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Methylation sequencing

Comprehensive exploration of methylation patterns with NGS

Scientists in lab reviewing data

Benefits of methylation sequencing

Methylation sequencing enables high-resolution mapping of DNA methylation, a key epigenetic event that regulates gene expression and cellular identity. Using next-generation sequencing (NGS), researchers can detect methylation at single-base resolution across the genome, uncovering patterns in CpG, CHH, and CHG contexts.*

*CpG: Genomic region where cytosine is directly followed by guanine; CHH and CHG: Other genomic regions where methylation can occur, defined by a cytosine that is immediately followed by any nucleotide other than guanine (H = A, T, or C)

Specific advantages of methylation sequencing:
Genome-wide coverage

View cytosine methylation in the genome across most species at the genome-wide level

Sensitivity

Capture full sample diversity with small amounts of DNA

Targeted insight

Cover emerging regions of interest in the human genome, such as those identified by ENCODE, FANTOM5, and the Epigenomics RoadMap Consortium

Advancing colon cancer research with methylation sequencing

Dr Daniel Wise introduces a transformative methylation sequencing–based research approach to colorectal cancer detection with rectal mucus analysis.

How does methylation sequencing work?

Methylation sequencing can detect methylated cytosines across the genome. Traditional methods, such as bisulfite conversion, chemically modify unmethylated cytosines to uracil, which are read as thymine during sequencing. While effective, bisulfite treatment can degrade DNA and reduce sequence complexity.

Innovative approaches, such as enzymatic conversion, now offer gentler, more efficient alternatives. For example, the Illumina 5-base solution uses a proprietary enzymatic method to convert 5-methylcytosine (5mC) to thymine, preserving DNA integrity and nucleotide diversity. This enables simultaneous detection of genetic variants and methylation marks in a single assay, streamlining workflows and enhancing data quality.

Learn more about genome and methylome sequencing

Choosing the right approach: whole-genome vs targeted methylation sequencing

Whole-genome methylation sequencing

Whole-genome methylation sequencing offers comprehensive coverage of methylation across the entire genome at single-base resolution and is ideal for discovery research, epigenome-wide association studies, and complex disease modeling. This approach captures both known and novel methylation sites, providing a more complete epigenetic landscape.

Targeted methylation sequencing

Targeted methylation sequencing focuses on specific genomic regions of interest, such as promoters, enhancers, or CpG islands. This method is highly cost-effective and suitable for cancer research applications, minimal residual disease (MRD) detection studies, and circulating tumor DNA (ctDNA) monitoring research. Targeted panels can be customized for high-throughput screening or focused biomarker validation studies.

Common methylation sequencing methods

Methylation sequencing is commonly performed using chemical or enzymatic conversion methods. These methods can be performed with either whole-genome or targeted sequencing approaches. See the table to identify the features of these methods to best suit your research needs.

Comparison of methylation sequencing methods

  Bisulfite sequencing
Sodium bisulfite converts unmethylated cytosines to uracil; methylated cytosines remain unchanged		 Illumina 5-base solution
An enzyme engineered by Illumina converts methylated cytosines (5mC) to thymine while preserving unmethylated cytosines		 C-to-T enzymatic sequencing
Uses enzymes to convert unmethylated cytosines to thymine without harsh chemicals
DNA damage High (bisulfite conversion causes fragmentation) Minimal (gentle single-step enzymatic conversion) Low (gentle enzymatic conversion)
Nucleotide diversity Low High Low
Workflow complexity High Low High
Readout C-to-T conversion indicates unmethylated cytosines 5mC-to-T conversion allows for simultaneous methylation and DNA variant detection C-to-T conversion indicates unmethylated cytosines
Methylation detection accuracy High High High
DNA variant detection accuracy Low High Low
Input DNA requirement High Low Low
Advantages Established method, widely used Easy workflow, dual detection of methylation and DNA variants, 5mC-to-T conversion is nondamaging and preserves library diversity for high accuracy Less damaging than bisulfite, works with degraded samples
Decoding complex pathways with 5-base analysis for multiomic insights

In this eBook, discover the value of sequencing both genetic variants and DNA methylation, together, to unravel the mechanisms of genetic disease, cancer, and other complex diseases.

Featured methylation sequencing workflows

The streamlined 5-base workflow includes optimized whole-genome library prep that takes less than a day from DNA to sequencing system and offers easy-to-use analysis for dual DNA and methylation annotations and visualizations.

1
Library prep

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.

2
Sequencing

NovaSeq 6000 System

Powerful high-throughput sequencing studies for exceptional sequencing performance. Match data output and time to results to project needs.

NovaSeq X Series

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

3
Data analysis & interpretation

DRAGEN secondary analysis

Accurate, comprehensive, and efficient secondary analysis of next-generation sequencing (NGS) data.

