User Interaction, Validation and Troubleshooting
This section explains how to validate the installation of the Illumina MiSeq i100 Series 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 Sequencing v1.0 workflow.
Running libraries through the MiSeq i100 Series Sequencing v1.0 workflow validates the following items:
Successful sequential step advancement of samples through the steps of the workflow.
Automated creation of a planned run. The control software retrieves the planned run for both Cloud and Hybrid run modes. Sample sheet is automatically generated and attached to Load to Dry Cartridge step.
Automated tracking of the MiSeq i100 Series sequencing run and parsing of run metrics from BaseSpace Sequence Hub to Clarity LIMS.
Automated tracking of the MiSeq i100 Series analysis run from BaseSpace Sequence Hub to Clarity LIMS.
The validation steps assume that the following conditions have been met:
The MiSeq i100 Series Integration v1.0.0 has a valid BaseSpace Sequence Hub account with an Enterprise or Professional subscription.
MiSeq i100 Series Integration Package v1.0.0 is installed and you have imported the default Clarity LIMS configuration.
Analysis configuration templates (ACTs) are created in BaseSpace Sequence Hub (BaseSpace Sequence Hub) for your run.
The index adapter kit label group in the selected ACT is created in Clarity LIMS and used in the Run Library Prep Validation v2.3.5 step to index the samples.
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.
Analysis Configuration Template Creation in BaseSpace Sequence Hub
You must create the Analysis Configuration Templates (ACTs) that are required for configuring secondary analysis in the MiSeq i100 Series Sequencing v1.0 workflows. Create and delete ACTs in BaseSpace Sequence Hub. For instructions, refer to the BaseSpace Sequence Hub Online Help on the 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 Sequencing v1.0 workflows.
On the Configuration tab, under Workflows, activate both the Library Prep Validation v2.3.5 and MiSeq i100 Series 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 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 exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the MiSeq i100 Series Sequencing v1.0 workflow, Assign Analysis Configuration Template (MiSeq i100 Series Sequencing v1.0).
Protocol: MiSeq i100 Series Sequencing v1.0
The MiSeq i100 Series Sequencing v1.0 protocol consists of the following steps:
Assign Analysis Configuration Template (MiSeq i100 Series Sequencing v1.0)
Make Bulk Pool (MiSeq i100 Series Sequencing v1.0)
Load To Dry Cartridge (MiSeq i100 Series Sequencing v1.0)
AUTOMATED - Sequencing Run (MiSeq i100 Series Sequencing v1.0)
AUTOMATED - Analysis Run (MiSeq i100 Series Sequencing v1.0)
Each step contains an optional script to register the start time (upon step entry) and the time that the step is completed (upon step exit). This information is published to CLPA through ICA. These scripts are used only for CLPA support and can be incorporated as part of an automation that performs other functions.
Step 1: Assign Analysis Configuration Template (MiSeq i100 Series Sequencing v1.0)
In Lab View, locate the MiSeq i100 Series Sequencing v1.0 protocol. The samples are queued for the Assign Analysis Configuration Template (MiSeq i100 Series Sequencing v1.0) step.
Add the samples to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work to start the Validate Sample Names and Retrieve Analysis Configuration Template List and Register Step Started automation.
The automation does the following:
checks that the sample names do not use restricted characters and are within character limits,
populates the Analysis Configuration Template dropdown options,
registers the start time of the step by publishing messages to CLPA through ICA. The part of the script for start time registration is used only for CLPA support.
Select the applicable ACT to assign to the samples. The index adapter kit specified by the ACT must correspond with the label group used in Library Prep Protocol.
[Optional] In Step Details, select Retrieve ACT Information to trigger the Retrieve ACT Information automation.
This automation retrieves ACT information (e.g., Library Prep Kit, Index Adapter Kit, Reference Genome, and so on) and populates the fields in Clarity LIMS. These details are saved to the ACTMetadata.csv file that you can download to view details of the selected ACT.
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 which does the following:
validates that the indexes applied to the libraries are valid for the selected ACT
assigns samples to the selected ACT
Select Finish Step.
Step 2: Make Bulk Pool (MiSeq i100 Series Sequencing v1.0)
In Lab View, locate the MiSeq i100 Series Sequencing v1.0 protocol. The samples are queued for the Make Bulk Pool (MiSeq i100 Series 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 have the sample type of analysis (Cloud or Local).
Pooled samples that have the same secondary analysis also have the same analysis version and settings.
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 required fields:
% 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.
[Optional] In the Sample Details table, select the pool next to the sample name to view details on the pool composition.
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 Sequencing v1.0) step.
Select Finish Step.
Step 4: Load to Dry Cartridge (MiSeq i100 Series Sequencing v1.0)
In Lab View, locate the MiSeq i100 Series Sequencing v1.0 protocol. The pool of samples are queued for the Load to Dry Cartridge (MiSeq i100 Series Sequencing v1.0) step.
Add the pools to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select the destination container type:
MiSeq i100 Series Dry Cartridge
Select Begin Work to trigger the Validate Single Pool and Register Step Started automations.
The Validate Single Pool automation ensures that only one pool is selected for loading to the dry cartridge in this step.
