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`Vol 437|15 September 2005|doi:10.1038/nature03959
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`Genome sequencing in microfabricated
`high-density picolitre reactors
`
`Marcel Margulies1*, Michael Egholm1*, William E. Altman1, Said Attiya1, Joel S. Bader1, Lisa A. Bemben1,
`Jan Berka1, Michael S. Braverman1, Yi-Ju Chen1, Zhoutao Chen1, Scott B. Dewell1, Lei Du1, Joseph M. Fierro1,
`Xavier V. Gomes1, Brian C. Godwin1, Wen He1, Scott Helgesen1, Chun He Ho1, Gerard P. Irzyk1,
`Szilveszter C. Jando1, Maria L. I. Alenquer1, Thomas P. Jarvie1, Kshama B. Jirage1, Jong-Bum Kim1,
`James R. Knight1, Janna R. Lanza1, John H. Leamon1, Steven M. Lefkowitz1, Ming Lei1, Jing Li1, Kenton L. Lohman1,
`Hong Lu1, Vinod B. Makhijani1, Keith E. McDade1, Michael P. McKenna1, Eugene W. Myers2,
`Elizabeth Nickerson1, John R. Nobile1, Ramona Plant1, Bernard P. Puc1, Michael T. Ronan1, George T. Roth1,
`Gary J. Sarkis1, Jan Fredrik Simons1, John W. Simpson1, Maithreyan Srinivasan1, Karrie R. Tartaro1,
`Alexander Tomasz3, Kari A. Vogt1, Greg A. Volkmer1, Shally H. Wang1, Yong Wang1, Michael P. Weiner4,
`Pengguang Yu1, Richard F. Begley1 & Jonathan M. Rothberg1
`
`The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to
`reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly
`greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of
`individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an
`approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an
`emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol
`optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness
`of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage
`at 99.96% accuracy in one run of the machine.
`
`DNA sequencing has markedly changed the nature of biomedical
`research and medicine. Reductions in the cost, complexity and time
`required to sequence large amounts of DNA, including improve
`ments in the ability to sequence bacterial and eukaryotic genomes,
`will have significant scientific, economic and cultural impact. Large
`scale sequencing projects, including whole genome sequencing, have
`usually required the cloning of DNA fragments into bacterial vectors,
`amplification and purification of individual templates, followed by
`Sanger sequencing1 using fluorescent chain terminating nucleotide
`analogues2 and either slab gel or capillary electrophoresis. Current
`estimates put the cost of sequencing a human genome between $10
`million and $25 million3. Alternative sequencing methods have been
`described4 8; however, no technology has displaced the use of
`bacterial vectors and Sanger sequencing as the main generators of
`sequence information.
`Here we describe an integrated system whose throughput routinely
`enables applications requiring millions of bases of sequence infor
`mation, including whole genome sequencing. Our focus has been on
`the co development of an emulsion based method9 11 to isolate and
`amplify DNA fragments in vitro, and of a fabricated substrate and
`instrument that performs pyrophosphate based sequencing (pyro
`sequencing5,12) in picolitre sized wells.
`In a typical run we generate over 25 million bases with a Phred
`quality score of 20 or better (predicted to have an accuracy of 99% or
`higher). Although this Phred 20 quality throughput is significantly
`
`higher than that of Sanger sequencing by capillary electrophoresis, it
`is currently at the cost of substantially shorter reads and lower
`average individual read accuracy. Sanger based capillary electrophor
`esis sequencing systems produce up to 700 bases of sequence
`information from each of 96 DNA templates at an average read
`accuracy of 99.4% in 1 h, or 67,000 bases per hour, with substantially
`all of the bases having Phred 20 or better quality23. We further
`characterize the performance of the system and demonstrate that it
`is possible to assemble bacterial genomes de novo from relatively
`short reads by sequencing a known bacterial genome, Mycoplasma
`genitalium (580,069 bases), and comparing our shotgun sequencing
`and de novo assembly with the results originally obtained for this
`genome13. The results of shotgun sequencing and de novo assembly of
`a larger bacterial genome, that of Streptococcus pneumoniae14
`(2.1 megabases (Mb)), are presented in Supplementary Table 4.
