New England Biolabs® Launches NEBNext® Enzymatic 5hmC-seq Kit, for enzyme-based 5hmC detection at single-base resolution


New bisulfite-free system allows scientists to accurately and sensitively discriminate 5hmC from un-modified cytosine and 5mC from a wide range of DNA inputs, advancing epigenetics research.

IPSWICH, MA (February 13, 2024) — New England Biolabs (NEB®) today announced the launch of the NEBNext Enzymatic 5hmC-seq Kit (E5hmC-seq), a novel enzyme-based method for the specific detection of 5hmC sites. The gentle, enzyme-based approach enables high yields and high-quality data, with an input range of 100 pg to 200 ng.

While the biological importance of 5hmC modification is less clear than that of 5mC, the abundance of 5hmC varies significantly between tissues, suggesting that it may play a critical role in gene regulation and other biological processes.

“So far, the study of 5hmC has been hampered by the lack of precise detection methods,” said Fiona Stewart, Associate Director, NEBNext Portfolio Management. “While NEBNext Enzymatic Methyl-seq (EM-seq), our gold standard for methylation detection, detects both 5mC and 5hmC, it does not distinguish between them. Additionally, bisulfite-based methods suffer from reduced data quality due to the sample fragmentation and loss of DNA that results from the damaging bisulfite treatment, thereby limiting their practical utility.

“To address these challenges, we developed the NEBNext Enzymatic 5hmC-seq Kit, which allows for the specific detection of 5hmC sites using a two-step enzymatic conversion workflow,” said Stewart. “The enzymatic method minimizes DNA damage and allows for the discrimination of 5hmC from unmodified cytosine and 5mC after Illumina® sequencing. Additionally, E5hmC-seq data can be subtracted from EM-seq data, allowing for precise determination of individual 5mC and 5hmC sites."

The kit includes the reagents required for E5hmC-seq conversion and library preparation compatible with Illumina sequencing; index primers for multiplexing are available separately. A conversion module is also available, for applications beyond library prep.

For more information, visit

About New England Biolabs:

Established in the mid 1970's, New England Biolabs, Inc. (NEB) is the industry leader in the discovery and production of enzymes for molecular biology applications and now offers the largest selection of recombinant and native enzymes for genomic research. NEB continues to expand its product offerings into areas related to PCR, gene expression, sample preparation for next generation sequencing, synthetic biology, glycobiology, epigenetics and RNA analysis. Additionally, NEB is focused on strengthening alliances that enable new technologies to reach key market sectors, including the development of molecular diagnostics, as well as nucleic acid vaccines. New England Biolabs is a privately held company, headquartered in Ipswich, MA, and has extensive worldwide distribution through a network of exclusive distributors, agents and subsidiaries located in Australia, Canada, China, France, Germany, Japan, New Zealand, Singapore South Korea and the UK. For more information about New England Biolabs visit


NEB®, NEW ENGLAND BIOLABS®, and NEBNext® are registered trademarks of New England Biolabs, Inc.

ILLUMINA® is a registered trademark of Illumina, Inc.


Contact information:


Deana D. Martin, Ph.D.

Marketing Communications Manager

New England Biolabs

240 County Road Ipswich, MA 01938

Tel: 978-380-7464




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