# MiniSeq v1.0 ## Overview The MiniSeq workflow includes the following functionality. * Preconfigured protocols that support the preparation of small genome, amplicon, targeted enrichment, and RNA sequencing using low library volumes for sequencing. * Automated calculation of sample and buffer volumes. * Automated calculation or display of reagents at every step in the protocol. * Automatic step transition when required. * Automatic placement of samples when necessary. * Automated assignment of QC Pass/Fail, based on user-selected threshold values. ## Protocol 1: Select Normalization Variation (MiniSeq v1.0) Protocol Type = Sequencing **Next Steps Configuration**
### Step 1: Select Normalization Variation (MiniSeq v1.0) * Master Step Name = Select Normalization Variation (MiniSeq v1.0) * Step Type = No Outputs #### Automations
Set Next Step - Remove * 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 %}
Routing Script for Normalization Variation * 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 'Normalization Variation' \ --FIELD_VALUE 'Standard Normalization' \ --WORKFLOW 'MiniSeq v1.0' \ --STEP 'Create Normalized Library Pool (MiniSeq v1.0)' \ --INPUTS_OR_OUTPUTS 'INPUTS' \ \ --FIELD_NAME 'Normalization Variation' \ --FIELD_VALUE 'Bead-Based Normalization' \ --WORKFLOW 'MiniSeq v1.0' \ --STEP 'Dilute Library Pool to Loading Concentration and Denature - Bead Based (MiniSeq v1.0)' \ --INPUTS_OR_OUTPUTS 'INPUTS' \ \ --FIELD_NAME 'Normalization Variation' \ --FIELD_VALUE 'AmpliSeq for Illumina Normalization' \ --WORKFLOW 'MiniSeq v1.0' \ --STEP 'Dilute Libraries - AmpliSeq (MiniSeq v1.0)' \ --INPUTS_OR_OUTPUTS 'INPUTS' \ \ --FIELD_NAME 'Normalization Variation' \ --FIELD_VALUE 'AmpliSeq Library Equalizer for Illumina Normalization' \ --WORKFLOW 'MiniSeq v1.0' \ --STEP 'Pool Libraries - AmpliSeq Equalizer (MiniSeq v1.0)' \ --INPUTS_OR_OUTPUTS 'INPUTS' " ``` {% endcode %} > ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.
Set Next Step - Advance * Trigger Location = Not Used {% code overflow="wrap" %} ```markup bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \ script:evaluateDynamicExpression \ -t false \ -h false \ -exp 'nextStep = ::ADVANCE::' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}
#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column * Sample Table (Column Headers) | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ----------------- | -------------- | ----------- | -------------------------------------------------------------------- | | Comment | Multiline Text | | | | Instruction Notes | Multiline Text | Read Only | Default = Select a Normalization Variation protocol for each sample. | * Step File Placeholders * Log - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Column * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ----------------------- | -------------- | -------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Normalization Variation | Text Dropdown | Required Field | | | Project | Project Name | Built-in | | | ## Protocol 2: Standard Normalization (MiniSeq v1.0) Protocol Type = Sequencing **Next Steps Configuration**
### Step 1: Create Normalized Library Pool (MiniSeq v1.0) * Master Step Name = Create Normalized Library Pool (MiniSeq v1.0) * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * Resuspension Buffer (RSB) #### Automations
Calculate Library & RSB Volumes - MiniSeq * 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.::Library (uL):: = (step.::Final Molarity (nM):: * step.::Final Volume (uL)::) / output.::Library Molarity (nM):: ; \ output.::RSB (uL):: = step.::Final Volume (uL):: - output.::Library (uL):: ; output.::Final Molarity (nM):: = step.::Final Molarity (nM)::' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}
Set Next Step - Advance * 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 %}
#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column * Sample Table (Column Headers) | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------------- | -------------- | ----------- | ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | Final Molarity (nM) | Numeric | | | | Final Volume (uL) | Numeric | | | | Instruction Notes | Multiline Text | Read Only |

Default =
- Enter in Final Molarity (nM), Final Volume (uL) and Library Molarity (nM) before clicking on calculation button.
- Add 10 uL of each 10 nM library to a new microcentrifuge tube.

