AmpliSeq for Illumina Focus Panel

Protocol 1: DNA Library Prep AmpliSeq for Illumina Focus Panel

Master Step 1: Dilute RNA (AmpliSeq for Illumina)

Step Type: Standard

Input = RNA with concentration

Output = Diluted RNA (1-100 ng)

Reagent Kits

Kit Name

Supplier

Cat. #

AmpliSeq Library PLUS for Illumina

Illumina

24 - 20019101; 96 - 200191102; 384 - 200191103
Placement

Setting

Value

Specification

Destination Containers

Tube

Configured on step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Desired Concentration (ng/µl)

Numeric

Range = 20–50
 

Multiline Text Fields

Directions

  

Default

Read Only

• If enough RNA is available, dilute to intermediate concentration ~20-50 ng/uL.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

Table Columns

Concentration

Numeric

Global field - derived sample
   

Conc. Units

Text

Global field - derived sample
   

Input Amount (ng)

Numeric

Global field - derived sample
   

Nuclease-free water (µl)

Numeric

Global field - derived sample
   

Sample Volume (µl)

Numeric

Global field - derived sample
   

Total Volume (µl)

Numeric

Global field - derived sample
   

Desired Concentration (ng/uL)

Numeric

Set by EPP
Automation

Automation Name

Setting

Value

Specification

Copy Concentration and Set Sample Volume

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

  
     

Command line

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

Trigger Location

Record Details

  
 

Trigger Style

Manual Button

  
     

Command line

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.::Sample Volume (uL):: ' \ -log {compoundOutputFileLuid0}"

Copy Desired Concentration

Trigger Location

Step

  
 

Trigger Style

Automatic upon exit

  
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Manual

Safe Stopping Point

Optional

• Qubit (Ampliseq for Illumina Focus Panel)
• Reverse Transcribe RNA (Ampliseq for Illumina Focus Panel)

Master Step 2: Qubit (AmpliSeq for Illumina)

Step Type: Standard QC

Input = Diluted RNA (1-100 ng)

Output = Measeurment

Placement

Setting

Value

Specification

Destination Containers

Tube

Configured on step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Criteria 1 - Source Data Field

Text

Default

• Concentration
   

Criteria 1 - Operator

Text

Default

• >=
   

Criteria 1 - Threshold Value

Numeric

 
   

Criteria 2 - Source Data Field

Text

Default

• Concentration
   

Criteria 2 - Operator

Text

Preset

• <=
   

Criteria 2 - Threshold Value

Numeric

 

Step File Placeholders

 

File Name

Log

Auto
     

QC Log File

Auto
     

QC Result File

Auto
     

Upload File

Manual

Sample Table

Table Columns

Concentration

Numeric

Global field - derived sample & measurement
   

Conc. Units

Text

Global field - derived sample & measurement
Automation

Automation Name

Setting

Value

Specification

Assign QC Flags

Trigger Location

Record Details

  
 

Trigger Style

Manual Button

  
     

Command line

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

  
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

  

Master Step 3: Dilute RNA (AmpliSeq for Illumina)

Step Type: Standard

Input = Diluted RNA with concentration

Output = Diluted RNA (1-100 ng)

Placement

Setting

Value

Specification

Destination Containers

Tube

Configured on step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Desired Concentration (ng/µl)

Numeric

Range = 20–50

Step File Placeholders

 

File Name

Log

Auto

Sample Table

Table Columns

Concentration

Numeric

Global field - derived sample
   

Conc. Units

Text

Global field - derived sample
   

Input Amount (ng)

Numeric

Global field - derived sample
   

Nuclease-free water (µl)

Numeric

Global field - derived sample
   

Sample Volume (µl)

Numeric

Global field - derived sample
   

Total Volume (µl)

Numeric

Global field - derived sample
Automation

Automation Name

Setting

Value

Specification

Copy Concentration and Set Sample Volume

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

  
     

Command line

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

Trigger Location

Record Details

  
 

Trigger Style

Manual Button

  
     

Command line

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.::Sample Volume (uL):: ' \ -log {compoundOutputFileLuid0}"

Set Next Step - Advance and Set Desired Conc

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

  
     

Command line

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

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

  

Master Step 4: Reverse Transcribe RNA (AmpliSeq for Illumina)

