The Illumina NovaSeq 6000 Integration v2.6.0 supports the integration between Clarity LIMS and the NovaSeq 6000 instrument. This integration also provides support for Clarity LIMS v6.2.0 and includes updates to the technology stack and third party libraries and utilities.

Clarity LIMS is automatically stopped during installation of the RPM and will need to be restarted. The file-based and API-based integrations cannot run at the same time. When the API-based integration has finished installing, shut down Clarity LIMS and uninstall the file-based integration.

This document provides instructions for installing NovaSeq 6000 Integration v2.6.0. It also describes the components that are installed in the default configuration.

Supporting Documentation

• For details on installed protocols and steps, automations, generated and captured files, and rules and constraints, refer to NovaSeq 6000 Integration v2.6.0 Configuration.

• For information on user interaction for each step, validating and troubleshooting the integration, refer to NovaSeq 6000 Integration v2.6.0 User Interaction, Validation and Troubleshooting.

Prerequisites

NovaSeq 6000 Integration v2.6.0 has the following prerequisites:

• Mount run data network-attached storage (NAS) share

• Secret Util is installed

• IPP is installed

Installation

NovaSeq 6000 Integration v2.6.0 supports both on-premise and cloud integrations. This integration is distributed as the following RPM packages:

• BaseSpaceLIMS-novaseq-extensions

• BaseSpaceLIMS-novaseq-sequencing-service (depends on BaseSpaceLIMS Automation Worker v1.x)

The BaseSpaceLIMS-novaseq-extensions RPM installs the following items:

• Protocols and steps

• Validation workflow

• Integration properties that configure the service

• novaseq-extensions.jar file that provides sample sheet generation and other utility scripts

The BaseSpaceLIMS-novaseq-sequencing-service RPM installs the following items:

• Bash scripts to run the novaseq_seqservice through systemctl

• novaseq-sequencing.jar

• novaseq-remote-extensions.jar file that contains scripts that need access to the run folders on the NAS

On-Premise Installation

Use the following instructions to install the BaseSpaceLIMS-novaseq-extensions and BaseSpaceLIMS-novaseq-sequencing-service RPMs on the Clarity LIMS server.

Cloud Installation

The BaseSpaceLIMS-novaseq-extensions RPM must be installed on the Clarity LIMS server. The BaseSpaceLIMS-novaseq-sequencing-service RPM can be installed remotely on another server within the network.

Configurable Properties

For more information on the configurable integration properties that enable capture and generation of files associated with the sequencing run, refer to Integration Properties Details

The sample sheet, run recipe, and run output folders can all be configured independently and do not have to be on the same mount or share. For examples of valid configuration mount points and properties, refer to Example Network Folder Structure and Database Properties.

Workflows, Protocols, and Steps Installed

NovaSeq 6000 v2.3 Workflow

NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) Workflow

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 v2.6.0 Configuration.

For instructions on using the NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) protocol, refer to NovaSeq 6000 Integration v2.6.0 User Interaction, Validation and Troubleshooting.

Instrument Software

The NovaSeq instrument software includes the following components:

• NovaSeq Control Software (NVCS) — Contains the user interface for setting up the sequencing run. Responsible for controlling the instrument and acquiring the images.

• Real-Time Analysis v3 (RTA3) — Takes the images generated by the first module, processes, and analyzes them. Makes sure that data files are created and copied to the final destination folder.

• Sequencing Analysis Viewer (SAV) — Displays the important quality metrics generated by the RTA3 software.

• 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).

Instrument Integration Configuration

For the NovaSeq API integration to work, the NovaSeq instrument must be able to communicate with Clarity LIMS through the API. Complete the following steps to configure the NVCS and confirm that you can access Clarity LIMS from the instrument.

1. Launch NVCS and wait for the initialization process to complete.

2. On the NVCS Settings page, do the following:

   1. Under Mode Selection, select File-Based.

   2. In the adjacent field, enter the network folder location that NVCS retrieves the JSON recipe from.

   3. In the Output Folder field, enter the default output folder location.

3. Select Save to complete the configuration.

4. On the NVCS home page, you can now select Sequence to log into Clarity LIMS and start a run.

Example Network Folder Structure and Database Properties

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 2.x.

The integration includes the following functionality:

• Preconfigured NovaSeq 6000 v2.3 workflow that maps to lab protocols and instrument runs.

• Preconfigured NovaSeq Standard (NovaSeq 6000 v2.3) protocol that supports the loading of pooled libraries into a library tube.

• Preconfigured NovaSeq Xp (NovaSeq 6000 v2.3) protocol that supports individual lane loading on the NovaSeq.

• Automated generation of a sample sheet for use with bcl2fastq2 v2.20 analysis software.

• Automatic generation of run recipe file (JSON format) that is automatically uploaded to the sequencing instrument and used to set up and initiate the run.

• Automated tracking of the NovaSeq sequencing run and parsing of run statistics (per run per lane) into Clarity LIMS, including:

  • Progress and metrics of sequencing run

  • Per instrument sequencing runs (tracked as part of the Run ID field value)

  • Sequencing run parameters

  • Real-Time Analysis v3 (RTA3) run directory location and other run-specific information

• Preconfigured NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) workflow used for validation purposes only. The workflow contains a single-step protocol that models the library prep required to produce normalized libraries that are ready for the NovaSeq 6000 v2.3 workflow. For details, refer to NovaSeq 6000 Integration v2.6.0 User Interaction, Validation and Troubleshooting.

This guide explains how to validate the installation of the Illumina NovaSeq 6000 Integration Package v2.5.0. The validation process involves the following actions:

• Running samples through the NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) workflow. The workflow contains a single-step protocol that models the library prep required to produce normalized libraries. At the end of the step, the normalized libraries are automatically advanced to the NovaSeq 6000 v2.3 workflow.

• Running normalized libraries through the NovaSeq 6000 v2.3 workflow. This process validates the following details:

  • Successful routing of samples from the Run Format (NovaSeq 6000 v2.3) step to the NovaSeq Standard (NovaSeq 6000 v2.3) or NovaSeq Xp (NovaSeq 6000 v2.3) step.

  • Automated generation of a sample sheet for use with bcl2fastq2 v2.20 analysis software.

  • Automated generation of a run recipe file (JSON file) with the library tube or flow cell barcode as the name (eg, NV1234567-LIB.json or H1234DRXX.json). This file is automatically uploaded to the sequencing instrument and used to set up and initiate the run.

  • Automated tracking of the NovaSeq sequencing run and parsing of run statistics (per run per lane) into Clarity LIMS.

Before executing the validation steps, make sure that you have installed the Illumina NovaSeq 6000 Integration Package v2.5.0 and have imported the default Clarity LIMS configuration.

Activate Workflow, Create Project, Add and Assign Samples

The following steps set up Clarity LIMS in preparation for running samples through the NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) and NovaSeq 6000 v2.3 workflows.

1. In the Clarity LIMS Configuration area, activate the NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) and NovaSeq 6000 v2.3 workflows.

2. On the Projects and Samples screen, create a project and add samples to it.

3. Assign the samples to the NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) workflow.

Library Prep Protocol: NovaSeq Validation Library Prep (NovaSeq 6000 v2.3)

This single-step protocol models the library prep required to produce normalized libraries that are ready for the NovaSeq 6000 v2.3 workflow.

• Label Group = TruSeq HT Adapters v2 (D7-D5)

• Sequencing Instrument = NovaSeq

On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the NovaSeq 6000 v2.3 workflow—Define Run Format (NovaSeq 6000 v2.3). This is the only step in Protocol 1: Run Format (NovaSeq 6000 v2.3).

