# Illumina RNA Prep with Enrichment (L) Tagmentation v1.1

## Overview

The Illumina RNA Prep with Enrichment (L) Tagmentation workflow includes the following functionality.

* Preset Illumina RNA Prep with Enrichment (L) Tagmentation protocols that support the generation of enriched libraries for dual-indexed, paired-end sequencing.
* 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: Prep Sample (Illumina RNA Prep with Enrichment (L) Tagmentation 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-5d970ef10487deffe97919be73a2884b275e79ab%2Frna-ltag-protocol1-next-step-config.png?alt=media" alt=""><figcaption></figcaption></figure>

### Step 1: Denature RNA (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

* Master Step Name = Denature RNA v1.0
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
  * Illumina RNA Prep with Enrichment (L) Tagmentation
    * Supplier = Illumina
    * Catalog Number = 16 - 20040536, 96 - 20040537
    * 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-553e866d041fea9f208818b35592a1dd7cde10b3%2Frna-ltag-protocol1-step1-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

<summary>Calculate Sample and Water Volume</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:http} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false \
-exp 'output.::SampleVolume (uL):: = (step.::Final Diluted Concentration (ng/uL):: * step.::Final Volume (uL)::) / output.::Starting Concentration (ng/uL):: ; output.::Nuclease-free Ultrapure Water (uL):: = step.::Final Volume (uL):: - output.::SampleVolume (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

* 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       |             |                                           |

#### Ice Bucket

* Defaults
  * Sample Grouping = Group by Containers
  * Well Sort Order = Column
* 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**                                                                                                                 |
  | ----------------------------------- | -------------- | -------------- | --------------------------------------------------------------------------------------------------------------------------------------------------------- |
  | Final Diluted Concentration (ng/uL) | Numeric        | Required Field | <ul><li>Default = 10</li><li>Range = 10 To 100</li></ul>                                                                                                  |
  | Final Volume (uL)                   | Numeric        |                | <ul><li>Default = 8.5</li></ul>                                                                                                                           |
  | Instruction Notes                   | Multiline Text | Read Only      | <ul><li>Default = Enter Final Diluted Concentration and Starting Concentration before clicking on the Calculate Sample and Water Volume button.</li></ul> |
  | Thermal Cycler Program              | Text           |                | <ul><li>Default = DEN\_RNA</li></ul>                                                                                                                      |
  | Thermal Cycler Program Notes        | Multiline Text | Read Only      | <ul><li>Default =<br>Choose the preheat lid option and set to 100°C<br>Reaction volume is 17 µl<br>65°C for 5 minutes<br>Hold at 4°C</li></ul>            |
* 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      | LIMS ID (Container)                | Built-in       |             |                                           |
    | Container      | Well                               | Built-in       |             |                                           |
    | Derived Sample | Nuclease-free Ultrapure Water (uL) | Numeric        |             | Decimal Places Displayed = 0              |
    | Derived Sample | Sample Name                        | Built-in       |             |                                           |
    | Derived Sample | SampleVolume (uL)                  | Numeric        |             | Decimal Places Displayed = 0              |
    | Derived Sample | Starting Concentration (ng/uL)     | Numeric        |             | Decimal Places Displayed = 0              |
    | Project        | Project Name                       | Built-in       |             |                                           |

### Step 2: Synthesize First Strand cDNA (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

* Master Step Name = Synthesize First Strand cDNA v1.0
* Step Type = No Outputs
* Reagent Kits
  * Illumina RNA Prep with Enrichment (L) Tagmentation
    * Supplier = Illumina
    * Catalog Number = 16 - 20040536, 96 - 20040537
    * 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-311a9440a72dcdfc90fc12b2305f29013b809d5c%2Frna-ltag-protocol1-step2-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

