routing Script automation expects this value and requires it to advance samples to the next step successfully.
On the Assign Next Steps screen, the Next Step for samples is prepopulated with Remove from workflow.
âš Do not change this value. If Next Step is not set to Remove from workflow, the routing script is not able to route samples correctly.
Select Finish Step.
âš Only one pool must be created.
Type a name for the pool or accept the default name Pool #1.
Select Record Details.
On exit from the Pooling screen, the Validate Inputs Flowcell Type and Single Pool is triggered. The automation checks the following details:
All samples in the pool have been assigned the same Flowcell Type.
Only one pool has been created.
On the Record Details screen in the Step Details area, complete the fields as follows.
Enter the value for the Number of Lanes to Sequence field.
This value is used in volume calculations to make sure that the volumes are sufficient for the number of times the pool is sequenced.
Enter the value for the Minimum Per Sample Volume (ul) field.
The value in this field is used to calculate how much of each sample is included in the pool. The field is prepopulated with the configured default value (5 µl), but the value can be edited if necessary.
If necessary, enter the value for the % PhiX (0.25 nM) Spike-In field.
The value in this field is used to calculate the volume of PhiX v3 control to be included in the pool for the given percentage of spike-in. The field is optional and can be edited.
ℹ Assuming the default Minimum Per Sample Volume (ul) value of 5, for a given batch:
If the smallest Per Sample Volume (ul) value is less than 5, the LIMS automatically assigns a value of 5 to the sample's Adjusted Per Sample Volume (ul) field.
Clarity LIMS then adjusts the Adjusted Per Sample Volume (ul) field value for all other samples in the batch, based on the ratio used to increase the lowest value to 5.
In the Sample Details table, select the pool icon to view details on the pool composition.
Select Calculate Volumes to trigger the Calculate Volumes automation. This automation performs calculations based on the selected Flowcell Type, then generates and attaches the Calculation File (CSV) to the step. This file contains volume information about the pool and the individual samples that it contains.
Select Next Steps to trigger the Set Next Steps automation.
This automation sets the next step for samples to ADVANCE, advancing them to the Dilute, Denature & ExAmp (NovaSeq 6000 v2.3) step in the protocol.
Select Finish Step.
NaOH Volume (ul)
Tris-HCl Volume (ul)
DPX1 Volume (ul)
DPX2 Volume (ul)
DPX3 Volume (ul)
Mastermix per Lane (ul)
The automation also generates the Calculation File (*csv) and attaches it to the step. This file contains information about the DPX Mastermix volume and the volume of Mastermix, NaOH, and Tris-HCI to add per working pool.
On the Record Details screen, the Reagent Lot Tracking section tracks the DPX1, DPX2, DPX3, NaOH, Resuspension Buffer, and Tris-HCI reagents used in the step. To add and activate reagent lots, refer to Add and Configure Reagent Kits and Lots in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
On the Record Details screen in the Reagent Lot Tracking section, select from the active lots displayed in each drop-down list.
In the Step Details area, the DPX1, DPX2, and DPX3 reagent volume values are already populated. These values are set by a script and are not editable while running the step.
In the Sample Details table, select the pool icon to view details on the working pool composition.
ℹ The BP Aliquot, Mastermix per lane, NaOH, and Tris-HCI volume values for each working pool are already populated. These values are set by a script and are not editable while running the step.
The working pool number is appended to the bulk pool name. This feature allows you to identify which working pools are derived from the same bulk pool quickly.
[Optional] Select the Calculation File (CSV) in the Files area to view details on the following volumes to add per working pool:
DPX Mastermix
Mastermix
NaOH
Tris-HCl
Select Next Steps.
On the Assign Next Steps screen, the next step is already set to Load to Flowcell (NovaSeq 6000 v2.3).
Select Finish Step.
Select Record Details.
On exit of the Placement screen, the Validate Flowcell Barcode automation validates the container barcode.
Flowcell Type
Barcode Mask
SP
[A-Z0-9]{5}DR[A-Z0-9]{2}
S1
[A-Z0-9]{5}DR[A-Z0-9]{2}
S2
[A-Z0-9]{5}DM[A-Z0-9]{2}
S4
[A-Z0-9]{5}DS[A-Z0-9]{2}
The fields displayed in the Step Details section are used to generate the sample sheet and run recipe files. Some of these fields are autopopulated and some must be completed manually. See the following table for details.
Fields Displayed on Record Details Screen of Load to Flowcell (NovaSeq 6000 v2.3) Step
Field
Value
Experiment Name
Enter the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. No spaces.
BaseSpace Sequence Hub Configuration
Presets
Not Used
Run Monitoring Only
Run Monitoring and Storage
Workflow
Select Generate Sample Sheet and Run Recipe. This selection triggers the automation script, which:
Generates and attaches the sample sheet and JSON files to the placeholders in the Files area of the Record Details screen.
Populates the Sample Sheet Path field with the path to the sample sheet file.
Select Next Steps.
This selection triggers the Prepare Files for NovaSeq automation, which copies the sample sheet and run recipe files to the location specified during installation. The NovaSeq instrument software uses these files to set up the run.
On the Assign Next Steps screen, make sure the Next Step for samples is prepopulated with Mark protocol as complete.
Select Finish Step.
If the error is related to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step, there are two places to check for log file information:
If the step does not start, check the NovaSeqIntegrator.log file written to:
This log file is written to this path wherever the remote extensions RPM was installed.
If the step starts but does not complete, open the step in Clarity LIMS (it can be found via a search). On the Record Details screen, the log file is attached to a placeholder called Log File. If you are unable to reach the Record Details screen, or if the file does not contain an error, review the NovaSeqIntegrator.log described previously.
Contact Illumina Support and supply the relevant information from the troubleshooting steps already performed.
type a value between 1 and 251 for the SP flow cell type or between 1 and 151 for all other types
Read 2 Cycles
Presets
251
151
101
51
type a value between 0 and 251 for the SP flow cell type or between 1 and 151 for all other types
Use Custom Read 1 Primer
Select if applicable.
Use Custom Read 2 Primer
Select if applicable.
Use Custom Index Read 1 Primer
Select if applicable.
Reverse Complement Workflow
Select Yes (by default) for v1.5 reagents in NovaSeq Control Software v1.7 and onwards. Select No for v1 reagents.
UMI - Read 1 Length
Enter the length of the Unique Molecular Identifiers (UMI) in Read 1. Leave blank if not applicable.
UMI - Read 2 Length
Enter the length of the Unique Molecular Identifiers (UMI) in Read 2. Leave blank if not applicable.
UMI - Read 1 Start From Cycle
Enter the cycle number that Read 1 starts from. Leave blank if not applicable.
UMI - Read 2 Start From Cycle
Enter the cycle number that Read 2 starts from. Leave blank if not applicable.
Sample Sheet Path
Automatically populated after the sample sheet has been generated.
Presets
GenerateFASTQ
Workflow Type
Presets
No Index
Single Index
Dual Index
Index Read 1
Presets
0
6
8
type a value between 0 and 20
Index Read 2
Presets
0
6
8
type a value between 0 and 20
Paired End
Presets
True
False
Read 1 Cycles
Presets
251
151
101
51
type a value between 1 and 251 for the SP flow cell type or between 1 and 151 for all other types
Read 2 Cycles
Presets
251
151
101
51
type a value between 0 and 251 for the SP flow cell type or between 1 and 151 for all other types
Use Custom Read 1 Primer
Select if applicable.
