Has PDF. Publication Type. More Filters. The Third Revolution in Sequencing Technology. Trends in genetics : TIG. Although next-generation sequencing NGS technology revolutionized sequencing, offering a tremendous sequencing capacity with groundbreaking depth and accuracy, it continues to demonstrate serious … Expand. Landscape of next-generation sequencing technologies. Analytical chemistry. Next-generation sequencing technologies and their impact on microbial genomics.
Briefings in functional genomics. View 1 excerpt, cites background. New sequencing technologies. Clinical and Translational Oncology. View 1 excerpt, cites methods. Highly Influenced. View 10 excerpts, cites background.
Next-Generation Sequencing. Computer Science, Biology. Next-generation DNA sequencing. Nature Biotechnology. Assembly of large genomes using second-generation sequencing. Genome research. Highly Influential. View 6 excerpts, references background. Single-molecule sequencing of an individual human genome. View 1 excerpt, references background. Finally, the massively high throughput achieved by SGS technologies per run generates mountains of highly informative data that challenge data storage and informatics operations, especially in light of the shorter reads compared with Sanger sequencing that make alignment and assembly processes challenging First-generation sequencing and SGS technologies have led the way in revolutionizing the field of genomics and beyond, motivating an astonishing number of scientific advances [for a comprehensive review of SGSs, see 11 ].
Nevertheless there are sequencing applications and aspects of genome biology that are presently beyond the reach of current sequencing technologies, leaving fertile ground for additional innovation in this space. There may not yet be consensus on what constitutes a third generation, or next—next-generation sequencing instrument, given advances are being made on rapid time scales that do not easily fit into generational time scales. However, for the purposes of this review article, we focus on SMS without the need to halt between read steps whether enzymatic or otherwise , where reads from SMS instruments represent sequencing of a single molecule of DNA.
SMS technologies that do not purposefully pause the sequencing reaction after each base incorporation represent the most thoroughly explored TGS approaches in hopes of increasing sequencing rates, throughput and read lengths, lowering the complexity of sample preparation and ultimately decreasing cost. However, as a result of using these criteria to define TGS, a number of exciting technologies do not fit neatly into this definition, but are nevertheless exciting in terms of how they complement current SGS technologies.
An interesting facet of Ion Torrent's sequencing instrument is that state-of-the-art semiconductor technology is employed to create a high-density array of micro-machined wells that carry out SBS by sensing the release of hydrogen ions as part of the base incorporation process.
This process eliminates the need for light, scanning and cameras to monitor the SBS process, thereby simplifying the overall sequencing process, dramatically accelerating the time to result, reducing the overall footprint of the instrument, and lowering cost to make DNA sequencing more generally accessible to all. As a result of this process, the overall read length is limited to that of current SGS systems, and ultimately, throughput is limited as well, compared with what SMS platforms will be capable of achieving.
The Helicos sequencing instrument works by imaging individual DNA molecules affixed to a planar surface as they are extended using a defined primer and a modified polymerase as well as proprietary fluorescently labeled nucleotide analogues, referred to as Virtual Terminator nucleotides, in which the dye is attached to the nucleotide via a chemically cleavable group that allows for step-wise sequencing to be carried out It can follow roughly one billion individual DNA molecules as they are sequenced over the course of many days.
Unlike SGS, these many hundreds of millions of sequencing reactions can be carried out asynchronously, a hallmark of TGS. Further, given individual monitoring of templates, the enzymatic incorporation step does not need to be driven to completion, which serves to reduce the overall mis-incorporation error rate.
As with the other TGS technologies discussed below, deletions and insertions are a significant issue. How third-generation DNA-sequencing technologies work. Third-generation DNA-sequencing technologies are distinguished by direct inspection of single molecules with methods that do not require wash steps during DNA synthesis. A DNA polymerase is confined in a zero-mode waveguide and base additions measured with florescence detection of gamma-labeled phosphonucleotides.
B Several companies seek to sequence DNA by direct inspection using electron microscopy similar to the Reveo technology pictured here, in which an ssDNA molecule is first stretched and then examined by STM.
C Oxford Nanopore technology for measuring translocation of nucleotides cleaved from a DNA molecule across a pore, driven by the force of differential ion concentrations across the membrane. D IBM's DNA transistor technology reads individual bases of ssDNA molecules as they pass through a narrow aperture based on the unique electronic signature of each individual nucleotide. Gold bands represent metal and gray bands dielectric layers of the transistor. The sample preparation part of this technology involves fragmenting genomic DNA into smaller pieces, adding a 3' poly A tail to the fragments, labeling and blocking by terminal transferase.
