RNA Library Prep v1.1

PreviousDNA Library Prep v1.1 NextEqualizer v1.1

Last updated 7 days ago

Overview

The RNA Library Prep AmpliSeq for Illumina Myeloid Panel is part of the AmpliSeq for Illumina Myeloid Panel protocols which include the following functionality:

Preconfigured AmpliSeq for Illumina Myeloid Panel protocol that supports the preparation of up to 96 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 Myeloid Panel v1.1)

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Dilute RNA (AmpliSeq for Illumina Myeloid 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 Myeloid 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 to Final Concentration (AmpliSeq for Illumina Myeloid 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 Myeloid 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 - Myeloid

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.::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}"

Calculate Input Amount - Myeloid 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):: < 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}"

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 - 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- 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

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.::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}"

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

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

AmpliSeq ERCC RNA Spike-in Volume (uL)

Numeric

Default = 0
Range = 0 To 2
Decimal Places Displayed = 2

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 Myeloid 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
- AmpliSeq Myeloid Panel for Illumina
  - Supplier = Illumina
  - Catalog Number = 20024478
  - Website = www.illumina.com

Automations

Set Sample Type and Calculate Master Mix - Myeloid 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.::Nuclease-free water (uL):: = 7.0 * 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 Samples - Myeloid 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 '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' "

ℹ️ 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 cDNA - 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 -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'"

ℹ️ 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' "

ℹ️ 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 - TCR beta-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 -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 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)

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

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