Nextera XT v2.0

Overview

The Nextera XT DNA includes the following functionality:

• Preconfigured Nextera XT DNA 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 v2.0

Protocol Type = Library Prep

Next Steps Configuration

Step 1: Tagment Genomic DNA (Nextera XT DNA v2.0)

• Master Step Name = Tagment v2.0

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {SubmittedSampleName}

• Reagent Kits

  • Nextera XT DNA Library Prep Kit, Box 1

    • Supplier = Illumina

    • Catalog Number = FC-131-1096

Automations

Normalize gDNA (Nextera XT)

• Trigger Location = Record Details

• Trigger Style = Manual button

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t true -h false -exp 'output.::Sample Volume (ul):: = (0.2 * 5) / input.::Concentration:: ; output.::Buffer Volume (ul):: = 5 - output.::Sample Volume (ul)::' -log {compoundOutputFileLuid0}"

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"

Normalize gDNA

• Trigger Location = Not Used

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t true -h false -exp 'output.::Sample Volume (ul):: = 50 / input.::Concentration:: ; output.::Buffer Volume (ul):: = 10 - output.::Sample Volume (ul)::' -log {compoundOutputFileLuid0}"

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Row

• Sample Table

  • Column Headers

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	Container Name	Built-in
    Container	Well	Built-in
    Derived Sample	Sample Name	Built-in
    Derived Sample	Waiting	Built-in

  • Expanded View Fields

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	LIMS ID (Container)	Built-in
    Project	Project Name	Built-in

Placement = Enabled

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Row

  • Placement Pattern = Row

• Destination Containers

  • 96 well plate

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Comment	Multiline Text
  Thermal Cycler Program	Text		Default = Tagmentation

• Step File Placeholders

  • Log File - Manually uploaded

• Sample Table

  • Sample Display Default = Expand

  • Well Sort Order = Row

  • Table Columns - Global Fields

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	Container Name	Built-in
    Container	LIMS ID (Container)	Built-in
    Container	Well	Built-in
    Derived Sample	Buffer Volume (ul)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Sample Name	Built-in
    Derived Sample	Sample Volume (ul)	Numeric		Decimal Places Displayed = 2
    Project	Project Name	Built-in

Step 2: Amplify Libraries (Nextera XT DNA v2.0)

• Master Step Name = Amplification with Labels v2.0

• Step Type = Add Labels

• Derived Sample Generation = Fixed, 1

• Naming Convention = {SubmittedSampleName}

• Reagent Kits

  • Nextera XT DNA Library Prep Kit, Box 1

    • Supplier = Illumina

    • Catalog Number = FC-131-1096

Automations

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"

Copy to Output

• Trigger Location = Not Used

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'output.::RNA Sample Type:: = input.::RNA Sample Type::' -log {compoundOutputFileLuid0}"

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Row

• Sample Table (Column Headers)

  Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
  Container	Container Name	Built-in
  Container	LIMS ID (Container)	Built-in
  Container	Well	Built-in
  Derived Sample	Sample Name	Built-in
  Derived Sample	Waiting	Built-in
  Project	Project Name	Built-in

Add Labels

• Label Groups

  • Nextera XT v2 Full Set

  • Nextera XT v2 Set A

  • Nextera XT v2 Set B

  • Nextera XT v2 Set C

  • Nextera XT v2 Set D

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Comment	Multiline Text
  Thermal Cycler Program	Text		Default = Amplify Libraries

• Step File Placeholders

  • Log File - Manually uploaded

• Sample Table

  • Sample Display Default = Collapse

  • Well Sort Order = Row

  • Table Columns - Global Fields

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	Container Name	Built-in
    Container	LIMS ID (Container)	Built-in
    Container	Well	Built-in
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Step 3: Clean Up Libraries (Nextera XT DNA v2.0)

• Master Step Name = Clean Up v2.0

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {InputItemName}

• Reagent Kits

  • AMPure XP Beads

    • Supplier = Beckman Coulter Genomics

    • Catalog Number = A63881

    • Website = https://www.beckmancoulter.com/wsrportal/wsr/research-and-discovery/products-and-services/nucleic-acid-sample-preparation/agencourt-ampure-xp-pcr-purification/index.htm

