# Nextera XT v2.0

## Overview

The Nextera XT DNA includes the following functionality:

* Preconfigured Nextera XT DNA protocol that converts the mRNA in total RNA into a library of template molecules of known strand origin. The library is suitable for subsequent cluster generation and DNA sequencing.
* Automated calculation of sample and buffer volumes.
* Automated calculation or display of reagents at every step in the protocol.
* Automatic step transition when required.
* Automatic placement of samples when necessary.
* Automated assignment of QC Pass/Fail, based on user-selected threshold values.
* A routing script that allows sequencing of libraries using any Illumina sequencing instrument.

## Protocol 1: Nextera XT DNA v2.0

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-03c973cf182b2d73b0790f581eea0ec8fc466129%2Fnextera-xt-dna-next-step-config.png?alt=media" alt=""><figcaption></figcaption></figure>

### Step 1: Tagment Genomic DNA (Nextera XT DNA v2.0)

* Master Step Name = Tagment v2.0
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {SubmittedSampleName}
* Reagent Kits
  * Nextera XT DNA Library Prep Kit, Box 1
    * Supplier = Illumina
    * Catalog Number = FC-131-1096

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-0a88292f53c83a318b484857a3e7c4c185621c0a%2Fnextera-xt-dna-step1-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

<summary>Normalize gDNA (Nextera XT)</summary>

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

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t true -h false -exp 'output.::Sample Volume (ul):: = (0.2 * 5) / input.::Concentration:: ; output.::Buffer Volume (ul):: = 5 - output.::Sample Volume (ul)::' -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

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

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

{% code overflow="wrap" %}

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

{% endcode %}

</details>

<details>

<summary>Normalize gDNA</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 'output.::Sample Volume (ul):: = 50 / input.::Concentration:: ; output.::Buffer Volume (ul):: = 10 - output.::Sample Volume (ul)::' -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

#### Queue/Ice Bucket

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

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

#### Placement = Enabled

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

#### 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 = Tagmentation                    |
* Step File Placeholders
  * Log File - Manually uploaded
* 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 | Buffer Volume (ul)  | Numeric        |             | Decimal Places Displayed = 2              |
    | Derived Sample | Sample Name         | Built-in       |             |                                           |
    | Derived Sample | Sample Volume (ul)  | Numeric        |             | Decimal Places Displayed = 2              |
    | Project        | Project Name        | Built-in       |             |                                           |

### Step 2: Amplify Libraries (Nextera XT DNA v2.0)

* Master Step Name = Amplification with Labels v2.0
* Step Type = Add Labels
* Derived Sample Generation = Fixed, 1
* Naming Convention = {SubmittedSampleName}
* Reagent Kits
  * Nextera XT DNA Library Prep Kit, Box 1
    * Supplier = Illumina
    * Catalog Number = FC-131-1096

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-7e7d63464fcc907bbb85a75403e831c4c5177aae%2Fnextera-xt-dna-step2-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

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

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

{% code overflow="wrap" %}

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

{% endcode %}

</details>

<details>

<summary>Copy to Output</summary>

* Trigger Location = Not Used

{% code overflow="wrap" %}

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

#### Add Labels

* Label Groups
  * Nextera XT v2 Full Set
  * Nextera XT v2 Set A
  * Nextera XT v2 Set B
  * Nextera XT v2 Set C
  * Nextera XT v2 Set D

#### 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 = Amplify Libraries               |
* Step File Placeholders
  * Log File - Manually uploaded
* Sample Table
  * Sample Display Default = Collapse
  * Well Sort Order = Row
  * Table Columns - Global Fields

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

### Step 3: Clean Up Libraries (Nextera XT DNA v2.0)

* Master Step Name = Clean Up v2.0
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
  * AMPure XP Beads
    * Supplier = Beckman Coulter Genomics
    * Catalog Number = A63881
    * Website = <https://www.beckmancoulter.com/wsrportal/wsr/research-and-discovery/products-and-services/nucleic-acid-sample-preparation/agencourt-ampure-xp-pcr-purification/index.htm>
  * Nextera XT DNA Library Prep Kit, Box 1
    * Supplier = Illumina
    * Catalog Number = FC-131-1096

