The NovaSeq X Series Sequencing v1.0 workflow uses analysis configuration templates (ACTs) to configure secondary analysis for planned runs. Required ACTs must be created using BSSH. The ACT names must be unique. The index adapter kit (the label group in Clarity LIMS) selected in the ACT is created in Clarity LIMS. The same label group must be used in the library preparation step. For more information, refer to NovaSeq X Series Integration v1.1.0 User Interaction, Validation and Troubleshooting.
Samples
Samples must go through the library preparation and quantification process before they enter the NovaSeq X Series Sequencing v1.0 workflow. It is assumed that the following steps have completed before samples are assigned to the workflow:
Samples have been accessioned into Clarity LIMS.
Samples have been run through QC and library prep.
Samples have the Molarity (nM) global field set to a value. This value is required for the Calculate Volumes automation in the Make Bulk Pool step. For more information on sample accessioning, refer to Sample Accessioning and Upload and Modify Samples in the Getting Started section of the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
You can assign samples to workflows automatically, using a routing script, or manually—from the Projects & Samples dashboard. Refer to Assign and Process Samples in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
Workflows, Protocols, and Steps
The NovaSeq X Series Integration Package v1.1.0 includes the following workflows:
Library Prep Validation v2.3.1 (optional, but recommended for validation purposes)
NovaSeq X Series Sequencing v1.0
The following describes the protocols and steps included in these workflows.
Library Prep Validation v2.3.1 Workflow
Protocol 1: Library Prep Validation v2.3.1
Purpose:
Included for validation purposes only, this protocol models the library prep steps required to advance samples to the NovaSeq X Series Sequencing v1.0 workflow.
The protocol contains a single step - Library Prep Validation v2.3.1. After this step, a routing script sends the samples to the first step of the NovaSeq X Series Sequencing v1.0 workflow (Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0)).
Steps:
Library Prep Validation v2.3.1
NovaSeq X Series Sequencing v1.0 Workflow
Protocol 1: NovaSeq X Series Sequencing v1.0
Purpose:
This protocol models the lab processes of run setup for starting a NovaSeq X Series sequencing run.
Steps:
Assign Analysis Configuration Template (NovaSeq X Series 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 so that samples use valid labels.
Make Bulk Pool (NovaSeq X Series Sequencing v1.0)
The Calculate Volumes script assists with pooling samples and adding resuspension buffers and reagents.
The Validate Analysis Configurations automation validates the ACT of pooled samples to make sure that the secondary analysis configurations remain valid after pooling.
Dilute and Denature (NovaSeq X Series Sequencing v1.0)
The Calculate Volumes script helps denature and dilute samples from step 2 to the final loading concentration.
Load To Library Tube Strip (NovaSeq X Series Sequencing v1.0)
The library pools from step 3 are ready to be loaded to the NovaSeq X Series library tube strip.
Validates run and analysis information.
Generates the sample sheet and creates the planned run, depending on the run mode.
AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0)
The step is fully automated and starts and completes automatically after the sequencing run is started and completed on the instrument side.
The sequencing run metadata (eg, run configuration, primary run metrics, and so on) are recorded automatically.
The integration does this step automatically. Do not add samples to the Ice Bucket or start or complete the step.
Step 1: Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0)
The Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0) step uses ACTs to configure secondary analysis for samples. Each ACT contains details related to the secondary analysis (eg, index adapter kit to use, reference genome setting, and so on). You can select an ACT from a list and assign samples to it. A planned run can have multiple ACTs and this step must be repeated for each ACT that is required for the run. For more information on creating ACTs and assigning samples to them, refer to NovaSeq X Series Integration v1.1.0 User Interaction, Validation and Troubleshooting.
The following automations are configured on this step:
Validate Sample Names and Retrieve Analysis Configuration Template List and Register Step Started²
Retrieve ACT Information
Validate Reagent Labels and Apply Selected ACT to Samples and Set Next Step
Register Step Completed¹
¹ These automations are required for CLPA support only.
