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Configuration

The Illumina NextSeq 1000/2000 Integration Package v2.5.0 supports the integration of Clarity LIMS to Illumina NextSeq 1000/2000 sequencing systems.

For instructions on validating and troubleshooting the NextSeq 1000/2000 Integration, refer to NextSeq 1000/2000 Integration v2.5.0 User Interaction, Validation and Troubleshooting.

The configuration provided in this integration has been established to support NextSeq 1000/2000 lab processes. Any configuration changes to protocols or workflows — including renaming protocols, steps, and fields — could break the process.

Prerequisites and Assumptions

The Illumina NextSeq 1000/2000 Integration Package v2.5.0 is compatible with Illumina cloud hosted deployments only.

This integration requires a Personal, Professional, or Enterprise BaseSpace Sequence Hub subscription.

It is assumed that samples entering the NextSeq 1000/2000 Sequencing v2.4 workflow have gone through library preparation and quantification processes. Before they are assigned to the workflow, samples have completed the following actions:

  • Samples have been accessioned into Clarity LIMS.

  • Samples have been run through QC and library prep.

  • Samples have the Molarity (nM) global field set to some value. The Calculate Volumes automation in the Library Pooling and Dilution step requires a value in the Molarity (nM) global field.

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 NextSeq 1000/2000 Integration Package v2.5.0 includes the following workflows:

  • NextSeq 1000/2000 Sequencing v2.4

  • [Optional] Library Prep Validation v2.3.3 (recommended for validation purposes)

The following protocols and steps are included in these workflows.

Library Prep Validation v2.3.3 Workflow

Protocol 1: Library Prep Validation v2.3.3

Purpose:

  • Included for validation purposes only, this protocol models the library prep steps required to advance samples to the NextSeq 1000/2000 Sequencing v2.4 workflow.

  • The protocol contains a single step—Library Prep Validation v2.3.3. At the end of this step, a routing script sends the samples to the first step of the NextSeq 1000/2000 Sequencing v2.4 workflow. The first step is Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4).

Steps:

  1. Library Prep Validation v2.3.3

NextSeq 1000/2000 Sequencing v2.4 Workflow

Protocol 1: NextSeq 1000/2000 Sequencing v2.4

Purpose:

  • This protocol models the lab processes of starting a NextSeq 1000/2000 sequencing run.

Steps:

  1. Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4)

    • Samples are pooled and diluted to the final loading concentration with the help of the Calculate Volume script.

  2. Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4)

    • The library pool from step 1 is then ready to be loaded to the NextSeq 1000/2000 reagent cartridge.

    • Run and analysis information is validated.

    • Sample sheet is generated and/or planned run is created.

  3. AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4)

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

    • All the metadata (for example, run configuration and primary run metrics) of the sequencing run are recorded automatically.

    ⚠ Do not add samples to the Ice Bucket or start or complete the step. The integration completes these actions automatically.

    • GenerateFastQ secondary analysis is planned by default for the run. Samples continues to the next step.

  4. Demultiplexing (NextSeq 1000/2000 Sequencing v2.4)

    • This step is a semi-automated step that is started automatically after the GenerateFastQ secondary analysis has started.

    ⚠ Do not add samples to the Ice Bucket or start or complete the step. The integration completes these actions automatically.

    • Demultiplexing result is parsed and recorded automatically.

    • User is required to assign QC label (Pass/Fail) to the individual library in the pool and complete the step.

Validation Workflow

The Library Prep Validation v2.3.3 workflow allows for validation of the system after installation is complete. For details, refer to NextSeq 1000/2000 Integration v2.5.0 User Interaction, Validation and Troubleshooting.

Step 1: Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4)

In this step, the addition of RSB dilutes pooled samples. Manually create a working pool based on the final loading concentration required.

Create one pool per step. The Calculate Volumes automation supports one pool only.

Register Step Started Automation¹

This automation registers the start time of the step by publishing messages to CLPA through Illumina Connected Analytics. This automation is automatically triggered on entry to the step.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/step_started.groovy'
Calculate Volumes Automation

This automation is triggered when you select Calculate Volumes on the Record Details screen. The automation completes the following actions:

  • Sets RSB Volume for Pool (ul) field value to 24 for calculation purpose of the 2 nM intermediate library pool.

