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Release Notes

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.

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Compatibility

Refer to Compatibility under Instruments & Integrations.

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New Features

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.

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Known Limitations

  • 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.

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Known Issues

  • 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.

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.

  • 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 for details.
  • The integration does not support pools with samples assigned with Analysis Configuration Templates from different instruments.

  • NovaSeq X Series On Prem Integration v1.0.0 Configuration

    NovaSeq X Series (On-Prem)

    The documents in this section support the Illumina NovaSeq X Series integration with on-premise Clarity LIMS setup.

    User Interaction, Validation and Troubleshooting

    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)

    The validation steps assume that the following conditions have been met:

    • Integration package has been successfully installed. Refer to .

    • 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.

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    Analysis Configuration Template Creation in Illumina Run Manager

    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.

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    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 NovaSeq X Series On-Prem Sequencing v1.0 workflows.

    1. 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.

    2. On the Projects and Samples screen, create a project and add samples to it.

      âš  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.

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    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 On-Prem Sequencing v1.0 workflow.

    Follow the steps in 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).

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    Protocol: 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)

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    Step 1: Assign Analysis Configuration Template (NovaSeq X Series On-Prem Sequencing v1.0)

    1. 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.

    2. 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.

    3. Select the connected NovaSeq X instrument to integrate with.

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    Step 2: Make Bulk Pool (NovaSeq X Series On-Prem Sequencing v1.0)

    1. 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.

    2. 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.

    1. 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.

    2. Select Finish Step.

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    Step 3: Dilute and Denature (NovaSeq X Series On-Prem Sequencing v1.0)

    1. 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.

    2. Add the pool to the Ice Bucket and select View Ice Bucket.

    3. [Optional] On the Ice Bucket screen, set the number of derivatives to create (placed into the library tube strip) and select Begin Work.

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    Step 4: Load to Library Tube Strip (NovaSeq X Series On-Prem Sequencing v1.0)

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    To perform multiple analyses in a single run, repeat Steps 1 to 3 for each pool and assign with different ACT.

    1. 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.

    2. Add the pools to the Ice Bucket and select View Ice Bucket.

    3. On the Ice Bucket screen, select one of the following destination container types:

    For more information on how to start the sequencing run, refer to .

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    Step 5: AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0)

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    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.

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    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.

    chevron-rightStep Details Sectionhashtag

    The following values populate the master step fields:

    • Run Name

    • Run Status

    chevron-rightSample Details Tablehashtag

    Summary metrics populate the following global container custom fields:

    • % Bases >=Q30 R1

    • % Bases >=Q30 R2

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    Step 6: AUTOMATED - Analysis Run (NovaSeq X Series On-Prem Sequencing v1.0)

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    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.

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    Review Analysis Data

    After the analysis run is complete, open the step and review the following values in the Step Details section:

    • Analysis Status - Status of the analysis run

    • Analysis Result Location - Location of the secondary analysis data. This field is empty if 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.

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    Troubleshooting

    If an automation trigger does not appear to run its corresponding scripts, refer to the Troubleshooting Automation section in the .

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    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. Refer to 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).

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    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.

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    Automated Step Does Not Start

    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.

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    Automated Step Starts, But Does Not Complete

    If the AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0) step starts, but does not complete, do as follows.

    1. Log in to the default user account and use one of the following methods to open the in progress step in Clarity LIMS:

      1. In Lab View, find the step in the Recent Activities pane.

      2. Search for the step in Clarity LIMS using the library tube strip barcode as the search term.

    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.

  • 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 Sequencesarrow-up-right.
  • 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.

  • Assign the samples to the Library Prep Validation workflow.
    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 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.

  • On the Ice Bucket screen, select Begin Work.
  • Create a pool of samples as follows.

    1. On the Pooling screen, create a pool by dragging samples into the Pool Creator.

    2. Enter a name for your pool or accept the default name (Pool #1).

    1. [Optional] If multiple pools are required, select the plus sign (+) next to Pool Creator to create a pool.

