AGI BUILDS ON BAC EXPERTISE AND SMRT® SEQUENCING
FOR RICE AND OTHER CROP GENOMES
At the University of Arizona, a leading genomics
research facility benefits from decades of BACbased 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
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ARIZONA GENOMICS INSTITUTE
Facility name:
Institution:
Staff size:
Year founded:
Investigators
served:
Arizona Genomics Institute
University of Arizona
About a dozen
2002
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 2014
installed:
Website:
www.genome.arizona.edu
Email:
[email protected]
A PacBio Certified Service Provider
biology, but also for the quality of many other
projects through his service facility. While Wing’s
efforts primarily focus on plant genomes, his PacBiocertified 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.
www.pacb.com/agbio
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“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 shortread 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.
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
“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.
“PacBio is revolutionizing
our approach for whole
genome shotgun, BAC,
and targeted sequencing.”
Uncovering Crop Genetic
Diversity to Feed Nine Billion
High-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.
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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
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-andtrue 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 wholegenome 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 wholeplant 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.
“We really want to have
genomes that are going
to be relevant for a long
time, without having to be
continuously improved.”
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 Organisms
Wing’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 highquality 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
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