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Illumina RNA Prep with Enrichment (L) Tagmentation v1.1

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Overview

The Illumina RNA Prep with Enrichment (L) Tagmentation workflow includes the following functionality.

Preset Illumina RNA Prep with Enrichment (L) Tagmentation protocols that support the generation of enriched libraries for dual-indexed, paired-end 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: Prep Sample (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

Protocol Type = Sample Prep

Next Steps Configuration

Step 1: Denature RNA (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

Master Step Name = Denature RNA v1.0
Step Type = Standard
Derived Sample Generation = Fixed, 1
Naming Convention = {InputItemName}
Reagent Kits
- Illumina RNA Prep with Enrichment (L) Tagmentation
  - Supplier = Illumina
  - Catalog Number = 16 - 20040536, 96 - 20040537
  - Website = www.illumina.com

Automations

Calculate Sample and Water Volume

Trigger Location = Record Details
Trigger Style = Manual button

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.::SampleVolume (uL):: = (step.::Final Diluted Concentration (ng/uL):: * step.::Final Volume (uL)::) / output.::Starting Concentration (ng/uL):: ; output.::Nuclease-free Ultrapure Water (uL):: = step.::Final Volume (uL):: - output.::SampleVolume (uL)::' \
-log {compoundOutputFileLuid0}"

Set Next Step - Advance

Trigger Location = Record Details
Trigger Style = Automatic upon exit

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}"

Queue

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

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 Diluted Concentration (ng/uL)
Numeric
Required Field
Default = 10
Range = 10 To 100
Final Volume (uL)
Numeric
Default = 8.5
Instruction Notes
Multiline Text
Read Only
Default = Enter Final Diluted Concentration and Starting Concentration before clicking on the Calculate Sample and Water Volume button.
Thermal Cycler Program
Text
Default = DEN_RNA
Thermal Cycler Program Notes
Multiline Text
Read Only
Default = Choose the preheat lid option and set to 100°C Reaction volume is 17 µl 65°C for 5 minutes Hold at 4°C
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
  Nuclease-free Ultrapure Water (uL)
  Numeric
  Decimal Places Displayed = 0
  Derived Sample
  Sample Name
  Built-in
  Derived Sample
  SampleVolume (uL)
  Numeric
  Decimal Places Displayed = 0
  Derived Sample
  Starting Concentration (ng/uL)
  Numeric
  Decimal Places Displayed = 0
  Project
  Project Name
  Built-in

Step 2: Synthesize First Strand cDNA (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

Master Step Name = Synthesize First Strand cDNA v1.0
Step Type = No Outputs
Reagent Kits
- Illumina RNA Prep with Enrichment (L) Tagmentation
  - Supplier = Illumina
  - Catalog Number = 16 - 20040536, 96 - 20040537
  - Website = www.illumina.com

Automations

Calculate First Strand Synthesis Master Mix

Trigger Location = Record Details
Trigger Style = Automatic upon entry

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.::FSA (uL):: = 9 * step.::Total Samples:: ; step.::RVT (uL):: = 1 * step.::Total Samples:: ' -log {compoundOutputFileLuid0}"

Set Next Step - Advance

Trigger Location = Record Details
Trigger Style = Automatic upon exit

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}"

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
FSA (uL)
Numeric
Decimal Places Displayed = 0
Instruction Notes
Multiline Text
Read Only
Default = Add 8 uL of First Strand Synthesis Master Mix to each sample.
RVT (uL)
Numeric
Decimal Places Displayed = 0
Thermal Cycler Program
Text
Default = FSS
Thermal Cycler Program Notes
Multiline Text
Read Only
Default = Choose the preheat lid option and set to 100°C Reaction volume is 25 µl 25°C for 10 minutes 42°C for 15 minutes 70°C for 15 minutes Hold at 4°C
Total Samples
Numeric
Default = 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 3: Synthesize Second Strand cDNA (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

Master Step Name = Synthesize Second Strand cDNA v1.0
Step Type = Standard
Derived Sample Generation = Fixed, 1
Naming Convention = {InputItemName}
Reagent Kits
- Agencourt AMPure XP, 5 ml
  - Supplier = Beckman Coulter
  - Catalog Number = A63880
- Illumina RNA Prep with Enrichment (L) Tagmentation
  - Supplier = Illumina
  - Catalog Number = 16 - 20040536, 96 - 20040537
  - Website = www.illumina.com

Automations

Set Next Step - Advance

Trigger Location = Record Details
Trigger Style = Automatic upon exit

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}"

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
Thermal Cycler Program
Text
Default = SSS
Thermal Cycler Program Notes
Multiline Text
Read Only
Default = Choose the preheat lid option and set to 40°C Reaction volume is 50 µl u 16°C for 1 hour Hold at 4°C
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 2: Library Prep (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Tagment cDNA (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

Master Step Name = Tagment cDNA v1.0
Step Type = No Outputs
Reagent Kits
- Illumina RNA Prep with Enrichment (L) Tagmentation
  - Supplier = Illumina
  - Catalog Number = 16 - 20040536, 96 - 20040537
  - Website = www.illumina.com

Automations

Calculate Tagmentation Master Mix - RNA Prep

Trigger Location = Record Details
Trigger Style = Automatic upon entry

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.::TB1 (uL):: = 11.5 * step.::Total Samples:: ; step.::EBLTL (uL):: = 11.5 * step.::Total Samples:: ; step.::Nuclease-free ultrapure water (uL):: = 14.5 * step.::Total Samples::' \
-log {compoundOutputFileLuid0}"

Set Next Step - Advance

Trigger Location = Record Details
Trigger Style = Automatic upon exit

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}"

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