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

    -exp 'step.::RSB Volume for Pool (ul):: = 24; output.::Final Loading Concentration (pM):: = step.::Final Loading Concentration (pM)::'

  • Calculate per sample volume required for each library to make the 2 nM intermediate library pool (24 µl in volume).

    -exp 'input.::Per Sample Volume (ul):: = ( 48/ input.::Molarity (nM):: ); step.::RSB Volume for Pool (ul):: -= input.::Per Sample Volume (ul)::'

  • Calculate the volume required of the 2 nM library pool to be diluted further to the Final Loading Concentration (pM) with the Final Loading Volume (ul).

    -exp 'step.::Library Pool Volume (ul):: = (step.::Final Loading Concentration (pM):: * step.::Final Loading Volume (ul)::/2000); output.::RSB Volume (ul):: = step.::Final Loading Volume (ul):: - step.::Library Pool Volume (ul)::'

  • Uses the NextSeq1K2K_Pool1.csv, NextSeq1K2K_Pool2.csv, and NextSeq1K2K_Pool3.csv template files to generate a single CSV file containing information about the pool and the samples it contains. The generated file is stored in the Calculation File placeholder, in the Files section, for download.

    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NextSeq1K2K_Pool1.csv \
-o 1.csv \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NextSeq1K2K_Pool2.csv \
-o 2.csv \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NextSeq1K2K_Pool3.csv \
-o 3.csv \
&& cat 1.csv 2.csv 3.csv > {compoundOutputFileLuid0}.csv
Set Next Step Automation

Automatically triggered on exit of the Record Details screen, this automation sets the next step for samples to ADVANCE, advancing them to the next step in the protocol. The next step is Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4):

nextStep = ::ADVANCE::
Register Pools & Register Step Completed Automation¹

This automation is automatically triggered on exit of the step and performs the following tasks:

  • 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 by publishing messages to CLPA through Illumina Connected Analytics.

This automation is only used for CLPA support.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/pooling.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/step_completed.groovy'

¹ These automations are required for CLPA support only.

Master Step Fields

The following fields are defined on the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4) step.

Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4) Master Step Field Configuration

Field Name

Field Type

Field Constraints/Options

Preset Values/Additional Options and Drop-down Items

Final Loading Concentration (pM)

Numeric Dropdown

  • Required Field

  • Custom Entries

Presets

  • 650

  • 750

  • 1000

  • 2000

Final Loading Volume (ul)

Numeric

  • Required Field

Default

  • 24

Library Pool Volume (ul)

Numeric

  • Read Only

Hidden

ℹ This field is used by the Calculate Volumes automation.</blockquote

RSB Volume for Pool (ul)

Numeric

  • Read Only

Hidden

ℹ This field is used by the Calculate Volumes automation.

Global Fields

The following table lists the global fields that are configured to display on the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4) step.

Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4) Global Field Configuration

Field Name

Field Type

Field Constraints/Options

Preset Values/Additional Options and Drop-down Items

Final Loading Concentration (pM)

Numeric Dropdown

  • Required Field

  • Custom Entries

  • Decimal places displayed: 0

  • Presets

    • 225

    • 400

RSB Volume (ul)

Numeric

  • Read Only

  • Decimal places displayed: 2

Step 2: Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4)

In this step, scan the reagent cartridge barcode into Clarity LIMS, then manually place the working pool into the reagent cartridge for the NextSeq 1000/2000 run. This step validates the run setup and analysis information and generates the sample sheet file and/or creates a planned run depending on the Run Mode selected.

Use alphanumeric, dash, or underscore characters only in the submitted sample names. Any other characters can cause sample sheet validation failure in the Load to Reagent Cartridge step. The NextSeq 1000/2000 Sequencing workflow handles these characters in the Load to Reagent Cartridge step by replacing them with an underscore in the submitted sample name. The Sample Details table in the Demultiplexing step reflects the modified submitted sample name.

The NextSeq 1000/2000 reagent cartridges support different read cycle numbers. Make sure that the read cycle values configured for the planned run are within the maximum allowable reads for the cartridge type.

