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
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.
Innovative 5mC-to-T base conversion and advanced analysis algorithms preserve DNA sequence complexity, enabling high alignment rates.
Simultaneously detect both methylation and genomic variants with high resolution and accuracy.
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
Illumina 5-Base DNA Prep
NovaSeq X Series
NovaSeq 6000 System
NovaSeq 6000Dx Instrument (RUO mode)
DRAGEN secondary analysis
Illumina Connected Multiomics
Project recommendations
Instrument: Recommended number of samples
NovaSeq X System: Samples per flow cell: 10B: 18, 25B: 48 (based on 500M paired-end reads, 35× coverage)
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)
Samples per flow cell: S4: 18 (based on 500M paired-end reads, 35× coverage)
NovaSeq 6000Dx in Research Mode: Samples per flow cell: S4: 18 (based on 500M paired-end reads, 35× coverage)
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.
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
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
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
Video resources
Illumina 5-base solution: A comprehensive Workflow Guide
Discover the benefits of high-accuracy dual-omic data with DRAGEN 5-base pipelines and gain insights into the reagents, equipment, and user-supplied consumables required for successful implementation.
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.
Other resources
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.
Validating methylation signatures and variants in pediatric cancer
Novel Illumina 5-base solution overcomes limitations associated with both chemical and enzymatic conversion
Illumina 5-base sequencing resolves complex central nervous system cancers and rare and undiagnosed genetic diseases
This poster highlights how Illumina 5‑base sequencing enables simultaneous detection of genomic variants and DNA methylation from a single, streamlined workflow. It demonstrates the power of this dual‑omic approach to resolve complex central nervous system tumors and rare or undiagnosed genetic diseases with greater accuracy and efficiency.
Demonstrating FFPE sample compatibility for Illumina 5-Base DNA Prep
TAn evaluation of Illumina 5-base sequencing for integrated methylation and variant analysis in FFPE samples and its potential for tumor-informed MRD applications.
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. View less
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Illumina 5-Base DNA Prep with NovaSeqX 25B (48 Samples)
20150505
Includes library prep and methylation detection reagents for preparing 48 libraries. Includes one NovaSeqX 25B reagent kits (300 cycle).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. View less
Illumina® Unique Dual Indexes, LT (48 Indexes, 48 Samples)
20098166
Illumina® Unique Dual Indexes, LT (48 Indexes, 48 Samples) View less
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. View less
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. View less
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. View less
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. View less
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How does the Illumina 5-Base DNA Prep work?
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.
Why use Illumina 5-Base DNA Prep?
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.
How do I analyze Illumina 5-Base DNA data?
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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