`
`Emulsion-based sample preparation
`We generate random libraries of DNA fragments by shearing an
`entire genome and isolating single DNA molecules by limiting
`dilution (Supplementary Methods). Specifically, we randomly frag
`ment the entire genome, add specialized common adapters to the
`fragments, capture the individual fragments on their own beads and,
`within the droplets of an emulsion, clonally amplify the individual
`fragment (Fig. 1a, b). Unlike in current sequencing technology, our
`approach does not require subcloning in bacteria or the handling of
`
`1454 Life Sciences Corp., 20 Commercial Street, Branford, Connecticut 06405, USA. 2University of California, Berkeley, California 94720, USA. 3Laboratory of Microbiology,
`The Rockefeller University, New York, New York 10021, USA. 4The Rothberg Institute for Childhood Diseases, 530 Whitfield Street, Guilford, Connecticut 06437, USA.
`*These authors contributed equally to this work.
`
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`individual clones; the templates are handled in bulk within the
`emulsions9 11.
`
`Sequencing in fabricated picolitre-sized reaction vessels
`We perform sequencing by synthesis simultaneously in open wells of
`a fibre optic slide using a modified pyrosequencing protocol that is
`designed to take advantage of the small scale of the wells. The fibre
`optic slides are manufactured by slicing of a fibre optic block that is
`obtained by repeated drawing and fusing of optic fibres. At each
`iteration, the diameters of the individual fibres decrease as they are
`hexagonally packed into bundles of increasing cross sectional sizes.
`Each fibre optic core is 44 m m in diameter and surrounded by
`2 3 m m of cladding; etching of each core creates reaction wells
`approximately 55 m m in depth with a centre to centre distance of
`50 m m (Fig. 1c), resulting in a calculated well size of 75 pl and a well
`density of 480 wells mm 2. The slide, containing approximately 1.6
`million wells15, is loaded with beads and mounted in a flow chamber
`designed to create a 300 m m high channel, above the well openings,
`through which the sequencing reagents flow (Fig. 2a, b). The
`unetched base of the slide is in optical contact with a second fibre
`optic imaging bundle bonded to a charge coupled device (CCD)
`
`sensor, allowing the capture of emitted photons from the bottom of
`each individual well (Fig. 2c; see also Supplementary Methods).
`We developed a three bead system, and optimized the components
`to achieve high efficiency on solid support. The combination of
`picolitre sized wells, enzyme loading uniformity allowed by the small
`beads and enhanced solid support chemistry enabled us to develop a
`method that extends the useful read length of sequencing by syn
`thesis to 100 bases (Supplementary Methods).
`In the flow chamber cyclically delivered reagents flow perpendi
`cularly to the wells. This configuration allows simultaneous exten
`sion reactions on template carrying beads within the open wells and
`relies on convective and diffusive transport to control the addition or
`removal of reagents and by products. The timescale for diffusion into
`and out of the wells is on the order of 10 s in the current configuration
`and is dependent on well depth and flow channel height. The
`timescales for the signal generating enzymatic reactions are on the
`order of 0.02 1.5 s (Supplementary Methods). The current reaction
`is dominated by mass transport effects, and improvements based on
`faster delivery of reagents are possible. Well depth was selected on the
`basis of a number of competing requirements: (1) wells need to be
`deep enough for the DNA carrying beads to remain in the wells in the
`presence of convective transport past the wells; (2) they must be
`sufficiently deep to provide adequate isolation against diffusion of
`by products from a well in which incorporation is taking place to a
`well where no incorporation is occurring; and (3) they must be
`shallow enough to allow rapid diffusion of nucleotides into the wells
`and rapid washing out of remaining nucleotides at the end of each
`flow cycle to enable high sequencing throughput and reduced reagent
`use. After the flow of each nucleotide, a wash containing apyrase is
`used to ensure that nucleotides do not remain in any well before the
`next nucleotide being introduced.
