Real-Time DNA Sequencing from Single Polymerase Molecules

Eid, John; Fehr, Adrian; Gray, Jeremy; Luong, Khai; Lyle, John; Otto, Geoff; Peluso, Paul; Rank, David R.; Baybayan, Primo; Bettman, Brad; Bibiłło, Arkadiusz; Bjornson, Keith P.; Chaudhuri, Bidhan; Christians, Frederick; Cicero, Ronald L.; Clark, Sonya; Dalal, Ravindra V.; deWinter, Alex D.; Dixon, J; Foquet, Mathieu; Gaertner, Alfred; Hardenbol, Paul; Heiner, Cheryl; Hester, Kevin; Holden, David W.; Kearns, Gregory L.; Kong, Xiangxu; Kuse, Ronald; Lacroix, Y.; Lin, Steven; Lundquist, P. M.; Ma, Congcong; Marks, Patrick; Maxham, Mark; Murphy, Devon; Park, Insil; Pham, Thang; Phillips, Michael; Roy, Joy; Sebra, Robert; Shen, Gene; Sorenson, Jon M.; Tomaney, A.; Travers, Kevin; Trulson, Mark O.; Vieceli, John; Wegener, Jeffrey; Wu, Dawn; Yang, Alicia; Zaccarin, D.; Zhao, Peter; Zhong, Frank; Korlach, Jonas; Turner, Stephen W.

doi:10.1126/science.1162986

Cited by 3,222 publications

(2,545 citation statements)

References 37 publications

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“…The principles behind these four systems are schematically depicted in Figure 3. Additional platforms exist, such as the Polonator[62], the Helicos Single Molecule Sequencer[63], and an in-house only nanoarray-based sequencing-by-ligation technology developed by Complete Genomics[64]. These latter systems remain quite limited in their use and will not be discussed.…”

Section: Massively Parallel Sequencingmentioning

confidence: 99%

“…Pacific Biosciences’ PacBio RS detects fluorescently labeled nucleotides in real time as they are incorporated during a second-strand synthesis (Fig. 3)[63, 120]. Oxford Nanopore’s sequencing platform uses an exonuclease cleavage reaction and a protein nanopore to sequence individual cleaved bases by a unique electrical signature as they are transported through the pore[121].…”

Section: Future Prospectsmentioning

confidence: 99%

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Direct mutation analysis by high-throughput sequencing: From germline to low-abundant, somatic variants

Gundry

Vijg

2012

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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DNA mutations are the source of genetic variation within populations. The majority of mutations with observable effects are deleterious. In humans mutations in the germ line can cause genetic disease. In somatic cells multiple rounds of mutations and selection lead to cancer. The study of genetic variation has progressed rapidly since the completion of the draft sequence of the human genome. Recent advances in sequencing technology, most importantly the introduction of massively parallel sequencing (MPS), have resulted in more than a hundred-fold reduction in the time and cost required for sequencing nucleic acids. These improvements have greatly expanded the use of sequencing as a practical tool for mutation analysis. While in the past the high cost of sequencing limited mutation analysis to selectable markers or small forward mutation targets assumed to be representative for the genome overall, current platforms allow whole genome sequencing for less than $5,000. This has already given rise to direct estimates of germline mutation rates in multiple organisms including humans by comparing whole genome sequences between parents and offspring. Here we present a brief history of the field of mutation research, with a focus on classical tools for the measurement of mutation rates. We then review MPS, how it is currently applied and the new insight into human and animal mutation frequencies and spectra that has been obtained from whole genome sequencing. While great progress has been made, we note that the single most important limitation of current MPS approaches for mutation analysis is the inability to address low-abundance mutations that turn somatic tissues into mosaics of cells. Such mutations are at the basis of intra-tumor heterogeneity, with important implications for clinical diagnosis, and could also contribute to somatic diseases other than cancer, including aging. Some possible approaches to gain access to low-abundance mutations are discussed, with a brief overview of new sequencing platforms that are currently waiting in the wings to advance this exploding field even further.

