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: