# RNA Library Prep v1.1

## Overview

The RNA Library Prep AmpliSeq for Illumina TCR beta-SR Panel is part of the AmpliSeq for Illumina TCR beta-SR Panel protocols which include the following functionality:

* Preconfigured AmpliSeq for Illumina TCR beta-SR Panel protocol that supports the preparation of up to 96 uniquely indexed libraries of 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.

## Protocol 1: RNA Library Prep (AmpliSeq for Illumina TCR beta-SR Panel v1.1)

Protocol Type = Sample 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-d67f57d4ed2c53c26ff3ca44b9916e30f2a3def0%2Fampliseq-tcr-rna-next-step-config.png?alt=media" alt=""><figcaption></figcaption></figure>

### Step 1: Dilute RNA (AmpliSeq for Illumina TCR beta-SR Panel v1.1)

* Master Step Name = Dilute RNA (AmpliSeq for Illumina v1.1)
* Step Type = Standard
* Derived Sample 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-2285c91fb038c31efbbe4f3ac7adc6b804a46cbc%2Fampliseq-tcr-rna-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 - RNA</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.::Nuclease-free water (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>Copy Desired Concentration</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 -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'output.::Desired Concentration (ng/uL):: = step.::Desired Concentration (ng/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

**Group of Defaults**

<details>

<summary>Immune Repertoire Panel</summary>

* Directions = If enough RNA is available, dilute to intermediate concentration \~20-1000 ng/uL.

</details>

<details>

<summary>TCR beta-SR Panel</summary>

* Default = If enough RNA is available, dilute to intermediate concentration \~20-1000 ng/uL.

</details>

* Step Data
  * Group of Defaults = TCR beta-SR Panel
  * Master Step Fields

    | **Field Name**                | **Field Type** | **Options** | **Additional Options and Dropdown Items**                                                 |
    | ----------------------------- | -------------- | ----------- | ----------------------------------------------------------------------------------------- |
    | Desired Concentration (ng/uL) | Numeric        |             | Range = 0 To 1000                                                                         |
    | Directions                    | Multiline Text | Read Only   | Default = If enough RNA is available, dilute to intermediate concentration \~20-50 ng/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** |
    | -------------- | ------------------------ | -------------- | -------------- | ----------------------------------------- |
    | 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 | Nuclease-free water (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              |

### Step 2: Qubit (AmpliSeq for Illumina TCR beta-SR 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-85a0083cc8fd43af70509d5566d07dc6e20ad101%2Fampliseq-tcr-rna-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** |
    | -------------- | -------------- | -------------- | -------------- | ----------------------------------------- |
    | 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           |                |                                           |

### Step 3: Dilute RNA (AmpliSeq for Illumina TCR beta-SR Panel v1.1)

* Master Step Name = Dilute RNA (AmpliSeq for Illumina v1.1)
* Step Type = Standard
* Derived Sample 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-104c59bc28394249d79bdf362a1fbc55b7ba3e00%2Fampliseq-tcr-rna-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 - RNA</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.::Nuclease-free water (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>

<details>

<summary>Copy Desired Concentration</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 -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'output.::Desired Concentration (ng/uL):: = step.::Desired Concentration (ng/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       |             |                                           |

#### Record Details

**Group of Defaults**

<details>

<summary>Immune Repertoire Panel</summary>

* Directions = If enough RNA is available, dilute to intermediate concentration \~20-1000 ng/uL.

</details>

<details>

<summary>TCR beta-SR Panel</summary>

* Default = If enough RNA is available, dilute to intermediate concentration \~20-1000 ng/uL.

</details>

* Step Data
  * Group of Defaults = TCR beta-SR Panel
  * Master Step Fields

    | **Field Name**                | **Field Type** | **Options** | **Additional Options and Dropdown Items**                                                 |
    | ----------------------------- | -------------- | ----------- | ----------------------------------------------------------------------------------------- |
    | Desired Concentration (ng/uL) | Numeric        |             | Range = 0 To 1000                                                                         |
    | Directions                    | Multiline Text | Read Only   | Default = If enough RNA is available, dilute to intermediate concentration \~20-50 ng/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** |
    | -------------- | ------------------------ | -------------- | -------------- | ----------------------------------------- |
    | 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 | Nuclease-free water (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              |

