Illumina Connected Software

Configuration

The Illumina NovaSeq 6000 Integration Package v3.6.0 supports the integration of Clarity LIMS to NovaSeq 6000 instruments. This documentation describes the integration and includes the following information:

  • Preconfigured workflows, protocols, steps, and automations

  • Installed components

  • Configuration requirements, rules, and constraints

For instructions on user interaction for each step, validating and troubleshooting the NovaSeq 6000 Integration, refer to NovaSeq 6000 Integration v3.6.0 User Interaction, Validation and Troubleshooting.

The configuration provided in this integration has been established to support NovaSeq 6000 lab processes. Any configuration changes to protocols or workflows - including renaming protocols, steps, and fields - could break process.

Prerequisites and Assumptions

It is assumed that samples enter the NovaSeq 6000 v3.8 workflow as normalized libraries. It is assumed that the following steps have completed before samples are assigned to the workflow:

  • Samples have been accessioned into the Clarity LIMS.

  • Samples have been run through QC and library prep.

  • Samples have been normalized, and the value is captured in a field called Normalized Molarity (nM).

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 Illumina NovaSeq 6000 Integration Package v3.6.0 includes the following workflows:

  • Library Prep Validation v2.3.1 (optional, but recommended for validation purposes)

  • NovaSeq 6000 v3.8

Library Prep Validation v2.3.1 Workflow

Protocol: 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 Run Format (NovaSeq 6000 v3.8) protocol.

  • 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 6000 v3.8 workflow - Define Run Format (NovaSeq 6000 v3.8).

Steps:

  1. Library Prep Validation v2.3.1

NovaSeq 6000 v3.8 Workflow

Protocol 1: Run Format (NovaSeq 6000 v3.8)

Purpose:

  • Allows for the assignment of per sample values for:

    • Loading Workflow Type: Select either NovaSeq Standard or NovaSeq Xp.

    • Normalized Molarity: Enter a value for each sample.

    • Flowcell Type: Select from options SP, S1, S2, or S4.

    • Final Loading Concentration (pM): Select from options 225 (PCR-free workflows) or 400 (Nano workflows), or enter a different value.

  • Compares the Normalized Molarity value of each sample with the Minimum Molarity value.

  • Routing script sends samples to the NovaSeq Standard or NovaSeq Xp protocol, according to the selected Loading Workflow Type.

  • Samples with Normalized Molarity less than Minimum Molarity are removed from the workflow.

Steps:

  1. Define Run Format (NovaSeq 6000 v3.8)

Protocol 2: NovaSeq Standard (NovaSeq 6000 v3.8)

Purpose:

  • Samples are pooled and added to the library tube in preparation for the NovaSeq run. The run setup information is validated and a sample sheet is generated.

  • Routing script sends the library tube to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) protocol.

Steps:

  1. Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8)

  2. Dilute and Denature (NovaSeq 6000 v3.8)

Protocol 3: NovaSeq Xp (NovaSeq 6000 v3.8)

Purpose:

  • Samples are pooled and added to lanes on the NovaSeq flow cell. The option selected in the Define Run Format (NovaSeq 6000 v3.8) step determines the flow cell type. The run setup information is validated and a sample sheet is generated.

  • Routing script sends flow cell to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) protocol.

Steps:

  1. Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8)

  2. Dilute, Denature & ExAmp (NovaSeq 6000 v3.8)

  3. Load to Flowcell (NovaSeq 6000 v3.8)

Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8)

Purpose:

  • All samples complete the workflow by going through this protocol.

  • This protocol contains one fully automated step.

    âš  Do not add samples to the Ice Bucket or start the step. The integration starts the step automatically.

Steps:

  1. AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8)

The Library Prep Validation v2.3.1 workflow allows for validation of the system after installation is complete. For details, refer to NovaSeq 6000 Integration v3.6.0 User Interaction, Validation and Troubleshooting.

Protocol 1: Run Format (NovaSeq 6000 v3.8)

This protocol sets the Loading Workflow Type and allows the choice of the appropriate Flowcell Type and Final Loading Concentration (pM). After the protocol, a routing script sends the normalized libraries to either the NovaSeq Standard (NovaSeq 6000 v3.8) or the NovaSeq Xp (NovaSeq 6000 v3.8) protocol.

This protocol contains one step: Define Run Format (NovaSeq 6000 v3.8).

Step 1. Define Run Format (NovaSeq 6000 v3.8)

  • Step input: NTP (normalized libraries)

  • Step output: None

Register Step Started Automation¹

Automatically triggered on entry to the step, this automation registers the start time of the step by publishing messages to Clarity LIMS Product Analytics (CLPA) through Illumina Connected Analytics:

This automation is used only for CLPA support.

Set Next Steps Automation

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

  • Sets the next step for samples to REMOVE:

  • Calculates the Minimum Molarity using the following formula:

  • Checks Normalized Molarity value. For samples with no Normalized Molarity value (e.g., empty value, not including 0), it generates an error message informing that the field cannot be empty:

  • Compares each sample Normalized Molarity value with the Minimum Molarity value. If the Normalized Molarity value is lower than the Minimum Molarity value, it sets the samples Loading Workflow Type to [Remove from workflow]. The automation also records a message in the Warning field for the sample:

At this point there are two options:

  • Correct the Normalized Molarity value on the Record Details screen. Edit the Loading Workflow Type field and set it to NovaSeq Standard or NovaSeq Xp, as applicable.

  • Complete the protocol without correcting the Normalized Molarity value. In this case, those samples are removed from the workflow.

Routing Script & Register Step Completed Automation²

Automatically triggered on exit of the step, this automation invokes the changeWorkflow script, which routes step inputs appropriately.

  • Samples with Loading Workflow Type field value = NovaSeq Standard are routed to the NovaSeq 6000 v3.8 workflow. Then, the samples are queued for the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8) step.

  • Samples with Loading Workflow Type field value = NovaSeq Xp are routed to the NovaSeq 6000 v3.8 workflow. Then, the samples are queued for the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8) step.

  • This automation also registers the completion time of the step by publishing messages to CLPA through Illumina Connected Analytics. This is only used for CLPA support.

    Default automation command line is as follows:

¹ This automation is for CLPA support only.

² This automation is required for the NovaSeq 6000 v3.8 workflow to function properly. This automation contains additional logic needed for CLPA support. If you would like to remove CLPA support, then contact Illumina Support.

Automations not identified with ¹ or ² are required for the NovaSeq 6000 v3.8 to work function properly.

Master Step Fields

The following table lists field configuration details defined on the Define Run Format (NovaSeq 6000 v3.8) master step.

Define Run Format (NovaSeq 6000 v3.8) Master Step Field Configuration

Field Name

Field Type

Options

Additional Options and Dropdown Items

Comment

Multiline Text

None

Flowcell Type

Text Dropdown

  • Required Field

  • Custom Entries

Presets

  • SP

  • S1

  • S2

  • S4

Instruction

Text

  • Read Only

Default

  • Add Flowcell Type and Loading Workflow Type below

Loading Workflow Type

Text Dropdown

  • Required Field

Presets

  • NovaSeq Standard

  • NovaSeq Xp

Global Fields

The following table lists the global custom fields that are configured to display on the Define Run Format (NovaSeq 6000 v3.8) step.

Define Run Format (NovaSeq 6000 v3.8) Global Field Configuration (Derived Sample)

Field Name

Field Type

Options

Additional Options and Dropdown Items

Adjusted Per Sample Volume (ul)

Numeric

  • Read Only

  • Decimal places displayed = 2

Final Loading Concentration (pM)

Numeric Dropdown

  • Required Field

  • Custom Entries

  • Decimal places displayed = 0

  • Presets

    • 225

    • 400

Flowcell Type

Text Dropdown

  • Required Field

  • Presets

    • SP

    • S1

    • S2

    • S4

Loading Workflow Type

Text Dropdown

  • Required Field

  • Presets

    • NovaSeq Standard

    • NovaSeq Xp

    • [Remove from workflow]

Minimum Molarity (nM)

Numeric

  • Decimal places displayed = 2

Normalized Molarity (nM)

Numeric

  • Decimal places displayed = 2

Per Sample Volume (ul)

Numeric

  • Read Only

  • Decimal places displayed = 2

Warning

Text Dropdown

  • Read Only

  • Custom Entries

  • Presets

    • The Normalized Molarity (nM) is too low.

