Product Class: Other

NEBNext® Enzymatic Methyl-seq v2 Kit

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 Kit has a wider input range (as low as 100 pg) and a faster, more streamlined workflow than the original EM-seq kit (NEB #E7120). 

The NEBNext Enzymatic Methyl-seq v2 Kit includes conversion reagents, library prep reagents and the EM-seq Adaptor. Multiple sets of the required index primers (NEBNext LV Unique Dual Index Primers) are available separately, enabling greater flexibility in multiplexing. 

  • Superior sensitivity of detection of 5mC and 5hmC 
  • 0.1 ng - 200 ng input range
  • Detection of more CpGs with fewer sequencing reads
  • Even GC coverage
  • High performance library preparation and larger library insert sizes
  • Index primers supplied separately
  • Enzymatic fragmentation of DNA compatible with EM-seq workflows can be achieved using NEBNext UltraShear® (NEB #M7634).

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

For conversion only, the NEBNext Enzymatic Methyl-seq v2 Conversion Module (NEB #E8020) is also available.

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

Bioz Badge Exists : True
Catalog # Size Concentration
E8015S 24 reactions
E8015L 96 reactions

Product Information

Description

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

NEBNext Enzymatic Methyl-seq is a high-performance enzyme-based alternative to bisulfite conversion for methylome analysis using Illumina® sequencing. With the expanded input range of the latest NEBNext Enzymatic Methyl-seq v2 Kit, as little as 100 pg of input DNA can be used. The protocol has also been streamlined to minimize cleanup steps and reduce workflow time. 

Libraries are prepared using the supplied NEBNext Ultra II reagents and the optimized EM-seq Adaptor. Index primers are available separately, as NEBNext LV Unique Dual Index Primers (NEB #E3390, E3392, E3400, E3402, E3404, E3406, E3408).

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. 

This is followed by amplification using a NEBNext master mix formulation of Q5U® (a modified version of Q5® High-Fidelity DNA Polymerase), and sequencing on the Illumina platform. 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.

Bioinformatic analysis tools used for bisulfite sequencing can also be used for EM-seq. 

NEBNext UltraShear (NEB #M7634) has been optimized for enzymatic fragmentation of DNA compatible with EM-seq workflows.

For specific detection of 5hmC, the NEBNext Enzymatic E5hmC-seq Kit (NEB #E3350) is now 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. The v2 workflow is more streamlined, has one fewer cleanup step and is faster. Note that NEBNext LV UDI primers are not included in the kit and are available separately.

Figure 2: EM-seq™ v2 exhibits high CpG coverage across a range of inputs

Line graph showing CpG coverage input range (number of CpGs versus observed coverage depth)
EM-seq™ v2 libraries were prepared from 200–0.1 ng of NA12878 DNA (sheared to 350 bp using Covaris® ME220), spiked with unmethylated lambda and CpG-methylated pUC19. Libraries were sequenced on an Illumina® NovaSeq® 6000 (2 x 150 bases). Approximately 910 million reads for each library were aligned to a composite human T2T, lambda and pUC19 reference genome using bwa-meth. The T2T genome covers a maximum of 67.8 million CpGs when the top and bottom strands are counted independently. EM-seq covered over 56 million CpG sites for 200–1 ng inputs and roughly 45 million CpG sites for 0.1 ng input libraries.

Figure 3: NEBNext EM-seq v2 identifies more CpGs than WGBS and the original EM-seq, at lower sequencing coverage depth

Two line graphs showing percent of CpGs covered (at 10X and 8X)
EM-seq v2 (NEB #E8015), EM-seq (NEB #E7120) and WGBS libraries were prepared from 200 ng and 10 ng of NA12878 DNA (sheared to ~350 bp), spiked with unmethylated lambda and CpG-methylated pUC19. Libraries were sequenced on an Illumina NovaSeq 6000. For accurate comparison of the original EM-seq and WGBS data with EM-seq v2 data, we evaluated data from approximately 625 million 100 base reads for each library aligned to a composite human T2T, lambda and pUC19 reference genome using bwa-meth.

The T2T genome covers a maximum of 67.8 million CpGs when the top and bottom strands are counted independently. EM-seq v2 and EM-seq covered over 54 million CpG sites for both 200 ng and 10 ng inputs; however, WGBS libraries covered only 46 million and 39 million for 200 ng and 10 ng inputs respectively at 1X coverage. The dashed lines represent coverage of (A) 10X and (B) 8X. The table lists the percentage of CpG sites covered by different libraries at (A) 10X and (B) 8X coverage level.

Figure 4: EM-seq™ v2 produces high library yields across a broad input range
Bar graph showing yield (library yield versus DNA input)

200–0.1 ng of NA12878 genomic DNA, sheared to 350 bp (Covaris® ME220) was used as input into the EM-seq™ v2 protocol, using the number of PCR cycles shown. Library yields were determined using the Agilent® TapeStation® with High Sensitivity D1000 reagents. Values shown are the average of two technical replicates and error bars show standard deviation. EM-seq v2 consistently produces high-yield libraries across a wide range of inputs.