Use Custom Read 2 Primer
Select if applicable.
Use Custom Index Read 1 Primer
Select if applicable.
Reverse Complement Workflow
Select Yes (by default) for v1.5 reagents in NovaSeq Control Software v1.7 and onwards. Select No for v1 reagents.
UMI - Read 1 Length
Enter the length of the Unique Molecular Identifiers (UMI) in Read 1. Leave blank if not applicable.
UMI - Read 2 Length
Enter the length of the Unique Molecular Identifiers (UMI) in Read 2. Leave blank if not applicable.
UMI - Read 1 Start From Cycle
Enter the cycle number that Read 1 starts from. Leave blank if not applicable.
UMI - Read 2 Start From Cycle
Enter the cycle number that Read 2 starts from. Leave blank if not applicable.
Sample Sheet Path
Automatically populated after the sample sheet has been generated.
Library Tube Barcode
Scan the library tube barcode.
Configuration
The Illumina NovaSeq 6000 Integration Package v2.6 supports the integration of Clarity LIMS to NovaSeq 6000 instruments.
This document describes the integration and includes information about the preconfigured protocols, steps, automations, installed components, configuration requirements, rules, and constraints.
The configuration provided in this integration has been established to support NovaSeq 6000 lab processes. Any configuration changes to protocols or workflows—including renaming protocols, steps, and fields—could break the process.
Prerequisites and Assumptions
It is assumed that samples enter the NovaSeq 6000 v2.3 workflow as normalized libraries. That is, before they are assigned to the workflow the following actions occur:
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
The Illumina NovaSeq 6000 Integration Package v2.6.0 includes two workflows:
Included for validation purposes only, this protocol models the library prep steps required to advance samples to the Run Format (NovaSeq 6000 v2.3) protocol.
NovaSeq 6000 v2.3 Workflow
Protocol 1: Run Format (NovaSeq 6000 v2.3)
Purpose:
Allows for the assignment of per sample values for Loading Workflow Type, Normalized Molarity, Flowcell Type, and Final Loading Concentration (pM).
Protocol 2: NovaSeq Standard (NovaSeq 6000 v2.3)
Purpose:
Samples are pooled and added to the library tube in preparation for the NovaSeq run. Sample sheet and run recipe files are generated.
Protocol 3: NovaSeq Xp (NovaSeq 6000 v2.3)
Purpose:
Samples are pooled and added to lanes on the NovaSeq flow cell. The flow cell type is determined by the option selected in the Define Run Format (NovaSeq 6000 v2.3) step. Sample sheet and run recipe files are generated.
Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq6000 v2.3)
Purpose:
All samples complete the workflow by going through this protocol.
This protocol contains one fully automated step.
Validation Workflow
The NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) workflow allows for validation of the system after installation is complete. For details, refer to .
Protocol 1: Run Format (NovaSeq 6000 v2.3)
This protocol sets the Loading Workflow Type and allows you to choose the appropriate Flowcell Type and Final Loading Concentration(pM). At the end of the protocol, a routing script sends the normalized libraries to either the NovaSeq Standard (NovaSeq 6000 v2.3) or the NovaSeq Xp (NovaSeq 6000 v2.3) protocol.
This protocol contains one step: Define Run Format (NovaSeq 6000 v2.3).
Step 1: Define Run Format (NovaSeq 6000 v2.3)
Step input: NTP (normalized libraries)
Step output: None
Set Next Steps Automation
Automatically triggered on exit of the Record Details screen, this automation does the following actions:
Sets the next step for samples to REMOVE with
Calculates the Minimum Molarity using the following formula:
Routing Script Automation
Automatically triggered on exit of the step, this automation invokes the changeWorkflow script, which routes step inputs appropriately.
Samples with Loading Workflow Type field value of NovaSeq Standard are routed to the NovaSeq 6000 v2.3 workflow and queued for the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3) step.
Master Step Fields
The following table provides field configuration details for the fields defined on the Define Run Format (NovaSeq 6000 v2.3) step.
Define Run Format (NovaSeq 6000 v2.3) Master Step Field Configuration
Global Fields
The following table lists the global fields that are configured to display on the Define Run Format (NovaSeq 6000 v2.3) step.
Global Field Configuration (Derived Sample)
Protocol 2: NovaSeq Standard (NovaSeq 6000 v2.3)
Samples are routed to this protocol if the Loading Workflow Type value is set to NovaSeq Standard. Samples are pooled and added to a library tube in preparation for the NovaSeq run.
At the end of this protocol, a routing script sends the library tube to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) protocol.
This protocol contains two steps:
Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3)
Dilute and Denature (NovaSeq 6000 v2.3)
Step 1: Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3)
In this step, manually place libraries into a single pool. Resuspension buffer and reagents are added.
Only create one pool per step.
Step input: NTP (normalized libraries)
Step output: Bulk pool
The following automations are configured on the step (in the order they are triggered at run time).
Validate Inputs Flowcell Type and Single Pool Automation
Automatically triggered on exit of the Pooling screen, this automation checks the following information:
All samples in the pool have the same Flowcell Type assigned to them.
Calculate Volumes Automation
Due to the 4000-character limit, part of the script definition of Calculate Volumes has been moved into the Calculate Volumes Script field. If this part of the script needs to be modified, you can make the changes to the default value of the Calculate Volumes Script field. The parts of the script that were moved into the Calculate Volumes Script field are identified below.
Automatically triggered when the Calculate Volumes button on the Record Details screen is selected, this automation does the following actions:
Calculates the number of samples in the pool:
Set Next Step Automation
Automatically triggered on exit of the Record Details screen, the following automation sets the next step for samples to ADVANCE, advancing them to the Dilute and Denature (NovaSeq 6000 v2.3) step in the protocol:
Validate Unique Indexes Automation
ℹ Not used. This functionality is handled by the Clarity LIMS configuration for pooling
Master Step Fields
The following table provides field configuration details for the fields defined on the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3) step.
Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3) Master Step Field Configuration
Calculate Volumes Script
Global Fields
The following table lists the global fields that are configured to display on the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3) step.
Global Field Configuration (Derived Sample)
Step 2: Dilute and Denature (NovaSeq 6000 v2.3)
In this step, pooled samples are denatured and diluted by the addition of NaOH, Tris-HCl, and Resuspension Buffer (RSB). Samples are manually placed into the library tube that is used in the NovaSeq run. In addition, this step generates the sample sheet file and the run recipe (*json file) needed to start the NovaSeq run. The step input is Bulk pool, and the step output is Library tube.
Validate Single Input Automation
Automatically triggered at the beginning of the step, the following automation checks that there is only one container input to the step:
Validate Library Tube Barcode Automation
Automatically triggered on exit of the Placement screen, the following automation validates the library tube barcode to make sure it conforms to the barcode mask [A-Z]{2}[0-9]{7}-[A-Z]{3}:
Generate Sample Sheet & Run Recipe Automation
Automatically triggered when a button on the Record Details screen is selected, this automation does the following actions:
Copies the Flowcell Type from the step input to the step output:
Prepare Files for NovaSeq Automation
Automatically triggered on exit of the Record Details screen, this automation invokes the copy_attachments_to_network_folder script, which is included in the novaseq-remote-extensions.jar file. This script places the generated sample sheet and run recipe (*json file) into the appropriate folder on the NAS, where the instrument uses them to start the run.
