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EpiNext High-Sensitivity Bisulfite-Seq Kit (Illumina)

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For streamlined bisulfite sequencing NGS library preparation from low input DNA

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Suggested Workflow
DNA Isolation
 
 
Bisulfite Conversion & Library Prep
 
Workflow of the EpiNext™ High-Sensitivity Bisulfite-Seq Kit (Illumina).
The EpiNext™ High-Sensitivity Bisulfite-Seq Kit (Illumina) was used to bisulfite convert DNA and prepare a library to be sequencing on an Illumina HiSeq 2500. Data tracks were aligned to a 30 kb region of chromosome 1 and showed unmethylated (blue) and methylated (red) areas and the corresponding gene regions in treated and control samples.
Size distribution of library fragments. Post-bisulfite DNA libraries were prepared from human placenta DNA using the EpiNext™ High-Sensitivity Bisulfite-Seq Kit (Illumina): A: 10 ng; B: 50 ng.
Input Type: DNA
Research Area: DNA Methylation, Next Gen Sequencing
Target Application: Library Construction, Sample Modification
Vessel Format: Columns/Tubes
100% Guarantee: 6 months
Catalog No.SizePriceQty
P-1056A-1212 reactions $524.00 
P-1056A-2424 reactions $999.00 
Availability: Usually Ships In 1-2 Days 
Product Overview

The EpiNext™ High-Sensitivity Bisulfite-Seq Kit (Illumina) is designed to bisulfite-convert DNA and prepare an Illumina-based library for bisulfite sequencing, all in one kit. Intended applications include whole genome (WGBS), oxidative (oxBS-seq), reduced representation (RRBS), and other bisulfite-next generation sequencing. The optimized protocol and components of the kit allow subnanogram amounts of DNA to be  bisulfite converted and fragmented simultaneously  followed by quick non-barcoded (singleplexed) and barcoded (multiplexed) library construction in less than 7 hours. 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 currently used WGBS and RRBS methods.
  • Fast and streamlined procedure - The procedure from DNA bisulfite treatment to ready-to-use library DNA can be completed within 6 hours and 30 minutes.
  • Complete conversion - Completely converts unmethylated cytosine into uracil (>99%) with negligible inappropriate- or error-conversions of methylcytosine to thymine. 
  • High sensitivity and efficiency - Innovative adaptor ligation of bisulfite DNA eliminates loss of fragments and selection bias, which enables input DNA to be as low as 0.2 ng, making it ideal for methylation profiling of precious, limited samples. The kit can be used for both non-barcoded (singleplexed) and barcoded (multiplexed) DNA library preparation.
  • Extremely convenient - The kit contains all required components for each step of DNA library preparation, 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 such as fresh and frozen tissue, cultured cells from a flask or microplate, microdissection samples, paraffin-embedded tissue, biopsy, 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 be also used as starting material.

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 successfully preparing a library directly using bisulfite DNA generated from a tiny amount of input DNA. In this preparation, DNA is simultaneously bisulfite converted and fragmented to the appropriate length during the bisulfite process. The bisulfite-treated DNA, which is in single stranded form, is then simultaneously converted to dsDNA and adaptor ligated. The ligated 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 tissue, cultured cells from a flask or microplate, microdissection samples, FFPE tissue, 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 material. 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 0.2 ng to 200 ng. For optimal preparation, the input amount should be 10-50 ng.


Fig. 1.  Workflow of the EpiNext High-Sensitivity Bisulfite-Seq Kit (Illumina).

Fig. 2. The EpiNext High-Sensitivity Bisulfite-Seq Kit (Illumina) was used to bisulfite convert DNA and prepare a library to be sequencing on an Illumina HiSeq 2500. Data tracks were aligned to a 30 kb region of chromosome 1 and showed unmethylated (blue) and methylated (red) areas and the corresponding gene regions in treated and control samples.

Fig. 3. Size distribution of library fragments. Post-bisulfite DNA libraries were prepared from human placenta DNA using the EpiNext High-Sensitivity Bisulfite-Seq Kit (Illumina): A: 10 ng; B: 50 ng.

Product Components

Modification Buffer
Modification Powder
DNA Binding Solution
Desulphonation Solution
Elution Solution
F-Spin Column
F-Collection Tube
5X Conversion Buffer*
Conversion Enzyme Mix*
Conversion Primer*
10X End Repair Buffer*
End Repair Enzyme Mix*
10X dA-Tailing Buffer*
Klenow Fragment (3’-5’ exo-)*
2X Ligation Buffer*
T4 DNA Ligase*
Adaptors (50 µM)*
MQ Binding Beads*
2X HiFi PCR Master Mix*
Primer U (10 µM)*
Primer I (10 µM)*
Elution Buffer*
User Guide

*Spin the solution down to the bottom prior to use.

User Guide & MSDS

[User Guide]*
*Always use the actual User Guide that shipped with your product. Is the above file locked? You can also request user guides by emailing info@epigentek.com along with your contact information and institution name.

Product Citations

Liu Y et. al. (November 2016). Methylation-sensitive enrichment of minor DNA alleles using a double-strand DNA-specific nuclease. Nucleic Acids Res.

Customer Reviews

Rating by t***********@duke.edu Verified Purchase Reviewed on: Thursday 29 December, 2016
Application Description
This kit was used to prepare bisulfite libraries for next generation sequencing. Samples consisted of a library of plasmids with inserts from a human cell line (GM12878). Samples were divided into two conditions 1) methylated using CpG Methylase and 2) unmethylated. The sequences generated will be compared between these two conditions.

Pros: The low input feature is fantastic, as we generally have very little input DNA from our samples of interest and often only have “one shot” at a successful library prep. This is also a relatively short protocol compared to some of the other kits we have tried.

Cons: The final cleanup does not eliminate leftover adaptors/primers. We have always had to do a second cleanup with a smaller ratio of SPRI beads to eliminate these fragments from our final library.

Other Thoughts
Great product overall. For the bead cleanups, we’ve seen better results using AMPure XP beads with 80% ethanol than we've seen with the beads that are included in the kit (which are then washed with 90% ethanol), but these differences are minor.


Bioanalyzer traces of some of our bisulfite seq libraries (before we added the additional (2nd) final cleanup at the end...)
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