<summary>Calculate First Strand Synthesis Master Mix</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:http} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'step.::Total Samples:: = step.::Total Samples:: + 1 ; step.::FSA (uL):: = 9 * step.::Total Samples:: ; step.::RVT (uL):: = 1 * step.::Total Samples:: ' -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 = Column
* 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**                                                                                                                                          |
  | ---------------------------- | -------------- | ----------- | ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
  | FSA (uL)                     | Numeric        |             | Decimal Places Displayed = 0                                                                                                                                                       |
  | Instruction Notes            | Multiline Text | Read Only   | Default = Add 8 uL of First Strand Synthesis Master Mix to each sample.                                                                                                            |
  | RVT (uL)                     | Numeric        |             | Decimal Places Displayed = 0                                                                                                                                                       |
  | Thermal Cycler Program       | Text           |             | Default = FSS                                                                                                                                                                      |
  | Thermal Cycler Program Notes | Multiline Text | Read Only   | <p>Default =<br>Choose the preheat lid option and set to 100°C<br>Reaction volume is 25 µl<br>25°C for 10 minutes<br>42°C for 15 minutes<br>70°C for 15 minutes<br>Hold at 4°C</p> |
  | Total Samples                | Numeric        |             | Default = 0                                                                                                                                                                        |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Collapse
  * 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      | LIMS ID (Container) | Built-in       |             |                                           |
    | Container      | Well                | Built-in       |             |                                           |
    | Derived Sample | Sample Name         | Built-in       |             |                                           |
    | Project        | Project Name        | Built-in       |             |                                           |

### Step 3: Synthesize Second Strand cDNA (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

* Master Step Name = Synthesize Second Strand cDNA v1.0
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
  * Agencourt AMPure XP, 5 ml
    * Supplier = Beckman Coulter
    * Catalog Number = A63880
  * Illumina RNA Prep with Enrichment (L) Tagmentation
    * Supplier = Illumina
    * Catalog Number = 16 - 20040536, 96 - 20040537
    * 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-002e6426d5b0ffc5597c474912dba7515a562248%2Frna-ltag-protocol1-step3-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 = Column
* 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**                                                                                      |
  | ---------------------------- | -------------- | ----------- | ------------------------------------------------------------------------------------------------------------------------------ |
  | 80% EtOH Prep Date           | Date           |             |                                                                                                                                |
  | Thermal Cycler Program       | Text           |             | Default = SSS                                                                                                                  |
  | Thermal Cycler Program Notes | Multiline Text | Read Only   | <p>Default =<br>Choose the preheat lid option and set to 40°C<br>Reaction volume is 50 µl u 16°C for 1 hour<br>Hold at 4°C</p> |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Collapse
  * 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      | LIMS ID (Container) | Built-in       |             |                                           |
    | Container      | Well                | Built-in       |             |                                           |
    | Derived Sample | Sample Name         | Built-in       |             |                                           |
    | Project        | Project Name        | Built-in       |             |                                           |

## Protocol 2: Library Prep (Illumina RNA Prep with Enrichment (L) Tagmentation 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-ea17d5df78b666877d2278c24ce523ec4f191533%2Frna-ltag-protocol2-next-step-config.png?alt=media" alt=""><figcaption></figcaption></figure>

### Step 1: Tagment cDNA (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

* Master Step Name = Tagment cDNA v1.0
* Step Type = No Outputs
* Reagent Kits
  * Illumina RNA Prep with Enrichment (L) Tagmentation
    * Supplier = Illumina
    * Catalog Number = 16 - 20040536, 96 - 20040537
    * 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-44efc1915562dc72e23b7a68c61539cb7c00886b%2Frna-ltag-protocol2-step1-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

<summary>Calculate Tagmentation Master Mix - RNA Prep</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:http} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false \
-exp 'step.::Total Samples:: = step.::Total Samples:: + 1 ; step.::TB1 (uL):: = 11.5 * step.::Total Samples:: ; step.::EBLTL (uL):: = 11.5 * step.::Total Samples:: ; step.::Nuclease-free ultrapure water (uL):: = 14.5 * step.::Total Samples::' \
-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 = Column
* 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**                                                                                           |
  | ---------------------------------- | -------------- | ----------- | ----------------------------------------------------------------------------------------------------------------------------------- |
  | EBLTL (uL)                         | Numeric        |             |                                                                                                                                     |
  | Instruction Notes                  | Multiline Text | Read Only   | Default = Add 32.5 µl Tagmentation Master Mix to each well.                                                                         |
  | Nuclease-free ultrapure water (uL) | Numeric        |             |                                                                                                                                     |
  | TB1 (uL)                           | Numeric        |             |                                                                                                                                     |
  | Thermal Cycler Program             | Text           |             | Default = TAG                                                                                                                       |
  | Thermal Cycler Program Notes       | Multiline Text | Read Only   | <p>Default =<br>Choose the preheat lid option and set to 100°C<br>Reaction volume is 50 µl u 55°C for 5 minutes<br>Hold at 10°C</p> |
  | Total Samples                      | Numeric        |             | Default = 0                                                                                                                         |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Collapse
  * 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      | LIMS ID (Container) | Built-in       |             |                                           |
    | Container      | Well                | Built-in       |             |                                           |
    | Derived Sample | Sample Name         | Built-in       |             |                                           |
    | Project        | Project Name        | Built-in       |             |                                           |

