SCIENCE CHINA

Life Sciences
January 2010 Vol.53 No.1: 44–57
Celebrating Scientia Sinica doi: 10.1007/s11427-010-0023-6
(SCIENCE CHINA)’S the 60th Anniverasry

· REVIEW ·

The next-generation sequencing technology: A technology review

and future perspective
ZHOU XiaoGuang1†*, REN LuFeng1†, LI YunTao2, ZHANG Meng1, YU YuDe2 & YU Jun1*
1
Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100029, China;
2
Institute of Semiconductor, Chinese Academy of Sciences, Beijing 100083, China

Received December 8, 2009; accepted December 16, 2009

As one of the most powerful tools in biomedical research, DNA sequencing not only has been improving its productivity in an
exponential growth rate but also been evolving into a new layout of technological territories toward engineering and physical
disciplines over the past three decades. In this technical review, we look into technical characteristics of the next-gen sequenc-
ers and provide prospective insights into their future development and applications. We envisage that some of the emerging
platforms are capable of supporting the $1000 genome and $100 genome goals if given a few years for technical maturation.
We also suggest that scientists from China should play an active role in this campaign that will have profound impact on both
scientific research and societal healthcare systems.

genomics, DNA sequencing, next generation sequencing technologies, sequencer

Citation: Zhou X G, Ren L F, Li Y T, et al. The next-generation sequencing technology: A technology review and future perspective. Sci China Life Sci, 2010,
53: 44–57, doi: 10.1007/s11427-010-0023-6

1 Introduction propel creation and development of other branches of ge-

nomic studies such as comparative genomics and bioinfor-
matics as well as closely related fields such as systems bi-
DNA sequencing technology has played an essential role in
ology and synthetic biology. In a way, technological ad-
the advancement of molecular biology ever since its inven-
vancement in DNA sequencing has transformed the study of
tion [1]. From early manual sequencing operation developed
fundamental element of life – from individual, localized
by Frederick Sanger, first-generation automated sequencer
genes or fragment of genes to whole genomes, which in turn
driven by Sanger chemistry, to present next-gen sequencing
demands more competent sequencing technology. The syn-
platforms, we have witnessed tremendous changes in this
ergetic relationship between sequencing and its applications
field [2]. Some even liken this change in genomic sequencing
ensures that the trend will continue in foreseeable future and
to the evolution of semiconductor technology [3]. This is not
even accelerate due to the promise of and drive for person-
totally unfounded – the speed of sequencing has improved
alized medicine in disease diagnosis and treatment. Here,
exponentially every few years over the last few decades,
we provide a review of sequencing technology evolution,
similar to what semiconductor industry has experienced
summary of generational advancements with their merits
under the Moore’s law [4]. This rapid transformation is
and drawbacks, and prediction of possible direction of the
captured in Figure 1 and has fundamentally changed the
field. For ease of discussion, we categorize the progress of
way we can examine the blue-print of all life and helps to
sequencing technology into three generations with sub-
† Contributed equally to this work
*Corresponding author (email:junyu@big.ac.cn; joezhou@big.ac.cn)
© Science China Press and Springer-Verlag Berlin Heidelberg 2010 life.scichina.com www.springerlink.com
 Zhou XiaoGuang, et al. Sci China Life Sci January (2010) Vol.53 No.1 45

time, to study in depth the genetic code of life.

The original method was primarily a manual endeavor
and hard to automate. For one, it utilized isotopic radioac-
tive labeling of primer for DNA ladder imaging, making the
sequencing process non-user friendly. The requirement of
four separate chain-termination reactions with dideoxynu-
cleotides (ddNTPs) and subsequent slab-gel based separa-
tion of chain-terminated products on four individual elec-
trophoretic lanes are both time- and reagent-consuming. All
these severely limited the overall throughput of sequencing-
hence, the desire to develop non-radioactive based 1st gen-
eration sequencing technology.

Figure 1 Sequencing technology timeline
generations as illustrated in Table 1. The designation of each
state of advancement is somewhat arbitrary but nevertheless
it captures the key delineation of technological advances of
each period.
2 A review of the technology and its recent de-
velopments
2.1 1st generation technology – fluorescently labeled
sanger method
Before the appearance of first automated DNA sequencing
platform, widely accepted DNA sequencing method of
choice had been the Sanger’s chain-termination method
developed in the mid 1970s, for which Sanger was awarded
the Nobel Chemistry Prize in 1980 [5]. His invention
opened a realm of possibility for researchers, for the first
2.1.1 G1.1
The initial version of 1st generation sequencer first appeared
in mid 80s and developed in Leroy Hood’s laboratory at Cal
Tech [6]. It made possible through modifications to the
Sanger’s method. The key improvement includes the use of
color fluorescent dyes to replace radioactive labeling - four
dideoxynucleotide terminators are tagged with differently
colored fluorophores. Furthermore, the tag is attached to the
terminator molecule (ddNTPs) instead of the primer as in
the case of original Sanger’s method. The color-coded
scheme made it possible to perform all four chain-termina-
tion reactions in one tube. Polyacrylamide gel analysis of
ladder fragments can be performed through computerized
fluorescence detection system. This greatly enhanced the
overall sequencing speed and reduced manual intervention
required by operator during sequencing run.
In the following year, ABI introduced its first semi-
automated DNA sequencing platform, e.g. ABI 370 Se-
quencer, based on the technology from Leroy Hood’s lab [7].
In the subsequent two decades, we had experienced a rapid
change and improvement of its performance. But the under-

Table 1 Roadmap of sequencing technologya)