Validate Single Input & Register Step Started Automation²

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

  • Checks that there is only one container input to the step. This script is required for the NextSeq 1000/2000 Sequencing v2.4 workflow.

    script:validateSampleCount -min 1 -max 1

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

    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/step_started.groovy'
Validate Reagent Cartridge Barcode Automation

Automatically triggered on exit of the Placement screen, the following automation validates the reagent cartridge barcode to make sure it conforms to the barcode mask [A-Z]{2}[0-9]{7}-[A-Z0-9]{4}:

if (!output.container.name.matches(::[A-Z]{2}[0-9]{7}-[A-Z0-9]{4}::)){
    fail (::Invalid Reagent Cartridge Barcode. Please verify and try again.::) 
}
Retrieve Analysis Workflow Versions Automation

Automatically triggered upon entry of the Record Details screen, this automation fetches the available analysis workflow versions and updates the preset values of both of the Local Analysis Workflow Versions and Cloud Analysis Workflow Versions custom fields.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/SIS/SISServices/extensions/automation/nextseq1k2k-automation.jar script:retrieve_analysis_workflow_versions -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid2}

⚠ Do not modify or disable the Retrieve Analysis Workflow Versions automation script. Modifying or disabling the script breaks the integration.

Validate Run Setup and Create Planned Run Automation

Automatically triggered when a button on the Record Details screen is selected, this automation validates the parameters entered on the Record Details screen. These parameters are used to set up the run and generate the sample sheet file and/or create the planned run.

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

    if (!step.::Run Name::.matches(::[a-zA-Z0-9-_.]+::)){ 
    fail(::Run Name contains prohibited characters. Allowed characters are: a-z, A-Z, 0-9, -, _, and .::) 
}

  • Run Name must not exceed 255 characters.

    if (step.::Run Name::.length() > 255) { 
    fail(::Run Name shall not exceed 255 characters.::) 
};

  • Checks the Index Reads, Index Read 1, and Index Read 2 field values.

    • If Index Reads is No Index, Index Read 1 and Index Read 2 values must be 0 (error results if it is otherwise).

    • If Index Reads is Single Index, Index Read 1 value must be greater than 0, and Index Read 2 values must be 0 (error results if it is otherwise).

    • If Index Reads is Dual Index, Index Read 1 and Index Read 2 values must be greater than 0 (error results if it is otherwise).

    if (step.::Index Reads:: == ::No Index::){ 
    if (step.::Index Read 1:: != 0 || step.::Index Read 2:: != 0) { 
        fail(::Index Read 1 and Index Read 2 must be 0 if the Index Reads is No Index.::) 
    } 
} 
else{ 
    if (step.::Index Reads:: == ::Single Index::){ 
        if (step.::Index Read 1:: == 0 || step.::Index Read 2:: != 0) { 
            fail(::Index Read 1 must be greater than 0 and Index Read 2 must be 0 if the Index Reads is Single Index.::) 
        } 
    }
    else{ 
        if (step.::Index Read 1:: == 0 || step.::Index Read 2:: == 0) { 
            fail(::Index Read 1 and Index Read 2 must be greater than 0 if the Index Reads is Dual Index.::) 
        } 
    } 
}

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

    • If Paired End is set to True and Read 2 Cycles value is 0, an error is generated.

    • If Paired End is set to False and Read 2 Cycles or Index Read 2 values are greater than 0, an error is generated.

    if (step.::Paired End::.toBoolean()){ 
    if (step.::Read 2 Cycles:: == 0) { 
        fail(::Read 2 Cycles must not be zero if it is Paired End read.::) 
    }
}
else{
    if (step.::Read 2 Cycles:: != 0 || step.::Index Read 2:: != 0) {
        fail(::Read 2 Cycles and Index 2 Cycles must be 0 if it is not Paired End Read.:: )
    }
}

  • Checks the Adapter Sequence Read 1 and Adapter Sequence Read 2 field values.

