Long-Read Metagenomics to Retrieve High-Quality Metagenome-Assembled Genomes from Canine Feces

Abstract

Background. Metagenomics is a powerful and rapidly developing approach that provides new biological insights into the microbes inhabiting underexplored environments, such as canine fecal microbiome. We investigate long-read metagenomics with Nanopore sequencing to profile the fecal microbiome and to retrieve high-quality metagenome-assembled genomes (HQ MAGs) from a healthy dog.Results. More than 99% of total classified reads corresponded to Bacteria. The most abundant phylum was Bacteroidetes (~80% of total reads), followed by Firmicutes, Proteobacteria, and Fusobacteria. Prevotella (>50%) and Bacteroides (>20%) are the more abundant genera, followed by Fusobacterium, Megamonas, Sutterella, and other fecal-related genera, (each representing <5% of the total bacterial composition). We retrieved eight single-contig HQ MAGs and three medium-quality MAGs, after combining several metagenome dataset assemblies. The HQ MAGs corresponded to Succinivibrio, Sutterella, Prevotellamassilia, Phascolarctobacterium, Enterococcus, Blautia, and Catenibacterium genera. Succinivibrio HQ MAG represents a novel candidate bacterial species. Sutterella HQ MAG is potentially the first reported genome assembly for Sutterella stercoricanis, as assigned by 16S rRNA gene similarity. Prevotellamassilia, Phascolarctobacterium, Catenibacterium, and Blautia sp900541345 HQ MAGs improved the contiguity of previously reported genome assemblies in their respective genera, and the number of rRNA genes and tRNA genes. Finally, Enterococcus hirae and Blautia sp003287895 HQ MAGs represented species that already have a complete reference genome. At the technical level, we demonstrated that a high-molecular weight DNA extraction improved the taxonomic classification of the raw unassembled reads, the metagenomics assembly contiguity, and the retrieval of longer and circular contigs, which are potential HQ MAGs. Conclusions. Long-read metagenomics allowed us to recover HQ MAGs from canine feces of a healthy dog. The high-molecular weight DNA extraction to improve contiguity and the correction of the insertions and deletions to reduce frameshift errors ensure the retrieval of complete single-contig HQ MAGs.

High molecular weight DNA extraction strategies for long‐read sequencing of complex metagenomes

“…Next, we sequenced the DNA from GT, PC and AE using two MinION flow cells to ensure the replicates of the same protocol were sequenced on different runs (Run 1: GT_1, PC_1 and AE_1; Run 2: GT_2, PC_2 and AE_2). The sequencing runs generated 4.84 and 7.79 Gbp, respectively, which were within the expected range of MinION sequencing output (Cusco et al, 2020; Moss et al, 2020). The increase in the output in the second run could be attributed to less sample loss during library preparation and subsequent input of three times more DNA (142.8 vs. 466.2 ng) into the flow cell.…”

“…Next, we sequenced the DNA from GT, PC and AE using two MinION flow cells to ensure the replicates of the same protocol were sequenced on different runs (Run 1: GT_1, PC_1 and AE_1; Run 2: GT_2, PC_2 and AE_2). The sequencing runs generated 4.84 and 7.79 Gbp, respectively, which were within the expected range of MinION sequencing output (Cusco et al, 2020;Moss et al, 2020).…”

“…Nanopore sequencing, which resolves the identity and order of nucleotides based on changes in ionic current as a single‐stranded RNA or DNA molecule passes through a tiny pore (Kasianowicz et al, 1996), has rapidly gained popularity among researchers (Wang et al, 2014) thanks to its availability through affordable and easy to operate sequencing devices, such as MinION by Oxford Nanopore Technologies (“The Long View on Sequencing”, 2018). Despite the high error rates and relatively lower sequencing depth, long reads from nanopore sequencing of metagenomes led to key insights from challenging systems (Pessi et al, 2020; Reveillaud et al, 2019) and enabled the recovery of circular, complete genomes from metagenomes (Cusco et al, 2020; Moss et al, 2020; Nicholls et al, 2019; Sanderson et al, 2018; Singleton et al, 2021; Somerville et al, 2019).…”

Oxford Nanopore R10.4 long-read sequencing enables near-perfect bacterial genomes from pure cultures and metagenomes without short-read or reference polishing

“…Interestingly, the estimated reduction in mismatches (at least 20 %) was less pronounced, as it was observed in 6 of 18 Nanopore R9 bins and 8 of 15 for R10, presumably due to Nanopore R10 bins featuring overall lower long read coverage (Fig 2d). Hence, even though it is possible to acquire HQ MAGs from Nanopore-only assemblies, as demonstrated in this and other studies (47,48), supplemental…”