Illumina Stranded mRNA Prep Ligation 1.1

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	Read Only	Default = Choose the preheated lid option and set to 100°C Reaction volume is 38 µl 30°C for 10 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 3: Clean Up Fragments (Illumina Stranded mRNA Prep Ligation v1.1)

• Master Step Name = Clean Up Fragments - RNA Prep 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
• Step File Placeholders

  • Log - Automatically attached

• Sample Table

  • Sample Display Default = Collapse

  • Well Sort Order = Column

  • Table Columns - Global Fields

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

Step 4: Amplify Library (Illumina Stranded mRNA Prep Ligation v1.1)

• Master Step Name = Amplify Library - RNA Prep v1.0

• Step Type = Add Labels

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}-{AppliedReagentLabels}

• 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

Add Labels

• Label Groups

  • IDT for Illumina RNA UD Indexes Set A Ligation

  • IDT for Illumina RNA UD Indexes Set A-D Ligation

  • IDT for Illumina RNA UD Indexes Set B Ligation

  • IDT for Illumina RNA UD Indexes Set C Ligation

  • IDT for Illumina RNA UD Indexes Set D Ligation

  • Illumina RNA UD Indexes Set A B C D Ligation

  • Illumina RNA UD Indexes Set A Ligation

  • Illumina RNA UD Indexes Set B Ligation

  • Illumina RNA UD Indexes Set C Ligation

  • Illumina RNA UD Indexes Set D Ligation

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  PCR Cycles	Text Dropdown	Custom Entries	Presets 15 13 10 Default = 15
  Recommendations for PCR Cycle Number	Multiline Text	Read Only	Default = Use 15 cycles for 10 ng of input DNA Use 13 cycles for 100 ng of input DNA Use 10 cycles for 1000 ng of input DNA
  Thermal Cycler Program	Text		Default = PCR
  Thermal Cycler Program Notes	Multiline Text	Read Only	Default = Choose the preheat lid option and set to 100°C Reaction volume is 50 µl u 98°C for 30 seconds X cycles of: 98°C for 10 seconds 60°C for 30 seconds 72°C for 30 seconds 72°C for 5 minutes Hold at 4°C for ≤ 16 hours
• 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 5: Clean Up Library (Illumina Stranded mRNA Prep Ligation v1.1)

• Master Step Name = Clean Library 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
• 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