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The Illumina NovaSeq 6000 instrument integration provides preconfigured workflows that map to established lab protocols and steps used with NovaSeq instruments. The integration also supports the associated library prep kits, reagent kits, and assays used in these protocols.
The documents in this section support Illumina NovaSeq 6000 Integration version 3.x.
The Illumina NovaSeq 6000 Integration v3.7.0 supports the integration between Clarity LIMS (v6.3 and later) and the NovaSeq 6000 instrument. Refer to NovaSeq 6000 Integration v3.7.0 Release Notes for updates to this integration.
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.
For details on installed protocols and steps, automations, generated and captured files, and rules and constraints, refer to NovaSeq 6000 Integration v3.7.0 Configuration.
For information on user interaction for each step, validating and troubleshooting the integration, refer to NovaSeq 6000 Integration v3.7.0 User Interaction, Validation and Troubleshooting.
For information on the NovaSeq 6000 instrument system, refer to the NovaSeq 6000 System Support Resources.
NovaSeq 6000 Integration v3.7.0 has the following prerequisites:
NovaSeq Integration v3.7.0 is distributed as the BaseSpaceLIMS-sequencer-api RPM package. This package must be installed on the Clarity LIMS server.
The BaseSpaceLIMS-sequencer-api RPM installs the following files and scripts:
Sequencer API WAR file
application.yml configuration file
configure_sequencer_api_proxy.sh configuration script
configure_sequencer_api_env.sh configuration script
configure_sequencer_api_application.sh configuration script
The RPM installs the Sequencer API WAR file into the same Tomcat container as Clarity LIMS. The WAR file is at
The version number can differ.
The WAR file is deployed automatically when Clarity LIMS launches. Because the RPM installs a new WAR file, Clarity LIMS is automatically stopped during installation of the RPM. Restart Clarity LIMS before installing the NovaSeq and Library Prep Validation workflow configuration from the IPP.
The application.yml configuration file is installed at
As part of the installation process, the file is configured with one of the configuration scripts.
For a complete list of all properties included in the application.yml file and the properties installed into database, refer to Integration Properties Details.
The scripts in the following table are installed at the
During installation, the RPM runs these scripts as they are needed.
If running the sequencing service and Automation Worker on the same instance, the instance must also be running a compatible version of Oracle Linux. For compatibility, refer to NovaSeq 6000 Integration v3.7.0 Release Notes.
NovaSeq 6000 Integration v3.7.0 includes the workflows, protocols, and steps listed in the following table. The workflows are found in IPP v2.9.0 package.
For detailed descriptions of the steps and automations included in each protocol, and details on other components in the configuration, refer to NovaSeq 6000 Integration v3.7.0 Configuration.
For instructions on using the Library Prep Validation v2.3.4 protocol, refer to NovaSeq 6000 Integration v3.7.0 User Interaction, Validation and Troubleshooting.
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.
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).
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 Server-Based.
In the adjacent field, enter the Clarity LIMS URL (e.g., https://customerserver.claritylims.com).
[Optional] In the Output Folder field, enter the default output folder location. You can also specify the output folder location when setting up a run in NVCS.
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.
This section explains how to validate the installation of the Illumina NovaSeq 6000 Integration Package v3.7.0.
The validation process involves the following items:
Running samples through the Library Prep Validation 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 advance to the workflow selected.
Running normalized libraries through the NovaSeq 6000 v3.8 workflow validates the following items:
Successful routing of samples from the Run Format (NovaSeq 6000 v3.8) step to the NovaSeq Standard (NovaSeq 6000 v3.8) or NovaSeq Xp (NovaSeq 6000 v3.8) step.
Automated generation of v1 sample sheet for use with bcl2fastq2 v2.20 analysis software or v2 sample sheet. This file automatically uploads to the sequencing system via the Sequencer API.
Automatic validation of run setup information. Information is uploaded to the NovaSeq Control Software (NVCS) via the Sequencer API and is used to create the run recipe and initiate the run.
Automated tracking of the NovaSeq sequencing run and parsing of run statistics into Clarity LIMS, via the Sequencer API.
The validation steps assume that the Illumina NovaSeq 6000 Integration Package v3.7.0 is installed and you have imported the default Clarity LIMS configuration.
The following steps set up Clarity LIMS in preparation for running samples through the Library Prep Validation and NovaSeq 6000 v3.8 workflows.
On the Configuration tab, under Workflows, activate both the Library Prep Validation and NovaSeq 6000 v3.8 workflows.
Library Prep Validation v2.3.4 is available with Illumina Preset Protocols (IPP) v2.9.
On the Projects and Samples screen, create a project and add samples to it.
Assign the samples to the Library Prep Validation workflow.
This single-step protocol models the library prep required to produce normalized libraries that are ready for the NovaSeq 6000 v3.8 workflow.
Follow the steps in Library Prep Validation Protocol to run the Library Prep Validation workflow with the following:
Label Group = TruSeq HT Adapters v2 (D7-D5)
Sequencing Instrument = NovaSeq 3.0
On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the NovaSeq 6000 v3.8 workflow, Define Run Format (NovaSeq 6000 v3.8). This step is the only one in Protocol 1: Run Format (NovaSeq 6000 v3.8).
This protocol includes a single step, Define Run Format (NovaSeq 6000 v3.8). 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.
In Lab View, locate the Run Format (NovaSeq 6000 v3.8) protocol. The samples are queued for the Define Run Format (NovaSeq 6000 v3.8) step.
Add the samples to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
On the Record Details screen in the Sample Details table, populate the following fields (values can vary across samples):
Loading Workflow Type — Select NovaSeq Standard or NovaSeq Xp from the drop-down list.
Flowcell Type — Select SP, S1, S2, or S4.
Final Loading Concentration (pM) — Select from the two preset options: 225 (for PCR-free workflows), 400 (for Nano workflows), or enter a different value.
Normalized Molarity (nM) — These values are copied over from the previous step. If this column is not populated during library prep, enter the values here.
Select Next Steps, which triggers the Set Next Steps automation and 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.
Calculates the Minimum Molarity.
Checks Normalized Molarity value. For samples with no Normalized Molarity value (an empty value not including 0), generates an error message notifying that the field cannot be empty.
Compares each sample Normalized Molarity value with the Minimum Molarity value.
On the Assign Next Steps screen:
In the Sample Details table, the Next Step for all samples is prepopulated with Remove from workflow, regardless of the Loading Workflow Type.
For samples whose Normalized Molarity value is lower than the Minimum Molarity value, the Loading Workflow Type is set to Remove from workflow. Also, a message is recorded in the Warning field for the sample.
At this point, there are two options:
Return to the Record Details screen and adjust the Normalized Molarity value so that it equals or exceeds the Minimum Molarity value. The Loading Workflow Type must be set to NovaSeq Standard or NovaSeq Xp, as applicable.
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 & Register Step Completed automation is triggered:
Samples whose Loading Workflow Type is set to Remove from workflow are removed from the Clarity LIMS workflow. (Occurs when the Normalized Molarity value is lower than the Minimum Molarity.)
Samples whose Loading Workflow Type is set to NovaSeq Standard are routed to the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8) step. This step is the first one in the NovaSeq Standard protocol.
Samples whose Loading Workflow Type is set to NovaSeq Xp are routed to the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8) step. This step is the first one in the NovaSeq Xp protocol.
In this protocol, samples are pooled and added to the library tube in preparation for the NovaSeq run.
In Lab View, locate the NovaSeq Standard (NovaSeq 6000 v3.8) protocol. Samples for the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8) step are queued.
On the Queue screen, add samples of the same Flowcell Type to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
On the Pooling screen:
Create a pool by dragging samples into the Pool Creator.
Enter a name for the pool or accept the default name (Pool #1).
Select Record Details.
After exiting from the Pooling screen, the Validate Inputs Flowcell Type and Single Pool automation is triggered. The automation verifies the following items:
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 two required fields and one optional field, as needed:
Number of Flowcells to Sequence — Used in volume calculations, to make sure that the volumes are sufficient for the number of times the pool is sequenced.
Minimum Per Sample Volume (ul) — 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 can be edited.
% PhiX (2.5 nM) Spike-In — Used to calculate the volume of PhiX v3 control included in the pool for the given percentage of spike-in. The field is optional and can be edited.
If the smallest Per Sample Volume (ul) value is < 5, Clarity LIMS automatically assigns a value of 5 to the Adjusted Per Sample Volume (ul) field.
Clarity LIMS changes 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 and advances them to the next step in the protocol, Dilute, and Denature (NovaSeq 6000 v3.8).
On the Assign Next Steps screen, the next step for samples is set to the next step in the workflow: Dilute and Denature (NovaSeq 6000 v3.8).
Select Finish Step.
At the end of this step, the pool of samples automatically advances to the Dilute and Denature (NovaSeq 6000 v3.8) step.
In Lab View, locate the NovaSeq Standard (NovaSeq 6000 v3.8) protocol. The pool of samples queued for the Dilute and Denature (NovaSeq 6000 v3.8) step are listed.
Add the samples to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work. The Validate Single Input & Register Step Started automation is triggered. This automation verifies that only one container is input for the step.
On the Placement screen:
Drag the pool into the library tube in the Placed Samples area.
Scan or type the barcode of the library tube into the Library Tube field.
Select Record Details.
After exiting the Placement screen, the Validate Library Tube Barcode automation makes sure that the library tube barcode conforms to the barcode mask [A-Z]{2}[0-9]{7}-[A-Z]{3}. If it does not, an error message displays. The automation also copies the Flowcell Type and Loading Workflow Type values from step inputs to outputs.
On the Record Details screen, the Reagent Lot Tracking section lets you tracks the NaOH, Resuspension Buffer, and Tris-HCl reagents used in the step. Add and activate lots for these reagents. For more information on creating a new lot, 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 on the Record Details screen are used to set up the run and generate the sample sheet. Some of these fields are autopopulated and some must be completed manually.
Fields Displayed on Record Details Screen of Dilute and Denature (NovaSeq 6000 v3.8)
On the Record Details screen, select Validate Run Setup and Generate Sample Sheet. This selection triggers the automation script, which performs the following actions:
Validates the parameters entered on the Record Details screen.
Generates the sample sheet and attaches it to the placeholder in the Files area of the Record Details screen.
Select Next Steps.
On the Assign Next Steps screen, the Next Step for samples is prepopulated with Remove from workflow. The Routing Script automation expects this value, and requires the value to advance samples to the next step.
Select Finish Step.
After exiting the step, the following actions occur:
The Routing Script & Register Step Completed automation is triggered and samples are routed to AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8).
In Lab View, the pool of samples is queued for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step.
At this point in the workflow, the user interaction ends. Proceed to AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8).
In this protocol, samples are pooled and added to lanes on the NovaSeq flow cell. The option selected in the Define Run Format (NovaSeq 6000 v3.8) step determines the flow cell type.
In Lab View, locate the NovaSeq Xp (NovaSeq 6000 v3.8) protocol. The samples are queued for the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8) step.
On the Queue screen, add samples of the same Flowcell Type to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
On the Pooling screen, create and name a pool as follows.
Create a pool by dragging samples into the Pool Creator.
Enter a name for the pool or accept the default name (Pool #1).
Select Record Details.
On exiting the Pooling screen, the Validate Inputs Flowcell Type and Single Pool automation is triggered. The automation verifies the following items:
All samples in the pool are assigned the same Flowcell Type.
Only one pool was created.
On the Record Details screen in the Step Details area, complete the two required fields and one optional field, as needed:
Number of Lanes to Sequence — Used in volume calculations to make sure that volumes are sufficient for the number of times the pool is sequenced.
Minimum Per Sample Volume (ul) — 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 can be edited.
% PhiX (0.25nM) Spike-In — 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.
If the smallest Per Sample Volume (ul) value is < 5, Clarity LIMS automatically assigns a value of 5 to the samples 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. This adjustment is 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. This selection triggers 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 performs 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 Dilute, Denature & ExAmp (NovaSeq 6000 v3.8) step in the protocol.
On the Assign Next Steps screen, the next step for samples is set to the Dilute, Denature & ExAmp (NovaSeq 6000 v3.8) step in the workflow.
Select Finish Step.
At the end of this step, the pool of samples automatically advances to the Dilute, Denature & ExAmp (NovaSeq 6000 v3.8) step.
In Lab View, locate the NovaSeq Xp (NovaSeq 6000 v3.8) protocol. The pool of samples queued for the Dilute, Denature & ExAmp (NovaSeq 6000 v3.8) step displays.
Add the pool to the Ice Bucket and select View Ice Bucket.
[Optional] On the Ice Bucket screen, set the number of derivatives to create (placed into the flow cell lanes) and select Begin Work.
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:
BP Aliquot Volume (ul)
NaOH Volume (ul)
Tris-HCl Volume (ul)
DPX1 Volume (ul)
DPX2 Volume (ul)
DPX3 Volume (ul)
Mastermix per Lane (ul)
The automation also populates the Flowcell Type and Loading Workflow Type columns of the Sample Details table.
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-HCl to add per working pool (see the next step).
On the Record Details screen, the Reagent Lot Tracking section tracks the DPX1, DPX2, DPX3, NaOH, Resuspension Buffer, and Tris-HCl reagents used in the step. These reagents must be added. 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 prepopulated. The script sets the values and they are not editable.
In the Sample Details table, select the pool icon to view details on the working pool composition.
The BP Aliquot, Mastermix per lane, NaOH, and Tris-HCl volume values for each working pool are populated. The script sets the values and they are not editable.
The Flowcell Type and Loading Workflow Type columns are populated.
The working pool number is appended to the bulk pool name, to identify which working pools are derived from the same bulk pool.
In the Files area, select the Calculation File (CSV) to open it and view details on the DPX Mastermix volume and the volume of Mastermix, NaOH, and Tris-HCl to add per working pool.
Select Next Steps. On the Assign Next Steps screen, the next step is already set to Load to Flowcell (NovaSeq 6000 v3.8).
Select Finish Step.
On the Ice Bucket screen:
In the Container Options panel, select the appropriate flow cell type from the Destination Container drop-down list.
Select Begin Work.
