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Illumina Stranded mRNA Prep Ligation 1.1

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Last updated 2 months ago

Overview

The Illumina Stranded mRNA Prep Ligation workflow includes the following functionality.

Preconfigured protocols that convert the messenger (mRNA) in total RNA into up to 384 dual-indexed libraries. Depletion reagents bind and deplete abundant transcripts from total RNA. Reverse transcription then converts the remaining RNA into cDNA, while subsequent ligation and amplification steps add adapters for clustering and sequencing on an Illumina system.
Automated calculation of sample and buffer volumes.
Automated calculation or display of reagents at every step in the protocol.
Automatic step transition when required.
Automatic placement of samples when necessary.
Automated assignment of QC Pass/Fail, based on user-selected threshold values.
A routing script that allows sequencing of libraries using any Illumina sequencing instrument.

Protocol 1: Prep Sample (Illumina Stranded mRNA Prep Ligation v1.1)

Protocol Type = Sample Prep

Next Steps Configuration

Step 1: Deplete mRNA (Illumina Stranded mRNA Prep Ligation v1.1)

Master Step Name = Deplete mRNA v1.0
Step Type = Standard
Derived Sample Generation = Fixed, 1
Naming Convention = {InputItemName}
Reagent Kits
- Illumina Stranded mRNA Prep, Ligation
  - Supplier = Illumina
  - Website = www.illumina.com

Automations

Calculate Fragmentation 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.::Nuclease-free ultrapure water (uL):: = 10.5 * step.::Total Samples:: ; step.::EPH3 (uL):: = 10.5 * step.::Total Samples::' \
-log {compoundOutputFileLuid0}"

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/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
EPH3 (uL)
Numeric
Final Diluted Concentration (ng/uL)
Numeric
Required Field
Default = 10
Range = 10 To 1000
Final Volume (uL)
Numeric
Required Field
Default = 25
Instruction Notes
Multiline Text
Read Only
Default = Final Diluted Concentration should be between 10-1000 ng/uL. Enter in Starting Concentration before clicking on Calculate Sample and Water Volumes. Add 19 µl Fragmentation Master Mix to each well.
Nuclease-free ultrapure water (uL)
Numeric
Decimal Places Displayed = 0
Thermal Cycler Program for Capture
Text
Default = mRNA_CAP
Thermal Cycler Program for Elute
Text
Default = mRNA_ELT
Thermal Cycler Program for Fragment and Denature mRNA
Text
Default = DEN94_8
Thermal Cycler Program Notes
Multiline Text
Read Only
Default = mRNA_CAP program on the thermal cycler: Choose the preheat lid option and set to 100°C Reaction volume is 50 µl u 65°C for 5 minutes 4°C for 30 seconds 23° for 5 minutes Hold at 23°C mRNA_ELT program on the thermal cycler: Choose the preheat lid option and set to 100°C Reaction volume is 25 µl 80°C for 2 minutes Hold at 25°C DEN94_8 program on the thermal cycler: Choose the preheat lid option and set to 100°C Reaction volume is 19 µl 94°C for 8 minutes Hold at 4°C
Total Samples
Numeric
Default = 0
Decimal Places Displayed = 0
Step File Placeholders
- Log - Automatically attached
Sample Table
- Sample Display Default = Expand
- Well Sort Order = Column
- Table Columns - Global Fields
  Category
  Field Name
  Field Type
  Options
  Additional Options and Dropdown Items
  Container
  Container Name
  Built-in
  Container
  LIMS ID (Container)
  Built-in
  Container
  Well
  Built-in
  Derived Sample
  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 Stranded mRNA Prep Ligation v1.1)

Master Step Name = Synthesize First Strand cDNA v1.0
Step Type = No Outputs
Reagent Kits
- Illumina Stranded mRNA Prep, Ligation
  - Supplier = Illumina
  - 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 Stranded mRNA Prep Ligation 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 Stranded mRNA Prep, Ligation
  - Supplier = Illumina
  - 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 Stranded mRNA Prep Ligation v1.1)

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Adenylate 3ʹ Ends (Illumina Stranded mRNA Prep Ligation v1.1)

Master Step Name = Adenylate 3ʹ Ends - RNA Prep v1.0
Step Type = No Outputs
Reagent Kits
- Illumina Stranded mRNA Prep, Ligation
  - Supplier = Illumina
  - 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
Instruction Notes
Multiline Text
Read Only
Default = Add 12.5 µl ATL4 to each well.
Thermal Cycler Program
Text
Default = ATAIL
Thermal Cycler Program Notes
Multiline Text
Read Only
Default = Choose the preheat lid option and set to 100°C Reaction volume is 30 µl 37°C for 30 minutes 70°C for 5 minutes 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

Step 2: Ligate Anchors (Illumina Stranded mRNA Prep Ligation v1.1)

Master Step Name = Ligate Anchors - RNA Prep v1.0
Step Type = No Outputs
Reagent Kits
- Illumina Stranded mRNA Prep, Ligation
  - Supplier = Illumina
  - 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
Instruction Notes
Multiline Text
Read Only
Default = Add 5 µl STL to each well.
Thermal Cycler Program
Text
Default = LIG
Thermal Cycler Program Notes
Multiline Text