Generation 1st-G 2nd-G 3rd-G

Version 1.1 1.2 2.1 2.2 2.3 3.1 3.2
ABI/GenoME
Sanger ABI
MS
SBS Illumina
Complete Ge-
SBL ABI/Polonator G.007
nomics
SBP Roche
FD Helicos
Platform SM-SBS Pacific Biosciences/ ?
FE
VisiGen
SM-SBL
SM-SBP
Pore PoC
Nano Nife PoC
Graphene PoC
a) SBS, sequence-by-synthesis; SBL, sequence-by-ligation; SBP: sequence-by- pyrosequencing; SM: single molecule, FD: fixed DNA; FE: fixed en-
zyme, PoC: proof-of-concept; ?: expected technology
46 Zhou XiaoGuang, et al. Sci China Life Sci
pinning working principle has not changed until very re-
cently.
2.2.2 G1.2
Toward the end of last century, the second version of the
1st-generation technology appeared. With it, we see further
enhancement in the speed and quality of DNA sequencing.
This was mainly achieved through improvements in two
areas. First, slab-gel based separation was replaced by cap-
illary-electrophoresis, and second, number of concurrent
samples that can be analyzed was increased through higher
parallelism. The use of capillary instead slab-gel eliminated
sample loading, reduced the reagent consumption and sped
up analysis. Further, the compact form of capillary device
makes it easier to parallelize multiple sequencing runs, re-
sulting in higher instrument throughput; 96 samples on ABI
3730 platform and 384 samples on Amersham MegaBACE
could be achieved in one run. This generation of sequencers
played a pivotal role in DNA sequence production at later
stage of the Human Genome Project and helped to accele-
rate the project completion. They have been continuously
used until this day due to key advantages in its raw data
accuracy and sequence read length.
Through decades of gradual improvement, 1st-generation
sequencer can be applied to achieve sequencing length up to
1000 bp, with raw accuracy as high as 99.999%, at a cost as
little as $0.50/kilobase and throughput close to 600000
bp/day. However impressive those numbers may seem, the
1st-gen technology has reached its pinnacle both in terms of
speed and cost. Reliance on electrophoretic separation has
rendered this approach difficult to further increase analysis
speed, to achieve higher degree of parallelism, and to re-
duce sequencing cost through miniaturization; hence, the
need for development of a completely new generation of
approaches that overcome those limitations.
That said, the 1st generation technology is not going to
disappear any time soon. It will co-exist with next-genera-
Figure 2 Workflow of next-gen sequencing
January (2010) Vol.53 No.1
tions of sequencing platform. The reliability, raw accuracy,
scalability of the tried-and-true method will continue to play
an important role, especially in sequencing PCR products
and clone-ends of plasmids and bacterial artificial chromo-
somes as well as genotyping for STR markers.
2.2 2nd Generation technologies – cyclic array sequenc-
ing by Synthesis
The so-called next generation sequencing methods encom-
pass a myriad of approaches based on different technology.
Although utilized quite diverse techniques and biochemistry
in each step from template library preparation, fragment
amplification, to sequencing, they all adopted a massive
matrix configuration popularized by microarray analysis –
DNA samples on the array are simultaneously analyzed in
parallel. Furthermore, sequencing is carried out by observ-
ing and recording optical events through microscopic
apparatus during iterative sequencing cycles - a serial ex-
tension of primed template by either DNA polymerase [8]
or ligase [9].
Several key characteristics can be easily observed based
on the general description. First, massive parallelism can be
achieved through ordered or disordered array configuration
that offers high degree of information density. Theoretically,
this is only limited by the diffraction limit of light (i.e., half
of the wavelength used for detection of independent optical
events). This dramatically increases the overall throughput
of the sequencing operation. Second, no electrophoresis is
used, resulting in ease of miniaturization and less sam-
ple/reagent consumption over the 1st-generation technology.
2.2.1 Next-gen Sequencer
All next-gen sequencing platforms follow a similar work
flow as outlined in Figure 2 and require clonal amplification
to enhance optical detection for sequencing. Three widely
used next-gen commercial platforms are Illumina Genome
 Zhou XiaoGuang, et al. Sci China Life Sci
Analyzer, Roche 454 Genome Sequencer, and Life Tech-
nologies SOLiD System. They were all invented and de-
veloped towards the end of 1990s and commercialized in
the mid of first decade of the century. A relatively new
comer in this arena is the Polonator G.007, initially devel-
oped in George Church’s lab at Harvard and now manufac-
tured by Dover Systems. Complete Genomics has recently
introduced its sequencing service platform based on its pro-
prietary sequencing technology although it has not indicated
its intention to market this instrument. All of them are util-
ized sequencing-by-synthesis with variation in DNA array
formation, cluster amplification, and enzyme-based se-
quencing biochemistry.
First, DNA template library is constructed. DNA library
fragments are prepared from either randomly sheared ge-
nomic DNA (10 s to 100s bp in size) or alternatively
pair-end fragments with controlled distance distribution.
The double-stranded fragments are ligated with adaptor
sequences at both ends and denatured. The resulting sin-
gle-stranded template library is created and immobilized on
a solid surface, either a planar surface or supporting beads,
and clonally amplified by one of several means, e.g. bridge
PCR [10], emulsion PCR [11], or in situ polonies [12]. DNA
clusters or amplified beads form an array of DNA clusters
on a slide, which then undergo cyclic manipulation through
enzyme such as polymerase or ligase. Optical events gener-
ated from the cyclic chain extension process are monitored
by microscopic detection system, and images recorded
through CCD camera. Sequential analysis of array image
yields DNA fragment sequences, which are assembled into
larger sequence contigs by computer algorithm.
(1) Illumina Genome Analyzer. The amplification of sin-
gle-stranded library fragments is carried out through a
process coined “bridge amplification” [13]. On an oligo-
derived flow-cell surface, consisting eight independent
lanes, single-strand DNAs flanked by asymmetrical adap-
tors form an oligo-bridges from both ends. After multiple
PCR thermal cycles, thousands of copies of DNA, ampli-
cons, based on one single-strand of DNA fragment are cre-
ated and clustered on the surface to a single physical loca-
tion. Millions of such clusters can be produced in each one
of the eight independent flow cell channels (as such, eight
independent libraries can be analyzed in parallel during one
instrument run). A sequencing primer is then hybridized to a
universal sequence in amplicons to start the sequencing run.
Illumina’s GA utilizes sequencing-by-synthesis with
fluorescently labeled nucleotides and reversible terminators.
In each cycle of sequence interrogation, four distinctly la-
beled nucleotides are added simultaneously to the flow cell
channel together with DNA polymerase to give rise to DNA
chain extension according to base pairing rule. Each nucleo-
tide is 3′-OH blocked to prevent further addition. A fluores-
cence image is acquired. The base-unique fluorescence re-
veals the identity of newly incorporated nucleotide for each
cluster and, therefore, sequence of corresponding template
January (2010) Vol.53 No.1 47
DNA. The 3′ end is then unblocked to allow next cycle of
extension to occur. This process repeats multiple times, up
to 50 cycles, to yield DNA read length of 50 bp.
Throughput of the platform can be thousand times higher
than that of the conventional sequencer platform, i.e. 1st-gen
platform. The main drawback is its relative short read length
contributed by optical signal decay and dephasing. Since
optical signal is acquired on each DNA cluster, it is critical
that all strands of DNA in an ensemble grow in unison.
However, each step of sequencing chemistry could fail, e.g.
failing to cleave fluorescent tag and/or remove blocking
group. This leads to some DNA strands extend out of synch
with other strands in the ensemble or fail to extend all to-
gether, causing signal decay or fluorescent signal dephased.
Furthermore, the error rate is accumulative, i.e. increasing
as DNA strand gets longer, limiting the length of sequen-
cing read.
(2) Roche 454 Genome Sequencer. The 454 Sequencer
utilizes emulsion PCR to yield amplicons used for the se-
quencing procedure [14]. Tiny paramagnetic beads coated
with DNA primers are mixed with single-stranded template
DNA library together with components necessary for PCR
reaction. Proportional amount of beads and library frag-
ments are mixed to ensure most beads carry no more than
one ssDNA molecule. The aqueous solution is mixed with
oil to form emulsion where each water compartment forms
an independent micro-reactor for subsequent PCR chemistry.
After multiple rounds of thermo-cycling, each bead is
coated with thousands of copies of DNA of the same se-
quence. Beads are further enriched, transferred, and depos-
ited on a picotiter plate fabricated in organized array of tiny
wells with each hole occupied by only one bead. The pico-
titer plate is engineered as part of flow cell for sequencing
chemistry on one side and bounded with optic fibers as part
of CCD based optical detection system on the other.
The base interrogation operation is sequencing-by-
synthesis that taps into pyrophosphate chemistry to produce
optical signal for detection [15]. The pyosequencing, as
often called, relies on enzymes ATP sulfurylase and
luciferase. Release of pyrophosphate, during nucleotide
triphosphate incorporation into the DNA chain, triggers a
cascade of biochemical reaction via ATP sulfurylase and
luciferase, resulting in a burst of biochemiluminescent light
being emitted. Sequencing is achieved by sequentially in-
troducing each of the four dNTPs into the flow cell. Pres-
ence or absence of light burst of each picotiter well indi-
cates the incorporation or not of corresponding nucleotide
and, therefore, reveals the identity of complementary base
on the template DNA in that well.
