Product Class: Kit

NEBNext® Microbiome DNA Enrichment Kit

Product Introduction

The NEBNext® Microbiome DNA Enrichment Kit facilitates enrichment of microbial DNA from samples containing methylated host DNA (including human), by selective binding and removal of the CpG-methylated host DNA. Importantly, microbial diversity remains intact after enrichment.

  • Effective enrichment of microbial genomic DNA from samples contaminating host DNA
  • Fast, simple protocol
  • Enables microbiome whole genome sequencing, even for samples with high levels of host DNA
  • Compatible with downstream applications including next generation sequencing on all platforms, qPCR and end point PCR
  • Suitable for a wide range of sample types
  • No requirement for live cells
  • Optional protocol to retain separated host DNA
  • Also effective for separation of organelle DNA (e.g. mitochondria, chloroplast) from eukaryote nuclear DNA
Catalog # Size Concentration
E2612S 6.0 reactions
E2612L 24.0 reactions

Product Information

Description

 

In human DNA, 4–6% of cytosines are methylated, and 60–90% of these methylated cytosines are at CpG sites (1,2). In contrast, methylation at CpG sites in microbial species is rare. The NEBNext Microbiome DNA Enrichment Kit uses a simple and fast magnetic bead-based method to selectively bind and remove CpG-methylated host DNA. The method uses the MBD2-Fc protein, which is composed of the methylated CpG-specific binding protein MBD2, fused to the Fc fragment of human IgG. The Fc fragment binds readily to Protein A, enabling effective attachment to Protein A-bound magnetic beads. The MBD2 domain of this protein binds specifically and tightly to CpG methylated DNA. Application of a magnetic field then pulls out the CpG-methylated (eukaryotic) DNA, leaving the non-CpG-methylated (microbial) DNA in the supernatant (3). If desired, the host DNA captured in the magnetic bead pellet can be eluted, and a protocol is provided for this.

The NEBNext Microbiome DNA Enrichment Kit is suitable for a wide range of sample types, including samples with high levels of contaminating host DNA, (3,4) and is also effective for separation of organelle DNA (e.g. mitochondria, chloroplast) from eukaryote nuclear DNA (5). The kit is compatible with downstream applications including next generation sequencing on all platforms, qPCR and end-point PCR.


Figure 1. Separation workflow
Figure 1. Separation workflow

Salivary Microbiome DNA Enrichment
Salivary Microbiome DNA Enrichment
DNA was purified from pooled human saliva DNA (Innovative Research) and enriched using the NEBNext Microbiome DNA Enrichment Kit. Libraries were prepared from unenriched and enriched samples and sequenced on the SOLiD 4 platform. The graph shows percentages of 500M-537M SOLiD4 50bp reads that mapped to either the Human reference sequence (hg19) or to a microbe listed in Human Oral Microbiome Database (HOMD)[1]. (Because the HOMD collection is not comprehensive, ~80% of reads in the enriched samples do not map to either database.) Reads were mapped using Bowtie 0.12.7[2] with typical settings (2 mismatches in a 28 bp seed region, etc.).
Microbiome Diversity is Retained after Enrichment with the NEBNext Microbiome DNA Enrichment Kit
Microbiome Diversity is Retained after Enrichment with the NEBNext Microbiome DNA Enrichment Kit
DNA was purified from pooled human saliva DNA (Innovative Research) and enriched using the NEBNext Microbiome DNA Enrichment Kit. Libraries were prepared from unenriched and enriched samples, followed by sequencing on the SOLiD 4 platform. The graph shows a comparison between relative abundance of each bacterial species listed in HOMD[1] before and after enrichment with the NEBNext Microbiome DNA Enrichment Kit. Abundance is inferred from the number of reads mapping to each species as a percentage of all reads mapping to HOMD. High concordance continues even to very low abundance species (inset). We compared 501M 50bp SOliD4 reads in the enriched dataset to 537M 50bp SOLiD4 reads in the unenriched dataset. Reads were mapped using Bowtie 0.12.7[2] with typical settings (2 mismatches in a 28bp seed region, etc).

* Neisseria flavescens – This organism may have unusual methylation density, allowing it to bind the enriching beads at a low level. Other Neisseria species (N. mucosa, N. sicca and N. elongata) are represented, but do not exhibit this anomalous enrichment.


 
Each kit contains sufficient reagents for the effective separation of CpG methylated DNA from a mixed pool containing microbial or viral DNA. If starting with 1 μg of input DNA per experiment, the volumes provided are sufficient for preparations of up to 6 reactions (NEB #E2612S) and 24 reactions (NEB #E2612L). All reactions should be stored at -20°.

Box 1: Store at -20°C.
Box 2: Store at 4°C. Do not freeze.

