We all remember this experiment from freshman biology: Take a sterile swab, rub it along the inside of your cheek, and transfer the collected microbes to a sterile agar plate.
After incubating the plate overnight at 37°C, the agar will be awash in bacterial colonies, a glossy and somewhat smelly testament to the fact that humans, fundamentally, are giant microbial buses.
Yet that picture, it turns out, is woefully incomplete. The human microbial complement, or “microbiome,” comprises “at least 10 times more bacteria than the number of human cells in the body” (1)—trillions upon trillions of cells representing thousands of microbial strains. And yet this is merely the snowflake atop the iceberg that is the global microbial population. Many strains have never even been seen before, and the vast majority, millions perhaps, simply will not grow in the lab. They are, in the parlance of the microbiologists who would study them, “uncultured.”
Uncultured, but not impenetrable.
A decade ago those microbes might have been known only by the arrangement of their 16S ribosomal RNA signatures on a phylogenetic tree. Today these same organisms are being probed in astonishing detail thanks in large part to next-generation DNA sequencing and cell separation technologies—not to mention an ever-expanding database of annotated genes against which to compare them.
It is, says J. Craig Venter Institute professor Roger Lasken, “a fantastic time” to be a microbiologist.The “new” microbiology
Single-cell genomics is the latest in a string of techniques microbiologists have used to delve into the worlds of unculturable microbes. The first approach, “molecular phylogenomics”, was spearheaded by Carl Woese and others in the late 1970s. Relying on PCR-amplified, Sanger-sequenced 16S ribosomal RNA genes to estimate evolutionary relationships among different organisms, the end result was a phylogenetic “tree of life.” Yet this was a tree where most leaves are mere placeholders, stubs that offer no insight into the biology of the organisms they represent, says Paul Blainey, a core faculty member at the Broad Institute in Cambridge, Massachusetts who develops technology to study unculturables.
“We know there's some organism there that we haven't sequenced, but we don't really get much useful information in terms of understanding that organism from these [16S] sequences,” Blainey explains. That's because researchers know what 16S rRNA does—it's involved in protein translation—and that isn't what makes most microbes interesting.
The next step in unculturable microbiology was metagenomics. In metagenomics, environmental samples are shotgun sequenced en masse, detailing the genetic and metabolic potential of samples as a whole — for instance, that something in the population can fix carbon, while another can extract energy from sulfur metabolism. The approach has been the subject of some seriously big science in recent years, from J. Craig Venter's Sorcerer II Global Ocean Sampling Expedition (GOS) to the $157-million NIH funded Human Microbiome Project.
Such studies produce voluminous data, to be sure. Yet metagenomics researchers often struggle to assign specific sequences to particular organisms. The result is a massive collection of DNA fragments, which may or may not belong to any particular microbe —the only way to know for certain is if both an interesting function and an identifying marker, such as a 16S rRNA, exist on the same fragment. Reassembling these sequences is like an archeologist trying to piece together thousands of different pottery shards, without knowing how many objects they represent or even what they looked like whole.
This is where single-cell genomics enters the picture. In single-cell genomics, researchers physically separate cells prior to sequencing. The resulting genomes may not be intact, says Tanja Woyke, microbial genomics program lead at the DOE Joint Genome Institute, but at least all the pieces come from one organism.
The process is simple is theory, complex in practice. Cells are separated and placed in individual reaction chambers, either by flow cytometry, micromanipulation, or microfluidics. They are then lysed to release nucleic acids, which are amplified to produce single amplified genomes, or SAGs. The SAGs are typically screened to identify samples for further analysis by sequencing 16S rRNA or some other gene of interest.