General FAQ for the Illumina 5 Base WGS and Enrichment kits

How does the Illumina 5-Base solution work?

• While conventional methylation conversion technologies convert unmethylated cytosine to thymine, Illumina proprietary conversion chemistry selectively converts methylated cytosine to thymine. As the majority of cytosines in a healthy human genome are unmethylated, selective conversion of methylated cytosine produces higher complexity libraries and, when combined with novel informatics, provides comprehensive, cost-effective 5-Base insights. The 5-Base assay can replace two separate genetic and epigenetic sequencing assays, saving time and budgets, with minimal incremental sequencing for methylation insights.

• The novel chemistry allows direct conversion of 5mC to T. The top row, below, represents input DNA, and the bottom row represents the converted DNA:

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Which types of modifications does the Illumina 5-Base solution detect?

• The Illumina 5-Base solution detects 5-methylcytosine (5mC) along with unmodified bases, adenine (A), thymine (T), guanine (G), and cytosine (C). DNA methylation is a key epigenetic modification to regulate gene expression.

How does the Illumina 5-Base solution compare to other NGS methylation solutions?

• Common methods for detecting DNA methylation use bisulfite or enzymes to convert unmethylated C to T. This reduces nucleotide diversity, making reads harder to align. Bisulfite treatment can also damage DNA, leaving data gaps. Illumina 5-Base chemistry directly converts only 5mC to T in a simple, single step, which is nondamaging to DNA and retains library complexity.

What benefits does this method have over bisulfite conversion?

• Since 5-Base conversion is enzymatic, not chemical, the DNA is not damaged or degraded.

• This allows for methylation and variant calling in the same assay.

• With this method, the base diversity of the library is also preserved.

• The workflow is simpler and shorter than most on-market methylation-only products.

How do whole-genome and target enrichment sequencing strategies for methylation studies differ?

• Whole-genome sequencing covers the entire genome, while targeted enrichment sequencing focuses on select genomic regions. Both strategies can detect DNA methylation at single-base resolution.

How complex is the 5-Base data analysis solution, given it provides dual insights?

• The 5-Base bioinformatics solution does not require advanced expertise. It offers highly accurate variant and methylation detection. This is a simple, powerful, and easy-to-use multiomic analysis solution. Insights from Illumina Connected Multiomics are powered by DRAGEN analysis.

What percent of cytosines are methylated in the human genome?

• ~95% of Cytosines in the human genome are unmethylated, ~5% are methylated.

Are there any limitations for CG-rich samples?

• Illumina has not observed detection bias related to CG content in the human genome, either high or low.

For any feedback or questions regarding this article (Illumina Knowledge Article #9939), contact Illumina Technical Support [email protected].