Protocol 1: Run Format (NovaSeq 6000 v2.3)

This protocol includes the Define Run Format (NovaSeq 6000 v2.3) step. The step allows for the assignment of per sample values for Loading Workflow Type, Normalized Molarity, Flowcell Type, and Final Loading Concentration (pM). At the end of the step, samples are routed to the NovaSeq Standard or NovaSeq Xp protocol, according to the selected Loading Workflow Type.

Step 1: Run Define Run Format (NovaSeq 6000 v2.3)

1. In Lab View, locate the Run Format (NovaSeq 6000 v2.3) protocol. The samples are queued for the Define Run Format (NovaSeq 6000 v2.3) step.

2. Add the samples to the Ice Bucket and select View Ice Bucket.

3. On the Ice Bucket screen, select Begin Work.

4. On the Record Details screen, specify the Sample Details.

   • Loading Workflow Type — Select NovaSeq Standard or NovaSeq Xp from the drop-down list.

   • Normalized Molarity — Enter values or use values copied from the previous step.

5. Populate the following fields:

   • Flowcell Type — Select SP, S1, S2, or S4.

   • Final Loading Concentration (pM) — Select from the two preset options — 225 (for PCR-free workflows) or 400 (for Nano workflows) — or enter a different value.

6. Select Next Steps to trigger the Set Next Steps automation, which does the following actions:

   • Sets the value of the next step (for all samples) to Remove from workflow. The Routing Script automation expects this value and requires it to advance samples to the next step successfully.

   • Calculates the Minimum Molarity.

   • Checks Normalized Molarity value. For samples with no Normalized Molarity value (eg, an empty value, not including 0), the automation generates an error message stating that the field cannot be empty.

   • Compares the Normalized Molarity value of each sample with the Minimum Molarity value.

7. On the Assign Next Steps screen, review the Sample Details table and make sure that the Next Step for all samples is prepopulated with Remove from workflow.

   Next Step must be set to Remove from workflow, regardless of the Loading Workflow Type.

8. Review the Warning field entries for the samples. If the field indicates that the Normalized Molarity is too low, continue with one of the following options:

   • Return to the Record Details screen and adjust the Normalized Molarity value so that it equals or exceeds the Minimum Molarity value. Set the Loading Workflow Type to NovaSeq Standard or NovaSeq Xp, as applicable.

   • Alternatively, complete the protocol without correcting the Normalized Molarity value. In this case, the samples in question are removed from the Clarity LIMS workflow.
9. Select Finish Step.

   The Routing Script automation is triggered if:

   • Samples with a Loading Workflow Type of Remove from workflow (ie, where Normalized Molarity value is lower than the Minimum Molarity) are removed from the Clarity LIMS workflow.

   • Samples with a Loading Workflow Type of NovaSeq Standard are routed to the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3) step. This step is the first of two steps in Protocol 2: NovaSeq Standard (NovaSeq 6000 v2.3).

   • Samples with a Loading Workflow Type of NovaSeq Xp are routed to the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3) step. This step is the first of three steps in Protocol 3: NovaSeq Xp (NovaSeq 6000 v2.3).

Protocol 2: NovaSeq Standard (NovaSeq 6000 v2.3)

In this protocol, samples are pooled and added to the library tube in preparation for the NovaSeq run. The protocol contains two steps:

1. Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3)

2. Dilute and Denature (NovaSeq 6000 v2.3)

Step 1: Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3)

1. In Lab View, locate the NovaSeq Standard (NovaSeq 6000 v2.3) protocol. The samples are queued for the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3) step.

2. On the Queue screen, add samples of the same flow cell type to the Ice Bucket and select Begin Work.
3. On the Pooling screen, create a pool by dragging samples into the Pool Creator.

   1. Type a name for the pool or accept the default name (Pool #1).

   1. Select Record Details.

4. On exit from the Pooling screen, the Validate Inputs Flowcell Type and Single Pool is triggered. The automation checks the following information:

   • All samples in the pool have been assigned the same Flowcell Type.

   • Only one pool has been created.

5. On the Record Details screen in the Step Details area, complete the fields as follows.

   1. Enter the value for the Number of Flowcells to Sequence field.

   This value is used in volume calculations to make sure that the volumes are sufficient for the number of times the pool is sequenced.

   1. Enter the value for the Minimum Per Sample Volume (ul) field.

   The value in this field is used to calculate how much of each sample is included in the pool. The field is prepopulated with the configured default value (5 µl), but the value can be edited if necessary.

   1. If necessary, enter the value for the % PhiX (2.5 nM) Spike-In field.

   The value in this field is used to calculate the volume of PhiX v3 control to be included in the pool for the given percentage of spike-in. The field is optional and can be edited.

   • If the smallest Per Sample Volume (ul) value is less than 5, the LIMS automatically assigns a value of 5 to the sample's Adjusted Per Sample Volume (ul) field.

   • Clarity LIMS then adjusts the Adjusted Per Sample Volume (ul) field value for all other samples in the batch, based on the ratio used to increase the lowest value to 5.

6. In the Sample Details table, select the pool icon to view details on the pool composition.
8. Select Next Steps to trigger the Set Next Steps automation.

   This automation sets the next step for samples to ADVANCE, advancing them to the Dilute and Denature (NovaSeq 6000 v2.3) step in the protocol.

9. Select Finish Step.

At the end of this step, the pool of samples automatically advances to the Dilute and Denature (NovaSeq 6000 v2.3) step.

Step 2: Dilute and Denature (NovaSeq 6000 v2.3)

1. In Lab View, locate the NovaSeq Standard (NovaSeq 6000 v2.3) protocol. The pool of samples is queued for the Dilute and Denature (NovaSeq 6000 v2.3) step.

2. Add the samples to the Ice Bucket and select Begin Work.

   At the beginning of the step, the Validate Single Input automation is triggered. This automation checks that there is only one container input to the step.

3. On the Placement screen, drag the pool into the library tube in the Placed Samples area.

   1. Scan or type the barcode of the library tube into the Library Tube field.

   1. Select Record Details.

   On exit of the Placement screen, the Validate Library Tube Barcode automation checks that the library tube barcode conforms to the barcode mask [A-Z]{2}[0-9]{7}-[A-Z]{3}. If not, an error message displays.

5. In the Reagent Lot Tracking section, select from the active lots displayed in each drop-down list. The fields displayed in the Step Details section are used to generate the sample sheet and run recipe files. Some of these fields are autopopulated and some must be completed manually. Refer to the following table for details.

   Fields Displayed on Record Details Screen of Dilute and Denature (NovaSeq 6000 v2.3) Step
6. On the Record Details screen, select Generate Sample Sheet & Run Recipe. This selection triggers the automation script: The sample sheet and JSON files are generated and attached to the placeholders in the Files area of the Record Details screen. The Sample Sheet Path field is populated with the path to the sample sheet file.

7. Select Next Steps. This selection triggers the Prepare Files for NovaSeq automation, which does the following:

   • Copies the sample sheet and run recipe files to the location specified during installation. The NovaSeq instrument software uses these files to set up the run.

   • Sets the value of the next step to Remove from workflow. The Routing Script automation expects this value and requires it to advance samples to the next step successfully.

8. On the Assign Next Steps screen, the Next Step for samples is prepopulated with Remove from workflow.

9. Select Finish Step.

On exit from the step, the Routing Script automation is triggered and samples are routed to Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3).

In Lab View, the pool of samples is queued for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step.

Protocol 3: NovaSeq Xp (NovaSeq 6000 v2.3)

In this protocol, samples are pooled and added to lanes on the NovaSeq flow cell. The flow cell type is determined by the option selected in the Define Run Format (NovaSeq 6000 v2.3) step.

