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Journal Article•DOI•

Sequencing technologies-the next generation

Michael L. Metzker1•
Baylor College of Medicine1
01 Jan 2010-Nature Reviews Genetics (Nature Publishing Group)-Vol. 11, Iss: 1, pp 31-46
TL;DR: A technical review of template preparation, sequencing and imaging, genome alignment and assembly approaches, and recent advances in current and near-term commercially available NGS instruments is presented.
Abstract: Demand has never been greater for revolutionary technologies that deliver fast, inexpensive and accurate genome information. This challenge has catalysed the development of next-generation sequencing (NGS) technologies. The inexpensive production of large volumes of sequence data is the primary advantage over conventional methods. Here, I present a technical review of template preparation, sequencing and imaging, genome alignment and assembly approaches, and recent advances in current and near-term commercially available NGS instruments. I also outline the broad range of applications for NGS technologies, in addition to providing guidelines for platform selection to address biological questions of interest.

Summary (1 min read)

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Summary

  • DNA sequencing is one of the most important platforms for study in biological systems today.
  • The high-throughput-next generation sequencing technologies delivers fast, inexpensive, and accurate genome information.
  • Next generation sequencing can produce over 100 times more data than methods based on Sanger Sequencing.
  • The next generation sequencing technologies offered from Illumina / Solexa, ABI/SOLiD, 454/Roche, and Helicos has provided unprecedented opportunity for high-throughput functional genomic research.
  • Next generation sequence technologies offer novel and rapid ways for genome-wide characterization and profiling of mRNA's, transcription factor regions, and DNA patterns.

