The Illumina NovaSeq X Series On-Prem Integration Package v1.0.0 supports the integration of Clarity LIMS to NovaSeq X Series instruments that are on-premise via Illumina Run Manager.
Steps:
Install / Upgrade Clarity LIMS
Install and Configure Illumina Run Manager Integration Service
Install NovaSeq X Series On-Prem Integration RPM
Install Illumina Preset Protocols (IPP) v2.10 RPM and Import Required IPP Workflows.
Install or upgrade to Clarity LIMS v6.2 or later. If you are using an older version of Clarity LIMS, please contact Illumina Support (techsupport@illumina.com) to schedule for upgrade.
Install and configure Illumina Run Manager Integration Service v1.0, please refer to Illumina Run Manager v1.0.0 Installation and User Interaction for details.
Install ClarityLIMS-NovaSeqXSeries-OnPremise RPM using yum or your preferred package manager.
Example with YUM:
Example with RPM:
Install BaseSpaceLIMS-Illumina-Preset-Protocols RPM using yum or your preferred package manager.
Example with YUM:
Example with RPM:
Upon successful installation of IPP v2.10.0, install the following workflow configurations:
NovaSeq X Series On-Prem Sequencing v1.0 workflow
(Optional) Library Prep Validation v2.3.5 workflow
For detailed options and steps on how to install IPP workflows and protocols, see Installer Parameters and Operations section of Illumina Preset Protocols v2.10 Installation and User Configuration.
Last Updated: December 2024
Release Date: December 2024
These Release Notes describe the key changes to software components for the Clarity LIMS NovaSeq X Series On Premise Integration Package version 1.0.0.
Refer to Compatibility under Instruments & Integrations.
Features provided with this release of the integration package are as follows.
Provides integration between the NovaSeq X Series instrument and on-premise Clarity LIMS.
Supports automated tracking of the sequencing run and parsing of run metrics to on-premise Clarity LIMS.
Supports creation of planned run with secondary analysis configuration and generation of v2 sample sheet via Illumina Run Manager on instrument.
Supports automated tracking of the sequencing run and parsing of run metrics to on-premise Clarity LIMS.
Supports automated tracking of local analysis for analysis status and demultiplexing results.
New NovaSeq X Series On-Prem Sequencing v1.0 workflow available from Illumina Preset Protocols v2.10.
Tracks only the analysis configured with the planned run. Does not track analysis requeue or analysis triggered externally with the same planned run.
The Assign Analysis Configuration Template step does not support pooled libraries.
The integration does not support --bcl-sampleproject-subdirectories option of BCL Convert.
The integration requires secondary analysis files to be present on the instrument for proper functioning of the AUTOMATED - Analysis Run step. The following instrument setting must be disabled: Permanently delete secondary analysis files from the instrument after they are transferred to the external storage and/or cloud. Refer to NovaSeq X Series On Prem Integration v1.0.0 Configuration for details.
The integration does not support pools with samples assigned with Analysis Configuration Templates from different instruments.
On the Make Bulk Pool step, the log displays a warning when the Calculate Volume automation is triggered and at least one pool consists of multiple inputs. This issue is caused by the output custom field being reset multiple times at the end of the automation. This issue does not affect the Calculate Volume automation functionality.
The integration includes the following:
Preconfigured NovaSeq X Series On-Prem Sequencing v1.0 workflow that maps to lab protocols and instrument runs.
Preconfigured protocols:
Assign Analysis Configuration Template (NovaSeq X Series On-Prem Sequencing v1.0)
Make Bulk Pool (NovaSeq X Series On-Prem Sequencing v1.0)
Dilute and Denature (NovaSeq X Series On-Prem Sequencing v1.0)
Load to Library Tube Strip (NovaSeq X Series On-Prem Sequencing v1.0)
AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0)
AUTOMATED - Analysis Run (NovaSeq X Series On-Prem Sequencing v1.0)
Automatic validation of run setup information before sample sheet generation and planned run creation.
Automated planned run creation on NovaSeq X Series instrument via Illumina Run Manager. The planned run created will be retrieved by NovaSeq X Series Control Software and used to start a sequencing run.
Automated tracking of the NovaSeq X Series sequencing run status and parsing of run metrics.
Automated tracking of the NovaSeq X Series analysis run status, result summary and demultiplexing result files.