`
`Base calling of individual reads
`Nucleotide incorporation is detected by the associated release of
`inorganic pyrophosphate and the generation of photons5,12. Wells
`containing template carrying beads are identified by detecting a
`known four nucleotide ‘key’ sequence at the beginning of the read
`
`Figure 1 | Sample preparation. a, Genomic DNA is isolated, fragmented,
`ligated to adapters and separated into single strands (top left). Fragments are
`bound to beads under conditions that favour one fragment per bead, the
`beads are captured in the droplets of a PCR reaction mixture in oil
`emulsion and PCR amplification occurs within each droplet, resulting in
`beads each carrying ten million copies of a unique DNA template (top right).
`The emulsion is broken, the DNA strands are denatured, and beads carrying
`single stranded DNA clones are deposited into wells of a fibre optic slide
`(bottom right). Smaller beads carrying immobilized enzymes required for
`pyrophosphate sequencing are deposited into each well (bottom left).
`b, Microscope photograph of emulsion showing droplets containing a bead
`and empty droplets. The thin arrow points to a 28 m m bead; the thick arrow
`points to an approximately 100 m m droplet. c, Scanning electron
`micrograph of a portion of a fibre optic slide, showing fibre optic cladding
`and wells before bead deposition.
`
`Figure 2 | Sequencing instrument. The sequencing instrument consists of
`the following major subsystems: a fluidic assembly (a), a flow chamber that
`includes the well containing fibre optic slide (b), a CCD camera based
`imaging assembly (c), and a computer that provides the necessary user
`interface and instrument control.
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`(Supplementary Methods). Raw signals are background subtracted,
`normalized and corrected. The normalized signal intensity at each
`nucleotide flow, for a particular well, indicates the number of
`nucleotides, if any, that were incorporated. This linearity in signal
`is preserved to at least homopolymers of length eight (Supplemen
`tary Fig. 6). In sequencing by synthesis a very small number of
`templates on each bead lose synchronism (that is, either get ahead of,
`or fall behind, all other templates in sequence16). The effect is
`primarily due to leftover nucleotides in a well (creating ‘carry
`forward’) or to incomplete extension. Typically, we observe a carry
`forward rate of 1 2% and an incomplete extension rate of 0.1 0.3%.
`Correction of these shifts is essential because the loss of synchronism
`is a cumulative effect that degrades the quality of sequencing at
`longer read lengths. We have developed algorithms, based on detailed
`models of the underlying physical phenomena, that allow us to
`determine, and correct for, the amounts of carry forward and
`incomplete extension occurring in individual wells (Supplementary
`Methods). Figure 3 shows the processed result, a 113 bases long read
`generated in the M. genitalium run discussed below. To assess
`sequencing performance and the effectiveness of the correction
`algorithms, independently of artefacts introduced during the emul
`sion based sample preparation, we created test fragments with
`difficult to sequence stretches of identical bases of increasing length
`(homopolymers) (Supplementary Methods and Supplementary Fig.
`4). Using these test fragments, we have verified that at the individual
`read level we achieve base call accuracy of approximately 99.4%, at
`read lengths in excess of 100 bases (Table 1).
`
`High-quality reads and consensus accuracy
`Before base calling or aligning reads, we select high quality reads
`without relying on a priori knowledge of the genome or template
`being sequenced (Supplementary Methods). This selection is based
`on the observation that poor quality reads have a high proportion of
`signals that do not allow a clear distinction between a flow during
`which no nucleotide was incorporated and a flow during which one
`or more nucleotide was incorporated. When base calling individual
`reads, errors can occur because of signals that have ambiguous values
`(Supplementary Fig. 5). To improve the usability of our reads, we also
`developed a metric that allows us to estimate ab initio the quality (or
`probability of correct base call) of each base of a read, analogous to
`the Phred score17 used by current Sanger sequencers (Supplementary
`Methods and Supplementary Fig. 8).
