i n s t ru m e n t a l s

Faster, cheaper
DNA sequencing
S

eeing his newborn son gasp for breath in a few hours instead of a few months, One of these enzymes is the firefly enzyme luciferase. When a base is incorpoRothberg says.
in an intensive care unit pushed JonaTo speed up sequencing, the research- rated to extend the DNA sequence, a
than Rothberg into the DNA sequencpyrophosphate is liberated. Another eners fashioned optical fibers into a chip
ing business. Wishing he could analyze
zyme converts the pyrophosphate to
the size of a credit card. The fibers creNoahs genome for insight into the
adenosine triphosphate, which enables
ate a honeycomb of wells so small that
problem, he thought, If only we could
luciferase to generate a visible light sigfour can fit on the end of a human hair.
put sequencing on a chip, it would get
nal by oxidizing the pigment luciferin.
Centrifuging the chip with the emulsion
twice as powerful and twice as fast every
Users place the chip into the
year, like computers.
Genome Sequencer 20 as if they
Fortunately, Noah did not
were inserting a CD into a drive.
have a genetic disease, but his faThe top of the chip is exposed to
ther still pursued sequencing on
computer-controlled cycles of
a chip. The company Rothberg
fluid, each providing one of the
founded and now chairs, 454
four deoxyribonucleotides. The
Life Sciences Corp., makes ma(b)
base of the chip sits on a camera
chines that sequence DNA 100
that captures the photons emitted
faster and 10100 cheaper than
from the wells as pyrosequencing
conventional technology, which
proceeds. For example, if guanine
is based on the Sanger method.
is provided in a cycle, the light
Developed by Rothberg and colsignals reveal which wells have cyleagues at 454 Life Sciences; the
tosine at that position on their
University of California, BerkeDNA template.
ley; the Rockefeller University;
(a)
One sequencing run involving
and the Rothberg Institute for
(c)
50100 cycles (enough to seChildhood Diseases, the Genome
quence a large bacterium) takes
Sequencer 20 can sequence 20
~4 h and costs $5000 for the reamillion base pairs per hour (Nagents and a chip. The machine itture 2005, 437, 376380).
self costs $500,000. When readCloning DNA in bacteria has
ing >100 bases in test fragments
always been a bottleneck in seof DNA, the researchers obtained
quencing. Rothberg sidestepped
an accuracy of ~99.4% for individthe problem by drawing on the
ual bases. With 4-fold coverage,
work of Andrew Griffiths, who
the consensus accuracy was 99.99%.
conducted billions of separate
The 454 Life Sciences Corp. sequencing instrument consists
Two potential sources of inacexperiments at the same time by
of (a) a CCD imaging system, (b) a flow chamber with a fibercuracy are that nucleotides someusing emulsion microdroplets as
optic slide, and (c) a fluidic system. (Adapted with permistimes remain in wells for more
test tubes (Nat. Biotechnol. 1998, sion. Copyright 2005 Nature Publishing Group.)
than one cycle and that some new
16, 652656). Rothberg and
fills each well with a bead. To limit cross- chains of DNA stop extending premacolleagues nebulize a genome into
talk, only about one-third of the 1.6 mil- turely. However, the company develpieces, attach each fragment to a bead,
oped algorithms to correct for these
and cocoon each bead in an emulsion of lion wells on a chip are filled; this perproblems and devised a program to asmits >400,000 pieces of DNA to be
oil, water, and detergent. After PCR is
sess base-call quality.
sequenced simultaneously. Smaller beads
performed, each bead carries 10 million
To test the technology on a small
carrying the enzymes needed for pyrosecopies of a unique piece of DNA. So,
bacterium, the researchers sequenced
quencing are also loaded into the wells.
you have a complete amplified genome
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Do-it-yourself sequencing
Another new sequencing method, which
deciphers very short pieces of DNA, was
recently described by Jay Shendure, Gregory Porreca, and colleagues at Harvard
Medical School and Washington University in St. Louis (Science 2005, 309,
17281732). The researchers developed
multiplex polony sequencing, which also
amplifies DNA fragments on beads in
emulsion droplets. But an epifluorescence microscope is used to analyze the

JAY SHENDURE AND GREGORY PORRECA

the Mycoplasma genitalium genome.

