From bugs and drugs to embryonic stem cells, high-throughput sequencing is enabling scientists to ask a wide range of new research questions.

Jillian Lokere

Over the last five years, several new DNA-sequencing machines have hit the market, dramatically lowering the time and cost associated with genome-wide sequencing. Whereas traditional machines can read about two million bases per day, newer technology, such as so-called sequencing-by-synthesis or sequencing-by-hybridization marketed by companies like 454 Life Sciences, Illumina, and Complete Genomics, can produce upwards of 300 million bases per day at a mere tenth of the cost.

The new technology was recently used to sequence James Watson’s genome (but not Craig Venter’s), but what can it do for the average laboratory? We talked with three researchers from the Boston area who have incorporated high-speed sequencing into their work. From characterizing large microbial populations to battling antibiotic resistance and understanding the genomic architecture in embryonic stem cells, these groups are just beginning to answer questions that, until now, hung far out of reach.

Fishing for bacterial DNA in seawater

Mitchell Sogin’s laboratory at the Marine Biological Laboratory (MBL) in Woods Hole, MA studies complex microbial populations, like those in seawater, to better understand biodiversity and evolution. The researchers focus on rapidly evolving regions in ribosomal RNA (rRNA) genes, which are unique to each species. By collecting and sequencing many rRNA gene sequences from a microbial population sample, they can determine the relative abundance of each species in that sample. The group is particularly interested in identifying low-abundance species, because these can eventually become dominant in response to environmental or climate change.

Using traditional sequencing, the researchers would have been able to fully sequence only a couple of thousand rRNA genes, says Sogin. “It’s limiting, because then you are really only able to detect the most abundant organisms in your sample, not the more interesting low-abundance species,” says Sogin, director of the Josephine Bay Paul Center for Comparative Molecular Biology and Evolution at MBL. With the new technology, the group has increased its yield to between several hundred thousand and a million sequences per sample. And with that higher yield, they recently showed that the biodiversity in seawater, for example, is at least ten times greater than previously reported.

The group is now exploring other microbial populations, such as waters polluted by fecal matter. Identifying the microbial makeup of polluted water could help scientists determine, for example, the source of the pollution: animal or human. Rather than just looking at a handful of “fecal indicator organisms” for clues, the Sogin laboratory has characterized the entire population of organisms in polluted water, providing a more precise understanding of its origin. “It’s exciting—our precision is just orders of magnitude greater with the new technology,” says Sogin.

Better antibiotic design through fast sequencing

Antibiotic resistance has been a continual challenge, not only for physicians but also for the researchers who study it. “It’s not trivial to figure out what’s happening to the bacterium’s genome to make it resistant to a particular drug,” says Deborah Hung of the Broad Institute and Massachusetts General Hospital. Her lab seeks to identify the changes in bacterial DNA that lead to drug resistance. This information can clue researchers into biochemical pathways important for infection that could be new targets for better antibiotics.

Pinpointing these genetic changes has been difficult with traditional sequencing methods. For example, despite years of work, no one has figured out how the DNA has changed in 30 percent of the drug-resistant strains of tuberculosis-causing bacteria. Sequencing the genome of even one species of bacteria has been a large and expensive undertaking.

Now, with high-throughput sequencing, the Hung laboratory is able to sequence the entire genome of a bacterium very accurately with just a single run, allowing researchers to determine precisely what genetic changes are conferring antibiotic resistance. They are beginning to use the technology to map the genetic changes in antibiotic-resistant strains of the bacteria that cause tuberculosis and cholera. “High-throughput sequencing changes everything,” says Hung. “Figuring out any single DNA mutation has now become trivial.”

How wound up is stem-cell DNA?

Bradley Bernstein’s laboratory at Massachusetts General Hospital is using high-throughput sequencing to study the dense package of DNA, called chromatin, in the cell’s nucleus. Increasing evidence over the last several years suggests that the way chromatin is organized encodes heritable “epigenetic” information that helps guide embryonic development and may also contribute to disease. Bernstein’s group looks at the chromatin in embryonic stem (ES) cells. “Our view is that the initial chromatin state in ES cells really underlies the unique capacity of these cells to form any tissue in the body,” says Bernstein.

To understand chromatin structures, the Bernstein lab focuses on histones: the proteins that bind to DNA and wind it up into chromatin. Certain kinds of histones package up the DNA into very tight structures, while others produce a looser chromatin structure. Genes in the loosely wound-up parts, dubbed “active” chromatin, tend to be more active than the ones in the tightly packaged “inactive” parts. The group is figuring out which kinds of histones are bound to which parts of the genome so that they can predict which genes are active in embryonic stem cells and other cell types, including diseased cells.

To do this, the researchers had been using DNA microarrays, which are very expensive, require a large amount of sample DNA, and do not allow the entire genome to be analyzed.

With the new sequencing technology, however, they have been able to generate highly accurate maps of active and inactive chromatin in embryonic stem cells across the whole genome using just a few nanograms of sample DNA. The next step will be to create chromatin maps of embryonic cells as they develop and specialize to see how the chromatin architecture changes. “It’s a whole new type of genome project. One can now sequence the different cell types in the body, or different disease states, and obtain unprecedented insight into their regulatory state and developmental potential,” says Bernstein.