The Validate Inputs and Selected Container & Register Step Started automation checks the following:
The Flowcell Type field is set to a valid value (SP, S1, S2, or S4) and that each input has the same value for the field.
The container type selected matches the value in the Flowcell Type field.
The number of outputs matches the number of lanes on the selected flow cell type. If validation fails, an error message reports the number of working pools does not match the number of lanes available on the flow cell.
On the Placement screen:
Drag one or more 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 Flowcell field.
Select Record Details.
After exiting the Placement screen, the Validate Flowcell Barcode automation makes sure that the container barcode conforms to the barcode mask for the selected Flowcell Type (as listed in the following table). This automation also copies the Flowcell Type and Loading Workflow Type field values from step inputs to outputs.
The fields displayed on the Record Details screen are used to set up the run and generate the sample sheet. Some of these fields are autopopulated and some must be entered manually. For details, refer to the following table:
Fields Displayed on Record Details Screen of Load to Flowcell (NovaSeq 6000 v3.8) Step
Select Validate Run Setup and Generate Sample Sheet to trigger the automation script, which does the following:
Validates the parameters entered on the Record Details screen.
Generates the sample sheet and attaches it to the placeholder in the Files area on the Record Details screen.
Select Next Steps.
On the Assign Next Steps screen, the Next Step field for samples is prepopulated with Mark protocol as complete.
Select Finish Step.
At this point in the workflow, the user interaction ends. The flow cell is queued for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step.
This protocol contains a single fully automated step - AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8).
The integration starts the step automatically and data from the run is parsed back into Clarity LIMS. User interaction is not required, but the various stages can be reviewed in Clarity LIMS under Review Run Data.
Automations
The NovaSeq Run (NovaSeq 6000 v3.8) step contains two automations:
Set Next Steps — This automation is disabled (Set to Not Used). By default, the Sequencer API is used to set the next step for output samples.
Update Lane Number — This automation is triggered automatically upon entry to the Record Details screen.
Read summary metrics are recorded for the library pool. After the run is complete, open the step and review these metrics on the Next Steps screen in the Step Data section and the Sample Details table.
Step Data Section
The following values populate in the master step fields:
Run ID
Run Status
Output Folder
Current Cycle
Current Read
Loading Workflow Type
Flow Cell ID
Flow Cell Side
Flow Cell Mode
Flow Cell Part Number
Flow Cell Lot Number
Flow Cell Expiration Date
Instrument ID
Instrument Type
Instrument Control Software Version
Firmware Version
RTA Version
Sequencing Log
Sample Details Table
Summary metrics populate the global fields listed. Values are aggregated across all lanes. Some values (for example Yield PF (Gb) R1) are summed, while others are averaged.
Yield PF (Gb) R1
Yield PF (Gb) R2
Reads PF (M) R1
Reads PF (M) R2
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 R2
% Error Rate R1
% Error Rate R2
The following steps summarize how the Sequencer API integration works. For details on sample sheet generation and the sequence of events involved in the NovaSeq run, refer to NovaSeq 6000 Integration v3.7.0 Configuration.
When setting up a run on the NovaSeq 6000 instrument, NVCS sends a request for the run recipe.
The Sequencer API validates that samples and containers are correctly queued for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step.
Clarity LIMS sends a JSON response to NVCS. The response includes the run recipe information and a link to download the sample sheet (used with analysis software such as bcl2fastq2).
When the run starts, NVCS sends a RunStarted run status request. The Sequencer API then does the following:
Validates reagent kit information in the RunStarted request:
Verifies that the reagent kit exists. If the reagent kit does not exist, the Sequencer API creates and enables it on the master step.
Verifies that the reagent kit is activated. If the reagent kit is not active, the Sequencer API activates it.
Initiates the step for the queued samples. The step produces one output file placeholder per lane of the flow cell in use, based on the flow cell type.
Records all relevant information from the RunStarted request on the step (such as reagent lots and step fields).
At the end of the run, NVCS sends one of the following run status requests:
RunCompletedSuccessfully
RunEndedByUSer
RunErroredOut
The Sequencer API updates the step with any new information, which includes only the status and cycle/read information.
After primary analysis completes, NVCS sends a request containing the parsed run metrics. NVCS sends a request only when the run completed successfully, but the API does not make this assumption and accepts the request regardless of status. The Sequencer API then does the following actions:
Records the metrics into the fields on file placeholder outputs in the LIMS.
Completes the step in Clarity LIMS when the status is RunCompletedSuccessfully. For all other status options, the step remains in progress. This action is the default autocomplete step behavior. For details, refer to NovaSeq 6000 Integration v3.7.0 Configuration.
Sample sheet generation and contents
NovaSeq 6000 run
User authentication and login
Run recipe contents
If an automation trigger does not appear to run its corresponding scripts, refer to the following sections in the Clarity LIMS API documentation:
Troubleshooting Automated Worker in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
Troubleshooting Automation in the Clarity LIMS (API & Database) documentation.
If an error occurs that does not provide direction on how to proceed, complete the following steps:
Confirm the version of the installed Illumina NovaSeq 6000 Integration Package by running the following command on the Clarity LIMS server command line:
If the error is related to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step, review the log file information. For details, refer to Logging.
If the automated run step starts but does not complete, proceed as follows.
Use one of the following methods to open the in progress step in Clarity LIMS:
Log in to the default user account.
In Lab View, find the step in the Recent Activities pane.
Search for the step in Clarity LIMS, using either the Library Tube, or Flowcell barcode as the search term. The steps depend on whether the search is a NovaSeq Standard or NovaSeq Xp run.
On the Record Details screen, the Sequencing Log multiline text field contains logging information. If you are unable to reach the Record Details screen, or if the Sequencing Log field does not contain enough information to resolve the issue, review the sequencer-api.log file. For details, refer to Logging.
Contact Illumina Support. Provide the relevant information from the troubleshooting steps already performed.
In addition to updating the Sequencing Log multiline text field on the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step, the Sequencer API writes a detailed log file to the following location:
Log messages include the Library Tube ID and Flowcell ID whenever the messages are related to a sequencing run-related request. In the LIMS workflows, these IDs are recorded as container names.
Some log messages are not directly related to a sequencing run request, such as downloading the sample sheet. In this case, the file LIMS ID and file name are included in log messages. The sample sheet downloaded by the sequencing system has a name that includes the sequencing container ID (Library Tube for Standard or Flowcell for Xp). The relevant container name search finds most the log messages for this request. However, if all messages for downloading a file are found, the LIMS ID of the file must be known. The LIMS ID is found in the Clarity LIMS API and, for sample sheets used in a run, in the recipe response (also included in the log file).
Example lines from the log file:
The Illumina NovaSeq 6000 Integration Package v3.7.0 supports the integration of Clarity LIMS to NovaSeq 6000 instruments. This documentation describes the integration and includes the following information:
Preconfigured workflows, protocols, steps, and automations
Installed components
Configuration requirements, rules, and constraints
For instructions on user interaction for each step, validating and troubleshooting the NovaSeq 6000 Integration, refer to .
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 process.
Importing either the Library Prep Validation v2.3.4 workflow or the NovaSeq 6000 v3.8 workflows provides a global field named Illumina Universal Sample Identifier. This is a text field that is reserved for CLPA support and is optional. A value is not required for this integration.
It is assumed that samples enter the NovaSeq 6000 v3.8 workflow as normalized libraries. It is assumed that the following steps have completed before samples are assigned to the workflow:
Samples have been accessioned into the 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 .
The Illumina NovaSeq 6000 Integration Package v3.7.0 includes the following workflows:
Library Prep Validation v2.3.4 (optional, but recommended for validation purposes)
NovaSeq 6000 v3.8
This protocol sets the Loading Workflow Type and allows the choice of the appropriate Flowcell Type and Final Loading Concentration (pM). After the protocol, a routing script sends the normalized libraries to either the NovaSeq Standard (NovaSeq 6000 v3.8) or the NovaSeq Xp (NovaSeq 6000 v3.8) protocol.
This protocol contains one step: Define Run Format (NovaSeq 6000 v3.8).
Step input: NTP (normalized libraries)
Step output: None
¹ This automation is for CLPA support only.
² This automation is required for the NovaSeq 6000 v3.8 workflow to function properly. This automation contains additional logic needed for CLPA support. If you would like to remove CLPA support, then contact Illumina Support.
Automations not identified with ¹ or ² are required for the NovaSeq 6000 v3.8 to work function properly.
The following table lists field configuration details defined on the Define Run Format (NovaSeq 6000 v3.8) master step.
Define Run Format (NovaSeq 6000 v3.8) Master Step Field Configuration
The following table lists the global custom fields that are configured to display on the Define Run Format (NovaSeq 6000 v3.8) step.
Define Run Format (NovaSeq 6000 v3.8) Global Field Configuration (Derived Sample)
Samples are routed to this protocol when their 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 v3.8) protocol.
This protocol contains two steps:
Step 1: Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8)
Step 2: Dilute and Denature (NovaSeq 6000 v3.8)
In this step, libraries are placed manually into a single pool. Resuspension buffer and reagents are added.
Step input: NTP (normalized libraries)
Step output: Bulk pool
¹ These automations are for CLPA support only.
Automations not identified with ¹ are required for the NovaSeq 6000 v3.8 to work function properly.
The following table lists configuration details defined on the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8) step.
Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8) Master Step Field Configuration
The following table lists the global custom fields that are configured to display on the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8) step.
Global Custom Fields Configuration (Derived Sample)
In this step, the addition of NaOH, Tris-HCl, and Resuspension Buffer (RSB) denatures and dilutes the pooled samples. Manually place the pooled samples into the library tube for the NovaSeq run.
In addition, this step validates the run setup information and generates the sample sheet file.
Step input: Bulk pool
Step output: Library tube
¹ These automations are required for the NovaSeq 6000 v3.8 workflow to function properly. These automations contain additional logic needed for CLPA support. If you would like to remove CLPA support, then contact Illumina Support.
Automations not identified with ¹ are required for the NovaSeq 6000 v3.8 to work function properly.
The following table lists field configuration details defined on the Dilute and Denature (NovaSeq 6000 v3.8) step. These fields are required for sample sheet and JSON file generation.
Dilute and Denature (NovaSeq 6000 v3.8) Master Step Field Configuration
The following table lists the global custom fields that are configured to display on the Dilute and Denature (NovaSeq 6000 v3.8) step.
Global Field Configuration (Derived Sample)
Samples are routed to this protocol when their 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 v3.8) step. At the end of this protocol, the flow cell is sent to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) protocol.
This protocol contains the following three steps:
Step 1: Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8)
Step 2: Dilute, Denature & ExAmp (NovaSeq 6000 v3.8)
Step 3: Load to Flowcell (NovaSeq 6000 v3.8)
Manually place libraries into a pool.
Step input: NTP (normalized libraries)
Step output: Bulk pool
¹ These automations are for CLPA support only.
Automations not identified with ¹ are required for the NovaSeq 6000 v3.8 to work function properly.
The following table lists field configuration details defined on the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8) step.
Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8) Master Step Field Configuration
The following table lists the global custom fields that are configured to display on the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8) step.
Global Field Configuration (Derived Sample)
In this step, the addition of DPX, NaOH, Tris-HCl, and RSB denatures and dilutes the pooled samples. Manually create working pools based on the number of lanes that you want to sequence.
Step input: Bulk pool
Step output: Working pool - variable number, choose how many working pools to create per bulk pool
¹ These automations are for CLPA support only.
² These automations are required for the NovaSeq 6000 v3.8 workflow to function properly. These automations contain additional logic needed for CLPA support. If you would like to remove CLPA support, then contact Illumina Support.
Automations not identified with ¹ or ² are required for the NovaSeq 6000 v3.8 to work function properly.
The following table lists field configuration details defined on the Load to Flowcell (NovaSeq 6000 v3.8) step. A script sets these field values. The values are not editable while running the step.
Dilute, Denature & ExAmp (NovaSeq v3.8) Master Step Field Configuration
The following table lists the global custom fields that are configured to display on the Dilute, Denature & ExAmp (NovaSeq 6000 v3.8) step.
Global Field Configuration (Derived Sample)
In this step, scan the flow cell barcode into the Clarity LIMS. Then, manually place the working pools into the lanes of the flow cell for the NovaSeq run. This step validates the run setup information and generates the sample sheet file.
Step input: Working pool
Step output: Flow cell (output containers: SP, S1, and S2 with 2 lanes, and S4 with 4 lanes)
¹ These automations are for CLPA support only.
² These automations are required for the NovaSeq 6000 v3.8 workflow to function properly. These automations contain additional logic needed for CLPA support. If you would like to remove CLPA support, then contact Illumina Support.
Automations not identified with ¹ or ² are required for the NovaSeq 6000 v3.8 to work function properly.
The following table lists the field configuration details defined on the Load to Flowcell (NovaSeq 6000 v3.8) step.
Load to Flowcell (NovaSeq 6000 v3.8) Master Step Field Configuration
The following table lists the global custom fields that are configured to display on the Load to Flowcell (NovaSeq 6000 v3.8) step.
Global Field Configuration (Derived Sample)
This final protocol contains one fully automated step, AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8), described in the following section.
Step input: Library tube from NovaSeq Standard or flow cell from NovaSeq Xp protocol
Step output: Result file/measurement
In this step, pooled samples are sequenced on the NovaSeq instrument and the run metrics are recorded in Clarity LIMS.
¹ These automations are for CLPA support only.
² Not used. By default, the Sequencer API determines this functionality. If necessary, enable the automation and use it to override the next step behavior used by the Sequencer API.
Automations not identified with ¹ are required for the NovaSeq 6000 v3.8 to work function properly.
