sequencing
Fakrudin B.
sequencing instrument companies will continue to develop more
user-friendly and cheaper technology, focused on the benchtop and
clinical markets. Manufacturers will also continue to form
partnerships and make acquisitions that place heavy bets on completely
novel, potentially disruptive sequencing technologies.
On a worldwide basis, life science research along with drug discovery
and development applications currently comprise the two largest
sequencing markets. They together accounted for about $920 million in
2010 and are expected to reach nearly $1.7 billion in 2015 with a
compound annual growth of 13%.
By far the largest market opportunity, though, is in emerging
applications of personal genomics and clinical diagnostics. These
segments are expected to account for $541 million by 2015 from $15.5
million in 2010, representing a CAGR of 103.5%.
Recent advancements in the field of next-generation sequencing have
resulted in the advent of so-called personal genome machines (PGMs),
smaller-scale, benchtop genome sequencers marketed by Illumina
(MiSeq), Life Technologies (Ion Torrent), and Roche 454 (GS Junior).
PGMs bring DNA sequencing directly into individual laboratories and
will impact the high-throughput sequencing (HTS) market in the
process. Companies will continue to advance these machines, but those
aiming at the clinical space will need to gain regulatory approval for
their use in clinical laboratory diagnostics.
Life Technologies’ Investments
A key disruptive technology introduced to the market in 2010 by Life
Technologies is the Ion Torrent DNA sequencer. It has set a completely
new competitive bar in PGMs. This technology eliminates the need for
optical readout, instead gathering sequence data by directly sensing
hydrogen ions produced by template-directed DNA synthesis.
In less than a year since Life Technologies first commercially
launched its PGM, the semiconductor-based instrument became the
best-selling sequencing machine in the world. The technology provides
low cost and scalable sequencing on a massively parallel
semiconductor-sensing device, or ion chip. Reactions are performed
using all natural nucleotides, and the individual ion-sensitive chips
are disposable and inexpensive. The instrument combines fluidics,
micromachining, and semiconductor technology, allowing direct
translation of genetic information to digital information.
The firm has ambitious plans for the magic sequencing machine. Last
October, the company announced that it will seek FDA 510(k)-clearance
for the Ion PGM™ sequencer in 2012 in order to expand it into the
clinical setting. The company’s R&D plans will also certainly focus on
kits to accompany this PGM, including its AmpliSeq™ Cancer Panel. It
is the first product utilizing Ion AmpliSeq technology and covering
oncogenes and tumor suppressor genes.
Additionally, Life Technologies has placed a big bet on another
potentially disruptive technology, privately held Genia’s biological
nanopore technology. In April 2011, Genia closed a strategic
investment with Life Technologies. The technology comprises an
engineered pore protein embedded in a lipid bilayer membrane.
Single-stranded DNA (ssDNA) with its double-stranded end inside the
vestibule of the nanopore and single-stranded end threaded through the
transmembrane of the nanopore travels through the central pore of the
protein.
As the ssDNA travels through the pore, it attenuates the current
traveling through the membrane in a sequence-dependent manner, each of
the four bases interacting with the nanopore recognition site
differently and partially blocking the ion current by a specific
amount characteristic of that base’s unique electrochemical
interactions with the nanopore recognition site. DNA sequences are
computed from the residual currents flowing through the nanopore/DNA
complex.
Genia co-founder and CEO, Stefan Roever, commented that the platform
can actively control the DNA template, moving it back-and-forth
through the nanopore multiple times if needed. "We can oversample,
rewind, and read again. You change the applied voltage and the DNA
goes backward. If you capture the DNA in the pore, you can ‘dental
floss’ it; you can read it 10–20 times." Roever would not detail the
read-out mechanism, other than to say, "our approach relies on some IT
to reassemble those sequences.
"If Ion Torrent—electrical detection but requiring amplification—and
Pacific Biosciences—single-molecule but optical—are third-generation
sequencing technologies, then we’re fourth generation: single
molecule, electrical detection," said Roever. "That’s the holy grail
because it combines low-cost instruments with simple sample prep. So
we’d like to think of it as last-gen!"
Illumina’s Strategy
Illumina is planning to extend the clinical reach of its MiSeq for
diagnosing infectious diseases. The firm says that through its
November 2011 alliance with Siemens Healthcare Diagnostics, it will
also apply the technology to identify potential treatment paths for
these diseases. The companies said they plan to make existing Siemens
molecular HIV tests compatible with MiSeq.
