1 At the University of Arizona, a leading genomics research facility benefits from decades of BAC- based sequencing expertise, original studies of crop genomes, and a unique emphasis on high molecular weight DNA. For Rod Wing, genome sequencing has come full circle. He was one of the pioneers in building plant bacterial artificial chromosome (BAC) libraries and BAC-based reference genomes in the ’90s, and today that carefully honed expertise in isolating large DNA fragments gives him and his lab a real advantage for making the most of long-read sequencing. As founding director of the Arizona Genomics Institute (AGI) and a professor in the School of Plant Sciences, Ecology & Evolutionary Biology at the University of Arizona, Wing is responsible not only for his own boundary-pushing research in genome biology, but also for the quality of many other projects through his service facility. While Wing’s efforts primarily focus on plant genomes, his PacBio- certified service facility performs genomic studies on a wide variety of organisms for investigators at the university and around the world. Much of his work centers on rice, which Wing sees as a crucial component in solving the challenge of increasing the food supply for a global population expected to reach 9 billion by the year 2050. “Our larger goal is to discover the natural variation ‘hidden’ in landraces and rice wild relatives, and translate that into helping solve the 9 billion people question, which is one of the biggest challenges that faces our society right now,” he says. “Rice will play a key role in that. It already feeds half the planet, and it’s that half that’s going to be expanding in population fastest over the next 30 or 35 years.” To meet his goal of building high-quality reference genomes for every species of rice, 23 in total, Wing chose Single Molecule, Real-Time (SMRT) Sequencing. “The highest-quality genomes out there are BAC-based, and I think that’s going to get even better now with PacBio,” Wing says. “PacBio is revolutionizing our approach for whole-genome shotgun, BAC, and targeted sequencing.” Beyond the highly accurate, long-read sequence data he obtains from SMRT Sequencing, Wing also likes the PacBio® System for full-length transcript isoform sequencing and its ability to characterize the methylome. For his customers, those capabilities are welcome additions to an already highly regarded core facility. BAC Expertise Builds a Strong Foundation Wing began his genomics career in the early days of the Human Genome Project, when the first efforts to sequence whole genomes relied on BAC-based physical maps and laborious but high-quality Sanger sequencing. His experience in that environment gave him a long-term respect for finished reference genomes. AGI BUILDS ON BAC EXPERTISE AND SMRT ® SEQUENCING FOR RICE AND OTHER CROP GENOMES Facility name: Arizona Genomics Institute Institution: University of Arizona Staff size: About a dozen Year founded: 2002 Investigators served: The facility serves scientists around the world, at academic or commercial labs, studying virtually any kind of organism Differentiator: AGI scientists are experts in building BAC libraries and physical maps, as well as isolating extremely high molecular weight DNA PacBio System installed: 2014 Website: www.genome.arizona.edu Email: [email protected]HELPINGSOLVETHE9BILLION-PEOPLEQUESTION ARIZONA GENOMICS INSTITUTE A PacBio Certified Service Provider www.pacb.com/agbio
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At the University of Arizona, a leading genomics research facility benefits from decades of BAC-based sequencing expertise, original studies of crop genomes, and a unique emphasis on high molecular weight DNA.
For Rod Wing, genome sequencing has come full circle. He was one of the pioneers in building plant bacterial artificial chromosome (BAC) libraries and BAC-based reference genomes in the ’90s, and today that carefully honed expertise in isolating large DNA fragments gives him and his lab a real advantage for making the most of long-read sequencing.
As founding director of the Arizona Genomics Institute (AGI) and a professor in the School of Plant Sciences, Ecology & Evolutionary Biology at the University of Arizona, Wing is responsible not only for his own boundary-pushing research in genome
biology, but also for the quality of many other projects through his service facility. While Wing’s efforts primarily focus on plant genomes, his PacBio-certified service facility performs genomic studies on a wide variety of organisms for investigators at the university and around the world.
Much of his work centers on rice, which Wing sees as a crucial component in solving the challenge of increasing the food supply for a global population expected to reach 9 billion by the year 2050. “Our larger goal is to discover the natural variation ‘hidden’ in landraces and rice wild relatives, and translate that into helping solve the 9 billion people question, which is one of the biggest challenges that faces our society right now,” he says. “Rice will play a key role in that. It already feeds half the planet, and it’s that half that’s going to be expanding in population fastest over the next 30 or 35 years.”
