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 . Only Clarity LIMS v6.2 or later is supported in MiSeqDx Integration Package v1.11.0 or in subsequent releases.
Instructions for configuring sample sheet generation are provided in .
The following protocols are included in MiSeqDx Integration Package v1.11.0.
Library Prep Protocols:
CF 139-Variant Assay Library Prep 1.2
CF Clinical Sequencing Assay Library Prep 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)
Library Prep Protocols
This section discusses the Library Prep protocols included in the MiSeqDx v1.11.0 Integration Package.
Step 1: Hybridization of Oligo Pool (CF 139-Variant Assay) 1.2
The following process is used to hybridize the oligo pool.
Add samples to Ice Bucket.
Add Negative Control and Positive Control samples to Ice Bucket.
ℹ 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.
Step 2: Removal of Unbound Oligos (CF 139-Variant Assay) 1.2
The process below is used to remove unbound oligos.
Add samples to Ice Bucket.
On the Placement screen, select all samples and place in container.
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.
Add samples to Ice Bucket. Select Begin Work.
On the Placement screen, select all samples and place in container.
Step 4: PCR Amplification (CF 139-Variant Assay) 1.2
Add samples to Ice Bucket.
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.
Step 5: PCR Clean-Up (CF 139-Variant Assay) 1.2
Add samples to Ice Bucket.
On the Placement screen, select all samples and place in container. Select Record Details.
MiSeqDx Validation Protocol
Follow the following steps to validate auto-placement of indexes and sample sheet generation
Activate Workflow, Add and Assign Samples
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
Extension-Ligation of Bound Oligos Step
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.
Add the samples to the Ice Bucket.
On the Placement screen, place the samples into the output container.
PCR Amplification Step
In Lab View, locate the MiSeqDx Validation protocol. You will see your samples queued for the PCR Amplification step.
Add the samples to the Ice Bucket.
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).
Library Pooling (MiSeqDx) Step
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.
Add samples to the Ice Bucket.
In the Add Control Samples panel, select PhiX Internal Control. Select Begin Work.
Denature, Dilute and Load Sample Step
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.
Place the pooled samples as follows.
Add the pool to the Ice Bucket.
Validation of auto-placement of indexes and sample sheet generation is completed at the end of this step.
MiSeqDx Run (MiSeqDx) Step
On the Record Details screen of the MiSeqDx Run (MiSeqDx) 1.2 step:
Run the instrument. The read-only field values are automatically populated as the instrument runs.
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.
Variant Calling (MiSeqDx) Step
On the Record Details screen of the Variant Calling (MiSeqDx) 1.2 step:
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.
After secondary analysis has completed, the following files are attached to the step:
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
On the Record Details screen, enter values for the following items:
Library volume (µl) transferred to destination plate
Library Pooling (MiSeqDx) Step
Refer to under MiSeqDx Validation Protocol for details.
Denature, Dilute and Load Sample Step
On the Placement screen, set up the reagent cartridge as follows.
Using an RFID scanner, scan the RFID of the MiSeqDx reagent cartridge into the Container Name field.
Place the pool of samples into the reagent cartridge.
Refer to 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 under MiSeqDx Validation Protocol for details.
Variant Calling (MiSeqDx) Step
Refer to 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.
In C:\Illumina\gls, double-click gls_event_mos_rta.bat.
Confirm that an empty event file appears in the configured DESTINATION_PATH.
Manually invoke the gls_event_mos.bat file.
The file produces an output similar to the following example:
Troubleshooting
If an automation trigger does not appear to run its corresponding scripts, see:
Troubleshooting Automation Worker in the .
Troubleshooting Automation in the .
If an error occurs that does not provide direction on how to proceed, troubleshoot the error as follows.
Confirm the version of the installed Illumina MiSeqDx Integration Package by running the following command from the server console.
If the error is related to retrieving run results data, review the MiseqDxIntegrator.log.
Contact the Clarity LIMS Support team, supplying the relevant information from the troubleshooting already performed.