`
`Figure 3 | Flowgram of a 113-bases read from an M. genitalium
`run. Nucleotides are flowed in the order T, A, C, G. The sequence is shown
`above the flowgram. The signal value intervals corresponding to the various
`homopolymers are indicated on the right. The first four bases (in red, above
`the flowgram) constitute the ‘key’ sequence, used to identify wells containing
`a DNA carrying bead.
`
`Higher quality sequence can be achieved by taking advantage of
`the high over sampling that our system affords and building a
`consensus sequence. Sequences are aligned to one another using
`the signal strengths at each nucleotide flow, rather than individual
`base calls, to determine optimal alignment (Supplementary
`Methods). The corresponding signals are then averaged, after
`which base calling is performed. This approach greatly improves
`the accuracy of the sequence (Supplementary Fig. 7) and provides an
`estimate of the quality of the consensus base. We refer to that quality
`measure as the Z score
`it is a measure of the spread of signals in all
`the reads at one location and the distance between the average signal
`and the closest base calling threshold value. In both re sequencing
`and de novo sequencing, as the minimum Z score is raised the
`consensus accuracy increases, while coverage decreases; approxi
`mately half of the excluded bases, as the Z score is increased, belong
`to homopolymers of length four and larger. Sanger sequencers
`usually require a depth of coverage at any base of three or more in
`order to achieve a consensus accuracy of 99.99%. To achieve a
`minimum of threefold coverage of 95% of the unique portions of a
`typical genome requires approximately seven to eightfold over
`sampling. Owing to our higher error rate, we have observed that
`comparable consensus accuracies, over a similar fraction of a gen
`ome, are achieved with a depth of coverage of four or more, requiring
`approximately ten to twelve times over sampling.
`
`Mycoplasma genitalium
`Mycoplasma genomic DNA was fragmented and prepared into a
`sequencing library as described above. (This was accomplished by a
`single individual in 4 h.) After emulsion polymerase chain reaction
`
`(PCR) and bead deposition onto a 60 £ 60 mm2 fibre optic slide, a
`
`process which took one individual 6 h, 42 cycles of four nucleotides
`were flowed through the sequencing system in an automated 4 h run
`of the instrument. The results are summarized in Table 2. In order to
`measure the quality of individual reads, we aligned each high quality
`read to the reference genome at 70% stringency using flow space
`mapping and criteria similar to those used previously in assessing the
`accuracy of other base callers17. When assessing sequencing quality,
`only reads that mapped to unique locations in the reference genome
`were included. Because this process excludes repeat regions (parts of
`the genome for which corresponding flowgrams are 70% similar to
`one another), the selected reads did not cover the genome comple
`tely. Figure 4a illustrates the distribution of read lengths for this run.
`The average read length was 110 bases, the resulting over sample 40
`fold, and 84,011 reads (27.4%) were perfect. Figure 4b summarizes
`the average error as a function of base position. Coverage of non
`repeat regions was consistent with the sample preparation and
`emulsion not being biased (Supplementary Fig. 8). At the individual
`read level, we observe an insertion and deletion error rate of
`approximately 3.3%; substitution errors have a much lower rate,
`on the order of 0.5%. When using these reads without any Z score
`restriction, we covered 99.94% of the genome in ten contiguous
`regions with a consensus accuracy of 99.97%. The error rate in
`homopolymers is significantly reduced in the consensus sequence
`(Supplementary Fig. 7). Of the bases not covered by this consensus
`
`Table 1 | Summary of sequencing statistics for test fragments
`60 £ 60 mm2
`Size of fibre optic slide
`Run time/number of cycles
`243 min/42
`Test fragment reads
`497,893
`Average read length (bases)
`108
`Number of bases in test fragments
`53,705,267
`Bases with a Phred score of 20 and above
`47,181,792
`Individual read insertion error rate
`0.44%
`Individual read deletion error rate
`0.15%
`Individual read substitution error rate
`0.004%
`All errors
`0.60%
`
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`sequence (366 bases), all belonged to excluded repeat regions. Setting
`a minimum Z score equal to 4, coverage was reduced to 98.1% of the
`genome, while consensus accuracy increased to 99.996%. We further
`demonstrated the reproducibility of the system by repeating the
`whole genome sequencing of M. genitalium an additional eight
`times, achieving a 40 fold coverage of the genome in each of the
`eight separate instrument runs (Supplementary Table 3).
