User Interaction, Validation and Troubleshooting
This section explains how to validate the installation of the Illumina NovaSeq X Series Integration Package v1.2.0.
The validation process involves the following actions:
Running samples through the Library Prep Validation v2.3.2 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 NovaSeq X Series Sequencing v1.1 workflow.
Running libraries through the NovaSeq X Series Sequencing v1.1 workflow validates the following items:
Successful sequential step advancement of samples through the steps of the workflow.
Automatic validation of run setup information before sample sheet generation or planned run creation.
Automated sample sheet generation. This sample sheet is used to start the sequencing run on the NovaSeq X Series Control Software through the Local run mode.
Automated creation of a planned run in Illumina Connected Analytics ( ICA). The control software retrieves the planned run. The run is started through the Cloud run mode.
Automated tracking of the NovaSeq X Series sequencing run and parsing of run statistics from BaseSpace Sequence Hub to Clarity LIMS.
Automated tracking of the NovaSeq X Series analysis run and high level analysis summary from BaseSpace Sequence Hub to Clarity LIMS.
The validation steps assume that the following conditions have been met:
The NovaSeq X Series Integration v1.2.0 has a valid BaseSpace Sequence Hub account with an Enterprise or Professional subscription.
NovaSeq X Series Integration Package v1.2.0 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. Make sure that the index adapter kit label group is created in Clarity LIMS before selecting it in the ACT. This same label group is used in the Run Library Prep Validation v2.3.2 step. 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 NovaSeq X Series Sequencing v1.1 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 v2.3.2 and NovaSeq X Series Sequencing v1.1 workflows.
On the Configuration tab, under Workflows, activate both the Library Prep Validation v2.3.2 and NovaSeq X Series Sequencing v1.1 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 Library Tube Strip 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 NovaSeq X Series Sequencing v1.1 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 = NovaSeq X Series
On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the NovaSeq X Series Sequencing v1.1 workflow, Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1).
Protocol: NovaSeq X Series Sequencing v1.1
The NovaSeq X Series Sequencing v1.1 protocol consists of the following steps:
Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1)
Make Bulk Pool (NovaSeq X Series Sequencing v1.1)
Dilute and Denature (NovaSeq X Series Sequencing v1.1)
Load To Library Tube Strip (NovaSeq X Series Sequencing v1.1)
AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1)
AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1)
Each step contains a 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 (NovaSeq X Series Sequencing v1.1)
In Lab View, locate the NovaSeq X Series Sequencing v1.1 protocol. The samples are queued for the Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1) 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 checks that the sample names do not use restricted characters and are within character limits and retrieves the list of ACTs that you can select. The automation also 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. If you are not sure of the analysis configuration before starting the sequencing and analysis run, refer to the details in the file.
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 validates that the indexes applied to the libraries are valid for the selected ACT and assigns samples to it. All samples added to the Ice Bucket (mentioned above) are assigned to this ACT.
Select Finish Step.
Step 2: Make Bulk Pool (NovaSeq X Series Sequencing v1.1)
In Lab View, locate the NovaSeq X Series Sequencing v1.1 protocol. The samples are queued for the Make Bulk Pool (NovaSeq X Series Sequencing v1.1) step.
Add the samples to the Ice Bucket and select View Ice Bucket.
ℹ️ If PhiX control sample is required for the run, the configuration setting shall be performed in the Step 3: Dilute and Denature with the 1–2% PhiX Spike-In toggle switch.
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.
If a control sample was added, it appears under the Sample List and can be pulled into the pool that needs the control.
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.
[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:
Number of Lanes to Sequence — Used in volume calculations, to make sure that the volumes are sufficient for the number of times the pool is sequenced. This field is applied to all pools in the step.
Final Loading Concentration (pM) — The final loading concentration of the pool in the flow cell.
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. This selection affects the computation of volumes in the Calculate Volumes automation that generates the calculation file. Select from the following options:
1.5B
10B
25B
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.
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 Dilute and Denature (NovaSeq X Series Sequencing v1.1) step.
Select Finish Step.
Step 3: Dilute and Denature (NovaSeq X Series Sequencing v1.1)
In Lab View, locate the NovaSeq X Series Sequencing v1.1 protocol. The pool of samples queued for the Dilute and Denature (NovaSeq X Series Sequencing v1.1) displays.
Add the pool to the Ice Bucket and select View Ice Bucket.
[Optional] On the Ice Bucket screen, set the number of derivatives to create (placed into the library tube strip) and select Begin Work.
On entry to the step, the Validate Inputs Flowcell Type and Register Step Started automation is triggered. This automation makes sure that the configured flow cell type is valid. The 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.
If PhiX is used for the run, navigate to the Record Details screen and set 1–2% PhiX Spike-In to Yes.
Select Calculate Volumes to trigger the Calculate Volume automation. This automation sets the following values:
BP Aliquot Volume (ul)
RSB Volume (ul)
NaOH Volume (ul)
TT2 Volume (ul)
PhiX Volume (ul) and Concentration (pM) (if there is a PhiX spike-in)
The automation also generates the calculation file (CSV) and attaches it to the step. This file contains information about the volume of RSB, NaOH, and TT2 to add per working pool. If there is a PhiX spike-in, the file also contains information on the PhiX volume and concentration.
On the Record Details screen, the Reagent Lot Tracking section tracks the NaOH, Resuspension Buffer, and TT2 reagents used in the step. To add and activate reagent lots, refer to Add and Configure Reagent Kits and Lots in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
On the Record Details screen, perform the following actions:
In the Reagent Lot Tracking section, select from the active lots displayed in each drop-down list.
