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Nextera XT DNA Library Preparation Kit

Offers a 90-min workflow to prepare DNA libraries for small genomes, PCR amplicons, plasmids, and cDNA sequencing, with a low DNA input requirement.

~5.5 hr from DNA extraction to normalized library. (Library prep time: ~90 minutes).

Assay time

15 min

Hands-on time

1 ng DNA

Input quantity

See full details in the specifications table

We recommend ordering the Illumina DNA/RNA UD Indexes Sets A-D (20091654, 20091656, 20091658, 20091660). The IDT for Illumina DNA/RNA UD Indexes Sets A-D (20027213, 20027214,20042666, 20042667) are being discontinued; the last order date is January 30th, 2025.

Overview

The Nextera XT DNA Library Preparation Kit offers a fast and easy workflow for preparing, quantifying, and normalizing libraries for sequencing small genomes, PCR amplicons, plasmids, microbial genomes, concatenated amplicons, and double-stranded cDNA from a low amount of input DNA (1 ng), making this kit amenable for use with precious samples available in limited quantity.

Key features

  • Provides rapid library preparation in as little as 90 min with only 15 min of hands-on time

  • Eliminates library quantification before sample pooling and sequencing 

  • Delivers quick results in under 8 hr using Illumina sequencing systems

  • Achieves high, even coverage and accurate calls for PCR amplicons

  • Produces high-quality de novo assembly of small genomes

  • Allows scalability by processing and uniquely barcoding up to 384 samples in a single experiment

Specifications

Assay time ~5.5 hr from DNA extraction to normalized library. (Library prep time: ~90 minutes).
Automation capability Liquid handling robot(s)
Automation details Explore available automation methods
Description Prepare sequencing libraries for small genomes, PCR amplicons, and plasmids in 90 min, with a low DNA input requirement.
Hands-on time 15 min
Input quantity 1 ng DNA
Instruments MiSeq System, iSeq 100 System, NextSeq 550 System, NextSeq 2000 System, NextSeq 1000 System, MiniSeq System, NextSeq 500 System
Mechanism of action Enzymatic fragmentation
Method Amplicon sequencing, 16s rRNA sequencing, De novo sequencing, Whole-genome sequencing, Shotgun sequencing
Multiplexing Up to 384 uniquely indexed samples may be pooled and sequenced together.
Nucleic acid type DNA
Specialized sample types Not FFPE-compatible, Low-input samples, Single cells
Species category Drosophila, Any species, Mammalian, Mouse, Yeast, Zebrafish, Fungal, Human, Rat, Plant, Virus, Nematode, Bacteria
Species details Compatible with any species
Target insert size 300 bp–1.5 kb
Technology Sequencing
Variant class Single nucleotide polymorphisms (SNPs), Structural variants

Required products

Required products:

Illumina Purification Beads for purifying amplified libraries.

One of the following index kits:

Bead-based normalization:

  • Nextera XT V2 Index Kit Set A, B, C, or D 
  • Nextera XT Index Kit (FC-131-1001)

Standard normalization:

  • Illumina DNA/RNA UD Indexes Set A, B, C, or D
/ Results

Applications

The Nextera XT DNA Library Preparation Kit supports a low DNA input (1 ng) and offers high-quality sequencing-ready libraries for small genomes, PCR amplicons, and plasmids, in as little as 90 minutes.

Example workflow

1
Prep

Nextera XT DNA Library Prep Kit

2
Sequence
3
Analyze

Project recommendations

Instrument Recommended number of samples Read length
MiSeq System

Up to 384 samples per run

Up to 2 × 300 bp
NextSeq 550 System

Up to 384 samples per run

Up to 2 × 150 bp

Related applications and methods

Microbial sequencing methods

NGS-based microbial sequencing methods include shotgun metagenomics, 16S rRNA sequencing, whole-genome and de novo sequencing, and transcriptomics.

Whole-genome sequencing (WGS)

Whole-genome sequencing delivers a comprehensive view, ideal for discovery applications. Newer genome sequencers perform WGS more rapidly than ever.

Single-cell and ultra-low-input RNA-Seq

With single-cell RNA-Seq, you can study cellular differences often masked by bulk sampling. Explore high- and low-throughput single-cell sequencing methods.

