Overcoming challenges in methylation sequencing with the Illumina 5-base solution

Introduction

DNA methylation is a key epigenetic modification that regulates gene expression and plays an important role in development, aging, and disease.¹ As methylation sequencing is applied to increasingly complex biological questions, researchers need methods that provide accurate, comprehensive methylation information while preserving valuable genomic information and supporting a range of sample types and study designs.² Different methylation sequencing approaches offer distinct capabilities and tradeoffs in sequencing performance, the information generated, and the complexity of laboratory and analysis workflows.

The Illumina 5-base solution combines advances in methylation chemistry and bioinformatics to enable integrated analysis of DNA methylation and genetic variation from the same sequencing data. This unified approach maximizes sequencing performance, streamlines data analysis, and supports diverse applications. Here, we compare key features and tradeoffs of different methylation sequencing approaches and show how the Illumina 5-base solution addresses limitations of alternative technologies across methylation coverage, variant calling, challenging sample analysis, and workflow flexibility.

Comparison of methylation sequencing methods

Methylation sequencing can be performed using conventional bisulfite and enzymatic conversion methods, as well as newer technologies that use alternative conversion strategies.³ These approaches differ in how methylation information is detected and the workflows used for sequencing and analysis (Table 1).

All these approaches have enabled important advances in epigenetics research, but their different technical characteristics can have important implications for sequencing and analysis, including:

• Reduced sequence complexity: conventional bisulfite and enzymatic conversion methods that convert unmodified cytosines to thymine create a three-base genome that can decrease mapping efficiency and make genetic variation more challenging, particularly for C>T mutations^3,6
• DNA damage: bisulfite conversion can damage and degrade DNA, potentially reducing coverage and sequencing performance, particularly for challenging sample types such as formalin-fixed, paraffin-embedded (FFPE) tissue and cell-free (cfDNA)^7–11
• Fragment-dependent methylation errors: some ten-eleven translocation (TET)‑based enzymatic conversion methods can introduce fragment-dependent methylation errors in cfDNA, potentially affecting fragment-level methylation analysis¹²
• Workflow complexity: multistep conversion methods require multiple protection and bead cleanup steps, increasing turnaround time and the risk of sample loss
• Separate analysis pipelines: many existing methylation sequencing methods require separate pipelines for methylation analysis and genetic variant calling, increasing computational burden and requiring downstream reconciliation of independent data sets^13,14

An integrated solution for methylation and genomic analysis

The Illumina 5-base solution combines two complementary technologies: the Illumina 5‑base enzyme and the DRAGEN™ 5-base analysis pipeline. During library preparation, the 5-base enzyme selectively converts methylated cytosines to thymines while leaving unmethylated cytosines unchanged, preserving DNA integrity and sequence complexity. The DRAGEN 5-base analysis pipeline simultaneously identifies DNA methylation and genetic variants from the same sequencing data within a single integrated analysis, eliminating the need for separate bioinformatics pipelines. Together, these technologies enable integrated methylation and genomic analysis. The resulting workflow improves sequencing performance, reduces turnaround time, and offers flexibility across a broad range of study designs and sample types (Table 2).

Illumina 5-base enzyme	DRAGEN 5-base analysis
Specifically converts 5-methylcytosine (5mC) while preserving the four-base genome	Integrates methylation calling and variant analysis within a single analysis pipeline
Supports efficient read mapping and accurate variant detection	Eliminates the need to reconcile separate methylation and variant analysis workflows
Uses a rapid single-step conversion workflow with fewer cleanup steps than conventional approaches	Reduces analysis time and computational burden with a streamlined workflow
Delivers accurate, unbiased methylation measurements across sequence contexts while avoiding limitations associated with conventional conversion methods	Provides an established, fully supported analysis workflow, reducing reliance on implementing and maintaining open-source pipelines

Table 2: Features of the Illumina 5-base enzyme and DRAGEN 5-base analysis pipeline

Broader and more uniform CpG coverage

Comprehensive and uniform CpG coverage is essential for accurate methylation analysis. Compared with bisulfite and enzymatic conversion–based methods, the Illumina 5-base solution captures a greater number of CpG sites at higher sequencing depths while maintaining consistently uniform coverage across the full range of methylation levels (Figure 1). This improved coverage enables more complete methylation profiling and reduces methylation-dependent bias. While some enzymatic conversion approaches exhibit coverage bias in highly methylated or unmethylated regions, the Illumina 5-base solution maintains uniform coverage across methylation levels, supporting accurate and sensitive methylation detection. 

Because coverage is maintained more efficiently across the genome, researchers can achieve equivalent coverage in highly methylated regions with up to two times less sequencing than alternative enzymatic conversion techniques. This improves sequencing efficiency while reducing sequencing costs.

Comparable variant calling performance

Accurate identification of genetic variants is critical for many methylation sequencing applications, particularly studies that seek to characterize both genetic and epigenetic alterations from the same sample.¹⁵

The Illumina 5-base solution preserves variant calling performance comparable to whole-genome sequencing (WGS) while simultaneously enabling comprehensive DNA methylation profiling. For example, the Illumina 5-base solution achieves recall and precision scores for single nucleotide polymorphisms (SNPs) and insertions and deletions (indels) that are comparable to those of PCR-free WGS and substantially higher than those of other conversion-based approaches (Figure 2). By combining accurate variant detection with methylation profiling, researchers can generate integrated genomic and epigenetic data sets without requiring separate sequencing workflows.