  • Nextera XT DNA Library Prep Kit, Box 1

    • Supplier = Illumina

    • Catalog Number = FC-131-1096

Automations

Set Next Step - Advance

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} \
      script:evaluateDynamicExpression \
      -t false \
      -h false \
      -exp 'nextStep = ::ADVANCE::' \
      -log {compoundOutputFileLuid0}"

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Row

• Sample Table

  • Column Headers

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	Container Name	Built-in
    Container	Well	Built-in
    Derived Sample	Sample Name	Built-in
    Derived Sample	Waiting	Built-in

  • Expanded View Fields

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	LIMS ID (Container)	Built-in
    Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Comment	Multiline Text
  80% EtOH Prep Date	Date

• Step File Placeholders

  • Log File - Automatically attached

• Sample Table

  • Sample Display Default = Expand

  • Well Sort Order = Row

  • Table Columns - Global Fields

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	Container Name	Built-in
    Container	LIMS ID (Container)	Built-in
    Container	Well	Built-in
    Derived Sample	Sample Name	Built-in
    Project	Project Name	Built-in

Step 4: Bioanalyzer QC (Library Validation) (Nextera XT DNA v2.0)

• Master Step Name = Bioanalyzer QC (Library Validation) v2.0

• Step Type = Standard QC

• Measurement Generation = Fixed, 1

• Naming Convention = {InputItemName} Bioanalyzer

Automations

Generate Bioanalyzer Input file

• Trigger Location = Record Details

• Trigger Style = Automatic upon entry

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/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

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 = Record Details

• Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t true -h false -excludeControls true -exp 'if (output.QC == true) { nextStep = ::ADVANCE:: } else { nextStep = ::ESCALATE:: }' -log {compoundOutputFileLuid0}"

Parse Bioanalyzer XML and assign QC flags

• Trigger Location = Not Used

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {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}"

Parse Bioanalyzer XML, Assign QC flags, and Copy Concentrations

• Trigger Location = Not Used

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {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} && /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.::Concentration:: = output.::Region 1 Conc.:: ; input.::Concentration:: = output.::Concentration:: ; output.::Conc. Units:: = ::ng/ul:: ; input.::Conc. Units:: = output.::Conc. Units::' -log {compoundOutputFileLuid8}"

Parse Bioanalyzer XML, Calculate nM and assign QC flags

• Trigger Location = Not Used

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {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 'output.::Concentration:: = output.::Region 1 Conc.:: ; output.::Molarity (nM):: = (output.::Concentration:: * 1000000) / (660 * output.::Region 1 Average Size - bp::) ; input.::Molarity (nM):: = output.::Molarity (nM):: ; output.::Conc. Units:: = ::ng/ul::' -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}"

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Row

• Sample Table (Column Headers)

  Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
  Container	Container Name	Built-in
  Container	LIMS ID (Container)	Built-in
  Container	Well	Built-in
  Derived Sample	Sample Name	Built-in
  Derived Sample	Waiting	Built-in
  Project	Project Name	Built-in

Placement = Enabled

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Column

  • Placement Pattern = Column

• Destination Containers

  • BioAnalyzer DNA High Sensitivity Chip

  

  • BioAnalyzer DNA 1000 Chip

  