| * Step File Placeholders * Log - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Column * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | --------------------- | -------------- | -------------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Final Molarity (nM) | Numeric | | | | Derived Sample | Library Molarity (nM) | Numeric | Required Field | | | Derived Sample | Library (uL) | Numeric | | Decimal Places Displayed = 0 | | Derived Sample | RSB (uL) | Numeric | | Decimal Places Displayed = 0 | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 2: Dilute Library Pool (MiniSeq v1.0) * Master Step Name = Dilute Library Pool (MiniSeq v1.0) * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * Resuspension Buffer (RSB) #### Automations
Copy Library Pool Molarity * Trigger Location = Record Details * Trigger Style = Automatic upon entry {% code overflow="wrap" %} ```markup bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2:http} -u {username} -p {password} script:evaluateDynamicExpression -t true -h false \ -exp 'output.::Library Pool (nM):: = input.::Final Molarity (nM)::' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}
Calculate Library Pool & RSB Volumes - MiniSeq * 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.::Library Pool (uL):: = (step.::Final Molarity (nM):: * step.::Final Volume (uL)::) / output.::Library Pool (nM):: ; \ output.::RSB (uL):: = step.::Final Volume (uL):: - output.::Library Pool (uL)::' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}
Set Next Step - Advance * 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 %}
#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column * Sample Table (Column Headers) | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------------- | -------------- | ----------- | ---------------------------------------------------------------------------------------------------- | | Final Molarity (nM) | Numeric | | Default = 1 | | Final Volume (uL) | Numeric | | Default = 100 | | Instruction Notes | Multiline Text | Read Only | Default = Enter in Final Molarity (nM), and Final Volume (uL) before clicking on calculation button. | * Step File Placeholders * Log - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Column * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Library Pool (nM) | Numeric | | Decimal Places Displayed = 0 | | Derived Sample | Library Pool (uL) | Numeric | | Decimal Places Displayed = 0 | | Derived Sample | RSB (uL) | Numeric | | Decimal Places Displayed = 0 | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 3: Denature Library Pool (MiniSeq v1.0) * Master Step Name = Denature Library Pool (MiniSeq v1.0) * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * 0.1 N NaOH * 200 mM Tris-HCl, pH 7.0 #### Automations
Set Next Step - Advance * 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 %}
#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column * Sample Table (Column Headers) | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ----------------- | -------------- | ----------- | ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | Instruction Notes | Multiline Text | Read Only |

Default =
- Combine 5 uL of 1 nM Library Pool and 5 uL of 0.1 N NaOH in a microcentridge tube.
- Vortex briefly and then centrifuge at 280 × g for 1 minute.
- Incubate at room temperature for 5 minutes.
- Add 5 µl 200 mM Tris-HCl, pH 7.0. Vortex briefly and then centrifuge at 280 × g for 1 minute.