The Register Step Started automation also registers the start time of the step by publishing messages to CLPA through ICA. The portion of the script used for start time registration is used only for CLPA support.
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 is from the ACT associated with the samples and no further analysis configuration is required. Refer to the following table for details.
Fields Displayed on Record Details Screen of Load to Dry Cartridge (MiSeq i100 Series 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
Cloud¹
Hybrid²
⚠ The selected Run Mode must correspond to the ACT type.
Read 1 Cycles
Presets
151
101
51
ℹ Allows custom entry
Read 2 Cycles
Presets
151
101
51
ℹ Allows custom entry
Index 1 Cycles
Presets
0
6
8
ℹ Allows custom entry³
Index 2 Cycles
Presets
0
6
8
ℹ Allows custom entry³
¹ Planned run is created and available on BaseSpace Sequence Hub. This configuration is downloaded to the MiSeq i100 Series instrument to start the run. The downstream secondary analysis is done in the cloud.
² Planned run is created and available on BaseSpace Sequence Hub. This configuration is downloaded to the MiSeq i100 Series instrument to start the run. The downstream secondary analysis is done using the on-premise DRAGEN module.
³ The custom value must correspond to the longest index sequence of the samples in the pools in the library tube strip.
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.
Generates the sample sheet and attaches it to the placeholder in the Files area on the Record Details screen.
Select Next Steps.
Select Finish Step to advance the pooled samples to the AUTOMATED - Sequencing Run (MiSeq i100 Series Sequencing v1.0) step.
For more information on how to start the sequencing run for different run modes, refer to MiSeq i100 Series Integration v1.0.0 Configuration.
For subsequent steps to successfully track the sequencing and analysis run, the Load to Dry Cartridge step must be completed and samples must be queued to the AUTOMATED - Sequencing Run step before starting the sequencing run on the instrument.
Step 5: AUTOMATED - Sequencing Run (MiSeq i100 Series Sequencing v1.0)
Do not add samples to the Ice Bucket or start or complete the AUTOMATED - Sequencing Run (MiSeq i100 Series Sequencing v1.0) step. The integration does this automatically.
The integration starts the step automatically and data from the run is parsed back into Clarity LIMS. 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 Sequencing v1.0)
This protocol contains the AUTOMATED - Analysis Run (MiSeq i100 Series Sequencing v1.0) step. This step is fully automated.
Do not add samples to the Ice Bucket or start or complete the AUTOMATED - Analysis Run (MiSeq i100 Series Sequencing v1.0) step. The integration does this automatically.
The integration starts the step automatically and parses run data from BaseSpace Sequence Hub back into Clarity LIMS. User interaction is not required, but you can review the stages of the step in Clarity LIMS. If the run analysis 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
Analysis Result Location
Log
After the analysis is complete, download the demultiplexing results from the file placeholders. Retrieve detailed analysis results from ICA.
The demultiplexing results file contains the following demultiplexing statistics for each sample:
Number of reads
Number of perfect index reads
Number of mismatch index reads
At this point, the entire MiSeq i100 Series 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:
ACTs of samples in the same pool or planned run must have the same analysis mode (Local or Cloud).
Resolve this error as follows.
Abort the step and remove samples with ACTs that have conflicting run modes from the Ice Bucket.
Make sure that all remaining samples in the Ice Bucket have ACTs with the same run modes.
Select Begin Work to continue the step.
ACTs of samples in the same pool or planned run that have the same secondary analysis must have the same analysis version.
Resolve this error as follows.
If the error occurs on the Make Bulk Pool step, pool the samples again. Make sure that samples with the same secondary analysis, but conflicting analysis versions, are in different pools.
If the error occurs on the Load to Library Tube step, reorganize the samples for the planned run. Make sure that samples with the same secondary analysis, but conflicting analysis versions, are in different runs.
ACTs of samples in the same pool or planned run that have the same secondary analysis must have the same analysis settings. The analysis setting fields can differ for a different secondary analysis and are configured in BaseSpace Sequence Hub when the ACT is created.
Resolve this error as follows.
Pool the samples again. Make sure that samples with the same secondary analysis, but conflicting 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 and only select the ACTs that are compatible with the planned run.
Automated Step Does Not Start
If the AUTOMATED - Sequencing Run (MiSeq i100 Series Sequencing v1.0) step does not start, Clarity LIMS is likely not receiving sequencing run events from BaseSpace Sequence Hub correctly. Check for the following issues:
Make sure that the MiSeq i100 Control Software is configured properly as follows.
Before you start the run, in the MiSeq i100 Control Software, select the appropriate BaseSpace Sequence Hub region in the Hosting location drop-down list.
Under Run Settings, check the boxes next to Cloud run monitoring and Cloud run storage.
If there are issues after using the configuration settings on the MiSeq i100 Control Software, contact Illumina Support.
Automated Step Starts, But Does Not Complete
If the AUTOMATED - Sequencing Run (MiSeq i100 Series Sequencing v1.0) step starts, but does not complete, do as follows.
Log in to the default user account and use one of the following methods to open the in progress step in Clarity LIMS:
In Lab View, find the step in the Recent Activities pane.
Search for the step in Clarity LIMS using the library tube strip barcode 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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