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  • NEBNext® Microbiome DNA Enrichment Kit

    Description






    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.

    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 compatible with downstream applications including next generation sequencing on all platforms, qPCR and end-point PCR.

    Method Overview:

    Step I—Prepare Genomic DNA
    DNA should be free of proteins, proteinase A, SDS and organic solvents; size should be ≥ 15 kb for optimal performance.

    Step II—Combine MBD2-Fc and Magnetic Beads in 1X Bind/wash Buffer.
    Incubate the reaction for 10 minutes at room temperature. Wash beads two times in Bind/wash Reaction Buffer.

    Step III—Add DNA to MBD2-Fc Magnetic Beads.
    Incubate the reaction for 15 minutes at room temperature with gentle mixing.

    Step IV—Collect Supernatant Fraction containing enriched microbial DNA and Bead Fraction-Containing Host DNA.

    Figure 1. Separation workflow

    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
    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).
    * Niesseria flavescens – This organism may have unusual methylation density, allowing it to bind the enriching beads at a low level. Other Niesseria species (N. mucosa, N. sicca and N. elognata) 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 kit provides sufficient reagents for 6 reactions (6.25 μg total input).

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

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBNext MBD2-Fc Protein-20
    NEBNext Bind/wash Buffer-205X
    16s rRNA Universal Gene Bacteria Control Primers-20
    NEBNext Protein A Magnetic Beads4
    RPL30 Human DNA Control Primers-20

    Properties and 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
    • TE Buffer, pH 7.5
    • Agencourt® AMPure® XP Beads (Beckman Coulter, Inc. #A63881) or Ethanol for purifying DNA
    • DNA Low Bind Microcentrifuge Tubes
    • 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.

    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?

    Protocols

    1. DNA Preparation and Quantitation (E2612)
    2. Prebind MBD2-Fc Protein to Magnetic Beads (E2612)
    3. Capture Methylated Host DNA (E2612)
    4. Collect Enriched Microbial DNA (E2612)
    5. Agencourt AMPure XP Bead Clean-up (E2612)
    6. Ethanol Precipitation (E2612)
    7. Optional Protocol for Eluting Captured Host DNA (E2612)
    8. Control Reaction (E2612)
    9. Downstream Analysis (E2612)

    Manuals

    The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.