Single-cell Genome Sequencing Research Articles

UGA is an additional glycine codon in uncultured SR1 bacteria from the human microbiota

The composition of the human microbiota is recognized as an important factor in human health and disease. Many of our cohabitating microbes belong to phylum-level divisions for which there are no cultivated representatives and are only represented by small subunit rRNA sequences. For one such taxon (SR1), which includes bacteria with elevated abundance in periodontitis, we provide a single-cell genome sequence from a healthy oral sample. SR1 bacteria use a unique genetic code. In-frame TGA (opal) codons are found in most genes (85%), often at loci normally encoding conserved glycine residues. UGA appears not to function as a stop codon and is in equilibrium with the canonical GGN glycine codons, displaying strain-specific variation across the human population. SR1 encodes a divergent tRNA(Gly)UCA with an opal-decoding anticodon. SR1 glycyl-tRNA synthetase acylates tRNA(Gly)UCA with glycine in vitro with similar activity compared with normal tRNA(Gly)UCC. Coexpression of SR1 glycyl-tRNA synthetase and tRNA(Gly)UCA in Escherichia coli yields significant β-galactosidase activity in vivo from a lacZ gene containing an in-frame TGA codon. Comparative genomic analysis with Human Microbiome Project data revealed that the human body harbors a striking diversity of SR1 bacteria. This is a surprising finding because SR1 is most closely related to bacteria that live in anoxic and thermal environments. Some of these bacteria share common genetic and metabolic features with SR1, including UGA to glycine reassignment and an archaeal-type ribulose-1,5-bisphosphate carboxylase (RubisCO) involved in AMP recycling. UGA codon reassignment renders SR1 genes untranslatable by other bacteria, which impacts horizontal gene transfer within the human microbiota.

The microbial diversity of Polar environments is a fertile ground for bioprospecting

The term bioprospecting has been adopted for systematic searches in nature for new bioactive compounds, genes, proteins, microorganisms and other products with potential for commercial use. Much effort has been focused on microorganisms able to thrive under harsh conditions, including the Polar environments. Both the lipid and protein cellular building blocks of Polar microorganisms are shaped by their adaptation to the permanently low temperatures. In addition, strongly differing environments, such as permafrost, glaciers and sea ice, have contributed to additional functional diversity. Emerging massive-parallel sequencing technologies have revealed the existence of a huge, hitherto unseen diversity of low-abundance phylotypes – the rare biosphere – even in the Polar environments. This realization has further strengthened the need to employ cultivation-independent approaches, including metagenomics and single-cell genomic sequencing, to get comprehensive access to the genetic diversity of microbial communities for bioprospecting purposes. In this review, we present an updated snapshot of recent findings on the molecular basis for adaptation to the cold and the phylogenetic diversities of different Polar environments. Novel approaches in bioprospecting are presented and we conclude by showing recent bioprospecting outcomes in terms of new molecules patented or applied by some biotech companies.

A programmable droplet-based microfluidic device applied to multiparameter analysis of single microbes and microbial communities

We present a programmable droplet-based microfluidic device that combines the reconfigurable flow-routing capabilities of integrated microvalve technology with the sample compartmentalization and dispersion-free transport that is inherent to droplets. The device allows for the execution of user-defined multistep reaction protocols in 95 individually addressable nanoliter-volume storage chambers by consecutively merging programmable sequences of picoliter-volume droplets containing reagents or cells. This functionality is enabled by "flow-controlled wetting," a droplet docking and merging mechanism that exploits the physics of droplet flow through a channel to control the precise location of droplet wetting. The device also allows for automated cross-contamination-free recovery of reaction products from individual chambers into standard microfuge tubes for downstream analysis. The combined features of programmability, addressability, and selective recovery provide a general hardware platform that can be reprogrammed for multiple applications. We demonstrate this versatility by implementing multiple single-cell experiment types with this device: bacterial cell sorting and cultivation, taxonomic gene identification, and high-throughput single-cell whole genome amplification and sequencing using common laboratory strains. Finally, we apply the device to genome analysis of single cells and microbial consortia from diverse environmental samples including a marine enrichment culture, deep-sea sediments, and the human oral cavity. The resulting datasets capture genotypic properties of individual cells and illuminate known and potentially unique partnerships between microbial community members.