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ABSTRACT
Conclusion and Future Work
Next Generation Sequencing
CONTACT INFO
Data Analysis Comparisons
Downstream Analysis
REFERENCES
DNA sequencing is one of the most important platforms for
study in biological systems today. The high-throughput-next
generation sequencing technologies delivers fast,
inexpensive, and accurate genome information. Next
generation sequencing can produce over 100 times more data
than methods based on Sanger Sequencing. The next
generation sequencing technologies offered from Illumina /
Solexa, ABI/SOLiD, 454/Roche, and Helicos has provided
unprecedented opportunity for high–throughput functional
genomic research. Next generation sequence technologies
offer novel and rapid ways for genome-wide characterization
and profiling of mRNA’s, transcription factor regions, and DNA
patterns.
Fig. 7) This is a plot of the frequency of each percentage covered for all nodes.
BLAST is in blue, MUMmer is in green.
Sequencing Technologies – the Next Generation,
Micahel L. Metzkerh
Next Generation Sequencing Pipeline Development and Data Analysis
Fig. 9) This is a plot of the coverage of each Node. BLAST points are blue,
MUMmer points are red.
Fig. 6) This is a plot of the frequency of each percentage covered for all contigs.
BLAST is in blue, MUMmer is in green.
•  454/Roche – 454 Life Sciences is a Biotechnology company
that is a part of Roche and based in Branford, Connecticut.
The center develops ultra-fast high-throughput DNA
sequencing methods and tools.
•  Illumina/Solexa– Illumina is a company that develops and
manufactures integrated systems for the analysis of gene
variation. Solexa was founded to develop genome
sequencing technology.
•  ABI/SOLiD - (Sequencing by Oligonucleotide Ligation and
Detection) is a next-generation DNA sequencing technology
developed by Life Technologies and has been commercially
available since 2006. This next generation technology
generates hundreds of millions to billions of small sequence
reads at one time.
•  Helicos - Helicos's technology images the extension of
individual DNA molecules using a defined primer and
individual fluorescently labeled nucleotides, which contain a
"virtual terminator" preventing incorporation of multiple
nucleotides per cycle.
Julian Pierre
1
, Jordan Taylor
2
, Amit Upadhyay
3
, Bhanu Rekepalli
3
Fig. 8) This is a plot of the coverage of each Contig. BLAST points are blue,
MUMmer points are red.
Using the coverage of
each individual contig
ID, the results for both
BLAST and MUMmer
were plotted. While
BLAST hit more contigs,
there are more contigs
with a higher coverage
that were hit by
MUMmer.
Using the data gathered
from both BLAST and
MUMmer, the frequency
of the amount covered
for each contig was
plotted. From Fig 6), it
can be inferred that
MUMmer hit more
accurately for contigs.
Fig 4) from main.g2.bx.psu.edu
Once the results were found using both the BLAST and
MUMmer search tools, we created a program to see which
sequencing tool had the most hits per contig. The total
number of contigs in the database file is 160,749 and the
total number of nodes in the query file is 552,305. BLAST
returned a total of 123,070 hits and MUMmer returned a
total of 121,829 hits. From the results, MUMmer hit more
accurately than BLAST while BLAST hit more contigs than
MUMmer.
In Next-Generation Sequencing, data analysis is one of the
most expensive processes. While the cost of genome
sequencing goes down, the cost of analyzing data is still
expensive. In the future, the “$1,000 genome will come with
a $20,000 analysis price tag.”
The same process was
done with the Nodes.
From Fig 7), it can be
inferred that BLAST hit
more accurately with
nodes. However, there
are more BLAST results
with lower coverage.
The future of next generation sequencing can be broken
down into a variety of categories such as personalized
medicine, bio fuels, climate change, and other life science
fields.
•  Personalized Medicine is a medical model that proposes
the customization of medical decision to tailor an
individual
•  Bio Fuels present a source of alternative energy.
Microalgal biofuels use algae to synthesize the fuel. In
order to optimize the process, an understanding of the
gene-function relationship of algae would prove helpful.
•  Climate change is the active study of past and future
theoretical models which uses the past climate data to
make future projections.
In conclusion, we hope to contribute the knowledge we
have gained to contribute to fields such as these.
The same process was
done with the Nodes.
While BLAST hit more
Nodes, there are more
Nodes that hit with a
lower coverage using
BLAST.
1 Texas Southern University, 2 Austin Peay State University, 3 University of Tennessee
Next Gen Sequencing uses a wide array of tools to obtain results based
on the genome sequence. The most widely used Tools are BLAST,
HMMER, and MUMmer.
•  BLAST (Basic Local Alignment Search Tool) is a multi-sequence
alignment tool developed by NIH (National Institute of Health). It is
used find similar regions in different sequences and then compare
their similarities.
•  MUMmer (Maximum Unique Matches) is a rapid alignment system
used for rapidly aligning entire genomes. It can also align incomplete
genomes and can easily handle 1000’s of contigs from a shotgun
sequencing project.
•  HMMER (Hidden Markov Modeler) is used for searching sequence
databases for homologs of protein sequences, and for making protein
sequence alignments. It implements methods using probabilistic
models called profile hidden Markov models (HMMs)
Genome Assembly
Sequence Analysis refers to
the process of subjecting a
DNA, RNA or peptide
sequence to a wide range of
analytical methods to:
•  Compare sequences to find
similarities and infer if they
are Homologous
•  To identify the features of
the sequence such as gene
structure, distribution,
introns and exons, and
regulation of gene
expression
•  Identify Sequence
differences and variations
such as mutations
Fig. 1) This is figure shows three different Next Generation Sequencing methods. [2]
Fig. 2) Taken from A Hitchhiker’s Guide to Next-Generation Sequencing, by Gabe Rudy
Fig. 3) Taken from bio.davidson.edu/courses. Shows alignment results for yeast.
Fig 5) from jcvi.org shows the mapping of chr6 of a Human Genome
Julian Pierre – julz_pierre@yahoo.com
Jordan Taylor – jtaylor74@my.apsu.edu
Amit Upadhyay – aupadhy1@utk.edu
Bhanu Rekepalli – brekapal@utk.edu
•  http://www.roche.com/research_and_development/r_d_overview/
r_d_sites.htm?id=18
•  http://www.pnas.org/content/99/6/3712/F1.expansion.html
•  http://www.yerkes.emory.edu/nhp_genomics_core/Services/
Sequencing.html
•  http://www.illumina.com/technology/solexa_technology.ilmn
•  http://blast.ncbi.nlm.nih.gov/Blast.cgi
•  https://main.g2.bx.psu.edu/u/dan/p/fastq
•  http://ori.dhhs.gov/education/products/n_illinois_u/datamanagement/
datopic.htmll
•  http://www.jcvi.org/medicago/include/images/chr6.BamHI.maps.jpg
•  Gabe Rudy, (2010) A Hitchhiker’s Guide to Next-Generation
Sequencing, :1-9, Golden Helix
•  [1] John D. McPherson, (2009) Next-Generation Gap, 6:1-4, Nature
Methods Supplement
•  [2]Michael L. Metzker, (2010) Sequencing Technologies, - the next
generation, 11:1-5, Nature Reviews
•  Md. Fakruddin,Khanjada Shahnewaj Bin mannan, (2012) Next
Generation sequencing technologies – Principles and prospects,
6:1-9, Research and Reviews in Biosciences
•  Misra N., Panda P. K., Parida B. K., Mishra B. K., (2012)
Phylogenomic Study of Lipid Genes Involved in Mocroalgal Biofuel
Production – Candidate Gene Mining and Metabolic Pathway
Analyses, Evolutionary Bioinformatics 8:545-564, doi: 10.4137/
EBO.S10159
Galaxy is an open, web-based
platform for data intensive
biomedical research. It can be
used on its own free public
server where you can perform,
reproduce, and share complete
analyses.
An example of how Galaxy
reflects its data is shown in Fig 5.
Two FASTA files related to the same nucleotide sequence
were input into both BLAST and MUMmer and the results
were parsed into tables. Then, the coverage of all hit contigs
and nodes from both programs was found.
Citations
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Journal Article•DOI•
Diversity in global maize germplasm: Characterization and utilization

