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.

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.