Illumina Preset Protocol - Nextera Flex for Enrichment

Protocol 1: Prep Sample (Nextera Flex for Enrichment)

Master Step 1: Sort Sample (Nextera Flex for Enrichment)

Master Step type: No outputs

Input = Blood, Saliva, gDNA, FFPE DNA

Output = Blood Lysis, Saliva Lysis, gDNA, FFPE DNA

Placement = none

Record Details

Section

Subsection

Setting

Value

Specification

Step File Placeholders

 

File Name

Log

  

Sample Table

Table Columns

Sample Type

Text Dropdown
• Blood
• Saliva
• gDNA
• FFPE DNA
Automation

Automation Name

Setting

Value

Specification

Sort and Set Next Step

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 'if (input.::NFE Sample Type:: == ::Blood:: ) {nextStep=::Blood Lysis (Nextera Flex for Enrichment v1.0):: } ; if (input.::NFE Sample Type:: == ::Saliva:: ) {nextStep=::Saliva Lysis (Nextera Flex for Enrichment v1.0):: } ; if (input.::NFE Sample Type:: == ::gDNA:: ) {nextStep=::Qubit gDNA QC (Nextera Flex for Enrichment v1.0):: } ; if (input.::NFE Sample Type:: == ::FFPE DNA::) {nextStep=::Qubit FFPE DNA QC (Nextera Flex for Enrichment v1.0):: }' \ -log {compoundOutputFileLuid0}"

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 2: Blood Lysis (Nextera Flex for Enrichment)

This protocol is expected to generate > 100 ng of DNA output at the end of the blood lysis step.

Step type: Standard

Input = Blood

Output = Blood Lysis

Reagent Kits

Kit Name

Supplier

Cat. #

Flex Lysis Reagent Kit

Illumina

20015884

Agencourt AMPure XP beads, 5 ml

Beckman Coulter

A63880
Placement

Setting

Value

Destination Containers

96 well plate

Placement Pattern

Column

Placement Screen

Manual
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

Text

Preset

• BLP
   

Total Samples

Numeric

Calculated - Total Samples + 1
   

BLB (uL)

Numeric

Calculated = Total Samples * 7 * 1.1
   

PK1 (uL)

Numeric

Calculated = Total Samples * 2 * 1.1
   

Water (uL)

Numeric

Calculated = Total Samples * 31 * 1.1
   

80% EtOH Prep Date

Date

  

Step File Placeholders

 

File Name

Log

  

Sample Table

Table Columns

NFE Sample Type

Text

Copy from previous step
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 'output.::NFE Sample Type:: = input.::NFE Sample Type:: ; \step.::Total Samples:: = step.::Total Samples:: + 1 ; \step.::BLB (uL):: = step.::Total Samples:: * 1.1 * 7 ; \step.::PK1 (uL):: = step.::Total Samples:: * 1.1 * 2 ; \step.::Water (uL):: = step.::Total Samples:: * 1.1 * 31' \-log {compoundOutputFileLuid0}["]

Set Next Step - Remove and Copy NFE Sample Type

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 = ::REMOVE:: ; \output.::NFE Sample Type:: = input.::NFE Sample Type::' \-log {compoundOutputFileLuid0}["]

Route NFE - Blood

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 'NFE Sample Type' \--FIELD_VALUE 'Blood' \--WORKFLOW 'Nextera Flex for Enrichment v1.0' \--STEP 'Tagment Genomic DNA (Nextera Flex for Enrichment v1.0)' \--INPUTS_OR_OUTPUTS 'OUTPUTS' ["]

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 3: Saliva Lysis (Nextera Flex for Enrichment)

Master Step Type: Standard

Input = Saliva

Output = Saliva Lysis

Reagent Kits

Kit Name

Supplier

Cat. #

Agencourt AMPure XP beads, 5 ml

Beckman Coulter

A63880
Placement

Setting

Value

Specification

Destination Containers

96 well plate

Configured on step

Placement Pattern

Column

  

Placement Screen

Manual

  
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

80% EtOH Prep

Date

  

Step File Placeholders

 

File Name

Log

Auto

Sample Table

Table Columns

NFE Sample Type

Text

Copy from previous step
   

Sample Volume (uL)

Numeric

Preset

• 30
   

80% EtOH (uL)

