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Configuration

The Illumina NovaSeq 6000 Integration Package v2.6 supports the integration of Clarity LIMS to NovaSeq 6000 instruments.

This document describes the integration and includes information about the preconfigured protocols, steps, automations, installed components, configuration requirements, rules, and constraints.

For instructions on user interaction for each step, validating and troubleshooting the Illumina NovaSeq 6000 Integration, refer to NovaSeq 6000 Integration v2.6.0 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 the process.

Prerequisites and Assumptions

It is assumed that samples enter the NovaSeq 6000 v2.3 workflow as normalized libraries. That is, before they are assigned to the workflow the following actions occur:

  • Samples have been accessioned into 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 v2.6.0 includes two workflows:

  • NovaSeq Validation Library Prep (NovaSeq 6000 v2.3)

  • NovaSeq 6000 v2.3

NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) Workflow

Protocol: NovaSeq Validation Library Prep (NovaSeq 6000 v2.3)

Purpose:

  • Included for validation purposes only, this protocol models the library prep steps required to advance samples to the Run Format (NovaSeq 6000 v2.3) protocol.

  • The protocol contains a single step: NovaSeq Validation Library Prep (NovaSeq 6000 v2.3). At the end of this step, a routing script sends the samples to the first step of the NovaSeq 6000 v2.3 workflow, which is Define Run Format (NovaSeq 6000 v2.3).

Steps:

  1. NovaSeq Validation Library Prep (NovaSeq 6000 v2.3)

NovaSeq 6000 v2.3 Workflow

Protocol 1: Run Format (NovaSeq 6000 v2.3)

Purpose:

  • Allows for the assignment of per sample values for Loading Workflow Type, Normalized Molarity, Flowcell Type, and Final Loading Concentration (pM).

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

    • Normalized Molarity: Enter a value for each sample.

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

    • Final Loading Concentration (pM): Select from the options 225 (PCR-free workflows) or 400 (Nano workflows). Alternatively, 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 v2.3)

Protocol 2: NovaSeq Standard (NovaSeq 6000 v2.3)

Purpose:

  • Samples are pooled and added to the library tube in preparation for the NovaSeq run. Sample sheet and run recipe files are generated.

  • Routing script sends library tube to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) protocol.

Steps:

  1. Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3)

  2. Dilute and Denature (NovaSeq 6000 v2.3)

Protocol 3: NovaSeq Xp (NovaSeq 6000 v2.3)

Purpose:

  • Samples are pooled and added to lanes on the NovaSeq flow cell. The flow cell type is determined by the option selected in the Define Run Format (NovaSeq 6000 v2.3) step. Sample sheet and run recipe files are generated.

  • Samples are queued for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) protocol.

Steps:

  1. Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3)

  2. Dilute, Denature & ExAmp (NovaSeq 6000 v2.3)

  3. Load to Flowcell (NovaSeq 6000 v2.3)

Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq6000 v2.3)

Purpose:

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

  • This protocol contains one fully automated step.

    ⚠ Do not add samples to the Ice Bucket or start the step. The integration does this action automatically.

Steps:

  1. AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3)

Validation Workflow

The NovaSeq Validation Library Prep (NovaSeq 6000 v2.3) workflow allows for validation of the system after installation is complete. For details, refer to NovaSeq 6000 Integration v2.6.0 User Interaction, Validation and Troubleshooting.

Protocol 1: Run Format (NovaSeq 6000 v2.3)

This protocol sets the Loading Workflow Type and allows you to choose the appropriate Flowcell Type and Final Loading Concentration(pM). At the end of the protocol, a routing script sends the normalized libraries to either the NovaSeq Standard (NovaSeq 6000 v2.3) or the NovaSeq Xp (NovaSeq 6000 v2.3) protocol.

This protocol contains one step: Define Run Format (NovaSeq 6000 v2.3).