Step Type: Standard

Input = RNA with concentration

Output = Reverse Transcribed RNA

Reagent Kits

Kit Name

Supplier

Cat. #

AmpliSeq cDNA Synthesis for Illumina

Illumina

20022654
Placement

Setting

Value

Specification

Destination Containers

96 well plate

Configured on step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

Text

Preset

• RT
   

5X AmpliSeq cDNA Reaction Mix (µl)

Numeric

  
   

10X AmpliSeq RT Enzyme Mix (µl)

Numeric

  
   

Nuclease-free water (µl)

Numeric

  
   

Prep Input Volume (µl)

Numeric

Calculated
   

Prep Input Amount (ng)

Numeric

Required

• User input
• Range = 1–100
   

Total Samples

Numeric

Default

• 0

Step File Placeholders

 

File Name

Log

Auto

Sample Table

Table Columns

Concentration

Numeric

Global field - derived sample
   

Conc. Units

Text

Global field - derived sample
   

Total Volume (µl)

Numeric

Global field - derived sample
   

Input Amount (ng)

Numeric
• Global field - derived sample
Automation

Automation Name

Setting

Value

Specification

Copy Concentration, Total Volume and Input Amount - Childhood Cancer Panel

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

  
     

Command line

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}"

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

  
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

  

Master Step 5: Amplify cDNA Targets Standard (AmpliSeq for Illumina)

Step Type: standard

Input = Reverse Transcribed RNA

Output = cDNA

Reagent Kits

Kit Name

Supplier

Cat. #

AmpliSeq Library PLUS for Illumina

Illumina

24 - 20019101; 96 - 200191102; 384 - 200191103

AmpliSeq Focus Panel for Illumina

Illumina

20019164
Placement

Setting

Value

Specification

Destination Containers

96 well plate

Configured on step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

Text

Default

• AMP_RNA
   

5X AmpliSeq HiFi Mix (uL)

Numeric

Decimal Places = 2
   

Nuclease-free water (µl)

Numeric

Decimal Places = 2
   

PCR Cycles

Numeric

  

Step File Placeholders

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Calculate Total Samples and Master Mix

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

  
     

Command line

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.::5x AmpliSeq Focus RNA 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

  
     

Command line

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}["]

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 6: Partially Digested Amplicons (AmpliSeq for Illumina)

Step Typ = No Outputs

Input = Pooled samples

Output = Partially Digested Amplicons

Reagent Kits

Kit Name

Supplier

Cat. #

AmpliSeq Library PLUS for Illumina

Illumina

24 - 20019101; 96 - 200191102; 384 - 200191103
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

Text

Preset

• FUPA

Step File Placeholders

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

  
     

Command line

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

  
     

Command line

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 v1.0' \--STEP 'Transfer RNA and DNA Amplicons (AmpliSeq for Illumina Focus Panel v1.0)' \--INPUTS_OR_OUTPUTS 'OUTPUTS' ["]

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Protocol 2: DNA Library Prep (AmpliSeq for Focus Panel)

Master Step 1: Dilute DNA (AmpliSeq for Illumina)

Step Type: Standard

Input = DNA with concentration

Output = Diluted DNA (1-100 ng)

Reagent Kits

Kit Name

Supplier

Cat. #

AmpliSeq Library PLUS for Illumina

Illumina

24 - 20019101; 96 - 200191102; 384 - 200191103
Placement

Setting

Value

Specification

Destination Containers

Tube

Configured on step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Desired Concentration (ng/µl)

Numeric

Range = 20–50
 

Multiline Text Fields

Directions

Multiline Text

Default

Read Only

• If enough DNA is available, dilute to intermediate concentration ~20–50 ng/µl.

