Jessica Jarett

Minimum information about a single amplified genome (MISAG) and a metagenome-assembled genome (MIMAG) of bacteria and archaea

We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a Metagenome-Assembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Gene Sequence (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.

Differential depth distribution of microbial function and putative symbionts through sediment-hosted aquifers in the deep terrestrial subsurface

An enormous diversity of previously unknown bacteria and archaea has been discovered recently, yet their functional capacities and distributions in the terrestrial subsurface remain uncertain. Here, we continually sampled a CO2-driven geyser (Colorado Plateau, Utah, USA) over its 5-day eruption cycle to test the hypothesis that stratified, sandstone-hosted aquifers sampled over three phases of the eruption cycle have microbial communities that differ both in membership and function. Genome-resolved metagenomics, single-cell genomics and geochemical analyses confirmed this hypothesis and linked microorganisms to groundwater compositions from different depths. Autotrophic Candidatus “Altiarchaeum sp.” and phylogenetically deep-branching nanoarchaea dominate the deepest groundwater. A nanoarchaeon with limited metabolic capacity is inferred to be a potential symbiont of the Ca. “Altiarchaeum”. Candidate Phyla Radiation bacteria are also present in the deepest groundwater and they are relatively abundant in water from intermediate depths. During the recovery phase of the geyser, microaerophilic Fe- and S-oxidizers have high in situ genome replication rates. Autotrophic Sulfurimonas sustained by aerobic sulfide oxidation and with the capacity for N2 fixation dominate the shallow aquifer. Overall, 104 different phylum-level lineages are present in water from these subsurface environments, with uncultivated archaea and bacteria partitioned to the deeper subsurface.Analysis of a CO2-driven geyser over a complete eruption cycle showed temporal changes in microbial community composition and function, associated with eruption phase and aquifer water depth, and revealed a putative archaeal symbiosis.

Function-driven single-cell genomics

Even prior to observing the great plate anomaly, one of the most enduring questions in microbial ecology has been, ‘who is there, and what are they doing?’. While we are still far from getting a true handle on microbial diversity on this planet, the ‘who’ has become easier to address, with the rise of environmental surveys of 16S ribosomal RNA sequences in the 1990s, allowing charting of community structure in seemingly countless ecosystems. The advent of shotgun metagenomics in the early 2000s enabled the latter question to be tackled by providing access to the functional potential of uncultivated microbes (‘what could they be doing?’). Next-generationto be tackled to be tackled sequencing emerged shortly thereafter, and the resulting exponential increase in sequencing throughput opened the floodgates to the cultivation-independent, sequence-based exploration of the microbial world. Transitions from 16S rRNA gene diversity surveys, to metagenomics and more recently single-cell genomics (SCG), were paralleled and to a large extent fuelled by similar technological advancements in sequencing methodologies, enabling megabase-to eventually terabase- scale surveys. Within the cultivation-independent genomics toolkit, SCG has unique advantages, as one can access the functional potential of completely unknown microorganisms without having to rely on accurate assembly and binning methods for metagenome data. One of the primary benefits of SCG is that it enables interrogation of samples at the basic unit of life, which is individual cells, allowing access to all of the nucleic acids and linkage of features like plasmids and cell-contained phages to the genome. This technology has become a standard approach, and single-cell genomes are now routinely sequenced and populate the public databases, just as metagenomes did 10 years ago. Howard Ochmans 2007 Crystal Ball prediction became reality, although isolating the single cells in the morning and obtaining their genome after lunch (‘complete, gap-free and annotated’) will still require further technology development, even 7 years later. Perhaps the continued development of single-molecule sequencing technologies such as single-molecule real-time sequencing and nanopore-based single-molecule sequencing may obviate the need to amplify a single cells genome and eventually allow the direct sequencing of the DNA contained within an individual environmental cell, including its epigenome, thus enabling this ‘after-lunch’ single-cell genome. While all these sequence-based methods have enabled tremendous progress in microbial ecology, they have also inadvertently given rise to an era of homology creep; one of todays key challenges is the slow pace of functional validation, which continues to lag further and further behind the rate of sequencing. Glancing into the future, it seems clear that annotating gene function and phenotype based on experimental evidence will be critical to understand this flood of environmental sequence data. Multi-omics approaches applied to communities encompassing genomics, transcriptomics, proteomics and metabolomics will greatly improve our basic understanding of microbial ecology and assist us in uncovering novel solutions for biotechnological applications. While multi-omics will be one strategy, we envision that these efforts will be heavily complemented with single-cell-based approaches. Thus, what our crystal ball reveals is the union of single-cell sequencing with an array of methodologies that enable functional or phenotypic characterization on a single-cell level prior to sequencing, and the high-throughput implementation of some of these methods. So what exactly might such a future look like? We currently possess and are actively improving the ability to handle single-micrometre scale cells using microfluidics and microdroplets as well as the ability to generate sequencing libraries from sub-nanogram quantities of DNA; these techniques can be combined with new and evolving methods that provide functional information at single-cell resolution and are non-destructive to nucleic acids. Some methodologies for functional screening of single cells already exist, such as Fluorescent Substrate Single Amplified Genome Analysis (FS-SAGA) (Martinez-Garcia et al., 2012). FS-SAGA utilizes fluorescently labelled substrates of interest, such as polysaccharides, which bind to cells in a microbial population, thereby labelling the cells and enabling selective fluorescence-activated cell sorting combined with single-cell sequencing. Raman microspectroscopy can rapidly and non-destructively reveal the composition of specific molecules in a cell providing a molecular fingerprint, and can be combined with SCG, fluorescent in situ hybridization (FISH), isotope labelling and other techniques (Wagner, 2009). Activity-based protein probes can be used to label active enzymes with fluorophores or other molecules (Cravatt et al., 2008), allowing sorting of cells containing an enzyme of interest. While trying to understand microbial life one cell at a time may seem rather absurd today, much like attempting to understand the universe ‘star by star,’ linking phenotypic traits and sequence information on the single-cell level, in conjunction with multi-omics approaches and across different spatial scales, will certainly bring us one step closer to understanding microbial communities and complex microbial systems. Importantly, it will allow us to zoom in on a biological or biogeochemical process of interest and capture the cells that are involved in this process, separating them from their often complex community. The most exciting potential that function-driven SCG has to offer relates to uncovering the ‘unknown unknowns’. An example of such ‘unknown unknowns’ is the recent discovery that the candidate phylum ‘Tectomicrobia’ is involved in the synthesis of bioactive metabolites previously thought to be rather limited in taxonomic range (Wilson et al., 2014). In our view, now and in the next several years to come, function-driven SCG will be another powerful genomics tool along with next-generation sequencing technologies, providing a glimpse into more than ‘just’ the coding potential of the microbial world. Eventually, this synthesis of sequence and functional information will move us from mere description to unifying theories and predictions in microbial ecology, called for by Tom Curtis in his 2007 Crystal Ball.