Documentation

Product literature
Support documentation

Compare

Nextera XT DNA Library Preparation Kit Illumina DNA Prep
Assay time ~5.5 hr from DNA extraction to normalized library. (Library prep time: ~90 minutes). ~3-4 hr (from DNA extraction to normalized library)
Automation capability Liquid handling robot(s) Liquid handling robot(s)
Automation details Explore available automation methods Explore available automation methods
Description Prepare sequencing libraries for small genomes, PCR amplicons, and plasmids in 90 min, with a low DNA input requirement. A fast, flexible library prep workflow that accommodates an assortment of sample types and DNA input amounts, allowing access to a wide range of applications, from human to microbial whole-genome sequencing and more. 
Hands-on time 15 min 1-1.5 hr
Input quantity 1 ng DNA 1-500 ng DNA for small genomes (e.g. microbial), or 100-500 ng DNA for large genomes (e.g. human). (For blood and saliva, see the reference guide).
Instruments MiSeq System, iSeq 100 System, NextSeq 550 System, NextSeq 2000 System, NextSeq 1000 System, MiniSeq System, NextSeq 500 System MiSeq System, iSeq 100 System, NextSeq 550 System, NextSeq 2000 System, NextSeq 1000 System, MiSeqDx in Research Mode, MiniSeq System, NextSeq 550Dx in Research Mode, NovaSeq X System, NovaSeq 6000 System, NovaSeq X Plus System, MiSeq i100 System, MiSeq i100 Plus System
Mechanism of action Enzymatic fragmentation Bead-linked transposome
Method Amplicon sequencing, 16s rRNA sequencing, De novo sequencing, Whole-genome sequencing, Shotgun sequencing Amplicon sequencing, De novo sequencing, Whole-genome sequencing, Shotgun sequencing
Multiplexing Up to 384 uniquely indexed samples may be pooled and sequenced together. Up to 384 unique dual (UD) combinations and 96 combinatorial dual (CD) combinations
Nucleic acid type DNA DNA
Specialized sample types Not FFPE-compatible, Low-input samples, Single cells Blood, Not FFPE-compatible, Saliva
Species category Drosophila, Any species, Mammalian, Mouse, Yeast, Zebrafish, Fungal, Human, Rat, Plant, Virus, Nematode, Bacteria Drosophila, Any species, Mammalian, Mouse, Yeast, Zebrafish, Human, Rat, Plant, Virus, Nematode, Bacteria
Species details Compatible with any species Compatible with any species
Target insert size 300 bp–1.5 kb ~350 bp
Technology Sequencing Sequencing
Variant class Single nucleotide polymorphisms (SNPs), Structural variants Single nucleotide polymorphisms (SNPs), Gene fusions, Loss of heterozygosity (LOH), Somatic variants, Chromosomal abnormalities, Germline variants, Structural variants, Insertions-deletions (indels), Copy number variants (CNVs)

Selection Tools:

Library Prep and Array Kit Selector

Find the right sequencing library preparation kit or microarray for your needs. Filter by method, species, and more. Compare, share, and order kits.

DesignStudio Custom Assay Design Tool

DesignStudio is a web-based assay design tool to help scientists design and order custom sequencing probes or create custom genotyping array assays.

Figures

High coverage, accurate calls

Nextera XT kit produces high and even coverage across a range of amplicon sizes with accurate variant calls as confirmed within the dbSNP database. 

Even coverage across large amplicons

Nextera XT kit shows (A) high sequencing coverage (> 1000×) and (B) accurate variant calling along positions of a large amplicon (5.1 kb) in a highly variable non-coding region of the human genome.

High-quality de novo assembly of small genomes

Nextera XT kit produces high-quality de novo assembly of Escherichia coli genome with excellent N50 scores and coverage from 1 ng of genomic DNA.

Resources

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Best practices for Nextera library prep

This video reviews the key steps in the Nextera library preparation process and provides some tips and tricks on how to achieve optimal results.

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Nextera XT: Sample preparation and usage

This webinar on the Nextera XT sample preparation process will cover the protocol, best practices for critical steps, and recommended applications.

Library prep (2)

Nextera XT DNA Library Preparation Kit (24 samples)

FC-131-1024

Includes reagents for preparing 24 libraries. Purchase index adapters and purification beads separately.

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Nextera XT DNA Library Preparation Kit (96 samples)

FC-131-1096

Includes reagents for preparing 96 libraries. Purchase index adapters and purification beads separately.

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Illumina Advantage Products (2)

TG Nextera® XT Index Kit v2 Set A (96 Indices, 384 Samples)

TG-131-2001

Version 2 of 96 indexes for Nextera XT sample prep (Set A).