    • n/a

Protocol 2: NovaSeq Standard (NovaSeq 6000 v3.8)

Samples are routed to this protocol when their Loading Workflow Type value is set to NovaSeq Standard. Samples are pooled and added to a library tube in preparation for the NovaSeq run.

At the end of this protocol, a routing script sends the library tube to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) protocol.

This protocol contains two steps:

  • Step 1: Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8)

  • Step 2: Dilute and Denature (NovaSeq 6000 v3.8)

Step 1: Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8)

In this step, libraries are placed manually into a single pool. Resuspension buffer and reagents are added.

Create only one pool per step.

  • Step input: NTP (normalized libraries)

  • Step output: Bulk pool

Validate Unique Indexes automation is not used. The Clarity LIMS configuration for pooling handles this functionality.

Register Step Started Automation¹

Automatically triggered on entry to the step, this automation registers the start time of the step by publishing messages to CLPA through Illumina Connected Analytics:

This automation is used only for CLPA support.

Validate Inputs Flowcell Type and Single Pool Automation

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

  • Checks that all samples in the pool have the same Flowcell Type assigned to them.

  • Checks that only one pool has been created.

  • Changes the value of error and logging messages to reference the type of pool passed as bulk pool.

Calculate Volumes Automation

Automatically triggered when selecting the Calculate Volumes button on the Record Details screen, this automation completes the following actions:

  • Calculates the number of samples in the pool.

  • Sets the value of the Bulk Pool Volume (ul), and PhiX Volume (ul) field based on the selected Flowcell Type.

  • Calculates the Per Sample Volume (ul) to be added to the pool.

    âš  For accurate pipetting of each sample in a pool, the Per Sample Volume (ul) value must be greater than or equal to the Minimum Per Sample Volume (ul). The default value (set at 5) can be edited.

    Assuming the default Minimum Per Sample Volume (ul) value of 5, completes the following steps for a given batch:

    1. If the smallest Per Sample Volume (ul) value is less than 5, automatically assigns a value of 5 to the Adjusted Per Sample Volume (ul) field.

    2. Adjusts the Adjusted Per Sample Volume (ul) field value for all other samples in the batch. This adjustment is based on the ratio used to increase the lowest value to 5.

  • Calculates the Total Sample Volume (ul) field value.

  • If the Total Sample Volume is less than the Bulk Pool Volume, the automation calculates the RSB Volume (ul) field value.

  • Copies the Flowcell Type and Loading Workflow Type values from the step inputs to the step outputs.

  • Sets the Volume of Pool to Denature (ul) value and calculates the NaOH Volume (ul) and Tris-HCl Volume (ul) values based on the Flowcell Type:

  • Uses the NovaSeq_Standard_Bulk_Pool1.csv, NovaSeq_Standard_Bulk_Pool2.csv, and NovaSeq_Standard_Bulk_Pool3.csv template files to generate a single CSV file containing information about the pool and the samples it contains. The generated file is available for download on the Step Setup screen of Dilute and Denature (NovaSeq 6000 v3.8).

  • Resets the Total Sample Volume (ul) and Number of Samples in Pool field values so that the automation is idempotent.

Set Next Step Automation

Automatically triggered on exit of the Record Details screen, the following automation sets the next step for samples to ADVANCE. The samples advance to the Dilute and Denature (NovaSeq 6000 v3.8) step:

Register Pools & Register Step Completed Automation¹

Automatically triggered on exit of the step, the following automation completes the following actions:

  • Registers the pool and library information used by the Clarity LIMS workflow. Samples in the pool are assumed to have gone through library preparation.

  • Registers the completion time of the step. These actions are done by publishing messages to CLPA through Illumina Connected Analytics. This automation is used only for CLPA support.

¹ These automations are for CLPA support only.

Automations not identified with ¹ are required for the NovaSeq 6000 v3.8 to work function properly.

Master Step Fields

The following table lists configuration details defined on the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8) step.

Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8) Master Step Field Configuration

Field Name

Field Type

Options

Additional Options and Dropdown Items

% PhiX (2.5 nM) Spike-In

Numeric

  • Range = 0–100

Bulk Pool Volume (ul)

ℹ for calculation purposes, not displayed

Numeric

  • Decimal places displayed = 2

Number of Flowcells to Sequence

Numeric

Required Field

  • Range = 1–10

  • Decimal places displayed = 0

Minimum Per Sample Volume (ul)

Numeric

Required Field

  • Decimal places displayed = 2

  • Default

    • 5

Number of Samples in Pool

ℹ for calculation purposes, not displayed

Numeric

  • Decimal places displayed = 0

  • Default

    • 0

PhiX Volume (ul)

ℹ for calculation purposes, not displayed

Numeric

  • Decimal places displayed = 2

Total Sample Volume (ul)

ℹ for calculation purposes, not displayed

Numeric

  • Decimal places displayed = 2

  • Default

    • 0

Global Fields

The following table lists the global custom fields that are configured to display on the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8) step.

Global Custom Fields Configuration (Derived Sample)

Field Name

Field Type

Options

Additional Options and Dropdown Items

Flowcell Type

Text Dropdown

Required Field

Presets

  • SP

  • S1

  • S2

  • S4

Loading Workflow Type

Text Dropdown

Required Field

Presets

  • NovaSeq Standard

  • NovaSeq Xp

  • [Remove from workflow]

NaOH Volume (ul)

Numeric

Read Only

Decimal places displayed = 2

RSB Volume (ul)

Numeric

Read Only

Decimal places displayed = 2

Tris-HCl Volume (ul)

Numeric

Read Only

Decimal places displayed = 2

Volume of Pool to Denature (ul)

ℹ Used in Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8) step only. Displays on Record Details screen and in the generated CSV file.

Numeric

Read Only

Decimal places displayed = 0

Step 2: Dilute and Denature (NovaSeq 6000 v3.8)

In this step, the addition of NaOH, Tris-HCl, and Resuspension Buffer (RSB) denatures and dilutes the pooled samples. Manually place the pooled samples into the library tube for the NovaSeq run.

In addition, this step validates the run setup information and generates the sample sheet file.

  • Step input: Bulk pool

  • Step output: Library tube

Validate Single Input & Register Step Started Automation¹

Automatically triggered at the beginning of the step, this automation completes the following actions:

  • Checks that there is only one container input to the step.

  • Registers the start time of the step by publishing messages to CLPA through Illumina Connected Analytics. This script is used only for CLPA support.

Validate Library Tube Barcode Automation

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

  • Validates the library tube barcode to ensure it conforms to the barcode mask [A-Z]{2}[0-9]{7}-[A-Z]{3}:

  • Copies Flowcell Type and Loading Workflow Type field values from step inputs to outputs.

Validate Run Setup and Generate Sample Sheet Automation

ℹ The script definition portion of Validate Run Setup has been moved into the Validation Script custom field due to the 4000 character limit.

  • If modification is required on Validate Run Setup script, perform the changes directly on the Validation Script custom field default value.

  • The script definition portion of the Generate Sample Sheet remains untouched.

Triggered by a button on the Record Details screen, this automation completes the following actions:

  • Copies the Flowcell Type from the step input to the Run Mode field (hidden):

  • Validates the parameters entered on the Record Details screen, which are used to set up the run and generate the sample sheet file. For more information, refer to the Respond to Validation / Recipe Request Call from Instrument section of NovaSeq 6000 Run.

    • Experiment Name can only contain alphanumeric, dash, or underscore characters. Spaces are not permitted.

    • When Workflow Type = No Index, both Index Read 1 and Index Read 2 must be zero.

    • When Workflow Type = Single Index, Index Read 1 must be greater than zero, and Index Read 2 must be zero.

