Illumina 5-Base DNA Prep

A single assay for comprehensive discovery of the 5-base genome (A, T, G, C, and 5mC), providing dual insights into the whole genome and methylome.

Illumina 5-Base DNA Prep
Data sheet PDF 7 MB

< 3 days DNA to data insights Library prep time* ~8 hr gDNA; ~6 hr cell-free DNA * Based on processing 24 samples manually; includes DNA shearing (if applicable), and library quantification. Normalization and pooling time not included.

Assay time

1–20 ng cell-free DNA 50–100 ng genomic DNA

Input quantity

See full details in the specifications table

Limited time offer: 25% off first kit with promo code ILMN5B25
Now through May 2026: receive 25% off your first library prep kit.
Limited quantity: 1

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Overview

Illumina 5-Base DNA Prep offers simultaneous discovery of genomic variants and methylation events across the whole genome. Maximize insights from every sequencing read with an easy-to-use, cost-effective multiomic assay.

Dual detection of 5-methylcytosine (5mC) and DNA variants from a single sample, library prep, sequencing run, and analysis workflow
Two-for-one assay with easy workflow and integrated analysis
Streamlined, single-day library preparation
High-quality data enabled by optimized library prep, novel base conversion, Illumina sequencing, and advanced analysis methods
Cost-effective whole-genome multiomic discovery
Integrated analysis with DRAGEN Germline and Somatic pipelines and Illumina Connected Multiomics

Simple, fast workflow

Go from sample to results in under three days with library prep completed in less than one day. Streamlined secondary analysis using 5-base DRAGEN pipelines delivers a dual readout in under an hour for a 30× genome.

Accurate, reproducible dual detection

Innovative 5mC-to-T base conversion and advanced analysis algorithms preserve DNA sequence complexity, enabling high alignment rates.

Dual insights, one workflow

Simultaneously detect both methylation and genomic variants with high resolution and accuracy.

Specifications

Assay time	< 3 days DNA to data insights Library prep time* ~8 hr gDNA; ~6 hr cell-free DNA * Based on processing 24 samples manually; includes DNA shearing (if applicable), and library quantification. Normalization and pooling time not included.
Automation capability	Liquid handling robot(s)
Automation details	Automation protocols are in development and will be added to the automation page when available.
Description	One streamlined assay for comprehensive multiomic discovery of genomic variants and methylation events across the whole genome. High-quality data provides 5-base insights from the same sequencing reads, enabled by a novel enzymatic method and optimized analysis.
Input quantity	1–20 ng cell-free DNA 50–100 ng genomic DNA
Instruments	NovaSeq X System, NovaSeq 6000Dx in Research Mode, NovaSeq 6000 System, NovaSeq X Plus System
Mechanism of action	Enzymatic conversion of 5-methylcytosine to thymine, ligation-based addition of adapters and indexes, mechanical shearing of genomic DNA
Method	Methylation sequencing, Whole-genome sequencing
Multiplexing	Up to 384-plex
Nucleic acid type	DNA
Specialized sample types	Circulating tumor DNA, Blood, Low-input samples, Cell-free DNA
Species category	Any species, Human
Species details	Illumina 5-base analysis software enables custom reference for analysis with species of interest. Users can upload custom reference genome to run Illumina 5-base assay/analysis. Not all species have been tested and specifications are only applicable to human samples.
Target insert size	450 bp for gDNA
Variant class	Somatic variants, Differentially methylated cytosines, Germline variants, Single nucleotide variants (SNVs), Insertions-deletions (indels), Copy number variants (CNVs)

Required products

Illumina 5-Base DNA Prep requires the use of DRAGEN secondary analysis. DRAGEN analysis can be accessed via a local DRAGEN server or the cloud through Illumina Connected Analytics or BaseSpace Sequence Hub. Illumina Connected Multiomics is available for data exploration and visualization, including differential methylation, clustering, and biological interpretation. Index adapters are sold separately through Illumina.

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Applications

Illumina 5-Base DNA Prep delivers high-resolution, whole-genome detection of both DNA variants and methylation events in a single, cost-effective assay, while enabling streamlined library prep, integrated analysis, and customizable dual-omic insights from every sequencing read.

Example workflow

Prep

Illumina 5-Base DNA Prep

Sequence

Analyze

Project recommendations

Instrument	Recommended number of samples	Read length
NovaSeq X System	Samples per flow cell: 10B: 18, 25B: 48 (based on 500M paired-end reads, 35× coverage)	2 × 151 bp
NovaSeq 6000 System	Samples per flow cell: S4: 18 (based on 500M paired-end reads, 35× coverage)	2 × 151 bp
NovaSeq 6000Dx in Research Mode	Samples per flow cell: S4: 18 (based on 500M paired-end reads, 35× coverage)	2 × 151 bp

Related applications and methods

Genome and methylome sequencing

Achieve simultaneous high-accuracy genome and methylome discovery in a single readout with the Illumina 5-base genome solution.