### Step 4: Reverse Transcribe (AmpliSeq for Illumina TCR beta-SR Panel v1.1)

* Master Step Name = Reverse Transcribe RNA (AmpliSeq for Illumina v1.1)
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
  * AmpliSeq cDNA Synthesis for Illumina
    * Supplier = Illumina
    * Catalog Number = 20022654
    * 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-a28ea942b9cc3ca7bae33979d43a6f2eb84e3f3e%2Fampliseq-tcr-rna-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 'step.::Total Samples:: = step.::Total Samples:: + 1' \
-log {compoundOutputFileLuid0} && /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):: ; \
step.::Desired Concentration (ng/uL):: = input.::Desired Concentration (ng/uL)::' \
-log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Calculate Input Amount - TCRbeta-SR</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 'step.::Prep Input Volume (uL):: = step.::Prep Input Amount (ng):: / step.::Desired Concentration (ng/uL):: ; \
if (step.::Prep Input Volume (uL):: < 7) {step.::Nuclease-free water (uL):: = (7 - step.::Prep Input Volume (uL)::) * step.::Total Samples::*1.1} else {step.::Nuclease-free water (uL):: = 0} ; \
step.::5X AmpliSeq cDNA Reaction Mix (uL):: = step.::Total Samples:: * 1.1 * 2 ; \
step.::10X AmpliSeq RT Enzyme Mix (uL):: = step.::Total Samples:: * 1.1 * 1' \
-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 Input Amount - Transcriptome Human Genome Expression 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 true \
-h false \
-exp 'step.::Prep Input Volume (uL):: = step.::Prep Input Amount (ng):: / step.::Desired Concentration (ng/uL):: ; \
if (step.::Prep Input Volume (uL):: < 3.5) {step.::Nuclease-free water (uL):: = (3.5 - (step.::Prep Input Volume (uL):: + output.::AmpliSeq ERCC RNA Spike-in Volume (uL)::)) * step.::Total Samples::*1.1} else {step.::Nuclease-free water (uL):: = 0} ; \
step.::5X AmpliSeq cDNA Reaction Mix (uL):: = step.::Total Samples:: * 1.1 * 1 ; \
step.::10X AmpliSeq RT Enzyme Mix (uL):: = step.::Total Samples:: * 1.1 * 0.5 ; \
step.::AmpliSeq ERCC RNA Spike-in Mix (uL):: = step.::Total Samples:: * output.::AmpliSeq ERCC RNA Spike-in Volume (uL):: *1.1' \
-log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Calculate Input Amount</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 true \
-h false \
-exp 'step.::Prep Input Volume (uL):: = step.::Prep Input Amount (ng):: / step.::Desired Concentration (ng/uL):: ; \
if (step.::Prep Input Volume (uL):: < 7) {step.::Nuclease-free water (uL):: = (7 - step.::Prep Input Volume (uL)::) * step.::Total Samples::*1.1} else {step.::Nuclease-free water (uL):: = 0} ; \
step.::5X AmpliSeq cDNA Reaction Mix (uL):: = step.::Total Samples:: * 1.1 * 2 ; \
step.::10X AmpliSeq RT Enzyme Mix (uL):: = step.::Total Samples:: * 1.1 * 1' \
-log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Calculate Input Amount - 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 -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t true \
-h false \
-exp 'step.::Prep Input Volume (uL):: = step.::Prep Input Amount (ng):: / step.::Desired Concentration (ng/uL):: ; \
if (step.::Prep Input Volume (uL):: < 7) {step.::Nuclease-free water (uL):: = (7 - step.::Prep Input Volume (uL)::) * step.::Total Samples::*1.1} else {step.::Nuclease-free water (uL):: = 0} ; \
step.::5X AmpliSeq cDNA Reaction Mix (uL):: = step.::Total Samples:: * 1.1 * 2; \
step.::10X AmpliSeq RT Enzyme Mix (uL):: = step.::Total Samples:: * 1.1 * 1' \