    • When Workflow Type = Dual Index, both Index Read 1 and Index Read 2 must be greater than zero.

  • Checks the Paired End and Read 2 Cycles field values.

    • If Paired End = True and Read 2 Cycles value is zero, generates an error.

    • If Paired End = False, Read 2 Cycles must be zero and UMI - Read 2 Length and UMI - Read 2 Start From Cycle must be empty.

  • Checks the Flowcell field value.

    • If Flowcell Type value is not SP, checks that the values of Read 1 Cycles and Read 2 Cycles are each 151 or less. If the value is greater than 151, an error message is generated.

  • Validates UMI Settings.

    • If UMI - Read 1 Length has value, then UMI - Read 1 Start From Cycle must have value.

    • If UMI - Read 1 Length has no value, then UMI - Read 1 Start From Cycle must not have value.

    • If UMI - Read 2 Length has value, then UMI - Read 2 Start From Cycle must have value.

    • If UMI - Read 2 Length has no value, then UMI - Read 2 Start From Cycle must not have value.

    • If UMI - Read 1 Length has no value, then UMI - Read 2 Length must not have value.

    • If UMI - Read 1 Start From Cycle has no value, then UMI - Read 2 Start From Cycle must not have value.

  • Validate Analysis Software Version to ensure it is a valid version string.

  • Validate Override Cycles value to contain only Y, N, I, U, 0-9, and semicolon characters.

  • Outputs the Settings Header in sample sheet when UMI - Read 1 Length or UMI - Read 2 Length have value.

  • Sets the next step for samples to REMOVE.

  • Generates the sample sheet and attaches it to the step. For more information, refer to Sample Sheet Generation. Sample sheet template used depends on the selected value of Samplesheet Format and/or Reverse Complement Workflow.

Routing Script & Register Step Completed Automation¹

Automatically triggered on exit of the step, this automation completes the following actions:

  • Invokes the changeWorkflow script. This script routes step outputs to the NovaSeq 6000 v3.8 workflow and queues them for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step.

  • Registers the completion time of the step by publishing messages to CLPA through Illumina Connected Analytics. This script is used only for CLPA support.

¹ These automations are required for the NovaSeq 6000 v3.8 workflow to function properly. These automations contain additional logic needed for CLPA support. If you would like to remove CLPA support, then contact Illumina Support.

Automations not identified with ¹ are required for the NovaSeq 6000 v3.8 to work function properly.

Master Step Fields

The following table lists field configuration details defined on the Dilute and Denature (NovaSeq 6000 v3.8) step. These fields are required for sample sheet and JSON file generation.

Dilute and Denature (NovaSeq 6000 v3.8) Master Step Field Configuration

Field Name

Field Type

Options

Additional Options and Dropdown Items

Analysis Software Version

Text

BaseSpace Sequence Hub Configuration

Text Dropdown

  • Required Field

  • Presets

    • Not Used

    • Run Monitoring Only

    • Run Monitoring and Storage

Custom Recipe Path

Text

  • Not applicable

Experiment Name

Text

  • Required Field

  • Not applicable

Index Read 1

Numeric Dropdown

  • Required Field

  • Custom Entries

  • Range = 0–20

  • Decimal places displayed = 0

  • Presets

    • 0

    • 6

    • 8

Index Read 2

Numeric Dropdown

  • Required Field

  • Custom Entries

  • Range = 0–20

  • Decimal places displayed = 0

  • Presets

    • 0

    • 6

    • 8

Output Folder

Text

  • Required Field

  • Not applicable

Override Cycles

Text

  • Not applicable

Paired End

Text Dropdown

  • Required Field

  • Presets

    • True

    • False

Read 1 Cycle

Numeric Dropdown

  • Required Field

  • Custom Entries

  • Range = 1–251

  • Decimal places displayed = 0

  • Presets

    • 251

    • 151

    • 101

    • 51

    ℹ Value of 251 is only supported for SP flow cell type. For all other cell types, maximum value is 151.

Read 2 Cycle

Numeric Dropdown

  • Required Field

  • Custom Entries

  • Range = 0–251

  • Decimal places displayed = 0

  • Presets

    • 251

    • 151

    • 101

    • 51

    ℹ Value of 251 is only supported for SP flow cell type. For all other cell types, maximum value is 151.

Reverse Complement Workflow

Toggle Switch

  • Required Field

  • Default

    • Yes

Run Mode

ℹ Not displayed in user interface

Text Dropdown

  • Read Only

  • Presets

    • SP

    • S1

    • S2

    • S4

Samplesheet Format

Text Dropdown

  • Required Field

  • Presets

    • V1 (default)

    • V2

Settings Header

ℹ Not displayed in user interface

Text

  • Read Only

  • Not applicable

UMI—Read 1 Length

Numeric

  • Range = 1

UMI—Read 1 Start From Cycle

Numeric

  • Range = 1

UMI—Read 2 Length

Numeric

  • Range = 1

UMI—Read 2 Start From Cycle

Numeric

  • Range = 1

Use Custom Index Read 1 Primer

Toggle Switch

  • Default

    • None Set

Use Custom Read 1 Primer

Toggle Switch

  • Default

    • None Set

Use Custom Read 2 Primer

Toggle Switch

  • Default

    • None Set

Use Custom Recipe

Toggle Switch

  • Required Field

  • Default

    • No

Validation Script

Multiline Text

  • Required Field

  • Read Only

  • Default value is provided in the drop-down section that follows the table.

    âš  Do not remove this field as it is used by Validate Run Setup and Generate Sample Sheet automation script.

Workflow

Text

  • Read Only

  • Default

    • GenerateFASTQ

Workflow Type

Text Dropdown

  • Required Field

  • Presets

    • No Index

    • Single Index

    • Dual Index

    • Custom

Validation Script

Global Fields

The following table lists the global custom fields that are configured to display on the Dilute and Denature (NovaSeq 6000 v3.8) step.

Global Field Configuration (Derived Sample)

Field Name

Field Type

Options

Additional Options and Dropdown Items

Flowcell Type

Text Dropdown

Required Field

Presets

  • SP

  • S1

  • S2

  • S4

Loading Workflow Type

Text Dropdown

Required Field

Presets

  • NovaSeq Standard

  • NovaSeq Xp

  • [Remove from workflow]

Protocol 3: NovaSeq Xp (NovaSeq 6000 v3.8)

Samples are routed to this protocol when their Loading Workflow Type value is set to NovaSeq Xp.

Samples are pooled and added to lanes on the NovaSeq flow cell type selected in the Define Run Format (NovaSeq 6000 v3.8) step. At the end of this protocol, the flow cell is sent to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) protocol.

This protocol contains the following three steps:

  • Step 1: Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8)

  • Step 2: Dilute, Denature & ExAmp (NovaSeq 6000 v3.8)

  • Step 3: Load to Flowcell (NovaSeq 6000 v3.8)

Step 1: Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8)

Manually place libraries into a pool.

Create only one pool per step.

  • Step input: NTP (normalized libraries)

  • Step output: Bulk pool

Validate Unique Indexes automation is not used. The Clarity LIMS configuration for pooling handles this functionality.

Register Step Started Automation¹

Automatically triggered on entry to the step, this automation registers the start time of the step by publishing messages to CLPA through Illumina Connected Analytics:

This automation is used only for CLPA support.

Validate Inputs Flowcell Type and Single Pool Automation

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

  • Checks that all samples in the pool have the same Flowcell Type assigned to them.

  • Checks that only one pool has been created.

  • Changes the value of error and logging messages to reference the type of pool passed as 'bulk' pool.

Calculate Volumes Automation

Triggered when the Calculate Volumes button is selected on the Record Details screen, this automation completes the following actions:

  • Calculates the number of samples in the pool.

  • Sets the value of the Bulk Pool Volume (ul) and PhiX Volume (ul) field, based on the selected Flowcell Type.

  • Calculates the Per Sample Volume (ul) to be added to the pool.

    âš  For accurate pipetting of each sample in a pool, the Per Sample Volume (ul) value must be greater than or equal to the Minimum Per Sample Volume (ul). The default value (set at 5) can be edited.

