User Interaction, Validation and Troubleshooting
This section explains how to validate the installation of the Illumina MiSeq i100 Series On-Prem Integration Package v1.0.0.
The validation process involves the following actions:
Running samples through the Library Prep Validation v2.3.5 workflow.
The workflow contains a single-step protocol that models the library prep required to produce libraries tagged with index sequences. At the end of the step, these libraries are routed to MiSeq i100 Series On-Prem Sequencing v1.0 workflow.
Running libraries through the MiSeq i100 Series On-Prem Sequencing v1.0 workflow validates the following items:
Successful sequential step advancement of samples through the steps of the workflow.
Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0)
Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0)
Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0)
AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0)
AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0)
Automated creation of a planned run on Illumina Run Manager (IRM).
Automatic validation of run setup information before planned run creation.
Automated generation of sample sheet as part of the planned run creation.
Automated tracking of the MiSeq i100 Series sequencing run and parsing of run statistics from Illumina Run Manager into the LIMS.
Automated tracking of the MiSeq i100 Series analysis run and demultiplexing result files from Illumina Run Manager into the LIMS.
The validation steps assume the following conditions have been met:
Integration package has been successfully installed. Refer to MiSeq i100 Series On-Prem Integration v1.0.0 Installation.
Analysis configuration templates (ACTs) are created in Illumina Run Manager for your run.
Index Adapter Kit used in selected ACT is created as label group in Clarity LIMS.
ℹ For more information on creating a reagent label group, refer to Add and Configure Labels and Label Groups in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation. For the adapter sequences for your library prep kits, refer to Illumina Adapter Sequences.
This same label group must be used in the Library Prep Validation v2.3.5 step or your custom library prep step.
Required DRAGEN analysis application is installed locally on instrument.
The MiSeq i100 Series instrument to be integration is connected successfully via Illumina Run Manager UI.
Analysis Configuration Template Creation in Illumina Run Manager
MiSeq i100 Series On-Prem Sequencing v1.0 workflow requires Analysis Configuration Templates (ACTs) for configuring secondary analysis.
Create these ACTs in Illumina Run Manager (IRM) UI:
Select Analysis Configuration Templates from the dropdown list of the IRM UI menu icon
Select Add analysis template to create and add a new ACT.
Fill up details of the ACT and select Save.
For details, refer to the Illumina Run Manager help documentation on Illumina support site.
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 v2.3.5 and MiSeq i100 Series On-Prem Sequencing v1.0 workflows.
On the Configuration tab, under Workflows, activate both the Library Prep Validation v2.3.5 and MiSeq i100 Series On-Prem Sequencing v1.0 workflows.
On the Projects and Samples screen, create a project and add samples to it.
⚠ Sample and library names must use only alphanumeric, dash, or underscore characters. Invalid characters can cause a sample sheet validation failure in the Load to Dry Cartridge step.
Assign the samples to the Library Prep Validation workflow.
Library Prep Protocol
This single-step protocol models the library prep required to produce libraries tagged with index sequences that are ready for the MiSeq i100 Series On-Prem Sequencing v1.0 workflow.
Follow the steps in Library Prep Validation Protocol to run the Library Prep Validation workflow with the following:
Label Group = same as the Index Adapter Kit selected in the ACT that is being used
Sequencing Instrument = MiSeq i100 Series On-Prem
On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the MiSeq i100 Series On-Prem Sequencing v1.0 workflow, Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0).
Protocol: MiSeq i100 Series On-Prem Sequencing v1.0
The MiSeq i100 Series On-Prem Sequencing v1.0 protocol consists of the following steps:
Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0)
Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0)
Load To Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0)
AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0)
AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0)
Step 1: Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0)
In Lab View, locate the MiSeq i100 Series On-Prem Sequencing v1.0 protocol. The samples are queued for the Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0) step.
Add the samples to the Ice Bucket and start the step. The Validate Sample Names automation is triggered upon step started, and validates the sample names.
Select the connected MiSeq i100 instrument to integrate with.
On Record Details screen, select 1. Retrieve ACT List to retrieve the list of ACTs created on Illumina Run Manager.
Select the applicable ACT to assign to the samples. The samples must be indexed with the index adapter kit configured in the selected ACT.
[Optional] Select 2. Retrieve ACT Information to trigger the automation.
