SAG-RAD: A Method for Single-Cell Population Genomics of Unicellular Eukaryotes.

Sequencing of reduced representation libraries enables genotyping of many individuals for population genomic studies. However, high amounts of DNA are required, and the method cannot be applied directly on single cells, preventing its use on most microbes. We developed and implemented the analysis of single amplified genomes followed by restriction-site-associated DNA sequencing to bypass labor-intensive culturing and to avoid culturing bias in population genomic studies of unicellular eukaryotes. This method thus opens the way for addressing important questions about the genetic diversity, gene flow, adaptation, dispersal, and biogeography of hitherto unexplored species.

Peeling off the layers from microbial dark matter (MDM): recent advances, future challenges, and opportunities.

Microbes represent the most common organisms on Earth; however, less than 2% of microbial species in the environment can undergo cultivation for study under laboratory conditions, and the rest of the enigmatic, microbial world remains mysterious, constituting a kind of "microbial dark matter" (MDM). In the last two decades, remarkable progress has been made in culture-dependent and culture-independent techniques. More recently, studies of MDM have relied on culture-independent techniques to recover genetic material through either unicellular genomics or shotgun metagenomics to construct single-amplified genomes (SAGs) and metagenome-assembled genomes (MAGs), respectively, which provide information about evolution and metabolism. Despite the remarkable progress made in the past decades, the functional diversity of MDM still remains uncharacterized. This review comprehensively summarizes the recently developed culture-dependent and culture-independent techniques for characterizing MDM, discussing major challenges, opportunities, and potential applications. These activities contribute to expanding our knowledge of the microbial world and have implications for various fields including Biotechnology, Bioprospecting, Functional genomics, Medicine, Evolutionary and Planetary biology. Overall, this review aims to peel off the layers from MDM, shed light on recent advancements, identify future challenges, and illuminate the exciting opportunities that lie ahead in unraveling the secrets of this intriguing microbial realm.

Uncultured prokaryotic genomes in the spotlight: An examination of publicly available data from metagenomics and single-cell genomics.

Owing to the ineffectiveness of traditional culture techniques for the vast majority of microbial species, culture-independent analyses utilizing next-generation sequencing and bioinformatics have become essential for gaining insight into microbial ecology and function. This mini-review focuses on two essential methods for obtaining genetic information from uncultured prokaryotes, metagenomics and single-cell genomics. We analyzed the registration status of uncultured prokaryotic genome data from major public databases and assessed the advantages and limitations of both the methods. Metagenomics generates a significant quantity of sequence data and multiple prokaryotic genomes using straightforward experimental procedures. However, in ecosystems with high microbial diversity, such as soil, most genes are presented as brief, disconnected contigs, and lack association of highly conserved genes and mobile genetic elements with individual species genomes. Although technically more challenging, single-cell genomics offers valuable insights into complex ecosystems by providing strain-resolved genomes, addressing issues in metagenomics. Recent technological advancements, such as long-read sequencing, machine learning algorithms, and in silico protein structure prediction, in combination with vast genomic data, have the potential to overcome the current technical challenges and facilitate a deeper understanding of uncultured microbial ecosystems and microbial dark matter genes and proteins. In light of this, it is imperative that continued innovation in both methods and technologies take place to create high-quality reference genome databases that will support future microbial research and industrial applications.

Industrial Biotechnology Based on Enzymes From Extreme Environments.

Biocatalysis is crucial for a green, sustainable, biobased economy, and this has driven major advances in biotechnology and biocatalysis over the past 2 decades. There are numerous benefits to biocatalysis, including increased selectivity and specificity, reduced operating costs and lower toxicity, all of which result in lower environmental impact of industrial processes. Most enzymes available commercially are active and stable under a narrow range of conditions, and quickly lose activity at extremes of ion concentration, temperature, pH, pressure, and solvent concentrations. Extremophilic microorganisms thrive under extreme conditions and produce robust enzymes with higher activity and stability under unconventional circumstances. The number of extremophilic enzymes, or extremozymes, currently available are insufficient to meet growing industrial demand. This is in part due to difficulty in cultivation of extremophiles in a laboratory setting. This review will present an overview of extremozymes and their biotechnological applications. Culture-independent and genomic-based methods for study of extremozymes will be presented.

