A Method for Selectively Enriching Microbial and Organellar DNA from Contaminating Eukaryotic Host DNA

Recent discoveries have implicated the microbiome of plants and animals as playing a role in health, disease states, and even animal behavior. The majority of microbiome DNA studies to date have employed 16S analysis, but these provide very little information regarding function. In contrast, sequencing of the total DNA of a microbiome sample provides a broader range of information including genes, variants, polymorphisms, and putative functional information. However, many samples contain both host and microbial DNA. In some cases, as low as 1% of sequencing reads may pertain to the microbes of interest and a large percentage of sequencing reads that are associated with the host genome must be discarded, making such experiments impractical. 

To address this issue, we developed an enrichment method to enrich for microbial DNA using methyl-CpG binding domain (MBD) to separate methylated host DNA from microbial DNA. Importantly, microbial diversity and relative abundance is maintained after enrichment. This simple magnetic bead-based method was used to remove fish host DNA from bacterial and protistan DNA.  We describe the enrichment of DNA samples from a black molly fish, followed by next generation sequencing.  Sequence reads aligning to the host genome were reduced approximately 50-fold, while the percentage of sequence reads corresponding to microbial sequences increased approximately 10-fold.  This new method for microbiome enrichment holds promise for use with a variety of sample types, enabling enrichment while accurately reflecting the diversity of the original sample.

Additionally, this enrichment method can be used to enrich for organellar DNA, such as mitochondrial and chloroplast DNA, which is of great interest for population genetics and disease studies. Organellar DNA is found in much lower abundance than nuclear DNA, so it is difficult to study in the absence of an enrichment method. As a demonstration of the efficacy of this methodology, DNA samples from two different plant species were enriched and sequenced. With an Arabidopsis thaliana total gDNA sample, we observed a 4.3 fold increase in chloroplast DNA, from 17% to 73% of the total reads, and 3.6 fold increase in mitochondrial DNA, from 1.8% to 6.5% of the total reads. We observed similar cpDNA enrichment in the plant Leucaena leucocephala.