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User Interaction, Validation and Troubleshooting

This section explains how to use the MiSeqDx Validation workflow that is included in the Illumina MiSeqDx Integration Package. The workflow allows for simple validation of the following functionality:

  • Auto-placement of reagent indexes on the PCR Amplification step

  • Generation of a sample sheet file for use with the MiSeqDx Operating Software (MOS) on the Denature, Dilute and Load Sample step

In addition, this section provides tips to help you troubleshoot the system.

All validation steps assume that you have installed the MiSeqDx Integration Package and have imported the default Clarity LIMS configuration. The validation steps are based on MiSeqDx Validation (CF 139-Variant Assay) 1.2, where the autoplacement of indexes validation occurs in Run PCR Amplification step and sample sheet generation validation occurs in Run Denature, Dilute and Load Samples step.

For compatibility information, refer to MiSeqDx Integration v1.10.0 Release Notes. Only Clarity LIMS v6.2 or later is supported in MiSeqDx Integration Package v1.10.0 or in subsequent releases.

Instructions for configuring sample sheet generation are provided in MiSeqDx Integration v1.10.0 Configuration.

Protocols

The following protocols are included in MiSeqDx Integration Package v1.10.0.

  • Library Prep Protocols:

    • CF 139-Variant Assay Library Prep 1.2

    • CF Clinical Sequencing Assay Library Prep 1.2

    • Universal Kit Library Prep 1.2

  • Illumina SBS MiSeqDx Protocols:

    • Illumina SBS MiSeqDx (CF 139-Variant Assay) 1.2

    • Illumina SBS MiSeqDx (CF Clinical Sequencing Assay) 1.2

    • Illumina SBS MiSeqDx (Universal Kit) 1.2

  • Validation Protocols:

    • MiSeqDx Validation (CF 139-Variant Assay) 1.2

    • MiSeqDx Validation (CF Clinical Sequencing Assay) 1.2

    • MiSeqDx Validation (Universal Kit) 1.2

Each validation protocol includes the following steps from the Library Prep and Illumina SBS MiSeqDx protocols:

  • Extension-Ligation of Bound Oligos (Library Prep step)

  • PCR Amplification (Library Prep step)

  • Library Pooling (MiSeqDx) (Illumina SBS MiSeqDx step)

  • Denature, Dilute and Load Sample (Illumina SBS MiSeqDx step)

  • MiSeqDx Run (MiSeqDx) (Illumina SBS MiSeqDx step)

  • Variant Calling (MiSeqDx) (Illumina SBS MiSeqDx step)

Library Prep Protocols

This section discusses the Library Prep protocols included in the MiSeqDx v1.10.0 Integration Package.

Step 1: Hybridization of Oligo Pool (CF 139-Variant Assay) 1.2

The following process is used to hybridize the oligo pool.

  1. Add samples to Ice Bucket.

  2. Add Negative Control and Positive Control samples to Ice Bucket.

    ℹ️ Good laboratory practices mandate that a positive control DNA sample and a negative (no-template) control sample are included in every run. The positive control DNA sample should be a well-characterized sample with a known CFTR mutation.

  3. Select Begin Work.

  4. On the Placement screen, select all samples and place in container. Select Record Details.

  5. On the Record Details screen, under Reagent Lot Tracking, set the following values:

    • gDNA Concentration (ng/uL): 50 ng/ul

    • gDNA Volume (uL): 5 uL

    • Select the reagent lots for CF 139-Variant Assay-Oligo Pool and Hybridization Buffer.

  6. Select Next Steps.

  7. Select Removal of Unbound Oligos (CF 139-Variant Assay) 1.2 as the next step.

  8. Select Apply.

  9. Select Finish Step.

Step 2: Removal of Unbound Oligos (CF 139-Variant Assay) 1.2

The process below is used to remove unbound oligos.

  1. Add samples to Ice Bucket.

  2. Select Begin Work.

  3. On the Placement screen, select all samples and place in container.

  4. Select Record Details.

  5. On the Record Details screen, under Reagent Lot Tracking, select the reagent lots for Stringent Wash Buffer and Universal Wash Buffer.

  6. Select Next Steps.

  7. Select Extension-Ligation of Bound Oligos (CF 139-Variant Assay) 1.2 as the next step.

  8. Select Apply.

  9. Select Finish Step.

Step 3: Extension-Ligation of Bound Oligos (CF 139-Variant Assay) 1.2

The process below is used to add reagents to the Extension-Ligation Mix field.

  1. Add samples to Ice Bucket. Select Begin Work.

  2. On the Placement screen, select all samples and place in container.

  3. Select Record Details.

  4. On the Record Details screen, under Reagent Lot Tracking, select the reagent lots for Extension-Ligation Mix.

  5. Select Next Steps.

  6. Select PCR Amplification (CF 139-Variant Assay) 1.2 as the next step.

  7. Select Apply.

  8. Select Finish Step.

Step 4: PCR Amplification (CF 139-Variant Assay) 1.2

  1. Add samples to Ice Bucket.

  2. Select one of the following Reagent Label Options:

    • If you are working with eight samples or less, select CF 139-Variant Assay 8-Sample Indexes.

    • If you are working with more than 8 samples, select CF 139-Variant Assay Indexes.

  3. Select Begin Work.

  4. On the Placement screen, select all samples and place in container. Select Add Reagents.

    On entry to the Add Reagents screen, the Auto Place Indexes automation is invoked and the reagents are placed on the samples.

  5. Once placement is complete, a message displays indicating that the index pattern has been applied successfully. Select OK.

  6. On Add Reagents screen, reagents are already placed. Select Record Details.

  7. On exit from the Add Reagents screen, the index placement is validated. Once validation is complete, a message displays indicating that the index pattern has been validated on all samples successfully. Select OK.

  8. On the Record Details screen, under Reagent Lot Tracking, enter the lots for the following fields:

    • Index Primers

    • NaOH

    • PCR Master Mix

    • PCR Polymerase

  9. Select Next Steps.

  10. Select PCR Clean-Up (CF 139-Variant Assay) 1.2 as the next step.

  11. Select Apply.

  12. Select Finish Step.

Step 5: PCR Clean-Up (CF 139-Variant Assay) 1.2

  1. Add samples to Ice Bucket.

  2. Select Begin Work.

  3. On the Placement screen, select all samples and place in container. Select Record Details.

  4. On the Record Details screen, under Reagent Lot Tracking, select the reagent lots for the following:

    • Elution Buffer

    • EtOH

    • PCR Clean-up Beads

  5. Select Next Steps.

  6. Select Mark protocol as complete as the next step. Select Apply.

  7. Select Finish Step.

MiSeqDx Validation Protocol

Follow the following steps to validate auto-placement of indexes and sample sheet generation

Activate Workflow, Add and Assign Samples

  1. In the Clarity LIMS web interface, on the Configuration > Workflows screen, activate one of the three Validation workflows:

    • MiSeqDx Validation (CF 139-Variant Assay) 1.2

    • MiSeqDx Validation (CF Clinical Sequencing Assay) 1.2

    • MiSeqDx Validation (Universal Kit) 1.2

    ℹ️ The validation steps are based on MiSeqDx Validation (CF 139-Variant Assay) 1.2

  2. On the Projects and Samples screen, create a project and assign samples as follows.

    1. Create a test project and add samples to it.

      ⚠️ The project must contain between 9 and 96 samples.

    2. Assign your samples to the MiSeqDx Validation workflow.

Extension-Ligation of Bound Oligos Step

  1. In Lab View, locate the MiSeqDx Validation protocol. You will see your samples queued for the Extension-Ligation of Bound Oligos step. Prepare the samples as follows.

  2. Add the samples to the Ice Bucket.

  3. On the Placement screen, place the samples into the output container.

  4. Select Record Details.

  5. On the Record Details screen, under Reagent Lot Tracking, select the Extension-Ligation Mix reagent lot used in the step.

    If required, activate the lot on the Reagents / Reagents and Controls screen.

  6. Select Next Steps.

  7. On the Assign Next Steps screen, assign the samples to the PCR Amplification step.

  8. Select Finish Step.

PCR Amplification Step

  1. In Lab View, locate the MiSeqDx Validation protocol. You will see your samples queued for the PCR Amplification step.

  2. Add the samples to the Ice Bucket.

  3. In the Reagent Label Options panel, in the Group of labels list, select CF 139-Variant Assay Indexes (or the equivalent for the workflow you activated).

  4. Select Begin Work.

  5. On the Placement screen, place the samples into the output container.

  6. Select Add Reagents.

    On entry to the Add Reagents screen, the Auto Place Indexes automation is automatically invoked and the reagents are placed on the samples.

  7. After placement is complete, a message displays indicating that the index pattern has been applied successfully. Select OK.

  8. On the Add Reagents screen, reagents are already placed. Select Record Details.

    On exit from the Add Reagents screen, the Validate Index Placement automation is automatically invoked and index placement is validated.

  9. When validation is complete, a message displays indicating that the index pattern has been validated on all samples successfully. Select OK.

  10. On the Record Details screen, under Reagent Lot Tracking, select the lots for the following items:

    • Index Primers

    • NaOH

    • PCR Master Mix

    • PCR Polymerase

  11. Select Next Steps.

  12. On the Assign Next Steps screen, assign samples to the Library Pooling (MiSeqDx) 1.2 step.

  13. Select Finish Step.

Library Pooling (MiSeqDx) Step

  1. Return to Lab View and locate the MiSeqDx Validation protocol. You will see your samples queued for the Library Pooling (MiSeqDx) 1.2 step.

    Prepare the samples as follows.

  2. Add samples to the Ice Bucket.

  3. In the Add Control Samples panel, select PhiX Internal Control. Select Begin Work.

  4. On the Pool Samples screen, create a pool of samples. Select Place Samples.

  5. On the Placement screen, place the pool into the output container. Select Record Details.

  6. Under Reagent Lot Tracking, select the reagent lot used in the step.

  7. Under Step Details, enter the concentration value in the Normalized conc. (nM) field.

  8. Select Next Steps.

  9. On the Assign Next Steps screen, assign the pool to the Denature, Dilute and Load Samples step.

Denature, Dilute and Load Sample Step

  1. Return to Lab View and locate the MiSeqDx Validation protocol.

    You will see your pooled samples queued for the Denature, Dilute and Load Sample step.

  2. Place the pooled samples as follows.

    1. Add the pool to the Ice Bucket.

      On the Place Samples screen, the Validate Single Input script runs and validates only one pool that is entered into this step as the input sample. If validation fails, Clarity LIMS prompts you that it is only able to proceed with the step with one pool.

    2. On the Placement screen, place the pool into a MiSeqDx reagent cartridge.

    3. Select Record Details.

      On entry to the Record Details screen, a script validates the Container Name and prompts you to scan the reagent cartridge barcode.

  3. On the Record Details screen of the Denature, Dilute and Load Sample step, generate the sample sheet as follows.

    ℹ️ All read-only files are autopopulated.

    1. Enter the folder path for the Reference Genomes that will be used for secondary analysis in the GenomeFolder field.

    2. Under Reagent Lot Tracking, select the reagent lots used in the step.

    3. Enter the lots for MiSeqDx Flow Cell - CF 139-Variant Assay and MiSeqDx SBS Solution (PR2) - CF 139-Variant Assay.

    4. In the Step Details section, review the step parameters.

    5. Select Generate MiSeqDx Sample Sheet to generate the MiSeqDx sample sheet.

    6. Clarity LIMS attaches the generated sample sheet and log file to placeholders in the Files area of the Record Details screen. Download the files and validate their format and content.

    7. [Optional] In the Files section, upload the Lab Tracking Form to the appropriate placeholder.

  4. Select Next Steps.

  5. On the Assign Next Steps screen, assign the samples to the MiSeqDx Run (MiSeqDx) 1.2 step.

Validation of auto-placement of indexes and sample sheet generation is completed at the end of this step.

MiSeqDx Run (MiSeqDx) Step

ℹ️ On the Record Details screen, do not select the AUTOMATED - Run Report Generation button. The run report is generated and attached to the step automatically. This process may take a while.

On the Record Details screen of the MiSeqDx Run (MiSeqDx) 1.2 step:

  1. Run the instrument. The read-only field values are automatically populated as the instrument runs.

  2. After the run has completed, observe the Record Details screen of the MiSeqDx Run (MiSeqDx) 1.2 step for the following.

