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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 -
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 -
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 -
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 -
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 -
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 -
Genomics, Proteomics & Bioinformatics Oct 2016The revolution of genome sequencing is continuing after the successful second-generation sequencing (SGS) technology. The third-generation sequencing (TGS) technology,... (Review)
Review
The revolution of genome sequencing is continuing after the successful second-generation sequencing (SGS) technology. The third-generation sequencing (TGS) technology, led by Pacific Biosciences (PacBio), is progressing rapidly, moving from a technology once only capable of providing data for small genome analysis, or for performing targeted screening, to one that promises high quality de novo assembly and structural variation detection for human-sized genomes. In 2014, the MinION, the first commercial sequencer using nanopore technology, was released by Oxford Nanopore Technologies (ONT). MinION identifies DNA bases by measuring the changes in electrical conductivity generated as DNA strands pass through a biological pore. Its portability, affordability, and speed in data production makes it suitable for real-time applications, the release of the long read sequencer MinION has thus generated much excitement and interest in the genomics community. While de novo genome assemblies can be cheaply produced from SGS data, assembly continuity is often relatively poor, due to the limited ability of short reads to handle long repeats. Assembly quality can be greatly improved by using TGS long reads, since repetitive regions can be easily expanded into using longer sequencing lengths, despite having higher error rates at the base level. The potential of nanopore sequencing has been demonstrated by various studies in genome surveillance at locations where rapid and reliable sequencing is needed, but where resources are limited.
Topics: Genome, Human; High-Throughput Nucleotide Sequencing; Humans; Nanopores; Repetitive Sequences, Nucleic Acid; Sequence Analysis, DNA
PubMed: 27646134
DOI: 10.1016/j.gpb.2016.05.004 -
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 -
Journal of Clinical Microbiology Dec 2019Metagenomic sequencing for infectious disease diagnostics is an important tool that holds promise for use in the clinical laboratory. Challenges for implementation so... (Review)
Review
Metagenomic sequencing for infectious disease diagnostics is an important tool that holds promise for use in the clinical laboratory. Challenges for implementation so far include high cost, the length of time to results, and the need for technical and bioinformatics expertise. However, the recent technological innovation of nanopore sequencing from Oxford Nanopore Technologies (ONT) has the potential to address these challenges. ONT sequencing is an attractive platform for clinical laboratories to adopt due to its low cost, rapid turnaround time, and user-friendly bioinformatics pipelines. However, this method still faces the problem of base-calling accuracy compared to other platforms. This review highlights the general challenges of pathogen detection in clinical specimens by metagenomic sequencing, the advantages and disadvantages of the ONT platform, and how research to date supports the potential future use of nanopore sequencing in infectious disease diagnostics.
Topics: Clinical Laboratory Services; Clinical Laboratory Techniques; Communicable Diseases; High-Throughput Nucleotide Sequencing; Humans; Nanopore Sequencing
PubMed: 31619531
DOI: 10.1128/JCM.01315-19 -
Archiwum Medycyny Sadowej I Kryminologii 2015The DNA analysis is a cornerstone in contemporary forensic sciences. DNA sequencing technologies are powerful tools that enrich molecular sciences in the past based on... (Review)
Review
The DNA analysis is a cornerstone in contemporary forensic sciences. DNA sequencing technologies are powerful tools that enrich molecular sciences in the past based on Sanger sequencing and continue to glowing these sciences based on Next generation sequencing (NGS). Next generation sequencing has excellent potential to flourish and increase the molecular applications in forensic sciences by jumping over the pitfalls of the conventional method of sequencing. The main advantages of NGS compared to conventional method that it utilizes simultaneously a large number of genetic markers with high-resolution of genetic data. These advantages will help in solving several challenges such as mixture analysis and dealing with minute degraded samples. Based on these new technologies, many markers could be examined to get important biological data such as age, geographical origins, tissue type determination, external visible traits and monozygotic twins identification. It also could get data related to microbes, insects, plants and soil which are of great medico-legal importance. Despite the dozens of forensic research involving NGS, there are requirements before using this technology routinely in forensic cases. Thus, there is a great need to more studies that address robustness of these techniques. Therefore, this work highlights the applications of forensic sciences in the era of massively parallel sequencing.
Topics: Forensic Genetics; High-Throughput Nucleotide Sequencing; Humans; Microsatellite Repeats; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Sequence Analysis, DNA
PubMed: 27543959
DOI: 10.5114/amsik.2015.61029