| * Step File Placeholders * Log - Automatically attached * Sample Table * Sample Display Default = Collapse * Well Sort Order = Column * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 4: Dilute to Loading Concentration (MiniSeq v1.0) * Master Step Name = Dilute to Loading Concentration (MiniSeq v1.0) * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * Hybridization Buffer * Supplier = Illumina; part of MiniSeq High Output Reagent Kit * Catalog Number = 75 cycles - FC-420-1001; 150 cycles - FC-420-1002; 300 cycles - FC-420-1003 * Website = [www.illumina.com](https://www.illumina.com) #### Automations
Set Next Step - Advance * 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 %}
Routing Script for MiniSeq * Trigger Location = Step * 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 -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputLuid0} script:changeWorkflow \ \ --FIELD_NAME 'Add PhiX Control' \ --FIELD_VALUE 'No' \ --WORKFLOW 'MiniSeq v1.0' \ --STEP 'Run Setup (MiniSeq v1.0)' \ --INPUTS_OR_OUTPUTS 'OUTPUTS' \ \ --FIELD_NAME 'Add PhiX Control' \ --FIELD_VALUE 'Yes' \ --WORKFLOW 'MiniSeq v1.0' \ --STEP 'Dilute Stock PhiX (MiniSeq v1.0)' \ --INPUTS_OR_OUTPUTS 'OUTPUTS'" ``` {% endcode %} > ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.
#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column * Sample Table (Column Headers) | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------------------- | -------------- | -------------- | ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | Add PhiX Control | Text Dropdown | Required Field | | | Instruction Notes | Multiline Text | Read Only | | | Loading Concentration (pM) | Numeric | | | | Total Volume (uL) | Numeric | | | * Step File Placeholders * Log - Automatically attached * Sample Table * Sample Display Default = Collapse * Well Sort Order = Column * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ## Protocol 3: Bead-Based Normalization (MiniSeq v1.0) Protocol Type = Sequencing **Next Steps Configuration**
### Step 1: Dilute Library Pool to Loading Concentration and Denature - Bead Based (MiniSeq v1.0) * Master Step Name = Dilute Library Pool to Loading Concentration and Denature - Bead Based (MiniSeq v1.0) * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * Hybridization Buffer * Supplier = Illumina; part of MiniSeq High Output Reagent Kit * Catalog Number = 75 cycles - FC-420-1001; 150 cycles - FC-420-1002; 300 cycles - FC-420-1003 * Website = [www.illumina.com](https://www.illumina.com) #### Automations
Calculate Prechilled Hybridization Buffer Volumes - MiniSeq * 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.::Prechilled Hybridization Buffer (uL):: = output.::Final Volume (uL):: - output.::Library Pool (uL)::' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}
Set Next Step - Advance * 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 %}
Routing Script for MiniSeq * Trigger Location = Step * 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 -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputLuid0} script:changeWorkflow \ \ --FIELD_NAME 'Add PhiX Control' \ --FIELD_VALUE 'No' \ --WORKFLOW 'MiniSeq v1.0' \ --STEP 'Run Setup (MiniSeq v1.0)' \ --INPUTS_OR_OUTPUTS 'OUTPUTS' \ \ --FIELD_NAME 'Add PhiX Control' \ --FIELD_VALUE 'Yes' \ --WORKFLOW 'MiniSeq v1.0' \ --STEP 'Dilute Stock PhiX (MiniSeq v1.0)' \ --INPUTS_OR_OUTPUTS 'OUTPUTS'" ``` {% endcode %} > ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.
#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column * Sample Table (Column Headers) | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ----------------- | -------------- | -------------- | -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | Add PhiX Control | Text Dropdown | Required Field | | | Instruction Notes | Multiline Text | Read Only |

Default =
- Enter in Library Pool and Final Volume before clicking on calculation button.
- Vortex briefly and then centrifuge at 280 × g for 1 minute.
- Transfer 250 µl diluted library to a new microcentrifuge tube.
- Add 250 µl prechilled Hybridization Buffer.
- Vortex briefly and then centrifuge at 280 × g for 1 minute.
- To denture the diluted library pool, place the tube in the incubator for 2 minutes. Immediately cool on ice and leave it on ice for 5 minutes.