Major advantages of pyrosequencing are its speed and
read length-up to 500 bp. Unlike other next-generation
technologies discussed here, pyrosequencing does not need
to carry out extra chemistries to the extending DNA chain
beyond normal biochemical process by DNA polymerase,
e.g . no need of removing label moiety and/or de-block ter-
48 Zhou XiaoGuang, et al. Sci China Life Sci
minator. This reduces the chances of mishap in chemical
reaction and, therefore, less chance for premature chain
termination or out of sync extension which is major cause of
dephasing. However, this asynchronous processing renders
a limitation for pyrosequencing when going through ho-
mopolymer region, e.g. GGGGGG in a row, where no
terminating moiety can stop the extension run. Length of the
homopolymer has to be inferred from optical signal inten-
sity which is prone to error. As a consequence, dominant
error type on this platform is insertion-deletion rather than
substitution. Another drawback for 454 is its relative high
cost for reagents, comparing with other next-gen sequencer,
due to its reliance on a set of enzymes for pyrophosphate
detection chemistry.
(3) Life Technologies SOLiD System. Like in the case of
454, SOLiD system also employs emulsion PCR as a DNA
template amplification scheme with paramagnetic beads.
After breaking the emulsion, amplified beads are collected,
enriched, and fixed on a flat glass substrate to create a dis-
order array.
Its sequencing-by-synthesis is driven by ligation rather
than polymerization as in previous platforms [16]. Further-
more, it employs a dual-base encoding scheme in the proc-
ess to assist error detection. A universal primer complemen-
tary to the adaptor region of template library DNA on the
bead is hybridized. A serial ligation cycles follow - each
ligation occurs between the extending chain and a pool of
fluorescently labeled (at the 8th position) degenerate oc-
tamer probes. The octamer pool is structured such that oli-
gos with bases at the probing positions, e.g. position 1 and 2,
correlate with specific fluorescent color. After ligation, a
fluorescent image is acquired. The octamer is, then, chemi-
cally cleaved between position 5 and 6 to remove the last
three bases together with the fluorescent label. Progressive
rounds of ligation enable interrogation of every 1th and 2th
positions along the extended chain (i.e., 1-2, 6-7, 11-12,
16-17, 21-22, 26-27, and 31-32). After 7 rounds of ligation
cycle, the extended chain is lifted away and system is reset.
A second primer, set back by one base, is annealed to the
adaptor region. This is followed by another 7 rounds of
ligation/interrogation cycles as described above. This en-
ables interrogation of a new set of positions at 0-1, 5-6 …
etc. This process continues with successive reset with
one-base-shortened primers to and followed by ligation cy-
cles until entire sequencing region is covered. This approach
sounds complicated on paper. In reality, it’s all driven by
computerized system and fully automated. Since each base
is measured twice, i.e. in two separate ligation cycles, this
approach has an added advantage of identify miscall during
sequencing [16]. The major limitation is its relatively short
sequence read length, also caused by dephasing in an en-
semble.
(4) Polonator G.007. Polonator is another next-genera-
tion sequencing platform that utilizes sequencing-by-liga-
tion. Its implementation uses single base probe instead of
January (2010) Vol.53 No.1
dual-base encoding approach as described above. Sequenc-
ing assay is carried out through a serial of ligation reactions
between a universal primer and a nanomer probe on emul-
sion PCR amplified DNA cluster [9]. Each pool of nanomer
probes, consisting of degenerate oligonucleotides with
fluorescent label correlating to one query position (i.e. fluo-
rescence color corresponds to the base at interrogation posi-
tion), is successively introduced together with DNA ligase
to carry out primer-probe ligation. After each ligation cycle,
a fluorescent image is taken. The extended primer-probe
chain is then denatured away to reset the system. Ligation
between primer and the second pool of degenerate oligo-
probes for next query position occurs. This reset-ligation-
imagining process repeats until all positions are interro-
gated.
In this system, since no consecutive ligation is required
after each reset, sequencing error is not accumulative. This
is one of the advantages of the system. However, this does
limit the reach of query position from each primer location
and, therefore, result in a shorter read length. This short-
coming can be somewhat mitigated by using multiple an-
chor locations in library sequence to extend the reach. The
Polonator is substantially lower in instrument price than
other commercially available next-generation systems. It is
also an open-source platform, which means it potentially
allows sequencing operation and/or chemistry of the in-
strument to be altered and enhanced by end users.
(5) Complete Genomics. Complete Genomics utilizes
sequencing by ligation approach in much the same way as
the Polonator does. However, it incorporates a unique crea-
tion to increase the density of DNA clusters on slide surface
and to reduce reagent consumption [17]. To increase the
read length, multiple adaptors (four) flanking genomic
fragments are inserted to form a circular DNA template
through sequence manipulation [18]. The template sequence
is then multiplied through circular PCR to make concate-
mers containing two hundred copies of the original template
sequence. The concatemer is folded into a ball structure
called DNA nano-ball (DNB). Each ball then self-assembles
onto a planar substrate surface patterned with sticky (or
activated) spots to form a dense array of DNBs. DNBs do
not stick to areas between activated spot on the slide, leav-
ing an orderly-arranged matrix of DNBs. This provides
much denser array on the surface than those created through
clusters or deposited beads because DNA nano-balls utilize
more effectively three-dimensional spaces. It also eliminates
the use of flow cell as the rest of the next-gen sequencing
machines all do [19].
The sequencing assay is carried out in a similar fashion
as the Polonator, i.e. sequencing by ligation with single
query position – coined as cPAL (combinatorial probe-
anchor ligation). cPAL sequencing uses pools of fluores-
cently labeled degenerate oligo-probes with four distinct
colors correlating to four types of base at a given position.
There is a separate pool for each reading position. A given
 Zhou XiaoGuang, et al. Sci China Life Sci
pool of probes is ligated with anchor according to base
pairing rule at the query position with fluorescent color of
the probe correlating to the query base. After each read,
probe-anchor complex is washed away. Another anchor hy-
bridized and next pool of probes for a different position is
cycled in. The process repeats until all positions are read. A
recent publication from Complete Genomics has shown its
sequence accuracy and cost-effectiveness based on the se-
quences of three human genomes [20].
There is no chaining of consecutive probes as ABI
SOLiD System does. This offers a couple of advantages.
First, there is no memory effect. Any error made in prior
ligation cycle does not carry forward, resulting in better
fault tolerance. Furthermore, ligation yield of each cycle
does not have to be high, reducing the amount of reagents,
e.g. probe and anchor, needed for assay. However, the read
length from each anchor position is still short due to limited
length of oligo-probes (9-mers). The overall read length is
extended by using four separate anchor locations in each
library fragment.
The next-generation sequencing platforms based on se-
quencing by synthesis shown above dramatically increase
the speed and reduce sequencing cost per base over the
1st-generation platforms. It is common to see these plat-
forms to churn out Giga-base of sequence data per day per
instrument at a cost only a fraction of a cent per kilobase.
However, their short sequence reads is an Archille’s heel for
all next-gen platforms except that of Roche 454 Sequencer.
This is mainly attributed to the dephasing problem of opti-
cal signal in DNA sequencing cluster. One solution to rem-
edy this would be to eliminate the ensemble effect all to-
gether – sequencing on single molecule.
2.2.2 Single Molecule Sequencing (SMS)
To overcome one of the major drawbacks of next-gen se-
quencing technology, relatively short read, efforts have been
made to develop single molecule sequencing platforms –
where sequencing by synthesis is performed on an array of
single DNA molecule. Single molecule also helps to in-
crease the number of DNA fragments that can be independ-
ently analyzed in a given surface area and, therefore,
achieves much higher level of throughput. Of course, it also
means no costly cluster amplification step is required, fur-
ther reducing sequencing cost. This, however, introduces a
new set of challenges, mostly in the area of optical signal
detection of single-molecule event. The major issue is to
reduce non-assay-specific fluorescent background interfer-
ence, e.g. free floating fluorescent molecules which are not
participants of the actual chemical reaction. Several ap-
proaches have been implemented in attempt to address this
challenge. The underpinning principle of all is to limit the
volume of detection close to actual site of sequencing reac-
tion, such as through evanescent electromagnetic wave. In
next few sections, we will take a look at a few platforms
that have been developed or are currently under develop-
January (2010) Vol.53 No.1 49
ment.
(1) Helicos HeliScope. The HeliScope Genetic Analysis
System by Helicos Biosciences, based on the work from
Quake’s group [21), is the first single molecule sequencing
system appeared on market very recently. It utilizes se-
quencing by synthesis on single molecule. Constructed sin-
gle-stranded DNA library is disorderly arrayed on a flat
substrate without any amplification. At each cycle of se-
quencing, DNA polymerase and one of four fluorescently
labeled nucleotides are flowed in, resulting in template-
dependent extension of DNA strands. Strands in the array
that have undergone base extension light up by fluorescent