Numeric

Preset

• 150
   

Water for Resuspension (uL) numeric

Numeric

Preset

• 30
Automation

Automation Name

Setting

Value

Specification

Copy NFE Sample Type

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.::NFE Sample Type:: = input.::NFE Sample Type::' \-log {compoundOutputFileLuid0}["]

Set Next Step - Remove & Copy NFE Sample Type

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 = ::REMOVE:: ; \output.::NFE Sample Type:: = input.::NFE Sample Type::' \-log {compoundOutputFileLuid0}["]

Route NFE - Saliva

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 'NFE Sample Type' \--FIELD_VALUE 'Saliva' \--WORKFLOW 'Nextera Flex for Enrichment v1.0' \--STEP 'Tagment Genomic DNA (Nextera Flex for Enrichment v1.0)' \--INPUTS_OR_OUTPUTS 'OUTPUTS' ["]

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 4: Qubit gDNA QC (Nextera Flex for Enrichment)

Master Step Type: Standard QC

Input = gDNA ( 10–1000 ng)

Output = Measurement

Placement

Setting

Value

Destination Containers

Tube

Placement Screen

Auto
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Criteria 1 - Source Data Field

  

Concentration
   

Criteria 1 - Operator

  

>=
   

Criteria 1 - Threshold Value

  

 
   

Criteria 2 - Source Data Field

  

Concentration
   

Criteria 2 - Operator

  

<=
   

Criteria 2 - Threshold Value

  

 

Step File Placeholders

 

File Name

Log

  
     

QC Log File

  
     

QC Result File

  
     

Upload File

  

Sample Table

Table Columns

Concentration

Numeric

  
   

Conc. Units

Text

  
   

NFE Sample Type

Text

Copy from previous step
Automation

Automation Name

Setting

Value

Specification

Assign QC Flags

Trigger Location

    
 

Trigger Style

    
     

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

    
 

Trigger Style

    
     

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 5: Dilute gDNA (Nextera Flex for Enrichment)

Master Step Type: Standard

Input = gDNA with concentration

Output = Diluted DNA (10-1000 ng)

Reagent Kits

Kit Name

Supplier

Cat. #

Nextera DNA Flex Pre-Enrichment Library Prep and Enrichment Reagents

Illumina

16 - 20025523; 96 - 20025524
Placement

Setting

Value

Destination Containers

96 well plate

Placement Pattern

Column

Placement Screen

Manual
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Desired gDNA Input (ng)

  

10-1000
   

Dilution Buffer

Text Dropdown

• 10 mM Tris-HCI, pH 7.5–8.5
• RSB
   

Sample Volume (uL)Numeric

  

Preset

• 5
   

Notes

Multiline Text

Preset

• For human gDNA samples and other large complex genomes, the recommended minimum gDNA input is 50 ng.

Step File Placeholders

 

File Name

Log

  

Sample Table

Table Columns

Concentration

Numeric

Value from previous step
   

Conc. Units (ng/uL)

Text

  
   

Total Volume (uL)

Numeric

Calculated = (Concentration * Sample Volume (uL)) / Desired gDNA Input (ng)
   

Dilution Volume (uL)

Numeric

Calculated = Total Volume (uL) - Sample Volume (uL)
Automation

Automation Name

Setting

Value

Specification

Copy Concentration

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::' \-log {compoundOutputFileLuid0}["]

Calculate Dilution Volume and Total Volume

Trigger Location

Record Details

  
 

Trigger Style

Manual

  
     

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.::Total Volume (uL):: = (output.::Concentration:: * step.::Sample Volume (uL)::) / step.::Desired gDNA Input (ng):: ; \output.::Dilution Volume (uL):: = output.::Total Volume (uL):: - step.::Sample Volume (uL)::' \-log {compoundOutputFileLuid0}["]

Set Next Step - Remove and Copy NFE Sample Type

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 = ::REMOVE:: ; \output.::NFE Sample Type:: = input.::NFE Sample Type::' \-log {compoundOutputFileLuid0}["]

Route NFE - gDNA

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 -u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \ \--FIELD_NAME 'NFE Sample Type' \--FIELD_VALUE 'gDNA' \--WORKFLOW 'Nextera Flex for Enrichment v1.0' \--STEP 'Tagment DNA (Nextera Flex for Enrichment v1.0)' \--INPUTS_OR_OUTPUTS 'OUTPUTS' ["]

