Detection of rare plasmid hosts using a targeted Hi-C approach

Despite the significant role plasmids play in microbial evolution, there is limited knowledge of their ecology, evolution, and transfer in microbial communities. This is partly due to the limitations of current methods in associating a plasmid with its host in microbiomes. To address this knowledge gap, we developed and implemented a novel approach to identify rare plasmid hosts by combining Hi-C, a proximity ligation method, with enrichment for plasmid-specific DNA. We hereafter refer to this approach as Hi-C+. We applied Hi-C and Hi-C+ to soil microbial communities in which we mimicked increasingly rare transfer of an antimicrobial resistance plasmid from a donor to a recipient. This was achieved by inoculating agricultural soil with mixtures of known plasmid-containing and plasmid-free cells at different proportions. We demonstrated that Hi-C can link a plasmid to its host in soil when the relative abundance of that plasmid-host pair is as low as 0.001%. Hi-C+ further improved the detection limit of Hi-C 100-fold and allowed the identification of plasmid hosts at the genus level. As a culture-independent approach, Hi-C+ will significantly improve our understanding of the range and frequency of spread of antibiotic resistance and other genes that are introduced into soil and other microbiomes.