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 v2.3.4. 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 v2.3.4

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:

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  nextStep = ::REMOVE::

• Calculates the Minimum Molarity using 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), it 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 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:

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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:: = ::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:

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  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'"