Improved confidence in liquid biopsy analysis

Liquid biopsy applications rely on preserving the biological characteristics of cfDNA, including fragment length, fragment end motifs, and methylation patterns. Conversion-induced changes to these characteristics can affect the biological information available for downstream analysis, making their preservation particularly important studies investigating cancer-associated biomarkers and tissue of origin.¹⁶

The Illumina 5-base solution preserves accurate fragment-level methylation information by avoiding the systematic, fragment-dependent methylation errors observed with some TET-based enzymatic conversion methods (Figure 3). This improves confidence in fragment-level methylation analysis for liquid biopsy applications.

The Illumina 5-base solution also preserves native cfDNA fragment length distributions that closely match WGS, whereas bisulfite conversion alters fragment length profiles (Figure 4). Preserving native fragment length and end motifs alongside accurate DNA methylation measurements enables researchers to extract complementary biological information from the same cfDNA molecules.

Faster, streamlined methylation sequencing workflows

As methylation sequencing becomes more widely adopted, researchers need workflows that minimize laboratory effort while delivering results quickly and efficiently. Conventional approaches can require multiple conversion, cleanup, and analysis steps, increasing hands-on time, extending turnaround time, and adding computational complexity.

The Illumina 5-base solution streamlines the complete workflow from library preparation through data analysis, enabling researchers to go from DNA to biological insights up to two times faster than alternative approaches (Figure 5A). The library prep is also designed for simplicity and efficiency, with fewer treatment and cleanup steps, reducing hands-on time and enabling laboratories to process samples more efficiently without compromising data quality (Figure 5B).

Consistent performance across challenging sample types

Many methylation studies rely on challenging sample types, including low-input DNA and degraded FFPE tissue.¹¹ These samples can reduce library yield and sequencing performance, and conversion methods that further damage DNA can compound these challenges. Preserving DNA integrity and genomic information is particularly important when both methylation and genetic variant information are needed from these valuable samples.

For the Illumina 5-base solution, high library yields can be achieved from as little as 1 ng of input DNA, while mapping rates remain greater than or equal to 97% across FFPE samples representing multiple cancer types and varying levels of DNA degradation (Figure 6). By preserving DNA integrity during conversion, the Illumina 5-base solution supports robust methylation sequencing across challenging sample inputs and DNA qualities.

The Illumina 5-base solution also enables high-accuracy somatic variant calling alongside methylation analysis. In FFPE-treated tumor–normal cell lines, rates for detection of somatic small nucleotide variants (SNVs) and indels were comparable to those observed with nonconverted WGS. This demonstrates that genomic variant information can be retained while simultaneously assessing DNA methylation.¹⁷ 

Flexible whole-genome and targeted methylation sequencing 

Different research questions require different sequencing strategies. The Illumina 5-base solution supports both whole-genome and targeted methylation sequencing using the same core sequencing workflow and analysis pipeline, with the addition of enrichment steps for targeted sequencing. While whole-genome methylation sequencing provides comprehensive genome-wide coverage, targeted enrichment focuses sequencing on specific regions of interest. Unlike conventional bisulfite and enzymatic conversion methods that convert unmethylated cytosines and reduce sequence complexity, the Illumina 5-base solution converts only methylated cytosines, preserving higher-complexity sequences for target enrichment probe design.¹⁸ High on-target enrichment efficiency and strong concordance between methylation measurements before and after enrichment demonstrate that targeted capture preserves methylation information while efficiently enriching regions of interest (Figure 7). This flexibility enables researchers to move between whole-genome and targeted study designs while maintaining a consistent 5-base sequencing and analysis framework.

Sensitive detection of low-frequency variants

Targeted sequencing applications often require sensitive detection of low-frequency variants while simultaneously characterizing DNA methylation patterns. Targeted enrichment workflows using the Illumina 5-base solution achieve high detection rates for low-frequency SNVs in both cfDNA and genomic DNA (gDNA) across a range of variant allele frequencies (Figure 8). By preserving sequence complexity, the 5-base approach supports sensitive low-frequency variant detection alongside comprehensive methylation profiling. This enables simultaneous analysis of genetic and epigenetic alterations from the same targeted sequencing workflow.

Summary

The Illumina 5-base solution integrates 5-base library preparation chemistry with the DRAGEN 5-base analysis pipeline to deliver comprehensive methylation profiling and variant calling comparable to WGS from a single workflow. By preserving sequence complexity and DNA integrity, streamlining laboratory and bioinformatics workflows, and supporting whole-genome and targeted applications across challenging sample types, the Illumina 5-base solution addresses key tradeoffs of alternative methylation sequencing approaches while enabling integrated genetic and epigenetic analysis.

Key takeaways

• Generate comprehensive methylation and genetic data from a single workflow
• Capture more CpGs with uniform coverage across methylation levels
• Preserve comparable WGS SNV and indel calling performance 
• Preserve cfDNA fragment characteristics and avoid fragment-dependent methylation errors observed with some TET-based approaches
• Streamline laboratory and analysis workflows across whole-genome and targeted research applications and challenging sample types

Learn more

Illumina 5-base solution

References

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