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JGI Focuses on New Technology; Moves Beyond Sequencing to Functional Annotation, Synthetic Biology

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NEW YORK (GenomeWeb) – The Department of Energy's Joint Genome Institute continues to test new next-generation sequencing technologies, develop techniques and methods to improve on existing technologies, and aims to move beyond sequencing into functional genomics and synthetic biology, JGI officials said at last week's annual user meeting in Walnut Creek, Calif.

Although next-gen sequencing continues to be a focus of the JGI, last year the institute said it was moving beyond being a sequencing facility and working on technologies at the front and back ends as well as on basic science itself. 

At this year's meeting, JGI officials presented on their advances in generating reference genomes, technology that the center is developing for sample prep and single-cell sequencing, functional genomics studies, and a new program in synthetic biology.

"JGI has and always will be a powerhouse of reference genome sequencing," Chia-Lin Wei, JGI's group lead of sequencing technologies, said in a presentation. And indeed, it expects to generate 120 Tb of sequence data this year, up from around 100 Tb the previous year.

Much of that data comes from de novo assemblies. According to Alicia Clum, genome assembly developer, JGI expects to de novo assemble around 1,000 microbial genomes, 110 fungal genomes, 20 plant genomes, and 580 metagenomes this year.

Nonetheless, said Lin, "we're pushing from genome sequencing to function" by developing technologies to characterize and understand the transcriptome, transcription regulation, epigenetic modification, and metabolomics.

NGS technology

JGI is an early-access user of Oxford Nanopore's MinIon and has been testing it for the last six months. The technology is working, Len Pennacchio, deputy of genomic technologies at JGI, told GenomeWeb, but he declined to provide additional details, citing a confidentiality agreement with the company.

JGI was also an early adopter of Pacific Biosciences' technology, and it now focuses on improving applications for the RS II instrument. It uses its two systems for de novo genome assembly, epigenetic profiling, and sequencing gene transcripts and isoforms.

Wei said using the RS II for de novo assembly has enabled 88 percent of microbial genomes to be assembled in five or fewer contigs. By comparison, with short-read technology, 82 percent of genomes are assembled in more than 20 contigs.

Wei noted that JGI has also been using the technology for epigenetics and sequencing gene transcripts. A feature of PacBio's sequencing technology is that it detects base modifications directly through the system's kinetics — there is a detectable pause at the site of a modification.

In the future, Wei said, as NGS technology matures and sequencing costs continue to decline, "the cutting edge will be shifted from NGS to sample prep." JGI is taking a two-pronged approach to sample prep, she said, working with commercial sources and also developing its own technology through academic collaborations.

For instance, she said, the JGI has been collaborating with Paul Blainey's lab at the Broad Institute and Adam Abate's lab at the University of California, San Francisco on microfluidics technology and droplet-based sample prep, respectively. Blainey previously presented on the microfluidics-based sample prep technology that his lab is developing. Essentially, the group is aiming to make a chip that costs less than $1 and includes nanoliter-sized reactors in which all of the library construction steps take place. At last week's meeting, Soohong Kim at the Broad Institute gave an update on the technology, saying that the research team has shown that the chip is reusable and that libraries can be made and sequenced with 100 pg of Escherichia coli DNA.

Pennacchio said JGI began collaborating with Blainey last fall and has replicated the prototype microfluidics device he developed at the Broad and is now looking to scale it up so it can handle production level library construction in an automated fashion. If successful, "it should save us money and decrease the amount of nucleic acid we need from users," he said.

Single-cell sequencing

Single-cell sequencing continues to be a growing application at JGI. Users requested 1,200 single cell genomes in 2014, up from around 700 in 2013. Pennacchio said that single-cell sequencing is often used to complement broad 16S sequencing and metagenomic sequencing of mixed samples to pick out organisms for further study.

The technology has been improving, he said, and JGI is focusing on technology to enable more multiplexing as well as on technology that would enable a more unbiased approach to identifying the cells in the first place. For instance, he said, using a 16S sequencing approach first is inherently biased because it relies on using primers to amplify the 16S gene, and sometimes the 16S amplification does not work because the organism is too diverse.

One way of getting around that problem is by doing a metagenomic shotgun sequencing approach, but that has to be done deep enough to pick out the rare species.

In addition, he said that JGI is looking to develop microfluidic technologies and droplet-based technologies to help reduce inherent bias in single-cell sequencing from the whole-genome amplification step. A number of studies have shown that reducing the reaction volume and reagents used helps reduce the bias.

Functional genomics and synthetic biology

One of JGI's newest applications as it has evolved beyond being solely a sequencing center has been synthetic biology. DNA synthesis is "exciting and largely unexplored," Pennacchio said.

Yasuro Yoshikuni, program head of the DNA Synthesis Science Program, said in a presentation that JGI expects to synthesize 4 million bases in fiscal year 2015, about half of which will be done through the Community Science Program. Around 1.5 million bases is slated for the three DOE Bioenergy Research Centers — the BioEnergy Science Center, led by the Oak Ridge National Laboratory; the Great Lakes Bioenergy Research Center, led by the University of Wisconsin, Madison; and the Joint BioEnergy Institute, led by Lawrence Berkeley National Laboratory. The final 400,000 bases will go to internal projects at JGI.

JGI put out a call for proposals in the first half of the year for projects wanting up to 250,000 bases of synthesized DNA. In the second half of the year, it will issue a second request for proposals for both independent projects and those done by consortiums. Independent projects should be between 50,000 and 400,000 bases of DNA, while the consortia projects should be a minimum of 50,000 bases, a maximum of 1.2 million bases, and will require three primary investigators from different institutions, Yoshikuni said.

Yoshikuni outlined three initiatives of the DNA synthesis program. The first, microbes-to-biomes, relates to understanding microbes that are relevant to bioenergy, including a Lawrence Berkeley National Laboratories-wide initiative "designed to reveal, decode, and harness microbes relevant to bioenergy microbes," he said. JGI is interested in "working with users that have interesting biological assays in order to characterize plant-microbe and microbe-microbe interactions by secondary metabolites," he said. In addition, JGI is developing tools to create entire microbial strains using DNA synthesis techniques.

A second initiative is its genome to enzymes and pathways initiative, which focuses on "large-scale characterization" of enzymes and pathways, for instance, those involved in carbon fixation or breaking down plant material.

Its third goal is fast-paced metabolic engineering, where it is designing strains that produce chemicals of interest.

"Synthetic biology is critical to bridge gaps" between sequence data and function, Yoshikuni said.

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