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 NovaSeqDx v1.2 workflow. For more information, refer to . The Library Prep Validation v2.3.4 workflow is available in the Illumina Preset Protocols (IPP) v2.9 package.

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 NovaSeqDx v1.2 workflow. For more information, refer to . The Library Prep Validation v2.3.1 workflow is available in the Illumina Preset Protocols (IPP) v2.6 package.

Tenant DB Password

Clarity DB Password

FTP password

LDAP manager password (if LDAP is enabled)

LabLink DB password

Reporting DB password (if installed)

RabbitMQ password

Library Prep Validation configuration (optional, but recommended)

configure_sequencer_api_application.sh configuration script

Enter y to confirm that you want to proceed with the RPM and package installation.

Make sure that NGS Extension Package v5.23.0 is installed.

If this package is not installed, you must install it or upgrade the existing package.

As the glsjboss user, install the QC_Protocols.qc-protocols configuration from the following location:

Enter y to confirm that you want to proceed with the RPM installation.

If necessary, install the Secret Utility Package.

For more information on this package, refer to Prerequisite 1: Secret Utility Configuration.

As the glsjboss user, run configure_sequencer_api_application.sh from the following location:

Enter values for the presented as follows.

You can also press Enter to accept the default values in the application.yml configuration file. For more information about the application.yml properties, refer to NovaSeq 6000Dx Integration v1.2.0 Configuration.

• Would you like to use the apiuser user for communication with Clarity? [Y]

• Enter required value for Token Expiry (hours) [88]

Update the Clarity LIMS proxy for the Sequencer API as follows.

• For an on-premise system, run the following command:

• For an Illumina cloud hosted system, make sure that the proxy has been updated as follows. Place the sequencer-api.include file in /etc/httpd/conf.d/clarity.

Update the Clarity LIMS Tomcat configuration as follows.

1. Make sure that Secret Utility is installed and configured. For more information, refer to Prerequisite 1: Secret Utility Configuration.

2. Run the following command to add the Secret Utility configuration to Clarity LIMS:

   ℹ 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.

3. Use the following command to Start Clarity LIMS:

[Optional] Run the following command to install the Library Prep Validation v2.3.1 workflow:

This step is optional, but recommended. The Library Prep Validation v2.3.1 workflow allows you to validate the NovaSeqDx v1.2 workflow.

Enter sign in credentials.

Select Sign In. A Successfully Authenticated message displays.

Universal Copy Service — Copies output files to destinations such as final destination folder and/or BSSH (when the instrument is configured for use with BSSH).

If you selected IVD Only instrument mode, select the IVD Settings tab and complete the following steps:

1. Configure Run Setup as follows.

   • Select the Select runs from the LIMS server option.

   • Enter the Clarity LIMS URL (e.g., https://yourserver.claritylims.com).

2. [Optional] Under Output Location, enter the output folder location. You can also specify the output folder location when setting up a run in NVOS.

If you selected RUO Only instrument mode, select the RUO Settings tab and complete the following steps:

1. Configure Run Setup as follows.

   • Select the Select runs from the LIMS server option.

   • Enter the Clarity LIMS URL(e.g., https://yourserver.claritylims.com).

2. Configure Default Work Type as follows.

   • [Optional] If selection support between NovaSeq Xp or NovaSeq Standard is required for the workflow, select the Enable workflow selection checkbox.

   • Select the required default workflow type: NovaSeq Standard or NovaSeq Xp.

3. [Optional] Configure Output Location as follows.

   • [Optional] Enter the output folder location. You can also specify the output folder location when setting up a run in NVOS.

   • [Optional] To upload and store run data in the cloud server, select the BSSH checkbox. Select a Configuration option (Run Monitoring and Storage or Run Monitoring Only) and select your Hosting Location from the drop-down list.

Select Save to complete the configuration.

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

Tenant DB Password

Clarity DB Password

FTP password

LDAP manager password (if LDAP is enabled)

LabLink DB password

Reporting DB password (if installed)

RabbitMQ password

Library Prep Validation configuration (optional, but recommended)

configure_sequencer_api_application.sh configuration script

Enter y to confirm that you want to proceed with the RPM and package installation.

Make sure that NGS Extension Package v5.25.0 is installed.

If this package is not installed, you must install it or upgrade the existing package.

As the glsjboss user, install the QC_Protocols.qc-protocols configuration from the following location:

Enter y to confirm that you want to proceed with the RPM installation.

