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Researchers Report Sequencing Two Dozen Genomes at Marine Field Site

NEW YORK (GenomeWeb) – By modifying the methods and analytical approaches it used, a team of researchers was able to sequence 26 marine microbial genomes and two marine metagenomes while on a research vessel in the Pacific Ocean.

The researchers had previously traveled to the Southern Line Islands, a set of islands in the central Pacific, to study the coral reef there and how the organisms that inhabit it compete for resources. They were, though, stymied by having to collect samples and return to San Diego to analyze them and come up with new hypotheses to test.

"If only we had had that data out in the field, we could have asked those questions there and then," Rob Edwards, a San Diego State University computer scientist and senior author of the PeerJ article describing the researchers' oceanic sequencing approach, said in a statement.

Field areas, particularly isolated ones, present challenges for local genomic sequencing and analysis.

For instance, preventing cross-contamination in a small space such as a ship can be difficult, and connecting to the Internet for computational work is only possible at limited bandwidth and is expensive.

Additionally, Edwards added, "[p]eople are a little bit hesitant to take a half-million-dollar piece of equipment into the middle of the Pacific if you're not sure it's going to be coming back."

For a three-week expedition to five islands in 2013, Edwards and his colleagues did bring an Ion Torrent PGM platform to study the microbes and their role in coral reef ecology in this marine ecosystem.

They collected water samples from various spots near the five different islands to isolate bacteria living there.

But to adapt to the realities of the boat, Edwards and his colleagues had to be creative.

For instance, emulsion PCR using One-Touch was carried out in the laundry room of the ship, located near the ship's hull where the researchers suspected swaying would be at its minimum and have a more limited effect on centrifugation. Additionally, the touch screen on the machine was damaged in transport, necessitating one of the researchers to reverse engineer control of the instrument using the X11 interface and a Linux laptop.

The sequencer itself was housed in a room that doubled as a bedroom for one of the scientists.

Prior to embarking on the expedition, the researchers installed local bioinformatics capabilities on a compute server, though they ran into data corruption issues that they suspected could have been due to the motion of the boat or the uneven power supply there. The server, they noted, went through several compute cycles without a hitch after it returned to San Diego.

"There were numerous challenges to remote DNA sequencing and analysis, however, the end result — genome sequences generated at the remote central Pacific Atolls — allowed us to focus our research on questions relevant to the samples we collected," the researchers wrote in their paper.

Edwards and his colleagues used the Ion Torrent PGM to generate some 1.5 billion bases of high-quality sequence data from three Pseudoalteromonas, one Ruegeria, two Serratia, and 20 Vibrio isolates. The Vibrio isolates included V. harveyi, V. coralliitycus, V. alginolyticus, V. shilonii, and V. cyclitrophicus.

In total, culturing the samples, isolating DNA, constructing the library, and sequencing took five days to complete. Analysis of each quadrant of the sequence chips — the researchers broke chip analysis into quadrants to hasten processing — took five hours while sequence annotation took an hour.

From that annotation, the researchers found some 1,440 orthologous gene groups that were unique to the Vibrio-like genomes and 4,913 orthologous genomes unique to the bacteria that could grow on Zobell media.

They also reported evidence that the microbes living near the Southern Line islands are limited by phosphorous and iron. Based on their genomes, some of the bacteria are predicted to scavenge phosphorus from phosphonate and iron through a number of transporters and siderophores.

Additionally, about half of the microbes could grow on L-serine, converting it to methionine. While the researchers noted that there was no genus-specific preference for growing on L-serine, all those bacteria that could do so contained a specific biochemical pathway that transforms L-serine to methionine via cobalamine.

The researchers claimed that their effort was the first successful attempt to bring next-generation sequencing to a remote field site, and that they were able to use their real-time analyses to uncover unique metabolic processes that may help bacteria survive in the region.

"At the end of the day, we were able to come up with the data we needed," Edwards said. "But when we go back next time, we're going to be better prepared."

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