Long-read metagenomics retrieves complete single-contig bacterial genomes from canine feces

Abstract

Background Long-read sequencing in metagenomics facilitates the assembly of complete genomes out of complex microbial communities. These genomes include essential biologic information such as the ribosomal genes or the mobile genetic elements, which are usually missed with short-reads. We applied long-read metagenomics with Nanopore sequencing to retrieve high-quality metagenome-assembled genomes (HQ MAGs) from a dog fecal sample. Results We used nanopore long-read metagenomics and frameshift aware correction on a canine fecal sample and retrieved eight single-contig HQ MAGs, which were > 90% complete with < 5% contamination, and contained most ribosomal genes and tRNAs. At the technical level, we demonstrated that a high-molecular-weight DNA extraction improved the metagenomics assembly contiguity, the recovery of the rRNA operons, and the retrieval of longer and circular contigs that are potential HQ MAGs. These HQ MAGs corresponded to Succinivibrio, Sutterella, Prevotellamassilia, Phascolarctobacterium, Catenibacterium, Blautia, and Enterococcus genera. Linking our results to previous gastrointestinal microbiome reports (metagenome or 16S rRNA-based), we found that some bacterial species on the gastrointestinal tract seem to be more canid-specific –Succinivibrio, Prevotellamassilia, Phascolarctobacterium, Blautia_A sp900541345–, whereas others are more broadly distributed among animal and human microbiomes –Sutterella, Catenibacterium, Enterococcus, and Blautia sp003287895. Sutterella HQ MAG is potentially the first reported genome assembly for Sutterella stercoricanis, as assigned by 16S rRNA gene similarity. Moreover, we show that long reads are essential to detect mobilome functions, usually missed in short-read MAGs. Conclusions We recovered eight single-contig HQ MAGs from canine feces of a healthy dog with nanopore long-reads. We also retrieved relevant biological insights from these specific bacterial species previously missed in public databases, such as complete ribosomal operons and mobilome functions. The high-molecular-weight DNA extraction improved the assembly’s contiguity, whereas the high-accuracy basecalling, the raw read error correction, the assembly polishing, and the frameshift correction reduced the insertion and deletion errors. Both experimental and analytical steps ensured the retrieval of complete bacterial genomes.

Technical note: overcoming host contamination in bovine vaginal metagenomic samples with nanopore adaptive sequencing

“…Additionally, multiple genes and non-coding regions have often been deciphered in one single long read, thus revealing the actual genomic structures which consequently contributed to assembling the high-quality metagenome-assembled genomes (MAGs) from metagenomic samples ( Overholt et al, 2020 ; Xie et al, 2020 ; Ciuffreda et al, 2021 ). Complete and circular genomes were reported from long-read metagenomic studies, with and without coupling with short-read data for error-corrections ( Moss et al, 2020 ; Cuscó et al, 2021 ). The MAGs generated with long-read data were much longer, if not complete, hence recovering more genomic elements as well as the gene arrangements which are useful for accurate strain tracking and providing more meaningful insights to the functions of the metagenomes ( Quick, 2019 ; Haro-Moreno et al, 2020 ; Maguire et al, 2021 ).…”

Analyzing Modern Biomolecules: The Revolution of Nucleic-Acid Sequencing – Review

“…As with other genomic, transcriptomic and epigenomic areas, microbiome analyses (microbiomics) require appropriate bioinformatics tools [184]. TGS is particularly useful in metagenomic analyses, since it can be used to generate almost or even complete genomes with single reads, significantly reducing or not requiring contig assembly [185]. Therefore, TGS platforms are being used to find microorganisms present in human microbiomes, foodstuff and beverages like milk, aquaculture, soil and many other ecological niches, allowing to identify both beneficial and pathogenic microorganisms [186][187][188], including serotypes with closely related, or even the same, antigenic formulae [189].…”

Nanopore-Based Surveillance of Zoonotic Bacterial Pathogens in Farm-Dwelling Peridomestic Rodents

“…With respect to metabarcoding, the ONT MinION platform has been successfully applied in several studies, including the characterization of bacterial mock communities [25,27,28]; microbiota profiling of species and tissues such as dog skin [29], canine feces [30], equine gut [31], water buffalo milk [32], sea louse [33], and microalgae [34]; identification of fungi [35]; and characterization of plastic-associated species in the Mediterranean sea [36]. Additionally, metagenetic analyses of environmental samples obtained from glacial regions [37], aquatic environments (e.g., ocean water column [38], river water [39], wastewater [40], and freshwater [41]), building dust [22], and the International Space Station [42] demonstrate the potential and applicability of nanopore sequencing for microorganism detection across diverse environments and field settings.…”