    • Adapter Sequence Read 1 and Adapter Sequence Read 2 can only contain ACTG+ characters.

    if (step.hasValue(::Adapter Sequence Read 1::) && !step.::Adapter Sequence Read 1::.matches(::[ACTG+]+::)) { 
    fail(::Adapter Sequence Read 1 contains prohibited characters. Allowed characters are: ACTG+::) 
};
if (step.hasValue(::Adapter Sequence Read 2::) && !step.::Adapter Sequence Read 2::.matches(::[ACTG+]+::)) { 
    fail(::Adapter Sequence Read 2 contains prohibited characters. Allowed characters are: ACTG+::) 
}

  • Checks Override Cycles field value.

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

    if (step.hasValue(::Override Cycles::) && !step.::Override Cycles::.matches(::[YNIU0-9;]+::)){ 
    fail(::Override Cycles contains prohibited characters. Allowed characters are: Y, N, I, U, 0-9 and ;. Example: N1Y150;I8;I7N1;Y141U10.::) 
}

  • When the Run Mode is Cloud, the script does the following actions:

    • Sets Local Analysis Workflow Versions to None.

    • If the Cloud Analysis Workflow Versions is None, the script displays the following error: Invalid option selected for Cloud Analysis Workflow Versions field.

    if (step.::Run Mode:: == ::Cloud::) { 
    step.::Local Analysis Workflow Versions:: = ::None::; 
    if (step.::Cloud Analysis Workflow Versions:: == ::None::) { 
        fail(::Invalid option selected for Cloud Analysis Workflow Versions field.::); 
    }
};

    • Due to automation script limitation, a validation rule resides in the backend service to perform the following action: If Cloud Analysis Workflow Versions is 3.7.4 or later, set FASTQ Compression Format to gzip.

  • When the Run Mode is Local or Hybrid, the script does the following actions:

    • Sets Cloud Analysis Workflow Versions to None.

    • If Local Analysis Workflow Versions is None, the script displays the following error message: Invalid option selected for Local Analysis Workflow Versions field.

    if (step.::Run Mode:: == ::Local:: || step.::Run Mode:: == ::Hybrid::) { 
    step.::Cloud Analysis Workflow Versions:: = ::None::; 
    if (step.::Local Analysis Workflow Versions:: == ::None::) { 
        fail(::Invalid option selected for Local Analysis Workflow Versions field.::); 
    }
};

    • Due to automation script limitations, a validation rule resides in the backend service. If Local Analysis Workflow Versions is 3.7.4 or later, an error occurs if FASTQ Compression Format is None.

  • Generates the sample sheet and/or creates the planned run.

    • Sample sheet is attached to the step.

    /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/SIS/SISServices/extensions/automation/nextseq1k2k-automation.jar
script:generate_v2_sample_sheet -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid2} -c {compoundOutputFileLuid0}
Set Next Step Automation

Sets the next step for samples to ADVANCE, advancing them to the next step in the protocol—AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4). The automation is automatically triggered on exit of the Record Details screen.

nextStep = ::ADVANCE::
Register Step Completed¹

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 only used for CLPA support.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/step_completed.groovy'

¹ These automations are required for CLPA support only.

² These automations are required for the NextSeq 1000/2000 Sequencing v2.4 workflows and contain additional logic needed for CLPA support. If you would like to remove CLPA support, contact Illumina Support.

Master Step Fields

There are 24 fields defined on the Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4) step. These fields are required for sample sheet generation and planned run creation.

Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4) Master Step Fields Configuration