    1. [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

  • 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.

  • 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.

  • 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

    • 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.

    1. Drag the pools into the Placed Samples area on the right.

    2. Scan or type the barcode of the library tube strip into the container name field.

    3. 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

    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.

  • Output Folder

  • Current Read

  • Current Cycle

  • Library Tube Barcode

  • Flow Cell ID

  • Flow Cell Side

  • Flow Cell Type

  • Flow Cell Part Number

  • Flow Cell Lot Number

  • Flow Cell Expiration Date

  • Instrument ID

  • Instrument Type

  • Instrument Control Software Version

  • Sequencing Log

  • Refer to NovaSeq X Series On-Prem Integration v1.0.0 Configuration for details of each field.

    % Error Rate R1

  • % Error Rate R2

  • Yield (Gb) R1

  • Yield (Gb) R2

  • Reads PF

  • % PF

  • % Aligned R1

  • % Aligned R2

  • % Occupied

  • % Phasing R1

  • % Phasing R2

  • % Prephasing R1

  • % Prephasing R2

  • Intensity Cycle 1 R1

  • Intensity Cycle 1 R2

  • 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.

    1. Abort the step and remove samples with ACTs that have conflicting run modes from the Ice Bucket.

    2. Make sure that all remaining samples in the Ice Bucket have ACTs with the same run modes.

    3. 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.

  • 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.

    NovaSeq X Series On-Prem Integration v1.0.0 Installation
    Library Prep Validation Protocol
    NovaSeq X Series On-Prem Integration v1.0.0 Configuration
    Clarity LIMS (API & Database) documentation
    Set Up Secondary Analysis in NovaSeq X Series documentationarrow-up-right
    25B

    Library 2-tube Strip

    2

    10B

    Library 8-tube Strip

    8

    25B

    Library 8-tube Strip

    8

    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

    Configuration

    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 NovaSeq X Series On-Premise Integration v1.0.0 User Interaction, Validation and Troubleshooting.

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    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.

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    Prerequisites and Assumptions

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    The NovaSeq X Series On-Premise Integration v1.0.0 requires NovaSeq X Control Software version v1.3 and above.

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    Analysis Configuration Template

    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).

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    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.

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    Samples

    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.

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    Workflows, Protocols, and Steps

    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.

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    Library Prep Validation v2.3.5 Workflow

    chevron-rightProtocol 1: Library Prep Validation v2.3.5hashtag

    Purpose:

    • Included for validation purposes only, this protocol models the library preparation steps required to advance samples to the NovaSeq X Series On-Prem Sequencing v1.0 workflow.

      âš  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

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    NovaSeq X Series On-Prem Sequencing v1.0 Workflow

    chevron-rightProtocol 1: NovaSeq X Series On-Prem Sequencing v1.0hashtag

    Purpose:

    • This protocol models the lab processes of run setup for starting a NovaSeq X Series sequencing and secondary analysis run on premise.

    Steps:

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    Validation Workflow

    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.

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    Step 1: Assign Analysis Configuration Template (NovaSeq X Series On-Prem Sequencing v1.0)

    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 .

    chevron-rightValidate Sample Names Automationhashtag

    Automatically triggered on entry of the step, this automation validates that the sample names contain only alphanumeric, dash and underscore characters.

    chevron-right1. Retrieve ACT List Automationhashtag

    Triggered when you select '1. Retrieve ACT List' button on the Record Details screen, this automation retrieves the list of ACTS that has been created using Illumina Run Manager UI.

    chevron-right2. Retrieve ACT Information Automationhashtag

    Triggered when you select '2. Retrieve ACT Information' button on the Record Details screen, this automation:

    • Retrieves the details of the selected ACT and populates the step UDFs (e.g., Analysis Version, Library Prep Kit, Index Adapter Kit, Reference Genome)

    chevron-rightApply Selected ACT to Samples and Set Next Step Automationhashtag

    Automatically triggered on exit of the Record Details screen, this automation:

