# DNA Library Prep v1.1

## Overview

The DNA Library Prep AmpliSeq for Illumina Childhood Cancer Panel is part of the AmpliSeq for Illumina Childhood Cancer Panel protocols that include the following functionality:

* Preconfigured AmpliSeq for Illumina Childhood Cancer Panel protocol that supports the preparation of up to 384 uniquely indexed libraries of genomic DNA or total RNA using the AmpliSeq for Illumina workflow.
* Automated calculation of sample and buffer volumes.
* Automated calculation or display of reagents at every step in the protocol.
* Automatic step transition when required.
* Automatic placement of samples when necessary. Automated assignment of QC Pass/Fail, based on user-selected threshold values.
* There is no extraction protocol in this workflow because the samples are already extracted.

## Protocol 1: DNA Library Prep (AmpliSeq for Illumina Childhood Cancer Panel v1.1)

Protocol Type = Library Prep

**Next Steps Configuration**

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-fe909e827011652a832fd2e1ce6796b2847d82cd%2Fampliseq-childhood-dna-next-step-config.png?alt=media" alt=""><figcaption></figcaption></figure>

### Step 1: Dilute DNA (AmpliSeq for Illumina Childhood Cancer Panel v1.1)

* Master Step Name = Dilute DNA (AmpliSeq for Illumina v1.1)
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
  * AmpliSeq Library PLUS for Illumina
    * Supplier = Illumina
    * Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103
    * Website = [www.illumina.com](https://www.illumina.com)

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-4869a0df20b0664da8adb722ab61c736984713ec%2Fampliseq-childhood-dna-step1-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

<summary>Copy Concentration and Set Sample Volume</summary>

* Trigger Location = Record Details
* Trigger Style = Automatic upon entry

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'output.::Concentration:: = input.::Concentration:: ; output.::Conc. Units:: = input.::Conc. Units:: ; output.::Sample Volume (uL):: = 5' \
-log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Calculate Input Amount</summary>

* Trigger Location = Record Details
* Trigger Style = Manual button

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
        -t true \
        -h false \
        -exp 'output.::Total Volume (uL):: = (output.::Concentration::/step.::Desired Concentration (ng/uL)::) * output.::Sample Volume (uL):: ; \
output.::Low TE (uL):: = output.::Total Volume (uL):: - output.::Sample Volume (uL):: ; \
output.::Input Amount (ng):: = step.::Desired Concentration (ng/uL):: * output.::Total Volume (uL):: ' \
        -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Set Next Step - Advance</summary>

* Trigger Location = Not Used

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

#### Queue/Ice Bucket

* Defaults
  * Sample Grouping = Group by Containers
  * Well Sort Order = Row
* Sample Table (Column Headers)

  | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
  | Container      | Container Name      | Built-in       |             |                                           |
  | Container      | LIMS ID (Container) | Built-in       |             |                                           |
  | Container      | Well                | Built-in       |             |                                           |
  | Derived Sample | Sample Name         | Built-in       |             |                                           |
  | Derived Sample | Waiting             | Built-in       |             |                                           |
  | Project        | Project Name        | Built-in       |             |                                           |

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**                | **Field Type** | **Options** | **Additional Options and Dropdown Items**                                                                      |
  | ----------------------------- | -------------- | ----------- | -------------------------------------------------------------------------------------------------------------- |
  | Desired Concentration (ng/uL) | Numeric        |             | <ul><li>Range = 20 To 50</li><li>Decimal Places Displayed = 0</li></ul>                                        |
  | Directions                    | Multiline Text | Read Only   | <ul><li>Default = If enough DNA is available, dilute to intermediate concentration of \~20-50 ng/uL.</li></ul> |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Expand
  * Well Sort Order = Row
  * Table Columns - Global Fields

