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From the Deep

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The ocean teems with life. Some of it is large, like the sperm whale, but other species are much, much smaller, and are often comprised of just one cell. Much of the diversity of marine microorganisms has been out of reach for researchers, as few of the microorganisms living beneath the waves can be easily cultured, leading genomics researchers to turn to metagenomic profiling of ribosomal DNA and, more recently, to sequencing single cells from those microorganisms.

Using a single-cell approach developed by Ramunas Stepanauskas at the Bigelow Laboratory for Ocean Sciences in West Boothbay Harbor, Maine, a group of researchers aims to sequence 60 new marine bacterioplankton in conjunction with the US Department of Energy's Joint Genome Institute. That way, there will be better reference genomes available for researchers studying marine microorganisms, to help them interpret metagenomic, metatranscriptomic, and other community-level data.

"I think that at the end, we will have a much better understanding of how the abundant microorganisms that are driving marine ecosystems actually look like," says Stepanauskas, who is leading this effort. "I think we are going to gain quite a bit of information about their geographic distribution and we may also get first glimpses into the population structure of some of these groups, getting preliminary data about population-level processes like gene exchange and selective pressures."

The approach

The project builds off of a methodology developed by Stepanauskas to peer into the genomes of single cells. "The first challenge was the one that Ramunas and his team faced, which was getting the technology to work in a high-throughput way," says Stephen Giovannoni, a professor at Oregon State University who is part of the project.

In the approach Stepanauskas uses, cells are separated using flow cytometry, and then the genomic DNA from each cell is amplified using multiple displacement amplification. This amplification, an approach pioneered by Roger Lasken, uses phi29 DNA polymerase and random primers to amplify DNA, and it can increase a single genome 10,000-fold in a few hours. The amplified DNA can then be sequenced.

The first complete single cell genome sequenced, reported last year by a JGI-led group, was that of an uncultured single cell of Candidatus Sulcia muelleri. That team used a similar approach to Stepanauskas, employing multiple displacement amplification followed by Illumina and 454 sequencing. That group, however, isolated the cells through micro-manipulation.

Stepanauskas has since ramped up his approach to be high throughput, and has established a single cell genomics center at the Bigelow lab that serves internal and external users. "It's been extremely successful, producing a lot of material, so we have now analyzed over 150,000 single cells," he says, adding that some had their genomes analyzed and others their 16S ribosomal gene.

The group

The approach, Stepanauskas says, is an opportunity for marine researchers to learn more about elusive microorganisms. There are many groups of microorganisms "that have been known to be dominating the oceans and other environments and the driving biogeochemical processes in different environments, but there was no way to access their genomic information. Now we certainly have this access," he says.
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So he contacted other researchers in the field to bring this project together. Stepanauskas' collaborators have been focusing their research on different taxonomic groups. "[I] asked if they would be interested in working together with us and also the Joint Genome Institute to use this material in a more efficient way to create reference genomes, which can be used in many ways," he says. Oregon State's Giovannoni, and a number of others from a variety of institutions, joined up.

Giovannoni's lab studies a particular clade of bacteria, called SAR11. The clade contains the most abundant group of ocean bacteria and Giovannoni's group has five of its genomes already sequenced — but, he says, those sequences don't even begin to cover the diversity found within the group. His lab runs a high-throughput cultivation lab, which he says may house the largest collection of marine oligotrophic bacteria — those that only need a low level of nutrients to survive. "We have a lot of successes, but there are also a lot of parts of marine microbial diversity where we have not been successful culturing cells, so single genome amplification is a very attractive way to study that topic," he says. By looking at the 16S genes from some of the single amplified genomes, he adds, the group to be sequenced includes diversity that his team hasn't been able to sample.

The genomes included in the project were chosen to be representative of what's out there in the ocean, but also to be different from what can be cultured. "We didn't pick anything that is closely related to cultures — but that's not difficult because there are very few cultures of marine micro-organisms, which are clearly representing abundant microorganisms in the ocean. Usually there is a big difference between what you find using culture and better techniques which are more reflective of the real situation out there and what you can actually recover by cultivation," Stepanauskas says.

With advice from Giovannoni and the other collaborators, candidates to be sequenced have been selected from various taxonomic groups, and the amplified genomes are ready to be sent to JGI for sequencing.

The hard part

This sequencing effort is just the beginning, however. The next challenge will be slogging through all the data that JGI returns. One question, Giovannoni says, is what the quality of the amplified genomes will be. "If they are nearly complete, that will be wonderful. If they are very fragmentary, that will be less useful," he says.

Then, Stepanauskas adds, the team has to begin "reconstructing the metabolic pathways and get into bio-geography questions and potentially evolutionary questions," he says.

Giovannoni's group will be focusing on the evolution of the SAR11 group of bacteria, and specifically whether niche specialization has occurred within the group. "We use comparative genomics to identify genes that are shared by all of them and ones that are unique to the different branches to help explain how those branches adapted," he says, adding that this project is "filling in the blanks" of their previous comparative genomics studies.

"For the individual research groups, it's a big boost in the knowledge about these taxonomic groups, something that's been limited to a few cultures so far, and, in some cases that we know, no culture at all," Stepanauskas says. "This will give a much more representative sample of microorganisms within each of these taxonomic groups, so it'll benefit everybody involved."

The Breakdown
Participants: Bigelow Laboratory for Ocean Sciences, Massachusetts Institute of Technology, Oregon State University, University of Georgia, University of New South Wales, and the Institute of Marine Sciences in Barcelona, Spain
Funding: US Department of Energy's Joint Genome Institute
Timeframe: 2010 through 2015

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