Support for configuration of multiple secondary analyses with multiple library prep kit (LPK) and index adapter kit (IAK) for the planned run. The analysis is performed by DRAGEN onboard.
(Optional) Preconfigured Library Prep Validation v2.3.5 workflow used for validation purposes only. The workflow contains a single-step protocol that models the library prep workflow required to produce libraries tagged with index sequences. At the end of the step, these libraries are routed to NovaSeq X Series On-Prem Sequencing v1.0 workflow.
This section explains how to validate the installation of the Illumina NovaSeq X 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 NovaSeq X Series On-Prem Sequencing v1.0 workflow.
Running libraries through the NovaSeq X 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 (NovaSeq X Series On-Prem Sequencing v1.0)
Make Bulk Pool (NovaSeq X Series On-Prem Sequencing v1.0)
Dilute and Denature (NovaSeq X Series On-Prem Sequencing v1.0)
Load to Library Tube Strip (NovaSeq X Series On-Prem Sequencing v1.0)
AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0)
AUTOMATED - Analysis Run (NovaSeq X 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 NovaSeq X Series sequencing run and parsing of run statistics from Illumina Run Manager into the LIMS.
Automated tracking of the NovaSeq X Series analysis run, high level analysis summary and demultiplexing result files from Illumina Run Manager into the LIMS.
The validation steps assume that the following conditions have been met:
Integration package has been successfully installed. Refer to NovaSeq X Series On-Prem Integration v1.0.0 Installation.
The NovaSeq X instrument to be integration is connected successfully via Illumina Run Manager UI.
Analysis configuration templates (ACTs) are created in Illumina Run Manager.
Index adapter kit configured in the ACT is created as label group in Clarity LIMS. 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 is used in the Library Prep Validation v2.3.5 step or your custom library preparation step.
Required DRAGEN analysis application is installed locally on instrument.
You must create the Analysis Configuration Templates (ACTs) that are required for configuring secondary analysis in the NovaSeq X Series On-Prem Sequencing v1.0 workflows. Create and delete ACTs in Illumina Run Manager. For instructions, refer to the Illumina Run Manager Online Help on the Illumina support site.
The following steps set up Clarity LIMS in preparation for running samples through the Library Prep Validation v2.3.5 and NovaSeq X Series On-Prem Sequencing v1.0 workflows.
On the Configuration tab, under Workflows, activate both the Library Prep Validation v2.3.5 and NovaSeq X 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 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 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 = NovaSeq X Series On-Prem
On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the NovaSeq X Series On-Prem Sequencing v1.0 workflow, Assign Analysis Configuration Template (NovaSeq X Series On-Prem Sequencing v1.0).
The NovaSeq X Series On-Prem Sequencing v1.0 protocol consists of the following steps:
Assign Analysis Configuration Template (NovaSeq X Series On-Prem Sequencing v1.0)
Make Bulk Pool (NovaSeq X Series On-Prem Sequencing v1.0)
Dilute and Denature (NovaSeq X Series On-Prem Sequencing v1.0)
Load To Library Tube Strip (NovaSeq X Series On-Prem Sequencing v1.0)
AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0)
AUTOMATED - Analysis Run (NovaSeq X Series On-Prem Sequencing v1.0)
In Lab View, locate the NovaSeq X Series On-Prem Sequencing v1.0 protocol. The samples are queued for the Assign Analysis Configuration Template (NovaSeq X Series On-Prem Sequencing v1.0) step.
Add the samples to the Ice Bucket and start the step.
The Validate Sample Names automation is started. It checks that the sample names do not use restricted characters and are within character limits.
Select the connected NovaSeq X instrument to integrate with.
In Step Details, select 1. Retrieve ACT List to retrieve a list of ACTs created on Illumina Run Manager.
Select the applicable ACT to assign to the samples. The index adapter kit specified by the ACT must correspond to the label group used in Library Prep Protocol.
[Optional] Select 2. Retrieve ACT Information.
The Retrieve ACT Information automation does the following:
retrieves information such as Library Prep Kit, Index Adapter Kit, Reference Genome, etc.
populates the fields in Record Details milestone.
saves ACT details to ACTMetadata.csv file for download.
Select Next Steps to assign the ACT to the samples.
This action triggers the Apply Selected ACT to Samples and Set Next Step automation. All samples in this step are assigned to the selected ACT.
Select Finish Step.
