# Configuration Last Updated: July 2025 Release Date: September 2023 Document Version: 2 {% hint style="danger" %} BSSH subscription (personal/professional/enterprise) is required for NextSeq 1000/2000 Integration v2.4.0 {% endhint %} The Illumina NextSeq 1000/2000 Integration Package v2.4.0 supports the integration of Clarity LIMS to Illumina NextSeq 1000/2000 sequencing systems. For instructions on user interaction for each step, validating and troubleshooting the NextSeq 1000/2000 Integration, refer to [NextSeq 1000/2000 Integration v2.4.0 User Interaction, Validation and Troubleshooting](https://help.claritylims.illumina.com/instruments-and-integrations/nextseq1k2k/nextseq1k2k-v2.4.0/nextseq1k2k-v2.4.0-user-interaction-validation-and-troubleshooting). {% hint style="danger" %} 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 (global or master step) — could break the process. {% endhint %} ## **Prerequisites and Assumptions** The Illumina NextSeq 1000/2000 Integration Package v2.4.0 is compatible with Illumina cloud hosted deployments only. It is assumed that samples entering the NextSeq 1000/2000 Sequencing v2.3 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](https://help.claritylims.illumina.com/instruments-and-integrations/nextseq1k2k/nextseq1k2k-v2.4.0/broken-reference). 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](https://help.claritylims.illumina.com/instruments-and-integrations/nextseq1k2k/nextseq1k2k-v2.4.0/broken-reference). ## **Workflows, Protocols, and Steps** The NextSeq 1000/2000 Integration Package v2.4.0 includes the following workflows: * \[Optional] Library Prep Validation v2.3.1 (recommended for validation purposes). * NextSeq 1000/2000 Sequencing v2.3 > ℹ Samples are routed to this protocol after they have gone through Library Prep Validation v2.3.1 (or any other library prep workflow). Samples are pooled, diluted to final loading concentration, and added to a reagent cartridge in preparation for the sequencing run. > ℹ The workflow configuration includes a global field named Illumina Universal Sample Identifier. This text field is reserved for Clarity LIMS Product Analytics support. The field is optional and not required for this integration. ### Library Prep Validation v2.3.1 Workflow
Protocol 1: Library Prep Validation v2.3.1 **Purpose**: * Included for validation purposes only, this protocol models the library prep steps required to advance samples to the NextSeq 1000/2000 Sequencing v2.3 workflow. * The protocol contains a single step—Library Prep Validation v2.3.1. At the end of this step, a routing script sends the samples to the first step of the NextSeq 1000/2000 Sequencing v2.3 workflow. The first step is Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3). **Steps**: 1. Library Prep Validation v2.3.1
### NextSeq 1000/2000 Sequencing v2.3 Workflow
Protocol 1: NextSeq 1000/2000 Sequencing v2.3 **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 v2.3) * 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 v2.3) * 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 v2.3) * 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 (E.g., run configuration, primary run metrics, etc.) of the sequencing run are recorded automatically. * GenerateFastQ secondary analysis is planned by default for the run. Samples continues to the next step. > ⚠ Do not add samples to the Ice Bucket or start or complete the step. The integration does this action automatically. 4. Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) * This step is a semi-automated step that is started automatically after the GenerateFastQ secondary analysis has started. * 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. > ⚠ Do not add samples to the Ice Bucket or start or complete the step. The integration does this action automatically.
### **Validation Workflow** The Library Prep Validation v2.3.1 workflow allows for validation of the system after installation is complete. For details, refer to [NextSeq 1000/2000 Integration v2.4.0 User Interaction, Validation and Troubleshooting](https://help.claritylims.illumina.com/instruments-and-integrations/nextseq1k2k/nextseq1k2k-v2.4.0/nextseq1k2k-v2.4.0-user-interaction-validation-and-troubleshooting). ## Step 1: Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) In this step, the addition of RSB dilutes pooled samples. Manually create a working pool based on the final loading concentration required. {% hint style="danger" %} Create one pool per step. The Calculate Volumes automation supports one pool only. {% endhint %}
Register Step Started¹ 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. {% code overflow="wrap" %} ```markup /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' ``` {% endcode %}
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 (µl) 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 (µl):: = 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 (24µl in volume). 
-exp 'input.::Per Sample Volume (µl):: = (48/ input.::Molarity (nM)::); step.::RSB Volume for Pool (µl):: -= input.::Per Sample Volume (µl)::'
* 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 (µl). 
-exp 'step.::Library Pool Volume (µl):: = (step.::Final Loading Concentration (pM):: * step.::Final Loading Volume (µl)::/2000); output.::RSB Volume (µl):: = step.::Final Loading Volume (µl):: - step.::Library Pool Volume (µl)::'
* 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 v2.3): {% code overflow="wrap" %} ```markup nextStep = ::ADVANCE:: ``` {% endcode %}
Register Pools & Register Step Completed¹ 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. {% code overflow="wrap" %} ```markup /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' ``` {% endcode %}
¹ These automations are required for Clarity LIMS Product Analytics support only. ### Master Step Fields The following fields are defined on the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) step. *Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) 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 | |