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Master Step 6: Qubit FFPE DNA QC (Nextera Flex for Enrichment)

Step Type: Standard QC

Input = FFPE DNA (5 0–1000 ng)

Output = Measurement

Standard QC

Placement

Setting

Value

Destination Containers

Tube
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Criteria 1 - Source Data Field

  

Concentration
   

Criteria 1 - Operator

  

>=
   

Criteria 1 - Threshold Value

  

 
   

Criteria 2 - Source Data Field

  

Concentration
   

Criteria 2 - Operator

  

<=
   

Criteria 2 - Threshold Value

  

 

Step File Placeholders

 

File Name

Log

  
     

QC Log File

  
     

QC Result File

  
     

Upload File

  

Sample Table

Table Columns

Concentration

Numeric

Global field - derived sample & measurement
   

Conc. Units

Text

Global field - derived sample & measurement
   

NFE Sample type

Text

Copy from previous step
Automation

Automation Name

Setting

Value

Specification

Assign QC Flags

Trigger Location

    
 

Trigger Style

    
     

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

    
 

Trigger Style

    
     

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 7: Dilute FFPE DNA (Nextera Flex for Enrichment)

Input = FFPE DNA

Output = Diluted DNA (50-1000 ng)

Reagent Kits

Kit Name

Supplier

Cat. #

Nextera DNA Flex Pre-Enrichment Library Prep and Enrichment Reagents

Illumina

16 - 20025523; 96 - 20025524
Placement

Setting

Value

Destination Containers

96 well plate
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Desired gDNA Input (ng)

  

50-1000
   

Dilution Buffer

Text Dropdown

• 10 mM Tris-HCI, pH 7.5–8.5
• RSB
   

Sample Volume (uL)

Numeric

Preset

• 5

Step File Placeholders

 

File Name

Log

  

Sample Table

Table Columns

Concentration

Numeric

Value from previous step
   

Conc. Units (ng/uL)

Text

  
   

Total Volume (uL)

Numeric

Calculated = (Concentration * Sample Volume (uL)) / (Desired gDNA Input (ng) * 5)
   

Dilution Buffer (uL)

Numeric

Calculated = Total Volume (uL) - Sample Volume (uL)
Automation

Automation Name

Setting

Value

Specification

Copy Concentration

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::' \-log {compoundOutputFileLuid0}["]

Calculate Dilution Volume and Total Volume

Trigger Location

Record Details

  
 

Trigger Style

Manual

  
     

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.::Total Volume (uL):: = (output.::Concentration:: * step.::Sample Volume (uL)::) / step.::Desired gDNA Input (ng):: ; \output.::Dilution Volume (uL):: = output.::Total Volume (uL):: - step.::Sample Volume (uL)::' \-log {compoundOutputFileLuid0}["]

Set Next Step - Advance and Copy NFE Sample Type

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.::NFE Sample Type:: = input.::NFE Sample Type::' \-log {compoundOutputFileLuid0}["]

Next Step

Setting

Value

Start Next Step

Automatic

Assign Next Step

Automatic

Must move forward

 

Protocol 2: Library Prep (Nextera Flex for Enrichment)

Master Step 1: Tagment Genomic DNA (Nextera Flex for Enrichment)

Input = DNA, Blood Lysis, Saliva Lysis

Output = Tagmented DNA

Reagent Kits

Kit Name

Supplier

Cat. #

Nextera DNA Flex Pre-Enrichment Library Prep and Enrichment Reagents

Illumina

16 - 20025523; 96 - 20025524
Placement

Setting

Value

Destination Containers

96 well plate
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

  

Default

• TAG
   

Total Samples

 

Preset

• 0
• Calculated = Total Samples + 1
   

eBLT (uL)

Numeric

Calculated = Total Samples * 11.5
   

TB1 (uL)

Numeric

Calculated = Total Samples * 11.5
   

Desired Input Amount (ng)

Numeric

Range = 50 - 1000 ng
   

Total Volume (uL)

Numeric

Preset

• 30
 

Multiline Text Fields

Notes

Multiline Text

Desired Input Amount should be between 50-1000 ng.