| * Step File Placeholders * Log - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Column * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------------------------ | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Final Volume (uL) | Numeric | | Decimal Places Displayed = 0 | | Derived Sample | Library Pool (uL) | Numeric | | Decimal Places Displayed = 0 | | Derived Sample | Prechilled Hybridization Buffer (uL) | Numeric | | Decimal Places Displayed = 0 | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ## Protocol 4: AmpliSeq for Illumina Normalization (MiniSeq v1.0) Protocol Type = Sequencing **Next Steps Configuration**
### Step 1: Dilute Libraries - AmpliSeq (MiniSeq v1.0) * Master Step Name = Dilute Libraries - AmpliSeq (MiniSeq v1.0) * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * Low TE * Supplier = Supplier - Illumina; part of AmpliSeq Library Plus Kit * Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103 * Website = [www.illumina.com](https://www.illumina.com) #### Automations
Copy Final Molarity * 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.::Final Molarity (nM):: = step.::Final Molarity (nM)::' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}
Calculate Starting Molarity and Library & Low TE Volumes - MiniSeq * Trigger Location = Record Details * Trigger Style = Manual button {% code overflow="wrap" %} ```markup bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2:http} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false \ -exp 'output.::Starting Molarity (nM):: = (output.::Concentration:: * 1000000) / (660 * output.::Average Library Size (bp)::) ; \ output.::Library (uL):: = (output.::Final Molarity (nM):: * step.::Final Volume (uL)::) / output.::Starting Molarity (nM):: ; \ output.::Low TE (uL):: = step.::Final Volume (uL):: - output.::Library (uL)::' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}
Set Next Step - Advance * 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 %}
#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column * Sample Table (Column Headers) | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------------- | -------------- | ----------- | -------------------------------------------------------------------------------------------------------- | | Final Molarity (nM) | Numeric | | Default = 2 | | Final Volume (uL) | Numeric | | Default = 50 | | Instruction Notes | Multiline Text | Read Only | Default = Enter in the concentration and average library size before clicking on the calculation button. | * Step File Placeholders * Log - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Column * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------------- | -------------- | -------------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Average Library Size (bp) | Numeric | | Default = 350 | | Derived Sample | Concentration | Numeric | Required Field | Decimal Places Displayed = 2 | | Derived Sample | Conc. Units | Text | Required Field | | | Derived Sample | Final Molarity (nM) | Numeric | | | | Derived Sample | Library (uL) | Numeric | | Decimal Places Displayed = 0 | | Derived Sample | Low TE (uL) | Numeric | | Decimal Places Displayed = 2 | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Starting Molarity (nM) | Numeric | | Decimal Places Displayed = 0 | | Project | Project Name | Built-in | | | ### Step 2: Pool Libraries - AmpliSeq (MiniSeq v1.0) * Master Step Name = Pool Libraries - AmpliSeq (MiniSeq v1.0) * Step Type = Pooling * Aliquot Generation = Fixed, 1 * Naming Convention = {PoolName} * Reagent Kits * Low TE * Supplier = Supplier - Illumina; part of AmpliSeq Library Plus Kit * Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103 * Website = [www.illumina.com](https://www.illumina.com) #### Automations
Copy Molarity - MiniSeq * 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.::Molarity (nM):: = input.::Final Molarity (nM)::' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}
Calculate DNA to RNA - MiniSeq * 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 'if (step.::AmpliSeq for Illumina Panel:: == ::Myeloid Panel::) {step.::DNA to RNA Ratio:: = ::8:1::} ; \ if (step.::AmpliSeq for Illumina Panel:: == ::Childhood Cancer Panel::) {step.::DNA to RNA Ratio:: = ::5:1::} ; \ if (step.::AmpliSeq for Illumina Panel:: == ::Focus Panel::) {step.::DNA to RNA Ratio:: = ::7:3::} ; \ if (step.::AmpliSeq for Illumina Panel:: == ::Comprehensive Panel v3::) {step.::DNA to RNA Ratio:: = ::25:1::}' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}
Set Next Step - Advance * 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 %}
#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column * Sample Table (Column Headers) | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | | Project | Project Name | Built-in | | | #### Pooling * Label Uniqueness = On * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ---------------------------- | -------------- | -------------- | -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | AmpliSeq for Illumina Panel | Text Dropdown | Required Field | | | DNA to RNA Ratio | Text | | | | Instruction Notes | Multiline Text | Read Only |

Default =
- Select the AmpliSeq for Illumina Panel before clicking on Calculate DNA to RNA ratio button.
- Combine equal library volumes to a 1.5 mL LoBind tube.
- User different tubes for DNA and RNA Libraries.
- Combine DNA to RNA ratio in a new tube.

| | Transfer Library Volume (uL) | Numeric | | | * Step File Placeholders * Log - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Column * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Molarity (nM) | Numeric | | Decimal Places Displayed = 2 | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 3: Denature Libraries - AmpliSeq (MiniSeq v1.0) * Master Step Name = Denature Libraries - AmpliSeq (MiniSeq v1.0) * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * Low TE * Supplier = Supplier - Illumina; part of AmpliSeq Library Plus Kit * Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103 * Website = [www.illumina.com](https://www.illumina.com) #### Automations
Copy Molarity - MiniSeq * 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.::Molarity (nM):: = input.::Molarity (nM)::' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}
Set Next Step - Advance * 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 %}
#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column * Sample Table (Column Headers) | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ----------------------- | -------------- | ----------- | ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | 0.2 N NaOH | Numeric | | Default = 10 | | 0.2 N NaOH Prep Date | Date | | | | 200 mM Tris-HCl, pH 7.0 | Numeric | | Default = 10 | | Instruction Notes | Multiline Text | Read Only |

Default =
- Combine Pool Libraries and 0.2 N NaOH volumes.
- Vortex briefly. Incubate at room temp for 5 mins.
- Add 10 µl 200 mM Tris-HCl, pH 7.0 to the tube. Vortex briefly.