### Step 2: Amplify Tagmented cDNA (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

* Master Step Name = Amplify Tagmented cDNA v1.0
* Step Type = Add Labels
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}-{AppliedReagentLabels}

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-bd6e26c0f82caad8b71f59b01327f18edab412ad%2Frna-ltag-protocol2-step2-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

<summary>Calculate PCR Master Mix - RNA Prep</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:http} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false \
-exp 'step.::Total Samples:: = step.::Total Samples:: + 1 ; step.::EPM (uL):: = 23 * step.::Total Samples:: ; step.::Nuclease-free ultrapure water (uL):: = 23 * step.::Total Samples::' \
-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 = Column
* 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 = Column
  * Placement Pattern = Column
* Destination Containers
  * 96 well plate

#### Add Labels

* Label Groups
  * IDT for Illumina DNA-RNA UD Indexes Set A Tagmentation
  * IDT for Illumina DNA-RNA UD Indexes Set B Tagmentation
  * IDT for Illumina DNA-RNA UD Indexes Set C Tagmentation
  * IDT for Illumina DNA-RNA UD Indexes Set D Tagmentation
  * Illumina DNA-RNA UD Indexes Set A Tagmentation
  * Illumina DNA-RNA UD Indexes Set B Tagmentation
  * Illumina DNA-RNA UD Indexes Set C Tagmentation
  * Illumina DNA-RNA UD Indexes Set D Tagmentation

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**                     | **Field Type** | **Options**    | **Additional Options and Dropdown Items**                                                                                                                                                                                                                      |
  | ---------------------------------- | -------------- | -------------- | -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
  | EPM (uL)                           | Numeric        |                | Decimal Places Displayed = 0                                                                                                                                                                                                                                   |
  | Instruction Notes                  | Multiline Text | Read Only      | Default = Add 40 µl PCR Master Mix to each well.                                                                                                                                                                                                               |
  | Nuclease-free ultrapure water (uL) | Numeric        |                | Decimal Places Displayed = 0                                                                                                                                                                                                                                   |
  | PCR Cycles                         | Text Dropdown  | Custom Entries | <p>Presets</p><ul><li>14 cycles for High-quality RNA with DV200 > 80%</li><li>17 cycles for FFPE and RNA with DV200 < 80%</li></ul>                                                                                                                            |
  | Thermal Cycler Program             | Text           |                | Default = TAG\_PCR                                                                                                                                                                                                                                             |
  | Thermal Cycler Program Notes       | Multiline Text | Read Only      | <p>Default =<br>Choose the preheat lid option and set to 100°C<br>Reaction volume is 50 µl u 72°C for 3 minutes u 98°C for 3 minutes<br>X cycles of: u 98°C for 20 seconds u 60°C for 30 seconds<br>72°C for 1 minute u 72°C for 3 minutes<br>Hold at 10°C</p> |
  | Total Samples                      | Numeric        |                | Default = 0                                                                                                                                                                                                                                                    |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Collapse
  * 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      | LIMS ID (Container) | Built-in       |             |                                           |
    | Container      | Well                | Built-in       |             |                                           |
    | Derived Sample | Sample Name         | Built-in       |             |                                           |
    | Project        | Project Name        | Built-in       |             |                                           |

### Step 3: Clean Library (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

* Master Step Name = Clean Library (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
  * Agencourt AMPure XP, 5 ml
    * Supplier = Beckman Coulter
    * Catalog Number = A63880
  * Illumina RNA Prep with Enrichment (L) Tagmentation
    * Supplier = Illumina
    * Catalog Number = 16 - 20040536, 96 - 20040537
    * 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-993fde0bb5b8a25ec4e3a36abb03a87b037bc695%2Frna-ltag-protocol2-step3-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 = Column
* 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** |
  | ------------------ | -------------- | ----------- | ----------------------------------------- |
  | 80% EtOH Prep Date | Date           |             |                                           |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Collapse
  * 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      | LIMS ID (Container) | Built-in       |             |                                           |
    | Container      | Well                | Built-in       |             |                                           |
    | Derived Sample | Sample Name         | Built-in       |             |                                           |
    | Project        | Project Name        | Built-in       |             |                                           |