In Lab View, locate the NovaSeq X Series On-Prem Sequencing v1.0 protocol. The samples are queued for the Make Bulk Pool (NovaSeq X Series On-Prem Sequencing v1.0) step.
Add the samples to the Ice Bucket and select View Ice Bucket.
ℹ 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.
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 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).
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.
[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 Dilute and Denature (NovaSeq X Series On-Prem Sequencing v1.0) step.
Select Finish Step.
In Lab View, locate the NovaSeq X Series On-Prem Sequencing v1.0 protocol. The pool of samples queued for the Dilute and Denature (NovaSeq X Series On-Prem 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 automation is triggered. This automation makes sure that the configured flow cell type is valid.
On the Record Details screen, the Reagent Lot Tracking section tracks the NaOH, Resuspension Buffer, and TT2 reagents used in the step. Select from the active lots displayed in each drop-down list.
[Optional] To add and activate reagent lots, refer to Add and Configure Reagent Kits and Lots in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
[Optional] If PhiX is used for the run, set 1–2% PhiX Spike-In to Yes.
Select Calculate Volumes to trigger the Calculate Volume automation. This automation does the following
Computes BP Aliquot Volume (ul), RSB Volume (ul), NaOH Volume (ul), and TT2 Volume (ul) and set these values in the Sample Details table.
Sets PhiX Volume (ul) and Concentration (pM) (if there is a PhiX spike-in)
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.
[Optional] In the Sample Details table, select the pool icon to view details of the working pool composition.
Select Next Steps.
Select Finish Step.
To perform multiple analyses in a single run, repeat Steps 1 to 3 for each pool and assign with different ACT.
In Lab View, locate the NovaSeq X Series On-Prem Sequencing v1.0 protocol. The pool of samples are queued for the Load to Library Tube Strip (NovaSeq X Series On-Prem Sequencing v1.0) 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 Analysis Configurations automation.
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.
Flowcell Types
Compatible Container
No. of Pools Expected
1.5B
Library 2-tube Strip
2
10B
Library 8-tube Strip
8
25B
Library 8-tube Strip
8
The Validate Analysis Configurations automation checks that analysis configuration for the run is within the maximum configuration limit.
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.
Upon exiting the Placement screen, the Validate Library Strip Tube Barcode automation makes sure that the library tube strip barcode conforms to the barcode mask.
Library 8-tube Strip barcode mask: LC[0-9]{7}-L[A-Z]{1}1
Library 2-tube Strip barcode mask: 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. Analysis-related configuration is retrieved from the ACT associated with the sample.
Refer to the following table for details.
Fields Displayed on Record Details Screen of Load to Library Tube Strip (NovaSeq X Series On-Prem Sequencing v1.0) Step
Field
Description
Run Name
Enter the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. No spaces.
Run Mode
Presets
Local
Read 1 Cycles
Presets
151
101
51
Accepts custom value
Read 2 Cycles
Presets
151
101
51
Accepts custom value
Index 1 Cycles
Presets
0
6
8
Accepts custom value¹
Index 2 Cycles
Presets
0
6
8
Accepts custom value¹
¹ 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 a 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 (NovaSeq X Series On-Prem Sequencing v1.0) step.
Select Finish Step to advance the pooled samples to the AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0) step.
For more information on how to start the sequencing run, refer to NovaSeq X Series On-Prem Integration v1.0.0 Configuration.
Do not add samples to the Ice Bucket or start or complete the AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem 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.
Do not add samples to the Ice Bucket or start or complete the AUTOMATED - Analysis Run (NovaSeq X Series On-Prem 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. If the run analysis tracking is successful, the integration completes the step automatically.
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 if transfer of secondary analysis data to an external location is disabled.
Log - Log message when integration handling the event from IRM
File placeholders:
Demultiplexing results file - demultiplexing statistics for each sample
Analysis results summary files - summary of analysis workflow performed and number of samples analyzed
At this point, the entire NovaSeq X Series Integration workflow is fully validated.
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.
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. Refer to Set Up Secondary Analysis in NovaSeq X Series documentation for limits of the number of analysis application and reference genome combinations supported.
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).
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.
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.
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 or 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 versions or analysis settings, are in different 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.
If the AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0) step does not start, Clarity LIMS is likely not receiving sequencing run events from Illumina Run Manager correctly.