    Assuming the default Minimum Per Sample Volume (ul) value of 5, completes the following steps for a given batch:

    1. If the smallest Per Sample Volume (ul) value is less than 5, automatically assigns a value of 5 to the sample Adjusted Per Sample Volume (ul) field.

    2. Adjusts the Adjusted Per Sample Volume (ul) field value for all other samples in the batch. This adjustment is based on the ratio used to increase the lowest value to 5.

  • Calculates the Total Sample Volume (ul) field value.

  • If the Total Sample Volume is less than the Bulk Pool Volume, calculates the RSB Volume (ul) field value.

  • Copies the Flowcell Type and Loading Workflow Type values from the step inputs to the step outputs.

  • Uses the NovaSeq_Xp_Bulk_Pool.csv and NovaSeq_Xp_Bulk_Pool2.csv template files to generate a single CSV file containing information about the bulk pool and the samples it contains. The generated file is available for download on the Step Setup screen of the Dilute, Denature & ExAmp (NovaSeq 6000 v3.8) step.

  • Resets the Total Sample Volume (ul) and Number of Samples in Pool field values so that the automation is idempotent.

Set Next Step Automation

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

  • Copies the Flowcell Type values from the step inputs to the step outputs.

  • Sets the next step for samples to ADVANCE, advancing them to the next step in the protocol.

Register Pools & Register Step Completed Automation¹

Automatically triggered on exit of the step, the following automation completes the following actions:

  • Registers the pool and library information used by the Clarity LIMS workflow. Samples in the pool are assumed to have gone through library preparation.

  • Registers the completion time of the step. These actions are done by publishing messages to CLPA through Illumina Connected Analytics. This automation is used only for CLPA support.

¹ These automations are for CLPA support only.

Automations not identified with ¹ are required for the NovaSeq 6000 v3.8 to work function properly.

Master Step Fields

The following table lists field configuration details defined on the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8) step.

Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8) Master Step Field Configuration

Field Name

Field Type

Options

Additional Options and Dropdown Items

% PhiX (0.25nM) Spike-in

Numeric

  • Range = 0–100

Bulk Pool Volume (ul)

ℹ For calculation purposes, not displayed

Numeric

  • Decimal places displayed = 2

Minimum Per Sample Volume (ul)

Numeric

Required Field

  • Decimal places displayed = 2

  • Default

    • 5

Number of Lanes to Sequence

Numeric

Required Field

  • Decimal places displayed = 0

Number of Samples in Pool

ℹ For calculation purposes, not displayed

Numeric

  • Decimal places displayed = 0

  • Default

    • 0

PhiX Volume (ul)

ℹ For calculation purposes, not displayed

Numeric

  • Decimal places displayed = 2

Total Sample Volume (ul)

ℹ For calculation purposes, not displayed

Numeric

  • Decimal places displayed = 0

  • Default

    • 0

Global Fields

The following table lists the global custom fields that are configured to display on the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8) step.

Global Field Configuration (Derived Sample)

Field Name

Field Type

Options

Additional Options and Dropdown Items

RSB Volume (ul)

Numeric

Read Only

Decimal places displayed = 2

Flowcell Type

Text Dropdown

Required Field

Presets

  • SP

  • S1

  • S2

  • S4

Loading Workflow Type

Text Dropdown

Required Field

Presets

  • NovaSeq Standard

  • NovaSeq Xp

  • [Remove from workflow]

Step 2: Dilute, Denature & ExAmp (NovaSeq 6000 v3.8)

In this step, the addition of DPX, NaOH, Tris-HCl, and RSB denatures and dilutes the pooled samples. Manually create working pools based on the number of lanes that you want to sequence.

  • Step input: Bulk pool

  • Step output: Working pool - variable number, choose how many working pools to create per bulk pool

Validate Inputs Flowcell Type & Register Step Started Automation²

Automatically triggered at the beginning of the step, this automation invokes the validate_flowcell_for_input_pools script. This script completes the following actions:

  • Checks the inputs to the step and validates that the Flowcell Type field is set to a valid value (SP, S1, S2, or S4). The automation also validates that all inputs have the same value for this field.

  • Checks that the container type selected matches the value in the Flowcell Type field.

  • Registers the start time of the step by publishing messages to CLPA through Illumina Connected Analytics:

    This automation is used only for CLPA support.

Calculate Volumes Automation

Automatically triggered on entry to of the Record Details screen, this automation sets the value of the following fields based on the Flowcell Type:

  • BP Aliquot Volume (ul)

  • NaOH Volume (ul)

  • Tris-HCl Volume (ul)

  • DPX1 Volume (ul)

  • DPX2 Volume (ul)

  • DPX3 Volume (ul)

  • Mastermix per Lane (ul)

The automation also completes the following actions:

  • Copies the Flowcell Type and Loading Workflow Type values from the step inputs to the step outputs.

  • Uses the NovaSeq_Xp_Working_Pool.csv and NovaSeq_Xp_Working_Pool2.csv template files to generate a single CSV file. This file contains information about the DPX volume and the volume of BP Aliquot, Mastermix, NaOH, and Tris-HCI to add per working pool. The generated file is available for download on the Step Setup screen of the Load to Flowcell (NovaSeq 6000 v3.8) step.

Set Next Step Automation

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

  • Copies the Flowcell Type values from the step inputs to the step outputs.

  • Sets the next step for samples to ADVANCE, advancing them to the next step in the protocol:

Register Step Completed Automation¹

Automatically triggered on exit of the step, this automation registers the completion time of the step by publishing messages to CLPA through Illumina Connected Analytics. This script is used only for CLPA support.

¹ These automations are for CLPA support only.

² These automations are required for the NovaSeq 6000 v3.8 workflow to function properly. These automations contain additional logic needed for CLPA support. If you would like to remove CLPA support, then contact Illumina Support.

Automations not identified with ¹ or ² are required for the NovaSeq 6000 v3.8 to work function properly.

Master Step Fields

The following table lists field configuration details defined on the Load to Flowcell (NovaSeq 6000 v3.8) step. A script sets these field values. The values are not editable while running the step.

Dilute, Denature & ExAmp (NovaSeq v3.8) Master Step Field Configuration

Field Name

Field Type

Options

Additional Options and Dropdown Items

DPX1 Volume (ul)

Numeric

Read Only

Decimal places displayed = 0

DPX2 Volume (ul)

Numeric

Read Only

Decimal places displayed = 0

DPX3 Volume (ul)

Numeric

Read Only

Decimal places displayed = 0

Global Fields

The following table lists the global custom fields that are configured to display on the Dilute, Denature & ExAmp (NovaSeq 6000 v3.8) step.

Global Field Configuration (Derived Sample)

Field Name

Field Type

Options

Additional Options and Dropdown Items

BP Aliquot Volume (ul)

Numeric

Read Only

Decimal places displayed = 0

Flowcell Type

Text Dropdown

Required Field

Presets

  • SP

  • S1

  • S2

  • S4

Loading Workflow Type

Text Dropdown

Required Field

Presets

  • NovaSeq Standard

  • NovaSeq Xp

  • [Remove from workflow]

Mastermix per Lane (ul)

Numeric

Read Only

Decimal places displayed = 0

NaOH Volume (ul)

Numeric

Read Only

Decimal places displayed = 2

Tris-HCl Volume (ul)

Numeric

Read Only

Decimal places displayed = 2

Step 3: Load to Flowcell (NovaSeq 6000 v3.8)

In this step, scan the flow cell barcode into the Clarity LIMS. Then, manually place the working pools into the lanes of the flow cell for the NovaSeq run. This step validates the run setup information and generates the sample sheet file.

  • Step input: Working pool

  • Step output: Flow cell (output containers: SP, S1, and S2 with 2 lanes, and S4 with 4 lanes)

Validate Inputs and Selected Container & Register Step Started Automation²

Automatically triggered at the beginning of the step, this automation invokes the validate_flowcell_for_input_pools and validateSelectedContainer scripts. These scripts validate the step inputs and the container selected by the user, as follows.