label, which are recorded with CCD camera. After washing,
fluorescent labels on the extended strands are chemically
cleaved and removed. Another cycle of single-base exten-
sion, label-cleave and imaging can begin. As in pyrose-
quencing, each iterative cycle is asynchronous - some
strands in the array may pull ahead, fall behind, or com-
pletely fail to extend all together. Since each strand operates
in independence, there is no dephasing issue to concern.
This does mean, however, homopolymer run could be an
issue as in the case for pyrosequencing. But, unlike Roche
454 platform, single molecule affords us mitigation by
playing trick with enzyme kinetics to slow down the rate of
chain extension so to reduce the chance of two consecutive
base incorporations before dNTP being washed away [22].
As mentioned above, a key challenge with SMS is its
detection. HeliScope utilizes a fluorescent microscopic
technique called the Total Internal Reflection Microscopy
(TIRM) – where only fluorophores within a very thin layer
of reaction volume on the surface of a flow cell can be ex-
cited by evanescent wave to produce fluorescence [23]. This
helps to reduce fluorescent background. But even with the
state-of-art optical system, it is still often a challenge to
capture single-molecule event. Therefore, raw sequencing
accuracy of the platform suffers in comparing with ensem-
ble-detection-based predecessors with dominant error type
being deletion. However, a two-pass strategy can substan-
tially improve the accuracy. Single molecule means we can
now reset the tethered template DNA to its original state by
lifting off the extended chain after one run of sequencing.
This affords us to carry out another sequencing pass in op-
posite direction from distal adaptor, yielding a second se-
quence of the same template. Duplicated sequences can then
be used to average out detection errors and, thus, give rise
to much higher accuracy than single pass.
(2) VisiGen. VisiGen Biotechnologies, now part of Life
Technologies, has also been working on an implementation
of single molecule sequencing by synthesis [24]. In a nut-
shell, they engineered a protein nano-device to observe and
record the DNA synthesis process by DNA polymerase in
real-time. This is achieved through FRET (Fluorescence
Resonance Energy Transfer) between fluorescence donor
and receptor. In FRET, receptor molecule does not emit
fluorescent light when excited except when there is a energy
50 Zhou XiaoGuang, et al. Sci China Life Sci
donor nearby. In their setup, each dNTP is attached with a
fluorescent receptor moiety of distinct color at gamma-
phosphate and DNA polymerase is engineered to carry a
fluorescent donor moiety close to its active site. During
DNA base extension, a matching dNTP is grabbed by DNA
polymerase and brought fluorescent receptor close to the
donor group. Fluorescent energy transfer occurs, giving off
fluorescence of correlating color. Once done, fluorescent
moiety as part of pyrophosphate is released by DNA poly-
merase. This, in essence, creates a short burst of fluores-
cent light in concert with nucleotide incorporation. By
tracking and analyzing sequential light burst, the DNA se-
quence can be constructed. Please note that there is no
pause in the process to remove fluorescent label or to cleave
blocking group - it is a true real-time process. This means it
can be done at tremendous speed, given that optical re-
cording apparatus can keep up. To further reduce back-
ground interference, they also apply TIRM as its fluores-
cence detection setup. Furthermore, unlike Helicos’ plat-
form, this system fixes DNA polymerase on a substrate sur-
face, instead of DNA strand - extending DNA strand grows
untethered. The benefit of immobilized enzyme instead of
DNA comes from keeping nucleotide incorporation event
close to the detection volume as DNA extends, i.e. fluores-
cence not out of sight as DNA chain grows. Although Life
Technologies has not said much about instrument’s per-
formance, we can surmise it is going to be fast and to give
long read length. Theoretically, the read length is only lim-
ited by the processivity of DNA polymerase. In reality, it
might be restricted by other factors such as photo-bleaching
of fluorescence donor molecule attached to DNA poly-
merase. It has been reported that Life Technologies is
working on a kind of quantum-dot fluorescent label to
overcome this problem.
(3) Pacific Biosciences. Pacific Biosciences is another
company that has been working to develop a new genera-
tion of sequencing technology, the SMRT (Single Molecule
Real Time) technology [25]. Its single-molecule sequenc-
ing-by-synthesis relies on a nano-structure called Zero
Mode Waveguide (ZMW) for real-time observation of DNA
polymerization [26]. It consists of thousands upon thou-
sands of sub-wavelength holes, tens of nanometers in di-
ameter, fabricated by perforating a thin metal film supported
by transparent substrate. When illuminated from the side of
glass, light cannot penetrate through the hole because the
dimension of each hole is less than the wavelength of illu-
minating light. This leaves an exponentially-decayed eva-
nescent wave at very bottom of each hole, creating a very
small volume of detection. During sequencing assay, dou-
ble-stranded DNA is synthesized from single-stranded tem-
plate by polymerase planted at the bottom of each
waveguide, one per hole. Each time a base being added,
polymerase locks on a fluorescently labeled dNTP (also
attached to gamma-phosphate position) and brings it to the
detection volume, creating a burst of light. This color-coded
January (2010) Vol.53 No.1
fluorescence burst reveals the identity of the complementary
base on template DNA. By continuously following the
bursts of fluorescence of each waveguide in real-time, se-
quence of template DNA in each hole can be rapidly deter-
mined.
The PacificBio technology has the great potential for
high speed sequencing with long read length and low se-
quencing cost. But, error stem from challenges of real-time
single-molecule detection might put a damper on its per-
formance in raw accuracy as with other SMS platforms.
This can be mitigated through multiple runs after reset on
same samples as described in previous section. Also, current
CCD chip technology has limited the maximum area of si-
multaneously observable ZMWs. Low yield ratio (~30%) of
polymerase-occupied ZMWs also put a limit on the number
of useable waveguides [27]. All these have limited the
higher throughput potential of the SMRT technology. Even
with these limitations, the first version of the instrument,
when introduced in 2010, is promised to have read length of
no less than 1500 bp, at a speed of 15 min per run, and with
reagent cost no more than $60 per run. It is anticipated that
future version of this platform, after those technical issues
are resolved, could churn out 100 gigabases of data per day
with read length up to 100000 bp.
(4) Mobious Nexus I. Other than announcing its intention
to develop a single molecule sequencing platform, Mobious
Biosystems has not said much about the inner workings of
its technology - the Polykinetic Sequencing [28]. This
technology exploits the natural chemical behavior of poly-
merase during DNA synthesis. For a polymerase to incor-
porate nucleotide to a growing DNA chain based on its
template, it needs to test a nucleotide from solution to de-
termine its complementarity. If the nucleotide not a match,
it is released immediately. If it is a match, polymerase will
hold on to it and continue time-consuming steps to add the
nucleotide to growing chain. It is this time difference for
each nucleotide which is exploited in Mobious’s sequenc-
ing-by-synthesis approach. Four nucleotides are sequen-
tially introduced into the reaction volume one at a time. By
measuring the time DNA polymerase (fixed on substrate
surface) hold on to the nucleotide to complete polymeriza-
tion, matching or non-matching nucleotide can be inferred.
Detection methods which can capture polymerase’s con-
formational change, for instance, can be used in this regard.
Fluorescence resonance energy transfer (FRET) with pair of
donor and receptor strategically mounted on DNA poly-
merase can certainly be used for this purpose as VisiGen
does. The key problem with fluorescence, however, is
photo-bleaching of fluorophore. To get around this problem,
Mobious exploits electromagnetic property variation as en-
zyme conformation changes, through plasmon resonance
spectroscopy, nuclear magnetic resonance, etc. [29]. One
added advantage of this approach, besides those with sin-
gle-molecule sequencing, is no fluorescently labeled nu-
cleotides are used - a potential reagent cost reduction.
 Zhou XiaoGuang, et al. Sci China Life Sci
2.3 Generation Technologies – Direct Sequencing
In all abovementioned 2nd-generation technologies, se-
quence is determined by indirect interrogation of nucleo-
base incorporation with either DNA polymerase or DNA
ligase through optical events generated during synthesis,
often assisted by fluorescence or chemiluminescence. Be-
sides requiring expensive optical detection system, large
numbers of optical images have to be recorded, stored, and
analyzed, adding to the complexity and cost of sequencing.
Reliance on biochemical reactions for base interrogation
further adds to the cost of consumables which account for a
substantial fraction of current sequencing expense. Direct
sequence interrogation, where no chemistry is required, is
highly desirable to further reduce sequencing cost. What is
going to happen next is hard to predict with a great certainty
in a field that has been experiencing changes with neck-
breaking speed. But, several areas of recent research with
great amount of activities offer us some hint on potential
technologies from which future generation of sequencing
platform may emerge.
2.3.1 Non-optical Microscopic Imaging
As the saying goes “picture speaks a thousand words”. One
of the most direct ways to determine DNA sequence is to
visualize the nucleotides’ (mostly the bases) linear arrange-
ment in space. If a picture of DNA strand can be taken with
enough resolution to distinguish four bases along a DNA
chain, sequence can be readily read off. This is precisely