[...]

Boddupalli M. Prasanna1•
International Maize and Wheat Improvement Center1
23 Oct 2012-Journal of Biosciences
TL;DR: ‘Seeds of Discovery’ (SeeD), a novel initiative by CIMMYT with financial support from the Mexican Government for generating international public goods, has initiated intensive exploration of phenotypic and molecular diversity of maize germplasm conserved in the CIMmyT Gene Bank, expected to aid in effective identification and use of novel alleles and haplotypes for maize improvement.
Abstract: Maize (Zea mays L.) is not only of worldwide importance as a food, feed and as a source of diverse industrially important products, but is also a model genetic organism with immense genetic diversity. Although it was first domesticated in Mexico, maize landraces are widely found across the continents. Several studies in Mexico and other countries highlighted the genetic variability in the maize germplasm. Applications of molecular markers, particularly in the last two decades, have led to new insights into the patterns of genetic diversity in maize globally, including landraces as well as wild relatives (especially teosintes) in Latin America, helping in tracking the migration routes of maize from the centers of origin, and understanding the fate of genetic diversity during maize domestication. The genome sequencing of B73 (a highly popular US Corn Belt inbred) and Palomero (a popcorn landrace in Mexico) in the recent years are important landmarks in maize research, with significant implications to our understanding of the maize genome organization and evolution. Next-generation sequencing and high-throughput genotyping platforms promise to further revolutionize our understanding of genetic diversity and for designing strategies to utilize the genomic information for maize improvement. However, the major limiting factor to exploit the genetic diversity in crops like maize is no longer genotyping, but high-throughput and precision phenotyping. There is an urgent need to establish a global phenotyping network for comprehensive and efficient characterization of maize germplasm for an array of target traits, particularly for biotic and abiotic stress tolerance and nutritional quality. ‘Seeds of Discovery’ (SeeD), a novel initiative by CIMMYT with financial support from the Mexican Government for generating international public goods, has initiated intensive exploration of phenotypic and molecular diversity of maize germplasm conserved in the CIMMYT Gene Bank; this is expected to aid in effective identification and use of novel alleles and haplotypes for maize improvement. Multi-institutional efforts are required at the global level to systematically explore the maize germplasm to diversify the genetic base of elite breeding materials, create novel varieties and counter the effects of global climate changes.

168 citations

Cites background from "Sequencing technologies-the next ge..."

  • ...Another important recent development is the availability of platforms for undertaking next-generation sequencing and high-density genotyping (Metzker 2010)....

    [...]

Journal Article•DOI•
High Throughput Sequencing For Plant Virus Detection and Discovery

[...]

D E V Villamor1, Thien Ho1, M. Al Rwahnih2, Robert R. Martin3, Ioannis E. Tzanetakis1 •
University of Arkansas System1, University of California, Davis2, United States Department of Agriculture3
01 Apr 2019-Phytopathology
TL;DR: A description of the process of plant virus detection and discovery from HTS data is presented, providing a summary of the different pipelines available for scientists' utility in their research.
Abstract: Over the last decade, virologists have discovered an unprecedented number of viruses using high throughput sequencing (HTS), which led to the advancement of our knowledge on the diversity of viruses in nature, particularly unraveling the virome of many agricultural crops. However, these new virus discoveries have often widened the gaps in our understanding of virus biology; the forefront of which is the actual role of a new virus in disease, if any. Yet, when used critically in etiological studies, HTS is a powerful tool to establish disease causality between the virus and its host. Conversely, with globalization, movement of plant material is increasingly more common and often a point of dispute between countries. HTS could potentially resolve these issues given its capacity to detect and discover. Although many pipelines are available for plant virus discovery, all share a common backbone. A description of the process of plant virus detection and discovery from HTS data are presented, providing a summary of the different pipelines available for scientists' utility in their research.