The Retrieve ACT Information automation retrieves the details of the selected ACT. High level information e.g., Library Prep Kit, Index Adapter Kit, Reference Genome etc. are populated on the step fields. For more details, refer to the ACTMetadata.csv file in the file placeholder section.
Select Next Steps to assign the ACT to the samples.
This action triggers the Validate Reagent Labels and Apply Selected ACT to Samples and Set Next Step automation. This automation
ensures every sample has Molarity (nM) UDF value greater than zero
validates that the indexes applied to the libraries are valid for the selected ACT
assigns all samples in this step to the selected ACT
Select Finish Step.
Step 2: Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0)
In Lab View, locate the MiSeq i100 Series On-Prem Sequencing v1.0 protocol. The samples are queued for the Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0) step.
Add the samples to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
Create a pool of samples as follows.
On the Pooling screen, create a pool by dragging samples into the Pool Creator.
Enter a name for your pool or accept the default name (Pool #1).
[Optional] If multiple pools are required, select the plus sign (+) next to Pool Creator to create a pool.
ℹ Only a single pool is allowed in the Load to Dry Cartridge step for each run. Create multiple pools if required for subsequent runs.
[Optional] To remove a pool, select the X in the top right corner of the pool.
Select Record Details to trigger the Validate Analysis Configurations automation.
This automation performs the following checks on the analysis configuration for each pool:
Pooled samples are within the maximum configuration limit.
Pooled samples assigned with the same secondary analysis application must have the same analysis version (e.g. v3.8.4) and settings (e.g. Map/Align Output Format = CRAM).
[Optional] In the Sample Details table, select the pool next to the sample name to view details on the pool composition.
On the Record Details screen, navigate to the Reagent Lot Tracking section to track the lot information used in the step.
[Optional] Create a new lot. For more information, refer to Add and Configure Reagent Kits and Lots in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
In the Step Details area, complete the following fields:
[Optional] % PhiX (100pM) Spike-In — The percentage of PhiX spike-in.
Final Loading Concentration (pM) — The final loading concentration of the pool in the flow cell.
Final Loading Volume (ul) — The final loading volume of the pool in the flow cell.
Bulk Pool Volume (ul) — The bulk pool volume is used in the Calculate Volumes script to determine RSB volumes required for pooling.
Minimum Per Sample Volume (ul) — The minimum volume for each sample. This field is prepopulated with the configured default value (2 µl) and can be edited. If the per sample volume is below the value set in this field, the Calculate Volumes script applies a rounding factor to affected samples so that the volume reaches the minimum volume.
Flowcell Type — The flow cell type that the run uses. Select from the following options:
5M
25M
Select Calculate Volumes to trigger the Calculate Volumes automation.
This automation calculates the volumes required for each library to form a pool that has the concentration and volume specified in the Step Details field. It also generates the calculation file in a CSV format and attaches it to the step. Select the file name to download it.
Select Next Steps to trigger the Set Next Step automation.
This automation sets the next step for samples to ADVANCE, which moves them to the Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0) step.
Select Finish Step.
Step 4: Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0)
In Lab View, locate the MiSeq i100 Series On-Prem Sequencing v1.0 protocol. The pool of samples are queued for the Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0) step.
Add the pool to the Ice Bucket and select View Ice Bucket.
⚠ Only single pool is allowed at this step.
On the Ice Bucket screen, select the destination container type:
MiSeq i100 Series Dry Cartridge
Select Begin Work to trigger the Validate Single Pool automation.
The Validate Single Pool automation ensures that only one pool is selected for loading to the dry cartridge in this step.
On the Placement screen, do as follows.
Drag the pool into the Placed Samples area on the right.
Scan or type the barcode of the dry cartridge into the container name field.
Select Record Details.
After exiting the Placement screen, the Validate Dry Cartridge Barcode automation makes sure that the dry cartridge barcode conforms to the barcode mask. The barcode mask is SC[0-9]{7}-SC3.
The fields displayed on the Record Details screen are used to create the planned run and generate the sample sheet.
The analysis related information for the planned run comes from the ACT associated with the samples. Refer to the following table for details.
Fields Displayed on Record Details Screen of Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0) Step
Field
Description
Run Name
Experiment name.
Only alphanumeric characters, dashes, periods, spaces and underscores are permitted.