These templates are then captured onto a surface with covalently bound 5' dT 50 oligonucleotides via hybridization The surface is then imaged using charge-coupled device CCD sensors, where those templates that have been appropriately captured are identified and then tracked for SBS. The dye—nucleotide linker is then cleaved to release the dye, and this process is repeated. Instead, each RNA molecule is polyadenylated and 3'-blocked and captured on a surface coated with dT 50 oligonucleotides, similar to the DNA sequencing process.
In addition to direct RNA sequencing, the Helicos platform can carry out other sequencing-based assays such as chromatin profiling While the Helicos SMS technology has been successfully deployed, representing the first example of true SMS, with many significant advantages over SGS technologies, it has many of the characteristics of SGS technologies and so has had a more difficult time clearly differentiating itself from SGS with respect to read lengths, throughput and run times, all of which are similar to leading SGS technologies.
When combined with a higher raw read error rate requiring repetitive sequencing to overcome , the end result is a higher sequencing cost compared with leading SGS technologies. While the Helicos technology may struggle to clearly differentiate itself from SGS in some respects, the direct RNA-sequencing application is the type of advance that will come to clearly distinguish this technology from SGS.
SMS technologies can roughly be binned into three different categories: i SBS technologies in which single molecules of DNA polymerase are observed as they synthesize a single molecule of DNA; ii nanopore-sequencing technologies in which single molecules of DNA are threaded through a nanopore or positioned in the vicinity of a nanopore, and individual bases are detected as they pass through the nanopore; and iii direct imaging of individual DNA molecules using advanced microscopy techniques.
Each of these technologies provides novel approaches to sequencing DNA and has advantages and disadvantages with respect to specific applications. These technologies are at varying stages of development, making the writing of a review on TGS difficult given there is still much to prove regarding the utility of many of the TGS technologies.
However, if the full potential of these technologies is realized, in several years time, whole genome sequencing will likely be fast enough and inexpensive enough to resequence genomes as needed for any application.
Here we discuss many of the emerging TGS technologies that have the potential to make such stunning advances possible. The single-molecule real-time SMRT sequencing approach developed by Pacific Biosciences is the first TGS approach to directly observe a single molecule of DNA polymerase as it synthesizes a strand of DNA, directly leveraging the speed and processivity of this enzyme to address many of the shortcomings of SGS 14 , Given that a single DNA polymerase molecule is of the order of 10 nm in diameter, two important obstacles needed to be overcome to enable direct observation of DNA synthesis as it occurs in real time are: i confining the enzyme to an observation volume that was small enough to achieve the signal-to-noise ratio needed to accurately call bases as they were incorporated into the template of interest; and ii labeling the nucleotides to be incorporated in the synthesis process such that the dye—nucleotide linker is cleaved after completion of the incorporation process so that a natural strand of DNA remains for continued synthesis and so that multiple dyes are not held in the confinement volume at a time something that would destroy the signal-to-noise ratio.
The problem of observing a DNA polymerase working in real time, detecting the incorporation of a single nucleotide taken from a large pool of potential nucleotides during DNA synthesis, was solved using zero-mode waveguide ZMW technology Fig. The principle employed is similar to that employed in the protective screen in a microwave oven door. The screen is perforated with holes that are much smaller than the wavelength of the microwaves. Because of their relative size, the holes prevent the much longer microwaves from passing through and penetrating the glass.
However, the much smaller wavelengths of visible light are able to pass through the holes in the screen, allowing food to be visible as it is cooked. A ZMW is a hole, tens of nanometers in diameter, fabricated in a nm metal film deposited on a glass substrate.
Rather than passing through, the light exponentially decays as it enters the ZMW. Therefore, by shining laser illumination up through the glass into the ZMW, only the bottom 30 nm of the ZMW becomes illuminated. Nucleotides, each type labeled with a different colored fluorophore, are then flooded above an array of ZMWs at the required concentration.
Diffusion at the nanoscale occurs in microseconds, so that labeled nucleotides travel down into the ZMW, surround the DNA polymerase, then diffuse back up and exit the hole. As no laser light penetrates up through the holes to excite the fluorescent labels, the labeled nucleotides above the ZMWs do not contribute to the measured signals.