    • Checks that the molarity of all samples are specified

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    Master Step Fields

    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

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    Global Fields

    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

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    Step 2: Make Bulk Pool (NovaSeq X Series On-Prem Sequencing v1.0)

    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.

    chevron-rightValidate Analysis Configurations Automationhashtag

    Automatically triggered on exit of the Pooling screen, this automation performs analysis configuration validity and checks each pool for the following characteristics:

    • Pooled samples are within the maximum configuration limit. See for more details.

    chevron-rightCalculate Volumes Automationhashtag

    Triggered when you select 'Calculate Volumes' on the Record Details screen, this automation completes the following actions:

    • Checks to ensure all samples have molarity values assigned.

    • Uses Number of Samples in Pool to calculate the volumes needed for the 2 nM intermediate library pools.

    chevron-rightSet Next Step Automationhashtag

    Automatically triggered on exit of the Record Details screen, this automation completes the following actions:

    • Proceeds only if the sample has a molarity value.

    • Copies the Run Mode from input to output.

    hashtag
    Master Step Fields

    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

    hashtag
    Global Fields

    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

    hashtag
    Step 3: Dilute and Denature (NovaSeq X Series On-Prem Sequencing v1.0)

    The Dilute and Denature (NovaSeq X Series On-Prem Sequencing v1.0) step allows you to dilute pooled samples with the addition of RSB.

    chevron-rightValidate Inputs Flowcell Type Automationhashtag

    Automatically triggered on entry of the step, this automation checks the a NovaSeq X Flowcell Type has been assigned to each input.

    chevron-rightCalculate Volumes Automationhashtag

    Triggered when you select 'Calculate Volumes' on the Record Details screen, this automation completes the following actions based on the NovaSeq X Flowcell Type selected:

    • Sets the NaOH Volume (µl) and TT2 Volume (µl) field values.

    chevron-rightSet Next Step Automationhashtag

    Automatically triggered on exit of the Record Details screen, this automation completes the following actions:

    • Copies the Run Mode, NovaSeq X Flowcell Type and Instrument ID from input to output.

    • Sets the next step for samples to ADVANCE, advancing them to the Load to Library Tube Strip (NovaSeq X Series On-Prem Sequencing v1.0) step.

    hashtag
    Master Step Fields

    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

    hashtag
    Global Fields

    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)

    hashtag
    Step 4: Load to Library Tube Strip (NovaSeq X Series On-Prem Sequencing v1.0)

    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.

    chevron-rightValidate Flowcell Inputs and Analysis Configurations Automationhashtag

    Automatically triggered on entry to the step, this automation completes the following actions:

    • Checks that the selected destination container type at Load to Library Tube Strip step matches the selected flowcell type of the samples. 1.5B flowcell is compatible with Library 2-tube Strip while 10B and 25B flowcells are compatible with Library 8-tube Strip.

    chevron-rightValidate Library Tube Strip Barcode Automationhashtag

    Automatically triggered on exit of the Placement screen, this automation validates the library tube strip barcode to ensure it conforms to the barcode mask LC[0-9]{7}-L[A-Z]1 for Library 8-tube Strip and LC[0-9]{7}-L[A-Z]2 for Library 2-tube Strip.

    chevron-rightValidate Run Setup and Create Planned Run Automationhashtag

    Triggered when you select 'Validate Run Setup and Create Planned Run' button on the Record Details screen, this automation completes the following actions:

    • Validates the parameters entered on the Record Details screen. These parameters are used to set up the run, generate the sample sheet file, and create the planned run on Illumina Run Manager.

    chevron-rightSet Next Step Automationhashtag

    Automatically triggered on exit of the Record Details screen, this automation completes the following actions:

    • Sets the next step for samples to ADVANCE, advancing them to the AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0) step.

    hashtag
    Master Step Fields

    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

    hashtag
    Step 5: AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0)

    triangle-exclamation

    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.

    hashtag
    Master Step Fields

    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.

    hashtag
    Global Fields

    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

    At the end of the step, the pools of samples are automatically route to next step.