Step File Placeholders

 

File Name

Log

  

Sample Table

Table Columns

Concentration

Numeric

Value from previous step
   

Conc. Units (ng/uL)

Text

  
   

Sample Volume (uL)

Numeric

Calculated = Concentration / Desired Input Amount (ng)
   

Nuclease-free water (uL) numeric

Numeric

Calculated = Total Volume (uL) - Sample Volume (uL)
   

NFE Sample Type

Text

Copy from previous step
Automation

Automation Name

Setting

Value

Specification

Calculate Total Samples and Master Mix & Copy Conc. & NFE Sample Type

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.::NFE Sample Type:: = input.::NFE Sample Type:: ; \step.::Total Samples:: = step.::Total Samples:: + 1 ; \step.::eBLT (uL):: = step.::Total Samples:: * 1.1 * 11.5 ; \step.::TB1 (uL):: = step.::Total Samples:: * 1.1 * 11.5 ; \if (output.::NFE Sample Type::.contains(::Saliva::) || output.::NFE Sample Type::.contains(::Blood::)) {output.::Concentration:: = 100 ; output.::Conc. Units:: = ::ng/uL::} ; \if (input.hasValue(::Concentration::)) {output.::Concentration:: = input.::Concentration:: ; output.::Conc. Units:: = input.::Conc. Units::}' \-log {compoundOutputFileLuid0}"

Calculate Sample Volume and Water

Trigger Location

Record Details

  
 

Trigger Style

Manual

  
     

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.::Sample Volume (uL):: = step.::Desired Input Amount (ng):: / output.::Concentration:: ; \output.::Nuclease-free water (uL):: = step.::Total Volume (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 2: Post Tagmentation Cleanup (Nextera Flex for Enrichment)

Step Type: No Outputs

Input = Tagmented DNA

Output = Cleaned Up Tagmented DNA

Placement = none

Reagent Kits

Kit Name

Supplier

Cat. #

Nextera DNA Flex Pre-Enrichment Library Prep and Enrichment Reagents

Illumina

16 - 20025523; 96 - 20025524
Record Details

Section

Subsection

Setting

Value

Specification

Step File Placeholders

 

File Name

Log

  
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 3: Amplify Tagmented DNA (Nextera Flex for Enrichment)

Step Type: Add Labels

Input = Cleaned up Tagmented DNA

Output = Amplifed Tagmented DNA

Placement = none, using the sample 96 well plate as the previous step.

Reagent Kits

Kit Name

Supplier

Cat. #

Nextera DNA Flex Pre-Enrichment Library Prep and Enrichment Reagents

Illumina

16 - 20025523; 96 - 20025524
Add Labels

Setting

Value

Label Groups

IDT for Illumina Nextera DNA UD Indexes Set A (96 Indexes, 96 Samples)
 

IDT for Illumina Nextera DNA UD Indexes Set B (96 Indexes, 96 Samples)
 

IDT for Illumina Nextera DNA UD Indexes Set C (96 Indexes, 96 Samples)
 

IDT for Illumina Nextera DNA UD Indexes Set D (96 Indexes, 96 Samples)
 

IDT for Illumina Nextera DNA UD Indexes Set A, B, C, D (384 Indexes, 384 Samples)

Location of indexes = UDP Run Setup Resources

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

Text

Preset

• eBLT PCR
   

PCR Cycles

Numeric

Preset

• 9 and 12
   

Total Samples

Numeric

Preset

• 0
• Calculated = Total Samples + 1

 
   

EPM (uL)

Numeric

Calculated = Total Samples * 23 *1.1
   

Nuclease-free water (uL)

Numeric

Calculated = Total Samples * 23 *1.1

Step File Placeholders

 

File Name

Log

  
Automation

Automation Name

Setting

Value

Specification

Calculate Total Samples & Master Mix and Copy NFE Sample Type - NFE Amplify

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.::NFE Sample Type:: = input.::NFE Sample Type:: ; \step.::Total Samples:: = step.::Total Samples:: + 1 ; \step.::EPM (uL):: = step.::Total Samples:: * 1.1 * 23 ; \step.::Nuclease-free water (uL):: = step.::Total Samples:: * 1.1 * 23' \-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 4: Clean Up Libraries (Nextera Flex for Enrichment)

Step Type: Standard

Input = Amplifed Tagmented DNA

Output = Cleaned up libraries

Reagent Kits

Kit Name

Supplier

Cat. #

Agencourt AMPure XP purification beads

Beckman Coulter

A63880

Nextera DNA Flex Pre-Enrichment Library Prep and Enrichment Reagents

Illumina

16 - 20025523; 96 - 20025524
Placement

Setting

Value

Destination Containers

96 well plate
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

80% EtOH prep date

Date

  

