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}
Automations
Normalize gDNA (Nextera XT)
Trigger Location = Record Details
Trigger Style = Manual button
Set Next Step - Advance
Trigger Location = Record Details
Trigger Style = Automatic upon exit
Normalize gDNA
Trigger Location = Not Used
Queue/Ice Bucket
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Row
Placement = Enabled
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Row
Record Details
Step Data (Master Step Fields)
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
Automations
Set Next Step - Advance
Trigger Location = Record Details
Trigger Style = Automatic upon exit
Copy to Output
Trigger Location = Not Used
Queue/Ice Bucket
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Row
Add Labels
Label Groups
Nextera XT v2 Full Set
Nextera XT v2 Set A
Record Details
Step Data (Master Step Fields)
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}
Automations
Set Next Step - Advance
Trigger Location = Record Details
Trigger Style = Automatic upon exit
Queue/Ice Bucket
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Row
Record Details
Step Data (Master Step Fields)
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
Automations
Generate Bioanalyzer Input file
Trigger Location = Record Details
Trigger Style = Automatic upon entry
Parse Bioanalyzer XML, Copy nM and Assign QC flags
Trigger Location = Record Details
Trigger Style = Manual button
Set Next Step - Output PASS/FAIL
Trigger Location = Record Details
Trigger Style = Automatic upon exit
Parse Bioanalyzer XML and assign QC flags
Trigger Location = Not Used
Parse Bioanalyzer XML, Assign QC flags, and Copy Concentrations
Trigger Location = Not Used
Parse Bioanalyzer XML, Calculate nM and assign QC flags
Trigger Location = Not Used
Queue/Ice Bucket
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Row
Placement = Enabled
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Column
Record Details
Group of Defaults
Nextera DNA Flex Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Peak 2 Size - bp
Nextera Mate Pair Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Region 1 Average Size - bp
Nextera XT DNA Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Peak 2 Size - bp
NRCC Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Peak 2 Size - bp
TruSeq ChIP-Seq Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Region 1 Average Size - bp
TruSeq Exome Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Peak 2 Size - bp
TruSeq Methyl Capture EPIC Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Peak 2 Size - bp
TruSeq Rapid Exome Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Peak 2 Size - bp
TruSeq RNA Access Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Peak 2 Size - bp
TruSeq RNA Exome Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Peak 2 Size - bp
TruSeq Small RNA Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Region 1 Average Size - bp
TruSeq Stranded mRNA Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Region 1 Average Size - bp
TruSeq Stranded Total RNA Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Region 1 Average Size - bp
TruSeq Targeted RNA Expression Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Peak 2 Size - bp
TruSight Myeloid Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Region 1 Average Size - bp
TruSight RNA Fusion Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Region 1 Average Size - bp
TSCA Library Validation
Criteria 1 - Operator = >=
Criteria 1 - Source Data Field = Region 1 Average Size - bp
Step Data
Group of Defaults = Nextera XT DNA Library Validation Validation
Master Step Fields
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
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
Routing script - Normalize Libraries
Trigger Location = Step
Trigger Style = Automatic upon exit
Queue/Ice Bucket
Defaults
Sample Grouping = Group by Containers
Well Sort Order = Row
Record Details
Step Data (Master Step Fields)
Reagent Kits
Nextera XT DNA Library Prep Kit, Box 1
Supplier = Illumina
Catalog Number = FC-131-1096
Sample Table
Column Headers
Category
Field Name
Field Type
Options
Additional Options and Dropdown Items
Container
Container Name
Built-in
Expanded View Fields
Placement Pattern = Row
Destination Containers
96 well plate
Default = Tagmentation
Step File Placeholders
Log File - Manually uploaded
Sample Table
Sample Display Default = Expand
Well Sort Order = Row
Table Columns - Global Fields
Naming Convention = {SubmittedSampleName}
Reagent Kits
Nextera XT DNA Library Prep Kit, Box 1
Supplier = Illumina
Catalog Number = FC-131-1096
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
Nextera XT v2 Set B
Nextera XT v2 Set C
Nextera XT v2 Set D
Default = Amplify Libraries
Step File Placeholders
Log File - Manually uploaded
Sample Table
Sample Display Default = Collapse
Well Sort Order = Row
Table Columns - Global Fields
Reagent Kits
AMPure XP Beads
Supplier = Beckman Coulter Genomics
Catalog Number = A63881
Website =
Nextera XT DNA Library Prep Kit, Box 1
Supplier = Illumina
Catalog Number = FC-131-1096
Sample Table
Column Headers
Category
Field Name
Field Type
Options
Additional Options and Dropdown Items
Container
Container Name
Built-in
Expanded View Fields
Step File Placeholders
Log File - Automatically attached
Sample Table
Sample Display Default = Expand
Well Sort Order = Row
Table Columns - Global Fields
Naming Convention = {InputItemName} Bioanalyzer
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
Placement Pattern = Column
Destination Containers
BioAnalyzer DNA High Sensitivity Chip
BioAnalyzer DNA 1000 Chip
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
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
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
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
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
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
Criteria 1 - Threshold Value = 200.00
Criteria 2 - Operator = <=
Criteria 2 - Source Data Field = Peak 2 Size - bp
Criteria 2 - Threshold Value = 300.00
Criteria 1 - Threshold Value = 200.00
Criteria 2 - Operator = <=
Criteria 2 - Source Data Field = Peak 2 Size - bp
Criteria 2 - Threshold Value = 500.00
Criteria 1 - Threshold Value = 200.00
Criteria 2 - Operator = <=
Criteria 2 - Source Data Field = Peak 2 Size - bp
Criteria 2 - Threshold Value = 320.00
Criteria 1 - Threshold Value = 200.00
Criteria 2 - Operator = <=
Criteria 2 - Source Data Field = Peak 2 Size - bp
Criteria 2 - Threshold Value = 320.00
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
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
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
Criteria 1 - Threshold Value = 100.00
Criteria 2 - Operator = <=
Criteria 2 - Source Data Field = Peak 2 Size - bp
Criteria 2 - Threshold Value = 300.00
Criteria 1 - Threshold Value = 150.00
Criteria 2 - Operator = <=
Criteria 2 - Source Data Field = Region 1 Size - bp
Criteria 2 - Threshold Value = 400.00
Criteria 1 - Threshold Value = 160.00
Criteria 2 - Operator = <=
Criteria 2 - Source Data Field = Region 1 Size - bp
Criteria 2 - Threshold Value = 700.00
Criteria 1 - Threshold Value = 300.00
Criteria 2 - Operator = <=
Criteria 2 - Source Data Field = Region 1 Size - bp