Only when they diffuse through the bottom 30 nm of the ZMW do they fluoresce. When the correct nucleotide is detected by the polymerase, it is incorporated into the growing DNA strand in a process that takes milliseconds, approximately three orders of magnitude longer than simple diffusion.
This difference in time results in higher signal intensity for incorporated versus unincorporated nucleotides, which creates a high signal-to-noise ratio. While held by the polymerase, the fluorescent label emits colored light. The sequencing instrument detects this as a flash whose color corresponds to the base identity.
Following incorporation, the signal immediately returns to baseline and the process repeats, with the DNA polymerase continuing to incorporate multiple bases per second. Thus, the ZMW has the ability to detect a single incorporation event against the background of fluorescently labeled nucleotides at biologically relevant concentrations. ZMWs overcome the first obstacle, but not the second. This is problematic for any system attempting to observe DNA synthesis in real time because the dye's large size relative to the DNA can interfere with the activity of the DNA polymerase.
Typically, a DNA polymerase can incorporate only a few base-labeled nucleotides before it halts. The SMRT sequencing approach instead attaches the fluorescent dye to the phosphate chain of the nucleotide rather than to the base. As a natural step in the synthesis process, the phosphate chain is cleaved when the nucleotide is incorporated into the DNA strand. Thus, upon incorporation of a phospholinked nucleotide, the DNA polymerase naturally frees the dye molecule from the nucleotide when it cleaves the phosphate chain.
Upon cleaving, the label quickly diffuses away, leaving a completely natural piece of DNA with no evidence of labeling remaining. The SMRT sequencing platform requires minimal amounts of reagent and sample preparation to carry out a run, and there are no time-consuming scanning and washing steps, enabling time to result in a matter of minutes as opposed to days Sample preparation processes for SGS technology often involve costly additional capital equipment, reagents, supplies and physical space.
The sample preparation process for SGS can take multiple days. However, with SMRT sequencing, the sample preparation consists of fragmenting the DNA into desired lengths, blunting the ends, ligating hairpin adaptors and then sequencing This provides for considerable flexibility in configuring the system for different applications.
One of the more interesting features of SMRT sequencing is the ability to observe and capture kinetic information. The ability to observe the activity of DNA polymerase in real time allows for the collection, measurement and assessment of the dynamics and timing of enzymatic incorporation, referred to as kinetics.
Via the SMRT sequencing process, changes in the kinetics of incorporation associated with chemical modifications to bases, such as methylation, can be detected in the normal course of collecting sequence data 2.
Beyond DNA sequencing, the SMRT sequencing instrument is flexible and should lead to a number of applications that are presently not approachable by any existing technology.
For example, one recently published application of the SMRT technology demonstrated direct, real-time observation of the ribosome as it translated mRNA Direct observation of other enzymes, like RNA-dependent polymerases and reverse transcriptase for RNA-sequencing applications, should also be possible. Despite the many potential advantages of SMRT sequencing, a number of challenges remain.
While ultimately the potential exists to observe many ZMWs in parallel, the first version released will be capable of only up to 75 ZMWs. Finally, as expected to be the case for most TGS technologies, SMRT sequencing data are different in form from SGS data, hence they are amenable to more advanced probabilistic modeling that has the potential to exploit more information about the chemical and structural nature of nucleotide sequences than previous sequencing technologies as discussed below for most TGS technologies.
Other SMS SBS technologies are in development, but little data are available to assess where they are at in development and when they are likely to be released. VisiGen Biotechnologies had one of the more promising approaches to SMS whereby the DNA polymerase is tagged with a fluorophore that when brought into close proximity to a nucleotide, tagged with an acceptor fluorophore, would emit a fluorescence resonance energy transfer FRET signal.
After incorporation, the fluorophore label on the nucleotide can be released. This type of approach could be considered an improvement over the Helicos technology, and has the potential to move at millions of bases per second, given potential for high multiplex.
Visigen Biotechnologies was acquired by Life Technologies recently, and the FRET technology seems to have become one of centerpieces of their SMS efforts, but presently it is hard to gauge progress. Halcyon Molecular is pioneering an SMS approach using transmission electron microscopy TEM to directly image and chemically detect atoms that would uniquely identify the nucleotides comprising a DNA template.
