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

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Last updated 1 month ago

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)

Ice Bucket

Defaults
- Sample Grouping = Group by Containers
- Well Sort Order = Column
Sample Table (Column Headers)

Record Details

Step Data (Master Step Fields)
Sample Table
- Sample Display Default = Expand
- Well Sort Order = Column
- Table Columns - Global Fields

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)

Record Details

Step Data (Master Step Fields)
Step File Placeholders
- Log - Automatically attached
Sample Table
- Sample Display Default = Collapse
- Well Sort Order = Column
- Table Columns - Global Fields

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)

Record Details

Step Data (Master Step Fields)
Step File Placeholders
- Log - Automatically attached
Sample Table
- Sample Display Default = Collapse
- Well Sort Order = Column
- Table Columns - Global Fields

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)

Record Details

Step Data (Master Step Fields)
Step File Placeholders
- Log - Automatically attached
Sample Table
- Sample Display Default = Collapse
- Well Sort Order = Column
- Table Columns - Global Fields

Step 2: Amplify Tagmented cDNA (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

Master Step Name = Amplify Tagmented cDNA v1.0
Step Type = Add Labels
Derived Sample Generation = Fixed, 1
Naming Convention = {InputItemName}-{AppliedReagentLabels}

Automations

Calculate PCR 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.::EPM (uL):: = 23 * step.::Total Samples:: ; step.::Nuclease-free ultrapure water (uL):: = 23 * 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)

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 DNA-RNA UD Indexes Set A Tagmentation
- IDT for Illumina DNA-RNA UD Indexes Set B Tagmentation
- IDT for Illumina DNA-RNA UD Indexes Set C Tagmentation
- IDT for Illumina DNA-RNA UD Indexes Set 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)
Step File Placeholders
- Log - Automatically attached
Sample Table
- Sample Display Default = Collapse
- Well Sort Order = Column
- Table Columns - Global Fields

Step 3: Clean Library (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

Master Step Name = Clean Library (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)
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)

Record Details

Step Data (Master Step Fields)
Step File Placeholders
- Log - Automatically attached
Sample Table
- Sample Display Default = Collapse
- Well Sort Order = Column
- Table Columns - Global Fields

Protocol 3: Normalize Libraries (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Qubit Library QC (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

Master Step Name = Qubit Library QC v1.0
Step Type = Standard QC
Measurement Generation = Fixed, 1
Naming Convention = {InputItemName}

Automations

Assign QC flags

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 -u {username} -p {password} \
      script:assignQC \
        -i {processURI:v2} \
        -log {compoundOutputFileLuid1} \
        -qcResult {compoundOutputFileLuid2}"

Set Next Step - Advance

Trigger Location = Not Used

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)

Record Details

Step Data (Master Step Fields)
Step File Placeholders
- Log - Automatically attached
- QC Result File - Manually uploaded
Sample Table
- Enable QC Flags = Yes
- Sample Display Default = Expand
- Well Sort Order = Column
- File Column Options
  - File Column Display = Show
  - File Attachment Method = Manual
- Table Columns - Global Fields

Step 2: BioAnalyzer Library QC (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

Master Step Name = BioAnalyzer Library QC v1.0
Step Type = Standard QC
Measurement Generation = Fixed, 1
Naming Convention = {InputItemName}

Automations

Generate Bioanalyzer Input file

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/DriverFileGenerator.jar script:driver_file_generator -i {processURI:v2} -u {username} -p {password} -t /opt/gls/clarity/extensions/ngs-common/v5/EPP/conf/readonly/bioA_driver_file_template.csv -o {compoundOutputFileLuid0}.csv -l {compoundOutputFileLuid1}  && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar script:addBlankLines -i {stepURI:v2} -u {username} -p {password} -f {compoundOutputFileLuid0}.csv -l {compoundOutputFileLuid1} -sep COMMA -b ',False,' -h 1 -c LIMSID -pre 'Sample '"

Parse Bioanalyzer XML and assign QC flags

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 -u {username} -p {password} -i {processURI:v2} \
      script:parseBioAnalyzer \
        -inputFile {compoundOutputFileLuid2} \
        -log {compoundOutputFileLuid5} \
        -configFile '/opt/gls/clarity/extensions/conf/v5/bioanalyzer/defaultBioAnalyzerDNAConfig.groovy' \
      script:assignQC \
        -log {compoundOutputFileLuid6} \
        -qcResult {compoundOutputFileLuid7}"

Set Next Step - Advance

Trigger Location = Not Used

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)

Placement = Enabled

Defaults
- Sample Grouping = Group by Containers
- Well Sort Order = Column
- Placement Pattern = Column
Destination Containers
- BioAnalyzer DNA High Sensitivity Chip
- BioAnalyzer DNA 1000 Chip

Record Details

Step Data (Master Step Fields)
Step File Placeholders
- Bioanalyzer Driver File - Automatically attached
- Bioanalyzer Driver File Generation Log File - Automatically attached
- Bioanalyzer XML Result File (required) - Manually uploaded
- Result File (optional) - Manually uploaded
- PDF Summary File (optional) - Manually uploaded
- Bioanalyzer XML Parsing Log File - Automatically attached
- QC Assignment Log File - Automatically attached
- QC Assignment Report - Automatically attached
Sample Table
- Enable QC Flags = Yes
- Sample Display Default = Expand
- Well Sort Order = Column
- File Column Options
  - File Column Display = Show
  - File Attachment Method = Manual
- Table Columns - Global Fields

Step 3: Dilute Libraries (Illumina RNA Prep Tagmentation (L) with Enrichment v1.0)

Master Step Name = Dilute Libraries 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, RSB, & Final Volumes

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):: * output.::Final Volume (uL)::) / output.::Starting Concentration (ng/uL):: ; if (step.::Enrichment Plexity:: == 1) {output.::Final Volume (uL):: = 7.5} else {output.::Final Volume (uL):: = 2.5} ; step.::RSB (uL):: = output.::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)

Record Details

Step Data (Master Step Fields)
Step File Placeholders
- Log - Automatically attached
Sample Table
- Sample Display Default = Expand
- Well Sort Order = Column
- Table Columns - Global Fields

Step 4: Hybridize Probes (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

Master Step Name = Hybridize Probes (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)
Step Type = Standard
Derived Sample Generation = Fixed, 1
Naming Convention = {InputItemName}
Reagent Kits
- Illumina Exome Panel
  - Supplier = Illumina
  - Catalog Number = 20020183
  - Website = www.illumina.com
- 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)

Record Details

Step Data (Master Step Fields)
Step File Placeholders
- Log - Manually uploaded
Sample Table
- Sample Display Default = Collapse
- Well Sort Order = Row
- Table Columns - Global Fields

Step 5: Capture Hybridize Probes (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

Master Step Name = Capture Hybridize Probes 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 Elution 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.::EEL (uL):: = 28.5 * step.::Total Samples:: ; step.::HP3 (uL):: = 1.5 * step.::Total Samples::' \
-log {compoundOutputFileLuid0}"

Set Next Step - Advance

Trigger Location = Record Details
Trigger Style = Automatic upon exit