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TG Nextera® XT Index Kit v2 Set B (96 Indices, 384 Samples)

TG-131-2002

Version 2 of 96 indexes for Nextera XT sample prep (Set B).

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Index adapters (9)

Illumina® DNA/RNA UD Indexes Set A, Tagmentation (96 Indexes, 96 Samples)

20091654

Illumina® DNA/RNA UD Indexes Set A, Tagmentation (96 Indexes, 96 Samples) Includes 96, 10 bp indexes sufficient for labeling 96 samples. Purchase library prep and enrichment reagents and probe panels separately.

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Illumina® DNA/RNA UD Indexes Set B, Tagmentation (96 Indexes, 96 Samples)

20091656

Illumina® DNA/RNA UD Indexes Set B, Tagmentation (96 Indexes, 96 Samples) Includes 96, 10 bp indexes sufficient for labeling 96 samples. Purchase library prep and enrichment reagents and probe panels separately.

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Illumina® DNA/RNA UD Indexes Set C, Tagmentation (96 Indexes, 96 Samples)

20091658

Illumina® DNA/RNA UD Indexes Set C, Tagmentation (96 Indexes, 96 Samples) Includes 96, 10 bp indexes sufficient for labeling 96 samples. Purchase library prep and enrichment reagents and probe panels separately.

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Illumina® DNA/RNA UD Indexes Set D, Tagmentation (96 Indexes, 96 Samples)

20091660

Illumina® DNA/RNA UD Indexes Set D, Tagmentation (96 Indexes, 96 Samples) Includes 96, 10 bp indexes sufficient for labeling 96 samples. Purchase library prep and enrichment reagents and probe panels separately.

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Nextera XT Index Kit v2 Set A (96 indexes, 384 samples)

FC-131-2001

Includes 96, 8 bp indexes sufficient for labeling 384 samples. Purchase library prep kit and purification beads separately.

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Nextera XT Index Kit v2 Set B (96 indexes, 384 samples)

FC-131-2002

Includes 96, 8 bp indexes sufficient for labeling 384 samples. Purchase library prep kit and purification beads separately.

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Nextera XT Index Kit v2 Set C (96 indexes, 384 samples)

FC-131-2003

Includes 96, 8 bp indexes sufficient for labeling 384 samples. Purchase library prep kit and purification beads separately.

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Nextera XT Index Kit v2 Set D (96 indexes, 384 samples)

FC-131-2004

Includes 96, 8 bp indexes sufficient for labeling 384 samples. Purchase library prep kit and purification beads separately.

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Nextera XT Index Kit (24 indexes, 96 samples)

FC-131-1001

Includes 24, 8 bp indexes sufficient for labeling 96 samples. Purchase library prep and purification beads separately.

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Accessory products (2)

Illumina Purification Bead, 100mL

20060057

Includes one, 100-ml container of beads for library clean-up and size selection. Purchase library prep and enrichment reagents, probe panels, and indexes separately.

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Illumina Purification Bead, 400mL

20060058

Includes four, 100-ml container of beads for library clean-up and size selection. Purchase library prep and enrichment reagents, probe panels, and indexes separately.

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FAQs

Nextera tagmentation uses in vitro transposition to prepare sequence-ready libraries from genomic DNA for all Illumina sequencing platforms. The tagmentation technology simultaneously fragments and tags DNA with sequencing adapters in a single-tube enzymatic reaction. 

Nextera chemistry uses transposon-based DNA fragmentation whereby a transposon cleaves and tags DNA with adapters, while TruSeq chemistry follows an alternate enrichment protocol that uses mechanical shearing to fragment the DNA.

A maximum of 384 samples can be multiplexed per Nextera XT library.
/ Results

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Illumina DNA Prep

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MiSeq Reagent Kits v2

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MiSeq Reagent Kit v3

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TruSight Hereditary Cancer Panel

Targeted sequencing panel that assesses germline mutations across 113 genes and 125 SNPs associated with genetic cancer risk predisposition.

Illumina Performance Qualification

Verify instrument performance and obtain an audit-ready PQ report from certified Illumina engineers to help meet regulatory requirements. 

Nextera DNA Indexes

Nextera DNA Indexes enable preparation of high-quality libraries that can be multiplexed for increased throughput.

iSeq 100 Reagents

Reagents for the iSeq 100 System feature a ready-to-use cartridge with one-channel SBS chemistry and CMOS technology for simple, low-cost sequencing.

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