# TruSeq DNA PCR-Free v2.0

## Protocol 1: TruSeq DNA PCR-Free v2.0

Protocol Type = Library Prep

**Next Steps Configuration**

<figure><img src="/files/36wqdt2UoD7qWmIVlXwY" alt=""><figcaption></figcaption></figure>

### Step 1: Fragment DNA (TruSeq DNA PCR-Free v2.0)

* Master Step Name = Fragment DNA v1.0
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
  * TruSeq DNA PCR-Free Sample Prep Kit - Set A, B or HT Box
    * Supplier = Illumina
    * Catalog Number = Set A: FC-121-3001, Set B: FC-121-3002, HT: FC-121-3003

#### Automations

<figure><img src="/files/KeBw7gCCT3lPaa1C5K9N" alt=""><figcaption></figcaption></figure>

<details>

<summary>Normalize gDNA (PCR Free)</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 'if ( step.::Target Insert Size (bp):: == 350 ) { output.::Sample Volume (ul):: = 1000 / output.::Concentration:: } ; if ( step.::Target Insert Size (bp):: == 550 ) { output.::Sample Volume (ul):: = 2000 / output.::Concentration:: } ; output.::RSB Volume (ul):: = 55 - output.::Sample Volume (ul):: ; output.::Target Insert Size (bp):: = step.::Target Insert Size (bp)::' -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>Normalize gDNA (Nano)</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:http} -u {username} -p {password} script:evaluateDynamicExpression -t true -h false -exp 'if ( step.::Target Insert Size (bp):: == 350 ) { output.::Sample Volume (ul):: = 100 / output.::Concentration:: } ; if ( step.::Target Insert Size (bp):: == 550 ) { output.::Sample Volume (ul):: = 200 / output.::Concentration:: } ; output.::RSB Volume (ul):: = 52.5 - output.::Sample Volume (ul):: ; output.::Target Insert Size (bp):: = step.::Target Insert Size (bp)::' -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      | Well           | Built-in       |             |                                           |
    | Derived Sample | Sample Name    | Built-in       |             |                                           |
    | Derived Sample | Waiting        | Built-in       |             |                                           |
  * Expanded View Fields

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

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**           | **Field Type**   | **Options**                                             | **Additional Options and Dropdown Items**                                                                                                       |
  | ------------------------ | ---------------- | ------------------------------------------------------- | ----------------------------------------------------------------------------------------------------------------------------------------------- |
  | Comment                  | Multiline Text   |                                                         |                                                                                                                                                 |
  | Cycles/Burst             | Numeric Dropdown | <ul><li>Custom Entries</li></ul>                        | <ul><li><p>Presets</p><ul><li>200</li></ul></li></ul>                                                                                           |
  | Duration (seconds)       | Numeric Dropdown | <ul><li>Custom Entries</li></ul>                        | <ul><li><p>Presets</p><ul><li>65</li><li>50</li><li>45</li><li>25</li></ul></li><li>Decimal Places Displayed = 0</li></ul>                      |
  | Duty Factor (%)          | Numeric Dropdown | <ul><li>Custom Entries</li></ul>                        | <ul><li><p>Presets</p><ul><li>20</li><li>5</li><li>10</li></ul></li><li>Decimal Places Displayed = 0</li></ul>                                  |
  | Intensity                | Numeric Dropdown | <ul><li>Custom Entries</li></ul>                        | <ul><li><p>Presets</p><ul><li>5</li><li>2</li></ul></li><li>Decimal Places Displayed = 1</li></ul>                                              |
  | Mode                     | Text Dropdown    | <ul><li>Custom Entries</li></ul>                        | <ul><li><p>Presets</p><ul><li>Frequency sweeping</li><li>-</li></ul></li></ul>                                                                  |
  | Peak/Displayed Power (W) | Numeric Dropdown | <ul><li>Custom Entries</li></ul>                        | <ul><li><p>Presets</p><ul><li>50</li><li>175</li><li>23</li><li>14</li><li>9</li><li>7</li></ul></li><li>Decimal Places Displayed = 0</li></ul> |
  | Target Insert Size (bp)  | Numeric Dropdown | <ul><li>Required Field</li><li>Custom Entries</li></ul> | <ul><li><p>Presets</p><ul><li>350</li><li>550</li></ul></li><li>Decimal Places Displayed = 0</li></ul>                                          |
  | Temperature (oC)         | Numeric Dropdown | <ul><li>Custom Entries</li></ul>                        | <ul><li><p>Presets</p><ul><li>20</li><li>6</li></ul></li></ul>                                                                                  |
  | 80% EtOH Prep Date       | Date             |                                                         |                                                                                                                                                 |
* Step File Placeholders
  * Log file - 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 | Concentration           | Numeric          | Required Field | <ul><li>Decimal Places Displayed = 2</li></ul>                                                         |
    | Derived Sample | Conc. Units             | Text             | Required Field |                                                                                                        |
    | Derived Sample | RSB Volume (ul)         | Numeric          | Read Only      | <ul><li>Decimal Places Displayed = 2</li></ul>                                                         |
    | Derived Sample | Sample Name             | Built-in         |                |                                                                                                        |
    | Derived Sample | Sample Volume (uL)      | Numeric          |                | <ul><li>Decimal Places Displayed = 2</li></ul>                                                         |
    | Derived Sample | Target Insert Size (bp) | Numeric Dropdown | Custom Entries | <ul><li><p>Presets</p><ul><li>350</li><li>550</li></ul></li><li>Decimal Places Displayed = 0</li></ul> |
    | Project        | Project Name            | Built-in         |                |                                                                                                        |

