Configuration

Purpose:

• Included for validation purposes only, this protocol models the library prep steps required to advance samples to the Run Format (NovaSeqDx v1.2) protocol.

• The protocol contains a single step - Library Prep Validation v2.3.4. After this step, a routing script sends the samples to the first step of the NovaSeqDx v1.2 workflow - Define Run Format (NovaSeqDx v1.2).

Steps:

1. Library Prep Validation v2.3.4

Purpose:

• Allows for the assignment of per sample values for the following properties:

  • NovaSeqDx Run Mode: Select either RUO or DX.

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

  • Normalized Molarity (nM): 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.

• Validates user selection on NovaSeqDx Run Mode, Loading Workflow Type and Flowcell Type.

• Routing script sends samples to the NovaSeqDx Standard or NovaSeqDx 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 (NovaSeqDx v1.2)

Purpose:

• Samples are pooled and added to the library tube in preparation for the NovaSeq 6000Dx run. The run setup information is validated and a sample sheet is generated.

• Routing script sends the library tube to the AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2) protocol.

Steps:

1. Make Bulk Pool for NovaSeqDx Standard (NovaSeqDx v1.2)

2. Dilute and Denature (NovaSeqDx v1.2)

Purpose:

• Samples are pooled and added to lanes on the NovaSeq 6000Dx flow cell. The option selected in the Define Run Format (NovaSeqDx v1.2) 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 - NovaSeqDx Run (NovaSeqDx v1.2) protocol.

Steps:

1. Make Bulk Pool for NovaSeqDx Xp (NovaSeqDx v1.2)

2. Dilute, Denature & ExAmp (NovaSeqDx v1.2)

3. Load to Flowcell (NovaSeqDx v1.2)

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 - NovaSeqDx Run (NovaSeqDx v1.2)

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

• Sets the next step for samples to REMOVE:

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  nextStep = ::REMOVE::

• Checks NovaSeqDx Run Mode, Loading Workflow Type, and Flowcell Type. Only the NovaSeq Standard with the S2/S4 flow cell is supported for the NovaSeqDx Run Mode:

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  if (input.::NovaSeqDx Run Mode:: == ::DX::) {
      if (input.::Loading Workflow Type:: == ::NovaSeq Xp::) {
          fail(::Invalid option selected. NovaSeq Standard shall be selected for DX mode.::);
      };
      if (input.::Flowcell Type:: == ::SP:: || input.::Flowcell Type:: == ::S1::) {
          fail(::Invalid option selected. Only S2 or S4 flowcell shall be selected for DX mode.::);
      };
  };

• Calculates the Minimum Molarity with the following formula:

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  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), generates an error message informing that the field cannot be empty:

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

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

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

  At this point, the following options are available:

  • Correct the NovaSeqDx Run Mode and 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 NovaSeqDx v1.2 workflow. Then, the samples are queued for the Make Bulk Pool for NovaSeqDx Standard (NovaSeqDx v1.2) step.

• Samples with Loading Workflow Type field value = NovaSeq Xp are routed to the NovaSeqDx v1.2 workflow. Then, the samples are queued for the Make Bulk Pool for NovaSeqDx Xp (NovaSeqDx v1.2) step.

Default automation command line is as follows.

bash -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar
-u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \
\
--FIELD_NAME 'Loading Workflow Type' \
--FIELD_VALUE 'NovaSeq Standard' \
--WORKFLOW 'NovaSeqDx v1.2' \
--STEP 'Make Bulk Pool for NovaSeqDx Standard (NovaSeqDx v1.2)' \
--INPUTS_OR_OUTPUTS 'INPUTS' \
\
--FIELD_NAME 'Loading Workflow Type' \
--FIELD_VALUE 'NovaSeq Xp' \
--WORKFLOW 'NovaSeqDx v1.2' \
--STEP 'Make Bulk Pool for NovaSeqDx Xp (NovaSeqDx v1.2)' \
--INPUTS_OR_OUTPUTS 'INPUTS'"

The Validate Single NovaSeqDx Run Mode automation is triggered on entry of the step. The following automation checks that all input samples have the same NovaSeqDx Run Mode assigned:

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar \
script:validate_same_analyte_udf_value_for_all_inputs \
-i {stepURI:v2} \
-u {username} \
-p {password} \
-l {compoundOutputFileLuid1} \
-f 'NovaSeqDx Run Mode'"

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 you select the Calculate Volumes button on the Record Details screen, this automation completes the following actions:

• Calculates the number of samples in the pool.

