RNA Library Prep v1.1

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

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Dilute RNA (AmpliSeq for Illumina Focus 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

Group of Defaults

Immune Repertoire Panel

Directions = If enough RNA is available, dilute to intermediate concentration ~20-1000 ng/uL.

TCR beta-SR Panel

Default = If enough RNA is available, dilute to intermediate concentration ~20-1000 ng/uL.

Step Data

Group of Defaults = Immune Repertoire Panel

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

Desired Concentration (ng/uL)

Numeric

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 Focus 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 Focus 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 Focus 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
- AmpliSeq Focus Panel for RNA
  - Supplier = Illumina
  - Catalog Number = 20019164

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 - Focus Panel

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

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 = Row
Destination Containers
- 96 well plate

Record Details

Group of Defaults

Childhood Cancer Panel

AmpliSeq ERCC RNA Spike-in Mix = No
Directions = If RNA was prepared from FFPE tissue and never heat-treated, heat at 80°C for 10 minutes and then cool to room temperature before proceeding to reverse transcription.
Prep Input Amount (ng) = 10
Recommended Prep Input (ng) = Each reaction requires 1-100 ng of DNase-treated total RNA. The recommended input is 10 ng RNA.
Thermal Cycler Program = RT
Total Samples = 0

Comprehensive Panel v3

AmpliSeq ERCC RNA Spike-in Mix = No
Directions = If RNA was prepared from FFPE tissue and never heat-treated, heat at 80°C for 10 minutes and then cool to room temperature before proceeding to reverse transcription.
Prep Input Amount (ng) = 20
Recommended Prep Input (ng) = Each reaction requires 1-100 ng of DNase-treated total RNA per pool. The recommended input is 10 ng RNA per pool.
Thermal Cycler Program = RT
Total Samples = 0

Focus Panel

AmpliSeq ERCC RNA Spike-in Mix = No
Directions = If RNA was prepared from FFPE tissue and never heat-treated, heat at 80°C for 10 minutes and then cool to room temperature before proceeding to reverse transcription.
Prep Input Amount (ng) = 10
Recommended Prep Input (ng) = Each reaction requires 1-100 ng of DNase-treated total RNA. The recommended input is 10 ng RNA.
Thermal Cycler Program = RT

Immune Repertoire Panel

AmpliSeq ERCC RNA Spike-in Mix = No
Directions = If RNA was prepared from FFPE tissue and never heat-treated, heat at 80°C for 10 minutes and then cool to room temperature before proceeding to reverse transcription.
Prep Input Amount (ng) = 1000
Recommended Prep Input (ng) = Each reaction requires 10-1000 ng of DNase-treated total RNA. If RNA is not limiting, the recommended input is 1000 ng RNA. High RNA input amounts are recommended for rare clones.
Thermal Cycler Program = RT

Immune Response Panel

AmpliSeq ERCC RNA Spike-in Mix = No
Directions = If RNA was prepared from FFPE tissue and never heat-treated, heat at 80°C for 10 minutes and then cool to room temperature before proceeding to reverse transcription.
Prep Input Amount (ng) = 10
Recommended Prep Input (ng) = Each reaction requires 1-100 ng of DNase-treated total RNA. The recommended input is 10 ng RNA.
Thermal Cycler Program = RT
Total Samples = 0

Myeloid Panel

AmpliSeq ERCC RNA Spike-in Mix = No
Directions = If RNA was prepared from FFPE tissue and never heat-treated, heat at 80°C for 10 minutes and then cool to room temperature before proceeding to reverse transcription.
Prep Input Amount (ng) = 10
Recommended Prep Input (ng) = Each reaction requires 10-100 ng of DNase-treated total RNA. The recommended input is 10 ng RNA.
Thermal Cycler Program = RT
Total Samples = 0

