FEMS Microbiology Reviews, fux009 doi: 10.1093/femsre/fux009 Review Article REVIEW ARTICLE Novel approaches in function-driven single-cell genomics Devin F. R. Doud and Tanja Woyke∗ DOE Joint Genome Institute, Walnut Creek, CA 94598, USA ∗Corresponding author: DOE Joint Genome Institute, 2800 Mitchell Dr., Walnut Creek, CA 94598, USA. Tel: (925) 296-5838; E-mail: [email protected] One sentence summary: This review highlights the use of function-driven single-cell genomics for the targeted recovery of single-cell genomes from uncultivated or uncharacterized microbes implicated with a specific function or phenotype. Editor: Antoine Danchin ABSTRACT Deeper sequencing and improved bioinformatics in conjunction with single-cell and metagenomic approaches continue to illuminate undercharacterized environmental microbial communities. This has propelled the ‘who is there, and what might they be doing’ paradigm to the uncultivated and has already radically changed the topology of the tree of life and provided key insights into the microbial contribution to biogeochemistry. While characterization of ‘who’ based on marker genes can describe a large fraction of the community, answering ‘what are they doing’ remains the elusive pinnacle for microbiology. Function-driven single-cell genomics provides a solution by using a function-based screen to subsample complex microbial communities in a targeted manner for the isolation and genome sequencing of single cells. This enables single-cell sequencing to be focused on cells with specific phenotypic or metabolic characteristics of interest. Recovered genomes are conclusively implicated for both encoding and exhibiting the feature of interest, improving downstream annotation and revealing activity levels within that environment. This emerging approach has already improved our understanding of microbial community functioning and facilitated the experimental analysis of uncharacterized gene product space. Here we provide a comprehensive review of strategies that have been applied for function-driven single-cell genomics and the future directions we envision. Keywords: function-driven sequencing; single-cell genomics; microbial dark matter; single-cell activity INTRODUCTION depends on homology-based computational tools to infer function with varying degrees of ‘homology creep’ (Woyke and Metagenomics has evolved through the need to better character- Jarett 2015), and provides no information regarding the activity ize our world by studying the composition and coding potential of that organism within the environment unless coupled with of complex microbial communities where a majority of the experimental approaches like metatranscriptomics, proteomics diversity is not yet cultivated. Facilitated by next-generation or stable isotope probing. Even when an ‘all of the above’ sequencing platforms, metagenomics can provide incredible approach is taken, the final result is the average of a bulk popu- sequence output from a minimally disturbed complex environ- lation, masking the large variability in functional activity due to mental sample, making it the gold standard for recovering DNA spatial microenvironments or phenotypic noise known to drive sequences from samples of interest. Despite the magnitude of divergent functions even in clonal populations (Ackermann the output, the logistics of metagenome assembly are compli- 2013). Thus, the strength of metagenomics for its indiscriminate cated and many community features can be lost within the capture of all DNA from a heterogeneous sample is also its sequence data. Furthermore, annotating assembled sequences pitfall in that the complex output can confound the recovery of Received: 28 October 2016; Accepted: 21 February 2017 C FEMS 2017. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, pro- vided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact [email protected] 1 2 FEMS Microbiology Reviews complete genomes, obscure genomic population hetero- have previously utilized clone libraries to heterologously ex- geneities (Engel, Stepanauskas and Moran 2014), overlook rare press environment-derived DNA fragments in a gain-of-function organisms (Ainsworth et al. 2015), miss ecological relationships, approach (Daniel 2005). Escherichia coli, the workhorse of het- blend spatially divergent populations and diminish perspective erologous screening approaches, was computationally deter- for the interpretation of recovered sequences of unknown minedtobeabletotranscribe∼40% of genes from well-known function. cultivated lineages of microbes with the typical expression li- Single-cell genomics offers