| Decimal Places Displayed = 2 |
| Derived Sample | Conc. Units | Text |
Required Field
| |
| Derived Sample | Prep Input Type | Text | | |
| Derived Sample | Sample Name | Built-in | | |
| Derived Sample | Sample Type | Text Dropdown |
Required Field
Custom Entries
|
Presets
DNA
RNA
|
| Measurement | Concentration | Numeric | | Decimal Places Displayed = 2 |
| Measurement | Conc. Units | Text | | |
| Measurement | Prep Input Type | Text | | |
| Measurement | Sample Type | Text | | |
| Project | Project Name | Built-in | | |
### Step 4: Whole Blood Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Master Step Name = Whole Blood Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
* Illumina Lysis Reagent Kit
* Supplier = Illumina
* Catalog Number = 20042221
* Website = [www.illumina.com](https://www.illumina.com)
{% hint style="info" %}
The version of Whole Blood Lysis master step name may be different depending on the version of IPP installed.
{% endhint %}
#### Automations
Calculate Whole Blood Lysis Master Mix & Copy Sample Type
* 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 ; \
step.::MLB (uL):: = step.::Total Samples:: * 30 *1.1 ; \
step.:: PK1 (uL):: = step.::Total Samples:: * 2 * 1.1 ; \
step.:: Nuclease-free water (uL):: = step.::Total Samples:: * 248 * 1.1 ; \
output.::Sample Type:: = input.::Sample Type for DNA PCR-Free Lib Prep::' \
-log {compoundOutputFileLuid0}"
```
{% endcode %}
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 - Whole Blood Lysis
* 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 {compoundOutputFileLuid0} script:changeWorkflow \
\
--FIELD_NAME 'Sample Type' \
--FIELD_VALUE 'Whole Blood' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'Tagment Genomic DNA - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"
```
{% endcode %}
> ℹ The actual version of the workflow and step names for the routing script may be different depending on the version of IPP installed.
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 | | |
#### Placement = Enabled
* Defaults
* Sample Grouping = Group by Containers
* Well Sort Order = Column
* Placement Pattern = Column
* Destination Containers
* 96 well plate
#### Record Details
* Step Data (Master Step Fields)
| **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
| ------------------------ | -------------- | ----------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ |
| 80% EtOH Prep Date | Date | | |
| Instruction Notes | Multiline Text | Read Only |
Default = 1. Combine MLB, PK1 and Nuclease-free water to create the Lysis Master Mix in a 15 mL tube. 2. Add 280 µl of lysis master mix to a new 2 mL tube. 3. Add 20 uL to each 2 mL tube, mix by pipetting then vortex briefly and centrifuge. 4. Incubate and shake at 1000 rpm for 15 mins then centrifuge briefly. 5. Add 135 uL of IPB, invert tube, vortex for 15 secs to mix. 6. Incubate at RT for 5 mins. 7. Place tube on magnetic stand for 5 mins then remove and discard supernatant. 8. Wash beads 2x with 500 uL of fresh 80% EtOH for each wash. 9. Air dry on magnetic stand for 5 mins. 10. Add 35 uL of RSB to each tube and vortex to mix. 11. Label a new 96 well PCR plate with LP1 and add 32 uL of supernatant of each sample to a new well.
|
| MLB (uL) | Numeric | Read Only |
Decimal Places Displayed = 0
|
| Nuclease-free water (uL) | Numeric | Read Only |
|
| Project | Project Name | Built-in | | |
### Step 5: Dried Blood Spot Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Master Step Name = Dried Blood Spot Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
* Illumina Lysis Reagent Kit
* Supplier = Illumina
* Catalog Number = 20042221
* Website = [www.illumina.com](https://www.illumina.com)
{% hint style="info" %}
The version of Dried Blood Spot Lysis master step name may be different depending on the version of IPP installed.
{% endhint %}
#### Automations
Calculate Dried Blood Lysis Master Mix
* 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 false -h false \
-exp 'step.::Total Samples:: = step.::Total Samples:: + 1 ; \
step.::10x Lysis Buffer (uL):: = step.::Total Samples:: * 30 *1.1 ; \
step.:: PK1 (uL):: = step.::Total Samples:: * 2 * 1.1 ; \
step.:: Nuclease-free water (uL):: = step.::Total Samples:: * 268 * 1.1 ; \
output.::Sample Type:: = input.::Sample Type for DNA PCR-Free Lib Prep::' \
-log {compoundOutputFileLuid0}"
```
{% endcode %}
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 - Dried Blood Spot Lysis
* 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 {compoundOutputFileLuid0} script:changeWorkflow \
\
--FIELD_NAME 'Sample Type' \
--FIELD_VALUE 'Dried Blood Spot' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'Tagment Genomic DNA - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"
```
{% endcode %}
> ℹ The actual version of the workflow and step names for the routing script may be different depending on the version of IPP installed.