Illumina Connected Multiomics

A cloud-based multiomic analysis and interpretation software enabling sample to insight workflow with visualizations, scalability, and secure data management.

The 5-base enrichment workflow includes optimized target enrichment library prep and easy-to-use analysis for dual DNA and methylation annotations and visualizations of target genes.

1
Library prep

Illumina 5-Base DNA Prep with Enrichment

A single assay for targeted detection of five DNA bases (A, T, G, C, and 5mC), providing dual insights into genomic variants and methylation events.

2
Sequencing

NextSeq 1000 and 2000 Systems

Expansive application breadth, operational simplicity, high flexibility, scalability for proven sequencing performance.

NovaSeq 6000 System

Powerful high-throughput sequencing studies for exceptional sequencing performance. Match data output and time to results to project needs.

NovaSeq X Series

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

3
Data analysis & interpretation

DRAGEN secondary analysis

Accurate, comprehensive, and efficient secondary analysis of next-generation sequencing (NGS) data.

Illumina Connected Multiomics

A cloud-based multiomic analysis and interpretation software enabling sample to insight workflow with visualizations, scalability, and secure data management.

Methylation sequencing data analysis process and tools

Alignment and methylation calling

After sequencing, reads are aligned to a reference genome using methylation-aware algorithms. Illumina DRAGEN secondary analysis accelerates this step with optimized pipelines for bisulfite, enzymatic, and 5-base workflows. DRAGEN performs single-pass alignment and methylation calling at single-base resolution, generating outputs in standard formats with Bismark-compatible tags (XR, XG, XM) for seamless integration with downstream tools. Reports include genome-wide cytosine methylation metrics and quality assessments.

Learn more about DRAGEN secondary analysis

Differential methylation analysis

To uncover biologically relevant changes, differential methylation analysis compares CpG sites or regions across experimental groups (eg, healthy vs disease). Illumina Connected Software enables intuitive tertiary analysis, applying robust statistical models to identify differentially methylated loci (DML) and regions (DMR) with associated p-values and false discovery rates. Interactive visualizations and pathway enrichment tools help researchers interpret methylation changes in the context of gene regulation and multiomic data.

Together, DRAGEN and Connected Software deliver an end-to-end solution from raw reads to secondary analysis within the Illumina ecosystem.

Learn more about Illumina Connected Software

Related content

Cancer epigenetics research

Studies of aberrant methylation, altered transcription factor binding, and other epigenetic alterations can provide insight into important tumorigenic pathways.

Complex disease genomics

Genome-wide methylation sequencing can help researchers understand the functional mechanisms at work in complex neurological, immunological, and other diseases.

Cellular and molecular biology

Broaden cell and molecular biology research beyond the conventional methods of protein interaction and single gene functional studies.

Additional resources

Advancing DNA methylation and gene expression studies

This eBook discusses gene expression and regulation research. See how scientists use methylation sequencing and arrays to accelerate their research.

FAQ

Generally, DNA methylation results in decreasing gene expression. Methylation of promoters and enhancers usually blocks binding of transcriptional machinery.1 However, methylation within coding regions can enhance transcriptional activity.2

Key targets include CpG islands, promotor regions, miRNA promoter regions, enhancers, and DNase hypersensitivity sites. For more information, download our DNA methylation infographic.

Methods such as bisulfite or enzymatic methylation sequencing (EM-Seq) can introduce bias and reduce sequence complexity, making alignment and variant calling more challenging. Newer methods, such as the Illumina 5-base solution, maintain nucleotide diversity and enable simultaneous methylation and variant analysis, improving overall data quality. When using Illumina software analysis solutions such as DRAGEN Germline, DRAGEN Somatic, and DRAGEN Enrichment pipelines, methylation reporting features can be easily activated with a check box.

Key considerations include methylation detection accuracy, DNA input requirements, workflow time, and compatibility with existing sequencing platforms. Labs already using Illumina systems may find the Illumina 5-base solution to be a particularly efficient option as it integrates variant and methylation detection capabilities into a single standard workflow and readout.

Learn more about the Illumina 5-base solution

Yes, methylation sequencing can be scaled up. While some traditional methods can be time-intensive, newer technologies such as the Illumina 5-base solution are designed for scalability, offering faster library prep and streamlined analysis for high-throughput projects.

Learn more about the Illumina 5-base solution

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References

  1. Miller JL, Grant PA. The role of DNA methylation and histone modifications in transcriptional regulation in humans. Subcell Biochem. 2013;61:289-317. doi:10.1007/978-94-007-4525-4_13
  2. Jones PA. The DNA methylation paradox. Trends Genet. 1999;15(1):34-37. doi:10.1016/s0168-9525(98)01636-9