168 citations

Cites background from "Sequencing technologies-the next ge..."

  • ...…how these different HTS platforms perform the sequencing and the amount and reliability of data that each generates (Rothberg and Leamon 2008; Metzker 2010; Glenn 2011; Mardis 2013, 2008; Buermans and den Dunnen 2014; Feng et al. 2015; Reuter et al. 2015; Rhoads and Au 2015; Goodwin et al.…...

    [...]

  • ...Several reviews have been published that describe how these different HTS platforms perform the sequencing and the amount and reliability of data that each generates (Rothberg and Leamon 2008; Metzker 2010; Glenn 2011; Mardis 2013, 2008; Buermans and den Dunnen 2014; Feng et al. 2015; Reuter et al. 2015; Rhoads and Au 2015; Goodwin et al. 2016; Heather and Chain, 2016; Levy and Myers 2016; Lu et al. 2016;Mardis 2017) and therefore this aspect will not be discussed here....

    [...]

Journal Article•DOI•
Identification by whole-genome resequencing of gene defect responsible for severe hypercholesterolemia

[...]

Jonathan J. Rios1, Evan Stein, Jay Shendure2, Helen H. Hobbs1, Jonathan C. Cohen1 •
University of Texas Southwestern Medical Center1, University of Washington2
15 Nov 2010-Human Molecular Genetics
TL;DR: The genome of an 11-month-old breast-fed girl with xanthomas and very high plasma cholesterol levels is sequences to demonstrate that whole-genome (or exome) sequencing can be a valuable aid to diagnose genetic diseases, even in individual patients.
Abstract: Whole-genome sequencing is a potentially powerful tool for the diagnosis of genetic diseases. Here, we used sequencing-by-ligation to sequence the genome of an 11-month-old breast-fed girl with xanthomas and very high plasma cholesterol levels (1023 mg/dl). Her parents had normal plasma cholesterol levels and reported no family history of hypercholesterolemia, suggesting either an autosomal recessive disorder or a de novo mutation. Known genetic causes of severe hypercholesterolemia were ruled out by sequencing the responsible genes (LDLRAP, LDLR, PCSK9, APOE and APOB), and sitosterolemia was ruled out by documenting a normal plasma sitosterol:cholesterol ratio. Sequencing revealed 3 797 207 deviations from the reference sequence, of which 9726 were nonsynonymous single-nucleotide substitutions. A total of 9027 of the nonsynonymous substitutions were present in dbSNP or in 21 additional individuals from whom complete exonic sequences were available. The 699 novel nonsynonymous substitutions were distributed among 604 genes, 23 of which were single-copy genes that each contained 2 nonsynonymous substitutions consistent with an autosomal recessive model. One gene, ABCG5, had two nonsense mutations (Q16X and R446X). This finding indicated that the infant has sitosterolemia. Thus, whole-genome sequencing led to the diagnosis of a known disease with an atypical presentation. Diagnosis was confirmed by the finding of severe sitosterolemia in a blood sample obtained after the infant had been weaned. These findings demonstrate that whole-genome (or exome) sequencing can be a valuable aid to diagnose genetic diseases, even in individual patients.

168 citations

Cites background from "Sequencing technologies-the next ge..."

  • ...The use of massively parallel DNA sequencing technologies (1) has the potential to identify the causative mutation by resequencing the whole genome (or exome) of affected individuals....

    [...]

Journal Article•DOI•
Accelerating the design of biomimetic materials by integrating RNA-seq with proteomics and materials science

[...]

