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 v2.3.1. 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 v2.3.1

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'"