# Standard v1.1

## Overview

The Standard Workflow AmpliSeq for Illumina BRCA Panel is part of the preconfigured AmpliSeq for Illumina BRCA Panel protocols supporting the preparation of uniquely indexed libraries of genomic DNA using the AmpliSeq for Illumina workflow.

This protocol includes the following:

* Preconfigured AmpliSeq for Illumina BRCA Panel protocol that supports the preparation of uniquely indexed libraries of genomic DNA using the AmpliSeq for Illumina workflow.
* Automated calculation of sample and buffer volumes.
* Automated calculation or display of reagents at every step in the protocol.
* Automatic step transition when required.
* Automatic placement of samples when necessary.
* Automated assignment of QC Pass/Fail, based on user-selected threshold values.
* A routing script that allows sequencing of libraries using any Illumina sequencing instrument.

## Protocol 1: Standard Workflow (AmpliSeq for Illumina BRCA Panel v1.1)

Protocol Type = Library Prep

**Next Steps Configuration**

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-317e522c1d7a6df5cd059aa1200d0653c1388ed4%2Fampliseq-brca-std-next-step-config.png?alt=media" alt=""><figcaption></figcaption></figure>

### Step 1: Clean Up Library (AmpliSeq for Illumina BRCA Panel v1.1)

* Master Step Name = Clean Up Library (AmpliSeq for Illumina v1.1)
* Step Type = No Outputs
* Reagent Kits
  * Agencourt AMPure XP Beads
    * Supplier = Thermo
    * Catalog Number = NC9959336; NC9933872

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-d26a97bf3c6b89b9a78c2d286e3c03de3e7c3a8a%2Fampliseq-brca-std-step1-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      | LIMS ID (Container) | Built-in       |             |                                           |
  | Container      | Well                | Built-in       |             |                                           |
  | Derived Sample | Sample Name         | Built-in       |             |                                           |
  | Derived Sample | Waiting             | Built-in       |             |                                           |
  | Project        | Project Name        | Built-in       |             |                                           |

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**     | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | ------------------ | -------------- | ----------- | ----------------------------------------- |
  | 70% EtOH Prep Date | Date           |             |                                           |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Expand
  * Well Sort Order = Row
  * Table Columns - Global Fields

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

### Step 2: Amplify Library (AmpliSeq for Illumina BRCA Panel v1.1)

* Master Step Name = Amplify Library (AmpliSeq for Illumina v1.1)
* Step Type = No Outputs
* Reagent Kits
  * Agencourt AMPure XP Beads
    * Supplier = Thermo
    * Catalog Number = NC9959336; NC9933872
  * AmpliSeq Library PLUS for Illumina
    * Supplier = Illumina
    * Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103
    * Website = [www.illumina.com](https://www.illumina.com)

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-f3383c7feac298c129c7d7ba660ac0ff2c17e518%2Fampliseq-brca-std-step2-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

<details>

<summary>Count Samples and Calculate Master Mix</summary>

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

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'step.::Total Samples:: = step.::Total Samples:: + 1' \
      -log {compoundOutputFileLuid0} && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'step.::1X Lib AMP Mix (uL):: = step.::Total Samples:: * 1.1 * 45 ; \
step.::10X Library Amp Primers (uL):: = step.::Total Samples:: * 1.1 * 5' \
      -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

<details>

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

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

{% code overflow="wrap" %}

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

{% endcode %}

</details>

<details>

<summary>Calculate Total Samples and Calculate Master Mix</summary>

* Trigger Location = Not Used

{% code overflow="wrap" %}

```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \ script:evaluateDynamicExpression \ -t false \ -h false \ -exp 'step.::Total Samples:: = step.::Total Samples:: + 1' \ -log {compoundOutputFileLuid0} && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \ script:evaluateDynamicExpression \ -t false \ -h false \ -exp 'step.::1X Lib Amp Mix:: = step.::Total Samples:: * 1.1 * 45 ; \ step.::10X Library Amp Primers:: = step.::Total Samples:: * 1.1 * 5' \ -log {compoundOutputFileLuid0}"
```

{% endcode %}

</details>

#### Queue/Ice Bucket

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

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

#### Record Details

**Group of Defaults**

<details>

<summary>AmpliSeq for CHS V2</summary>

* Thermal Cycler Program = AMP

</details>

<details>

<summary>Equalizer Workflow</summary>

* Thermal Cycler Program = EQUAL

</details>

<details>

<summary>Standard Workflow</summary>

* Thermal Cycler Program = AMP\_7

</details>

* Step Data
  * Group of Defaults = Standard Workflow
  * Master Step Fields

    | **Field Name**               | **Field Type** | **Options**    | **Additional Options and Dropdown Items**                        |
    | ---------------------------- | -------------- | -------------- | ---------------------------------------------------------------- |
    | 1X Lib AMP Mix (uL)          | Numeric        |                | Decimal Places Displayed = 2                                     |
    | 10X Library Amp Primers (uL) | Numeric        |                | Decimal Places Displayed = 2                                     |
    | Thermal Cycler Program       | Text Dropdown  | Custom Entries | <p>Presets</p><ul><li>AMP\_7</li><li>EQUAL</li><li>AMP</li></ul> |
    | Total Samples                | Numeric        |                | Default = 0                                                      |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Expand
  * Well Sort Order = Row
  * Table Columns - Global Fields

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

### Step 3: Perform Second Clean Up (AmpliSeq for Illumina BRCA Panel v1.1)

* Master Step Name = Perform Second Clean Up (AmpliSeq for Illumina v1.1)
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
  * Agencourt AMPure XP Beads
    * Supplier = Thermo
    * Catalog Number = NC9959336; NC9933872
  * AmpliSeq Library PLUS for Illumina
    * Supplier = Illumina
    * Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103
    * Website = [www.illumina.com](https://www.illumina.com)

#### Automations

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-8d00cfdff11cc2af2523953fd7ff116d6f3116e7%2Fampliseq-brca-std-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      | LIMS ID (Container) | Built-in       |             |                                           |
  | Container      | Well                | Built-in       |             |                                           |
  | Derived Sample | Sample Name         | Built-in       |             |                                           |
  | Derived Sample | Waiting             | Built-in       |             |                                           |
  | Project        | Project Name        | Built-in       |             |                                           |

#### Record Details

* Step Data (Master Step Fields)

  | **Field Name**     | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
  | ------------------ | -------------- | ----------- | ----------------------------------------- |
  | 70% EtOH Prep Date | Date           |             |                                           |
* Step File Placeholders
  * Log - Automatically attached
* Sample Table
  * Sample Display Default = Expand
  * Well Sort Order = Row
  * Table Columns - Global Fields

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