Configuration

Purpose:

• Included for validation purposes only, this protocol models the library prep steps required to advance samples to the Run Format (NovaSeq 6000 v3.8) protocol.

• The protocol contains a single step - Library Prep Validation v2.3.1. After this step, a routing script sends the samples to the first step of the NovaSeq 6000 v3.8 workflow - Define Run Format (NovaSeq 6000 v3.8).

Steps:

1. Library Prep Validation v2.3.1

Purpose:

• Allows for the assignment of per sample values for:

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

  • Normalized Molarity: Enter a value for each sample.

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

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

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

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

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

Steps:

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

Purpose:

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

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

Steps:

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

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

Purpose:

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

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

Steps:

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

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

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

Purpose:

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

• This protocol contains one fully automated step.

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

Steps:

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

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

/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'

This automation is used only for CLPA support.

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

• Sets the next step for samples to REMOVE:

  Copy

  nextStep = ::REMOVE::

• Calculates the Minimum Molarity using the following formula:

  Copy

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

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

  Copy

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

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

  Copy

  else if (input.::Normalized Molarity (nM):: < input.::Minimum Molarity (nM)::) {
      input.::Warning:: = ::The Normalized Molarity is too low.:: ; 
      input.::Loading Workflow Type:: = ::[Remove from workflow]:: ; }
  else { input.::Warning:: = ::not applicable:: }

At this point there are two options:

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

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

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

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

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

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

  Default automation command line is as follows:

  Copy

  bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \
  \
  --FIELD_NAME 'Loading Workflow Type' \
  --FIELD_VALUE 'NovaSeq Standard' \
  --WORKFLOW 'NovaSeq 6000 v3.8' \
  --STEP 'Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.8)' \
  --INPUTS_OR_OUTPUTS 'INPUTS' \
  \
  --FIELD_NAME 'Loading Workflow Type' \
  --FIELD_VALUE 'NovaSeq Xp' \
  --WORKFLOW 'NovaSeq 6000 v3.8' \
  --STEP 'Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.8)' \
  --INPUTS_OR_OUTPUTS 'INPUTS' \
  && /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'"

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

/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'

This automation is used only for CLPA support.

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

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

• Checks that only one pool has been created.

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

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar \
script:validate_flowcell_for_input_pools \
-i {stepURI:v2} \
-u {username} \
-p {password} \
-l {compoundOutputFileLuid1} \
-validateSingleOutput true \
-poolType bulk"

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

• Calculates the number of samples in the pool.

  Copy

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

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

  Copy

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

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

  Copy

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

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

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

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

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

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

  Copy

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

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

  Copy

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

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

  Copy

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

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

  Copy

  if ( input.::Flowcell Type:: == ::SP:: ) { output.::Volume of Pool to Denature (ul):: = 100 ; output.::NaOH Volume (ul):: = 25 ; output.::Tris-HCl Volume (ul):: = 25 } ;
  if ( input.::Flowcell Type:: == ::S1:: ) { output.::Volume of Pool to Denature (ul):: = 100 ; output.::NaOH Volume (ul):: = 25 ; output.::Tris-HCl Volume (ul):: = 25 } ;
  if ( input.::Flowcell Type:: == ::S2:: ) { output.::Volume of Pool to Denature (ul):: = 150 ; output.::NaOH Volume (ul):: = 37 ; output.::Tris-HCl Volume (ul):: = 38 } ;
  if ( input.::Flowcell Type:: == ::S4:: ) { output.::Volume of Pool to Denature (ul):: = 310 ; output.::NaOH Volume (ul):: = 77 ; output.::Tris-HCl Volume (ul):: = 78 }

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

  Copy

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

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

  Copy

  step.::Number of Samples in Pool:: = step.::Number of Samples in Pool:: - 1;
  step.::Total Sample Volume (ul):: = step.::Total Sample Volume (ul):: - input.::Adjusted Per Sample Volume (ul)::

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

nextStep = ::ADVANCE::

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

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

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

/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'

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

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

  Copy

  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 used only for CLPA support.

  Copy

  /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'

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

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

  Copy

  if (!output.container.name.matches(::[A-Z]{2}[0–9]{7}-[A-Z]{3}::)) {
      fail(::Invalid Library Tube Barcode. Verify and try again.::)
  }

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

  Copy

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

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

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

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

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

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

  Copy

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

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

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

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

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

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

  Copy

  if(!step.::Experiment Name::.matches(::[a-zA-Z0-9-_]+::)) {
      fail(::Experiment Name contains prohibited characters. Allowed characters are: a-z, A-Z, 0-9, -, and _::)
  };
  if (step.::Workflow Type:: == ::No Index::) {
      if (step.::Index Read 1:: != 0 || step.::Index Read 2:: != 0) {
          fail(::Index Read 1 and Index Read 2 must be 0 if No Index is selected.::)
      }
  } else if (step.::Workflow Type:: == ::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 Single Index is selected.::)
      }
  } 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 Dual Index is selected.::)
      }
  };

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

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

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

  Copy

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

• Checks the Flowcell field value.

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

  Copy

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

• Validates UMI Settings.

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

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

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

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

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

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

  Copy

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

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

  Copy

  if (step.hasValue(::Analysis Software Version::) && !step.::Analysis Software Version::.matches(::^(\\d+)(\\.\\d+)*$::)) {
      fail(::Analysis Software Version contains prohibited characters. Allowed characters are 0-9 and period. It shall start and end with numbers and separated by single period e.g. 3.8.4::)
  };

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

  Copy

  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.::)
  };

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

  Copy

  step.::Settings Header:: = step.hasValue(::UMI - Read 1 Length::) || step.hasValue(::UMI - Read 2 Length::) ? ::[Settings]:: : ::::;

• Sets the next step for samples to REMOVE.

  Copy

  nextStep = ::REMOVE::

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

  Copy

  && if [ '{udf:Samplesheet Format}' == 'V2' ]; then \
  templateFilename='NovaSeq_BCL2FASTQ_Samplesheet_v2.csv'; \
  elif [ '{udf:Reverse Complement Workflow}' == 'true' ]; then \
  templateFilename='NovaSeq_BCL2FASTQ_Reverse_Complement_Samplesheet.csv'; \
  elif [ '{udf:Reverse Complement Workflow}' == 'false' ]; then \
  templateFilename='NovaSeq_BCL2FASTQ_Samplesheet.csv'; \
  else echo 'UDF Reverse Complement Workflow is empty or undefined.'; exit -1; fi \
  && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} \
  script:driver_file_generator \
  -t /opt/gls/clarity/extensions/conf/driverfiletemplates/$templateFilename \
  -o {compoundOutputFileLuid0}.csv \
  -q true \
  -destLIMSID {compoundOutputFileLuid0} \
  -l {compoundOutputFileLuid1}"

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

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

  Copy

  script:changeWorkflow \
  --FIELD_NAME 'N/A' \
  --FIELD_VALUE 'N/A' \
  --WORKFLOW 'NovaSeq 6000 v3.8' \
  --STEP 'AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.8)' \
  --INPUTS_OR_OUTPUTS 'OUTPUTS' \

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

  Copy

  /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'

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

/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'

This automation is used only for CLPA support.

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

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

• Checks that only one pool has been created.

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

/opt/gls/clarity/bin/java \
-jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar \
script:validate_flowcell_for_input_pools \
-i {stepURI:v2} \
-u {username} \
-p {password} \
-l {compoundOutputFileLuid1} \
-validateSingleOutput true \
-poolType bulk"

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

• Calculates the number of samples in the pool.

  Copy

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

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

  Copy

  step.::PhiX Volume (ul):: = !step.hasValue(::% PhiX (0.25nM) Spike-In::) ? 0 : step.::% PhiX (0.25nM) Spike-In::;
  if (input.::Flowcell Type:: == ::SP::) {
      step.::Bulk Pool Volume (ul):: = step.'Number of Lanes to Sequence' * 18;
      step.::PhiX Volume (ul):: *= 0.7;
  };
  if (input.::Flowcell Type:: == ::S1::) {
      step.::Bulk Pool Volume (ul):: = step.'Number of Lanes to Sequence' * 18;
      step.::PhiX Volume (ul):: *= 0.7;
  };
  if (input.::Flowcell Type:: == ::S2::) {
      step.::Bulk Pool Volume (ul):: = step.'Number of Lanes to Sequence' * 22;
      step.::PhiX Volume (ul):: *= 0.8;
  };
  if (input.::Flowcell Type:: == ::S4::) {
      step.::Bulk Pool Volume (ul):: = step.'Number of Lanes to Sequence' * 30;
      step.::PhiX Volume (ul):: *= 1.1;
  };
  if (step.::PhiX Volume (ul):: == 0) {
      step.::PhiX Volume (ul):: = ::::;
  };

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

  Copy

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

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

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

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

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

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

  Copy

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

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

  Copy

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

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

  Copy

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

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

  Copy

  && /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/NovaSeq_Xp_Bulk_Pool.csv \
  -o {compoundOutputFileLuid0}.csv \ script:driver_file_generator \
  -t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeq_Xp_Bulk_Pool2.csv \
  -o append.csv \
  && cat append.csv >> {compoundOutputFileLuid0}.csv"

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

  Copy

  step.::Number of Samples in Pool:: = step.::Number of Samples in Pool:: - 1;
  step.::Total Sample Volume (ul):: = step.::Total Sample Volume (ul):: - input.::Adjusted Per Sample Volume (ul)::

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

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

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

output.::Flowcell Type:: = input.::Flowcell Type:: ; nextStep = ::ADVANCE::

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

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

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

/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'

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

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

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

  Copy

  script:validate_flowcell_for_input_pools \
  -i {stepURI:v2} \
  -u {username} \
  -p {password} \
  -l {compoundOutputFileLuid1}" \

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

  Copy

  /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'

  This automation is used only for CLPA support.