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  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.

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  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.

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  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 for sequencing, the Per Sample Volume (ul) value must be equal to or higher than 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, this automation 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 of the sample.

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

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

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  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, this automation calculates the RSB Volume (ul) field value.

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  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.

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

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

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  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 in the Files section of the Record Details milestone in the Make Bulk Pool for NovaSeqDx Standard (NovaSeqDx v1.2) step.

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  && /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.

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  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, advancing them to the Dilute and Denature (NovaSeqDx v1.2) step in the protocol:

nextStep = ::ADVANCE::

The following automation copies the NovaSeqDx Run Mode values from the step inputs to the step outputs:

output.::NovaSeqDx Run Mode:: = input.::NovaSeqDx Run Mode::

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

script:validateSampleCount -min 1 -max 1

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 following barcode masks:

  • DX mode: DX[0-9]{7}-[A-Z]{3}

  • RUO mode: [A-Z]{2}[0-9]{7}-[A-Z]{3}

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  if ((input.::NovaSeqDx Run Mode:: == ::DX:: && !output.container.name.matches(::^DX[0-9]{7}-[A-Z]{3}$::)) || (input.::NovaSeqDx Run Mode:: == ::RUO:: && !output.container.name.matches(::[A-Z]{2}[0-9]{7}-[A-Z]{3}::))) {
      fail(::Invalid Library Tube Barcode. Please verify and try again.::)
  }

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

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

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

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.

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

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

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

  • Experiment Name only contains 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.

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  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 as follows.

  • If Paired End = True, both Read 1 Cycles and Read 2 Cycles must be greater than 0.

  • If Paired End = False, Read 2 Cycles must be zero.

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  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.::)
      }
  };

• Checks the Flowcell field value as follows.

  • 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.

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  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::)
  };

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

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  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.

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

• Sets the next step for samples to REMOVE:

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  nextStep = ::REMOVE::

• Generates the V2 sample sheet and attaches it to the step. For more information, refer to Sample Sheet Generation.

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  /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/NovaSeqDx_BCL2FASTQ_Samplesheet_v2.csv \
  -o {compoundOutputFileLuid0}.csv \
  -q true \
  -destLIMSID {compoundOutputFileLuid0} \
  -l {compoundOutputFileLuid1}"

Automatically triggered on exit of the step, this automation invokes the changeWorkflow script, which routes step outputs to the NovaSeqDx v1.2 workflow, and queues them for the AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2) step.

The default automation command line is as follows.

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar
-u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid1} script:changeWorkflow \
--FIELD_NAME 'N/A' \
--FIELD_VALUE 'N/A' \
--WORKFLOW 'NovaSeqDx v1.2' \
--STEP 'AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"

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:

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  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:

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  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:

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  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::

  ⚠ To ensure accurate pipetting of each sample in a pool for sequencing, the Per Sample Volume (ul) value must be equal to or higher than the Minimum Per Sample Volume (ul). The default value (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, based on the ratio used to increase the lowest value to 5.

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

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  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, this automation calculates the RSB Volume (ul) field value:

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  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:

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  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 in the Files segment of the Make Bulk Pool for NovaSeqDx Xp (NovaSeqDx v1.2) step.

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  && /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.

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  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 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). This automation also checks that all inputs have the same value for this field.

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

bash -l -c "/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}"

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:

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  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 in the Files segment of the Dilute, Denature & ExAmp (NovaSeqDx v1.2) step.

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  /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:

• 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 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.

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  bash -l -c "/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} \
  -validateSelectedContainer true"

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:

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  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:

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  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:

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

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

• Validates the parameters entered on the Record Details screen, which is used to set up the run and generate the sample sheet file.

