Product Class: Kit

NEBNext® Enzymatic Methyl-seq v2 Conversion Module


Catalog #E8020

Product Introduction

NEBNext Enzymatic Methyl-seq (EM-seq™) is a high-performance enzyme-based alternative to bisulfite conversion for the identification of 5mC and 5hmC. Unlike bisulfite conversion, this highly efficient method minimizes DNA damage, resulting in superior detection of methylated cytosines, with fewer sequencing reads.

The new NEBNext Enzymatic Methyl-seq v2 Conversion Module has a wider input range (as low as 100 pg) than the original EM-seq Conversion Module (NEB #E7125).

For use in Illumina® sequencing workflows, we recommend use of the NEBNext Enzymatic Methyl-seq v2 Kit (NEB #E8015) instead of this module. The full kit includes the EM-seq Adaptor, which has been optimized for high performance in the EM-seq workflow, as well as the high-efficiency NEBNext Ultra™ II library prep reagents, and these components are not included in the Conversion Module.

For specific detection of 5hmC, the NEBNext Enzymatic E5hmC-seq Conversion Module (NEB #E3365) is also available.

View or download extensive EM-seq v2 performance data in our Data Supplement.

 

Product Information

Description

View or download extensive EM-seq v2 performance data in our Data Supplement.

The NEBNext Enzymatic Methyl-seq v2 Conversion Module provides a high-performance enzyme-based alternative to bisulfite conversion for methylome analysis, with as little as 100 pg of input DNA.
EM-seq is a two-step enzymatic conversion process to detect modified cytosines. In the first step, TET2 and T4-BGT protect modified cytosines from downstream deamination. TET2 enzymatically oxidizes 5mC and 5hmC, and T4-BGT glucosylates 5hmC.

 

The second enzymatic step uses APOBEC to deaminate unmodified cytosines to uracils, but 5mC and 5hmC protected in the first step are not deaminated.

For specific detection of 5hmC, the NEBNext Enzymatic E5hmC-seq Conversion Module (NEB #E3365) is also available.

Figure 1: EM-seq™ conversion method

Diagram showing sodium bisulfite conversion method versus EM-seq conversion method

The EM-seq™ v2 workflow accommodates a wider input range than the original EM-seq workflow, with a 100-fold lower minimum input amount.

 
This product is related to the following categories:
Enzymatic Conversion for DNA Methylation Analysis,
Methylome Analysis Products,
Epigenetics,
DNA Methylation Analysis,
Next Generation Sequencing Library Preparation Products,

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E8020S     -20    
      Control DNA CpG methylated pUC19 E7122AVIAL -20 1 x 0.024 ml 0.1 ng/µl
      Control DNA CpG unmethylated Lambda E7123AVIAL -20 1 x 0.024 ml 2 ng/µl
      NEBNext® Carrier DNA E3351AVIAL -20 1 x 0.024 ml Not Applicable
      Elution Buffer E7124AVIAL -20 1 x 2.1 ml Not Applicable
      TET2 Reaction Buffer E7126AVIAL -20 1 x 0.3 ml Not Applicable
      TET2 Reaction Buffer Supplement (x 1) E8013AVIAL -20 3 x 100 µl Not Applicable
      UDP-Glucose E3353AVIAL -20 1 x 0.024 ml Not Applicable
      DTT E7139AAVIAL -20 1 x 0.5 ml 100 mM
      T4-BGT E3354AVIAL -20 1 x 0.024 ml Not Applicable
      T4-BGT Diluent E8014AVIAL -20 1 x 0.216 ml Not Applicable
      TET2 E7130AVIAL -20 1 x 0.096 ml Not Applicable
      Fe (II) Solution E7131AVIAL -20 1 x 0.024 ml Not Applicable
      Stop Reagent E7132AVIAL -20 1 x 0.024 ml Not Applicable
      APOBEC E7133AVIAL -20 1 x 0.024 ml Not Applicable
      Deamination Reaction Buffer E3356AVIAL -20 1 x 0.096 ml Not Applicable
      Recombinant Albumin E3357AVIAL -20 1 x 0.024 ml Not Applicable
  • E8020L     -20    
      Control DNA CpG methylated pUC19 E7122AAVIAL -20 1 x 0.096 ml 0.1 ng/µl
      Control DNA CpG unmethylated Lambda E7123AAVIAL -20 1 x 0.096 ml 2 ng/µl
      NEBNext® Carrier DNA E3351AAVIAL -20 1 x 0.096 ml Not Applicable
      Elution Buffer E7124AAVIAL -20 1 x 8.6 ml Not Applicable
      TET2 Reaction Buffer E7126AAVIAL -20 1 x 1.2 ml Not Applicable
      TET2 Reaction Buffer Supplement (x 1) E8013AAVIAL -20 3 x 400 µl Not Applicable
      UDP-Glucose E3353AAVIAL -20 1 x 0.096 ml Not Applicable
      DTT E7139AAVIAL -20 1 x 0.5 ml 100 mM
      T4-BGT E3354AAVIAL -20 1 x 0.096 ml Not Applicable
      T4-BGT Diluent E8014AAVIAL -20 1 x 0.864 ml Not Applicable
      TET2 E7130AAVIAL -20 1 x 0.384 ml Not Applicable
      Fe (II) Solution E7131AAVIAL -20 1 x 0.096 ml Not Applicable
      Stop Reagent E7132AAVIAL -20 1 x 0.096 ml Not Applicable
      APOBEC E7133AAVIAL -20 1 x 0.096 ml Not Applicable
      Deamination Reaction Buffer E3356AAVIAL -20 1 x 0.384 ml Not Applicable
      Recombinant Albumin E3357AAVIAL -20 1 x 0.096 ml Not Applicable