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-4e9ef0d569c922ed7b5bfb368f1df761b96d1652%2Fnextera-xt-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 = 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 |             |                                           |
  | 80% EtOH Prep Date | Date           |             |                                           |
* 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       |             |                                           |
    | Container      | LIMS ID (Container) | Built-in       |             |                                           |
    | Container      | Well                | Built-in       |             |                                           |
    | Derived Sample | Sample Name         | Built-in       |             |                                           |
    | Project        | Project Name        | Built-in       |             |                                           |

### Step 4: Bioanalyzer QC (Library Validation) (Nextera XT DNA v2.0)

* Master Step Name = Bioanalyzer QC (Library Validation) v2.0
* Step Type = Standard QC
* Measurement Generation = Fixed, 1
* Naming Convention = {InputItemName} Bioanalyzer

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-e8310afbd43f48f195d081417357050e0072a917%2Fnextera-xt-dna-step4-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

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

{% endcode %}

</details>

<details>

<summary>Parse Bioanalyzer XML, Copy nM and Assign QC flags</summary>

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

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {processURI:v2} -u {username} -p {password} script:parseBioAnalyzer -inputFile {compoundOutputFileLuid2} -log {compoundOutputFileLuid5} -configFile '/opt/gls/clarity/extensions/conf/v5/bioanalyzer/defaultBioAnalyzerDNAConfig.groovy' && /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.::Conc. Units::.contains(::pg::)) {output.::Molarity (nM):: = output.::Region 1 Molarity:: / 1000} else {output.::Molarity (nM):: = output.::Region 1 Molarity::} ; (input.::Molarity (nM):: = output.::Molarity (nM)::) ' -log {compoundOutputFileLuid8} && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {processURI:v2} -u {username} -p {password} script:assignQC -log {compoundOutputFileLuid6} -qcResult {compoundOutputFileLuid7}"
```

{% endcode %}

</details>

<details>

<summary>Set Next Step - Output 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 (output.QC == true) { nextStep = ::ADVANCE:: } else { nextStep = ::ESCALATE:: }' -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

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

{% endcode %}

</details>

<details>

<summary>Parse Bioanalyzer XML, Assign QC flags, and Copy Concentrations</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 {processURI:v2} -u {username} -p {password} script:parseBioAnalyzer -inputFile {compoundOutputFileLuid2} -log {compoundOutputFileLuid5} -configFile '/opt/gls/clarity/extensions/conf/v5/bioanalyzer/defaultBioAnalyzerDNAConfig.groovy' script:assignQC -log {compoundOutputFileLuid6} -qcResult {compoundOutputFileLuid7} && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t true -h false -exp 'output.::Concentration:: = output.::Region 1 Conc.:: ; input.::Concentration:: = output.::Concentration:: ; output.::Conc. Units:: = ::ng/ul:: ; input.::Conc. Units:: = output.::Conc. Units::' -log {compoundOutputFileLuid8}"
```