Record Details

Group of Defaults

Nextera DNA Flex Library Validation

• Criteria 1 - Operator = >=

• Criteria 1 - Source Data Field = Peak 2 Size - bp

• Criteria 1 - Threshold Value = 150.00

• Criteria 2 - Operator = <=

• Criteria 2 - Source Data Field = Peak 2 Size - bp

• Criteria 2 - Threshold Value = 1,500.00

Nextera Mate Pair Library Validation

• Criteria 1 - Operator = >=

• Criteria 1 - Source Data Field = Region 1 Average Size - bp

• Criteria 1 - Threshold Value = 150.00

• Criteria 2 - Operator = <=

• Criteria 2 - Source Data Field = Region 1 Average Size - bp

• Criteria 2 - Threshold Value = 400.00

Nextera XT DNA Library Validation

• Criteria 1 - Operator = >=

• Criteria 1 - Source Data Field = Peak 2 Size - bp

• Criteria 1 - Threshold Value = 250.00

• Criteria 2 - Operator = <=

• Criteria 2 - Source Data Field = Peak 2 Size - bp

• Criteria 2 - Threshold Value = 1,000.00

NRCC Library Validation

• Criteria 1 - Operator = >=

• Criteria 1 - Source Data Field = Peak 2 Size - bp

• Criteria 1 - Threshold Value = 350.00

• Criteria 2 - Operator = <=

• Criteria 2 - Source Data Field = Peak 2 Size - bp

• Criteria 2 - Threshold Value = 1,000.00

TruSeq ChIP-Seq Library Validation

• Criteria 1 - Operator = >=

• Criteria 1 - Source Data Field = Region 1 Average Size - bp

• Criteria 1 - Threshold Value = 150.00

• Criteria 2 - Operator = <=

• Criteria 2 - Source Data Field = Region 1 Average Size - bp

• Criteria 2 - Threshold Value = 400.00

TruSeq Exome Library Validation

• Criteria 1 - Operator = >=

• Criteria 1 - Source Data Field = Peak 2 Size - bp

• Criteria 1 - Threshold Value = 150.00

• Criteria 2 - Operator = <=

• Criteria 2 - Source Data Field = Peak 2 Size - bp

• Criteria 2 - Threshold Value = 1,000.00

TruSeq Methyl Capture EPIC Library Validation

• Criteria 1 - Operator = >=

• Criteria 1 - Source Data Field = Peak 2 Size - bp

• Criteria 1 - Threshold Value = 200.00

• Criteria 2 - Operator = <=

• Criteria 2 - Source Data Field = Peak 2 Size - bp

• Criteria 2 - Threshold Value = 300.00

TruSeq Rapid Exome Library Validation

• Criteria 1 - Operator = >=

• Criteria 1 - Source Data Field = Peak 2 Size - bp

• Criteria 1 - Threshold Value = 200.00

• Criteria 2 - Operator = <=

• Criteria 2 - Source Data Field = Peak 2 Size - bp

• Criteria 2 - Threshold Value = 500.00

TruSeq RNA Access Library Validation

• Criteria 1 - Operator = >=

• Criteria 1 - Source Data Field = Peak 2 Size - bp

• Criteria 1 - Threshold Value = 200.00

• Criteria 2 - Operator = <=

• Criteria 2 - Source Data Field = Peak 2 Size - bp

• Criteria 2 - Threshold Value = 320.00

TruSeq RNA Exome Library Validation

• Criteria 1 - Operator = >=

• Criteria 1 - Source Data Field = Peak 2 Size - bp

• Criteria 1 - Threshold Value = 200.00

• Criteria 2 - Operator = <=

• Criteria 2 - Source Data Field = Peak 2 Size - bp

• Criteria 2 - Threshold Value = 320.00

TruSeq Small RNA Library Validation

• Criteria 1 - Operator = >=

• Criteria 1 - Source Data Field = Region 1 Average Size - bp

• Criteria 1 - Threshold Value = 100.00

• Criteria 2 - Operator = <=

• Criteria 2 - Source Data Field = Region 1 Average Size - bp

• Criteria 2 - Threshold Value = 200.00

TruSeq Stranded mRNA Library Validation

• Criteria 1 - Operator = >=

• Criteria 1 - Source Data Field = Region 1 Average Size - bp

• Criteria 1 - Threshold Value = 250.00

• Criteria 2 - Operator = <=