Single-cell genomics for resolution of conserved bacterial genes and mobile genetic elements of the human intestinal microbiota using flow cytometry.

As our understanding of the importance of the human microbiota in health and disease grows, so does our need to carefully resolve and delineate its genomic content. 16S rRNA gene-based analyses yield important insights into taxonomic composition, and metagenomics-based approaches reveal the functional potential of microbial communities. However, these methods generally fail to directly link genetic features, including bacterial genes and mobile genetic elements, to each other and to their source bacterial genomes. Further, they are inadequate to capture the microdiversity present within a genus, species, or strain of bacteria within these complex communities. Here, we present a method utilizing fluorescence-activated cell sorting for isolation of single bacterial cells, amplifying their genomes, screening them by 16S rRNA gene analysis, and selecting cells for genomic sequencing. We apply this method to both a cultured laboratory strain of Escherichia coli and human stool samples. Our analyses reveal the capacity of this method to provide nearly complete coverage of bacterial genomes when applied to isolates and partial genomes of bacterial species recovered from complex communities. Additionally, this method permits exploration and comparison of conserved and variable genomic features between individual cells. We generate assemblies of novel genomes within the Ruminococcaceae family and the Holdemanella genus by combining several 16S rRNA gene-matched single cells, and report novel prophages and conjugative transposons for both Bifidobacterium and Ruminococcaceae. Thus, we demonstrate an approach for flow cytometric separation and sequencing of single bacterial cells from the human microbiota, which yields a variety of critical insights into both the functional potential of individual microbes and the variation among those microbes. This method definitively links a variety of conserved and mobile genomic features, and can be extended to further resolve diverse elements present in the human microbiota.

Printing Microbial Dark Matter: Using Single Cell Dispensing and Genomics to Investigate the Patescibacteria/Candidate Phyla Radiation.

As of today, the majority of environmental microorganisms remain uncultured. They are therefore referred to as "microbial dark matter." In the recent past, cultivation-independent methods like single-cell genomics (SCG) enabled the discovery of many previously unknown microorganisms, among them the Patescibacteria/Candidate Phyla Radiation (CPR). This approach was shown to be complementary to metagenomics, however, the development of additional and refined sorting techniques beyond the most commonly used fluorescence-activated cell sorting (FACS) is still desirable to enable additional downstream applications. Adding image information on the number and morphology of sorted cells would be beneficial, as would be minimizing cell stress caused by sorting conditions such as staining or pressure. Recently, a novel cell sorting technique has been developed, a microfluidic single-cell dispenser, which assesses the number and morphology of the cell in each droplet by automated light microscopic processing. Here, we report for the first time the successful application of the newly developed single-cell dispensing system for label-free isolation of individual bacteria from a complex sample retrieved from a wastewater treatment plant, demonstrating the potential of this technique for single cell genomics and other alternative downstream applications. Genome recovery success rated above 80% with this technique-out of 880 sorted cells 717 were successfully amplified. For 50.1% of these, analysis of the 16S rRNA gene was feasible and led to the sequencing of 50 sorted cells identified as Patescibacteria/CPR members. Subsequentially, 27 single amplified genomes (SAGs) of 15 novel and distinct Patescibacteria/CPR members, representing yet unseen species, genera and families could be captured and reconstructed. This phylogenetic distinctness of the recovered SAGs from available metagenome-assembled genomes (MAGs) is accompanied by the finding that these lineages-in whole or in part-have not been accessed by genome-resolved metagenomics of the same sample, thereby emphasizing the importance and opportunities of SCGs.