Paul A. Guerette1, Shawn Hoon2, Shawn Hoon1, Yiqi Seow2, Manfred Raida2, Admir Masic3, Fong Tian Wong2, Vincent H.B. Ho2, Kiat Whye Kong2, Melik C. Demirel4, Abdon Pena-Francesch4, Shahrouz Amini1, Gavin Z. Tay1, Dawei Ding1, Ali Miserez1 •
Nanyang Technological University1, Agency for Science, Technology and Research2, Max Planck Society3, Pennsylvania State University4
01 Oct 2013-Nature Biotechnology
TL;DR: It is shown that biomimetic engineering can be accelerated by integrating high-throughput RNA-seq with proteomics and advanced materials characterization, and can be applied to a broad range of systems, as illustrated by investigating diverse high-performance biological materials involved in embryo protection, adhesion and predation.
Abstract: Efforts to engineer new materials inspired by biological structures are hampered by the lack of genomic data from many model organisms studied in biomimetic research. Here we show that biomimetic engineering can be accelerated by integrating high-throughput RNA-seq with proteomics and advanced materials characterization. This approach can be applied to a broad range of systems, as we illustrate by investigating diverse high-performance biological materials involved in embryo protection, adhesion and predation. In one example, we rapidly engineer recombinant squid sucker ring teeth proteins into a range of structural and functional materials, including nanopatterned surfaces and photo-cross-linked films that exceed the mechanical properties of most natural and synthetic polymers. Integrating RNA-seq with proteomics and materials science facilitates the molecular characterization of natural materials and the effective translation of their molecular designs into a wide range of bio-inspired materials.

168 citations

Journal Article•DOI•
The Past, Present, and Future of Immune Repertoire Biology – The Rise of Next-Generation Repertoire Analysis

[...]

Adrien Six1, Maria Encarnita Mariotti-Ferrandiz1, Wahiba Chaara1, Susana Magadán2, Hang-Phuong Pham3, Marie-Paule Lefranc4, Thierry Mora5, Véronique Thomas-Vaslin1, Aleksandra M. Walczak5, Pierre Boudinot2 •
French Institute of Health and Medical Research1, Institut national de la recherche agronomique2, Centre national de la recherche scientifique3, University of Montpellier4, École Normale Supérieure5
27 Nov 2013-Frontiers in Immunology
TL;DR: A broad range of available methodological tools developed in the past decades, each of which answering different questions and showing complementarity for progressive identification of the level of repertoire alterations, can be found in this article.
Abstract: T and B cell repertoires are collections of lymphocytes, each characterised by its antigen-specific receptor. We review here classical technologies and analysis strategies developed to assess Immunoglobulin (IG) and T cell receptor (TR) repertoire diversity, and describe recent advances in the field. First, we describe the broad range of available methodological tools developed in the past decades, each of which answering different questions and showing complementarity for progressive identification of the level of repertoire alterations: global overview of the diversity by flow cytometry, IG repertoire descriptions at the protein level for the identification of IG reactivities, IG/TR CDR3 spectratyping strategies, and related molecular quantification or dynamics of T/B cell differentiation. Additionally, we introduce the recent technological advances in molecular biology tools allowing deeper analysis of IG/TR diversity by next-generation sequencing (NGS), offering systematic and comprehensive sequencing of IG/TR transcripts in a short amount of time. NGS provides several angles of analysis such as clonotype frequency, CDR3 diversity, CDR3 sequence analysis, V allele identification with a quantitative dimension, therefore requiring high-throughput analysis tools development. In this line, we discuss the recent efforts made for nomenclature standardisation and ontology development. We then present the variety of available statistical analysis and modelling approaches developed with regards to the various levels of diversity analysis, and reveal the increasing sophistication of modelling approaches. To conclude, we provide some examples of recent mathematical modelling strategies and perspectives that illustrate the active rise of a “next-generation” of repertoire analysis.

168 citations

Cites background from "Sequencing technologies-the next ge..."

  • ...Metzker thoroughly reviewed their principles and properties (134)....

    [...]

  • ...Pyrosequencing currently has a 500 bp (GS Junior) to 700 bp (GS FLX) sequencing capacity with a respective deepness of 150,000–3,000,000 reads per run (134)....

    [...]

  • ...Protected fluorescent nucleotides are added, imaged, delabeled, and deprotected cyclically (134), providing a deeper sequencing (from 15 to 6 billion reads per run for the MiSeq to the HiSeq2500/2000) of shorter reads (100–250 bp for the very recent MiSeq) with the possibility to perform pairend sequencing (two-side sequencing) to increase the read length after aligning the generated complementary sequences....

    [...]

References
More filters
Journal Article•DOI•
RNA-Seq: a revolutionary tool for transcriptomics

[...]

Zhong Wang1, Mark Gerstein1, Michael Snyder1•
Yale University1
01 Jan 2009-Nature Reviews Genetics
TL;DR: The RNA-Seq approach to transcriptome profiling that uses deep-sequencing technologies provides a far more precise measurement of levels of transcripts and their isoforms than other methods.
Abstract: RNA-Seq is a recently developed approach to transcriptome profiling that uses deep-sequencing technologies. Studies using this method have already altered our view of the extent and complexity of eukaryotic transcriptomes. RNA-Seq also provides a far more precise measurement of levels of transcripts and their isoforms than other methods. This article describes the RNA-Seq approach, the challenges associated with its application, and the advances made so far in characterizing several eukaryote transcriptomes.