² These automations are required for the NovaSeq X Series Sequencing v1.0 workflows and contain additional logic needed for CLPA support. If you would like to remove support for CLPA, contact Illumina Support.
All remaining automations are required for the NovaSeq X Series Sequencing v1.0 workflows.
Validate Sample Names and Retrieve Analysis Configuration Template List and Register Step Started Automation
Automatically triggered on entry to the step, this automation completes the following actions:
Checks that submitted sample names contain only alphanumeric, dash, and underscore characters.
/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}
Retrieves the list of ACTs that were created in BSSH.
The following table lists the configuration details for fields that are defined on the Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0) step.
Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0) Master Step Field Configuration
Global Fields
The following table lists the global custom fields that are configured to display on the Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.0) step.
Global Field Configuration (Configured on Derived Sample)
Step 2: Make Bulk Pool (NovaSeq X Series Sequencing v1.0)
Samples are pooled in the Make Bulk Pool (NovaSeq X Series Sequencing v1.0) step. You can manually create working pools based on the final loading concentration required.
The following automations are configured on this step:
Register Step Started¹
Validate Analysis Configurations
Calculate Volumes
Set Next Step
Register Pools & Register Step Completed¹
¹ These automations are required for CLPA support only.
All remaining automations are required for the NovaSeq X Series Sequencing v1.0 workflows.
Register Step Started Automation
Automatically triggered on entry to the step, this automation registers the start time of the step by publishing messages to CLPA through ICA.
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.
Pooled samples have the same type of analysis (eg, Cloud or Local).
Pooled samples that have the same secondary analysis also have the same analysis version (eg, v3.8.4) and analysis settings (eg, Map/Align Output Format = CRAM).
Automatically triggered when you select Calculate Volumes on the Record Details screen, this automation completes the following actions:
Proceeds if the sample has molarity values.
if (!input.hasValue(::Molarity (nM)::)) { return; };
Uses Number of Samples in Pool to calculate the volumes needed for the 2 nM intermediate library pools.
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
Uses the Bulk Pool Volume (ul) field and Number of Lanes to Sequence (x100) for the B3 flow cell type 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.
if (step.::Flowcell Type:: == ::B3::) { 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)::;
Calculates the per sample volume required for each library to make a 2 nM intermediate library pool.
input.::Per Sample Volume (ul):: = 2 * output.::Bulk Pool Volume (ul):: / input.::Molarity (nM):: / output.::Number of Samples in Pool::;
If PhiX is used for a planned run (eg, a value for % PhiX (2.0 nM) Spike-In is specified), sets the PhiX Volume (ul).
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.
This automation completes this action by publishing messages to CLPA through ICA.
Master Step Fields
The following table lists configuration details for the fields that are defined on the Make Bulk Pool (NovaSeq X Series Sequencing v1.0) step.
Make Bulk Pool (NovaSeq X Series Sequencing v1.0) Master Step Field Configuration
Global Fields
The following table lists the global custom fields that are configured to display on the Make Bulk Pool (NovaSeq X Series Sequencing v1.0) step.
Global Custom Fields Configuration (Configured on Derived Sample)
Step 3: Dilute and Denature (NovaSeq X Series Sequencing v1.0)
The Dilute and Denature (NovaSeq X Series Sequencing v1.0) step allows you to dilute pooled samples with the addition of RSB.
The following automations are configured on this step:
Validate Inputs Flowcell Type and Register Step Started²
Calculate Volumes
Set Next Step
Register Step Completed¹
¹ These automations are required for CLPA support only.
² These automations are required for the NovaSeq X Series Sequencing v1.0 workflows and contain additional logic needed for CLPA support. If you would like to remove support for CLPA, contact Illumina Support.
All remaining automations are required for the NovaSeq X Series Sequencing v1.0 workflows.
Validate Inputs Flowcell Type and Register Step Started
Automatically triggered on entry to the step, this automation completes the following actions:
Checks that the specified flow cell type is valid.
/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}
Registers the start time of the step by publishing messages to CLPA through ICA.