Step 1: Define Run Format (NovaSeq 6000 v2.3)

  • Step input: NTP (normalized libraries)

  • Step output: None

Set Next Steps Automation

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

  • Sets the next step for samples to REMOVE with

    nextStep = ::REMOVE::

  • Calculates the Minimum Molarity using the following formula:

    input.::Minimum Molarity (nM):: = (5 * input.::Final Loading Concentration (pM)::)/1000

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

    if (!input.hasValue(::Normalized Molarity (nM)::)) { fail(::The Normalized Molarity cannot be empty.::) ; }

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

    else if (input.::Normalized Molarity (nM):: < input.::Minimum Molarity (nM)::) { input.::Warning:: = ::The Normalized Molarity is too low.:: ; input.::Loading Workflow Type:: = ::[Remove from workflow]:: ; } else { input.::Warning:: = ::n/a:: }

    At this point, there are two options:

    • Correct the Normalized Molarity value on the Record Details screen. Also, 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 Automation

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 of NovaSeq Standard are routed to the NovaSeq 6000 v2.3 workflow and queued for the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3) step.

  • Samples with Loading Workflow Type field value of NovaSeq Xp are routed to the NovaSeq 6000 v2.3 workflow and queued for the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3) step.

The default automation command line is as follows.

bash -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar
-u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \
\
--FIELD_NAME 'Loading Workflow Type' \
--FIELD_VALUE 'NovaSeq Standard' \
--WORKFLOW 'NovaSeq 6000 v2.3' \
--STEP 'Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3)' \
--INPUTS_OR_OUTPUTS 'INPUTS' \
\
--FIELD_NAME 'Loading Workflow Type' \
--FIELD_VALUE 'NovaSeq Xp' \
--WORKFLOW 'NovaSeq 6000 v2.3' \
--STEP 'Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v2.3)' \
--INPUTS_OR_OUTPUTS 'INPUTS'"

Master Step Fields

The following table provides field configuration details for the fields defined on the Define Run Format (NovaSeq 6000 v2.3) step.

Define Run Format (NovaSeq 6000 v2.3) Master Step Field Configuration

Field Name

Field Type

Field Constraints/Options

Preset Values/Additional Options and Drop-Down Items

Comment

Multiline Text

None

Instruction

Text

  • Read Only

Default

  • Add Flowcell Type and Loading Workflow Type below

Flowcell Type

Text Dropdown

  • Required Field

  • Custom Entries

ℹ (does not show on Record Details screen)

Presets

  • S1

  • S2

  • S3

  • S4

Loading Workflow Type

Text Dropdown

  • Required Field

ℹ Hidden (does not show on Record Details screen)

Presets

  • NovaSeq Standard

  • NovaSeq Xp

Global Fields

The following table lists the global fields that are configured to display on the Define Run Format (NovaSeq 6000 v2.3) step.

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

  • Presets

    • 225

    • 400

  • Decimal places displayed = 0

Flowcell Type

Text Dropdown

  • Required Field

  • Presets

    • S1

    • S2

    • S4

    • SP

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 v2.3)

Samples are routed to this protocol if the 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 v2.3) protocol.

This protocol contains two steps:

  1. Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3)

  2. Dilute and Denature (NovaSeq 6000 v2.3)

Step 1: Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3)

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

Only create one pool per step.

  • Step input: NTP (normalized libraries)

  • Step output: Bulk pool

The following automations are configured on the step (in the order they are triggered at run time).

Validate Inputs Flowcell Type and Single Pool Automation

Automatically triggered on exit of the Pooling screen, this automation checks the following information:

  • All samples in the pool have the same Flowcell Type assigned to them.

  • Only one pool has been created.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/novaseq/novaseq-extensions.jar script:validate_flowcell_for_input_pools -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -validateSingleOutput true -poolType bulk
Calculate Volumes Automation

Due to the 4000-character limit, part of the script definition of Calculate Volumes has been moved into the Calculate Volumes Script field. If this part of the script needs to be modified, you can make the changes to the default value of the Calculate Volumes Script field. The parts of the script that were moved into the Calculate Volumes Script field are identified below.