### Step 2: Repair Ends and Select Library Size (TruSeq DNA PCR-Free v2.0)

* Master Step Name = Repair Ends v1.0
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {SubmittedSampleName}
* Reagent Kits
  * TruSeq DNA PCR-Free Sample Prep Kit - Set A, B or HT Box
    * Supplier = Illumina
    * Catalog Number = Set A: FC-121-3001, Set B: FC-121-3002, HT: FC-121-3003

#### Automations

<figure><img src="/files/r3ruiXiBRHaShqHwaqf3" alt=""><figcaption></figcaption></figure>

<details>

<summary>SPB Dilution</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 Number of Samples:: = step.::Total Number of 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 'if(input.::Target Insert Size (bp):: == 350) { step.::SPB (ml):: = (step.::Total Number of Samples:: * 109.25) / 1000} ; if(input.::Target Insert Size (bp):: == 350) { step.::PCR Grade Water (ml):: = (step.::Total Number of Samples:: * 74.75) / 1000 } ; if(input.::Target Insert Size (bp):: == 550) { step.::SPB (ml):: = (step.::Total Number of Samples:: * 92) / 1000} ; if(input.::Target Insert Size (bp):: == 550) { step.::PCR Grade Water (ml):: = (step.::Total Number of Samples:: * 92) / 1000 } ; output.::Target Insert Size (bp):: = input.::Target Insert Size (bp)::' -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      | Well                    | Built-in         |                |                                                                                                        |
    | Derived Sample | Sample Name             | Built-in         |                |                                                                                                        |
    | Derived Sample | Target Insert Size (bp) | Numeric Dropdown | Custom Entries | <ul><li><p>Presets</p><ul><li>350</li><li>550</li></ul></li><li>Decimal Places Displayed = 0</li></ul> |
    | Derived Sample | Waiting                 | Built-in         |                |                                                                                                        |
  * Expanded View Fields