The approach being pursued has been shown to reliably detect atoms in a non-periodic material on a planar surface, using annular dark-field imaging in an aberration-corrected scanning TEM This approach harkens back to a lecture Richard Feynman gave in at the annual meeting of the American Physical Society at Caltech where he indicated the easiest way to study important biomolecules like DNA, RNA and proteins was to look at them directly.
Beyond the TEM technology, Halcyon is developing a number of supporting technologies required to carry out TEM-based DNA sequencing, like the use of functionalized needles to attach stretched molecules of DNA to a substrate for the direct imaging procedure. As of the writing of this review, no publications have appeared demonstrating this procedure for DNA sequencing, but if successfully implemented, the chief advantage of the technology would be very long read lengths potential into the many millions of bases at low cost.
With this technology, labeled atoms within the nucleotides of DNA are imaged using a high-resolution sub-angstrom electron microscope where individual bases are detected and identified based on their size and intensity differences between the different labeled bases. While no proof of concept studies have yet been published regarding this technology, ZS Genetics claims that the technology is capable of producing 10 —20 base reads at a rate of 1.
Like most of the other TGS technologies, read length and reduced costs are expected to be the chief advantages. Reveo is developing a technology related to IBM's DNA transistor approach see subsequently in which DNA is placed on a conductive surface to detect bases electronically using scanning tunneling microscope STM tips and tunneling current measurements The STM tips are knife-edge shaped and have nanoscale dimensions Fig.
The aim in applying this technology to SMS is to stretch and confine a molecule of DNA such that tunneling current measurements can be taken to identify individual bases.
The procedure for linearizing and depositing DNA sequences on a conductive surface for this application has not yet been described. No proof of concept study for DNA sequencing has been published, but the advantages of this type of technology are expected again to be speed, very long read lengths and a significant reduction in cost, given labeling can be avoided. Most nanopore sequencing technologies rely on transit of a DNA molecule or its component bases through a hole and detecting the bases by their effect on an electric current or optical signal.
Because this type of technology uses single molecules of unmodified DNA, they have the potential to work quickly on extremely small amounts of input material. Both biological nanopores constructed from engineered proteins and entirely synthetic nanopores are under development.
In particular, there is potential to use atomically thin sheets of grapheme as a matrix supporting nanopores 35 and also carbon nanotubes Oxford Nanopore is commercializing a system for DNA sequencing based on three natural biological molecules that have been engineered to work as a system Fig. A synthetic cyclodextrin sensor is also covalently attached to the inside surface of the nanopore. This system is contained in a synthetic lipid bilayer so that when DNA is loaded onto its exonuclease-containing face and a voltage is applied across the bilayer by changing the concentration of salt, the exonuclease can cleave off individual nucleotides.
The individual nucleotides are detected once they are cleaved based on their characteristic disruption of the ionic current flowing through the pore. Reliable throughput at high multiplex may be difficult to achieve with this system using natural lipid bilayers, but synthetic membranes and solid-state nanopores, if developed, may help overcome this challenge. Like many of the other TGS technologies in this category, the advantages are expected to be long read length and high scalability at low cost, given the technology is driven by electronics, not optics.
Another approach aims to use a biological nanopore directly on intact DNA. Unlike Oxford Nanopore, which addresses axial resolution limitations in the alpha-haemolysin pore by disassembling the DNA molecule, in this case, the Mycobacterium smegmatis Porin A MspA protein, which has a shorter blockade region and thus a better resolution, is used as the pore and the effect of a linear molecule of single-stranded DNA ssDNA on the current transiting the pore is measured To slow the transit of the ssDNA through the pore to a level allowing detection of individual bases as they interrupt current transiting the pore, a region of double-stranded DNA dsDNA is introduced.
The ability of this method to directly measure ssDNA in a processive fashion is attractive, but the complexity of introducing the needed dsDNA break on the pore transit velocity appears to be a significant obstacle at this point to an efficient large-scale laboratory workflow for routine DNA sequencing. One significant challenge in nanopore-based sequencing lies in the need for simultaneously monitoring a large number of nanopores.
The first parallel readout of any nanopore-based method has recently been demonstrated through the use of optical multipore detection In this approach, the contrast between the four bases is first increased off-line through a biochemical process that converts each base in the DNA into a specific, ordered pair of concatenated oligonucleotides. Subsequently, two different fluorescently labeled molecular beacons are hybridized to the converted DNA. The beacons are then sequentially unzipped from the DNA molecules as they are translocated through a nanopore.