For more information, refer to .
Routing Script Automation
Automatically triggered on exit of the step, this automation invokes the changeWorkflow script, which routes step outputs to the NovaSeq 6000 v2.3 workflow, and queues them for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step.
The default automation command line is as follows.
Master Step Fields
The following table provides field configuration details fields defined on the Dilute and Denature (NovaSeq 6000 v2.3) step. These fields are required for sample sheet and JSON file generation.
Dilute and Denature (NovaSeq 6000 v2.3) Master Step Field Configuration
Global Step Fields
The following table lists the global step fields that are configured to display on the Dilute and Denature (NovaSeq 6000 v2.3) step.
Global Field Configuration (Derived Sample)
Protocol 3: NovaSeq Xp (NovaSeq 6000 v2.3)
Samples are routed to this protocol if the Loading Workflow Type value is set to NovaSeq Xp.
Samples are pooled and added to lanes on the NovaSeq flow cell type selected in the Define Run Format (NovaSeq 6000 v2.3) step.
At the end of the protocol, a routing script sends the flow cell to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) protocol.
This protocol contains three steps:
Step 1: Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3)
Step 1: Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3)
In this step, the libraries are manually placed into a pool. The step input is NTP (normalized libraries) and the step output is Bulk pool.
Only create one pool per step.
These automations are described in the order in which they are triggered at run time.
Validate Inputs Flowcell Type and Single Pool Automation
Automatically triggered on exit of the Pooling screen, this automation checks the following information:
All samples in the pool have the same Flowcell Type assigned to them.
Calculate Volumes Automation
Automatically triggered when the Calculate Volumes button on the Record Details screen is selected, this automation:
Calculates the number of samples in the pool:
Sets the value of the Bulk Pool Volume (ul) and PhiX Volume (ul) field, based on the selected Flowcell Type:
Calculates the Per Sample Volume (ul) to be added to the pool:
ℹ To ensure accurate pipetting of each sample in a pool for sequencing, the Per Sample Volume (ul) value must be equal to or higher than the Minimum Per Sample Volume (ul). The default value set at 5 and can be edited.
Assuming the default Minimum Per Sample Volume (ul) value of 5, for a given batch:
If the smallest Per Sample Volume (ul) value is less than 5, Clarity LIMS automatically assigns a value of 5 to the sample's Adjusted Per Sample Volume (ul) field.
Set Next Step Automation
Automatically triggered on exit of the Record Details screen, this automation does the following actions:
Copies the Flowcell Type values from the step inputs to the step outputs.
Sets the next step for samples to ADVANCE, advancing them to the next step in the protocol:
Validate Unique Indexes Automation
ℹ Not used. This functionality is handled by the Clarity LIMS configuration for pooling.
Master Step Fields
The following table provides field configuration details for the fields defined on the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3) step.
Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3) Master Step Field Configuration
Global Fields
The following table lists the global fields that are configured to display on the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3) step.
In this step, pooled samples are denatured and diluted by the addition of DPX, NaOH, Tris-HCl, and RSB. Manually create working pools based on the number of lanes you want to sequence. The step input is Bulk pool and the step output is Working pool. Working pool is a variable number for how many working pools need to be created per bulk pool.
These automations are described in the order in which they are triggered at run time.
Validate Inputs Flowcell Type Automation
Automatically triggered at the beginning of the step, this automation invokes the validate_flowcell_for_input_pools script. This script does the following actions:
Checks the inputs to the step and validates that the Flowcell Type field has been set to a valid value (SP, S1, S2, or S4). The automation also checks that all inputs have the same value.
Calculate Volumes Automation
Automatically triggered on entry to of the Record Details screen, this automation sets the value of the following fields, based on the Flowcell Type:
BP Aliquot Volume (ul)
NaOH Volume (ul)
Set Next Step Automation
Automatically triggered on exit of the Record Details screen, this automation:
Copies the Flowcell Type values from the step inputs to the step outputs.
Sets the next step for samples to ADVANCE, advancing them to the next step in the protocol:
Master Step Fields
The following table provides field configuration details for the fields defined on Dilute, Denature & ExAmp (NovaSeq 6000 v2.3) step. These field values are set by a script and are not editable while running the step.
The following table lists the global fields that are configured to display on the Dilute, Denature & ExAmp (NovaSeq 6000 v2.3) step.
Global Field Configuration (Derived Sample)
Step 3: Load to Flowcell (NovaSeq 6000 v2.3)
In this step, scan the flow cell barcode into Clarity LIMS, then manually place the working pools into the lanes of the flow cell that is used in the NovaSeq run. The step input is Working pool and the step output is Flow cell (output containers: S1 and S2 with 2 lanes, and S4 with 4 lanes).
These automations are described in the order in which they are triggered at run time.
Validate Inputs and Selected Container Automation
Automatically triggered at the beginning of the step, this automation invokes the validate_flowcell_for_input_pools and validateSelectedContainer scripts. These scripts validate the step inputs and the container selected, as follows.
Check that the Flowcell Type field has been set to a valid value (S1, S2, S4, or SP) and that all inputs have the same value.
Validate Flowcell Barcode Automation
Automatically triggered on exit of the Placement screen, this automation validates the flow cell barcode scanned into Clarity LIMS, using the following logic:
Generate Sample Sheet & Run Recipe Automation
Automatically triggered when a button on the Record Details screen is selected, this automation:
Copies the Flowcell Type from the step input to the Run Mode field (hidden):
Prepare Files for NovaSeq Automation
Automatically triggered on exit of the Record Details screen, this automation invokes the copy_attachments_to_network_folder script, which is included in the novaseq-remote-extensions.jar file. This script places the generated sample sheet and run recipe (*json file) into the appropriate folder on the NAS, where the instrument uses them to start the run.
For more information, refer to .
Master Step Fields
The following table provides field configuration details for the fields defined on the Load to Flowcell (NovaSeq 6000 v2.3) step.
Load to Flowcell (NovaSeq 6000 v2.3) Master Step Field Configuration
Global Fields
The following table lists the global fields (derived samples) that are configured to display on the Load to Flowcell (NovaSeq 6000 v2.3) step.
Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3)
This final protocol contains one fully automated step, AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3).
Step 1: AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3)
Step input: Library tube from the NovaSeq Standard protocol or flow cell from the NovaSeq Xp protocol.
Step output: Result file and measurement.
Do not add samples to the Ice Bucket or start the step manually. This is a fully automated step. The sequencing service may not update samples correctly if they have been manually started.
In this step, pooled samples are sequenced on the NovaSeq instrument. The run data network folder is monitored by the sequencing service and automation worker to determine when the sequencing run begins and ends.
When a run is initiated on the NovaSeq instrument, the NovaSeq Control Software (NVCS) looks for the sample sheet and run recipe (*.json file) on the shared network drive.