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

• BP Aliquot Volume (ul)

• NaOH Volume (ul)

• Tris-HCl Volume (ul)

• DPX1 Volume (ul)

• DPX2 Volume (ul)

• DPX3 Volume (ul)

• Mastermix per Lane (ul)

if ( input.::Flowcell Type:: == ::SP:: ) {
    output.::BP Aliquot Volume (ul):: = 18 ;
    output.::NaOH Volume (ul):: = 4;
    output.::Tris-HCl Volume (ul):: = 5;
    step.::DPX1 Volume (ul):: = 126 ;
    step.::DPX2 Volume (ul):: = 18 ;
    step.::DPX3 Volume (ul):: = 66 ;
    output.::Mastermix per Lane (ul):: = 63
};
if (input.::Flowcell Type:: == ::S1::) {
    output.::BP Aliquot Volume (ul):: = 18;
    output.::NaOH Volume (ul):: = 4;
    output.::Tris-HCl Volume (ul):: = 5;
    step.::DPX1 Volume (ul):: = 126;
    step.::DPX2 Volume (ul):: = 18;
    step.::DPX3 Volume (ul):: = 66;
    output.::Mastermix per Lane (ul):: = 63
};
if (input.::Flowcell Type:: == ::S2::) {
    output.::BP Aliquot Volume (ul):: = 22;
    output.::NaOH Volume (ul):: = 5;
    output.::Tris-HCl Volume (ul):: = 6;
    step.::DPX1 Volume (ul):: = 126;
    step.::DPX2 Volume (ul):: = 18;
    step.::DPX3 Volume (ul):: = 66;
    output.::Mastermix per Lane (ul):: = 77
};
if (input.::Flowcell Type:: == ::S4::) {
    output.::BP Aliquot Volume (ul):: = 30;
    output.::NaOH Volume (ul):: = 7;
    output.::Tris-HCl Volume (ul):: = 8;
    step.::DPX1 Volume (ul):: = 315;
    step.::DPX2 Volume (ul):: = 45;
    step.::DPX3 Volume (ul):: = 165;
    output.::Mastermix per Lane (ul):: = 105
};

The automation also completes the following actions:

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

  Copy

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

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

  Copy

  /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/NovaSeq_Xp_Working_Pool.csv \
  -o {compoundOutputFileLuid0}.csv \
  script:driver_file_generator \
  -t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeq_Xp_Working_Pool2.csv \
  -o append.csv \
  && cat append.csv >> {compoundOutputFileLuid0}.csv"

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

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

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

output.::Flowcell Type:: = input.::Flowcell Type:: ; nextStep = ::ADVANCE::

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

/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'

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

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

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

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

  • SP: 2 working pools

  • S1: 2 working pools

  • S2: 2 working pools

  • S4: 4 working pools

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

  Copy

  script:validate_flowcell_for_input_pools \
  -i {stepURI:v2} \
  -u {username} \
  -p {password} \
  -l {compoundOutputFileLuid1} \
  -validateSelectedContainer true \

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

  Copy

  /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'

  This automation is used only for CLPA support.

Automatically triggered on exit of the Placement screen, this automation

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

  Copy

  if (input.::Flowcell Type:: == ::SP:: && !output.container.name.matches( ::[A-Z0-9]{5}DR[A-Z0-9]{2}:: ) ) {
      fail ( ::Invalid Flowcell Barcode. Please verify and try again.:: )
  };
  if (input.::Flowcell Type:: == ::S1:: && !output.container.name.matches( ::[A-Z0-9]{5}DR[A-Z0-9]{2}:: ) ) {
      fail ( ::Invalid Flowcell Barcode. Please verify and try again.:: )
  };
  if (input.::Flowcell Type:: == ::S2:: && !output.container.name.matches( ::[A-Z0-9]{5}DM[A-Z0-9]{2}:: ) ) {
      fail ( ::Invalid Flowcell Barcode. Please verify and try again.:: )
  };
  if (input.::Flowcell Type:: == ::S4:: && !output.container.name.matches( ::[A-Z0-9]{5}DS[A-Z0-9]{2}:: ) ) {
      fail ( ::Invalid Flowcell Barcode. Please verify and try again.:: )
  };

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

  Copy

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

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

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

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

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

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

  Copy

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

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

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

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

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

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

  Copy

  if(!step.::Experiment Name::.matches(::[a-zA-Z0-9-_]+::)) {
      fail(::Experiment Name contains prohibited characters. Allowed characters are: a-z, A-Z, 0-9, -, and _::)
  };
  if (step.::Workflow Type:: == ::No Index::) {
      if (step.::Index Read 1:: != 0 || step.::Index Read 2:: != 0) {
          fail(::Index Read 1 and Index Read 2 must be 0 if No Index is selected.::)
      }
  } else if (step.::Workflow Type:: == ::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 Single Index is selected.::)
      }
  } 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 Dual Index is selected.::)
      }
  };

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

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

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

  Copy

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

• Checks the Flowcell field value.

