User Interaction, Validation and Troubleshooting

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.

Follow the steps in Library Prep Validation Protocol to run the Library Prep Validation workflow with the following:

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

• Sequencing Instrument = NovaSeq

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:

   ℹ Values can vary across samples.

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

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

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.

   ⚠ If the Next Step value is not set to Remove from workflow, the routing script automation cannot advance to the next step.

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.

   ⚠ Only one pool must be created.

   

   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.

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

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

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

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

   
7. 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.

   
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.

4. On the Record Details screen, the Reagent Lot Tracking section tracks the NaOH, Resuspension Buffer, and Tris-HCI reagents used in the step. To add and activate reagent lots, refer to Add and Configure Reagent Kits and Lots in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
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

   Field	Value
   Experiment Name	Enter the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. No spaces.
   BaseSpace Sequence Hub Configuration	Presets Not Used Run Monitoring Only Run Monitoring and Storage
   Workflow	Presets GenerateFASTQ
   Workflow Type	Presets No Index Single Index Dual Index
   Index Read 1	Presets 0 6 8 type a value between 0 and 20
   Index Read 2	Presets 0 6 8 type a value between 0 and 20
   Paired End	Presets True False
   Read 1 Cycles	Presets 251 151 101 51 type a value between 1 and 251 for the SP flow cell type or between 1 and 151 for all other types
   Read 2 Cycles	Presets 251 151 101 51 type a value between 0 and 251 for the SP flow cell type or between 1 and 151 for all other types
   Use Custom Read 1 Primer	Select if applicable.
   Use Custom Read 2 Primer	Select if applicable.
   Use Custom Index Read 1 Primer	Select if applicable.
   Reverse Complement Workflow	Select Yes (by default) for v1.5 reagents in NovaSeq Control Software v1.7 and onwards. Select No for v1 reagents.
   UMI - Read 1 Length	Enter the length of the Unique Molecular Identifiers (UMI) in Read 1. Leave blank if not applicable.
   UMI - Read 2 Length	Enter the length of the Unique Molecular Identifiers (UMI) in Read 2. Leave blank if not applicable.
   UMI - Read 1 Start From Cycle	Enter the cycle number that Read 1 starts from. Leave blank if not applicable.
   UMI - Read 2 Start From Cycle	Enter the cycle number that Read 2 starts from. Leave blank if not applicable.
   Sample Sheet Path	Automatically populated after the sample sheet has been generated.

   
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.

   ⚠ 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.

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.

Do not add samples to the Ice Bucket or start the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step. The integration does this action automatically.

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

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

The protocol contains three steps:

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.

   ⚠ Only one pool must be created.

   

   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.

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

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

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

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

   
6. 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.

   
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.

4. On the Record Details screen, the Reagent Lot Tracking section tracks the DPX1, DPX2, DPX3, NaOH, Resuspension Buffer, and Tris-HCI reagents used in the step. To add and activate reagent lots, refer to Add and Configure Reagent Kits and Lots in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
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.

   

   ℹ The BP Aliquot, Mastermix per lane, NaOH, and Tris-HCI volume values for each working pool are already populated. These values are set by a script and are not editable while running the step.

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

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.

   Flowcell Type	Barcode Mask
   SP	[A-Z0-9]{5}DR[A-Z0-9]{2}
   S1	[A-Z0-9]{5}DR[A-Z0-9]{2}
   S2	[A-Z0-9]{5}DM[A-Z0-9]{2}
   S4	[A-Z0-9]{5}DS[A-Z0-9]{2}

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

   Field	Value
   Experiment Name	Enter the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. No spaces.
   BaseSpace Sequence Hub Configuration	Presets Not Used Run Monitoring Only Run Monitoring and Storage
   Workflow	Presets GenerateFASTQ
   Workflow Type	Presets No Index Single Index Dual Index
   Index Read 1	Presets 0 6 8 type a value between 0 and 20
   Index Read 2	Presets 0 6 8 type a value between 0 and 20
   Paired End	Presets True False
   Read 1 Cycles	Presets 251 151 101 51 type a value between 1 and 251 for the SP flow cell type or between 1 and 151 for all other types
   Read 2 Cycles	Presets 251 151 101 51 type a value between 0 and 251 for the SP flow cell type or between 1 and 151 for all other types
   Use Custom Read 1 Primer	Select if applicable.
   Use Custom Read 2 Primer	Select if applicable.
   Use Custom Index Read 1 Primer	Select if applicable.
   Reverse Complement Workflow	Select Yes (by default) for v1.5 reagents in NovaSeq Control Software v1.7 and onwards. Select No for v1 reagents.
   UMI - Read 1 Length	Enter the length of the Unique Molecular Identifiers (UMI) in Read 1. Leave blank if not applicable.
   UMI - Read 2 Length	Enter the length of the Unique Molecular Identifiers (UMI) in Read 2. Leave blank if not applicable.
   UMI - Read 1 Start From Cycle	Enter the cycle number that Read 1 starts from. Leave blank if not applicable.
   UMI - Read 2 Start From Cycle	Enter the cycle number that Read 2 starts from. Leave blank if not applicable.
   Sample Sheet Path	Automatically populated after the sample sheet has been generated.
   Library Tube Barcode	Scan the library tube barcode.

   
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).

Do not add samples to the Ice Bucket or start the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step. The integration does this action automatically.

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

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

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

Refer to Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) in NovaSeq 6000 Integration v2.6.0 Configuration for how the integration works, and for details on the automations.

Troubleshooting

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

• Troubleshooting Automated Worker in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
• Troubleshooting Automation in the Clarity LIMS (API & Database) documentation.

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

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

   rpm -qa | grep -i novaseq

   • 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:

   /opt/gls/clarity/extensions/novaseq/SequencingService/NovaSeqIntegrator.log

   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.