Step File Placeholders

 

File Name

Log

Auto

Sample Table

Table Columns

Concentration

Numeric

Global field - derived sample
   

Conc. Units

Text

Global field - derived sample
   

Input Amount (ng)

Numeric

Global field - derived sample
   

Low TE (µl)

Numeric

Global field - derived sample
   

Sample Volume (µl)

Numeric

Global field - derived sample
   

Total Volume (µl)

Numeric

Global field - derived sample
Automation

Automation Name

Setting

Value

Specification

Copy Concentration and Set Sample Volume

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

  
     

Command line

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

Trigger Location

Record Details

  
 

Trigger Style

Manual Button

  
     

Command line

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.::Low TE (uL):: = output.::Total Volume (uL):: - output.::Sample Volume (uL):: ; \ output.::Input Amount (ng):: = step.::Desired Concentration (ng/uL):: * output.::Sample Volume (uL):: ' \ -log {compoundOutputFileLuid0}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Manual

Safe Stopping Point

Optional

• Qubit (Ampliseq for Illumina Focus Panel)
• Amplify Targets (Ampliseq for Illumina Focus Panel)

Master Step 2: Qubit (AmpliSeq for Illumina)

Step Type: Standard QC

Input = Diluted DNA (1-100 ng)

Output = Measurement

Placement

Setting

Value

Specification

Destination Containers

Tube

Configured on step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Criteria 1 - Source Data Field

Text

Default

• Concentration
   

Criteria 1 - Operator

Text

Default

• >=
   

Criteria 1 - Threshold Value

Numeric

 
   

Criteria 2 - Source Data Field

Text

Default

• Concentration
   

Criteria 2 - Operator

Text

Default

• <=
   

Criteria 2 - Threshold Value

Numeric

 

Step File Placeholders

 

File Name

Log

Auto
     

QC Log File

Auto
     

QC Result File

Auto
     

Upload File

Manual

Sample Table

Table Columns

Concentration

Numeric

Global field - derived sample
   

Conc. Units

Text

Global field - derived sample
Automation

Automation Name

Setting

Value

Specification

Assign QC Flags

Trigger Location

Record Details

  
 

Trigger Style

Manual Button

  
     

Command line

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

  
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Manual

Safe Stopping Point

  

Master Step 3: Dilute DNA (AmpliSeq for Illumina)

Step Type: Standard

Input = DNA with concentration

Output = Diluted DNA (1-100 ng)

Reagent Kits

Kit Name

Supplier

Cat. #

AmpliSeq Library PLUS for Illumina

Illumina

24 - 20019101; 96 - 200191102; 384 - 200191103
Placement

Setting

Value

Specification

Destination Containers

Tube

Configured on step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Desired Concentration (ng/µl)

  

Range = 20–50

Step File Placeholders

 

File Name

Log

Auto

Sample Table

Table Columns

Concentration

Numeric

Global field - derived sample
   

Conc. Units

Text

Global field - derived sample
   

Input Amount (ng)

Numeric

Global field - derived sample
   

Low TE (µl)

Numeric

Global field - derived sample
   

Sample Volume (µl)

Numeric

Global field - derived sample
   

Total Volume (µl)

Numeric

Global field - derived sample
Automation

Automation Name

Setting

Value

Specification

Copy Concentration and Set Sample Volume

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

  
     

Command line

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

Trigger Location

Record Details

  
 

Trigger Style

Manual Button

  
     

Command line

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.::Low TE (uL):: = output.::Total Volume (uL):: - output.::Sample Volume (uL):: ; \ output.::Input Amount (ng):: = step.::Desired Concentration (ng/uL):: * output.::Sample Volume (uL):: ' \ -log {compoundOutputFileLuid0}"

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

  
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 4: Amplify Targets Duplicates (AmpliSeq for Illumina)

Step Type: Standard

Input = Diluted DNA (1-100 ng)

Output = Amplified DNA

Sample Name = {InputItemName}

Reagent Kits

Kit Name

Supplier

Cat. #

AmpliSeq Library PLUS for Illumina

Illumina

24 - 20019101; 96 - 200191102; 384 - 200191103

AmpliSeq Focus Panel for Illumina

Illumina

20019164
Placement

Setting

Value

Specification

Destination Containers

96 well plate

Configured on step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

Text

Default

• AMP
   

PCR Cycles

Numeric

  

Step File Placeholders

 

File Name

Log

Auto

Sample Table

Table Columns

5X AmpliSeq HiFi Mix (µl)

Numeric

Global field - derived sample
   

Concentration

Numeric

Global field - derived sample
   

Conc. Units

Text

Global field - derived sample
   

Nuclease-free water (µl)

Numeric
• Global field - derived sample
• Calculated
   

Prep Input Volume (µl)

Numeric
• Global field - derived sample
• Calculated
   

Prep Input Amount (ng)