The following fields are configured on the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step in Clarity LIMS:
Current Cycle
Current Read
Firmware Version
Flow Cell Expiration Date
Flow Cell ID
Flow Cell Lot Number
Flow Cell Mode
Flow Cell Part Number
Flow Cell Side
Instrument Control Software Version
Instrument ID
Instrument Type
Lane Counter
Loading Workflow Type
Output Folder
RTA Version
Run Completion Date
Run ID
Run Status
Sequencing Log
The following fields are used to capture the run metrics in Clarity LIMS:
% Aligned R1
% Aligned R2
% Bases >=Q30 R1
% Bases >=Q30 R2
% Error Rate R1
% Error Rate R2
% Phasing R1
% Phasing R2
% Prephasing R1
% Prephasing R2
%PF R1
%PF R2
Cluster Density (K/mm^2) R1
Cluster Density (K/mm^2) R2
Intensity Cycle 1 R1
Intensity Cycle 1 R2
Reads PF (M) R1
Reads PF (M) R2
Yield PF (Gb) R1
Yield PF (Gb) R2
Note the following details:
Values are aggregated across all lanes. Some values (e.g., Yield PF (Gb) R1) are summed while others are averaged.
The names listed previously are the default global custom field names installed with the NovaSeq Integration v3.8 configuration provided in the Illumina Preset Protocols (IPP) v2.9 or later.
All global configuration fields are configured on the Container entity.
All field names are configurable through the Custom Fields screen in the Global Fields tab.
All run metrics tracked in Clarity LIMS are overall metrics for the run. There are currently no per-lane metrics provided by NovaSeq Control Software.
The sample sheet is generated on the step before the run. This step is Dilute and Denature (NovaSeq 6000 v3.8) in the NovaSeq Standard protocol or Load to Flowcell (NovaSeq 6000 v3.8) in the NovaSeq Xp protocol. This step places samples on the library tube or flow cell that are loaded in the NovaSeq 6000 instrument.
In the default configuration, the Validate Run Setup and Generate Sample Sheet automation generates one CSV format sample sheet file. The bcl2fastq v2.20 downstream analysis uses this file.
The sample sheet is uploaded to the NVCS via the /Illumina/Sequencer/v2/sequencing-run/files endpoint to the Sequencer API. The file endpoint allows for a file to be downloaded from the Clarity LIMS using OAuth (instead of Basic Authentication), which is required for the NovaSeq 6000 instrument.
The run recipe response sets the sample sheet URL to the link to download the file from this endpoint and sets the sampleSheetRequiresOAuth value to true.
The following steps outline the sequence of events that occurs when a flow cell is loaded onto the NovaSeq 6000 instrument.
The following sections describe the components (files, properties, reagent categories/label groups, reagent kits, and containers) that are installed by default as part of this integration.
Illumina NovaSeq 6000 Integration v3.7.0 is distributed as the BaseSpaceLIMS-sequencer-api RPM package. This RPM package must be installed on the Clarity LIMS server.
The BaseSpaceLIMS-sequencer-api RPM installs the following items:
Sequencer API WAR file
application.yml configuration file
Two configuration scripts:
configure_sequencer_api_proxy.sh
configure_sequencer_api_application.sh
If NGS Extensions Package v5.25.0 or later is already installed, the upgrade is not forced or required.
The following table lists the components installed by the RPM package.
TruSeq HT Adapters v2 (D7-D5)
Buffer Cartridge
Cluster Cartridge
DPX1
DPX2
DPX3
NaOH
Resuspension Buffer (RSB)
SBS Cartridge
Tris HCl
Library Tube
SP
S1
S2
S4
This integration supports the following items:
Library tube with barcode provided in the format [A-Z]{2}[0-9]{7}-[A-Z]{3} (eg, AB1234567-XYZ)
SP, S1, S2, or S4 flow cell with barcode provided in one of the following formats:
SP and S1 flow cell: [A-Z0-9]{5}DR[A-Z0-9]{2}
S2 flow cell: [A-Z0-9]{5}DM[A-Z0-9]{2}
S4 flow cell: [A-Z0-9]{5}DS[A-Z0-9]{2}
Example: H1991DMXX
For details on configuring NVCS for integration with Clarity LIMS, contact the Clarity LIMS Support team.
The requirements for the routing script functionality are as follows.
On the steps that use the routing script (Define Run Format (NovaSeq 6000 v3.8) and Dilute and Denature (NovaSeq 6000 v3.8)), the Next Step for all samples must be set to Remove from workflow. A script sets this value. The value must not change in the Assign Next Steps screen.
In the Next Steps section of the protocol configuration screen, the method of assigning the next step must be set to Automation for the last step of the protocol.
The integration.sequencer_api.v2.run.autoComplete and integration.sequencer_api.v2.run.autoCompleteOnlyAtSuccess properties determine the conditions under which the last step of the NovaSeq 6000 workflow (AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8)) is automatically completed when the run status is "RunCompletedSuccessfully" (default behavior) or regardless of the run status.
By default, both properties are set to true and the step only completes when the run is successful. If the run fails or is aborted, manually complete the step in Clarity LIMS.
For the step to autoComplete regardless of the result of the sequencing run, change the autoCompleteOnlyAtSuccess property value to false.
Enabling/Disabling autoComplete Properties
Edit the value of integration.sequencer_api.v2.run.autoCompleteOnlyAtSuccess property as required using the omxProps-ConfigTool utility.
Save the file.
The following table shows how the combined value of integration.sequencer_api.v2.run.autoComplete and integration.sequencer_api.v2.run.autoCompleteOnlyAtSuccess properties affects the autoComplete behavior of the sequencing step.
run.autoComplete and run.autoCompleteOnlyAtSuccess Value Matrix
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 determine the following information:
Which samples, and how many, can enter each step together.
Which samples, and how many, can be pooled together.
The library tube ID must be unique. There must not be multiple library tube containers in the system with the same name.
Reagent labels (indexes) must be unique.
Only controls are permitted as unindexed samples; all other unindexed samples and pools are not permitted.
Do not manually start the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step. This step is a fully automated and the sequencing service may not update samples correctly if they have been manually started.
For the automated run to start successfully, Validate Run Setup and Generate Sample Sheet must be selected.
Last Updated: November 2024
Release Date: July 2024
Document Version: 2
These release notes describe the key changes to software components for the Clarity LIMS NovaSeq 6000 Integration Package v3.7.0.
Refer to under Instruments & Integrations.
Some integration properties are moved to database and can now be accessed and updated via System Setting in Clarity v6.3.
This change requires updates to the configure_sequencer_api_application.sh script.
Refer to for configurable properties.
Fixed the denial of service (CVE-2024-30172) vulnerability.
None
The Illumina NovaSeq 6000 Integration v3.6.0 supports the integration between Clarity LIMS (v6.2 and later) and the NovaSeq 6000 instrument. This integration also includes updates to the technology stack and third party libraries and utilities. The key changes from v3.5.1 are as follows.
Updated the preconfigured NovaSeq 6000 workflow to include Clarity LIMS Product Analytics (CLPA) automation scripts by default.
Compatibility with Oracle Linux. For compatibility, refer to .
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 v3.6.0. It also describes the components that are installed in the default configuration.
If you are installing the NovaSeq 6000 integration for the first time, follow the instructions provided in this document.
If NovaSeq 6000 Integration v3.5.1 is already installed, with only minor changes to the default configuration, follow the instructions provided in this document and then duplicate the configuration changes from the v3.5.1 workflow.
If there have been significant changes to the default configuration, follow the instructions provided in .
For details on installed protocols and steps, automations, generated and captured files, and rules and constraints, refer to .
For information on validating and troubleshooting the integration, refer to .
For information on the NovaSeq 6000 instrument system, refer to the .
NovaSeq 6000 Integration v3.6.0 has the following prerequisites:
NovaSeq Integration v3.6.0 is distributed as the BaseSpaceLIMS-sequencer-api RPM package. This package must be installed on the Clarity LIMS server.
The BaseSpaceLIMS-sequencer-api RPM installs the following files and scripts:
Sequencer API WAR file
application.yml configuration file
configure_sequencer_api_proxy.sh configuration script
configure_sequencer_api_env.sh configuration script
configure_sequencer_api_application.sh configuration script
The RPM installs the Sequencer API WAR file into the same Tomcat container as Clarity LIMS. The WAR file is at /opt/gls/clarity/tomcat/current/webapps/Illumina#Sequencer#v2##2.6.0.0.war. The version number can differ.
The WAR file is deployed automatically when Clarity LIMS launches. Because the RPM installs a new WAR file, Clarity LIMS is automatically stopped during installation of the RPM. Restart Clarity LIMS before installing the NovaSeq and Library Prep Validation workflow configuration from the IPP.
The application.yml configuration file is installed at /opt/gls/clarity/extensions/sequencer-api/application.yml. As part of the installation process, the file is configured with one of the configuration scripts.
The scripts in the following table are installed at the /opt/gls/clarity/config/ folder. During installation, the RPM runs these scripts as they are needed.
NovaSeq 6000 Integration v3.6.0 includes the workflows, protocols, and steps listed in the following table. The workflows are found in IPP v2.6.0 package.
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.
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).
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 Server-Based.
In the adjacent field, enter the Clarity LIMS URL (e.g., https://customerserver.claritylims.com).
[Optional] In the Output Folder field, enter the default output folder location. You can also specify the output folder location when setting up a run in NVCS.
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.
The integration includes the following features:
Preconfigured NovaSeq 6000 v3.8 workflow that maps to lab protocols and instrument runs.
The following preconfigured protocols:
Run Format (NovaSeq 6000 v3.8)
NovaSeq Standard (NovaSeq 6000 v3.8) (supports the loading of pooled libraries into a library tube)
NovaSeq Xp (NovaSeq 6000 v3.8) (supports individual lane loading on the NovaSeq)
AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8)
Automated generation of v1 sample sheet for use with bcl2fastq2 v2.20 analysis software or v2 sample sheet. This file is automatically uploaded to the instrument through the API.
Automatic validation of run setup information. This information is used to create the run recipe and initiate the run.
Automated tracking of the NovaSeq sequencing run and parsing of run statistics into Clarity LIMS, including the following features:
Progress and metrics of sequencing run
Per-instrument sequencing runs (tracked as part of the Run ID field value)
Sequencing run parameters
Sequencing run data directory location
Real-Time Analysis v3 (RTA3) version and other run-specific information
[Optional] Preconfigured Library Prep Validation v2.3.4 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 v3.8 workflow. For more information, refer to . The Library Prep Validation v2.3.4 workflow is available in IPP v2.9 package.
This section explains how to validate the installation of the Illumina NovaSeq 6000 Integration Package v3.6.0.
The validation process involves the following items:
Running samples through the Library Prep Validation 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 advance to the workflow selected.
Running normalized libraries through the NovaSeq 6000 v3.8 workflow validates the following items:
Successful routing of samples from the Run Format (NovaSeq 6000 v3.8) step to the NovaSeq Standard (NovaSeq 6000 v3.8) or NovaSeq Xp (NovaSeq 6000 v3.8) step.
Automated generation of v1 sample sheet for use with bcl2fastq2 v2.20 analysis software or v2 sample sheet. This file automatically uploads to the sequencing system via the Sequencer API.
Automatic validation of run setup information. Information is uploaded to the NovaSeq Control Software (NVCS) via the Sequencer API and is used to create the run recipe and initiate the run.
Automated tracking of the NovaSeq sequencing run and parsing of run statistics into Clarity LIMS, via the Sequencer API.
The validation steps assume that the Illumina NovaSeq 6000 Integration Package v3.6.0 is installed and you have imported the default Clarity LIMS configuration.
The following steps set up Clarity LIMS in preparation for running samples through the Library Prep Validation and NovaSeq 6000 v3.8 workflows.
On the Configuration tab, under Workflows, activate both the Library Prep Validation and NovaSeq 6000 v3.8 workflows.
Library Prep Validation v2.3.1 is available with Illumina Preset Protocols (IPP) v2.6.
On the Projects and Samples screen, create a project and add samples to it.
Assign the samples to the Library Prep Validation workflow.
This single-step protocol models the library prep required to produce normalized libraries that are ready for the NovaSeq 6000 v3.8 workflow.
Label Group = TruSeq HT Adapters v2 (D7-D5)
Sequencing Instrument = NovaSeq 3.0
On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the NovaSeq 6000 v3.8 workflow, Define Run Format (NovaSeq 6000 v3.8). This step is the only one in Protocol 1: Run Format (NovaSeq 6000 v3.8).
This protocol includes a single step, Define Run Format (NovaSeq 6000 v3.8). 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.
In Lab View, locate the Run Format (NovaSeq 6000 v3.8) protocol. The samples are queued for the Define Run Format (NovaSeq 6000 v3.8) step.
Add the samples to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
On the Record Details screen in the Sample Details table, populate the following fields (values can vary across samples):
Loading Workflow Type — Select NovaSeq Standard or NovaSeq Xp from the drop-down list.
Flowcell Type — Select SP, S1, S2, or S4.
Final Loading Concentration (pM) — Select from the two preset options: 225 (for PCR-free workflows), 400 (for Nano workflows), or enter a different value.
Normalized Molarity (nM) — These values are copied over from the previous step. If this column is not populated during library prep, enter the values here.
Select Next Steps, which triggers the Set Next Steps automation and 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.
Calculates the Minimum Molarity.
Checks Normalized Molarity value. For samples with no Normalized Molarity value (an empty value not including 0), generates an error message notifying that the field cannot be empty.
Compares each sample Normalized Molarity value with the Minimum Molarity value.
On the Assign Next Steps screen:
In the Sample Details table, the Next Step for all samples is prepopulated with Remove from workflow, regardless of the Loading Workflow Type.
For samples whose Normalized Molarity value is lower than the Minimum Molarity value, the Loading Workflow Type is set to Remove from workflow. Also, a message is recorded in the Warning field for the sample.
At this point, there are two options:
Return to the Record Details screen and adjust the Normalized Molarity value so that it equals or exceeds the Minimum Molarity value. The Loading Workflow Type must be set to NovaSeq Standard or NovaSeq Xp, as applicable.
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 & Register Step Completed automation is triggered:
Samples whose Loading Workflow Type is set to Remove from workflow are removed from the Clarity LIMS workflow. (Occurs when the Normalized Molarity value is lower than the Minimum Molarity.)
Samples whose Loading Workflow Type is set to NovaSeq Standard are routed to the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8) step. This step is the first one in the NovaSeq Standard protocol.
Samples whose Loading Workflow Type is set to NovaSeq Xp are routed to the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8) step. This step is the first one in the NovaSeq Xp protocol.