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 | | |
#### Placement = Enabled
* Defaults
* Sample Grouping = Group by Containers
* Well Sort Order = Column
* Placement Pattern = Column
* Destination Containers
* 96 well plate
#### Record Details
* Step Data (Master Step Fields)
| **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
| ------------------------ | -------------- | ----------- | ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| 10x Lysis Buffer (uL) | Numeric | Read Only |
Decimal Places Displayed = 0
|
| 80% EtOH Prep Date | Date | | |
| Instruction Notes | Multiline Text | Read Only |
Default = 1. Combine 10x Lysis Buffer, PK1 and Nuclease-free water to create the Lysis Master Mix in a 15 mL tube. 2. Add 300 uL of lysis master mix to a new 2 mL tube. 3. Add 6 x 3 mm² punches to a each 2 ml tube, mix by pipetting then vortex briefly and centrifuge. 4. Incubate and shake at 1000 rpm for 15 mins then centrifuge briefly. 5. Without removing the punches, transfer all supernatant from each tube to a new 2 ml tube. 6. Add 135 uL of IPB, invert tube, vortex for 15 secs to mix. 7. Incubate at RT for 5 mins. 8. Place tube on magnetic stand for 5 mins then remove and discard supernatant. 9. Wash beads 2x with 500 uL of fresh 80% EtOH for each wash. 10. Air dry on magnetic stand for 5 mins. 11. Add 35 uL of RSB to each tube and vortex to mix. 12. Incubate at RT for 2 mins then on the magnetic stand until the liquid turns clear. 13. Label a new 96 well PCR plate with LP1 and add 32 uL of supernatant of each sample to a new well.
|
| Nuclease-free water (uL) | Numeric | Read Only |
|
| Project | Project Name | Built-in | | |
### Step 6: Saliva Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Master Step Name = Saliva Lysis (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
* Illumina Lysis Reagent Kit
* Supplier = Illumina
* Catalog Number = 20042221
* Website = [www.illumina.com](https://www.illumina.com)
{% hint style="info" %}
The version of Saliva Lysis master step name may be different depending on the version of IPP installed.
{% endhint %}
#### Automations
Copy Sample Type for DNA PCR-Free Lib Prep
* 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.::Sample Type:: = input.::Sample Type for DNA PCR-Free Lib Prep:: ' \
-log {compoundOutputFileLuid0}"
```
{% endcode %}
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 - Saliva Lysis
* 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 {compoundOutputFileLuid0} script:changeWorkflow \
\
--FIELD_NAME 'Sample Type' \
--FIELD_VALUE 'Saliva' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'Tagment Genomic DNA - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"
```
{% endcode %}
> ℹ The actual version of the workflow and step names for the routing script may be different depending on the version of IPP installed.
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 | | |
#### Placement = Enabled
* Defaults
* Sample Grouping = Group by Containers
* Well Sort Order = Column
* Placement Pattern = Column
* Destination Containers
* 96 well plate
#### Record Details
* Step Data (Master Step Fields)
| **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
| ------------------ | -------------- | ----------- | ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| 80% EtOH Prep Date | Date | | |
| Instruction Notes | Multiline Text | Read Only |
Default = 1. Incubate saliva overnight at 50°C in a water bath or air incubator. 2. Add 250 uL of Nuclease-Free Water to a new 2 mL tube. 3. Invert each heat-treated saliva collection tube 5 times to mix, add 50 uL of Saliva to each 2 mL tube, pipette to mix, vortex briefly and then centrifuge. 4. Add 135 uL of IPB, invert tube, vortex for 15 secs to mix. 5. Incubate at RT for 5 mins. 6. Place tube on magnetic stand for 5 mins then remove and discard supernatant. 7. Wash beads 2x with 500 uL of fresh 80% EtOH for each wash. 8. Air dry on magnetic stand for 5 mins. 9. Add 35 uL of RSB to each tube and vortex to mix. 10. Incubate at RT for 2 mins then on the magnetic stand until the liquid turns clear. 11. Label a new 96 well PCR plate with LP1 and add 32 uL of supernatant of each sample to a new well.
|
| Project | Project Name | Built-in | | |
## Protocol 2: Library Prep - Hybex (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
Protocol Type = Library Prep
**Next Steps Configuration**
### Step 1: Tagment Genomic DNA (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Master Step Name = Tagment Genomic DNA (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
* Illumina DNA PCR-Free Library Prep
* Supplier = Illumina
* Catalog Number = 24 - 20041794; 96 - 20041795
* Website = [www.illumina.com](https://www.illumina.com)
{% hint style="info" %}
The version of Tagment Genomic DNA master step name may be different depending on the version of IPP installed.
{% endhint %}
#### Automations
Copy Concentration
* 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.::Concentration:: = input.::Concentration:: ; output.::Conc. Units:: = input.::Conc. Units::' \
-log {compoundOutputFileLuid0}"
```
{% endcode %}
Calculate DNA Volume & Copy Desired DNA Input
* 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.::DNA (uL):: = (step.::Desired DNA Input (ng):: / output.::Concentration::) ; \
output.::RSB (uL):: = (25 - output.::DNA (uL)::) ; \
output.::Desired DNA Input (ng):: = step.::Desired DNA Input (ng)::' \
-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 | | |
#### Placement = Enabled
* Defaults
* Sample Grouping = Group by Containers
* Well Sort Order = Column
* Placement Pattern = Column
* Destination Containers
* 96 well plate
#### Record Details
* Step Data (Master Step Fields)
| **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
| ---------------------- | -------------- | ----------- | --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| 80% EtOH Prep Date | Date | | |
| Desired DNA Input (ng) | Numeric | |
Range = 300 To 2000
Decimal Places Displayed = 0
|
| Instruction Notes | Multiline Text | Read Only |
Default = 1. Label a new 96 well MIDI plate with LP1 and add the calculated DNA and RSB volumes of each sample to a new well on the plate. 2. Add 10 uL of TB1 to each well. 3. Add 15 uL of BLT-PF to each well. 4. Seal and shake at 1800 rpm for 1 minute. 5. Incubate in pre-heated Hybex for 8 minutes.
|
| Derived Sample | DNA (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: Post Tagmentation Cleanup (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Master Step Name = Post Tagmentation Cleanup (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Step Type = No Outputs
* Reagent Kits
* Illumina DNA PCR-Free Library Prep
* Supplier = Illumina
* Catalog Number = 24 - 20041794; 96 - 20041795
* Website = [www.illumina.com](https://www.illumina.com)
{% hint style="info" %}
The version of Post Tagmentation Cleanup master step name may be different depending on the version of IPP installed.