-log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Calculate Input Amount - 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 -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t true \
-h false \
-exp 'step.::Prep Input Volume (uL):: = step.::Prep Input Amount (ng):: / step.::Desired Concentration (ng/uL):: ; \
if (step.::Prep Input Volume (uL):: < 3.5) {step.::Nuclease-free water (uL):: = (3.5 - step.::Prep Input Volume (uL)::) * step.::Total Samples::*1.1} else {step.::Nuclease-free water (uL):: = 0} ; \
step.::5X AmpliSeq cDNA Reaction Mix (uL):: = step.::Total Samples:: * 1.1 * 1 ; \
step.::10X AmpliSeq RT Enzyme Mix (uL):: = step.::Total Samples:: * 1.1 * 0.5' \
-log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Calculate Input Amount - 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 -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t true \
-h false \
-exp 'step.::Prep Input Volume (uL):: = step.::Prep Input Amount (ng):: / step.::Desired Concentration (ng/uL):: ; \
if (step.::Prep Input Volume (uL):: < 7.0) {step.::Nuclease-free water (uL):: = (7.0 - (step.::Prep Input Volume (uL):: + output.::AmpliSeq ERCC RNA Spike-in Volume (uL)::)) * step.::Total Samples::*1.1} else {step.::Nuclease-free water (uL):: = 0} ; \
step.::5X AmpliSeq cDNA Reaction Mix (uL):: = step.::Total Samples:: * 1.1 * 1 ; \
step.::10X AmpliSeq RT Enzyme Mix (uL):: = step.::Total Samples:: * 1.1 * 0.5 ; \
step.::AmpliSeq ERCC RNA Spike-in Mix (uL):: = step.::Total Samples:: * output.::AmpliSeq ERCC RNA Spike-in Volume (uL):: *1.1' \
-log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Calculate Input Amount - 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 true \
-h false \
-exp 'step.::Prep Input Volume (uL):: = step.::Prep Input Amount (ng):: / step.::Desired Concentration (ng/uL):: ; \
if (step.::Prep Input Volume (uL):: < 3.5) {step.::Nuclease-free water (uL):: = (3.5 - step.::Prep Input Volume (uL)::) * step.::Total Samples::*1.1} else {step.::Nuclease-free water (uL):: = 0} ; \
step.::5X AmpliSeq cDNA Reaction Mix (uL):: = step.::Total Samples:: * 1.1 * 1 ; \
step.::10X AmpliSeq RT Enzyme Mix (uL):: = step.::Total Samples:: * 1.1 * 0.5' \
-log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Calculate Input Amount- Custom RNA 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 true \
-h false \
-exp 'step.::Prep Input Volume (uL):: = step.::Prep Input Amount (ng):: / step.::Desired Concentration (ng/uL):: ; \
if (step.::Prep Input Volume (uL):: < 7) {step.::Nuclease-free water (uL):: = (7 - step.::Prep Input Volume (uL)::) * step.::Total Samples::*1.1} else {step.::Nuclease-free water (uL):: = 0} ; \
step.::5X AmpliSeq cDNA Reaction Mix (uL):: = step.::Total Samples:: * 1.1 * 2; \
step.::10X AmpliSeq RT Enzyme Mix (uL):: = step.::Total Samples:: * 1.1 * 1' \
-log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Copy Concentration, Total Volume and Input Amount - Myeloid</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 'step.::Total Samples:: = step.::Total Samples:: + 1' \
-log {compoundOutputFileLuid0} && /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.::Desired Concentration (ng/uL):: ; \
output.::Conc. Units:: = input.::Conc. Units:: ; \
output.::Total Volume (uL):: = input.::Total Volume (uL):: ; \
output.::Input Amount (ng):: = input.::Input Amount (ng):: ; \
step.::Desired Concentration (ng/uL):: = input.::Desired Concentration (ng/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 = Column
* Destination Containers
  * 96 well plate