    hashtag
    Step 6: AUTOMATED - Analysis Run (NovaSeq X Series On-Prem Sequencing v1.0)

    triangle-exclamation

    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.

    hashtag
    Master Step Fields

    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.

    hashtag
    How the Integration Works

    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.

    triangle-exclamation

    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.

    hashtag
    Configuration of Instrument Setting

    The integration requires secondary analysis files to be present on the instrument for proper functioning of the AUTOMATED - Analysis Run step.

    triangle-exclamation

    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.

    hashtag
    Start a Sequencing Run on NovaSeq X Series Instrument

    To start a planned run, please refer to .

    hashtag
    Enabling Planned Run Generation for Samples Having Duplicate Name with Different Indexes

    The library preparation workflow of the samples must be before routing the samples through the library preparation workflow.

    hashtag
    Components Installed

    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

    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

    hashtag
    Rules and Constraints

    • 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.

    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 protocol contains a single step - Library Prep Validation v2.3.5. After this step, a routing script sends the 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)).

  • Steps:

    1. Library Prep Validation v2.3.5

    Assign Analysis Configuration Template (NovaSeq X Series On-Prem Sequencing v1.0)

    • The secondary analysis of samples is configured using the ACT.

    • The labels applied to the samples are validated against the selected ACT to ensure that samples use valid labels.

  • Make Bulk Pool (NovaSeq X Series On-Prem Sequencing v1.0)

    • Samples are pooled and resuspension buffers and reagents are added with the help of the 'Calculate Volumes' automation.

    • ACT of pooled samples are validated with Validate Analysis Configurations automation to ensure that the secondary analysis configurations remain valid after pooling.

  • Dilute and Denature (NovaSeq X Series On-Prem Sequencing v1.0)

    • Samples from step 2 are denatured and diluted to the final loading concentration with the help of the 'Calculate Volumes' automation.

  • Load To Library Tube Strip (NovaSeq X Series On-Prem Sequencing v1.0)

    • The library pools from step 3 are now ready to be loaded to the NovaSeq X Series library tube strip.

    • Run and analysis information is validated.

    • Samplesheet is generated and planned run is created on NovaSeq X Illumina Run Manager.

  • AUTOMATED - Sequencing Run (NovaSeq X Series On-Prem Sequencing v1.0)

    • This is a fully-automated step that is started and completed automatically once the sequencing run is started and completed on the instrument side.

    • All the sequencing run metadata e.g. run configuration, primary run metrics etc. are recorded automatically.

    âš  Samples should be queued at the step. The user must not add samples to the Ice Bucket or start or complete the step. The integration will do this automatically.

  • AUTOMATED - Analysis Run (NovaSeq X Series On-Prem Sequencing v1.0)

    • This is a fully-automated step that is started and completed automatically once the analysis run is started and completed on DRAGEN on-board the instrument.

    • All the analysis run metadata e.g. status and high level analysis summary and demultiplexing result files are recorded automatically.

    âš  Samples should be queued at the step. The user must not add samples to the Ice Bucket or start or complete the step. The integration will do this automatically.

  • Saves the details into a file stored in the Analysis Configuration Metadata file placeholder, which is available for user to download.
    Checks that all samples are indexed, indexes are the same as the selected ACT.
  • Assign the ACT ID, ACT Name, Run Mode and Instrument ID to all the samples in the step.

  • Sets the next step for samples to ADVANCE, advancing them to the next step in the protocol - Make Bulk Pool (NovaSeq X Series On-Prem Sequencing v1.0).

  • Text

    Read Only

    Reference Genome

    Text

    Read Only

    Secondary Analysis Mode

    Text

    Read Only

    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).
  • Uses the Bulk Pool Volume (ul) field and Number of Lanes to Sequence to calculate volumes needed for the 2 nM intermediate library pools.

  • Copies the Final Loading Concentration (pM) and Flowcell Type from the step inputs to the step outputs.

  • Calculates the per sample volume required for each library to make a 2 nM intermediate library pool and sets the total sample volume to zero.

