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Nature Biotechnology May 2024
Topics: Nanopore Sequencing; Humans; Nanopores; High-Throughput Nucleotide Sequencing; Sequence Analysis, DNA
PubMed: 38760549
DOI: 10.1038/s41587-024-02231-1 -
The Veterinary Clinics of North... Aug 2020The sequencing and assembly of a reference genome for the horse has been revolutionary for investigation of horse health and performance. Next-generation sequencing... (Review)
Review
The sequencing and assembly of a reference genome for the horse has been revolutionary for investigation of horse health and performance. Next-generation sequencing (NGS) methods represent a second revolution in equine genomics. Researchers can align and compare DNA and RNA sequencing data to the reference genome to explore variation that may contribute or be attributed to disease. NGS has also facilitated the translation of research discovery to clinically relevant applications. This article discusses the history and development of NGS, details some of the available sequencing platforms, and describes currently available applications in the context of both discovery and clinical settings.
Topics: Animals; Genomics; High-Throughput Nucleotide Sequencing; Horse Diseases; Horses; Sequence Analysis, DNA
PubMed: 32654781
DOI: 10.1016/j.cveq.2020.03.002 -
Advances in Food and Nutrition Research 2020Massive parallel sequencing (High-Throughput Sequencing, HTS) permits reading of sequenced millions to billions short DNAs in parallel (reads) and is revolutionizing... (Review)
Review
Massive parallel sequencing (High-Throughput Sequencing, HTS) permits reading of sequenced millions to billions short DNAs in parallel (reads) and is revolutionizing microbiology and food safety research from the laboratory methods to computational analysis, with the inevitable use of Bioinformatics. The time and cost reduction of microbiota, microbiome and metagenome studies allows the rapid progress in diagnosis, taxonomy, epidemiology, comparative genomics, virulence, discovery of genes or variants of interest and the association of microorganisms with food spoilage and foodborne infections.
Topics: Bacteria; Food Microbiology; Genomics; High-Throughput Nucleotide Sequencing
PubMed: 32035598
DOI: 10.1016/bs.afnr.2019.10.003 -
Journal of Chemical Neuroanatomy Oct 2019Mapping brain connectivity at single cell resolution is critical for understanding brain structure. For decades, such mapping has been principally approached with... (Review)
Review
Mapping brain connectivity at single cell resolution is critical for understanding brain structure. For decades, such mapping has been principally approached with microscopy techniques, aiming to visualize neurons and their connections. However, these techniques are often very labor intensive and do not scale well to the complexity of mammalian brains. We recently leveraged the speed and parallelization of DNA sequencing to map the projections of thousands of single neurons in single experiments, and to map cortical mesoscale connectivity in single mice. Here, I review the state of sequencing-based neuroanatomy, and discuss future directions in synaptic connectivity mapping and comparative connectomics.
Topics: Animals; Connectome; High-Throughput Nucleotide Sequencing; Humans; Neuroanatomy
PubMed: 31173871
DOI: 10.1016/j.jchemneu.2019.101653 -
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 -
Experimental & Molecular Medicine Oct 2022To date, more than 170 chemical modifications have been characterized in RNA, providing a new layer of gene expression regulation termed the 'epitranscriptome'. RNA... (Review)
Review
To date, more than 170 chemical modifications have been characterized in RNA, providing a new layer of gene expression regulation termed the 'epitranscriptome'. RNA modification detection methods and tools advance the functional studies of the epitranscriptome. According to the detection throughput and principles, existing RNA modification detection technologies can be categorized into four classes, including quantification methods, locus-specific detection methods, next-generation sequencing-based detection technologies and nanopore direct RNA sequencing-based technologies. In this review, we summarize the current knowledge about these RNA modification detection technologies and discuss the challenges for the existing detection tools, providing information for a comprehensive understanding of the epitranscriptome.
Topics: RNA; High-Throughput Nucleotide Sequencing; Sequence Analysis, RNA; Gene Expression Regulation
PubMed: 36266445
DOI: 10.1038/s12276-022-00821-0 -
Nature Genetics Nov 2022
Topics: RNA-Seq; Sequence Analysis, RNA; High-Throughput Nucleotide Sequencing; Gene Expression Profiling
PubMed: 36333507
DOI: 10.1038/s41588-022-01217-6 -
Methods in Molecular Biology (Clifton,... 2023Pyrosequencing is a DNA sequencing-by-synthesis technique that can quantitatively detect single-nucleotide polymorphisms (SNPs). With pyrosequencing, the level of DNA...