{% endhint %}
#### 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 %}
Copy Desired DNA Input
* 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.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng)::' \
-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 = 1. Add 10 µl ST2 to each well then seal and shake at 1800 rpm for 1 minute. 2. Place on the magnetic stand and wait until the liquid is clear (\~2 minutes). 3. Without disturbing the bead pellet, remove and discard all supernatant from each well. 5. Add 150 μl TWB to each well then seal and shake at 1800 rpm for 1 minute. 6. Place on the magnetic stand and wait until the liquid is clear (\~2 minutes).
|
| Derived Sample | Sample Name | Built-in | | |
| Project | Project Name | Built-in | | |
### Step 3: Ligate Indexes (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Master Step Name = Ligate Indexes (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Step Type = Add Labels
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}-{AppliedReagentLabels}
* Reagent Kits
* Illumina DNA PCR-Free Library Prep
* Supplier = Illumina
* Catalog Number = 24 - 20041794; 96 - 20041795
* Website = [www.illumina.com](https://www.illumina.com)
{% hint style="info" %}
The version of Ligate Indexes master step name may be different depending on the version of IPP installed.
{% endhint %}
#### Automations
Calculate Dilution for HP3 & Copy Desired DNA Input
* 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.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng):: ; \
step.::Total Samples:: = step.::Total Samples:: + 1 ; \
step.::HP3 (uL):: = step.::Total Samples:: * 6 ; \
step.::Nuclease-free water (uL):: = step.::Total Samples:: * 54' \
-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 = 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
* 96 well plate
#### Add Labels
* Label Groups
* IDT for Illumina Nextera DNA UD Indexes Set A for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
* IDT for Illumina Nextera DNA UD Indexes Set A-D for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
* IDT for Illumina Nextera DNA UD Indexes Set B for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
* IDT for Illumina Nextera DNA UD Indexes Set C for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
* IDT for Illumina Nextera DNA UD Indexes Set D for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
* Illumina DNA-RNA UD Indexes Set A B C D Tagmentation
* Illumina DNA-RNA UD Indexes Set A Tagmentation
* Illumina DNA-RNA UD Indexes Set B Tagmentation
* Illumina DNA-RNA UD Indexes Set C Tagmentation
* Illumina DNA-RNA UD Indexes Set D Tagmentation
#### Record Details
* Step Data (Master Step Fields)
| **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
| ------------------------------------ | -------------- | ----------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| HP3 (uL) | Numeric | Read Only |
Decimal Places Displayed = 0
|
| Instruction Notes for Hybex Protocol | Multiline Text | Read Only |
Default = 1. Remove and discard all supernatant. 2. Add 45 µl ELM to each well. 3. Add 5 µl index adapters to each well then seal and shake at 1800 rpm for 1 min. 4. Incubate in the preheated Hybex for 8 mins. 5. Place on the magnetic stand and wait until the liquid is clear (\~2 minutes). 6. Remove and discard all supernatant from each well. 7. Add 75 µl TWB onto the beads in each well then seal and shake at 1800 rpm for 1 minute. 8. Place on the magnetic stand and wait until the liquid is clear (\~2 minutes). 9. Remove and discard all supernatant from each well. 10. Without disturbing the bead pellet, use a 20 µl pipette to remove and discard residual TWB from each well. 11. Add 45 µl diluted HP3 to each well then seal and shake at 1800 rpm for 1 minute.
|
| Nuclease-free water (uL) | Numeric | Read Only |
|
| Derived Sample | Sample Name | Built-in | | |
| Project | Project Name | Built-in | | |
### Step 4: Clean Up Libraries (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Master Step Name = Clean Up Libraries (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
* Illumina DNA PCR-Free Library Prep
* Supplier = Illumina
* Catalog Number = 24 - 20041794; 96 - 20041795
* Website = [www.illumina.com](https://www.illumina.com)
{% hint style="info" %}
The version of Clean Up Libraries master step name may be different depending on the version of IPP installed.
{% endhint %}
#### Automations
Copy Desired DNA Input
* 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.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng)::' \
-log {compoundOutputFileLuid0}"
```
{% endcode %}
Set Next Step-Remove & Prep Input Type
* 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 -exp 'nextStep = ::REMOVE:: ; \
output.::Prep Input Type:: = ::Standard::' -log {compoundOutputFileLuid0}"
```
{% endcode %}
Routing - Quantify & Pool Libraries
* 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 'Prep Input Type' \
--FIELD_VALUE 'Standard' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'Pool Samples - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Prep Input Type' \
--FIELD_VALUE 'Low' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'KAPA Sample Prep (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"
```
{% endcode %}
> ℹ The actual version of the workflows and steps in the routing automation script may be different depending on the version of IPP installed.