• Criteria 2 - Source Data Field = Region 1 Average Size - bp

• Criteria 2 - Threshold Value = 275.00

TruSeq Stranded Total RNA Library Validation

• Criteria 1 - Operator = >=

• Criteria 1 - Source Data Field = Region 1 Average Size - bp

• Criteria 1 - Threshold Value = 250.00

• Criteria 2 - Operator = <=

• Criteria 2 - Source Data Field = Region 1 Average Size - bp

• Criteria 2 - Threshold Value = 275.00

TruSeq Targeted RNA Expression Library Validation

• Criteria 1 - Operator = >=

• Criteria 1 - Source Data Field = Peak 2 Size - bp

• Criteria 1 - Threshold Value = 100.00

• Criteria 2 - Operator = <=

• Criteria 2 - Source Data Field = Peak 2 Size - bp

• Criteria 2 - Threshold Value = 300.00

TruSight Myeloid Library Validation

• Criteria 1 - Operator = >=

• Criteria 1 - Source Data Field = Region 1 Average Size - bp

• Criteria 1 - Threshold Value = 150.00

• Criteria 2 - Operator = <=

• Criteria 2 - Source Data Field = Region 1 Size - bp

• Criteria 2 - Threshold Value = 400.00

TruSight RNA Fusion Library Validation

• Criteria 1 - Operator = >=

• Criteria 1 - Source Data Field = Region 1 Average Size - bp

• Criteria 1 - Threshold Value = 160.00

• Criteria 2 - Operator = <=

• Criteria 2 - Source Data Field = Region 1 Size - bp

• Criteria 2 - Threshold Value = 700.00

TSCA Library Validation

• Criteria 1 - Operator = >=

• Criteria 1 - Source Data Field = Region 1 Average Size - bp

• Criteria 1 - Threshold Value = 300.00

• Criteria 2 - Operator = <=

• Criteria 2 - Source Data Field = Region 1 Size - bp

• Criteria 2 - Threshold Value = 400.00

• Step Data

  • Group of Defaults = Nextera XT DNA Library Validation Validation

  • Master Step Fields

    Field Name	Field Type	Options	Additional Options and Dropdown Items
    Criteria 1 - Operator	Text Dropdown	Custom Entries	Presets >= <= = !=
    Criteria 1 - Source Data Field	Text Dropdown		Presets Concentration Conc. Units Number of Peaks found Peak 1 Size - bp Peak 1 Conc. Peak 1 Molarity Peak 2 Size - bp Peak 2 Conc. Peak 2 Molarity Peak 3 Size - bp Peak 3 Conc. Peak 3 Molarity Peak 4 Size - bp Peak 4 Conc. Peak 4 Molarity Peak 5 Size - bp Peak 5 Conc. Peak 5 Molarity Number of Regions found Region 1 Average Size - bp Region 1 Conc. Region 1 Molarity Region 2 Average Size - bp Region 2 Conc. Region 2 Molarity Region 3 Average Size - bp Region 3 Conc. Region 3 Molarity Region 4 Average Size - bp Region 4 Conc. Region 4 Molarity Region 5 Average Size - bp Region 5 Conc. Region 5 Molarity
    Criteria 1 - Threshold Value	Numeric		Decimal Places Displayed = 2
    Criteria 2 - Operator	Text Dropdown	Custom Entries	Presets >= <= = !=
    Criteria 2 - Source Data Field	Text Dropdown		Presets Concentration Conc. Units Number of Peaks found Peak 1 Size - bp Peak 1 Conc. Peak 1 Molarity Peak 2 Size - bp Peak 2 Conc. Peak 2 Molarity Peak 3 Size - bp Peak 3 Conc. Peak 3 Molarity Peak 4 Size - bp Peak 4 Conc. Peak 4 Molarity Peak 5 Size - bp Peak 5 Conc. Peak 5 Molarity Number of Regions found Region 1 Average Size - bp Region 1 Conc. Region 1 Molarity Region 2 Average Size - bp Region 2 Conc. Region 2 Molarity Region 3 Average Size - bp Region 3 Conc. Region 3 Molarity Region 4 Average Size - bp Region 4 Conc. Region 4 Molarity Region 5 Average Size - bp Region 5 Conc. Region 5 Molarity
    Criteria 2 - Threshold Value	Numeric		Decimal Places Displayed = 2
    Use strict matching for Bioanalyzer results	Toggle Switch		Default = None Set

