RNA Library Prep v1.1

Overview

The RNA Library Prep AmpliSeq for Illumina TCR beta-SR Panel is part of the AmpliSeq for Illumina TCR beta-SR Panel protocols which include the following functionality:

Preconfigured AmpliSeq for Illumina TCR beta-SR Panel protocol that supports the preparation of up to 96 uniquely indexed libraries of 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.

Protocol 1: RNA Library Prep (AmpliSeq for Illumina TCR beta-SR Panel v1.1)

Protocol Type = Sample Prep

Next Steps Configuration

Step 1: Dilute RNA (AmpliSeq for Illumina TCR beta-SR 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)

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 = TCR beta-SR Panel
- Master Step Fields
Step File Placeholders
- Log - Automatically attached
Sample Table
- Sample Display Default = Expand
- Well Sort Order = Row
- Table Columns - Global Fields

Step 2: Qubit (AmpliSeq for Illumina TCR beta-SR 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)

Record Details

Step Data (Master Step Fields)
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

Step 3: Dilute RNA (AmpliSeq for Illumina TCR beta-SR 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)

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 = TCR beta-SR Panel
- Master Step Fields
Step File Placeholders
- Log - Automatically attached
Sample Table
- Sample Display Default = Expand
- Well Sort Order = Row
- Table Columns - Global Fields

Step 4: Reverse Transcribe (AmpliSeq for Illumina TCR beta-SR 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 - TCRbeta-SR

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 - 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

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 - 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- 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)

Placement = Enabled

Defaults
- Sample Grouping = Group by Containers
- Well Sort Order = Row
- Placement Pattern = Column
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 = TCR beta-SR panel
- Master Step Fields
Step File Placeholders
- Log - Automatically attached
Sample Table
- Sample Display Default = Expand
- Well Sort Order = Row
- Table Columns - Global Fields

Step 5: Amplify cDNA Targets (AmpliSeq for Illumina TCR beta-SR Panel v1.1)

Master Step Name = Amplify cDNA Targets Standard (AmpliSeq for Illumina v1.1)
Step Type = Standard
Derived Sample Generation = Fixed, 1
Naming Convention = {InputItemName}
Reagent Kits
- 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 - TCR beta-SR Panel

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):: = 4.0 * step.::Total Samples:: * 1.1 ; \
step.::5X AmpliSeq TCR beta-SR Panel (uL):: = 4.0 * step.::Total Samples:: * 1.1 ; \
step.::Nuclease-free water (uL):: = 2.0 * 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 cDNA - TCRbeta SR

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 TCR beta-SR Panel v1.1' \
--STEP 'Transfer RNA and DNA Amplicons (AmpliSeq for Illumina TCR beta-SR Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"

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

Copy Desired RNA Conc. & Calculate Master Mix - Custom RNA Pool 1

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 'output.::Diluted RNA Conc. (ng/uL):: = input.::Desired Final Concentration (ng/uL):: ; \
output.::Master Mix (uL):: = 10 ; \
step.::Total Samples:: = step.::Total Samples:: + 1 ; \
step.::5X AmpliSeq HiFi Mix (uL):: = step.::Total Samples:: *1.1* 4 ; \
step.::5X AmpliSeq Custom RNA Panel (uL):: = step.::Total Samples:: * 1.1 * 4 ; \
output.::RNA Volume (uL):: = 10 / output.::Diluted RNA Conc. (ng/uL):: ; \
if (output.::RNA Volume (uL):: < 10) {output.::Nuclease-free water (uL):: = 20 -(output.::Master Mix (uL):: + output.::RNA Volume (uL)::)}' \
-log {compoundOutputFileLuid0}"

Copy Desired RNA Conc. & Calculate Master Mix - Custom RNA Pool 2

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 'output.::Diluted RNA Conc. (ng/uL):: = input.::Desired Final Concentration (ng/uL):: ; \
output.::Master Mix (uL):: = 10 ; \
step.::Total Samples:: = step.::Total Samples:: + 1 ; \
step.::5X AmpliSeq HiFi Mix (uL):: = step.::Total Samples:: *1.1* 4.5 ; \
step.::5X AmpliSeq Custom RNA Panel (uL):: = step.::Total Samples:: * 1.1 * 4 ; \
output.::RNA Volume (uL):: = 10 / output.::Diluted RNA Conc. (ng/uL):: ; \
if (output.::RNA Volume (uL):: < 10) {output.::Nuclease-free water (uL):: = 20 -(output.::Master Mix (uL):: + output.::RNA Volume (uL)::)}' \
-log {compoundOutputFileLuid0}"

Routing Samples - 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 -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 Myeloid Panel v1.1' \
--STEP 'Transfer RNA and DNA Amplicons (AmpliSeq for Illumina Myeloid Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' "

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

Routing Samples - TCRbeta SR 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 -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 TCR beta-SR Panel v1.1' \
--STEP 'Transfer RNA and DNA Amplicons (AmpliSeq for Illumina TCR beta-SR Panel v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' "

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

Set Sample Type and Calculate Master Mix - Custom RNA

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 Custom RNA Panel (uL):: = 4.0 * step.::Total Samples:: * 1.1 ; \
step.::Nuclease-free water (uL):: = 2.0 * 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}"

Set Sample Type and Calculate Master Mix - 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 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.::Nuclease-free water (uL):: = 2.0 * step.::Total Samples:: * 1.1 ; \
step.::5x AmpliSeq Immune Response Panel (uL):: = 2.0 * step.::Total Samples:: * 1.1' \
-log {compoundOutputFileLuid0}"

Set Sample Type and Calculate Master Mix - 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 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.::Nuclease-free water (uL):: = 7.0 * step.::Total Samples:: * 1.1' \
-log {compoundOutputFileLuid0}"

Set Sample Type and Calculate Master Mix - TGx 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.::Nuclease-free water (uL):: = 3.0 * step.::Total Samples:: * 1.1' \
-log {compoundOutputFileLuid0}"

Queue/Ice Bucket

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

Record Details

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