RNA Library Prep v1.1

Overview

The RNA Library Prep AmpliSeq for Illumina Childhood Cancer Panel is part of the AmpliSeq for Illumina Childhood Cancer Panel protocols that include the following functionality:

• Preconfigured AmpliSeq for Illumina Childhood Cancer Panel protocol that supports the preparation of up to 384 uniquely indexed libraries of genomic DNA or total RNA using the AmpliSeq for Illumina workflow.

• 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.

• There is no extraction protocol in this workflow because the samples are already extracted.

Protocol 1: RNA Library Prep (AmpliSeq for Illumina Childhood Cancer Panel v1.1)

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Dilute RNA (AmpliSeq for Illumina Childhood Cancer Panel v1.1)

• Master Step Name = Dilute RNA (AmpliSeq for Illumina v1.1)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

Automations

Copy Concentration and Set Sample Volume

• 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 'output.::Concentration:: = input.::Concentration:: ; output.::Conc. Units:: = input.::Conc. Units:: ; output.::Sample Volume (uL):: = 5' \
-log {compoundOutputFileLuid0}"

Calculate Input Amount - RNA

• 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} -u {username} -p {password} \
      script:evaluateDynamicExpression \
        -t true \
        -h false \
        -exp 'output.::Total Volume (uL):: = (output.::Concentration::/step.::Desired Concentration (ng/uL)::) * output.::Sample Volume (uL):: ; \
output.::Nuclease-free water (uL):: = output.::Total Volume (uL):: - output.::Sample Volume (uL):: ; \
output.::Input Amount (ng):: = step.::Desired Concentration (ng/uL):: * output.::Total Volume (uL):: ' \
        -log {compoundOutputFileLuid0}"

Copy Desired Concentration

• Trigger Location = Step

• 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 'output.::Desired Concentration (ng/uL):: = step.::Desired Concentration (ng/uL)::' \
-log {compoundOutputFileLuid0}"

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 = 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

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Desired Concentration (ng/uL)	Numeric		Range = 0 To 1000
  Directions	Multiline Text	Read Only	Default = If enough RNA is available, dilute to intermediate concentration ~20-50 ng/uL.

• Step File Placeholders

  • Log - 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
    Derived Sample	Concentration	Numeric	Required Field	Decimal Places Displayed = 2
    Derived Sample	Conc. Units	Text	Required Field
    Derived Sample	Input Amount (ng)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Nuclease-free water (uL)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Sample Name	Built-in
    Derived Sample	Sample Volume (uL)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Total Volume (uL)	Numeric		Decimal Places Displayed = 2

Step 2: Qubit (AmpliSeq for Illumina Childhood Cancer Panel v1.1)

• Master Step Name = Qubit (AmpliSeq for Illumina v1.1)

• Step Type = Standard QC

• Measurement Generation = Fixed, 1

• Naming Convention = {InputItemName}

Automations

Assign QC flags (Qubit QC)

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

Set Next Step and Copy Concentration

• 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:: ; input.::Concentration:: = output.::Concentration:: ; input.::Conc. Units:: = output.::Conc. Units::' -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	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
  Criteria 2 - Operator	Text		Default = <=
  Criteria 2 - Source Data Field	Text		Default = Concentration
  Criteria 2 - Threshold Value	Numeric

• Step File Placeholders

  • Log - Automatically attached

  • QC Log File - Automatically attached

  • QC Result File - Automatically attached

  • Upload File - Manually uploaded

• Sample Table

  • Enable QC Flags = Yes

  • Sample Display Default = Expand

  • Well Sort Order = Row

  • File Column Options

    • File Column Display = Hide

    • File Attachment Method = Auto

  • Table Columns - Global Fields

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    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

Step 3: Dilute RNA (AmpliSeq for Illumina Childhood Cancer Panel v1.1)

• Master Step Name = Dilute RNA (AmpliSeq for Illumina v1.1)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

Automations

Copy Concentration and Set Sample Volume

• 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 'output.::Concentration:: = input.::Concentration:: ; output.::Conc. Units:: = input.::Conc. Units:: ; output.::Sample Volume (uL):: = 5' \
-log {compoundOutputFileLuid0}"