## Protocol 3: Normalize Libraries (Illumina RNA Prep with Enrichment (L) Tagmentation 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-a7a01451f8075b13e362355696055475eebb8e6e%2Frna-ltag-protocol3-next-step-config.png?alt=media" alt=""><figcaption></figcaption></figure>

### Step 1: Qubit Library QC (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

* Master Step Name = Qubit Library QC v1.0
* 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-680fb42966742ed7c1e8eb7c51be9b6875c6b66d%2Frna-ltag-protocol3-step1-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

<summary>Assign QC flags</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 -u {username} -p {password} \
      script:assignQC \
        -i {processURI:v2} \
        -log {compoundOutputFileLuid1} \
        -qcResult {compoundOutputFileLuid2}"
```

{% 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 = Column
* 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        |             | Default = 200                             |
  | Criteria 2 - Operator          | Text           |             | Default = <=                              |
  | Criteria 2 - Source Data Field | Text           |             | Default = Concentration                   |
  | Criteria 2 - Threshold Value   | Numeric        |             | Default = 1500                            |
* Step File Placeholders
  * Log - Automatically attached
  * QC Result File - Manually uploaded
* Sample Table
  * Enable QC Flags = Yes
  * Sample Display Default = Expand
  * Well Sort Order = Column
  * File Column Options
    * File Column Display = Show
    * File Attachment Method = Manual
  * 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 2: BioAnalyzer Library QC (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

* Master Step Name = BioAnalyzer Library QC v1.0
* 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-bff630b13449a167db7b3f702c225856b55cc3a4%2Frna-ltag-protocol3-step2-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

<summary>Generate Bioanalyzer Input file</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/DriverFileGenerator.jar script:driver_file_generator -i {processURI:v2} -u {username} -p {password} -t /opt/gls/clarity/extensions/ngs-common/v5/EPP/conf/readonly/bioA_driver_file_template.csv -o {compoundOutputFileLuid0}.csv -l {compoundOutputFileLuid1}  && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar script:addBlankLines -i {stepURI:v2} -u {username} -p {password} -f {compoundOutputFileLuid0}.csv -l {compoundOutputFileLuid1} -sep COMMA -b ',False,' -h 1 -c LIMSID -pre 'Sample '"
```

