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

Abstract

BackgroundLong-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.ResultsWe 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 gain biological insights that are otherwise missed in short-read MAGs catalogs, as shown by the mobilome functions detected in the long-read HQ MAGs.ConclusionsWe 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 insertions and deletion errors. Both experimental and analytical steps ensured the retrieval of complete bacterial genomes.

Taxonomic, genomic, and functional variation in the gut microbiomes of wild spotted hyenas across two decades of study

“…Of the high-quality MAGs in our dataset, the one with the highest relative abundance across samples was classified as Prevotellamassilia sp. (# 386) (Table S10), a taxon that is closely related to an Alloprevotella isolated from jackal guts ( Canis mesomelas ) (85) and a Prevotella isolated from the canine GI tract (86) (Fig S6). A lactating, 8 year-old hyena from matriline 3 (G3) harbored the highest relative abundance of this MAG, with over 10% of their metagenomic reads mapping to this particular MAG (Table S10).…”

“…This same hyena also had the highest relative abundance of an Enterococcus hirae MAG (# 89) and a Vagococcus lutrae MAG (#228), both of which were rare in all other samples (Table S10). MAGs assigned to E. hirae bacterial species were also recovered from the GI microbiome of healthy dogs (86).…”