The protocol contains three steps:

1. Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3)

2. Dilute, Denature & ExAmp (NovaSeq 6000 v2.3)

3. Load to Flowcell (NovaSeq 6000 v2.3)

Step 1: Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3)

1. In Lab View, locate the NovaSeq Xp (NovaSeq 6000 v2.3) protocol. The samples are queued for the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3) step.

2. On the Queue screen, add samples of the same Flowcell Type to the Ice Bucket and select Begin Work.
3. On the Pooling screen, create a pool by dragging samples into the Pool Creator.

   1. Type a name for the pool or accept the default name Pool #1.

   1. Select Record Details.

   On exit from the Pooling screen, the Validate Inputs Flowcell Type and Single Pool is triggered. The automation checks the following details:

   • All samples in the pool have been assigned the same Flowcell Type.

   • Only one pool has been created.

4. On the Record Details screen in the Step Details area, complete the fields as follows.

   1. Enter the value for the Number of Lanes to Sequence field.

   This value is used in volume calculations to make sure that the volumes are sufficient for the number of times the pool is sequenced.

   1. Enter the value for the Minimum Per Sample Volume (ul) field.

   The value in this field is used to calculate how much of each sample is included in the pool. The field is prepopulated with the configured default value (5 µl), but the value can be edited if necessary.

   1. If necessary, enter the value for the % PhiX (0.25 nM) Spike-In field.

   The value in this field is used to calculate the volume of PhiX v3 control to be included in the pool for the given percentage of spike-in. The field is optional and can be edited.

   • If the smallest Per Sample Volume (ul) value is less than 5, the LIMS automatically assigns a value of 5 to the sample's Adjusted Per Sample Volume (ul) field.

   • Clarity LIMS then adjusts the Adjusted Per Sample Volume (ul) field value for all other samples in the batch, based on the ratio used to increase the lowest value to 5.

5. In the Sample Details table, select the pool icon to view details on the pool composition.
7. Select Next Steps to trigger the Set Next Steps automation.

   This automation sets the next step for samples to ADVANCE, advancing them to the Dilute, Denature & ExAmp (NovaSeq 6000 v2.3) step in the protocol.

8. Select Finish Step.

At the end of this step, the pool of samples automatically advances to the Dilute, Denature & ExAmp (NovaSeq 6000 v2.3) step.

Step 2: Dilute, Denature & ExAmp (NovaSeq 6000 v2.3)

1. In Lab View, locate the NovaSeq Xp (NovaSeq 6000 v2.3) protocol. The pool of samples is queued for the Dilute, Denature & ExAmp (NovaSeq 6000 v2.3) step. Add the pool to the Ice Bucket.

2. On the Ice Bucket screen, set the number of derivatives to create (they are placed into the flow cell lanes) and select Begin Work.

   The Validate Inputs Flowcell Type automation checks that there is only one container input to the step.

3. 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:

   • NaOH Volume (ul)

   • Tris-HCl Volume (ul)

   • DPX1 Volume (ul)

   • DPX2 Volume (ul)

   • DPX3 Volume (ul)

   • Mastermix per Lane (ul)

   • The automation also generates the Calculation File (*csv) and attaches it to the step. This file contains information about the DPX Mastermix volume and the volume of Mastermix, NaOH, and Tris-HCI to add per working pool.

5. On the Record Details screen in the Reagent Lot Tracking section, select from the active lots displayed in each drop-down list.

   In the Step Details area, the DPX1, DPX2, and DPX3 reagent volume values are already populated. These values are set by a script and are not editable while running the step.

6. In the Sample Details table, select the pool icon to view details on the working pool composition.

   The working pool number is appended to the bulk pool name. This feature allows you to identify which working pools are derived from the same bulk pool quickly.

7. [Optional] Select the Calculation File (CSV) in the Files area to view details on the following volumes to add per working pool:

   • DPX Mastermix

   • Mastermix

   • NaOH

   • Tris-HCl
8. Select Next Steps.

   On the Assign Next Steps screen, the next step is already set to Load to Flowcell (NovaSeq 6000 v2.3).

9. Select Finish Step.

Step 3: Run Load to Flowcell (NovaSeq 6000 v2.3)

1. On the Ice Bucket screen in the Container Options panel, select the appropriate flow cell type from the Destination Container drop-down list and select Begin Work.
2. On the Placement screen, drag the pools from the left of the screen over into the Placed Samples area on the right.

   1. Scan or type the barcode of the flow cell into the Flow Cell field.

   1. Select Record Details.

   On exit of the Placement screen, the Validate Flowcell Barcode automation validates the container barcode.
3. The fields displayed in the Step Details section are used to generate the sample sheet and run recipe files. Some of these fields are autopopulated and some must be completed manually. See the following table for details.

   Fields Displayed on Record Details Screen of Load to Flowcell (NovaSeq 6000 v2.3) Step
4. Select Generate Sample Sheet and Run Recipe. This selection triggers the automation script, which:

   • Generates and attaches the sample sheet and JSON files to the placeholders in the Files area of the Record Details screen.

   • Populates the Sample Sheet Path field with the path to the sample sheet file.

5. Select Next Steps.

   This selection triggers the Prepare Files for NovaSeq automation, which copies the sample sheet and run recipe files to the location specified during installation. The NovaSeq instrument software uses these files to set up the run.

6. On the Assign Next Steps screen, make sure the Next Step for samples is prepopulated with Mark protocol as complete.

7. 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 v2.3) step.

Proceed to Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3).

Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3)

This protocol contains a single fully automated step - AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3).

The integration starts the step automatically and data from the run is parsed back into Clarity LIMS. No user interaction is required. However, you can open and review the various stages of the step in Clarity LIMS.

Troubleshooting

If an automation trigger does not appear to run its corresponding scripts, see the following sections in the Clarity LIMS documentation:

If an error occurs that does not provide direction on how to proceed, complete the following steps:

1. Confirm the version of the installed Illumina NovaSeq Integration Package by running the command as follows:

   • For an on-premise deployment, run the command on Clarity LIMS server command line. This command retrieves the version of both RPMs installed.

   • For a cloud hosted deployment, run the command on the local server installation. This command retrieves the version of the remote extensions RPM installed.

2. If the error is related to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step, there are two places to check for log file information:

   • If the step does not start, check the NovaSeqIntegrator.log file written to:

   This log file is written to this path wherever the remote extensions RPM was installed.

   • If the step starts but does not complete, open the step in Clarity LIMS (it can be found via a search). On the Record Details screen, the log file is attached to a placeholder called Log File. If you are unable to reach the Record Details screen, or if the file does not contain an error, review the NovaSeqIntegrator.log described previously.

3. Contact Illumina Support and supply the relevant information from the troubleshooting steps already performed.

This guide explains how to validate the installation of the Illumina NovaSeq 6000 Integration Package v2.6.0. The validation process involves the following actions:

• Running samples through the NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) workflow. The workflow contains a single-step protocol that models the library prep required to produce normalized libraries. At the end of the step, the normalized libraries are automatically advanced to the NovaSeq 6000 v2.3 workflow.

• Running normalized libraries through the NovaSeq 6000 v2.3 workflow. This process validates the following details:

  • Successful routing of samples from the Run Format (NovaSeq 6000 v2.3) step to the NovaSeq Standard (NovaSeq 6000 v2.3) or NovaSeq Xp (NovaSeq 6000 v2.3) step.

  • Automated generation of a sample sheet for use with bcl2fastq2 v2.20 analysis software.

  • Automated generation of a run recipe file (JSON file) with the library tube or flow cell barcode as the name (eg, NV1234567-LIB.json or H1234DRXX.json). This file is automatically uploaded to the sequencing instrument and used to set up and initiate the run.

