DNA methylation is the most studied form of epigenetic modification of the genome. Thought primarily to be related to the regulation of gene expression via the inhibition of transcription factor binding, analysis of methylated cytosines within a genome, and across a species’ genomes, can shed light on which genes and regulatory elements are required, or not, at a given point in time. Therefore, the methylome – a genome’s collection of methyl marks – is a changeable snapshot of an organism’s (or a population’s) genomic response to its environment.
Historically, whole genome bisulfite sequencing (WGBS) has been the gold standard technique for methyl sequencing, but it relies on harsh chemical treatments at high temperatures that were extremely damaging to DNA. In the wake of bisulfite conversion, the DNA sample was often so damaged that the resulting libraries were significantly shortened and more difficult to sequence. It was these challenges with bisulfite sequencing that led to the development of the NEBNext® Enzymatic Methyl-seq (EM-seq™) Kit.
This animated workflow will introduce the NEBNext Enzymatic Methyl-seq Kit’s gentler alternative to bisulfite-based cytosine conversion.
The EM-seq kit is a combination of new reagents for enzymatic cytosine protection and conversion and the trusted Ultra® II DNA library prep reagents. The addition of Q5U®, a modified version of Q5® High-Fidelity DNA Polymerase, enables amplification through the uracil-containing converted DNA. The result is superior detection of CpGs with fewer sequencing reads.
For a closer look at the extensive performance data behind EM-seq, download our technical note.
The EpiMark® suite of products were the first generation of New England Biolabs’ epigenetics offering. You can find the current list of available EpiMark products here.
- Is it normal if the FE(II) solution from the EM-seq product is yellow or a color change is observed?
- Can the EM-seq Adaptor be substituted with another adaptor?
- Can the EM-seq Adaptor be used for bisulfite sequencing?
- What is the expected size of an EM-seq library?
- Can enzymatically sheared DNA be used in EM-seq?
- Can EM-seq libraries be prepared from FFPE DNA?
- How should EM-seq libraries be sequenced?
- What is the concentration of the EM-seq Adaptor and EM-seq Index Primers?
- Are EM-seq libraries directional or non-directional?
- How should EM-seq sequencing data be analyzed?
- What is the concentration of the EM-seq Adaptor and Index Primers?
- What levels of conversion are typical with the control DNAs supplied in the EM-seq kit?
- Can other buffers be used in place of the supplied EM-seq Elution Buffer?
- How are EM-seq libraries prepared from cell-free DNA (cfDNA)?
- Why, at some stages of the EM-seq protocol, do the NEBNext Sample Purification Beads behave differently when cleaning up the sample?
- Are Sample Sheets available for use with the NEBNext® Multiplex Oligos for EM-seq?
- Can I use the NEBNext Ultra II FS DNA PCR-free Library Prep Kit for bisulfite conversion or EM-seq™ workflows?
- What is the difference between the NEBNext Enzymatic Methyl-seq Kit and the Enzymatic Methyl-seq Conversion Module?
- What is provided in the NEBNext Multiplex Oligos for Enzymatic Methyl-seq?
- Do I need to spike in custom sequencing primers when sequencing libraries made with NEBNext® Multiplex Oligos for Enzymatic Methyl-seq on Illumina® sequencing instruments?
- Are the NEBNext® Multiplex Oligos for Enzymatic Methyl-seq the same as NEBNext® Multiplex Oligos for Illumina®?
- What buffers are recommended for shearing DNA?
Enzymatic Methyl-seq: The Next Generation of Methylome Analysis
Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email firstname.lastname@example.org.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
Behind the Paper: Using the Human Methyl-Binding Domain to Partition Genomic DNA Derived from Plant Tissues
Can microbiome-enrichment techniques be used to partition plant DNA? Erbay describes the findings of his recent paper.
This video walks you through DNA Library Preparation using the NEBNext Ultra II DNA Library Prep Kit. The video also includes tips for optimization as well as safe stopping points.
Get a quick overview of NGS library preparation from an NEB scientist.
Optimizing DNA inputs for NGS library prep is an important step, if you want to ensure a high-quality library. Start by following these tips for DNA quantification and assessing purity.
These 12 quick tips for NGS library preparation are a great refresher if you're a seasoned pro, or a great introduction if you're a beginner.