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** |
| ------------------ | -------------- | ----------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ |
| 80% EtOH Prep Date | Date | | |
| Instruction Notes | Multiline Text | Read Only |
Default = 1. Add 36 µl IPB to each well then seal and shake at 1800 rpm for 1 minute. 2. Incubate at room temperature for 2 minutes. 3. Place on the magnetic stand and wait until the liquid is clear (\~5 minutes). 4. Label a new 96-well MIDI plate LP2. 5. Add 42 µl IPB to each well of LP2. 6. Without disturbing the bead pellet, transfer 76 µl supernatant from each well of LP1 to the corresponding well of LP2 then seal and shake at 1800 rpm for 1 minute. 7. Discard LP1 and incubate LP2 at room temperature for 2 minutes. 8. Place on the magnetic stand and wait until the liquid is clear (\~5 minutes). 9. Without disturbing the bead pellet, remove and discard all supernatant from each well. 10. Wash beads 2x with 180 uL of fresh 80% EtOH for each wash. 11. Using a 20 µl pipette, remove residual EtOH from each well. 12. Air-dry on the magnetic stand (\~4 minutes). 13. Add 22 µl of RSB onto the beads in each well then seal and shake at 1800 rpm for 1 minute. 14. Incubate at room temperature for 2 minutes. 15. Place on the magnetic stand and wait until the liquid is clear (\~2 minutes). 16. Label a new PCR plate FLP. 17. Transfer 20 µl supernatant from each well of LP2 to the corresponding well of FLP.
|
| Derived Sample | Prep Input Type | Text | | |
| Derived Sample | Sample Name | Built-in | | |
| Project | Project Name | Built-in | | |
## Protocol 3: Library Prep - Thermal Cycler Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
Protocol Type = Library Prep
**Next Steps Configuration**
### Step 1: Tagment Genomic DNA - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Master Step Name = Tagment Genomic DNA - Standard Input v1.0.2
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
* Illumina DNA PCR-Free Library Prep
* Supplier = Illumina
* Catalog Number = 24 - 20041794; 96 - 20041795
* Website = [www.illumina.com](https://www.illumina.com)
{% hint style="info" %}
The version of Tagment Genomic DNA - Standard Input master step name may be different depending on the version of IPP installed.
{% endhint %}
#### Automations
Copy Sample Type & Concentration
* 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.::Sample Type:: = input.::Sample Type:: ; \
if (output.::Sample Type:: == ::gDNA::) {output.::Concentration:: = input.::Concentration::} else {output.::Concentration:: = 0} ; \
if (output.::Sample Type:: == ::gDNA::) {output.::Conc. Units:: = input.::Conc. Units::} else {output.::Conc. Units:: = ::NA::}' \
-log {compoundOutputFileLuid0}"
```
{% endcode %}
Calculate Dilution & BLT-PF Volumes and Copy Desired DNA Input
* 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.::Desired DNA Input (ng):: = step.::Desired DNA Input (ng):: ; \
if (output.::Sample Type:: == ::gDNA::) {output.::DNA (uL):: = output.::Desired DNA Input (ng):: / output.::Concentration::} ; \
if (output.::Sample Type:: == ::Whole Blood::) {output.::DNA (uL):: = 30} ; \
if (output.::Sample Type:: == ::Dried Blood Spot::) {output.::DNA (uL):: = 30} ; \
if (output.::Sample Type:: == ::Saliva::) {output.::DNA (uL):: = 30} ; \
if (output.::Sample Type:: == ::gDNA::) {output.::RSB (uL):: = 25 - output.::DNA (uL)::} ; \
if (output.::Sample Type:: == ::Whole Blood::) {output.::RSB (uL):: = 0} ; \
if (output.::Sample Type:: == ::Dried Blood Spot::) {output.::RSB (uL):: = 0} ; \
if (output.::Sample Type:: == ::Saliva::) {output.::RSB (uL):: = 0} ; \
if (output.::Sample Type:: == ::gDNA::) {output.::BLT-PF (uL):: = 15} ; \
if (output.::Sample Type:: == ::Whole Blood::) {output.::BLT-PF (uL):: = 10} ; \
if (output.::Sample Type:: == ::Dried Blood Spot::) {output.::BLT-PF (uL):: = 10} ; \
if (output.::Sample Type:: == ::Saliva::) {output.::BLT-PF (uL):: = 10}' \
-log {compoundOutputFileLuid0}"
```
{% endcode %}
Set Next Step-Post TAG
* 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 \
-exp 'if (input.::Sample Type:: == ::Whole Blood::) {nextStep = ::Post Tagmentation Cleanup (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::} ; \
if (input.::Sample Type:: == ::Dried Blood Spot::) {nextStep = ::Post Tagmentation Cleanup (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::} ; \
if (input.::Sample Type:: == ::Saliva::) {nextStep = ::Post Tagmentation Cleanup (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::} ; \
if (input.::Sample Type:: == ::gDNA::) {nextStep = ::Post Tagmentation Cleanup (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::}' \
-log {compoundOutputFileLuid0}"
```
{% endcode %}
> ℹ The actual version of the nextStep step names may be different depending on the version of IPP installed.
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 | | |
#### Placement = Enabled
* Defaults
* Sample Grouping = Group by Containers
* Well Sort Order = Column
* Placement Pattern = Column
* Destination Containers
* 96 well plate
#### Record Details
* Step Data (Master Step Fields)
| **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
| ---------------------------- | -------------- | -------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| 80% EtOH Prep Date | Date | | |
| Desired DNA Input (ng) | Numeric | Required Field | Range = 100 To 2000 |
| Instruction Notes | Multiline Text | Read Only |
Default = 1. Label a new 96 well PCR plate LP1. 2. Add the calculated DNA, and RSB volumes to the LP1 plate and then pipette to mix. 3. Add the calculated BLT-PF to each well and pipette to mix. 4. Add 10 uL of TB1 to each well, pipette to mix and then seal. 5. Place the LP1 plate on the thermo cycler and run the TAG program.
|
| Thermal Cycler Program | Text | | Default = TAG |
| Thermal Cycler Program Notes | Multiline Text | Read Only |
Default = 1. Choose the preheat lid option and set to 100°C 2. Set the reaction volume to 50 µl u 41°C for 5 minutes.