If necessary, install the Secret Utility Package.

For more information on this package, refer to Prerequisite 1: Secret Utility Configuration.

As the glsjboss user, run configure_sequencer_api_application.sh from the following location:

Enter values for the presented as follows.

You can also press Enter to accept the default values in the application.yml configuration file. For more information about the application.yml properties, refer to Integration Properties Details.

• Would you like to use the apiuser user for communication with Clarity? [Y]

• Enter required value for Token Expiry (hours) [88]

Update the Clarity LIMS proxy for the Sequencer API as follows.

• For an on-premise system, run the following command:

• For an Illumina cloud hosted system, make sure that the proxy has been updated as follows. Place the sequencer-api.include file in /etc/httpd/conf.d/clarity.

Update the Clarity LIMS Tomcat configuration as follows.

1. Make sure that Secret Utility is installed and configured. For more information, refer to Prerequisite 1: Secret Utility Configuration.

2. Run the following command to add the Secret Utility configuration to Clarity LIMS:

   ℹ 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.

3. Use the following command to Start Clarity LIMS:

[Optional] Run the following command to install the Library Prep Validation v2.3.4 workflow:

This step is optional, but recommended. The Library Prep Validation v2.3.4 workflow allows you to validate the NovaSeqDx v1.2 workflow.

Enter sign in credentials.

Select Sign In. A Successfully Authenticated message displays.

Universal Copy Service — Copies output files to destinations such as final destination folder and/or BSSH (when the instrument is configured for use with BSSH).

If you selected IVD Only instrument mode, select the IVD Settings tab and complete the following steps:

1. Configure Run Setup as follows.

   • Select the Select runs from the LIMS server option.

   • Enter the Clarity LIMS URL (e.g., https://yourserver.claritylims.com).

2. [Optional] Under Output Location, enter the output folder location. You can also specify the output folder location when setting up a run in NVOS.

If you selected RUO Only instrument mode, select the RUO Settings tab and complete the following steps:

1. Configure Run Setup as follows.

   • Select the Select runs from the LIMS server option.

   • Enter the Clarity LIMS URL(e.g., https://yourserver.claritylims.com).

2. Configure Default Work Type as follows.

   • [Optional] If selection support between NovaSeq Xp or NovaSeq Standard is required for the workflow, select the Enable workflow selection checkbox.

   • Select the required default workflow type: NovaSeq Standard or NovaSeq Xp.

3. [Optional] Configure Output Location as follows.

   • [Optional] Enter the output folder location. You can also specify the output folder location when setting up a run in NVOS.

   • [Optional] To upload and store run data in the cloud server, select the BSSH checkbox. Select a Configuration option (Run Monitoring and Storage or Run Monitoring Only) and select your Hosting Location from the drop-down list.

Select Save to complete the configuration.

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

Automated tracking of the NovaSeq 6000Dx sequencing run and parsing of run statistics into Clarity LIMS, via the Sequencer API.

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.

• Checks that the NovaSeqDx Run Mode value is compatible with the Loading Workflow Type and Flowcell Type. This automation also generates an error message if the Loading Workflow Type or the Flowcell Type are incompatible.

• Calculates the Minimum Molarity.

• Checks Normalized Molarity value. For samples with no Normalized Molarity value (eg, 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.

  ⚠ Do not change this value. If Next Step is not set to Remove from workflow, the routing script is not routed samples correctly.

• For samples whose Normalized Molarity value is lower than the Minimum Molarity value, the Loading Workflow Type is set to Remove from workflow. A message is recorded in the Warning field for the sample.

  To proceed, choose 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. 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 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 NovaSeqDx Standard (NovaSeqDx v1.2) step. This step is the first step in the NovaSeqDx Standard protocol.

• Samples whose Loading Workflow Type is set to NovaSeq Xp are routed to the Make Bulk Pool for NovaSeqDx Xp (NovaSeqDx v1.2) step. This step is the first step in the NovaSeqDx Xp protocol.

On the Pooling screen, perform the following actions:

1. Create a pool by dragging samples into the Pool Creator.

   ⚠ Only create one pool.
2. Enter a name for the pool or accept the default name (eg, Pool #1).
3. Select Record Details.

   After exiting the Pooling screen, the Validate Inputs Flowcell Type and Single Pool 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.

