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Illumina DNA Prep

A fast, integrated workflow for preparing libraries for use in a wide range of sequencing applications.

~3-4 hr (from DNA extraction to normalized library)

Assay time

1-1.5 hr

Hands-on time

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).

Input quantity

See full details in the specifications table

Nextera DNA Flex products are now called Illumina DNA Prep. The (M) designation denotes a bead-linked transposome that generates an insert size of ~350bp. 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

Illumina DNA Prep, previously known as Nextera DNA Flex, offers a fast, robust, and flexible workflow for preparing normalized, sequencing-ready libraries from a wide range of DNA input types and amounts facilitating an array of applications, from human whole-genome sequencing to sequencing amplicons, plasmids, and microbial species.1

  • Delivers even genome coverage and exceptional data quality 

  • Enhances library preparation efficiency with integrated DNA extraction protocols 

  • Provides a fast, user-friendly workflow that reduces total library prep time to ~3.5 hr 

Obtain reliable results

Illumina DNA Prep workflow delivers consistent insert sizes, uniform coverage, and optimized performance, regardless of DNA input amount or genome size. The bead-based technology minimizes bias and opportunities for error, resulting in highly reproducible sequencing data. 

Access flexible throughput options

The Illumina DNA/RNA UD Indexes Sets offer up to 384 unique dual indexes, enabling accurate assignment of reads and efficient use of the flow cell. 

Learn more about multiplexing

Illumina Advantage product availability

This product is also available as an Illumina Advantage (TG) product. Illumina Advantage large-scale sequencing products feature lot-specific shipments and testing, extended shelf life, and advanced change notifications for greater laboratory efficiency.

Illumina DNA Prep product line

Find fast, optimized sequencing library preparation solutions for use in a wide range of applications.

Illumina DNA Prep

A fast, integrated workflow for preparing libraries for use in a wide range of sequencing applications.

Illumina DNA Prep with Enrichment

A complete targeted resequencing solution for a wide range of applications, with fast integrated workflow and options for custom and fixed enrichment panels.

Illumina DNA PCR-Free Prep

An efficient, fast, and integrated library preparation workflow that yields highly accurate data for sensitive sequencing applications.

Learn more about the Illumina DNA Prep product line

Specifications

Assay time ~3-4 hr (from DNA extraction to normalized library)
Automation capability Liquid handling robots
Automation details Explore available automation methods
Description 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 1-1.5 hr
Input quantity 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, MiSeqDx in Research Mode, MiniSeq System, NextSeq 550Dx in Research Mode, NovaSeq X System, NovaSeq 6000 System, NovaSeq X Plus System
Mechanism of action Bead-linked transposome
Method Amplicon sequencing, Whole-genome sequencing, De novo sequencing, Shotgun sequencing
Multiplexing Up to 384 unique dual (UD) combinations and 96 combinatorial dual (CD) combinations
Nucleic acid type DNA
Sample type details Not validated for use with FFPE samples 
Specialized sample types Blood, Not FFPE-compatible, Saliva
Species category Drosophila, Any species, Mammalian, Mouse, Yeast, Zebrafish, Human, Rat, Plant, Virus, Nematode, Bacteria
Species details Compatible with any species
Target insert size ~350 bp
Technology Sequencing
Variant class 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)

Required products

Each Illumina DNA Prep kit requires one of the index kits to complete the protocol, regardless of the sample pooling level used for sequencing. We encourage customers to use unique dual indexing whenever possible.  Learn more about  choosing the right indexing option for your application. 

The index kits are NOT interchangeable with the Nextera DNA or Nextera XT Index Kits. 

The Flex Lysis Kit is a separate product that complements the blood DNA extraction aspect of Illumina DNA Prep. 

Illumina purification beads are included in the Illumina DNA Prep kit and do not need to be purchased separately.  

/ Results

Applications

Illumina DNA Prep supports a broad DNA input range (1–500 ng) and offers high-quality libraries for sequencing samples ranging from large whole genomes to amplicons, plasmids, and microbial species, in as little as ~3.5 hours. 

