Illumina DNA PCR-Free Prep

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

~1.5 hr

Assay time

~45 min

Hands-on time

25 ng to 300 ng

Input quantity

See full details in the specifications table

Overview

Illumina DNA PCR-Free Prep offers a rapid and flexible workflow for preparing libraries for use in sensitive whole-genome sequencing applications, such as human whole-genome sequencing (WGS), de novo assembly of microbial genomes, and tumor–normal variant calling.

  • Reduces cost by eliminating pre- and post-library quantification steps

  • Provides highly uniform coverage across repetitive or uneven genome regions

  • Delivers fast, automation-compatible workflow in 90 min

Simplify lab operations

The Illumina DNA PCR-Free Prep workflow supports a broad DNA input range, multiple sample types, and both small and large genomes. The workflow includes DNA extraction from blood, saliva, or dried blood spots.

Obtain reliable results

On-bead tagmentation chemistry combined with PCR-free library preparation eliminates PCR-induced bias and diminishes opportunities for error, providing superior and even coverage across high-GC or -AT regions. Learn more about on-bead tagmentation.

Access flexible throughput

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.


Illumina DNA Prep product line

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


Specifications


Required products

Purchase the Illumina DNA PCR-Free Sequencing and Indexing Primer (cat. no. 20041797) for all instruments except the NovaSeq 6000 System.

Index adapters are optional but recommended for multiplexing. Choose the number of indexes needed to match your sample throughput. 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.

Learn more about multiplexing.

The Illumina Lysis Kit is required only for processing blood samples.

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Applications

The Illumina DNA PCR-Free Prep delivers PCR-free sequencing-ready libraries for sensitive applications such as tumor–normal variant identification or human whole-genome sequencing (WGS) in approximately 1.5 hours.

Example workflow



Project recommendations

Instrument Recommended number of samples Read length
NovaSeq 6000 System

S1: 3 (based on 607M reads, 40× coverage, or > 160 Gb of total data passing filter)

S2: 6 (based on 663M reads, 40× coverage, or > 160 Gb of total data passing filter)

S4: 16 (based on 669M reads, 40× coverage, or > 160 Gb of total data passing filter)

2 × 150 bp


Related applications and methods

Documentation

Product literature

Compare

Illumina DNA PCR-Free Prep Illumina DNA Prep TruSeq DNA PCR-Free TruSeq DNA Nano
Assay time ~1.5 hr ~3-4 hr (from DNA extraction to normalized library) 5 hr total assay time

~6 hr total assay time

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

Prepare sequencing libraries for sensitive applications using a unique combination of on-bead tagmentation with PCR-free chemistry which simultaneously eliminates PCR-induced bias and reduces total library prep time to 90 minutes.

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.
Hands-on time ~45 min 1-1.5 hr 4 hr ~4 hr
Input quantity 25 ng to 300 ng 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
Mechanism of action Bead-linked transposome 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.
Method Whole-Genome Sequencing, De Novo Sequencing Shotgun Sequencing, Whole-Genome Sequencing, De Novo Sequencing, Amplicon Sequencing Shotgun Sequencing, Whole-Genome Sequencing, Genotyping by Sequencing Shotgun Sequencing, Whole-Genome Sequencing, Genotyping by Sequencing
Multiplexing Up to 384-plex 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
Specialized sample types Not FFPE-Compatible Blood, Not FFPE-Compatible, Saliva Not FFPE-Compatible Not FFPE-Compatible, Low-Input Samples
Species category Any species, Human 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
Target insert size 450 bp +/- 75 bp ~350 bp 350 bp or 550 bp 350 bp or 550 bp
Variant class Short tandem repeats (STRs), Single nucleotide polymorphisms (SNPs), 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), Gene fusions, Loss of heterozygosity (LOH), Somatic variants, Chromosomal abnormalities, Germline variants, Structural variants, Insertions-deletions (indels), Copy number variants (CNVs)

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Figures

Illumina DNA PCR-Free Prep chemistry

A unique combination of on-bead tagmentation with a PCR-free workflow in a single, rapid reaction.

Comparison of read coverage across GC-rich regions

The Illumina DNA PCR-Free Prep kit provides superior read coverage across the GC-rich promoter region of the human RNPEPL1 gene, as compared to TruSeq DNA PCR-Free and TruSeq DNA Nano Library Prep Kits.

Illumina DNA PCR-Free Prep coverage uniformity

The Illumina DNA PCR-Free Prep provides uniform coverage across a range of GC content in the human genome.

Illumina DNA PCR-Free Prep performance across a range of DNA inputs

Illumina DNA PCR-Free Prep libraries prepared from a range of DNA inputs demonstrate (A) passing quality specifications for all DNA inputs and (B) equivalent callability performance.

Resources

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This webinar discusses the library preparation workflow and sequencing considerations and provides tips for successful next-generation sequencing projects.

Library prep (2)

Illumina® DNA PCR-Free Prep, Tagmentation (24 Samples)

20041794

Includes reagents and Illumina Purification Beads for preparing 24 libraries. Purchase index adapters separately.

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Illumina® DNA PCR-Free Prep, Tagmentation (96 Samples)

20041795

Includes reagents and Illumina Purification Beads for preparing 96 libraries. Purchase index adapters separately.

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Index adapters (8)

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 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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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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Services (1)

Illumina DNA PCR-Free Prep-Customer Site

20046010

1-day customer-site training for Illumina DNA PCR-Free Prep. Hands-on training includes quantification of DNA input as well as best practices, troubleshooting tips, and training on Illumina-supported analysis tools specific to the application workflow.

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Accessory products (2)

Illumina® DNA PCR-Free Sequencing and Indexing Primer

20041797

Includes two, 7.5-ml tubes of VP10 custom read 1 primer and two, 7.5-ml tubes of VP14 custom index 2 primer for use on the iSeq 100, MiniSeq, NextSeq 500/550, NextSeq 1000/2000, and HiSeq 3000/4000 Systems.

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Illumina® Lysis Kit

20042221

Includes one lysis kit to process blood samples with the Illumina DNA PCR-Free Prep. Purchase library prep and index adapters separately.

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Selection summary

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FAQs

PCR-free library preparation is the construction of a DNA library without the use of PCR amplification. Illumina DNA PCR-Free chemistry eliminates PCR-induced bias and provides highly accurate sequence information for sensitive applications.

PCR-free library preparation uses a bead-linked transposome complex to tag genomic DNA by fragmenting and adding adapter tag sequences in a single and rapid reaction step. The single reaction step is called on-bead tagmentation. After the bead-linked transposomes are saturated with DNA, no additional tagmentation can occur, delivering consistent library yield and insert sizes.  Learn more about on-bead tagmentation

 

PCR-free sequencing eliminates PCR amplification steps, significantly reducing typical PCR-induced bias and uneven amplification.

Illumina DNA PCR-Free Prep workflow uses on-bead tagmentation, allowing for consistent insert sizes, uniform coverage, and enhanced performance, regardless of DNA input amount or genome size in a single, rapid reaction. On-bead tagmentation eliminates the need for PCR amplification steps, minimizing biased base composition and opportunities for error, making this kit ideal for sensitive sequencing applications.

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Illumina DNA Prep

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

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