  • Calculates the adjusted per sample volume for the pools if any of the calculated volume is lesser than the value specified in Minimum Per Sample Volume (ul) step UDF.

  • Uses the Total Sample Volume (ul) to calculate the RSB volume (ul) required to top up the pools that are needed to create the 2 nM intermediate library pools. The RSB volume is saved to the file in the Calculation File placeholder in the Files section.

  • Uses the NovaSeqXSeries_Bulk_Pool1.csv and NovaSeqXSeries_Bulk_Pool2.csv template files to generate a single CSV file. This file contains information about the pools and the samples that they contain. The generated file is stored in the Calculation File placeholder in the Files section.

  • Resets the Number of Samples in Pool, Total Sample Volume (ul), and Bulk Pool Volume (ul) to null before exiting the step.

  • Sets the next step for samples to ADVANCE, which advances them to the Dilute and Denature (NovaSeq X Series On-Prem Sequencing v1.0) step.

    Number of Lanes to Sequence

    Numeric

    • Required Field

    • Range: 1–10

    Computes the BP Aliquot Volume (µl) and RSB Volume (µl) required for the dilution of pools to the required final loading concentration.
  • Sets the PhiX Volume (µl) and PhiX Concentration (µl) if there is a PhiX spike-in.

  • Generates a single CSV file containing information about the reagents volume required to dilute the working pools. The generated file is stored in the Calculation File placeholder, in the Files section, for user to download.

  • Performs basic checks on the secondary analysis configuration of the samples in the same planned run. The following checks are included in this script:
    • Secondary analysis configuration of samples in a planned run is within maximum configuration limit. See herearrow-up-right for more details.

    • Samples in the same pools that have 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).

    âš  This script is required for the NovaSeq X Series On-Prem Sequencing v1.0 workflow to function properly.

    Run Name may only contain alphanumeric, dash, underscore or period characters. Spaces are not permitted.

  • Run Name shall not exceed 255 characters.

  • Checks the Index 1 Cycles and Index 2 Cycles field values. If Index 2 Cycles is greater than 0, the Index 1 Cycles value must be greater than 0 or an error can occur.

  • Generates the sample sheet and creates the planned run on Illumina Run Manager. The sample sheet is attached to the step.

  • Planned Run ID

    Text

    • Read Only

    • Hidden

    Read 1 Cycles

    Numeric Dropdown

    • Required Field

    • Allow Custom Entries

    • Range = 1–251

    • Presets

      • 51

    Read 2 Cycles

    Numeric Dropdown

    • Required Field

    • Allow Custom Entries

    • Range = 0–251

    • Presets

      • 51

    Run Mode

    Text

    • Read Only

    • Default: Local

    Run Name

    Text

    • Required Field

    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.

    % 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

  • 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.

  • 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

    Numeric

    • Read Only

    • Decimal Places Displayed: 2

    Final Loading Concentration (pM)

    Numeric Dropdown

    • Required

    • Preset:

      • 225

    Instrument ID

    Text

    • Read Only

    Molarity (nM)

    Numeric

    • Decimal Places Displayed: 2

    NovaSeq X Flowcell Type

    Text Dropdown

    • Required

    • Preset:

      • 1.5B

    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

    Resuspension Buffer (RSB)

  • TT2

  • 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:

    1. select Validate Run Setup and Create Planned Run in the Load to Library Tube Strip step and the automation must be ran successfully.

    2. complete the Load to Library Tube Strip step. The libraries must be queued at the AUTOMATED - Sequencing Run step.

  • 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

    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

    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

    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.