    | **Category**   | **Field Name**      | **Field Type** | **Options**    | **Additional Options and Dropdown Items** |
    | -------------- | ------------------- | -------------- | -------------- | ----------------------------------------- |
    | Container      | Container Name      | Built-in       |                |                                           |
    | Container      | LIMS ID (Container) | Built-in       |                |                                           |
    | Container      | Well                | Built-in       |                |                                           |
    | Derived Sample | Concentration       | Numeric        | Required Field | Decimal Places Displayed = 2              |
    | Derived Sample | Conc. Units         | Text           | Required Field |                                           |
    | Derived Sample | Input Amount (ng)   | Numeric        |                | Decimal Places Displayed = 0              |
    | Derived Sample | Low TE (uL)         | Numeric        |                | Decimal Places Displayed = 2              |
    | Derived Sample | Sample Name         | Built-in       |                |                                           |
    | Derived Sample | Sample Volume (uL)  | Numeric        |                | Decimal Places Displayed = 2              |
    | Derived Sample | Total Volume (uL)   | Numeric        |                | Decimal Places Displayed = 2              |
    | Project        | Project Name        | Built-in       |                |                                           |

### Step 2: Qubit (AmpliSeq for Illumina Childhood Cancer Panel v1.1)

* Master Step Name = Qubit (AmpliSeq for Illumina v1.1)
* Step Type = Standard QC
* Measurement Generation = Fixed, 1
* Naming Convention = {InputItemName}

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-9445a8d3ad3bd6753de597a2923e435b9afdb56e%2Fampliseq-childhood-dna-step2-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

