Illumina Preset Protocol - Illumina DNA Prep (M) Tagmentation

Overview

The Illumina DNA Prep (M) Tagmentation workflow includes the following functionality.

•

Preset Illumina DNA Prep (M) Tagmentation protocols that support the generation of enriched libraries for dual-indexed, paired-end sequencing.

•

Automated calculation of sample and buffer volumes.

•

Automated calculation or display of reagents at every step in the protocol.

•

Automatic step transition when required.

•

Automatic placement of samples when necessary.

•

Automated assignment of QC Pass/Fail, based on user-selected threshold values.

Protocol 1: Prep Sample (Illumina DNA Prep (M) Tagmentation)

Step 1: Sort Sample Type (Illumina DNA Prep (M) Tagmentation)

Master Step 1: Sort Sample

Step Type: No Output

Input = Small Genomes, Large Complex Genomes, Blood or Saliva

Output = Small Genomes, Large Complex Genomes, Blood or Saliva

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Multiline Text Fields

Comment

Multiline Text

Step File Placeholders

File Name

Log

Auto

Sample Table

Table Columns

Prep Sample Type - Tagmentation

Text Dropdown

Required

Presets

•

Blood

•

Saliva

•

Large Complex Genomes

•

Small Genomes

Automation
Automation Name	Setting	Value	Specification
Set Next Step - Tagmentation	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 true -h false -exp 'if (input.::Prep Sample Type - Tagmentation:: == ::Large Complex Genomes::) {nextStep = ::REMOVE::} ; \` `if (input.::Prep Sample Type - Tagmentation:: == ::Small Genomes::) {nextStep = ::DNA Quantification (Illumina DNA Prep (M) Tagmentation v1.0)::} ; \` `if (input.::Prep Sample Type - Tagmentation:: == ::Saliva::) {nextStep = ::Saliva Lysis (Illumina DNA Prep (M) Tagmentation v1.0)::} ; \` `if (input.::Prep Sample Type - Tagmentation:: == ::Blood::) {nextStep = ::Blood Lysis (Illumina DNA Prep (M) Tagmentation v1.0)::} ' -log {compoundOutputFileLuid0}"`
Route Samples - Large Complex Genome	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 'Prep Sample Type - Tagmentation' \` `--FIELD_VALUE 'Large Complex Genomes' \` `--WORKFLOW 'Illumina DNA Prep (M) Tagmentation v1.0' \` `--STEP 'Tagment Genomic DNA (Illumina DNA Prep (M) Tagmentation v1.0)' \` `--INPUTS_OR_OUTPUTS 'INPUTS' "`

Next Step

Setting

Value

Start Next Step

Manual

Available next steps:

•

Step 2: DNA Quantification (Illumina DNA Prep (M) Tagmentation)

•

Step 3: Blood Lysis (Illumina DNA Prep (M) Tagmentation)

•

Step 4: Saliva Lysis (Illumina DNA Prep (M) Tagmentation)

Assign Next Step

Automatic

Safe Stopping Point

Step 2: DNA Quantification (Illumina DNA Prep (M) Tagmentation)

Master Step 2: DNA Quantification (Illumina DNA Prep (M) Tagmentation)

(This is the Drop-down text)

Step Type: Standard QC

Input = Small Genomes

Output = QC'd Small Genomes

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Multiline Text Fields

Comment

Multiline Text

Step File Placeholders

File Name

Log

Auto

Sample Table

Table Columns

Concentration (ng/µl)

Numeric

•

Global field - measurement & derived sample

Sample Volume (µl)

Required

•

Global field - measurement

DNA Amount (ng)