Each unzipping event unquenches a new fluorophore, resulting in a series of dual-color fluorescence pulses that are detected by a high-speed CCD camera with a conventional total internal reflection fluorescence microscopy setup. The unzipping process is slowed down by adjusting the voltage governing DNA translocation through the nanopore to a speed compatible with single-molecule optical detection. With the feasibility of the components of this approach demonstrated, it will be interesting to see whether the potential of extremely high throughput can be achieved through faithful and unbiased biochemical conversion, and accurate, long-read sequencing with high parallelism.
IBM is developing a nanostructured sequencing device capable of electronically detecting individual bases in a single molecule of DNA Fig. The nanostructures are nanometer-sized pores. The surface of the pores consists of axially stratified, alternating layers of metal and dielectric material like a transistor.
Single DNA molecules can then be passed through the pores, controlling the motion of the DNA through the pores by appropriately modulating the current in the electrodes of the transistor. Speed, read length and low cost are again the chief advantages of this type of approach. In fact, the speed of sequencing could be very dramatically increased with this approach, given the theoretical limit has been computed to be bases read per transistor per second.
In addition, like other TGS technologies in this category, the assay would be label free and require no optics, again greatly diminishing cost. While the original DNA transistor idea proposed was based on theoretical calculations and molecular dynamic simulations 41 , IBM recently published a solution to one of the two technical challenges facing this approach: modulation of the speed with which the DNA molecule is passed through the nanopore to enable optimal base orientation as well as sufficient sampling of a base as it passes through the nanopore The other challenge remaining for this approach is demonstrating that the signal for a single base can be distinguished from the signals of nearby bases.
A recent publication related to this challenge indicates via simulation that factors such as ionic motion in the nanopore may not necessarily affect the desired signal of an individual base In particular, IBM's approach will not have the same issues with respect to spatial resolution and sensitivity, which are issues with Oxford Nanopore's approach However, an advantage of Oxford Nanopore over the DNA transistor is that it requires less detection sensitivity, given it is detecting cleaved bases, not intact DNA molecules.
The informatics challenges with SGS technologies are largely due to the short reads that are characteristic of these technologies The short-read nature of SGS makes it difficult, even with paired-end reads, to assemble complete genomes de novo. Indeed, nearly all human genomes sequenced to date have been assembled using reference-based mapping algorithms While this assembly approach is efficient for accurately identifying SNPs in the human genome 44 , 45 , it does not enable a thorough characterization of structural variations, insertions and deletions.
Only de novo assembly of individual genomes can accomplish this feat. While reasonable assemblies are now feasible using state-of-the-art SGS technologies and algorithms, they are still not capable of achieving the assembly qualities that can be achieved using first-generation Sanger sequencing, with hybrid sequencing approaches that include data generated from multiple technologies now becoming a more standard way to enhance the quality of assemblies Most of the TGS technologies discussed address or have the potential to address the limitations of SGS technologies with respect to assembly quality, given the read lengths and mate-pair distances in TGS are not only significantly beyond those realized with SGS, but with Sanger sequencing as well.
Longer reads can span repeat regions that make assembly difficult and can obviate the need for more complex mate-pair strategies required to scaffold SGS reads. As an example, depicted in Figure 3 A are seven contigs assembled using Abyss 54 applied to short read data generated from the genome of Rhodopseudomonas palustris using the Illumina GA platform. Because the six blue contigs are overlapping, the red contig representing a 1. However, in Figure 3 B, we depict just three molecules of long-range sequencing data Figure 3 , legend from the TGS SMRT sequencing platform that span the repeat and unambiguously resolve how the contigs should be ordered with respect to one another.
Long reads span long repeats to unambiguously orient contigs. TGS Technologies are capable of generating long reads that are critical for de novo assembly of genomes. A Contigs assembled from short read data alone cannot be unambiguously ordered because they overlap but do not span a repeat region. B Depicted here by colored traces are individual single-molecule sequences that span several thousand bases, including a copy of the repeat region, overlapping the flanking contigs to unambiguously resolve the contig order.
