-
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 -
Electrophoresis Jan 2023The recent development of small, single-amplicon-based benchtop systems for pyrosequencing has opened up a host of novel procedures for applications in forensic science.... (Review)
Review
The recent development of small, single-amplicon-based benchtop systems for pyrosequencing has opened up a host of novel procedures for applications in forensic science. Pyrosequencing is a sequencing by synthesis technique, based on chemiluminescent inorganic pyrophosphate detection. This review explains the pyrosequencing workflow and illustrates the step-by-step chemistry, followed by a description of the assay design and factors to keep in mind for an exemplary assay. Existing and potential forensic applications are highlighted using this technology. Current applications include identifying species, identifying bodily fluids, and determining smoking status. We also review progress in potential applications for the future, including research on distinguishing monozygotic twins, detecting alcohol and drug abuse, and other phenotypic characteristics such as diet and body mass index. Overall, the versatility of the pyrosequencing technologies renders it a useful tool in forensic genomics.
Topics: Forensic Medicine; Genomics; Forensic Sciences; High-Throughput Nucleotide Sequencing; Forensic Genetics
PubMed: 36168852
DOI: 10.1002/elps.202200177 -
Human Immunology Nov 2021Since the days of Sanger sequencing, next-generation sequencing technologies have significantly evolved to provide increased data output, efficiencies, and applications.... (Review)
Review
Since the days of Sanger sequencing, next-generation sequencing technologies have significantly evolved to provide increased data output, efficiencies, and applications. These next generations of technologies can be categorized based on read length. This review provides an overview of these technologies as two paradigms: short-read, or "second-generation," technologies, and long-read, or "third-generation," technologies. Herein, short-read sequencing approaches are represented by the most prevalent technologies, Illumina and Ion Torrent, and long-read sequencing approaches are represented by Pacific Biosciences and Oxford Nanopore technologies. All technologies are reviewed along with reported advantages and disadvantages. Until recently, short-read sequencing was thought to provide high accuracy limited by read-length, while long-read technologies afforded much longer read-lengths at the expense of accuracy. Emerging developments for third-generation technologies hold promise for the next wave of sequencing evolution, with the co-existence of longer read lengths and high accuracy.
Topics: DNA Probes, HLA; Genotyping Techniques; HLA Antigens; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA
PubMed: 33745759
DOI: 10.1016/j.humimm.2021.02.012 -
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 -
Trends in Genetics : TIG Sep 2018Forty years ago the advent of Sanger sequencing was revolutionary as it allowed complete genome sequences to be deciphered for the first time. A second revolution came... (Review)
Review
Forty years ago the advent of Sanger sequencing was revolutionary as it allowed complete genome sequences to be deciphered for the first time. A second revolution came when next-generation sequencing (NGS) technologies appeared, which made genome sequencing much cheaper and faster. However, NGS methods have several drawbacks and pitfalls, most notably their short reads. Recently, third-generation/long-read methods appeared, which can produce genome assemblies of unprecedented quality. Moreover, these technologies can directly detect epigenetic modifications on native DNA and allow whole-transcript sequencing without the need for assembly. This marks the third revolution in sequencing technology. Here we review and compare the various long-read methods. We discuss their applications and their respective strengths and weaknesses and provide future perspectives.
Topics: DNA; Genome; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA; Exome Sequencing
PubMed: 29941292
DOI: 10.1016/j.tig.2018.05.008 -
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 -
Methods in Molecular Biology (Clifton,... 2022With the ability to obtain several millions of reads per sample, high-throughput RNA sequencing (RNA-Seq) enables investigation of any transcriptome at a fine...
With the ability to obtain several millions of reads per sample, high-throughput RNA sequencing (RNA-Seq) enables investigation of any transcriptome at a fine resolution. Not just the messenger RNA (mRNA), but a wide variety of different RNA populations (e.g., total RNA, microRNA, long ncRNA, pre-mRNA) can also be investigated using RNA-Seq. While facilitating accurate quantification of gene expression, RNA-Seq offers the opportunity to estimate abundance of isoforms and find novel transcripts and allele-specific transcripts. In this chapter, we describe a protocol to construct an RNA-Seq library for sequencing on Illumina NGS platforms and a computational pipeline to perform RNA-Seq data analysis. The protocols described in this chapter can be applied to the analysis of differential gene expression in control versus 17β-estradiol treatment of in vivo or in vitro systems.
Topics: Data Analysis; High-Throughput Nucleotide Sequencing; RNA-Seq; Transcriptome
PubMed: 35119677
DOI: 10.1007/978-1-0716-1920-9_22 -
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 -
Methods in Molecular Biology (Clifton,... 2018MiSeq, Illumina's integrated next generation sequencing instrument, uses reversible-terminator sequencing-by-synthesis technology to provide end-to-end sequencing... (Review)
Review
MiSeq, Illumina's integrated next generation sequencing instrument, uses reversible-terminator sequencing-by-synthesis technology to provide end-to-end sequencing solutions. The MiSeq instrument is one of the smallest benchtop sequencers that can perform onboard cluster generation, amplification, genomic DNA sequencing, and data analysis, including base calling, alignment and variant calling, in a single run. It performs both single- and paired-end runs with adjustable read lengths from 1 × 36 base pairs to 2 × 300 base pairs. A single run can produce output data of up to 15 Gb in as little as 4 h of runtime and can output up to 25 M single reads and 50 M paired-end reads. Thus, MiSeq provides an ideal platform for rapid turnaround time. MiSeq is also a cost-effective tool for various analyses focused on targeted gene sequencing (amplicon sequencing and target enrichment), metagenomics, and gene expression studies. For these reasons, MiSeq has become one of the most widely used next generation sequencing platforms. Here, we provide a protocol to prepare libraries for sequencing using the MiSeq instrument and basic guidelines for analysis of output data from the MiSeq sequencing run.
Topics: Animals; Genome, Human; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA
PubMed: 29423801
DOI: 10.1007/978-1-4939-7471-9_12