Calculate Input Amount - RNA

• 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} -u {username} -p {password} \
      script:evaluateDynamicExpression \
        -t true \
        -h false \
        -exp 'output.::Total Volume (uL):: = (output.::Concentration::/step.::Desired Concentration (ng/uL)::) * output.::Sample Volume (uL):: ; \
output.::Nuclease-free water (uL):: = output.::Total Volume (uL):: - output.::Sample Volume (uL):: ; \
output.::Input Amount (ng):: = step.::Desired Concentration (ng/uL):: * output.::Total Volume (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}"

Copy Desired Concentration

• Trigger Location = Step

• 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 'output.::Desired Concentration (ng/uL):: = step.::Desired Concentration (ng/uL)::' \
-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	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
  Desired Concentration (ng/uL)	Numeric		Range = 0 To 1000
  Directions	Multiline Text	Read Only	Default = If enough RNA is available, dilute to intermediate concentration ~20-50 ng/uL.

• Step File Placeholders

  • Log - 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
    Derived Sample	Concentration	Numeric	Required Field	Decimal Places Displayed = 2
    Derived Sample	Conc. Units	Text	Required Field
    Derived Sample	Input Amount (ng)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Nuclease-free water (uL)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Sample Name	Built-in
    Derived Sample	Sample Volume (uL)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Total Volume (uL)	Numeric		Decimal Places Displayed = 2

Step 4: Reverse Transcribe (AmpliSeq for Illumina Childhood Cancer Panel v1.1)

• Master Step Name = Reverse Transcribe RNA (AmpliSeq for Illumina v1.1)

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • AmpliSeq cDNA Synthesis for Illumina

    • Supplier = Illumina

    • Catalog Number = 20022654

    • Website = www.illumina.com

Automations

Copy Concentration, Total Volume and Input Amount

• 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 'output.::Concentration:: = input.::Concentration:: ; \
output.::Conc. Units:: = input.::Conc. Units:: ; \
output.::Total Volume (uL):: = input.::Total Volume (uL):: ; \
output.::Input Amount (ng):: = input.::Input Amount (ng):: ; \
step.::Desired Concentration (ng/uL):: = input.::Desired Concentration (ng/uL)::' \
-log {compoundOutputFileLuid0}"

Calculate Input Amount

• 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} -u {username} -p {password} \
script:evaluateDynamicExpression \
-t true \
-h false \
-exp 'step.::Prep Input Volume (uL):: = step.::Prep Input Amount (ng):: / step.::Desired Concentration (ng/uL):: ; \
if (step.::Prep Input Volume (uL):: < 7) {step.::Nuclease-free water (uL):: = (7 - step.::Prep Input Volume (uL)::) * step.::Total Samples::*1.1} else {step.::Nuclease-free water (uL):: = 0} ; \
step.::5X AmpliSeq cDNA Reaction Mix (uL):: = step.::Total Samples:: * 1.1 * 2 ; \
step.::10X AmpliSeq RT Enzyme Mix (uL):: = step.::Total Samples:: * 1.1 * 1' \
-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}"

Calculate Input Amount - Focus Panel

• 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 true \
-h false \
-exp 'step.::Prep Input Volume (uL):: = step.::Prep Input Amount (ng):: / step.::Desired Concentration (ng/uL):: ; \
if (step.::Prep Input Volume (uL):: < 7) {step.::Nuclease-free water (uL):: = (7 - step.::Prep Input Volume (uL)::) * step.::Total Samples::*1.1} else {step.::Nuclease-free water (uL):: = 0} ; \
step.::5X AmpliSeq cDNA Reaction Mix (uL):: = step.::Total Samples:: * 1.1 * 2; \
step.::10X AmpliSeq RT Enzyme Mix (uL):: = step.::Total Samples:: * 1.1 * 1' \
-log {compoundOutputFileLuid0}"

Calculate Input Amount - Immune Repertoire Panel

• 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 true \
-h false \
-exp 'step.::Prep Input Volume (uL):: = step.::Prep Input Amount (ng):: / step.::Desired Concentration (ng/uL):: ; \
if (step.::Prep Input Volume (uL):: < 3.5) {step.::Nuclease-free water (uL):: = (3.5 - step.::Prep Input Volume (uL)::) * step.::Total Samples::*1.1} else {step.::Nuclease-free water (uL):: = 0} ; \
step.::5X AmpliSeq cDNA Reaction Mix (uL):: = step.::Total Samples:: * 1.1 * 1 ; \
step.::10X AmpliSeq RT Enzyme Mix (uL):: = step.::Total Samples:: * 1.1 * 0.5' \
-log {compoundOutputFileLuid0}"