Figure 5: EM-seq™ v2 provides even GC coverage

Line graph showing GC coverage (normalized coverage versus GC content)
EM-seq™ v2 libraries were prepared from 200–0.1 ng of NA12878 DNA (sheared to 350 bp using Covaris® ME220), spiked with unmethylated lambda and CpG-methylated pUC19. Libraries were sequenced on an Illumina® NovaSeq® 6000 (2 x 150 bases). Approximately 910 million reads for each library were aligned to a composite human T2T, lambda and pUC19 reference genome using bwa-meth. GC coverage was analyzed using Picard and the distribution of normalized coverage across different GC contents of the genome (0–100%) was plotted for reads mapping to human genome. EM-seq v2 libraries have uniform GC coverage across the input range.
This product is related to the following categories:
Library Preparation for Ultima Genomics,
Enzymatic Conversion for DNA Methylation Analysis,
Methylome Analysis,
DNA Methylation Analysis,
Epigenetics,
Next Generation Sequencing Library Preparation,

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • NEB # Stored at (°C)
    E8015S Multi-temperature
    Component Name Component # Stored at (°C) Amount Concentration
    NEBNext® Enzymatic Methyl-seq v2 Kit E8015-2 -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 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
    NEBNext® Carrier DNA E3351AVIAL -20 1 x 0.024 ml Not Applicable
    NEBNext® EM-seq™ Adaptor E7165AVIAL -20 1 x 0.06 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 E8013AVIAL -20 3 x 100 µl Not Applicable
    UDP-Glucose E3353AVIAL -20 1 x 0.024 ml Not Applicable
    100mM 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
    NEBNext® Q5U®  Master Mix E3369AVIAL -20 1 x 1.08 ml Not Applicable
    NEBNext® Sample Purification Beads E3355S 25 1 x 24 reactions Not Applicable
    NEBNext® Sample Purification Beads E3355AVIAL 25 1 x 5.2 ml Not Applicable
  • NEB # Stored at (°C)
    E8015L Multi-temperature
    Component Name Component # Stored at (°C) Amount Concentration
    NEBNext® Enzymatic Methyl-seq v2 Kit E8015-3 -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 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
    NEBNext® Carrier DNA E3351AAVIAL -20 1 x 0.096 ml Not Applicable
    NEBNext® EM-seq™ Adaptor E7165AAVIAL -20 1 x 0.24 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 E8013AAVIAL -20 3 x 400 µl Not Applicable
    UDP-Glucose E3353AAVIAL -20 1 x 0.096 ml Not Applicable
    100mM 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
    NEBNext® Q5U®  Master Mix E3369AAVIAL -20 1 x 4.32 ml Not Applicable
    NEBNext® Sample Purification Beads E3355L 25 1 x 96 reactions Not Applicable
    NEBNext® Sample Purification Beads E3355AAVIAL 25 1 x 20.64 ml Not Applicable

Properties & Usage

Materials Required but not Supplied

  • NEBNext UltraShear® (NEB #M7634) or Covaris® instrument and the required tubes or other fragmentation equipment
  • PCR strip tubes or 96-well plates
  • 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

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  1. What is the difference between the NEBNext® Enzymatic Methyl-seq v2 Kit (NEB #E8015) and the original NEBNext Enzymatic Methyl-seq Kit (NEB #E7120)?
  2. What is the difference between the NEBNext® Enzymatic Methyl-seq v2 Kit (NEB #E8015) and the Enzymatic Methyl-seq v2 Conversion Module (NEB #E8020)?
  3. 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)?
  4. 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)?
  5. What buffers are recommended for shearing DNA in NEBNext® Enzymatic Methyl-seq (EM-seq™) workflows?
  6. What is the concentration of the EM-seq™ Adaptor and the NEBNext® LV Unique Dual Index Primers?
  7. Can the “active” TET2 Buffer be stored longer than 4 months?
  8. Can the freshly diluted T4-BGT be stored long term?
  9. What is the expected size of an EM-seq™ v2 (NEB #E8015) 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 libraries be prepared from FFPE DNA using the NEBNext® EM-seq v2 Kit? 
  17. What conversion levels are typical with the control DNAs supplied in the EM-seq™ v2 kits?
  18. Can enzymatically fragmented DNA be used as input material for EM-seq™ v2?
  19. Are Sample Sheets available with the NEBNext® LV Unique Dual Index Primers?
  20. Can I use NaOH (sodium hydroxide) instead of formamide to denature my DNA before the deamination reaction?
  21. Are dual-indexed libraries compatible with single-end sequencing?
  22. Is it normal if the Fe(II) solution from the EM-seq™ product is yellow or a color change is observed?
  23. What percentage of PhiX is needed for EM-seq™ libraries?
  24. 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?
  25. How should controls be diluted for applications involving shallow sequencing?
  26. What should I do if I observe adaptor-dimers in my EM-seq™ v2 libraries?
  27. Can the freshly diluted Fe(II) Solution be stored long term?
  28. Does the kit include primers?

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]

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 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.

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. 10,227,646, 10,260,088, 10,619,200, 11,001,876, and 11,124,825; European Pat. No. EP3368688 and CN108699598.

This product is licensed for research purposes only, worldwide, under the patent family that includes U.S. Patent No. 9,115,386 and related patents, including any domestic and foreign counterparts.

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