  ℹ Assuming the default Minimum Per Sample Volume (ul) value of 5, for a given batch:

  • If the smallest Per Sample Volume (ul) value is < 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 Step automation. This automation sets the next step for samples to ADVANCE and advances them to the next step in the protocol, Dilute, and Denature (NovaSeqDx v1.2). The automation also copies the NovaSeqDx Run Mode value from the input pool to the output pool.

On the Assign Next Steps screen, the next step for samples is set to the next step in the workflow: Dilute and Denature (NovaSeqDx v1.2).

Select Finish Step.

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. To add and activate reagent lots, refer to Add and Configure Reagent Kits and Lots in 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 (NovaSeqDx v1.2)

On the Record Details screen, select Validate Run Setup and Generate Sample Sheet. This triggers the automation script, which performs the following actions:

• Validates the parameters entered on the Record Details screen.

• Generates the v2 sample sheet and attaches it to the placeholder in the Files area of the Record Details screen.

  ℹ 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.

• 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.

⚠ Do not change this value. If Next Step is not set to Remove from workflow, the routing script is not able to route samples correctly.

Select Finish Step.

After exiting the step, the following actions occur:

• The Routing Script automation is triggered and samples are routed to AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2).

• In Lab View, the pool of samples is queued for the AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2) step.

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.

  ℹ Assuming the default Minimum Per Sample Volume (ul) value of 5, for a given batch:

  • If the smallest Per Sample Volume (ul) value is < 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, based on the ratio used to increase the lowest value to 5.

[Optional] 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 Step 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 next step in the protocol—Dilute, Denature & ExAmp (NovaSeqDx v1.2).

On the Assign Next Steps screen, the next step for samples is set to Dilute, Denature & ExAmp (NovaSeqDx v1.2).

Select Finish 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, perform the following actions:

1. In the Reagent Lot Tracking section, select from the active lots displayed in each drop-down list.

2. In the Step Details section, verify that the DPX1, DPX2, and DPX3 reagent volume values are populated. The script sets these values and they cannot be edited.

3. [Optional] In the Sample Details table, select the pool icon to view details of the working pool composition.

1. Make sure that the following values and columns are populated:

   • BP Aliquot

   • Mastermix per lane

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.

On the Placement screen, do as follows.

1. Drag 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 Flowcell field.

1. 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. This automation also copies the Flowcell Type and Loading Workflow Type field values from step inputs to outputs. The following table shows the barcode mask information.

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. Refer to the following table for details.

Fields Displayed on Record Details Screen of Load to Flowcell (NovaSeqDx v1.2) Step

Select Validate Run Setup and Generate Sample Sheet to trigger the automation script. The script 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 on the Record Details screen.

ℹ 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.

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.

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

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

At the end of the run, NVOS 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, NVOS sends a request containing the parsed run metrics. NVOS sends a request only when the run completes successfully. However, the API does not make this assumption and accepts the request regardless of status. The Sequencer API then does the following:

1. Records the metrics into the fields on file placeholder outputs in Clarity LIMS.

2. Completes the step in Clarity LIMS if the status is RunCompletedSuccessfully. For all other status options, the step remains in progress. This action is the default autocomplete step behavior. For more information, refer to NovaSeq 6000Dx Integration v1.2.0 Configuration.

ℹ provides detailed information on how the following components work with the Sequencer API integration:

• Sample sheet generation and contents

• NovaSeq 6000Dx run

• User authentication and login

On the Record Details screen, the Sequencing Log multiline text field contains logging information.

If 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 the Clarity LIMS support team. Provide the relevant information from the troubleshooting steps already performed.

Tris-HCl

Flowcell Type

Loading Workflow Type

Automated tracking of the NovaSeq 6000Dx sequencing run and parsing of run statistics into Clarity LIMS, via the Sequencer API.

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.

• Checks that the NovaSeqDx Run Mode value is compatible with the Loading Workflow Type and Flowcell Type. This automation also generates an error message if the Loading Workflow Type or the Flowcell Type are incompatible.

• Calculates the Minimum Molarity.

• Checks Normalized Molarity value. For samples with no Normalized Molarity value (eg, 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.

  ⚠ Do not change this value. If Next Step is not set to Remove from workflow, the routing script is not routed samples correctly.

• For samples whose Normalized Molarity value is lower than the Minimum Molarity value, the Loading Workflow Type is set to Remove from workflow. A message is recorded in the Warning field for the sample.

  To proceed, choose 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. 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 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 NovaSeqDx Standard (NovaSeqDx v1.2) step. This step is the first step in the NovaSeqDx Standard protocol.

