Metagenomics are effective tools for studying microbial communities, but it is difficult to "classify" sequences to the species and strain levels in community composition. Similar to the principle of Hi-C technology applied to eukaryotic chromosome-level reference genome-assisted assembly, Hi-C/3C technology can be applied to the assembly of metagenomes to improve the results of metagenome assembly.
Based on the principle that DNA molecules from the same cell (microorganism) interact more strongly with each other than with different cells (microorganisms), the contigs sequences from the same microorganism can be clustered into the same groups and the groups can be identified as species.
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Hi-C not only clusters metagenomic results to species and strain levels, but also associates plasmids, phages and host bacteria in microbial communities. Horizontal gene transfer between strains is mediated by mobile genetic elements (such as plasmids, integrons, phages, transposons, etc.) of various drug-resistant genes that can be passed between bacterial strains of the same and different species, thus allowing rapid transmission of bacterial resistance, with recipient bacteria exhibiting multi-drug resistance. Dissecting host-plasmid and host-phage associations is critical to understanding the ecology of antibiotic resistance and the pathways of resistance gene transmission in the clinic, and is a prerequisite for designing and implementing interventions to reduce the threat of bacterial infection.
The 3C technology, the predecessor of Hi-C technology, Meta 3C can also be used to associate mobile genetic elements (resistance genes, plasmids and integrons) with the host genome. Importantly, Meta 3C is used to construct three 3C libraries directly for each sample by three four-base restriction endonucleases (HpaII, MluCI, Sau3AI) without labeling biotin, making the experimental process relatively simple.
Moreover, it can better guarantee the GC coverage of various microorganisms in the flora, and is more suitable for metagenomic assembly, which can make up for the defect that simple metagenomic sequencing is difficult to "classify" sequences to the species and strain levels in the community composition. The Meta Hi-C clustering is less contaminating and is suitable for biological problems such as plasmids and mobile elements.
1. Metagenome assembly contigs are clustered to the microbial species level;
2. Associating mobile genetic elements (resistance genes, plasmids, and integrons) with the host genome.
|Category||Microorganism||Library Sequencing||Sample Requirement|
|Metagenome Hi-C||Microbial community||Intestinal and other samples, 40G/sample;
Complex samples such as soil silt, 60G/sample;
|20-30g soil after removal of visible impurities; 1-5g intestinal contents or human feces|
|Metagenome 3C||Construct 3 3C libraries, the enzymes are Hapll, Sau3Al, MluCl, 20G for each library|
There are molecular interactions between DNA in cells (cells), and their spatial conformations are close to each other. Hi-C technology is used to cross-link and fix the DNA conformation in cells, so that sequences from the same cell can be determined. After the cells (cells) are lysed, the cross-linked and fixed DNA is obtained for library construction and sequencing. Using the cross-linking relationship, the sequence from the same cell is determined, and the genome of a single bacteria is constructed. Near-complete genomes can be obtained to deepen metagenomic research.
Three enzymes (HpaII: AATT; MluCl: GGCC; Sau3AI: GATC) are used in Meta 3C to better ensure the GC coverage of various microorganisms in the flora.