#### Record Details

**Group of Defaults**

<details>

<summary>Childhood Cancer Panel</summary>

* AmpliSeq ERCC RNA Spike-in Mix = No
* Directions = If RNA was prepared from FFPE tissue and never heat-treated, heat at 80°C for 10 minutes and then cool to room temperature before proceeding to reverse transcription.
* Prep Input Amount (ng) = 10
* Recommended Prep Input (ng) = Each reaction requires 1-100 ng of DNase-treated total RNA. The recommended input is 10 ng RNA.
* Thermal Cycler Program = RT
* Total Samples = 0

</details>

<details>

<summary>Comprehensive Panel v3</summary>

* AmpliSeq ERCC RNA Spike-in Mix = No
* Directions = If RNA was prepared from FFPE tissue and never heat-treated, heat at 80°C for 10 minutes and then cool to room temperature before proceeding to reverse transcription.
* Prep Input Amount (ng) = 20
* Recommended Prep Input (ng) = Each reaction requires 1-100 ng of DNase-treated total RNA per pool. The recommended input is 10 ng RNA per pool.
* Thermal Cycler Program = RT
* Total Samples = 0

</details>

<details>

<summary>Focus Panel</summary>

* AmpliSeq ERCC RNA Spike-in Mix = No
* Directions = If RNA was prepared from FFPE tissue and never heat-treated, heat at 80°C for 10 minutes and then cool to room temperature before proceeding to reverse transcription.
* Prep Input Amount (ng) = 10
* Recommended Prep Input (ng) = Each reaction requires 1-100 ng of DNase-treated total RNA. The recommended input is 10 ng RNA.
* Thermal Cycler Program = RT

</details>

<details>

<summary>Immune Repertoire Panel</summary>

* AmpliSeq ERCC RNA Spike-in Mix = No
* Directions = If RNA was prepared from FFPE tissue and never heat-treated, heat at 80°C for 10 minutes and then cool to room temperature before proceeding to reverse transcription.
* Prep Input Amount (ng) = 1000
* Recommended Prep Input (ng) = Each reaction requires 10-1000 ng of DNase-treated total RNA. If RNA is not limiting, the recommended input is 1000 ng RNA. High RNA input amounts are recommended for rare clones.
* Thermal Cycler Program = RT

</details>

<details>

<summary>Immune Response Panel</summary>

* AmpliSeq ERCC RNA Spike-in Mix = No
* Directions = If RNA was prepared from FFPE tissue and never heat-treated, heat at 80°C for 10 minutes and then cool to room temperature before proceeding to reverse transcription.
* Prep Input Amount (ng) = 10
* Recommended Prep Input (ng) = Each reaction requires 1-100 ng of DNase-treated total RNA. The recommended input is 10 ng RNA.
* Thermal Cycler Program = RT
* Total Samples = 0

</details>

<details>

<summary>Myeloid Panel</summary>

* AmpliSeq ERCC RNA Spike-in Mix = No
* Directions = If RNA was prepared from FFPE tissue and never heat-treated, heat at 80°C for 10 minutes and then cool to room temperature before proceeding to reverse transcription.
* Prep Input Amount (ng) = 10
* Recommended Prep Input (ng) = Each reaction requires 10-100 ng of DNase-treated total RNA. The recommended input is 10 ng RNA.
* Thermal Cycler Program = RT
* Total Samples = 0

</details>

<details>

<summary>TCR beta-SR panel</summary>

* AmpliSeq ERCC RNA Spike-in Mix = No
* Directions = If RNA was prepared from FFPE tissue and never heat-treated, heat at 80°C for 10 minutes and then cool to room temperature before proceeding to reverse transcription.
* Prep Input Amount (ng) = 1000
* Recommended Prep Input (ng) = Each reaction requires 50-1000 ng of DNase-treated total RNA. If RNA is not limited, the recommended input is 1000 ng RNA
* Thermal Cycler Program = RT

</details>

<details>

<summary>Transcriptome HGx</summary>

* AmpliSeq ERCC RNA Spike-in Mix = No
* Directions = If RNA was prepared from FFPE tissue and never heat-treated, heat at 80°C for 10 minutes and then cool to room temperature before proceeding to reverse transcription.
* Prep Input Amount (ng) = 10
* Recommended Prep Input (ng) = Each reaction requires 1-100 ng of DNase-treated total RNA. The recommended input is 10 ng RNA.
* Thermal Cycler Program = RT
* Total Samples = 0