Calculate Input Amount - Immune Response Panel

• 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 true \
-h false \
-exp 'step.::Prep Input Volume (uL):: = step.::Prep Input Amount (ng):: / step.::Desired Concentration (ng/uL):: ; \
if (step.::Prep Input Volume (uL):: < 7.0) {step.::Nuclease-free water (uL):: = (7.0 - (step.::Prep Input Volume (uL):: + output.::AmpliSeq ERCC RNA Spike-in Volume (uL)::)) * step.::Total Samples::*1.1} else {step.::Nuclease-free water (uL):: = 0} ; \
step.::5X AmpliSeq cDNA Reaction Mix (uL):: = step.::Total Samples:: * 1.1 * 1 ; \
step.::10X AmpliSeq RT Enzyme Mix (uL):: = step.::Total Samples:: * 1.1 * 0.5 ; \
step.::AmpliSeq ERCC RNA Spike-in Mix (uL):: = step.::Total Samples:: * output.::AmpliSeq ERCC RNA Spike-in Volume (uL):: *1.1' \
-log {compoundOutputFileLuid0}"

Calculate Input Amount - Myeloid Panel

• 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 true \
-h false \
-exp 'step.::Prep Input Volume (uL):: = step.::Prep Input Amount (ng):: / step.::Desired Concentration (ng/uL):: ; \
if (step.::Prep Input Volume (uL):: < 3.5) {step.::Nuclease-free water (uL):: = (3.5 - step.::Prep Input Volume (uL)::) * step.::Total Samples::*1.1} else {step.::Nuclease-free water (uL):: = 0} ; \
step.::5X AmpliSeq cDNA Reaction Mix (uL):: = step.::Total Samples:: * 1.1 * 1 ; \
step.::10X AmpliSeq RT Enzyme Mix (uL):: = step.::Total Samples:: * 1.1 * 0.5' \
-log {compoundOutputFileLuid0}"

Calculate Input Amount - TCRbeta-SR

• 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 true \
-h false \
-exp 'step.::Prep Input Volume (uL):: = step.::Prep Input Amount (ng):: / step.::Desired Concentration (ng/uL):: ; \
if (step.::Prep Input Volume (uL):: < 7) {step.::Nuclease-free water (uL):: = (7 - step.::Prep Input Volume (uL)::) * step.::Total Samples::*1.1} else {step.::Nuclease-free water (uL):: = 0} ; \
step.::5X AmpliSeq cDNA Reaction Mix (uL):: = step.::Total Samples:: * 1.1 * 2 ; \
step.::10X AmpliSeq RT Enzyme Mix (uL):: = step.::Total Samples:: * 1.1 * 1' \
-log {compoundOutputFileLuid0}"

Calculate Input Amount - Transcriptome Human Genome Expression Panel

• 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 true \
-h false \
-exp 'step.::Prep Input Volume (uL):: = step.::Prep Input Amount (ng):: / step.::Desired Concentration (ng/uL):: ; \
if (step.::Prep Input Volume (uL):: < 3.5) {step.::Nuclease-free water (uL):: = (3.5 - (step.::Prep Input Volume (uL):: + output.::AmpliSeq ERCC RNA Spike-in Volume (uL)::)) * step.::Total Samples::*1.1} else {step.::Nuclease-free water (uL):: = 0} ; \
step.::5X AmpliSeq cDNA Reaction Mix (uL):: = step.::Total Samples:: * 1.1 * 1 ; \
step.::10X AmpliSeq RT Enzyme Mix (uL):: = step.::Total Samples:: * 1.1 * 0.5 ; \
step.::AmpliSeq ERCC RNA Spike-in Mix (uL):: = step.::Total Samples:: * output.::AmpliSeq ERCC RNA Spike-in Volume (uL):: *1.1' \
-log {compoundOutputFileLuid0}"

Calculate Input Amount- Custom RNA Panel

• 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 true \
-h false \
-exp 'step.::Prep Input Volume (uL):: = step.::Prep Input Amount (ng):: / step.::Desired Concentration (ng/uL):: ; \
if (step.::Prep Input Volume (uL):: < 7) {step.::Nuclease-free water (uL):: = (7 - step.::Prep Input Volume (uL)::) * step.::Total Samples::*1.1} else {step.::Nuclease-free water (uL):: = 0} ; \
step.::5X AmpliSeq cDNA Reaction Mix (uL):: = step.::Total Samples:: * 1.1 * 2; \
step.::10X AmpliSeq RT Enzyme Mix (uL):: = step.::Total Samples:: * 1.1 * 1' \
-log {compoundOutputFileLuid0}"