Pyrosequencing is a DNA sequencing-by-synthesis technique that can quantitatively detect single-nucleotide polymorphisms (SNPs). With pyrosequencing, the level of DNA methylation can be calculated according to the ratio of artificial cytosine/thymine SNPs produced by bisulfite conversion at each CpG site. This analysis method provides a reproducible and accurate measurement of methylation levels at CpG sites near sequencing primers with high quantitative resolution. DNA methylation plays an important role in mammalian development and cellular physiology; alterations in DNA methylation patterns have been implicated in several common diseases as well as cancers and imprinting disorders. Evaluating DNA methylation levels via pyrosequencing is useful for identifying biomarkers that could help with the diagnosis, prognosis, treatment selection, and onset risk assessment for several diseases. We describe the principles of pyrosequencing and detail a bisulfite pyrosequencing protocol based on our experience and the PyroMark Q24 User Manual.
Topics: Animals; CpG Islands; Cytosine; DNA Methylation; DNA Primers; High-Throughput Nucleotide Sequencing; Mammals; Polymorphism, Single Nucleotide; Sequence Analysis, DNA; Sulfites; Thymine
PubMed: 36173562
DOI: 10.1007/978-1-0716-2724-2_1 -
Trends in Biotechnology Sep 2019A substantial amount of structural variation in the human genome remains uninvestigated due to the limitations of existing technologies, the presence of repetitive... (Review)
Review
A substantial amount of structural variation in the human genome remains uninvestigated due to the limitations of existing technologies, the presence of repetitive sequences, and the complexity of a diploid genome. New technologies have been developed, increasing resolution and appreciation of structural variation and how it affects human diversity and disease. The genetic etiology of most patients with complex disorders such as neurodegenerative brain diseases is not yet elucidated, complicating disease diagnosis, genetic counseling, and understanding of underlying pathological mechanisms needed to develop therapeutic interventions. Here, we focus on innovative progress and opportunities provided by the newest methods such as linked read sequencing, strand-specific sequencing, and long-read sequencing. Finally, we describe a strategy for generating a comprehensive catalog of structural variations across populations.
Topics: Base Sequence; Genetic Variation; Genome, Human; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA
PubMed: 30902345
DOI: 10.1016/j.tibtech.2019.02.003 -
Clinical Chemistry Jan 2020The newest advances in DNA sequencing are based on technologies that perform massively parallel sequencing (MPS). Since 2006, the output from MPS platforms has increased... (Review)
Review
BACKGROUND
The newest advances in DNA sequencing are based on technologies that perform massively parallel sequencing (MPS). Since 2006, the output from MPS platforms has increased from 20 Mb to >7 Tb. First-generation MPS platforms amplify individual DNA molecules to multiple copies and then interrogate the sequence of those molecules. Second-generation MPS analyzes single unamplified molecules to generate much longer sequence reads but with less output than first-generation MPS and lower first-pass accuracy. With MPS technologies, it is now possible to analyze genomes, exomes, a defined subset of genes, transcriptomes, and even methylation across the genome. These technologies have and will continue to completely transform the clinical practice.
CONTENT
The major first- and second-generation MPS platforms and how they are used in clinical practice are discussed.
SUMMARY
The ability to sequence terabases of DNA per run on an MPS platform will dramatically change how DNA sequencing is used in clinical practice. Currently, MPS of targeted gene panels is the most common use of this technology clinically, but as the cost for genome sequencing inches downward to $100, this may soon become the method of choice (with the caveat that, at least in the near term, clinical-grade genome sequencing with interpretation may cost much more than $100). Other uses of this technology include sequencing of a mixture of bacterial and viral species (metagenomics), as well as the characterization of methylation across the genome.
Topics: DNA; DNA Methylation; Genome, Human; High-Throughput Nucleotide Sequencing; Humans; Metagenomics; Nanostructures; Polymerase Chain Reaction; Sequence Analysis, DNA
PubMed: 31811004
DOI: 10.1373/clinchem.2019.303305