Product Class: Kit

NEBNext® Enzymatic Methyl-seq Kit

For improved performance, NEBNext Enzymatic Methyl-seq v2 Kit (NEB #E8015) is our recommended kit. For enzymatic fragmentation of DNA for EM-seq, NEBNext UltraShear™ (NEB #M7634) is now available.

Product Introduction

NEBNext Enzymatic Methyl-seq (EM-seq™) is a new method for identification of 5-mC and 5-hmC.

While bisulfite sequencing has been the gold standard for methylome analysis, this conversion treatment damages DNA, resulting in fragmentation, loss and bias. 

In contrast, the highly effective enzyme-based conversion in the NEBNext Enzymatic Methyl-seq Kit minimizes damage to DNA and, with the supplied NEBNext Ultra™ II library preparation workflow reagents, produces high quality libraries that enable superior detection of 5-mC and 5-hmC from fewer sequencing reads.

  • Superior sensitivity of detection of 5-mC and 5-hmC 
  • Greater mapping efficiency
  • More uniform GC coverage 
  • Detection of more CpGs with fewer sequence reads
  • Uniform dinucleotide distribution
  • High-efficiency library preparation, with larger library insert sizes
  • Conversion module also available separately

Enzymatic fragmentation of DNA compatible with EM-seq workflows can be achieved using NEBNext UltraShear (NEB #M7634).

View or download extensive EM-seq performance data in our Technical Note.

For specific detection of 5hmC, the NEBNext Enzymatic E5hmC-seq Kit (NEB #E3350) is now also available.

Catalog # Size Concentration
E7120S 24 reactions
E7120L 96 reactions

Product Information

Description

View or download extensive performance data in our Technical Note.

The NEBNext Enzymatic Methyl-seq Kit provides a high-performance enzyme-based alternative to bisulfite conversion for methylome analysis using Illumina® sequencing. 

Libraries are prepared using as little as 10 ng input DNA and the supplied NEBNext Ultra II reagents and the optimized EM-seq Adaptor. TET2 then oxidizes 5-mC and 5-hmC, providing protection from deamination by APOBEC in the next step. In contrast, unmodified cytosines are deaminated to uracils.  Libraries are then amplified using a NEBNext master mix formulation of Q5U® (a modified version of Q5® High-Fidelity DNA Polymerase), and sequenced using Illumina instrumentation.

The consistently high conversion performance and minimized DNA damage with the EM-seq protocol, in combination with highly efficient Ultra II library prep, result in superior detection of CpGs with fewer sequencing reads.

NEBNext UltraShear (NEB #M7634) has been optimized for enzymatic fragmentation of DNA compatible with EM-seq workflows, and protocols are available in the NEBNext UltraShear product manual.

Features:

  • Superior sensitivity of detection of 5-mC and 5-hmC 
  • Greater mapping efficiency
  • More uniform GC coverage 
  • Detection of more CpGs with fewer sequence reads
  • Uniform dinucleotide distribution
  • High-efficiency library preparation, with larger library insert sizes
  • Conversion module also available separately

For specific detection of 5hmC, the NEBNext Enzymatic E5hmC-seq Kit (NEB #E3350) is now also available.

NEBNext UltraShear can be used for enzymatic DNA fragmentation in the EM-seq workflow.



Figure 1: NEBNext Enzymatic Methyl-seq and Sodium Bisulfite Conversion Methods





Figure 2: NEBNext Enzymatic Methyl-seq identifies more CpGs than WGBS, at lower sequencing coverage depth

A.

B. 

10, 50 and 200 ng Human NA12878 genomic DNA was sheared to 300 bp using the Covaris® S2 instrument and used as input into EM-seq and WGBS protocols. For WGBS, NEBNext Ultra II DNA was used for library construction, followed by the Zymo Research EZ DNA Methylation-Gold™ Kit for bisulfite conversion. Libraries were sequenced on an Illumina NovaSeq® 6000 (2 x 100 bases). Reads were aligned to hg38 using bwa-meth 0.2.2. Coverage of CpGs with EM-seq and WGBS libraries was analyzed using 324 million paired end reads.

A: Each top and bottom strand CpGs were counted independently, yielding a maximum of 56 million possible CpG sites. EM-seq identifies more CpGs at lower depth of sequencing.

B:The number of unique and common CpGs identified by EM-seq and WGBS at 1x and 8x minimum coverage for each input amount are shown. EM-seq covers at least 20% more CpGs than WGBS at 1x minimum coverage threshold. The difference in CpG coverage increases to two-fold at 8x minimum coverage threshold.

View additional performance data in our technical note.


