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  • NEBNext® Microbiome DNA Enrichment Kit

    Description

    The NEBNext Microbiome DNA Enrichment Kit will effectively remove up to 98% of methylated host DNA, such as human DNA (4–6% of all the cytosines are methylated, 60–90% of which is at CpG sites) (1,2) from a microbial DNA pool (non-CpG methylated or partially CpG methylated at 0.1% of the genome) in under 1 hour.

    Methylated host DNA is removed from a DNA mixture by binding to the methyl-CpG binding domain of human MBD2-Fc protein (MBD2-Fc refers to the MBD domain of the MBD2a protein fused to the Fc portion of a human antibody heavy chain). This stable complex will selectively bind double-stranded methylated CpG-containing DNA. After capture, the microbial DNA which is not CpG methylated, or is minimally CpG methylated, remains in the supernatant and is recovered by gentle aspiration with minimal sample loss. If desired, the host DNA captured in the pellet can be easily eluted in a small volume of TE and Proteinase K by incubation at 75°C. The samples are immediately ready for downstream analysis by a variety of methods including:

    • Library preparation for Next-Gen Sequencing
    • Endpoint and real-time PCR assays
    • DNA microarray analysis

    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
    NEBNext Bind/wash Buffer5X
    IMR90/E. coli (10:1) DNA Mixture
    16s rRNA Gene Universal Bacteria Control Primers
    NEBNext Protein A Magnetic Beads
    RPL30 Human DNA Control Primers

    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.

    Supporting Documents

    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.

    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].
    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?
    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)

    Feature Articles

    NEB Publications

    • George R. Feehery, Erbay Yigit, Samuel O. Oyola, Bradley W. LanghorstVictor T. Schmidt, Fiona J. Stewart, Eileen T. Dimalanta, Linda A. Amaral-Zettler, Theodore Davis, Michael A. Quail, Sriharsa Pradhan (2013) PLOS ONE