Example workflow

1
Prep

Illumina DNA Prep 

2
Sequence
3
Analyze

Featured application notes

Automated Illumina DNA Prep workflow for high-throughput metagenomics
Nextera crude lysate protocol for metagenomic next-generation sequencing
Direct bacterial colony sequencing with the Nextera DNA Flex library preparation kit
Fast, flexible, and self-normalizing library prep with Nextera Flex chemistry
Nextera DNA Flex library preparation for soil shotgun metagenomics analysis
Microbial WGS with Illumina DNA Prep

Project recommendations

Instrument Recommended number of samples Read length
NextSeq 550 System

1 sample per run (high output; based on 30× coverage of a human genome)

Up to 2 × 150 bp
NovaSeq 6000 System

2–10 samples per run  (dual flow cell; based on 30× coverage of a human genome)

Up to 2 × 125 bp (rapid run) 
Up to 2 × 150 bp (high output)

Related applications and methods

Microbial whole-genome sequencing

Use microbial whole-genome sequencing to map genomes of novel organisms, finish genomes of known organisms, or compare genomes across multiple samples.

Large whole-genome sequencing

Large whole-genome sequencing informs disease research and population genomics studies and reveals disease-associated alleles.

Metagenomic sequencing

Comprehensively sample all genes in all organisms present in a given complex sample to evaluate bacterial diversity and detect unculturable microorganisms.

Documentation

Product literature
  • Application note PDF < 1 MB
    2 versions
  • Application note PDF < 1 MB
    2 versions
Support documentation

Compare

Illumina DNA Prep TruSeq DNA PCR-Free TruSeq DNA Nano Nextera XT DNA Library Preparation Kit
Assay time ~3-4 hr (from DNA extraction to normalized library) 5 hr total assay time ~6 hr total assay time ~5.5 hr from DNA extraction to normalized library. (Library prep time: ~90 minutes).
Automation capability Liquid handling robots Liquid handling robots Liquid handling robots Liquid handling robots
Automation details Explore available automation methods Explore available automation methods Explore available automation methods Explore available automation methods
Description 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.  A simple, all-inclusive PCR-free prep for whole-genome sequencing studies with the ability to sequence through challenging regions of the genome. A low-input research method that delivers high genome coverage quality and reduced bias. Prepare sequencing libraries for small genomes, PCR amplicons, and plasmids in 90 min, with a low DNA input requirement.
Hands-on time 1-1.5 hr 4 hr ~4 hr 15 min
Input quantity 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). 1 ug DNA 100 ng genomic DNA 1 ng DNA
Instruments 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 System, NextSeq 550 System, NextSeq 2000 System, NextSeq 1000 System, MiSeqDx in Research Mode, MiniSeq System, NovaSeq X System, NextSeq 500 System, NovaSeq 6000 System, NovaSeq X Plus System MiSeq System, NextSeq 550 System, NextSeq 2000 System, MiSeqDx in Research Mode, MiniSeq System, NovaSeq X System, NextSeq 500 System, NovaSeq 6000 System, NovaSeq X Plus System MiSeq System, iSeq 100 System, NextSeq 550 System, NextSeq 2000 System, NextSeq 1000 System, MiniSeq System, NextSeq 500 System
Mechanism of action Bead-linked transposome Mechanical DNA fragmentation and adapter ligation. Workflow is PCR-free and gel-free. Mechanical DNA fragmentation and adapter ligation. Workflow uses reduced-bias PCR and is gel-free. Enzymatic fragmentation
Method Amplicon sequencing, Whole-genome sequencing, De novo sequencing, Shotgun sequencing Genotyping by sequencing, Whole-genome sequencing, Shotgun sequencing Genotyping by sequencing, Whole-genome sequencing, Shotgun sequencing Amplicon sequencing, Whole-genome sequencing, 16s rRNA sequencing, De novo sequencing, Shotgun sequencing
Multiplexing Up to 384 unique dual (UD) combinations and 96 combinatorial dual (CD) combinations Up to 24 single, 96 combinatorial (CD) dual, 24 unique dual and 96 unique dual (UD) combinations Up to 24 single, 96 combinatorial (CD) dual, 24 unique dual and 96 unique dual (UD) combinations Up to 384 uniquely indexed samples may be pooled and sequenced together.
Nucleic acid type DNA DNA DNA DNA
Sample type details Not validated for use with FFPE samples 
Specialized sample types Blood, Not FFPE-compatible, Saliva Not FFPE-compatible Not FFPE-compatible, Low-input samples Not FFPE-compatible, Low-input samples, Single cells
Species category Drosophila, Any species, Mammalian, Mouse, Yeast, Zebrafish, Human, Rat, Plant, Virus, Nematode, Bacteria Other, Mammalian, Mouse, Human, Rat, Plant Other, Mammalian, Mouse, Human, Rat, Plant Drosophila, Any species, Mammalian, Mouse, Yeast, Zebrafish, Fungal, Human, Rat, Plant, Virus, Nematode, Bacteria
Species details Compatible with any species Compatible with most large DNA genomes. Compatible with most large DNA genomes. Compatible with any species
Target insert size ~350 bp 350 bp or 550 bp 350 bp or 550 bp 300 bp–1.5 kb
Technology Sequencing Sequencing Sequencing Sequencing
Variant class 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) 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) 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) Single nucleotide polymorphisms (SNPs), Structural variants