a complimentary approach to brary approach (Gabor, Alkema and Janssen 2004). Consider- metagenomics by capturing and amplifying DNA from a sin- ing downstream incompatibilities such as codon bias (Tuller gle isolated cell, as opposed to many cells in metagenomics, et al. 2010), strategic rare codon utilization (Komar et al. 1999), and is routinely capable of producing partial (Zhang et al. 2006), required metabolite pools, post-translational modification, ac- and even complete (Woyke et al. 2010), non-composite genomes. cessory proteins, apoprotein activation, secretion and even the This approach thus addresses some of the caveats of metage- availability of a compatible read-out assay, it becomes appar- nomics at the sacrifice of throughput. Although technical lim- ent that only a small fraction of functional space is accessible itations and inherent biases currently exist for single-cell ap- through this approach. Furthermore, when introducing more di- proaches (Lasken 2012;Gawad,KohandQuake2016), within vergent DNA sequences, such as those from candidate phyla, the microbiology single-cell genomics has demonstrated itself as a success rate for accessing the vast functional diversity that ex- powerful tool by generating genomes from rare (Martijn et al. ists with current tools is expected to rapidly diminish. Thus, the 2015), symbiotic (Siegl et al. 2011) and previously uncharacter- majority of unknown functions from uncultivated organisms re- ized microbial lineages (Marcy et al. 2007; Blainey et al. 2011; main obscured within their native expression hosts. Rinke et al. 2013). In addition, auxiliary DNA from viruses, or- Due to the increasing recovery and accumulation of se- ganelles, plasmids, transformed DNA and symbionts is also cap- quences of unknown function, a context-driven approach to tured and sequenced along with the host DNA due to its colo- investigate the roles of these genes within their native hosts calization with the cell (Stepanauskas 2015). Thus, associations and environment is required. This will facilitate improved pro- between these genetic elements and the host organism of in- tein annotation, interpretation of microbial networks and un- terest are maintained with high resolution, allowing character- derstanding of microbial influences on biogeochemistry (Hicks ization of subtle ecological interactions (Yoon et al. 2011; Roux and Prather 2014; Woyke and Jarett 2015). Thus, an increased et al. 2014). When coupled with microscopy, single-cell genomics focus on characterizing the functional roles and activity levels enables spatiotemporal characterization of microscale environ- of uncultivated organisms in conjunction with downstream ge- ments, improving the resolution at which our understanding nomic sequencing has motivated the development of methods of microbial ecology takes place (Landry et al. 2013). Single-cell to ascertain both functional trait and/or phenotype, and corre- genomics has identified a novel genetic code (Campbell et al. sponding coding potential from these organisms at the resolu- 2013), uncovered unexplored protein sequence space relevant tion of a single cell. Here we present the approaches and lim- for biotechnology and human medicine (Wilson et al. 2014), and itations of pioneering studies focused on developing a suite of facilitated the expansion of novel branches in the bacterial and methods for the function-driven single-cell identification, iso- archaeal tree of life and improved phylogenetic read anchoring lation and sequencing of uncultivated environmental microbes. for metagenomic data sets (Rinke et al. 2013). With these efforts, Though this review focuses on these approaches from a largely pipelines for the single-cell isolation and sequencing of environ- bacterial and archaeal perspective, many of the same strate- mental microbes have optimized their efficiency and through- gies can be adopted to small environmental eukaryotic cells, and put (Hutchison et al. 2005;Woykeet al. 2011;Rinkeet al. 2014), some are being applied in human cells to address human health thereby advancing single-cell genomics methods to the next issues (Gao et al. 2004;Yuet al. 2011). level. The major outstanding limitation in metagenomic and single-cell approaches is the overreliance on using predicted FUNCTION-DRIVEN APPROACHES protein function as a proxy for functional activity in the envi- Label-free ronment. These approaches lack the ability to discern which re- covered populations are active, potentially overestimating the The earliest ‘targeted’ single-cell screens took advantage of pre- importance of abundant organisms while