# TruSeq Nano DNA v1.0

## Overview

The TruSeq Nano DNA includes the following functionality:

* Preconfigured TruSeq Nano DNA protocol that explains how to prepare up to 96 uniquely indexed paired-end libraries of genomic DNA (gDNA) using Illumina® TruSeq® Nano DNA Library Prep kits.
* 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.
* A routing script that allows sequencing of libraries using any Illumina sequencing instrument.

## Protocol 1: TruSeq Nano DNA v1.0

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-9e731a5fe62bc16a16a2114810d12f4cff66b515%2Ftruseq-nano-dna-next-step-config.png?alt=media" alt=""><figcaption></figcaption></figure>

### Step 1: Fragment DNA (TruSeq Nano DNA v1.0)

* Master Step Name = Fragment DNA v1.0
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
  * TruSeq Nano DNA LT Library Prep Kit - Set A, Set B
    * Supplier = Illumina
    * Catalog Number = Set A: FC-121-4001, Set B: FC-121-4002

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-757241b2f1db6c4f8b83d1b3a7c75bb5fc9ac41a%2Ftruseq-nano-dna-step1-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

<summary>Normalize gDNA (Nano)</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 '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>

<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 (PCR Free)</summary>

* Trigger Location = Not Used

{% code overflow="wrap" %}

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

#### 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 | 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>                                                                                  |
* Step File Placeholders
  * Log file - 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         |                |                                                 |
    | Derived Sample | Concentration           | Numeric          | Required Field | Decimal Places Displayed = 2                    |
    | Derived Sample | Conc. Units             | Text             | Required Field |                                                 |
    | Derived Sample | RSB Volume (ul)         | Numeric          | Read Only      | Decimal Places Displayed = 2                    |
    | Derived Sample | Sample Name             | Built-in         |                |                                                 |
    | Derived Sample | Sample Volume (ul)      | Numeric          |                | Decimal Places Displayed = 2                    |
    | Derived Sample | Target Insert Size (bp) | Numeric Dropdown | Custom Entries | <p>Presets</p><ul><li>350</li><li>550</li></ul> |
    | Project        | Project Name            | Built-in         |                |                                                 |

### Step 2: Repair Ends and Select Library Size (TruSeq Nano DNA v1.0)

* Master Step Name = Repair Ends v1.0
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {SubmittedSampleName}
* Reagent Kits
  * TruSeq Nano DNA LT Library Prep Kit - Set A, Set B
    * Supplier = Illumina
    * Catalog Number = Set A: FC-121-4001, Set B: FC-121-4002

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-d31719c44de81d1db30b6eac505a2085d26a7861%2Ftruseq-nano-dna-step2-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

<summary>SPB Dilution</summary>

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

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp '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 = 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 |             |                                           |
  | PCR Grade Water (ml)   | Numeric        |             | Decimal Places Displayed = 2              |
  | SPB (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 = Row
  * 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 | <p>Presets</p><ul><li>350</li><li>550</li></ul> |
    | Project        | Project Name            | Built-in         |                |                                                 |

### Step 3: Adenylate 3' Ends (TruSeq Nano DNA v1.0)

* Master Step Name = Adenylate 3' Ends v2.0
* Step Type = No Outputs
* Reagent Kits
  * TruSeq Nano DNA LT Library Prep Kit - Set A, Set B
    * Supplier = Illumina
    * Catalog Number = Set A: FC-121-4001, Set B: FC-121-4002

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-9113f89a90d6f5b5635b33510ccea2432759522a%2Ftruseq-nano-dna-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      | 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 | <p>Presets</p><ul><li>350</li><li>550</li></ul> |
    | Project        | Project Name            | Built-in         |                |                                                 |

### Step 4: Ligate Adapters (TruSeq Nano DNA v1.0)

* Master Step Name = Ligate Adapters v2.0
* Step Type = Add Labels
* Derived Sample Generation = Fixed, 1
* Naming Convention = {SubmittedSampleName}
* Reagent Kits
  * TruSeq Nano DNA LT Library Prep Kit - Set A, Set B
    * Supplier = Illumina
    * Catalog Number = Set A: FC-121-4001, Set B: FC-121-4002

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-f89e48104d6866f7e902cc523bddcd7d88ed78a2%2Ftruseq-nano-dna-step4-automation.png?alt=media" 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 - 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>Set Next Step &#x26; Copy to Input</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:: ; output.::Target Insert Size (bp):: = input.::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 | 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 = ATAIL70                         |
  | 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 | <p>Presets</p><ul><li>350</li><li>550</li></ul> |
    | Project        | Project Name            | Built-in         |                |                                                 |

### Step 5: PCR Amplification (TruSeq Nano DNA v1.0)

* Master Step Name = PCR Amplification (TruSeq Nano DNA v1.0.10)
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {SubmittedSampleName}
* Reagent Kits
  * TruSeq Nano DNA LT Library Prep Kit - Set A, Set B
    * Supplier = Illumina
    * Catalog Number = Set A: FC-121-4001, Set B: FC-121-4002

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

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-332ad3bd64c33b7e4dd500b8d3ab50906945dc20%2Ftruseq-nano-dna-step5-automation.png?alt=media" 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 - 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 |             |                                           |
  | Thermal Cycler Program | Text           |             | Default = PCRNano                         |
  | 80% EtOH Prep Date     | Date           |             |                                           |
* Step File Placeholders
  * Log File - 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         |                |                                                 |
    | Derived Sample | Target Insert Size (bp) | Numeric Dropdown | Custom Entries | <p>Presets</p><ul><li>350</li><li>550</li></ul> |
    | Project        | Project Name            | Built-in         |                |                                                 |

### Step 6: Validate Libraries (TruSeq Nano DNA v1.0)

* Master Step Name = Validate Libraries (TruSeq Nano DNA v1.0.10)
* 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="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-6d681dd1dfe2f835df3a135028cfa521aa550d3a%2Ftruseq-nano-dna-step6-automation.png?alt=media" 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):: >= 10 && output.::Molarity (nM):: <= 250) { output.QC = true } else { output.QC = false } ; if (input.::Molarity (nM):: >= 10 && input.::Molarity (nM):: <= 250) { 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 = 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 |             |                                           |
* Step File Placeholders
  * Log File - Manually uploaded
* Sample Table
  * Enable QC Flags = Yes
  * Sample Display Default = Expand
  * Well Sort Order = Column
  * File Column Options
    * File Column Display = Hide
    * 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      | Well               | Built-in       |             |                                           |
    | Derived Sample | Molarity (nM)      | Numeric        |             | Decimal Places Displayed = 2              |
    | Derived Sample | Sample Name        | Built-in       |             |                                           |
    | Measurement    | Concentration (pM) | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Dilution Factor    | Text           | Read Only   |                                           |
    | Measurement    | Exclude            | Toggle Switch  |             | Default = None Set                        |
    | Measurement    | Molarity (nM)      | Numeric        |             | Decimal Places Displayed = 2              |
    | Project        | Project Name       | Built-in       |             |                                           |

### Step 7: Normalize Libraries (TruSeq Nano DNA v1.0)

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

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

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-72c2c331b75c7a3ffbcc14a2b535d3f79de06b2e%2Ftruseq-nano-dna-step7-automation.png?alt=media" 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>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>

<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 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 = 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 |                |                                                                     |
  | 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 = 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 | Buffer Volume (ul)       | Numeric        |                | Decimal Places Displayed = 2                                                                                                                                                                         |
    | Derived Sample | Molarity (nM)            | 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       |                |                                                                                                                                                                                                      |