The instrument must be reflected as Connected and Active in the Illumina Run Manager Integration UI. Reconnect the instrument if the status reflects otherwise.
If the issues remain, contact Illumina Support.
If the AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem 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.
The Illumina NovaSeq X Series On-Prem Integration Package v1.0.0 supports the integration of Clarity LIMS to Illumina NovaSeq X Series sequencer connected to the Illumina Run Manager platform on premise.
For instructions on validating and troubleshooting the Illumina NovaSeq X Series On-Premise Integration, refer to .
The configuration provided in this integration has been established to support NovaSeq X Series lab processes. Any configuration changes to protocols or workflows (including renaming protocols, steps, and fields) could break the integration.
The NovaSeq X Series On-Premise Integration v1.0.0 requires NovaSeq X Control Software version v1.3 and above.
The NovaSeq X Series On-Prem Sequencing v1.0 workflow uses analysis configuration templates (ACT) to configure secondary analysis for the planned runs. It is assumed that the required ACTs for your run has been pre-created using Illumina Run Manager UI. Please make sure that the index adapter kit (label group on Clarity) selected in the ACT has already been created on Clarity LIMS. The same label group shall be used in the library preparation step. Names of ACT must be unique. Please refer to the for details on creating ACT on Illumina Run Manager User Interface (UI).
Clarity LIMS trims all leading and trailing spaces from the ACT names and ACTs created in Illumina Run Manager UI with names containing leading and/or trailing spaces may not be recognized properly on Clarity. Please ensure that the ACT do not have leading and/or trailing spaces.
It is assumed that samples that enter the NovaSeq X Series On-Prem Sequencing v1.0 workflow have gone through library preparation and quantification processes before they are assigned to the workflow:
Samples have been accessioned into Clarity LIMS.
Sample and library names must contain only alphanumeric, dash, or underscore characters.
Samples have been run through QC and library prep.
Samples have the Molarity (nM) global field set to a valid value (required for the 'Calculate Volumes' automation in the Make Bulk Pool step).
The NovaSeq X Series On-Premise Integration Package v1.0.0 includes the following workflows:
NovaSeq X Series On-Prem Sequencing v1.0
Library Prep Validation v2.3.5 (optional, but recommended for validation purposes)
The following describes the protocols and steps included in these workflows.
The following table lists the configuration details for custom fields that are defined on the Assign Analysis Configuration Template (NovaSeq X Series On-Prem Sequencing v1.0) step.
Assign Analysis Configuration Template (NovaSeq X Series On-Prem Sequencing v1.0) Master Step Field Configuration
The following lists the global custom fields that are configured to display on the Assign Analysis Configuration Template (NovaSeq X Series On-Prem Sequencing v1.0) step.
Molarity (nM)
ACT Name
Samples are pooled in the Make Bulk Pool (NovaSeq X Series On-Prem Sequencing v1.0) step. You can manually create working pools based on the final loading concentration required.
The following table lists configuration details for the custom fields that are defined on the Make Bulk Pool (NovaSeq X Series On-Prem Sequencing v1.0) step.
Make Bulk Pool (NovaSeq X Series On-Prem Sequencing v1.0) Master Step Field Configuration
The following lists the global custom fields that are configured to display on the Make Bulk Pool (NovaSeq X Series On-Prem Sequencing v1.0) step.
Final Loading Concentration (pM)
RSB Volume (ul)
NovaSeq X Flowcell Type
The Dilute and Denature (NovaSeq X Series On-Prem Sequencing v1.0) step allows you to dilute pooled samples with the addition of RSB.
The following table lists configuration details for the custom fields that are defined on the Dilute and Denature (NovaSeq X Series On-Prem Sequencing v1.0) step.
Dilute and Denature (NovaSeq X Series On-Prem Sequencing v1.0) Master Step Field Configuration
The following lists the global custom fields that are configured to display on the Dilute and Denature (NovaSeq X Series On-Prem Sequencing v1.0) step.
BP Aliquot Volume (ul)
NaOH Volume (ul)
RSB Volume (ul)
TT2 Volume (ul)
In this step, the user scans the library tube strip barcode into the LIMS, and then manually places the working pools into the library tube strip to be used in the NovaSeq X Series run. In addition, this step validates the run setup and analysis information and generates samplesheet file and/or creates a planned run on instrument's Illumina Run Manager UI.