• Step File Placeholders

  • Bioanalyzer Input File - Automatically attached

  • Bioanalyzer Input File Generation Log File - Automatically attached

  • Bioanalyzer XML Result File (required) - Manually uploaded

  • Result File (optional) - Manually uploaded

  • PDF Summary File (optional) - Manually uploaded

  • Bioanalyzer XML Parsing Log File - Automatically attached

  • QC Assignment Log File - Automatically attached

  • QC Assignment Report - Automatically attached

• Sample Table

  • Enable QC Flags = Yes

  • Sample Display Default = Expand

  • Well Sort Order = Column

  • File Column Options

    • File Column Display = Hide

    • File Attachment Method = Auto

  • Table Columns - Global Fields

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Derived Sample	Molarity (nM)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Sample Name	Built-in
    Measurement	BA Sample Name	Text
    Measurement	Concentration	Numeric		Decimal Places Displayed = 2
    Measurement	Conc. Units	Text
    Measurement	Molarity (nM)	Numeric		Decimal Places Displayed = 2
    Measurement	Number of Peaks found	Numeric		Decimal Places Displayed = 0
    Measurement	Number of Regions found	Numeric		Decimal Places Displayed = 0
    Measurement	Peak 1 Conc.	Numeric		Decimal Places Displayed = 2
    Measurement	Peak 1 Molarity	Numeric		Decimal Places Displayed = 2
    Measurement	Peak 1 Size - bp	Numeric		Decimal Places Displayed = 0
    Measurement	Peak 2 Conc.	Numeric		Decimal Places Displayed = 2
    Measurement	Peak 2 Molarity	Numeric		Decimal Places Displayed = 2
    Measurement	Peak 2 Size - bp	Numeric		Decimal Places Displayed = 0
    Measurement	Peak 3 Conc.	Numeric		Decimal Places Displayed = 2
    Measurement	Peak 3 Molarity	Numeric		Decimal Places Displayed = 2
    Measurement	Peak 3 Size - bp	Numeric		Decimal Places Displayed = 0
    Measurement	Peak 4 Conc.	Numeric		Decimal Places Displayed = 2
    Measurement	Peak 4 Molarity	Numeric		Decimal Places Displayed = 2
    Measurement	Peak 4 Size - bp	Numeric		Decimal Places Displayed = 0
    Measurement	Peak 5 Conc.	Numeric		Decimal Places Displayed = 2
    Measurement	Peak 5 Molarity	Numeric		Decimal Places Displayed = 2
    Measurement	Peak 5 Size - bp	Numeric		Decimal Places Displayed = 2
    Measurement	Region 1 Average Size - bp	Numeric		Decimal Places Displayed = 0
    Measurement	Region 1 Conc.	Numeric		Decimal Places Displayed = 2
    Measurement	Region 1 Molarity	Numeric		Decimal Places Displayed = 2
    Measurement	Region 2 Average Size - bp	Numeric		Decimal Places Displayed = 0
    Measurement	Region 2 Conc.	Numeric		Decimal Places Displayed = 2
    Measurement	Region 2 Molarity	Numeric		Decimal Places Displayed = 2
    Measurement	Region 3 Average Size - bp	Numeric		Decimal Places Displayed = 0
    Measurement	Region 3 Conc.	Numeric		Decimal Places Displayed = 2
    Measurement	Region 3 Molarity	Numeric		Decimal Places Displayed = 2
    Measurement	Region 4 Average Size - bp	Numeric		Decimal Places Displayed = 0
    Measurement	Region 4 Conc.	Numeric		Decimal Places Displayed = 2
    Measurement	Region 4 Molarity	Numeric		Decimal Places Displayed = 2
    Measurement	Region 5 Average Size - bp	Numeric		Decimal Places Displayed = 0
    Measurement	Region 5 Conc.	Numeric		Decimal Places Displayed = 2
    Measurement	Region 5 Molarity	Numeric		Decimal Places Displayed = 2

Step 5: Normalize Libraries (Nextera XT DNA v2.0)

• Master Step Name = Normalize Libraries 3 v2.0.10

• Step Type = Standard

• Derived Sample Generation = Fixed, 1

• Naming Convention = {SubmittedSampleName}

• Reagent Kits

  • Nextera XT DNA Library Prep Kit, Box 1

    • Supplier = Illumina

    • Catalog Number = FC-131-1096

  • Nextera XT DNA Library Prep Kit, Box 1

    • Supplier = Illumina

    • Catalog Number = FC-131-1096

    • Website = www.illumina.com

ℹ️ The version of Normalized Libraries 3 master step name may be different depending on the version of IPP installed.