The human gut microbiome: are we our enterotypes?

Blood groups, finger prints, iris scans and DNA bar codes are several ways to distinguish or identify individual humans. According to Arumugam and colleagues (2011) we can now also be distinguished by the microbial communities in our faeces, our ‘enterotypes’. The human gut is one of the most densely populated ecosystems known, and although this ecosystem contains members of the three domains of life – bacteria, archaea and eucarya (Finegold et al., 1983) – it is dominated by bacteria. They are essential in digesting the food we eat and in keeping us healthy by stimulating our immune system and fighting off pathogenic bacteria. It is estimated that 1013–1014 microbes inhabit our gastrointestinal tract (GIT), with the greatest number residing in the distal gut, where they synthesize essential vitamins and process otherwise indigestible components of our diet such as plant polysaccharides (Backhed et al., 2005). Early studies used 16S ribosomal DNA (rDNA) analysis to type and enumerate the distal gut and faecal microbiota. More than 90% of the bacterial phylotypes present in the intestinal microbiota in healthy humans are members of only three bacterial divisions: the Bacteroidetes, the Firmicutes and the Actinobacteria (Zoetendal et al., 2006). Nevertheless, each healthy adult's gut appears to have a unique and relatively stable microbiota (Zoetendal et al., 1998; Turnbaugh et al., 2007), which is a reflection of the numerous different phylogenetic clusters among the Firmicutes, Clostridium clusters IV, IX and XIVa, including the predominant genera Clostridium, Eubacterium, Roseburia and Ruminococcus. Furthermore, the Actinobacteria that encompass mainly the genera Bifidobacterium and Atopobium also represent important members of the gut microbial community (Harmsen et al., 2002; Turroni et al., 2008). Notably, recent estimates of the diversity of the human gut microbial ecosystem indicate it may encompass more than 1000 species and a multitude of strains (Backhed et al., 2005; Blaut and Clavel, 2007; Rajilic‐Stojanovic et al., 2007). Microbiota composition studies in humans have discovered that aberrations in the microbiome composition is present in obese individuals (Ley et al., 2006; Turnbaugh et al., 2009) as well as in individuals with a variety of other diseases (Zoetendal et al., 2008). Since the largest part (∼80%) of gut microbes remains uncultured, metagenomics analysis has become fashionable in the past 5 years to estimate the types, relative abundance and genome content of microbes in various parts of the GIT. The first metagenomics analysis from just two faecal samples (Gill et al., 2006) led to early insight into enrichment of genes encoding specific metabolic pathways, including metabolism associated with glycans, amino acids and xenobiotics, but also methanogenesis and biosynthesis of vitamins and isoprenoids. However, this early study provided only a very fragmented view due to limitations in sample size, sequencing technology, number of bases sequenced and availability of suitable reference genomes of gut inhabitants.

Single Cell Genome Amplification Accelerates Identification of the Apratoxin Biosynthetic Pathway from a Complex Microbial Assemblage

Filamentous marine cyanobacteria are extraordinarily rich sources of structurally novel, biomedically relevant natural products. To understand their biosynthetic origins as well as produce increased supplies and analog molecules, access to the clustered biosynthetic genes that encode for the assembly enzymes is necessary. Complicating these efforts is the universal presence of heterotrophic bacteria in the cell wall and sheath material of cyanobacteria obtained from the environment and those grown in uni-cyanobacterial culture. Moreover, the high similarity in genetic elements across disparate secondary metabolite biosynthetic pathways renders imprecise current gene cluster targeting strategies and contributes sequence complexity resulting in partial genome coverage. Thus, it was necessary to use a dual-method approach of single-cell genomic sequencing based on multiple displacement amplification (MDA) and metagenomic library screening. Here, we report the identification of the putative apratoxin. A biosynthetic gene cluster, a potent cancer cell cytotoxin with promise for medicinal applications. The roughly 58 kb biosynthetic gene cluster is composed of 12 open reading frames and has a type I modular mixed polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) organization and features loading and off-loading domain architecture never previously described. Moreover, this work represents the first successful isolation of a complete biosynthetic gene cluster from Lyngbya bouillonii, a tropical marine cyanobacterium renowned for its production of diverse bioactive secondary metabolites.

