EpiNext Bisulfite Sequencing Kit (Illumina)

We recommend a newer version of this kit: EpiNext High-Sensitivity Bisulfite-Seq Kit (Illumina)

The EpiNext™ Bisulfite-Seq Kit (Illumina) is designed to carry out bisulfite conversion, followed by a "post-bisulfite" library preparation process for Illumina platform-based bisulfite sequencing, all in one kit. Intended applications include whole genome bisulfite sequencing, oxidative bisulfite sequencing, reduced representative bisulfite sequencing, and various other bisulfite-based next generation sequencing techniques. The optimized protocol and components of the kit allow the DNA to be bisulfite converted and fragmented simultaneously followed by quick non-barcoded (singleplexed) and barcoded (mutilplexed) library construction using sub-nanogram quantities of bisulfite converted DNA. The kit has the following advantages:

• Innovative method - Allows for simultaneous bisulfite conversion and size-appropriate DNA fragmentation. The bisulfite DNA can be directly ligated to adaptors thereby eliminating the possibility of breaking adaptor-ligated fragments, which often occurs with traditional WGBS and RRBS methods.
  
• Fast and streamlined procedure - the procedure from DNA bisulfte treatment to PCR amplification can be finished within the same day (<8 h). Gel-free size selection/purification saves time and prevents handling errors, as well as loss of valuable samples.
  
• Complete conversion - The innovation reagent composition converts unmethylated cytosine into uracil at a level greater than 99.9%, with negligible inappropriate- or error-conversions of methylcytosine to thymine (<0.1%).
  
• High sensitivity, efficiency and flexibility- Adaptor ligation after bisulfite treatment eliminates loss of fragments and selection bias, which enables input DNA to be as low as 0.5 ng. The kit can be used for both non-barcoded (singleplexed) and barcoded (multiplexed) DNA library preparation.
  
• Extremely convenient - the kit contains all the required components for each step of the DNA library preparation process, which are sufficient for bisulfite conversion, ligation, clean-up, size selection, and library amplification, thereby allowing the bisulfite DNA library preparation to be streamlined for the most reliable and consistent results.
  
• Minimal bias - Ultra HiFi amplification enables achievement of reproducibly high yields of DNA libraries with minimal sequence bias and low error rates.
  
• Broad sample suitability - Starting materials can be genomic DNA isolated from various tissue/cell samples such as fresh and frozen tissues, cultured cells from a flask or microplate, microdissection samples, paraffin-embedded tissues, biopsy samples, embryonic cells, plasma/serum samples, and body fluid samples, etc. DNA enriched from various enrichment reactions such as ChIP, MeDIP/hMeDIP, or exon capture may also be used as starting materials.

Background Information
DNA methylation occurs by the covalent addition of a methyl group (CH3) at the 5-carbon of the cytosine ring, resulting in 5-methylcytosine (5-mC). DNA methylation is essential in regulating gene expression in nearly all biological processes including development, growth, and differentiation. Alterations in DNA methylation have been demonstrated to cause a change in gene expression. For example, hypermethylation leads to gene silencing or decreased gene expression while hypomethylation activates genes or increases gene expression. Aberrant DNA methylation is also associated with pathogenesis of diseases such as cancer, autoimmune disorders, and schizophrenia. Thus genome-wide analysis of DNA methylation could provide valuable information for discovering epigenetic markers used for disease diagnosis, and potential targets used for therapeutics.

Principle & Procedure
This kit includes all reagents required for a successful preparation of a DNA library by directly using bisulfite-converted DNA generated from a small amount of input DNA (500 pg to 500 ng). In this preparation, DNA is bisulfite converted and fragmented to the appropriate length simultaneously during the bisulfite process. The bisulfite-treated DNA, which is in single stranded form, is then converted to dsDNA and directly used for ligation with BisDNA-specific adaptors that are necessary for amplification and sequencing. The fragments are size selected and purified using MQ binding beads, which allows for quick and precise size selection of DNA. Size-selected DNA fragments are amplified using a high-fidelity PCR Mix which ensures maximum yields from minimum amounts of starting material and provides highly accurate amplification of library DNA with low error rates and minimal bias.

Starting Materials
Starting materials can be genomic DNA isolated from various tissue/cell samples such as fresh and frozen tissues, cultured cells from a flask or microplate, microdissection samples, FFPE tissues, plasma/serum, and body fluid samples, etc. DNA enriched from various enrichment reactions such as ChIP, MeDIP/hMeDIP, or exon capture may also be used as starting materials.  DNA should be without any previous restriction digestion step. Plasmid DNA can be used for bisulfite treatment with or without previous linearization, as the kit allows for DNA denaturation status to remain during the entire DNA bisulfite conversion process and direct ligation of adaptors to bisulfite DNA. Input amount of DNA can be from 0.5 ng to 1 ug. For optimal preparation, the input amount should be 100 ng to 200 ng.

Fig. 1.  Workflow of the EpiNext™ Bisulfite-Seq Kit

Fig. 2. Size distribution of library fragments: Post-bisulfite DNA library was prepared  from 10 ng of input DNA using the EpiNext™ Bisulfite-Seq Kit.