Automations

Set Next Step - Remove

• Trigger Location = Record Details

• Trigger Style = Automatic upon exit

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'nextStep = ::REMOVE::' -log {compoundOutputFileLuid0}"

Routing script - Normalize Libraries

• Trigger Location = Step

• Trigger Style = Automatic upon exit

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 v3.2' \
--STEP 'Library Pooling (MiSeq v3.2)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Sequencing Instrument' \
--FIELD_VALUE 'NextSeq' \
--WORKFLOW 'NextSeq 500/550 Sequencing v1.2' \
--STEP 'Library Pooling (NextSeq 500/550 v1.2)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Sequencing Instrument' \
--FIELD_VALUE 'NovaSeq 2.0' \
--WORKFLOW 'NovaSeq 6000 v2.3' \
--STEP 'Define Run Format (NovaSeq 6000 v2.3)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Sequencing Instrument' \
--FIELD_VALUE 'NovaSeq 3.0' \
--WORKFLOW 'NovaSeq 6000 v3.8' \
--STEP 'Define Run Format (NovaSeq 6000 v3.8)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Sequencing Instrument' \
--FIELD_VALUE 'NovaSeqDx' \
--WORKFLOW 'NovaSeqDx v1.2' \
--STEP 'Define Run Format (NovaSeqDx v1.2)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Sequencing Instrument' \
--FIELD_VALUE 'NextSeq 1000/2000' \
--WORKFLOW 'NextSeq 1000/2000 Sequencing v2.4' \
--STEP 'Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Sequencing Instrument' \
--FIELD_VALUE 'NovaSeq X Series' \
--WORKFLOW 'NovaSeq X Series v1.1' \
--STEP 'Assign Analysis Configuration Template (NovaSeq X Series Sequencing v1.1)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS' \
\
--FIELD_NAME 'Sequencing Instrument' \
--FIELD_VALUE 'NextSeq 1000/2000 On-Prem' \
--WORKFLOW 'NextSeq 1000/2000 On-Prem Sequencing v1.0' \
--STEP 'Library Pooling and Dilution (NextSeq 1000/2000 On-Prem Sequencing v1.0)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"

ℹ️ The field name, workflow version and step version for the Sequencing Instrument may vary depending on the version of the IPP.

Queue/Ice Bucket

• Defaults

  • Sample Grouping = Group by Containers

  • Well Sort Order = Row

• Sample Table

  • Column Headers

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	Container Name	Built-in
    Container	Well	Built-in
    Derived Sample	Sample Name	Built-in
    Derived Sample	Waiting	Built-in

  • Expanded View Fields

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	LIMS ID (Container)	Built-in
    Project	Project Name	Built-in

Record Details

• Step Data (Master Step Fields)

  Field Name	Field Type	Options	Additional Options and Dropdown Items
  Comment	Multiline Text
  0.1 N NaOH Prep Date	Date

• Step File Placeholders

  • Log File - Automatically attached

• Sample Table

  • Sample Display Default = Expand

  • Well Sort Order = Row

  • Table Columns - Global Fields

    Category	Field Name	Field Type	Options	Additional Options and Dropdown Items
    Container	Container Name	Built-in
    Container	LIMS ID (Container)	Built-in
    Container	Well	Built-in
    Derived Sample	Normalized Molarity (nM)	Numeric		Decimal Places Displayed = 2
    Derived Sample	Sample Name	Built-in
    Derived Sample	Sequencing Instrument	Text Dropdown	Required Field	Presets MiSeq NextSeq NextSeq 1000/2000 NextSeq 1000/2000 On-Prem NovaSeq 2.0 NovaSeq 3.0 NovaSeq X Series NovaSeqDx
    Project	Project Name	Built-in

    ℹ️ The preset options for Derived Sample Sequencing Instrument may vary depending on the version of the IPP.