Obtaining high-quality draft genomes from uncultured microbes by cleaning and co-assembly of single-cell amplified genomes

Kogawa, Masato; Hosokawa, Masahito; Nishikawa, Yohei; Mori, Kazuki; Takeyama, Haruko

doi:10.1038/s41598-018-20384-3

Cited by 51 publications

(66 citation statements)

References 35 publications

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“…2a), mouse intestinal microbes were captured in agarose gel beads by microfluidic droplet generator. Cell lysis with an enzyme cocktail, genomic DNA purification [21], and subsequent whole-genome amplification [22,23] were all performed in the agarose gel matrix. After sorting the single beads, which contain single-cell amplified DNA, into standard PCR plates using a cell sorter, the DNA samples were re-amplified as a single-cell amplified genome (SAG) library.…”

Section: Single-cell Resolution Genome Analysis Of the Fecal Microbiomentioning

confidence: 99%

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

et al. 2020

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Background: The 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 single-cell 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. Results: Inulin-feeding changed the mouse fecal microbiome composition to increase Bacteroides spp., resulting in the production of abundant succinate 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 2 phyla, 4 classes, 7 orders, and 14 families, and 31 different strains of SAGs were graded as highand 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. Conclusions: Our single-cell genomics approach generated a massive amount of SAGs, enabling a functional analysis of uncultured bacteria in the intestinal microbiome. This enabled us to estimate metabolic lineages involved in the bacterial 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.

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Section: Single-cell Resolution Genome Analysis Of the Fecal Microbiomentioning

confidence: 99%

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

et al. 2020

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“…Using the single-cell sequencing based on SAG-gel platform (Figure 2a), mouse intestinal microbes were captured in agarose gel beads by microfluidic droplet generator. Cell lysis with an enzyme cocktail, genomic DNA purification [21], and subsequent whole genome amplification [22,23] were all performed in the agarose gel matrix. After the collection of beads contain single-cell amplified DNA, the DNA samples were re-amplified as a single-cell amplified genome (SAG) library.…”

Section: Single-cell Resolution Genome Analysis Of the Fecal Microbiomentioning

confidence: 99%

“…In 16S rRNA gene homology analysis, a considerable number of SAGs showed low similarity to reference genomes, suggesting that these SAGs contain the genomes of species currently undescribed in public databases. Furthermore, by acquiring duplicate SAG data from identical strains, we could assemble the draft genomes using a cleaning and co-assembly strategy [22,23]. For example, IMSAGC_001 and IMSAGC_004 were duplicated 41 and 12 times, respectively, allowing the construction of composite draft genomes, which are very advantageous for obtaining genomes of medium or high quality.…”

Section: Microbiome Composition Monitoring Demonstrated Rapid Responsmentioning

confidence: 99%

“…According to the definition used in Almeida et al [16], 40% of the draft genomes of quality higher than medium were qualified as near-complete (> 90% completeness and < 5% contamination, Additional file 1: Table S5). Hence, SAG-gel platform can provide increased numbers of draft genomes from single microbial cells, with 14 higher completeness than other single-cell genome sequencing techniques [22,23,[29][30][31][32].…”

Section: Microbiome Composition Monitoring Demonstrated Rapid Responsmentioning

confidence: 99%

“…Composite draft genomes were constructed by co-assembly of SAG groups with ≥ 99% identical marker genes detected by CheckM and ≥ 95% ANI. When an assembled composite draft genome showed ≥ 10% completeness, the composite draft was reassembled from the same SAG group by ccSAG [23]. CDS, rRNAs, and tRNAs were extracted from SAG contigs or composite draft genomes by prokka 1.13 [38], and metabolic functions were estimated by Genomaple 2.3.2 [39].…”

Section: Single-cell Genome Sequencing Analysismentioning

confidence: 99%

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

Chijiiwa

Hosokawa

Kogawa

et al. 2019

Preprint

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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 ...

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Anwendungen von Einzelzellmethoden in der mikrobiellen Naturstoffforschung

Cahn

Piel

2021

Angewandte Chemie

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Die diversen Mikroorganismen, die Naturstoffe produzieren, sind eine wichtige Quelle für neuartige Therapeutika, Arzneimittelkandidaten und wissenschaftliche Hilfsmittel. Der allergrößte Teil der mikrobiellen Vielfalt konnte jedoch nicht axenisch kultiviert werden und gehört komplexen Lebensgemeinschaften an. Während meta′omische Methoden wie Metagenomik, ‐transkriptomik und ‐proteomik kollektive molekulare Merkmale der “mikrobiellen dunklen Materie” identifizieren, kann die Untersuchung einzelner Mikrobiom‐Bakterien eine Herausforderung sein. Um diese Limitierungen zu überwinden, wurden in den letzten eineinhalb Jahrzehnten mehrere Techniken für das Studium einzelner Bakterienzellen entwickelt. Während verschiedene dieser Methoden in der mikrobiellen Ökologie Verbreitung finden, werden sie bisher weniger häufig in der Naturstoffforschung eingesetzt. In diesem Aufsatz stellen wir die verfügbaren und neu aufkommenden Techniken für die gezielte Einzelzellanalyse vor, mit einem besonderen Schwerpunkt auf Anwendungen in der Entdeckung und Untersuchung von Naturstoffen.

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Obtaining high-quality draft genomes from uncultured microbes by cleaning and co-assembly of single-cell amplified genomes

Cited by 51 publications

References 35 publications

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

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

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

Anwendungen von Einzelzellmethoden in der mikrobiellen Naturstoffforschung

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