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Section: Massively Parallel Sequencingmentioning

confidence: 99%

Section: Future Prospectsmentioning

confidence: 99%

Direct mutation analysis by high-throughput sequencing: From germline to low-abundant, somatic variants

Gundry

Vijg

2012

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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“…However, to improve accuracy in such endeavors, there is a need for genomic resources with far more contiguous sequence and less pervasive gaps. The acquisition of such contiguous sequences is possible now with third generation sequencing technologies, such as Single Molecule Real Time (SMRT) sequencing technology [Eid et al, 2009], which provide mean read lengths that are more than an order of magnitude larger than earlier sequencing technologies. The reads can span repetitive elements, resulting in longer contigs and minimal gaps within scaffolds [Bickhart et al, 2017], [Jiao et al, 2017], [Gordon et al, 2016].…”

Section: Introductionmentioning

confidence: 99%

A reference genome of the Chinese hamster based on a hybrid assembly strategy

Rupp

MacDonald

et al. 2018

Biotech & Bioengineering

103

121

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Accurate and complete genome sequences are essential in biotechnology to facilitate genome-based cell engineering efforts. The current genome assemblies for Cricetulus griseus, the Chinese hamster, are fragmented and replete with gap sequences and misassemblies, consistent with most short-read based assemblies. Here, we completely resequenced C. griseus using Single Molecule Real Time (SMRT) sequencing and merged this with Illumina-based assemblies. This generated a more contiguous and complete genome assembly than either technology alone, reducing the number of scaffolds by >28-fold, with 90% of the sequence in the 122 longest scaffolds. Most genes are now found in single scaffolds, including up- and downstream regulatory elements, enabling improved study of noncoding regions. With >95% of the gap sequence filled, important CHO cell mutations have been detected in draft assembly gaps. This new assembly will be an invaluable resource for continued basic and pharmaceutical research.

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“…Determination of DNA sequences from a single DNA can result in an ultra-high-throughput sequencing of DNA or RNA without any amplification, thus reducing cost and increasing accuracy compared to the conventional Sanger sequencing techniques. Recently these optical (Eid et al, 2009) or electrochemical (Rothberg et al, 2011) methods for singlemolecule DNA sequencing have been commercialized. Furthermore, Zong et al (2012) identified single-nucleotide and copy-number variations via sequencing single cell genome without error-prone exponential amplification.…”

Section: Dna Sequencingmentioning

confidence: 99%

Recent Advances in Nanobiotechnology and High-Throughput Molecular Techniques for Systems Biomedicine

Kim

Ahn

Chung

et al. 2013

Molecules and Cells

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Nanotechnology-based tools are beginning to emerge as promising platforms for quantitative high-throughput analysis of live cells and tissues. Despite unprecedented progress made over the last decade, a challenge still lies in integrating emerging nanotechnology-based tools into macroscopic biomedical apparatuses for practical purposes in biomedical sciences. In this review, we discuss the recent advances and limitations in the analysis and control of mechanical, biochemical, fluidic, and optical interactions in the interface areas of nanotechnologybased materials and living cells in both in vitro and in vivo settings.

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Real-Time DNA Sequencing from Single Polymerase Molecules

Abstract: We report here on the genome sequence of Pasteurella multocida Razi 0002 of avian origin, isolated in Iran. The genome has a size of 2,289,036 bp, a GC content of 40.3%, and is predicted to contain 2,079 coding sequences.

Cited by 3,222 publications

References 37 publications

Direct mutation analysis by high-throughput sequencing: From germline to low-abundant, somatic variants

Direct mutation analysis by high-throughput sequencing: From germline to low-abundant, somatic variants

A reference genome of the Chinese hamster based on a hybrid assembly strategy

Recent Advances in Nanobiotechnology and High-Throughput Molecular Techniques for Systems Biomedicine

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