•

Global field - measurement

•

Calculated

Prep Sample Type - Tagmentation

•

Global field - measurement & derived sample

•

Copied in

Automation
Automation Name	Setting	Value	Specification
Calculate DNA Amount & Assign QC	Trigger Location	Record Details
	Trigger Style	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.::DNA Amount (ng):: = output.::Concentration (ng/ul):: * output.::Sample Volume (ul)::' -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 true -h false -exp 'if (output.::DNA Amount (ng):: >= 0.33 && output.::DNA Amount (ng):: <= 100) { output.QC = true } else { output.QC = false } ; input.::DNA Amount (ng):: = output.::DNA Amount (ng):: ; input.::Concentration (ng/ul):: = output.::Concentration (ng/ul):: ; output.::Prep Sample Type - Tagmentation:: = input.::Prep Sample Type - Tagmentation::' -log {compoundOutputFileLuid0}"
Set Next Step - Remove	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::' -log {compoundOutputFileLuid0}"`
Route Samples - Small Genomes	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 'Prep Sample Type - Tagmentation' \` `--FIELD_VALUE 'Small Genomes' \` `--WORKFLOW 'Illumina DNA Prep (M) Tagmentation v1.0' \` `--STEP 'Tagment Genomic DNA (Illumina DNA Prep (M) Tagmentation v1.0)' \` `--INPUTS_OR_OUTPUTS 'INPUTS' "`

Next Step
Setting	Value
Start Next Step	Manual
Assign Next Step	Automatic
Safe Stopping Point

Step 3: Blood Lysis (Illumina DNA Prep (M) Tagmentation)

Master Step 3: Blood Lysis (Illumina DNA Prep (M) Tagmentation)

Step Type: Standard

Input = Blood Samples

Output = Lysed Blood Samples

Reagent Kit
Kit Name	Supplier
Illumina DNA (M) Tagmentation Library Prep Kit 48/96, Box 1	Illumina
Illumina DNA (M) Tagmentation Library Prep Kit 48/96, Flex Lysis Reagent Kit	Illumina

Placement

Setting

Value

Specification

Destination Containers

•

96 well plate

•

Tube

Configured on step

Record Details
Section	Subsection	Setting	Value	Specification
Step Data	Master Step Fields	80% Ethanol Prep Date	Date
		Total Samples	Numeric	Default = 0
		BLB (µl)	Numeric	Calculated
		PK1 (µl)	Numeric	Calculated
		Nuclease-free water (µl)	Numeric	Calculated
		Thermal Cycler Program	Text	Default = BLP
	Multiline Text Fields	Comment	Multiline Text
Step File Placeholders		File Name	Log	Auto
Sample Table	Table Columns	Prep Sample Type - Tagmentation	Text	Copied in

Automation
Automation Name	Setting	Value	Specification
Count 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 'output.::Prep Sample Type - Tagmentation:: = input.::Prep Sample Type - Tagmentation:: ; \ `step.::BLB (uL):: = step.::Total Samples:: * 7 ; \` `step.::PK1 (uL):: = step.::Total Samples:: * 2 ; \` `step.::Nuclease-free water (uL):: = step.::Total Samples:: * 31' -log {compoundOutputFileLuid0}"`
Set Next Step - Remove	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::' -log {compoundOutputFileLuid0}"`
Route Samples - 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 'Prep Sample Type - Tagmentation' \` `--FIELD_VALUE 'Blood' \` `--WORKFLOW 'Illumina DNA Prep (M) Tagmentation v1.0' \` `--STEP 'Tagment Genomic DNA (Illumina DNA Prep (M) Tagmentation v1.0)' \` `--INPUTS_OR_OUTPUTS 'OUTPUTS' "`

Next Step
Setting	Value
Start Next Step	Manual
Assign Next Step	Automatic
Safe Stopping Point

Step 4: Saliva Lysis (Illumina DNA Prep (M) Tagmentation)

Master Step 4: Saliva Lysis (Illumina DNA Prep (M) Tagmentation)

Step Type: Standard

Input = Saliva Samples

Output = Lysed Saliva Samples

Reagent Kit
Kit Name	Supplier
Illumina DNA (M) Tagmentation Library Prep Kit 48/96, Box 1	Illumina