Cancer epigenetics

Explore the benefits of next-generation sequencing and microarrays for analyzing altered methylation patterns and other epigenetic changes in cancer.

Epigenomics

Illumina offers solutions for epigenetic analysis. Use our tools to study epigenetic modifications and their impact on gene regulation.

Documentation

Product literature

Illumina 5-base solution for methylation and variant detection
flyer PDF < 1 MB

Illumina 5-Base DNA Prep
Data sheet PDF 7 MB

Support documentation

Illumina 5-Base DNA Prep Sample Sheet Templates Documentation

Index Adapters Pooling Guide Documentation

Denature and Dilute Protocol Generator Documentation

Illumina 5-Base DNA Prep Product Documentation

All Illumina 5-Base DNA Prep Support

Compare

	Illumina 5-Base DNA Prep	Infinium MethylationEPIC v2.0 Kit	Illumina DNA Prep	Illumina DNA PCR-Free Prep
Assay time	< 3 days DNA to data insights Library prep time* ~8 hr gDNA; ~6 hr cell-free DNA * Based on processing 24 samples manually; includes DNA shearing (if applicable), and library quantification. Normalization and pooling time not included.		~3-4 hr (from DNA extraction to normalized library)	~1.5 hr
Automation capability	Liquid handling robot(s)	Automated array loader	Liquid handling robot(s)	Liquid handling robot(s)
Automation details	Automation protocols are in development and will be added to the automation page when available.		Explore available automation methods	Explore available automation methods
Description	One streamlined assay for comprehensive multiomic discovery of genomic variants and methylation events across the whole genome. High-quality data provides 5-base insights from the same sequencing reads, enabled by a novel enzymatic method and optimized analysis.	The Infinium MethylationEPIC v2.0 BeadChip is a genome-wide methylation analysis tool with enhanced, expert-selected functional content. The kit is processed on the iScan or NextSeq 550 Systems and follows a straightforward, user-friendly workflow that does not require sample pooling and indexing.	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.	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.
Input quantity	1–20 ng cell-free DNA 50–100 ng genomic DNA	250 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).	25 ng to 300 ng
Instruments	NovaSeq X System, NovaSeq 6000Dx in Research Mode, NovaSeq 6000 System, NovaSeq X Plus System	NextSeq 550 System, NextSeq 550Dx in Research Mode, iScan 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	MiSeq System, NextSeq 550 System, NextSeq 2000 System, NextSeq 1000 System, MiSeqDx in Research Mode, NextSeq 550Dx in Research Mode, NovaSeq X System, NextSeq 500 System, NovaSeq 6000 System, NovaSeq X Plus System
Mechanism of action	Enzymatic conversion of 5-methylcytosine to thymine, ligation-based addition of adapters and indexes, mechanical shearing of genomic DNA		Bead-linked transposome	Bead-linked transposome
Method	Methylation sequencing, Whole-genome sequencing	Methylation array	Amplicon sequencing, Whole-genome sequencing, De novo sequencing, Shotgun sequencing	Whole-genome sequencing, De novo sequencing
Multiplexing	Up to 384-plex		Up to 384 unique dual (UD) combinations and 96 combinatorial dual (CD) combinations	Up to 384-plex
Nucleic acid type	DNA	DNA	DNA	DNA
Specialized sample types	Circulating tumor DNA, Blood, Low-input samples, Cell-free DNA	Blood, FFPE tissue	Blood, Not FFPE-compatible, Saliva	Blood, Not FFPE-compatible, Low-input samples, Saliva, Not FFPE-compatible
Species category	Any species, Human	Human	Drosophila, Any species, Mammalian, Mouse, Yeast, Zebrafish, Human, Rat, Plant, Virus, Nematode, Bacteria	Any species, Human
Species details	Illumina 5-base analysis software enables custom reference for analysis with species of interest. Users can upload custom reference genome to run Illumina 5-base assay/analysis. Not all species have been tested and specifications are only applicable to human samples.		Compatible with any species
Target insert size	450 bp for gDNA		~350 bp	450 bp +/- 75 bp
Variant class	Somatic variants, Differentially methylated cytosines, Germline variants, Single nucleotide variants (SNVs), Insertions-deletions (indels), Copy number variants (CNVs)	Differentially methylated cytosines	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)	Short tandem repeats (STRs), Single nucleotide polymorphisms (SNPs), Somatic variants, Chromosomal abnormalities, Germline variants, Structural variants, Insertions-deletions (indels), Copy number variants (CNVs)

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Figures

Exceptional sequencing performance with Illumina 5-Base DNA Prep

(A) High coverage of cytosine-guanine dinucleotides (CpGs) and (B) uniform coverage across a range of GC content using Illumina 5-Base DNA Prep. Input amounts are 1–20 ng cfDNA from healthy donors and 50–100 ng gDNA from human reference sample NA12878.