  • Checks that the Flowcell Type field has been set to a valid value (SP, S1, S2, or S4) and that all inputs have the same value.

  • Checks that the number of outputs matches the number of lanes on the selected flow cell type. If validation fails, an error message informs that the number of working pools does not match the number of lanes available on the flow cell.

    The following defines the number of samples allowed for different flow cell types:

    • SP: 2 working pools

    • S1: 2 working pools

    • S2: 2 working pools

    • S4: 4 working pools

  • Checks that the container type selected on entry to the Placement screen matches the value in the Flowcell Type field. If validation fails, an error message displays.

  • Registers the start time of the step by publishing messages to CLPA through Illumina Connected Analytics:

    This automation is used only for CLPA support.

Validate Flowcell Barcode Automation

Automatically triggered on exit of the Placement screen, this automation

  • Validates the flow cell barcode scanned into the Clarity LIMS by the user, using the following logic:

  • Copies the Flowcell Type and Loading Workflow Type field values from step inputs to outputs:

Validate Run Setup and Generate Sample Sheet Automation

ℹ The script definition portion of Validate Run Setup has been moved into the Validation Script custom field due to the 4000 character limitation.

  • If modification is required to the Validate Run Setup script, perform the changes directly on Validation Script custom field default value.

  • The script definition portion of Generate Sample Sheet is unchanged.

Automatically triggered by selecting a button on the Record Details screen, this automation completes the following actions:

  • Copies the Flowcell Type from the step input to the Run Mode field (hidden):

  • Validates the parameters entered on the Record Details screen, which are used to set up the run and generate the sample sheet file. For more information, refer to the Respond to Validation / Recipe Request Call from Instrument section of NovaSeq 6000 Run.

    • Experiment Name can only contain alphanumeric, dash, or underscore characters. Spaces are not permitted.

    • When Workflow Type = No Index, both Index Read 1 and Index Read 2 must be zero.

    • When Workflow Type = Single Index, Index Read 1 must be greater than zero and Index Read 2 must be zero.

    • When Workflow Type = Dual Index, both Index Read 1 and Index Read 2 must be greater than zero.

  • Checks the Paired End and Read 2 Cycles field values.

    • If Paired End = True and Read 2 Cycles value is 0, an error displays.

    • If Paired End = False, Read 2 Cycles must be zero and UMI - Read 2 Length and UMI - Read 2 Start From Cycle must be empty.

  • Checks the Flowcell field value.

    If Flowcell Type value is not SP, checks that the value of both Read 1 Cycles and Read 2 Cycles is 151 or less. If value is greater than 151, the automation generates an error message.

  • Validates UMI Settings:

    • If UMI - Read 1 Length has value, UMI - Read 1 Start From Cycle must have value.

    • If UMI - Read 1 Length has no value, UMI - Read 1 Start From Cycle must not have value.

    • If UMI - Read 2 Length has value, UMI - Read 2 Start From Cycle must have value.

    • If UMI - Read 2 Length has no value, UMI - Read 2 Start From Cycle must not have value.

    • If UMI - Read 1 Length has not value, UMI - Read 2 Length must not have value.

    • If UMI - Read 1 Start From Cycle has no value, UMI - Read 2 Start From Cycle must not have value.

  • Validates Analysis Software Version to make sure it is a valid version string.

  • Checks Override Cycles field value. This value can contain only Y, N, I, U, 0–9, and semicolon characters.

  • Outputs the Settings Header in sample sheet when UMI - Read 1 Length or UMI - Read 2 Length have values.

  • Sets the next step for samples to ADVANCE, which advances to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step.

  • Generates the sample sheet and attaches it to the step. The sample sheet template depends on the selected value for Samplesheet Format and/or Reverse Complete Workflow. For more information, refer to Sample Sheet Generation.

Register Step Completed Automation¹

Registers the completion time of the step by publishing messages to CLPA through Illumina Connected Analytics. This script is used only for CLPA support.

¹ These automations are for CLPA support only.

² These automations are required for the NovaSeq 6000 v3.8 workflow to function properly. These automations contain additional logic needed for CLPA support. If you would like to remove CLPA support, then contact Illumina Support.

Automations not identified with ¹ or ² are required for the NovaSeq 6000 v3.8 to work function properly.

Master Step Fields

The following table lists the field configuration details defined on the Load to Flowcell (NovaSeq 6000 v3.8) step.

Load to Flowcell (NovaSeq 6000 v3.8) Master Step Field Configuration

Field Name

Field Type

Options

Additional Options and Dropdown Items

Analysis Software Version

Text

  • Not applicable

BaseSpace Sequence Hub Configuration

Text Dropdown

  • Required Field

  • Presets

    • Not Used

    • Run Monitoring Only

    • Run Monitoring and Storage

Custom Recipe Path

Text

  • Not applicable

Experiment Name

Text

  • Required Field

Index Read 1

Numeric Dropdown

  • Required Field

  • Custom Entries

  • Range = 0–20

  • Decimal places displayed = 0

  • Presets

    • 0

    • 6

    • 8

Index Read 2

Numeric Dropdown

  • Required Field

  • Custom Entries

  • Range = 0–20

  • Decimal places displayed = 0

  • Presets

    • 0

    • 6

    • 8

Library Tube Barcode

Text

  • Required Field

  • Not applicable

Output Folder

Text

  • Required Field

  • Not applicable

Override Cycles

Text

  • Not applicable

Paired End

Text Dropdown

  • Required Field

  • Presets

    • True

    • False

Read 1 Cycle

Numeric Dropdown

  • Required Field

  • Custom Entries

  • Range = 1–251

  • Decimal places displayed = 0

  • Presets

    • 251

    • 151

    • 101

    • 51

    ℹ Value of 251 is only supported for SP flow cell type. For all other cell types, maximum value is 151.

Read 2 Cycle

Numeric Dropdown

  • Required Field

  • Custom Entries

  • Range = 0–251

  • Decimal places displayed = 0

  • Presets

    • 251

    • 151

    • 101

    • 51

    ℹ Value of 251 is only supported for SP flow cell type. For all other cell types, maximum value is 151.

Reverse Complement Workflow

Toggle Switch

  • Required Field

  • Default

    • Yes

Run Mode

ℹ Not displayed in user interface

Text Dropdown

  • Read Only

  • Presets

    • SP

    • S1

    • S2

    • S4

Samplesheet Format

Text Dropdown

  • Required Field

  • Presets

    • V1 (default)

    • V2

Settings Header

ℹ Not displayed in user interface

Text

  • Read Only

  • Not applicable

UMI—Read 1 Length

Numeric

  • Range = 1

UMI—Read 1 Start From Cycle

Numeric

  • Range = 1

UMI—Read 2 Length

Numeric

  • Range = 1

UMI—Read 2 Start From Cycle

Numeric

  • Range = 1

Use Custom Index Read 1 Primer

Toggle Switch

  • Default

    • None Set

Use Custom Read 1 Primer

Toggle Switch

  • Default

    • None Set

Use Custom Read 2 Primer

Toggle Switch

  • Default

    • None Set

Use Custom Recipe

Toggle Switch

  • Required Field

  • Default

    • No

Validation Script

Multiline Text

  • Required Field

  • Read Only

  • Default value is provided in the drop-down section that follows the table.

    âš  Do not remove this field as it is used by Validate Run Setup and Generate Sample Sheet automation script.

Workflow

Text

  • Read Only

  • Default

    • GenerateFASTQ

Workflow Type

Text Dropdown

  • Required Field

  • Presets

    • No Index

    • Single Index

    • Dual Index

    • Custom

Validation Script

Global Fields

The following table lists the global custom fields that are configured to display on the Load to Flowcell (NovaSeq 6000 v3.8) step.

Global Field Configuration (Derived Sample)

Field Name

Field Type

Options

Additional Options and Dropdown Items

Flowcell Type

Text Dropdown

Required Field

Presets

  • SP

  • S1

  • S2

  • S4

Loading Workflow Type

Text Dropdown

Required Field

Presets

  • NovaSeq Standard

  • NovaSeq Xp

  • [Remove from workflow]

Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8)

This final protocol contains one fully automated step, AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8), described in the following section.