  • Automated tracking of the NovaSeq sequencing run and parsing of run statistics (per run per lane) into Clarity LIMS.

Before executing the validation steps, make sure that you have installed the Illumina NovaSeq 6000 Integration Package v2.6.0 and have imported the default Clarity LIMS configuration.

Activate Workflow, Create Project, Add and Assign Samples

The following steps set up Clarity LIMS in preparation for running samples through the NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) and NovaSeq 6000 v2.3 workflows.

1. In the Clarity LIMS Configuration area, activate the NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) and NovaSeq 6000 v2.3 workflows.

2. On the Projects and Samples screen, create a project and add samples to it.

3. Assign the samples to the NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) workflow.

Library Prep Protocol: NovaSeq Validation Library Prep (NovaSeq 6000 v2.3)

This single-step protocol models the library prep required to produce normalized libraries that are ready for the NovaSeq 6000 v2.3 workflow.

• Label Group = TruSeq HT Adapters v2 (D7-D5)

• Sequencing Instrument = NovaSeq

On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the NovaSeq 6000 v2.3 workflow—Define Run Format (NovaSeq 6000 v2.3). This is the only step in Protocol 1: Run Format (NovaSeq 6000 v2.3).

Protocol 1: Run Format (NovaSeq 6000 v2.3)

This protocol includes the Define Run Format (NovaSeq 6000 v2.3) step. The step allows for the assignment of per sample values for Loading Workflow Type, Normalized Molarity, Flowcell Type, and Final Loading Concentration (pM). At the end of the step, samples are routed to the NovaSeq Standard or NovaSeq Xp protocol, according to the selected Loading Workflow Type.

Step 1: Run Define Run Format (NovaSeq 6000 v2.3)

1. In Lab View, locate the Run Format (NovaSeq 6000 v2.3) protocol. The samples are queued for the Define Run Format (NovaSeq 6000 v2.3) step.

2. Add the samples to the Ice Bucket and select View Ice Bucket.

3. On the Ice Bucket screen, select Begin Work.

4. On the Record Details screen, specify the Sample Details.

   • Loading Workflow Type — Select NovaSeq Standard or NovaSeq Xp from the drop-down list.

   • Normalized Molarity — Enter values or use values copied from the previous step.

5. Populate the following fields:

   • Flowcell Type — Select SP, S1, S2, or S4.

   • Final Loading Concentration (pM) — Select from the two preset options—225 (for PCR-free workflows) or 400 (for Nano workflows)—or enter a different value.

6. Select Next Steps to trigger the Set Next Steps automation, which does the following actions:

   • Sets the value of the next step (for all samples) to Remove from workflow. The Routing Script automation expects this value and requires it to advance samples to the next step successfully.

   • Calculates the Minimum Molarity.

   • Checks Normalized Molarity value. For samples with no Normalized Molarity value (eg, an empty value, not including 0), the automation generates an error message stating that the field cannot be empty.

   • Compares the Normalized Molarity value of each sample with the Minimum Molarity value.

7. On the Assign Next Steps screen, review the Sample Details table and make sure that the Next Step for all samples is prepopulated with Remove from workflow.

   Next Step must be set to Remove from workflow, regardless of the Loading Workflow Type.

8. Review the Warning field entries for the samples. If the field indicates that the Normalized Molarity is too low, continue with one of the following options:

   • Return to the Record Details screen and adjust the Normalized Molarity value so that it equals or exceeds the Minimum Molarity value. Set the Loading Workflow Type to NovaSeq Standard or NovaSeq Xp, as applicable.

   • Alternatively, complete the protocol without correcting the Normalized Molarity value. In this case, the samples in question are removed from the Clarity LIMS workflow.
9. Select Finish Step.

   The Routing Script automation is triggered if:

   • Samples with a Loading Workflow Type of Remove from workflow (ie, where Normalized Molarity value is lower than the Minimum Molarity) are removed from the Clarity LIMS workflow.

   • Samples with a Loading Workflow Type of NovaSeq Standard are routed to the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3) step. This step is the first of two steps in Protocol 2: NovaSeq Standard (NovaSeq 6000 v2.3).

   • Samples with a Loading Workflow Type of NovaSeq Xp are routed to the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3) step. This step is the first of three steps in Protocol 3: NovaSeq Xp (NovaSeq 6000 v2.3).

Protocol 2: NovaSeq Standard (NovaSeq 6000 v2.3)

In this protocol, samples are pooled and added to the library tube in preparation for the NovaSeq run. The protocol contains two steps:

1. Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3)

2. Dilute and Denature (NovaSeq 6000 v2.3)

Step 1: Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3)

1. In Lab View, locate the NovaSeq Standard (NovaSeq 6000 v2.3) protocol. The samples are queued for the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3) step.

2. On the Queue screen, add samples of the same flow cell type to the Ice Bucket and select Begin Work.
3. On the Pooling screen, create a pool by dragging samples into the Pool Creator.

   1. Type a name for the pool or accept the default name (Pool #1).

   1. Select Record Details.

4. On exit from the Pooling screen, the Validate Inputs Flowcell Type and Single Pool is triggered. The automation checks the following information:

   • All samples in the pool have been assigned the same Flowcell Type.

   • Only one pool has been created.

5. On the Record Details screen in the Step Details area, complete the fields as follows.

   1. Enter the value for the Number of Flowcells to Sequence field.

   This value is used in volume calculations to make sure that the volumes are sufficient for the number of times the pool is sequenced.

   1. Enter the value for the Minimum Per Sample Volume (ul) field.

   The value in this field is used to calculate how much of each sample is included in the pool. The field is prepopulated with the configured default value (5 µl), but the value can be edited if necessary.

   1. If necessary, enter the value for the % PhiX (2.5 nM) Spike-In field.

   The value in this field is used to calculate the volume of PhiX v3 control to be included in the pool for the given percentage of spike-in. The field is optional and can be edited.

   • If the smallest Per Sample Volume (ul) value is less than 5, the LIMS automatically assigns a value of 5 to the sample's Adjusted Per Sample Volume (ul) field.

   • Clarity LIMS then adjusts the Adjusted Per Sample Volume (ul) field value for all other samples in the batch, based on the ratio used to increase the lowest value to 5.

6. In the Sample Details table, select the pool icon to view details on the pool composition.
8. Select Next Steps to trigger the Set Next Steps automation.

   This automation sets the next step for samples to ADVANCE, advancing them to the Dilute and Denature (NovaSeq 6000 v2.3) step in the protocol.

9. Select Finish Step.

At the end of this step, the pool of samples automatically advances to the Dilute and Denature (NovaSeq 6000 v2.3) step.

Step 2: Dilute and Denature (NovaSeq 6000 v2.3)

1. In Lab View, locate the NovaSeq Standard (NovaSeq 6000 v2.3) protocol. The pool of samples is queued for the Dilute and Denature (NovaSeq 6000 v2.3) step.

2. Add the samples to the Ice Bucket and select Begin Work.

   At the beginning of the step, the Validate Single Input automation is triggered. This automation checks that there is only one container input to the step.

3. On the Placement screen, drag the pool into the library tube in the Placed Samples area.

   1. Scan or type the barcode of the library tube into the Library Tube field.

   1. Select Record Details.

   On exit of the Placement screen, the Validate Library Tube Barcode automation checks that the library tube barcode conforms to the barcode mask [A-Z]{2}[0-9]{7}-[A-Z]{3}. If not, an error message displays.

5. In the Reagent Lot Tracking section, select from the active lots displayed in each drop-down list. The fields displayed in the Step Details section are used to generate the sample sheet and run recipe files. Some of these fields are autopopulated and some must be completed manually. Refer to the following table for details.