<summary>Assign QC flags (Qubit QC)</summary>

* Trigger Location = Record Details
* Trigger Style = Manual button

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {processURI:v2} -u {username} -p {password} script:assignQC -log {compoundOutputFileLuid1} -qcResult {compoundOutputFileLuid2}"
```

{% endcode %}

</details>

<details>

<summary>Set Next Step and Copy Concentration</summary>

* Trigger Location = Record Details
* Trigger Style = Automatic upon exit

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'nextStep = ::ADVANCE:: ; input.::Concentration:: = output.::Concentration:: ; input.::Conc. Units:: = output.::Conc. Units::' -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

#### Queue/Ice Bucket

* Defaults
  * Sample Grouping = Group by Containers
  * Well Sort Order = Row
* Sample Table (Column Headers)

  | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
  | Container      | Container Name      | Built-in       |             |                                           |
  | Container      | LIMS ID (Container) | Built-in       |             |                                           |
  | Container      | Well                | Built-in       |             |                                           |
  | Derived Sample | Sample Name         | Built-in       |             |                                           |
  | Derived Sample | Waiting             | Built-in       |             |                                           |
  | Project        | Project Name        | Built-in       |             |                                           |

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**                 | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | ------------------------------ | -------------- | ----------- | ----------------------------------------- |
  | Criteria 1 - Operator          | Text           |             | Default = >=                              |
  | Criteria 1 - Source Data Field | Text           |             | Default = Concentration                   |
  | Criteria 1 - Threshold Value   | Numeric        |             |                                           |
  | Criteria 2 - Operator          | Text           |             | Default = <=                              |
  | Criteria 2 - Source Data Field | Text           |             | Default = Concentration                   |
  | Criteria 2 - Threshold Value   | Numeric        |             |                                           |
* Step File Placeholders
  * Log - Automatically attached
  * QC Log File - Automatically attached
  * QC Result File - Automatically attached
  * Upload File - Manually uploaded
* Sample Table
  * Enable QC Flags = Yes
  * Sample Display Default = Expand
  * Well Sort Order = Row
  * File Column Options
    * File Column Display = Hide
    * File Attachment Method = Auto
  * Table Columns - Global Fields

    | **Category**   | **Field Name**      | **Field Type** | **Options**    | **Additional Options and Dropdown Items** |
    | -------------- | ------------------- | -------------- | -------------- | ----------------------------------------- |
    | Container      | Container Name      | Built-in       |                |                                           |
    | Container      | LIMS ID (Container) | Built-in       |                |                                           |
    | Container      | Well                | Built-in       |                |                                           |
    | Derived Sample | Concentration       | Numeric        | Required Field | Decimal Places Displayed = 2              |
    | Derived Sample | Conc. Units         | Text           | Required Field |                                           |
    | Derived Sample | Sample Name         | Built-in       |                |                                           |
    | Measurement    | Concentration       | Numeric        |                | Decimal Places Displayed = 2              |
    | Measurement    | Conc. Units         | Text           |                |                                           |
    | Project        | Project Name        | Built-in       |                |                                           |

### Step 3: Dilute DNA to Final Concentration (AmpliSeq for Illumina Childhood Cancer Panel v1.1)

* Master Step Name = Dilute DNA (AmpliSeq for Illumina v1.1)
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
  * AmpliSeq Library PLUS for Illumina
    * Supplier = Illumina
    * Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103
    * Website = [www.illumina.com](https://www.illumina.com)

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-4d263d71f9b913d00a22445f05d1fef4898b007b%2Fampliseq-childhood-dna-step3-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

<summary>Copy Concentration and Set Sample Volume</summary>

* Trigger Location = Record Details
* Trigger Style = Automatic upon entry

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'output.::Concentration:: = input.::Concentration:: ; output.::Conc. Units:: = input.::Conc. Units:: ; output.::Sample Volume (uL):: = 5' \
-log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Calculate Input Amount</summary>

* Trigger Location = Record Details
* Trigger Style = Manual button

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
        -t true \
        -h false \
        -exp 'output.::Total Volume (uL):: = (output.::Concentration::/step.::Desired Concentration (ng/uL)::) * output.::Sample Volume (uL):: ; \
output.::Low TE (uL):: = output.::Total Volume (uL):: - output.::Sample Volume (uL):: ; \
output.::Input Amount (ng):: = step.::Desired Concentration (ng/uL):: * output.::Total Volume (uL):: ' \
        -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Set Next Step - Advance</summary>

* Trigger Location = Record Details
* Trigger Style = Automatic upon exit

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

#### Queue/Ice Bucket

* Defaults
  * Sample Grouping = Group by Containers
  * Well Sort Order = Row
* Sample Table (Column Headers)

  | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
  | Container      | Container Name      | Built-in       |             |                                           |
  | Container      | LIMS ID (Container) | Built-in       |             |                                           |
  | Container      | Well                | Built-in       |             |                                           |
  | Derived Sample | Sample Name         | Built-in       |             |                                           |
  | Derived Sample | Waiting             | Built-in       |             |                                           |
  | Project        | Project Name        | Built-in       |             |                                           |

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**                | **Field Type** | **Options** | **Additional Options and Dropdown Items**                               |
  | ----------------------------- | -------------- | ----------- | ----------------------------------------------------------------------- |
  | Desired Concentration (ng/uL) | Numeric        |             | <ul><li>Range = 20 To 50</li><li>Decimal Places Displayed = 0</li></ul> |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Expand
  * Well Sort Order = Row
  * Table Columns - Global Fields

    | **Category**   | **Field Name**      | **Field Type** | **Options**    | **Additional Options and Dropdown Items** |
