This new enzyme-based system allows scientists to accurately and sensitively assess DNA methylation without bisulfite treatment, for epigenetics research.
IPSWICH, Mass. /PRNewswire/ -- New England Biolabs (NEB®) today announced the launch of NEBNext® Enzymatic Methyl-seq (EM-seq™), an enzyme-based alternative to bisulfite sequencing for methylation analysis.
Bisulfite treatment has long been the gold standard for methylome analysis, specifically the identification of 5mC and 5hmC. However, this harsh chemical treatment damages DNA, resulting in DNA fragmentation and loss. Sensitivity of detection and confidence in results are also reduced by the bias introduced, including in GC coverage and over-representation of methylated regions.
In response to the need for an alternative, NEB has developed NEBNext EM-seq, a conversion method that utilizes enzymatic treatment and minimizes damage to DNA. When combined with the supplied NEBNext Ultra™ II DNA Library Prep reagents, EM-seq produces high-quality libraries for next generation sequencing that enable superior detection of 5mC and 5hmC from fewer sequencing reads. Additionally, the EM-seq method results in the same sequence conversion as whole genome bisulfite sequencing (WGBS), and so the same data analysis pipelines can be used.
"We've been testing EM-seq on a variety of inputs, platforms and samples," says Christopher Mason, Ph.D., Associate Professor of Weill Cornell Medical College. "It shows more even coverage across CpG islands, the whole genome, and also greater detection of CpG sites across the genome vs. WGBS."
The novel two-step enzymatic conversion involves:
- Protection of 5mC and 5hmC from deamination, by TET2 and an Oxidation Enhancer;
- APOBEC deamination of unprotected cytosines, converting them to uracils, while the protected 5mC and 5hmC bases are not deaminated.
Because EM-seq uses enzymes, as opposed to harsh sodium bisulfite, it results in larger library insert sizes, minimal GC bias, uniform dinucleotide distribution, improved CpG coverage and greater mapping efficiency, all with a wide range of DNA inputs.
"In addition to the user-friendliness of EM-seq, the system has enabled us to precisely determine the cytosine methylation status of even low integrity DNA," said Vladimir Benes, Head of Genomics Core Facility at the European Molecular Biology Laboratory, where a course on EM-seq is scheduled for later this year. "If bisulfite conversion were the only approach to apply, we would definitely fail to generate relevant results."
The kit includes all of the reagents required for EM-seq conversion and library preparation suitable for Illumina® sequencing. For other applications, the conversion module is available separately. For more information, visit NEBNext.com.
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