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 workflowSalivary Microbiome DNA EnrichmentDNA 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 KitDNA 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.
This product is related to the following categories:
Microbiome DNA Enrichment,
Next Generation Sequencing Library Preparation
品牌介绍
NEB公司成立于二十世纪七十年代中期,拥有众多经验丰富的科学家,是生产生命科学试剂的领导者。目前,NEB为基因组研究提供最齐全的重组酶和天然酶,并且公司业务范围已延伸至蛋白质组学和药品开发领域。回顾三十余年来的历程,NEB公司作为先驱公司之一,为促进生物科技工业的发展做出了巨大的贡献。NEB美国总部乔 迁新址后拥有最尖端的设备,有一座现代化的发酵中心及设备齐全的实验室,这些实验室主要用于产品生产、质量监控、 产品开发和基础科研之用。作为首批以商业规模生产限制性内切酶的公司之一,NEB一直专注于内切酶的研究,并保持 业内领先水平。NEB公司一贯坚持以科学为本的原则,公司生产的试剂因其高质量、高性价比享誉世界。NEB(北京)有限公司 New England Biolabs (Beijing) LTD. NEB(北京)有限公司New England Biolabs (Beijing) LTD. 为美国New England Biolabs, Inc.在华投资兴建的独资子公司,负责处理其在中国内地及香港地区的全部业务,同时承担部分新产品的研发工作。纽英伦生物技术(北京)有限公司成立于2001年11月,为中国生命科学研究者提供一流的产品和专业的技术服务,促进国内外生命科学学术交流,与中国科研工作者一起将中国推入到二十一世纪生命科学的前沿中去。