    | -------------- | ------------------- | -------------- | -------------- | ----------------------------------------- |
    | Container      | Container Name      | Built-in       |                |                                           |
    | Container      | LIMS ID (Container) | Built-in       |                |                                           |
    | Container      | Well                | Built-in       |                |                                           |
    | Derived Sample | Concentration       | Numeric        | Required Field | Decimal Places Displayed = 2              |
    | Derived Sample | Conc. Units         | Text           | Required Field |                                           |
    | Derived Sample | Input Amount (ng)   | Numeric        |                | Decimal Places Displayed = 0              |
    | Derived Sample | Low TE (uL)         | Numeric        |                | Decimal Places Displayed = 2              |
    | Derived Sample | Sample Name         | Built-in       |                |                                           |
    | Derived Sample | Sample Volume (uL)  | Numeric        |                | Decimal Places Displayed = 2              |
    | Derived Sample | Total Volume (uL)   | Numeric        |                | Decimal Places Displayed = 2              |
    | Project        | Project Name        | Built-in       |                |                                           |

### Step 4: Amplify DNA Targets (AmpliSeq for Illumina Childhood Cancer Panel v1.1)

* Master Step Name = Amplify Targets Duplicates (AmpliSeq for Illumina v1.1)
* Step Type = Standard
* Derived Sample Generation = Fixed, 2
* Naming Convention = {InputItemName}\_{OutputItemSubsetNumber}
* Reagent Kits
  * AmpliSeq Childhood Cancer Panel for Illumina
    * Supplier = Illumina
    * Catalog Number = 20028446
    * Website = [www.illumina.com](https://www.illumina.com)
  * AmpliSeq Library PLUS for Illumina
    * Supplier = Illumina
    * Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103
    * Website = [www.illumina.com](https://www.illumina.com)

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-d7623a99ceb7c1d525319e8f7f68dd4d50f7bdb7%2Fampliseq-childhood-dna-step4-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

<summary>Copy Concentration, Total Volume and Input Amount</summary>

* Trigger Location = Record Details
* Trigger Style = Automatic upon entry

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
        -t false \
        -h false \
        -exp 'output.::Concentration:: = input.::Concentration:: ; output.::Conc. Units:: = input.::Conc. Units:: ; output.::Total Volume (uL):: = input.::Total Volume (uL):: ; output.::Input Amount (ng):: = input.::Input Amount (ng):: ; output.::5X AmpliSeq HiFi Mix (uL):: = 4.5'\
        -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Calculate Prep Input - CCP</summary>

* Trigger Location = Record Details
* Trigger Style = Manual button

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
        -t false \
        -h false \
        -exp 'output.::Prep Input Volume (uL):: = output.::Prep Input Amount (ng):: / output.::Concentration:: ; \
if (output.::Prep Input Volume (uL):: < 12.5) {output.::Nuclease-free water (uL):: = 12.5 - output.::Prep Input Volume (uL)::} '\
        -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Set Next Step - Advance</summary>

* Trigger Location = Record Details
* Trigger Style = Automatic upon exit

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Calculate Prep Input - Myeloid Panel</summary>

* Trigger Location = Not Used

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
        -t false \
        -h false \
        -exp 'output.::Prep Input Volume (uL):: = output.::Prep Input Amount (ng):: / output.::Concentration:: ; \
if (output.::Prep Input Volume (uL):: < 13.5) {output.::Nuclease-free water (uL):: = 13.5 - output.::Prep Input Volume (uL)::} '\
        -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

#### Queue/Ice Bucket

* Defaults
  * Sample Grouping = Group by Containers
  * Well Sort Order = Row
* Sample Table (Column Headers)

  | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
  | Container      | Container Name      | Built-in       |             |                                           |
  | Container      | LIMS ID (Container) | Built-in       |             |                                           |
  | Container      | Well                | Built-in       |             |                                           |
  | Derived Sample | Sample Name         | Built-in       |             |                                           |
  | Derived Sample | Waiting             | Built-in       |             |                                           |
  | Project        | Project Name        | Built-in       |             |                                           |

#### Placement = Enabled

* Defaults
  * Sample Grouping = Group by Containers
  * Well Sort Order = Row
  * Placement Pattern = Row
* Destination Containers
  * 96 well plate

#### Record Details

**Group of Defaults**

<details>

<summary>Childhood Cancer Panel</summary>

* Recommended Prep Input (ng) = This protocol supports 1-100 ng per pool. The recommended input is 10 ng high-quality DNA per pool.
* Thermal Cycler Program = AMP

</details>

<details>

<summary>Myeloid Panel</summary>

* Recommended Prep Input (ng) = This protocol supports 10-100 ng per pool. The recommended input is 10 ng high-quality DNA per pool.
* Thermal Cycler Program = AMP

</details>

* Step Data
  * Group of Defaults = Childhood Cancer Panel
  * Master Step Fields

    | **Field Name**              | **Field Type** | **Options** | **Additional Options and Dropdown Items**                                                                      |
    | --------------------------- | -------------- | ----------- | -------------------------------------------------------------------------------------------------------------- |
    | PCR Cycles                  | Numeric        |             |                                                                                                                |
    | Recommended Prep Input (ng) | Multiline Text | Read Only   | Default = This protocol supports 10-100 ng per pool. The recommended input is 10 ng high-quality DNA per pool. |
    | Thermal Cycler Program      | Text           |             | Default = AMP                                                                                                  |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Expand
  * Well Sort Order = Row
  * Table Columns - Global Fields

    | **Category**   | **Field Name**            | **Field Type** | **Options**    | **Additional Options and Dropdown Items**                               |
    | -------------- | ------------------------- | -------------- | -------------- | ----------------------------------------------------------------------- |
    | Container      | Container Name            | Built-in       |                |                                                                         |
    | Container      | LIMS ID (Container)       | Built-in       |                |                                                                         |
    | Container      | Well                      | Built-in       |                |                                                                         |