To meet his goal of building high-quality reference genomes for every species of rice, 23 in total, Wing chose Single Molecule, Real-Time (SMRT) Sequencing. “The highest-quality genomes out there are BAC-based, and I think that’s going to get even better now with PacBio,” Wing says. “PacBio is revolutionizing our approach for whole-genome shotgun, BAC, and targeted sequencing.”
Beyond the highly accurate, long-read sequence data he obtains from SMRT Sequencing, Wing also likes the PacBio® System for full-length transcript isoform sequencing and its ability to characterize the methylome. For his customers, those capabilities are welcome additions to an already highly regarded core facility.
BAC Expertise Builds a Strong FoundationWing began his genomics career in the early days of the Human Genome Project, when the first efforts to sequence whole genomes relied on BAC-based physical maps and laborious but high-quality Sanger sequencing. His experience in that environment gave him a long-term respect for finished reference genomes.
AGI BUILDS ON BAC EXPERTISE AND SMRT® SEQUENCING FOR RICE AND OTHER CROP GENOMES
Facility name: Arizona Genomics InstituteInstitution: University of ArizonaStaff size: About a dozenYear founded: 2002Investigators served:
The facility serves scientists around the world, at academic or commercial labs, studying virtually any kind of organism
Differentiator: AGI scientists are experts in building BAC libraries and physical maps, as well as isolating extremely high molecular weight DNA
“We were the first lab in the world to develop plant BAC libraries back in 1994,” Wing says. They went on to develop the majority of plant BAC libraries globally, which are now stored in freezers in Tucson and available to investigators who request them. After successfully generating the BACs, Wing’s team built physical maps for rice, maize, and other organisms and conducted a large amount of the sequencing and assembly for the original reference genomes. Since 2002 his lab has been involved in more than 30 genome sequencing projects, both plant and animal.
Wing is concerned about the state of genome sequencing today and its reliance on short-read technologies for de novo genome assembly. “One of the big issues now is there are a lot of genomes being developed, but I don’t really like to call them genomes,” he says. “They’re not the whole genome; they’re full of hot air. This hurts researchers because if you want to do a large investigation of the genome, you need to have the whole genome, not just pieces of it, for the best representation of an organism.”
His own team applies various technologies to ensure that their genome assemblies are as close to finished as possible. “Our emphasis has really been to build high-quality reference genomes. We have always tried to have BAC libraries and physical maps. When we make a genome sequence, we align it to our physical map so we have as much of a representation of the genome as we can get,” Wing says.
Uncovering Crop Genetic Diversity to Feed Nine BillionHigh-quality reference genomes are imperative for the future of research. In Wing’s case, the rice genome assemblies his team is building will be essential to improving rice crops for higher yield, expanded growing areas, and stress tolerance.
“Our goal is to understand the wild relatives of rice because they have a virtually untapped reservoir of genes that can be used for crop improvement,” Wing says. “They have a number of biotic and abiotic stress response traits and genes and can grow in extreme environments — there’s even one strain that can grow in salt water.” Once these species are better characterized, the information can be used with traditional plant crossing schemes to introduce new features to cultivated species of rice.
Wing was a leader of the original rice genome project for Oryza sativa, but the community now understands that having one reference assembly is not sufficient for crop improvement to solve the 9 billion people challenge. “A single reference
genome is really not enough to help capture all the diversity out there,” he says. “You need 20 or 30 high-quality reference genomes because there’s so much variation.” His team is part of a consortium that has assembled 11 of the 23 Oryza genomes for this effort. New rice varieties created from this information should require less water, yield more food, grow on marginal lands, be more nutritious, and reduce greenhouse gas emissions.
The initiative to build so many high-quality reference genomes led Wing to the PacBio platform. His lab, which has adopted every major new sequencing technology since Sanger, tested SMRT Sequencing with 10 rice BACs previously finished with Sanger. “We put those in a pool and sequenced them in a single SMRT Cell,” Wing says. “All 10 BACs recircularized with 99.99 percent accuracy. It was absolutely amazing.”