    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

    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

    NovaSeq X Series On-Premise Integration v1.0.0 User Interaction, Validation and Troubleshooting
    NovaSeq X Series On-Premise Integration v1.0.0 User Interaction, Validation and Troubleshooting
    routing script
    NovaSeq X Series On-Premise Integration v1.0.0 User Interaction, Validation and Troubleshooting
    herearrow-up-right
    NovaSeq X - Start a Planned Runarrow-up-right
    configured to ensure unique derived sample names

    Library Prep Kit

    Flow Cell Side

    % Error Rate R1

    Bulk Pool Volume (ul)

    NovaSeq X Series On-Premise Integration v1.0.0 User Interaction, Validation and Troubleshooting
    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/novaseqxseries-onprem/automation/novaseqxseries-onprem-automation.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -a '{udf:Analysis Configuration Template}' -n 'INSTRUMENT_USED' --derivedSampleInstrumentIdUdf 'Instrument ID' script:validate_indexes script:assign_act
    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'nextStep = ::ADVANCE::' -log {compoundOutputFileLuid1}
    output.::Bulk Pool Volume (ul):: = 100 * step.::Number of Lanes to Sequence::; output.::NovaSeq X Flowcell Type:: = step.::Flowcell Type::; output.::Final Loading Concentration (pM):: = step.::Final Loading Concentration (pM)::;
    input.::Per Sample Volume (ul):: = 2 * output.::Bulk Pool Volume (ul):: / input.::Molarity (nM):: / output.::Number of Samples in Pool::;output.::Total Sample Volume (ul):: = 0;' -t true
    script:calculate_multipool_adjusted_per_sample_volume -t
    script:evaluateDynamicExpression \
      -exp 'if (output.hasValue(::Total Sample Volume (ul)::)) { output.::Total Sample Volume (ul):: = output.::Total Sample Volume (ul):: + input.::Adjusted Per Sample Volume (ul)::; } else { output.::Total Sample Volume (ul):: = input.::Adjusted Per Sample Volume (ul)::; }' -t true \
      script:evaluateDynamicExpression \
      -exp 'if (output.::Total Sample Volume (ul):: > output.::Bulk Pool Volume (ul)::) { output.::RSB Volume (ul):: = 0 } else { output.::RSB Volume (ul):: = output.::Bulk Pool Volume (ul):: - output.::Total Sample Volume (ul):: }' -t true
    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} script:driver_file_generator \
            -t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeqXSeries_Bulk_Pool1.csv -o 1.csv \
            script:driver_file_generator \
            -t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeqXSeries_Bulk_Pool2.csv -o 2.csv \
            && cat 1.csv 2.csv > {compoundOutputFileLuid0}.cs
    -exp 'output.::Number of Samples in Pool:: = ::::; output.::Total Sample Volume (ul):: = ::::; output.::Bulk Pool Volume (ul):: = ::::;' -t true
    bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} -log {compoundOutputFileLuid1} \
      script:evaluateDynamicExpression \
      -exp '
      if (input.::NovaSeq X Flowcell Type:: == ::10B:: || input.::NovaSeq X Flowcell Type:: == ::1.5B::) {
          output.::NaOH Volume (ul):: = 8.5;
          output.::TT2 Volume (ul):: = 127.5;
          output.::BP Aliquot Volume (ul):: = input.::Final Loading Concentration (pM):: * 170 / (2 * 1000);
          output.::RSB Volume (ul):: = 34 - output.::BP Aliquot Volume (ul)::;
          if (step.::1-2% PhiX Spike-In::) { 
              output.::PhiX Volume (ul):: = 1; output.::PhiX Concentration (pM):: = 300; 
          }else { 
              output.::PhiX Volume (ul):: = ::::; output.::PhiX Concentration (pM):: = ::::; 
          };
      } else {
          output.::NaOH Volume (ul):: = 14;
          output.::TT2 Volume (ul):: = 210;
          output.::BP Aliquot Volume (ul):: = input.::Final Loading Concentration (pM):: * 280 / (2 * 1000);
          output.::RSB Volume (ul):: = 56 - output.::BP Aliquot Volume (ul)::;
          if (step.::1-2% PhiX Spike-In::) { 
              output.::PhiX Volume (ul):: = 1.6; output.::PhiX Concentration (pM):: = 300; 
          }
          else { 
              output.::PhiX Volume (ul):: = ::::; output.::PhiX Concentration (pM):: = ::::; 
          };
      }'
    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} \
      script:driver_file_generator \
      -t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeqXSeries_Dilute_Denature_Calculate_Volumes.csv -o {compoundOutputFileLuid0}.csv -q true -destLIMSID {compoundOutputFileLuid0} -l {compoundOutputFileLuid1} \
      && echo; echo 'Calculate Volumes completed successfully.'"