   Fields Displayed on Record Details Screen of Dilute and Denature (NovaSeq 6000 v2.3) Step
6. On the Record Details screen, select Generate Sample Sheet & Run Recipe. This selection triggers the automation script: The sample sheet and JSON files are generated and attached to the placeholders in the Files area of the Record Details screen. The Sample Sheet Path field is populated with the path to the sample sheet file.

7. Select Next Steps. This selection triggers the Prepare Files for NovaSeq automation, which does the following:

   • Copies the sample sheet and run recipe files to the location specified during installation. The NovaSeq instrument software uses these files to set up the run.

   • Sets the value of the next step to Remove from workflow. The Routing Script automation expects this value and requires it to advance samples to the next step successfully.

8. On the Assign Next Steps screen, the Next Step for samples is prepopulated with Remove from workflow.

9. Select Finish Step.

On exit from the step, the Routing Script automation is triggered and samples are routed to Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3).

In Lab View, the pool of samples is queued for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step.

Protocol 3: NovaSeq Xp (NovaSeq 6000 v2.3)

In this protocol, samples are pooled and added to lanes on the NovaSeq flow cell. The flow cell type is determined by the option selected in the Define Run Format (NovaSeq 6000 v2.3) step.

The protocol contains three steps:

1. Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3)

2. Dilute, Denature & ExAmp (NovaSeq 6000 v2.3)

3. Load to Flowcell (NovaSeq 6000 v2.3)

Step 1: Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3)

1. In Lab View, locate the NovaSeq Xp (NovaSeq 6000 v2.3) protocol. The samples are queued for the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3) step.

2. On the Queue screen, add samples of the same Flowcell Type to the Ice Bucket and select Begin Work.
3. On the Pooling screen, create a pool by dragging samples into the Pool Creator.

   1. Type a name for the pool or accept the default name Pool #1.

   1. Select Record Details.

   On exit from the Pooling screen, the Validate Inputs Flowcell Type and Single Pool is triggered. The automation checks the following details:

   • All samples in the pool have been assigned the same Flowcell Type.

   • Only one pool has been created.

4. On the Record Details screen in the Step Details area, complete the fields as follows.

   1. Enter the value for the Number of Lanes to Sequence field.

   This value is used in volume calculations to make sure that the volumes are sufficient for the number of times the pool is sequenced.

   1. Enter the value for the Minimum Per Sample Volume (ul) field.

   The value in this field is used to calculate how much of each sample is included in the pool. The field is prepopulated with the configured default value (5 µl), but the value can be edited if necessary.

   1. If necessary, enter the value for the % PhiX (0.25 nM) Spike-In field.

   The value in this field is used to calculate the volume of PhiX v3 control to be included in the pool for the given percentage of spike-in. The field is optional and can be edited.

   • If the smallest Per Sample Volume (ul) value is less than 5, the LIMS automatically assigns a value of 5 to the sample's Adjusted Per Sample Volume (ul) field.

   • Clarity LIMS then adjusts the Adjusted Per Sample Volume (ul) field value for all other samples in the batch, based on the ratio used to increase the lowest value to 5.

5. In the Sample Details table, select the pool icon to view details on the pool composition.
7. Select Next Steps to trigger the Set Next Steps automation.

   This automation sets the next step for samples to ADVANCE, advancing them to the Dilute, Denature & ExAmp (NovaSeq 6000 v2.3) step in the protocol.

8. Select Finish Step.

At the end of this step, the pool of samples automatically advances to the Dilute, Denature & ExAmp (NovaSeq 6000 v2.3) step.

Step 2: Dilute, Denature & ExAmp (NovaSeq 6000 v2.3)

1. In Lab View, locate the NovaSeq Xp (NovaSeq 6000 v2.3) protocol. The pool of samples is queued for the Dilute, Denature & ExAmp (NovaSeq 6000 v2.3) step. Add the pool to the Ice Bucket.

2. On the Ice Bucket screen, set the number of derivatives to create (they are placed into the flow cell lanes) and select Begin Work.

   The Validate Inputs Flowcell Type automation checks that there is only one container input to the step.

3. 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:

   • NaOH Volume (ul)

   • Tris-HCl Volume (ul)

   • DPX1 Volume (ul)

   • DPX2 Volume (ul)

   • DPX3 Volume (ul)

   • Mastermix per Lane (ul)

   • The automation also generates the Calculation File (*csv) and attaches it to the step. This file contains information about the DPX Mastermix volume and the volume of Mastermix, NaOH, and Tris-HCI to add per working pool.

5. On the Record Details screen in the Reagent Lot Tracking section, select from the active lots displayed in each drop-down list.

   In the Step Details area, the DPX1, DPX2, and DPX3 reagent volume values are already populated. These values are set by a script and are not editable while running the step.

6. In the Sample Details table, select the pool icon to view details on the working pool composition.

   The working pool number is appended to the bulk pool name. This feature allows you to identify which working pools are derived from the same bulk pool quickly.

7. [Optional] Select the Calculation File (CSV) in the Files area to view details on the following volumes to add per working pool:

   • DPX Mastermix

   • Mastermix

   • NaOH

   • Tris-HCl
8. Select Next Steps.

   On the Assign Next Steps screen, the next step is already set to Load to Flowcell (NovaSeq 6000 v2.3).

9. Select Finish Step.

Step 3: Run Load to Flowcell (NovaSeq 6000 v2.3)

1. On the Ice Bucket screen in the Container Options panel, select the appropriate flow cell type from the Destination Container drop-down list and select Begin Work.
2. On the Placement screen, drag the pools from the left of the screen over into the Placed Samples area on the right.

   1. Scan or type the barcode of the flow cell into the Flow Cell field.

   1. Select Record Details.

   On exit of the Placement screen, the Validate Flowcell Barcode automation validates the container barcode.
3. The fields displayed in the Step Details section are used to generate the sample sheet and run recipe files. Some of these fields are autopopulated and some must be completed manually. See the following table for details.

   Fields Displayed on Record Details Screen of Load to Flowcell (NovaSeq 6000 v2.3) Step
4. Select Generate Sample Sheet and Run Recipe. This selection triggers the automation script, which:

   • Generates and attaches the sample sheet and JSON files to the placeholders in the Files area of the Record Details screen.

   • Populates the Sample Sheet Path field with the path to the sample sheet file.

5. Select Next Steps.

   This selection triggers the Prepare Files for NovaSeq automation, which copies the sample sheet and run recipe files to the location specified during installation. The NovaSeq instrument software uses these files to set up the run.

6. On the Assign Next Steps screen, make sure the Next Step for samples is prepopulated with Mark protocol as complete.

7. 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 v2.3) step.

Proceed to Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3).

Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3)

This protocol contains a single fully automated step - AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3).

The integration starts the step automatically and data from the run is parsed back into Clarity LIMS. No user interaction is required. However, you can open and review the various stages of the step in Clarity LIMS.

Troubleshooting

If an automation trigger does not appear to run its corresponding scripts, see the following sections in the Clarity LIMS documentation:

If an error occurs that does not provide direction on how to proceed, complete the following steps:

1. Confirm the version of the installed Illumina NovaSeq Integration Package by running the command as follows:

   • For an on-premise deployment, run the command on Clarity LIMS server command line. This command retrieves the version of both RPMs installed.

   • For a cloud hosted deployment, run the command on the local server installation. This command retrieves the version of the remote extensions RPM installed.

2. If the error is related to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step, there are two places to check for log file information:

   • If the step does not start, check the NovaSeqIntegrator.log file written to:

   This log file is written to this path wherever the remote extensions RPM was installed.