Field Name

Field Type

Field Constraints/Options

Preset Values/Additional Options and Drop-down Items

Run Name

Text

  • Required Field

Instrument Type

Text Dropdown

  • Required Field

Presets

  • NextSeq1000

  • NextSeq2000

Run Mode

Text Dropdown

  • Required Field

Presets

  • Local

  • Hybrid

  • Cloud

Paired End

Text Dropdown

  • Required Field

Presets

  • True

  • False

Read 1 Cycles

Numeric Dropdown

  • Required Field

  • Custom Entries

Presets

  • 301

  • 151

  • 101

  • 51

Read 2 Cycles

Numeric Dropdown

  • Required Field

  • Custom Entries

Presets

  • 301

  • 151

  • 101

  • 51

Index Reads

Text Dropdown

  • Required Field

Presets

  • No Index

  • Single Index

  • Dual Index

Index Read 1

Numeric Dropdown

  • Required Field

  • Custom Entries

Presets

  • 0

  • 6

  • 8

Index Read 2

Numeric Dropdown

  • Required Field

  • Custom Entries

Presets

  • 0

  • 6

  • 8

Analysis Workflow

Text

  • Required Field

  • Read Only

Default

  • GenerateFASTQ

Adapter Sequence Read 1

Text

Adapter Sequence Read 2

Text

Barcode Mismatches Index 1

Numeric Dropdown

Presets

  • 0

  • 1

  • 2

Barcode Mismatches Index 2

Numeric Dropdown

Presets

  • 0

  • 1

  • 2

Override Cycles

Text

Cloud Run ID

Text

  • Read Only

Hidden

ℹ Used in creating planned run.

Use Custom Index Read 1 Primer

Toggle Switch

Default

  • None Set

Hidden

⚠ Custom primer option is currently not supported. Do not enable as it will break the integration.

Use Custom Index Read 2 Primer

Toggle Switch

Default

  • None Set

Hidden

⚠ Custom primer option is currently not supported. Do not enable as it will break the integration.

Use Custom Read 1 Primer

Toggle Switch

Default

  • None Set

Hidden

⚠ Custom primer option is currently not supported. Do not enable as it will break the integration.

Use Custom Read 2 Primer

Toggle Switch

Default

  • None Set

Hidden

Custom primer option is currently not supported. Do not enable as it will break the integration.

Instructions

Multiline Text

  • Read Only

Default

  • If GenerateFASTQ analysis is required, select the DRAGEN version in Cloud Analysis Workflow Versions (for Cloud run mode) or Local Analysis Workflow Versions (for Local/Hybrid run mode) custom fields.

Local Analysis Workflow Versions

Text Dropdown

Default

  • None

Cloud Analysis Workflow Versions

Text Dropdown

Default

  • None

FASTQ Compression Format

Text Dropdown

  • Required Field

Presets

  • gzip

  • DRAGEN

  • None

Default

  • gzip

Step 3: AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4)

This step is fully automated.

The integration starts and completes the step automatically. Data from the run is parsed back to Clarity LIMS. No user interaction is required. In this step, the pooled samples in the reagent cartridge are sequenced on the NextSeq 1000/2000 instrument.

Do not add samples to the Ice Bucket or start or complete the step. The integration completes these actions automatically.

Register Step Started & Register NextSeq1k2k Run & Register NextSeq1k2k Run Association & Register NextSeq1k2k RunStarted Automation¹

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

  • Registers the start time of the step.

  • Registers the NextSeq 1000/2000 sequencing run configuration.

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

  • Registers the time that the sequencing run starts.

The automation completes these actions by publishing messages to CLPA through Illumina Connected Analytics. This automation is only used for CLPA support.


/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid0} -s 'com/illumina/upa/scripts/common/step_started.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid0} -s 'com/illumina/upa/scripts/nextseq1k2k/register_nextseq1k2k_run.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid0} -s 'com/illumina/upa/scripts/nextseq1k2k/nextseq1k2k_associate_seqrun.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid0} -s 'com/illumina/upa/scripts/nextseq1k2k/nextseq1k2k_seqrun_started.groovy'
Register NextSeq1k2k RunCompleted & Register Step Completed Automation¹

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

  • Registers the sequencing run completion time, run status, and run metrics. This script requires the Cloud Run ID field from the Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4) step.

  • Registers the completion time of the step.

The automation completes these actions by publishing messages to CLPA through Illumina Connected Analytics. This automation is only used for CLPA support.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid0} -s 'com/illumina/upa/scripts/nextseq1k2k/nextseq1k2k_seqrun_completed.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid0} -s 'com/illumina/upa/scripts/common/step_completed.groovy'

¹ These automations are required for CLPA support only.

Master Step Fields

There following fields are defined on the AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4) step. These fields are used to display the run status and sequencing run and analysis configuration parsed from the RunParameters.xml file of the sequencing run.