Presets

| | Final Loading Volume (ul) | Numeric | |

Default

| | Library Pool Volume (ul) | Numeric | |

Hidden

⚠ This field is used by the Calculate Volumes automation. Removing, renaming, or modifying the field will break the automation.

| | RSB Volume for Pool (ul) | Numeric | |

Hidden

⚠ This field is used by the Calculate Volumes automation. Removing, renaming, or modifying the field will break the 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 v2.3) step. *Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) Global Field Configuration* | **Field Name** | **Field Type** | **Field Constraints/Options** | **Preset Values/Additional Options and Drop-down Items** | | -------------------------------- | ---------------- | ------------------------------------------------------- | ------------------------------------------------------------------------------- | | Final Loading Concentration (pM) | Numeric Dropdown | |

Decimal places displayed = 0
Presets

| | RSB Volume (µl) | Numeric | | Decimal places displayed = 2 | ## Step 2: Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3) 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 on Illumina Connected Analytics, depending on the Run Mode selected. {% hint style="info" %} 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. {% endhint %}
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 v2.3 workflow. 
script:validateSampleCount -min 1 -max 1
* Registers the start time of the step by publishing messages to Clarity LIMS Product Analytics through Illumina Connected Analytics. This script is only used for Clarity LIMS Product Analytics 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-Za-z]{2}\[0-9]{7}-\[A-Za-z0-9]{4}: {% code overflow="wrap" %} ```markup if (!output.container.name.matches(::[A-Za-z]{2}[0-9]{7}-[A-Za-z0-9]{4}::)){ fail (::Invalid Reagent Cartridge Barcode. Please verify and try again.::) } ``` {% endcode %}
Retrieve Analysis Workflow Versions Automation Automatically triggered upon entry of the Record Details screen, this automation completes the following actions: * Fetches the available analysis workflow versions from Illumina Connected Analytics. * Updates the preset values of both Local Analysis Workflow Versions & Cloud Analysis Workflow Versions fields. {% code overflow="wrap" %} ```markup /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} ``` {% endcode %} > ⚠ 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 does the following actions: * 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 on Illumina Connected Analytics. * 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 limitation, 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 on Illumina Connected Analytics. * 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}
* There is backend validation of the sample sheet content. This validation makes sure that the sample sheet is valid to set up a Local Mode run or is ready for DRAGEN applications. Refer to [NextSeq 1000/2000 Integration v2.4.0 User Interaction, Validation and Troubleshooting](https://help.claritylims.illumina.com/instruments-and-integrations/nextseq1k2k/nextseq1k2k-v2.4.0/nextseq1k2k-v2.4.0-user-interaction-validation-and-troubleshooting) for submitted sample name character restrictions.
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 v2.3). The automation is automatically triggered on exit of the Record Details screen. {% code overflow="wrap" %} ```markup nextStep = ::ADVANCE:: ``` {% endcode %}
Register Step Completed¹ Automatically triggered on exit of the step, this automation registers the completion time of the step by publishing messages to Clarity LIMS Product Analytics through Illumina Connected Analytics. This script is only used for Clarity LIMS Product Analytics support. {% code overflow="wrap" %} ```markup /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' ``` {% endcode %}
¹ These automations are required for Clarity LIMS Product Analytics support only. ² These automations are required for the NextSeq 1000/2000 Sequencing v2.3 workflows and contain additional logic needed for Clarity LIMS Product Analytics support. If you would like to remove Clarity LIMS Product Analytics support, contact Illumina Support. ### Master Step Fields There are 24 fields defined on the Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3) step. These fields are required for sample sheet generation and planned run creation on Illumina Connected Analytics (ICA). *Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3) Master Step Fields Configuration* | **Field Name** | **Field Type** | **Field Constraints/Options** | **Preset Values/Additional Options and Drop-down Items** | | ------------------------------------------------------------------------------------------ | ---------------- | ------------------------------------------------------- | ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | Run Name | Text | | | | Instrument Type | Text Dropdown | |