{% endcode %}

</details>

<details>

<summary>Parse Bioanalyzer XML, Calculate nM and assign QC flags</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 {processURI:v2} -u {username} -p {password} script:parseBioAnalyzer -inputFile {compoundOutputFileLuid2} -log {compoundOutputFileLuid5} -configFile '/opt/gls/clarity/extensions/conf/v5/bioanalyzer/defaultBioAnalyzerDNAConfig.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t true -h false -exp 'output.::Concentration:: = output.::Region 1 Conc.:: ; output.::Molarity (nM):: = (output.::Concentration:: * 1000000) / (660 * output.::Region 1 Average Size - bp::) ; input.::Molarity (nM):: = output.::Molarity (nM):: ; output.::Conc. Units:: = ::ng/ul::' -log {compoundOutputFileLuid8} && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {processURI:v2} -u {username} -p {password} script:assignQC -log {compoundOutputFileLuid6} -qcResult {compoundOutputFileLuid7}"
```

{% endcode %}

</details>

#### Queue/Ice Bucket

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

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

#### Placement = Enabled

* Defaults
  * Sample Grouping = Group by Containers
  * Well Sort Order = Column
  * Placement Pattern = Column
* Destination Containers

  * BioAnalyzer DNA High Sensitivity Chip

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

  * BioAnalyzer DNA 1000 Chip

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

#### Record Details

**Group of Defaults**

<details>

<summary>Nextera DNA Flex Library Validation</summary>

* Criteria 1 - Operator = >=
* Criteria 1 - Source Data Field = Peak 2 Size - bp
* Criteria 1 - Threshold Value = 150.00
* Criteria 2 - Operator = <=
* Criteria 2 - Source Data Field = Peak 2 Size - bp
* Criteria 2 - Threshold Value = 1,500.00

</details>

<details>

<summary>Nextera Mate Pair Library Validation</summary>

* Criteria 1 - Operator = >=
* Criteria 1 - Source Data Field = Region 1 Average Size - bp
* Criteria 1 - Threshold Value = 150.00
* Criteria 2 - Operator = <=
* Criteria 2 - Source Data Field = Region 1 Average Size - bp
* Criteria 2 - Threshold Value = 400.00

</details>

<details>

<summary>Nextera XT DNA Library Validation</summary>

* Criteria 1 - Operator = >=
* Criteria 1 - Source Data Field = Peak 2 Size - bp
* Criteria 1 - Threshold Value = 250.00
* Criteria 2 - Operator = <=
* Criteria 2 - Source Data Field = Peak 2 Size - bp
* Criteria 2 - Threshold Value = 1,000.00

</details>

<details>

<summary>NRCC Library Validation</summary>

* Criteria 1 - Operator = >=
* Criteria 1 - Source Data Field = Peak 2 Size - bp
* Criteria 1 - Threshold Value = 350.00
* Criteria 2 - Operator = <=
* Criteria 2 - Source Data Field = Peak 2 Size - bp
* Criteria 2 - Threshold Value = 1,000.00

</details>

<details>

<summary>TruSeq ChIP-Seq Library Validation</summary>

* Criteria 1 - Operator = >=
* Criteria 1 - Source Data Field = Region 1 Average Size - bp
* Criteria 1 - Threshold Value = 150.00
* Criteria 2 - Operator = <=
* Criteria 2 - Source Data Field = Region 1 Average Size - bp
* Criteria 2 - Threshold Value = 400.00

</details>

<details>

<summary>TruSeq Exome Library Validation</summary>

* Criteria 1 - Operator = >=
* Criteria 1 - Source Data Field = Peak 2 Size - bp
* Criteria 1 - Threshold Value = 150.00
* Criteria 2 - Operator = <=
* Criteria 2 - Source Data Field = Peak 2 Size - bp
* Criteria 2 - Threshold Value = 1,000.00

</details>

<details>

<summary>TruSeq Methyl Capture EPIC Library Validation</summary>

* Criteria 1 - Operator = >=
* Criteria 1 - Source Data Field = Peak 2 Size - bp
* Criteria 1 - Threshold Value = 200.00
* Criteria 2 - Operator = <=
* Criteria 2 - Source Data Field = Peak 2 Size - bp
* Criteria 2 - Threshold Value = 300.00

</details>

<details>

<summary>TruSeq Rapid Exome Library Validation</summary>

* Criteria 1 - Operator = >=
* Criteria 1 - Source Data Field = Peak 2 Size - bp
* Criteria 1 - Threshold Value = 200.00
* Criteria 2 - Operator = <=
* Criteria 2 - Source Data Field = Peak 2 Size - bp
* Criteria 2 - Threshold Value = 500.00

</details>

<details>

<summary>TruSeq RNA Access Library Validation</summary>

* Criteria 1 - Operator = >=
* Criteria 1 - Source Data Field = Peak 2 Size - bp
* Criteria 1 - Threshold Value = 200.00
* Criteria 2 - Operator = <=
* Criteria 2 - Source Data Field = Peak 2 Size - bp
* Criteria 2 - Threshold Value = 320.00

</details>

<details>

<summary>TruSeq RNA Exome Library Validation</summary>

* Criteria 1 - Operator = >=
* Criteria 1 - Source Data Field = Peak 2 Size - bp