Copy Concentration, Total Volume and Input Amount - Myeloid

• 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 '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 'output.::Concentration:: = input.::Desired Concentration (ng/uL):: ; \
output.::Conc. Units:: = input.::Conc. Units:: ; \
output.::Total Volume (uL):: = input.::Total Volume (uL):: ; \
output.::Input Amount (ng):: = input.::Input Amount (ng):: ; \
step.::Desired Concentration (ng/uL):: = input.::Desired Concentration (ng/uL)::' \
-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	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 = Row

  • Placement Pattern = Column

• Destination Containers

  • 96 well plate

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  5X AmpliSeq cDNA Reaction Mix (uL)	Numeric		Decimal Places Displayed = 1
  10X AmpliSeq RT Enzyme Mix (uL)	Numeric		Decimal Places Displayed = 1
  Desired Concentration (ng/uL)	Numeric	Read Only
  Nuclease-free water (uL)	Numeric		Decimal Places Displayed = 1
  Prep Input Amount (ng)	Numeric		Default = 2 Range = 2 To 1000
  Prep Input Volume (uL)	Numeric		Decimal Places Displayed = 2
  Thermal Cycler Program	Text	Read Only	Default = RT
  Total Samples	Numeric		Default = 0 Decimal Places Displayed = 0

• Step File Placeholders

  • Log - 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
    Derived Sample	Concentration	Numeric	Required Field	Decimal Places Displayed = 2
    Derived Sample	Conc. Units	Text	Required Field
    Derived Sample	Input Amount (ng)	Numeric		Decimal Places Displayed = 0
    Derived Sample	Sample Name	Built-in
    Derived Sample	Total Volume (uL)	Numeric		Decimal Places Displayed = 2

Step 5: Amplify cDNA Targets (AmpliSeq for Illumina Childhood Cancer Panel v1.1)

• Master Step Name = Amplify cDNA Targets (AmpliSeq for Illumina v1.1)

• Step Type = Standard

• Derived Sample Generation = Fixed, 2

• Naming Convention = {InputItemName}_{OutputItemSubsetNumber}

• Reagent Kits

  • AmpliSeq Childhood Cancer Panel for Illumina

    • Supplier = Illumina

    • Catalog Number = 20028446

    • Website = www.illumina.com

  • AmpliSeq Library PLUS for Illumina

    • Supplier = Illumina

    • Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103

    • Website = www.illumina.com

Automations

Set Sample Type and 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 'output.::Sample Type:: = ::cDNA:: ; \
step.::5X AmpliSeq HiFi Mix (uL):: = 4.5 * step.::Total Samples:: * 1.1 ; \
step.::Nuclease-free water (uL):: = 3.5 * step.::Total Samples:: * 1.1' \
-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}"

Routing Samples

• Trigger Location = Step

• 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 -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \
\
--FIELD_NAME 'Sample Type' \
--FIELD_VALUE 'cDNA' \
--WORKFLOW 'DNA Library Prep AmpliSeq for Illumina Childhood Cancer Panel v1.1' \
--STEP 'Transfer RNA and DNA Amplicons (AmpliSeq for Illumina Childhood Cancer Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' "

ℹ The workflow version and step version for the routing script may vary depending on the version of the IPP.

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	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
  5X AmpliSeq HiFi Mix (uL)	Numeric		Decimal Places Displayed = 2
  Instructions - Childhood Cancel Panel	Multiline Text		Default = Add 5 µl 2X AmpliSeq Comprehensive Cancer Panel Pool 1 (blue cap) to the first well, and 5 µl 2X AmpliSeq Comprehensive Cancer Panel Pool 2 (blue cap) to the second. Add 5 µl 2X AmpliSeq Comprehensive Cancer Panel Pool 3 (blue cap) to the third well, and 5 µl 2X AmpliSeq Comprehensive Cancer Panel Pool 4 (blue cap) to the fourth.
  Nuclease-free water (uL)	Numeric		Decimal Places Displayed = 2
  PCR Cycles	Text
  Thermal Cycler Program	Text		Default = AMP_RNA
  Total Samples	Numeric		Default = 0

• Step File Placeholders

  • Log - 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