Placement

Setting

Value

Specification

Destination Containers

•

96 well plate

•

Tube

Configured on step

Record Details
Section	Subsection	Setting	Value	Specification
Step Data	Master Step Fields	80% Ethanol Prep Date	Date
	Multiline Text Fields	Comment	Multiline Text
Step File Placeholders		File Name	Log	Auto
Sample Table	Table Columns	Prep Sample Type - Tagmentation	Text	Copied in

Automation
Automation Name	Setting	Value	Specification
Copy Prep Sample Type - Tagmentation	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.::Prep Sample Type - Tagmentation:: = input.::Prep Sample Type - Tagmentation::' -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

Protocol 2: Library Prep (Illumina DNA Prep (M) Tagmentation)

Step 1: Tagment Genomic DNA (Illumina DNA Prep (M) Tagmentation)

Master Step 1: Tagment Genomic DNA

Step Type: Standard

Input = Small Genomes, Large Complex Genomes, Lysed Salvia or Lysed Blood

Output = Tagmented DNA

Reagent Kit
Kit Name	Supplier
Illumina DNA (M) Tagmentation Library Prep Kit 48/96, Box 2	Illumina
Illumina DNA (M) Tagmentation Library Prep Kit 48/96, Box 3	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 = TAG

Desired DNA Amount (ng)

Numeric

Calculated

Multiline Text Fields

Directions

If Sample Type is Small Genome, enter a Desired DNA Amount (ng) at the top of the screen and press the blue button. If Sample Type is Large Complex Genome, enter Sample Volume (µl).

Comment

Step File Placeholders

File Name

Log

Auto

Sample Table

Table Columns

Prep Sample Type - Tagmentation

Text

•

Global field - derived sample

•

Copied in

Sample Volume (µl)

Numeric

•

Global field - derived sample

•

Calculated for small genome samples

Nuclease-free water (µl)

Numeric

•

Global field - derived sample

•

Calculated for small genome samples

Automation
Automation Name	Setting	Value	Specification
Copy Prep Sample Type - Tagmentation, DNA Amount & Set Blood and Saliva 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.::Prep Sample Type - Tagmentation:: = input.::Prep Sample Type - Tagmentation:: ; \` `if (input.::Prep Sample Type - Tagmentation:: == ::Blood:: \|\| input.::Prep Sample Type - Tagmentation:: == ::Saliva::) {output.::Sample Volume (ul):: = 30} ; \` `if (input.hasValue(::DNA Amount (ng)::)) {output.::DNA Amount (ng):: = input.::DNA Amount (ng)::}' -log {compoundOutputFileLuid0}"`
Calculate DNA Amount for SMALL GENOMES	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 true -h false -exp 'if (output.::Prep Sample Type - Tagmentation:: == ::Small Genomes:: && !step.hasValue(::Desired DNA Amount (ng)::)) {fail(::Please enter Desired DNA Amount (ng)::)} else {if (output.::Prep Sample Type - Tagmentation:: == ::Small Genomes:: && step.hasValue(::Desired DNA Amount (ng)::)) {output.::Sample Volume (ul):: = step.::Desired DNA Amount (ng):: / input.::Concentration (ng/ul):: ; output.::Nuclease-free water (ul):: = 30 - 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

Step 2: Post Tagmentation Clean Up (Illumina DNA Prep (M) Tagmentation)

Master Step 2: Post Tagmentation Clean Up

Step Type: Standard

Input = Tagmented DNA

Output = Clean up tagmented DNA

Reagent Kit
Kit Name	Supplier
Illumina DNA (M) Tagmentation Library Prep Kit 48/96, Box 1	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

Default = PTC

Multiline Text Fields

Comment

Step File Placeholders

File Name

Log

Auto

Sample Table

Table Columns

Prep Sample Type - Tagmentation

Text

•

Global field - derived sample

•

Copied in

Automation
Automation Name	Setting	Value	Specification
Copy Prep Sample Type - Tagmentation & DNA Amount	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.::Prep Sample Type - Tagmentation:: = input.::Prep Sample Type - Tagmentation:: ; \` `if (input.hasValue(::DNA Amount (ng)::)) {output.::DNA Amount (ng):: = input.::DNA Amount (ng)::}' -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