|
| Desired DNA Input (ng) | Numeric | Required Field |
Default = 25
Range = 25 To 99\
|
| Instruction Notes | Multiline Text | Read Only |
Default = 1. Label a new 96 well MIDI plate LP1. 2. Combine the calculated BLT-PF and TB1 volumes into a 1.5 mL tube to prepare the Tagmentation Master Mix. 3. Add the calculated DNA, and RSB volumes to the LP1 plate and then pipette to mix. 4. Add 20 µl Tagmentation Master Mix to each well, pipette to mix and then seal. 5. Place the LP1 plate on the thermo cycler and run the TAG program.
|
| TB1 (uL) | Numeric | Read Only |
Decimal Places Displayed = 0
|
| Thermal Cycler Program | Text | |
Default = TAG
|
| Thermal Cycler Program Notes | Multiline Text | Read Only |
Default = 1. Choose the preheat lid option and set to 100°C 2. Set the reaction volume to 50 µl u 41°C for 5 minutes.
|
| Project | Project Name | Built-in | | |
### Step 3: Post Tagmentation Cleanup (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Master Step Name = Post Tagmentation Cleanup v1.0.1
* Step Type = No Outputs
* Reagent Kits
* Illumina DNA PCR-Free Library Prep
* Supplier = Illumina
* Catalog Number = 24 - 20041794; 96 - 20041795
* Website = [www.illumina.com](https://www.illumina.com)
{% hint style="info" %}
The version of Post Tagmentation Cleanup master step name may be different depending on the version of IPP installed.
{% endhint %}
#### 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 %}
Copy Desired DNA Input & Sample Type
* 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.::Sample Type:: = input.::Sample Type:: ; \
output.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng)::' \
-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 = 1. Add 10 µl ST2 to each well, seal and then shake at 1800 rpm for 1 minute. 2. Place on the magnetic stand and wait until the liquid is clear (\~2 minutes). 3. Without disturbing the bead pellet, remove and discard all supernatant from each well. 4. Add 150 μl TWB to each well, seal and shake at 1800 rpm for 1 minute. 5. Place on the magnetic stand and wait until the liquid is clear (\~2 minutes).
|
| Derived Sample | Sample Name | Built-in | | |
| Derived Sample | Sample Type | Text Dropdown |
Requird Field
Custom Entries
|
Presets
DNA
RNA
|
| Project | Project Name | Built-in | | |
### Step 4: Ligate Indexes - Thermal Cycler (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Master Step Name = Ligate Indexes - Thermal Cycler (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Step Type = Add Labels
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}-{AppliedReagentLabels}
* Reagent Kits
* Illumina DNA PCR-Free Library Prep
* Supplier = Illumina
* Catalog Number = 24 - 20041794; 96 - 20041795
* Website = [www.illumina.com](https://www.illumina.com)
{% hint style="info" %}
The version of Ligate Indexes - Thermal Cycler (Illumina DNA PCR-Free Library Prep Manual) master step name may be different depending on the version of IPP installed.
{% endhint %}
#### Automations
Calculate Dilution for HP3 & Copy Desired DNA Input
* 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.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng):: ; \
step.::Total Samples:: = step.::Total Samples:: + 1 ; \
step.::HP3 (uL):: = step.::Total Samples:: * 6 ; \
step.::Nuclease-free water (uL):: = step.::Total Samples:: * 54' \
-log {compoundOutputFileLuid0}"
```
{% endcode %}
Set Next Step - Clean Up Libraries
* 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 \
-exp 'if (output.::Desired DNA Input (ng):: >= 100) {nextStep = ::Clean Up Libraries - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::} ; \
if (output.::Desired DNA Input (ng):: <= 99) {nextStep = ::Clean Up Libraries - Low Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)::}' \
-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 | | |
#### Add Labels
* Label Groups
* IDT for Illumina Nextera DNA UD Indexes Set A for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
* IDT for Illumina Nextera DNA UD Indexes Set A-D for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
* IDT for Illumina Nextera DNA UD Indexes Set B for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
* IDT for Illumina Nextera DNA UD Indexes Set C for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
* IDT for Illumina Nextera DNA UD Indexes Set D for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000
* Illumina DNA-RNA UD Indexes Set A B C D Tagmentation
* Illumina DNA-RNA UD Indexes Set A Tagmentation
* Illumina DNA-RNA UD Indexes Set B Tagmentation
* Illumina DNA-RNA UD Indexes Set C Tagmentation
* Illumina DNA-RNA UD Indexes Set D Tagmentation
#### Record Details
* Step Data (Master Step Fields)
| **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
| ---------------------------- | -------------- | ----------- | ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| HP3 (uL) | Numeric | Read Only |
Decimal Places Displayed = 0
|
| Instruction Notes | Multiline Text | Read Only |
Default = 1. Remove and discard all supernatant. 2. Add 45 µl ELM to each well and pipette to mix. 3. Add 5 µl index adapters to each well then seal and shake at 1800 rpm for 1 min. 4. Place on the preprogrammed thermal cycler and run the ELM program. 5. Place on the magnetic stand and wait until the liquid is clear (\~2 minutes). 6. Remove and discard all supernatant from each well. 7. Add 75 µl TWB onto the beads in each well then pipette to mix. 8. Place on the magnetic stand and wait until the liquid is clear (\~2 minutes). 9. Remove and discard all supernatant from each well. 10. Seal and centrifuge at 280 x g for 10 seconds and then place on magnetic stand. 11. Without disturbing the bead pellet, remove and discard residual TWB from each well. 12. Remove the plate from the magnetic stand. 13. Add 45 µl diluted HP3 to each well, pipette to mix and then incubate at RT for 2 mins.