* Criteria 1 - Threshold Value = 200.00
* Criteria 2 - Operator = <=
* Criteria 2 - Source Data Field = Peak 2 Size - bp
* Criteria 2 - Threshold Value = 320.00

</details>

<details>

<summary>TruSeq Small RNA Library Validation</summary>

* Criteria 1 - Operator = >=
* Criteria 1 - Source Data Field = Region 1 Average Size - bp
* Criteria 1 - Threshold Value = 100.00
* Criteria 2 - Operator = <=
* Criteria 2 - Source Data Field = Region 1 Average Size - bp
* Criteria 2 - Threshold Value = 200.00

</details>

<details>

<summary>TruSeq Stranded mRNA Library Validation</summary>

* Criteria 1 - Operator = >=
* Criteria 1 - Source Data Field = Region 1 Average Size - bp
* Criteria 1 - Threshold Value = 250.00
* Criteria 2 - Operator = <=
* Criteria 2 - Source Data Field = Region 1 Average Size - bp
* Criteria 2 - Threshold Value = 275.00

</details>

<details>

<summary>TruSeq Stranded Total RNA Library Validation</summary>

* Criteria 1 - Operator = >=
* Criteria 1 - Source Data Field = Region 1 Average Size - bp
* Criteria 1 - Threshold Value = 250.00
* Criteria 2 - Operator = <=
* Criteria 2 - Source Data Field = Region 1 Average Size - bp
* Criteria 2 - Threshold Value = 275.00

</details>

<details>

<summary>TruSeq Targeted RNA Expression Library Validation</summary>

* Criteria 1 - Operator = >=
* Criteria 1 - Source Data Field = Peak 2 Size - bp
* Criteria 1 - Threshold Value = 100.00
* Criteria 2 - Operator = <=
* Criteria 2 - Source Data Field = Peak 2 Size - bp
* Criteria 2 - Threshold Value = 300.00

</details>

<details>

<summary>TruSight Myeloid Library Validation</summary>

* Criteria 1 - Operator = >=
* Criteria 1 - Source Data Field = Region 1 Average Size - bp
* Criteria 1 - Threshold Value = 150.00
* Criteria 2 - Operator = <=
* Criteria 2 - Source Data Field = Region 1 Size - bp
* Criteria 2 - Threshold Value = 400.00

</details>

<details>

<summary>TruSight RNA Fusion Library Validation</summary>

* Criteria 1 - Operator = >=
* Criteria 1 - Source Data Field = Region 1 Average Size - bp
* Criteria 1 - Threshold Value = 160.00
* Criteria 2 - Operator = <=
* Criteria 2 - Source Data Field = Region 1 Size - bp
* Criteria 2 - Threshold Value = 700.00

</details>

<details>

<summary>TSCA Library Validation</summary>

* Criteria 1 - Operator = >=
* Criteria 1 - Source Data Field = Region 1 Average Size - bp
* Criteria 1 - Threshold Value = 300.00
* Criteria 2 - Operator = <=
* Criteria 2 - Source Data Field = Region 1 Size - bp
* Criteria 2 - Threshold Value = 400.00

</details>

* Step Data
  * Group of Defaults = Nextera XT DNA Library Validation Validation
  * Master Step Fields

    | **Field Name**                              | **Field Type** | **Options**    | **Additional Options and Dropdown Items**                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |
    | ------------------------------------------- | -------------- | -------------- | --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
    | Criteria 1 - Operator                       | Text Dropdown  | Custom Entries | <p>Presets</p><ul><li>>=</li><li><=</li><li>=</li><li>!=</li></ul>                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |
    | Criteria 1 - Source Data Field              | Text Dropdown  |                | <p>Presets</p><ul><li>Concentration</li><li>Conc. Units</li><li>Number of Peaks found</li><li>Peak 1 Size - bp</li><li>Peak 1 Conc.</li><li>Peak 1 Molarity</li><li>Peak 2 Size - bp</li><li>Peak 2 Conc.</li><li>Peak 2 Molarity</li><li>Peak 3 Size - bp</li><li>Peak 3 Conc.</li><li>Peak 3 Molarity</li><li>Peak 4 Size - bp</li><li>Peak 4 Conc.</li><li>Peak 4 Molarity</li><li>Peak 5 Size - bp</li><li>Peak 5 Conc.</li><li>Peak 5 Molarity</li><li>Number of Regions found</li><li>Region 1 Average Size - bp</li><li>Region 1 Conc.</li><li>Region 1 Molarity</li><li>Region 2 Average Size - bp</li><li>Region 2 Conc.</li><li>Region 2 Molarity</li><li>Region 3 Average Size - bp</li><li>Region 3 Conc.</li><li>Region 3 Molarity</li><li>Region 4 Average Size - bp</li><li>Region 4 Conc.</li><li>Region 4 Molarity</li><li>Region 5 Average Size - bp</li><li>Region 5 Conc.</li><li>Region 5 Molarity</li></ul> |
    | Criteria 1 - Threshold Value                | Numeric        |                | Decimal Places Displayed = 2                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |
    | Criteria 2 - Operator                       | Text Dropdown  | Custom Entries | <p>Presets</p><ul><li>>=</li><li><=</li><li>=</li><li>!=</li></ul>                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |
    | Criteria 2 - Source Data Field              | Text Dropdown  |                | <p>Presets</p><ul><li>Concentration</li><li>Conc. Units</li><li>Number of Peaks found</li><li>Peak 1 Size - bp</li><li>Peak 1 Conc.</li><li>Peak 1 Molarity</li><li>Peak 2 Size - bp</li><li>Peak 2 Conc.</li><li>Peak 2 Molarity</li><li>Peak 3 Size - bp</li><li>Peak 3 Conc.</li><li>Peak 3 Molarity</li><li>Peak 4 Size - bp</li><li>Peak 4 Conc.</li><li>Peak 4 Molarity</li><li>Peak 5 Size - bp</li><li>Peak 5 Conc.</li><li>Peak 5 Molarity</li><li>Number of Regions found</li><li>Region 1 Average Size - bp</li><li>Region 1 Conc.</li><li>Region 1 Molarity</li><li>Region 2 Average Size - bp</li><li>Region 2 Conc.</li><li>Region 2 Molarity</li><li>Region 3 Average Size - bp</li><li>Region 3 Conc.</li><li>Region 3 Molarity</li><li>Region 4 Average Size - bp</li><li>Region 4 Conc.</li><li>Region 4 Molarity</li><li>Region 5 Average Size - bp</li><li>Region 5 Conc.</li><li>Region 5 Molarity</li></ul> |
    | Criteria 2 - Threshold Value                | Numeric        |                | Decimal Places Displayed = 2                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |
    | Use strict matching for Bioanalyzer results | Toggle Switch  |                | Default = None Set                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |
* Step File Placeholders
  * Bioanalyzer Input File - Automatically attached
  * Bioanalyzer Input File Generation Log File - Automatically attached
  * Bioanalyzer XML Result File (required) - Manually uploaded
  * Result File (optional) - Manually uploaded
  * PDF Summary File (optional) - Manually uploaded
  * Bioanalyzer XML Parsing Log File - Automatically attached
  * QC Assignment Log File - Automatically attached
  * QC Assignment Report - Automatically attached
* Sample Table
  * Enable QC Flags = Yes
  * Sample Display Default = Expand
  * Well Sort Order = Column
  * File Column Options
    * File Column Display = Hide
    * File Attachment Method = Auto
  * Table Columns - Global Fields

    | **Category**   | **Field Name**             | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
    | -------------- | -------------------------- | -------------- | ----------- | ----------------------------------------- |
    | Derived Sample | Molarity (nM)              | Numeric        |             | Decimal Places Displayed = 2              |
    | Derived Sample | Sample Name                | Built-in       |             |                                           |
    | Measurement    | BA Sample Name             | Text           |             |                                           |
    | Measurement    | Concentration              | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Conc. Units                | Text           |             |                                           |
    | Measurement    | Molarity (nM)              | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Number of Peaks found      | Numeric        |             | Decimal Places Displayed = 0              |
    | Measurement    | Number of Regions found    | Numeric        |             | Decimal Places Displayed = 0              |
    | Measurement    | Peak 1 Conc.               | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Peak 1 Molarity            | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Peak 1 Size - bp           | Numeric        |             | Decimal Places Displayed = 0              |
    | Measurement    | Peak 2 Conc.               | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Peak 2 Molarity            | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Peak 2 Size - bp           | Numeric        |             | Decimal Places Displayed = 0              |
    | Measurement    | Peak 3 Conc.               | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Peak 3 Molarity            | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Peak 3 Size - bp           | Numeric        |             | Decimal Places Displayed = 0              |
    | Measurement    | Peak 4 Conc.               | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Peak 4 Molarity            | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Peak 4 Size - bp           | Numeric        |             | Decimal Places Displayed = 0              |