The following table shows the master step fields that are configured on the Load to Library Tube Strip (NovaSeq X Series On-Prem Sequencing v1.0) step. These fields are required for sample sheet generation and planned run creation in Illumina Run Manager.
Load to Library Tube Strip (NovaSeq X Series On-Prem Sequencing v1.0) Master Step Field Configuration
Do not add samples to the Ice Bucket, start or complete the AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0) step. The integration will do this automatically.
In this step, the pooled samples in the library tube strip are sequenced on the NovaSeq X Series instrument.
There are no automation associated to this step.
The following tables show the master step fields that are configured on the AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0) step.
Clarity LIMS Master Step Field Information
All the fields above are Read-Only Text field, except Sequencing Log is a Read-Only Multiline Text.
The following global custom fields are used to capture the run metrics in Clarity LIMS:
% Aligned R1
% Aligned R2
% Bases >=Q30 R1
% Bases >=Q30 R2
% Error Rate R1
% Error Rate R2
% Occupied
% Phasing R1
% Phasing R2
% Prephasing R1
% Prephasing R2
% PF
Intensity Cycle 1 R1
Intensity Cycle 1 R2
Reads PF
Yield (Gb) R1
Yield (Gb) R2
At the end of the step, the pools of samples are automatically route to next step.
Do not add samples to the Ice Bucket or start and complete the AUTOMATED - Analysis Run (NovaSeq X Series On-Prem Analysis v1.0) step. The integration does this automatically.
Data from the analysis is parsed back to Clarity LIMS. In this step, the secondary analysis configured using the Analysis Configuration Template (ACT) is performed on DRAGEN on premise.
There are no automation associated to this step.
The following tables show the master step fields that are configured on the AUTOMATED - Analysis Run (NovaSeq X Series On-Prem Sequencing v1.0) step.
AUTOMATED - Analysis Run (NovaSeq X Series On-Prem Sequencing v1.0) Master Step Field Information
All the fields above are Read-Only Text field, except Log is a Read-Only Multiline Text.
The following information summarizes how the NovaSeq X Series On-Premise Integration works.
After the 'Validate Run Setup and Create Planned Run' automation is triggered on the Load to Library Tube Strip (NovaSeq X Series On-Prem Sequencing v1.0) step, the run parameters entered in the Run Details screen and the sample informations are sent to Illumina Run Manager. Illumina Run Manager validates the run and analysis configuration before the planned run is created, the automation also generates the sample sheet and attaches it to the step. In the event of incomplete information or misconfiguration on the planned run, an error will be displayed on the automation banner and details of the error will be logged in the automation log file.
When the sequencing run starts on the instrument, the NovaSeq X Series Control Software notifies the Illumina Run Manager integration. The events are processed and the integration service retrieves the run information from NovaSeq X Illumina Run Manager. This information is used to populate the custom fields in the AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0) step.
Other run events follow the same information flow. When sequencing is complete, the control software uploads the sequencing run data (primary metrics). Then, Illumina Run Manager integration retrieves the primary metrics and uses them to populate the fields in the Sample Details table (e.g., % Error Rate R1). The custom fields (e.g., Run Status, Current Read, and so on) on the AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0) step are updated using the run related information. If the sequencing run is successfully completed, the step automatically completes.
The integration tracks the analysis events and results in the AUTOMATED - Analysis Run (NovaSeq X Series On-Prem Sequencing v1.0) step. The secondary analysis results are in the external storage configured in Illumina Run Manager. The external storage information is found in the External Storage for Analysis Results configuration settings in Illumina Run Manager.
If a planned run with the same sample name and project name (case-insensitive) has been created previously in Illumina Run Manager, the sample sheet generated from the 'Validate Run Setup and Create Planned Run' automation can reflect the original case of the previous sample name. This can cause validation errors for analysis configurations with sample-level settings. To resolve this issue, change the sample name or the project name on Clarity LIMS and run the automation again.
The integration requires secondary analysis files to be present on the instrument for proper functioning of the AUTOMATED - Analysis Run step.
The following instrument setting must be disabled:
Permanently delete secondary analysis files from the instrument after they are transferred to the external storage and/or cloud.
The following sections describe the components (files, properties, reagent categories / label groups, reagent kits, and containers) that are installed by default as part of this integration.