In this protocol, samples are pooled and added to the library tube in preparation for the NovaSeq run.
In Lab View, locate the NovaSeq Standard (NovaSeq 6000 v3.8) protocol. Samples for the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8) step are queued.
On the Queue screen, add samples of the same Flowcell Type to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
On the Pooling screen:
Create a pool by dragging samples into the Pool Creator.
Enter a name for the pool or accept the default name (Pool #1).
Select Record Details.
After exiting from the Pooling screen, the Validate Inputs Flowcell Type and Single Pool automation is triggered. The automation verifies the following items:
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 two required fields and one optional field, as needed:
Number of Flowcells to Sequence — Used in volume calculations, to make sure that the volumes are sufficient for the number of times the pool is sequenced.
Minimum Per Sample Volume (ul) — 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 can be edited.
% PhiX (2.5 nM) Spike-In — Used to calculate the volume of PhiX v3 control included in the pool for the given percentage of spike-in. The field is optional and can be edited.
If the smallest Per Sample Volume (ul) value is < 5, Clarity LIMS automatically assigns a value of 5 to the Adjusted Per Sample Volume (ul) field.
Clarity LIMS changes 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 Next Steps to trigger the Set Next Steps automation. This automation sets the next step for samples to ADVANCE and advances them to the next step in the protocol, Dilute, and Denature (NovaSeq 6000 v3.8).
On the Assign Next Steps screen, the next step for samples is set to the next step in the workflow: Dilute and Denature (NovaSeq 6000 v3.8).
Select Finish Step.
At the end of this step, the pool of samples automatically advances to the Dilute and Denature (NovaSeq 6000 v3.8) step.
In Lab View, locate the NovaSeq Standard (NovaSeq 6000 v3.8) protocol. The pool of samples queued for the Dilute and Denature (NovaSeq 6000 v3.8) step are listed.
Add the samples to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work. The Validate Single Input & Register Step Started automation is triggered. This automation verifies that only one container is input for the step.
On the Placement screen:
Drag the pool into the library tube in the Placed Samples area.
Scan or type the barcode of the library tube into the Library Tube field.
Select Record Details.
After exiting the Placement screen, the Validate Library Tube Barcode automation makes sure that the library tube barcode conforms to the barcode mask [A-Z]{2}[0-9]{7}-[A-Z]{3}. If it does not, an error message displays. The automation also copies the Flowcell Type and Loading Workflow Type values from step inputs to outputs.
In the Reagent Lot Tracking section, select from the active lots displayed in each drop-down list.
The fields displayed on the Record Details screen are used to set up the run and generate the sample sheet. Some of these fields are autopopulated and some must be completed manually.
Fields Displayed on Record Details Screen of Dilute and Denature (NovaSeq 6000 v3.8)
On the Record Details screen, select Validate Run Setup and Generate Sample Sheet. This selection triggers the automation script, which performs the following actions:
Validates the parameters entered on the Record Details screen.
Generates the sample sheet and attaches it to the placeholder in the Files area of the Record Details screen.
Select Next Steps.
On the Assign Next Steps screen, the Next Step for samples is prepopulated with Remove from workflow. The Routing Script automation expects this value, and requires the value to advance samples to the next step.
Select Finish Step.
After exiting the step, the following actions occur:
The Routing Script & Register Step Completed automation is triggered and samples are routed to AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8).
In Lab View, the pool of samples is queued for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step.
At this point in the workflow, the user interaction ends. Proceed to AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8).
In this protocol, samples are pooled and added to lanes on the NovaSeq flow cell. The option selected in the Define Run Format (NovaSeq 6000 v3.8) step determines the flow cell type.
In Lab View, locate the NovaSeq Xp (NovaSeq 6000 v3.8) protocol. The samples are queued for the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8) step.
On the Queue screen, add samples of the same Flowcell Type to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
On the Pooling screen, create and name a pool as follows.
Create a pool by dragging samples into the Pool Creator.
Enter a name for the pool or accept the default name (Pool #1).
Select Record Details.
On exiting the Pooling screen, the Validate Inputs Flowcell Type and Single Pool automation is triggered. The automation verifies the following items:
All samples in the pool are assigned the same Flowcell Type.
Only one pool was created.
On the Record Details screen in the Step Details area, complete the two required fields and one optional field, as needed:
Number of Lanes to Sequence — Used in volume calculations to make sure that volumes are sufficient for the number of times the pool is sequenced.
Minimum Per Sample Volume (ul) — 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 can be edited.
% PhiX (0.25nM) Spike-In — 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.
If the smallest Per Sample Volume (ul) value is < 5, Clarity LIMS automatically assigns a value of 5 to the samples 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. This adjustment is 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 Next Steps to trigger the Set Next Steps automation. This automation performs 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 Dilute, Denature & ExAmp (NovaSeq 6000 v3.8) step in the protocol.
On the Assign Next Steps screen, the next step for samples is set to the Dilute, Denature & ExAmp (NovaSeq 6000 v3.8) step in the workflow.
Select Finish Step.
At the end of this step, the pool of samples automatically advances to the Dilute, Denature & ExAmp (NovaSeq 6000 v3.8) step.
In Lab View, locate the NovaSeq Xp (NovaSeq 6000 v3.8) protocol. The pool of samples queued for the Dilute, Denature & ExAmp (NovaSeq 6000 v3.8) step displays.
Add the pool to the Ice Bucket and select View Ice Bucket.
[Optional] On the Ice Bucket screen, set the number of derivatives to create (placed into the flow cell lanes) and select Begin Work.
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:
BP Aliquot Volume (ul)
NaOH Volume (ul)
Tris-HCl Volume (ul)
DPX1 Volume (ul)
DPX2 Volume (ul)
DPX3 Volume (ul)
Mastermix per Lane (ul)
The automation also populates the Flowcell Type and Loading Workflow Type columns of the Sample Details table.
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-HCl to add per working pool (see the next step).
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 prepopulated. The script sets the values and they are not editable.
In the Sample Details table, select the pool icon to view details on the working pool composition.
The BP Aliquot, Mastermix per lane, NaOH, and Tris-HCl volume values for each working pool are populated. The script sets the values and they are not editable.
The Flowcell Type and Loading Workflow Type columns are populated.
The working pool number is appended to the bulk pool name, to identify which working pools are derived from the same bulk pool.
In the Files area, select the Calculation File (CSV) to open it and view details on the DPX Mastermix volume and the volume of Mastermix, NaOH, and Tris-HCl to add per working pool.
Select Next Steps. On the Assign Next Steps screen, the next step is already set to Load to Flowcell (NovaSeq 6000 v3.8).
Select Finish Step.
On the Ice Bucket screen:
In the Container Options panel, select the appropriate flow cell type from the Destination Container drop-down list.
Select Begin Work.
The Validate Inputs and Selected Container & Register Step Started automation checks the following:
The Flowcell Type field is set to a valid value (SP, S1, S2, or S4) and that each input has the same value for the field.
The container type selected matches the value in the Flowcell Type field.
The number of outputs matches the number of lanes on the selected flow cell type. If validation fails, an error message reports the number of working pools does not match the number of lanes available on the flow cell.
On the Placement screen:
Drag one or more 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 Flowcell field.
Select Record Details.
After exiting the Placement screen, the Validate Flowcell Barcode automation makes sure that the container barcode conforms to the barcode mask for the selected Flowcell Type (as listed in the following table). This automation also copies the Flowcell Type and Loading Workflow Type field values from step inputs to outputs.
The fields displayed on the Record Details screen are used to set up the run and generate the sample sheet. Some of these fields are autopopulated and some must be entered manually. For details, refer to the following table:
Fields Displayed on Record Details Screen of Load to Flowcell (NovaSeq 6000 v3.8) Step
Select Validate Run Setup and Generate Sample Sheet to trigger the automation script, which does the following:
Validates the parameters entered on the Record Details screen.
Generates the sample sheet and attaches it to the placeholder in the Files area on the Record Details screen.
Select Next Steps.
On the Assign Next Steps screen, the Next Step field for samples is prepopulated with Mark protocol as complete.
Select Finish Step.
At this point in the workflow, the user interaction ends. The flow cell is queued for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step.
This protocol contains a single fully automated step - AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8).
The integration starts the step automatically and data from the run is parsed back into Clarity LIMS. User interaction is not required, but the various stages can be reviewed in Clarity LIMS under Review Run Data.
Automations
The NovaSeq Run (NovaSeq 6000 v3.8) step contains two automations:
Set Next Steps — This automation is disabled (Set to Not Used). By default, the Sequencer API is used to set the next step for output samples.
Update Lane Number — This automation is triggered automatically upon entry to the Record Details screen.
Read summary metrics are recorded for the library pool. After the run is complete, open the step and review these metrics on the Next Steps screen in the Step Data section and the Sample Details table.
Step Data Section
The following values populate in the master step fields:
Run ID
Run Status
Output Folder
Current Cycle
Current Read
Loading Workflow Type
Flow Cell ID
Flow Cell Side
Flow Cell Mode
Flow Cell Part Number
Flow Cell Lot Number
Flow Cell Expiration Date
Instrument ID
Instrument Type
Instrument Control Software Version
Firmware Version
RTA Version
Sequencing Log
Sample Details Table
Summary metrics populate the global fields listed. Values are aggregated across all lanes. Some values (for example Yield PF (Gb) R1) are summed, while others are averaged.
Yield PF (Gb) R1
Yield PF (Gb) R2
Reads PF (M) R1
Reads PF (M) R2
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 R2
% Error Rate R1
% Error Rate R2
When setting up a run on the NovaSeq 6000 instrument, NVCS sends a request for the run recipe.
The Sequencer API validates that samples and containers are correctly queued for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step.
Clarity LIMS sends a JSON response to NVCS. The response includes the run recipe information and a link to download the sample sheet (used with analysis software such as bcl2fastq2).
When the run starts, NVCS sends a RunStarted run status request. The Sequencer API then does the following:
Validates reagent kit information in the RunStarted request:
Verifies that the reagent kit exists. If the reagent kit does not exist, the Sequencer API creates and enables it on the master step.
Verifies that the reagent kit is activated. If the reagent kit is not active, the Sequencer API activates it.
Initiates the step for the queued samples. The step produces one output file placeholder per lane of the flow cell in use, based on the flow cell type.
Records all relevant information from the RunStarted request on the step (such as reagent lots and step fields).
At the end of the run, NVCS sends one of the following run status requests:
RunCompletedSuccessfully
RunEndedByUSer
RunErroredOut
The Sequencer API updates the step with any new information, which includes only the status and cycle/read information.
After primary analysis completes, NVCS sends a request containing the parsed run metrics. NVCS sends a request only when the run completed successfully, but the API does not make this assumption and accepts the request regardless of status. The Sequencer API then does the following actions:
Records the metrics into the fields on file placeholder outputs in the LIMS.
Sample sheet generation and contents
NovaSeq 6000 run
User authentication and login
Run recipe contents
If an automation trigger does not appear to run its corresponding scripts, refer to the following sections in the Clarity LIMS API documentation:
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 6000 Integration Package by running the following command on the Clarity LIMS server command line:
If the automated run step starts but does not complete, proceed as follows.
Use one of the following methods to open the in progress step in Clarity LIMS:
Log in to the default user account.
In Lab View, find the step in the Recent Activities pane.
Search for the step in Clarity LIMS, using either the Library Tube, or Flowcell barcode as the search term. The steps depend on whether the search is a NovaSeq Standard or NovaSeq Xp run.
Contact Illumina Support. Provide the relevant information from the troubleshooting steps already performed.
In addition to updating the Sequencing Log multiline text field on the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step, the Sequencer API writes a detailed log file to the following location:
Log messages include the Library Tube ID and Flowcell ID whenever the messages are related to a sequencing run-related request. In the LIMS workflows, these IDs are recorded as container names.
Some log messages are not directly related to a sequencing run request, such as downloading the sample sheet. In this case, the file LIMS ID and file name are included in log messages. The sample sheet downloaded by the sequencing system has a name that includes the sequencing container ID (Library Tube for Standard or Flowcell for Xp). The relevant container name search finds most the log messages for this request. However, if all messages for downloading a file are found, the LIMS ID of the file must be known. The LIMS ID is found in the Clarity LIMS API and, for sample sheets used in a run, in the recipe response (also included in the log file).
Example lines from the log file:
This script modifies /opt/gls/clarity/tomcat/current/bin/setenv.sh for Clarity LIMS Tomcat to load the Secret Utility environment variable file (/etc/profile.d/claritysecret.sh) during startup.
The installer validates the import of the workflow and provides the Warning/Error details in STDOUT, which allows you to either proceed with the import or cancel it.
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 process.
Do not change this value. If Next Step is not set to Remove from workflow, the routing script is not routed samples correctly.
Only create one pool.
Assuming the default Minimum Per Sample Volume (ul) value of 5, for a given batch:
The v2 sample sheet generated does not include Cloud and Analysis sections. Contact Illumina Support for information on generating v2 sample sheets with those sections.
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.
Do not add samples to the Ice Bucket or start the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step. The integration adds samples automatically.
Create only one pool.
Assuming the default Minimum Per Sample Volume (ul) value of 5, for a given batch:
The v2 sample sheet generated does not include Cloud and Analysis sections. Contact Illumina Support for information on generating v2 sample sheets with these sections.
Do not add samples to the Ice Bucket or start the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step. The integration adds samples automatically.
The Illumina Preset Protocols (IPP) include a Set Next Steps automation configured on the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step. You can use this automation to override the default next step behavior. For details, refer to NovaSeq 6000 Integration v3.7.0 Configuration for details.
Do not disable or modify this automation to make sure that the Lane Number displays properly.
NovaSeq 6000 Integration v3.7.0 Configuration provides detailed information on how the following components work with the Sequencer API integration:
Do not add samples to the Ice Bucket or start the step. The integration starts the step automatically.
The Library Prep Validation v2.3.4 workflow allows for validation of the system after installation is complete. For details, refer to .
Create only one pool per step.
Validate Unique Indexes automation is not used. The Clarity LIMS configuration for pooling handles this functionality.
