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NEBNext® Enzymatic Methyl-seq Kit

Catalog # Concentration Size List Price Quantity Your Price
E7120L 96 reactions $4,921.00
$4,428.90
E7120S 24 reactions $1,311.00
$1,179.90
Catalog # Size List Price Your Price
E7120L 96 reactions $4,921.00
$4,428.90
E7120S 24 reactions $1,311.00
$1,179.90
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

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.

Featured Videos
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    NEBNEXT® Enzymatic Methyl-seq Workflow
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    Highly multiplexed capture of genomic markers for high throughput NGS based genotyping
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    NEB® TV Ep. 28 – Advances in Methylome Analysis
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    New Frontiers in Epigenomic Analysis - from Probing Disease Progression to Enhancing Sequencing Methodologies

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.
 
 
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  NEBNext® Enzymatic Methyl-seq Kit E7120-2 -20 1 x 24 reactions
  NEBNext® Multiplex Oligos for Enzymatic Methyl-seq (Unique Dual Index Primer Pairs) E7140S -20 1 x 24 reactions
  NEBNext® Sample Purification Beads E7137S 25 1 x 24 reactions
  NEBNext® Enzymatic Methyl-seq Kit E7120-3 -20 1 x 96 reactions
  NEBNext® Multiplex Oligos for Enzymatic Methyl-seq (Unique Dual Index Primer Pairs) E7140L -20 1 x 96 reactions
  NEBNext® Sample Purification Beads E7137L 25 1 x 96 reactions
An exception occurred during the operation, making the result invalid. Check InnerException for exception details.

Notes
  • It is possible for FE(II) Solution to have a yellow color. Testing has shown no difference in performance based on color.
FAQs
Citations
Additional Citations
  • Morrison J, et al. (2021) Evaluation of whole-genome DNA methylation sequencing library preparation protocols. Epigenet Chromatin PREPRINT (version 1) available at Research Square, DOI: 10.21203/rs.3.rs-249202/v1
  • Erger F, et al. (2020) cfNOMe - A Single Assay for Comprehensive Epigenetic Analyses of Cell-Free DNA. Genome Med 12(1):54,PubMedID: 32580754, DOI: 10.1186/s13073-020-00750-5
  • Feng S, et al. (2020) Efficient and accurate determination of genome-wide DNA methylation patterns in Arabidopsis with enzymatic methyl sequencing. Epigenet Chromatin DOI: 10.21203/rs.3.rs-41816/v1
  • Sun Z, et al. (2019) Non-destructive enzymatic deamination enables single molecule long read sequencing for the determination of 5-methylcytosine and 5-hydroxymethylcytosine at single base resolution. bioRxiv DOI: 10.1101/2019.12.20.885061
  • Vaisvila R, et al. (2021) Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA. Genome ResPubMedID: 34140313, DOI: 10.1101/gr.266551.120
Publications
  • Vaisvila R, et al. (2021). Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA. Genome Res.PubMedID: 34140313, DOI: 10.1101/gr.266551.120
Quality Control Assay
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
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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 Data Sheets
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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.

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. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645, 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 is licensed from University of Newcastle Upon Tyne under EPO Patent No. 1463809B1 and Australian Patent No. AU2003226543.

Nucleic acid-based aptamers for use with thermophilic DNA polymerases are licensed exclusively by New England Biolabs, Inc. from SomaLogic, Inc. New England Biolabs, Inc. gives the Buyer/User a non-exclusive license to use the NEBNext® Enzymatic Methyl-seq Kit for Research Use Only (RUO). Commercial use of this product may require a license from New England Biolabs, Inc. For additional information or to inquire about commercial use, please contact busdev@neb.com.

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