11,528 citations

"Sequencing technologies-the next ge..." refers background in this paper

  • ...For example, in gene-expression studies microarrays are now being replaced by seq-based methods , which can identify and quantify rare transcripts without prior knowledge of a particular gene and can provide information regarding alternative splicing and sequence variation in identified gene...

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Journal Article•DOI•
Velvet: Algorithms for de novo short read assembly using de Bruijn graphs

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Daniel R. Zerbino1, Ewan Birney1•
European Bioinformatics Institute1
01 May 2008-Genome Research
TL;DR: Velvet represents a new approach to assembly that can leverage very short reads in combination with read pairs to produce useful assemblies and is in close agreement with simulated results without read-pair information.
Abstract: We have developed a new set of algorithms, collectively called "Velvet," to manipulate de Bruijn graphs for genomic sequence assembly. A de Bruijn graph is a compact representation based on short words (k-mers) that is ideal for high coverage, very short read (25-50 bp) data sets. Applying Velvet to very short reads and paired-ends information only, one can produce contigs of significant length, up to 50-kb N50 length in simulations of prokaryotic data and 3-kb N50 on simulated mammalian BACs. When applied to real Solexa data sets without read pairs, Velvet generated contigs of approximately 8 kb in a prokaryote and 2 kb in a mammalian BAC, in close agreement with our simulated results without read-pair information. Velvet represents a new approach to assembly that can leverage very short reads in combination with read pairs to produce useful assemblies.

9,389 citations

Journal Article•DOI•
Genome sequencing in microfabricated high-density picolitre reactors

[...]

Marcel Margulies, Michael Egholm, William E. Altman, Said Attiya, Joel S. Bader, Lisa A. Bemben, Jan Berka, Michael S. Braverman, Yi-Ju Chen, Zhoutao Chen, Scott Dewell, Lei Du, J. M. Fierro, Xavier V. Gomes, Brian C. Godwin, Wen He, Scott Edward Helgesen, Chun Heen Ho, Gerard P. Irzyk, Szilveszter C. Jando, Maria L. I. Alenquer, Thomas P. Jarvie, Kshama B. Jirage, Jong-Bum Kim, James R. Knight, Janna R. Lanza, John H. Leamon, Steven Lefkowitz, Ming Lei, Jing Li, Kenton Lohman, Hong Lu, Vinod Makhijani, Keith Mcdade, Michael P. McKenna, Eugene W. Myers1, Elizabeth Nickerson, John Nobile, Ramona Plant, Bernard P. Puc, Michael T. Ronan, George T. Roth, Gary J. Sarkis, Jan Fredrik Simons, John Simpson, Maithreyan Srinivasan, Karrie R. Tartaro, Alexander Tomasz2, Kari A. Vogt, Greg A. Volkmer, Shally H. Wang, Yong Wang, Michael P. Weiner3, Pengguang Yu, Richard F. Begley, Jonathan M. Rothberg •
University of California, Berkeley1, Rockefeller University2, Rothberg Institute For Childhood Diseases3
15 Sep 2005-Nature
TL;DR: A scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments with 96% coverage at 99.96% accuracy in one run of the machine is described.
Abstract: 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.

8,434 citations

Journal Article•DOI•
Origins and Mechanisms of miRNAs and siRNAs

[...]

Richard W. Carthew1, Erik J. Sontheimer1•
Northwestern University1
20 Feb 2009-Cell
TL;DR: This work has revealed unexpected diversity in their biogenesis pathways and the regulatory mechanisms that they access, which has direct implications for fundamental biology as well as disease etiology and treatment.

4,490 citations

"Sequencing technologies-the next ge..." refers background in this paper

  • ...and to elucidate the role of non-coding RNAs in health and diseas...

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Journal Article•DOI•
Evolution and functions of long noncoding RNAs

[...]

Chris P. Ponting1, Peter L. Oliver1, Wolf Reik2•
University of Oxford1, University of Cambridge2
20 Feb 2009-Cell
TL;DR: The evolution of long noncoding RNAs and their roles in transcriptional regulation, epigenetic gene regulation, and disease are reviewed.

4,277 citations

"Sequencing technologies-the next ge..." refers background in this paper

  • ...and to elucidate the role of non-coding RNAs in health and diseas...

    [...]

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