Automatically triggered when the Calculate Volumes button on the Record Details screen is selected, this automation does the following actions:

  • Calculates the number of samples in the pool:

    step.::Number of Samples in Pool:: = step.::Number of Samples in Pool:: + 1

  • Sets the value of the Bulk Pool Volume (ul) and PhiX Volume (ul) fields, based on the selected Flowcell Type. The following part of the automation is found in the Calculate Volumes Script field:

    step.::PhiX Volume (ul):: = !step.hasValue(::% PhiX (2.5nM) Spike-In::) ? 0 : step.::% PhiX (2.5nM) Spike-In::;
if (input.::Flowcell Type:: == ::SP::) {
    step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 100;
    step.::PhiX Volume (ul):: *= 0.6
};
if (input.::Flowcell Type:: == ::S1::) {
    step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 100;
    step.::PhiX Volume (ul):: *= 0.6
};
if (input.::Flowcell Type:: == ::S2::) {
    step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 150;
    step.::PhiX Volume (ul):: *= 0.9
};
if (input.::Flowcell Type:: == ::S4::) {
    step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 310;
    step.::PhiX Volume (ul):: *= 1.9
};
if (step.::PhiX Volume (ul):: == 0) {
    step.::PhiX Volume (ul):: = ::::
};

  • Calculates the Per Sample Volume (ul) to be added to the pool. The following part of the automation is found in the Calculate Volumes Script field:

    input.::Per Sample Volume (ul):: = ( ( (input.::Final Loading Concentration (pM):: * 5 / 1000) / input.::Normalized Molarity (nM):: ) * step.::Bulk Pool Volume (ul):: ) / step.::Number of Samples in Pool::

    ℹ To ensure accurate pipetting of each sample in a pool for sequencing, the Per Sample Volume (ul) value must be equal to or higher than the Minimum Per Sample Volume (ul). The default value is set at 5, which can be edited. Assuming the default Minimum Per Sample Volume (ul) value of 5, for a given batch:

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

    2. Clarity LIMS then adjusts the Adjusted Per Sample Volume (ul) field value for all other samples in the batch, based on the ratio used to increase the lowest value to 5.

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

    step.::Total Sample Volume (ul):: = step.::Total Sample Volume (ul):: + input.::Adjusted Per Sample Volume (ul)::

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

    if (step.::Total Sample Volume (ul):: >= step.::Bulk Pool Volume (ul)::) {output.::RSB Volume (ul):: = 0} else {output.::RSB Volume (ul):: = step.::Bulk Pool Volume (ul):: - step.::Total Sample Volume (ul)::} ;

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

    output.::Flowcell Type::= input.::Flowcell Type::;output.::Loading Workflow Type::= input.::Loading Workflow Type::;

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

    step.::PhiX Volume (ul):: = !step.hasValue('% PhiX (2.5nM) Spike-In') ? 0 : step.'% PhiX (2.5nM) Spike-In';
if (input.::Flowcell Type:: == ::SP::) {
    output.::Volume of Pool to Denature (ul):: = 100;
    output.::NaOH Volume (ul):: = 25;
    output.::Tris-HCl Volume (ul):: = 25;
}
if (input.::Flowcell Type:: == ::S1::) {
    output.::Volume of Pool to Denature (ul):: = 100;
    output.::NaOH Volume (ul):: = 25;
    output.::Tris-HCl Volume (ul):: = 25;
}
if (input.::Flowcell Type:: == ::S2::) {
    output.::Volume of Pool to Denature (ul):: = 150;
    output.::NaOH Volume (ul):: = 37;
    output.::Tris-HCl Volume (ul):: = 38;
}
if (input.::Flowcell Type:: == ::S4::) {
    output.::Volume of Pool to Denature (ul):: = 310;
    output.::NaOH Volume (ul):: = 77;
    output.::Tris-HCl Volume (ul):: = 78;
}

  • 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 the following step: Dilute and Denature (NovaSeq 6000 v2.3).

    script:driver_file_generator \ -t /opt/gls/clarity/extensions/novaseq/templates/NovaSeq_Standard_Bulk_Pool1.csv \ -o 1.csv \ script:driver_file_generator \ -t /opt/gls/clarity/extensions/novaseq/templates/NovaSeq_Standard_Bulk_Pool2.csv \ -o 2.csv \ script:driver_file_generator \ -t /opt/gls/clarity/extensions/novaseq/templates/NovaSeq_Standard_Bulk_Pool3.csv \ -o 3.csv \&& cat 1.csv 2.csv 3.csv > {compoundOutputFileLuid0}.csv