Step 3: Amplify Tagmented DNA (Illumina DNA Prep (M) Tagmentation)

Master Step 3: Amplify Tagmented DNA

Step Type: Add Labels

Input = Clean up tagmented DNA

Output = Amplified tagmented DNA

Reagent Kit
Kit Name	Supplier
Illumina DNA (M) Tagmentation Library Prep Kit 48/96, Box 2	Illumina

Add Labels
Setting	Value
Label Groups	IDT for Illumina Nextera DNA UD Indexes Set A for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000-label-sheet.xlsx
	IDT for Illumina Nextera DNA UD Indexes Set A-D for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000-label-sheet.xlsx
	IDT for Illumina Nextera DNA UD Indexes Set B for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000-label-sheet.xlsx
	IDT for Illumina Nextera DNA UD Indexes Set C for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000-label-sheet.xlsx
	IDT for Illumina Nextera DNA UD Indexes Set D for NovaSeq, MiSeq, HiSeq 2500 and HiSeq 2000-label-sheet.xlsx
	IDT for Illumina DNA-RNA UD Indexes Set A Tagmentation-label-sheet.xlsx
	IDT for Illumina DNA-RNA UD Indexes Set A-D Tagmentation-label-sheet.xlsx
	IDT for Illumina DNA-RNA UD Indexes Set B Tagmentation-label-sheet.xlsx
	IDT for Illumina DNA-RNA UD Indexes Set C Tagmentation-label-sheet.xlsx
	IDT for Illumina DNA-RNA UD Indexes Set D Tagmentation-label-sheet.xlsx

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 = BLT PCR

Multiline Text Fields

Comment

Multiline Text

Step File Placeholders

File Name

Log

Auto

Sample Table

Table Columns

Prep Sample Type - Tagmentation

Text

•

Global field - derived sample

•

Copied in

PCR Cycles

Numeric

Calculated via script

Automation
Automation Name	Setting	Value	Specification
Set Prep Sample Type - Tagmentation & Set PCR Cycles	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.::Prep Sample Type - Tagmentation:: = input.::Prep Sample Type - Tagmentation:: ; \` `if (input.::Prep Sample Type - Tagmentation:: == ::Small Genomes::) {if (input.::DNA Amount (ng):: >= 1 && input.::DNA Amount (ng):: <= 9) {output.::PCR Cycles:: = 12} ; \` `if (input.::DNA Amount (ng):: >=10 && input.::DNA Amount (ng):: <= 24) {output.::PCR Cycles:: = 8} ; \` `if (input.::DNA Amount (ng):: >=25 && input.::DNA Amount (ng):: <= 49) {output.::PCR Cycles:: = 6} ; \` `if (input.::DNA Amount (ng):: >=50 && input.::DNA Amount (ng):: <= 100) {output.::PCR Cycles:: = 5}} else {output.::PCR Cycles:: = 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

Step 4: Clean Up Libraries (Illumina DNA Prep (M) Tagmentation)

Master Step 4: Clean Up Libraries (Illumina DNA Prep (M) Tagmentation)

Step Type: Standard

Input = Amplified tagmented DNA

Output = Cleaned up amplified tagmented DNA

Reagent Kit
Kit Name	Supplier
Illumina DNA (M) Tagmentation Library Prep Kit 48/96, Box 1	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

80% EtOH Prep Date

Date

Total Samples

Numeric

•

Default = 0

•

Calculated

SPB (µl)

Numeric

•

2 decimal places

•

Calculated

Nuclease-free water (µl)

Numeric

•

2 decimal places

•

Calculated

Multiline Text Fields

Comment

Multiline Text

Step File Placeholders

File Name

Log

Auto

Sample Table

Table Columns

Prep Sample Type - Tagmentation

Text

•

Global field - derived sample

•

Copied in

Pool Samples?