    | Derived Sample | 5X AmpliSeq HiFi Mix (uL) | Numeric        |                | <ul><li>Decimal Places Displayed = 1</li></ul>                          |
    | Derived Sample | Concentration             | Numeric        | Required Field | <ul><li>Decimal Places Displayed = 2</li></ul>                          |
    | Derived Sample | Conc. Units               | Text           | Required Field |                                                                         |
    | Derived Sample | Nuclease-free water (uL)  | Numeric        |                | <ul><li>Decimal Places Displayed = 2</li></ul>                          |
    | Derived Sample | Prep Input Amount (ng)    | Numeric        | Required Field | <ul><li>Range = 2 To 200</li><li>Decimal Places Displayed = 0</li></ul> |
    | Derived Sample | Prep Input Volume (uL)    | Numeric        |                | <ul><li>Decimal Places Displayed = 2</li></ul>                          |
    | Derived Sample | Sample Name               | Built-in       |                |                                                                         |
    | Derived Sample | Total Volume (uL)         | Numeric        |                | <ul><li>Decimal Places Displayed = 2</li></ul>                          |
    | Project        | Project Name              | Built-in       |                |                                                                         |

### Step 5: Transfer RNA and DNA Amplicons (AmpliSeq for Illumina Childhood Cancer Panel v1.1)

* Master Step Name = Transfer RNA and DNA Amplicons (AmpliSeq for Illumina v1.1)
* Step Type = Pooling
* Aliquot Generation = Fixed, 1
* Naming Convention = {PoolName}

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-1a2db56e5d706511ac28ea150815cab9388ac594%2Fampliseq-childhood-dna-step5-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

<summary>Set Next Step - Advance</summary>

* Trigger Location = Record Details
* Trigger Style = Automatic upon exit

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

#### Queue/Ice Bucket

* Defaults
  * Sample Grouping = Group by Containers
  * Well Sort Order = Row
* Sample Table (Column Headers)

  | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
  | Container      | Container Name      | Built-in       |             |                                           |
  | Container      | LIMS ID (Container) | Built-in       |             |                                           |
  | Container      | Well                | Built-in       |             |                                           |
  | Derived Sample | Sample Name         | Built-in       |             |                                           |
  | Derived Sample | Waiting             | Built-in       |             |                                           |
  | Project        | Project Name        | Built-in       |             |                                           |

#### Pooling

* Label Uniqueness = Off
* Defaults
  * Sample Grouping = Group by Containers
  * Well Sort Order = Row

#### Placement = Enabled

* Defaults
  * Well Sort Order = Row
  * Placement Pattern = Column
* Destination Containers
  * 96 well plate

#### Record Details

* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Expand
  * Well Sort Order = Column
  * Table Columns - Global Fields

    | **Category**   | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
    | -------------- | -------------- | -------------- | ----------- | ----------------------------------------- |
    | Container      | Container Name | Built-in       |             |                                           |
    | Container      | Well           | Built-in       |             |                                           |
    | Derived Sample | Sample Name    | Built-in       |             |                                           |

### Step 6: Partially Digested Amplicons (AmpliSeq for Illumina Childhood Cancer Panel v1.1)

* Master Step Name = Partially Digested Amplicons (AmpliSeq for Illumina v1.1)
* Step Type = No Outputs
* Reagent Kits
  * AmpliSeq Library PLUS for Illumina
    * Supplier = Illumina
    * Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103
    * Website = [www.illumina.com](https://www.illumina.com)

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-788669bf35cc6fb2e11dfe7a86f8f5108e7059f4%2Fampliseq-childhood-dna-step6-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

<summary>Set Next Step - Advance</summary>

* Trigger Location = Record Details
* Trigger Style = Automatic upon exit

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

#### Queue/Ice Bucket

* Defaults
  * Sample Grouping = Group by Containers
  * Well Sort Order = Row
* Sample Table (Column Headers)

  | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
  | Container      | Container Name      | Built-in       |             |                                           |
  | Container      | LIMS ID (Container) | Built-in       |             |                                           |
  | Container      | Well                | Built-in       |             |                                           |
  | Derived Sample | Sample Name         | Built-in       |             |                                           |
  | Derived Sample | Waiting             | Built-in       |             |                                           |
  | Project        | Project Name        | Built-in       |             |                                           |

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**         | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | ---------------------- | -------------- | ----------- | ----------------------------------------- |
  | Thermal Cycler Program | Text           |             | Default = FUPA                            |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Expand
  * Well Sort Order = Row
  * Table Columns - Global Fields

    | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
    | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
    | Container      | Container Name      | Built-in       |             |                                           |
    | Container      | LIMS ID (Container) | Built-in       |             |                                           |
    | Container      | Well                | Built-in       |             |                                           |
    | Derived Sample | Sample Name         | Built-in       |             |                                           |
    | Project        | Project Name        | Built-in       |             |                                           |

### Step 7: Ligate Indexes (AmpliSeq for Illumina Childhood Cancer Panel v1.1)

* Master Step Name = Ligate Indexes (AmpliSeq for Illumina v1.1)
* Step Type = Add Labels
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
  * AmpliSeq Library PLUS for Illumina
    * Supplier = Illumina
    * Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103
    * Website = [www.illumina.com](https://www.illumina.com)

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-4f1a2ea234c73b77dc4f9e704fc5c0c2ce9d5f03%2Fampliseq-childhood-dna-step7-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

<summary>Set Next Step - Advance</summary>

* Trigger Location = Record Details
* Trigger Style = Automatic upon exit

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Route AmpliSeq Samples - Childhood Cancer Panel</summary>

* Trigger Location = Step
* Trigger Style = Automatic upon exit

{% code overflow="wrap" %}

