# 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.

**Does this product convert and detect both 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC)?**

* The Illumina enzyme is very specific to converting 5mC, and has minimal activity on 5hmC.

**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.

**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.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #9939), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000009939%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |