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TruSeq Small RNA v1.0

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Last updated 16 days ago

Overview

TruSeq Small RNA v1.0 includes the following functionality:

Preconfigured TruSeq Small RNA v1.0 protocol that explains how to prepare total RNA or purified small RNA using a Illumina® TruSeq® Small RNA Library Prep Kit.
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.

It is not required to have the total RNA samples quantified prior to starting this protocol, however it is highly recommended to ensure the starting number of samples.

Protocol 1: TruSeq Small RNA v1.0

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Ligate Adapters (TruSeq Small RNA v1.0)

Master Step Name = Ligate Adapters (TruSeq Small RNA v1.0.10)
Step Type = Standard
Derived Sample Generation = Fixed, 1
Naming Convention = {SubmittedSampleName}
Reagent Kits
- T4 RNA Ligase 2, Deletion Mutant
  - Supplier = Epicentre
  - Catalog Number = LR2D1132K; LR2D11310K
- TruSeq Small RNA Library Prep Kit, Core Solutions
  - Supplier = Illumina
  - Catalog Number = RS-200-0012; RS-200-0024; RS-200-0036; RS-200-0048
  - Website = https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/truseq-small-rna.html
- TruSeq Small RNA Library Prep Kit, Indices A, B, C or D
  - Supplier = Illumina
  - Catalog Number = RS-200-0012; RS-200-0024; RS-200-0036; RS-200-0048
  - Website = https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/truseq-small-rna.html
Control Types
- Human Brain Total RNA
  - Supplier = Thermo Fisher Scientific
  - Catalog Number = AM7962

The version of Ligate Adapters master step name may be different depending on the version of IPP installed.

Automations

Calculate 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} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp '(step.::Total samples:: = step.::Total samples:: + 1)' -log {compoundOutputFileLuid0} &&  /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.::HML Volume (ul):: = step.::Total samples:: * 2.2) ; (step.::RNase Inhibitor Volume (ul):: = step.::Total samples:: * 1.1) ; (step.::T4 RNA Ligase 2, Depletion Mutant Volume (ul):: = step.::Total samples:: * 1.1) ; (step.::RA5 Volume (ul):: = step.::Total samples:: * 1.21) ; (step.::10mM ATP Volume (ul):: = step.::Total samples:: * 1.21) ; (step.::T4 RNA Ligase Volume (ul):: = step.::Total samples:: * 1.21)' -log {compoundOutputFileLuid1}"

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 = Row
Sample Table
- Column Headers
  Category
  Field Name
  Field Type
  Options
  Additional Options and Dropdown Items
  Container
  Container Name
  Built-in
  Container
  Well
  Built-in
  Derived Sample
  Sample Name
  Built-in
  Derived Sample
  Waiting
  Built-in
- Expanded View Fields
  Category
  Field Name
  Field Type
  Options
  Additional Options and Dropdown Items
  Container
  LIMS ID (Container)
  Built-in
  Project
  Project Name
  Built-in

Record Details

Step Data (Master Step Fields)
Field Name
Field Type
Options
Additional Options and Dropdown Items
Comment
Multiline Text
HML Volume (ul)
Numeric
Decimal Places Displayed = 2
RA5 Volume (ul)
Numeric
Decimal Places Displayed = 2
RNase Inhibitor Volume (ul)
Numeric
Decimal Places Displayed = 2
T4 RNA Ligase 2, Depletion Mutant Volume (ul)
Numeric
Decimal Places Displayed = 2
T4 RNA Ligase Volume (ul)
Numeric
Decimal Places Displayed = 2
10mM ATP Volume (ul)
Numeric
Decimal Places Displayed = 2
Step File Placeholders
- Log File - Automatically attached
- Log File - Automatically attached
Sample Table
- Sample Display Default = Expand
- Well Sort Order = Row
- 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: Perform Reverse Transcription (TruSeq Small RNA v1.0)

Master Step Name = Perform Reverse Transcription (TruSeq Small RNA v1.0.10)
Step Type = Standard
Derived Sample Generation = Fixed, 1
Naming Convention = {SubmittedSampleName}
Reagent Kits
- SuperScript II Reverse Transcriptase
  - Supplier = Life Technologies
  - Catalog Number = 18064-014
  - Website = http://www.lifetechnologies.com/order/catalog/product/18064014
- TruSeq Small RNA Library Prep Kit, Core Solutions
  - Supplier = Illumina
  - Catalog Number = RS-200-0012; RS-200-0024; RS-200-0036; RS-200-0048
  - Website = https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/truseq-small-rna.html
- TruSeq Small RNA Library Prep Kit, Indices A, B, C or D
  - Supplier = Illumina
  - Catalog Number = RS-200-0012; RS-200-0024; RS-200-0036; RS-200-0048
  - Website = https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/truseq-small-rna.html

The version of Perform Reverse Transcription master step name may be different depending on the version of IPP installed.