Generates a single CSV file that contains 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.
The following table lists the global custom fields that are configured to display on the Dilute and Denature (NovaSeq X Series Sequencing v1.0) step.
Global Field Configuration (Configured on Derived Sample)
Step 4: Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0)
The Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0) step allows you to scan the library tube strip barcode into Clarity LIMS. Then, you can place the working pools into the library tube strip used in the NovaSeq X Series run. This step also does the following actions:
Validates the run setup and analysis information.
Generates the sample sheet file.
Creates a planned run on ICA, depending on the selected run mode.
Validate Input Count and Validate Analysis Configurations and Register Step Started¹
Automatically triggered on entry to the step, this automation completes the following actions:
Checks that the number of input pools is the same as the number of tubes in the library tube strip.
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.
Samples in a planned run have the same type of analysis (eg, Cloud or Local).
Samples in the same pools that have the same secondary analysis (eg, Illumina DRAGEN Enrichment) also have the same analysis settings (eg, Map/Align Output Format = CRAM) and the same analysis version (eg, v3.8.4).
Automatically triggered on the Placement screen, this automation validates the library tube strip barcode to make sure that it conforms to the barcode mask LC[0-9]{7}-LC1.
Validate Run Setup and Create Planned Run Automation
Automatically triggered after a selection 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 in ICA, depending on the run mode.
Run Name can only contain alphanumeric, dash, underscore, or period characters. Do not use spaces.
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::);}
Run Name cannot exceed 255 characters.
if (step.::Run Name::.length() > 255) { fail(::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.
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::); };
Generates the sample sheet. If you are in Cloud run mode, the automation creates the planned run in ICA. The sample sheet is attached to the step.
There is a backend validation of the sample sheet content in ICA. This validation makes sure that the sample sheet is valid to set up a Local mode run and that it is ready for use with DRAGEN applications.
¹ These automations are required for the NovaSeq X Series Sequencing v1.0 workflows and contain additional logic needed for CLPA support. If you would like to remove support for CLPA, contact Illumina Support.
² These automations are required for CLPA support only.
All remaining automations are required for the NovaSeq X Series Sequencing v1.0 workflows.
Master Step Fields
The following table shows the master step fields that are configured on the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0) step. These fields are required for sample sheet generation and planned run creation in ICA.
Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0) Master Step Field Configurationn
Step 5: AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0)
The AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0) step is fully automated. The integration starts the step, adds samples to the Ice Bucket, and completes the step. Data from the run is parsed back to Clarity LIMS. In this step, pooled samples in the library tube strip are sequenced on the NovaSeq X Series instrument.
Register Step Started & Register NovaSeq X Run & Register NovaSeq X Run Association & Register NovaSeq X RunStarted Automation¹
Automatically triggered at the beginning of the step, this automation completes the following actions:
Registers the start time of the step.
Registers the NovaSeq X Series sequencing run configuration.
Registers the association of the sequencing run with the samples by linking the Instrument Run ID with the sample ID.
Registers the time that the sequencing run started. This automation completes these actions by publishing messages to CLPA through ICA.
Register NovaSeq X RunCompleted & Register Step Completed Automation¹
Automatically triggered on exit of the step, this automation completes the following actions:
Registers the sequencing run completion time, run status, and run metrics. This script requires the Cloud Run ID custom field from the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0) step.
Registers the completion time of the step. This automation completes these actions by publishing messages to CLPA through ICA.
¹ These automations are required for CLPA support only.
All remaining automations are required for the NovaSeq X Series Sequencing v1.0 workflows.
Master Step Fields
The following tables show the master step fields that are configured on the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0) step.
Clarity LIMS Master Step Field Information
Other Clarity LIMS Master Step Fields
Global Fields
The following global custom fields are used to capture the run metrics in Clarity LIMS:
% Bases >=Q30 R1
% Bases >=Q30 R2
% Error Rate R1
% Error Rate R2
Yield (Gb) R1
Yield (Gb) R2
Reads PF R1
Reads PF R2
%PF R1
%PF R2
% Aligned R1
% Aligned R2
% Phasing R1
% Phasing R2
% Prephasing R1
% Prephasing R2
Intensity Cycle 1 R1
Intensity Cycle 1 R2
Cluster Density R1
Cluster Density R2
At the end of the step, the pools of samples are automatically removed from the step. The step completes automatically when Run Status is RunCompletedSuccessfully.