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

  • When Workflow Type = No Index, both Index Read 1 and Index Read 2 must be 0.

  • When Workflow Type = Single Index, Index Read 1 must be greater than 0 and Index Read 2 must be 0.

  • When Workflow Type = Dual Index, both Index Read 1 and Index Read 2 must be greater than 0.

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  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.

  • When Paired End = True, both Read 1 Cycles and Read 2 Cycles must be greater than 0.

  • When Paired End = False, Read 2 Cycles must be 0.

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  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.::)
      }
  };

• 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 the value is greater than 151, an error message displays.

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  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::)
  }

• Validate Analysis Software Version

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  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 field contains only Y, N, I, U, 0–9, and semicolon characters.

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  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.::)
  };

• Sets the next step for samples to ADVANCE, advancing them to the AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2) step.

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  nextStep = ::ADVANCE::

• Generates the sample sheet and attaches it to the step. For more information, refer to Sample Sheet Generation.

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  /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/NovaSeqDx_BCL2FASTQ_Samplesheet_v2.csv \
  -o {compoundOutputFileLuid0}.csv \
  -q true \
  -destLIMSID {compoundOutputFileLuid0} \
  -l {compoundOutputFileLuid1}

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 integration.sequencer_api.v2.run.autoComplete and integration.sequencer_api.v2.run.autoCompleteOnlyAtSuccess properties. For details, refer to Configure autoComplete Properties.

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

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

The following step custom fields that display in the Step Details area of the Record Details screen control the sample sheet content:

• Experiment Name

• Read 1 Cycles

• Read 2 Cycles

• Index 1 Cycles

• Index 2 Cycles

• Analysis Software Version

• Override Cycles

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 6000Dx instrument is achieved with the getSequencerLoginURL endpoint (/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 6000Dx Operating Software (NVOS) with a URL for the Clarity LIMS login screen. The NVOS displays this screen to the user. Log in to authorize NVOS to use your account to access Clarity LIMS. This authorization is automatic.

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

3. The user is redirected to the Sequencer login page at

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   /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 redirecting to

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     /Illumina/Sequencer/v2/sequencer_login/Success

     This redirect does not reload the page, but allows the NovaSeqDx to continue with run setup.

   • If authentication fails, the login screen does not change and a warning message displays. A background redirect to

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     /Illumina/Sequencer/v2/sequencer_login/Error

     informs the NovaSeq 6000Dx of the failure.

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

   • LibraryContainerId, FlowCellId, and reagents are provided by the NovaSeq 6000Dx.

   • 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 integration.sequencer_api.v2.novaseq.sequenceStepNames property. The property is a list and each 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 Properties section of Components Installed.

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

   Note the following details:

   • 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 (NovaSeqDx v1.2) or Load to Flowcell (NovaSeqDx v1.2) step, depending on workflow.

   • The default field values match the names of the fields in the NovaSeq 6000Dx v1.2 configuration included in IPP. The field names are configurable through the integration.sequencer_api.v2.recipe.udfNames.* properties. Each property 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 is configured through the integration.sequencer_api.v2.recipe.sampleSheet.outputName property (Sample Sheet by default). If no matching output is found, or no file was attached to the output, the value of the integration.sequencer_api.v2.recipe.sampleSheet.notAvailableValue property (Not Available by default) is used in the recipe. For more information, refer to the Properties section of Components Installed.

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 contents and the fields configured on the Dilute and Denature (NovaSeqDx v1.2) and Load to Flowcell (NovaSeqDx v1.2) steps control the contents of the run recipe.

Example of run recipe content is as follows.

{
    "run_name": "NVOS16027_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 is thrown 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, and 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, and 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.

   • You can look up existing reagent lots 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.

   • 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.

• Finds 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. The Sequencing Container matches the Information, 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:

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

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

5. By default, 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. The Sequencing Container matches the Information, 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:

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

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

If the user ends a sequencing run, the API call for Sequencing RunEndedByUser is received. The Sequencing Container matches the Information, 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:

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   "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.