The NVCS copies the sample sheet to the run directory.
The NVCS uses the run recipe to start the sequencing run.
Parse Run Parameters and Generate Link to Run Folder Automation
Automatically triggered on entry to the Record Details screen, this automation does the following actions:
Parses the RunParameters.xml file attached on the Step Setup screen.
Read InterOp Metrics Automation
Automatically triggered when the sequencing service detects the CopyComplete.txt file has been created, this automation parses the InterOp data generated by the run.
Read summary metrics are recorded on the library pool. These values are aggregated across all lanes. Some values — for example Yield PF (Gb) — are summed, while others are averaged.
Set Next Steps Automation
Automatically triggered on exit of the Record Details screen, the following automation sets the next step to ADVANCE and the samples complete the protocol:
Update Lane Number Automation
Automatically triggered on exit of the Step Setup screen, this automation updates the lane number so that Read InterOp Metrics automation script can populate lane metric to the correct lane.
âš Do not disable Update Lane Number automation as it causes run metric parsing to work improperly.
Master Step Fields
The fields defined on the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step are listed in the Clarity LIMS Configuration area (Custom Fields > Master Step Fields tab).
The following table shows how the parsed RunParameters.xml fields map to step fields configured on the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step in Clarity LIMS.
Fields Parsed by parse_novaseq_run_parameters script
Global Fields
The global fields that are configured to support the parsing of key metrics back into Clarity LIMS are as follows.
Yield PF (Gb) R1
Yield PF (Gb) R2
Reads PF (M) R1
Reads PF (M) R2
Components Installed
The following sections describe the various components that are installed by default as part of this integration. These components include files, properties, reagent categories/label groups, reagent kits, and containers. Information on installed workflows, protocols, steps, and automation points is provided in the previous section.
The Illumina NovaSeq 6000 Integration v2.6.0 Package is distributed as two RPM packages:
BaseSpaceLIMS-novaseq-extensions
BaseSpaceLIMS-novaseq-sequencing-service
The RPM packages install the components listed in the following table.
The NovaSeq 6000 Integration requires installation of NGS Extensions Package v5.25.0 or later.
If the NGS Extensions Package is not already installed, or if a version earlier than v5.25.0 is installed, the latest version is installed by default with the NovaSeq integration. For details, refer to .
If NGS Extensions Package v5.25.0 is already installed, upgrade is not forced or required.
Secret Util Package is required for the NGS Extensions Package v5.25.0.
Refer to for the properties installed with the integration package.
Label Groups, Reagent Kits, and Containers
The NovaSeq 6000 Integration v2.6.0 Package installs the following label groups, reagent kits, and container types.
Label Groups
TruSeq HT Adapters v2 (D7-D5)
Reagent Kits
DPX1
DPX2
DPX3
Resuspension Buffer (RSB)
Container Types
Library Tube
S1
S2
S4
Instrument Integration
The following are instructions for configuring the NovaSeq instrument for the Clarity LIMS integration:
Launch NovaSeq Control Software.
On the home screen, select the menu (top left) and select Settings.
Select File-Based and enter the network folder location that NovaSeq Control Software retrieves the JSON recipe from.
For more information, refer to the NovaSeq 6000 Sequencing System Guide at .
Routing Script Requirements
The requirements for the routing script functionality are as follows.
On the steps that use the routing script — Define Run Format (NovaSeq 6000 v2.3) and Dilute and Denature (NovaSeq 6000 v2.3) — the Next Step for all samples must be set to Remove from workflow. This value is set by a script. Do not change this value in the Assign Next Steps screen.
In the protocol configuration screen, the following setting is required and must not be changed.
In the Next Steps section for the last step in the protocol, the method of assigning the next step must be set to Automatic.
Sample Sheet and Run Recipe File Generation
Sample sheet and run recipe file generation occurs on the step before the run—Dilute and Denature (NovaSeq 6000 v2.3) in the NovaSeq Standard protocol or Load to Flowcell (NovaSeq 6000 v2.3) in the NovaSeq Xp protocol. This step is where samples are placed on the library tube or flow cell that is loaded in the NovaSeq instrument.
In the default configuration, the Generate Sample Sheet & Run Recipe automation generates the following:
One CSV format sample sheet file for use bcl2fastq v2.20 downstream analysis
One JSON format run recipe file
The Prepare Files for NovaSeq automation places the generated files on the NAS where the instrument uses them to start the run.
How It Works
The Prepare Files for NovaSeq automation command line invokes the copy_attachments_to_network_folder script in novaseq-remote-extensions.jar. The script can be used on a remote AI node/automation worker, in case the Clarity LIMS server does not have direct access to the NAS.
The output folder for the run recipe file is found in the novaseq.runSetupFolder property.
Sample Sheet Contents
The contents of the sample sheet are controlled by the following fields configured on the Dilute and Denature (NovaSeq 6000 v2.3) and Load to Flowcell (NovaSeq 6000 v2.3) steps:
Experiment Name
Read 1 Cycles
Run Recipe Contents
The contents of the run recipe are controlled by the following fields. These fields are configured on the Dilute and Denature (NovaSeq 6000 v2.3) and Load to Flowcell (NovaSeq 6000 v2.3) steps.
Experiment Name
Run Mode
Rules and Constraints
The sample sheet and run recipe files are mandatory inputs for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step. For the automated run to start successfully, select the Generate Sample Sheet & Run Recipe button to generate these files.
The output folders must exist on the system where the script runs (can be a remote AI node/automation worker).
Container Types
The integration supports the following container types:
Library tube with barcode provided in the following format:
[A-Z]{2}[0-9]{7}-[A-Z]{3}
Example — NV1234567-LIB
Rules and Constraints
The workflow configuration contains several validation checks. To make sure that the calculations work properly, it is important that you do not disable any of this validation logic. The validation checks the following information:
Which samples, and how many, can enter each step together.
Which samples, and how many, can be pooled together.
The protocol contains a single step: NovaSeq Validation Library Prep (NovaSeq 6000 v2.3). At the end of this step, a routing script sends the samples to the first step of the NovaSeq 6000 v2.3 workflow, which is Define Run Format (NovaSeq 6000 v2.3).
Loading Workflow Type: Select from NovaSeq Standard or NovaSeq Xp.
Normalized Molarity: Enter a value for each sample.
Flowcell Type: Select from options S1, S2, S4, and SP.
Final Loading Concentration (pM): Select from the options 225 (PCR-free workflows) or 400 (Nano workflows). Alternatively, enter a different value.
Compares the Normalized Molarity value of each sample with the Minimum Molarity value.
Routing script sends samples to the NovaSeq Standard or NovaSeq Xp protocol, according to the selected Loading Workflow Type. Samples with Normalized Molarity less than Minimum Molarity are removed from the workflow.
Steps:
Define Run Format (NovaSeq 6000 v2.3)
Routing script sends library tube to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) protocol.
Steps:
Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3)
Dilute and Denature (NovaSeq 6000 v2.3)
Samples are queued for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) protocol.
Steps:
Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3)
Dilute, Denature & ExAmp (NovaSeq 6000 v2.3)
Load to Flowcell (NovaSeq 6000 v2.3)
âš Do not add samples to the Ice Bucket or start the step. The integration does this action automatically.
