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Current Protocols in Molecular Biology Apr 2018High throughput DNA sequencing methodology (next generation sequencing; NGS) has rapidly evolved over the past 15 years and new methods are continually being... (Review)
Review
High throughput DNA sequencing methodology (next generation sequencing; NGS) has rapidly evolved over the past 15 years and new methods are continually being commercialized. As the technology develops, so do increases in the number of corresponding applications for basic and applied science. The purpose of this review is to provide a compendium of NGS methodologies and associated applications. Each brief discussion is followed by web links to the manufacturer and/or web-based visualizations. Keyword searches, such as with Google, may also provide helpful internet links and information. © 2018 by John Wiley & Sons, Inc.
Topics: Genome; High-Throughput Nucleotide Sequencing; Humans
PubMed: 29851291
DOI: 10.1002/cpmb.59 -
Journal of Applied Genetics Nov 2011The high-throughput - next generation sequencing (HT-NGS) technologies are currently the hottest topic in the field of human and animals genomics researches, which can... (Review)
Review
The high-throughput - next generation sequencing (HT-NGS) technologies are currently the hottest topic in the field of human and animals genomics researches, which can produce over 100 times more data compared to the most sophisticated capillary sequencers based on the Sanger method. With the ongoing developments of high throughput sequencing machines and advancement of modern bioinformatics tools at unprecedented pace, the target goal of sequencing individual genomes of living organism at a cost of $1,000 each is seemed to be realistically feasible in the near future. In the relatively short time frame since 2005, the HT-NGS technologies are revolutionizing the human and animal genome researches by analysis of chromatin immunoprecipitation coupled to DNA microarray (ChIP-chip) or sequencing (ChIP-seq), RNA sequencing (RNA-seq), whole genome genotyping, genome wide structural variation, de novo assembling and re-assembling of genome, mutation detection and carrier screening, detection of inherited disorders and complex human diseases, DNA library preparation, paired ends and genomic captures, sequencing of mitochondrial genome and personal genomics. In this review, we addressed the important features of HT-NGS like, first generation DNA sequencers, birth of HT-NGS, second generation HT-NGS platforms, third generation HT-NGS platforms: including single molecule Heliscope™, SMRT™ and RNAP sequencers, Nanopore, Archon Genomics X PRIZE foundation, comparison of second and third HT-NGS platforms, applications, advances and future perspectives of sequencing technologies on human and animal genome research.
Topics: Animals; Biomarkers, Tumor; Epigenomics; Genetic Variation; Genome; High-Throughput Nucleotide Sequencing; Humans; Mutation; Neoplasms; Sequence Analysis, DNA; Sequence Analysis, RNA
PubMed: 21698376
DOI: 10.1007/s13353-011-0057-x -
Cold Spring Harbor Perspectives in... Jul 2019More than a decade ago, the term "next-generation" sequencing was coined to describe what was, at the time, revolutionary new methods to sequence RNA and DNA at a faster... (Review)
Review
More than a decade ago, the term "next-generation" sequencing was coined to describe what was, at the time, revolutionary new methods to sequence RNA and DNA at a faster pace and cheaper cost than could be performed by standard bench-top protocols. Since then, the field of DNA sequencing has evolved at a rapid pace, with new breakthroughs allowing capacity to exponentially increase and cost to dramatically decrease. As genome-scale sequencing has become routine, a paradigm shift is occurring in genomics, which uses the power of high-throughput, rapid sequencing power with large-scale studies. These new approaches to genetic discovery will provide direct impact to fields such as personalized medicine, evolution, and biodiversity. This work reviews recent technology advances and methods in next-generation sequencing and highlights current large-scale sequencing efforts driving the evolution of the genomics space.
Topics: Genome, Human; Genomics; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA
PubMed: 30323017
DOI: 10.1101/cshperspect.a025791 -
Archives of Pathology & Laboratory... Sep 2013DNA sequencing is critical to identifying many human genetic disorders caused by DNA mutations, including cancer. Pyrosequencing is less complex, involves fewer steps,... (Review)
Review
CONTEXT
DNA sequencing is critical to identifying many human genetic disorders caused by DNA mutations, including cancer. Pyrosequencing is less complex, involves fewer steps, and has a superior limit of detection compared with Sanger sequencing. The fundamental basis of pyrosequencing is that pyrophosphate is released when a deoxyribonucleotide triphosphate is added to the end of a nascent strand of DNA. Because deoxyribonucleotide triphosphates are sequentially added to the reaction and because the pyrophosphate concentration is continuously monitored, the DNA sequence can be determined.