This step is fully automated. Do not add samples to the Ice Bucket or start the step manually. The sequencing service may not update samples correctly when they have been manually started.

Step 1: AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8)

  • Step input: Library tube from NovaSeq Standard or flow cell from NovaSeq Xp protocol

  • Step output: Result file/measurement

In this step, pooled samples are sequenced on the NovaSeq instrument and the run metrics are recorded in Clarity LIMS.

Register Step Started & Register NovaSeq Run & Register NovaSeq Run Association & Register NovaSeq Run Started 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 6000 sequencing run configuration

  • Registers the association of the sequencing run with the actual samples by linking the Instrument Run ID with samples ID

  • Registers the time that the sequencing run started

These actions are done by publishing messages to CLPA through Illumina Connected Analytics. This automation is used only for CLPA support.

Set Next Steps Automation²

By default, the Set Next Steps automation is disabled (set to Not Used) and the Sequencer API sets the next step for samples.

If necessary, override the default next step behavior used by the Sequencer API by performing the following steps:

  1. Enable the automation and use it to set custom next step behavior and generate a log file. By default, the automation sets the next step to ADVANCE and the samples complete the protocol.

  2. Configure the automation to trigger automatically on exit of the Record Details screen.

  3. Disable or configure auto-complete behavior by setting the values of the run.autoComplete and autoCompleteOnlyAtSuccess properties. For details, refer to Configuring autoComplete Properties.

Update Lane Number Automation

This automation triggers automatically upon entry to the Record Details screen.

âš  Do not disable or modify this automation to ensure the Lane Number displays properly.

Register NovaSeq RunCompleted & Register Step Completed Automation¹

Automatically triggered upon exiting the step, this automation registers the sequencing run completion time, run status, and run metrics.

These actions are done by publishing messages to CLPA through Illumina Connected Analytics. This automation is used only for CLPA support.

¹ These automations are for CLPA support only.

² Not used. By default, the Sequencer API determines this functionality. If necessary, enable the automation and use it to override the next step behavior used by the Sequencer API.

Automations not identified with ¹ are required for the NovaSeq 6000 v3.8 to work function properly.

Master Step Fields

The following fields are configured on the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step in Clarity LIMS:

  • Current Cycle

  • Current Read

  • Firmware Version

  • Flow Cell Expiration Date

  • Flow Cell ID

  • Flow Cell Lot Number

  • Flow Cell Mode

  • Flow Cell Part Number

  • Flow Cell Side

  • Instrument Control Software Version

  • Instrument ID

  • Instrument Type

  • Lane Counter

  • Loading Workflow Type

  • Output Folder

  • RTA Version

  • Run Completion Date

  • Run ID

  • Run Status

  • Sequencing Log

Global Fields

The following fields are used to capture the run metrics in Clarity LIMS:

  • % Aligned R1

  • % Aligned R2

  • % Bases >=Q30 R1

  • % Bases >=Q30 R2

  • % Error Rate R1

  • % Error Rate R2

  • % Phasing R1

  • % Phasing R2

  • % Prephasing R1

  • % Prephasing R2

  • %PF R1

  • %PF R2

  • Cluster Density (K/mm^2) R1

  • Cluster Density (K/mm^2) R2

  • Intensity Cycle 1 R1

  • Intensity Cycle 1 R2

  • Reads PF (M) R1

  • Reads PF (M) R2

  • Yield PF (Gb) R1

  • Yield PF (Gb) R2

Note the following details:

  • Values are aggregated across all lanes. Some values (e.g., Yield PF (Gb) R1) are summed while others are averaged.

  • The names listed previously are the default global custom field names installed with the NovaSeq Integration v3.8 configuration provided in the Illumina Preset Protocols (IPP) v2.6 or later.

  • All global configuration fields are configured on the Container entity.

  • All field names are configurable through the Custom Fields screen in the Global Fields tab.

  • If field names are changed in Clarity LIMS, they must also be changed through the run.metricUdfNames properties file in the application.yml file for the Sequencer API. For more information, refer to the application.yml Properties section in Components Installed. All fields are configured to be visible in the Sample Details table on the Record Details screen for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step.

These global custom fields are configured on the Container entity, visible on each input, but they should not be confused with per-lane metrics.

All run metrics tracked in Clarity LIMS are overall metrics for the run. There are currently no per-lane metrics provided by NovaSeq Control Software.

Sample Sheet Generation

The sample sheet is generated on the step before the run. This step is Dilute and Denature (NovaSeq 6000 v3.8) in the NovaSeq Standard protocol or Load to Flowcell (NovaSeq 6000 v3.8) in the NovaSeq Xp protocol. This step places samples on the library tube or flow cell that are loaded in the NovaSeq 6000 instrument.

In the default configuration, the Validate Run Setup and Generate Sample Sheet automation generates one CSV format sample sheet file. The bcl2fastq v2.20 downstream analysis uses this file.

The sample sheet is uploaded to the NVCS via the /Illumina/Sequencer/v2/sequencing-run/files endpoint to the Sequencer API. The file endpoint allows for a file to be downloaded from the Clarity LIMS using OAuth (instead of Basic Authentication), which is required for the NovaSeq 6000 instrument.

The run recipe response sets the sample sheet URL to the link to download the file from this endpoint and sets the sampleSheetRequiresOAuth value to true.

Sample Sheet Content

The following master step fields control the sample sheet content. These fields display in the Step Details area of the Record Details screen.

  • Experiment Name

  • Read 1 Cycles

  • Read 2 Cycles

  • Workflow

  • Reverse Complement Workflow

  • Samplesheet Format

Depending on Samplesheet Format field value selection, a v1 or v2 sample sheet is generated. For detailed information on the following items, refer to the Bcl2fastq2 Sample Sheet Generation section of Illumina Instrument Sample Sheets (NGS v5.17 and later):

  • Sample sheet generation script parameters and usage

  • Sample sheet data and configuration options

  • Enabling unique FASTQ file names per sequencing run

NovaSeq 6000 Run

The following steps outline the sequence of events that occurs when a flow cell is loaded onto the NovaSeq 6000 instrument.

User Authentication and Login

User authentication and login to the NovaSeq instrument is achieved via the getSequencerLoginURL endpoint (found at /Illumina/Sequencer/v2/sequencing-run/login). This endpoint returns the URL that the instrument uses to get an access token.

  1. The Sequencer API service provides the NovaSeq Control Software (NVCS) with a URL for the Clarity LIMS login screen and the NVCS displays this screen to the user. Log in to authorize the NVCS to use your account automatically to access Clarity LIMS.

  2. Enter credentials:

    • Username and password are passed to the Clarity LIMS server to make sure that the access is valid.

    • By default, the Sequencer API service tries to contact the Clarity LIMS at http://localhost:9080/clarity/. The installation script sets this URL, but it can be configured with the clarity.url property in the application.yml file. For more information, refer to the application.yml Properties section of Components Installed.

  3. The user is redirected to the Sequencer login page at /Illumina/Sequencer/v2/sequencer_login.

    • If authentication is successful, the screen displays a success message. The Clarity LIMS server provides the OAuth token and username by returning them in redirect to /Illumina/Sequencer/v2/sequencer_login/Success. This redirect does not reload the page, but allows the NovaSeq to continue with run setup.

    • If authentication fails, the user is left on the login screen, and a warning message informs them of the failure. A background redirect to /Illumina/Sequencer/v2/sequencer_login/Error informs the NovaSeq 6000 of the failure.

Respond to Validation / Recipe Request Call from Instrument

  1. The sequencing-run/recipe/novaseq endpoint consumes a POST request with a LibraryContainerId, FlowCellId, and reagents information.

    • LibraryContainerId, FlowCellId, and reagent information is provided by the NovaSeq 6000.

    • LibraryContainerId and FlowCellId can match either the library tube ID or flow cell ID of the corresponding containers in Clarity LIMS.