Do not modify the master step field names. This action causes the integration to break.

Run Parameters and Corresponding Clarity LIMS Step Fields

The following table shows how some of the step fields map to the fields on the RunParameters.xml file, and whether the field is visible on the Record Details screen.

Master Step Field

RunParameters.xml Field

On Record Details Screen

Current Cycle

Calculated based on CompletedCycles Field

Visible

Current Read

Calculated based on CompletedCycles field against PlannedCycles

Visible

Flow Cell ID

FlowCellSerialNumber

Visible

Flow Cell Lot Number

FlowCellLotNumber

Visible

Instrument Control Software Version

ApplicationVersion

Visible

Instrument ID

InstrumentSerialNumber

Visible

Output Folder

OutputFolder

Visible

Reagent Cartridge ID

CartridgeSerialNumber

Visible

Reagent Cartridge Lot Number

CartridgeLotNumber

Visible

RTA Version

RtaVersion

Visible

Run Name

ExperimentName

Visible

Secondary Analysis Workflow

SecondaryAnalysisWorkflow

Visible

Flow Cell Mode

FlowCellMode

Hidden

Instrument Run ID

Derived from OutputFolder

Hidden

Run End Time

RunEndTime

Hidden

Run Start Time

RunStartTime

Hidden

Secondary Analysis Mode

SecondaryAnalysisMode

Hidden

Secondary Analysis Platform Version

SecondaryAnalysisPlatformVersion

Hidden

SkipObdd

SkipObdd

Hidden

Additional Master Step Fields and Values

The following table shows how the other step fields derive their values, and whether the step field is visible on the Record Details screen.

Master Step Field

Description

On Record Details Screen

Instrument Platform

  • NextSeq 1000/2000

  • Constant value

Visible

Instrument Type

  • One of the following options:

    • NextSeq1000

    • NextSeq2000

  • Determined from Instrument Type step field from previous Load to Reagent Cartridge step.

Visible

Run Status

  • One of the following options:

    • RunStarted

    • RunCompletedSuccessfully

    • RunAbortedByUser

    • RunErroredOut

  • Set by the integration service¹

Visible

Sequencing Log

  • Filled in by the integration service¹ as the sequencing run proceeds

Visible

BaseSpace Run ID

  • Received from SQS message in the integration service¹

Hidden

¹ For information on how the integration works, refer to Run Status, Primary Metrics, and Analysis Results Parsing and Recording in How the NextSeq 1000/2000 Integration Works.

Global Fields

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

  • % Bases >=Q30 R1

  • % Bases >=Q30 R2

  • % Error Rate R1

  • % Error Rate R2

  • Yield (Gb) R1

  • Yield (Gb) R2

  • Reads PF R1

  • Reads PF R2

  • %PF R1

  • %PF R2

  • % Aligned R1

  • % Aligned R2

  • % Phasing R1

  • % Phasing R2

  • % Prephasing R1

  • % Prephasing R2

  • Intensity Cycle 1 R1

  • Intensity Cycle 1 R2

  • Cluster Density R1

  • Cluster Density R2

At the end of this step, the pool of samples is automatically advanced to (and queued for) the Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) step.

Step 4: Demultiplexing (NextSeq 1000/2000 Sequencing v2.4)

This step is a semi-automated step.

The integration starts the step automatically and demultiplexing data from the GenerateFASTQ secondary analysis is parsed back to Clarity LIMS. The lab scientist reviews the demultiplexing result parsed into Clarity LIMS, assigns QC flags, and completes the step.

Do not add samples to the Ice Bucket or start or complete the step. The integration completes these actions automatically.

Register Step Started Automation¹

Automatically triggered at the beginning of the step, this automation registers the start time of the step by publishing messages to CLPA through Illumina Connected Analytics. This automation is only used for CLPA support.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/step_started.groovy'
Assign Demultiplexing QC Flags Automation

Automatically triggered when you select a button on the Record Details screen, this automation assigns QC flags based on the criteria set in the step fields.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} script:assignQCForDemuxStep -i {processURI:v2} -log {compoundOutputFileLuid1} -qcResult {compoundOutputFileLuid2}
Set Next Step Automation

Automatically triggered on exit of the Record Details screen, this automation sets the next step to Advance and the samples to complete the protocol.

nextStep = ::ADVANCE::
Register Analysis Completed & Register Step Completed Automation¹

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

  • Registers the NextSeq 1000/2000 analysis status and result.