    • The autopopulated read-only fields.

    • The generated and attached Illumina Run Report and Log File.

    • The attached Run Parameters and Run Info files.

    • The generated and attached Link to Run Folder.

    • QC flags set on each lane.

  3. [Optional] In the Files section, upload the Lab Tracking Form.

  4. Select Next Steps.

    The Verify Report Status automation should run and allow you to go to the Assign Next Steps screen.

  5. On the Assign Next Steps screen, assign the samples to the Variant Calling (MiSeqDx) 1.2 step.

  6. Select Finish Step.

Variant Calling (MiSeqDx) Step

On the Record Details screen of the Variant Calling (MiSeqDx) 1.2 step:

  1. Make sure that the VCF has the correct name based on the sample sheet.

    The file-naming format for the VCF file is SampleName_S#.vcf. In this format, the SampleName is the value in the Sample_Name column of the sample sheet, and # is the sample number determined by the order in which samples are listed in the sample sheet.

  2. After secondary analysis has completed, the following files are attached to the step:

    • Combined Variant Call File — a compressed VCF file (zip file) containing all the variant call files, including the CFTR specific VCF files.

    • Combined Output Text File — a combined text file containing a summary of all the sample metrics.

    • Variant Call File per individual sample

  3. QC flags are set on each lane.

Illumina SBS MiSeqDx Protocol

Sort MiSeqDx Samples (MiSeqDx) Step

This step routes your samples to the appropriate next step. Assign each sample to one of the following steps:

  • Library Normalization (MiSeqDx)

  • Library Pooling (MiSeqDx)

  • Denature, Dilute and Load Sample

Library Normalization (MiSeqDx) Step

  1. On the Record Details screen, enter values for the following items:

    • Library volume (µl) transferred to destination plate

    • Normalized conc (nM)

    • Maximum destination container volume (µl)

    • Any other required fields

  2. Select Create Normalization CSV to start the automation.

    When the normalizationBufferVolumes script has completed, the Normalization buffer volumes CSV file is generated and attached to the step. This file is in the Files area of the Record Details screen.

Library Pooling (MiSeqDx) Step

Refer to Library Pooling (MiSeqDx) step under MiSeqDx Validation Protocol for details.

Denature, Dilute and Load Sample Step

  1. On the Placement screen, set up the reagent cartridge as follows.

    1. Using an RFID scanner, scan the RFID of the MiSeqDx reagent cartridge into the Container Name field.

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

  2. Select Record Details.

Refer to Denature, Diluate and Load Sample step under MiSeqDx Validation Protocol for more details.

On exit from the Placement screen, the automation is automatically triggered and validates the Container Name.

MiSeqDx Run (MiSeqDx) Step

Refer to MiSeqDx Run (MiSeqDx) step under MiSeqDx Validation Protocol for details.

Variant Calling (MiSeqDx) Step

Refer to Variant Calling (MiSeqDx) step under MiSeqDx Validation Protocol for details.

Validate Creation of Event Files

Validating the creation of the event files confirms the following:

  • 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.

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.

  1. In C:\Illumina\gls, double-click gls_event_mos_rta.bat.

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

  3. Manually invoke the gls_event_mos.bat file.

    The file produces an output similar to the following example:

    cycleNumber = 318
runFolder = "D:\Illumina\MiSeqTemp\161030_M99999_0056_FC1234567-ABCDE"
netFolder = "D:\Illumina\MiSeqOutput\161030_M99999_0056_FC1234567-ABCDE"
readType = 4
eventType = EndRun
softwareType = MOS
finishDate = 2016-10-30

Troubleshooting

If an automation trigger does not appear to run its corresponding scripts, see:

If an error occurs that does not provide direction on how to proceed, troubleshoot the error as follows.

  1. Confirm the version of the installed Illumina MiSeqDx Integration Package by running the following command from the server console.

    rpm -qa | grep -i miseqdx

  2. If the error is related to retrieving run results data, review the MiseqDxIntegrator.log.

  3. Contact the Clarity LIMS Support team, supplying the relevant information from the troubleshooting already performed.

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