|
| Nuclease-free water (uL) | Numeric | Read Only |
Decimal Places Displayed = 0
|
| Thermal Cycler Program | Text | |
Default = ELM
|
| Thermal Cycler Program Notes | Multiline Text | Read Only |
Default = ELM Program 1. Choose the preheat lid option and set to 100°C 2. Set the reaction volume to 50 μl 3. Run for 37°C for 5 minutes 4. Run for 50°C for 5 minutes
|
| Derived Sample | Sample Name | Built-in | | |
| Project | Project Name | Built-in | | |
### Step 5: Clean Up Libraries - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Master Step Name = Clean Up Libraries - Thermal Cycler v1.0.2
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
* Illumina DNA PCR-Free Library Prep
* Supplier = Illumina
* Catalog Number = 24 - 20041794; 96 - 20041795
* Website = [www.illumina.com](https://www.illumina.com)
{% hint style="info" %}
The version of Clean Up Libraries - Thermal Cycler master step name may be different depending on the version of IPP installed.
{% endhint %}
#### Automations
Copy Desired DNA Input
* 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.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng)::' \
-log {compoundOutputFileLuid0}"
```
{% endcode %}
Set Next Step-Remove & Prep Input Type
* 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 -exp 'nextStep = ::REMOVE:: ; \
if (output.::Desired DNA Input (ng):: >= 300) {output.::Prep Input Type:: = ::Standard::} else {output.::Prep Input Type:: = ::Low::}' \
-log {compoundOutputFileLuid0}"
```
{% endcode %}
Routing - Quantify & Pool Libraries
* 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 'Prep Input Type' \
--FIELD_VALUE 'Standard' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'Pool Samples - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Prep Input Type' \
--FIELD_VALUE 'Low' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'KAPA Sample Prep (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"
```
{% endcode %}
> ℹ The actual version of the workflow and step names for the routing script may be different depending on the version of IPP installed.
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** |
| ------------------------------------ | -------------- | ----------- | --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| 80% EtOH Prep Date | Date | | |
| Instruction Notes for Standard Input | Multiline Text | Read Only |
Default = 1. Add 36 µl IPB to each well containing BLT-PF beads and pipette to mix. 2. Incubate at room temperature for 2 minutes. 3. Place on the magnetic stand and wait until the liquid is clear (\~5 minutes). 4. Label a new 96-well PCR plate LP2. 5. Add 42 µl IPB to each well of LP2. 6. Without disturbing the bead pellet, transfer 76 µl supernatant from each well of LP1 to the corresponding well of the LP2, pipette to mix and remove LP1 from the magnetic stand, and then discard. 7. Incubate LP2 at room temperature for 2 minutes. 8. Place on the magnetic stand and wait until the liquid is clear (\~5 minutes). 9. Without disturbing the bead pellet, remove and discard all supernatant from each well. 10. Wash beads 2x with 180 µl fresh 80% ethanol per well for each wash. 11. Seal and then centrifuge 280 x g for 10 seconds. 12. Place on the magnetic stand, and then wait 10 seconds. 13. Remove residual EtOH from each well, air-dry on the magnetic stand (\~2 minutes) then remove from the magnetic stand. 14. Add 22 µl RSB onto the beads in each well and pipette to mix. 15. Incubate at room temperature for 2 minutes. 16. Place on the magnetic stand and wait until the liquid is clear (\~2 minutes). 17. Label a new PCR plate FLP. 18. Transfer 20 µl supernatant from each well of LP2 to the corresponding well of FLP.
|
| Derived Sample | Prep Input Type | Text | | |
| Derived Sample | Sample Name | Built-in | | |
| Project | Project Name | Built-in | | |
### Step 6: Clean Up Libraries - Low Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Master Step Name = Clean Up Libraries - Thermal Cycler v1.0.2
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
* Illumina DNA PCR-Free Library Prep
* Supplier = Illumina
* Catalog Number = 24 - 20041794; 96 - 20041795
* Website = [www.illumina.com](https://www.illumina.com)
{% hint style="info" %}
The version of Clean Up Libraries - Thermal Cycler master step name may be different depending on the version of IPP installed.
{% endhint %}
#### Automations
Copy Desired DNA Input
* 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.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng)::' \
-log {compoundOutputFileLuid0}"
```
{% endcode %}
Set Next Step-Remove & Prep Input Type
* 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 -exp 'nextStep = ::REMOVE:: ; \
if (output.::Desired DNA Input (ng):: >= 300) {output.::Prep Input Type:: = ::Standard::} else {output.::Prep Input Type:: = ::Low::}' \
-log {compoundOutputFileLuid0}"
```
{% endcode %}
Routing - Quantify & Pool Libraries
* 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 'Prep Input Type' \
--FIELD_VALUE 'Standard' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'Pool Samples - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Prep Input Type' \
--FIELD_VALUE 'Low' \
--WORKFLOW 'Illumina DNA PCR-Free Library Prep Manual v1.0.3' \
--STEP 'KAPA Sample Prep (Illumina DNA PCR-Free Library Prep Manual v1.0.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"
```
{% endcode %}
> ℹ The actual version of the workflow and step names for the routing script may be different depending on the version of IPP installed.