The average length of a sequence was
110 bases, and the DNA was oversampled (obtaining redundant sequences to
reduce errors) 40-fold. For 10 contiguous stretches covering 99.94% of the
genome, the consensus accuracy was
99.97%. With stricter quality criteria,
98.1% of the genome was covered with
a consensus accuracy of 99.996%.
George Weinstock at Baylor College
of Medicine says that the main advantage of the Genome Sequencer 20 is
its tremendous throughput: >200,000
samples in a 4-h run versus 96 samples/h with conventional technology.
Elaine Mardis at Washington University
in St. Louis agrees; she adds that skipping the bacterial cloning step not only
saves time but also removes cloning bias,
which is the production of amplified
DNA that lacks some pieces of the
genome because bacteria cant copy
them. Clones can be useful, however,
because they can be sequenced from
both ends, so Weinstock is trying to find
other ways to incorporate that function.
The main disadvantages of the new
approach, Weinstock says, are the lower
accuracy rates for individual reads and
the short read length, which makes it difficult to read across repeated sequences.
Therefore, he says that scientists may
want to hang onto their current DNA
sequencers so that they can identify human mutations and polymorphisms.
Nevertheless, short read lengths will
be perfectly adequate for many other
applications, such as sequencing copies
of exons, Weinstock adds. Mardis is already using the machine for digital karyotyping, which counts how many times
a snippet of DNA occurs in a genomic
region. With conventional methods, researchers must sequence large amounts
of DNA to cull such snippets. But that
isnt a problem if you can produce 200,000
to 400,000 in a single run on the 454
machine, Mardis says.
Meanwhile, 454 Life Sciences has
sequenced >100 genomes of bacteria,
fungi, and plants. The company now
has perfect reads on fragments longer
than 500 bases and has sequenced >100
million bases in a single run, according
to Rothberg.

False-color image of multiplex polony

sequencing on beads.

beads in a layer of acrylamide gel on a

microscope slide. This system can be
built by anyone, Porreca says. All the
components are off the shelf, and the
reagents are standard.
The setup includes a syringe pump,
autosampler, flow cell, epifluorescence
microscope, and modules for temperature control and automated washing.
Assembling one costs ~$140,000. The
upfront cost is ~$20,000 for fluorescently labeled oligonucleotides.
First, researchers shear a genome and
select 1-kb fragments. Using an endonuclease, they insert universal sequences
between and flanking two unique 1718
bp pieces, or tags, from each fragment.
The resulting constructs are amplified
on beads. After the beads are immobilized in a flow cell on a microscope slide,
an anchor for DNA ligase is hybridized
to the base immediately before or after
one of the two tags. To the beads, the
researchers add the ligase and a mixture
of nonamers, each color-coded with a
fluorescent dye that reveals the identity
of the base at a chosen position. For example, nonamers color-coded according

A N A LY T I C A L C H E M I S T R Y / N O V E M B E R 1 , 2 0 0 5

to the fifth of the nine bases identify a

base five positions away from the anchor
primer.
Rastering over the slide, the microscope collects bright-field and fluorescent images for each of the four fluorophores. Image analysis determines
which color, and therefore which complementary base, hybridized to the first
base of a tag on a particular bead. The
next cycle identifies the second base in
that tag, and so on. Because it is possible to sequence 7 bases from one end of
a tag and 6 bases from the other, the 2
tags on each bead generate 26 bases of
noncontiguous sequence. Furthermore,
a microscope slide can hold millions of
beads.
Shendure and Porreca tested the
technology by sequencing an E. coli
mutant and comparing it with the
known wild-type sequence. They located ~1.16 million reads, covering ~30.1
million bases, on the reference sequence
with a median raw accuracy of 99.7%.
A consensus sequence for 70.5% of the
genome revealed 6 discrepancies between the mutant and reference genomes, which were confirmed as bona
fide mutations. Preparing and arraying
the beads took 2 days, and the 26 runs
took 60 h. The cost per raw kilobase
was 11, which included 3/kb for library construction.
Multiplex polony sequencing wasnt
designed to replace Sanger sequencing,
Porreca explains. The method delivers
short sequences at low cost and with
high accuracy, he says. So the best applications right now are bacterial resequencing and applications such as
SAGE [serial analysis of gene expression, in which] you need to sequence
bar codes. Although critics say that the
method cannot detect mutations such as
insertions and large deletions, Church
asserts that it can. Porreca adds that the
technologys primary advantages are its
low cost and high consensus accuracy.
Weinstock predicts an explosion of
sequencing technologies in the next few
years: I suspect a fair number of those
will succeed, and one will use different
sequencing approaches for different apa
plications.a
Linda Sage