Step File Placeholders

 

File Name

Log

  
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: Bioanalyzer QC Qualify Pre-Enriched Libraries (Nextera Flex for Enrichment)

Step Type: Standard QC

Input = Enriched DNA Fragments

Output = Result File

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Criteria 1 - Source Data Field

  

Preset

• Peak 2 Size - bp
   

Criteria 1 - Operator

  

Preset

• >=
   

Criteria 1 - Threshold

  

Preset

• 300
   

Criteria 1 - Source Data Field

  

Preset

• Peak 2 Size - bp
   

Criteria 1 - Operator

  

Preset

• <=
   

Criteria 1 - Threshold

  

Preset

• 400
   

Use strict matching for Bioanalyzer results

  

Preset

• Nextera Flex for Enrichment Validation

Step File Placeholders

 

File Name

Placeholder Bioanalyzer Input File

Auto
     

Bioanalyzer Input File Generation

 
     

Log File

Auto
     

Bioanalyzer XML Result File (required)

Manual
     

Result File (optional)

Manual
     

PDF Summary File (optional)

Manual
     

Bioanalyzer XML Parsing Log File

Auto
     

QC Assignment Log File

Auto
     

QC Assignment Report

Auto
Automation

Automation Name

Setting

Value

Specification

Generate Bioanalyzer Input File

Trigger Location

    
 

Trigger Style

    
     

Command line

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar script:driver_file_generator -i {processURI:v2} -u {username} -p {password} -t /opt/gls/clarity/extensions/ngs-common/v5/EPP/conf/readonly/bioA_driver_file_template.csv -o {compoundOutputFileLuid0}.csv -l {compoundOutputFileLuid1}  && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar script:addBlankLines -i {stepURI:v2} -u {username} -p {password} -f {compoundOutputFileLuid0}.csv -l {compoundOutputFileLuid1} -sep COMMA -b ',False,' -h 1 -c LIMSID -pre 'Sample '"

Parse Bioanalyzer XML and Assign QC flags

Trigger Location

    
 

Trigger Style

    
     

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:parseBioAnalyzer -inputFile {compoundOutputFileLuid2} -log {compoundOutputFileLuid5} -configFile '/opt/gls/clarity/extensions/conf/v5/bioanalyzer/defaultBioAnalyzerDNAConfig.groovy' script:assignQC -log {compoundOutputFileLuid6} -qcResult {compoundOutputFileLuid7}"

Next Step

Setting

Value

Start Next Step

Manual

Safe Stopping Point

Optional

• Step 6: Pool Pre-Enriched Libraries by Volume (Nextera Flex for Enrichment)
• Step 7: Qubit (Nextera Flex for Enrichment)

Master Step 6: Pool Pre-Enriched Libraries (Nextera Flex for Enrichment)

Step Type: Standard

Input = Diluted Pre-Enriched Libraries

Output = Pooled Pre-Enriched Libraries

Reagent Kits

Kit Name

Supplier

Cat. #

Agencourt AMPure XP purification beads

Beckman Coulter

A63880

Nextera DNA Flex Pre-Enrichment Library Prep and Enrichment Reagents

Illumina

16 - 20025523; 96 - 20025524
Placement

Setting

Value

Destination Containers

Tube
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Library Pool Plexity

Text DropDown

• 1-plex
• 12-plex
   

Total Pooled Volume (uL)

Numeric

Preset

Read Only

• 30

Step File Placeholders

 

File Name

Log

  

Sample Table

Table Columns

Pre-Enriched Library Volume (uL)

Numeric

Calculated = if Library Pool Plexity = 1-plex then Pre-Enriched Library Volume (uL) = 14 otherwise pre-enriched library volume (uL) = 2.5
   

RSB Volume (uL)

Numeric

Calculated = Total Pooled Volume (uL) - Pre-Enriched Library Volume (uL)
Automation

Automation Name

Setting

Value

Specification

Calculate Pre-Enriched Library Volume and RSB Volume

Trigger Location

    
 

Trigger Style

    
     

