scispace - formally typeset
Search or ask a question
Journal ArticleDOI

Sequencing technologies-the next generation

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)

Jump to:  and [Summary]

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.

Did you find this useful? Give us your feedback

Content maybe subject to copyright    Report

TEMPLATE DESIGN © 2008
www.PosterPresentations.com
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 mRNAs, 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 Hitchhikers 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
More filters
Journal ArticleDOI
Xin Qi1, Shaojun Xie1, Yuwei Liu1, Fei Yi1, Jingjuan Yu1 
TL;DR: A deep sequencing approach was used to generate a genome-wide transcriptome of foxtail millet after exposure to simulated drought stress, and it was found that the reduced levels of 24-nt siRNA flanking genes were associated, for the most part, with proximal up-regulated genes, indicating a potential effect of 24.nt siRNAs on drought-regulated gene expression.
Abstract: Drought is a major abiotic stress that affects plant growth, production, and survival. Plants have evolved sophisticated and highly complex reactions to drought stress, including large-scale transcriptome reconfiguration. Foxtail millet (Setaria italica) is a member of the Poaceae family. Because of its outstanding tolerance to drought stress foxtail millet has the potential to become a new model organism. To enrich our knowledge of the processes that contribute to drought resistance, we have used a deep sequencing approach to generate a genome-wide transcriptome of foxtail millet after exposure to simulated drought stress. A large number of differentially expressed genes were characterized; in particular, we examined the roles of small interfering RNAs (siRNAs) and long noncoding RNAs (lncRNAs) in response to a water-deficit condition. These RNAs have remained largely unexplored in previous studies of stress-induced transcriptomes. We found that the reduced levels of 24-nt siRNA flanking genes were associated, for the most part, with proximal up-regulated genes, indicating a potential effect of 24-nt siRNAs on drought-regulated gene expression. Several lncRNAs that responded to the simulated drought stress were also identified, and we found that one of them shared sequence conservation and colinearity with its counterpart in sorghum (Sorghum bicolor). Our findings provide new insights into drought-induced changes in the foxtail millet transcriptome.

157 citations


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

  • ...Here, we used deep sequencing technology (Nobuta et al. 2007; Wang et al. 2009; Li et al. 2010; Lu et al. 2010; Metzker 2010; Trapnell et al. 2010; Wang et al. 2010a; Kakumanu et al. 2012) to investigate the genomewide transcriptome reconfiguration of foxtail millet challenged by polyethylene glycol-simulated drought stress in a high-throughput manner....

    [...]

  • ...Here, we used deep sequencing technology (Nobuta et al. 2007; Wang et al. 2009; Li et al. 2010; Lu et al. 2010; Metzker 2010; Trapnell et al. 2010; Wang et al. 2010a; Kakumanu et al. 2012) to investigate the genomewide transcriptome reconfiguration of foxtail millet challenged by polyethylene…...

    [...]

Journal ArticleDOI
TL;DR: The signatures that demographic history imparts on patterns of DNA sequence variation, statistical methods that have been developed to leverage information contained in genome-scale data sets and insights gleaned from these studies are reviewed.
Abstract: The rapid accumulation and increasing quality of human DNA sequence-variation data brought about by advances in genome-scale sequencing present opportunities to investigate human evolution. The authors discuss the statistical methods and models that can be used to gain insight into the evolution of human populations from analyses of large-scale genomic data sets, as well as the challenges associated with these approaches.

157 citations

Journal ArticleDOI
TL;DR: NGS is an extremely powerful tool with which to investigate previously inaccessible aspects of viral dynamics, such as the contribution of different viral reservoirs to replicating virus in the course of the natural history of the infection, co-receptor usage in minority viral populations harboured by different cell lineages, the dynamics of development of drug resistance, and the re-emergence of hidden genomes after treatment interruptions.

157 citations


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

  • ...Current NGS methods use a three-step sequencing process: library preparation, DNA capture and enrichment, and sequencing/detection [1]....