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

    step.'Number of Samples in Pool' = step.'Number of Samples in Pool' - 1;
step.'Total Sample Volume (ul)' = step.'Total Sample Volume (ul)' - input.'Adjusted Per Sample Volume (ul)'
Set Next Step Automation

Automatically triggered on exit of the Record Details screen, the following automation sets the next step for samples to ADVANCE, advancing them to the Dilute and Denature (NovaSeq 6000 v2.3) step in the protocol:

nextStep = ::ADVANCE::
Validate Unique Indexes Automation

ℹ Not used. This functionality is handled by the Clarity LIMS configuration for pooling

Master Step Fields

The following table provides field configuration details for the fields defined on the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3) step.

Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3) Master Step Field Configuration

Field Name

Field Type

Options

Additional Options and Drop-Down Items

% PhiX (2.5nM) Spike-In

Numeric

  • Range = 1–100

  • Decimal places displayed = 0

Bulk Pool Volume (ul)

ℹ For calculation purposes, not displayed

Numeric

  • Decimal places displayed = 2

Minimum Per Sample Volume (uL)

Numeric

  • Required Field

  • Default

    • 5

  • Decimal places displayed = 2

Number of Flowcells to Sequence

Numeric

  • Required Field

  • Range = 1–10

  • Decimal places displayed = 0

Number of Samples in Pool

ℹ For calculation purposes, not displayed

Numeric

  • Default

    • 0

  • Decimal places displayed = 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

  • Default

    • 0

  • Decimal places displayed = 2

Calculate Volumes Script

Text

  • Required Field

  • Read Only

  • Default value provided in the drop-down section following the table.

⚠ Do not remove this field. It is used by the Calculate Volumes automation script.

Calculate Volumes Script
step.::PhiX Volume (ul):: = !step.hasValue(::% PhiX (2.5nM) Spike-In::) ? 0 : step.::% PhiX (2.5nM) Spike-In::; if (input.::Flowcell Type:: == ::SP::) { step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 100; step.::PhiX Volume (ul):: *= 0.6 }; if (input.::Flowcell Type:: == ::S1::) { step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 100; step.::PhiX Volume (ul):: *= 0.6 }; if (input.::Flowcell Type:: == ::S2::) { step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 150; step.::PhiX Volume (ul):: *= 0.9 }; if (input.::Flowcell Type:: == ::S4::) { step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 310; step.::PhiX Volume (ul):: *= 1.9 }; if (step.::PhiX Volume (ul):: == 0) { step.::PhiX Volume (ul):: = :::: }; input.::Per Sample Volume (ul):: = (((input.::Final Loading Concentration (pM):: * 5 / 1000) / input.::Normalized Molarity (nM)::) * step.::Bulk Pool Volume (ul)::) / step.::Number of Samples in Pool::

Global Fields

The following table lists the global fields that are configured to display on the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v2.3) step.

Global Field Configuration (Derived Sample)

Field Name

Field Type

Options

Additional Options and Drop-Down Items

Flowcell Type

Text Dropdown

Required Field

Presets

  • S1

  • S2

  • S4

  • SP

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 v2.3) 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 v2.3)

In this step, pooled samples are denatured and diluted by the addition of NaOH, Tris-HCl, and Resuspension Buffer (RSB). Samples are manually placed into the library tube that is used in the NovaSeq run. In addition, this step generates the sample sheet file and the run recipe (*json file) needed to start the NovaSeq run. The step input is Bulk pool, and the step output is Library tube.