Numeric

Required

• User input
• Range = 1–100
Automation

Automation Name

Setting

Value

Specification

Concentration, Total Volume and Input Amount- Focus Panel

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

  
     

Command line

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.::Total Volume (uL):: = input.::Total Volume (uL):: ; output.::Input Amount (ng):: = input.::Input Amount (ng):: ; output.::5X AmpliSeq HiFi Mix (uL):: = 4.0 ; output.::5x AmpliSeq Focus Panel (uL):: = 4.0'\

-log {compoundOutputFileLuid0}["]

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

  
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 6: Partially Digested Amplicons (AmpliSeq for Illumina)

Step Type: No Outputs

Input = Pooled samples

Output = Partially Digested Amplicons

Reagent Kits

Kit Name

Supplier

Cat. #

AmpliSeq Library PLUS for Illumina

Illumina

24 - 20019101; 96 - 200191102; 384 - 200191103
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

Text

Default

• FUPA

Step File Placeholders

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

  
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 5: Transfer RNA and DNA Amplicons (AmpliSeq for Illumina)

Step Type: Standard

Input = cDNA and DNA amplicon samples

Output = cDNA and DNA samples

Placement

Setting

Value

Specification

Destination Containers

96 well plate

Configured on step

Proof of concept on API page for automatic plating.

Record Details

Section

Subsection

Setting

Value

Specification

Step File Placeholders

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

  
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 6: Ligate Indexes (AmpliSeq for Illumina)

Step Type: Add Labels

Input = Partially Digested Amplicons

Output = Ligated Libraries

Reagent Kits

Kit Name

Supplier

Cat. #

AmpliSeq Library PLUS for Illumina

Illumina

24 - 20019101; 96 - 200191102; 384 - 200191103
Placement

Setting

Value

Destination Containers

96 well plate
Add Labels

Setting

Value

Label Groups

AmpliSeq CD Indexes Set A for Illumina - AmpliSeq CD Indexes Set A for Illumina-label-sheet.xlsx
 

AmpliSeq CD Indexes Set B for Illumina - AmpliSeq CD Indexes Set B for Illumina-label-sheet.xlsx
 

AmpliSeq CD Indexes Set C for Illumina - AmpliSeq CD Indexes Set C for Illumina-label-sheet.xlsx
 

AmpliSeq CD Indexes Set D for Illumina - AmpliSeq CD Indexes Set D for Illumina-label-sheet.xlsx
 

AmpliSeq UD Indexes for Illumina (24 Indexes) - AmpliSeq UD Indexes for Illumina (24 Indexes)-label-sheet.xlsx
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

Text

Preset

• LIGATE

Step File Placeholders

 

File Name

Log

Auto

Sample Table

Table Columns

AmpliSeq Workflow

Text Dropdown

Required

• Standard Workflow or Equalizer Workflow (preset)
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

  
     

Command line

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}"

Route AmpliSeq Samples - Focus Panel

Trigger Location

Step

  
 

Trigger Style

Automatic upon exit

  
     

Command line

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 'AmpliSeq Workflow' \

--FIELD_VALUE 'Standard Workflow' \

--WORKFLOW 'Standard Workflow AmpliSeq for Illumina Focus Panel v1.0' \

--STEP 'Clean Up Library (AmpliSeq for Illumina Focus Panel v1.0)' \

--INPUTS_OR_OUTPUTS 'OUTPUTS' \

\

--FIELD_NAME 'AmpliSeq Workflow' \

--FIELD_VALUE 'Equalizer Workflow' \

--WORKFLOW 'Equalizer AmpliSeq for Illumina Focus Panel v1.0' \

--STEP 'Clean Up Library (AmpliSeq for Illumina Focus Panel v1.0)' \

--INPUTS_OR_OUTPUTS 'OUTPUTS'["]

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Protocol 3: Standard Workflow (AmpliSeq for Focus Panel)

Master Step 1: Clean Up Library (AmpliSeq for Illumina)

Step Type: No Outputs

Input = Ligated Libraries

Output = Cleaned Up Libraries

Reagent Kits

Kit Name

Supplier

Cat. #

Agencourt AMPure XP Beads

Thermo

NC9959336; NC9933872
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

70% EtOH Prep Date

Date

  

