TECHNOLOGIES

DRUG DISCOVERY

TODAY

Next generation sequencingtechnologiesThomas Jarvie454 Life Sciences, 20 Commercial Street, Branford, CT 06405, USA

Drug Discovery Today: Technologies Vol. 2, No. 3 2005

Editors-in-Chief

Kelvin Lam – Pfizer, Inc., USA

Henk Timmerman – Vrije Universiteit, The Netherlands

Emerging technologies

Section Editors:Steve Gullans – RxGen, Inc., New Haven, CT, USARobert Zivin – Johnson and Johnson, New Brunswick, NJ,USA

From the investigation of disease-associated loci in

humans, to monitoring the changing genomes of

pathogenic viruses and bacteria, sequencing is a power-

ful and versatile tool. A new generation of sequencing

technologies will increase the speed and lower the cost

of sequencing, and promises to expand the utility of

sequencing in drug discovery and development.

Introduction

DNA sequencing is a central technology in our understanding

of biology and plays a significant, supporting role in drug

discovery and development. The Human Genome Project

and the resequencing of selected regions of the human

genome in disease association studies have contributed to

a refined understanding of the molecular basis of many

diseases. Sequencing of pathogenic microbes and drug resis-

tant strains has aided in our understanding of drug resistance,

the development of drug resistance over time and the

mechanism of action of new drugs. Additionally, the sequen-

cing of full viral genomes, or a subset of the genomes, derived

from clinical samples provides a picture of the course of

infection over time, response to antiviral therapies and an

insight into possible strategies for further drug development.

This review focuses on the next generation of sequencing

technologies and the potential for these technologies to

revolutionize pharmaceutical development.

The need for new sequencing methods

Electrophoresis-based, Sanger sequencing technology is the

most commonly used technology for sequencing and was the

E-mail address: T. Jarvie (tjarvie@454.com)

1740-6749/$ � 2005 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddtec.2005.08.003

mainstay of the Human Genome Project. A look into the Gold

Database (http://www.genomesonline.org/) shows that San-

ger-based sequencing has build a solid foundation of genomic

sequence that the next generation of technologies can build

upon through resequencing and comparative genomics stu-

dies. In addition to the whole genome sequencing, Sanger-

based sequencing has been used to sequence countless ampli-

cons for applications such as verification of clones, searching

for SNPs, forensic analysis and resequencing. Over the past 10

years, significant improvements in Sanger technology have

cut the cost of sequencing from �$10/kb to �$1/kb. Over the

same period of time, the throughput for a state of the art

instrument has increased from <10 kb/h to �100 kb/h. The

standard method of sequencing, however, might be nearing

the end of the line for dramatic cost reductions and through-

put increases.

The most high profile example of the drive to lower

sequencing cost is the goal of a $1000 human genome, a

goal that would enable the sequencing of individual human

genomes as a component of diagnostic and preventative

medicine, or personalized medicine. In addition to human

genome resequencing, several other applications for low cost,

high-throughput sequencing are discussed in the literature. A

few of the applications with pharmaceutical relevance are

sequencing of multiple strains of pathogenic bacteria to

monitor drug resistance and pathogenicity in bacteria, rese-

quencing selected regions to search for human variation in

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Drug Discovery Today: Technologies | Emerging technologies Vol. 2, No. 3 2005

populations [1,2], monitoring the onset of drug resistance in

HIV [3,4] or HCV, profiling tumors to guide cancer therapies

[5] and discerning the mechanism of action of antibiotics [6].

For sequence-based studies to play a more central role in

pharmaceutical research, the cost and time associated with

sequencing must be reduced. The flexibility in experimental

design afforded by quicker and more cost efficient sequencing

holds the promise of not only making the current experi-

ments more feasible, but will generate new sequence based

experiments.

Several academic labs, start-up companies and large instru-

ment companies are all developing a variety of technologies

aimed at lowering the cost and increasing the throughput of

sequencing. In October of 2004, The US National Human

Genome Research Institute (NHGRI) awarded $38 million to

18 companies and academic laboratories to develop the next

generation sequencing technologies (http://www.genome.-

gov/12513162). This government money along with private

investment has led to a range of technologies, some of them

usable today and some still in the R&D phase of their devel-

opment. Some of the new sequencing technologies are well

suited to resequencing, whereas others are more flexible and

suitable for resequencing and de novo sequencing. In rese-

quencing, one is performing a sequence-based comparison of

an entire genome or a subset of the genome and looking for

differences as compared to a previously determined sequence.