OBJECTIVE
To demonstrate the fundamental principles of pyrosequencing.
DATA SOURCES
Salient features of pyrosequencing are demonstrated using the free software program Pyromaker ( http://pyromaker.pathology.jhmi.edu ), through which users can input DNA sequences and other pyrosequencing parameters to generate the expected pyrosequencing results.
CONCLUSIONS
We demonstrate how mutant and wild-type DNA sequences result in different pyrograms. Using pyrograms of established mutations in tumors, we explain how to analyze the pyrogram peaks generated by different dispensation sequences. Further, we demonstrate some limitations of pyrosequencing, including how some complex mutations can be indistinguishable from single base mutations. Pyrosequencing is the basis of the Roche 454 next-generation sequencer and many of the same principles also apply to the Ion Torrent hydrogen ion-based next-generation sequencers.
Topics: Base Sequence; DNA Mutational Analysis; DNA, Neoplasm; Diphosphates; Genotype; High-Throughput Nucleotide Sequencing; Humans; Mutation; Neoplasms; Polymerase Chain Reaction; Sequence Analysis, DNA; Software
PubMed: 23991743
DOI: 10.5858/arpa.2012-0463-RA -
Genomics Jan 2016Determining the order of nucleic acid residues in biological samples is an integral component of a wide variety of research applications. Over the last fifty years large... (Review)
Review
Determining the order of nucleic acid residues in biological samples is an integral component of a wide variety of research applications. Over the last fifty years large numbers of researchers have applied themselves to the production of techniques and technologies to facilitate this feat, sequencing DNA and RNA molecules. This time-scale has witnessed tremendous changes, moving from sequencing short oligonucleotides to millions of bases, from struggling towards the deduction of the coding sequence of a single gene to rapid and widely available whole genome sequencing. This article traverses those years, iterating through the different generations of sequencing technology, highlighting some of the key discoveries, researchers, and sequences along the way.
Topics: High-Throughput Nucleotide Sequencing; History, 20th Century; History, 21st Century; Nanotechnology; Sequence Analysis, DNA
PubMed: 26554401
DOI: 10.1016/j.ygeno.2015.11.003 -
Nature Reviews. Genetics May 2011Comparisons of human genomes show that more base pairs are altered as a result of structural variation - including copy number variation - than as a result of point... (Review)
Review
Comparisons of human genomes show that more base pairs are altered as a result of structural variation - including copy number variation - than as a result of point mutations. Here we review advances and challenges in the discovery and genotyping of structural variation. The recent application of massively parallel sequencing methods has complemented microarray-based methods and has led to an exponential increase in the discovery of smaller structural-variation events. Some global discovery biases remain, but the integration of experimental and computational approaches is proving fruitful for accurate characterization of the copy, content and structure of variable regions. We argue that the long-term goal should be routine, cost-effective and high quality de novo assembly of human genomes to comprehensively assess all classes of structural variation.
Topics: DNA Copy Number Variations; Genetic Variation; Genome, Human; Genotype; High-Throughput Nucleotide Sequencing; Humans; Oligonucleotide Array Sequence Analysis; Polymorphism, Single Nucleotide; Sequence Analysis, DNA
PubMed: 21358748
DOI: 10.1038/nrg2958 -
Archives of Pathology & Laboratory... Nov 2017- Next-generation sequencing (NGS) is a technology being used by many laboratories to test for inherited disorders and tumor mutations. This technology is new for many... (Review)
Review
CONTEXT
- Next-generation sequencing (NGS) is a technology being used by many laboratories to test for inherited disorders and tumor mutations. This technology is new for many practicing pathologists, who may not be familiar with the uses, methodology, and limitations of NGS.
OBJECTIVE
- To familiarize pathologists with several aspects of NGS, including current and expanding uses; methodology including wet bench aspects, bioinformatics, and interpretation; validation and proficiency; limitations; and issues related to the integration of NGS data into patient care.
DATA SOURCES
- The review is based on peer-reviewed literature and personal experience using NGS in a clinical setting at a major academic center.
CONCLUSIONS
- The clinical applications of NGS will increase as the technology, bioinformatics, and resources evolve to address the limitations and improve quality of results. The challenge for clinical laboratories is to ensure testing is clinically relevant, cost-effective, and can be integrated into clinical care.