   • If the step starts but does not complete, open the step in Clarity LIMS (it can be found via a search). On the Record Details screen, the log file is attached to a placeholder called Log File. If you are unable to reach the Record Details screen, or if the file does not contain an error, review the NovaSeqIntegrator.log described previously.

3. Contact Illumina Support and supply the relevant information from the troubleshooting steps already performed.

Last Updated: November 2024

Release Date: September 2023

Document Version: 2

These release notes describe the key changes to software components for NovaSeq 6000 Integration Package v2.5.0.

Compatibility

Refer to under Instruments & Integrations.

New Features

• Updates Java and third-party dependency libraries.

• Updates Groovy to v3.0.7.

Defects Fixed

None

Known Issues

• Log file name is missing the -Logfile.html suffix and the content is missing the sample sheet generation logs.

Revision History

The integration includes the following functionality:

• Preconfigured NovaSeq 6000 v2.3 workflow that maps to lab protocols and instrument runs.

• Preconfigured NovaSeq Standard (NovaSeq 6000 v2.3) protocol that supports the loading of pooled libraries into a library tube.

• Preconfigured NovaSeq Xp (NovaSeq 6000 v2.3) protocol that supports individual lane loading on the NovaSeq.

• Automated generation of a sample sheet for use with bcl2fastq2 v2.20 analysis software.

• Automatic generation of run recipe file (JSON format) that is automatically uploaded to the sequencing instrument and used to set up and initiate the run.

• Automated tracking of the NovaSeq sequencing run and parsing of run statistics (per run per lane) into Clarity LIMS, including:

  • Progress and metrics of sequencing run

  • Per instrument sequencing runs (tracked as part of the Run ID field value)

  • Sequencing run parameters

  • Real-Time Analysis v3 (RTA3) run directory location and other run-specific information

• Preconfigured NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) workflow used for validation purposes only. The workflow contains a single-step protocol that models the library prep required to produce normalized libraries that are ready for the NovaSeq 6000 v2.3 workflow. For details, refer to NovaSeq 6000 Integration v2.5.0 User Interaction, Validation and Troubleshooting.

Last Updated: November 2024

Release Date: July 2024

Document Version: 2

These release notes describe the key changes to software components for NovaSeq 6000 Integration Package v2.6.0.

Compatibility

Refer to Compatibility under Instruments & Integrations.

New Features

• Some integration properties can now be accessed and updated via System Setting in Clarity v6.3. Refer to NovaSeq 6000 Integration v2.6.0 Configuration for configurable properties.

Defects Fixed

• Fix log file showing unnecesssary warning and error message when generating sample sheet.

Known Issues

• Log file name is missing the -Logfile.html suffix and the content is missing the sample sheet generation logs.

Revision History

The Illumina NovaSeq 6000 Integration Package v2.6 supports the integration of Clarity LIMS to NovaSeq 6000 instruments.

This document describes the integration and includes information about the preconfigured protocols, steps, automations, installed components, configuration requirements, rules, and constraints.

For instructions on user interaction for each step, validating and troubleshooting the Illumina NovaSeq 6000 Integration, refer to NovaSeq 6000 Integration v2.6.0 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 the process.

Prerequisites and Assumptions

It is assumed that samples enter the NovaSeq 6000 v2.3 workflow as normalized libraries. That is, before they are assigned to the workflow the following actions occur:

• Samples have been accessioned into Clarity LIMS.

• Samples have been run through QC and library prep.

• Samples have been normalized, and the value is captured in a field called Normalized Molarity (nM).

For more information on sample accessioning, refer to Sample Accessioning and Upload and Modify Samples in the Getting Started section of the 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.

Workflows, Protocols, and Steps

The Illumina NovaSeq 6000 Integration Package v2.6.0 includes two workflows:

• NovaSeq Validation Library Prep (NovaSeq 6000 v2.3)

• NovaSeq 6000 v2.3

NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) Workflow

NovaSeq 6000 v2.3 Workflow

Validation Workflow

The NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) workflow allows for validation of the system after installation is complete. For details, refer to NovaSeq 6000 Integration v2.6.0 User Interaction, Validation and Troubleshooting.

Protocol 1: Run Format (NovaSeq 6000 v2.3)

This protocol sets the Loading Workflow Type and allows you to choose the appropriate Flowcell Type and Final Loading Concentration(pM). At the end of the protocol, a routing script sends the normalized libraries to either the NovaSeq Standard (NovaSeq 6000 v2.3) or the NovaSeq Xp (NovaSeq 6000 v2.3) protocol.

This protocol contains one step: Define Run Format (NovaSeq 6000 v2.3).

Step 1: Define Run Format (NovaSeq 6000 v2.3)

• Step input: NTP (normalized libraries)

• Step output: None

bash -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 v2.3' \
--STEP 'Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3)' \
--INPUTS_OR_OUTPUTS 'INPUTS' \
\
--FIELD_NAME 'Loading Workflow Type' \
--FIELD_VALUE 'NovaSeq Xp' \
--WORKFLOW 'NovaSeq 6000 v2.3' \
--STEP 'Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3)' \
--INPUTS_OR_OUTPUTS 'INPUTS'"

Master Step Fields

The following table provides field configuration details for the fields defined on the Define Run Format (NovaSeq 6000 v2.3) step.

Define Run Format (NovaSeq 6000 v2.3) Master Step Field Configuration

Global Fields

The following table lists the global fields that are configured to display on the Define Run Format (NovaSeq 6000 v2.3) step.

Global Field Configuration (Derived Sample)

Protocol 2: NovaSeq Standard (NovaSeq 6000 v2.3)

Samples are routed to this protocol if the Loading Workflow Type value is set to NovaSeq Standard. Samples are pooled and added to a library tube in preparation for the NovaSeq run.

At the end of this protocol, a routing script sends the library tube to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) protocol.

This protocol contains two steps:

1. Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3)

2. Dilute and Denature (NovaSeq 6000 v2.3)

Step 1: Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3)

In this step, manually place libraries into a single pool. Resuspension buffer and reagents are added.

• Step input: NTP (normalized libraries)

• Step output: Bulk pool

The following automations are configured on the step (in the order they are triggered at run time).

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/novaseq/novaseq-extensions.jar script:validate_flowcell_for_input_pools -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -validateSingleOutput true -poolType bulk

nextStep = ::ADVANCE::

Master Step Fields

The following table provides field configuration details for the fields defined on the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3) step.

Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3) Master Step Field Configuration

step.::PhiX Volume (ul):: = !step.hasValue(::% PhiX (2.5nM) Spike-In::) ? 0 : step.::% PhiX (2.5nM) Spike-In::; if (input.::Flowcell Type:: == ::SP::) { step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 100; step.::PhiX Volume (ul):: *= 0.6 }; if (input.::Flowcell Type:: == ::S1::) { step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 100; step.::PhiX Volume (ul):: *= 0.6 }; if (input.::Flowcell Type:: == ::S2::) { step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 150; step.::PhiX Volume (ul):: *= 0.9 }; if (input.::Flowcell Type:: == ::S4::) { step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 310; step.::PhiX Volume (ul):: *= 1.9 }; if (step.::PhiX Volume (ul):: == 0) { step.::PhiX Volume (ul):: = :::: }; input.::Per Sample Volume (ul):: = (((input.::Final Loading Concentration (pM):: * 5 / 1000) / input.::Normalized Molarity (nM)::) * step.::Bulk Pool Volume (ul)::) / step.::Number of Samples in Pool::

Global Fields

The following table lists the global fields that are configured to display on the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3) step.