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** |
| ------------------------------- | -------------- | ----------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ |
| 80% EtOH Prep Date | Date | | |
| Instruction Notes for Low Input | Multiline Text | Read Only |
Default = 1. Add 81 µl IPB to each well and pipette to mix. 2. Incubate at room temperature for 5 minutes. 3. Place on the magnetic stand and wait until the liquid is clear (\~5 minutes). 4. Remove and discard all supernatant from each well. 5. Wash beads 2x with 180 uL of fresh 80% EtOH for each wash. 6. Seal plate then centrifuge 280 x g for 10 seconds. 7. Place on the magnetic stand and wait until the liquid is clear (\~5 minutes). 8. Remove residual EtOH from each well and air-dry on the magnetic stand (\~2 minutes). 9. Remove the plate from the magnetic stand. 10. Add 15 µl RSB onto the beads in each well and pipette to resuspend. 11. Incubate at room temperature for 2 minutes. 12. Place on the magnetic stand and wait until the liquid is clear (\~2 minutes). 13. Transfer 14 µl supernatant from each well of the plate to the corresponding well of a new PCR plate.
|
| Derived Sample | Prep Input Type | Text | | |
| Derived Sample | Sample Name | Built-in | | |
| Project | Project Name | Built-in | | |
## Protocol 4: Quantify and Pool Libraries - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
Protocol Type = Library Prep
**Next Steps Configuration**
### Step 1: Pool Samples - Standard Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Master Step Name = Pool Samples (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Step Type = Pooling
* Aliquot Generation = Fixed, 1
* Naming Convention = {PoolName}
* Reagent Kits
* Illumina DNA PCR-Free Library Prep
* Supplier = Illumina
* Catalog Number = 24 - 20041794; 96 - 20041795
* Website = [www.illumina.com](https://www.illumina.com)
{% hint style="info" %}
The version of Pool Samples (Illumina DNA PCR-Free Library Prep Manual) master step name may be different depending on the version of IPP installed.
{% endhint %}
#### 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 | | |
#### 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** |
| ----------------- | -------------- | ----------- | -------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| Instruction Notes | Multiline Text | Read Only |
Default = 1. Combine 9 µl of each library in a 1.5 or 1.7 ml microcentrifuge tube. 2. Vortex to mix, and then centrifuge at 280 x 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 2: Qubit (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Master Step Name = Qubit (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Step Type = Standard QC
* Measurement Generation = Fixed, 1
* Naming Convention = {InputItemName}
{% hint style="info" %}
The version of Qubit (Illumina DNA PCR-Free Library Prep Manual) master step name may be different depending on the version of IPP installed.
{% endhint %}
#### Automations
Assign QC flags
* 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 -u {username} -p {password} \
script:assignQC \
-i {processURI:v2} \
-log {compoundOutputFileLuid1} \
-qcResult {compoundOutputFileLuid2}"
```
{% endcode %}
Set Next Step-Remove and Copy Concentration
* 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:: ; input.::Concentration:: = output.::Concentration:: ; input.::Conc. Units:: = output.::Conc. Units::' -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** |
| ------------------------------ | -------------- | ----------- | ----------------------------------------- |
| Criteria 1 - Operator | Text | | Default = >= |
| Criteria 1 - Source Data Field | Text | | Default = Concentration |
| Criteria 1 - Threshold Value | Numeric | | |
| Criteria 2 - Operator | Text | | Default = <= |
| Criteria 2 - Source Data Field | Text | | Default = Concentration |
| Criteria 2 - Threshold Value | Numeric | | |
* Step File Placeholders
* Log - Automatically attached
* QC Log File - Automatically attached
* QC Result File - Automatically attached
* Upload File - Manually uploaded
* Sample Table
* Enable QC Flags = Yes
* Sample Display Default = Expand
* Well Sort Order = Column
* File Column Options
* File Column Display = Hide
* File Attachment Method = Auto
* Table Columns - Global Fields
| **Category** | **Field Name** | **Field Type** | **Options** | **Additional Options and Dropdown Items** |
| -------------- | ------------------- | -------------- | -------------- | ----------------------------------------- |
| Container | Container Name | Built-in | | |
| Container | LIMS ID (Container) | Built-in | | |
| Container | Well | Built-in | | |
| Derived Sample | Concentration | Numeric | Required Field | Decimal Places Displayed = 2 |
| Derived Sample | Conc. Units | Text | Required Field | |
| Derived Sample | Sample Name | Built-in | | |
| Measurement | Concentration | Numeric | | Decimal Places Displayed = 2 |
| Measurement | Conc. Units | Text | | |
| Project | Project Name | Built-in | | |
## Protocol 5: Quantify and Pool Libraries - Low Input (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
Protocol Type = Library Prep
**Next Steps Configuration**
### Step 1: KAPA Sample Prep (Illumina DNA PCR-Free Library Prep Manual v1.0.3)
* Master Step Name = KAPA Sample Prep - Illumina/Universal v1.0.1
* Step Type = Standard
* Derived Sample Generation = Fixed, 1
* Naming Convention = {InputItemName}
* Reagent Kits
* 0.05% Tween® 20
* 10 mM Tris-HCl, pH 8.0 – 8.5 (25°C)
* Control Types
* KAPA DNA Standard 1
* Supplier = Roche
* Catalog Number = KK4828-07960166001
* Website = [www.roche.com](https://www.roche.com)
* Conc. = 20 pM
* Control Use
* Single step only = No
* KAPA DNA Standard 2
* Supplier = Roche
* Catalog Number = KK4828-07960166001
* Website = [www.roche.com](https://www.roche.com)
* Conc. = 2 pM
* Control Use
* Single step only = No
* KAPA DNA Standard 3
* Supplier = Roche
* Catalog Number = KK4828-07960166001
* Website = [www.roche.com](https://www.roche.com)
* Conc. = 0.2 pM
* Control Use
* Single step only = No
* KAPA DNA Standard 4
* Supplier = Roche
* Catalog Number = KK4828-07960166001
* Website = [www.roche.com](https://www.roche.com)
* Conc. = 0.02 pM
* Control Use
* Single step only = No
* KAPA DNA Standard 5
* Supplier = Roche
* Catalog Number = KK4828-07960166001
* Website = [www.roche.com](https://www.roche.com)
* Conc. = 0.002 pM
* Control Use
* Single step only = No
* KAPA DNA Standard 6
* Supplier = Roche
* Catalog Number = KK4828-07960166001
* Website = [www.roche.com](https://www.roche.com)
* Conc. = 0.0002
* Control Use
* Single step only = No
* KAPA Library Quantification Dilution Control
* Supplier = Roche
* Catalog Number = KK4906
* Website = [www.roche.com](https://www.roche.com)
* Conc. = 200 pM
* Control Use
* Single step only = No
* KAPA NTC
* Control Use
* Single step only = No
{% hint style="info" %}
The version of KAPA Sample Prep - Illumina/Universal master step name may be different depending on the version of IPP installed.