| | Pool Libraries (uL) | Numeric | | Default = 10 | * Step File Placeholders * Log - Automatically attached * Sample Table * Sample Display Default = Collapse * Well Sort Order = Column * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Molarity (nM) | Numeric | | Decimal Places Displayed = 2 | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 4: Dilute Denatured Libraries to Loading Concentration - AmpliSeq (MiniSeq v1.0) * Master Step Name = Dilute Denatured Libraries to Loading Concentration - AmpliSeq (MiniSeq v1.0) * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * HT1 * Supplier = Illumina; part of AmpliSeq Library Plus Kit * Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103 Website - [www.illumina.com](http://www.illumina.com) * Website = [www.illumina.com](https://www.illumina.com) #### Automations
Calculate Diluted Denatured Libraries & Prechilled HT1 Volumes - MiniSeq * 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 'output.::Diluted Denatured Libraries (uL):: = (step.::Loading Concentration (pM):: * step.::Final Volume (uL)::) / output.::Diluted Denatured Libraries (pM):: ; \ output.::Prechilled HT1(uL):: = step.::Final Volume (uL):: - output.::Diluted Denatured Libraries (uL)::' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}
Set Next Step - Advance * 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 %}
Routing Script for MiniSeq * Trigger Location = Step * 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 -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputLuid0} script:changeWorkflow \ \ --FIELD_NAME 'Add PhiX Control' \ --FIELD_VALUE 'No' \ --WORKFLOW 'MiniSeq v1.0' \ --STEP 'Run Setup (MiniSeq v1.0)' \ --INPUTS_OR_OUTPUTS 'OUTPUTS' \ \ --FIELD_NAME 'Add PhiX Control' \ --FIELD_VALUE 'Yes' \ --WORKFLOW 'MiniSeq v1.0' \ --STEP 'Dilute Stock PhiX (MiniSeq v1.0)' \ --INPUTS_OR_OUTPUTS 'OUTPUTS'" ``` {% endcode %} > ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.
#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column * Sample Table (Column Headers) | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------------------- | -------------- | -------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ | | 200 mM Tris-HCl, pH 7.0 | Numeric | | | | Add PhiX Control | Text Dropdown | Custom Entries | | | Final Volume (uL) | Numeric | | | | Instruction Notes | Multiline Text | Read Only | | | Loading Concentration (pM) | Numeric | | | * Step File Placeholders * Log - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Column * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | -------------------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Diluted Denatured Libraries (pM) | Numeric | | Default = 20 | | Derived Sample | Diluted Denatured Libraries (uL) | Numeric | | Decimal Places Displayed = 0 | | Derived Sample | Prechilled HT1(uL) | Numeric | | Decimal Places Displayed = 0 | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ## Protocol 5: AmpliSeq Library Equalizer for Illumina Normalization (MiniSeq v1.0) Protocol Type = Sequencing **Next Steps Configuration**
### Step 1: Pool Libraries - AmpliSeq Equalizer (MiniSeq v1.0) * Master Step Name = Pool Libraries - AmpliSeq (MiniSeq v1.0) * Step Type = Pooling * Aliquot Generation = Fixed, 1 * Naming Convention = {PoolName} * Reagent Kits * Low TE * Supplier = Supplier - Illumina; part of AmpliSeq Library Plus Kit * Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103 * Website = [www.illumina.com](https://www.illumina.com) #### Automations
Copy Molarity - MiniSeq * 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.::Molarity (nM):: = input.::Final Molarity (nM)::' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}
Calculate DNA to RNA - MiniSeq * 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 'if (step.::AmpliSeq for Illumina Panel:: == ::Myeloid Panel::) {step.::DNA to RNA Ratio:: = ::8:1::} ; \ if (step.::AmpliSeq for Illumina Panel:: == ::Childhood Cancer Panel::) {step.::DNA to RNA Ratio:: = ::5:1::} ; \ if (step.::AmpliSeq for Illumina Panel:: == ::Focus Panel::) {step.::DNA to RNA Ratio:: = ::7:3::} ; \ if (step.::AmpliSeq for Illumina Panel:: == ::Comprehensive Panel v3::) {step.::DNA to RNA Ratio:: = ::25:1::}' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}
Set Next Step - Advance * 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 %}
#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column * Sample Table (Column Headers) | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | | Project | Project Name | Built-in | | | #### Pooling * Label Uniqueness = On * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ---------------------------- | -------------- | -------------- | -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | AmpliSeq for Illumina Panel | Text Dropdown | Required Field | | | DNA to RNA Ratio | Text | | | | Instruction Notes | Multiline Text | Read Only |