Global Field Configuration (Derived Sample)

Step 2: Dilute and Denature (NovaSeq 6000 v2.3)

In this step, pooled samples are denatured and diluted by the addition of NaOH, Tris-HCl, and Resuspension Buffer (RSB). Samples are manually placed into the library tube that is used in the NovaSeq run. In addition, this step generates the sample sheet file and the run recipe (*json file) needed to start the NovaSeq run. The step input is Bulk pool, and the step output is Library tube.

script:validateSampleCount -min 1 -max 1

if (!output.container.name.matches(::[A-Z]{2}[0-9]{7}-[A-Z]{3}::)){
    fail(::Invalid Library Tube Barcode. Please verify and try again.::)
}

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 {compoundOutputFileLuid2} script:changeWorkflow \
\
--FIELD_NAME 'N/A' \
--FIELD_VALUE 'N/A' \
--WORKFLOW 'NovaSeq 6000 v2.3' \
--STEP 'AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"

Master Step Fields

The following table provides field configuration details fields defined on the Dilute and Denature (NovaSeq 6000 v2.3) step. These fields are required for sample sheet and JSON file generation.

Dilute and Denature (NovaSeq 6000 v2.3) Master Step Field Configuration

Global Step Fields

The following table lists the global step fields that are configured to display on the Dilute and Denature (NovaSeq 6000 v2.3) step.

Global Field Configuration (Derived Sample)

Protocol 3: NovaSeq Xp (NovaSeq 6000 v2.3)

Samples are routed to this protocol if the Loading Workflow Type value is set to NovaSeq Xp.

Samples are pooled and added to lanes on the NovaSeq flow cell type selected in the Define Run Format (NovaSeq 6000 v2.3) step.

At the end of the protocol, a routing script sends the flow cell to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) protocol.

This protocol contains three steps:

1. Step 1: Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3)

2. Step 2: Dilute, Denature & ExAmp (NovaSeq 6000 v2.3)

3. Step 3: Load to Flowcell (NovaSeq 6000 v2.3)

Step 1: Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3)

In this step, the libraries are manually placed into a pool. The step input is NTP (normalized libraries) and the step output is Bulk pool.

These automations are described in the order in which they are triggered at run time.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/novaseq/novaseq-extensions.jar script:validate_flowcell_for_input_pools
-i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -validateSingleOutput true

• Calculates the Per Sample Volume (ul) to be added to the pool:

  Copy

  input.::Per Sample Volume (ul)::= (((input.::Final Loading Concentration (pM)::* 5/ 1000) / input.::Normalized Molarity (nM)::) * step.::Bulk Pool Volume (ul)::) / step.::Number of Samples in Pool::

  1. If the smallest Per Sample Volume (ul) value is less than 5, Clarity LIMS automatically assigns a value of 5 to the sample's Adjusted Per Sample Volume (ul) field.

  2. Clarity LIMS then adjusts the Adjusted Per Sample Volume (ul) field value for all other samples in the batch, based on the ratio used to increase the lowest value to 5.

• Calculates the Total Sample Volume (ul) field value:

  Copy

  step.::Total Sample Volume (ul):: = step.::Total Sample Volume (ul):: + input.::Adjusted Per Sample Volume (ul)::

• If the Total Sample Volume is less than the Bulk Pool Volume, calculates the RSB Volume (ul) field value:

  Copy

  if (step.::Total Sample Volume (ul):: >= step.::Bulk Pool Volume (ul)::) {output.::RSB Volume (ul):: = 0} else {output.::RSB Volume (ul):: = step.::Bulk Pool Volume (ul):: - step.::Total Sample Volume (ul)::} ;

• Copies the Flowcell Type and Loading Workflow Type values from the step inputs to the step outputs:

  Copy

  output.::Flowcell Type::= input.::Flowcell Type::;
  output.::Loading Workflow Type::= input.::Loading Workflow Type::;

• Uses the NovaSeq_Xp_Bulk_Pool.csv and NovaSeq_Xp_Bulk_Pool2.csv template files to generate a single CSV file containing information about the bulk pool and the samples it contains. The generated file is available for download on the Step Setup screen of the following step—Dilute, Denature & ExAmp (NovaSeq 6000 v2.3).

• Resets the Total Sample Volume (ul) and Number of Samples in Pool field values so that the automation is idempotent:

  Copy

  step.'Number of Samples in Pool' = step.'Number of Samples in Pool' - 1;
  step.'Total Sample Volume (ul)' = step.'Total Sample Volume (ul)' - input.'Adjusted Per Sample Volume (ul)'

output.::Flowcell Type:: = input.::Flowcell Type:: ; nextStep = ::ADVANCE::

Master Step Fields

The following table provides field configuration details for the fields defined on the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3) step.

Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3) Master Step Field Configuration

Global Fields

The following table lists the global fields that are configured to display on the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3) step.

Global Field Configuration (Derived Sample)

Step 2: Dilute, Denature & ExAmp (NovaSeq 6000 v2.3)

In this step, pooled samples are denatured and diluted by the addition of DPX, NaOH, Tris-HCl, and RSB. Manually create working pools based on the number of lanes you want to sequence. The step input is Bulk pool and the step output is Working pool. Working pool is a variable number for how many working pools need to be created per bulk pool.

These automations are described in the order in which they are triggered at run time.

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/novaseq/novaseq-extensions.jar script:validate_flowcell_for_input_pools -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1}"

if (input.::Flowcell Type:: == ::SP::) {
    output.::BP Aliquot Volume (ul):: = 18;
    output.::NaOH Volume (ul):: = 4;
    output.::Tris-HCl Volume (ul):: = 5;
    step.::DPX1 Volume (ul):: = 126;
    step.::DPX2 Volume (ul):: = 18;
    step.::DPX3 Volume (ul):: = 66;
    output.::Mastermix per Lane (ul):: = 63
};
if (input.::Flowcell Type:: == ::S1::) {
    output.::bp Aliquot Volume (ul):: = 18;
    output.::NaOH Volume (ul):: = 4;
    output.::Tris-HCl Volume (ul):: = 5;
    step.::DPX1 Volume (ul):: = 126;  
    step.::DPX2 Volume (ul):: = 18;    
    step.::DPX3 Volume (ul):: = 66;    
    output.::Mastermix per Lane (ul):: = 63
};
if (input.::Flowcell Type:: == ::S2::) {
    output.::bp Aliquot Volume (ul):: = 22;    
    output.::NaOH Volume (ul):: = 5;    
    output.::Tris-HCl Volume (ul):: = 6;
    step.::DPX1 Volume (ul):: = 126;
    step.::DPX2 Volume (ul):: = 18;  
    step.::DPX3 Volume (ul):: = 66;  
    output.::Mastermix per Lane (ul):: = 77
};
if (input.::Flowcell Type:: == ::S4::) {
    output.::bp Aliquot Volume (ul):: = 30;  
    output.::NaOH Volume (ul):: = 7;  
    output.::Tris-HCl Volume (ul):: = 8;    
    step.::DPX1 Volume (ul):: = 315;  
    step.::DPX2 Volume (ul):: = 45;  
    step.::DPX3 Volume (ul):: = 165;    
    output.::Mastermix per Lane (ul):: = 105
};

output.::Flowcell Type:: = input.::Flowcell Type:: ; nextStep = ::ADVANCE::

Master Step Fields

The following table provides field configuration details for the fields defined on Dilute, Denature & ExAmp (NovaSeq 6000 v2.3) step. These field values are set by a script and are not editable while running the step.

Dilute, Denature & ExAmp (NovaSeq 6000 v2.3) Master Step Field Configuration

Global Fields

The following table lists the global fields that are configured to display on the Dilute, Denature & ExAmp (NovaSeq 6000 v2.3) step.