what researchers in microscopy community have been at-
tempting to do. The idea is to tap into powerful non-optical
microscopes with resolving power down to atomic level,
such as scanning tunneling microscopy (STM), atomic force
microscopy (AFM), etc [30,31]. With admittedly limited
success so far, progress has been made. Recently, research-
ers from a Japanese group at Osaka University showed that
the scanning tunneling microscope imaging can be use to
distinguish guanine from other three bases with its distinct
electronic fingerprint along a real stretch of DNA molecule
[32]. Other group has been actively working on atomic
force microscope to read off the distinct force required to
pull the tightly fitted ring structure of each base [33]. Yet,
ZS Genetics is working on an electron microscopy directed
approach to sequence DNA. To address insufficient contrast
under electron microscope of natural DNA molecule, they
attempt to use nucleotides labeled with heavier elements to
synthesize a new DNA strand by polymerase from the tem-
plate DNA molecule [34]. The obtained heavier DNA strand
can then be visualized under electron microscope to con-
struct nucleobase sequence.
2.3.2 Nanopore
Another area we have seen flurry of activities is nanopore
structure for DNA sequencing. Nanopore, as its name im-
plies, is a tiny hole with a diameter in the range of nanome-
January (2010) Vol.53 No.1 51
ters (1-2 nanometers), usually made of membrane of
solid-state material or biological molecules with perforated
hole. The idea is that when threading a DNA strand through
the pore, driven electropheritically, one can read off the
bases as they pass through pore opening by some electro-
physical means. Various groups and organizations around
the globe are exploring this idea: Agilent, DNA Electronics,
IBM, NabSys, Oxford Nanopore Technologies, Sequenom,
etc., just name a few.
Two key challenges [35] facing all nanopore-based ap-
proaches are (i) distinguish four nucleotides in time com-
mensurate with the travel rate of passing DNA strand, (ii)
control the speed of DNA translocation through the pore.
Initial attempts to measure the fluctuation of ionic current -
variation in electric potentials across the nanopore upon
blockage - as the single-stranded DNA passes through the
hole have so far yielded little success. Calculation and ex-
periment have shown measurement of ionic conductivity
across the nanopore alone is unlikely to provide the required
resolution to discern each nucleotide in DNA molecule [36].
The channel length of nanopore is usually more than 5 nm,
spanning over a dozen nucleobases which is too long to
offer single base current resolution needed for sequencing.
Even though the ionic current measurement is not dis-
tinguishable for individual base, it can readily discern sin-
gle-stranded vs. double-stranded DNA [37]. NABsys in
collaboration with a research group at Brown University
exploited this capability and is developing a sequencing-by-
hybridization technology [38] - the Hybridization Assisted
Nanopore Sequencing (HANS). Genomic DNA is ran-
domly cut to fragments of about 100 kb in length, made
single-stranded and hybridized with 6-mer oligonucleotide
probe. Genomic library fragments bound with probes are
then driven through an addressable nanopore array device.
Ionic current across each pore is independently measured to
create a current tracing that shows the precise positions of
hybridized probes on each genomic fragment. Overlapping
probe regions of genomic fragments are used to align frag-
ment library to create a probe map of the genome. This is
repeated for all hybridization probes in turn, creating a
complete set of probe maps of the genome. Using computer
algorithm, the entire genomic DNA sequence can then be
constructed. The precision and consistency of this
nanopore-based measurement of hybridized location, how-
ever, still need to be demonstrated.
To enhance the sensitivity of nanopore to reveal features
of nucleobases, research groups are working on other ap-
proaches, including embedded electrical probes inside
nanopore structure [39]. They hope that the pair of tunnel-
ing electrodes abutted on opposite side of the nanopore can
register characteristic tunneling current as each base being
driven through pore. Both computer simulation and experi-
ence with scanning tunneling microscopy which has been
successfully used to reveal atomic-scale features give us
optimism for this to work. But fabricating a nano-device of
52 Zhou XiaoGuang, et al. Sci China Life Sci
this scale is not a walk in the park. Another creative solution
to the problem is to develop chemically functionalized
nanopore. Instead of embedding solid-state electrodes,
Lindsay and colleagues [40] have proposed to use two
chemical probes to form hydrogen bonds with phosphate
group (the grabber) and base moiety (the base reader) re-
spectively, as nucleobase pass through. Four different reader
probes would be required for four nucleotides in this ap-
proach.
Yet, other groups are working on alternatives of solid-
state nanopore - biopore. One group uses engineered MspA
protein to construct bio-nanopore for analysis of ssDNA [41].
They demonstrate that ssDNA can be threaded through this
biopore, a potential for single molecule sequencing. Oxford
Nanopore Technologies, collaborating with University of
Oxford, is working on another protein engineered nanopore.
Through genetic engineering, they have been able to con-
struct a biochemical nanopore by covalently attaching the
aminocyclodextrin adaptor within the α-hemolysin pore in a
lipid-bilayer membrane [42]. Recently, they demonstrated
that by driving four nucleotide monophosphates (dNMPs)
through the pore the ionic current is reduced to one of four
levels, each of which correlates to one nucleotide [43].
Coupling this discovery with successive release of nucleo-
tide from DNA chain, which can be achieved through ex-
onuclease, offers us another potential nanopore-based single
molecule sequencing technology. To make it happen, it is
important to demonstrate the exonuclease moiety can be
mounted in a way to ensure delivery of released nucleotide
monophosphates into and through the pore in strict sin-
gle-file.
Aside from base detection, controlling the motion and
speed of DNA translocation through a nanopore is also im-
portant and challenging. The speedy translocation of DNA
through a nanopore holds the promise for ultra-fast se-
quencing. But, if the DNA strand passes through the pore
too fast, it might give little time for each base to be accu-
rately determined. The situation is made worse by stochastic
motion of DNA molecule and non-specific interaction of
bases with the nanopore surface [44]. All these add to the
uncertainty to the rate of DNA molecule translocating
through the nanopore. Although the travel speed of DNA
can be reduced by lowering temperature, increasing solvent
viscosity, and decreasing potential bias across nanapore,
variation of velocity is still problematic. There have been
various attempts and proposals to overcome this difficulty.
One such idea [45] is to tap into processive enzyme of some
sort to bind with traversing DNA strand; this should sub-
stantially reduce the rate of translocation. More recently, a
group at IBM announced their idea based on a nanopore-
device they termed DNA transistor [46] – a nanopore em-
bedded with metal layers, forming metal-dielectric structure
which can be modulated to trap DNA molecule inside the
nanopore. By cyclically turning the gate potential on and off,
their computer simulation result shows that it would be pos-
January (2010) Vol.53 No.1
sible to move DNA molecule one base at a time through the
nanopore. This would give them ample time to interrogate
each passing nucleobase.
2.3.3 Graphene and Carbon Nanotube
Graphene is a single layer carbon arranged in a sheet. It is
very strong and has great electrical conductivity, a very
good candidate for making electrode used for nucleobase
interrogation. One idea [47] that has been proposed is to
create a gap, about 1 nm wide, on the graphene sheet. DNA
molecule is guided vertically through the gap. As the DNA
passes through the gap, two edges of the graphene sheet can
act as electrodes to interrogate nucleobase for sequencing.
Besides challenge for making such a small gap on a gra-
phene sheet, controlling the orientation, motion, and speed
of DNA translocation through the gap is also an issue.
Carbon nanotube (CNT) has been promised to have great
potential to play an important role in rapid DNA sequencing
because of its unique electro-physical properties and
nanometric dimension, albeit no working device has yet
been fabricated so far. It has been shown that surface of
CNT is highly interactive with DNA molecule, even in se-
quence specific way [48]. Long genomic ssDNA can wrap
around a single-walled CNT to form a tight, stable DNA-
CNT complex [49]. Computer simulation has demonstrated
that four types of nucleotides introduce distinct characteris-
tic features in the local density of states [50], making CNT a
good candidate for the development of electronic sequenc-
ing strategy on its own or in combination with other tech-
nology. Most of those ideas are still in proof- of-concept
phase.
3 Future Perspectives
After providing the overview of sequencing technology
development over three decades and potential new break-
throughs that may follow, it begs the question of what is
going to happen in the years to come. What is the outlook of
technical parameters of sequencing, where is the thousand
or the hundred dollar genomes ($1000 genome or TDG and
$100 genome or HDG; the two goals assume the total cost
for sequencing a human genome and the way to analyze it is
reference-based) technology going to come from, and when
will it happen? In this section, we will try to provide our
answers to some of these questions.
3.1 Technology Convergence
We do not know for sure which game changing idea or dis-
ruptive technology would ultimately bring us to the promise
land of the $1000 genome or the eventual $100 genome.
One thing is strikingly clear when we look at the
technological progression of three generations of sequenc-
ing platforms – the marriage of solid-state technology and
 Zhou XiaoGuang, et al. Sci China Life Sci January (2010) Vol.53 No.1 53