The known sequence is either used as a reference, or in

sequencing by hybridization, is used as the basis of the

resequencing technology. In de novo sequencing a new gen-

ome (or other sequence) is sequenced and assembled without

direct comparison against a known sequence. As a result, de

novo sequencing technologies are suitable for new genetic

material and genetic material that differs markedly from a

previously sequenced strain [7,8]. All de novo technologies

can be used for resequencing. This paper discusses four broad

classes of new sequencing technologies that are all capable of

de novo sequencing: microelectrophoretic methods, sequen-

cing by hybridization, real time detection of single molecules

and cyclic-array sequencing.

New methods for sequencing

Microelectrophoretic methods

Microelectrophoretic methods have the advantage of

employing and building upon the existing capillary electro-

phoresis, Sanger sequencing technologies. The advance of

microelectrophoretic technology, as compared to the com-

mercially available capillary sequencing technologies, come

from scaling down the size of the electrophoresis platform

(and therefore the cost of reagents and potentially, capital

equipment) and, frequently, scaling up of the number of

lanes used in the electrophoresis [9–11]. Additional efforts

have integrated the sample preparation and sequencing pro-

cesses onto one single microfabricated device [12,13]. The

256 www.drugdiscoverytoday.com

majority of the work to date has been in academic labs,

although Shimadzu Biotech (http://www.shimadzu-biotech.-

net/) has plans to introduce a commercially available instru-

ment in the near future, and Microchip Biotechnologies

(http://microchipbiotech.com/) received a large NHGRI

grant to develop an instrument.

Sequencing by hybridization

Sequencing by hybridization utilizes the microarray technol-

ogies that are the basis of much of the gene expression work

commonly performed as a part of drug development. Hybri-

dization sequencing works by hybridizing single-stranded

sample DNA to a microfabricated array of DNA oligonucleo-

tide probes. Each base in a sequence is queried by changing

the middle base in the oligonucleotide probe to all four

possibilities (A, C, G and T) while keeping the remaining

sequence unchanged. The sequence of the DNA is determined

by which of the four probe oligonucleotide yields the stron-

gest hybridization signal. Although the amount of sequence

that can be generated is high, the readlength is limited to the

length of the oligonucleotide probe. Additionally, although

investigation of single nucleotide changes are straightfor-

ward, more complex changes in a genome, such as insertion

or deletion of a codon (or codons), multiple point mutations

within close proximity of one another, and insertion or

deletion of large segments of genetic material (such as entire

ORFs) are challenging. Although this technology has been

applied to both resequencing and de novo sequencing [14–19],

the strength of the technology and its greatest potential is in

the massive resequencing of a limited number of genomic

positions. Several companies such as Illumina (http://www.il-

lumina.com/), Perlegen (http://perlegen.com/), Nimblegen

(http://nimblegen.com/) and Parellele (http://www.paralle-

lebio.com/) (recently purchased by Affymetrix http://affyme-

trix.com/index.affx) offer instruments and/or services.

Real-time detection of single molecules

The most elegant of sequencing technology, should it ever

become viable, is the direct detection of single molecules.

This technology would allow for fast sequencing of small

quantities of DNA. Nanopore sequencing and directly mon-

itoring the incorporation of nucleotides by a polymerase are

two fundamental approaches that are under consideration for

direct single molecule detection.

Direct monitoring of nucleotide incorporation operates

on the principal of watching an engineered polymerase

synthesize the second strand of DNA. The nucleotides are

distinguished from one another by differing fluorescent

labels. Among the challenges in the direct monitoring

technology is one of achieving sufficient signal from single

nucleotide incorporation events in a background of labeled

nucleotides and capturing all of the nucleotide incorpo-

ration events. Visigen (http://www.visigenbio.com/) and

Vol. 2, No. 3 2005 Drug Discovery Today: Technologies | Emerging technologies

LI-COR (http://www.licor.com/) are two companies work-

ing on this technology.

In the nanopore sequencing methodology, DNA is mon-

itored as it passes through a nanometer scale surface pore. The

sequencing process relies upon the ability to translate the

differing chemical and physical properties of each base into

electrical signals as the nucleotides pass through the nano-

pore [20,21]. To date, the promise of this technology is still

speculative. Various academic groups have reported the abil-

ity to monitor fragments as they pass through the pores,

although single base sequencing is still elusive. Extensive

work is underway on improved nanopores and detection

schemes [22,23]. Agilent (http://www.chem.agilent.com/

Scripts/Phome.asp) is developing the technology.

Cyclic-array sequencing

The category of cyclic-array sequencing is composed of sev-

eral different approaches. All of the various approaches are

broadly classified into either methods that sequence ampli-

fied molecules or those that sequence from single molecules.