Wing’s team now routinely uses the PacBio RS II, both for whole-genome and for targeted sequencing projects. In recent work to sequence the African
Rod Wing and his team plan to build high-quality reference genomes for all 23 species of rice, creating new resources for the development of improved varieties. Image by Norma Jean Gargasz/UANews
“PacBio is revolutionizing our approach for whole genome shotgun, BAC,
and targeted sequencing.”
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rice genome, a few highly repetitive and rearranged BACs were particularly challenging to sequence with other platforms. “It was taking us months to try to get through this one region,” Wing recalls. Using SMRT Sequencing, he produced the full sequence of this region “in a nice single piece within a couple of days.” For particularly difficult regions like that one, PacBio is a very powerful targeted approach, he adds. With a few other rice genomes, his team is pooling BACs, 32 at a time, in individual SMRT Cells. “Around 85 to 90 percent of those BACs are completely circularized,” he says. “It’s pretty easy to go through a genome using this approach.”
While he still likes the tried-and-true BAC approach, Wing says the PacBio platform has convinced him to switch to a shotgun approach. “As the read lengths are getting longer, we’re going to use a hybrid approach where we do whole-genome shotgun assembly and then, in regions that are difficult to get through, we use targeted BAC sequencing,” he notes. Working with the International Rice Research Institute, his team aims to build another 20 reference genomes to serve as the foundation for resequencing the institute’s germplasm bank of 125,000 accessions. “PacBio is going to help us do that very quickly,” he says.
“We think this approach is going to give us the best genomes available for long-term analysis,” Wing adds. “We really want to have genomes that are going to be relevant for a long time, without having to be continuously improved.”
Wing is already looking beyond rice DNA to other important biological mechanisms. His team is working with full-length transcript isoform sequencing on the PacBio System and is excited about the potential of whole-genome methylome analyses
from the SMRT Sequencing data. “One of the goals to overcome the 9 billion people question project is to identify the functions of all the genes in rice. One thing we can do now is take rice tissues at several developmental stages and under many different environmental conditions, isolate RNA, and do Iso-Seq™ analysis on these samples to enable whole-plant transcriptome analysis,” Wing says. This could help the community map gene networks and pinpoint the biological mechanisms behind traits such as bigger leaves, water uptake, and more.
He also aims to study ecosystem genomics to better understand the impact of microbes on crop health and productivity. “We want to use the PacBio platform to deeply explore the microbiome of optimal soils in which rice will grow,” Wing says. “The hope is to identify a set of bacterial and fungal genomes that are optimal when paired with certain genotypes of rice.”
Using SMRT Sequencing for All OrganismsWing’s track record of success in genome biology translates to his service facility, where his team uses protocols and strategies honed in their own research efforts. They’ve been at the leading edge of sequencing and assembly since the mid-’90s.
Thanks to this experience, AGI can add considerable expert insight to clients’ projects, from sample prep and sequencing to assembly and annotation. Wing’s team is one of the few with the expertise
to build BAC libraries and physical maps, which can still be important components in generating a high-quality reference genome and in targeted sequencing for larger, more complex genomes. AGI’s service facility accepts any type of organism for sequencing, not just plants.
“Because we’ve had so much experience working with BACs, we’re one of the few labs in the world that really knows how to isolate ‘big’ DNA — very high molecular weight DNA,” Wing says. “Pretty much any molecular biology lab can clone a 5 kb fragment, but can they clone a 150 kb fragment? Probably not.” For example, his team was tapped to provide high molecular weight DNA to the Joint Genome Institute for several genome projects.
That expertise really comes in handy for SMRT Sequencing, which works optimally with very long DNA fragments. “As sequencing read lengths get longer and longer from PacBio, people are going to need that skill, and we already have it,” Wing says.
While his team currently has a heavy focus on rice to demonstrate the power of SMRT Sequencing, Wing says the PacBio platform is useful for customers interested in sequencing any kind of crop, as well as animals and other organisms. “If your genome is littered with repetitive elements that are highly similar, PacBio allows you to get through those elements and back into some more unique sequence for a better assembly,” he notes. “Our facility can work on pretty much any organism — we just have to have some good DNA.”
To learn how the Wing lab’s proven track record can be applied to your research, visit AGI’s website: www.genome.arizona.edu
“We really want to have genomes that are going to be relevant for a long
time, without having to be continuously improved.”
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