User Interaction, Validation and Troubleshooting

This guide explains how to validate the installation of the Illumina NextSeq 500/550 Integration Package v2.3.0.

The validation process involves:

• Validating the creation of event files.

• Running samples through the Library Prep Validation v2.3.1 workflow. This workflow is installed by Illumina Preset Protocols (IPP) v2.6 and 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 NextSeq 500/550 Sequencing v1.1 workflow.

• Running normalized libraries through the NextSeq 500/550 Sequencing v1.1 workflow. This validates the automated generation of a sample sheet for use with bcl2fastq2 v2.20.0 analysis software. It also validates the automated tracking of the NextSeq sequencing run and parsing of run statistics into Clarity LIMS, including:

  • Run status and metrics of sequencing run

  • Sequencing run parameters

  • Real Time Analysis v2 (RTA2) run directory location and other run specific information

Activate Workflow, Create Project, Add and Assign Samples

The following steps set up Clarity LIMS in preparation for running samples through the Library Prep Validation v2.3.1 and NextSeq 500/550 Sequencing v1.1 workflows.

1. In the Clarity LIMS configuration area, activate the Library Prep Validation v2.3.1 and NextSeq 500/550 Sequencing v1.1 workflows.

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

3. Assign your samples to the Library Prep Validation v2.3.1 workflow.

Library Prep Validation v2.3.1 Protocol

This single-step protocol models the library prep required to produce normalized libraries that are ready for the NextSeq 500/550 Sequencing v1.1 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 = NextSeq

On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the NextSeq 500/550 Sequencing v1.1 workflow, which is Library Pooling (NextSeq 500/550 v1.1) step.

NextSeq 500/550 Sequencing v1.1 Protocol

Step 1: Library Pooling (NextSeq 500/550 v1.1)

1. In Lab View, locate the NextSeq 500/550 Sequencing v1.1 protocol. You will see your samples queued for the Library Pooling (NextSeq 500/550 v1.1) step.

   Select the step to proceed to the Queue screen.

2. On the Queue screen, add samples as follows.

   1. Add the samples to the Ice Bucket. In the Add Control Samples panel, add the PhiX v3 control sample to the Ice Bucket.

   

   1. Select View Ice Bucket.

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

4. On the Pool Samples screen, create a pool of samples as follows.

   1. Drag samples into the Pool Creator.

   

   1. Name the pool. You can also accept the default name Pool #1.

   

   1. Select Place Samples.

5. On the Placement screen, move the pool to the container as follows.

   1. Select the pool in the Samples to be Placed area on the left. Drag it over to the container on the right.

   

   1. Select Record Details.

6. On the Record Details screen, select Next Steps.

   On the Assign Next Steps screen, the next step is already set to Denature and Dilute (NextSeq 500/550 v1.1).

7. Select Finish Step.

Step 2: Denature and Dilute (NextSeq 500/550 v1.1)

1. In Lab View, locate the NextSeq 500/550 Sequencing v1.1 protocol. You will see your pooled samples queued for the Denature and Dilute (NextSeq 500/550 v1.1) step.

   Select the step to proceed to the Queue screen.

2. On the Queue screen, add the pool to the Ice Bucket. Select View Ice Bucket.

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

4. On the Placement screen, move the pool to the reagent cartridge as follows.

   1. Scan the NextSeq reagent cartridge barcode into the NextSeq Reagent Cartridge field.

   2. Place the pool of samples into the reagent cartridge.

   

   1. Select Record Details.

5. On the Record Details screen, create the sample sheet as follows.

   1. Under Reagent Lot Tracking, select the reagent lot used in the step. You may need to add/activate the lot on the Reagents and Controls screen.

   2. In the Step Details table, populate the fields as appropriate.

      • The Workflow and Read 1 Cycles are required fields.

      • In the Samplesheet Template drop-down list, the reverse complement template is selected by default. Do not change this value.

   

   1. In the Sample Details table, enter the Final Loading Concentration. You may select preset 225 (for PCR-free workflows) or preset 400 (for Nano workflows). You can also enter a different value.