Steps:
AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3)
Checks Normalized Molarity value. For samples with no Normalized Molarity value (e.g., an empty value, not including 0), the automation generates an error message stating that the field cannot be empty:
Compares the Normalized Molarity value of each sample with the Minimum Molarity value. If Normalized Molarity value is lower than the Minimum Molarity value, sets the Loading Workflow Type of the sample to [Remove from workflow] and records a message in the Warning field:
At this point, there are two options:
Correct the Normalized Molarity value on the Record Details screen. Also, edit the Loading Workflow Type field and set it to NovaSeq Standard or NovaSeq Xp, as applicable.
Complete the protocol without correcting the Normalized Molarity value. In this case, those samples are removed from the workflow.
Samples with Loading Workflow Type field value of NovaSeq Xp are routed to the NovaSeq 6000 v2.3 workflow and queued for the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3) step.
The default automation command line is as follows.
Loading Workflow Type
Text Dropdown
Required Field
ℹ Hidden (does not show on Record Details screen)
Presets
NovaSeq Standard
NovaSeq Xp
Loading Workflow Type
Text Dropdown
Required Field
Presets
NovaSeq Standard
NovaSeq Xp
Minimum Molarity (nM)
Numeric
Decimal places displayed = 2
Normalized Molarity (nM)
Numeric
Decimal places displayed = 2
Per Sample Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Warning
Text Dropdown
Read Only
Custom Entries
Presets
The Normalized Molarity (nM) is too low.
n/a
Only one pool has been created.
Sets the value of the Bulk Pool Volume (ul) and PhiX Volume (ul) fields, based on the selected Flowcell Type. The following part of the automation is found in the Calculate Volumes Script field:
Calculates the Per Sample Volume (ul) to be added to the pool. The following part of the automation is found in the Calculate Volumes Script field:
ℹ To ensure accurate pipetting of each sample in a pool for sequencing, the Per Sample Volume (ul) value must be equal to or higher than the Minimum Per Sample Volume (ul). The default value is set at 5, which can be edited.
Assuming the default Minimum Per Sample Volume (ul) value of 5, for a given batch:
If the smallest Per Sample Volume (ul) value is less than 5, Clarity LIMS automatically assigns a value of 5 to the Adjusted Per Sample Volume (ul) field.
Clarity LIMS then adjusts the Adjusted Per Sample Volume (ul) field value for all other samples in the batch, based on the ratio used to increase the lowest value to 5.
Calculates the Total Sample Volume (ul) field value:
If the Total Sample Volume is less than the Bulk Pool Volume, calculates the RSB Volume (ul) field value:
Copies the Flowcell Type and Loading Workflow Type values from the step inputs to the step outputs:
Sets the Volume of Pool to Denature (ul) value and calculates NaOH Volume (ul) and Tris-HCl Volume (ul) values, based on the Flowcell Type:
Uses the NovaSeq_Standard_Bulk_Pool1.csv, NovaSeq_Standard_Bulk_Pool2.csv, and NovaSeq_Standard_Bulk_Pool3.csv template files to generate a single CSV file containing information about the pool and the samples it contains. The generated file is available for download on the Step Setup screen of the following step: Dilute and Denature (NovaSeq 6000 v2.3).
Resets the Total Sample Volume (ul) and Number of Samples in Pool field values so that the automation is idempotent:
Number of Flowcells to Sequence
Numeric
Required Field
Range = 1–10
Decimal places displayed = 0
Number of Samples in Pool
ℹ For calculation purposes, not displayed
Numeric
Default
0
Decimal places displayed = 0
PhiX Volume (ul)
ℹ For calculation purposes, not displayed
Numeric
Decimal places displayed = 2
Total Sample Volume (ul)
ℹ For calculation purposes, not displayed
Numeric
Default
0
Decimal places displayed = 2
Calculate Volumes Script
Text
Required Field
Read Only
Default value provided in the drop-down section following the table.
âš Do not remove this field. It is used by the Calculate Volumes automation script.
RSB Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Tris-HCl Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Volume of Pool to Denature (ul)
ℹ Used in Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3) step only. Displays on Record Details screen and in the generated CSV file.
Numeric
Read Only
Decimal places displayed = 0
Copies the Flowcell Type from the step input to the Run Mode field (hidden):
Copies the Loading Workflow Type values from the step inputs to the step outputs:
Validates the parameters entered on the Record Details screen:
Experiment Name can only contain alphanumeric, dash, or underscore characters. Spaces are not permitted.
When Workflow Type is No Index, Index Read 1 must be zero. For any other Workflow Type, Index Read 1 must be greater than zero.
When Paired End is True, Index Read 1 and Index Read 2 must be greater than zero.
When Paired End is False, Index Read 2 must be zero, Reverse Complement Workflow must be false, UMI - Read 2 Length must not have a value, and UMI - Read 2 Start from Cycle must not have a value.
Validates allowed read cycles to be not greater than 151 if the Flowcell Type is not SP.
Validates allowed value for UMI - Read 1 Length, UMI - Read 2 Length, UMI - Read 1 From Cycle, UMI - Read 2 From Cycle.
Sets the next step for samples to REMOVE.
Generates the sample sheet and the run recipe file and attaches them to the step. For details on how the files are generated and their contents, refer to Sample Sheet and Run Recipe File Generation.
Index Read 2
Numeric Dropdown
Required Field
Custom Entries
Range = 0–20
Decimal places displayed = 0
Presets
Paired End
Text Dropdown
Required Field
Presets
True
False
Read 1 Cycles
Numeric Dropdown
Required Field
Custom Entries
Range = 1–251
Decimal places displayed = 0
Presets
Read 2 Cycles
Numeric Dropdown
Required Field
Custom Entries
Range = 0–251
Decimal places displayed = 0
Presets
Reverse Complement Workflow
Toggle Switch
Required Field
Default
Yes
Run Mode
ℹ Not displayed in user interface
Text Dropdown
Read Only
Presets
SP
S1
Sample Sheet Path
Text
Read Only
UMI - Read 1 Length
Numeric
Range = 1
Decimal places displayed = 0
UMI - Read 1 Start From Cycle
Numeric
Range = 1
Decimal places displayed = 0
UMI - Read 2 Length
Numeric
Range = 1
Decimal places displayed = 0
UMI - Read 2 Start From Cycle
Numeric
Range = 1
Decimal places displayed = 0
Use Custom Index Read 1 Primer
Toggle Switch
Default
No
Use Custom Read 1 Primer
Toggle Switch
Default
No
Use Custom Read 2 Primer
Toggle Switch
Default
No
Workflow
Text
Read Only
Default
GenerateFASTQ
Workflow Type
Text Dropdown
Required Field
Presets
No Index
Single Index
Only one pool has been created.
Clarity LIMS then adjusts the Adjusted Per Sample Volume (ul) field value for all other samples in the batch, based on the ratio used to increase the lowest value to 5.
Calculates the Total Sample Volume (ul) field value:
If the Total Sample Volume is less than the Bulk Pool Volume, calculates the RSB Volume (ul) field value:
Copies the Flowcell Type and Loading Workflow Type values from the step inputs to the step outputs:
Uses the NovaSeq_Xp_Bulk_Pool.csv and NovaSeq_Xp_Bulk_Pool2.csv template files to generate a single CSV file containing information about the bulk pool and the samples it contains. The generated file is available for download on the Step Setup screen of the following step—Dilute, Denature & ExAmp (NovaSeq 6000 v2.3).