Validate Single Input Automation

Automatically triggered at the beginning of the step, the following automation checks that there is only one container input to the step:

script:validateSampleCount -min 1 -max 1
Validate Library Tube Barcode Automation

Automatically triggered on exit of the Placement screen, the following automation validates the library tube barcode to make sure it conforms to the barcode mask [A-Z]{2}[0-9]{7}-[A-Z]{3}:

if (!output.container.name.matches(::[A-Z]{2}[0-9]{7}-[A-Z]{3}::)){
    fail(::Invalid Library Tube Barcode. Please verify and try again.::)
}
Generate Sample Sheet & Run Recipe Automation

Automatically triggered when a button on the Record Details screen is selected, this automation does the following actions:

  • Copies the Flowcell Type from the step input to the step output:

    output.::Flowcell Type:: = input.::Flowcell Type::

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

    step.::Run Mode:: = input.::Flowcell Type::

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

    output.::Loading Workflow Type:: = input.::Loading Workflow Type::;

  • Validates the parameters entered on the Record Details screen:

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

    • When Workflow Type is No Index, Index Read 1 must be zero. For any other Workflow Type, Index Read 1 must be greater than zero.

    if(!step.::Experiment Name::.matches(::[a-zA-Z0-9-_]+::)) {
    fail(::Experiment Name contains prohibited characters. Allowed characters are: a-z, A-Z, 0-9, -, and _::)
}
if(step.::Workflow Type::== ::No Index::) {
    if(step.::Index Read 1::!= 0) {
        fail(::Index Read 1 must be 0 if the Workflow Type is No Index.::)
    }
} else {
    if(step.::Index Read 1::== 0) {
        fail(::Index Read 1 must be greater than 0 if the Workflow Type is ::+ step.::Workflow Type::+ ::.::)
    }
}

  • When Paired End is True, Index Read 1 and Index Read 2 must be greater than zero.

  • When Paired End is False, Index Read 2 must be zero, Reverse Complement Workflow must be false, UMI - Read 2 Length must not have a value, and UMI - Read 2 Start from Cycle must not have a value.

    if (step.::Paired End::.toBoolean()) {
    if (step.::Read 1 Cycles:: == 0 || step.::Read 2 Cycles:: == 0) {
        fail(::Read 1 Cycles and Read 2 Cycles must be greater than 0 if Paired End is True.::);
    }
} else {
    if (step.::Read 2 Cycles:: != 0) {
        fail(::Read 2 Cycles must be 0 if Paired End is False.::);
    }
    if (step.hasValue(::UMI - Read 2 Length::) || step.hasValue(::UMI - Read 2 Start From Cycle::)) {
        fail(::UMI - Read 2 Length and UMI - Read 2 Start From Cycle cannot be defined if Paired End is False.::);
    }
}

  • Validates allowed read cycles to be not greater than 151 if the Flowcell Type is not SP.

    if (input.::Flowcell Type:: != ::SP:: && step.::Read 1 Cycles:: > 151) {
    fail(::Read 1 Cycles must not be larger than 151 if it is not SPrime Flowcell.::);
}
if (input.::Flowcell Type:: != ::SP:: && step.::Read 2 Cycles:: > 151) {
    fail(::Read 2 Cycles must not be larger than 151 if it is not SPrime Flowcell.::);
}

  • Validates allowed value for UMI - Read 1 Length, UMI - Read 2 Length, UMI - Read 1 From Cycle, UMI - Read 2 From Cycle.

    if (step.hasValue(::UMI - Read 1 Length::) && !step.hasValue(::UMI - Read 1 Start From Cycle::)) {
    fail(::UMI - Read 1 Start From Cycle must be greater than 0 if UMI - Read 1 Length is greater than 0.::);
}
if (!step.hasValue(::UMI - Read 1 Length::) && step.hasValue(::UMI - Read 1 Start From Cycle::)) {
    fail(::UMI - Read 1 Length must be greater than 0 if UMI - Read 1 Start From Cycle is greater than 0.::);
}
if (step.hasValue(::UMI - Read 2 Length::) && !step.hasValue(::UMI - Read 2 Start From Cycle::)) {
    fail(::UMI - Read 2 Start From Cycle must be greater than 0 if UMI - Read 2 Length is greater than 0.::);
}
if (!step.hasValue(::UMI - Read 2 Length::) && step.hasValue(::UMI - Read 2 Start From Cycle::)) {
    fail(::UMI - Read 2 Length must be greater than 0 if UMI - Read 2 Start From Cycle is greater than 0.::);
}
if (!step.hasValue(::UMI - Read 1 Length::) && step.hasValue(::UMI - Read 2 Length::)) {
    fail(::UMI - Read 1 Length must be greater than 0 if UMI - Read 2 Length is greater than 0.::);
}
if (!step.hasValue(::UMI - Read 1 Start From Cycle::) && step.hasValue(::UMI - Read 2 Start From Cycle::)) {
    fail(::UMI - Read 1 Start From Cycle must be greater than 0 if UMI - Read 2 Start From Cycle is greater than 0.::);
}