High yield and alignment rates using Illumina 5-Base DNA Prep

(A) Library yields and (B) alignment rates (percent mapped reads) using Illumina 5-Base DNA Prep for 1–20 ng inputs of cfDNA from healthy donors and 50–100 ng inputs of gDNA from human reference sample NA12878.

Methylation conversion and detection with Illumina 5-Base DNA Prep

(A) Reproducible methylation detection for all CpG island regions. Highly selective 5mC conversion measured by small genome control spike-ins (B) methylated pUC and (C) unmethylated lambda. Input amounts are 1–20 ng cfDNA from healthy donors and 50–100 ng gDNA from human reference sample NA12878.

High-accuracy germline variant calling with Illumina 5-Base DNA Prep

(A) Single nucleotide variant (SNV) and (B) insertion-deletion (indel) germline variant calling using Illumina 5-Base DNA Prep for 50–100 ng inputs of gDNA from human reference sample NA12878. (C) Germline CNV calling accuracy by variant size from Genome in A Bottle sample HG002.

Advantages of Illumina 5-Base DNA Prep

Traditional methylation sequencing methods	Illumina 5-base solution
Challenges	Benefits
Limited mapping efficiency Low variant detection accuracy Chemical conversion that damages DNAa Multistep conversion	High mapping efficiency High-accuracy variant detection Enzymatic conversion that is nondamaging to DNA Single-step conversion

Resources

Decode DNA and methylation together with the Illumina 5-base solution

Join Fiona Kaper, VP and Head of Assay R&D at Illumina, as she discusses the innovative 5-base solution that simplifies and scales multiomic analysis. Discover how Illumina 5-base chemistry enables dual genomic and epigenomic insights in one assay, providing genetic variant and DNA methylation data with exceptional accuracy, simplicity, and scale.

An introduction to the Illumina 5-base solution

A streamlined, end-to-end NGS library prep and analysis workflow for simultaneous methylation profiling and high-accuracy genetic variant calling.

Library prep (1)

Illumina® 5-Base DNA Prep (24 Samples)

20140364

Includes library prep and methylation detection reagents for preparing 24 libraries manually. Analyze data on the cloud using DRAGEN apps on Illumina Connected Analytics or BaseSpace Sequence Hub or on-premise with DRAGEN Server. Perform data visualization using Illumina connected Multiomics (option). Purchase indexes separately.

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Index adapters (5)

Illumina® Unique Dual Indexes, LT (48 Indexes, 48 Samples)

20098166

Illumina® Unique Dual Indexes, LT (48 Indexes, 48 Samples)

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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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FAQs

Illumina 5-Base DNA Prep uses a one-step enzymatic process to convert 5mC to T, yielding greater nucleotide diversity than traditional bisulfite treatment. This enhanced diversity, combined with optimized ligation-based library preparation and specialized analysis methods, enable effective detection of both 5mC and genomic variants in a single assay.

Illumina 5-Base DNA Prep enables comprehensive methylation profiling and variant detection in one simple, scalable, and affordable workflow, making it ideal for whole-genome discovery.

For methylation-focused applications, the Illumina 5-base solution requires less sequencing than standard methylation assays due to greater mapping efficiency and CpG coverage per run.

DRAGEN secondary analysis uses innovative 5-base algorithms to provide accurate annotation of both genome and methylome from a single dataset. An easy checkbox option activates methylation reporting features within DRAGEN Germline and Somatic pipelines. Illumina Connected Multiomics directly imports output files from DRAGEN secondary analysis and offers industry-proven statistical analysis methods and visualization tools for easy data exploration and deeper insights.

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Illumina 5-Base DNA Prep

< 3 days DNA to data insights Library prep time* ~8 hr gDNA; ~6 hr cell-free DNA * Based on processing 24 samples manually; includes DNA shearing (if applicable), and library quantification. Normalization and pooling time not included.

1–20 ng cell-free DNA 50–100 ng genomic DNA