The Evolutionary Origins of Extreme Halophilic Archaeal Lineages.

Interest and controversy surrounding the evolutionary origins of extremely halophilic Archaea has increased in recent years, due to the discovery and characterization of the Nanohaloarchaea and the Methanonatronarchaeia. Initial attempts in explaining the evolutionary placement of the two new lineages in relation to the classical Halobacteria (also referred to as Haloarchaea) resulted in hypotheses that imply the new groups share a common ancestor with the Haloarchaea. However, more recent analyses have led to a shift: the Nanohaloarchaea have been largely accepted as being a member of the DPANN superphylum, outside of the euryarchaeota; whereas the Methanonatronarchaeia have been placed near the base of the Methanotecta (composed of the class II methanogens, the Halobacteriales, and Archaeoglobales). These opposing hypotheses have far-reaching implications on the concepts of convergent evolution (distantly related groups evolve similar strategies for survival), genome reduction, and gene transfer. In this work, we attempt to resolve these conflicts with phylogenetic and phylogenomic data. We provide a robust taxonomic sampling of Archaeal genomes that spans the Asgardarchaea, TACK Group, euryarchaeota, and the DPANN superphylum. In addition, we assembled draft genomes from seven new representatives of the Nanohaloarchaea from distinct geographic locations. Phylogenies derived from these data imply that the highly conserved ATP synthase catalytic/noncatalytic subunits of Nanohaloarchaea share a sisterhood relationship with the Haloarchaea. We also employ a novel gene family distance clustering strategy which shows this sisterhood relationship is not likely the result of a recent gene transfer. In addition, we present and evaluate data that argue for and against the monophyly of the DPANN superphylum, in particular, the inclusion of the Nanohaloarchaea in DPANN.

Single cell ecogenomics reveals mating types of individual cells and ssDNA viral infections in the smallest photosynthetic eukaryotes.

Planktonic photosynthetic organisms of the class Mamiellophyceae include the smallest eukaryotes (less than 2 µm), are globally distributed and form the basis of coastal marine ecosystems. Eight complete fully annotated 13-22 Mb genomes from three genera, Ostreococcus, Bathycoccus and Micromonas, are available from previously isolated clonal cultured strains and provide an ideal resource to explore the scope and challenges of analysing single cell amplified genomes (SAGs) isolated from a natural environment. We assembled data from 12 SAGs sampled during the Tara Oceans expedition to gain biological insights about their in situ ecology, which might be lost by isolation and strain culture. Although the assembled nuclear genomes were incomplete, they were large enough to infer the mating types of four Ostreococcus SAGs. The systematic occurrence of sequences from the mitochondria and chloroplast, representing less than 3% of the total cell's DNA, intimates that SAGs provide suitable substrates for detection of non-target sequences, such as those of virions. Analysis of the non-Mamiellophyceae assemblies, following filtering out cross-contaminations during the sequencing process, revealed two novel 1.6 and 1.8 kb circular DNA viruses, and the presence of specific Bacterial and Oomycete sequences suggests that these organisms might co-occur with the Mamiellales. This article is part of a discussion meeting issue 'Single cell ecology'.

Optofluidic cell selection from complex microbial communities for single-genome analysis.

Genetic analysis of single cells is emerging as a powerful approach for studies of heterogeneous cell populations. Indeed, the notion of homogeneous cell populations is receding as approaches to resolve genetic and phenotypic variation between single cells are applied throughout the life sciences. A key step in single-cell genomic analysis today is the physical isolation of individual cells from heterogeneous populations, particularly microbial populations, which often exhibit high diversity. Here, we detail the construction and use of instrumentation for optical trapping inside microfluidic devices to select individual cells for analysis by methods including nucleic acid sequencing. This approach has unique advantages for analyses of rare community members, cells with irregular morphologies, small quantity samples, and studies that employ advanced optical microscopy.