Resets the Total Sample Volume (ul) and Number of Samples in Pool field values so that the automation is idempotent:
Number of Lanes to Sequence
Numeric
Required Field
Decimal places displayed = 0
Number of Sample in Pool
ℹ For calculation purposes, not displayed
Numeric
Default
0
Decimal places displayed = 0
PhiX Volume (ul)
ℹ For calculation purposes, not displayed
Numeric
Decimal places displayed = 2
Total Sample Volume (ul)
ℹ For calculation purposes, not displayed
Numeric
Default
0
Decimal places displayed = 0
Validates that the number of outputs matches the number of lanes on the selected flow cell type. If validation fails, an error message state that the number of working pools does not match the number of lanes available on the flow cell.
Tris-HCl Volume (ul)
DPX1 Volume (ul)
DPX2 Volume (ul)
DPX3 Volume (ul)
Mastermix per Lane (ul)
Copies the Flowcell Type and Loading Workflow Type values from the step inputs to the step outputs:
Uses the NovaSeq_Xp_Working_Pool.csv and NovaSeq_Xp_Working_Pool2.csv template files to generate a single CSV file containing information about the DPX volume and the volume of BP Aliquot, Mastermix, NaOH, and Tris-HCI to add per working pool. The generated file is available for download on the Step Setup screen of the following step—Load to Flowcell (NovaSeq 6000 v2.3).
Mastermix per Lane (ul)
Numeric
Read Only
Decimal places displayed = 0
NaOH Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Tris-HCl Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Check that the number of outputs matches the number of lanes on the selected flow cell type. If validation fails, an error message states that the number of working pools does not match the number of lanes available on the flow cell.
The following defines the number of samples allowed for different flow cell types:
SP: 2 working pools
S1: 2 working pools
S2: 2 working pools
S4: 4 working pools
Check that the container type selected on entry to the Placement screen matches the value in the Flowcell Type field. If validation fails, an error message displays.
Validates the parameters entered on the Record Details screen:
Experiment Name can only contain alphanumeric, dash, or underscore characters. Spaces are not permitted.
When Workflow Type is No Index, Index Read 1 must be zero. For any other Workflow Type, Index Read 1 must be greater than zero.
When Paired End is True, Index Read 1 and Index Read 2 must be greater than zero.
When Paired End = False, Index Read 2 must be zero. Reverse Complement Workflow must be false. UMI - Read 2 Length and UMI - Read 2 Start from Cycle must not have a value.
Validates allowed read cycles to be not greater than 151 if the Flowcell Type is not SP.
Validates allowed value for UMI - Read 1 Length, UMI - Read 2 Length, UMI - Read 1 From Cycle, UMI - Read 2 From Cycle.
Copies the Flowcell Type value from the step inputs to the step outputs:
Sets the next step for samples to ADVANCE, advancing them to the next step in the protocol—AUTOMATED - Run (NovaSeq 6000) v2.3.
Generates the sample sheet and the run recipe file and attaches them to the step. For details on how the files are generated, and their contents, refer to Sample Sheet and Run Recipe File Generation.
Index Read 2
Numeric Dropdown
Required Field
Custom Entries
Range = 0–20
Decimal places displayed = 0
Presets
Library Tube Barcode
Text
Required Field
Paired End
Text Dropdown
Required Field
Presets
True
False
Read 1 Cycles
Numeric Dropdown
Required Field
Custom Entries
Range = 1–251
Decimal places displayed = 0
Presets
Read 2 Cycles
Numeric Dropdown
Required Field
Custom Entries
Range = 0–251
Decimal places displayed = 0
Presets
Reverse Complement Workflow
Toggle Switch
Required Field
Default
Yes
Run Mode
ℹ Not displayed in user interface
Text Dropdown
Read Only
Presets
SP
S1
Sample Sheet Path
Text
Read Only
UMI - Read 1 Length
Numeric
Range = 1
Decimal places displayed = 0
UMI - Read 1 Start From Cycle
Numeric
Range = 1
Decimal places displayed = 0
UMI - Read 2 Length
Numeric
Range = 1
Decimal places displayed = 0
UMI - Read 2 Start From Cycle
Numeric
Range = 1
Decimal places displayed = 0
Use Custom Index Read 1 Primer
Toggle Switch
Default
No
Use Custom Read 1 Primer
Toggle Switch
Default
No
Use Custom Read 2 Primer
Toggle Switch
Default
No
Workflow
Text
Read Only
Default
GenerateFASTQ
Workflow Type
Text Dropdown
Required Field
Presets
No Index
Single Index
When the run starts:
The instrument software creates a new run folder (named <libraryID>) on the shared network drive and copies the following files into that folder:
RunParameters.xml
RunInfo.xml
LIMS/<libraryID>.json file (run recipe)
The Real-Time Analysis v3 (RTA3) software creates the InterOp folder.
The sequencing service detects the presence of the RunParameters, RunInfo, and JSON files and starts the AUTOMATED- NovaSeq Run (NovaSeq 6000 v2.3) step in Clarity LIMS.
As the run progresses, the InterOp data files are filled in.
At the end of the run, the NovaSeq:
Copies the run data files to the InterOp folder.
Creates the CopyComplete.txt file in the run folder. This step indicates to the sequencing service that the run has completed and the data files are ready.
The sequencing service triggers the Read InterOp Metrics automation. This automation reads the files, records the parsed metrics into Clarity LIMS, and finally completes the step in Clarity LIMS.
Populates the master and global step fields with extracted values.
If there is an instrument in Clarity LIMS that matches the instrument name in the file (InstrumentName field), the script associates this instrument with the run.
Generates and attaches a link to the run folder using the run output folder parsed from RunParameters.xml.
The following rules and constraints are present:
If there is no InstrumentName value in the file, the script continues, but it logs a warning.
If there is an InstrumentName value in the file and multiple instruments with the same name exist in Clarity LIMS, the script fails and logs an error.
If the RunParameters.xml file is not attached to the step during step setup, the script fails.
Every RunParameters.xml field that has an equivalent step UDF/custom field in Clarity LIMS is required — except for InstrumentName. If a field is not present in the RunParameters.xml file, Clarity LIMS logs an error to this effect.
Per lane metrics are recorded on the measurement outputs.
The read_interop_metrics script loads data from the InterOp files and records summary metrics into derived sample UDFs/custom fields in Clarity LIMS. If any field is not configured or cannot be written to, the script fails.