In the Sample Details table, make sure that the BP aliquot, RSB, NaOH, and TT2 reagent volume values are populated. The script sets these values and they cannot be edited.
[Optional] In the Sample Details table, select the pool icon to view details of the working pool composition.
In the Files area, select the Calculation File (CSV) to open it and view details on the volumes for each reagent and sample pool used for the dilute and denature process.
Select Next Steps.
Select Finish Step.
Step 4: Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1)
In Lab View, locate the NovaSeq X Series Sequencing v1.1 protocol. The pool of samples are queued for the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1) step.
Add the pools to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select one of the following destination container types:
Library 8-tube Strip
Library 2-tube Strip
Select Begin Work to trigger the Validate Flowcell Inputs and Validate Analysis Configurations and Register Step Started automations.
The Validate Flowcell Inputs automation ensures that the correct container is selected for the flow cell type and that the number of inputs is the same as the number of available wells on the library tube strip. The Validate Analysis Configurations automation is similar to the automation used in the Make Bulk Pool 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.
To use multiple ACTs in a single run, repeat Steps 1–4 for each ACT.
The pool of samples for each ACT is then queued for the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.1) step to be added to the Ice Bucket.
On the Placement screen, do as follows.
Drag the pools into the Placed Samples area on the right.
Scan or type the barcode of the library tube strip into the container name field.
Select Record Details.
After exiting the Placement screen, the Validate Library Strip Tube Barcode automation makes sure that the library tube strip barcode conforms to the barcode mask. For the Library 8-tube Strip, the barcode mask is LC[0-9]{7}-L[A-Z]{1}1 and an error message displays if the barcode does not match. For the Library 2-tube Strip, the barcode mask is LC[0-9]{7}-L[A-Z]{1}2.
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 Library Tube Strip (NovaSeq X Series Sequencing v1.1) Step
Field
Description
Run Name
Enter the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. No spaces.
Run Mode
Presets
Local¹
Cloud²
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 value³
Index 2 Cycles
Presets
0
6
8
Type a custom value³
¹ The Local run mode generates the sample sheet. The Run/Analysis configuration is imported manually to the NovaSeq X Series instrument through the sample sheet. The sample sheet is downloaded and imported manually by the user on NovaSeq X Control Software to start the run. The downstream secondary analysis is done using the onboard DRAGEN module.
² The Cloud run mode sends the Run/Analysis configuration to ICA. This configuration is downloaded to the NovaSeq X Series instrument through ICA to start the run. The downstream secondary analysis is done in the cloud.
³ 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 and sends it to ICA when the Run Mode is Cloud.
Generates the sample sheet and attaches it to the placeholder in the Files area on the Record Details screen for all Run Mode types.
Select Next Steps.
On the Assign Next Steps screen, the next step for the pooled samples is set to the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step.
Select Finish Step to advance the pooled samples to the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step. For more information on how to start the sequencing run for different run modes, refer to NovaSeq X Series Integration v1.2.0 Configuration.
Step 5: AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1)
This protocol contains the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) step. This step is fully automated.
⚠️ Do not add samples to the Ice Bucket or start or complete the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) 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 (NovaSeq X Series Sequencing v1.1)
This protocol contains the AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1) step. This step is fully automated.
⚠️ Do not add samples to the Ice Bucket or start or complete the AUTOMATED - Analysis Run (NovaSeq X Series Sequencing v1.1) 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. 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 and analysis results summary files 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
The analysis result summary file provides a summary of what analysis workflows were performed. This file also identifies the total number of samples analyzed and how many were completed or failed for each analysis.
At this point, the entire NovaSeq X 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.
Automated Step Does Not Start
If the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) 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 NovaSeq X Control Software is configured properly as follows.
Before you start the run, in the NovaSeq X 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 NovaSeq X Control Software, contact Illumina Support.
Automated Step Starts, But Does Not Complete
If the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.1) 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.
Error When Creating Planned Runs
If you receive an error when creating a planned run, check the log message in the Load to Library Tube Strip 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.
Validate Analysis Configuration Automation Check Fails
The Validate Analysis Configuration automation check is in the Make Bulk Pool (upon pooling) and Load to Library Tube Strip (upon step entry) steps. If a failure happens, an error message displays and the step can be aborted, or you might be prevented from continuing the step. For NovaSeq X Series Integration v1.2, the configuration limits for the Cloud and Local modes are as follows.
Cloud — Maximum of seven applications and genome configurations (and, if needed, the BCL Convert Only application). Cloud mode also allows up to eight different library prep kits for each application and genome configuration.
Local — Maximum of three applications and genome configurations (and, if needed, the BCL Convert Only application). Local mode also allows up to eight different library prep kits for each application and genome configuration.
This check can fail for the following reasons:
The analysis configuration after library pooling or during the planned run creation exceeds the configuration limit.
Resolve this error as follows.
If the error occurs on the Make Bulk Pool step, reduce the number of analysis configurations for a pool. To reduce the number, reorganize the samples for the pooling step (eg, by pooling samples that have similar configurations together).
If the error occurs on the Load to Library Tube step, reduce the number of analysis configurations for the planned run by reorganizing the samples for multiple runs instead of a single run.
ACTs of samples in the same pool or planned run must have the same run 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.
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 settings, 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 settings, are in different runs.
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