biochemistry. And furthermore, technological convergence and processivity, respectively, of DNA polymerase. Future
is shifting toward physical interrogation from biochemical generation of sequencing technology based on nanopore has
or chemical approaches as illustrated Table 2. We believe also been promised to achieve long read as nucleotides are
this trend will continue and future generation of sequencing determined, one by one, while DNA threads through the
technology could eliminate biochemistry all together; nanopore structure. In principle, the length of DNA that can
nanotechnology will play a bigger role. be read in a single swoop is only limited by the practicali-
ties of threading a very long DNA strand through the pore
3.2 Sequencing Throughput and Read Length without shearing. It has been demonstrated that ssDNA up to
5.4-kb can be threaded through a solid-state nanopore [37].
Throughput and read length have been a dichotomy for se- It can, therefore, readily anticipate that newer generation of
quencing platform selection. From the first-generation to the sequencers will be capable of super-high throughput with
next-gen sequencing technology, we have seen a dra- read length surpassing Sanger instruments.
matic improvement in sequencing throughput but at the
suffering of read length. As stated in previous sections, this
is primarily the result of out-of-synch sequencing chemistry 3.3 Sequencing Cost and Productivity-the Experience
for extending DNA strands in an ensemble - the dephasing Curve
effect. Users are left with a choice between sequencing
platforms of longer read-length but low-throughput, i.e. the Over last three decades, we have seen the cost of sequenc-
first-gen, and that of short read and high throughput, i.e. the ing dropped precipitously while sequencing throughput (or
next-gen. The situation will change with single molecule productivity) increased exponentially. Some has likened
sequencing, e.g. PacBio’s ZMW technology, which prom- the dramatic change to that of IT industry as abovemen-
ises super-fast throughput with multi-kilobase read length. tioned. In fact, the speed of change in DNA sequencing has
Assuming uninhibited optical detection capability, through- beaten the Moore’s law of semiconductor industry in certain
put and read length are only limited by the synthesis rate aspect. Figure 3 shows plots of change over the years in log
scale for sequencing cost (in US dollar per human genome)
and productivity (in nucleotides per day per instrument).
Table 2 Technology convergence
A couple of observations can be made in these plots. First,
Key technology 1st Gen
2.1-2.2th 2.3th
3rd Gen
as expected, cost of sequencing and throughput has fol-
Gen Gen lowed pretty much exponential drop and uptake, respec-
DNA hybridization √ √
tively, over three decades. Closer examination reveals an-
DNA polymerase √ √ √
other interesting point - the slope of both curves increased
PCR √ √
Electrophoresis √
with an inflection point right around the year of 2005,
Optoelectronics √ √ √ meaning the rate of cost reduction and throughput en-
Microfluidics √ √ hancement has accelerated since then. This is the time when
Micro/Nano-fab √ √ √ next-gen sequencing platforms started to make their ways
Single molecular
√ √
into laboratories. The near linear slopes on both sides of the
detection inflection point reflect the two distinct drivers for sequenc-