    [...]

Journal ArticleDOI
19 Oct 2011-PLOS ONE
TL;DR: A low-cost and robust protocol to produce Illumina-compatible (GAIIx and HiSeq2000 platforms) RNA-seq libraries by combining several recent improvements is developed and significance tests for determining differential gene expression and intron retention events are applied.
Abstract: The emergence of NextGen sequencing technology has generated much interest in the exploration of transcriptomes. Currently, Illumina Inc. (San Diego, CA) provides one of the most widely utilized sequencing platforms for gene expression analysis. While Illumina reagents and protocols perform adequately in RNA-sequencing (RNA-seq), alternative reagents and protocols promise a higher throughput at a much lower cost. We have developed a low-cost and robust protocol to produce Illumina-compatible (GAIIx and HiSeq2000 platforms) RNA-seq libraries by combining several recent improvements. First, we designed balanced adapter sequences for multiplexing of samples; second, dUTP incorporation in 2(nd) strand synthesis was used to enforce strand-specificity; third, we simplified RNA purification, fragmentation and library size-selection steps thus drastically reducing the time and increasing throughput of library construction; fourth, we included an RNA spike-in control for validation and normalization purposes. To streamline informatics analysis for the community, we established a pipeline within the iPlant Collaborative. These scripts are easily customized to meet specific research needs and improve on existing informatics and statistical treatments of RNA-seq data. In particular, we apply significance tests for determining differential gene expression and intron retention events. To demonstrate the potential of both the library-construction protocol and data-analysis pipeline, we characterized the transcriptome of the rice leaf. Our data supports novel gene models and can be used to improve current rice genome annotation. Additionally, using the rice transcriptome data, we compared different methods of calculating gene expression and discuss the advantages of a strand-specific approach to detect bona-fide anti-sense transcripts and to detect intron retention events. Our results demonstrate the potential of this low cost and robust method for RNA-seq library construction and data analysis.

156 citations


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

  • ...com) [1] and its latest sequencing machine Hiseq2000 (first commercialized during mid-2010) is capable of producing approximately 200 million clusters per lane that typically yield 180–190 million sequencing reads post filtering (TruSeq Cluster Kit v3)....

    [...]

  • ...The advent of ultra-high-throughput sequencing (UHTS) technology has invoked a paradigm shift in the field of genomics and transcriptomics [1,2]....

    [...]

Journal ArticleDOI
TL;DR: A review of the technologies used to detect SARS-CoV-2 in clinical laboratories as well as advances made for molecular, antigen-based, and immunological point-of-care testing, including recent developments in sensor and biosensor devices is presented in this paper.
Abstract: The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory disease coronavirus 2 (SARS-CoV-2), has led to millions of confirmed cases and deaths worldwide. Efficient diagnostic tools are in high demand, as rapid and large-scale testing plays a pivotal role in patient management and decelerating disease spread. This paper reviews current technologies used to detect SARS-CoV-2 in clinical laboratories as well as advances made for molecular, antigen-based, and immunological point-of-care testing, including recent developments in sensor and biosensor devices. The importance of the timing and type of specimen collection is discussed, along with factors such as disease prevalence, setting, and methods. Details of the mechanisms of action of the various methodologies are presented, along with their application span and known performance characteristics. Diagnostic imaging techniques and biomarkers are also covered, with an emphasis on their use for assessing COVID-19 or monitoring disease severity or complications. While the SARS-CoV-2 literature is rapidly evolving, this review highlights topics of interest that have occurred during the pandemic and the lessons learned throughout. Exploring a broad armamentarium of techniques for detecting SARS-CoV-2 will ensure continued diagnostic support for clinicians, public health, and infection prevention and control for this pandemic and provide advice for future pandemic preparedness.

156 citations

References
More filters
Journal ArticleDOI
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...

    [...]

Journal ArticleDOI
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 ArticleDOI
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 ArticleDOI
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...

    [...]

Journal ArticleDOI
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...

    [...]