Illumina Preset Protocol - Nextera XT

Overview

The Nextera XT includes the following functionality:

•

Preconfigured Nextera XT protocol that converts the mRNA in total RNA into a library of template molecules of known strand origin. The library is suitable for subsequent cluster generation and DNA 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.

•

A routing script that allows sequencing of libraries using any Illumina sequencing instrument.

Protocol 1: Nextera XT DNA

Step 1: Tagment Genomic DNA (Nextera XT DNA)

Master Step 1: Tagment

Step Type:

Input = gDNA

Output = Tagmented and neutralized gDNA

SharedResultFile = Log File

Reagent Kits
Kit Name	Supplier
Nextera XT DNA Library Prep Kit, Box 1 (FC-131-1096) ATM
Nextera XT DNA Library Prep Kit, Box 2 (FC-131-1096) NT

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

Text

Group of Defaults

•

Nextera XT

–

TAG

Multiline Text Fields

Comment

Multiline Text

Automation
Automation Name	Setting	Specification
Normalize gDNA (Nextera XT)	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 true -h false -exp 'output.::Sample Volume (ul):: = (0.2 * 5) / input.::Concentration:: ; output.::Buffer Volume (ul):: = 5 - output.::Sample Volume (ul)::' -log {compoundOutputFileLuid0}"`
Next Step = 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 {outputFileLuid0}["]`

Step 2: Amplify Libraries (Nextera XT DNA)

Master Step 2: Amplification

(This is the Drop-down text)

Input = Tagmented and neutralized gDNA

Output = Library DNA

SharedResultFile = Log File

Place Samples = Autoplacement into 96 well plate

Reagent Kits
Kit Name	Supplier
Nextera XT DNA Library Prep Kit, Box 1 (FC-131-1096)
NPM

Add Labels
Setting	Value
Label Groups (for 5.x)	Nextera XT v2 Full Set.xlsx
	Nextera XT v2 Set A.xlsx
	Nextera XT v2 Set B.xlsx
	Nextera XT v2 Set C.xlsx
	Nextera XT v2 Set D.xlsx

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

Thermal Cycler Program

Text

Group of Defaults

•

Nextera XT

–

NXT PCR

Multiline Text Fields

Comment

Multiline Text

Step File Placeholders

File Name

Log

Auto

Automation
Automation Name	Setting	Specification
Step = 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}["]`

Step 3: Clean Up Libraries (Nextera XT DNA)

Master Step 3: Clean Up

Input = Amplified Libraries

Output = Purified DNA Libraries

SharedResultFile = Log File

Place Samples = Auto placement into 96 well plate

Reagent Kits
Kit Name	Supplier
AMPure XP beads
Nextera XT DNA Library Prep Kit, Box 1 (FC-131-1096)

Record Details
Section	Subsection	Setting	Value	Specification
Step Data	Master Step Fields	80% EtOH Prep Date	Date
	Multiline Text Fields	Comment	Multiline Text
Step File Placeholders		File Name	Log	Auto

Automation
Automation Name	Setting	Specification
Next Step = 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}["]`

Step 4: Bioanalyzer QC (Library Validation)

Master Step 4: Bioanalyzer QC (Library Validation)

Input = Purified DNA Libraries

Output = Result File

SharedResultFile =

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 Value

Preset

•

250

Criteria 2 - Source Data Field

Preset

•

Peak 2 Size - bp

Criteria 2 - Operator

Preset

•

Criteria 2 - Threshold Value

Preset

•

1000

BA Instrument

Preset

•

Nextera XT DNA Library Validation

Automation
Automation Name	Setting	Specification
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' && /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 - Output PASS/FAIL	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 true -h false -excludeControls true -exp 'if (output.QC == true) { nextStep = ::ADVANCE:: } else { nextStep = ::ESCALATE:: }' -log {compoundOutputFileLuid0}"`

Step 5: Normalize Libraries (Nextera XT DNA)

Master Step 5: Normalize Libraries

Input = Purified DNA Libraries

Output = Normalized Libraries

SharedResultFile = Log File

Place Samples = Automatic placement into 96 well plate

Reagent Kits
Kit Name	Supplier
Nextera XT DNA Library Prep Kit, Box 1 (FC-131-1096)


Nextera XT DNA Library Prep Kit, Box 2 (FC-131-1096)

Record Details

Section

Subsection

Setting

Value

Specification

Step Data

Master Step Fields

0.1 N NaOH Prep Date

Date

Multiline Text Fields

Comment

Multiline Text

Step File Placeholders

File Name

Log

Auto

Sample Table

Table Columns

Normalized Molarity (nM)

Numeric

Required

Sequencing Instrument

Text

Preset

Required

•

MiSeq

•

NextSeq

•

HiSeq2500

•

HiSeq3000/4000

•

NovaSeq

•

HiSeqX

Automation
Automation Name	Setting	Specification
Next Step = Set Next Step - Remove	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 = ::REMOVE::' -log {compoundOutputFileLuid0} ["]`
Finish Step = Routing script - Normalize Libraries	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 -u {username} -p {password} -i { stepURI:v2 } -l {compoundOutputFileLuid0} script:changeWorkflow \` \ --FIELD_NAME ' Sequencing Instrument ' \--FIELD_VALUE 'MiSeq' \--WORKFLOW 'MiSeq Sequencing v1.0' \--STEP 'Library Pooling (MiSeq v1.0)' \--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' \--WORKFLOW 'NovaSeq 5000/6000 v2.0' \--STEP 'Define Run Format (NovaSeq 5000/6000 v2.0)' \--INPUTS_OR_OUTPUTS 'OUTPUTS'["]