`We assembled the M. genitalium reads from a single run into 25
`contigs with an average length of 22.4 kb. One of these contigs was
`misassembled due to a collapsed tandem repeat region of 60 bases,
`and was corrected by hand. The original sequencing of M. genitalium
`resulted in 28 contigs before directed sequencing used for finishing
`the sequence13. Our assembly covered 96.54% of the genome and
`attained a consensus accuracy of 99.96%. Non resolvable repeat
`regions amount to 3% of the genome: we therefore covered 99.5%
`of the unique portions of the genome. Sixteen of the breaks between
`contigs were due to non resolvable repeat regions, two were due to
`missed overlapping reads (our read filter and trimmer are not perfect
`and the algorithms we use to perform the pattern matching of
`flowgrams occasionally miss valid overlaps) and the remainder to
`thin read coverage. Setting a minimum Z score of 4, coverage was
`reduced to 95.27% of the genome (98.2% of the resolvable part of the
`genome) with the consensus accuracy increasing to 99.994%.
`
`Discussion
`We have demonstrated the simultaneous acquisition of hundreds of
`thousands of sequence reads, 80 120 bases long, at 96% average
`accuracy in a single run of the instrument using a newly developed in
`vitro sample preparation methodology and sequencing technology.
`With Phred 20 as a cutoff, we show that our instrument is able to
`produce over 47 million bases from test fragments and 25 million
`bases from genomic libraries. We used test fragments to de couple
`our sample preparation methodology from our sequencing technol
`ogy. The decrease in single read accuracy from 99.4% for test
`fragments to 96% for genomic libraries is primarily due to a lack
`of clonality in a fraction of the genomic templates in the emulsion,
`and is not an inherent limitation of the sequencing technology. Most
`of the remaining errors result from a broadening of signal distri
`butions, particularly for large homopolymers (seven or more),
`leading to ambiguous base calls. Recent work on the sequencing
`
`Table 2 | Summary statistics for M. genitalium
`Sequencing summary
`Number of instrument runs
`Size of fibre optic slide
`Run time/number of cycles
`High quality reads
`Average read length (bases)
`Number of bases in high quality reads
`Bases with a Phred score of 20 and above
`Re sequencing
`Reads mapped to single locations
`Number of bases in mapped reads
`Individual read insertion error rate
`Individual read deletion error rate
`Individual read substitution error rate
`Re sequencing consensus
`Average over sampling
`Coverage, all (Z $ 4)
`Consensus accuracy, all (Z $ 4)
`Consensus insertion error rate, all (Z $ 4)
`Consensus deletion error rate, all (Z $ 4)
`Consensus substitution error rate, all (Z $ 4)
`Number of contigs
`De novo assembly
`Coverage, all (Z $ 4)
`Consensus accuracy, all (Z $ 4)
`Number of contigs
`Average contig size (kb)
`
`1
`60 £ 60 mm2
`243 min/42
`306,178
`110
`33,655,553
`26,753,540
`
`238,066
`27,687,747
`1.67%
`1.60%
`0.68%
`£ 40
`99.9% (98.2%)
`99.97% (99.996%)
`0.02% (0.003%)
`0.01% (0.002%)
`0.001% (0.0003%)
`10
`
`96.54% (95.27%)
`99.96% (99.994%)
`25
`22.4
`
`The individual read error rates are referenced to the total number of bases in mapped reads.
