User Interaction, Validation and Troubleshooting

The following sections explain how to validate the installation of the Illumina MiSeq Integration Package v8.3.0.

The validation involves the following processes:

• Running samples through the Library Prep Validation v2.3.4 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 automatically advance to the MiSeq Sequencing v3.2 workflow.

• Running normalized libraries through the MiSeq Sequencing v3.2 workflow. This process validates the following information:

  • Automated generation of a sample sheet for use with the MiSeq Control Software (MCS) and Local Run Manager (LRM).

  • Automated tracking of the MiSeq sequencing run and parsing of run statistics into Clarity LIMS, including the following information:

    • Run status and metrics of sequencing run

    • Sequencing run parameters

    • Real-Time Analysis (RTA) v1 run directory location and other run-specific information

Activate Workflow, Create Project, Add and Assign Samples

The following steps prepare Clarity LIMS to run samples through the Library Prep Validation and MiSeq Sequencing v3.2 workflows.

1. In the Clarity LIMS Configuration area, select each workflow and change the Workflow Status toggle to Active.

2. On the Projects & Samples screen, create a project. Add samples to the project.

3. Assign the samples to the Library Prep Validation workflow.

Library Prep Validation Protocol

This single-step protocol models the library prep required to produce normalized libraries that are ready for the MiSeq Sequencing v3.2 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 = MiSeq

On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the MiSeq Sequencing v3.2 workflow, which is Library Pooling (MiSeq v3.2) step.

MiSeq Sequencing v3.2 Protocol

Run Library Pooling (MiSeq v3.2)

1. In Lab View, locate the MiSeq Sequencing v3.2 protocol. Samples are queued for the Library Pooling (MiSeq v3.2) step. Select the step to proceed to the Queue screen.

2. On the Queue screen, do as follows.

   1. Add the samples to the Ice Bucket.

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

      
   3. Select View Ice Bucket.

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

   The Validate Maximum Number of Samples automation script runs. The maximum number of samples allowed is 1536.

4. On the Pool Samples screen, do as follows.

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

      
   2. Name the pool or accept the default name (Pool #1).

      
   3. Select Place Samples.

5. On the Placement screen, do as follows.

   1. Select the pool from the Samples to be Placed area and drag it to the container.

      
   2. Select Record Details.

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

   On the Assign Next Steps screen, next step is automatically set to Denature and Dilute (MiSeq v3.2).

7. Select Finish Step.

Run Denature and Dilute (MiSeq v3.2)

1. In Lab View, locate the MiSeq Sequencing v3.2 protocol. The pooled samples are queued for the Denature and Dilute (MiSeq v3.2) step. Select the step to proceed to the Queue screen.

2. On the Queue screen, add the pool to the Ice Bucket, and then select View Ice Bucket.

   ⚠️ Only one pool is allowed as an input.

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

   The Validate Single Input automation script runs.

4. On the Placement screen, do as follows.

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

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

      
   3. Select Record Details.

5. On the Record Details screen, do as follows.

   1. In Reagent Lot Tracking, select the reagent lot used in the step.

      If the reagent lot is not listed, add/activate the lot on the Reagents and Controls screen.

   2. Use the Preset drop-down list to help populate the fields in Step Details, as needed.

      For more information on presets, refer to Using Step Details Presets. Workflow, Experiment Name, and Read 1 Cycles are required fields.

      
   3. In the Sample Details table, enter the Final Loading Concentration. Select from the following preset options or enter a different value.

      • 225 (for PCR-free workflows)

      • 400 (for Nano workflows)

   4. Select Validate Run Setup and Generate MiSeq 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.

      
   5. Download the files and validate the format and content.

6. Select Next Steps.

   On the Assign Next Steps screen, samples are automatically assigned to the MiSeq Run (MiSeq v3.2) step.

7. Select Finish Step.

Run MiSeq Run (MiSeq v3.2)

1. In Lab View, locate the MiSeq Sequencing v3.2 protocol. The pool of samples is queued for the MiSeq Run (MiSeq v3.2) step. Select the step to proceed to the Queue screen.

2. On the Queue screen, add the pool to the Ice Bucket, and then select View Ice Bucket.

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

4. On the Record Details screen, the fields are read-only.

   ⚠️ If the MiSeq Run master step is configured to track instruments, select the applicable instrument from the Instrument Used dropdown on Record Details screen and select Save.

   

   For more information on adding instruments, refer to Add and Configure Instruments in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

5. When the run completes, the integration automatically performs the following actions:

   • Populates the fields.

   • Attaches files to the Illumina Run Report, Link to Run Folder, Run Parameters, and Run Info placeholders.

   • Populates the fields in the Sample Details table. For details, refer to MiSeq Integration v8.3.0 Configuration.

   

   The Log File is attached after the next step for samples is assigned by the Next Step - Advance automation.

   ⚠️ Make sure the Illumina Run Report has been attached before continuing.

   
6. Select Next Steps.

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

7. Select Finish Step.

Using Step Details Presets

Most of the custom fields in Step Details are analysis settings used by Local Run Manager (LRM) and are for advanced users only. The following fields are exceptions:

• Workflow

• Experiment Name

• Description

• Read 1 Cycles

• Read 2 Cycles

• Custom Primers

To use the default analysis settings, select the analysis module from the Preset drop-down list.

The default values populate the respective fields and indicate the following actions are needed:

• Required — The field is required for the specific analysis module selected. Replace the word Required with a proper value. For example, the Genome Folder field should contain the directory to the genome folder.

• None — The field is not applicable for the specific analysis module selected. Leave the field as is.

For a list of fields that are applicable for the analysis module selected, refer to the Applicable analysis fields for the selected Workflow field in Step Details.

Validating Creation of Event Files

This validation checks the following information:

• The 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.

Use the following steps 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.

1. In C:\Illumina\gls, double-click the batch file gls_event_mcs_rta_lrm.bat.

2. Confirm that an empty event file appears in the configured DESTINATION_PATH.

Manual execution of the batch file produces an output resembling the following example:

Filename:
event-EndRun-07295667.txt
Contents:
cycleNumber =
runFolder =
netFolder =
readType =
eventType = EndRun
softwareType = MCS
finishDate = 2021-01-25

Troubleshooting

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

• Troubleshooting Automation 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, confirm the version of the installed MiSeq Integration Package. To confirm the version, run the

rpm -qa | grep -i miseq

command from the server console.

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

/opt/gls/clarity/extensions/Illumina_MiSeq/v8/SequencingService

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

If you are unable to resolve the issue, contact the Clarity LIMS support team. Supply the relevant information from the troubleshooting already performed.