NEBNext® Microbiome DNA Enrichment Kit

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

Ordering Information

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  • 6 reactions
    $210.00
  • 24 reactions
    $715.00
  • Product Information





    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).
    * 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 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.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C) Concentration
    NEBNext MBD2-Fc Protein -20
    NEBNext Protein A Magnetic Beads 4
    NEBNext Bind/wash Buffer -20 5 X
    16s rRNA Universal Gene Bacteria Control Primers -20
    RPL30 Human DNA Control Primers -20
    Product Categories:
    Targeted Enrichment,
    Microbiome DNA Enrichment
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