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Sequencing Coverage Calculator

Calculator to help determine the reagents and sequencing runs needed to arrive at desired coverage for your experiment.

Figures

Illumina bead-linked transposome chemistry

(A) Bead-linked transposomes mediate the simultaneous fragmentation of gDNA and the addition of Illumina sequencing primers. (B) Reduced-cycle PCR amplification amplifies sequencing ready DNA fragments and adds indexes and adapters. (C) Sequencing-ready fragments are washed and pooled.

Illumina DNA Prep delivers the fastest Illumina workflow

Comparison of the total workflow time from DNA extraction to library normalization and pooling. Illumina DNA Prep produces a workflow with the least number of steps and the fastest total workflow time. 

Improved coverage uniformity

(A) Illumina DNA Prep delivers uniform coverage across the genome comparable to the TruSeq DNA Nano kit. (B) Coverage is shown for microorganisms with extremely high- or low-GC content. Due to improved on-bead tagmentation library prep chemistry, Illumina DNA Prep shows more even coverage than Nextera XT.

Resources

Illumina DNA Prep: A robust and self-normalizing enzymatic library prep technology

Library prep innovation pipeline at Illumina

Learn about Illumina technologies designed to offer easy-to-use, fast, and simple solutions for both DNA and RNA library preparation.

Illumina DNA Prep: Introduction

An introductory presentation on the Illumina DNA Prep kit.

How on-bead tagmentation works

On-bead tagmentation library prep chemistry integrates DNA extraction, fragmentation, library prep, and library normalization.

Library prep (2)

Illumina DNA Prep, (M) Tagmentation (24 Samples, IPB)

20060060

Includes reagents for preparing 24 libraries. Illumina Purification Beads are included. Purchase enrichment probe panel and index adapters separately.

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Illumina DNA Prep, (M) Tagmentation (96 Samples, IPB)

20060059

Includes reagents for preparing 96 libraries. Illumina Purification Beads are included. Purchase enrichment probe panel and index adapters separately.

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

20027213

Includes 96, 10 bp indexes sufficient for labeling 96 samples. Purchase library prep and enrichment reagents and probe panels separately.

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

20027214

Includes 96, 10 bp indexes sufficient for labeling 96 samples. Purchase library prep and enrichment reagents and probe panels separately.

