Restriction Endonuclease-Based Modification-Dependent Enrichment (REMoDE) of DNA for Metagenomic Sequencing

Abstract

Metagenomic sequencing is a swift and powerful tool to ascertain the presence of an organism of interest in a sample. However, sequencing coverage of the organism of interest can be insufficient due to an inundation of reads from irrelevant organisms in the sample. Here, we report a nuclease-based approach to rapidly enrich for DNA from certain organisms, including enterobacteria, based on their differential endogenous modification patterns. We exploit the ability of taxon-specific methylated motifs to resist the action of cognate methylation-sensitive restriction endonucleases that thereby digest unwanted, unmethylated DNA. Subsequently, we use a distributive exonuclease or electrophoretic separation to deplete or exclude the digested fragments, thus enriching for undigested DNA from the organism of interest. As a proof of concept, we apply this method to enrich for the enterobacteria Escherichia coli and Salmonella enterica by 11- to 142-fold from mock metagenomic samples and validate this approach as a versatile means to enrich for genomes of interest in metagenomic samples. IMPORTANCE Pathogens that contaminate the food supply or spread through other means can cause outbreaks that bring devastating repercussions to the health of a populace. Investigations to trace the source of these outbreaks are initiated rapidly but can be drawn out due to the labored methods of pathogen isolation. Metagenomic sequencing can alleviate this hurdle but is often insufficiently sensitive. The approach and implementations detailed here provide a rapid means to enrich for many pathogens involved in foodborne outbreaks, thereby improving the utility of metagenomic sequencing as a tool in outbreak investigations. Additionally, this approach provides a means to broadly enrich for otherwise minute levels of modified DNA, which may escape unnoticed in metagenomic samples.

Keywords: DNA; foodborne outbreaks; metagenomics; methylation; modifications; restriction endonuclease; sequencing.

FIG 1

Schematic of the pipeline for endonuclease- and exonuclease-based enrichment of methylated DNA. A metagenomic sample containing DNA that is and is not Dam and Dcm methylated is treated with methylation-sensitive enzymes. The unmethylated DNA is digested to short fragments while the methylated DNA remains long and intact. Size selection for longer fragments is performed with either electrophoretic separation or a distributive exonuclease (which preferentially degrades short fragments). The enriched sample is then sequenced.

FIG 2

Methylation-sensitive endonucleases and T5 exonuclease enrich for E. coli DNA in an E. coli and C. elegans DNA mixture. (A) Gel showing the susceptibility of either E. coli DNA or C. elegans DNA to PspGI, MboI and EcoRII separately and all together. The genomic high-molecular-weight C. elegans band disappears when the endonuclease is applied. (B) Gel showing time points of T5 exonuclease treatment when applied to a 1:3 mixture of E. coli-to-C. elegans DNA treated with the corresponding endonucleases. Notice the disappearance of the low-molecular-weight smear (C. elegans DNA) with longer T5 exonuclease incubation. (C) Paired-end sequencing data from untreated and treated samples. In blue are the proportion of reads in that sample that map to the E. coli genome and in yellow are the proportion that map to the C. elegans genome. Any reads that do not map to either or have chimeric paired reads are colored gray. The C. elegans-only sample contains a certain amount of E. coli DNA likely due to the fact that the worms are fed E. coli. Shown below is the relative enrichment of E. coli DNA calculated as the ratio of the number of E. coli reads to C. elegans reads divided by the ratio in the untreated control. (D) For each T5 exonuclease time point, all C. elegans reads that remained were mapped to the length of the theoretical fragment size that they would be found in an in silico digestion of the C. elegans genome. A cumulative density plot of these fragments is shown to ascertain whether remaining C. elegans reads originate from long fragments or short fragments.

FIG 3

Methylation-sensitive endonucleases and various size-selection approaches enrich for E. coli and S. enterica DNA in the Zymo mix. (A) Paired-end sequencing data from untreated, endonuclease-only treated and endonuclease- as well as T5 exonuclease-treated DNA. In blue are reads that map to genomes that are Dam and Dcm methylated. In yellow are reads that map to genomes that are not Dam and Dcm methylated. Mean proportions of two biological replicates were plotted. Relative enrichment of E. coli and S. enterica shown below were calculated from the mean proportions. The raw enrichment values for each replicate are as follows (1.0, 0.87, 9.72) and (1.0, 0.76, 12.74) from left to right. (B) Paired-end sequencing data from untreated and endonuclease-treated DNA size-selected through gel electrophoresis (see Fig. S3 in the supplemental material). Mean proportions of two biological duplicates were plotted. Relative enrichments of E. coli and S. enterica shown below were calculated from mean proportions. The raw enrichment values for both duplicates were (1.0, 141.8) from left to right.

FIG 4

Dynamic range of enrichment on various amounts and ratios of methylated DNA. (A) Paired-end sequencing data from either half, one-tenth, or one-hundredth the amount of Zymo mix DNA used in the standard protocol following otherwise the same enzyme concentrations. In blue are reads that map to genomes that are Dam and Dcm methylated. In yellow are reads that map to genomes that are not Dam and Dcm methylated. Relative enrichment is shown below. (B) Paired end sequencing data from Zymo mix DNA mixed with S. cerevisiae DNA in 1:1, 1:9, and 1:99 ratios with the total amount remaining 75 ng. Mean proportions of two biological duplicates were plotted for panel B. Relative enrichment of E. coli and S. enterica shown below were calculated from the mean proportions. The raw enrichment values for each replicate are (1.0, 62.9, 1.0, 45.0, 1.0, 28.6) and (1.0, 81.8, 1.0, 65.1, 1.0, 35.8) from left to right. In red are reads that map to the T4 phage. T4 phage DNA sequences represent a population of DNA fortuitously included with the yeast DNA material used in these assays. Notably, this DNA is enriched in parallel to the modified bacterial DNAs, a behavior that is both of interest and expected as a consequence of the known modification of T4 DNA. Thus, this population serves as a fortuitous positive control on the enrichment observed. Mean relative enrichment of T4 phage, E. coli, and S. enterica, together, is (1.0, 89.5, 1.0, 193.4, 1.0, 307.8) from left to right.

FIG 5

DpnI and T5 exonuclease treatment enriches for DNA that is not Dam methylated. Paired-end sequencing data for Zymo mix DNA treated with DpnI, which only cuts at methylated Dam sites. Relative enrichments are shown below.