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

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**         | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | ---------------------- | -------------- | ----------- | ----------------------------------------- |
  | Comment                | Multiline Text |             |                                           |
  | SPB (ml)               | Numeric        |             | Decimal Places Displayed = 2              |
  | PCR Grade Water (ml)   | Numeric        |             | Decimal Places Displayed = 2              |
  | Thermal Cycler Program | Text           |             | Default = ERP                             |
* Step File Placeholders
  * Next Step Log - Automatically attached
* Sample Table
  * Sample Display Default = Expand
  * Well Sort Order = Column
  * Table Columns - Global Fields

    | **Category**   | **Field Name**          | **Field Type**   | **Options**    | **Additional Options and Dropdown Items**                                                              |
    | -------------- | ----------------------- | ---------------- | -------------- | ------------------------------------------------------------------------------------------------------ |
    | Container      | Container Name          | Built-in         |                |                                                                                                        |
    | Container      | Well                    | Built-in         |                |                                                                                                        |
    | Derived Sample | Sample Name             | Built-in         |                |                                                                                                        |
    | Derived Sample | Target Insert Size (bp) | Numeric Dropdown | Custom Entries | <ul><li><p>Presets</p><ul><li>350</li><li>550</li></ul></li><li>Decimal Places Displayed = 0</li></ul> |
    | Project        | Project Name            | Built-in         |                |                                                                                                        |

### Step 3: Adenylate 3' Ends (TruSeq DNA PCR-Free v2.0)

* Master Step Name = Adenylate 3' Ends v2.0
* Step Type = No Outputs
* Reagent Kits
  * TruSeq DNA PCR-Free Sample Prep Kit - Set A, B or HT Box
    * Supplier = Illumina
    * Catalog Number = Set A: FC-121-3001, Set B: FC-121-3002, HT: FC-121-3003

#### Automations

<figure><img src="/files/yHyV845XyStH5xwhgOEJ" alt=""><figcaption></figcaption></figure>

<details>

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

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

{% code overflow="wrap" %}

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

{% endcode %}

</details>

#### Queue/Ice Bucket

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

    | **Category**   | **Field Name**          | **Field Type**   | **Options**    | **Additional Options and Dropdown Items**                                                              |
    | -------------- | ----------------------- | ---------------- | -------------- | ------------------------------------------------------------------------------------------------------ |
    | Container      | Container Name          | Built-in         |                |                                                                                                        |
    | Container      | Well                    | Built-in         |                |                                                                                                        |
    | Derived Sample | Sample Name             | Built-in         |                |                                                                                                        |
    | Derived Sample | Target Insert Size (bp) | Numeric Dropdown | Custom Entries | <ul><li><p>Presets</p><ul><li>350</li><li>550</li></ul></li><li>Decimal Places Displayed = 0</li></ul> |
    | Derived Sample | Waiting                 | Built-in         |                |                                                                                                        |
  * Expanded View Fields

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

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**         | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | ---------------------- | -------------- | ----------- | ----------------------------------------- |
  | Comment                | Multiline Text |             |                                           |
  | Thermal Cycler Program | Text           |             | Default = ATAIL70                         |
* Step File Placeholders
  * Log File - 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 | Sample Name             | Built-in         |                |                                                                                                        |
    | Derived Sample | Target Insert Size (bp) | Numeric Dropdown | Custom Entries | <ul><li><p>Presets</p><ul><li>350</li><li>550</li></ul></li><li>Decimal Places Displayed = 0</li></ul> |
    | Project        | Project Name            | Built-in         |                |                                                                                                        |

### Step 4: Ligate Adapters (TruSeq DNA PCR-Free v2.0)

* Master Step Name = Ligate Adapters v2.0
* Step Type = Add Labels
* Derived Sample Generation = Fixed, 1
* Naming Convention = {SubmittedSampleName}
* Reagent Kits
  * TruSeq DNA PCR-Free Sample Prep Kit - Set A, B or HT Box
    * Supplier = Illumina
    * Catalog Number = Set A: FC-121-3001, Set B: FC-121-3002, HT: FC-121-3003

#### Automations

<figure><img src="/files/LBkNFBwi8Z0dbb5iXJq2" alt=""><figcaption></figcaption></figure>