  • Registers the completion time of the step.

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

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/step_completed.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/nextseq1k2k/nextseq1k2k_analysisrun_completed.groovy'

¹ These automations are required for CLPA support only.

Master Step Fields

The following table lists the master step fields that are configured on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) master step.

Master Step Field Configuration for Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) Step

Field Name

Field Type

Field Constraints/Options

Preset Values/Additional Options and Drop-down Items

Criteria 1 - Source Data Field

Text Dropdown

Custom Entries

Presets

  • # Reads

  • # Perfect Index Reads

  • # One Mismatch Index Reads

Criteria 2 - Source Data Field

Text Dropdown

Custom Entries

Presets

  • # Reads

  • # Perfect Index Reads

  • # One Mismatch Index Reads

Criteria 1 - Operator

Text Dropdown

Custom Entries

Presets

  • >=

  • <=

  • =

  • !=

Criteria 2 - Operator

Text Dropdown

Custom Entries

Presets

  • >=

  • <=

  • =

  • !=

Criteria 1 - Threshold Value

Numeric

Valid integer value

Criteria 2 - Threshold Value

Numeric

Valid integer value

Log

Multiline Text

Read Only

Analysis Completed Tim

Text

Read Only

Hidden

Analysis Started Time

Text

Read Only

Hidden

Analysis Status

Text

Read Only

BSSH Analysis ID

Text

Read Only

Hidden

Reagent Cartridge ID

Text

Read Only

Hidden

ℹ Used for parsing demultiplexing results

Global Fields

The following table lists the global fields that are configured on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) step. These fields are used to display the demultiplexing result metrics for individual library in the sample pool.

The demultiplexing statistics are aggregated for multi-lane flow cells. For each demultiplexing field (for example, # Reads) displayed in the Sample Details table for the multi-lane flow cell (for example, P3), the displayed value is the sum of the statistics across all lanes.

Global Field Configuration for Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) Step

Field Name

Field Type

Field Constraints/Options

Index Sequence

Text

Read Only

# One Mismatch Index Reads

Numeric

Read Only

# Perfect Index Reads

Numeric

Read Only

# Reads

Numeric

Read Only

How the NextSeq 1000/2000 Integration Works

Sample Sheet Generation and Planned Run Creation

On the Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4) step, when the Validate Run Setup and Create Planned Run automation is triggered, the run and analysis parameters entered in the Run Details screen are used to create a planned run. Then, the run and analysis configuration is validated. If the validation fails, an error message is sent to Clarity LIMS. If the validation passes, the sample sheet is generated (and sent back to Clarity LIMS) and/or a planned run is created depending on the Run Mode selected.

  • Local run mode: Only the sample sheet is generated and stored on Clarity LIMS. The sample sheet contains all the run and analysis configuration required to start the run on the instrument.

  • Hybrid/Cloud run mode: A planned run is created. This planned run contains all the run and analysis configuration required to start the run on the instrument. The sample sheet is also generated and stored on Clarity LIMS for reference purposes.

For details on how to start a run using different run modes, refer to Start A Sequencing Run On Instrument With Different Run Modes.

If a planned run with the same sample and project name has been created previously, the original case with the previous sample name can be reflected in the sample sheet generated from the Validate Run Setup and Create Planned Run automation. To resolve this issue, change the sample or project name in Clarity LIMS and run the automation again.

Run Status, Primary Metrics, and Analysis Results Parsing and Recording

After the sequencing run is started on the instrument, NextSeq 1000/2000 Control Software (NCS) notifies BaseSpace Sequence Hub that the sequencing run has started.

BaseSpace Sequence Hub then does the following actions:

  • Local run mode: Creates a run on the BaseSpace Sequence Hub database with the run configuration information received from NCS.

  • Hybrid/Cloud run mode: Creates a run on the BaseSpace Sequence Hub database by copying the run configuration information from the planne