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

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	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
  EBLTL (uL)	Numeric
  Instruction Notes	Multiline Text	Read Only	Default = Add 32.5 µl Tagmentation Master Mix to each well.
  Nuclease-free ultrapure water (uL)	Numeric
  TB1 (uL)	Numeric
  Thermal Cycler Program	Text		Default = TAG
  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 55°C for 5 minutes Hold at 10°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 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)

  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

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)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  EPM (uL)	Numeric		Decimal Places Displayed = 0
  Instruction Notes	Multiline Text	Read Only	Default = Add 40 µl PCR Master Mix to each well.
  Nuclease-free ultrapure water (uL)	Numeric		Decimal Places Displayed = 0
  PCR Cycles	Text Dropdown	Custom Entries	Presets 14 cycles for High-quality RNA with DV200 > 80% 17 cycles for FFPE and RNA with DV200 < 80%
  Thermal Cycler Program	Text		Default = TAG_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 72°C for 3 minutes u 98°C for 3 minutes X cycles of: u 98°C for 20 seconds u 60°C for 30 seconds 72°C for 1 minute u 72°C for 3 minutes Hold at 10°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: 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)

  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

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)

  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
  Criteria 1 - Operator	Text		Default = >=
  Criteria 1 - Source Data Field	Text		Default = Concentration
  Criteria 1 - Threshold Value	Numeric		Default = 200
  Criteria 2 - Operator	Text		Default = <=
  Criteria 2 - Source Data Field	Text		Default = Concentration
  Criteria 2 - Threshold Value	Numeric		Default = 1500

• 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

    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	Concentration	Numeric	Required Field	Decimal Places Displayed = 2
    Derived Sample	Conc. Units	Text	Required Field
    Derived Sample	Sample Name	Built-in
    Measurement	Concentration	Numeric		Decimal Places Displayed = 2
    Measurement	Conc. Units	Text
    Project	Project Name	Built-in

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)

  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

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)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Criteria 1 - Operator	Text Dropdown	Custom Entries	Presets >= <= = !=
  Criteria 1 - Source Data Field	Text Dropdown	Custom Entries	Presets Peak 2 Size - bp Peak 2 Conc. Peak 2 Molarity
  Criteria 1 - Threshold	Numeric
  Criteria 2 - Operator	Text Dropdown	Custom Entries	Presets >= <= = !=
  Criteria 2 - Source Data Field	Text Dropdown	Custom Entries	Presets Peak 2 Size - bp Peak 2 Conc. Peak 2 Molarity
  Criteria 2 - Threshold	Numeric
  Use strict matching for Bioanalyzer results	Toggle Switch		Default = None Set

• 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

    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: 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)

  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
  Enrichment Plexity	Numeric Dropdown	Required Field Custom Entries	Presets 1 3
  Final Diluted Concentration (ng/uL)	Numeric		Default = 200
  Instruction Notes	Multiline Text	Read Only	Default = In one well of a new PCR plate, combine the applicable number of 200 ng libraries: If Enrichment Plexity is 1 then add 7.5 ul of the sample. If Enrichment Plexity is 3 then add 2.5 ul of each sample to one well with a final volume of 7.5 ul.

• 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	Final Volume (uL)	Numeric		Decimal Places Displayed = 0
    Derived Sample	RSB (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 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)

  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
  Thermal Cycler Program	Text		Default = HYB
  Thermal Cycler Program Notes	Multiline Text	Read Only	Default = Choose the preheat lid option and set to 100°C Reaction volume is 25 µl u 95°C for 5 minutes 18 cycles of 1 minute each: u 94°C for the first cycle Decrease 2°C per subsequent cycle 58°C for 90 minutes Hold at 58°C for ≤ 24 hours

• Step File Placeholders

  • Log - Manually uploaded

• 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 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

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
  EEL (uL)	Numeric		Decimal Places Displayed = 0
  HP3 (uL)	Numeric		Decimal Places Displayed = 0
  Instruction Notes	Multiine Text	Read Only	Default = Add 23 µl Elution Master Mix to each well.
  Thermal Cycler Program Notes	Multiline Text	Read Only	Default = Choose the preheat lid option and set to 70°C Reaction volume is 100 µl Hold at 58°C
  Total Samples	Numeric		Default = 0 Decimal Places Displayed = 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 6: Amplify Enriched Library (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

• Master Step Name = Amplify Enriched Library 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

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
  Thermal Cycler Program	Text		Default = AMP
  Thermal Cycler Program Notes	Multiline Text	Read Only	Default = Choose the preheat lid option and set to 100°C Reaction volume is 50 µl 98°C for 30 seconds 14 cycles of: 98°C for 10 seconds 60°C for 30 seconds 72°C for 30 seconds 72°C for 5 minutes Hold at 10°C

• Step File Placeholders

  • Log - Manually uploaded

• 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 7: Clean Up Enriched Library (Illumina RNA Prep with Enrichment (L) Tagmentation v1.1)

• Master Step Name = Clean Up Enriched 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 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

• Step File Placeholders

  • Log - 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