Figure 3: NEBNext Enzymatic Methyl-seq has superior uniformity of GC coverage



10, 50 and 200 ng Human NA12878 genomic DNA was sheared to 300 bp using the Covaris S2 instrument and used as input into EM-seq and WGBS protocols. For WGBS, NEBNext Ultra II DNA was used for library construction, followed by the Zymo Research EZ DNA Methylation-Gold Kit for bisulfite conversion. Libraries were sequenced on an Illumina NovaSeq 6000 (2 x 100 bases). Reads were aligned to hg38 using bwa-meth 0.2.2. GC coverage was analyzed using Picard 2.17.2 and the distribution of normalized coverage across different GC contents of the genome (0-100%) was plotted. EM-seq libraries have significantly more uniform GC coverage, and lack the AT over-representation and GC under-representation typical of WGBS libraries.

View additional performance data in our technical note.


Figure 4: NEBNext Enzymatic Methyl-seq Libraries have Larger Insert Sizes



50 ng Human NA12878 genomic DNA was sheared to 300 bp using the Covaris S2 instrument and used as input into EM-seq and WGBS protocols. For WGBS, NEBNext Ultra II DNA was used for library construction, followed by the Zymo Research EZ DNA Methylation-Gold kit for bisulfite conversion. Libraries were sequenced on an Illumina MiSeq® (2 x 76 bases) and insert sizes were determined using Picard 2.18.14. The normalized frequency of each insert size was plotted, illustrating that library insert sizes are larger for EM-seq than for WGBS, and indicating that EM-seq does not damage DNA as bisulfite treatment does in WGBS.

View additional performance data in our technical note.
 
 
This product is related to the following categories:
Enzymatic Conversion for DNA Methylation Analysis,
Automation for NEBNext® NGS Library Prep,
Methylome Analysis,
DNA Methylation Analysis,
Epigenetics,

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E7120S     Multi-temperature    
      NEBNext® Enzymatic Methyl-seq Kit E7120-2 -20 1 x 24 reactions Not Applicable
      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® Ultra II End Prep Reaction Buffer E7647AVIAL -20 1 x 0.168 ml Not Applicable
      NEBNext® Ultra II End Prep Enzyme Mix E7646AVIAL -20 1 x 0.072 ml Not Applicable
      NEBNext® Ultra™ II Ligation Master Mix E7648AVIAL -20 1 x 0.72 ml Not Applicable
      NEBNext® Ligation Enhancer E7374AVIAL -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
      Oxidation Supplement E7128AVIAL -20 1 x 0.024 ml Not Applicable
      Oxidation Enhancer E7129AVIAL -20 1 x 0.024 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
      APOBEC Reaction Buffer E7134AVIAL -20 1 x 0.24 ml Not Applicable
      BSA E7135AVIAL -20 1 x 0.024 ml Not Applicable
      NEBNext® Q5U®  Master Mix E7136AVIAL -20 1 x 0.6 ml Not Applicable
      100mM DTT E7139AAVIAL -20 1 x 0.5 ml 100 mM
      TET2 Reaction Buffer Supplement (x 1) E8013AVIAL -20 3 x 100 µl Not Applicable
      NEBNext® EM-seq™ Adaptor E7165AVIAL -20 1 x 0.06 ml Not Applicable
      NEBNext® Multiplex Oligos for Enzymatic Methyl-seq (Unique Dual Index Primer Pairs) E7140S -20 1 x 24 reactions Not Applicable
      EM-seq™ Index Primer 1 (S) E7141AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 2 (S) E7142AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 3 (S) E7143AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 4 (S) E7144AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 5 (S) E7145AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 6 (S) E7146AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 7 (S) E7147AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 8 (S) E7148AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 9 (S) E7149AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 10 (S) E7150AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 11 (S) E7151AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 12 (S) E7152AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 13 (S) E7153AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 14 (S) E7154AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 15 (S) E7155AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 16 (S) E7156AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 17 (S) E7157AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 18 (S) E7158AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 19 (S) E7159AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 20 (S) E7160AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 21 (S) E7161AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 22 (S) E7162AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 23 (S) E7163AVIAL -20 1 x 0.005 ml 10 µM
      EM-seq™ Index Primer 24 (S) E7164AVIAL -20 1 x 0.005 ml 10 µM
      NEBNext® EM-seq™ Adaptor E7165AVIAL -20 1 x 0.06 ml Not Applicable
      NEBNext® Sample Purification Beads E7137S 25 1 x 24 reactions Not Applicable
      NEBNext® Sample Purification Beads E7137AVIAL 25 1 x 8.6 ml Not Applicable
  • E7120L     Multi-temperature    
      NEBNext® Enzymatic Methyl-seq Kit E7120-3 -20 1 x 96 reactions Not Applicable
      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® Ultra II End Prep Reaction Buffer E7647AAVIAL -20 1 x 0.672 ml Not Applicable
      NEBNext® Ultra II End Prep Enzyme Mix E7646AAVIAL -20 1 x 0.288 ml Not Applicable
      NEBNext® Ultra™ II Ligation Master Mix E7648AAVIAL -20 3 x 0.96 ml Not Applicable
      NEBNext® Ligation Enhancer E7374AAVIAL -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
      Oxidation Supplement E7128AAVIAL -20 1 x 0.096 ml Not Applicable
      Oxidation Enhancer E7129AAVIAL -20 1 x 0.096 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
      APOBEC Reaction Buffer E7134AAVIAL -20 1 x 0.96 ml Not Applicable
      BSA E7135AAVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext® Q5U®  Master Mix E7136AAVIAL -20 2 x 1.2 ml Not Applicable
      100mM DTT E7139AAVIAL -20 1 x 0.5 ml 100 mM
      TET2 Reaction Buffer Supplement (x 1) E8013AAVIAL -20 3 x 400 µl Not Applicable
      NEBNext® EM-seq™ Adaptor E7165AAVIAL -20 1 x 0.24 ml Not Applicable
      NEBNext® Multiplex Oligos for Enzymatic Methyl-seq (Unique Dual Index Primer Pairs) E7140L -20 1 x 96 reactions Not Applicable
      NEBNext® 96 Unique Dual Index Primer Pairs Plate (L) E7166AVIAL -20 1 x 5 μl/well 10 µM
      NEBNext® EM-seq™ Adaptor E7165AAVIAL -20 1 x 0.24 ml Not Applicable
      NEBNext® Sample Purification Beads E7137L 25 1 x 96 reactions Not Applicable
      NEBNext® Sample Purification Beads E7137AAVIAL 25 1 x 34.6 ml Not Applicable

Properties & Usage

Materials Required but not Supplied

  • Covaris® S2 instrument or other fragmentation equipment
  • PCR strip tubes
  • Formamide (Sigma #F9037-100 ml) or 0.1 N NaOH
  • 80% Ethanol • 0.1X TE, pH 8.0
  • Nuclease-free Water
  • Magnetic rack/stand, such as NEBNext Magnetic Separation Rack (NEB #S1515)
  • PCR machine
  • Bioanalyzer®, TapeStation® and associated consumables or other fragment analyzer

Product Notes

  1. It is possible for FE(II) Solution to have a yellow color. Testing has shown no difference in performance based on color.

Protocols, Manuals & Usage

Protocols

  1. Where can I find protocols for using the NEBNext® Enzymatic Methyl-seq Kit? (NEB #E7120, E7125, E7140)

Manuals

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

Usage & Guidelines

FAQs & Troubleshooting

FAQs

  1. What types of sample can be processed using the NEBNext® Enzymatic Methyl-seq Kit and the Enzymatic Methyl-seq Conversion Module?
  2. What are the recommended inputs for the NEBNext® Enzymatic Methyl-seq Kit and the Enzymatic Methyl-seq Conversion Module?
  3. What is the difference between the NEBNext Enzymatic Methyl-seq Kit and the Enzymatic Methyl-seq Conversion Module?
  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 EM-seq 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. Why, at some stages of the EM-seq protocol, do the NEBNext Sample Purification Beads behave differently when cleaning up the sample?
  9. What is the expected size of an EM-seq library?
  10. How should EM-seq libraries be sequenced?
  11. How should EM-seq sequencing data be analyzed?
  12. Can the EM-seq™ Adaptor be substituted with another adaptor?
  13. Are EM-seq™ libraries directional or non-directional?
  14. Can other buffers be used in place of the supplied EM-seq Elution Buffer?
  15. How are EM-seq libraries prepared from cell-free DNA (cfDNA)?
  16. Can EM-seq libraries be prepared from FFPE DNA?
  17. What levels of conversion are typical with the control DNAs supplied in the EM-seq kit?
  18. Are Sample Sheets available for use with the NEBNext® Multiplex Oligos for EM-seq?
  19. Can enzymatically fragmented DNA be used as input material for EM-seq™?
  20. How should controls be diluted for applications involving shallow sequencing?
  21. Can I use NaOH (sodium hydroxide) instead of formamide to denature my DNA before the deamination reaction?
  22. Are dual-indexed libraries compatible with single-end sequencing?
  23. Is it normal if the Fe(II) solution from the EM-seq™ product is yellow or a color change is observed?
  24. What percentage of PhiX is needed for EM-seq™ libraries?

Troubleshooting

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]

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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