{% endcode %}

</details>

<details>

<summary>Parse Bioanalyzer XML and assign QC flags</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 -u {username} -p {password} -i {processURI:v2} \
      script:parseBioAnalyzer \
        -inputFile {compoundOutputFileLuid2} \
        -log {compoundOutputFileLuid5} \
        -configFile '/opt/gls/clarity/extensions/conf/v5/bioanalyzer/defaultBioAnalyzerDNAConfig.groovy' \
      script:assignQC \
        -log {compoundOutputFileLuid6} \
        -qcResult {compoundOutputFileLuid7}"
```

{% 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 = Column
* 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 = Column
  * Placement Pattern = Column
* Destination Containers

  * BioAnalyzer DNA High Sensitivity Chip

  <figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-b01a3d1277eb9352528522ca613f83cae1d7c7c6%2Fbioanalyzer-dna-high-sensitivity-chip-container.png?alt=media" alt=""><figcaption></figcaption></figure>

  * BioAnalyzer DNA 1000 Chip

  <figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-28c00d95372efbcc2c4cb08969972a094491630e%2Fbioanalyzer-dna-1000-chip-container.png?alt=media" alt=""><figcaption></figcaption></figure>

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**                              | **Field Type** | **Options**    | **Additional Options and Dropdown Items**                                                     |
  | ------------------------------------------- | -------------- | -------------- | --------------------------------------------------------------------------------------------- |
  | Criteria 1 - Operator                       | Text Dropdown  | Custom Entries | <p>Presets</p><ul><li>>=</li><li><=</li><li>=</li><li>!=</li></ul>                            |
  | Criteria 1 - Source Data Field              | Text Dropdown  | Custom Entries | <p>Presets</p><ul><li>Peak 2 Size - bp</li><li>Peak 2 Conc.</li><li>Peak 2 Molarity</li></ul> |
  | Criteria 1 - Threshold                      | Numeric        |                |                                                                                               |
  | Criteria 2 - Operator                       | Text Dropdown  | Custom Entries | <p>Presets</p><ul><li>>=</li><li><=</li><li>=</li><li>!=</li></ul>                            |
  | Criteria 2 - Source Data Field              | Text Dropdown  | Custom Entries | <p>Presets</p><ul><li>Peak 2 Size - bp</li><li>Peak 2 Conc.</li><li>Peak 2 Molarity</li></ul> |
  | Criteria 2 - Threshold                      | Numeric        |                |                                                                                               |
  | Use strict matching for Bioanalyzer results | Toggle Switch  |                | Default = None Set                                                                            |
* Step File Placeholders
  * Bioanalyzer Driver File - Automatically attached
  * Bioanalyzer Driver File Generation Log File - Automatically attached
  * Bioanalyzer XML Result File (required) - Manually uploaded
  * Result File (optional) - Manually uploaded
  * PDF Summary File (optional) - Manually uploaded
  * Bioanalyzer XML Parsing Log File - Automatically attached
  * QC Assignment Log File - Automatically attached
  * QC Assignment Report - Automatically attached
* Sample Table
  * Enable QC Flags = Yes
  * Sample Display Default = Expand
  * Well Sort Order = Column
  * File Column Options
    * File Column Display = Show
    * File Attachment Method = Manual
  * 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 3: Dilute Libraries (Illumina RNA Prep Tagmentation (L) with Enrichment v1.0)

* Master Step Name = Dilute Libraries v1.0
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
  * Illumina RNA Prep with Enrichment (L) Tagmentation
    * Supplier = Illumina
    * Catalog Number = 16 - 20040536, 96 - 20040537
    * 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-44443156143daba072893710df42dcad5b526216%2Frna-ltag-protocol3-step3-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

<summary>Calculate Sample, RSB, &#x26; Final Volumes</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:http} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false \
-exp 'output.::SampleVolume (uL):: = (step.::Final Diluted Concentration (ng/uL):: * output.::Final Volume (uL)::) / output.::Starting Concentration (ng/uL):: ; if (step.::Enrichment Plexity:: == 1) {output.::Final Volume (uL):: = 7.5} else {output.::Final Volume (uL):: = 2.5} ; step.::RSB (uL):: = output.::Final Volume (uL):: - output.::SampleVolume (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 = Column
* 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**                                                                                                                                                                                                                                   |
  | ----------------------------------- | ---------------- | ------------------------------------------------------- | --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
  | Enrichment Plexity                  | Numeric Dropdown | <ul><li>Required Field</li><li>Custom Entries</li></ul> | <p>Presets</p><ul><li>1</li><li>3</li></ul>                                                                                                                                                                                                                                 |
  | Final Diluted Concentration (ng/uL) | Numeric          |                                                         | Default = 200                                                                                                                                                                                                                                                               |
  | Instruction Notes                   | Multiline Text   | <ul><li>Read Only</li></ul>                             | <p>Default =<br>In one well of a new PCR plate, combine the applicable number of 200 ng libraries:<br>If Enrichment Plexity is 1 then add 7.5 ul of the sample.<br>If Enrichment Plexity is 3 then add 2.5 ul of each sample to one well with a final volume of 7.5 ul.</p> |
* 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      | LIMS ID (Container)            | Built-in       |             |                                           |
    | Container      | Well                           | Built-in       |             |                                           |
    | Derived Sample | Final Volume (uL)              | Numeric        |             | Decimal Places Displayed = 0              |
    | Derived Sample | RSB (uL)                       | Numeric        |             | Decimal Places Displayed = 0              |
    | Derived Sample | Sample Name                    | Built-in       |             |                                           |
    | Derived Sample | SampleVolume (uL)              | Numeric        |             | Decimal Places Displayed = 0              |