How the Integration Works
The following information summarizes how the NovaSeq X Series integration works.
After the Validate Run Setup and Create Planned Run automation is triggered on the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0) step, the run parameters entered in the Run Details screen are sent to ICA. The analysis configuration parameters from the selected ACTs are also sent to ICA. ICA validates the run and analysis configuration. If the validation fails, ICA sends an error message to Clarity LIMS. If the validation passes, ICA generates the sample sheet and sends it back to Clarity LIMS. ICA also creates the planned run based on the selected run mode (eg, Local or Cloud).
For the Local run mode, the sample sheet is generated and stored in Clarity LIMS. This sample sheet contains the run and analysis configuration information required to start the run on the NovaSeq X Series instrument.
For the Cloud run mode, the planned run is created in ICA. The planned run contains the run and analysis configuration information required to start the run on the instrument. The sample sheet is generated and stored in Clarity LIMS for reference purposes.
When the sequencing run starts on the instrument, the NovaSeq X Series Control Software notifies BSSH. BSSH monitors the run and uses the Stratus Integration Service (SIS) to notify Clarity LIMS. The events are processed and the integration service retrieves the run information from BSSH. This information is used to populate the custom fields in the AUTOMATED - Sequencing Run (NovaSeq X Series 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) and associated files to ICA. Then, Clarity LIMS retrieves the primary metrics and uses them to populate the fields in the Sample Details table (eg, % Error Rate R1). The custom fields (eg, Run Status, Current Read, and so on) on the AUTOMATED - Sequencing Run (NovaSeq X Series Sequencing v1.0) step are updated using the run related information. If the sequencing run is successfully completed, the step automatically completes.
The analysis results are not tracked. You must log in to BSSH to retrieve the cloud analysis results. For local analysis, the secondary analysis results are found 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.
Start a Sequencing Run on NovaSeq X Series Instrument
The following sections outline the steps to start a Clarity LIMS cloud or local run on the NovaSeq X Series instrument.
Start a Sequencing Run in Local Run or Cloud Run Modes
You can start the sequencing run in BSSH for both the Local and Cloud modes. Make sure that you log in using the account that was used during the NovaSeq X Series integration package setup. You must also import the sample sheet created from the Load to Library Tube Strip (NovaSeq X Series Sequencing v1.0) step for the Local Run Mode.
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.
Reagent Kits
Buffer Cartridge
Lyophilization Cartridge
NaOH
Reagent Cartridge
Resuspension Buffer (RSB)
TT2
Container Types
Library 8-Tube Strip
Tube This integration supports the library 8-tube strip with the barcode in the LC[0-9]{7}-LC1 format (eg, LC1234567-LC1).
Control Types
PhiX v3
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.
All submitted samples must have an associated secondary analysis that is configured using the analysis configuration template (ACT). The ACT must be configured on BSSH before starting the Assign Analysis Configuration Template step. The ACT names must be unique.
The library tube strip barcode must be unique. There must not be multiple library tube strip containers with the same name in the 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 Sequencing v1.0) step must not be manually started or completed. This step is 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 step.
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 process.
The Illumina Universal Sample Identifier is a global field that is imported with the Library Prep Validation v2.3.1 and NovaSeq X Series Sequencing v1.0 workflows. Illumina Universal Sample Identifier is a text field that is reserved for CLPA support. The field is optional and not required for this integration.
Make sure that ACT names created in BaseSpace Sequence Hub do not have leading or trailing spaces. Otherwise, Clarity LIMS can have issues with recognizing the ACT names.
If a planned run with the same sample name and project name (case-insensitive) has been created previously in ICA, 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.