    • If there are no containers or multiple containers, an error is generated.

  2. All populated containers whose name matches either the LibraryContainerId or FlowCellId are validated as follows.

    • All samples in the container must be queued for a sequencing run step.

    • All samples must be queued for only one sequencing run step.

    • All samples must be queued for the same sequencing run step.

    ℹ Configure the steps that are considered sequencing run steps in the novaseq.sequenceStepNames property in the application.yml file. The property is a list and each list entry must be the exact name of a sequencing step, not the name of the underlying master step/process type. For more information, refer to the application.yml Properties section of Components Installed.

  3. After a single container has passed validation, the run setup information (run recipe request) is returned to the NVCS via the API. Then, the run recipe is generated.

    • The run recipe information is read from fields configured on the parent process/master step of the samples in the container. The workflow must include recipe definition in that step.

    • By default, the parent process/master step is the Dilute and Denature (NovaSeq 6000 v3.8) or Load to Flowcell (NovaSeq 6000 v3.8) step, depending on workflow.

    • The default field values match the names of the fields in the NovaSeq v3.8 workflow configuration included in IPP. The field names are configurable through the recipe.udfNames.* properties. Each of these properties corresponds to an entry in the recipe. The value of a property defines the name of the field that is used to populate that entry in the recipe.

    • It is assumed that this parent process/master step removes any illegal characters from the Experiment Name field.

    • The Clarity LIMS download link to the sample sheet created on the run setup step is used for the SampleSheet value in the recipe. The name of the output of the sample sheet on the setup step can be configured through the recipe.sampleSheet.outputName property (Sample Sheet by default) in the application.yml file. For more information, refer to the application.yml Properties section of Components Installed. If no matching output is found, or no file was attached to the output, then the value of the recipe.sampleSheet.notAvailableValue property (Not Available by default) is used in the recipe.

  4. After the recipe is generated, it is returned as a *.json file in the response, in the form defined by the Swagger definition for the API.

Run Recipe Content

The sample sheet content and the fields configured on the Dilute and Denature (NovaSeq 6000 v3.8) and Load to Flowcell (NovaSeq 6000 v3.8) steps control the content of the run recipe.

The following example shows the run recipe content:

Respond to Sequencing Started Call from Instrument

  1. Matching container for the run (library tube or flow cell) is found, and contents are validated to be queued for the correct step.

    ℹ If no matching container is found, or if the samples in the container are incorrectly queued, an error occurs and the step is not started. This validation is also done during the run recipe request immediately prior, so this error case should not be encountered.

  2. Reagent kits that are configured for the Sequencer API are checked against the configuration for the step to be run:

    • If a kit is configured for the API but does not exist in Clarity LIMS, it is created.

    • If a kit exists, but is not enabled on the step to be run, the step configuration is updated to enable it.

      • If a kit that is to be enabled is Archived, it is set to Active.

      • If a kit that is to be enabled is in Pending status, it is set to Active.

    • If any kits enabled on the step are not configured for the Sequencer API, a warning is logged. The API cannot complete the step without user intervention.

    • If any kits provided in the recipe request are not configured for the Sequencer API, they are ignored. A warning is logged during lot tracking.

  3. The type of flow cell being used in the run determines the number of replicates for the step.

    • For Standard mode, this information is based on the number of lanes in the flow cell. This information is stored as part of the Sequencer API configuration.

    • For XP mode, there is always one replicate, because there is already one input tracked for each lane.

  4. The automated sequencing step is started.

  5. Reagent lots are attached to the step. The Flow Cell and Library Tube reagents in the request are ignored. Information from these reagents is tracked in Clarity LIMS as containers and custom fields.

    Any other reagents in the request are handled as follows.

    • If the reagent is not configured to be tracked, a warning is logged, and the reagent is ignored.

    • Existing reagent lots are looked up by kit name (determined by Sequencer API configuration), name (the serial number in the request), and lot number (the lot number in the request).

    • If one or more matching reagent lots exist in Clarity LIMS, the newest one is used.

    • If the expiry date does not match the expiry in the request, or if the status is not set to Active, the lot is updated to have the correct expiry date and an Active status.

    Otherwise a new lot is created with the following configuration:

    • Reagent Kit — Matching kit as determined from Sequencer API configuration and step 2 above.

    • Name — Serial number in request

    • Lot Number — Lot number in request

    • Expiry — Expiry date in request

    • Status — Active

    If any reagents are expected to be in the request but are not, these reagents are logged. This action prevents the step from being completed without user intervention.

  6. Master step custom fields are updated with the run information parsed from the request. For more information, refer to Respond to Record InterOps Metrics Call from Instrument.

  7. Any important events during this request are logged into the Sequencer API log file and in the Sequencing Log multiline global custom field on the step (assuming the step was started successfully). For more information, refer to Respond to Record InterOps Metrics Call from Instrument.

Respond to Record InterOps Metrics Call from Instrument

When the metrics endpoint receives a POST, it completes the following actions:

  • Looks for a matching library tube or flow cell in Clarity LIMS.

  • Find the in-progress sequencing step for that container.

When the step is found, the run metrics in the request are written to container global custom fields in Clarity LIMS.

Respond to Sequencing Completed Call from Instrument

If the sequencing run is successful, after the metrics are recorded, the API call for Sequencing Completed is received. Information is matched by Sequencing Container and the following occurs in Clarity LIMS:

  1. The Current Cycle, Current Read, and Status master step custom fields are updated.

  2. The multiline Sequencing Log master step field is updated.

  3. A success message is recorded in the log file at the info level:

  4. The Clarity LIMS waits for any automations that are triggered automatically as it advances the step.

  5. By default, the Clarity LIMS assigns the next steps on the outputs to move them on to the first configured next step. If there are no next steps, the protocol is completed.

Respond to Sequencing Errored Call from Instrument

If the sequencing run is unsuccessful, the API call for Sequencing Error is received. Information is matched by Sequencing Container and the following occurs in Clarity LIMS:

  1. The Current Cycle, Current Read, and Status master step custom fields are updated.

  2. The multiline Sequencing Log master step field is updated.

  3. A failure message is recorded in the log file at the error level:

  4. The step is left at the Record Details screen.

Respond to Sequencing RunEndedByUser Call from Sequencer

If you end a sequencing run, the API call for Sequencing RunEndedByUser is received. Information is matched by Sequencing Container and the following occurs in Clarity LIMS:

  1. The Current Cycle, Current Read, and Status master fields are updated.

  2. The multiline Sequencing Log master step field is updated.

  3. The following message is recorded in the log file, at the warning level:

  4. The step is left at the Record Details screen.

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.

Illumina NovaSeq 6000 Integration v3.6.0 is distributed as the BaseSpaceLIMS-sequencer-api RPM package. This RPM package must be installed on the Clarity LIMS server.

The BaseSpaceLIMS-sequencer-api RPM installs the following items:

  • Sequencer API WAR file

  • application.yml configuration file

  • Two configuration scripts:

    • configure_sequencer_api_proxy.sh

    • configure_sequencer_api_application.sh

The NovaSeq 6000 v3 configuration is delivered through Illumina Preset Protocols (IPP) v2.6, which requires installation of the NGS Extensions Package v5.23.0 or later.

  • If the NGS Extensions Package is not already installed, or if a version earlier than v5.23.0 is installed, the latest version is installed by default with the NovaSeq 6000 integration. For more information, refer to NovaSeq 6000 Integration v3.6.0 Release Notes.

  • If NGS Extensions Package v5.23.0 or later is already installed, the upgrade is not forced or required.

NovaSeq 6000 Integration RPM Components

The following table lists the components installed by the RPM package.

Files Installed

Location

Description

Illumina#Sequencer#v2.war

/opt/gls/clarity/tomcat/current/webapps

War file for Sequencer API application.

configure_sequencer_api_application.sh

/opt/gls/clarity/config/

Script that configures the Sequencer API application through its external application.yml file.

configure_sequencer_api_env.sh

/opt/gls/clarity/config/

Script that configures the Clarity LIMS Tomcat configuration to include Secret Util settings.

configure_sequencer_api_proxy.sh

/opt/gls/clarity/config/

Script that configures the proxy to allow communication with the Sequencer API application.

sequencer-api.conf

/etc/httpd/clarity/

Proxy setting to communicate with the Clarity LIMS.

application.yml Properties

The application.yml file is at opt/gls/clarity/extensions/sequencer-api/.