• Samples whose Loading Workflow Type is set to NovaSeq Xp are routed to the Make Bulk Pool for NovaSeqDx Xp (NovaSeqDx v1.2) step. This step is the first step in the NovaSeqDx Xp protocol.

On the Pooling screen, perform the following actions:

1. Create a pool by dragging samples into the Pool Creator.

   ⚠ Only create one pool.
2. Enter a name for the pool or accept the default name (eg, Pool #1).
3. Select Record Details.

   After exiting the Pooling screen, the Validate Inputs Flowcell Type and Single Pool 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.

  ℹ Assuming the default Minimum Per Sample Volume (ul) value of 5, for a given batch:

  • If the smallest Per Sample Volume (ul) value is < 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 Step automation. This automation sets the next step for samples to ADVANCE and advances them to the next step in the protocol, Dilute, and Denature (NovaSeqDx v1.2). The automation also copies the NovaSeqDx Run Mode value from the input pool to the output pool.

On the Assign Next Steps screen, the next step for samples is set to the next step in the workflow: Dilute and Denature (NovaSeqDx v1.2).

Select Finish Step.

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. To add and activate reagent lots, refer to Add and Configure Reagent Kits and Lots in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

In the Reagent Lot Tracking section, select from the active lots displayed in each drop-down list.

The fields displayed 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 (NovaSeqDx v1.2)

On the Record Details screen, select Validate Run Setup and Generate Sample Sheet. This triggers the automation script, which performs the following actions:

• Validates the parameters entered on the Record Details screen.

• Generates the v2 sample sheet and attaches it to the placeholder in the Files area of the Record Details screen.

  ℹ 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.

• 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.

⚠ Do not change this value. If Next Step is not set to Remove from workflow, the routing script is not able to route samples correctly.

Select Finish Step.

After exiting the step, the following actions occur:

• The Routing Script automation is triggered and samples are routed to AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2).

• In Lab View, the pool of samples is queued for the AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2) step.

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.

  ℹ Assuming the default Minimum Per Sample Volume (ul) value of 5, for a given batch:

  • If the smallest Per Sample Volume (ul) value is < 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, based on the ratio used to increase the lowest value to 5.

[Optional] 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 Step 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 next step in the protocol—Dilute, Denature & ExAmp (NovaSeqDx v1.2).

On the Assign Next Steps screen, the next step for samples is set to Dilute, Denature & ExAmp (NovaSeqDx v1.2).

Select Finish 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, perform the following actions:

1. In the Reagent Lot Tracking section, select from the active lots displayed in each drop-down list.

2. In the Step Details section, verify that the DPX1, DPX2, and DPX3 reagent volume values are populated. The script sets these values and they cannot be edited.

3. [Optional] In the Sample Details table, select the pool icon to view details of the working pool composition.

1. Make sure that the following values and columns are populated:

   • BP Aliquot

   • Mastermix per lane

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.

On the Placement screen, do as follows.

1. Drag 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 Flowcell field.

1. 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. This automation also copies the Flowcell Type and Loading Workflow Type field values from step inputs to outputs. The following table shows the barcode mask information.

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. Refer to the following table for details.

Fields Displayed on Record Details Screen of Load to Flowcell (NovaSeqDx v1.2) Step

Select Validate Run Setup and Generate Sample Sheet to trigger the automation script. The script 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 on the Record Details screen.

ℹ 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.

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.

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

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

At the end of the run, NVOS 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, NVOS sends a request containing the parsed run metrics. NVOS sends a request only when the run completes successfully. However, the API does not make this assumption and accepts the request regardless of status. The Sequencer API then does the following:

1. Records the metrics into the fields on file placeholder outputs in Clarity LIMS.

2. Completes the step in Clarity LIMS if the status is RunCompletedSuccessfully. For all other status options, the step remains in progress. This action is the default autocomplete step behavior. For more information, refer to NovaSeq 6000Dx Integration v1.3.0 Configuration.

ℹ provides detailed information on how the following components work with the Sequencer API integration:

• Sample sheet generation and contents

• NovaSeq 6000Dx run

• User authentication and login

On the Record Details screen, the Sequencing Log multiline text field contains logging information.

If 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 the Clarity LIMS support team. Provide the relevant information from the troubleshooting steps already performed.

Tris-HCl

Flowcell Type

Loading Workflow Type