Figure 3 Sequencing cost and productivity

54 Zhou XiaoGuang, et al. Sci China Life Sci
ing production improvement: one being the productivity
increase and cost reduction within a technological genera-
tion and the other propelled by technological breakthrough.
Productivity and cost relationship can be modeled by
experience curve (aka the learning curve) effect. This effect
states that the more often a task is performed the lower will
be the cost of doing it. It was first successfully used by
Theodore Wright in mid of 1930s to quantify and project
decreases in cost as a function of increased airplane produc-
tion [51]. Since then, the model has been used to study pro-
ductivity and cost relation in many industries. If we plot the
sequencing cost since 2005 in log scale against sequencing
throughput (in log scale) over the same period, we obtain
the experience curve for the 2nd-gen sequencing technology
as shown in Figure 4. The linearity of the plot is a typical
experience curve effect – as productivity increases by a cer-
tain fold, the cost of doing it reduces by a certain percentage.
Occasionally the linearity of experience curve can stop
abruptly. The discontinuity reflects the obsolete technology
or process that has been replaced by newer one. This is what
we have observed between 1st-gen and 2nd-gen technologies
right around 2005 as previously discussed. In our current
generation, i.e. 2nd-gen, we have not seen the discontinuity
point yet. So far, an order of magnitude increase in se-
quencing throughput translates into 1.8 orders of magnitude
in sequencing cost reduction since 2005.
3.4 Thousand Dollar Genome (TDG) and Hundred
Dollar Genome (HDG)
Extending the trend of our experience curve as shown in
Figure 4, we can conclude that the TDG and the HDG goal
might be reached when we can produce genome sequences
at a rate of 20 Gb per day and 70 Gb per day per instrument,
respectively, based on the 2nd-generation technologies (in-
cluding SMS currently under development). For the TDG, it
could happen pretty soon, within a year or two, if current
trend remains. For the HDG, it might happen in two to three
years based on the same model.
One big caveat, of course, is that all these predictions are
based on our experience curve model. As we already know,
there are other factors that experience curve cannot predict
Figure 4 Experience curve of 2nd-generation
January (2010) Vol.53 No.1
such as disruptive technological breakthrough or lack of it.
If we hit a wall with current technology before reaching the
goals, all bets are off. Current model we use to make our
projection will become obsolete and a new one based on
replacing technology will have to be established. If hap-
pened, how long that is going to take is everybody’s guess.
But one thing is more certain – we should know the answer
in a year or two. Based on current trajectory of the experi-
ence curve, we project the TDG could be attainable within
2nd-gen technology based on cyclic array sequencing by
synthesis. This could come in a year or two. But for the
HDG, it is harder to project because current generation of
technology, even with its newer SMS, might not be techno-
logically sufficient to lower the cost to $100 dollar. If that is
the case, we may have to wait until a newer generation of
disruptive technology comes along, e.g. the 3rd-generation.
3.5 Cost of Sequencing to Customer
Are we really getting there this quickly? Are we really going
to see $1000 customer cost for human genome sequencing?
Not so fast. All recent sequencing cost data we rely our
analysis on are based on consumable or reagent cost in most
part. Even with the same instrument platform, a surprisingly
wide range of cost estimates can be generated [52]. One
frequently under-appreciated cost is the downstream infor-
matics analysis. All too often, people forget to include the
time-consuming and human-intensive analysis of sequenc-
ing data generated by instrument to give rise a well-
annotated genome data in the cost estimate or procla-mation.
If one factors in all the associated costs to generate quality
human genome data, e.g. consumables, machine amortiza-
tion, maintenance, labor, and computation, real cost could
run up many times higher than those numbers.
Besides the real cost of sequencing, we also have to con-
sider the market force and include business operation cost.
Even if we can get sequencing cost down to a thousand dol-
lar range, initial market demands of such service will prob-
ably keep the price higher than that – simple market supply
and demand. Adding all these up implies that we may not
see the $1000 genome in real sense of the word for some
time soon in the very near future.
The real cost structure currently is that the reagent cost is
artificially high since both library construction and se-
quencing reaction reagents are fully controlled by the com-
panies who provide the instruments. Until serious competi-
tors who challenge the reagent monopoly enter the market-
place, the situation is not going to change in favor of the
customers. Therefore, the customers’ job is to choose their
resources for sequencing with priority, starting from the top
of the to-be-done list, rather than to jump on the bandwagon
right away.
3.6 Sequencing Operation Model
Another factor we need to consider on the issue of sequenc-
 Zhou XiaoGuang, et al. Sci China Life Sci January (2010) Vol.53 No.1 55