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

20042666

Includes 96, 10 bp indexes sufficient for labeling 96 samples. Purchase library prep and enrichment reagents and probe panels separately.

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

20042667

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™ DNA CD Indexes (96 Indexes, 96 Samples)

20018708

Includes 20 indexes, eight of i500s and 12 of i700 indexes that together provide 96 dual indexing capability sufficient for labeling 96 samples. Indexes are provided in a 96 well format. Purchase library prep and probe panels separately.

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Services (1)

Illumina DNA Prep - Customer Site

20022900

A 1-day onsite training for the complete Illumina DNA Prep library preparation workflow. Participants will learn all steps of the workflow including sample input and library preparation, quality control, best practices, and troubleshooting, in addition to training on Illumina- supported sequencing and data analysis tools specific to the Illumina DNA Prep workflow.

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Accessory products (6)

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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Flex Lysis Reagent Kit (96 reactions)

20018706

Includes reagents for processing blood samples with the Illumina DNA Prep and Illumina DNA Prep with Enrichment. Purchase library prep, enrichment, enrichment probe panel, and index adapter reagents separately.

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Illumina® Free Adapter Blocking Reagent (12 Reactions)

20024144

This reagent is used during library preparation to reduce index hopping levels.

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Illumina® Free Adapter Blocking Reagent (48 Reactions)

20024145

This reagent is used during library preparation to reduce index hopping levels.

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NextSeq PhiX Control Kit

FC-110-3002

DNA control for the NextSeq Illumina sequencing platform. Compatible with single and paired-end reads up to 150 bp. 10 µl of a 10 nM PhiX solution.

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FAQs

Yes, Illumina has designed specific extraction protocols for both blood and saliva. The protocols can be found in the Illumina DNA Prep Reference Guide.

In general, Illumina DNA Prep is designed to be compatible with most automated liquid-handling systems. 

All Illumina automation partners are developing methods for at least one existing automation platform. Contact the individual partners for more information. 

Coverage of GC-rich regions can be impacted by the model, settings, and performance of the thermal cycler used. Illumina has validated the Bio-Rad DNA Engine Tetrad 2, the Bio-Rad S1000, the Bio-Rad C1000, and the MJ Research PTC-225 DNA Engine Tetrad thermal cyclers for use with Illumina DNA Prep. Other thermal cyclers may differ in their performance, which may impact genomic coverage.

Yes, the minimum input range (1–500 ng) of Illumina DNA Prep allows the use of DNA extracted from Dried Blood Spot (DBS) cards. 

/ Results

Order the NovaSeq X Series

Advanced chemistry, optics, and informatics combine to deliver exceptional speed and data quality, outstanding throughput and scalability.

MiSeq Reagent Kit v3

Optimized reagent kit that provides increased cluster density and read length, improving sequencing quality scores compared to earlier versions.

TruSight Oncology 500

Enable CGP with a large pan-cancer panel covering all major variant classes plus gene signatures (TMB, MSI, and HRD) from FFPE tissue.

Illumina DNA Prep

A fast, integrated workflow for preparing libraries for use in a wide range of sequencing applications.

Illumina DNA PCR-Free Prep

An efficient, fast, and integrated library preparation workflow that yields highly accurate data for sensitive sequencing applications.

NextSeq 1000/2000 Reagents

Reagents for the NextSeq 1000/2000 System feature easy-to-use cartridges and multiple flow cell configurations for flexible sequencing options.

Illumina Human Comprehensive Panel

Predesigned, optimized panel to provide enhanced coverage of challenging-to-map regions within protein-coding genes using Illumina Complete Long Reads with Enrichment library prep kit. Analyzed in combination with a required ≥ 30× standard short-read whole genome.

References

1. Bruinsma S, Burgess J, Schlingman D, et al. Bead-Linked Transposomes Enable a Normalization-Free Workflow for NGS Library Preparation. 2018;19:722. BMC Genomics. Published online 2018 Oct 1. doi: 10.1186/s12864-018-5096-9

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