Its strengths notwithstanding, TGS will come with its own set of challenges. Because a TGS system by definition assays a single molecule, there is no longer any safety in numbers to minimize raw read errors. For example, if unlabeled nucleotide were present at 0. Similarly, if a base fails to progress through a nanopore or a DNA transistor as intended and gets counted twice, there will be an insertion in the raw data.
Hence, the frequency of errors for raw reads will likely be greater, and the error profile of TGS will certainly differ from that of earlier technologies, so both will need to be accounted for in the algorithms that analyze TGS data. However, because the error profile may be less biased more uniform , the consensus accuracies have the potential to be significantly higher than that of SGS. While the longer read lengths of TGS will ease many of the informatics challenges relating to assembly now experienced by those focussed on SGS data, the increased information content will demand new types of mathematical models and algorithms to get the most from the data.
For example, real-time monitoring of SMS events can provide kinetic information that transforms one's ability to understand each base as it is incorporated e. In addition, because a single molecule at a time is being monitored, the error structure will be significantly different from the ensemble-based approach employed by SGS technologies, with higher error rates for raw reads, but then consensus sequences converging more rapidly to higher quality sequences, given significantly fewer biases in the distribution of errors 14 , Therefore, because this new generation of sequencing technologies provides for a significant shift in how sequencing is carried out, they demand a new generation of analysis tools to derive maximal information from the raw data.
As a result, for each cluster, there correspond four images per cycle, and the analysis proceeds by analyzing each of the images and quantitating the intensities for each cluster and selecting the dominant intensity to determine the most likely base for a given cluster at a given cycle.
The primary issues relating to analysis of these data such that the accuracy of the base calls is optimized are crosstalk, dephasing and chastity filtering In SMRT sequencing, for example, nucleotides are labeled with four different fluorescent dyes randomly diffusing throughout the sequencing chamber and illuminated by a laser only when the polymerase binds the nucleotide to incorporate it into the sequence being synthesized.
A camera monitors illumination events at a rate of frames per second over the course of a sequencing run typically 15 min , thereby producing a movie comprising 90 frames for a 15 min run. Primary analysis in this instance involves quantifying the intensities for each channel for each sequencing reaction, identifying the illumination events as pulses and then translating the pulses into base calls. While crosstalk between the different dyes is still an issue with this type of sequencing, the phasing correction and chastity filtering are no longer necessary, given the asynchronous nature of SMS.
However, given the stochastic fluctuations that result from interrogating a single-template DNA molecule, a number of issues arise that lead to uncertainty around the number and identity of bases read for a given template. For example, a given incorporation event may be missed because the number of photons emitted from the dye attached to the newly incorporated nucleotide could not be distinguished from the background noise, or the polymerase may fail to incorporate a nucleotide and try multiple times before succeeding, creating what appear to be multiple consecutive incorporation events for the same base There are well-established mathematical frameworks for modeling trace data so that inferences around the interpretation of those traces can be made with respect to the underlying DNA sequence any given trace represents.
Most generally, from a given sequencing trace, we detect pulses that represent contiguous time segments in which the intensity for a given channel goes from a background state to significantly above background over the period of time defined by the time segment.
A pulse represents an illumination event during the sequencing process that ideally signifies the incorporation of a specific nucleotide into the sequence being synthesized. Each o i can be a measurement or vector of measurements that characterize a given observation event.
In the first-generation sequencing and SGS contexts, each interpretation t i of observation o i represents either a single nucleotide base from the alphabet A , G , C , T or is empty. Because the components of T and O are random variables, the aim is to find the best sequence of interpretations given a sequence of observations.
While this approach has worked well for FGS and SGS, there are two significant issues relating to the nature of most TGS technologies that necessitate a more advanced formulation of the type of mathematical model just described. First, there are significant stochastic components of SMS that complicate the relationship between observations and interpretations of those observations. For example, pulses observed in SMRT-sequencing trace data will not perfectly convey the sequence of incorporation events—the random and exponentially distributed pulse widths and inter-pulse durations mean that in some instances pulses or gaps between pulses can go undetected.
As a result, a final output consisting of a single sequence representation of a given template will not fully reflect the likelihood any given base is correctly positioned, has been called correctly or has been missed or incorrectly inserted.
By appropriately characterizing this uncertainty and incorporating it into the analysis, the ability to appropriately map a given sequence to the correct region of a genome, provide for alternative base calls at any given position or identify more general structural variants is improved.
The second major issue relates to the amount of data that will ultimately be achievable with third-generation technologies.
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