For accurate pipetting of each sample in a pool, the Per Sample Volume (ul) value must be greater than or equal to the Minimum Per Sample Volume (ul). The default value (set at 5) can be edited.
The script definition portion of Validate Run Setup has been moved into the Validation Script custom field due to the 4000 character limit.
Validates the parameters entered on the Record Details screen, which are used to set up the run and generate the sample sheet file. For more information, refer to the Respond to Validation / Recipe Request Call from Instrument section of .
Generates the sample sheet and attaches it to the step. For more information, refer to . Sample sheet template used depends on the selected value of Samplesheet Format and/or Reverse Complement Workflow.
Create only one pool per step.
Validate Unique Indexes automation is not used. The Clarity LIMS configuration for pooling handles this functionality.
For accurate pipetting of each sample in a pool, the Per Sample Volume (ul) value must be greater than or equal to the Minimum Per Sample Volume (ul). The default value (set at 5) can be edited.
The script definition portion of Validate Run Setup has been moved into the Validation Script custom field due to the 4000 character limitation.
Validates the parameters entered on the Record Details screen, which are used to set up the run and generate the sample sheet file. For more information, refer to the Respond to Validation / Recipe Request Call from Instrument section of .
Generates the sample sheet and attaches it to the step. The sample sheet template depends on the selected value for Samplesheet Format and/or Reverse Complete Workflow. For more information, refer to .
This step is fully automated. Do not add samples to the Ice Bucket or start the step manually. The sequencing service may not update samples correctly when they have been manually started.
Disable or configure auto-complete behavior by setting the values of the integration.sequencer_api.v2.run.autoComplete and integration.sequencer_api.v2.run.autoCompleteOnlyAtSuccess properties. For details, refer to .
Do not disable or modify this automation to ensure the Lane Number displays properly.
If field names are changed in Clarity LIMS, they must also be changed for the integration.sequencer_api.v2.run.metricUdfNames property. For more information, refer to the Properties section in . All fields are configured to be visible in the Sample Details table on the Record Details screen for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step.
These global custom fields are configured on the Container entity, visible on each input, but they should not be confused with per-lane metrics.
Depending on Samplesheet Format field value selection, a v1 or v2 sample sheet is generated. For detailed information on the following items, refer to the Bcl2fastq2 Sample Sheet Generation section of :
By default, the Sequencer API service tries to contact the Clarity LIMS at http://localhost:9080/clarity/. The installation script sets this URL, but it can be configured with the clarity.url property in the application.yml file. For more information, refer to the Properties section of .
Configure the steps that are considered sequencing run steps in the integration.sequencer_api.v2.novaseq.sequenceStepNames property. The property is a list and each list entry must be the exact name of a sequencing step, not the name of the underlying master step/process type. For more information, refer to the Properties section of .
The Clarity LIMS download link to the sample sheet created on the run setup step is used for the SampleSheet value in the recipe. The name of the output of the sample sheet on the setup step can be configured through the integration.sequencer_api.v2.recipe.sampleSheet.outputName property (Sample Sheet by default). For more information, refer to the Properties section of . If no matching output is found, or no file was attached to the output, then the value of the integration.sequencer_api.v2.recipe.sampleSheet.notAvailableValue property (Not Available by default) is used in the recipe.
If no matching container is found, or if the samples in the container are incorrectly queued, an error occurs and the step is not started. This validation is also done during the run recipe request immediately prior, so this error case should not be encountered.
The NovaSeq 6000 v3 configuration is delivered through Illumina Preset Protocols (IPP) v2.9, which requires installation of the NGS Extensions Package 5.25.0 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 6000 integration. For more information, refer to .
Refer to for the properties installed with the integration package.
For sample sheet generation constraints, refer to the Bcl2fastq2 Sample Sheet Generation section in .
For a complete list of all properties included in the application.yml file, refer to .
If running the sequencing service and Automation Worker on the same instance, the instance must also be running a compatible version of Oracle Linux. For compatibility, refer to .
For more information on this package, refer to .
You can also accept the default values in the application.yml configuration file by pressing Enter. For more information about the application.yml properties, refer to .
Make sure that the Secret Util is installed and configured. For more information, refer to .
This script modifies /opt/gls/clarity/tomcat/current/bin/setenv.sh for Clarity LIMS Tomcat to load the Secret Utility environment variable file (/etc/profile.d/claritysecret.sh) during startup.
The installer validates the import of the workflow and provides the Warning/Error details in STDOUT, which allows you to either proceed with the import or cancel it.
For more information, refer to .
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 Library Prep Validation v2.3.1 protocol, refer to .
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 process.
Follow the steps in to run the Library Prep Validation workflow with the following:
Do not change this value. If Next Step is not set to Remove from workflow, the routing script is not routed samples correctly.
Only create one pool.
Assuming the default Minimum Per Sample Volume (ul) value of 5, for a given batch:
Select Calculate Volumes to trigger the . 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.
On the Record Details screen, the Reagent Lot Tracking section lets you tracks the NaOH, Resuspension Buffer, and Tris-HCl reagents used in the step. Add and activate lots for these reagents. For more information on creating a new lot, refer to Add and Configure Reagent Kits and Lots in the .
The v2 sample sheet generated does not include Cloud and Analysis sections. Contact Illumina Support for information on generating v2 sample sheets with those sections.
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.
Do not add samples to the Ice Bucket or start the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step. The integration adds samples automatically.
Create only one pool.
Assuming the default Minimum Per Sample Volume (ul) value of 5, for a given batch:
Select Calculate Volumes. This selection triggers the . 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.
On the Record Details screen, the Reagent Lot Tracking section tracks the DPX1, DPX2, DPX3, NaOH, Resuspension Buffer, and Tris-HCl reagents used in the step. These reagents must be added. To add and activate reagent lots, refer to Add and Configure Reagent Kits and Lots in the .
The v2 sample sheet generated does not include Cloud and Analysis sections. Contact Illumina Support for information on generating v2 sample sheets with these sections.
Proceed to .
Do not add samples to the Ice Bucket or start the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step. The integration adds samples automatically.
The Illumina Preset Protocols (IPP) include a Set Next Steps automation configured on the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step. You can use this automation to override the default next step behavior. For details, refer to for details.
Do not disable or modify this automation to make sure that the Lane Number displays properly.
The following steps summarize how the Sequencer API integration works. For details on sample sheet generation and the sequence of events involved in the NovaSeq run, refer to .
Completes the step in Clarity LIMS when the status is RunCompletedSuccessfully. For all other status options, the step remains in progress. This action is the default autocomplete step behavior. For details, refer to .
provides detailed information on how the following components work with the Sequencer API integration:
Troubleshooting Automated Worker in the .
Troubleshooting Automation in the .
If the error is related to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step, review the log file information. For details, refer to .
On the Record Details screen, the Sequencing Log multiline text field contains logging information. If you are unable to reach the Record Details screen, or if the Sequencing Log field does not contain enough information to resolve the issue, review the sequencer-api.log file. For details, refer to .
Field
Value
Experiment Name
Enter the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. Spaces are not permitted.
BaseSpace Sequence Hub Configuration
Presets
Not Used
Run Monitoring Only
Run Monitoring and Storage
Workflow
Preset
GenerateFASTQ
Workflow Type
Presets
No Index
Single Index
Dual Index
Custom
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
251¹
151
101
51
type a value between 1 and 251
¹The value of 251 is only supported for SP flow cell type. For all other flow cell types, the maximum value is 151.
Read 2 Cycles
Presets
251¹
151
101
51
type a value between 0 and 251
¹The value of 251 is only supported for SP flow cell type. For all other flow cell types, the maximum value is 151.
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.
Use Custom Recipe
Select if applicable.
Custom Recipe Path
If the Use Custom Recipe option is selected, enter the path to the custom recipe file to be used.
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 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.
Output Folder
Enter network path for sequencing run folder (e.g., \\networkshare\run_data)
Samplesheet Format
Presets
V1
V2
Analysis Software Version
Enter the software version for BCL Convert Analysis for v2 sample sheet. Leave blank if not applicable.
Override Cycles
Enter the Override Cycles value for v2 sample sheet. Leave blank if not applicable.
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}
Field
Value
Experiment Name
Enter the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. Spaces are not permitted.
BaseSpace Sequence Hub Configuration
Presets
Not Used
Run Monitoring Only
Run Monitoring and Storage
Workflow
Preset
GenerateFASTQ
Workflow Type
Presets
No Index
Single Index
Dual Index
Custom
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
251¹
151
101
51
type a value between 1 and 251
¹The value of 251 is only supported for SP flow cell type. For all other flow cell types, the maximum value is 151.
Read 2 Cycles
Presets
251¹
151
101
51
type a value between 0 and 251
¹The value of 251 is only supported for SP flow cell type. For all other flow cell types, the maximum value is 151.
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.
Use Custom Recipe
Select if applicable.
Custom Recipe Path
If you selected the Use Custom Recipe option, enter the path to the custom recipe file to be used.
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 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 1 starts from. Leave blank if not applicable.
Output Folder
Enter network path for sequencing run folder (eg, \networkshare\run_data)
Library Tube Barcode
Scan the library tube barcode.
Samplesheet Format
Presets
V1
V2
Analysis Software Version
Enter the software version for BCL Convert Analysis for v2 sample sheet. Leave blank if not applicable.
Override Cycles
Enter the Override Cycles value for v2 sample sheet. Leave blank if not applicable.
Field Name | Field Type | Options | Additional Options and Dropdown Items |
Comment | Multiline Text | None |
Flowcell Type | Text Dropdown |
| Presets
|
Instruction | Text |
| Default
|
Loading Workflow Type | Text Dropdown |
| Presets
|
Field Name | Field Type | Options | Additional Options and Dropdown Items |
Adjusted Per Sample Volume (ul) | Numeric |
|
|
Final Loading Concentration (pM) | Numeric Dropdown |
|
|
Flowcell Type | Text Dropdown |
|
|
Loading Workflow Type | Text Dropdown |
|
|
Minimum Molarity (nM) | Numeric |
|
Normalized Molarity (nM) | Numeric |
|
Per Sample Volume (ul) | Numeric |
|
|
Warning | Text Dropdown |
|
|
Field Name | Field Type | Options | Additional Options and Dropdown Items |
Flowcell Type | Text Dropdown | Required Field | Presets
|
Loading Workflow Type | Text Dropdown | Required Field | Presets
|
Field Name | Field Type | Options | Additional Options and Dropdown Items |
RSB Volume (ul) | Numeric | Read Only | Decimal places displayed = 2 |
Flowcell Type | Text Dropdown | Required Field | Presets
|
Loading Workflow Type | Text Dropdown | Required Field | Presets
|
Field Name | Field Type | Options | Additional Options and Dropdown 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 | Options | Additional Options and Dropdown Items |
BP Aliquot Volume (ul) | Numeric | Read Only | Decimal places displayed = 0 |
Flowcell Type | Text Dropdown | Required Field | Presets
|
Loading Workflow Type | Text Dropdown | Required Field | Presets
|
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 |
Field Name | Field Type | Options | Additional Options and Dropdown Items |
Flowcell Type | Text Dropdown | Required Field | Presets
|
Loading Workflow Type | Text Dropdown | Required Field | Presets
|
Files Installed | Location | Description |
Illumina#Sequencer#v2.war | /opt/gls/clarity/tomcat/current/webapps | War file for Sequencer API application. |
configure_sequencer_api_application.sh | /opt/gls/clarity/config/ | Script that configures the Sequencer API application through its external application.yml file. |
configure_sequencer_api_env.sh | /opt/gls/clarity/config/ | Script that configures the Clarity LIMS Tomcat configuration to include Secret Util settings. |
configure_sequencer_api_proxy.sh | /opt/gls/clarity/config/ | Script that configures the proxy to allow communication with the Sequencer API application. |
sequencer-api.conf | /etc/httpd/clarity/ | Proxy setting to communicate with the Clarity LIMS. |
run.autoComplete Property Value | run.autoCompleteOnlyAtSuccess Property Value | Outcome |
true | true |
|
true | false |
|
false | true |
|
false | false |
|
Version | Changes |
2 |
|
1 |
|
Field | Value |
Experiment Name | Enter the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. Spaces are not permitted. |
BaseSpace Sequence Hub Configuration | Presets
|
Workflow | Preset
|
Workflow Type | Presets
|
Index Read 1 | Presets
|
Index Read 2 | Presets
|
Paired End | Presets
|
Read 1 Cycles | Presets
¹The value of 251 is only supported for SP flow cell type. For all other flow cell types, the maximum value is 151. |
Read 2 Cycles | Presets
¹The value of 251 is only supported for SP flow cell type. For all other flow cell types, the maximum value is 151. |
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. |
Use Custom Recipe | Select if applicable. |
Custom Recipe Path | If the Use Custom Recipe option is selected, enter the path to the custom recipe file to be used. |
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 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. |
Output Folder | Enter network path for sequencing run folder (e.g., \\networkshare\run_data) |
Samplesheet Format | Presets
|
Analysis Software Version | Enter the software version for BCL Convert Analysis for v2 sample sheet. Leave blank if not applicable. |
Override Cycles | Enter the Override Cycles value for v2 sample sheet. Leave blank if not applicable. |
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} |
Field | Value |
Experiment Name | Enter the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. Spaces are not permitted. |
BaseSpace Sequence Hub Configuration | Presets
|
Workflow | Preset
|
Workflow Type | Presets
|
Index Read 1 | Presets
|
Index Read 2 | Presets
|
Paired End | Presets
|
Read 1 Cycles | Presets
¹The value of 251 is only supported for SP flow cell type. For all other flow cell types, the maximum value is 151. |
Read 2 Cycles | Presets
¹The value of 251 is only supported for SP flow cell type. For all other flow cell types, the maximum value is 151. |
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. |
Use Custom Recipe | Select if applicable. |
Custom Recipe Path | If you selected the Use Custom Recipe option, enter the path to the custom recipe file to be used. |
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 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 1 starts from. Leave blank if not applicable. |
Output Folder | Enter network path for sequencing run folder (eg, \networkshare\run_data) |
Library Tube Barcode | Scan the library tube barcode. |
Samplesheet Format | Presets
|
Analysis Software Version | Enter the software version for BCL Convert Analysis for v2 sample sheet. Leave blank if not applicable. |
Override Cycles | Enter the Override Cycles value for v2 sample sheet. Leave blank if not applicable. |
The integration includes the following features:
Preconfigured NovaSeq 6000 v3.8 workflow that maps to lab protocols and instrument runs.