`
`chemistry and algorithms that correct for crosstalk between wells
`suggests that the signal distributions will narrow, with an attendant
`reduction in errors and increase in read lengths. In preliminary
`experiments with genomic libraries that also include improvements
`in the emulsion protocol, we are able to achieve, using 84 cycles, read
`lengths of 200 bases with accuracies similar to those demonstrated
`here for 100 bases. On occasion, at 168 cycles, we have generated
`individual reads that are 100% accurate over greater then 400 bases.
`Using M. genitalium we demonstrate that short fragments a priori
`do not prohibit the de novo assembly of bacterial genomes. In fact, the
`larger over sampling afforded by the throughput of our system
`resulted in a draft sequence having fewer contigs than with Sanger
`reads, with substantially less effort. By taking advantage of the over
`sampling, consensus accuracies greater then 99.96% were achieved
`for this genome. Further quality filtering of the assembly results in
`the selection of a consensus sequence with accuracy exceeding
`99.99% while incurring only a minor loss of genome coverage.
`Comparable results were seen when we shotgun sequenced and de
`novo assembled the 2.1 Mb genome of Streptococcus pneumoniae14
`(Supplementary Table 4). The de novo assembly of genomes more
`complex than bacteria, including mammalian genomes, may require
`the development of methods similar to those developed for Sanger
`sequencing, to prepare and sequence paired end libraries that can
`span repeats in these genome. To facilitate the use of paired end
`libraries we have developed methods to sequence, in an individual
`well, from both ends of genomic template, and plan to add paired end
`read capabilities to our assembler (Supplementary Methods).
`Future increases in throughput, and a concomitant reduction in
`cost per base, may come from the continued miniaturization of the
`fibre optic reactors, allowing more sequence to be produced per unit
`area
`a scaling characteristic similar to that which enabled the
`
`Figure 4 | M. genitalium data. a, Read length distribution for the 306,178
`high quality reads of the M. genitalium sequencing run. This distribution
`reflects the base composition of individual sequencing templates. b, Average
`read accuracy, at the single read level, as a function of base position for the
`238,066 mapped reads of the same run.
`
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`prediction of significant improvements in the integrated circuit at the
`start of its development cycle18.
`
`Received 6 May; accepted 10 June 2005.
`Published online 31 July 2005.
`
`1.
`
`2.
`
`METHODS
`Emulsion based clonal amplification. The simultaneous amplification of frag
`ments is achieved by isolating individual DNA carrying beads in separate ,100
`m m aqueous droplets (on the order of 2 £ 106 ml
`1) made through the creation
`of a PCR reaction mixture in oil emulsion. (Fig. 1b; see also Supplementary
`Methods). The droplets act as separate microreactors in which parallel DNA
`amplifications are performed, yielding approximately 107 copies of a template
`per bead; 800 m l of emulsion containing 1.5 million beads are prepared in a
`standard 2 ml tube. Each emulsion is aliquoted into eight PCR tubes for
`amplification. After PCR, the emulsion is broken to release the beads, which
`include beads with amplified, immobilized DNA template and empty beads
`(Supplementary Methods). We then enrich for template carrying beads (Sup
`plementary Methods). Typically, about 30% of the beads will have DNA,
`producing 450,000 template carrying beads per emulsion reaction. The number
`of emulsions prepared depends on the size of the genome and the expected
`number of runs required to achieve adequate over sampling. The 580 kb M.
`
`genitalium genome, sequenced on one 60 £ 60 mm2 fibre optic slide, required
`
`1.6 ml of emulsion. A human genome, over sampled ten times, would require
`approximately 3,000 ml of emulsion.
