Product Class: Kit

NEBNext® Enzymatic Methyl-seq Kit
Item# Description List Price Web Price Qty
E7120L NEBNext® Enzymatic Methyl-seq Kit - 96 reactions $4,872.00
$4,384.80
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E7120S NEBNext® Enzymatic Methyl-seq Kit - 24 reactions $1,298.00
$1,168.20
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*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

    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

    View or download extensive performance data in our Technical Note.

    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.

    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


    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.
     
     

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C) Concentration
    Control DNA CpG methylated pUC19 -20
    Control DNA CpG unmethylated Lambda -20
    TET2 Reaction Buffer Supplement -20
    Oxidation Supplement -20
    Oxidation Enhancer -20
    TET2 -20
    Fe (II) Solution -20
    Stop Reagent -20
    APOBEC -20
    APOBEC Reaction Buffer -20
    BSA -20
    NEBNext® Q5U™ Master Mix -20
    NEBNext® Sample Purification Beads 25
    EM-seq™ Index Primer 1 -20
    EM-seq™ Index Primer 2 -20
    EM-seq™ Index Primer 3 -20

    EM-seq™ Index Primer 4

    -20
    EM-seq™ Index Primer 5 -20
    EM-seq™ Index Primer 6 -20
    EM-seq™ Index Primer 7 -20
    EM-seq™ Index Primer 8 -20

    EM-seq™ Index Primer 9

    -20
    EM-seq™ Index Primer 10 -20
    EM-seq™ Index Primer 11 -20
    EM-seq™ Index Primer 12 -20
    EM-seq™ Index Primer 13 -20
    EM-seq™ Index Primer 14 -20
    EM-seq™ Index Primer 15 -20
    EM-seq™ Index Primer 16 -20
    EM-seq™ Index Primer 17 -20
    EM-seq™ Index Primer 18 -20
    EM-seq™ Index Primer 19 -20
    EM-seq™ Index Primer 20 -20
    EM-seq™ Index Primer 21 -20
    EM-seq™ Index Primer 22 -20

    EM-seq™ Index Primer 23

    -20
    EM-seq™ Index Primer 24 -20
    NEBNext® EM-seq™ Adaptor -20
    NEBNext® 96 Unique Dual Index Primer Pairs Plate -20
    NEBNext® Ligation Enhancer -20
    NEBNext Ultra II End Prep Enzyme Mix -20
    NEBNext Ultra II End Prep Reaction Buffer -20
    NEBNext® Ultra II Ligation Master Mix -20
    Product Categories:
    Epigenetics Products,
    Sample Prep for NGS & Target Enrichment Products,
    Epigenetic Analysis
    ,
    DNA Library Prep Products
    Applications:
    DNA Methylation Analysis,
    Applications of Epigenetics,
    DNA for Illumina
    ,
    Methylated DNA Analysis Products
    ,
    DNA Library Preparation
    ,
    Sample Prep for NGS & Target Enrichment
    ,
    Illumina Library Preparation

    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

    Storage Temperature

    -20°C

    Protocols, Manuals & Usage

    Protocols

    1. Protocol for use with Standard Insert Libraries (370–420 bp) (NEB #E7120)
    2. Protocol for use with Large Insert Libraries (470–520 bp) (NEB #E7120)

    Manuals

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

    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?
    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)?

    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

    This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

    While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

    For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].

    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.

    Licenses

    This product is covered by one or more patents.  This product is licensed 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 for use only in:  (a) standard (not real-time) PCR in the research field only, but not real time PCR or digital PCR; (b) real-time PCR for use as a library preparation quantitation tool in Next Generation Sequencing workflows; (c) any in-vitro diagnostics applications, except for applications using real-time PCR or digital PCR; and (d) any non-PCR applications in DNA sequencing, isothermal amplification, and the production of synthetic DNA.

    This product is licensed from University of Newcastle Upon Tyne under EPO Patent No. 1463809B1 and Australian Patent No. AU2003226543.

    Notice to Purchaser: Nucleic acid-based aptamers for use with thermophilic DNA polymerases are licensed exclusively by New England Biolabs, Inc. from SomaLogic, Inc. (See Patent Nos. 5,475,096; 5,670,637; 5,696,249; 5,874,557; and 5,693,502). New England Biolabs, Inc. gives the Buyer/User a non-exclusive license to use the aptamer-based NEBNext Q5U Master Mix for RESEARCH PURPOSES ONLY. Commercial use of the aptamer-based NEBNext Q5U Master Mix requires a license from New England Biolabs, Inc. Please contact [email protected] for more information.