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}

  • 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 v2.5.0 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 v2.4). 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 planned run using a special Cloud Run ID received from NCS.

After the run is created on BaseSpace Sequence Hub, a run update event message that carries the RunStarted equivalent message is created on BaseSpace Sequence Hub and delivered to Clarity LIMS SQS queue service. The message is captured and processed by the integration.

Based on the reagent cartridge barcode information in the event message (refer to the following example), the integration service looks in the queue of AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4) 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 service then updates all the other step fields (for example, Current Read, Output Folder) by parsing the details from the RunParameters.xml uploaded to BaseSpace Sequence Hub by NCS.

#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"
}

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 v2.4)	Run Status field is updated to RunStarted. All other step fields (except Run End Time) are updated based on RunParameters.xml detail obtained from BaseSpace Sequence Hub, the SQS event message, and the step field value from the Load to Reagent Cartridge (NextSeq 1000/2000 v2.4) step. Reagent cartridge lot information received from BaseSpace Sequence Hub is compared against the details of the pre-accessioned reagent cartridge on Clarity LIMS. If the expiration date is different, it is updated to the date received from BaseSpace Sequence Hub. If reagent cartridge is not found, it is created and set to Active. If reagent cartridge is not in Active status, it is set to Active. Multiline Sequencing Log field is updated.
RunCompletedSuccessfully	AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4)	Run Status field is updated to RunCompletedSuccessfully. Current Read and Current Cycle fields are updated to the final cycle number and read number based on RunParameters.xml detail obtained from BaseSpace Sequence Hub. Run End Time field is updated based on RunParameters.xml detail obtained from BaseSpace Sequence Hub. Sequencing run result (primary metrics) is downloaded from BaseSpace Sequence Hub and the fields in Sample Details table (e.g., % Error Rate R1, % Phasing, etc.) are updated with the values. Multiline Sequencing Log field is updated. The integration automatically completes the step and routes the samples in the reagent cartridge container to the Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) step.
RunErroredOut	AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4)	Run Status field is updated to RunErroredOut. Current Read and Current Cycle fields are updated to the final cycle number and read number that the run stopped based on RunParameters.xml detail obtained from BaseSpace Sequence Hub. Run End Time field is updated based on RunParameters.xml detail obtained from BaseSpace Sequence Hub. Sequencing run result (primary metrics) is downloaded from BaseSpace Sequence Hub and the fields in Sample Details table (e.g., % Error Rate R1, % Phasing, etc.) are updated with the values. Multiline Sequencing Log field is updated. The integration does not automatically complete the step. Complete the step manually.
RunAbortedByUser	AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4)	Run Status field is updated to RunAbortedByUser. Current Read and Current Cycle fields are updated to the final cycle number and read number that was aborted based on RunParameters.xml details obtained from BaseSpace Sequence Hub. Run End Time field is updated based on RunParameters.xml detail obtained from BaseSpace Sequence Hub. Sequencing run result (primary metrics) is downloaded from BaseSpace Sequence Hub and the fields in Sample Details table (e.g., % Error Rate R1, % Phasing, etc.) are updated with the values. Multiline Sequencing Log field is updated. The integration does not automatically complete the step. Complete the step manually.
AnalysisStarted	Demultiplexing (NextSeq 1000/2000 Sequencing v2.4)	The integration starts the step automatically for the reagent cartridge container. Multiline Sequencing Log field is updated. Analysis Status is set to Started.
AnalysisCompleted	Demultiplexing (NextSeq 1000/2000 Sequencing v2.4)	The integration downloads the following metric files: Adapter_Metrics.csv Demultiplex_stats.csv Index_Hopping_Counts.csv Quality_Metrics.csv Top_Unknown_Barcodes.csv The metric files are placed in the Run_Metrics.zip folder that is attached to the file placeholder in the step. The Demultiplex_stats.csv file contains the demultiplexing results from BaseSpace Sequence Hub. These results are parsed and the details are recorded in the Sample Details table for each library in the library pool. For multi-lane flow cells, the demultiplexing stats are aggregated for each stat across all lanes. Hidden master step fields are updated with information from BaseSpace Sequence Hub. The Analysis Status field is updated to Complete. The Multiline Sequencing Log field is updated. The integration does not automatically complete the step. Assign the QC flag to the individual library and manually complete the step.
AnalysisFailed	Demultiplexing (NextSeq 1000/2000 Sequencing v2.4)	Hidden step fields are updated with information from BaseSpace Sequence Hub. The Analysis Status field is updated to either TimedOut or Aborted. The Multiline Sequencing Log field is updated. The integration does not automatically complete the step. Assign the QC flag to the individual library and manually complete the step.

Start A Sequencing Run On Instrument With Different Run Modes

Refer to the NextSeq 1000/2000 Product Documentation for the following information:

• Configuring a run with the Proactive, Run Monitory and Storage option. Local runs are done in Local mode, and cloud or hybrid runs are done in Cloud or Hybrid mode.

• Starting a local, cloud, or hybrid run.

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 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 (for example, appending LIMS ID to the default naming convention, like OutputItemLIMSID).

4. Select Save.

Components Installed

The following sections describe the various components that are installed by default as part of this integration. These components include the following items:

• Reagent categories/label groups

• Reagent kits

• Control types

• Containers

Information on installed workflows, protocols, steps, and automation points is provided in the Workflows, Protocols, and Steps section of NextSeq 1000/2000 Integration v2.5.0 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-Z]{2}[0-9]{7}-[A-Z0-9]{4} (for example, EC1234567-EC03).

Rules and Constraints

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.

• 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 v2.4) 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 v2.4) 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.