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 'if (step.::Library Pool Plexity:: == ::1-plex::) {output.::Pre-Enriched Library Volume (uL):: = 14} ; \if (step.::Library Pool Plexity:: == ::12-plex::) {output.::Pre-Enriched Library Volume (uL):: = 2.5} ; \output.::RSB Volume (uL):: = step.::Total Pooled Volume (uL):: - output.::Pre-Enriched Library Volume (uL)::' \-log {compoundOutputFileLuid0}"

Set Next Step - Advance

Trigger Location

    
 

Trigger Style

    
     

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

• Step 8: Hybridize Probes (Nextera Flex for Enrichment)

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 7: Qubit (Nextera Flex for Enrichment)

Step Type: Standard QC

Input = Cleaned Up Libraries

Output = Measurement

Placement

Setting

Value

Destination Containers

Tube
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Criteria 1 - Source Data Field

Text

Preset

• Concentration
   

Criteria 1 - Operator

Text

Preset

• >=
   

Criteria 1 - Threshold Value

Numeric

 
   

Criteria 2 - Source Data Field

Text

Preset

• Concentration
   

Criteria 2 - Operator

Text

Preset

• <=
   

Criteria 2 - Threshold Value

Numeric

 

Step File Placeholders

 

File Name

Log

  
     

QC Log File

  
     

QC Result File

  
     

Upload File

  

Sample Table

Table Columns

Concentration

Numeric

  
   

Conc. Units

Text

  
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

Automatic

Safe Stopping Point

 

Master Step 8: Pool Pre-Enriched Libraries (Nextera Flex for Enrichment)

(This is the Drop-down text)

Step Type: Standard

Input = Diluted Pre-Enriched Libraries

Output = Pooled Pre-Enriched Libraries

Reagent Kits

Kit Name

Supplier

Cat. #

Agencourt AMPure XP purification beads

Beckman Coulter

A63880

Nextera DNA Flex Pre-Enrichment Library Prep and Enrichment Reagents

Illumina

16 - 20025523; 96 - 20025524
Placement

Setting

Value

Destination Containers

Tube
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Library Pool

Text Dropdown

• 1
• 12
   

Pre-Enriched Library Sample (ng)

Numeric

Preset

Read Only

• 500
   

Total Pooled Volume (uL)

Numeric

Preset

Read Only

• 30

Step File Placeholders

 

File Name

Log

  

Sample Table

Table Columns

Concentration

Numeric

Copy from previous step
   

Conc. Unit

Text

Copy from previous step
   

Pre-Enriched Library Volume (uL)

Numeric

Calculated = Pre-Enriched Library Sample (ng)/Concentration
   

RSB Volume (uL)

Numeric

Calculated = Total Pooled Volume (uL) - Pre-Enriched Library Volume (uL)
Automation

Automation Name

Setting

Value

Specification

Copy Concentration

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::' \-log {compoundOutputFileLuid0}["]

Calculate Pre-Enriched Library Volume and RSB Volume

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 false \-h false \-exp 'if (step.::Library Pool Plexity:: = 1-plex) {output.::Pre-Enriched Library Volume (uL):: = 14} ; \if (step.::Library Pool Plexity:: = 12-plex) {output.::Pre-Enriched Library Volume (uL):: = 2.5} ; \output.::RSB Volume (uL):: = step.::Total Pooled Volume (uL):: - output.::Pre-Enriched Library 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

Manual

Assign Next Step

Automatic

Safe Stopping Point

 

Master Step 9: Hybridize Probes (Nextera Flex for Enrichment)

Input = Pooled Pre-Enriched Libraries

Output = Hybridized Probes

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina Exome Panel (8 Enrichment Reactions) (CEX)(optional)

Illumina

20020183

TruSight One (6 Enrichment Reactions) (TOO)(optional)

Illumina

20029227

TruSight One Expanded (6 Enrichment Reactions) (TOE)(optional)

Illumina

20029226

TruSight Cancer (8 Enrichment Reactions)(optional)

Illumina

FC-121-0202

TruSight Hereditary Cancer (8 Enrichment Reactions)(optional)

Illumina

20029551

TruSight Cardio (8 Enrichment Reactions)(optional)

Illumina

20029229

TruSeq Neurodegeneration (8 Enrichment Reactions)(optional)

Illumina

20029550

Illumina Custom Enrichment Panel via DesignStudio (8 Enrichment Reactions)(optional)