Presets

| | Run Mode | Text Dropdown | |

Presets

| | Paired End | Text Dropdown | |

Presets

| | Read 1 Cycles | Numeric Dropdown | |

Presets

| | Read 2 Cycles | Numeric Dropdown | |

Presets

| | Index Reads | Text Dropdown | |

Presets

| | Index Read 1 | Numeric Dropdown | |

Presets

| | Index Read 2 | Numeric Dropdown | |

Presets

| | Analysis Workflow | Text | |

Default

| | Adapter Sequence Read 1 | Text | | | | Adapter Sequence Read 2 | Text | | | | Barcode Mismatches Index 1 | Numeric Dropdown | |

Presets

| | Barcode Mismatches Index 2 | Numeric Dropdown | |

Presets

| | Override Cycles | Text | | | |

Cloud Run ID
(Used in creating planned run in ICA)

| Text | | Hidden | |

Use Custom Index Read 1 Primer
(Custom primer option is currently not supported)

| Toggle Switch | |

Default

Hidden

| |

Use Custom Index Read 2 Primer
(Custom primer option is currently not supported)

| Toggle Switch | |

Default

Hidden

| |

Use Custom Read 1 Primer
(Custom primer option is currently not supported)

| Toggle Switch | |

Default

Hidden

| |

Use Custom Read 2 Primer
(Custom primer option is currently not supported)

| Toggle Switch | |

Default

Hidden

| | Instructions | Multiline Text | |

Default

| | Local Analysis Workflow Versions | Text Dropdown | |

Default

| | Cloud Analysis Workflow Versions | Text Dropdown | |

Default

| | FASTQ Compression Format | Text Dropdown | |

Presets

Default

| ## Step 3: AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) 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. {% hint style="danger" %} Do not add samples to the Ice Bucket or start or complete the step. The integration does this action automatically. {% endhint %}
Register Step Started & Register NextSeq1k2k Run & Register NextSeq1k2k Run Association & Register NextSeq1k2k RunStarted¹ 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 Clarity LIMS Product Analytics through Illumina Connected Analytics. This automation is only used for Clarity LIMS Product Analytics support. {% code overflow="wrap" %} ```markup /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' ``` {% endcode %}
Register NextSeq1k2k RunCompleted & Register Step Completed¹ 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 v2.3) step. * Registers the completion time of the step. The automation completes these actions by publishing messages to Clarity LIMS Product Analytics through Illumina Connected Analytics. This automation is only used for Clarity LIMS Product Analytics support. {% code overflow="wrap" %} ```markup /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' ``` {% endcode %}
¹ These automations are required for Clarity LIMS Product Analytics support only. ### Master Step Fields There following fields are defined on the AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) 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. **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** | **RunParameters.xml Field** | **On Record Details Screen** | | --------------------- | -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | ---------------------------- | | Instrument Platform |

NextSeq 1000/2000
Constant value

| Visible | | Instrument Type |

One of the following options:

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

| Visible | | Run Status |

One of the following options:

Set by the integration service¹

| Visible | | Sequencing Log | Filled in by the integration service¹ as the sequencing run proceeds | Visible | | BaseSpace Run ID | Received as part of the event payload¹ | 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](#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 v2.3) step. ## Step 4: Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) This step is a semi-automated step. {% hint style="danger" %} Do not add samples to the Ice Bucket or start or complete the step. The integration does this action automatically. For the demultiplexing information in the Sample Details table to display correctly, the following conditions must be met: * The DRAGEN version must be earlier than v3.9.3. * The samples in the run must not repeat. * The run must be for the single lane flow cell (P1 or P2). {% endhint %} 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.
Register Step Started Automation Automatically triggered at the beginning of the step, this automation registers the start time of the step by publishing messages to Clarity LIMS Product Analytics through Illumina Connected Analytics. This automation is only used for Clarity LIMS Product Analytics support. {% code overflow="wrap" %} ```markup /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' ``` {% endcode %}
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. {% code overflow="wrap" %} ```markup /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} ``` {% endcode %}
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. {% code overflow="wrap" %} ```markup nextStep = ::ADVANCE:: ``` {% endcode %}
Register Analysis Completed & Register Step Completed Automation 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 Clarity LIMS Product Analytics through Illumina Connected Analytics. This automation is only used for Clarity LIMS Product Analytics support. {% code overflow="wrap" %} ```markup /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' ``` {% endcode %}
### Master Step Fields The following table lists the 12 master step fields that are configured on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) master step. *Master Step Field Configuration for Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) 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