Global Fields
Container Global Fields
Derived Sample Global Fields
Reagent Kits
Buffer Cartridge
Lyophilization Cartridge
NaOH
Reagent Cartridge
Resuspension Buffer (RSB)
TT2
Container Types
Library 2-Tube Strip
Library 8-Tube Strip
Tube
Instrument Types
NovaSeq X Series
Supported Library Tube Strip barcode formats by the integration
Library 2-Tube Strip - LC[0-9]{7}-L[A-Z]2
Library 8-Tube Strip - LC[0-9]{7}-L[A-Z]1
The workflow configuration contains several validation checks. To make sure that the calculations work properly, it is important that you do not disable any of this validation logic. The validation checks determine the following information:
Which samples, and how many, can enter each step together.
Which samples, and how many, can be pooled together.
All submitted samples must have an associated secondary analysis that is configured using the analysis configuration template (ACT). The ACT must be configured on NovaSeq X Illumina Run Manager before starting the Assign Analysis Configuration Template step. The ACT names must be unique.
Assign Analysis Configuration Template step does not support pool library as input.
The library tube strip barcode must be unique. There must not be multiple library tube strip containers with the same name in the Clarity LIMS system.
Reagent labels, or indexes, must be unique.
One library pool can only contain one library or control with no label/index.
The AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0) and AUTOMATED - Analysis Run (NovaSeq X Series On-Prem Sequencing v1.0) steps must not be manually started or completed. These steps are fully automated and the sequencing service does not update samples correctly if they have been manually started.
For the automated run to start successfully, you must:
select Validate Run Setup and Create Planned Run in the Load to Library Tube Strip step and the automation must be ran successfully.
complete the Load to Library Tube Strip step. The libraries must be queued at the AUTOMATED - Sequencing Run step.
⚠ The label group (index adapter kit) used for library preparation must be the same as the index adapter kit specified in the ACT that is being used. For more information, refer to .
The Library Prep Validation v2.3.5 workflow allows for validation of the system after installation is complete. This workflow can be replaced by other custom library preparation workflows. For details, refer to .
[Optional] Configure a custom library preparation workflow. Routing of libraries from the custom workflow to the NovaSeq X Series On-Premise Sequencing protocol can be enabled in the master step with the available in the NGS package.
In this step, secondary analyses are configured for the samples using ACTs. Each ACT contains details related to a particular secondary analysis (e.g., index adapter kit to use, reference genome setting). At this step, the user selects an ACT from a list of pre-constructed ACTs and assigns samples to it. A planned run can involve multiple ACTs and users have to repeat Step 1 for each ACT that is required for the planned run. For more information on creating ACTs and assigning samples to them, refer to .
Pooled samples are within the maximum configuration limit. See for more details.
Secondary analysis configuration of samples in a planned run is within maximum configuration limit. See for more details.
To start a planned run, please refer to .
The library preparation workflow of the samples must be before routing the samples through the library preparation workflow.
Field Name
Field Type
Options
Analysis Configuration Template
Text Dropdown
Allow Custom Entries
Application
Text
Read Only
Application Version
Text
Read Only
Index Adapter Kit
Text
Read Only
Library Prep Kit
Text
Read Only
Reference Genome
Text
Read Only
Secondary Analysis Mode
Text
Read Only
Field Name
Field Type
Options
Additional Options and Dropdown Items
Final Loading Concentration (pM)
Numeric Dropdown
Required Field
Allow Custom Entries
Dropdown Items
90
140
150
160
180
Flowcell Type
Text Dropdown
Required Field
Dropdown Items
1.5B
10B
25B
Minimum Per Sample Volume (ul)
Numeric
Required Field
Decimal places displayed: 2
Default: 2
Number of Lanes to Sequence
Numeric
Required Field
Range: 1–10
Field Name
Field Type
Options
Additional Options and Dropdown Items
1-2% Spike-In
Numeric
Required Field
Decimal places displayed: 1
PhiX Volume (ul)
Numeric
Optional Field
Decimal places displayed: 1
Field Name
Field Type
Options
Additional Options and Dropdown Items
Index 1 Cycles
Numeric Dropdown
Required Field
Allow Custom Entries
Range = 0–20
Presets
0
6
8
Index 2 Cycles
Numeric Dropdown
Required Field
Allow Custom Entries
Range = 0–20
Presets
0
6
8
Instrument
Text
Read Only
Planned Run ID
Text
Read Only
Hidden
Read 1 Cycles
Numeric Dropdown
Required Field
Allow Custom Entries
Range = 1–251
Presets
51
101
151
Read 2 Cycles
Numeric Dropdown
Required Field
Allow Custom Entries
Range = 0–251
Presets
51
101
151
Run Mode
Text
Read Only
Default: Local
Run Name
Text
Required Field
Field Name
Description
Current Cycle
Current completed cycle count.
