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JGI Aims to Move Beyond Sequencing, Become 'Cutting Edge' Genomics Facility

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The Department of Energy's Joint Genome Institute is looking to establish itself as more than just a sequencing service provider, but as a complete genomics facility that offers state-of-the art technologies in experimental data generation and biological data interpretation as well as user interactions to help solve genomic science problems, officials said at last week's annual JGI user meeting in Walnut Creek, Calif.

While next-gen sequencing will continue to play a major role at JGI — the facility expects to generate 80 terabases of sequence data in its fiscal year ending in October and up to 100Tb next year — JGI Director Eddy Rubin said in a presentation that the institute is "aimed at pushing new functional genomic capabilities," rather than being solely a genome sequencing center. As sequencing continues to become cheaper and cheaper, the limiting factor of genomics studies will not be the number of bases, but other components, such as the front end, the analysis, and even the basic science itself, Rubin said.

"We're interested in investing resources into continuing to be at the cutting edge," he said.

Representative of this goal, the institute this year changed the name of its Community Sequencing Program to Community Science Program.

Nonetheless, next-gen sequencing remains a core technology at JGI. It is currently equipped with eight of Illumina's HiSeq systems, five MiSeqs, and two of Pacific Biosciences' RS II machines. Looking ahead, JGI's deputy director of genomic technologies, Len Pennacchio, predicted that the HiSeq machines would be "superseded by the next generation of sequencers" within the next few years.

He cited Illumina's NextSeq 500 and Oxford Nanopore Technologies' MinIon as two up and coming NGS systems that JGI is interested in testing. It has no plans to purchase Illumina's HiSeq X Ten, however, since it does not do any human genome sequencing.

Among JGI's current NGS technologies, Pennacchio said that the biggest improvements have been with the PacBio RS II machine. "Read lengths have gone up tremendously," he said, averaging nearly 10,000 bases. The "long reads make it easier to put assemblies together."

Two years ago, the JGI was using a hybrid method with both the Illumina HiSeq and PacBio RS for de novo assembly, but now for all of its microbial de novo assemblies it is using only the PacBio machine, Pennacchio said.

Hybrid assemblies involved building multiple libraries, including jumping libraries, which "were not easy to make and take a lot of material," Pennacchio said. But now, "because PacBio reads are so long, we only have to build one library."

Aside from microbial genomes, the institute has also been successful in de novo PacBio assembly of fungal genomes, and is starting to move into plant genomes.

The institute is also using the RS machines to look for epigenetic marks in microbial genomes. "We've explored epigenomics in a large panel of microbes" and are currently in the process of summarizing the findings, Pennacchio said.

Single-cell genomics

One area in particular that has really taken off at JGI is single-cell genomics. One JGI project devoted to single-cell genomics is the Microbial Dark Matter Project, and a vast number of users' projects are also in the area of single-cell genomics.

In the first phase of the Microbial Dark Matter Project, researchers collected around 10,000 microbial cells from "places where evidence suggested that there were phyla that had not been sequenced," Rubin said during a presentation. The nine global sites included underwater hydrothermal vents, sediment samples, seawater, and brackish water.

The researchers isolated, whole-genome amplified, and did 16S rRNA sequencing on all the cells. The 16S rRNA sequencing results enabled the team to narrow down the number of whole genomes to sequence to unknown or rare, unculturable species. They chose 201 cells for whole-genome sequencing and published the results of the project last year in Nature.

Rubin said the team is now looking to begin phase two of the project, which will examine samples from 30 sites chose because there is evidence that those sites boast "a lot of funny life."

For its external single-cell sequencing projects, the JGI offers a similar protocol to the one it used in its Microbial Dark Matter project. From an environmental sample, it will isolate and sort single cells and the perform whole-genome amplification and 16S rRNA sequencing, using the MiSeq system, on all those cells. From that collection, users can choose which ones to pursue for whole-genome sequencing.

Because of the increased interest both internally and externally in single-cell sequencing, one of JGI's focuses is in bringing on board and developing technologies that improve on the whole-genome amplification step.

Currently, whole-genome single-cell sequencing yields only about half the genome, Pennacchio said, due primarily to amplification bias.

The JGI has been implementing technologies that enable the whole-genome amplification step to be performed in ever-smaller volumes, which helps reduce that bias. Presently, it is employing Labcyte's Echo Liquid Handler to enable reactions to be performed in smaller volumes and with fewer reagents, which helps reduce the bias. Eventually, Pennacchio said it would likely purchase Fluidigm's microfluidic-based C1 system, which would reduce the volumes, and subsequently, the amplification bias, even further.

Aside from introducing microfluidics to its single-cell sequencing protocol, Pennacchio said the institute is interested in implementing microfluidic technology for sample prep in general.

He said the JGI is interested in Illumina's NeoPrep system, a sample prep system based on digital microfluidic technology it acquired with Advanced Liquid Logic. Illumina plans to launch the system this summer. The NeoPrep is designed for "making libraries with very small amounts of DNA," he said. In addition, it owns several of NuGen's Mondrian SP systems, which also use digital microfluidic technology for sample preparation.

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