  • All properties are configured automatically during installation, but they can also be configured by editing the application.yml file and then restarting Tomcat.

  • Properties in the file represent all custom field names, both for recipe and run information.

  • For all custom fields, the names (values in this file) can be changed. However, the property names cannot be changed, removed, or added to. For example, Loading Workflow Type can be changed, but recipe.udfNames.sampleLoadingType cannot.

application.yml Properties Installed

Property

Description

Default Value

spring.profiles.active

Tells the application that it is running deployed in Tomcat.

  • tomcat

âš  Do not change

clarity.url

Base URL that the Sequencer API service uses to contact the Clarity LIMS. The installation script (configure_sequencer_api_application) prompts for this URL.

clarity.username

Username to be used when communicating with Clarity LIMS.

security.signing-key

Private key that is used when signing/validating OAuth tokens. Changing this key invalidates any issued tokens.

security.token-expiry

Specifies (in hours) the expiry period for login tokens issued by the Sequencer API.

  • 88

novaseq.sequenceStepNames

List of NovaSeq sequencing run step names for which the integration can find samples queued.

  • Names must be an exact match to the name of the step in Clarity LIMS (not the master step/process type).

  • Each line must be indented to the same point and start with a dash (-).

  • - "AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8)"

  • - "AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.1)"

novaseq.flowcells

List of supported NovaSeq flow cell types.

  • Type names exactly match the values used by the NVCS run status requests.

  • To add more types, provide both type and lanes properties, indented to the same point and starting with a dash (-).

  • - type: "SP" lanes: 2

  • - type: "S1" lanes: 2

  • - type: "S2" lanes: 2

  • - type: "S4" lanes: 4

novaseq.reagents

List of supported reagent kits to be tracked on sequencing steps.

  • lotName — The name of the lot as it appears in the run started request.

  • kitName — The name of the corresponding reagent kit in Clarity LIMS that the lot is tracked as.

To add more types, provide both type and lanes properties, indented to the same point and starting with a dash (-).

ℹ The name can differ in the NVCS request versus the Clarity LIMS configuration.

  • - lotName: "Buffer" kitName: "Buffer Cartridge"

  • - lotName: "Cluster" kitName: "Cluster Cartridge"

  • - lotName: "SBS" kitName: "SBS Cartridge"

recipe.udfNames

Configures the names of the fields used in the run recipe. For more information, refer to the Run Recipe Contents section of NovaSeq 6000 Run.

  • Each value must be enclosed in quotes (e.g., recipe.udfNames.runMode "Run Mode").

  • Value provided for sampleLoadingType must match the value provided in the run configuration (e.g., recipe.udfNames.sampleLoadingType "Loading Workflow Type").

recipe.sampleSheet.outputName recipe.sampleSheet.notAvailableValue

Configures the name of the sample sheet file placeholder on the step where run recipe information is populated, and the property value to use when the file is not found.

run.stepUdfNames

Configures the master step field names to be used when recording run results. For more information, refer to the Master Step Fields section of Step 1: AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.7) in Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8).

  • Each value must be enclosed in quotes (e.g., run.stepUdfNames.runID "Run ID")

  • Value provided for sampleLoadingType must match the value provided in the run configuration (e.g., run.stepUdfNames.sampleLoadingType "Loading Workflow Type"). It is expected that the run step field and the field on the input derived sample share a name.

run.metricUdfNames

Configures the result file custom fields/measurement global field names to be used when recording run results. These fields capture the run data. For more information, refer to the Global Fields section of Step 1: AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.7) in Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8).

run.autoCompleteOnlyAtSuccess

Determines which of the following options that the sequencing run step must be:

  • Autocompleted when the run status is RunCompletedSuccessfully (if autoCompleteOnlyAtSuccess is true)

  • Autocompleted regardless of the run status (if autoCompleteOnlyAtSuccess is false). For more information, refer to Configuring autoComplete Properties.

  • true

Reagent Categories/Label Groups

  • TruSeq HT Adapters v2 (D7-D5)

Reagent Kits

  • Buffer Cartridge

  • Cluster Cartridge

  • DPX1

  • DPX2

  • DPX3

  • NaOH

  • Resuspension Buffer (RSB)

  • SBS Cartridge

  • Tris HCl

Container Types

  • Library Tube

  • SP

  • S1

  • S2

  • S4

This integration supports the following items:

  • Library tube with barcode provided in the format [A-Z]{2}[0-9]{7}-[A-Z]{3} (eg, AB1234567-XYZ)

  • SP, S1, S2, or S4 flow cell with barcode provided in one of the following formats:

    • SP and S1 flow cell: [A-Z0-9]{5}DR[A-Z0-9]{2}

    • S2 flow cell: [A-Z0-9]{5}DM[A-Z0-9]{2}

    • S4 flow cell: [A-Z0-9]{5}DS[A-Z0-9]{2}

    Example: H1991DMXX

NVCS Configuration

For details on configuring NVCS for integration with Clarity LIMS, contact the Clarity LIMS Support team.

Routing Script Requirements

The requirements for the routing script functionality are as follows.

  1. On the steps that use the routing script (Define Run Format (NovaSeq 6000 v3.8) and Dilute and Denature (NovaSeq 6000 v3.8)), the Next Step for all samples must be set to Remove from workflow. A script sets this value. The value must not change in the Assign Next Steps screen.

  2. In the Next Steps section of the protocol configuration screen, the method of assigning the next step must be set to Automation for the last step of the protocol.

Configuring autoComplete Properties

The application.yml configuration file contains the following properties:

  • run.autoComplete

  • run.autoCompleteOnlyAtSuccess

These properties determine the conditions under which the last step of the NovaSeq 6000 workflow (AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8)) is automatically completed when the run status is "RunCompletedSuccessfully" (default behavior) or regardless of the run status.

By default, both properties are set to true and the step only completes when the run is successful. If the run fails or is aborted, manually complete the step in Clarity LIMS.

For the step to autoComplete regardless of the result of the sequencing run, change the autoCompleteOnlyAtSuccess property value to false.

Enabling/Disabling autoComplete Properties

  1. Open the application.yml file at /opt/gls/clarity/extensions/sequencer-api/application.yml:

  2. Edit the value of autoCompleteOnlyAtSuccess property as required.

  3. Save the file.

The following table shows how the combined value of run.autoComplete and run.autoCompleteOnlyAtSuccess properties affects the autoComplete behavior of the sequencing step.

run.autoComplete and run.autoCompleteOnlyAtSuccess Value Matrix

run.autoComplete Property Value

run.autoCompleteOnlyAtSuccess Property Value

Outcome

true

true

  • Step automatically completes only if sequencing run is successful.

  • If run is not successful, step does not automatically complete, and the run details are recorded in Clarity LIMS.

true

false

  • Step automatically completes, regardless of the run status.

false

true

  • Step does not automatically complete.

  • Run details are recorded in Clarity LIMS.

false

false

  • Step does not automatically complete.

  • Run details are recorded in Clarity LIMS.

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.

  • The library tube ID must be unique. There must not be multiple library tube containers in the system with the same name.

  • Reagent labels (indexes) must be unique.

  • Only controls are permitted as unindexed samples; all other unindexed samples and pools are not permitted.

  • For sample sheet generation constraints, refer to the Bcl2fastq2 Sample Sheet Generation section in Illumina Instrument Sample Sheets (NGS v5.17 & later).

  • Do not manually start the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8) step. This step is a fully automated and the sequencing service may not update samples correctly if they have been manually started.

  • For the automated run to start successfully, Validate Run Setup and Generate Sample Sheet must be selected.

PreviousInstallationNextUser Interaction, Validation and Troubleshooting

Last updated

Was this helpful?