Illumina, like Life Technologies, has also put a stake in the nanopore
sequencing space through its deal with Oxford Nanopore Technologies.
Oxford is developing its GridION system, which uses nanopores for the
direct, electronic analysis of single molecules including DNA, RNA,
proteins, and other molecules. The company says that its
nanopore-based method obviates the need for amplification or labeling
by detecting a direct electrical signal.
While the company has not disclosed key elements of the system’s
operation, a 2010 paper in Nature Nanotechnology described some
important aspects of the process. The paper was published by Oxford
scientists and their collaborators at the University of California,
Santa Cruz (UCSC).
It describes the passage of ssDNA as it translocates through a protein
nanopore. Movement of the ssDNA was controlled by
polymerase-facilitated replication of individual DNA molecules and
could be initiated under electronic control. Polymerase activity could
be blocked in solution when the ssDNA was not at the nanopore opening,
however, capture of the strand by the pore removes a blocking strand
of nucleotides and allows the polymerase to function on the trapped
strand.
Commenting on the technology, Jay Flatley, Illumina president and CEO
said, "Oxford Nanopore’s technology holds tremendous promise to
achieve the sub-$1,000 human genome. Making electrical measurements of
unmodified DNA removes the need for complex sample prep and the
high-performance optics found in today’s sequencing systems."
Agilent’s Pipeline
Agilent will continue to enhance the efficiency of its next-gen
sequencing technologies and has moved to augment its SureSelect
technology platform. SureSelect is the company’s front-end method for
isolating complex subsets used in targeted resequencing, for example.
SureSelect, the company has explained, replaces other labor-intensive
methods of targeted resequencing such as PCR techniques.
Eric Endicott, Agilent’s global public relations manager, life
sciences group, told GEN that the company’s November 2011 acquisition
of Halo Genomics’ technology will complement Aglient’s SureSelect
target-enrichment platform technology. Halo Genomics’ HaloPlex
technology, Endicott said, provides a high-performance solution for
small capture sizes, at a speed that specifically addresses the needs
of the desktop sequencing market.
In addition to expanding Agilent’s portfolio of solutions for the
rapidly growing next-generation sequencing market, Halo Genomics’
technology further pulls Agilent’s target capture solutions toward the
next-generation clinical sequencing market.
New Kid on the Block
Massachusetts 2010 startup Noblegen says its nanopore-based technology
usually requires complex instruments, but has the potential to deliver
high speed and low costs. Noblegen says its technology’s ability to
directly and rapidly read DNA sequences could make it economically
feasible to bring sequencing technology into clinical labs to diagnose
cancer and other diseases.
Noblegen’s technology works by first converting genomic DNA into a
synthetic version that’s labeled with four different fluorescent dyes,
one for each type of base. Each base in the original sequence is
represented by one fluorescently labeled segment in the synthetic one.
The synthetic sequences are then directly read out by Noblegen’s
relatively simple instrument based on a silicon chip with pores a few
nanometers in diameter and illuminated by an inexpensive laser. The
long, charged synthetic molecules are pulled through the hole by
electrostatic forces. As the DNA moves through the pore one segment at
a time, the labels become detached, creating a flash of light that is
then imaged by a CMOS sensor similar to those used in digital cameras.
NobleGen co-founder and CEO Frank Feist has said that the company’s
goal is to aggressively drive down the cost and increase the speed of
sequencing whole genomes to a point where it makes economic sense for
hospital labs in the next three or four years.
While the company won’t divulge details of its current instrument
prototypes, Feist noted that the technology could be scaled up to
arrays of 400 by 400 nanopores that sequence over 500 gigabases an
hour—or about one genome, covered 30 times, in 15 minutes.
All around, sequencing equipment manufacturers run some financial risk
from spending cutbacks by research labs for this year. But all are
focused on staying ahead of the technology curve through smaller,
faster, and less expensive instrumentation. They are also counting on
the future of clinical sequencing.
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B. Fakrudin, Ph.D.
Department of Biotechnology/IABT
University of Agricultural Sciences, Dharwad
Krishinagar, DHARWAD-580 005
Karnataka, INDIA
Phone: +91-836-2748624 (Direct); 2747627 (O); Mobile:919480369274
email:fak...@yahoo.com
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