Step File Placeholders

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

  
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 2: Amplify Library (AmpliSeq for Illumina)

Step Type: No Outputs

Input = Cleaned Up Libraries

Output = Amplified Libraries

Reagent Kits

Kit Name

Supplier

Cat. #

Agencourt AMPure XP Beads

Thermo

NC9959336; NC9933872

AmpliSeq Library PLUS for Illumina

Illumina

24 - 20019101; 96 - 200191102; 384 - 200191103
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

 

Group of Defaults

• Standard Workflow
– AMP_7
   

Total Samples

Numeric

Default

• 0
   

1X Lib AMP Mix (µl)

Numeric

  
   

10X Library Amp Primers (µl)

Numeric

  

Step File Placeholders

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Calculate Total Samples and Calculate Master Mix

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

  
     

Command line

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 'step.::1X Lib Amp Mix:: = step.::Total Samples:: * 1.1 * 45 ; \ step.::10X Library Amp Primers:: = step.::Total Samples:: * 1.1 * 5' \ -log {compoundOutputFileLuid0}"

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

  
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 3: Perform Second Clean Up (AmpliSeq for Illumina0)

Step Type: Standard

Input = Amplified Libraries

Output = Cleaned Up Amplified Libraries

Reagent Kits

Kit Name

Supplier

Cat. #

Agencourt AMPure XP Beads

Thermo

NC9959336; NC9933872

AmpliSeq Library PLUS for Illumina

Illumina

24 - 20019101; 96 - 200191102; 384 - 200191103
Placement

Setting

Value

Destination Containers

96 well plate
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

70% EtOH Prep Date

Date

  

Step File Placeholders

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

  
     

Command line

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}"

End of Standard Protocol

Protocol 4: Equalizer Workflow (Ampliseq for Illumina Focus Panel)

Master Step 1: Clean Up Library (AmpliSeq for Illumina)

Step Type: No Outputs

Input = Ligated Libraries

Output = Cleaned Up Libraries

Reagent Kits

Kit Name

Supplier

Cat. #

Agencourt AMPure XP Beads

Thermo

NC9959336; NC9933872
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

70% EtOH Prep Date

Date

  

Step File Placeholders

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

  
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 2: Amplify Library (AmpliSeq for Illumina)

Step Type: No Outputs

Input = Cleaned Up Libraries

Output = Amplified Libraries

Reagent Kits

Kit Name

Supplier

Agencourt AMPure XP Beads

  

AmpliSeq Library PLUS for Illumina

  
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

Text

Group of Defaults

• Equalizer Workflow
– EQUAL
   

Total Samples

Numeric

Default

• 0
   

1X Lib AMP Mix (µl)

Numeric

  
   

10X Library Amp Primers (µl)

Numeric

  

Step File Placeholders

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Calculate Total Samples and Calculate Master Mix

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon entry

  
     

Command line

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 'step.::1X Lib Amp Mix:: = step.::Total Samples:: * 1.1 * 45 ; \ step.::10X Library Amp Primers:: = step.::Total Samples:: * 1.1 * 5' \ -log {compoundOutputFileLuid0}"

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

  
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 3: Perform Capture and Clean Up (AmpliSeq for Illumina)

Step Type: Standard

Input = Amplified Libraries

Output = Cleaned Up Libraries

Reagent Kits

Kit Name

Supplier

AmpliSeq Library Equalizer for Illumina

Illumina
Placement

Setting

Value

Specification

Destination Containers

96 well plate

Configured on step
Record Details

Section

Subsection

Setting

Value

Specification

Step File Placeholders

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

  
     

Command line

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}"

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 4: Elute Library (AmpliSeq for Illumina)

Step Type: Standard

Input = Cleaned Up Libraries

Output = Final Library

Reagent Kits

Kit Name

Supplier

AmpliSeq Library Equalizer for Illumina

Illumina
Placement

Setting

Value

Specification

Destination Containers

96 well plate

Configured on step
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

Text

Default

• ELUTE

Step File Placeholders

 

File Name

Log

Auto
Automation

Automation Name

Setting

Value

Specification

Set Next Step - Advance

Trigger Location

Record Details

  
 

Trigger Style

Automatic upon exit

  
     

Command line

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}"

End of Equalizer ProtocoL