{% endhint %}
#### Automations
Copy Desired DNA Input for KAPA
* Trigger Location = Record Details
* Trigger Style = Automatic upon entry
{% code overflow="wrap" %}
```markup
bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t false \
-h false \
-exp 'if (output.name.contains(::KAPA DNA Standard 1::)) {output.::Desired DNA Input (ng):: = 0.005952} ; \
if (output.name.contains(::KAPA DNA Standard 2::)) {output.::Desired DNA Input (ng):: = 0.000595} ; \
if (output.name.contains(::KAPA DNA Standard 3::)) {output.::Desired DNA Input (ng):: = 0.00006} ; \
if (output.name.contains(::KAPA DNA Standard 4::)) {output.::Desired DNA Input (ng):: = 0.000006} ; \
if (output.name.contains(::KAPA DNA Standard 5::)) {output.::Desired DNA Input (ng):: = 0.0000006} ; \
if (output.name.contains(::KAPA DNA Standard 6::)) {output.::Desired DNA Input (ng):: = 0.00000006} ; \
if (output.name.contains(::KAPA Library Quantification Dilution Control::)) {output.::Desired DNA Input (ng):: = 0.059524} ; \
if (output.name.contains(::KAPA NTC::)) {output.::Desired DNA Input (ng):: = 0} ; \
if (!output.name.contains(::KAPA::)) {output.::Desired DNA Input (ng):: = input.::Desired DNA Input (ng)::}' \
-log {compoundOutputFileLuid0}"
```
{% endcode %}
Calculate Expected Concentration & Dilution Factor
* 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.::Expected Concentration (pM):: = output.::Desired DNA Input (ng):: * 3.36 * 1000 ; \
if (output.::Expected Concentration (pM):: >= 84000 && output.::Expected Concentration (pM):: <= 188000) {output.::Dilution Factor:: = 10000} ; \
if (output.::Expected Concentration (pM):: >= 189000 && output.::Expected Concentration (pM):: <= 333000) {output.::Dilution Factor:: = 20000} ; \
if (output.::Expected Concentration (pM):: <= 83000) {output.::Dilution Factor:: = 0}' \
-log {compoundOutputFileLuid0} && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2:http} -u {username} -p {password} script:evaluateDynamicExpression -t true -h false \
-exp 'if (output.::Expected Concentration (pM):: > 333000) {fail(::Expected Concentration is too high, please edit Dilution Factor and recalculate::)}' \
-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** |
| ----------------------------- | -------------- | ----------- | ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| DNA Dilution Buffer Prep Date | Date | | |
| Instruction Notes | Multiline Text | Read Only |
Default = - Prepare the appropriate library dilutions (using DNA dilution buffer). - Depending on the expected concentration of the library, 1:1,000 – 1:100,000 dilutions may be appropriate.
|
| Instruction Notes | Multiline Text | Read Only |
Default = 1. Set reaction volume and adding Rox? then click on calculate qPCR Master Mix and Sample Volume button. 2. Combine KAPA SYBR FAST qPCR Master Mix + Primer Premix, ROX and PCR-grade water to produce the qPCR Master Mix. 3. Add the qPCR Master Mix and Sample volume to a new 96 well PCR plate, seal the plate, vortex briefly to mix and then centrifuge. 4. Place the plate on the qPCR instrument. 5. Select Absolute Quantification and run the following cycling protocol: - Initial denaturation Temp. at 95C fro 5 mins for 1 cycle - Denaturation Temp. at 95C for 30 secs for 35 cycles - Annealing/Extension/Data acquisition Temp. at 95C for 45 sec for 35 cycles - Melt curve analysis (C) between 65C - 95C 6. Upload Cq values 7. Set Cq Thresholds then click on Assign QC Flags button. 8. Review Cq values and remove outliers. 9. Click on Calculate Average Cq.
|
| KAPA SYBR FAST qPCR Master Mix + Primer Premix (uL) | Numeric | Read Only |
Decimal Places Displayed = 2
|
| PCR-grade water (uL) | Numeric | Read Only |
Decimal Places Displayed = 2
|
| qPCR Master Mix with Primer Prep Date | Date | | |
| Reaction Volume (uL) | Numeric Dropdown | |
Default = 1. Enter the Slope and the Y-Intercept for your standard curve then click on the Calculate Concentration button. 2. Set your Concentration Threshold then click on the Assign QC Flogs button.