Global Field Configuration (Derived Sample)

Step 3: Load to Flowcell (NovaSeq 6000 v2.3)

In this step, scan the flow cell barcode into Clarity LIMS, then manually place the working pools into the lanes of the flow cell that is used in the NovaSeq run. The step input is Working pool and the step output is Flow cell (output containers: S1 and S2 with 2 lanes, and S4 with 4 lanes).

These automations are described in the order in which they are triggered at run time.

if (input.::Flowcell Type:: == ::SP:: && !output.container.name.matches(::[A-Z0-9]{5}DR[A-Z0-9]{2}::)) {
    fail(::Invalid Flowcell Barcode. Please verify and try again.::)
};
if (input.::Flowcell Type:: == ::S1:: && !output.container.name.matches(::[A-Z0-9]{5}DR[A-Z0-9]{2}::)) {
    fail(::Invalid Flowcell Barcode. Please verify and try again.::)
};
if (input.::Flowcell Type:: == ::S2:: && !output.container.name.matches(::[A-Z0-9]{5}DM[A-Z0-9]{2}::)) {
    fail(::Invalid Flowcell Barcode. Please verify and try again.::)
};
if (input.::Flowcell Type:: == ::S4:: && !output.container.name.    matches(::[A-Z0-9]{5}DS[A-Z0-9]{2}::)) {
    fail(::Invalid Flowcell Barcode. Please verify and try again.::)
};

Master Step Fields

The following table provides field configuration details for the fields defined on the Load to Flowcell (NovaSeq 6000 v2.3) step.

Load to Flowcell (NovaSeq 6000 v2.3) Master Step Field Configuration

Global Fields

The following table lists the global fields (derived samples) that are configured to display on the Load to Flowcell (NovaSeq 6000 v2.3) step.

Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3)

This final protocol contains one fully automated step, AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3).

Step 1: AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3)

• Step input: Library tube from the NovaSeq Standard protocol or flow cell from the NovaSeq Xp protocol.

• Step output: Result file and measurement.

In this step, pooled samples are sequenced on the NovaSeq instrument. The run data network folder is monitored by the sequencing service and automation worker to determine when the sequencing run begins and ends.

1. When a run is initiated on the NovaSeq instrument, the NovaSeq Control Software (NVCS) looks for the sample sheet and run recipe (*.json file) on the shared network drive.

   • The NVCS copies the sample sheet to the run directory.

   • The NVCS uses the run recipe to start the sequencing run.

2. When the run starts:

   • The instrument software creates a new run folder (named <libraryID>) on the shared network drive and copies the following files into that folder:

     • RunParameters.xml

     • RunInfo.xml

     • LIMS/<libraryID>.json file (run recipe)

   • The Real-Time Analysis v3 (RTA3) software creates the InterOp folder.

3. The sequencing service detects the presence of the RunParameters, RunInfo, and JSON files and starts the AUTOMATED- NovaSeq Run (NovaSeq 6000 v2.3) step in Clarity LIMS.

4. As the run progresses, the InterOp data files are filled in.

5. At the end of the run, the NovaSeq:

   • Copies the run data files to the InterOp folder.

   • Creates the CopyComplete.txt file in the run folder. This step indicates to the sequencing service that the run has completed and the data files are ready.

6. The sequencing service triggers the Read InterOp Metrics automation. This automation reads the files, records the parsed metrics into Clarity LIMS, and finally completes the step in Clarity LIMS.

nextStep = ::ADVANCE::

Master Step Fields

The fields defined on the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step are listed in the Clarity LIMS Configuration area (Custom Fields > Master Step Fields tab).

The following table shows how the parsed RunParameters.xml fields map to step fields configured on the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step in Clarity LIMS.

Fields Parsed by parse_novaseq_run_parameters script

Global Fields

The global fields that are configured to support the parsing of key metrics back into Clarity LIMS are as follows.

• Yield PF (Gb) R1

• Yield PF (Gb) R2

• Reads PF (M) R1

• Reads PF (M) R2

• Cluster Density (K/mm^2) R1

• Cluster Density (K/mm^2) R2

• %PF R1

• %PF R2

• % Bases >=Q30 R1

• % Bases >=Q30 R2

• Intensity Cycle 1 R1

• Intensity Cycle 1 R2

• % Phasing R1

• % Phasing R2

• % Prephasing R1

• % Prephasing R2

• % Aligned R1

• % Aligned R

• % Error Rate R1

• % Error Rate R2

Components Installed

The following sections describe the various components that are installed by default as part of this integration. These components include files, properties, reagent categories/label groups, reagent kits, and containers. Information on installed workflows, protocols, steps, and automation points is provided in the previous Workflows, Protocols, and Steps section.

The Illumina NovaSeq 6000 Integration v2.6.0 Package is distributed as two RPM packages:

• BaseSpaceLIMS-novaseq-extensions

• BaseSpaceLIMS-novaseq-sequencing-service

The RPM packages install the components listed in the following table.

The NovaSeq 6000 Integration requires installation of NGS Extensions Package v5.25.0 or later.

• If the NGS Extensions Package is not already installed, or if a version earlier than v5.25.0 is installed, the latest version is installed by default with the NovaSeq integration. For details, refer to NovaSeq 6000 Integration v2.6.0 Release Notes.

• If NGS Extensions Package v5.25.0 is already installed, upgrade is not forced or required.

• Secret Util Package is required for the NGS Extensions Package v5.25.0.

• If the Secret Util Package is not already installed, then Secret Util package is installed along with the installation of NGS Extension Package v5.25.0.

• If the Secret Util Package is already installed, upgrade is not forced or required.

Illumina NovaSeq 6000 Integration v2.6.0 RPM Components

Properties

Refer to Integration Properties Details for the properties installed with the integration package.

Label Groups, Reagent Kits, and Containers

The NovaSeq 6000 Integration v2.6.0 Package installs the following label groups, reagent kits, and container types.

Label Groups

• TruSeq HT Adapters v2 (D7-D5)

Reagent Kits

• DPX1

• DPX2

• DPX3

• Resuspension Buffer (RSB)

• NaOH

• Tris-HCl

Container Types

• Library Tube

• S1

• S2

• S4

• SP

Instrument Integration

The following are instructions for configuring the NovaSeq instrument for the Clarity LIMS integration:

1. Launch NovaSeq Control Software.

2. On the home screen, select the menu (top left) and select Settings.

3. Select File-Based and enter the network folder location that NovaSeq Control Software retrieves the JSON recipe from.

4. Enter the network output folder location.

5. Select Save.

For more information, refer to the NovaSeq 6000 Sequencing System Guide at support.illumina.com.

Routing Script Requirements

The requirements for the routing script functionality are as follows.

• On the steps that use the routing script — Define Run Format (NovaSeq 6000 v2.3) and Dilute and Denature (NovaSeq 6000 v2.3) — the Next Step for all samples must be set to Remove from workflow. This value is set by a script. Do not change this value in the Assign Next Steps screen.

• In the protocol configuration screen, the following setting is required and must not be changed.

  • In the Next Steps section for the last step in the protocol, the method of assigning the next step must be set to Automatic.

Sample Sheet and Run Recipe File Generation

Sample sheet and run recipe file generation occurs on the step before the run—Dilute and Denature (NovaSeq 6000 v2.3) in the NovaSeq Standard protocol or Load to Flowcell (NovaSeq 6000 v2.3) in the NovaSeq Xp protocol. This step is where samples are placed on the library tube or flow cell that is loaded in the NovaSeq instrument.

In the default configuration, the Generate Sample Sheet & Run Recipe automation generates the following:

• One CSV format sample sheet file for use bcl2fastq v2.20 downstream analysis

• One JSON format run recipe file

The Prepare Files for NovaSeq automation places the generated files on the NAS where the instrument uses them to start the run.