<details>

<summary>Copy to Output</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.::Target Insert Size (bp):: = input.::Target Insert Size (bp)::' -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Set Next Step &#x26; Copy to Input</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:: ; output.::Target Insert Size (bp):: = input.::Target Insert Size (bp)::'  -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 = Column
* Sample Table
  * Column Headers

    | **Category**   | **Field Name**          | **Field Type**   | **Options**    | **Additional Options and Dropdown Items**                                                              |
    | -------------- | ----------------------- | ---------------- | -------------- | ------------------------------------------------------------------------------------------------------ |
    | Container      | Container Name          | Built-in         |                |                                                                                                        |
    | Container      | Well                    | Built-in         |                |                                                                                                        |
    | Derived Sample | Sample Name             | Built-in         |                |                                                                                                        |
    | Derived Sample | Target Insert Size (bp) | Numeric Dropdown | Custom Entries | <ul><li><p>Presets</p><ul><li>350</li><li>550</li></ul></li><li>Decimal Places Displayed = 0</li></ul> |
    | Derived Sample | Waiting                 | Built-in         |                |                                                                                                        |
  * Expanded View Fields

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

#### Add Labels

* Label Groups
  * TruSeq DNA HT
  * TruSeq DNA LT

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**         | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | ---------------------- | -------------- | ----------- | ----------------------------------------- |
  | Comment                | Multiline Text |             |                                           |
  | Thermal Cycler Program | Text           |             | Default = LIG                             |
  | 80% EtOH Prep Date     | Date           |             |                                           |
* Step File Placeholders
  * Log File - 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 | Reagent Name            | Built-in         |                |                                                                                                        |
    | Derived Sample | Sample Name             | Built-in         |                |                                                                                                        |
    | Derived Sample | Target Insert Size (bp) | Numeric Dropdown | Custom Entries | <ul><li><p>Presets</p><ul><li>350</li><li>550</li></ul></li><li>Decimal Places Displayed = 0</li></ul> |
    | Project        | Project Name            | Built-in         |                |                                                                                                        |

### Step 5: Clean Up Ligated Fragments (TruSeq DNA PCR-Free v2.0)

* Master Step Name = Clean Up Ligated Fragments (TruSeq DNA PCR-Free v2.0.10)
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}

{% hint style="info" %}
The version of Clean Up Ligated Fragments master step name may be different depending on the version of IPP installed.
{% endhint %}

#### Automations

<figure><img src="/files/ERAy1SinMZJrSj313YMK" alt=""><figcaption></figcaption></figure>

<details>

<summary>Set Next Step &#x26; Copy to Input</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:: ; output.::Target Insert Size (bp):: = input.::Target Insert Size (bp)::'  -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

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

* Trigger Location = Not Used

{% code overflow="wrap" %}

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

{% endcode %}

</details>

#### Queue/Ice Bucket

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

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

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**     | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | ------------------ | -------------- | ----------- | ----------------------------------------- |
  | Comment            | Multiline Text |             |                                           |
  | 80% EtOH Prep Date | Date           |             |                                           |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Expand
  * Well Sort Order = Row
  * Table Columns - Global Fields

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

### Step 6: Validate Libraries (TruSeq DNA PCR-Free v2.0)

* Master Step Name = Validate Libraries (TruSeq DNA PCR-Free v2.0.10)
* Step Type = Standard QC
* Measurement Generation = Fixed, 4
* Naming Convention = {SubmittedSampleName}\_{OutputItemSubsetNumber} of {OutputItemSubsetTotal}

{% hint style="info" %}
The version of Validate Libraries master step name may be different depending on the version of IPP installed.
{% endhint %}

#### Automations

<figure><img src="/files/vDRyiygPo1GduLimMMR1" alt=""><figcaption></figcaption></figure>