    | Measurement    | Peak 5 Conc.               | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Peak 5 Molarity            | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Peak 5 Size - bp           | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Region 1 Average Size - bp | Numeric        |             | Decimal Places Displayed = 0              |
    | Measurement    | Region 1 Conc.             | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Region 1 Molarity          | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Region 2 Average Size - bp | Numeric        |             | Decimal Places Displayed = 0              |
    | Measurement    | Region 2 Conc.             | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Region 2 Molarity          | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Region 3 Average Size - bp | Numeric        |             | Decimal Places Displayed = 0              |
    | Measurement    | Region 3 Conc.             | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Region 3 Molarity          | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Region 4 Average Size - bp | Numeric        |             | Decimal Places Displayed = 0              |
    | Measurement    | Region 4 Conc.             | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Region 4 Molarity          | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Region 5 Average Size - bp | Numeric        |             | Decimal Places Displayed = 0              |
    | Measurement    | Region 5 Conc.             | Numeric        |             | Decimal Places Displayed = 2              |
    | Measurement    | Region 5 Molarity          | Numeric        |             | Decimal Places Displayed = 2              |

### Step 5: Normalize Libraries (Nextera XT DNA v2.0)

* Master Step Name = Normalize Libraries 3 v2.0.10
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {SubmittedSampleName}
* Reagent Kits
  * Nextera XT DNA Library Prep Kit, Box 1
    * Supplier = Illumina
    * Catalog Number = FC-131-1096
  * Nextera XT DNA Library Prep Kit, Box 1
    * Supplier = Illumina
    * Catalog Number = FC-131-1096
    * Website = [www.illumina.com](https://www.illumina.com)

{% hint style="info" %}
The version of Normalized Libraries 3 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-46951c6338593de894becd95aae2550eed53c307%2Fnextera-xt-dna-step5-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<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 name, workflow version and step version for the Sequencing Instrument may vary depending on the version of the IPP.

</details>

#### Queue/Ice Bucket

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

    | **Category**   | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
    | -------------- | -------------- | -------------- | ----------- | ----------------------------------------- |
    | Container      | Container Name | Built-in       |             |                                           |
    | Container      | 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 |             |                                           |
  | 0.1 N NaOH Prep Date | Date           |             |                                           |
* 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       |                |                                                                                                                                                                                                      |
    | Container      | LIMS ID (Container)      | Built-in       |                |                                                                                                                                                                                                      |
    | Container      | Well                     | Built-in       |                |                                                                                                                                                                                                      |
    | Derived Sample | Normalized Molarity (nM) | Numeric        |                | Decimal Places Displayed = 2                                                                                                                                                                         |
    | Derived Sample | Sample Name              | Built-in       |                |                                                                                                                                                                                                      |
    | 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.