Illumina

20025371
Placement

Setting

Value

Destination Containers

96 well plate
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

Text

Preset

• NF-HYB program
   

PCR Cycles

Numeric

  

Step File Placeholders

 

File Name

Log

  
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 10: Capture Hybridize Probes (Nextera Flex for Enrichment)

Step Type: Standard

Input = Pooled Pre-Enriched Libraries

Output = Hybridized Probes

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina Exome Panel (8 Enrichment Reactions) (CEX)(optional)

Illumina

20020183

TruSight One (6 Enrichment Reactions) (TOO)(optional)

Illumina

20029227

TruSight One Expanded (6 Enrichment Reactions) (TOE)(optional)

Illumina

20029226

TruSight Cancer (8 Enrichment Reactions)(optional)

Illumina

FC-121-0202

TruSight Hereditary Cancer (8 Enrichment Reactions)(optional)

Illumina

20029551

TruSight Cardio (8 Enrichment Reactions)(optional)

Illumina

20029229

TruSeq Neurodegeneration (8 Enrichment Reactions)(optional)

Illumina

20029550

Illumina Custom Enrichment Panel via DesignStudio (8 Enrichment Reactions)(optional)

Illumina

20025371
Placement

Setting

Value

Destination Containers
• 96 well plate
• Tube
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Total Samples

Numeric

Preset

• 0
• Calculated = Total Samples + 1
   

EE1 (uL)

Numeric

Calculated = Total Samples * 28.5 * 1.1
   

HP3 (uL)

Numeric

Calculated = Total Samples * 1.5 *1.1

Step File Placeholders

 

File Name

Log

  
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 ; \step.::EE1 (uL):: = step.::Total Samples:: * 1.1 * 28.5 ; \step.::HP3 (uL):: = step.::Total Samples:: * 1.1 * 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 11: Amplify Enriched Library (Nextera Flex for Enrichment)

Step Type: Standard

Input = Pooled Pre-Enriched Libraries

Output = Hybridized Probes

Reagent Kits

Kit Name

Supplier

Cat. #

Illumina Exome Panel (8 Enrichment Reactions) (CEX)(optional)

Illumina

20020183

TruSight One (6 Enrichment Reactions) (TOO)(optional)

Illumina

20029227

TruSight One Expanded (6 Enrichment Reactions) (TOE)(optional)

Illumina

20029226

TruSight Cancer (8 Enrichment Reactions)(optional)

Illumina

FC-121-0202

TruSight Hereditary Cancer (8 Enrichment Reactions)(optional)

Illumina

20029551

TruSight Cardio (8 Enrichment Reactions)(optional)

Illumina

20029229

TruSeq Neurodegeneration (8 Enrichment Reactions)(optional)

Illumina

20029550

Illumina Custom Enrichment Panel via DesignStudio (8 Enrichment Reactions)(optional)

Illumina

20025371
Placement

Setting

Value

Destination Containers
• 96 well plate
• Tube
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

Text

Preset

• AMP
   

PCR Cycles

Numeric

  

Step File Placeholders

 

File Name

Log

  
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 11: Clean Up Amplified Enriched Library (Nextera Flex for Enrichment)

Input = Amplified Enriched Libraries

Output = Cleaned up amplified enriched libraries

Reagent Kits

Kit Name

Supplier

Cat. #

Agencourt AMPure XP beads, 5 ml

Beckman Coulter

A63880

Illumina Exome Panel (8 Enrichment Reactions) (CEX)(optional)

Illumina

20020183

TruSight One (6 Enrichment Reactions) (TOO)(optional)

Illumina

20029227

TruSight One Expanded (6 Enrichment Reactions) (TOE)(optional)

Illumina

20029226

TruSight Cancer (8 Enrichment Reactions)(optional)

Illumina

FC-121-0202

TruSight Hereditary Cancer (8 Enrichment Reactions)(optional)

Illumina

20029551

TruSight Cardio (8 Enrichment Reactions)(optional)

Illumina

20029229

TruSeq Neurodegeneration (8 Enrichment Reactions)(optional)

Illumina

20029550

Illumina Custom Enrichment Panel via DesignStudio (8 Enrichment Reactions)(optional)

Illumina

20025371
Placement

Setting

Value

Destination Containers
• 96 well plate
• Tube
Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

80% EtOH prep Date

Date

  

Step File Placeholders

 

File Name

Log

  
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}["]

Safe Stopping Point