<details>

<summary>Set Dilution Factor</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 'if (output.name.contains(::_1 of 4::)) {output.::Dilution Factor:: = ::1:10000::} ; if (output.name.contains(::_2 of 4::)) {output.::Dilution Factor:: = ::1:10000::} ; if (output.name.contains(::_3 of 4::)) {output.::Dilution Factor:: = ::1:20000::} ; if (output.name.contains(::_4 of 4::)) {output.::Dilution Factor:: = ::1:20000::}' -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Calculate Molarity and Assign 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 {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t true -h false -exp ' if (output.::Dilution Factor:: == ::1:10000:: && input.::Target Insert Size (bp):: == 350) {output.::Molarity (nM):: = ((output.::Concentration (pM):: * 10000) * (452 / 470)) / 1000} ; if (output.::Dilution Factor:: == ::1:20000:: && input.::Target Insert Size (bp):: == 350) {output.::Molarity (nM):: = ((output.::Concentration (pM):: * 20000) * (452 / 470)) / 1000} ; if (output.::Dilution Factor:: == ::1:10000:: && input.::Target Insert Size (bp):: == 550) {output.::Molarity (nM):: = ((output.::Concentration (pM):: * 10000) * (452 / 670)) / 1000} ; if (output.::Dilution Factor:: == ::1:20000:: && input.::Target Insert Size (bp):: == 550) {output.::Molarity (nM):: = ((output.::Concentration (pM):: * 10000) * (452 / 670)) / 1000}' -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:computeReplicateAverage -src 'Molarity (nM)' -dest 'Molarity (nM)' -exclude 'Exclude' -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 true -h false -exp 'if (output.::Molarity (nM):: >= 2 && output.::Molarity (nM):: <= 75) { output.QC = true } else { output.QC = false } ; if (input.::Molarity (nM):: >= 2 && input.::Molarity (nM):: <= 75) { input.QC = true } else { input.QC = false }' -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Set Next Steps - PASS/FAIL</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 true -h false -excludeControls true -exp 'if (input.QC == true) { nextStep = ::ADVANCE:: } else { nextStep = ::ESCALATE:: }' -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 File Placeholders
  * Log 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 (pM)      | Numeric          |                                                         | <ul><li>Decimal Places Displayed = 2</li></ul>                                                         |
    | Derived Sample | Dilution Factor         | Text Dropdown    | <ul><li>Required Field</li><li>Custom Entries</li></ul> | <ul><li><p>Presets</p><ul><li>1:10000</li><li>1:20000</li></ul></li></ul>                              |
    | Derived Sample | Molarity (nM)           | Numeric          |                                                         | <ul><li>Decimal Places Displayed = 2</li></ul>                                                         |
    | Derived Sample | Sample Name             | Built-in         |                                                         |                                                                                                        |
    | Derived Sample | Target Insert Size (bp) | Numeric Dropdown | <ul><li>Custom Entries</li></ul>                        | <ul><li><p>Presets</p><ul><li>350</li><li>550</li></ul></li><li>Decimal Places Displayed = 0</li></ul> |
    | Measurement    | Concentration (pM)      | Numeric          |                                                         | <ul><li>Decimal Places Displayed = 2</li></ul>                                                         |
    | Measurement    | Dilution Factor         | Text             | <ul><li>Read Only</li></ul>                             |                                                                                                        |
    | Measurement    | Exclude                 | Toggle Switch    |                                                         | <ul><li>Default = None Set</li></ul>                                                                   |
    | Measurement    | Molarity (nM)           | Numeric          |                                                         | <ul><li>Decimal Places Displayed = 2</li></ul>                                                         |

### Step 7: Normalize Libraries (TruSeq DNA PCR-Free v2.0)

* Master Step Name = Normalize Libraries 2 v2.0.10
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}

{% hint style="info" %}
The version of Normalized Libraries 2 master step name may be different depending on the version of IPP installed.
{% endhint %}

#### Automations

<figure><img src="/files/3FEYfsbiMw6kkUREDkYw" alt=""><figcaption></figcaption></figure>

<details>

<summary>Normalization Calculations - Option 2</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 true -h false -exp 'output.::Molarity (nM):: = input.::Molarity (nM):: ; if (output.::Molarity (nM):: <= step.::Target Normalization (nM)::) {output.::Sample Volume (ul):: = step.::Sample Volume (ul):: ; output.::Buffer Volume (ul):: = 0 ; output.::Normalized Molarity (nM):: = output.::Molarity (nM)::} else {output.::Sample Volume (ul):: = step.::Sample Volume (ul):: ; output.::Buffer Volume (ul):: = ((output.::Molarity (nM):: * step.::Sample Volume (ul)::) / step.::Target Normalization (nM)::) - step.::Sample Volume (ul):: ; output.::Normalized Molarity (nM):: = step.::Target Normalization (nM)::}' -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Set Next Step - Remove</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 = ::REMOVE::' -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