  • Sets the next step for samples to REMOVE.

    nextStep = ::REMOVE::

  • Generates the sample sheet and the run recipe file and attaches them to the step. For details on how the files are generated and their contents, refer to Sample Sheet and Run Recipe File Generation.

Prepare Files for NovaSeq Automation

Automatically triggered on exit of the Record Details screen, this automation invokes the copy_attachments_to_network_folder script, which is included in the novaseq-remote-extensions.jar file. This script places the generated sample sheet and run recipe (*json file) into the appropriate folder on the NAS, where the instrument uses them to start the run.

For more information, refer to Sample Sheet and Run Recipe File Generation.

Routing Script Automation

Automatically triggered on exit of the step, this automation invokes the changeWorkflow script, which routes step outputs to the NovaSeq 6000 v2.3 workflow, and queues them for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3) step.

The default automation command line is as follows.

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid2} script:changeWorkflow \
\
--FIELD_NAME 'N/A' \
--FIELD_VALUE 'N/A' \
--WORKFLOW 'NovaSeq 6000 v2.3' \
--STEP 'AUTOMATED - NovaSeq Run (NovaSeq 6000 v2.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"

Master Step Fields

The following table provides field configuration details fields defined on the Dilute and Denature (NovaSeq 6000 v2.3) step. These fields are required for sample sheet and JSON file generation.

Dilute and Denature (NovaSeq 6000 v2.3) Master Step Field Configuration

Field Name

Field Type

Options

Additional Options and Drop-Down Items

BaseSpace Sequence Hub Configuration

Text Dropdown

  • Required Field

  • Presets

    • Not Used

    • Run Monitoring Only

    • Run Monitoring and Storage

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

Paired End

Text Dropdown

  • Required Field

  • Presets

    • True

    • False

Read 1 Cycles

Numeric Dropdown

  • Required Field

  • Custom Entries

  • Range = 1–251

  • Decimal places displayed = 0

  • Presets

    • 251

    • 151

    • 101

    • 51

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

Read 2 Cycles

Numeric Dropdown

  • Required Field

  • Custom Entries

  • Range = 0–251

  • Decimal places displayed = 0

  • Presets

    • 251

    • 151

    • 101

    • 51

    ℹ The value of 251 is only supported for SP flow cell type. For all other flow cell types, the 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

Sample Sheet Path

Text

  • Read Only

UMI - Read 1 Length

Numeric

  • Range = 1

  • Decimal places displayed = 0

UMI - Read 1 Start From Cycle

Numeric

  • Range = 1

  • Decimal places displayed = 0

UMI - Read 2 Length

Numeric

  • Range = 1

  • Decimal places displayed = 0

UMI - Read 2 Start From Cycle

Numeric

  • Range = 1

  • Decimal places displayed = 0

Use Custom Index Read 1 Primer

Toggle Switch

  • Default

    • No

Use Custom Read 1 Primer

Toggle Switch

  • Default

    • No

Use Custom Read 2 Primer

Toggle Switch

  • Default

    • No

Workflow

Text

  • Read Only

  • Default

    • GenerateFASTQ

Workflow Type

Text Dropdown

  • Required Field

  • Presets

    • No Index

    • Single Index

    • Dual Index

Global Step Fields

The following table lists the global step fields that are configured to display on the Dilute and Denature (NovaSeq 6000 v2.3) step.

Global Field Configuration (Derived Sample)

Field Name

Field Type

Options

Additional Options and Drop-Down Items

Flowcell Type