Single Virus Genomics: A New Tool for Virus Discovery

Whole genome amplification and sequencing of single microbial cells has significantly influenced genomics and microbial ecology by facilitating direct recovery of reference genome data. However, viral genomics continues to suffer due to difficulties related to the isolation and characterization of uncultivated viruses. We report here on a new approach called ‘Single Virus Genomics’, which enabled the isolation and complete genome sequencing of the first single virus particle. A mixed assemblage comprised of two known viruses; E. coli bacteriophages lambda and T4, were sorted using flow cytometric methods and subsequently immobilized in an agarose matrix. Genome amplification was then achieved in situ via multiple displacement amplification (MDA). The complete lambda phage genome was recovered with an average depth of coverage of approximately 437X. The isolation and genome sequencing of uncultivated viruses using Single Virus Genomics approaches will enable researchers to address questions about viral diversity, evolution, adaptation and ecology that were previously unattainable.

Genome of a Low-Salinity Ammonia-Oxidizing Archaeon Determined by Single-Cell and Metagenomic Analysis

Ammonia-oxidizing archaea (AOA) are thought to be among the most abundant microorganisms on Earth and may significantly impact the global nitrogen and carbon cycles. We sequenced the genome of AOA in an enrichment culture from low-salinity sediments in San Francisco Bay using single-cell and metagenomic genome sequence data. Five single cells were isolated inside an integrated microfluidic device using laser tweezers, the cells' genomic DNA was amplified by multiple displacement amplification (MDA) in 50 nL volumes and then sequenced by high-throughput DNA pyrosequencing. This microscopy-based approach to single-cell genomics minimizes contamination and allows correlation of high-resolution cell images with genomic sequences. Statistical properties of coverage across the five single cells, in combination with the contrasting properties of the metagenomic dataset allowed the assembly of a high-quality draft genome. The genome of this AOA, which we designate Candidatus Nitrosoarchaeum limnia SFB1, is ∼1.77 Mb with >2100 genes and a G+C content of 32%. Across the entire genome, the average nucleotide identity to Nitrosopumilus maritimus, the only AOA in pure culture, is ∼70%, suggesting this AOA represents a new genus of Crenarchaeota. Phylogenetically, the 16S rRNA and ammonia monooxygenase subunit A (amoA) genes of this AOA are most closely related to sequences reported from a wide variety of freshwater ecosystems. Like N. maritimus, the low-salinity AOA genome appears to have an ammonia oxidation pathway distinct from ammonia oxidizing bacteria (AOB). In contrast to other described AOA, these low-salinity AOA appear to be motile, based on the presence of numerous motility- and chemotaxis-associated genes in the genome. This genome data will be used to inform targeted physiological and metabolic studies of this novel group of AOA, which may ultimately advance our understanding of AOA metabolism and their impacts on the global carbon and nitrogen cycles.

Whole genome amplification and de novo assembly of single bacterial cells.

Background Single-cell genome sequencing has the potential to allow the in-depth exploration of the vast genetic diversity found in uncultured microbes. We used the marine cyanobacterium Prochlorococcus as a model system for addressing important challenges facing high-throughput whole genome amplification (WGA) and complete genome sequencing of individual cells.Methodology/Principal Findings We describe a pipeline that enables single-cell WGA on hundreds of cells at a time while virtually eliminating non-target DNA from the reactions. We further developed a post-amplification normalization procedure that mitigates extreme variations in sequencing coverage associated with multiple displacement amplification (MDA), and demonstrated that the procedure increased sequencing efficiency and facilitated genome assembly. We report genome recovery as high as 99.6% with reference-guided assembly, and 95% with de novo assembly starting from a single cell. We also analyzed the impact of chimera formation during MDA on de novo assembly, and discuss strategies to minimize the presence of incorrectly joined regions in contigs.Conclusions/SignificanceThe methods describe in this paper will be useful for sequencing genomes of individual cells from a variety of samples.