The following preconfigured protocols:
Run Format (NovaSeq 6000 v3.8)
NovaSeq Standard (NovaSeq 6000 v3.8) (supports the loading of pooled libraries into a library tube)
NovaSeq Xp (NovaSeq 6000 v3.8) (supports individual lane loading on the NovaSeq)
AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8)
Automated generation of v1 sample sheet for use with bcl2fastq2 v2.20 analysis software or v2 sample sheet. This file is automatically uploaded to the instrument through the API.
Automatic validation of run setup information. This information is used to create the run recipe and initiate the run.
Automated tracking of the NovaSeq sequencing run and parsing of run statistics into Clarity LIMS, including the following features:
Progress and metrics of sequencing run
Per-instrument sequencing runs (tracked as part of the Run ID field value)
Sequencing run parameters
Sequencing run data directory location
Real-Time Analysis v3 (RTA3) version and other run-specific information
[Optional] Preconfigured Library Prep Validation v2.3.1 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 v3.8 workflow. For more information, refer to NovaSeq 6000 Integration v3.6.0 User Interaction, Validation and Troubleshooting. The Library Prep Validation v2.3.1 workflow is available in IPP v2.6 package.
Script Name
User
Description
configure_sequencer_api_proxy.sh
root
Adds configuration to the proxy to allow communication with the Sequencer API after Tomcat has been started.
Run this script in the following scenarios:
When the Sequencer API is first installed.
When reconfiguring the Clarity LIMS proxy. This process happens until a version of Clarity LIMS that includes the necessary configuration files from /etc/httpd/clarity/*.conf is available.
configure_sequencer_api_env.sh
root
Adds Secret Management setting into the Clarity LIMS Tomcat configuration.
configure_sequencer_api_application.sh
glsjboss
Configures the installed application.yml file.
Sets the Clarity LIMS URL and the key to use when signing access tokens.
If you run the script multiple times, the script reconfigures each setting to its default.
The only exception is the signing key. For this setting, you are asked if you want a reset. If the reset is done, all previously issued tokens are no longer valid.
Field Name | Field Type | Options | Additional Options and Dropdown Items |
% PhiX (2.5 nM) Spike-In | Numeric |
|
Bulk Pool Volume (ul) | Numeric |
|
Number of Flowcells to Sequence | Numeric | Required Field |
|
Minimum Per Sample Volume (ul) | Numeric | Required Field |
|
Number of Samples in Pool | Numeric |
|
PhiX Volume (ul) | Numeric |
|
Total Sample Volume (ul) | Numeric |
|
Field Name | Field Type | Options | Additional Options and Dropdown Items |
Flowcell Type | Text Dropdown | Required Field | Presets
|
Loading Workflow Type | Text Dropdown | Required Field | Presets
|
NaOH Volume (ul) | Numeric | Read Only | Decimal places displayed = 2 |
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) | Numeric | Read Only | Decimal places displayed = 0 |
Field Name | Field Type | Options | Additional Options and Dropdown Items |
Analysis Software Version | Text |
BaseSpace Sequence Hub Configuration | Text Dropdown |
|
|
Custom Recipe Path | Text |
|
Experiment Name | Text |
|
Index Read 1 | Numeric Dropdown |
|
|
Index Read 2 | Numeric Dropdown |
|
|
Output Folder | Text |
|
|
Override Cycles | Text |
|
Paired End | Text Dropdown |
|
|
Read 1 Cycle | Numeric Dropdown |
|
|
Read 2 Cycle | Numeric Dropdown |
|
|
Reverse Complement Workflow | Toggle Switch |
|
|
Run Mode | Text Dropdown |
|
|
Samplesheet Format | Text Dropdown |
|
|
Settings Header | Text |
|
|
UMI—Read 1 Length | Numeric |
|
UMI—Read 1 Start From Cycle | Numeric |
|
UMI—Read 2 Length | Numeric |
|
UMI—Read 2 Start From Cycle | Numeric |
|
Use Custom Index Read 1 Primer | Toggle Switch |
|
Use Custom Read 1 Primer | Toggle Switch |
|
Use Custom Read 2 Primer | Toggle Switch |
|
Use Custom Recipe | Toggle Switch |
|
|
Validation Script | Multiline Text |
|
|
Workflow | Text |
|
|
Workflow Type | Text Dropdown |
|
|
Field Name | Field Type | Options | Additional Options and Dropdown Items |
% PhiX (0.25nM) Spike-in | Numeric |
|
Bulk Pool Volume (ul) | Numeric |
|
Minimum Per Sample Volume (ul) | Numeric | Required Field |
|
Number of Lanes to Sequence | Numeric | Required Field |
|
Number of Samples in Pool | Numeric |
|
PhiX Volume (ul) | Numeric |
|
Total Sample Volume (ul) | Numeric |
|
Field Name | Field Type | Options | Additional Options and Dropdown Items |
Analysis Software Version | Text |
|
BaseSpace Sequence Hub Configuration | Text Dropdown |
|
|
Custom Recipe Path | Text |
|
Experiment Name | Text |
|
|
Index Read 1 | Numeric Dropdown |
|
|
Index Read 2 | Numeric Dropdown |
|
|
Library Tube Barcode | Text |
|
|
Output Folder | Text |
|
|
Override Cycles | Text |
|
Paired End | Text Dropdown |
|
|
Read 1 Cycle | Numeric Dropdown |
|
|
Read 2 Cycle | Numeric Dropdown |
|
|
Reverse Complement Workflow | Toggle Switch |
|
|
Run Mode | Text Dropdown |
|
|
Samplesheet Format | Text Dropdown |
|
|
Settings Header | Text |
|
|
UMI—Read 1 Length | Numeric |
|
UMI—Read 1 Start From Cycle | Numeric |
|
UMI—Read 2 Length | Numeric |
|
UMI—Read 2 Start From Cycle | Numeric |
|
Use Custom Index Read 1 Primer | Toggle Switch |
|
Use Custom Read 1 Primer | Toggle Switch |
|
Use Custom Read 2 Primer | Toggle Switch |
|
Use Custom Recipe | Toggle Switch |
|
|
Validation Script | Multiline Text |
|
|
Workflow | Text |
|
|
Workflow Type | Text Dropdown |
|
|
Script Name | User | Description |
configure_sequencer_api_proxy.sh | root |
|
configure_sequencer_api_env.sh | root | Adds Secret Management setting into the Clarity LIMS Tomcat configuration. |
configure_sequencer_api_application.sh | glsjboss |
|
Last Updated: November 2024
Release Date: September 2023
Document Version: 2
These release notes describe the key changes to software components for the Clarity LIMS NovaSeq 6000 Integration Package v3.6.0.
Refer to Compatibility under Instruments & Integrations.
Updates Groovy to v3.0.7.
Replaces the non-CLPA enabled NovaSeq 6000 v3 workflow with the CLPA enabled workflow.
During configuration, you can now validate the existence of the input user instead of resetting the user and password.
None
The Illumina NovaSeq 6000 Integration Package v3.6.0 supports the integration of Clarity LIMS to NovaSeq 6000 instruments. This documentation describes the integration and includes the following information:
Preconfigured workflows, protocols, steps, and automations
Installed components
Configuration requirements, rules, and constraints
For instructions on user interaction for each step, validating and troubleshooting the NovaSeq 6000 Integration, refer to NovaSeq 6000 Integration v3.6.0 User Interaction, Validation and Troubleshooting.
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 process.
It is assumed that samples enter the NovaSeq 6000 v3.8 workflow as normalized libraries. It is assumed that the following steps have completed before samples are assigned to the workflow:
Samples have been accessioned into the 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 Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
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 Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
The Illumina NovaSeq 6000 Integration Package v3.6.0 includes the following workflows:
Library Prep Validation v2.3.1 (optional, but recommended for validation purposes)
NovaSeq 6000 v3.8
This protocol sets the Loading Workflow Type and allows the choice of the appropriate Flowcell Type and Final Loading Concentration (pM). After the protocol, a routing script sends the normalized libraries to either the NovaSeq Standard (NovaSeq 6000 v3.8) or the NovaSeq Xp (NovaSeq 6000 v3.8) protocol.
This protocol contains one step: Define Run Format (NovaSeq 6000 v3.8).
Step input: NTP (normalized libraries)
Step output: None
¹ This automation is for CLPA support only.
² This automation is required for the NovaSeq 6000 v3.8 workflow to function properly. This automation contains additional logic needed for CLPA support. If you would like to remove CLPA support, then contact Illumina Support.
Automations not identified with ¹ or ² are required for the NovaSeq 6000 v3.8 to work function properly.
The following table lists field configuration details defined on the Define Run Format (NovaSeq 6000 v3.8) master step.
Define Run Format (NovaSeq 6000 v3.8) Master Step Field Configuration
The following table lists the global custom fields that are configured to display on the Define Run Format (NovaSeq 6000 v3.8) step.
Define Run Format (NovaSeq 6000 v3.8) Global Field Configuration (Derived Sample)
Samples are routed to this protocol when their 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 v3.8) protocol.
This protocol contains two steps:
Step 1: Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8)
Step 2: Dilute and Denature (NovaSeq 6000 v3.8)
In this step, libraries are placed manually into a single pool. Resuspension buffer and reagents are added.
Step input: NTP (normalized libraries)
Step output: Bulk pool
¹ These automations are for CLPA support only.
Automations not identified with ¹ are required for the NovaSeq 6000 v3.8 to work function properly.
The following table lists configuration details defined on the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8) step.
Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8) Master Step Field Configuration
The following table lists the global custom fields that are configured to display on the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8) step.
Global Custom Fields Configuration (Derived Sample)
In this step, the addition of NaOH, Tris-HCl, and Resuspension Buffer (RSB) denatures and dilutes the pooled samples. Manually place the pooled samples into the library tube for the NovaSeq run.
In addition, this step validates the run setup information and generates the sample sheet file.
Step input: Bulk pool
Step output: Library tube
¹ These automations are required for the NovaSeq 6000 v3.8 workflow to function properly. These automations contain additional logic needed for CLPA support. If you would like to remove CLPA support, then contact Illumina Support.
Automations not identified with ¹ are required for the NovaSeq 6000 v3.8 to work function properly.
The following table lists field configuration details defined on the Dilute and Denature (NovaSeq 6000 v3.8) step. These fields are required for sample sheet and JSON file generation.
Dilute and Denature (NovaSeq 6000 v3.8) Master Step Field Configuration
The following table lists the global custom fields that are configured to display on the Dilute and Denature (NovaSeq 6000 v3.8) step.
Global Field Configuration (Derived Sample)
Samples are routed to this protocol when their 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 v3.8) step. At the end of this protocol, the flow cell is sent to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) protocol.
This protocol contains the following three steps:
Step 1: Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8)
Step 2: Dilute, Denature & ExAmp (NovaSeq 6000 v3.8)
Step 3: Load to Flowcell (NovaSeq 6000 v3.8)
Manually place libraries into a pool.
Step input: NTP (normalized libraries)
Step output: Bulk pool
¹ These automations are for CLPA support only.
Automations not identified with ¹ are required for the NovaSeq 6000 v3.8 to work function properly.
The following table lists field configuration details defined on the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8) step.
Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8) Master Step Field Configuration
The following table lists the global custom fields that are configured to display on the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8) step.
Global Field Configuration (Derived Sample)
In this step, the addition of DPX, NaOH, Tris-HCl, and RSB denatures and dilutes the pooled samples. Manually create working pools based on the number of lanes that you want to sequence.
Step input: Bulk pool
Step output: Working pool - variable number, choose how many working pools to create per bulk pool
¹ These automations are for CLPA support only.
² These automations are required for the NovaSeq 6000 v3.8 workflow to function properly. These automations contain additional logic needed for CLPA support. If you would like to remove CLPA support, then contact Illumina Support.
Automations not identified with ¹ or ² are required for the NovaSeq 6000 v3.8 to work function properly.
The following table lists field configuration details defined on the Load to Flowcell (NovaSeq 6000 v3.8) step. A script sets these field values. The values are not editable while running the step.
Dilute, Denature & ExAmp (NovaSeq v3.8) Master Step Field Configuration
The following table lists the global custom fields that are configured to display on the Dilute, Denature & ExAmp (NovaSeq 6000 v3.8) step.
Global Field Configuration (Derived Sample)
In this step, scan the flow cell barcode into the Clarity LIMS. Then, manually place the working pools into the lanes of the flow cell for the NovaSeq run. This step validates the run setup information and generates the sample sheet file.
Step input: Working pool
Step output: Flow cell (output containers: SP, S1, and S2 with 2 lanes, and S4 with 4 lanes)
¹ These automations are for CLPA support only.
² These automations are required for the NovaSeq 6000 v3.8 workflow to function properly. These automations contain additional logic needed for CLPA support. If you would like to remove CLPA support, then contact Illumina Support.
Automations not identified with ¹ or ² are required for the NovaSeq 6000 v3.8 to work function properly.
The following table lists the field configuration details defined on the Load to Flowcell (NovaSeq 6000 v3.8) step.
Load to Flowcell (NovaSeq 6000 v3.8) Master Step Field Configuration
The following table lists the global custom fields that are configured to display on the Load to Flowcell (NovaSeq 6000 v3.8) step.
Global Field Configuration (Derived Sample)
This final protocol contains one fully automated step, AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8), described in the following section.
Step input: Library tube from NovaSeq Standard or flow cell from NovaSeq Xp protocol
Step output: Result file/measurement
In this step, pooled samples are sequenced on the NovaSeq instrument and the run metrics are recorded in Clarity LIMS.
¹ These automations are for CLPA support only.
² Not used. By default, the Sequencer API determines this functionality. If necessary, enable the automation and use it to override the next step behavior used by the Sequencer API.
Automations not identified with ¹ are required for the NovaSeq 6000 v3.8 to work function properly.