    | Derived Sample | Starting Concentration (ng/uL) | Numeric        |             | Decimal Places Displayed = 0              |
    | Project        | Project Name                   | Built-in       |             |                                           |

### Step 4: Hybridize Probes (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

* Master Step Name = Hybridize Probes (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
  * Illumina Exome Panel
    * Supplier = Illumina
    * Catalog Number = 20020183
    * Website = [www.illumina.com](https://www.illumina.com)
  * Illumina RNA Prep with Enrichment (L) Tagmentation
    * Supplier = Illumina
    * Catalog Number = 16 - 20040536, 96 - 20040537
    * 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-e8aa1bcea205b723eb8f0ba93be4a5c5d07c64bf%2Frna-ltag-protocol3-step4-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 = Column
* 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 = HYB                                                                                                                                                                                                                                                            |
  | Thermal Cycler Program Notes | Multiline Text | Read Only   | <p>Default =<br>Choose the preheat lid option and set to 100°C<br>Reaction volume is 25 µl u 95°C for 5 minutes<br>18 cycles of 1 minute each: u 94°C for the first cycle<br>Decrease 2°C per subsequent cycle<br>58°C for 90 minutes<br>Hold at 58°C for ≤ 24 hours</p> |
* Step File Placeholders
  * Log - Manually uploaded
* Sample Table
  * Sample Display Default = Collapse
  * 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 5: Capture Hybridize Probes (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

* Master Step Name = Capture Hybridize Probes v1.0
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
  * Illumina RNA Prep with Enrichment (L) Tagmentation
    * Supplier = Illumina
    * Catalog Number = 16 - 20040536, 96 - 20040537
    * 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-95c857d4a565a807281b9769df1e9003ce9446b1%2Frna-ltag-protocol3-step5-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

<summary>Calculate Elution Master Mix</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:http} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false \
-exp 'step.::Total Samples:: = step.::Total Samples:: + 1 ; step.::EEL (uL):: = 28.5 * step.::Total Samples:: ; step.::HP3 (uL):: = 1.5 * step.::Total Samples::' \
-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 = Column
* 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**                                                                                 |
  | ---------------------------- | -------------- | ----------- | ------------------------------------------------------------------------------------------------------------------------- |
  | EEL (uL)                     | Numeric        |             | <ul><li>Decimal Places Displayed = 0</li></ul>                                                                            |
  | HP3 (uL)                     | Numeric        |             | <ul><li>Decimal Places Displayed = 0</li></ul>                                                                            |
  | Instruction Notes            | Multiine Text  | Read Only   | <ul><li>Default = Add 23 µl Elution Master Mix to each well.</li></ul>                                                    |
  | Thermal Cycler Program Notes | Multiline Text | Read Only   | <ul><li>Default =<br>Choose the preheat lid option and set to 70°C<br>Reaction volume is 100 µl<br>Hold at 58°C</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 = Collapse
  * 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      | LIMS ID (Container) | Built-in       |             |                                           |
    | Container      | Well                | Built-in       |             |                                           |
    | Derived Sample | Sample Name         | Built-in       |             |                                           |
    | Project        | Project Name        | Built-in       |             |                                           |

### Step 6: Amplify Enriched Library (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

* Master Step Name = Amplify Enriched Library v1.0
* Step Type = No Outputs
* Reagent Kits
  * Illumina RNA Prep with Enrichment (L) Tagmentation
    * Supplier = Illumina
    * Catalog Number = 16 - 20040536, 96 - 20040537
    * 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-7c9856d7212db72019993892b1f3584e4ed9e7b9%2Frna-ltag-protocol3-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 = Column
* 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 = AMP                                                                                                                                                                                                                                     |
  | Thermal Cycler Program Notes | Multiline Text | Read Only   | <p>Default =<br>Choose the preheat lid option and set to 100°C<br>Reaction volume is 50 µl<br>98°C for 30 seconds<br>14 cycles of:<br>98°C for 10 seconds<br>60°C for 30 seconds<br>72°C for 30 seconds<br>72°C for 5 minutes<br>Hold at 10°C</p> |
* Step File Placeholders
  * Log - Manually uploaded
* Sample Table
  * Sample Display Default = Collapse
  * 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      | LIMS ID (Container) | Built-in       |             |                                           |
    | Container      | Well                | Built-in       |             |                                           |
    | Derived Sample | Sample Name         | Built-in       |             |                                           |
    | Project        | Project Name        | Built-in       |             |                                           |

### Step 7: Clean Up Enriched Library (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

* Master Step Name = Clean Up Enriched Library v1.0
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
  * Agencourt AMPure XP, 5 ml
    * Supplier = Beckman Coulter
    * Catalog Number = A63880
  * Illumina RNA Prep with Enrichment (L) Tagmentation
    * Supplier = Illumina
    * Catalog Number = 16 - 20040536, 96 - 20040537
    * 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-bb9eda2b4b7a1dc5eee74f2c2e0e19c690795fcd%2Frna-ltag-protocol3-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>

#### Queue/Ice Bucket

* Defaults
  * Sample Grouping = Group by Containers
  * Well Sort Order = Column
* 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** |
  | ------------------ | -------------- | ----------- | ----------------------------------------- |
  | 80% EtOH Prep Date | Date           |             |                                           |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Collapse
  * 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       |             |                                           |


---

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