    if (!step.::Run Name::.matches(::[a-zA-Z0-9-_]+[a-zA-Z0-9-_. ]*::)) { fail(::Run Name contains prohibited characters. Please check to make sure Run Name only contains alphanumeric characters, spaces, dashes and underscores. Run Name must start with alphanumeric, a dash or an underscore::); }
    if (step.::Run Name::.length() > 255) { fail(::Run Name shall not exceed 255 characters.::); };
    if (step.::Index 2 Cycles:: > 0 && step.::Index 1 Cycles:: == 0) { fail(::Index 1 Cycles cannot be zero if Index 2 Cycles is non-zero::); };
    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/novaseqxseries-onprem/automation/novaseqxseries-onprem-automation.jar  script:generate_sample_sheet -i {stepURI:v2} -u {username} -p {password} -c {compoundOutputFileLuid0} -l {compoundOutputFileLuid1} -n 'INPUT.Instrument ID'
    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -exp 'submittedSamples.each { sample -> if ( !sample.name.matches( ::[A-Za-z0-9_-]+:: ) ) { fail ( ::Submitted sample :: + sample.name + :: should only have alphanumeric, dash, and underscore characters. Please rename and try again.:: ) } }' -log {compoundOutputFileLuid1}
    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/novaseqxseries-onprem/automation/novaseqxseries-onprem-automation.jar script:retrieve_act_list -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -n 'INSTRUMENT_USED' -t true
    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'if (!input.hasValue(::Molarity (nM)::)) { fail(::The Molarity cannot be empty.::); } else if (input.::Molarity (nM):: <= 0) { fail(::The Molarity cannot be negative or zero.::); }' -log {compoundOutputFileLuid1}
    if (!input.hasValue(::Molarity (nM)::)) { return; };
    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -h false -exp 'if (!input.hasValue(::NovaSeq X Flowcell Type::)){fail(::Invalid Flowcell Type custom field value. Please make sure all input samples have a value assigned for Flowcell Type custom field.::)}' -log {compoundOutputFileLuid1}
    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} \
      script:validate_same_udf_value_for_analytes -f 'Run Mode' -f 'NovaSeq X Flowcell Type' \
      script:validate_selected_container \
      -fn 'NovaSeq X Flowcell Type' -fv '1.5B' -ct 'Library 2-tube Strip' \
      -fn 'NovaSeq X Flowcell Type' -fv '10B' -ct 'Library 8-tube Strip' \
      -fn 'NovaSeq X Flowcell Type' -fv '25B' -ct 'Library 8-tube Strip'
    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:validate_output_containers -l {compoundOutputFileLuid1} -r 'Library 8-tube Strip:LC[0-9]{7}-L[A-Z]1' -r 'Library 2-tube Strip:LC[0-9]{7}-L[A-Z]2' -max 1
    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'nextStep = ::ADVANCE::' -log {compoundOutputFileLuid1}
    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/novaseqxseries-onprem/automation/novaseqxseries-onprem-automation.jar script:retrieve_act_info -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -a '{udf:Analysis Configuration Template}' -n 'INSTRUMENT_USED'
    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} script:validate_same_udf_value_for_analytes -f 'Instrument ID' -f 'Run Mode' -v 'perPool' && \
    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'output.::Instrument ID:: = input.::Instrument ID::' -log {compoundOutputFileLuid1} && \
    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/novaseqxseries-onprem/automation/novaseqxseries-onprem-automation.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -m PerPool -n 'INPUT.Instrument ID' script:validate_analysis_config script:validate_physical_logical_configurations
    if (output.hasValue(::Number of Samples in Pool::)) { output.::Number of Samples in Pool:: = output.::Number of Samples in Pool:: + 1; } else { output.::Number of Samples in Pool:: = 1; }' -t true
    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} -log {compoundOutputFileLuid1} \
    script:evaluateDynamicExpression -exp 'if (!input.hasValue(::Molarity (nM)::)){ return; }; output.::Run Mode:: = input.::Run Mode::; nextStep = ::ADVANCE::;'"
    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'output.::Run Mode:: = input.::Run Mode::; output.::NovaSeq X Flowcell Type:: = input.::NovaSeq X Flowcell Type::; output.::Instrument ID:: = input.::Instrument ID::; nextStep = ::ADVANCE::' -log {compoundOutputFileLuid1}
    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/novaseqxseries-onprem/automation/novaseqxseries-onprem-automation.jar  -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -m PerRun -n 'INPUT.Instrument ID' script:validate_analysis_config script:validate_physical_logical_configurations
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  • NovaSeq X Series On-Prem v1.0.0

