Novel canine high-quality metagenome-assembled genomes, prophages and host-associated plasmids provided by long-read metagenomics together with Hi-C proximity ligation

Abstract

The human gut microbiome has been extensively studied, yet the canine gut microbiome is still largely unknown. The availability of high-quality genomes is essential in the fields of veterinary medicine and nutrition to unravel the biological role of key microbial members in the canine gut environment. Our aim was to evaluate nanopore long-read metagenomics and Hi-C (high-throughput chromosome conformation capture) proximity ligation to provide high-quality metagenome-assembled genomes (HQ MAGs) of the canine gut environment. By combining nanopore long-read metagenomics and Hi-C proximity ligation, we retrieved 27 HQ MAGs and 7 medium-quality MAGs of a faecal sample of a healthy dog. Canine MAGs (CanMAGs) improved genome contiguity of representatives from the animal and human MAG catalogues - short-read MAGs from public datasets - for the species they represented: they were more contiguous with complete ribosomal operons and at least 18 canonical tRNAs. Both canine-specific bacterial species and gut generalists inhabit the dog's gastrointestinal environment. Most of them belonged to Firmicutes, followed by Bacteroidota and Proteobacteria. We also assembled one Actinobacteriota and one Fusobacteriota MAG. CanMAGs harboured antimicrobial-resistance genes (ARGs) and prophages and were linked to plasmids. ARGs conferring resistance to tetracycline were most predominant within CanMAGs, followed by lincosamide and macrolide ones. At the functional level, carbohydrate transport and metabolism was the most variable within the CanMAGs, and mobilome function was abundant in some MAGs. Specifically, we assigned the mobilome functions and the associated mobile genetic elements to the bacterial host. The CanMAGs harboured 50 bacteriophages, providing novel bacterial-host information for eight viral clusters, and Hi-C proximity ligation data linked the six potential plasmids to their bacterial host. Long-read metagenomics and Hi-C proximity ligation are likely to become a comprehensive approach to HQ MAG discovery and assignment of extra-chromosomal elements to their bacterial host. This will provide essential information for studying the canine gut microbiome in veterinary medicine and animal nutrition.

Keywords: Hi-C proximity ligation; canine metagenome; gut microbiome; long-read metagenomics; metagenome-assembled genomes; nanopore.

Fig. 1.

Number of ribosomal genes and contigs between long-read CanMAGs and representative genomes from public datasets. Boxplots represent the distribution of the number of ribosomal genes (left) and contigs (right) for the bacterial species identified in this study. Other quality parameters assessments are detailed in Table S1. For each bacterial species, the best genome assembly available on public datasets was included for comparison. Representative genomes available from public database were: (a) short-read MAGs for 19 bacterial species, (b) WGS assemblies for 12 bacterial species and (c) complete genome assemblies for 3 bacterial species.

Fig. 2.

CanMAGs overview: taxonomy, prevalence in canine gut, ARGs, bacteriophages and plasmids. Fu, Fusobacteriota ; Ac, Actinobacteriota ; Prot, Proteobacteria . Genome assemblies with a taxonomy of 'g__' are considered novel species by GTDB-tk. Those marked with an asterisk are MQ MAGs. A dark blue paw symbol indicates that the bacterial species has only been observed in dogs when assessing animal and human faecal MAG catalogues; a light blue paw symbol indicates that the bacterial species is more prevalent in dogs (see Table S3 for more details). All the predicted bacteriophages were integrated within the bacterial host chromosome. Plasmids were linked to the genome using Hi-C data. Cov., coverage; ID, identity. Coloured lines represent resistance to a specific antibiotic, as stated in the key.

Fig. 3.

Analysis of the 27 VCs that included CanMAG bacteriophages. Both parts of the figure contain data from the 33 clustered CanMAG bacteriophages and the representatives from GPD grouping together within the same VC. (a) Boxplots representing the bacteriophage genome sizes within each VC coloured by bacterial host phylum. (b) VCs network. For visualization purposes, each VC is coloured differently.

Fig. 4.

Heatmap hierarchical clustering of the most abundant COG functions for CanMAGs. The CanMAGs are divided in two main clusters driven by carbohydrate transport and metabolism relative abundances. Only the most abundant COG functions are represented in the plot, for detailed COG functions see Tables S6 and S7.