Next-gen DNA Sequencers, Changing the Face of Biotechnology
DNA Sequencers, Changing the Face of Biotechnology
P Karandikar, vice president, Applied BioSystems Division, Lab India
Since last couple of years, the rapid progress in several
next-generation sequencing technologies is driving down the cost of
genome sequencing and re-sequencing at an astonishing rate. It is a
writing on the wall that within next two to three years, the era of the
$1,000 genome will arrive. And the personalized medicines and early
prediction of susceptibility to diseases would be a foreseeable reality
in near future.
Major players in next-gen
By virtue of their
technological prowess, experience, and funding, today there are four
prominent players in the next-gen DNA sequencing space that include
Roche (454 Life Sciences), Illumina (Solexa), Applied Biosystems
(Agencourt), and Helicos.
Roche (454 Life
Roche GS20 is based on
pyrosequencing reaction, in which DNA is nebulized into tiny fragments
which are subjected to emulsion polymerase chain reaction (PCR). The
DNA is then attached to micron-sized beads, which are dropped into
octagonal wells etched into micro-titer plates. Then each DNA base (A,
C, T and G) is independently washed over the plate. CCD camera records
each base added by a pyrosequencing reaction.
In 2005, Roche GS20
managed to demonstrate its high throughput capabilities by publishing
‘Bacterial Whole Genome’ data in Nature. This was
the first commercially available next-gen platform in the market. Being
able to sequence just over 100 bases with sub-optimal accuracy, it was
still costlier than the scientist’s expectations. With every
major genome centers craving to acquire first next gene sequencers,
early entry in the market was a huge advantage to Roche with virtually
no competition to GS20!
In early 2007, Roche
introduced their second-generation system, the GS FLX, featuring
several major improvements over its predecessor, including improved
read length up from an average of 100 bases to 250 bases. The new
system also doubled the number of reads per run from 200,000 to just
over 400,000. Most importantly, one system could churn out 100MB data
in less than eight hours with greatly improved accuracy of 99.5
Towards the end of
2008, Roche launched its new titanium series reagents for their
existing ‘GS FLX systems’. The titanium improved
the performance of GS FLX to average throughput of 500MB/Run with the
read length of 400 bases with 99 percent accuracy at the 400th base and
higher for preceding bases. With this improved performance, despite
fierce competition, Roche remains the major player in the field of
next-gen sequencing space. It would be interesting to see how it fares
on important criteria of ‘cost per GB’ in the
presence of other alternatives like Illumina’s GAII and
Applied Biosystems SOLiD ver3.0!
Genome Analyzer (acquired from Solexa in 2006) is based on massively
parallel sequencing of millions of fragments using novel reversible
terminator-based sequencing chemistry. In this system, randomly
fragmented DNA is attached to an optically transparent surface.
Amplification step produces more than 10 million clusters, each
containing approximately 1,000 copies of template per square
centimeter. These templates are sequenced using a four-color
‘DNA sequencing-by-synthesis’ technology that
employs reversible terminators with removable fluorescence. Short
sequence reads of 25-base range were aligned against a reference genome
and genetic variants were highlighted using a unique software pipeline.
These short fragment reads lend the technology to re-sequencing
applications, gene expression, and small RNA analysis.
Although the read lengths were smaller than those of 454’s,
Illumina compensated that by using much smaller beads, producing a
roughly 1,000-fold increase in bead density compared to that of 454.
In early 2007, Solexa
began shipping its ‘groundbreaking’ 1G analyzer
system which can re-sequence one billion bases of human DNA in two
days. It raised lot of hopes to complete human genome at 15X coverage
on a single machine in approximately three months. Many scientists were
pleased with Illumina’s attractive price/GB compared to
454’s at that time.
In early 2009, with the
intention of taking the technology to its limits, Illumina officially
launched its new version of next-gen system GAII. With improvements in
optics, flow cell design and chemistry, the performance of GAII is
greatly improved. At present GAII performance specifications stands at
10GB/Run with read length of 2x75bp with paired end reads at
a price much lower than its earlier variant GI. Illumina has impressive
plans to improve its specifications even further in the years to come.
With the continuous
development in hardware, reagents and down-stream software pipeline
Illumina’s GA II is equipped to run wide range of
applications such as re-sequencing, gene expression, SNP Genotyping
etc. Being a relatively early entrant in the market, Illumina enjoys
largest market share in the next-gen sequencing space so far. With
strong players like AB, Helicos and others coming out with more options
in the market, competition will heat up in near future!
Applied Biosystems (AB)
was the late entrant in the next-gen sequencing market. After careful
due diligence of 40 odd technologies available in the market, in 2006
AB acquired Agencourt Personal Genomics (APG) from Beckman Coulter. By
the end of 2007, it introduced its first next-gen DNA sequencing
platform SOLiD in the market.
‘sequencing by synthesis’ approach, using ligases
enzyme coupled with emulsion PCR, SOLiD is designed with several
technological advantages. Use of ligases facilitates the interrogation
of two bases at a time and reading every base twice leading to 10 times
more accuracy than other DNA sequencing platform presently available in
the market. With the possibility to increase the density of beads per
run, SOLiD is capable of increasing throughput without change in the
hardware. With two completely independent flow cells, SOLiD offers much
needed flexibility to the scientists.
The first version of
SOLiD was introduced with throughput of 3.0GB/Run at 99.95 percent
accuracy and most importantly at the lowest cost/GB.
In 2008, AB introduced
SOLiD ver2.0 in the market with ‘free upgrade’ to
existing users. The upgrade consisted of change in software and
chemistry with no change in hardware. With the introduction of ver2.0,
SOLiD throughput went up from three to six GB/Run. That was the highest
throughput available in the market at that point in time. This created
a positive ripple in the market. No wonder SOLiD system won the
prestigious Life Science Innovation of the Year Award in 2008.
In February 2009, AB
introduced SOLiD ver3.0 with many more advancements both in terms of
automation and unprecedented throughput specifications of 20GB/Run of
mappable data within a week. In short, now SOLiD ver3.0 is equipped to
map the whole human genome with 15X coverage in less than two weeks at
a very reasonable price.
With three major
players constantly improving their product range and more companies
including Helicos eager to introduce their products in near future,
next-gen market space is still in its highly dynamic phase without any
one’s dominance so far.
Indian scientific community is highly enthusiastic about the next-gen
technology available in the market. Running such a state-of-the-art,
highly dynamic next-gen sequencing technology will always be a
challenge for any research institute unless it is supported well by the
suppliers in India. Possibility of having a ‘hands on
demo’ facility along with opportunity to run ‘proof
of concept’ projects would be of great interest to Indian
scientists before finalizing the next-gen projects. Needless to say,
high quality service and application support along with bio-informatics
backup would be the key to successful implementation of next-gen
sequencing systems in India.
Impact of next-gen sequencers
systems on other technologies
systems available in the market can offer 360 degree view of molecular
biology. Therefore arguably, these systems have already raised a big
question mark on the future of microarrays. With the availability of
newer systems to perform hypothesis-neutral evaluation of expressed
genes, it also allows researchers to explore the expression of genes
without having prior knowledge of DNA sequences of RNA transcripts or
genes. When compared to microarrays, the the next-gen sequencers have
the added benefit of being capable of high throughput genotyping. Once
researchers have identified expressed DNA sequences, they can use the
data set from the same experiments to explore mutations and coding
single-nucleotide polymorphisms (SNPs) within these sequences. With
wide variety of high throughput applications, next-gen systems are set
to replace some of the popular technologies like microarrays in near