   2. Select Generate bcl2fastq2 NextSeq SampleSheet. Clarity LIMS generates the sample sheet and attaches it and a log file to placeholders in the Files area of the Record Details screen.

   

   1. Download the files and validate their format and content.

   2. Select Next Steps.

6. On the Assign Next Steps screen, make sure samples are already assigned to the NextSeq 500/550 Run (NextSeq 500/550 v1.1) step.

7. Select Finish Step.

Step 3: NextSeq 500/550 Run (NextSeq 500/550 v1.1)

ℹ️ When the sequencing run is finished, the integration generates a run report file. The Instrument Details section is left blank by default, unless the NextSeq 500/500 Run master step is configured to track instruments created in Clarity LIMS. For more information, refer to Add and Configure Instruments from the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

1. In Lab View, locate the NextSeq 500/550 Sequencing v1.1 protocol. You will see your pool of samples queued for the NextSeq 500/550 Run (NextSeq 500/550 v1.1) step.

   Select the step to proceed to the Queue screen.

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

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

   On the Record Details screen, the fields in the table are read-only. Once the run completes, the integration will automatically populate the fields.

   

   The integration also attaches files to the Illumina Run Report, Link to Run Folder, Run Parameters, and Run Info placeholders. The Log File is attached once the next step for samples has been assigned by the Next Step - Advance automation. The integration also populates the fields in the Sample Details table. For details, refer to NextSeq 500/550 v2.3.0 Configuration.

   ⚠️ Do not continue to step 5 and complete the step until the Illumina Run Report has been attached.

   ⚠️ If the NextSeq 500/550 Run master step is configured to track instruments, select the appropriate instrument from the Instrument Used drop-down on Record Details screen, then select Save.

   

   For more detailed steps on adding instruments, refer to the Add and Configure Instruments section of the [Clarity LIMS (Clarity & LabLink Reference Guide) documentation](../../../clarity-lims/clarity-and-lablink.md).

4. Select Next Steps.

   On the Assign Next Steps screen, make sure the next step is already set to Mark protocol as complete.

5. Select Finish Step.

Validating Creation of Event Files

Follow the steps below to confirm that event files are created by the batch file in the destination path. The steps assume that the default configuration has been successfully imported, and support for NextSeq Control Software (NCS) v4.0 has been configured.

This test validates that:

• Your DESTINATION_PATH is correctly configured.

• The instrument computer can access and write to the DESTINATION_PATH.

• There are no syntax errors in the Clarity LIMS batch file.

You can validate the gls_event_ncs_rta.bat batch file by executing it manually as follows.

1. Edit gls_event_ncs_rta.bat using any text editor (Notepad++ recommended).

   By default, this file is installed to C:\Illumina\gls.

2. In the code line set DESTINATION_PATH=C:\Illumina\gls\Events, change C:\Illumina\gls\Events\ to the network path of the event files directory.

   Remember to include the trailing "" at the end of the file path.

3. Save your changes.

Manually executing the batch file will create a dummy EndRun event file in the directory that you defined in the previous step.

Troubleshooting

If an automation trigger does not appear to run its corresponding scripts, refer to the following topics:

• Troubleshooting Automation Worker in the Administration section of the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

• Troubleshooting Automation in the Automation section of the Clarity LIMS (API & Database) documentation.

If an error occurs that does not provide direction on how to proceed, confirm the version of the installed Illumina NextSeq Integration Package by running the following command from the server console

rpm -qa | grep -i nextseq

If the error is related to data parsing, retrieving run results data, or report values not appearing as expected, review the NextSeqIntegrator.log file. It is located at

/opt/gls/clarity/extensions/Illumina_NextSeq/v2/SequencingService

Additional troubleshooting information for this integration is provided on the Illumina Instrument Integrations FAQ page.

If you are unable to resolve the issue, contact the Clarity LIMS Support team. Make sure you supply the relevant information from the troubleshooting that was already performed.