Text Dropdown

Required

Presets

•

Yes

•

Automation
Automation Name	Setting	Value	Specification
Set Prep Sample Type - Tagmentation, Count Samples & 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 'output.::Prep Sample Type - Tagmentation:: = input.::Prep Sample Type - Tagmentation:: ; \ `step.::SPB (ul):: = step.::Total Samples:: * 45 ; \` `step.::Nuclease-free water (ul):: = step.::Total Samples:: * 40' -log {compoundOutputFileLuid0}"`
Set Next Step - Optional Pooling	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 (output.::Pool Samples?:: == ::YES::) {nextStep = ::Optional Pool Libraries (Illumina DNA Prep (M) Tagmentation v1.0)::} ; \` `if (output.::Pool Samples?:: == ::NO::) {nextStep = ::Bioanalyzer Library QC (Illumina DNA Prep (M) Tagmentation v1.0)::}' -log {compoundOutputFileLuid0}"`

Next Step

Setting

Value

Start Next Step

Manual

Assign Next Step

Automatic

Next steps:

•

Optional Pool Libraries (Illumina DNA Prep (M) Tagmentation v1.0)

•

Bioanalyzer (Illumina DNA Prep (M) Tagmentation v1.0)

Safe Stopping Point

Step 5: Optional Pool Libraries (Illumina DNA Prep (M) Tagmentation)

Master Step 5: Pool Libraries

Step Type: Pooling

Input = Cleaned up amplified tagmented DNA

Output = Pool

Placement

Setting

Value

Specification

Destination Containers

•

96 well plate

•

Tube

Configured on step

Record Details
Section	Subsection	Setting	Value	Specification
Step Data	Multiline Text Fields	Comment
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

Step 6: Bioanalyzer Library QC (Illumina DNA Prep (M) Tagmentation)

Master Step 6: Bioanalyzer QC (Library Validation)

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: 150
		Criteria 2 - Source Data Field		Preset: Peak 2 Size - bp
		Criteria 2 - Operator		Preset: <=
		Criteria 2 - Threshold Value		Preset: 1500
		BA Instrument
		Use strict matching for Bioanalyzer results
		Group of Defaults	Nextera DNA Flex Library Validation
Step File Placeholders		File Name	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	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/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, Copy nM and 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:parseBioAnalyzer -inputFile {compoundOutputFileLuid2} -log {compoundOutputFileLuid5} -configFile '/opt/gls/clarity/extensions/conf/v5/bioanalyzer/defaultBioAnalyzerDNAConfig.groovy' && /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 'if (output.::Conc. Units::.contains(::pg::)) {output.::Molarity (nM):: = output.::Region 1 Molarity:: / 1000} else {output.::Molarity (nM):: = output.::Region 1 Molarity::} ; (input.::Molarity (nM):: = output.::Molarity (nM)::) ' -log {compoundOutputFileLuid8} && /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 {compoundOutputFileLuid6} -qcResult {compoundOutputFileLuid7}"
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

Step 7: Normalize Libraries (Illumina DNA Prep (M) Tagmentation)

Master Step 7: Normalize Libraries 1

Input = Purified libraries

Output = Normalized libraries

Placement
Setting	Value	Specification
Destination Containers	96 well plate
Placement Screen	Auto

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Multiline Text Fields

Comment

Multiline Text

Target Normalization (nM)

Numeric

Required

Default = 2

Final Volume (µl)

Numeric

Required

Step File Placeholders

File Name

Log

Auto

Sample Table

Table Columns

Molarity (nM)

Numeric

•

Global field - derived sample

•

Copied in from input

Sample Volume (µl)

Numeric

•

Global field - derived sample

•

Copied down from step UDF Sample Volume (µl)

Buffer Volume (µl)

Numeric

•

Global field - derived sample

•

Calculated in automation

Normalized Molarity (nM)