The following fields are configured on the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step in Clarity LIMS:
Current Cycle
Current Read
Firmware Version
Flow Cell Expiration Date
Flow Cell ID
Flow Cell Lot Number
Flow Cell Mode
Flow Cell Part Number
Flow Cell Side
Instrument Control Software Version
Instrument ID
Instrument Type
Lane Counter
Loading Workflow Type
Output Folder
RTA Version
Run Completion Date
Run ID
Run Status
Sequencing Log
The following fields are used to capture the run metrics in Clarity LIMS:
% Aligned R1
% Aligned R2
% Bases >=Q30 R1
% Bases >=Q30 R2
% Error Rate R1
% Error Rate R2
% Phasing R1
% Phasing R2
% Prephasing R1
% Prephasing R2
%PF R1
%PF R2
Cluster Density (K/mm^2) R1
Cluster Density (K/mm^2) R2
Intensity Cycle 1 R1
Intensity Cycle 1 R2
Reads PF (M) R1
Reads PF (M) R2
Yield PF (Gb) R1
Yield PF (Gb) R2
Note the following details:
Values are aggregated across all lanes. Some values (e.g., Yield PF (Gb) R1) are summed while others are averaged.
The names listed previously are the default global custom field names installed with the NovaSeq Integration v3.8 configuration provided in the Illumina Preset Protocols (IPP) v2.6 or later.
All global configuration fields are configured on the Container entity.
All field names are configurable through the Custom Fields screen in the Global Fields tab.
If field names are changed in Clarity LIMS, they must also be changed through the run.metricUdfNames properties file in the application.yml file for the Sequencer API. For more information, refer to the application.yml Properties section in Components Installed. All fields are configured to be visible in the Sample Details table on the Record Details screen for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step.
All run metrics tracked in Clarity LIMS are overall metrics for the run. There are currently no per-lane metrics provided by NovaSeq Control Software.
The sample sheet is generated on the step before the run. This step is Dilute and Denature (NovaSeq 6000 v3.8) in the NovaSeq Standard protocol or Load to Flowcell (NovaSeq 6000 v3.8) in the NovaSeq Xp protocol. This step places samples on the library tube or flow cell that are loaded in the NovaSeq 6000 instrument.
In the default configuration, the Validate Run Setup and Generate Sample Sheet automation generates one CSV format sample sheet file. The bcl2fastq v2.20 downstream analysis uses this file.
The sample sheet is uploaded to the NVCS via the /Illumina/Sequencer/v2/sequencing-run/files endpoint to the Sequencer API. The file endpoint allows for a file to be downloaded from the Clarity LIMS using OAuth (instead of Basic Authentication), which is required for the NovaSeq 6000 instrument.
The run recipe response sets the sample sheet URL to the link to download the file from this endpoint and sets the sampleSheetRequiresOAuth value to true.
The following steps outline the sequence of events that occurs when a flow cell is loaded onto the NovaSeq 6000 instrument.
The following sections describe the components (files, properties, reagent categories/label groups, reagent kits, and containers) that are installed by default as part of this integration.
Illumina NovaSeq 6000 Integration v3.6.0 is distributed as the BaseSpaceLIMS-sequencer-api RPM package. This RPM package must be installed on the Clarity LIMS server.
The BaseSpaceLIMS-sequencer-api RPM installs the following items:
Sequencer API WAR file
application.yml configuration file
Two configuration scripts:
configure_sequencer_api_proxy.sh
configure_sequencer_api_application.sh
If the NGS Extensions Package is not already installed, or if a version earlier than v5.23.0 is installed, the latest version is installed by default with the NovaSeq 6000 integration. For more information, refer to NovaSeq 6000 Integration v3.6.0 Release Notes.
If NGS Extensions Package v5.23.0 or later is already installed, the upgrade is not forced or required.
The following table lists the components installed by the RPM package.
The application.yml file is at opt/gls/clarity/extensions/sequencer-api/.
All properties are configured automatically during installation, but they can also be configured by editing the application.yml file and then restarting Tomcat.
Properties in the file represent all custom field names, both for recipe and run information.
For all custom fields, the names (values in this file) can be changed. However, the property names cannot be changed, removed, or added to. For example, Loading Workflow Type can be changed, but recipe.udfNames.sampleLoadingType cannot.
application.yml Properties Installed
TruSeq HT Adapters v2 (D7-D5)
Buffer Cartridge
Cluster Cartridge
DPX1
DPX2
DPX3
NaOH
Resuspension Buffer (RSB)
SBS Cartridge
Tris HCl
Library Tube
SP
S1
S2
S4
This integration supports the following items:
Library tube with barcode provided in the format [A-Z]{2}[0-9]{7}-[A-Z]{3} (eg, AB1234567-XYZ)
SP, S1, S2, or S4 flow cell with barcode provided in one of the following formats:
SP and S1 flow cell: [A-Z0-9]{5}DR[A-Z0-9]{2}
S2 flow cell: [A-Z0-9]{5}DM[A-Z0-9]{2}
S4 flow cell: [A-Z0-9]{5}DS[A-Z0-9]{2}
Example: H1991DMXX
For details on configuring NVCS for integration with Clarity LIMS, contact the Clarity LIMS Support team.
The requirements for the routing script functionality are as follows.
On the steps that use the routing script (Define Run Format (NovaSeq 6000 v3.8) and Dilute and Denature (NovaSeq 6000 v3.8)), the Next Step for all samples must be set to Remove from workflow. A script sets this value. The value must not change in the Assign Next Steps screen.
In the Next Steps section of the protocol configuration screen, the method of assigning the next step must be set to Automation for the last step of the protocol.
The application.yml configuration file contains the following properties:
run.autoComplete
run.autoCompleteOnlyAtSuccess
These properties determine the conditions under which the last step of the NovaSeq 6000 workflow (AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8)) is automatically completed when the run status is "RunCompletedSuccessfully" (default behavior) or regardless of the run status.
By default, both properties are set to true and the step only completes when the run is successful. If the run fails or is aborted, manually complete the step in Clarity LIMS.
For the step to autoComplete regardless of the result of the sequencing run, change the autoCompleteOnlyAtSuccess property value to false.
Enabling/Disabling autoComplete Properties
Open the application.yml file at /opt/gls/clarity/extensions/sequencer-api/application.yml:
Edit the value of autoCompleteOnlyAtSuccess property as required.
Save the file.
The following table shows how the combined value of run.autoComplete and run.autoCompleteOnlyAtSuccess properties affects the autoComplete behavior of the sequencing step.
run.autoComplete and run.autoCompleteOnlyAtSuccess Value Matrix
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 determine the following information:
Which samples, and how many, can enter each step together.
Which samples, and how many, can be pooled together.
The library tube ID must be unique. There must not be multiple library tube containers in the system with the same name.
Reagent labels (indexes) must be unique.
Only controls are permitted as unindexed samples; all other unindexed samples and pools are not permitted.
For sample sheet generation constraints, refer to the Bcl2fastq2 Sample Sheet Generation section in Illumina Instrument Sample Sheets (NGS v5.17 & later).
Do not manually start the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step. This step is a fully automated and the sequencing service may not update samples correctly if they have been manually started.
For the automated run to start successfully, Validate Run Setup and Generate Sample Sheet must be selected.
NovaSeq Integration Package v3.6.0 contains modifications to the existing automations from NovaSeq Integration Package v3.5.1. The NovaSeq Sequencing v3.8 workflow and Illumina Preset Protocols (IPP) v2.6 are required for the integration package to work.
If you do not want to upgrade the existing NovaSeq Sequencing workflow to the latest version through IPP, you can update the workflow configuration manually from NovaSeq Integration package v3.5.1 to v3.6.0 by completing the following steps.
For manual upgrades from versions prior to v3.5.1, please refer to manual upgrade guides for those versions.
The key change between NovaSeq 6000 v3.7 workflow and NovaSeq 6000 v3.8 workflow that comes with NovaSeq 6000 Integration v3.6.0 is the addition of Clarity LIMS Product Analytics (CLPA) enabled automation scripts. If CLPA is not required, it is not necessary to perform this manual upgrade.
From Configuration, select the Automation tab.
Add the automations in the following table:
¹ This automation has -l {compoundOutputFileLuid0} in the log file output index.
² This automation has -l {compoundOutputFileLuid1} in the log file output index.
From Configuration, select the Automation tab.
Under the Step Automation tab, select the Routing Script automation that is enabled on the Define Run Format (NovaSeq 6000 v3.7) master step.
In the automation name field, change the name to Routing Script & Register Step Completed.
Update the existing command line as follows. Changes are in red.
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \ \ --FIELD_NAME 'Loading Workflow Type' \ --FIELD_VALUE 'NovaSeq Standard' \ --WORKFLOW 'NovaSeq 6000 v3.7' \ --STEP 'Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.7)' \ --INPUTS_OR_OUTPUTS 'INPUTS' \ \ --FIELD_NAME 'Loading Workflow Type' \ --FIELD_VALUE 'NovaSeq Xp' \ --WORKFLOW 'NovaSeq 6000 v3.7' \ --STEP 'Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.7)' \ --INPUTS_OR_OUTPUTS 'INPUTS' \ && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid0} -s 'com/illumina/upa/scripts/common/step_completed.groovy'"
Select Save.
From Configuration, select the Automation tab.
Under the Step Automation tab, select the Routing Script automation that is enabled on the Dilute and Denature (NovaSeq 6000 v3.7) master step.
In the automation name field, change the name to Routing Script & Register Step Completed.
Update the existing command line as follows. Changes are in red.
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \ \ --FIELD_NAME 'Loading Workflow Type' \ --FIELD_VALUE 'NovaSeq Standard' \ --WORKFLOW 'NovaSeq 6000 v3.7' \ --STEP 'Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.7)' \ --INPUTS_OR_OUTPUTS 'INPUTS' \ \ --FIELD_NAME 'Loading Workflow Type' \ --FIELD_VALUE 'NovaSeq Xp' \ --WORKFLOW 'NovaSeq 6000 v3.7' \ --STEP 'Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.7)' \ --INPUTS_OR_OUTPUTS 'INPUTS' \ && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid0} -s 'com/illumina/upa/scripts/common/step_completed.groovy'"
Select Save.
From Configuration, select the Automation tab.
Under the Step Automation tab, select the Validate Inputs and Selected Container automation that is enabled on the Load to Flowcell (NovaSeq 6000 v3.7) master step.
In the automation name field, change the name to Validate Inputs and Selected Container & Register Step Started.
Update the existing command line as follows. Changes are in red.
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar script:validate_flowcell_for_input_pools -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -validateSelectedContainer true && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/step_started.groovy'"
Select Save.
From Configuration, select the Automation tab.
Under the Step Automation tab, select the Validate Inputs Flowcell Type automation that is enabled on the Dilute, Denature & ExAmp (NovaSeq 6000 v3.7) master step.
In the automation name field, change the name to Validate Inputs Flowcell Type & Register Step Started.
Update the existing command line as follows. Changes are in red.
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar script:validate_flowcell_for_input_pools -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/step_started.groovy'"
Select Save.
From Configuration, select the Automation tab.
Under the Step Automation tab, select the Validate Single Input automation that is enabled on the Dilute and Denature (NovaSeq 6000 v3.7) master step.
In the automation name field, change the name to Validate Single Input & Register Step Started.
Update the existing command line as follows. Changes are in red.
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:validateSampleCount -min 1 -max 1 && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/step_started.groovy'"
Select Save.
From Configuration, select the Lab Work tab.
Change the trigger locations and styles for the automations in the following table:
Not required for Illumina cloud hosted installations.
Run this script when the Sequencer API is first installed.
For calculation purposes, not displayed
For calculation purposes, not displayed
For calculation purposes, not displayed
For calculation purposes, not displayed
Used in Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8) step only. Displays on Record Details screen and in the generated CSV file.
Value of 251 is only supported for SP flow cell type. For all other cell types, maximum value is 151.
Value of 251 is only supported for SP flow cell type. For all other cell types, maximum value is 151.
Not displayed in user interface
Not displayed in user interface
Do not remove this field as it is used by Validate Run Setup and Generate Sample Sheet automation script.
For calculation purposes, not displayed
For calculation purposes, not displayed
For calculation purposes, not displayed
For calculation purposes, not displayed
Value of 251 is only supported for SP flow cell type. For all other cell types, maximum value is 151.
Value of 251 is only supported for SP flow cell type. For all other cell types, maximum value is 151.
Not displayed in user interface
Not displayed in user interface
Do not remove this field as it is used by Validate Run Setup and Generate Sample Sheet automation script.
Not required for Illumina cloud hosted installations.
Run this script when the Sequencer API is first installed.
Do not add samples to the Ice Bucket or start the step. The integration starts the step automatically.
The Library Prep Validation v2.3.1 workflow allows for validation of the system after installation is complete. For details, refer to NovaSeq 6000 Integration v3.6.0 User Interaction, Validation and Troubleshooting.
Create only one pool per step.
Validate Unique Indexes automation is not used. The Clarity LIMS configuration for pooling handles this functionality.
For accurate pipetting of each sample in a pool, the Per Sample Volume (ul) value must be greater than or equal to the Minimum Per Sample Volume (ul). The default value (set at 5) can be edited.
The script definition portion of Validate Run Setup has been moved into the Validation Script custom field due to the 4000 character limit.
Create only one pool per step.
Validate Unique Indexes automation is not used. The Clarity LIMS configuration for pooling handles this functionality.
For accurate pipetting of each sample in a pool, the Per Sample Volume (ul) value must be greater than or equal to the Minimum Per Sample Volume (ul). The default value (set at 5) can be edited.
The script definition portion of Validate Run Setup has been moved into the Validation Script custom field due to the 4000 character limitation.
This step is fully automated. Do not add samples to the Ice Bucket or start the step manually. The sequencing service may not update samples correctly when they have been manually started.
Do not disable or modify this automation to ensure the Lane Number displays properly.
These global custom fields are configured on the Container entity, visible on each input, but they should not be confused with per-lane metrics.
Configure the steps that are considered sequencing run steps in the novaseq.sequenceStepNames property in the application.yml file. The property is a list and each list entry must be the exact name of a sequencing step, not the name of the underlying master step/process type. For more information, refer to the application.yml Properties section of Components Installed.