| | Criteria 2 - Source Data Field | Text Dropdown | Custom Entries |

Presets

| | 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 Time | Text | Read Only | Hidden | | Analysis Started Time | Text | Read Only | Hidden | | Analysis Status | Text | Read Only | Hidden | | BSSH Analysis ID | Text | Read Only | Hidden | |

Reagent Cartridge ID
(Used for parsing demultiplexing results)

| Text | Read Only | Hidden | ### Global Fields The following table lists the global fields that are configured on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) step. These fields are used to display the demultiplexing result metrics for individual library in the sample pool. *Global Field Configuration for Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) Step* | **Field Name** | **Field Type** | **Field Constraints/Options** | | -------------------------- | -------------- | ----------------------------- | | # of >= Q30 Bases (PF) | Numeric | Read Only | | # One Mismatch Index Reads | Numeric | Read Only | | # Perfect Index Reads | Numeric | Read Only | | # Reads | Numeric | Read Only | | Mean Quality Score (PF) | 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 v2.3) 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 sent to Illumina Connected Analytics (ICA). ICA validates the run and analysis configuration, and sends an error message to Clarity LIMS if the validation fails. If the validation passes, ICA generates the sample sheet (and sends back to Clarity LIMS) and/or creates a planned run depending on the [Run Mode](https://help.claritylims.illumina.com/references/terminology#run-mode) selected. NextSeq 1000/2000 Integration supports local, hybrid and cloud run mode. Refer to [Start A Sequencing Run On Instrument With Different Run Modes](#start-a-sequencing-run-on-instrument-with-different-run-modes) for details on how to start a run using different run mode. ### 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 (BSSH) that the sequencing run has started. BSSH then does the following actions: * Local run mode: Creates a run on the BSSH database with the run configuration information received from NCS. * Hybrid/Cloud run mode: Creates a run on the BSSH database by copying the run configuration information from the planned run created in ICA, using a special Cloud Run ID received from NCS. After the run is created on BSSH, a run update event message that carries the RunStarted equivalent message is created on BSSH and sent to the integration service which processes the message. Based on the reagent cartridge barcode information in the event message (refer to the following example), the integration looks in the queue of AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) step for a container with the same reagent cartridge barcode. When identified, the step is started automatically and the Run Status field on Record Details screen is updated to RunStarted. The integration then updates all the other step fields (eg, Current Read, Output Folder) by parsing the details from the RunParameters.xml uploaded to BSSH by NCS. {% code overflow="wrap" %} ```markup #Example of an run update event { "acl": ["wid:61dc14e8-d823-3bd1-bea3-66f9c8388254"], "apiUrl": "https://api.basespace.illumina.com/v2/runs/r.ZvTfDXsNhUyh2kA5S4Zewg", "dateModified": "2020-03-26T09:19:58.5546497Z", "flowcellBarcode": "11M11COMC", "gdsFolderPath": "/Runs/200326_MP2-15_87_11M11COMC_r.ZvTfDXsNhUyh2kA5S4Zewg", "gdsVolumeName": "bssh.61dc14e8d8233bd1bea366f9c8388254", "id": "r.ZvTfDXsNhUyh2kA5S4Zewg", "instrumentRunId": "200326_MP2-15_87_11M11COMC", "name": "200326_MP2-15_87_11M11COMC", "reagentBarcode": "EC0001990-EC5", "sampleSheetName": "SampleSheet.csv", "status": "Running", "v1pre3Id": "194424236" } ``` {% endcode %} ### How the Integration Service Handles Run Status The NextSeq 1000/2000 Control Software (NCS) continues to notify BaseSpace Sequence Hub (BSSH) of any run status change throughout the course of the sequencing run. The run update event delivery process continues as described in the previous section. The following table lists the run status details and how the integration service handles each status. | **Run Status** | **Shown in Step** | **Details** | | ------------------------ | -------------------------------------------------------------- | ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | RunStarted | AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) | | | RunCompletedSuccessfully | AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) | | | RunErroredOut | AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) | | | RunAbortedByUser | AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) | | | AnalysisStarted | Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) | | | AnalysisCompleted | Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) | | | AnalysisFailed | Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) | | ## Start A Sequencing Run On Instrument With Different Run Modes 1. Configure a run with the Proactive, Run Monitoring and Storage option. Local runs are done in Local mode, and cloud or hybrid runs are done in Cloud or Hybrid mode. {% hint style="danger" %} Runs with local analysis (i.e., Local and Hybrid run mode) requires ICA Run Storage setting in BaseSpace Sequence Hub to be set to Off for automatic tracking of status and results by Clarity integration. Only Workgroup owner has the permission to change this setting. {% endhint %}
2. Start the local, cloud, or hybrid run. Refer to the [NextSeq 1000/2000 Product Documentation](https://support.illumina.com/sequencing/sequencing_instruments/nextseq-1000-2000.html) for information on run configuration and starting a run. ## Components Installed This section describes the various components that are installed by default as part of this integration. Information on installed workflows, protocols, steps, and automation points is provided in the Workflows, Protocols, and Steps section of [NextSeq 1000/2000 Integration v2.4.0 User Interaction, Validation and Troubleshooting](https://help.claritylims.illumina.com/instruments-and-integrations/nextseq1k2k/nextseq1k2k-v2.4.0/nextseq1k2k-v2.4.0-user-interaction-validation-and-troubleshooting). **Reagent Categories/Label Groups** * TruSeq HT adapters v2 (D7-D5) **Reagent Kits** * Resuspension Buffer (RSB) * NextSeq 1000/2000 reagent cartridge **Container Types** * Tube * 96-well plate * NextSeq 1000/2000 reagent cartridge **Control Types** * PhiX v3 This integration supports the NextSeq 1000/2000 reagent cartridge with barcode provided in the format \[A-Za-z]{2}\[0-9]{7}-\[A-Za-z0-9]{4} (eg, EC1234567-EC03). ## Enabling Planned Run Generation for Samples Having Duplicate Name with Different Indexes The [library preparation workflow of the samples must be configured to ensure unique derived sample names](https://help.claritylims.illumina.com/instruments-and-integrations/references/configure-support-samples-duplicate-names-different-indexes) before routing the samples through the library preparation workflow. ## Re-queue Samples for Cloud Run Clarity LIMS requires unique library names when samples are re-queued to the workflow in the NextSeq 1000/2000 integration. Assign unique names to libraries after going through library preparation. The following steps are used to configure the library preparation workflow correctly before routing the samples for re-queuing: 1. From Configuration, select the Lab Work tab. 2. Search for the library preparation workflow used for the re-queued samples. 3. In the Master Step of the library preparation workflow, modify the naming convention under Step Type to generate unique library names (E.g., appending LIMS ID to the default naming convention, like OutputItemLIMSID). 4. Select **Save**.
## Rules and Constraints {% hint style="danger" %} The NextSeq 1000/2000 Reagent Cartridge barcode should not be modified after a successful validation. Modifications can cause issues when Clarity LIMS tries to update the status and sample details of subsequent steps. {% endhint %} * 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: * Which samples, and how many, can enter each step together. * Which samples, and how many, can be pooled together. * The NextSeq 1000/2000 reagent cartridge barcode must be unique. There should not be multiple NextSeq 1000/2000 reagent cartridge containers in the system with the same name. * Reagent labels (indexes) must be unique. * One library pool can only contain one library or control with no label (index). * Do not manually start or complete the AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) step. This step is a fully automated step, and the integration service does not update samples correctly if they have been manually started. * Do not manually start the Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) step. This step is semi-automated, and the integration service does not update the demultiplexing result correctly if they have been manually started. * For the automated run to start successfully, select **Validate Run Setup and Create Planned Run** in the Load to Reagent Cartridge step. * The NextSeq 1000/2000 Reagent Cartridge barcode should not be modified after it has been successfully validated. Modifying the barcode would result in Clarity failing to update subsequent steps and sample details properly. ## Revision History | **Version** | **Changes** | | ----------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | 2 | | | 1 | |