<summary>Routing script - Normalize Libraries</summary>

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

{% code overflow="wrap" %}

```markup
bash -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \
\
--FIELD_NAME 'Sequencing Instrument' \
--FIELD_VALUE 'MiSeq' \
--WORKFLOW 'MiSeq Sequencing v3.2' \
--STEP 'Library Pooling (MiSeq v3.2)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Sequencing Instrument' \
--FIELD_VALUE 'NextSeq' \
--WORKFLOW 'NextSeq 500/550 Sequencing v1.2' \
--STEP 'Library Pooling (NextSeq 500/550 v1.2)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Sequencing Instrument' \
--FIELD_VALUE 'NovaSeq 2.0' \
--WORKFLOW 'NovaSeq 6000 v2.3' \
--STEP 'Define Run Format (NovaSeq 6000 v2.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Sequencing Instrument' \
--FIELD_VALUE 'NovaSeq 3.0' \
--WORKFLOW 'NovaSeq 6000 v3.8' \
--STEP 'Define Run Format (NovaSeq 6000 v3.8)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Sequencing Instrument' \
--FIELD_VALUE 'NovaSeqDx' \
--WORKFLOW 'NovaSeqDx v1.2' \
--STEP 'Define Run Format (NovaSeqDx v1.2)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Sequencing Instrument' \
--FIELD_VALUE 'NextSeq 1000/2000' \
--WORKFLOW 'NextSeq 1000/2000 Sequencing v2.4' \
--STEP 'Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Sequencing Instrument' \
--FIELD_VALUE 'NovaSeq X Series' \
--WORKFLOW 'NovaSeq X Series v1.1' \
--STEP 'Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Sequencing Instrument' \
--FIELD_VALUE 'NextSeq 1000/2000 On-Prem' \
--WORKFLOW 'NextSeq 1000/2000 On-Prem Sequencing v1.0' \
--STEP 'Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"
```

{% endcode %}

> ℹ The field value and the actual version of the workflows and steps in the routing automation script may be different depending on the version of IPP installed.

</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**                           |
  | ------------------------- | -------------- | -------------- | ------------------------------------------------------------------- |
  | Comment                   | Multiline Text |                |                                                                     |
  | Sample Volume (ul)        | Numeric        | Required Field | <ul><li>Default = 10</li><li>Decimal Places Displayed = 0</li></ul> |
  | Target Normalization (nM) | Numeric        | Required Field | <ul><li>Default = 10</li><li>Decimal Places Displayed = 0</li></ul> |
* Step File Placeholders
  * Log File - 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 | Molarity (nM)            | Numeric        |                | Decimal Places Displayed = 2                                                                                                                                                                         |
    | Derived Sample | Buffer Volume (ul)       | Numeric        |                | Decimal Places Displayed = 2                                                                                                                                                                         |
    | Derived Sample | Normalized Molarity (nM) | Numeric        |                | Decimal Places Displayed = 2                                                                                                                                                                         |
    | Derived Sample | Sample Name              | Built-in       |                |                                                                                                                                                                                                      |
    | Derived Sample | Sample Volume (ul)       | Numeric        |                | Decimal Places Displayed = 2                                                                                                                                                                         |
    | Derived Sample | Sequencing Instrument    | Text Dropdown  | Required Field | <p>Presets</p><ul><li>MiSeq</li><li>NextSeq</li><li>NextSeq 1000/2000</li><li>NextSeq 1000/2000 On-Prem</li><li>NovaSeq 2.0</li><li>NovaSeq 3.0</li><li>NovaSeq X Series</li><li>NovaSeqDx</li></ul> |
    | Project        | Project Name             | Built-in       |                |                                                                                                                                                                                                      |

    > ℹ The preset options for Derived Sample Sequencing Instrument may vary depending on the version of the IPP.


---

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