```markup
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 'AmpliSeq Workflow' \
--FIELD_VALUE 'Standard Workflow' \
--WORKFLOW 'Standard Workflow AmpliSeq for Illumina Childhood Cancer Panel v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina Childhood Cancer Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'AmpliSeq Workflow' \
--FIELD_VALUE 'Equalizer Workflow' \
--WORKFLOW 'Equalizer Workflow AmpliSeq for Illumina Childhood Cancer Panel v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina Childhood Cancer Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"
```

{% endcode %}

> ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.

</details>

<details>

<summary>Route AmpliSeq Samples - Immune Repertoire Panel</summary>

* Trigger Location = Not Used

{% code overflow="wrap" %}

```markup
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 'AmpliSeq Workflow' \
--FIELD_VALUE 'Standard Workflow' \
--WORKFLOW 'Standard Workflow AmpliSeq for Illumina Immune Repertoire Panel v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina Immune Repertoire Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'AmpliSeq Workflow' \
--FIELD_VALUE 'Equalizer Workflow' \
--WORKFLOW 'Equalizer Workflow AmpliSeq for Illumina Immune Repertoire Panel v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina Immune Repertoire Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"
```

{% endcode %}

> ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.

</details>

<details>

<summary>Route AmpliSeq Samples - BRCA</summary>

* Trigger Location = Not Used

{% code overflow="wrap" %}

```markup
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 'AmpliSeq Workflow' \
--FIELD_VALUE 'Standard Workflow' \
--WORKFLOW 'Standard Workflow AmpliSeq for Illumina BRCA Panel v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina BRCA Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'AmpliSeq Workflow' \
--FIELD_VALUE 'Equalizer Workflow' \
--WORKFLOW 'Equalizer Workflow AmpliSeq for Illumina BRCA Panel v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina BRCA Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"
```

{% endcode %}

> ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.

</details>

<details>

<summary>Route AmpliSeq Samples - CHP v2</summary>

* Trigger Location = Not Used

{% code overflow="wrap" %}

```markup
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 'AmpliSeq Workflow' \
--FIELD_VALUE 'Standard Workflow' \
--WORKFLOW 'Standard Workflow AmpliSeq for Illumina Cancer HotSpot Panel V2 v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina Cancer HotSpot Panel V2 v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'AmpliSeq Workflow' \
--FIELD_VALUE 'Equalizer Workflow' \
--WORKFLOW 'Equalizer Workflow AmpliSeq for Illumina Cancer HotSpot Panel V2 v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina Cancer HotSpot Panel V2 v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"
```

{% endcode %}

> ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.

</details>

<details>

<summary>Route AmpliSeq Samples - CP</summary>

* Trigger Location = Not Used

{% code overflow="wrap" %}

```markup
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 'AmpliSeq Workflow' \
--FIELD_VALUE 'Standard Workflow' \
--WORKFLOW 'Standard Workflow AmpliSeq for Illumina Comprehensive Panel v3 v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina Comprehensive Panel v3 v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'AmpliSeq Workflow' \
--FIELD_VALUE 'Equalizer Workflow' \
--WORKFLOW 'Equalizer Workflow AmpliSeq for Illumina Comprehensive Panel v3 v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina Comprehensive Panel v3 v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"
```

{% endcode %}

> ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.

</details>

<details>

<summary>Route AmpliSeq Samples - Custom DNA Panel</summary>

* Trigger Location = Not Used

{% code overflow="wrap" %}

```markup
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 'AmpliSeq Workflow' \
--FIELD_VALUE 'Standard Workflow' \
--WORKFLOW 'Standard Workflow AmpliSeq for Illumina Custom DNA Panel v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina Custom DNA Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'AmpliSeq Workflow' \
--FIELD_VALUE 'Equalizer Workflow' \
--WORKFLOW 'Equalizer Workflow AmpliSeq for Illumina Custom DNA Panel v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina Custom DNA Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"
```

{% endcode %}

> ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.

</details>

<details>

<summary>Route AmpliSeq Samples - Focus Panel</summary>

* Trigger Location = Not Used

{% code overflow="wrap" %}

```markup
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 'AmpliSeq Workflow' \
--FIELD_VALUE 'Standard Workflow' \
--WORKFLOW 'Standard Workflow AmpliSeq for Illumina Focus Panel v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina Focus Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'AmpliSeq Workflow' \
--FIELD_VALUE 'Equalizer Workflow' \
--WORKFLOW 'Equalizer Workflow AmpliSeq for Illumina Focus Panel v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina Focus Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"
```

{% endcode %}

> ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.

</details>

<details>

<summary>Route AmpliSeq Samples - Immune Response Panel</summary>

* Trigger Location = Not Used

{% code overflow="wrap" %}

```markup
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 'AmpliSeq Workflow' \
--FIELD_VALUE 'Standard Workflow' \
--WORKFLOW 'Standard Workflow AmpliSeq for Illumina Immune Response Panel v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina Immune Response Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'AmpliSeq Workflow' \
--FIELD_VALUE 'Equalizer Workflow' \
--WORKFLOW 'Equalizer Workflow AmpliSeq for Illumina Immune Response Panel v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina Immune Response Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"
```

{% endcode %}

> ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.

</details>

<details>

<summary>Route AmpliSeq Samples - Myeloid Panel</summary>

* Trigger Location = Not Used

{% code overflow="wrap" %}

```markup
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 'AmpliSeq Workflow' \
--FIELD_VALUE 'Standard Workflow' \
--WORKFLOW 'Standard Workflow AmpliSeq for Illumina Myeloid Panel v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina Myeloid Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'AmpliSeq Workflow' \
--FIELD_VALUE 'Equalizer Workflow' \
--WORKFLOW 'Equalizer Workflow AmpliSeq for Illumina Myeloid Panel v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina Myeloid Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"
```

{% endcode %}

> ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.

</details>

<details>

<summary>Route AmpliSeq Samples -Transcriptome Human GEx Panel</summary>

* Trigger Location = Not Used

{% code overflow="wrap" %}

```markup
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 'AmpliSeq Workflow' \
--FIELD_VALUE 'Standard Workflow' \
--WORKFLOW 'Standard Workflow AmpliSeq for Illumina Transcriptome Human Gene Expression Panel v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina Transcriptome Human Gene Expression Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'AmpliSeq Workflow' \
--FIELD_VALUE 'Equalizer Workflow' \
--WORKFLOW 'Equalizer Workflow AmpliSeq for Illumina Transcriptome Human Gene Expression Panel v1.1' \
--STEP 'Clean Up Library (AmpliSeq for Illumina Transcriptome Human Gene Expression Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"
```

{% endcode %}

> ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.

</details>

#### Queue/Ice Bucket

* Defaults
  * Sample Grouping = Group by Containers
  * Well Sort Order = Row
* Sample Table (Column Headers)

  | **Category**   | **Field Name**      | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- |
  | Container      | Container Name      | Built-in       |             |                                           |
  | Container      | LIMS ID (Container) | Built-in       |             |                                           |
  | Container      | Well                | Built-in       |             |                                           |
  | Derived Sample | Sample Name         | Built-in       |             |                                           |
  | Derived Sample | Waiting             | Built-in       |             |                                           |
  | Project        | Project Name        | Built-in       |             |                                           |

#### Add Labels

* Label Groups
  * AmpliSeq CD Indexes Set A for Illumina
  * AmpliSeq CD Indexes Set B for Illumina
  * AmpliSeq CD Indexes Set C for Illumina
  * AmpliSeq CD Indexes Set D for Illumina
  * AmpliSeq UD Indexes for Illumina (24 Indexes)

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**         | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | ---------------------- | -------------- | ----------- | ----------------------------------------- |
  | Thermal Cycler Program | Text           |             | Default = LIGATE                          |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Expand
  * Well Sort Order = Row
  * Table Columns - Global Fields

    | **Category**   | **Field Name**      | **Field Type** | **Options**    | **Additional Options and Dropdown Items**                                   |
    | -------------- | ------------------- | -------------- | -------------- | --------------------------------------------------------------------------- |
    | Container      | Container Name      | Built-in       |                |                                                                             |
    | Container      | LIMS ID (Container) | Built-in       |                |                                                                             |
    | Container      | Well                | Built-in       |                |                                                                             |
    | Derived Sample | AmpliSeq Workflow   | Text Dropdown  | Required Field | <p>Preset</p><ul><li>Standard Workflow</li><li>Equalizer Workflow</li></ul> |
    | Derived Sample | Sample Name         | Built-in       |                |                                                                             |
    | Project        | Project Name        | Built-in       |                |                                                                             |