</details>

* Step Data
  * Group of Defaults = TCR beta-SR panel
  * Master Step Fields

    | **Field Name**                     | **Field Type** | **Options** | **Additional Options and Dropdown Items**                                                                                   |
    | ---------------------------------- | -------------- | ----------- | --------------------------------------------------------------------------------------------------------------------------- |
    | 5X AmpliSeq cDNA Reaction Mix (uL) | Numeric        |             | <ul><li>Decimal Places Displayed = 1</li></ul>                                                                              |
    | 10X AmpliSeq RT Enzyme Mix (uL)    | Numeric        |             | <ul><li>Decimal Places Displayed = 1</li></ul>                                                                              |
    | Desired Concentration (ng/uL)      | Numeric        | Read Only   |                                                                                                                             |
    | Nuclease-free water (uL)           | Numeric        |             | <ul><li>Decimal Places Displayed = 1</li></ul>                                                                              |
    | Prep Input Amount (ng)             | Numeric        |             | <ul><li>Default = 2</li><li>Range = 2 To 1000</li></ul>                                                                     |
    | Prep Input Volume (uL)             | Numeric        |             | <ul><li>Decimal Places Displayed = 2</li></ul>                                                                              |
    | Recommended Prep Input (ng)        | Multiline Text | Read Only   | <ul><li>Default = Each reaction requires 1-100 ng of DNase-treated total RNA. The recommended input is 10 ng RNA.</li></ul> |
    | Thermal Cycler Program             | Text           | Read Only   | <ul><li>Default = RT</li></ul>                                                                                              |
    | Total Samples                      | Numeric        |             | <ul><li>Default = 0</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** |
    | -------------- | ----------------- | -------------- | -------------- | ----------------------------------------- |
    | 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 | Sample Name       | Built-in       |                |                                           |
    | Derived Sample | Total Volume (uL) | Numeric        |                | Decimal Places Displayed = 2              |

### Step 5: Amplify cDNA Targets (AmpliSeq for Illumina TCR beta-SR Panel v1.1)

* Master Step Name = Amplify cDNA Targets Standard (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-0221546007f3cbd8e3b925c1b1cb954bece89344%2Fampliseq-tcr-rna-step5-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

<summary>Set Sample Type and Calculate Master Mix - TCR beta-SR Panel</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 'step.::Total Samples:: = step.::Total Samples:: + 1' \
-log {compoundOutputFileLuid0} && /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.::Sample Type:: = ::cDNA:: ; \
step.::5X AmpliSeq HiFi Mix (uL):: = 4.0 * step.::Total Samples:: * 1.1 ; \
step.::5X AmpliSeq TCR beta-SR Panel (uL):: = 4.0 * step.::Total Samples:: * 1.1 ; \
step.::Nuclease-free water (uL):: = 2.0 * step.::Total Samples:: * 1.1' \
-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>Routing cDNA - TCRbeta SR</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 'Sample Type' \
--FIELD_VALUE 'cDNA' \
--WORKFLOW 'DNA Library Prep AmpliSeq for Illumina TCR beta-SR Panel v1.1' \
--STEP 'Transfer RNA and DNA Amplicons (AmpliSeq for Illumina TCR beta-SR 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>Copy Desired RNA Conc. &#x26; Calculate Master Mix - Custom RNA Pool 1</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.::Diluted RNA Conc. (ng/uL):: = input.::Desired Final Concentration (ng/uL):: ; \
output.::Master Mix (uL):: = 10 ; \
step.::Total Samples:: = step.::Total Samples:: + 1 ; \
step.::5X AmpliSeq HiFi Mix (uL):: = step.::Total Samples:: *1.1* 4 ; \
step.::5X AmpliSeq Custom RNA Panel (uL):: = step.::Total Samples:: * 1.1 * 4 ; \
output.::RNA Volume (uL):: = 10 / output.::Diluted RNA Conc. (ng/uL):: ; \
if (output.::RNA Volume (uL):: < 10) {output.::Nuclease-free water (uL):: = 20 -(output.::Master Mix (uL):: + output.::RNA Volume (uL)::)}' \
-log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Copy Desired RNA Conc. &#x26; Calculate Master Mix - Custom RNA Pool 2</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.::Diluted RNA Conc. (ng/uL):: = input.::Desired Final Concentration (ng/uL):: ; \
output.::Master Mix (uL):: = 10 ; \
step.::Total Samples:: = step.::Total Samples:: + 1 ; \
step.::5X AmpliSeq HiFi Mix (uL):: = step.::Total Samples:: *1.1* 4.5 ; \
step.::5X AmpliSeq Custom RNA Panel (uL):: = step.::Total Samples:: * 1.1 * 4 ; \
output.::RNA Volume (uL):: = 10 / output.::Diluted RNA Conc. (ng/uL):: ; \
if (output.::RNA Volume (uL):: < 10) {output.::Nuclease-free water (uL):: = 20 -(output.::Master Mix (uL):: + output.::RNA Volume (uL)::)}' \
-log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Routing 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 'Sample Type' \
--FIELD_VALUE 'cDNA' \
--WORKFLOW 'DNA Library Prep AmpliSeq for Illumina Myeloid Panel v1.1' \
--STEP 'Transfer RNA and DNA Amplicons (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>Routing Samples - TCRbeta SR 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 'Sample Type' \
--FIELD_VALUE 'cDNA' \
--WORKFLOW 'DNA Library Prep AmpliSeq for Illumina TCR beta-SR Panel v1.1' \
--STEP 'Transfer RNA and DNA Amplicons (AmpliSeq for Illumina TCR beta-SR 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>Set Sample Type and Calculate Master Mix - Custom RNA</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 'step.::Total Samples:: = step.::Total Samples:: + 1' \
-log {compoundOutputFileLuid0} && /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.::Sample Type:: = ::cDNA:: ; \
step.::5X AmpliSeq HiFi Mix (uL):: = 4.0 * step.::Total Samples:: * 1.1 ; \
step.::5X AmpliSeq Custom RNA Panel (uL):: = 4.0 * step.::Total Samples:: * 1.1 ; \
step.::Nuclease-free water (uL):: = 2.0 * step.::Total Samples:: * 1.1' \
-log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Set Sample Type and Calculate Master Mix - 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 -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'step.::Total Samples:: = step.::Total Samples:: + 1' \
-log {compoundOutputFileLuid0} && /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.::Sample Type:: = ::cDNA:: ; \
step.::5X AmpliSeq HiFi Mix (uL):: = 4.0 * step.::Total Samples:: * 1.1 ; \
step.::5X AmpliSeq Immune Repertoire Panel (uL):: = 4.0 * step.::Total Samples:: * 1.1 ; \
step.::Nuclease-free water (uL):: = 7.0 * step.::Total Samples:: * 1.1' \
-log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Set Sample Type and Calculate Master Mix - 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 -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'step.::Total Samples:: = step.::Total Samples:: + 1' \
-log {compoundOutputFileLuid0} && /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.::Sample Type:: = ::cDNA:: ; \
step.::5X AmpliSeq HiFi Mix (uL):: = 4.0 * step.::Total Samples:: * 1.1 ; \
step.::Nuclease-free water (uL):: = 2.0 * step.::Total Samples:: * 1.1 ; \
step.::5x AmpliSeq Immune Response Panel (uL):: = 2.0 * step.::Total Samples:: * 1.1' \
-log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Set Sample Type and Calculate Master Mix - 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 'step.::Total Samples:: = step.::Total Samples:: + 1' \
-log {compoundOutputFileLuid0} && /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.::Sample Type:: = ::cDNA:: ; \
step.::5X AmpliSeq HiFi Mix (uL):: = 4.0 * step.::Total Samples:: * 1.1 ; \
step.::Nuclease-free water (uL):: = 7.0 * step.::Total Samples:: * 1.1' \
-log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Set Sample Type and Calculate Master Mix - TGx 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 'step.::Total Samples:: = step.::Total Samples:: + 1' \
-log {compoundOutputFileLuid0} && /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.::Sample Type:: = ::cDNA:: ; \
step.::5X AmpliSeq HiFi Mix (uL):: = 4.0 * step.::Total Samples:: * 1.1 ; \
step.::Nuclease-free water (uL):: = 3.0 * step.::Total Samples:: * 1.1' \
-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** |
  | ---------------------------------- | -------------- | ----------- | ----------------------------------------- |
  | 5X AmpliSeq HiFi Mix (uL)          | Numeric        |             | Decimal Places Displayed = 2              |
  | 5X AmpliSeq TCR beta-SR Panel (uL) | Numeric        |             | Decimal Places Displayed = 1              |
  | Nuclease-free water (uL)           | Numeric        |             | Decimal Places Displayed = 2              |
  | Thermal Cycler Program             | Text           |             | Default = AMP\_RNA                        |
* 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       |             |                                           |