`Bead loading into picolitre wells. The enriched template carrying beads are
`deposited by centrifugation into open wells (Fig. 1c), arranged along one face of a
`
`60 £ 60 mm2 fibre optic slide. The beads (diameter , 28 m m) are sized to ensure
`
`that no more than one bead fits in most wells (we observed that 2 5% of filled
`wells contain more than one bead). Loading 450,000 beads (from one emulsion
`
`preparation) onto each half of a 60 £ 60 mm2 plate was experimentally found to
`
`limit bead occupancy to approximately 35% of all wells, thereby reducing
`chemical and optical crosstalk between wells. A mixture of smaller beads that
`carry immobilized ATP sulphurylase and luciferase necessary to generate light
`from free pyrophosphate are also loaded into the wells to create the individual
`sequencing reactors (Supplementary Methods).
`Image capture. A bead carrying 10 million copies of a template yields
`approximately 10,000 photons at the CCD sensor, per incorporated nucleotide.
`The generated light is transmitted through the base of the fibre optic slide and
`
`detected by a large format CCD (4,095 £ 4,096 pixels). The images are processed
`
`to yield sequence information simultaneously for all wells containing template
`carrying beads. The imaging system was designed to accommodate a large
`number of small wells and the large number of optical signals being generated
`from individual wells during each nucleotide flow. Once mounted, the fibre
`optic slide’s position does not shift; this makes it possible for the image analysis
`software to determine the location of each well (whether or not it contains a
`DNA carrying bead), based on light generation during the flow of a pyropho
`sphate solution, which precedes each sequencing run. A single well is imaged by
`approximately nine 15 m m pixels. For each nucleotide flow, the light intensities
`collected by the pixels covering a particular well are summed to generate a signal
`for that particular well at that particular nucleotide flow. Each image captured by
`the CCD produces 32 megabytes of data. In order to perform all of the necessary
`signal processing in real time, the control computer is fitted with an accessory
`board (Supplementary Methods), hosting a 6 million gate Field Programmable
`Gate Array (FPGA)19,20.
`De novo shotgun sequence assembler. A de novo flow space assembler was
`developed to capture all of the information contained in the original flow based
`signal trace. It also addresses the fact that existing assemblers are not optimized
`for 80 120 bases reads, particularly with respect to memory management due to
`the increased number of sequencing reads needed to achieve equivalent genome
`coverage. (A completely random genome covered with 100 bases reads requires
`approximately 50% more reads to yield the same number of contiguous regions
`(contigs) as achieved with 700 bases reads, assuming the need for a 30 bases
`overlap between reads21.) This assembler consists of a series of modules: the
`Overlapper, which finds and creates overlaps between reads; the Unitigger, which
`constructs larger contigs of overlapping sequence reads; and the Multialigner,
`which generates consensus calls and quality scores for the bases within each
`contig (Supplementary Methods). (The names of the software modules are based
`on those performing related functions in other assemblers developed pre
`viously22.)
`
`5.
`
`6.
`
`7.
`
`8.
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`Ronaghi, M. et al. Real time DNA sequencing using detection of pyrophosphate
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`Jacobson, K. B. et al. Applications of mass spectrometry to DNA sequencing.
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`Bains, W. & Smith, G. C. A novel method for nucleic acid sequence
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`Jett, J. H. et al. High speed DNA sequencing: an approach based upon
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`Supplementary Information is linked to the online version of the paper at
`www.nature.com/nature.
`
`Acknowledgements We acknowledge P. Dacey and the support of the
`Operations groups of 454 Life Sciences. This research was supported in part by
`the US Department of Health and Human Services under NIH grants.
`
`Author Information Sequences for M. genitalium and S. pneumoniae are
`deposited at DDBJ/EMBL/GenBank under accession numbers AAGX01000000
`and AAGY01000000, respectively. Reprints and permissions information is
`available at npg.nat