Regardless of whether the sequencing will occur on a single

molecule or amplified molecules, all of the methods utilize

the physical separation of the DNA fragments to be

sequenced in an array and the multiple cycles of reagent

addition/enzymatic manipulation that are responsible for the

sequence generation. The set-up of the array can be either

ordered or random: the important point is the physical

separation of distinct fragments from one another. The

majority of the cyclic-array sequencing technologies are

based on a stepwise build-up of the sequence by a polymerase

(sequencing-by-synthesis, Fig. 1) coupled with a detection

mechanism, although one method, the ‘massively parallel

Figure 1. Sequencing-by-synthesis is the underlying method used by many of

built upon sequencing-by-synthesis are 454 Life Sciences, Agencourt, Genovoxx

number (either clonally amplified molecules or single molecules) and detection

nucleotides to a primed, single strand of DNA. The specific example in this fig

signature sequencing’, or MPPS [24] from Lynx (http://

www.lynxgen.com) employs cyclic restriction digestion

and ligation. Several companies are working on amplified

molecule cyclic-array sequencing-by-synthesis.

The first of the amplified molecule cyclic-array technolo-

gies to be commercialized is the sequencing-by-synthesis

method from 454 Life Sciences (http://www.454.com/)

[25]. This technology relies on the clonal amplification of

single molecules (either single-stranded or double-stranded

DNA) on capture beads isolated in an emulsion, and the

subsequent highly parallel sequencing of the clonally ampli-

fied DNA on beads deposited into the picoliter scale wells of a

PicoTiterPlateTM. The 454 sequencing instrument is currently

capable of sequencing a minimum of 20 Mb, or 200,000

fragments with a median 100 base-pair readlength, in a

4.5-h run. The detection scheme for the 454 Life Sciences

instrument is based on the conversion of pyrophosphate,

which is released by the polymerase mediated addition of a

nucleotide to the complimentary strand, into light via an

enzyme cascade. In May 2005, 454 Life Sciences entered into

a worldwide distribution deal for the sequencing instrument

and reagents with Roche Diagnostics (http://www.roche-

applied-science.com/). An overview of the sequencing pro-

cess is presented in Fig. 2.

Two other companies, Agencourt and Solexa, are promi-

nent in the amplified molecule cyclic-array field. Agencourt

(http://www.agencourt.com/), a sequencing service com-

pany, was purchased by Beckman Coulter (http://www.

beckmancoulter.com/) in late April 2005. As part of the

purchase, a new company, Agencourt Personal Genomics

was spun off to accelerate development of a new platform.

The platform is based upon the fluorescent detection of

the next generation sequencing technologies. The companies

, Helicos, Nanofluidics, Solexa and Visigen. Although the specifics of copy

schemes vary, all methods rely on polymerase-mediated addition of

ure is clonally amplified DNA fragments attached to a bead.

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Drug Discovery Today: Technologies | Emerging technologies Vol. 2, No. 3 2005

Figure 2. Schematic overview of the 454 Life Sciences instrument and sequencing process. The process begins with large DNA molecules

(such as genomic DNA) that are fragmented and subsequently ligated with universal adaptors before clonal amplification on beads, deposition on a

PicoTiterPlateTM, and pyrophosphate based sequencing-by-synthesis. A CCD camera captures the light generated from the sequencing reaction.

The resulting signals are converted into sequence. An alternative input to the process is small fragments, such as exons, that are amplified with

tailed-primers containing the 454 universal adaptors. These tailed-primer amplicons enter the process and the clonal amplification step.

single-nucleotide extensions of DNA fragments attached to

beads. Solexa (http://www.solexa.com) (merged in early 2005

with Lynx) is also working on fluorescent-based detection of

amplified DNA. Solexa has a planned instrument release

schedule of early 2006.

The cyclic-array, amplified molecule methods all rely upon

clonal amplification of the fragments before sequencing [26].

The clonal amplification is achieved either by isolation of the

molecules by means such as an emulsion or an acrylamide

matrix [27–29], or through tagging and subsequent separa-

tion of molecules. As a result, these methods, although not

strictly single-molecule detection methods, have the ability

to sequence from single molecules that originated in a com-

plex mixture.

A second class of cyclic array technologies is aimed at single

molecule detection. These methods directly sequence from

single molecules and thus avoid the cost associated with

258 www.drugdiscoverytoday.com

either cloning or PCR amplification. The sequencing

approach to cyclic-array single molecule sequencing is similar

to the amplified molecule sequencing-by-synthesis methods,

the difference is a more sensitive detection scheme that

avoids the need for multiple molecules to provide sufficient,

detectable signal. All of the single molecule methods, includ-

ing those being worked on by several academic labs and

companies such as Genovoxx (http://www.genovoxx.com/),

Nanofluidics (http://www.nanofluidics.com/) [30] and

Helicos (http://www.helicosbio.com/) [31], proceed by using

the step-wise incorporation of fluorescent nucleotides. The

difference between the methods lies in the different signal

detection schemes and the details of the biochemistry.