If no matching container is found, or if the samples in the container are incorrectly queued, an error occurs and the step is not started. This validation is also done during the run recipe request immediately prior, so this error case should not be encountered.
The NovaSeq 6000 v3 configuration is delivered through Illumina Preset Protocols (IPP) v2.6, which requires installation of the NGS Extensions Package v5.23.0 or later.
Field Name
Field Type
Options
Additional Options and Dropdown Items
Comment
Multiline Text
None
Flowcell Type
Text Dropdown
Required Field
Custom Entries
Presets
SP
S1
S2
S4
Instruction
Text
Read Only
Default
Add Flowcell Type and Loading Workflow Type below
Loading Workflow Type
Text Dropdown
Required Field
Presets
NovaSeq Standard
NovaSeq Xp
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
Decimal places displayed = 0
Presets
225
400
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]
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
Field Name
Field Type
Options
Additional Options and Dropdown Items
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 Dropdown Items
RSB Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
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 Dropdown 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
Options
Additional Options and Dropdown 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]
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
Field Name
Field Type
Options
Additional Options and Dropdown Items
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]
Files Installed
Location
Description
Illumina#Sequencer#v2.war
/opt/gls/clarity/tomcat/current/webapps
War file for Sequencer API application.
configure_sequencer_api_application.sh
/opt/gls/clarity/config/
Script that configures the Sequencer API application through its external application.yml file.
configure_sequencer_api_env.sh
/opt/gls/clarity/config/
Script that configures the Clarity LIMS Tomcat configuration to include Secret Util settings.
configure_sequencer_api_proxy.sh
/opt/gls/clarity/config/
Script that configures the proxy to allow communication with the Sequencer API application.
sequencer-api.conf
/etc/httpd/clarity/
Proxy setting to communicate with the Clarity LIMS.
run.autoComplete Property Value
run.autoCompleteOnlyAtSuccess Property Value
Outcome
true
true
Step automatically completes only if sequencing run is successful.
If run is not successful, step does not automatically complete, and the run details are recorded in Clarity LIMS.
true
false
Step automatically completes, regardless of the run status.
false
true
Step does not automatically complete.
Run details are recorded in Clarity LIMS.
false
false
Step does not automatically complete.
Run details are recorded in Clarity LIMS.
Version
Changes
2
Updated Compatibility section to reference Compatibility matrix table.
1
Initial release.
Field Name
Field Type
Options
Additional Options and Dropdown Items
% PhiX (2.5 nM) Spike-In
Numeric
Range = 0–100
Bulk Pool Volume (ul)
Numeric
Decimal places displayed = 2
Number of Flowcells to Sequence
Numeric
Required Field
Range = 1–10
Decimal places displayed = 0
Minimum Per Sample Volume (ul)
Numeric
Required Field
Decimal places displayed = 2
Default
5
Number of Samples in Pool
Numeric
Decimal places displayed = 0
Default
0
PhiX Volume (ul)
Numeric
Decimal places displayed = 2
Total Sample Volume (ul)
Numeric
Decimal places displayed = 2
Default
0
Field Name
Field Type
Options
Additional Options and Dropdown Items
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]
NaOH Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
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)
Numeric
Read Only
Decimal places displayed = 0
Field Name
Field Type
Options
Additional Options and Dropdown Items
Analysis Software Version
Text
BaseSpace Sequence Hub Configuration
Text Dropdown
Required Field
Presets
Not Used
Run Monitoring Only
Run Monitoring and Storage
Custom Recipe Path
Text
Not applicable
Experiment Name
Text
Required Field
Not applicable
Index Read 1
Numeric Dropdown
Required Field
Custom Entries
Range = 0–20
Decimal places displayed = 0
Presets
0
6
8
Index Read 2
Numeric Dropdown
Required Field
Custom Entries
Range = 0–20
Decimal places displayed = 0
Presets
0
6
8
Output Folder
Text
Required Field
Not applicable
Override Cycles
Text
Not applicable
Paired End
Text Dropdown
Required Field
Presets
True
False
Read 1 Cycle
Numeric Dropdown
Required Field
Custom Entries
Range = 1–251
Decimal places displayed = 0
Presets
251
151
101
51
Read 2 Cycle
Numeric Dropdown
Required Field
Custom Entries
Range = 0–251
Decimal places displayed = 0
Presets
251
151
101
51
Reverse Complement Workflow
Toggle Switch
Required Field
Default
Yes
Run Mode
Text Dropdown
Read Only
Presets
SP
S1
S2
S4
Samplesheet Format
Text Dropdown
Required Field
Presets
V1 (default)
V2
Settings Header
Text
Read Only
Not applicable
UMI—Read 1 Length
Numeric
Range = 1
UMI—Read 1 Start From Cycle
Numeric
Range = 1
UMI—Read 2 Length
Numeric
Range = 1
UMI—Read 2 Start From Cycle
Numeric
Range = 1
Use Custom Index Read 1 Primer
Toggle Switch
Default
None Set
Use Custom Read 1 Primer
Toggle Switch
Default
None Set
Use Custom Read 2 Primer
Toggle Switch
Default
None Set
Use Custom Recipe
Toggle Switch
Required Field
Default
No
Validation Script
Multiline Text
Required Field
Read Only
Default value is provided in the drop-down section that follows the table.
Workflow
Text
Read Only
Default
GenerateFASTQ
Workflow Type
Text Dropdown
Required Field
Presets
No Index
Single Index
Dual Index
Custom
Field Name
Field Type
Options
Additional Options and Dropdown Items
% PhiX (0.25nM) Spike-in
Numeric
Range = 0–100
Bulk Pool Volume (ul)
Numeric
Decimal places displayed = 2
Minimum Per Sample Volume (ul)
Numeric
Required Field
Decimal places displayed = 2
Default
5
Number of Lanes to Sequence
Numeric
Required Field
Decimal places displayed = 0
Number of Samples in Pool
Numeric
Decimal places displayed = 0
Default
0
PhiX Volume (ul)
Numeric
Decimal places displayed = 2
Total Sample Volume (ul)
Numeric
Decimal places displayed = 0
Default
0
Field Name
Field Type
Options
Additional Options and Dropdown Items
Analysis Software Version
Text
Not applicable
BaseSpace Sequence Hub Configuration
Text Dropdown
Required Field
Presets
Not Used
Run Monitoring Only
Run Monitoring and Storage
Custom Recipe Path
Text
Not applicable
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
Index Read 2
Numeric Dropdown
Required Field
Custom Entries
Range = 0–20
Decimal places displayed = 0
Presets
0
6
8
Library Tube Barcode
Text
Required Field
Not applicable
Output Folder
Text
Required Field
Not applicable
Override Cycles
Text
Not applicable
Paired End
Text Dropdown
Required Field
Presets
True
False
Read 1 Cycle
Numeric Dropdown
Required Field
Custom Entries
Range = 1–251
Decimal places displayed = 0
Presets
251
151
101
51
Read 2 Cycle
Numeric Dropdown
Required Field
Custom Entries
Range = 0–251
Decimal places displayed = 0
Presets
251
151
101
51
Reverse Complement Workflow
Toggle Switch
Required Field
Default
Yes
Run Mode
Text Dropdown
Read Only
Presets
SP
S1
S2
S4
Samplesheet Format
Text Dropdown
Required Field
Presets
V1 (default)
V2
Settings Header
Text
Read Only
Not applicable
UMI—Read 1 Length
Numeric
Range = 1
UMI—Read 1 Start From Cycle
Numeric
Range = 1
UMI—Read 2 Length
Numeric
Range = 1
UMI—Read 2 Start From Cycle
Numeric
Range = 1
Use Custom Index Read 1 Primer
Toggle Switch
Default
None Set
Use Custom Read 1 Primer
Toggle Switch
Default
None Set
Use Custom Read 2 Primer
Toggle Switch
Default
None Set
Use Custom Recipe
Toggle Switch
Required Field
Default
No
Validation Script
Multiline Text
Required Field
Read Only
Default value is provided in the drop-down section that follows the table.
Workflow
Text
Read Only
Default
GenerateFASTQ
Workflow Type
Text Dropdown
Required Field
Presets
No Index
Single Index
Dual Index
Custom
Property
Description
Default Value
spring.profiles.active
Tells the application that it is running deployed in Tomcat.
tomcat
clarity.url
Base URL that the Sequencer API service uses to contact the Clarity LIMS. The installation script (configure_sequencer_api_application) prompts for this URL.
clarity.username
Username to be used when communicating with Clarity LIMS.
security.signing-key
Private key that is used when signing/validating OAuth tokens. Changing this key invalidates any issued tokens.
security.token-expiry
Specifies (in hours) the expiry period for login tokens issued by the Sequencer API.
88
novaseq.sequenceStepNames
List of NovaSeq sequencing run step names for which the integration can find samples queued.
Names must be an exact match to the name of the step in Clarity LIMS (not the master step/process type).
Each line must be indented to the same point and start with a dash (-).
- "AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8)"
- "AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.1)"
novaseq.flowcells
List of supported NovaSeq flow cell types.
Type names exactly match the values used by the NVCS run status requests.
To add more types, provide both type and lanes properties, indented to the same point and starting with a dash (-).
- type: "SP" lanes: 2
- type: "S1" lanes: 2
- type: "S2" lanes: 2
- type: "S4" lanes: 4
novaseq.reagents
List of supported reagent kits to be tracked on sequencing steps.
lotName — The name of the lot as it appears in the run started request.
kitName — The name of the corresponding reagent kit in Clarity LIMS that the lot is tracked as.
To add more types, provide both type and lanes properties, indented to the same point and starting with a dash (-).
- lotName: "Buffer" kitName: "Buffer Cartridge"
- lotName: "Cluster" kitName: "Cluster Cartridge"
- lotName: "SBS" kitName: "SBS Cartridge"
recipe.udfNames
Configures the names of the fields used in the run recipe. For more information, refer to the Run Recipe Contents section of NovaSeq 6000 Run.
Each value must be enclosed in quotes (e.g., recipe.udfNames.runMode "Run Mode").
Value provided for sampleLoadingType must match the value provided in the run configuration (e.g., recipe.udfNames.sampleLoadingType "Loading Workflow Type").
recipe.sampleSheet.outputName recipe.sampleSheet.notAvailableValue
Configures the name of the sample sheet file placeholder on the step where run recipe information is populated, and the property value to use when the file is not found.
run.stepUdfNames
Configures the master step field names to be used when recording run results. For more information, refer to the Master Step Fields section of Step 1: AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.7) in Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8).
Each value must be enclosed in quotes (e.g., run.stepUdfNames.runID "Run ID")
Value provided for sampleLoadingType must match the value provided in the run configuration (e.g., run.stepUdfNames.sampleLoadingType "Loading Workflow Type"). It is expected that the run step field and the field on the input derived sample share a name.
run.metricUdfNames
Configures the result file custom fields/measurement global field names to be used when recording run results. These fields capture the run data. For more information, refer to the Global Fields section of Step 1: AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.7) in Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8).
run.autoCompleteOnlyAtSuccess
Determines which of the following options that the sequencing run step must be:
Autocompleted when the run status is RunCompletedSuccessfully (if autoCompleteOnlyAtSuccess is true)
Autocompleted regardless of the run status (if autoCompleteOnlyAtSuccess is false). For more information, refer to Configuring autoComplete Properties.
true
Automation Name
Command Line
Master Steps
Register NovaSeq RunCompleted & Register Step Completed
AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.7)
Register Pools & Register Step Completed
Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.7)
Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.7)
Register Step Completed
Dilute, Denature & ExAmp (NovaSeq 6000 v3.7)
Load to Flowcell (NovaSeq 6000 v3.7)
Register Step Started¹
Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.7)
Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.7)
Register Step Started²
Define Run Format (NovaSeq 6000 v3.7)
Register Step Started & Register NovaSeq Run & Register NovaSeq Run Association & Register NovaSeq RunStarted
AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.7)
Step Name
Automation Configuration
Define Run Format (NovaSeq 6000 v3.7)
Register Step Started
Trigger Location: Step
Trigger Style: Automatic upon entry
Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.7)
Register Step Started
Trigger Location: Step
Trigger Style: Automatic upon entry
Register Pools & Register Step Completed
Trigger Location: Step
Trigger Style: Automatic upon exit
Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.7)
Register Step Started
Trigger Location: Step
Trigger Style: Automatic upon entry
Register Pools & Register Step Completed
Trigger Location: Step
Trigger Style: Automatic upon exit
Dilute, Denature & ExAmp (NovaSeq 6000 v3.7)
Register Step Completed
Trigger Location: Step
Trigger Style: Automatic upon exit
Load to Flowcell (NovaSeq 6000 v3.7)
Register Step Completed
Trigger Location: Step
Trigger Styler: Automatic upon exit
AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.7)
Register Step Started & Register NovaSeq Run & Register NovaSeq Run Association & Register NovaSeq RunStarted
Trigger Location: Step
Trigger Style: Automatic upon entry
Register NovaSeq RunCompleted & Register Step Completed
Trigger Location: Step
Trigger Style: Automatic upon exit
for calculation purposes, not displayed
for calculation purposes, not displayed
for calculation purposes, not displayed
for calculation purposes, not displayed
Used in Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8) step only. Displays on Record Details screen and in the generated CSV file.
Value of 251 is only supported for SP flow cell type. For all other cell types, maximum value is 151.
Value of 251 is only supported for SP flow cell type. For all other cell types, maximum value is 151.
Not displayed in user interface
Not displayed in user interface
Do not remove this field as it is used by Validate Run Setup and Generate Sample Sheet automation script.
For calculation purposes, not displayed
For calculation purposes, not displayed
For calculation purposes, not displayed
For calculation purposes, not displayed
Value of 251 is only supported for SP flow cell type. For all other cell types, maximum value is 151.
Value of 251 is only supported for SP flow cell type. For all other cell types, maximum value is 151.
Not displayed in user interface
Not displayed in user interface
Do not remove this field as it is used by Validate Run Setup and Generate Sample Sheet automation script.
Do not change
The name can differ in the NVCS request versus the Clarity LIMS configuration.