TCR beta-SR panel

AmpliSeq ERCC RNA Spike-in Mix = No
Directions = If RNA was prepared from FFPE tissue and never heat-treated, heat at 80°C for 10 minutes and then cool to room temperature before proceeding to reverse transcription.
Prep Input Amount (ng) = 1000
Recommended Prep Input (ng) = Each reaction requires 50-1000 ng of DNase-treated total RNA. If RNA is not limited, the recommended input is 1000 ng RNA
Thermal Cycler Program = RT

Transcriptome HGx

AmpliSeq ERCC RNA Spike-in Mix = No
Directions = If RNA was prepared from FFPE tissue and never heat-treated, heat at 80°C for 10 minutes and then cool to room temperature before proceeding to reverse transcription.
Prep Input Amount (ng) = 10
Recommended Prep Input (ng) = Each reaction requires 1-100 ng of DNase-treated total RNA. The recommended input is 10 ng RNA.
Thermal Cycler Program = RT
Total Samples = 0

Step Data

Group of Defaults = Focus Panel

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

Recommended Prep Input (ng)

Multiline Text

Read Only

Default = Each reaction requires 1-100 ng of DNase-treated total RNA. The recommended input is 10 ng RNA.

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 Focus Panel v1.1)

Master Step Name = Amplify cDNA Targets (AmpliSeq for Illumina Focus Panel v1.1)
Step Type = Standard
Derived Sample Generation = Fixed, 1
Naming Convention = {InputItemName}
Reagent Kits
- AmpliSeq Focus Panel for RNA
  - Supplier = Illumina
  - Catalog Number = 20019164
- AmpliSeq Library PLUS for Illumina
  - Supplier = Illumina
  - Catalog Number = 24 - 20019101; 96 - 200191102; 384 - 200191103
  - Website = www.illumina.com

Automations

Sample Type and Calculate Master Mix - Focus

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 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 * step.::Total Samples:: * 1.1 ; \
step.::5x AmpliSeq Focus Panel (uL):: = 4 * step.::Total Samples:: * 1.1 ; \
step.::Nuclease-free water (uL):: = 2 * 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 = Not Used

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' "

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

Sample Type and Calculate Master Mix

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.::Sample Type:: = ::cDNA:: ; \
step.::5X AmpliSeq HiFi Mix (uL):: = 4 * step.::Total Samples:: * 1.1 ; \
step.::Nuclease-free water (uL):: = 2 * step.::Total Samples:: * 1.1' \
-log {compoundOutputFileLuid0}"

Set Sample Type and Calculate Master Mix - 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 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):: = 4.0 * step.::Total Samples:: * 1.1 ; \
step.::5X AmpliSeq Immune Repertoire Panel (uL):: = 4.0 * step.::Total Samples:: * 1.1 ; \
step.::Nuclease-free water (uL):: = 7.0 * step.::Total Samples:: * 1.1' \
-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

5x AmpliSeq Focus Panel (uL)

Numeric

Decimal Places Displayed = 1

5X AmpliSeq HiFi Mix (uL)

Numeric

Decimal Places Displayed = 1

Nuclease-free water (uL)

Numeric

Decimal Places Displayed = 1

PCR Cycles

Numeric

Decimal Places Displayed = 1

Thermal Cycler Program

Text

Default = AMP

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

Step 6: Partially Digest Amplicons (AmpliSeq for Illumina Focus Panel v1.1)

Master Step Name = Partially Digested Amplicons (AmpliSeq for Illumina Focus Panel 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

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 cDNA- Focus Panel

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 'Queue for Plate Transfer' \
--FIELD_VALUE 'Transfer Amplicons' \
--WORKFLOW 'DNA Library Prep AmpliSeq for Illumina Focus Panel v1.1' \
--STEP 'Transfer RNA and DNA Amplicons (AmpliSeq for Illumina Focus Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' "

ℹ️ 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
Thermal Cycler Program
Text
Default = FUPA
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

Queue for Plate Transfer

Text

Default = Transfer Amplicons

Derived Sample

Sample Name

Built-in

Project

Project Name

Built-in

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