Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
For all the bacterial cultures grown in microbiology laboratories around the world and over the decades, they represent precious few microbial species.
“It has been estimated that <1% of bacterial species have been axenically cultured, and fewer than half of the recognized bacterial phyla include cultivated representatives,” Stephen Quake and colleagues wrote in 2007 [1].
Most microorganisms, if they’re known at all, are known for their 16S ribosomal RNA alone. And that information gap represents a significant problem, as uncultivable organisms—what Quake’s team called “biology’s ‘dark matter’ problem”—almost certainly play key (and previously unrecognized) roles in the environments in which they live. On a more practical level, these cells also represent potential sources of novel enzymatic activities and biotechnologically or pharmaceutically useful molecules, such as antibiotics.
Researchers have devised several strategies for studying uncultivable microbes, including both 16S rRNA sequencing and metagenomics. But there also is another increasingly popular approach: single-cell genomics.
Single-cell genomics is exactly what it sounds like: Researchers isolate individual cells, lyse them and amplify and sequence their genomic DNA. The resulting assemblies are never complete—there’s exactly one DNA molecule in the tube to start with, and any loss or amplification bias essentially removes that sequence from the data—but they can establish which genetic material goes with which organism, information that cannot be obtained by other means.
The trick, of course, is isolating that single cell.
Single-cell isolation strategies
According to Tanja Woyke, microbial genomics program lead at the U.S. Department of Energy (DOE) Joint Genome Institute, researchers have a handful of methods they can use for single-cell isolation, each with its own pluses and minuses.
In his 2007 article, Quake used a custom microfluidics circuit to effect isolation of an uncultivated microbe called TM7. TM7 are rods, and so Quake and his team used their circuit specifically to capture rod-shaped bacteria from an oral microbial community. They then homed in on the desired cells, based on ribosomal RNA. Of 35 isolated rods, four turned out to be TM7, one of which the team sequenced.
The microfluidics approach has two key strengths, Woyke says. One is that users can visualize the cells they are isolating, enabling them to both select specific morphologies or phenotypes and correlate that information with genotype. The other strength relates to amplification efficiency. A single copy of a bacterial genome simply isn’t enough DNA to sequence, so whole-genome amplification is required. But not all segments of the genome amplify equivalently, and some may be lost. “Some studies show that if the reaction volume is smaller, as it is in microfluidics, the single-cell genome recovery is better and coverage more even,” Woyke says.
That said, microfluidics strategies typically are relatively low-throughput, and few labs have access to the equipment and expertise required to use them. One commercial option is the Fluidigm C1™ Single-Cell Auto Prep System, a microfluidics system that can simplify and automate the isolation and preparation of up to 96 single-cell sequencing libraries from mammalian cells.. (According to a company spokesperson, the C1 does “not yet” support bacterial isolation.)
Another approach to single-cell isolation is micromanipulation, in which researchers use a joystick-driven glass capillary to pick individual cells off a slide and transfer them, one by one, to a destination vessel.
Woyke says she occasionally uses micromanipulation in her lab, mostly when dealing with low-complexity samples. In one 2010 paper, she and her colleagues sequenced a bacterial symbiont of the green sharpshooter insect, isolating it from another symbiont based on its unique “strap-like shape” [2].
“That’s the key advantage,” Woyke says. “You can see the cells.”
But micromanipulator throughput also is very low, she says, and the technique itself is tricky, especially if the cells are highly motile or sticky.
Just the FACS
Two rarely used isolation approaches in the context of microbial single-cell genomics are laser-capture microdissection and serial dilution, the latter of which Woyke calls the “least expensive yet least accurate” option.
The most popular strategy is fluorescence-activated cell sorting (FACS). Ramunas Stepanauskas, director of the Bigelow Laboratory Single Cell Genomics Center, uses two FACS machines in his center, a Beckman Coulter MoFlo™ and a BD™ Influx™ from BD Biosciences. “It’s a very robust and high-throughput automated technology that’s been developed and improved for several decades,” he says of cell sorting. “And it allows us to work with thousands of individual cells in a matter of minutes.”
FACS offers two key advantages over other approaches. One is throughput—flow systems can rapidly sort single cells into 384-well plates, a pace that would be impossible to match manually. The other is that FACS can be programmed to select cells matching specific criteria, such as the presence of a particular pigment, nucleic acid or biochemical activity.
“Owing to its high speed and throughput and its ability to separate individual environmental cells on the basis of various cellular properties … FACS has become the preferred method for single-cell isolation in the context of [single-cell genomics],” Woyke, Stepanauskas and colleagues wrote in a 2014 article in Nature Protocols [3].
In 2012, Stepanauskas specifically isolated aquatic bacteria capable of degrading certain polysaccharides by incubating the sample with fluorescently labeled laminarin and xylan and gating on fluorescent cells [4]. The team also enriched for metabolically active cells using 5-cyano-2,3-ditolyltetrazolium chloride, which lights up cells with working electron transport systems.
On the other hand, it isn’t possible in traditional FACS to capture images of the individual cells you’re collecting. And with higher volumes than microfluidics approaches, amplification efficiency post-FACS can suffer, Woyke says. (Her solution to that problem has been to switch from traditional liquid-handling systems to the tipless Labcyte Echo®, which uses acoustic energy to dispense nanoliter volumes and minimize total reaction size.)
According to Robert Balderas, vice president of biological sciences at BD Biosciences, today’s commercial sorters can filter cells using up to 20 parameters, cell size and granularity, plus up to 18 colors.
Any sorter can handle single-cell work, Balderas says, though they differ in terms of capabilities. What matters, he says, are users’ needs. Do they need to isolate narrowly defined cell populations from complex mixtures, requiring more complex sorting strategies and thus more colors? Are the cells to be sorted pathogenic or infectious, in which case an instrument enclosed in a biosafety containment unit might make sense? “The most important thing is the science,” he says.
Keep it clean
According to Woyke, the key to successful single-cell genomics is cleanliness. “Contamination is our major enemy in single-cell genomics,” she says. Given the vanishingly small quantities of DNA being used, even trace contaminants can loom large following amplification, “so having a very clean process end-to-end, with a dedicated [work] area, pipettors and liquid-handling system, helps.”
Other than that, she says, it isn’t difficult—all users need do is “just follow the protocols.” Woyke notes, for instance, that some companies now offer ultrapure kits specifically designed for single-cell genome amplification, which should minimize contamination concerns. (Qiagen’s REPLI-g kit is one such product, and the company has produced a webinar on single-cell amplification using the kit, available here.)
Still, given the complexities, equipment and data-analysis issues involved, Stepanauskas says single-cell genomics neophytes might want to consider outsourcing instead. “You do need extensive, dedicated infrastructure and know-how to get it right,” he says. “I would not recommend somebody doing it all by themselves for a single research project.”
References
[1] Marcy, Y, et al., “Dissecting biological ‘dark matter’ with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth,” PNAS, 104:11889-94, 2007. [PubMed ID: 17620602]
[2] Woyke, T, et al., “One bacterial cell, one complete genome,” PLOS ONE, 5[4]:e10314, 2010. [PubMed ID: 20428247]
[3] Rinke, C, et al., “Obtaining genomes from uncultivated environmental microorganisms using FACS-based single-cell genomics,” Nat Protocols, 9[5]:1038-45, 2014. [PubMed ID: 24722403]
[4] Martinez-Garcia, M, et al., “Capturing single cell genomes of active polysaccharide degraders: An unexpected contribution of Verrucomicrobia,” PLOS ONE, 7[4]:e35314, 2012. [PubMed ID: 22536372]
Image: Courtesy of BD Biosciences.