Properties & Usage

Materials Required but not Supplied

  • NEBNext UltraShear® (M7634) or Covaris® instrument and the required tubes or other fragmentation equipment
  • PCR strip tubes or 96-well plates
  • Cleanup beads: SPRIselect Reagent Kit (Beckman Coulter®, Inc. #B23317), AMPure® XP beads (Beckman Coulter, Inc. #A63881) or preferred bead manufacturer
  • Hi-Di™ Formamide (Thermo Fisher Scientific® #4401457), Formamide (Sigma #F9037-100 ml), or 0.05 N NaOH. Formamide is preferred. If using NaOH, please see FAQ associated with NEB #E8015.
  • 80% Ethanol
  • 10 mM Tris-HCl pH 7.5 or 8.0 or low TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA)
  • Nuclease-free Water
  • Magnetic rack/stand, such as NEBNext Magnetic Separation Rack (NEB #S1515)
  • Metal cooling block, such as Diversified Biotech® (#CHAM-1000)
  • PCR machine

 

Protocols, Manuals & Usage

Protocols

  1. Where can I find protocols for use of the NEBNext® Enzymatic Methyl-seq v2 Conversion Module? (NEB #E8020)

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  1. What is the difference between the NEBNext® Enzymatic Methyl-seq v2 Kit (NEB #E8015) and the Enzymatic Methyl-seq v2 Conversion Module (NEB #E8020)?
  2. What types of samples can be processed using the NEBNext® Enzymatic Methyl-seq v2 Kit (NEB #E8015) and the Enzymatic Methyl-seq v2 Conversion Module (NEB #E8020)?
  3. What are the recommended inputs for the NEBNext® Enzymatic Methyl-seq v2 Kit (NEB #E8015) and the Enzymatic Methyl-seq v2 Conversion Module (NEB #E8020)?
  4. What buffers are recommended for shearing DNA in NEBNext® Enzymatic Methyl-seq (EM-seq™) workflows?
  5. What is the concentration of the EM-seq™ Adaptor and the NEBNext® LV Unique Dual Index Primers?
  6. Can the “active” TET2 Buffer be stored longer than 4 months?
  7. Can the freshly diluted Fe(II) Solution be stored long term?
  8. Can the freshly diluted T4-BGT be stored long term?
  9. What conversion levels are typical with the control DNAs supplied in the EM-seq™ v2 kits?
  10. Can I use NaOH (sodium hydroxide) instead of formamide to denature my DNA before the deamination reaction?
  11. Is it normal if the Fe(II) solution from the EM-seq™ product is yellow or a color change is observed?
  12. How can pre-shear spike-in controls be added to cfDNA or other pre-fragmented DNA (amplicons, restriction digested DNA, etc.) going into EM-seq™ workflow?
  13. How should controls be diluted for applications involving shallow sequencing?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

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 busdev@neb.com.

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.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Licenses

In the United States and Europe, this product cannot be used in, or incorporated into, any blood-based, sequencing-based screening diagnostic assay (including product or service-based offerings) for the early detection of colorectal, gastric, lung, liver, ovarian, breast, prostate, esophageal, thyroid, uterine, pancreatic, bladder or kidney cancer indications.

This product and/or its manufacture and/or its use either alone or in combination with other product(s) are protected by one or more of the following patents and related pending patents: U.S. Pat. Nos. 9,121,061, 9,896,726, 10,227,646, 10,260,088, 10,619,200, 11,001,876, and 11,124,825; European Pat. Nos. EP2825645 and EP3368688; JP6224689; and CN108699598.

This product is licensed for research and commercial use from Bio-Rad Laboratories, Inc., under U.S. Pat. No. 8,470,573 and corresponding patents in other countries. No rights are granted for use of the product for Digital PCR or real-time PCR applications, with the exception of quantification in Next Generation Sequencing workflows. For more information, please email busdev@neb.com.

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