This product is related to the following categories:
Microbiome DNA Enrichment Products,
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
  • E2612S     Multi-temperature    
      NEBNext® Microbiome DNA Enrichment Kit E2612-1 -20 1 x 6 reactions Not Applicable
      NEBNext MBD2-Fc Protein E2614AVIAL -20 1 x 0.1 ml Not Applicable
      NEBNext Bind/wash Buffer E2616AVIAL -20 1 x 12 ml 5 X
      16s rRNA Universal Gene Bacteria Control Primers E2618AVIAL -20 1 x 0.012 ml Not Applicable
      RPL30 Human DNA Control Primers E2619AVIAL -20 1 x 0.012 ml Not Applicable
      NEBNext® Microbiome DNA Enrichment Kit E2612-2 4 1 x 6 reactions Not Applicable
      NEBNext Protein A Magnetic Beads E2615AVIAL 4 1 x 1 ml Not Applicable
  • E2612L     Multi-temperature    
      NEBNext® Microbiome DNA Enrichment Kit E2612-3 -20 1 x 24 reactions Not Applicable
      NEBNext MBD2-Fc Protein E2614AAVIAL -20 1 x 0.4 ml Not Applicable
      NEBNext Bind/wash Buffer E2616AAVIAL -20 1 x 48 ml 5 X
      16s rRNA Universal Gene Bacteria Control Primers E2618AAVIAL -20 1 x 0.048 ml Not Applicable
      RPL30 Human DNA Control Primers E2619AAVIAL -20 1 x 0.048 ml Not Applicable
      NEBNext® Microbiome DNA Enrichment Kit E2612-4 4 1 x 24 reactions Not Applicable
      NEBNext Protein A Magnetic Beads E2615AAVIAL 4 1 x 4 ml Not Applicable

Properties & Usage

Materials Required but not Supplied

  • λ DNA-HindIII Digest (NEB #N3012)
  • 6-Tube Magnetic Separation Rack (NEB #S1506)
  • Gel Loading Dye Blue (6X) (NEB #B7021)
  • Nuclease-free water
  • 0.8% Agarose Gel
  • 1X TE Buffer, pH 7.5
  • Agencourt® AMPure® XP Beads (Beckman Coulter, Inc. #A63881) or Beckman SPRIselect Reagent Kit (Beckman Coulter, Inc. #B23317)
  • 100% Ethanol for Alcohol Precipitation,
  • 80% Ethanol for AMPure XP or SPRIselect
  • DNA Low Bind Microcentrifuge Tubes
  • Rotating Mixer
  • Gel running equipment
  • Heat block or Thermomixer
  • Microcentrifuge
  • Proteinase K, Molecular Biology Grade (optional; required for eluting captured host DNA) (NEB #P8107)

References

  1. Lister, et al. (2009). Nature. 462, 315-322.
  2. Tucker, K.L. (2001). Neuron. 30, 649-652.
  3. Feehery, G. R. et al. (2013). PLoS One. 8(10):e76096.,
  4. Zheng, Z. (2014). Genome Announcements. 2(2):e00273–14.,
  5. Yigit, E. et al. (2014). Appl. Plant Sci. 2 (11), 1400064.

Protocols, Manuals & Usage

Protocols

  1. Protocol for use with NEBNext Microbiome DNA Enrichment (E2612)

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. Can I incubate the DNA input sample with the MBD2-Fc-bound beads for a longer period of time?
  2. How important is the MBD2-Fc bead to DNA ratio?
  3. What is the maximum volume of input DNA that I can use per reaction?
  4. Will the procedure work on degraded DNA?
  5. What is the best method for purifying the DNA after the enrichment?
  6. If my sample DNA is particularly degraded, as is the case with fecal microbiome samples, are there any protocol modifications that will enhance the performance of the NEBNext Microbiome DNA Enrichment Kit with these samples?

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

MBD-Fc and MCiP was developed by Michael Rehli at the University of Regensburg to improve the sensitivity and specificity of conventional CpG binding techniques.

Limited Use Label License:

This product and its use is the subject of one or more issued and/or pending U.S. and foreign patent applications owned by SEQUENOM, Inc. The purchase of this product from NEB or its authorized distributors conveys to the buyer the non-tranferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for profit entity). The purchase of this product does not convey a license under any method claims in the foregoing patents or patent applications directed to producing the product. The buyer cannot sell or otherwise transfer this product or its components to a third party or otherwise use the product for the following COMMERCIAL PURPOSES: (1) use of the product or its components in manufacturing; (2) use of the product or its components for therapeutic, diagnostic or prophylactic purposes in humans or animals; or (3) resale of the product or its components, whether or not such product or its components are resold for use in research.

ACCEPTANCE OF THE FOREGOING LIMITED USE PROVISIONS: If the purchaser is not willing to accept the limitations of this limited use statement, NEB is willing to accept return of the product with a full refund. For information on purchasing a license to use this product for purposes other than those permitted above, contact the NEB Licensing Department at busdev@neb.com.

The MBD-Fc technology is licensed exclusively to New England Biolabs, Inc. from Sequenom, Inc.

Trademarks



NEW ENGLAND BIOLABS® and NEBNext® are registered trademarks of New England Biolabs, Inc.
ULTRA™ is a trademark of New England Biolabs, Inc.

AGENCOURT® and AMPure® are registered trademarks of Beckman Coulter, Inc.
ILLUMINA® is a registered trademark of Illumina, Inc.
NANODROP® is a registered trademark of Nanodrop Technologies, Inc.
QIAGEN® is a registered trademark of The Qiagen Group.
ION TORRENT™ and SOLID™ are trademarks of Life Technologies, Inc.

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