ing cost is economies of scale. In a way, this is factored in
by the experience curve effect abovementioned - the more
one does something the less costly it will become. This
raises another interesting question: which direction of the
sequencing business is going to go, production-oriented
center or distributed sequencing activity? We predict the
market needs will diversify the operation models.
There are two operational models for DNA sequencing:
as a routine laboratory technique or a high-throughput op-
eration. The formal has been obviously observed for capill-
ary-electrophoresis -based machines, such as ABI-3730 XL,
which are necessary for sequencing limited amount samples
and/or for special tasks. It is predictable that the current
next-gen sequencers will move to such a niche-based opera-
tion. For instance, we will soon see scaled-down versions of
the current machines, such as those of Roche/454 and Illu-
mina GA in the market, which will be used for small-scale
operations to satisfy individual labs’ needs that start from as
small as a few bacterial genomes in a single machine run to
as large as a human genome in a few runs. The large-scale
machines will be pushed to the capability of acquiring
20x-coverage sequence data for a haploid human genome,
equivalent to 100-150 Gb per run.
We believe that most of future large-scale DNA se-
quencing activities will shift to large commercial service
centers or providers rather than being done in individual
labs or even small institutions. As sequencing cost drops to
a level below $1000 per genome, if current promise holds
true, it becomes harder to justify the purchase of a half mil-
lion dollar instrument (assuming instrument price stays the
same as of now – a reasonable assumption based on what
we have experienced so far). To quickly recuperate the in-
strument cost and reach break-even point, it would require
large enough volume of sequencing jobs. Service-oriented
sequencing provider of efficient operation with large cus-
tomer base would certainly have the financial advantage.
Simply put it, it is the economies of scale. This is, in a way,
similar to the changes that we have seen in the IT industry –
large Web hosting service providers have taken over the
responsibility of hosting web sites for many organizations,
large or small. Genomic sequencing service providers of
such kind have already sprouted up with companies such as
Agencourt Bioscience, Cofactor Genomics, Complete Ge-
nomics, Knome, SeqWright, etc., just name a few. This
trend will continue. But eventually, many of them will con-
solidate into a few larger providers as the market becomes
mature.
3.7 Technology Coexistence
Generations of sequencing technology are not mutually ex-
clusive – emergence of a newer generation technology does
not mean older platforms become obsolete completely. They
often coexist due to complementary functionality of differ-
ent generations as illustrated in Table 3. A case in point, the
next-gen platform, which provided much higher throughput,
has not completely replaced the 1st-gen technology based on
Sanger’s method. Advantage of read length and raw se-
quencing accuracy attainable through Sanger’s method still
finds it a niche in small-scale sequencing projects and/or
provide complement to the next-gen systems in large scale
project.
3.8 What China Should Do?
Fierce competitions in developing newer generation of se-
quencing technologies among organizations in highly de-
veloped countries and regions, such as the US and EU, is
one of many indicators for its importance. For China, it is
still a great challenge to develop technology of this sophis-
tication. But, it is also a great opportunity.
Traditionally, China has been lacking financial support
and technical know-how to develop sophisticated analytical
instrument such as newer generation of DNA sequencer.
Most analytical equipments that have so far been developed
in China are limited to basic laboratory products such as
centrifuge, shaker, and power supply. For high-end analyti-
cal needs such as high-throughput DNA sequencing, all the

Table 3 Functionality of sequencing technologya)

Functionality
Technology Definition De novo
PCR Seq Re-Seq GT 1000/100
WGS CBC
1.1 Slab Gel + + + + + NA
1st-G
1.2 Cap-4color +++ ++ ++ ++ ++ NA
LR NA +++ NA ++ NA NA
2.1 emPCR
SR NA ++ NA +++ NA NA
2nd-G
2.2 HT/No Flow Cell NA +++ NA +++ NA NA
2.3 Dingle Molecule NA +++ NA +++ + 1000
3.1 Chem/Nanotech NA +++ NA +++ + 1000
3rd-G 3.2 Nanotech NA +++ NA +++ + 100
3.3 Nanotech NA +++ NA +++ + 100
a) WGS: short reads; LR, long reads; GT: genotyping, 1000/100: per genome costs; WGS, whole genome shotgun; CBC, clone; HT, high throughput; SM,
single molecule
56 Zhou XiaoGuang, et al. Sci China Life Sci
equipments are imported. This is understandable due to
China’s state of economy and technology in the past.
To develop an advanced analytical platform of this kind,
one needs technical and engineering infrastructure, collabo-
ration, and integration of multiple disciplines, e.g. biologi-
cal chemistry, semiconductor, electronic engineering, me-
chanical engineering, computing, etc. It also requires a huge
public or private investment organizationally and financially
in such an endeavor. But the potential benefit is huge.
First, through the development of an analytical system of
this kind, Chinese scientists and engineers alike not only
can contribute to human efforts in deciphering the secret
code of life but also can learn and acquire the technical and
organizational skills needed for such effort. Second, beyond
advancing technological, engineering, and scientific know-
how, it has huge economical potential as well. Advanced
genomic sequencing technology opens the door for person-
alized medicine. A corollary of this is the potential eco-
nomic benefit for ultimate healthcare delivery. If only ten
percent of population in China opt to have their genome
determined at a price of $1000 a piece, it would create 130
billion US dollar economy. Therefore, we argue that even
though the up-front investment for China to develop this
technology seems large but the potential loss of not doing it
is even greater.
4 Conclusions
Three decades’ innovation and development have ushered in
a new era for genomic sequencing. It has evolved from
manual and one-sample-at-a-time operation to a highly-
automated and massive array-based sequencing activity. We
have experienced an exponential increase in sequencing
throughput while seeing a precipitous reduction in its cost.
With current and newer generation of sequencing technolo-
gies in the horizon, the goal of reaching one thousand dollar
genome becomes more attainable. Ability engendered by
rapid and less expensive readout of sequence information
opens up a realm of possibility in comparative genomic
analysis, disease diagnosis, and ultimately personalized (or
individualized) medicine. With its huge potential benefit in
both scientific and financial terms, China should play a
greater role in this key technological invention of the cen-
tury and its in-depth application in biological research and
medicine. When genomes of all extant life forms and their
meaningful variations are thoroughly acquired and discov-
ered, the contributors of such an endeavor should be all very
proud of themselves.
This work was supported by the Chinese Academy of Sciences Scientific
Research Equipments (Grant No. YZ200823)
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