Automations

Calculate 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} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp '(step.::Total samples:: = step.::Total samples:: + 1)' -log {compoundOutputFileLuid0} && /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.::5X First Strand Buffer Volume (ul):: = step.::Total samples:: * 2.2) ; (step.::12.5 mM dNTP Mix Volume (ul):: = step.::Total samples:: * 0.55) ; (step.::100mM DTT Volume (ul):: = step.::Total samples:: * 1.1) ; (step.::RNase Inhibitor Volume (ul):: = step.::Total samples:: * 1.1) ; (step.::SuperScript II Reverse Transcriptase Volume (ul):: = step.::Total samples:: * 1.1)' -log {compoundOutputFileLuid1}"

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 = Row
Sample Table
- Column Headers
  Category
  Field Name
  Field Type
  Options
  Additional Options and Dropdown Items
  Container
  Container Name
  Built-in
  Container
  Well
  Built-in
  Derived Sample
  Sample Name
  Built-in
  Derived Sample
  Waiting
  Built-in
- Expanded View Fields
  Category
  Field Name
  Field Type
  Options
  Additional Options and Dropdown Items
  Container
  LIMS ID (Container)
  Built-in
  Project
  Project Name
  Built-in

Record Details

Step Data (Master Step Fields)
Field Name
Field Type
Options
Additional Options and Dropdown Items
Comment
Multiline Text
Dilute 25mM dNTP mix to 12.5mM (per library): 0.5ul of 25mM dNTP Mix and 0.5ul of Ultrapure water.
Toggle Switch
Default = None Set
RNase Inhibitor Volume (ul)
Numeric
Decimal Places Displayed = 2
SuperScript II Reverse Transcriptase Volume (ul)
Numeric
Decimal Places Displayed = 2
5X First Strand Buffer Volume (ul)
Numeric
Decimal Places Displayed = 2
12.5 mM dNTP Mix Volume (ul)
Numeric
Decimal Places Displayed = 2
100mM DTT Volume (ul)
Numeric
Decimal Places Displayed = 2
Step File Placeholders
- Log File - Automatically attached
- Log File - Automatically attached
Sample Table
- Sample Display Default = Expand
- Well Sort Order = Row
- 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: Amplify Libraries (TruSeq Small RNA v1.0)

Master Step Name = Amplify Libraries (TruSeq Small RNA v1.0.10)
Step Type = Add Labels
Derived Sample Generation = Fixed, 1
Naming Convention = {SubmittedSampleName}
Reagent Kits
- TruSeq Small RNA Library Prep Kit, Core Solutions
  - Supplier = Illumina
  - Catalog Number = RS-200-0012; RS-200-0024; RS-200-0036; RS-200-0048
  - Website = https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/truseq-small-rna.html
- TruSeq Small RNA Library Prep Kit, Indices A, B, C or D
  - Supplier = Illumina
  - Catalog Number = RS-200-0012; RS-200-0024; RS-200-0036; RS-200-0048
  - Website = https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/truseq-small-rna.html

The version of Amplify Libraries master step name may be different depending on the version of IPP installed.

Automations

Calculate 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} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp '(step.::Total samples:: = step.::Total samples:: + 1)' -log {compoundOutputFileLuid0} && /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.::Ultrapure water Volume (ul):: = step.::Total samples:: * 9.35) ; (step.::PML Volume (ul):: = step.::Total samples:: * 27.5) ; (step.::RP1 Volume (ul):: = (step.::Total samples:: * 2.2).round(2)) ' -log {compoundOutputFileLuid1}"

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

Placement = Enabled

Defaults
- Sample Grouping = Group by Containers
- Well Sort Order = Column
- Placement Pattern = Column
Destination Containers
- Tube

Add Labels

Label Groups
- TruSeq Small RNA

Record Details

Step Data (Master Step Fields)
Field Name
Field Type
Options
Additional Options and Dropdown Items
Comment
Multiline Text
Ultrapure water Volume (ul)
Numeric
Decimal Places Displayed = 2
PML Volume (ul)
Numeric
Decimal Places Displayed = 2
RP1 Volume (ul)
Numeric
Decimal Places Displayed = 2
Add 2ul of chosen RPIX to each sample.
Toggle Switch
Default = None Set
Step File Placeholders
- Log File - Automatically attached
- Log File - Automatically attached
Sample Table
- Sample Display Default = Collapse
- Well Sort Order = Row
- 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: Bioanalyzer QC (Library Validation) (TruSeq Small RNA v1.0)

Master Step Name = Bioanalyzer QC (Library Validation) v2.0
Step Type = Standard QC
Measurement Generation = Fixed, 1
Naming Convention = {InputItemName} Bioanalyzer

Automations

Generate Bioanalyzer Input file