User Interaction, Validation and Troubleshooting
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Last Updated: December 2024
Release Date: September 2023
Document Version: 2
This section explains the user interaction for each step and how to validate the installation of the Illumina NovaSeq X Series Integration Package v1.1.0.
The validation process involves the following actions:
Running samples through the Library Prep Validation v2.3.1 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 v1.0 workflow.
Running libraries through the NovaSeq X Series Sequencing v1.0 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 into Clarity LIMS.
The validation steps assume that the following conditions have been met:
NovaSeq X Series Integration Package v1.1.0 is installed and you have imported the default Clarity LIMS configuration.
Analysis configuration templates (ACTs) are created in 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 v2.3.1 step. For more information on creating a reagent label group, refer to Add and Configure Labels and Label Groups in the . For the adapter sequences for your library prep kits, refer to .
The following steps set up Clarity LIMS in preparation for running samples through the Library Prep Validation v2.3.1 and NovaSeq X Series Sequencing v1.0 workflows.
On the Configuration tab, under Workflows, activate both the Library Prep Validation v2.3.1 and NovaSeq X Series Sequencing v1.0 workflows.
On the Projects and Samples screen, create a project and add samples to it.
âš Only use alphanumeric, dash, or underscore characters in the submitted sample names. 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.
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 v1.0 workflow.
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 v1.0 workflow, Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0).
The NovaSeq X Series Sequencing v1.0 protocol consists of the following steps:
Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0)
Make Bulk Pool (NovaSeq X Series Sequencing v1.0)
Dilute and Denature (NovaSeq X Series Sequencing v1.0)
Load To Library Tube Strip (NovaSeq X Series Sequencing v1.0)
AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0)
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.
In Lab View, locate the NovaSeq X Series Sequencing v1.0 protocol. The samples are queued for the Assign Analysis Configuration Template (NovaSeq X 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 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.
[Optional] Under 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.
To use multiple ACTs in a single run, repeat 2–7 for each ACT. For step 2, only add samples that are being assigned to that ACT into the Ice Bucket.
In Lab View, locate the NovaSeq X Series Sequencing v1.0 protocol. The samples are queued for the Make Bulk Pool (NovaSeq X 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.
[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.
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.
Final Loading Volume (ul) — The final loading volume of the pool in the flow cell. This field is prepopulated with the configured default value (200 ul), but can be edited if more volume is required.
Minimum Per Sample Volume (ul) — The minimum volume for each sample. This field is prepopulated with the configured default value (2 ul) 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.
% PhiX (2.0nM) Spike-In — The value entered in this field is used to calculate the volume of PhiX spike-in required.
Flowcell Type — This is a read-only field configured with the default value of B3.
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 fields. The automation is applied to control samples when they are indexed and molarity is specified. It also generates the calculation file in a CSV format which is available for download under Files placeholder section below.
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 v1.0) step.
Select Finish Step.
In Lab View, locate the NovaSeq X Series Sequencing v1.0 protocol. The pool of samples queued for the Dilute and Denature (NovaSeq X Series Sequencing v1.0) 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.
On entry to the Record Details screen, the Calculate Volumes automation is triggered. This automation sets the following values:
BP Aliquot Volume (ul)
RSB Volume (ul)
NaOH Volume (ul)
TT2 Volume (ul)
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.
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, the BP aliquot, RSB, NaOH, and TT2 reagent volume values are auto populated and read-only.
[Optional] In the Sample Details table, select the pool icon to view details of the working pool composition.
The working pool number is appended to the bulk pool name so that you can identify which working pools are derived from the same bulk pool.
In the Files area, download the Calculation File (CSV) and open it and view details on the RSB, NaOH, and TT2 volumes to add per working pool.
Select Next Steps.
Select Finish Step.
In Lab View, locate the NovaSeq X Series Sequencing v1.0 protocol. The pool of samples are queued for the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0) step.
Add the pools to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work to trigger the Validate Analysis Configurations and Register Step Started automation.
Validate Analysis Configurations automation performs the following basic 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.
Register Step Started automation 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 Library 8-tube Strip in the Placed Samples area on the right.
Scan or type the barcode of the Library 8-tube Strip into the Library 8-tube Strip 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 LC[0-9]{7}-LC1.
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 v1.0) Step
Field
Value
Run Name
Enter the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. No spaces.
Run Mode
The selected Run Mode must correspond to the ACT type. 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 Run/Analysis configuration is imported manually to the NovaSeq X Series instrument through a sample sheet. The sample sheet generated on this step 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 planned run configuration is downloaded to the NovaSeq X Series Instrument Control Software to start the run. The downstream secondary analysis is done on 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.
Clarity consolidates sample/library information, run and analysis configuration details and send them to ICA to create a planned run and samplesheet for Cloud run mode.
Clarity consolidates sample/library information, run and analysis configuration details and send them to ICA to generate samplesheet for Local run mode.
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 v1.0) step.
To use multiple ACTs in a single run, repeat steps 1–3 for each ACT. The pool of samples (for different ACTs) are then queued for the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0) step to be added to the Ice Bucket.
This protocol contains the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0) 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 v1.0) 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.
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.
If the AUTOMATED - Sequencing Run (NovaSeq X 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 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 modifying the configuration settings on the NovaSeq X Control Software, contact Illumina Support.
If the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0) step starts, but does not complete, do as follows.
Log in to Clarity LIMS and use one of the following methods to open the in-progress step:
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.
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. 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 Strip 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.
Version
Changes
2
Updated Step 2: Make Bulk Pool (NovaSeq X Series Sequencing v1.1) to remove instructions on adding control. PhiX control should be added via the % PhiX (2.0nM) Spike-In step field.
1
Initial release.
You must create the Analysis Configuration Templates (ACTs) that are required for configuring secondary analysis in the NovaSeq X 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 .
Follow the steps in to run the Library Prep Validation workflow with the following:
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 .
[Optional] Create a new lot. For more information, refer to Add and Configure Reagent Kits and Lots in the .
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 .
Select Finish Step to advance the pooled samples to the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0) step. For more information on how to start the sequencing run for different run modes, refer to .
If an automation trigger does not appear to run its corresponding scripts, refer to the Troubleshooting Automation section in the .