Maximum of 255 characters
Run Mode
Presets
Local (Default)¹
Read 1 Cycles
Presets
151
101
51
Type a custom value
Read 2 Cycles
Presets
151
101
51
Type a custom value
Index 1 Cycles
Presets
0
6
8
Type a custom value²
Index 2 Cycles
Presets
0
6
8
Type a custom value²
¹ The Local run mode sends the Run/Analysis configuration to Illumina Run Manager. This configuration is downloaded to the MiSeq i100 Series instrument through Illumina Run Manager to start the run. The downstream secondary analysis is done on premise.
² The custom value must correspond to the longest index sequence of the samples in the pools in the library tube strip. Otherwise, the planned run creation fails and an error message displays.
Select Validate Run Setup and Create Planned Run to trigger the automation script. The script performs the following actions:
Validates the parameters entered on the Record Details screen.
Creates the planned run on Illumina Run Manager.
Generates the sample sheet and attaches it to the placeholder in the Files area on the Record Details screen.
Select Next Steps.
On the Assign Next Steps screen, the next step for the pooled samples is set to the AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) step.
Select Finish Step to advance the pooled samples to the AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) step. For more information on how to start the sequencing run for different run modes, refer to MiSeq i100 Series On-Prem Integration v1.0.0 Configuration.
Step 5: AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0)
Do not add samples to the Ice Bucket or start or complete the AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) step. The integration does this automatically.
User interaction is not required, but you can review the stages of the step in Clarity LIMS.
Review Run Data
Read summary metrics are recorded for the library pool. After the run is complete, open the step and review these metrics in the Step Details section and the Sample Details table.
Step 6: AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0)
Do not add samples to the Ice Bucket or start or complete the AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0) step. The integration does this automatically.
User interaction is not required, but you can review the stages of the step in Clarity LIMS. If the run analysis tracking is successful, the integration completes the step automatically.
Review Analysis Data
After the analysis run is complete, open the step and review the following values in the Step Details section:
Analysis Status - Status of the analysis run
Analysis Result Location - Location of the secondary analysis data. This field is empty if transfer of secondary analysis data to an external location is disabled.
Log - Log message when integration handling the event from IRM
File placeholder:
Demultiplexing results file - demultiplexing statistics for each sample
Retrieve detailed analysis results from Illumina Run Manager.
At this point, the entire MiSeq i100 Series On-Prem Integration workflow is fully validated.
Troubleshooting
If an automation trigger does not appear to run its corresponding scripts, refer to the Troubleshooting Automation section in the Clarity LIMS (API & Database) documentation.
Validate Analysis Configuration Automation Check Fails
The Validate Analysis Configuration automation check is in the Make Bulk Pool (upon pooling) step. If a failure happens, an error message displays and the step can be aborted, or you might be prevented from continuing the step.
This check can fail for the following reasons:
Pooled samples assigned with the same secondary analysis application must have the same analysis version (e.g. v3.8.4) and settings (e.g. Map/Align Output Format = CRAM).
Resolve this error as follows.
Pool the samples again. Make sure that samples with conflicting secondary analysis application, analysis versions or analysis settings, are in different pools.
Error When Creating Planned Runs
If you receive an error when creating a planned run, check the log message in the Load to Dry Cartridge step to identify the issue. If you cannot correct the issue, contact Illumina Support.
Incompatible Analyses in a Planned Run
Only compatible analysis versions should be combined in a single planned run. When incompatible analysis versions are combined, an error log message displays. An example of the error log message is shown below.
To resolve this error, check the ACTs that were used for the run. Create a new planned run with ACTs that have the same analysis and analysis versions.
Automated Step Does Not Start
If the AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) step does not start, Clarity LIMS is likely not receiving sequencing run events from Illumina Run Manager correctly. Check for the following issues:
The instrument must be reflected as Connected and Active in the Illumina Run Manager Integration UI. Reconnect the instrument if the status reflects otherwise.
The container must be queued at the AUTOMATED - Sequencing Run step. It should not be in the ice bucket or in the previous step.
If the issues remain, contact Illumina Support.
Automated Step Starts, But Does Not Complete
If the AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) step starts, but does not complete, do as follows.
Log in to the default user account and search for the in progress step in Clarity LIMS using the container name as the search term.
On the Record Details screen, the Sequencing Log multiline text field contains logging information.
If unable to reach the Record Details screen, or if the Sequencing Log field does not contain enough information to resolve the issue, contact Illumina Support. Provide the relevant information from the troubleshooting steps already performed.
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