Selective Microbial Genomic DNA Isolation Using Restriction Endonucleases

Abstract

To improve the metagenomic analysis of complex microbiomes, we have repurposed restriction endonucleases as methyl specific DNA binding proteins. As an example, we use DpnI immobilized on magnetic beads. The ten minute extraction technique allows specific binding of genomes containing the DpnI Gm6ATC motif common in the genomic DNA of many bacteria including γ-proteobacteria. Using synthetic genome mixtures, we demonstrate 80% recovery of Escherichia coli genomic DNA even when only femtogram quantities are spiked into 10 µg of human DNA background. Binding is very specific with less than 0.5% of human DNA bound. Next Generation Sequencing of input and enriched synthetic mixtures results in over 100-fold enrichment of target genomes relative to human and plant DNA. We also show comparable enrichment when sequencing complex microbiomes such as those from creek water and human saliva. The technique can be broadened to other restriction enzymes allowing for the selective enrichment of trace and unculturable organisms from complex microbiomes and the stratification of organisms according to restriction enzyme enrichment.

Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing

“…Additionally, previous studies using the MolYsis kit focused on improving techniques such as real-time PCR, whereas use of these methods for WGS, where microbial DNA enrichment could be even more useful, has been less studied (Votintseva et al, 2015). Other methods such as host cell lysis with detergents (Hasan et al, 2016) or ox bile (Zhou & Pollard, 2012) and immunoprecipitation of DNA with inactive methyl-specific restriction endonucleases (Barnes et al, 2014; Liu et al, 2016) have also been reported, but are not available as commercial products.…”

Epigenetic Segregation of Microbial Genomes from Complex Samples Using Restriction Endonucleases HpaII and McrB

“…coli genomic DNA into low molecular weight fragments (<500 bp) and showed little activity on human DNA ( S1 Fig ). To develop a magnetic bead based enrichment workflow, we removed magnesium ions from the reaction buffer which prevents digestion activity [ 12 ] but still enables HpaII to bind target DNA ( Fig 1A ). HpaII mediated enrichment conditions were optimized using selective qPCR assays on a predefined DNA mixture of Yersenia pestis and human genomes.…”

“…A volume of 0.5 ml ethanol was added and the solution was loaded on the spin column up to three times. DNA was eluted twice with 30 μl 1X Binding Buffer [ 12 ] at 60°C and the eluates were combined. The DNA yield was determined with the Qubit BR assay (LifeTechnologies).…”

“…For microbial taxa identification, Illumina data sets were analyzed by an automated pipeline (ZovaSeq from Zova Systems, LLC, San Diego CA)[ 12 ] in which identifying sequence reads are assigned to specific microbial taxa when a given read length is found to occur uniquely within the taxa as defined by the NCBI taxonomy database [ 20 , 21 ]. Relative abundance was calculated using two methods which gave equivalent results: tallying the number of ZovaSeq identifying reads or “microbial ID reads” for each bacterial taxa or by using Bowtie 1.0.0 to map reads to all identified organisms in the sample.…”

“…Nevertheless for most clinical sample DNA preparations, microbes, particularly pathogens, are typically present at trace levels resulting in inefficiently sequencing a vast majority of host DNA rather than the desired microbiome or causative pathogen. Techniques to improve targeted sequencing have been developed but recent epigenetic methods to segregate target genomes [ 12 – 14 ] have the advantage of enriching nearly whole genomes for sequencing. However, the epigenome of only a small number of bacterial species has been well defined [ 15 – 17 ], and epigenomes of protists, fungi and viruses remain poorly characterized.…”