PE Serial Barcode
ClusterPartNumber
PE Part Number
ClusterLotNumber
PE Lot Number
ClusterExpirationdate
PE Expiration Date
ClusterCycleKit
PE Cycle Kit
SbsSerialBarcode
SBS Serial Barcode
SbsPartNumber
SBS Part Number
SbsLotNumber
SBS Lot Number
SbsExpirationdate
SBS Expiration Date
SbsCycleKit
SBS Cycle Kit
BufferSerialBarcode
Buffer Serial Barcode
BufferPartNumber
Buffer Part Number
BufferLotNumber
Buffer Lot Number
BufferExpirationdate
Buffer Expiration
OutputRunFolder
Output Folder
WorkflowType
Loading Workflow Type
Cluster Density (K/mm^2) R1
Cluster Density (K/mm^2) R2
%PF R1
%PF R2
% Bases >=Q30 R1
% Bases >=Q30 R2
Intensity Cycle 1 R1
Intensity Cycle 1 R2
% Phasing R1
% Phasing R2
% Prephasing R1
% Prephasing R2
% Aligned R1
% Aligned R
% Error Rate R1
% Error Rate R2
If the Secret Util Package is not already installed, then Secret Util package is installed along with the installation of NGS Extension Package v5.25.0.
If the Secret Util Package is already installed, upgrade is not forced or required.
/opt/gls/clarity/extensions/novaseq/lib
Illumina shared library for parsing InterOp data files.
Sample sheet generation script parameters and usage
Sample sheet data and configuration options
Enabling unique FASTQ file names per sequencing run
Workflow Type
Sample Loading Type (populated based on Workflow field)
Paired End
Read 1
Read 2
Index Read 1
Index Read 2
Sample Sheet Path
BaseSpace Configuration
Use Custom Read 1 Primer
Use Custom Read 2 Primer
Use Custom Index Read 1 Primer
The following fields are also included in the run recipe:
Library Tube Barcode (librarytube_ID in JSON file)
NovaSeq Standard workflow — Library tube barcode is derived from the container name entered or scanned on the Placement screen.
NovaSeq Xp workflow — Library tube barcode is derived from the Library Tube Barcode field in the Load To Flowcell (NovaSeq 6000 v2.3) step.
Flowcell Barcode (flowcell_ID in JSON file)
NovaSeq Standard workflow — Library tube barcode is not output to JSON recipe file.
NovaSeq Xp workflow — Flow cell barcode is derived from the container name entered or scanned on the Placement screen.
Rehyb — preset to False.
Output Folder (output_folder in JSON file) — Derived from the system property novaseq.seqservice.netPathPrefixSearch.
Attachment — Derived from the system property novaseq.sampleSheetPathPrefixSearch with the name of the sample sheet added to the end of the path.
Use BaseSpace (use_basespace in JSON file) — Set to False if the BaseSpace Sequence Hub Configuration step field value is Not Used, and true otherwise.
The output folders must be writable by the glsai user.
Flow cell with barcode provided in one of the following formats:
For SP and S1 — [A-Z0-9]{5}DR[A-Z0-9]{2}
For S2 — [A-Z0-9]{5}DM[A-Z0-9]{2}
For S4 — [A-Z0-9]{5}DS[A-Z0-9]{2}
Example S2 flow cell barcode — H1234DMXX
Reagent labels (indexes) must be unique.
The library tube ID must be unique. There should not be multiple library tube containers in the system with the same name.
Only controls are permitted as unindexed samples. All other unindexed samples and pools are not permitted.
Sample sheet generation script parameters and usage
Sample sheet data and configuration options
Enabling unique FASTQ file names per sequencing run
Do not manually start the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step. This step is fully automated. If the samples have been manually started, the sequencing service may not update them correctly.
The output folder must be readable by the Clarity LIMS glsjboss account on the server.
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-Down Items
Comment
Multiline Text
None
Instruction
Text
Read Only
Default
Add Flowcell Type and Loading Workflow Type below
Flowcell Type
Text Dropdown
Required Field
Custom Entries
ℹ (does not show on Record Details screen)
Presets
S1
S2
S3
S4
Field Name
Field Type
Options
Additional Options and Dropdown Items
Adjusted Per Sample Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Final Loading Concentration (pM)
Numeric Dropdown
Required Field
Custom Entries
Presets
225
400
Decimal places displayed = 0
Flowcell Type
Text Dropdown
Required Field
Presets
S1
S2
S4
SP
Field Name
Field Type
Options
Additional Options and Drop-Down Items
% PhiX (2.5nM) Spike-In
Numeric
Range = 1–100
Decimal places displayed = 0
Bulk Pool Volume (ul)
ℹ For calculation purposes, not displayed
Numeric
Decimal places displayed = 2
Minimum Per Sample Volume (uL)
Numeric
Required Field
Default
5
Decimal places displayed = 2
Field Name
Field Type
Options
Additional Options and Drop-Down Items
Flowcell Type
Text Dropdown
Required Field
Presets
S1
S2
S4
SP
Loading Workflow Type
Text Dropdown
Required Field
Presets
NovaSeq Standard
NovaSeq Xp
[Remove from workflow]
NaOH Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Field Name
Field Type
Options
Additional Options and Drop-Down Items
BaseSpace Sequence Hub Configuration
Text Dropdown
Required Field
Presets
Not Used
Run Monitoring Only
Run Monitoring and Storage
Experiment Name
Text
Required Field
Index Read 1
Numeric Dropdown
Required Field
Custom Entries
Range = 0–20
Decimal places displayed = 0
Presets
0
6
8
Field Name
Field Type
Options
Additional Options and Drop-Down Items
Flowcell Type
Text Dropdown
Required Field
Presets
S1
S2
S4
SP
Loading Workflow Type
Text Dropdown
Required Field
Presets
NovaSeq Standard
NovaSeq Xp
[Remove from workflow]
Field Name
Field Type
Options
Additional Options and Drop-Down Items
% PhiX (0.25nM) Spike-In
Numeric
Range = 0–100
Decimal places displayed = 0
Bulk Pool Volume (ul)
ℹ For calculation purposes, not displayed
Numeric
Decimal places displayed = 2
Minimum Per Sample Volume (ul)
Numeric
Required Field
Default
5
Decimal places displayed = 2
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-Down Items
RSB Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Flowcell Type
Text Dropdown
Required Field
Presets
S1
S2
S4
SP
Loading Workflow Type
Text Dropdown
Required Field
Presets
NovaSeq Standard
NovaSeq Xp
[Remove from workflow]
Field Name
Field Type
Options
Additional Options and Drop-Down Items
DPX1 Volume (ul)
Numeric
Read Only
Decimal places displayed = 0
DPX2 Volume (ul)
Numeric
Read Only
Decimal places displayed = 0
DPX3 Volume (ul)
Numeric
Read Only
Decimal places displayed = 0
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-Down Items
BP Aliquot Volume (ul)
Numeric
Read Only
Decimal places displayed = 0
Flowcell Type
Text Dropdown
Required Field
Presets
SP
S1
S2
S4
Loading Workflow Type
Text Dropdown
Required Field
Presets
NovaSeq Standard
NovaSeq Xp
[Remove from workflow]
Field Name
Field Type
Options
Additional Options and Drop-Down Items
BaseSpace Sequence Hub Configuration
Text Dropdown
Required Field
Presets
Not Used
Run Monitoring Only
Run Monitoring and Storage
Experiment Name
Text
Required Field
Index Read 1
Numeric Dropdown
Required Field
Custom Entries
Range = 0–20
Decimal places displayed = 0
Presets
0
6
8
Field Name
Field Type
Options
Additional Options and Drop-Down Items
Flowcell Type
Text Dropdown
Required Field
Presets
S1
S2
S4
SP
RunParameters.xml Field
Field Name
FlowCellSerialBarcode
Flow Cell ID
FlowCellPartNumber
Flow Cell Part Number
FlowCellLotNumber
Flow Cell Lot Number
FlowCellExpirationdate
Flow Cell Expiration Date
FlowCellMode
Flow Cell Mode
RunId
Run ID
Files Installed
Location
Description
configure_extensions_novaseq.sh
/opt/gls/clarity/config/
Script that imports Clarity LIMS configuration into the application database and updates property values.