Default =
- Select the AmpliSeq for Illumina Panel before clicking on Calculate DNA to RNA ratio button.
- Combine equal library volumes to a 1.5 mL LoBind tube.
- User different tubes for DNA and RNA Libraries.
- Combine DNA to RNA ratio in a new tube.

| | Transfer Library Volume (uL) | Numeric | | | * Step File Placeholders * Log - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Column * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Molarity (nM) | Numeric | | Decimal Places Displayed = 2 | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 2: Denature Libraries - AmpliSeq Equalizer (MiniSeq v1.0) * Master Step Name = Denature Libraries - AmpliSeq (MiniSeq v1.0) * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * Low TE * Supplier = Supplier - Illumina; part of AmpliSeq Library Plus Kit * Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103 * Website = [www.illumina.com](https://www.illumina.com) #### Automations
Copy Molarity - MiniSeq * 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.::Molarity (nM):: = input.::Molarity (nM)::' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}
Set Next Step - Advance * 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 %}
#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column * Sample Table (Column Headers) | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ----------------------- | -------------- | ----------- | ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | 0.2 N NaOH | Numeric | | Default = 10 | | 0.2 N NaOH Prep Date | Date | | | | 200 mM Tris-HCl, pH 7.0 | Numeric | | Default = 10 | | Instruction Notes | Multiline Text | Read Only |

Default =
- Combine Pool Libraries and 0.2 N NaOH volumes.
- Vortex briefly. Incubate at room temp for 5 mins.
- Add 10 µl 200 mM Tris-HCl, pH 7.0 to the tube. Vortex briefly.

| | Pool Libraries (uL) | Numeric | | Default = 10 | * Step File Placeholders * Log - Automatically attached * Sample Table * Sample Display Default = Collapse * Well Sort Order = Column * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Molarity (nM) | Numeric | | Decimal Places Displayed = 2 | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 3: Dilute Denatured Libraries to Loading Concentration - AmpliSeq Equalizer (MiniSeq v1.0) * Master Step Name = Dilute Denatured Libraries to Loading Concentration - AmpliSeq (MiniSeq v1.0) * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * HT1 * Supplier = Illumina; part of AmpliSeq Library Plus Kit * Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103 Website - [www.illumina.com](http://www.illumina.com) * Website = [www.illumina.com](https://www.illumina.com) #### Automations
Calculate Diluted Denatured Libraries & Prechilled HT1 Volumes - MiniSeq * 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 'output.::Diluted Denatured Libraries (uL):: = (step.::Loading Concentration (pM):: * step.::Final Volume (uL)::) / output.::Diluted Denatured Libraries (pM):: ; \ output.::Prechilled HT1(uL):: = step.::Final Volume (uL):: - output.::Diluted Denatured Libraries (uL)::' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}
Set Next Step - Advance * 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 %}
Routing Script for MiniSeq * Trigger Location = Step * 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 -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputLuid0} script:changeWorkflow \ \ --FIELD_NAME 'Add PhiX Control' \ --FIELD_VALUE 'No' \ --WORKFLOW 'MiniSeq v1.0' \ --STEP 'Run Setup (MiniSeq v1.0)' \ --INPUTS_OR_OUTPUTS 'OUTPUTS' \ \ --FIELD_NAME 'Add PhiX Control' \ --FIELD_VALUE 'Yes' \ --WORKFLOW 'MiniSeq v1.0' \ --STEP 'Dilute Stock PhiX (MiniSeq v1.0)' \ --INPUTS_OR_OUTPUTS 'OUTPUTS'" ``` {% endcode %} > ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.
#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column * Sample Table (Column Headers) | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------------------- | -------------- | -------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ | | 200 mM Tris-HCl, pH 7.0 | Numeric | | | | Add PhiX Control | Text Dropdown | Custom Entries | | | Final Volume (uL) | Numeric | | | | Instruction Notes | Multiline Text | Read Only | | | Loading Concentration (pM) | Numeric | | | * Step File Placeholders * Log - Automatically attached * Sample Table * Sample Display Default = Expand * Well Sort Order = Column * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | -------------------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Diluted Denatured Libraries (pM) | Numeric | | Default = 20 | | Derived Sample | Diluted Denatured Libraries (uL) | Numeric | | Decimal Places Displayed = 0 | | Derived Sample | Prechilled HT1(uL) | Numeric | | Decimal Places Displayed = 0 | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ## Protocol 6: Add PhiX Control (MiniSeq v1.0) Protocol Type = Sequencing **Next Steps Configuration**
### Step 1: Dilute Stock PhiX (MiniSeq v1.0) * Master Step Name = Dilute Stock PhiX (MiniSeq v1.0) * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * Resuspension Buffer (RSB) * Control Types * PhiX v3 * Supplier = Illumina * Catalog Number = FC-110-3001 * Website = #### Automations
Calculate Stock PhiX and RSB Volumes * 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.::Stock PhiX Volume (uL):: = (step.::Final PhiX Molarity (nM):: * step.::Final PhiX Volume (uL)::) / step.::Stock PhiX Molarity (nM):: ; \ step.::RSB (uL):: = step.::Final PhiX Volume (uL):: - step.::Stock PhiX Volume (uL)::' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}
Set Next Step - Advance * 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 %}
#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column * Sample Table (Column Headers) | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------------------ | -------------- | ----------- | ----------------------------------------------------------------------------------------------------------------------- | | Final PhiX Molarity (nM) | Numeric | | Default = 4 | | Final PhiX Volume (uL) | Numeric | | Default = 25 | | Instruction Notes | Multiline Text | Read Only | Default = Enter Stock PhiX (nM), Final PhiX Molarity (nM), and Final Volume (uL) before clicking on calculation button. | | RSB (uL) | Numeric | | Decimal Places Displayed = 0 | | Stock PhiX Molarity (nM) | Numeric | | Default = 10 | | Stock PhiX Volume (uL) | Numeric | | Decimal Places Displayed = 0 | * Step File Placeholders * Log - Automatically attached * Sample Table * Sample Display Default = Collapse * Well Sort Order = Column * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 2: Denature PhiX (MiniSeq v1.0) * Master Step Name = Denature PhiX (MiniSeq v1.0) * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * 0.1 N NaOH * 200 mM Tris-HCl, pH 7.0 #### Automations
Set Next Step - Advance * 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 %}
#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column * Sample Table (Column Headers) | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | | Project | Project Name | Built-in | | | #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ----------------- | -------------- | ----------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ | | Instruction Notes | Multiline Text | Read Only |