Current Read
Current completed reads count.
Flow Cell Expiration Date
Flow Cell expiration data.
Flow Cell ID
Serial number of the Flow Cell.
Flow Cell Lot Number
Flow Cell lot number.
Flow Cell Part Number
Flow Cell part number.
Flow Cell Side
Flow Cell side. Value can be A or B.
Flow Cell Type
Flow Cell Type. Value can be 1.5B, 10B or 25B.
Instrument Control Software Version
Version of the instrument control software.
Instrument ID
Unique identification for the instrument.
Instrument Type
Type of the instrument, either NovaSeqX or NovaSeqXPlus.
Library Tube Strip Barcode
Library Tube Strip barcode.
Output Folder
Output folder path for the sequencing run data.
Run Name
Run name user configured for the sequencing run.
Run Status
Status of the sequencing run.
Sequencing Log
Log messages by the Illumina Run Manager integration while handling the sequencing event from instrument.
Field Name
Description
Analysis Result Location
Analysis files final copy out location.
Analysis Run ID
Run ID associate with the Analysis run. Hidden on Record Details screen by default.
Analysis Status
Status for the the analysis run.
Log
Log messages by the Illumina Run Manager integration while handling the analysis event from instrument.
Field Name
Field Type
Settings
% Aligned R1
Numeric
Read Only
Decimal Places Displayed: 2
% Aligned R2
Numeric
Read Only
Decimal Places Displayed: 2
% Bases >=Q30 R1
Numeric
Read Only
Decimal Places Displayed: 2
% Bases >=Q30 R2
Numeric
Read Only
Decimal Places Displayed: 2
% Error Rate R1
Numeric
Read Only
Decimal Places Displayed: 2
% Error Rate R2
Numeric
Read Only
Decimal Places Displayed: 2
% Occupied
Numeric
Read Only
Decimal Places Displayed: 2
% PF
Numeric
Read Only
Decimal Places Displayed: 2
% Phasing R1
Numeric
Read Only
Decimal Places Displayed: 3
% Phasing R2
Numeric
Read Only
Decimal Places Displayed: 3
% Prephasing R1
Numeric
Read Only
Decimal Places Displayed: 3
% Prephasing R2
Numeric
Read Only
Decimal Places Displayed: 3
Intensity Cycle 1 R1
Numeric
Read Only
Intensity Cycle 1 R2
Numeric
Read Only
Reads PF
Numeric
Read Only
Decimal Places Displayed: 2
Yield (Gb) R1
Numeric
Read Only
Decimal Places Displayed: 2
Yield (Gb) R2
Numeric
Read Only
Decimal Places Displayed: 2
Field Name
Field Type
Settings
ACT ID
Text
Read Only
ACT Name
Text
Read Only
Adjusted Per Sample Volume (ul)
Numeric
Read Only
Decimal Places Displayed: 2
BP Aliquot Volume (ul)
Numeric
Read Only
Decimal Places Displayed: 1
Bulk Pool Volume (ul)
Numeric
Read Only
Decimal Places Displayed: 2
Final Loading Concentration (pM)
Numeric Dropdown
Required
Preset:
225
400
Allow custom entries
Instrument ID
Text
Read Only
Molarity (nM)
Numeric
Decimal Places Displayed: 2
NovaSeq X Flowcell Type
Text Dropdown
Required
Preset:
1.5B
10B
25B
Number of Samples in Pool
Numeric
Per Sample Volume (ul)
Numeric
Read Only
Decimal Places Displayed: 2
PhiX Concentration (pM)
Numeric
PhiX Volume (ul)
Numeric
Decimal Places Displayed: 2
RSB Volume (ul)
Numeric
Read Only
Decimal Places Displayed: 2
Run Mode
Text
Read Only
Total Sample Volume (ul)
Numeric
Read Only
Decimal Places Displayed: 2
TT2 Volume (ul)
Numeric
Read Only
Decimal Places Displayed: 2
