Single-cell genomics of uncultured bacteria reveals dietary fiber responders in the mouse gut microbiota

Abstract

BackgroundThe gut microbiota can have dramatic effects on host metabolism; however, current genomic strategies for uncultured bacteria have several limitations that hinder their ability to identify responders to metabolic changes in the microbiota. In this study, we describe a novel singlecell genomic sequencing technique that can identify metabolic responders at the species level without the need for reference genomes, and apply this method to identify bacterial responders to an inulin-based diet in the mouse gut microbiota. ResultsInulin feeding changed the mouse fecal microbiome composition to increase Bacteroides spp., and increased microbial metabolic function resulted in increased succinate concentrations in the mouse intestine. Using our massively parallel single-cell genome sequencing technique, named SAG-gel platform, we obtained 346 single-amplified genomes (SAGs) from mouse gut microbes before and after dietary inulin supplementation. After quality control, the SAGs were classified as 267 bacteria, spanning two phyla, four classes, seven orders, and 14 families, and 31 different strains of SAGs were graded as high-and medium-quality draft genomes. From these, we have successfully obtained the genomes of the dominant inulin responders, Bacteroides spp., and identified their polysaccharide utilization loci and their specific metabolic pathways for succinate production. ConclusionsOur single-cell genomics approach generated a massive amount of SAGs, enabling functional analysis of uncultured bacteria in the intestinal microbiome and consideration of host responses. This enabled us to estimate metabolic lineages involved in the bacterial 2 fermentation of dietary fiber and metabolic outcomes such as short-chain fatty acid production in the intestinal environment based on the fibers ingested. The technique allows the in-depth isolation and characterization of uncultured bacteria with specific functions in the microbiota, and could be exploited to improve human and animal health. BackgroundThe gut microbiota plays a crucial role in the control of host physiology and metabolism through the release and transformation of metabolites like short-chain fatty acids (SCFAs) [1,2]. Many studies have demonstrated that dietary fiber supplementation can modulate gut microbiota composition and promote SCFA production [3][4][5]. This dietary fiber metabolism is particularly important for non-digestible inulin-type fructans classified as prebiotics, which are substrates that promote the growth of beneficial microorganisms in the gut [6][7][8][9][10][11][12]. Thus, to enhance the benefits of dietary fiber to the host, it is important to identify inulin responders in the complex microbiome community of the host intestine, and characterize their mechanisms of inulin fermentation and potential for SCFA production.Recently developed metagenomic approaches can provide comprehensive microbial profiling and putative functions using 16S rRNA gene amplicon sequencing and shotgun metagenome sequencing. These methods have enabled the ...

Recovery of high-quality assembled genomes via metagenome binning guided with single-cell amplified genomes

“…However, the number of constructed genomes was limited, and no tool has been developed to comprehensively obtain multispecies genomes at once, which is mostly due to the lack of technology that provides good quality SAGs as binning guides. In SIGMA, the qualities of SAGs obtained by our SAG-gel technology 22 were sufficiently high to prevent false-positive contigs in supervised contig identification. In addition, merging of SAGs with the metagenomic bin aided recovery of rRNA and tRNA sequences, which were frequently lacking in the MAGs obtained by conventional binners.…”

“…Single-cell genome sequencing was performed with single-cell whole genome amplification (WGA) using the SAG-gel platform according to our previous reports 22,26 . Following homogenization of human feces in GuSCN solution (500 μL), the supernatant was recovered by centrifugation at 2000 × g for 30 s, followed by filtration through 35-μm nylon mesh and centrifugation at 8,000 × g for 5 min.…”

“…Single-cell genome sequencing was performed with single-cell whole genome amplification (WGA) using the SAG-gel platform according to our previous reports 22,26 . Following homogenization of human feces in GuSCN solution (500 μL),…”

Extensive single-cell genomics reveals bacterial diversity and diverse phage host ranges in the area in and around the Red Sea

“…However, unlike the genomes of isolates, challenges in genome assembly are not fully resolved using SAG-gel platform alone, as WGA process generates numerous chimeric reads 32 , hampering the assembly of long contiguous sequences and randomly biased amplified genomes 11 , and causing incomplete genome coverage. By combining our methods with other tools including cleaning and co-assembly of SAGs from the same species, the number of chimeric contigs and genome incompleteness could be significantly reduced 26,33 . Draft genomes acquired by SAG-gel can also be combined with other sequence data sets including shotgun metagenome sequencing and long-read sequencing, all of which will promote genome finishing 34 .…”

Microbe-seq: high-throughput, single-microbe genomics with strain resolution, applied to a human gut microbiome

“…By contrast, high-quality strainresolved genomes of taxa from the human gut microbiome have been assembled from colonies cultured from individual microbes (Almeida et al, 2019;Browne et al, 2016;Poyet et al, 2019;Zhao et al, 2019); however, culturing colonies can be labor-intensive and biased toward easy-to-culture microbes. Alternatively, single-cell genomics relies upon isolating and lysing individual microbes in wells on a titer plate, and subsequently amplifying their DNA for sequencing (Chijiiwa et al, 2020;Lasken, 2007;Pachiadaki et al, 2019;Rinke et al, 2014;Xu et al, 2016); such approaches also yield strain-resolved genomes and have been used to probe the association between phages (Džunková et al, 2019) and bacteria in the human gut microbiome. For both culture and well-plate approaches, however, available resources severely limit the number of strain-resolved genomes that originate from the same community (Almeida et al, 2019;Poyet et al, 2019), thereby constraining our knowledge of the genomic structure and dynamics of the human gut microbiome of an individual.…”