    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.

    Installation

    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.

    hashtag
    Prerequisites

    chevron-rightPrerequisite 1: Clarity LIMS v6.2 or later with SecretUtil v1.5.0.12 or laterhashtag

    The NovaSeq X Series On-Prem integration v1.0 relies on secret management util version 1.5.0.12 or later to store the access token to Illumina Run Manager and its related secrets.

    ℹ If using Vault, ensure that the appropriate ACL policies are added.

    chevron-rightPrerequisite 2: Illumina Run Manager Integration Service v1.0.0 or laterhashtag

    Illumina Run Manager Integration Service v1.0 or later is required for this integration.

    chevron-rightPrerequisite 3: Illumina Preset Protocols (IPP) v2.10 with NGS Commons Package v5.25.0 or laterhashtag

    Illumina Preset Protocols (IPP) v2.10 package contains the workflow configuration and template files required to enable the NovaSeq X Series On-Prem workflow to run properly.

    Install the workflow configurations below on the same server on which you intend to install the NovaSeq X Series On-Prem integration.

    • NovaSeq X Series On-Prem Sequencing v1.0 workflow

    hashtag
    Installation Steps

    Steps:

    1. Install / Upgrade Clarity LIMS

    2. Install and Configure Illumina Run Manager Integration Service

    3. Install NovaSeq X Series On-Prem Integration RPM

    hashtag
    Step 1: Install / Upgrade Clarity LIMS

    Install or upgrade to Clarity LIMS v6.2 or later. If you are using an older version of Clarity LIMS, please contact Illumina Support ([email protected]) to schedule for upgrade.

    hashtag
    Step 2: Install and Configure Illumina Run Manager Integration Service

    Install and configure Illumina Run Manager Integration Service v1.0, please refer to for details.

    hashtag
    Step 3: Install NovaSeq X Series On-Prem Integration RPM

    Install ClarityLIMS-NovaSeqXSeries-OnPremise RPM using yum or your preferred package manager.

    Example with YUM:

    Example with RPM:

    hashtag
    Step 4: Install Illumina Preset Protocols (IPP) v2.10 and Import Required IPP Workflows

    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 .

    Library Prep Validation v2.3.5 workflow (optional, but recommended)

    NGS Commons Package v5.25.0 or later is required for Step Automation to work properly.

    Install Illumina Preset Protocols (IPP) v2.10 RPM and Import Required IPP Workflows.
    Illumina Run Manager v1.0.0 Installation and User Interaction
    Installer Parameters and Operations section of Illumina Preset Protocols v2.10 Installation and User Configuration
    yum install ClarityLIMS-NovaSeqXSeries-OnPremise
    rpm -i ClarityLIMS-NovaSeqXSeries-OnPremise-{version}.86_64.rpm
    yum install BaseSpaceLIMS-Illumina-Preset-Protocols
    rpm -i BaseSpaceLIMS-Illumina-Preset-Protocols-{version}.86_64.rpm