Rokubacteria: Genomic Giants among the Uncultured Bacterial Phyla

Recent advances in single-cell genomic and metagenomic techniques have facilitated the discovery of numerous previously unknown, deep branches of the tree of life that lack cultured representatives. Many of these candidate phyla are composed of microorganisms with minimalistic, streamlined genomes lacking some core metabolic pathways, which may contribute to their resistance to growth in pure culture. Here we analyzed single-cell genomes and metagenome bins to show that the “Candidate phylum Rokubacteria,” formerly known as SPAM, represents an interesting exception, by having large genomes (6–8 Mbps), high GC content (66–71%), and the potential for a versatile, mixotrophic metabolism. We also observed an unusually high genomic heterogeneity among individual Rokubacteria cells in the studied samples. These features may have contributed to the limited recovery of sequences of this candidate phylum in prior cultivation and metagenomic studies. Our analyses suggest that Rokubacteria are distributed globally in diverse terrestrial ecosystems, including soils, the rhizosphere, volcanic mud, oil wells, aquifers, and the deep subsurface, with no reports from marine environments to date.

Minimum information about a single amplified genome (MISAG) and a metagenome-assembled genome (MIMAG) of bacteria and archaea (vol 35, pg 725, 2017)

Nat. Biotechnol. 35, 725–731 (2017); published online 8 August 2017; corrected after print 29 November 2017; corrected after print 7 December 2017 In the version of this article initially published, the following acknowledgment was omitted: A.L. was supported by the Russian Science Foundation (grantnumber 14-50-00069).

Solagigasbacteria: Lone genomic giants among the uncultured bacterial phyla

Recent advances in single-cell genomic and metagenomic techniques have facilitated the discovery of numerous previously unknown, deep branches of the tree of life that lack cultured representatives. Many of these candidate phyla are composed of microorganisms with minimalistic, streamlined genomes lacking some core metabolic pathways, which may contribute to their resistance to growth in pure culture. Here we analyzed single-cell genomes and metagenome bins to show that the “Candidate phylum SPAM” represents an interesting exception, by having large genomes (6-8 Mbps), high GC content (66%-71%), and the potential for a versatile, mixotrophic metabolism. We also observed an unusually high genomic heterogeneity among individual SPAM cells in the studied samples. These features may have contributed to the limited recovery of sequences of this candidate phylum in prior metagenomic studies. Based on these observations, we propose renaming SPAM to “Candidate phylum Solagigasbacteria”. Current evidence suggests that Solagigasbacteria are distributed globally in diverse terrestrial ecosystems, including soils, the rhizosphere, volcanic mud, oil wells, aquifers and the deep subsurface, with no reports from marine environments to date.