Conclusion

Several promising technologies will revolutionize the role of

sequencing in pharmaceutical research over the next few

Vol. 2, No. 3 2005 Drug Discovery Today: Technologies | Emerging technologies

Table 1. Overview of next generation de novo sequencing technologies

Technology Microelectrophoretic

sequencing

Sequencing by

hybridization

Single molecule,

real-time detection

Cyclic-array sequencing

Company Shimadzu Biotech,

Microchip Biotechnologies

Perlegen, Parallele, Affymetrix,

Nimblegen, Illumina

Visigen, LI-COR, Agilent 454 Life Sciences, Agencourt,

Solexa, Genovoxx,

Nanofluidics, Helicos

Pros - Sequencing by

electrophoresis is well established

- Ideal for resequencing

of known point mutations

- Ability to detect single

molecules in complex mixture

- Ability to detect single

molecules in complex

mixture- Long readlengths - Commercially available - Suitable for resequencing

and de novo - Suitable for resequencing

and de novo

- Low error rate - Commercially available

Cons - Potentially not as high-throughput

as other methods

- Current readlengths less than

electrophoretic methods

- Still not a commercially

viable technology

- Current readlengths

less than electrophoretic

methods- Potentially not as low cost

as other methods

- De novo sequencing is slow

References [4,9–13] [14–19] [20–23] [6,24,26–32]

Outstanding issues

years (Table 1). The first of the new generation instruments is

commercially available from one cyclic-array sequencing

based company. Microarray companies are introducing an

increasing variety of microarrays for sequencing. Other man-

ufactures plan the commercial release of instruments over the

next few years. Additionally, access to several the low-cost

high-throughput technologies is already available as a service

from some companies (454 Life Sciences, Paralelle, Perlegen

and Solexa/Lynx).

The idea behind the drive for low cost, high-throughput

sequencing has been to lower the cost of sequencing enough

to enable personalized human genome sequencing. Along

the way to this lofty goal, many potential applications of the

technology are already available or will be enabled soon as the

bioinformatics development races to keep up with the large

quantities of data and the new possibilities that inexpensive

and quick sequencing allow. For example, affordable micro-

bial sequencing, either resequencing for SNP identification or

de novo sequencing of more variable strains, enables compara-

tive genomics on strains of varying virulence, drug resistance

and host species preference.

The molecular based cloning employed in the cyclic-array

methods and the real-time, single molecule methods, with

their inherent ability to sequence from single molecules in a

complex mixture, open up the possibility of massive over-

sampling of specific regions of interest or tagged sequences

and do so in a quick and cost effective manner. The first

Related articles

Shendure, J. et al. (2004) Advanced sequencing technologies: methods

and goals. Nat. Rev. 5, 335–344

Marziali, A. and Akeson, M. (2001) New DNA sequencing methods.

Ann. Rev. Biomed. Eng. 3, 195–223

demonstration of sequencing from complex mixtures has

sensitivity below 1% in a complex mixture of HIV quasispe-

cies [32]. This level of sensitivity is achievable by the Sanger

based methods only by cloning of fragments into bacteria.

Microarray based sequencing methods are not as sensitive or

as quantitative as the direct sequencing of clonally amplified

single molecules. Examples of the utility of this technology

are applications such as disease associated exons within a

population, investigation of viral quasi-species present

within a patient as a function of time and drug response,

deep sequencing of microRNAs, querying somatic mutations

in tumor samples, and monitoring pathogens for changes in

their genome as a function of drug resistance or changing

virulence.

The applications enabled by the next generation sequen-

cing technologies and their usefulness to the drug discovery

and development process are only beginning to be discov-

ered. Once the technology is widely available and the power/

promise of the technology is known, additional applications

will be developed. The ability of many of the technologies to

sequence de novo opens up a wide array of possibilities for new

discovery and creative approaches to important and unad-

dressed problems in pharmaceutical research and develop-

ment.

� Long readlengths, comparable to traditional electrophoretic

methods, are a challenge for some of the new technologies.

� The high volume of sequencing data that will result from low-cost

high-throughput technology presents demands on data handling and

bioinformatics/interpretation infrastructure.

� All new technologies are still too expensive and too time consuming

to enter into the range of personalized human genome sequencing

(the $1000 genome).

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Drug Discovery Today: Technologies | Emerging technologies Vol. 2, No. 3 2005

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