Direct Selection of Metagenomic DNA from Host DNA

Nucleic-acid based techniques such as hybridization, PCR, qPCR and next generation sequencing offer a rapid and highly sensitive option for direct metagenomic detection from specimens when compared with culture-based techniques. Currently, 16S rRNA gene sequencing is the method of choice for metagenomic studies. However, this approach lacks sensitivity to detect a rare member of the microbial community with divergent target sequences, and not adequate to detect virulence factors in individual strains. Aside from these inherent limitations of amplification and identification of biological samples, the non-microbial host genome itself may interfere with the detection and diagnosis of pathogens due to the higher percentage of host genomic DNA relative to the target microbiome. Therefore, analyses of a metagenome or microbiome directly from host samples by next generation sequencing or PCR are inefficient, difficult and time consuming. To address this problem, we have developed a unique method for separating large pieces of host DNA from microbial DNA using a methyl-CpG binding domain fused to the Fc portion of a human antibody heavy chain (MBD-Fc). This MBD-Fc protein is then bound to a protein A magnetic bead and used to separate methylated host DNA from unmethylated microbial DNA. As a demonstration of the efficacy of this methodology, DNA samples form various sources were enriched and sequenced on different next generation sequencing platforms. Sequencing data showed that non-microbial host DNA reads decreased 50-fold, whereas microbiome DNA reads increased 9-fold, corresponding to ~90-95% microbiome DNA in the enriched fraction. Importantly, microbiome diversity after the enrichment remained intact. This simple methodology can be used to analyze entire microbiomes in a cost-effective manner utilizing established next generation sequencing platforms, as well as newer single molecule sequencing technologies.