novaseq-extensions.jar
/opt/gls/clarity/extensions/novaseq
Jar file containing API-based Clarity LIMS extensions used throughout the protocols.
if(!step.::Experiment Name::.matches(::[a-zA-Z0-9-_]+::)) {
fail(::Experiment Name contains prohibited characters. Allowed characters are: a-z, A-Z, 0-9, -, and _::)
}
if(step.::Workflow Type::== ::No Index::) {
if(step.::Index Read 1::!= 0) {
fail(::Index Read 1 must be 0 if the Workflow Type is No Index.::)
}
} else {
if(step.::Index Read 1::== 0) {
fail(::Index Read 1 must be greater than 0 if the Workflow Type is ::+ step.::Workflow Type::+ ::.::)
}
}
if (step.::Paired End::.toBoolean()) {
if (step.::Read 1 Cycles:: == 0 || step.::Read 2 Cycles:: == 0) {
fail(::Read 1 Cycles and Read 2 Cycles must be greater than 0 if Paired End is True.::);
}
} else {
if (step.::Read 2 Cycles:: != 0) {
fail(::Read 2 Cycles must be 0 if Paired End is False.::);
}
if (step.hasValue(::UMI - Read 2 Length::) || step.hasValue(::UMI - Read 2 Start From Cycle::)) {
fail(::UMI - Read 2 Length and UMI - Read 2 Start From Cycle cannot be defined if Paired End is False.::);
}
}
if (input.::Flowcell Type:: != ::SP:: && step.::Read 1 Cycles:: > 151) {
fail(::Read 1 Cycles must not be larger than 151 if it is not SPrime Flowcell.::);
}
if (input.::Flowcell Type:: != ::SP:: && step.::Read 2 Cycles:: > 151) {
fail(::Read 2 Cycles must not be larger than 151 if it is not SPrime Flowcell.::);
}
if (step.hasValue(::UMI - Read 1 Length::) && !step.hasValue(::UMI - Read 1 Start From Cycle::)) {
fail(::UMI - Read 1 Start From Cycle must be greater than 0 if UMI - Read 1 Length is greater than 0.::);
}
if (!step.hasValue(::UMI - Read 1 Length::) && step.hasValue(::UMI - Read 1 Start From Cycle::)) {
fail(::UMI - Read 1 Length must be greater than 0 if UMI - Read 1 Start From Cycle is greater than 0.::);
}
if (step.hasValue(::UMI - Read 2 Length::) && !step.hasValue(::UMI - Read 2 Start From Cycle::)) {
fail(::UMI - Read 2 Start From Cycle must be greater than 0 if UMI - Read 2 Length is greater than 0.::);
}
if (!step.hasValue(::UMI - Read 2 Length::) && step.hasValue(::UMI - Read 2 Start From Cycle::)) {
fail(::UMI - Read 2 Length must be greater than 0 if UMI - Read 2 Start From Cycle is greater than 0.::);
}
if (!step.hasValue(::UMI - Read 1 Length::) && step.hasValue(::UMI - Read 2 Length::)) {
fail(::UMI - Read 1 Length must be greater than 0 if UMI - Read 2 Length is greater than 0.::);
}
if (!step.hasValue(::UMI - Read 1 Start From Cycle::) && step.hasValue(::UMI - Read 2 Start From Cycle::)) {
fail(::UMI - Read 1 Start From Cycle must be greater than 0 if UMI - Read 2 Start From Cycle is greater than 0.::);
}
if(!step.::Experiment Name::.matches(::[a-zA-Z0-9-_]+::)) {
fail(::Experiment Name contains prohibited characters. Allowed characters are: a-z, A-Z, 0-9, -, and _::)
};
if(step.::Workflow Type::== ::No Index::) {
if(step.::Index Read 1::!= 0) {
fail(::Index Read 1 must be 0 if the Workflow Type is No Index.::)
}
} else{
if(step.::Index Read 1::== 0) {
fail(::Index Read 1 must be greater than 0 if the Workflow Type is ::+ step.::Workflow Type::+ ::.::)
}
};
if (step.::Paired End::.toBoolean()) {if (step.::Read 1 Cycles:: == 0 || step.::Read 2 Cycles:: == 0) {fail(::Read 1 Cycles and Read 2 Cycles must be greater than 0 if Paired End is True.::);}} else {if (step.::Read 2 Cycles:: != 0) {fail(::Read 2 Cycles must be 0 if Paired End is False.::);}
if (step.hasValue(::UMI - Read 2 Length::) || step.hasValue(::UMI - Read 2 Start From Cycle::)) {fail(::UMI - Read 2 Length and UMI - Read 2 Start From Cycle cannot be defined if Paired End is False.::);}}
if (input.::Flowcell Type:: != ::SP:: && step.::Read 1 Cycles:: > 151) {
fail(::Read 1 Cycles must not be larger than 151 if it is not SPrime Flowcell.::);
}
if (input.::Flowcell Type:: != ::SP:: && step.::Read 2 Cycles:: > 151) {
fail(::Read 2 Cycles must not be larger than 151 if it is not SPrime Flowcell.::);
}
if (step.hasValue(::UMI - Read 1 Length::) && !step.hasValue(::UMI - Read 1 Start From Cycle::)) {
fail(::UMI - Read 1 Start From Cycle must be greater than 0 if UMI - Read 1 Length is greater than 0.::);
}
if (!step.hasValue(::UMI - Read 1 Length::) && step.hasValue(::UMI - Read 1 Start From Cycle::)) {
fail(::UMI - Read 1 Length must be greater than 0 if UMI - Read 1 Start From Cycle is greater than 0.::);
}
if (step.hasValue(::UMI - Read 2 Length::) && !step.hasValue(::UMI - Read 2 Start From Cycle::)) {
fail(::UMI - Read 2 Start From Cycle must be greater than 0 if UMI - Read 2 Length is greater than 0.::);
}
if (!step.hasValue(::UMI - Read 2 Length::) && step.hasValue(::UMI - Read 2 Start From Cycle::)) {
fail(::UMI - Read 2 Length must be greater than 0 if UMI - Read 2 Start From Cycle is greater than 0.::);
}
if (!step.hasValue(::UMI - Read 1 Length::) && step.hasValue(::UMI - Read 2 Length::)) {
fail(::UMI - Read 1 Length must be greater than 0 if UMI - Read 2 Length is greater than 0.::);
}
if (!step.hasValue(::UMI - Read 1 Start From Cycle::) && step.hasValue(::UMI - Read 2 Start From Cycle::)) {
fail(::UMI - Read 1 Start From Cycle must be greater than 0 if UMI - Read 2 Start From Cycle is greater than 0.::);
}