Default =
- Combine 5uL of 4 nM PhiX and 5 uL of 0.1 N NaOH.
- Vortex briefly and then pulse centrifuge.
- Incubate at room temperature for 5 minutes.
- Add 5 µl 200 mM Tris-HCl, pH 7.0.
- Vortex briefly and then centrifuge at 280 × g for 1 minute.

| * Step File Placeholders * Log - Automatically attached * Sample Table * Sample Display Default = Collapse * Well Sort Order = Column * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ### Step 3: Dilute PhiX to Loading Concentration and Combine Library & PhiX (MiniSeq v1.0) * Master Step Name = Dilute PhiX to Loading Concentration and Combine Library & PhiX (MiniSeq v1.0) * Step Type = Pooling * Aliquot Generation = Fixed, 1 * Naming Convention = {PoolName} * Reagent Kits * Hybridization Buffer \* Supplier = Illumina; part of MiniSeq High Output Reagent Kit \* Catalog Number = 75 cycles - FC-420-1001; 150 cycles - FC-420-1002; 300 cycles - FC-420-1003 \* Website = [www.illumina.com](https://www.illumina.com) #### Automations
Calculate Denatured PhiX and Prechilled Hybridization Buffer Volumes * 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.::Denatured PhiX Volume (uL):: = (step.::Final Denatured PhiX Molarity (pM):: * step.::Final Volume (uL)::) / step.::Denatured PhiX Molarity (pM):: ; \ step.::Prechilled Hybridization Buffer (uL):: = step.::Final Volume (uL):: - step.::Denatured PhiX Volume (uL)::' \ -log {compoundOutputFileLuid0}" ``` {% endcode %}
Set Next Step - Advance * 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 %}
#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column * Sample Table (Column Headers) | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | | Project | Project Name | Built-in | | | #### Pooling * Label Uniqueness = On * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column #### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ------------------------------------ | -------------- | ----------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ | | Denatured PhiX Molarity (pM) | Numeric | | Default = 20 | | Denatured PhiX Volume (uL) | Numeric | | Decimal Places Displayed = 0 | | Final Denatured PhiX Molarity (pM) | Numeric | | Default = 1.8 | | Final Volume (uL) | Numeric | | Default = 500 | | Instruction Notes | Multiline Text | Read Only |