Numeric

Required

Sequencing Instrument

Text Dropdown

Required

Read Only

Presets

•

MiSeq

•

NextSeq

•

HiSeq2500

•

HiSeq3000/4000

•

NovaSeq 2.0

•

NovaSeq 3.0

•

HiSeqX

•

NextSeq 1000/2000

Automation
Automation Name	Setting	Value	Specification
Normalization Calculations - Option 1			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.::Molarity (nM):: = input.::Molarity (nM):: ; if (output.::Molarity (nM):: <= step.::Target Normalization (nM)::) {output.::Sample Volume (ul):: = step.::Final Volume (ul):: ; output.::Buffer Volume (ul):: = 0 ; output.::Normalized Molarity (nM):: = output.::Molarity (nM)::} else {output.::Sample Volume (ul):: = (step.::Target Normalization (nM):: * step.::Final Volume (ul):: ) / input.::Molarity (nM):: ; output.::Buffer Volume (ul):: = step.::Final Volume (ul):: - output.::Sample Volume (ul):: ; output.::Normalized Molarity (nM):: = step.::Target Normalization (nM)::}' -log {compoundOutputFileLuid0}"
Next Step = Set Next Step - Remove			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::' -log {compoundOutputFileLuid0}"`
Finish Step = Routing script - Normalize Libraries			Command line `bash -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 'Sequencing Instrument' \` `--FIELD_VALUE 'MiSeq' \` `--WORKFLOW 'MiSeq Sequencing v2.0' \` `--STEP 'Library Pooling (MiSeq v2.0)' \` `--INPUTS_OR_OUTPUTS 'OUTPUTS' \` `\` `--FIELD_NAME 'Sequencing Instrument' \` `--FIELD_VALUE 'NextSeq' \` `--WORKFLOW 'NextSeq 500/550 Sequencing v1.0' \` `--STEP 'Library Pooling (NextSeq 500/550 v1.0)' \` `--INPUTS_OR_OUTPUTS 'OUTPUTS' \` `\` `--FIELD_NAME 'Sequencing Instrument' \` `--FIELD_VALUE 'HiSeq2500' \` `--WORKFLOW 'HiSeq 2500 Sequencing v1.0' \` `--STEP 'Library Pooling (HiSeq 2500 v1.0)' \` `--INPUTS_OR_OUTPUTS 'OUTPUTS' \` `\` `--FIELD_NAME 'Sequencing Instrument' \` `--FIELD_VALUE 'HiSeq3000/4000' \` `--WORKFLOW 'HiSeq 3000/4000 Sequencing v1.0' \` `--STEP 'Library Pooling (HiSeq 3000/4000 v1.0)' \` `--INPUTS_OR_OUTPUTS 'OUTPUTS' \` `\` `--FIELD_NAME 'Sequencing Instrument' \` `--FIELD_VALUE 'HiSeqX' \` `--WORKFLOW 'HiSeq X Sequencing v1.0' \` `--STEP 'Library Pooling (HiSeq X v1.0)' \` `--INPUTS_OR_OUTPUTS 'OUTPUTS' \` `\` `--FIELD_NAME 'Sequencing Instrument' \` `--FIELD_VALUE 'NovaSeq 2.0' \` `--WORKFLOW 'NovaSeq 5000/6000 v2.0' \` `--STEP 'Define Run Format (NovaSeq 5000/6000 v2.0)' \` `--INPUTS_OR_OUTPUTS 'OUTPUTS' \` `\` `--FIELD_NAME 'Sequencing Instrument' \` `--FIELD_VALUE 'NovaSeq 3.0' \` `--WORKFLOW 'NovaSeq 5000/6000 v3.1' \` `--STEP 'Define Run Format (NovaSeq 5000/6000 v3.1)' \` `--INPUTS_OR_OUTPUTS 'OUTPUTS'` `\` `--FIELD_NAME 'Sequencing Instrument' \` `--FIELD_VALUE 'NextSeq 1000/2000' \` `--WORKFLOW 'NextSeq 1000/2000 Sequencing v1.0' \` `--STEP 'Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v1.0)' \` `--INPUTS_OR_OUTPUTS 'OUTPUTS'"`