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

  Copy

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

• Validates UMI Settings:

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

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

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

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

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

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

  Copy

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

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

  Copy

  if (step.hasValue(::Analysis Software Version::) && !step.::Analysis Software Version::.matches(::^(\\d+)(\\.\\d+)*$::)) {
      fail(::Analysis Software Version contains prohibited characters. Allowed characters are 0-9 and period. It shall start and end with numbers and separated by single period e.g. 3.8.4::)
  };

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

  Copy

  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.::)
  };

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

  Copy

  step.::Settings Header:: = step.hasValue(::UMI - Read 1 Length::) || step.hasValue(::UMI - Read 2 Length::) ? ::[Settings]:: : ::::;

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

  Copy

  nextStep = ::ADVANCE::

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

  Copy

  && if [ '{udf:Samplesheet Format}' == 'V2' ]; then \
  templateFilename='NovaSeq_BCL2FASTQ_Samplesheet_v2.csv'; \
  elif [ '{udf:Reverse Complement Workflow}' == 'true' ]; then \
  templateFilename='NovaSeq_BCL2FASTQ_Reverse_Complement_Samplesheet.csv'; \
  elif [ '{udf:Reverse Complement Workflow}' == 'false' ]; then \
  templateFilename='NovaSeq_BCL2FASTQ_Samplesheet.csv'; \
  else echo 'UDF Reverse Complement Workflow is empty or undefined.'; exit -1; fi \
  && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} \
  script:driver_file_generator \
  -t /opt/gls/clarity/extensions/conf/driverfiletemplates/$templateFilename \
  -o {compoundOutputFileLuid0}.csv \
  -q true \
  -destLIMSID {compoundOutputFileLuid0} \
  -l {compoundOutputFileLuid1}"

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

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

• Registers the start time of the step

• Registers the NovaSeq 6000 sequencing run configuration

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

• Registers the time that the sequencing run started

bash -l -c "/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/novaseq6000/register_novaseq_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/novaseq6000/novaseq_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/novaseq6000/novaseq_seqrun_started.groovy'"

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

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

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

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

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

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

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

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

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

bash -l -c "/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/novaseq6000/novaseq_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'"

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

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

• Experiment Name

• Read 1 Cycles

• Read 2 Cycles

• Workflow

• Reverse Complement Workflow

• Samplesheet Format

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

• Sample sheet generation script parameters and usage

• Sample sheet data and configuration options

• Enabling unique FASTQ file names per sequencing run

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

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

2. Enter credentials:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The following example shows the run recipe content:

{
    "run_name": "NVCS16027_SPrime_2x250",
    "run_mode": "S4",
    "workflow_type": "DualIndex",
    "librarytube_id": "NV0025867-LIB",
    "flowcell_id": "",
    "sample_loading_type": "NovaSeqStandard",
    "rehyb": false,
    "paired_end": true,
    "read1": 151,
    "read2": 151,
    "index_read1": 8,
    "index_read2": 8,
    "output_folder": "\\\\network_path\\run_data",
    "samplesheet": "",
    "require_samplesheet_authentication": true,
    "usecustomrecipe": false,
    "customRecipe": null,
    "use_basespace": false,
    "basespace_mode": null,
    "use_custom_read1_primer": false,
    "use_custom_read2_primer": false,
    "use_custom_index_read1_primer": false
}

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

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

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

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

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

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

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

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

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

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

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

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

4. The automated sequencing step is started.

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

   Any other reagents in the request are handled as follows.

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

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

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

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

   Otherwise a new lot is created with the following configuration:

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

   • Name — Serial number in request

   • Lot Number — Lot number in request

   • Expiry — Expiry date in request

   • Status — Active

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

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

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

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

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

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

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

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

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

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

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

   Copy

   Sequencing run with Run ID {runId} of Step '{step.name}' ({step.limsid}) completed successfully.

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

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

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

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

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

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

   Copy

   Step '${Step.name}' (${Step.limsid}) failed with Run ID ${runId}.

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

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

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

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

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

   Copy

   "Sequencing run with Run ID ${status.runInfo.runId} of Step '${step.configuration.name}' (${step.limsid}) aborted by user ${status.runInfo.userName}."

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