Default =
- Enter Denatured PhiX Molarity (pM), Final Denatured PhiX Molarity (pM) and Final Volume (uL) before clicking on calculation button.
- Add Denatured PhiX and Prechilled Hybridization Buffer volumes too dilute the denatured PhiX.
- Invert to mix and then centrifuge at 280 × g for 1 minute. - Combine 5 uL of denatured and diluted PhiX control with 500 uL of denatured and diluted library pool.

| | Prechilled Hybridization Buffer (uL) | Numeric | | Decimal Places Displayed = 0 | * Step File Placeholders * Log - Automatically attached * Sample Table * Sample Display Default = Collapse * Well Sort Order = Column * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | ## Protocol 7: Run Setup (MiniSeq v1.0) Protocol Type = Sequencing **Next Steps Configuration**
### Step 1: Run Setup (MiniSeq v1.0) * Master Step Name = Run Setup (MiniSeq v1.0) * Step Type = Standard * Derived Sample Generation = Fixed, 1 * Naming Convention = {InputItemName} * Reagent Kits * MiniSeq High Output Reagent Kit * Supplier = Illumina * Catalog Number = 75 cycles - FC-420-1001; 150 cycles - FC-420-1002; 300 cycles - FC-420-1003 * Website = [www.illumina.com](https://www.illumina.com) #### Automations
1. miniseq sample sheet * Trigger Location = Record Details * Trigger Style = Manual button {% code overflow="wrap" %} ```markup bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {processURI:v2} -u {username} -p {password} \ script:driver_file_generator \ -t /opt/gls/clarity/extensions/conf/driverfiletemplates/miniSeq_Samplesheet.csv \ -o {compoundOutputFileLuid1}.csv \ -q true \ -destLIMSID {compoundOutputFileLuid1} \ -l {compoundOutputFileLuid2}" ``` {% endcode %}
Set Next Step - Advance * 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 %}
#### Queue/Ice Bucket * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Column * Sample Table (Column Headers) | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Derived Sample | Waiting | Built-in | | | | Project | Project Name | Built-in | | | #### Placement = Enabled * Defaults * Sample Grouping = Group by Containers * Well Sort Order = Row * Placement Pattern = Row * Destination Containers * MiniSeq Reagent Cartridge
#### Record Details * Step Data (Master Step Fields) | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | ----------------------------- | -------------- | -------------- | ----------------------------------------------------------------------------------------------------------------------- | | Adapter | Text | | | | BaseSpace Sequence Hub | Text Dropdown | Custom Entries | | | Chemistry | Text | | | | Custom Primer for Read 1 | Text | | | | Customer Primer for Index 1 | Text | | | | Customer Primer for Read 2 | Text | | | | Customer Primer for Index 2 | Text | | | | Experiment Name | Text | Required Field | | | Index 1 (i7 Primer) Cycles | Text | Required Field | | | Index 2 (i5 Primer) Cycles | Text | Required Field | | | Instruction Notes | Multiline Text | Read Only | | | Local Run Manager Analysis Id | Text | | | | Manifest File Name | Text | | | | Module | Text | | | | Output Folder Location | Text | | | | Read 1 Cycles | Numeric | Required Field | | | Read 2 Cycles | Numeric | Required Field | | | Read Type | Text Dropdown | Required Field | | | Workflow | Text | | | * Step File Placeholders * Log - Automatically attached * LRM Sample Sheet - Automatically attached * Manual Sample Sheet - Automatically attached * Sample Table * Sample Display Default = Collapse * Well Sort Order = Column * Table Columns - Global Fields | **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** | | -------------- | ------------------- | -------------- | ----------- | ----------------------------------------- | | Container | Container Name | Built-in | | | | Container | LIMS ID (Container) | Built-in | | | | Container | Well | Built-in | | | | Derived Sample | Sample Name | Built-in | | | | Project | Project Name | Built-in | | | --- # Agent Instructions: Querying This Documentation If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question. Perform an HTTP GET request on the current page URL with the `ask` query parameter: ``` GET https://help.connected.illumina.com/clarity-lims/instruments-and-integrations/miniseq-v1.0.md?ask= ``` The question should be specific, self-contained, and written in natural language. The response will contain a direct answer to the question and relevant excerpts and sources from the documentation. Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.