Topics: Computational Biology; DNA Mutational Analysis; Databases, Genetic; Genetic Diseases, Inborn; Genetic Testing; Health Care Costs; High-Throughput Nucleotide Sequencing; Humans; Laboratory Proficiency Testing; Mutation; Neoplasms; Systems Integration
PubMed: 28782984
DOI: 10.5858/arpa.2016-0501-RA -
Journal of Veterinary Diagnostic... Nov 2020Genetic sequencing, or DNA sequencing, using the Sanger technique has become widely used in the veterinary diagnostic community. This technology plays a role in...
Genetic sequencing, or DNA sequencing, using the Sanger technique has become widely used in the veterinary diagnostic community. This technology plays a role in verification of PCR results and is used to provide the genetic sequence data needed for phylogenetic analysis, epidemiologic studies, and forensic investigations. The Laboratory Technology Committee of the American Association of Veterinary Laboratory Diagnosticians has prepared guidelines for sample preparation, submission to sequencing facilities or instrumentation, quality assessment of nucleic acid sequence data performed, and for generating basic sequencing data and phylogenetic analysis for diagnostic applications. This guidance is aimed at assisting laboratories in providing consistent, high-quality, and reliable sequence data when using Sanger-based genetic sequencing as a component of their laboratory services.
Topics: Animal Diseases; Animals; Base Sequence; High-Throughput Nucleotide Sequencing; Humans; Laboratories; Phylogeny; Polymerase Chain Reaction; Sequence Analysis, DNA
PubMed: 32070230
DOI: 10.1177/1040638720905833 -
European Journal of Human Genetics :... Jan 2016We present, on behalf of EuroGentest and the European Society of Human Genetics, guidelines for the evaluation and validation of next-generation sequencing (NGS)...
We present, on behalf of EuroGentest and the European Society of Human Genetics, guidelines for the evaluation and validation of next-generation sequencing (NGS) applications for the diagnosis of genetic disorders. The work was performed by a group of laboratory geneticists and bioinformaticians, and discussed with clinical geneticists, industry and patients' representatives, and other stakeholders in the field of human genetics. The statements that were written during the elaboration of the guidelines are presented here. The background document and full guidelines are available as supplementary material. They include many examples to assist the laboratories in the implementation of NGS and accreditation of this service. The work and ideas presented by others in guidelines that have emerged elsewhere in the course of the past few years were also considered and are acknowledged in the full text. Interestingly, a few new insights that have not been cited before have emerged during the preparation of the guidelines. The most important new feature is the presentation of a 'rating system' for NGS-based diagnostic tests. The guidelines and statements have been applauded by the genetic diagnostic community, and thus seem to be valuable for the harmonization and quality assurance of NGS diagnostics in Europe.
Topics: Accreditation; Biomarkers; Databases, Genetic; Europe; Gene Expression; Genetic Diseases, Inborn; Genetic Testing; High-Throughput Nucleotide Sequencing; Humans; Incidental Findings; Information Dissemination; Informed Consent; Proteins; Research Design; Sensitivity and Specificity
PubMed: 26508566
DOI: 10.1038/ejhg.2015.226 -
Biomolecules Jul 2021Recent developments have revolutionized the study of biomolecules. Among them are molecular markers, amplification and sequencing of nucleic acids. The latter is... (Review)
Review
Recent developments have revolutionized the study of biomolecules. Among them are molecular markers, amplification and sequencing of nucleic acids. The latter is classified into three generations. The first allows to sequence small DNA fragments. The second one increases throughput, reducing turnaround and pricing, and is therefore more convenient to sequence full genomes and transcriptomes. The third generation is currently pushing technology to its limits, being able to sequence single molecules, without previous amplification, which was previously impossible. Besides, this represents a new revolution, allowing researchers to directly sequence RNA without previous retrotranscription. These technologies are having a significant impact on different areas, such as medicine, agronomy, ecology and biotechnology. Additionally, the study of biomolecules is revealing interesting evolutionary information. That includes deciphering what makes us human, including phenomena like non-coding RNA expansion. All this is redefining the concept of gene and transcript. Basic analyses and applications are now facilitated with new genome editing tools, such as CRISPR. All these developments, in general, and nucleic-acid sequencing, in particular, are opening a new exciting era of biomolecule analyses and applications, including personalized medicine, and diagnosis and prevention of diseases for humans and other animals.
Topics: Animals; Base Sequence; DNA; Genome; Genomics; High-Throughput Nucleotide Sequencing; History, 20th Century; History, 21st Century; Humans; RNA, Messenger; Sequence Analysis, DNA; Sequence Analysis, RNA; Whole Genome Sequencing
PubMed: 34439777
DOI: 10.3390/biom11081111